taxonomic study of the bulinus africanus … · 2 abstract certain species of the bulinus africanus...

TAXONOMIC STUDY OF THE BULINUS AFRICANUS GROUP IN KENYA

BY RICHARD ARCHER

A thesis submitted for the degree of Master of Philosophy from the University of London and the Diploma of Membership of Imperial College

Department of Pure and Applied Biology, Imperial College,London SW7 andExperimental Taxonomy Unit, Department of Zoology,British Museum (Natural History),London SW7 5BD.

2

ABSTRACT

Certain species of the Bulinus africanus group have been implicated in the transmission of Schistosoma haematobium in Kenya. Full taxonomic resolution of the group has previously been hampered by extensive intergradation of form between certain taxa. The major source of difficulty has been the inadequate quantification of traditional characters, particularly those on the shell. With the advent of various numerical and experimental techniques there is a need for re-assessment of shell morphology.

This study involved the morphological characterisation of populations from Western and Central Kenya, using new and traditional shell variables. These were standardised and quantified morphometrically, producing two data sets: PI, consisting of fourmorphologically distinct samples; and P2, consisting of 10 larger samples, less visually distinct. PI and P2 were subjected to multivariate analysis, principally Canonical Variates Analysis and Principal Components Analysis. This study suggests that Principal Components Analysis is of limited use in resolving samples using shell variables. Canonical Variate Analysis is a more useful technique since it minimises the effects of variation within each sample whilst maximising variation between samples. Numerical analysis suggests the presence of three morphotypes among these samples, corresponding to the b.africanus^B.nasutus and B.globosus/ugandae forms. The effects of isometric and allometric growth are discussed. The most useful discriminatory variables appear to be

suet-cthe\angles A, AAA, APA and BWSA. Other variables are discussed. Results from polyacrylamide gel isoelectrophoresis of four enzyme systems are also presented. These generally support the morphometric interpretation, as do the egg protein separations. A detailed description is given of shell microsculpture in the Bulinus africanus group in Western Kenya. Details of the parasite burdens of the wild caught snails are also documented.

pftfrS: CO N T E N T S

H C H A P T E R 1 INTRODUCTION

1! 1.1 G E N E R A L INTRODUCTION

14 1.2 TH E Bulinus africanus G R O U P IN K E N Y A

IS 1.3 HISTORICAL PERSPECTIVE TO THE STUDY O F Bulinus

n M PRESENT R E S E A R C H A N D PROBLEMS

2.1 C H A P T E R 2 HISTORY O F C H A R A C T E R S USED IN TH E T A X O N O M Y O F BULINUS

LI 2.1 SHELL M O R P H O L O G Y

IS 2.2 A N A T O M I C A L C H A R A C T E R S

17 2.3 BIOCHEMICAL C H A R A C T E R S

31 2M PARASITOLOGICAL STUDIES

31 2.5 ECOLOGICAL, BEHAVIOURAL A N D

PHYSIOLOGICAL STUDIES

34 CHAPTER 3 MATERIALS AND METHODS

34 3 .1 SNAIL CAPTURE, MAINTAINANCE AND PRESERVATION34 (a ) DETAILS OF PRESERVED MATERIAL AND FIELD COLLECTION3 C> (b ) FIELD CAPTURE AND PRESERVATION3T ( c) LABORATORY MAINTAINANCE3<1 (d ) SCHISTOSOME EXTRACTION34 ( e ) CLEANING AND PREPARATION OF PRESERVED SHELLS34 ( f ) EXAMINATION OF SHELLS4 0 ' (g ) SHELL ORIENTATION

3 .2 NUMERICAL ANALYSIS4fc (a ) INTRODUCTION4 (o (b ) PRELIMINARY ANALYSIS P i4fe (1 ) CHOICE OF VARIABLES4 6 (2 ) NUMBER OF INDIVIDUALS MEASUREDW (3 ) ASSESSMENT OF ALLOMETRY4 * (4 ) MULTIVARIATE ANALYSIS4? ( i ) ORDINATION/MULTIDIMENSIONAL SCALING

( i i ) HIERARCHICAL CLUSTERINGS3 ( c ) MAIN ANALYSIS P2

4PAGE :

Sty 3.3 BIOCHEMICAL ANALYSIS54- (a) ISOELECTRIC FOCUSING ( I EF)54- (1) INTRODUCTIONSt* (2) GEL PREPARATION AND RUNNING A SYSTEM5 2 (b) EGG PROTEIN ELECTROPHORESIS

s<\ 3.4 SCANNING ELECTRON MICROSCOPY

c>\ C H A P T E R 4 RESULTS

61 4.1 M O R P H O M E T R I C ANALYSIS

61 A. P R O G R A M M E PI

U (a) VISUAL INSPECTION OF SAMPLES

M (b) ASSESSMENT OF A L L O M E T R Y

6>3 (c) UNIVARIATE A N D BIVARIATE ANALYSIS

6>3 (d) PRINCIPAL C O M P O N E N T S ANALYSIS

CA (1) FULL D A T A SET

11 (2) SIZE-EFFECT R E M O V E D

1 ( (3) REDUCTION IN N U M B E R OF VARIABLES

1 5 (e) CA NO NI CA L VARIATES ANALYSIS

1 5 (f) PRINCIPAL CO-ORDINATES ANALYSIS

2 1 (g) HIERARCHICAL CLUSTERING ANALYSIS

B. P R O G R A M M E P2

W (a) VISUAL INSPECTION OF SAMPLES

84- (b) ASSESSMENT OF A L L O M E T R Y

98 (c) UNIVARIATE A N D BIVARIATE ANALYSIS

°1 0 (d) PRINCIPAL C O M P O N E N T S ANALYSIS

(1) FULL D A T A SET

5 3 (2) SIZE-EFFECT R E M O V E D

1 3 (3) REDUCTION IN N U M B E R OF VARIABLES

<?3 (4) INCLUSION OF VARIABLES F R O M 4TH & 5TH W H O R L S55 (e) CA NO NI CA L VARIATES ANALYSIS

5PAO?E :

105 4*. 2. ISOELECTRIC FOCUSING ELECTROPHORESIS105 (CL) ACID PHOSPHATASE109 (b) MALATE DEHYDROGENASE109 (c) GLUCOSE PHOSPHATE ISOMERASE|0<f W) HYDROXYBUTYRATE DEHYDROGENASE

us 4.3 EG G PROTEIN ELECTROPHORESIS

IIS 4.4 PARASITOLOGY

120 4.5 EXAMINATION OF SHELL MICROSCULPTURE

130 CHAPTER 5 DISCUSSION130 5.1 NUMERICAL DISCUSSION132. 5.2 VARIABLE ASSESSMENT133 5.3 ISOELECTROPHORESIS DISCUSSION

m 5.4 EGG PROTEINS DISCUSSION

135 5.5 PARASITOLOGICAL DISCUSSION13L 5.6 SUMMARY

13S ACKNOWLEDGEMENTS139 REFERENCES

150 APPENDIX -

151 D A T A F O R PRESERVED K E N Y A N MATERIALPI P R O G R A M M E

|57 D A T A F O R CO LL EC TE D FIELD MATERIAL 1985P 2 P R O G R A M M E

6Pflfrg: LIST O F FIGURES

12 FIG 1 - M A P OF K E N Y A

IX 2 - M A P OF COASTAL LOCALITIES VISITED

12 3 - M A P O F COLLECTION LOCALITIES N E A R LAKE VICTORIA

15 4 - DISTRIBUTION OF THE Bulinus afrlcanus G R O U P IN K E N Y A

24 5 - MAIN SHELL FEATURES O F Bulinus41 6.1 - PL AN VIEW OF SHELL APEX T H R O U G H GRATICULE

4-1 6.2 - DETAIL O F W H O R L M E A S U R E M E N T

4*1 6.3 - POSITIONING OF W H O R L ORIENTATED SHELL F O R D R A W I N G

41 6.4 - POINTS O F FOCUS (P & Q) F O R S T A N D A R D SHELL ORIENTATION

4 2 7.1 - WOV: SHELL M E A S U R E M E N T S USED IN PI A N D P2

4 2 7.2 - WOV: F U R T H E R SHELL M E A S U R E M E N T S USED IN PI A N D P2

43 8.1 - AOV: LINEAR SHELL M E A S U R E M E N T S USED IN PI A N D P2

43 8.2 - AOV: A N G U L A R SHELL M E A S U R E M E N T S USED IN PI A N D P2

4 5 8.3 - C O L L U M E L L A R VARIABLES USED IN PI A N D P2

<o% 9 - REPRESENTATIVE SHELLS F R O M THE F O U R PI SAMPLES:

<oZ 9.1 & 9.2 A R A M M A R K E T

Q>Z 9.3 <5c 9.4 MIWANI

Q>Z 9.5 & 9.6 KAPSIMOTWA D A M

Q>Z 9.7 - 9.9 KISUMU ABATTOIR

fafa 10 - CORRELATION MATRIX OF PI VARIABLES

£>g 11 - PI - PRINCIPAL C O M P O N E N T PLOTS FO R FULL D A T A SET:

11.1- PCI AGAINST PC2

eg 11.2- PCI AGAINST PC2 - A R E A OF DISTRIBUTION OF THE F O U R SAMPLES

61 11.3- PCI AGAINST PC3

c* 11.4- PC2 AGAINST PC3

1 2 12 - PI - PRINCIPAL C O M P O N E N T PLOTS FOR FULL D A T A SET, WITH ISOMETRIC SIZE EFFECT REMOVED:



13 12.3- PC2 AGAINST PC3

lb 13 - PI - PRINCIPAL C O M P O N E N T PLOTS F O R FULL D A T A SET WITH 6 VARIABLES REMOVED:

lb 13.1- PCI AGAINST PC2

n 13.2- PCI AGAINST PC3

TT 13.3- PC2 AGAINST PC3

n 14 - PI - CA NO NI CA L VARIATE ANALYSIS FOR FULL D/.TA SET:

11 14.1- CV1 AGAINST CV2

*0 14.2- CV2 AGAINST CV3

%0 14.3- CV1 AGAINST CV3

PflCrE

S3 FIG 15 - PI - PRINCIPAL COORDINATE PLOT A N D MINIMUM SPANNING TREE. PCI AGAINST PC2

S3 16 - PI - HIERARCHICAL CLUSTERING D E N D R O G R A M (SINGLE LINKAGE)

?S 17 - REPRESENTATIVE SHELLS O F ADDITIONAL P2 SAMPLES :

17.1-17.4 B.africanus F O R M (CVA SAMPLE A) KISIAN STREAM

SS- 17.5-17.8 B.nasutus F O R M (CVA SAMPLE S) KISIAN STREAM

st 17.9 & 17.10 B.africanus FORM, PAPONDITI

St 17.11 & 17.12 B.africanus/nasutus FORM, MIGOSI

SI 17.13 & 17.14 B.(?)globosus FORM, S H A M B A

SI 17.15 & 17.16 B.(?)ugandae FORM, A S E M B O B A Y

n 17.17 & 17.18 B.africanus/nasutus FORM, KINYUI

18 - BIVARIATE PLOT SEPARATING THE T W O KISIAN ST R E A M MORPHOTYPES:

UPPER SPIRE HEIGHT AGAINST SHELL LENGTH

19 - CORRELATION MATRIX OF P2 VARIABLES

K 20 - P2 - PRINCIPAL C O M P O N E N T PLOT F O R FULL D A T A SET PCI AGAINST PC2 O N L Y

% 21 - P2 - PRINCIPAL C O M P O N E N T PLOT F O R FULL D A T A SET, WITH ISOMETRIC SIZE-EFFECT R E M O V E D PCI AGAINST PC2 O N L Y

W 22 - P2 - PRINCIPAL C O M P O N E N T PLOT F O R FULL D A T A SET, WITH 7 VARIABLES REMOVED. PCI AGAINST PC2 O N L Y

qs 23 - P2 - PRINCIPAL C O M P O N E N T PLOT F O R OTUs WITH FULL D A T A SET A N D CO MP LE TE F O U R T H A N D FIFTH W H O R L MEASUREMENTS. PCI AGAINST PC2 O N L Y

loz 24 - P2 - CA NO NI CA L VARIATE ANALYSIS F O R FULL D A T A SET. CV1 AGAINST CV2 O N L Y

102- 25 - P2 - MAHALANOBIS DISTANCE D E N D R O G R A M (SINGLE - LINKAGE) F O R 11 SAMPLES

u z 26 - DI AG RA MM AT IC REPRESENTATION OF THE MAIN IEF BANDING PATTERNS:

u z 26.1 AcP

n z 26.2 M D H

u z 26.3 GPI

IIZ 26.4 H B D H

Pfl o e••

i n FIG 27 - RELATIVE E G G PROTEIN B A N D INTENSITY PROFILES A N D CELLULOSE ACETATE STRIPS F O R WILD C A U G H T SNAILS:

m 27.1 MARGIZA D A M

i n 27.2 MARIDZANI D A M

i n 27.3 KISIAN ST R E A M (POOL 4)

i n 2 1 A KISIAN ST R E A M (POOL 1)

u i 27.5 MIGOSI

u i 27.6 PAPONDITI

m 27.7 A S E M B O B A Y

i n 27.8 S H A M B A

i n 27.9 D U N G A B E A C H

i n 27.10 KINYUI

9PflOE:

LIST O F TABLES

13 TABLE 1 R E C E N T CLASSIFICATION OF THE GENUS Bulinus

lb 2 DETAILS OF THE FIVE NO MI NA L Bulinus africanus G R O U P SPECIES IN K E N Y A

11 3 DETAILS OF THE Bulinus africanus G R O U P SAMPLES

4 S U M M A R Y OF VARIABLES USED IN THE PI A N D P2 ANALYSES

si 5 E N Z Y M E ASSAY SOLUTIONS

5S 6 DETAILS OF THE SAMPLES AN AL YS ED B Y C ellulose acetate electrophoresi

bO 7 S U M M A R Y OF SAMPLE SIZE F O R E A C H ANALYSIS

10 8 PI - NORMALISED EIGENVECTOR ELEMENTS O N THE FIRST T H R E E AXES F R O M THE PRINCIPAL C O M P O N E N T ANALYSIS OF 228 OTUS A N D 20 VARIABLES

1 9 PI - NORMALISED EIGENVECTOR ELEMENTS O N THE FIRST TH R E E AXES F R O M THE PRINCIPAL C O M P O N E N T ANALYSIS OF 228 OTUS A N D 20 VARIABLES, WITH ISOMETRIC SIZE-EFFECT REMOVED.

SI 10 PI - MAHALANOBIS DISTANCES BETWEEN MULTIVARIATE SAMPLE ME AN S F R O M CANONICAL VARIATE ANALYSIS

I0H- 11 P2 - MAHALANOBIS DISTANCES BETWEEN MULTIVARIATE SAMPLE M E AN S F R O M CA NONICAL VARIATE ANALYSIS

113 12 E N Z Y M E BANDING PATTERNS OF THE Bulinus africanus G R O U P SAMPLES.

111 13 PARASITE INFECTIONS OF WILD C A U G H T SNAILS

Pft&e :

LIST O F PLATES

101 PLATES 1-4 REPRESENTATIVE IEF PATTERNS F O R THE F O U R E N Z Y M E SYSTEMS:

101 1 AcP

101 2 M D H

108 3 GPI

108 4 H B D H

114- PLATES 5-15 SEM P H O T O G R A P H S OF B.africanus G R O U P SHELLS

114- 5-9 B.africanus FORM, KAPS IM OT WA D A M

ui 10-12 B.nasutus FORM, MIWANI

m 13 B.globosus FORM, KISUMU ABATTOIR

\v\ 14 <5c 15 B.ugandae FORM, A R A M M A R K E T

1.1 G E N E R A L INTRODUCTION

This study was carried out in order to characterise selected populations of snails belonging to the Bulinus africanus group and to assess their taxonomic relationships. It was not intended to produce any formal classification from the results. All the material analysed was collected from Kenya (Figs 1 - 3 ) and no comparison was made with snails from other parts of Africa.

Bulinus Muller (Gastropoda: family Planorbidae) is a genus of freshwater basommatophoran snails, confined mainly to the tropical and sub-tropical zones of Africa. About forty nominal species are presently recognised (Table 1), covering a wide geographical and ecological range, in a wide variety of permanent and transient fresh water-bodies. The genus has been intensively studied (over the last thirty years in particular), since certain species transmit blood flukes of the genus Schistosoma. Bilharzia, or Schistosomiasis*, in man is thought to bring about heavy economic losses over many parts of its range. 200-250 million people are believed to carry bilharzia, with another 600 million living in areas where it could become established. S.bovis affects a number of domestic animals, particularly cattle, causing the loss of meat and milk production.The need for accurate identification of susceptible snails is crucial in the context of the epidemiology and control of schistosomiasis. Not only is this necessary to monitor the spread of these snails, but in the development of specific, effective control methods, and research into the general ecology, behaviour and physiology of the various species. By identifying the most vulnerable stages in the snail’s life cycle, for example, control measures can be applied at the most effective time, thereby maximising disruption of the snail population structure. This also makes the most economic use of limited resources and minimises disruption to other parts of the freshwater ecosystem.

* Schistosomiasis in man is caused by five species ( S.mansoni, S.japonicum,S.mekongi, S.haematobium and S.intercalatum).Bulinus is responsible for the transmission of species within the S.haematobium group - species for the most part characterised by terminal- spined eggs ( S.haematobium, S.intercalatum, S.curassoni, S.bovis, S.mattheei, S.margrebowiei and S.leiperi).

12

«. SO M A LIA T. T A N Z A N IA

5. L A K E V IC T O R IA

6. INDIAN OCEAN

FIG 1 - MAP OF KENYA

t. SUDAN

2. E T H IO P IA

3. UCANOA

■ Nimbodze AMarigiza□ Kiziamkala rMarizan iv Bovo

FIG 2 - MAP OF COASTAL LOCALITIES VISITED

FIG 3 - MAP OF COLLECTION LOCALITIES NEAR LAKE VICTORIA

1. Asembo Bay2. Kisian stream3. Shamba4. M igosi

5. Dunga Beach6. Paponditi7. Nandi H ills

TABLE 1 - RECENT CLASSIFICATION OF THE GENUS BULINUS (AFTER BROWN, 1980)

B .a fricanus group B .trunca tu s /trop icus group B .fo rska lii group B .re ticu la tus group

B .a fricanus * B .trunca tus * B .fo rska lii * B .re ticu la tusB.nasutus * B .trop icus * B .sca laris * B .w rig h tiB.giobosus * B.permembranaceus B .ba rth i *B.ugandae * B .transversa lis * B .browni *B .h igh ton i * B .trigonus * B.canescensB.abyssinicus B.angolensis B.senegalensisB.jousseaumei B .coulbo isi B.camerunensisB.obtusus B.depressus B .c rys ta llinusB .obtusisp ira B.guernei B .becca riiB .um b ilica tus B.hexaploidus

B .lira tusB.natalensisB.nyassanusB .octop lo idusB .roh lfs iB .succinoidesB.yemenensis (?)

B.cernicusB.bavayi

* Found in Kenya

1.2 THE BULINUS AFRICANUS GROUP IN KENYAAbout one million Kenyans are known to be infected with urinary schistosomiasis (Diesfeld and Hecklau, 197S). The disease shows a rather discontinuous distribution, which correlates well with that of certain snails of the Bulinus africanus group (Fig 4). The B.africanus group is thought to be responsible for all transmission of S.haematobium in Kenya. However, there is potential for considerable spread ot the parasite via B.truncatus (the primary host in North Africa) if a suitable strain of S.haematobium were to become established (Brown and Wright, 1974). Developing irrigation schemes and water conservation projects in particular, provide increasingly large areas of new aquatic habitat for these snails. The Ahero IS, near Kisumu and the Perkerra IS near Lake Baringo provide good examples of these. Both were visited by the author while in Kenya.Fourteen nominal species of Bulinus occur in Kenya, of which 5 belong to the B.africanus group (details of these are given in Table 2). This study was not concerned with B.hightoni, a species recorded only along the lower Tana river in north-east Kenya. The geographical range of the B.africanus group has been detailed by Brown et al (1980), shown in Fig 4. These snails are confined to the wetter provinces in the southern half of the country, where there are three main areas of distribution:-1) SW Kenya near Lake Victoria2) Central Kenya3) Coast and lower Tana river basin.Kenyan snails were chosen for this study because of the accessibility of known collection localities in these areas and because of the large amount of preserved material kept in the British Museum (Natural History) (BM(NH)).The group as a whole is characterised by a number of morphological and anatomical features. On the shell, the most distinctive feature is the truncated appearance of the columella margin, due to the presence of a ridge on the lip of the aperture (Fig 5). However, this is absent from B.hightoni and shows great variation in development in shells corresponding to B.ugandae and B.globosus. The group also has a characteristic nodular spiral sculpture on the upper whorls - this is discussed in greater detail in Section 4.5. Anatomically, the group is characterised by the presence of a renal ridge. Biochemical features of the B.africanus group are described in Section 2.A number of areas in the taxonomy of the Bulinus africanus group need particular attention. The definitions of B.globosus and B.ugandae need reassessment, since further evidence is required to validate their present status as separate species. The reported resistance of B.ugandae to S.haematobium needs re-examination. Another important problem is to redefine B.nasutus and B.africanus, particularly on the Kenyan coast, where B.nasutus has been implicated as a host to S.haematobium in certain areas, along with B.globosus (Dr. R F. Sturrock, pers. comm), but B.africanus has not.Further details concerning the traditional characters used to separate these 4 nominal species in Kenya from each other are given in Section 2.

FIG 4 - DISTRIBUTION OF THE BULINUS AFRICANUS GROUP IN KENYA (AFTER BROWN, DS, ET AL, 1981)

TABLE 2 - DETAILS OF THE FIVE NOMINAL BULINUS AFRICANUSGROUP SPECIES IN KENYA(a) B .a fricanus (K R A U S S , 1848, P H Y SO P SIS). M A N D A H L -B A R T H ,

1965, 1968. A Z E V E D O E T A L , 1961. BROW N 1966. VAN A A RD T & VAN E E D E N , 1969.

T Y P E L O C A L IT Y : SO U TH A F R IC A , P O R T N A T A L , (D U R BA N ).

(b) B.nasutus (M A R TEN S, 1979, P H Y SO P SIS). M A N D A H L-B A R T H , 1960.

(c) B.Rlobosus

T Y P E L O C A L IT Y : T A N G A N Y IK A , B A G A M O Y O

(M O R E L E T , 1866; 1868, P H Y S A ) M A N D A H L-B A R T H 1954, 1958. A Z E V E D O E T A L , 1961. W R IG H T, 1963a, BROW N, 1966.

T Y P E L O C A L IT Y : A N G O LA , D A N D E R IV E R (LU A N D A P R O V IN C E )

(d) B.ugandae M A N D A H L -B A R T H , 1954 (B.Rlobosus URandae);1958 (B.URandae). BROW N, 1965.

T Y P E L O C A L IT Y : U G A N D A , 3IN3A B A Y IN L A K E V IC T O R IA

(e) B .h iRhtoni BROW N A N D W R IG H T, 1978

T Y P E L O C A L IT Y : N E K E N Y A , A S E A S O N A L R A IN P O O L (O RM A K O T A ) N E A R H O L A ON T H E W ESTER N S ID E O F T H E T A N A R IV E R

17Much of the work on the B.africanus group has been done outside Kenya, particularly in South Africa, Nigeria and Ethiopia. In Kenya itself, most studies have been carried out on and around the Kano Plain, near Kisumu (Kinoti, 1971; Brown, 1975; Southgate and Knowles, 1975, 1977; Jelnes 1979b). On the coast, most work has been concerned with the population dynamics (Webbe and Msangi, 1958; Teesdale, 1962; O'Keeffe, 1985a & b). An ongoing study involving further population censusing and other work related to the epidemiology of S.haematobium, is being carried out at Msambweni (Sturrock, unpublished reports l98*f, 1985) (Fig 2).

1.3 HISTORICAL PERSPECTIVE TO THE STUDY OF BULINUSBetween 17^9 and 1753, the French botanist Michel Adanson, made the first recorded collection of Bulinus, in Senegal, West Africa. The vernacular name 'Le Bulin' was given to it, taken from ’bulle', meaning bubble, following observations on the distinctive habit of trapping air bubbles beneath the shell. This enables the snail to float on the surface, and happens especially after rain. Because Adanson’s description was pre-Linnean, the Danish biologist O F Muller is the acredited author. In 1782, he formally described B.senegalensis from the shells of Senegalese specimens. This became the type species of the genus.The nineteenth century saw a rapid expansion in the number of new species described, especially in the latter half. Many were described simply on the basis of novel shell forms. Further confusion was to arise when a number of these nominal species were placed in other genera, particularly the morphologically similar Physa, and Isidora (Brown 1980). The expansion continued until about 1915 when over 100 morphospecies had been described (Wright, 1971).In 1957, Mandahl-Barth published the first major revision of the genus, invoking characters taken from the anatomy of the soft parts, particularly the penal complex and radula. The species list was reduced to twenty, with ten subspecies. In the past 29 years since Mandahl-Barth’s publication, the status of certain nominal species has become unacceptable, particularly when these have hindered identification of the human schistosome transmitters. The concept of the biological species has gained general taxonomic acceptance, whereby any character may be incorporated into a taxonomic description, provided it can be standardised adequately. In Bulinus this has involved the exploration of cytological, immunological, electrophoretic and parasitogical techniques.Brown (1980) provisionally re-organised the genus Bulinus into 37 species and three more are possibly valid (Paggi et al, 1978; Jelnes, 1979a). The use of subgeneric nomenclature is now generally avoided (the B.africanus group was once the sub-genus Physopsis). Widespread confusion was caused in the past due to its misuse. It is practicable to divide the genus into the 4 species groups shown in Table 1.

1.4 PRESENT R E S E A R C H A N D P R O B L E M S

Present research takes the form of two main areas of study:1) Re-assessment of the old nominal species which persist in usage. This includes work by Brown et al (1982) on B.coulboisi from Lake Tanganyika, and Brown et al (1986) on B.guernei from West Africa. Using a wide variety of 'biological' variables, it is now thought that these two species are conspecific with B.truncatus. The present study concerned characterisation of snail populations which had been preliminarily identified according to their morphological similarities with the four nominal Kenyan species. It was hoped to provide a morphological basis, using numerical quantification of shell variables, on which to assess the status of the various Kenyan morphotypes.2) Description of new species, such as B.hightoni, described by Brown and Wright (1978). This is one of the most comprehensively described bulinids, having been characterised according to its chromosome number, digestive gland isozyme and egg protein electrophoresis patterns and its susceptibility to trematode infections. This is in addition to shell morphology and soft part anatomy.

Although the study of Bulinus has been intensive in certain areas over the last three decades, there are a great many unresolved taxonomic problems. This can be put down to three particular features of the genus:a. Very high morphological and anatomical variation exists within most of the currently accepted species. Geographical and ecophenotypic variation can be pronounced. This is exacerbated by a large number of overlapping generations within each deme, so that size-effects may also obscure the search for useful taxonomic characters. Interpopulational variation also occurs in biochemical characters so far studied, such as the digestive gland enzymes. Wright and Rollinson (1979) identified ten polymorphic forms of the enzyme acid phosphatase (AcP) in a survey of the B. africanus group in Africa. Specimens corresponding to B. africanus were polymorphic for four of these isozymes alone.Mandahl-Barth (1965) has tried to account for the wide variation seen in the genus. From a biogeographical viewpoint, freshwater habitats form discrete islands. After a period of isolation, a snail population will undergo a certain degree of genetic drift (depending on its initial size) and selection due to environmental and biotic constraints peculiar to each deme. Changes in allelic frequencies may lead to significant genetic divergence between populations, in accordance with the theory of allopatric speciation. Many African fresh water-bodies show seasonal instability during the rains - flooding can bring about a bottleneck phenomenon in which many snails may be washed away, leaving a small residual population of the survivors. A genetically diverse population is therefore decimated, and any increase in numbers which may follow, takes place in a very impoverished gene pool, since many alleles become fixed. Such populations often show distinct phenetic characteristics (due to this genetic divergence), even though they may belong to the same species. Periodic flooding can, however, also break down the physical barriers between demes so that they may merge. Since molluscs often require relatively long periods of genetic isolation in order to speciate, except in the case of of polyploidy which gives rise to 'instant speciation', these conditions overall favour the formation of numerous microgeographical races within a species. High intraspecific variation may, therefore, 'mask' interspecific differences and variation between species may overlap

extensively for many conveniently observable characteristics. All this makes species recognition very difficult in many cases. In extreme cases, such as the differentiation of B natalensis from B tropicus in SE Africa (Brown et al, 1971b), frequencies of various character states have to be employed to separate the two putative species.b. Bulinus is potentially a self-fertilising hermaphrodite, but can outcrossunder normal conditions. Following a population devastation, or when single snails colonise new freshwater sites, extensive self-fertilisation occurs (although a limited amount of sperm storage may occur during aestivation). Clonal populations of this sort appear very distinct and further confuse the taxonomic situation. Clonal populations can also arise when aphallicpopulations occur, even though the parental gene pool is relatively diverse. However, usually only a certain proportion of individuals in a deme are aphallic, leading to a situation where some gene flow does occur from euphallic individuals.c. Certain species in the B truncatus/tropicus complex are morphologically indistinguishable, yet are cytologically distinct due to polyploidy. This multiplication in the number of chromosomes produces immediate reproductive isolation, as in the case B permembranaceus from the CentralKenyan Highlands, which is morphologically indistinguishable from the diploid B tropicus (Brown, 1976).

212. HISTORY O F C H A R A C T E R S USED IN T H E T A X O N O M Y O F BULINUS

2.1 SHELL M O R P H O L O G Y

Few species of Bulinus can be easily identified from using shell characters alone. However, no species description is complete without them. Fig 5 shows the main shell features on Bulinus. The shift in emphasis away from the shell as the main source of characters is partly due to its great variability, but also because of the development of techniques to quantify other variables, particularly biochemical.The terminology used to describe shell characters suffers from an inadequacy in accounting for the more visible differences, such as shape. Terms such as 'relatively broad' to describe the shape of B reticulatus and 'globose' for B wrighti need improved definition if differences between them are to be clearly expressed.The use of ratios, as indices of shell or aperture shape has proved useful in some cases, although very little morphometric work of any sort has been carried out to try and separate species of the B africanus group. Brown et al (1971 a <5c b) found the shell length/aperture length ratio useful for separating B.natalensis from B.tropicus, although less success has been achieved in distinguishing shell morphotypes within species groups. Other ratios have had similar limited use such as the body whorl length/shell width.Mean shell length has provided a useful supplementary character for the separation of certain species, such as B hightoni which is conspicuously smaller than other members of the B africanus group. This is particularly useful in distinguishing it from the similar, but larger, B umbilicatus. Caution is needed in using this character since the growth characteristics of the snail may vary in different environments.The typical B africanus form* has a shell with a moderately high spire and rounded aperture, from which the typical B globosus shows no significant differences (Brown 1980). The typical B nasutus has a much more slender shell with a markedly higher spire, which may be half as high as the aperture. Shells representative of the nominal subspecies B n, productus, in Western Kenya have an even taller spire, which may exceed the height of the aperture. Coastal specimens of the B n nasutus form have relatively higher apertures compared to the spire. In certain areas, B nasutus and B africanus intergrade morphologically - Southgate and Knowles (1977) reported snails of intermediate form between the two types in the Kisat Stream, Western Kenya. B ugandae has a relatively squat shell, usually with a low spire. It intergrades with B globosus in many regions - in Kenya they overlap geographically near Lake Victoria.The unique shape of the B africanus group columellar margin has already been mentioned. A certain degree of twisting is seen in other species groups, although no ridge is present. The margin is usually straight in B truncatus and related species, and typically concave in B tropicus. Brown et at (1971a) successfully quantified the degree of reflection of the parietal margin whilst studying the B natalensis/tropicus complex, but found it to have no taxanomic value. The margin of the columella below the ridge is characteristically spout-

*For B africanus form, read 'shells showing morphological similarities to the B africanus type.1

22like in B nasutus and often develops as a very angular lip with the aperture (similar, but more pronounced than is shown in fig 9.3 o n ml).Umbilical size has frequently been used in shell descriptions, particularly for species in the B truncatus/tropicus complex. It varies from the closed state, as seen in B succinoides, to an open state in B hexaploidus and a widely open state in B umbilicatus. Brown et al (197 la)quantified the various states for snails in the B natalensis/tropicus complex. However, the various umbilical states are difficult to measure and will probably always have to be quantified by visual inspection. No mention has been found in any publication about the use of the umbilicus in separating the Bulinus africanus group snails involved in this study. Observations on the collected material suggested that differences between samples were insufficient to warrant its use.Apertural shape is one of the more obvious features of any shell and has been described by numerous authors. A whole array of terms has been used to describe a wide variety of forms - round, ovate, lanceolate and oval, for example (the latter describing B hexaploidus). The ratio of aperturelength/aperture width has been used in specific circumstances (such as resolution of the B natalensis/tropicus complex by Brown et al 1971a) although its use in separating other species has yet to be assessed. This also applies to snails in the B africanus group. Generally, B nasutus has a more slender aperture compared to the other three Kenyan species in this study. B ugandae

usually has the roundest.The shape of the outer lip of the aperture and the outline of the body whorl were analysed by Wu (1972). His terminology was not precise enough to account for the subtle differences in shell form, however. Curvature of the whorl when seen in profile (known as shouldering), has often proved a useful supplementary character. Species of the B forskalii group often have particularly convex shoulders, although, once again, the use of this character within the different species groups is diminished because no easy method of quantifying it has been found. This present study incorporated variables B*f, B5, H*f, H5, W4and_W5_in an attempt to account for these shape differences (fig 7.1 and 7.2)7 HoweverP(PA&t <*2 ) Ferson, Rohlf and Koehn (1985) were able to measure shape variation far more accurately using an Eye Com Scanner connected to a digitizer, to compare valve shape of Mysus edulis on the east coast of North America. Such a system, which can compare very subtle differences in curvature, requires access to intricate computer software.The shape of the shell apex has been described in the most distinctive species, such as B globosus. This has an obtuse apex, while B liratus, a similar species, has a sharply pointed apex. This character would seem to be due to a combination of spire height and the strength of the shoulders on the upper whorls. It has been used in the present study, since it appears to be a valuable character to separate B globosus and B ugandae from B nasutus.The significance of a whorl number appears to have been given little attention in past work probably because bulinids are difficult to age, due to uncertainties concerning their growth characteristics. As with maximum shell length, a whorl number provides no basis for comparison because Bulinus exhibits limitless growth. Gould (1969), working on the Bermudan lard snail, Poecilozonites, has shown that whorl number cannot be used to assess snail age where limitless growth occurs. In addition, Bulinus exhibits considerable ecological plasticity in its growth rate (Hubendick, 1958; Webbe, 1962). A certain consistency has been noted among large specimens of B africanus which

23complete five whorls, and large specimens of B nasutus which complete six. Whorl number was included in this study in order to compare the rate of whorl descent between B africanus forms and B nasutus forms.Shell colour in Bulinus is notoriously variable, even within a single population. It is also difficult to describe accurately. In B tropicus, for example, it is described as a 'dark brownish colour', whereas in B truncatus it is a 'light yellowish colour'. A standard colour chart could be used to quantify this character more accurately. However, the effects of age, nutritional state and weathering bring into question the value of shell colour as a useful taxonomic character. Within the B africanus group, no significant shell colour differences have been reported, and the character has been omitted from this study.Shell microsculptural characters have been used occasionally by previous authors, to provide supplementary evidence of a species affinity to a particular group. Apical microsculpture has received considerable attention in separating species within the africanus group (Mandahl-Barth, 1957, 1965), although the most intensive work has been carried out by Brown et al (1971 a and b) on the B natalensis/tropicus complex. This involved quantitative assessment of differences in the strength of the rib sculpturing (see section 4.5). However, no significant differences were found between different shell forms since 60% of the samples contained three of the four character states defined. It is clear that a more thorough assessment of the various microsculptural elements is required before their taxonomic significance within each species group can be determined. Within the B africanus group, only relatively vague differences have been reported. Spirally arranged nodules are found on B africanus and B nasutus, although they are more extensive in the latter, occurring even on the body whorl. In B ugandae, spiral sculpture is almost absent. The microsculpture of B globosus is described by Mandahl-Barth (1957, 1965) and discussed by Wright (1957). The present study includes a descriptive analysis of the microsculpture of these four nominal Kenyan species, using high magnification scanning electron photgraphs (Plates 5-19). The main problem with analysing shell microsculpture is that erosional wear and overzealous cleaning often erase large areas of surface detail. However, this is usually detectable since microsculpture elements usually remain undamaged near the sutures, even if the rest of the shell is smooth.It is clear that traditional shell characters need more adequate standardisation. Numerical quantification provides a useful way of doing this, to obtain a clearer view of the relationship between different shell forms, which may represent different taxa. This is particularly relevant to the four species in this present study, which show significant intergradation in shell morphology.

APEX

RIDGE

FIG 5 MAIN SHELL FEATURES OF Bulinus

2.2 ANATOMICAL CHARACTERSSince anatomical characters were omitted from this study only a brief description of the most useful ones are presented in this section.The presence or absence of the male copulatory organ has been well documented. The large majority of B truncatus populations, for example, are aphallic, whereas most B tropicus populations are phallic. Overall length of the copulatory organ has been used to separate similar species, notably B forskalii from B scalaris. The ratio of the preputium/penis sheath lengths have proved useful in separating B globosus from B africanus, the latter having a relatively smaller preputium (Brown, 1966). However, intermediate forms are known to exist (Wright, 1963a; Mandahl-Barth, 1968). Width ratios of the preputium and penis sheath have given useful information when the length ratios overlap (Brown 1966). The copulatory organ can undergo considerable distortion in alcohol, and size depends very much on the state of muscle contraction when the snail dies. Size is thought to correlate with the degree of trematode infection as well, so that the copulatory organ decreases in size as infection increases. Work by Brown on B globosus (1966) suggests that the relative lengths of the preputium and penis sheath remains unchanged however.Much attention has been given by various authors to differences in the structure of the radular teeth, especially the shape and size of the mesocone cusps of the first lateral tooth. Most work has been done on the B truncatus/tropicus group. Three types of mesocone cusps are presently recognised, each specimen being characterised by the percentage frequency of each type using a standard surveillance procedure. This is the most useful criterion for separating B natalensis from B tropicus at present (Brown et al, 1971a) : snails with greater than 50% angular mesocones are designated as B tropicus, and those with less as B natalensis (providing other characters do not contradict). Brown (1982), using scanning electron microscopy (SEM), supports the evidence for differences between angular and non-angular cusps, although pointing out that minor differences could be artefacts due to the narrow depth of field offered by a light microscope. Radular characters do have the advantage over other anatomical features in that they are heavily cuticularised and are not subject to muscular distortion. No results have been published concerning the four B africanus species involved in this present study.The renal ridge is a structure specific to the B africanus group and to some B tropicus populations. Just like the shell microsculpture of the group, interspecific variation has not yet been assessed.Body colour shows great intrapopulational variation which, like shell colour, may be related to the physiological state of the snail. Even so, it has been widely used as a supplementary character. For example, B tropicus and related species are often strongly pigmented, whereas B truncatus and its relatives are often whitish grey. No mention of significant body colour differences within the B africanus group have been published, although specimens conforming to the B ugandae form are often very dark in colour. Mantle pigmentation has been used as a taxonomic character by several authors (such as Wright 1957; Wu, 1972). Brown et al (1971a) tried to quantify the degree of pigmentation in the B natalensis/tropicus complex, but with little success.

26

Mean egg-length has been used by Brown and Wright (1972), as a supplementary character to separate a number of Ethiopian polyploids. B octoploidus was found to have significantly larger eggs than either diploids or tetraploids.

Very few authors have mentioned differences in foot shape which can be seen by observing captured snails on glass surface. B globosus often has a very tapered posterior foot margin, which is often more acute in young snails, trailing conspicuously beyond the shell apex. B africanus and B nasutus forms generally have similar foot shapes, narrower than B globosus and less pointed.

2.3 BIOCHEMICAL CHARACTERSCharacters derived from biochemical analysis are now widely used in the description of bulinid taxa. There are a number of advantages to be gained from using these new methods, which may account for their current popularity:

a. They give results that are precise and easy to quantify.

b. There is evidence that many characters derived from these methods (such as zymogram bands) are independent of age, size, nutrition and local environmental conditions (3elnes, 1979a and 1981; Henriksen and 3elnes 1980).

c. Many of the patterns obtained (especially zymograms) contain isolates whose production is thought by many authors (3elnes, 1985) to be under monogenic control and are therefore a more direct expression of the genotype than most morphological and anatomical characters, which may be controlled by many genes.These biochemical techniques provided a number of new avenues to search for useful new characters. However, many techniques have often proved difficult to standardise, particularly isoelectrophoresis (Ross, 1976). Although individual workers have obtained precise results using their own refined techniques it has proved difficult to relate their findings to that of other works. Because of this problem, few biochemical variables have been studied extensively (although up to 26 enzymes have been examined by some researchers).

There are very few populations of Bulinus which have been fully analysed using any of these techniques. It could be argued, therefore, that even if morphological and anatomical characters are under polymorphic control and influenced by many other variables, they do at least reflect a much larger part of the snail's genome. At present they still provide a more comprehensive description of the phenotype than do the few enzymological, immunological, or cytological characters so far described.

Paper disc chromatography of body surface mucus has had limited use in the characterisation of Bulinus, although Wright (1959) carried out extensive work on the British Lymnaea. Its application has provided useful supplementary evidence to separate B.obtusispira from the very similar B.liratus on Madagascar (Wright 1971 ) and in the description of B hightoni (Brown and Wright, 1978). Fluorescent patterns visible in ammonia vapour seem to be constant throughout life. Although polymorphic variation exists between different populations, these also appear to remain constant. Species group differences are well marked for this character: the B africanus group generally reveals no fluorescent substances at all; the B truncatus/tropicus complex gives a 3-5 band fluorescent pattern, and members of the B forskalii group give a single fluorescent band. Using this technique, B hightoni was shown to have affinities with the B africanus group, since it gave only a very pale blue band, with an r.f. value very different from those of the B truncatus/tropicus complex or B forskalii group. Similarly, B reticulatus was separated from the B.forskalii

group (Wright, 1971 ), since it gave no fluorescent bands. Work has also shown that some diploid snail populations, especially of B tropicus, give a distinct outer band when fumed with ammonia (Brown and Wright, 1972 - this analysis included some Kenyan snails). Further research concerning the frequency of the outer band in different populations may prove useful. The technique has been described in detail by Wright (1959 and 1964), and also by Brown and Wright (1972).

Electrophoresis of proteins is widely carried out in many taxonomic fields. For example in fish taxonomy Ferguson and Mason (1980) and Taggart et al (1980) used this technique to show that certain morphotypes of the Brown Trout (Salmo trutta) were genetically distinct in Lough Melvin, Ireland. Comparini and Rodino (1980) carried out similar work to separate the leptocephali of European and American Eels (Anguilla spp) in the Sargasso Sea. Electrophoresis of proteins is also widely used in molluscan taxonomy (Davis, 1978). Electrophoresis was originally carried out on cellulose acetate strips (Kohn 1957) to separate egg proteins. Wright and Ross (1965 and 1966) surveyed egg proteins of 60 populations of eighteen nominal Bulinus species using this technique. Although less informative than other types of electrophoresis, the technique is sensitive enough to separate populations from different geographical regions. This is shown by the work of Brown and Wright (1972) on the very similar Ethiopian polyploids, and by the description of new taxa, such as B hightoni (Brown and Wright, 1978).

Work by Brown et al (1982) on the egg protein banding patterns of B coulboisi gave additional evidence of the conspecificity of this species with B truncatus. Within the B africanus group, little has been published on the egg protein banding patterns of the four Kenyan species. However, Brown and Wright (1978) noted that the group as a whole showed a distinct banding pattern. Rollinson and Southgate (1979) also found snails with morphological similarities to B nasutus in Northern Tanzania giving banding patterns distinct from the other three B africanus group species.

As well as egg proteins, foot muscle esterases have also been separated (Burch and Lindsay (1967), and digestive gland esterases (Wright (1971), comparing B obtusus and B liratus from Madagascar). Wright and File (1968) compared digestive gland extracts of over 45 populations of Bulinus, representing 17 nominal species. They found starch gel electrophoresis to be useful in separating certain more distinct populations of snails. In general, although starch electrophoresis was sensitive to differences among these populations, it often failed to separate more similar populations, since pore size remained very variable, even when using standard gel concentrations. Wium-Andersen (1973, 1974) characterised the enzymes of Biomphalaria using starch electrophoresis as has Jelnes (1979a) with Bulinus. On the basis of differences in the mobilities of three enzymes he separated B barthi and B browni from B forskalii in Western Kenya using whole body homogenates. For these species there are no described morphological differences, and the validity of promoting these chemotypes to separate species caused some debate. A similar situation has arisen concerning the taxonomic status of chemotypes among certain fruticose lichens which also show a high degree of morphological variation, and where

particular lichen acids are associated with certain morphotypes especially in Ramalina and Usnea. The general view is that lichen chemotypes should be regarded as varieties until adequate morphological evidence can be found to separate them (Culberson, 1969). Jelnes (1979b) compared populations of B africanus group snails from the Kano Plain, Western Kenya. He noted differences in the mobility of phosphoglucose isomerase (PGI) between B africanus and B nasutus forms. B ugandae and B globosus forms also showed different allelic frequencies from B nasutus and B africanus forms for a number of polymorphic enzymes. Jelnes (1981) separated B nasutus and B africanus forms from each other on the basis of their xanthine dehydrogenase patterns, since B africanus forms showed a distinct extra band. The use of single enzymes to separate snail species is questionable (Rollinson, 1980). In view of the high degree of polymorphism exhibited by many proteins, consistent allozymic differences between limited samples of different populations should be treated with caution. In a wider taxonomic sense, the separation of any species should be based on as many characters as possible, especially where no obvious means of discrimination is found, such as in Bulinus,

With the advent of polyacrylamide gels, pore size can now be determined very precisely, giving better resolution than either cellulose acetate or starch gels. Polyacrylamide gels are very stable, both chemically and mechanically, providing a good medium for stabilising ampholines in IEF. They also respond less to the heating effects of an electric current and therefore help protect proteins from denaturing and precipitating.

IEF was introduced into bulinid taxonomy by Saladin et al (1976) to compare egg proteins of B obtusispira and B liratus, both from Madagascar. They found that B liratus gave a single-banded main fraction like B tropicus, while B obtusispira showed the B africanus pattern mentioned earlier. Brown and Rollinson (1982) used whole body homogenates to help separate B truncatus from other diploids such as B tropicus and B natalensis using two enzyme systems. Similarly, Wright and Rollinson (1981) succeeded in identifying four enzyme systems for which B permembranaceus showed different mobilities from B rohlfsi and other B truncatus/tropicus species. Rollinson and Southgate (1979) noted that large sample sizes are necessary to make full use of this technique, and that a greater number of populations need sampling, since enzyme profiles for only 180 populations of Bulinus have so far been produced, jelnes (1985) believes that thorough enzyme characterisation may provide a framework for future revision of the genus. Work on the IEF characteristics of the four B africanus species involved in the present study is included in the results.

Immunodiffusion experiments on egg proteins have been very useful in determining group affinities of problem species such as B obtusispira, B liratus and B.wriqhti . Most work of this sort has involved the Ouchterlony doublediffusion technique (Wright, 1971; Brown and Wright, 1978). Unfortunately, relatively little has been done in recent years, as a consequence of problems in purification, concentration and standardisation of the antisera and antigens. It is a very time consuming process, taking several months to produce adequate amounts of high quality rabbitantisera. Wright and Klein (1967) carried out the .~>itial work on immunodiffusion experiments in Bulinus. Using the Ouchterlony technique, they showed that it was possible to discriminate between different species of snail because of the high degree of selectivity by the antibodies of the albumen gland (Bulinus egg proteins have also been used, eg Brown and

30

Wright, 1978). A technique such as this has an advantage over other 'biochemical' techniques since different snails can be compared directly, one against the other, giving a good indication of their degree of relatedness.

The chomosome number represents one of the few distinct reliable differences found so far in Bulinus. It is known for 32 of the 40 taxa currently recognised. The common diploid number is 36, including all the B africanus group snails (134 populations studied). It has proved a valuable taxonomic character in separating the very similar polyploid bulinids of the B truncatus/tropicus complex in Ethiopia (Brown and Wright, 1972; Brown, 1978X B hexaploidus is the only known hexaploid in the genus (2n = 108) and B octoploidus from the same area, the only octoploid (2n = 144). Although there is no proof that polyploid bulinids cannot interbreed, the lack of triploids or other odd-numbered polyploids indicates that these cytologically distinct forms are reproductively isolated. Certain species of Bulinus, such as B truncatus and B permembranaceus are tetraploids (2n = 74). The latter species was separated from the diploid B tropicus using this character amongst others (Brown, 1976). Chromosome numbers are determined from diakinesis figures of the snail's ovotestis, following the technique of Burch (1960), or from whole crushed embryos (Claugher, 1971).

Research on the structure of chromosomes has been carried out concurrently with work on chromosome number. This was initiated by Claugher (1971), although most of the subsequent research has been carried out by Goldman et al (1980, 1983 , 1984). These authors have remarked on the greater diversity in the structure of Bulinus karyotypes compared to those of Biomphalaria, especially the number of metacentric and submetacentric chromosomes (Goldman et al, 1980). Much attention has been paid to the chromosome banding patterns, especially bands G and C (1984). Using these two bands for comparison between populations, they found that intrapopulational variation was often as great as that between species. Reanalysing Claugher's data, Goldman et al (1983 ) found that B obtusispira and B liratus were very similar karyologically, contradicting other evidence used to separate them into different species groups. Karyological studies on the problematic B natalensis/tropicus complex has given no useful characters for taxonomic purposes. Overall, insufficient research has been carried out on the karyology of Bulinus; in fact, only 0.5% of all molluscs have been analysed in this way (Patterson and Burch, 1978). Chromosomal and karyological analyses were omitted from this present study.

2 A PARASITOLOGICAL STUDIESA great deal has been published concerning the schistosome infections of certain snails. These results have been obtained not only from observations of wild populations but also laboratory trials by exposing unparasitised snails to the miracidia of various schistosome species (and different strains of the same species). It is important to distinguish laboratory results from field obsevations, since laboratory infection rates are invariably much higher. B wrighti, for example, has been found to be susceptible to all schistosoma species in the laboratory (Brown, 1980)^ It is a universal host and is often used to culture schistosome species in the laboratory. The only reports of natural infection come from South Yemen (S.haematobium) (Wright, 1963b). Conversely, B nasutus has proved difficult to infect in the laboratory, yet in East Africa, it is often found to be infected with S haematobium and S bovis (Southgate and Knowles, 1975 and 1977). Particular attention has been given to the B truncatus/tropicus complex, since B truncatus and related species transmit S haematobium in their natural habitats, particularly in North Africa and the Mediterranean. Conversely, B tropicus and related species do not transmit this disease although some success is reported in laboratory exposures (Lo, Burch & Schutte, 1970; Mandahl-Barth, 1976; Frandsen, 1979). So far field data has contributed little to the taxonomy of the group, since it is difficult to identify both the cercariae and the snails. A greater number of laboratory infection experiments are needed, using defined strains of schistosome miracidia and specific populations of snails. Frandsen (1980) has reviewed the appropriate methodologies. B ugandae is different from the other three Kenyan B africanus group species, since it has not been proved susceptible to infection by S haematobium, either in the natural habitat or in the laboratory. This is an important character in distinguishing it from other species (Southgate and Knowles, 1975). Although B africanus, B nasutus and B globosus are susceptible to Kenyan S haematobium (Southgate and Knowles, 1975 and 1977) the main transmitting species is thought to vary from region to region. This is discussed further on. B nasutus and B africanus forms have been found to transmit S bovis in their natural habitats (Kinoti, 1971; Southgate and Knowles, 1975).

Work has also been carried out to compile a list of non-schistosome Bulinus parasites (Porter, 1938; Loker et al, 1981).

-*•A l l S e n t S T o S o m e s -TETirvu n»a u _Y - S P injcTD EG O S , TH *vr I S ,

2.5 ECOLOGICAL, BEHAVIOURAL A N D PHYSIOLOGICAL STUDIES

As far back as 1934, Gordon et al studied the effects of temperature changes on populations of B globosus in Sierra Leone. In 1939, Mozeley described the breeding patterns of this species in Zanzibar.

During the 1950s, a number of basic biological studies were carried out, particularly on the B africanus group, in order to reveal vulnerable stages in the life cycle of the snails and also to improve methods of snail control. Webbe and Msangi (1958) carried out a study on B globosus, B nasutus and B africanus on the Tanzanian coast, and found that their numbers depended greatly on changes in rainfall and temperature, along with the ecological changes these bring about. Up until the 1960's, most publications were concerned with describing changes in population densities, and in establishing the relationship between the transmission rate of schistosomes and seasonal changes in snail population structure (Webbe, 1962b). Early work includes that by McCullough (1957) on seasonal changes in the density of B globosus in Ghana; that of Teesdale (1962) on the schistosome carriers in Kenya; that of Shiff (1964a)on the bionomics of B globosus in Rhodesia, and that of Hira and Muller (1966) on the ecology of schistosome carriers in Nigeria.

Over the last twenty years, some of this basic work has continued (Sturrock, 1973; Hira, 1975; Stiglingh and Van Eeden, 1977; Pretorius (l4)!®!); Pretorius, 1982). Over the last ten years however, much attention has been paid to the construction of theoretical models, designed to identify how the important variables affect population dynamics, and to identify more closely those parts of the snail's life cycle most prone to disruption (eg, MacDonald, 1965; Anderson and May, 1979; Barbour, 1982). Pretorius forexample formulated a model based on B africanus from South Africa, to express the biomass increase which occurs at different water temperatures within the inhabitable range. This study also centred on the dynamics of population fluctuation to see how various environmental factors determined the population size and age structure. They were able to make some predictions about populational changes under different climatic conditions. Detailed population studies have been carried out by O ' K e e f e (1985a 6c b), working on coastal populations of B globosus in Kenya. His work also used mathematical models, and he suggested that the intrinsic rate of natural increase (Rm) of the population is inversely related to the water temperature;’11’ also, that the carrying capacity of the habitat (K) is related to the amount of rainfall. His studies showed that R m increased fastest during heavy unseasonal rain in the coolest months. Hubendick (1958) noted the need for more detailed and accurate field data - this sentiment is still echoed in the 1980's by a number of authors, notably Anderson (1980) and Iarotski and Davis (1981).

Some work has been carried out on the general ecology of Bulinus, particularly in the last ten years. Smithers (1956) carried out work on schistosome carriers in The Gambia. Heeg (1975) studied the effects of dissolved solids on B africanus in South Africa; Appleton (1975)’ studied the distribution ofsnails of the B africanus group in relation to stream geology; Fashuyi(1981) worked on the ecology of B globosus in the rice swamps of Sierra Leone; De Kock (1982) looked at the role of temperature in affecting the geographical distribution of schistosome vectors, and Smith (1982) carried out work on their distribution in polluted water in Nigeria.

1 A lso Apple-tom amo S - t iu e s , R i k .

■* There is a parabolic relationship between Rm and temperature. Rm is greatest at about 25° centigrade (Smiff t

Work on the physiology and behaviour of the group has been sporadic and rather limited. The genus has provided suitable material for the study of basic physiological and behavioural phenomena. Behavioural work has included a study by Morgan and Last (1982) on the circadian profile of B africanus. These authors looked at the effects of different light regimens on six different behaviour patterns. Rudolph and White (1979) studied the egg laying behaviour of B octoploidus, whilst kuma (1975) examined the general behaviour of B globosus. Van Aardt and Frey (1979) looked at the response of B globosus to different oxygen concentrations. Very little physiological work has been done on Bulinus. Published studies include the work of Van Aardt and Frey (1981a) who recorded the oxygen binding characteristics of B globosus. In another paper, Van Aardt and Frey (1981b) examined the non-assimilation of Chorella by B globosus. Shiff and Coutts (1979) examined the effects of molluscicides on the transmission of S haematobium, whilst Banna and Plummer (1978) carried out a more general study on the effects of rescon on molluscan hearts. More research is clearly required in this area of biological study of the bulinid snails.

Shell growth rate studies have been few, but include work by Prinsloo and Van Eeden (1973), who examined the effects of temperature on the growth rate of B tropicus. Unfortunately, laboratory growth studies are seldom applicable to natural habitats (Shiff 1964 a), since considerable distortion may occur, because of differences in water quality, food and overcrowding (Wright, 1960).V J e n e .s e (I ' I b l b ) ALSO C*ftK.IED o v T f SomoE. >o«.k •*) "THI.S flE U D .

3. MATERIALS AND METHODS

3.1 SNAIL CAPTURE, MAINTAINANCE AND PRESERVATION(a) DETAILS OF PRESERVED MATERIAL AND FIELD COLLECTIONSummarised details of all the material examined in this study are given in Table3. A more detailed list of sample localities is given below.

All localities are in Kenya. Reference numbers are given to the preserved collection (PC) and livestock record (LR) in the Experimental Taxonomy Unit of the Zoology Department, British Museum (Natural History). Provisional identifications are given in brackets.

1. Nandi Hills, Kapsomtwa dam. An earth dam in a coffee estate (Fig 3) (B Africanus). G Bowyer, 17 August 1987. Nos PC3746, LR932. Approx 00°06'N 35 °1 1'E

2. 5 miles towards Miwani from the main road junction north of Kisumu on the road to Kakmega. A roadside ditch filled by recent rain (B nasutus). D S Brown, 9 May 1975. Nos PC2739, LR375, 00°03'S 34°51'E.

3. Marsh with Papyrus on the shore of Lake Victoria near Aram Market, Asembo Bay (Fig 3) (B ugandae). D S Brown, 6 April 1973. No PC2812. 00° 12’S 34°21°E.

4 Kisumu, abattoir on the shore of Lake Victoria. A drainage channel about 2 feet wide and cement-lined (B ?globosus). D S Brown, December 1975. Nos PC2842, LR401. 00°06'S 34°45°E.

5 Eastern tributary of the Kisian stream, 5 miles west of Tiengre (Fig 3). A series of steep-sided pools either side of the road, up to 3 feet deep (B africanus/nasutus). R C Archer, September 1985. No LR 1249-1 to 1249-4. 00°04'S 34°41'E.

6. Marsh with Papyrus on the shore of Lake Victoria near Aram Market, Asembo Bay (Fig 3) (B ugandae). R C Archer, September 1985. No LR1250 00° 12'S 34°21’E.

7. Dunga Beach. On boulders on the shore of Lake Victoria (Fig 3) (B ugandae). R C Archer, September 1985. No LR1251. 00°08'S 34°44'E.

8. Large rain filled channel, 2 km south of Migosi, on the eastern side of the main Kisumu to Kakamega road (Fig 3). (B africanus/nasutus) R C Archer, September 1985. NoLR1252. 00°04{'S 34°46 j'E.

9. Ministry of Agriculture Shamba (Cl5), j mile north of Kisumu, on the main Kisumu to Maseno road (Fig 3). A shallow irrigation channel. (B africanus/nasutus). R C Archer, October 1985. No LR1260. 00°18'S 34°44i'E.

10 Paponditi, North Nyakach (Fig 3). Roadside channels, up to 2 feet deep, 1 mile north of the village (B africanus). R C Arche-, October 1985. No LR1261. 00°18'S 34°56'E.

11. Kangundo, Machakos District. Roadside channels, 100 metres south of the Koptic Orthodox Church (B africanus/nasutus). R C Archer, September 1985. NoLR1254. 01°27'S 37°15’E.

3512. Margiza dam, I Msambweni (Fig 2). Large pond to the west of Margiza village, in a palm plantation. (B globosus/nasutus). R C Archer, October 1985. NoLR1263. 39°28'E 4°27?S.

13. Maridzani dam, Msambweni (Fig 2). Medium sized pond to the north-east of Ngaja village, in a palm plantation. (B globosus/nasutus). R C Archer, October 1985. No LR1264. 39°28'E 4°29'S.

14. Kiziamkala dam, Msambweni (Fig 2). Large pond to the northwest of Msambwe Hospital, in a palm plantation. (B globosus/nasutus). R C Archer, October 1985. NoLR1265. 39°28'E 4°29'S.

15. Nimbodze dam, Msambweni (Fig 2). Medium-sized pond to the west of Milalani village. (B globosus/nasutus). R C Archer, October 1985. No LR1266. 39°37'E 4°27'S.

16. Bovo dam, Msambweni (Fig 2). Small, dried-out pond to the west of the main Mombasa to Lungalunga road, 1 mile south-west of Bomani village. (B globosus/nasutus)., R C Archer, October 1985. 39°27'E 4°28'S.

17 Tiwi dam, Msambweni . Large, open pond 2 km off the A 14 on the Cl06 towards Kwale. (B globosus/nasutus). R C Archer, October 1985. 39°34fE 4°14'S.

18. Mazeras Botanical Gardens, 7 kms N W of Mombasa, on the road to Nairobi. A series of shallow, interconnected pools. (B globosus).

(b) FIELD C A P T U R E A N D PRESERVATION

Details of each collection are given in Table 3. Snails were caught using wire dip-nets with 4 ft handies for sweeping in deeper water or where access was difficult, such as the steep sided banks, along the Kisian Stream. An 8" plastic scoop was used to collect at shallow sites, especially in narrow ditches, such as Kinyui and the Shamba at Kisumu. As noted by Webbe (1963), it is almost impossible to obtain a sample with fair size representation of the wild population. Even using-mosquito gauze, a large proportion of the smaller snails may be missed. Using the plastic scoops, however, most snails over 2 m m length were caught after extensive searching. In certain sites, such as the Kisian Stream Sample 1249-4, most of the snails captured were found concentrated on rotting bullrush, making a more comprehensive collection more probable, since they were found to be laying eggs on the same material. In addition to the physical problem of locating different size snails, population structure may vary dramatically at various times of the year, particularly just before the rainy season begins, when many snails have hatched out and may be quite well grown. When the rains begin, extensive flooding often results in large numbers of smaller snails being washed away.

Snails were temporarily kept in the standard plastic trays used in the Experimental Taxonomy Unit at the BM(NH), (320 m m x 220 m m x 48mm). These were covered in transparent polythene to prevent the snails from escaping. Water was changed once a day, using collected pond water and the snails were fed on a mixture of vegetation taken from the original habitat and dried beans.

Specimens with broken shell apices were narcotised in closed containers by adding a few drops of ethanol containing a few crystals of dissolved menthol. After 2-3 hours, when the snails were completely relaxed, they were fixed in 10% formaldehyde solution at 60 degrees centrigrade and transferred to 70% ethanol for preservation. When dissecting the soft parts it is often important to be able to compare snails having relaxed organs, particularly if measurements are to be taken.

Snails with undamaged shell apices were dried and packed in plastic boxes lined with moist cotton wool to induce aestivating behaviour, according to the method described by Brown (1980, pp. 438-439). These were then posted to the UK, where they were unpacked; survivors were then kept in the laboratory.

TABLE 3 DETAILS OF Bulinus africanus GROUP SAMPLES

SA M PLEN OLIVE

STfloCK

ETUP R ESER V ED

C O L L E C T IO NNO

L O C A L IT Y PR ELIM IN A R YD EN TIFICA TIO N H A BITA T

1. L R 93 2 P C 3 7 4 6 K a p s im o tw a D am , N and i H ills (F ig 3)

B a f r ic a n u s E a r th d am in c o f f e e p la n ta t io n .

2 . L R 37 5 P C 2 7 3 9 M iw ani, N e a r K isum u

B n a s u tu s R o a d s id e d itc h , s e a s o n a lly f i l le d .

3 . P C 2 8 1 2 . A ra m M a rk e t, A sem b o B ay

B u g a n d a e M arsh a t sh o re o f L a k e V ic to r ia .

4 . L R 401 ' P C 28 42 K isum u A b a t to i r B g lo bo su s D ra in a g e c h a n n e l a b o u t 2 f e e t w id e c o n c re te - l in e d .

5 . L R 12 49 -1 L R 1249-2 L R 1249-3 L R 1 2 4 9 -4

K is ian S tr e a m , T ie n g re (F ig 3)

B a f r ic a n u s / n a s u tu s

S te e p -s id e d c a t t l e p o o ls b e s id e ro a d . S n a ils la rg e ly on r o t t in g S isa l.

6. L R 12 50 A ra m M a rk e t, A sem b o B ay

B u g a n d a e M arsh a t s h o re o f L a k e V ic to r ia .

7 . L R 1251 D un g a (F ig 3)

B u g a n d a e O n b o u ld e rs a lo n g sh o re o f L ak e V ic to r ia .

8. L R 12 52 M igosi, N e a r K isum u (F ig 3)


R a in f i l le d c h a n n e ls n e a r ro a d .

9 . L R 12 60 S h a m b a , K isum u (F ig 3)

B g lo bo su s O n d e t r i tu s a t b o t to m o f i r r ig a t io n d itc h .

10. L R 1261 P a p o n d iti , N o r th N y a k ach (F ig 3)

B a f r ic a n u s R a in f i l le d c h a n n e ls n e a r ro a d .

11. L R 12 54 K in y u i, M ach ak os D is t r i c t


S h a llo w , m uddy d i tc h e s b e s id e ro a d .

12. L R 12 63

_______

. iM a rg iza D am , M w asem b w en i (F ig 2)

B g lo bo su s L a rg e pond in p a lm p la n ta t io n .

TABLE 3 DETAILS OF Bullnus africanus GROUP SAMPLES (Cont'd)

SA M PLEN O

LIVESTO C K

N O

E TUP R ESE R V E D

C O L L E C T IO NNO

L O C A L IT Y PR EL IM IN A R YID EN TIFIC A TIO N H A B ITA T

13. L R 1264 M ari z an ! D am , M w asem b w en i (F ig 2)

B R lobosus L a rg e pond in p a lm p la n ta t io n . S n a ils on W a te r L ill ie s (N y m p h ae a sp).

14. L R 12 65 K iz ia m k a ia D am , M w asem b w en i (F ig 2)

B g lo bo su s L a rg e pond in p a lm p la n ta t io n . S n a ils on W a te r L ill ie s (N y m p h a e a S£).

15. L R 12 66 N im b o d ze D am M w asem b w en i (F ig 2)

-> M e d iu m -s iz e p ond s n a ils on W a te r L ill ie s (N y m p h ae a sp)

16. - B ovo D am (F ig 2)

D rie d p oo l b e s id e ro a d .

17. - T iw i D am ? L a rg e o pen pond .18. M a z e ra s B o ta n ic a l

G a rd e n s? S m all c o n n e c te d

p o o ls . S n a ils on d e t r i tu s .

I

39

(C) L A B O R A T O R Y MAINTENANCE

For the biometric study, sufficient material had already been preserved. Living specimens were brought back from Kenya to provide FI for future laboratory stocks and IEF work, whilst the egg capsules were used in the egg protein electrophoretic work. None of the coastal snails responded well to laboratory conditions, so FI from these snails were used in the isoelectrophoretic study.

Snails were kept at a constant temperature of 25 degrees centrigrade in glass- covered plastic trays. Water was stored in a large tank containing guppies'?

which help chlorine evaporation and provide a number of amino acids through their excrement. Snail water was changed once a week. A mixture of dried mud and Sycamore leaves (Acer pseudoplatanus) was provided as food. The leaves were pre-soaked for up to 12 hours to remove tannins. Polythene strips, 10mm* were provided as egg laying surfaces. A maximum snail density of 10 individuals per tray was maintained in order to minimise the effects of overcrowding (Wright, 1960).

(d) SCHISTOSOME EXTRACTION

Adult wild caught snails were screened once a week for evidence of cercarial shedding. Where this was found, hamsters and mice were exposed to 100-200 cercariae for 30 minutes. After a prepatent period of about 53 days, the hamster or mouse was killed in a chloroform chamber and the mesenteries perfused with 3% sodium citrate and 0.8% sodium chloride. The adult worms were collected and stored in liquid nitrogen at -180 degrees centigrade for up to 60 days.

(e.) CLEANING A N D PREPARATION OF PRESERVED SHELLS F O R EXAMINATION

In well relaxed snails the soft parts were easily removed by careful use of the forceps, then preserved separately in labelled tubes. Each shell was kept in a numbered pot prior to cleaning. Many shells were coated with a thick ferrous deposit, which often occurs on shells in tropical Africa, and is perhaps connected with the rich red soil found in Kenya. Originally, oxalic acid was used to remove these deposits. However, this was found to strip the microsculptural detail from the shell, so ordinary household bleach was used, diluted to 10%. Deposits were removed using a stiffened camel hair brush, after which the shell was dried and then numbered.

(f) EXAMINATION OF SHELLS

Originally many workers used sliding callipers to measure shells. However, Mayr et al (1953) found them unreliable for measuring shell variables less than 12mm long. Following the method adopted by Brown (1980), all shells were drawn using a camera lucida attachment on a Wild Heerbrugg stereo microscope, at magnifications of X 6, XI2, or X25 for the smallest shells. This gives a permanent record of the specimen, allowing the dimensions to be recorded more accurately, and provides a permanent reference, more convenient to examine than a shell which has to be set up again and reorientated. For ease of manipulation, each shell was kept in a watchglass containing charcoal granules, to avoid backscatter of light. Other workers have mounted shells on plasticine, although the shell is more likely to stick to the substrate and is therefore more likely to be damaged during manipulation.

* Lebistes reticulatus

(9 ) SHELL ORIENTATION

Many workers have found that whorls preceding the final, body whorl have often provided a number of useful variables. Gould et al (1985), working on the West Indian pulmonate snail Cerion, was able to incorporate measurements on the height and width of the fourth whorl, as well as characterise the rib sculpture on it. Cerion is an ideal animal for biometric investigation since it has a clearly recognisable protoconch providing a recognisable origin from which to number whorls. In addition, it reaches a final adult size, at which a permanent, thickened adult lip is laid down around the aperture margin. This present study on the Bulinus africanus group includes a detailed study to look for a distinct protoconch margin. Bulinus shows an indeterminate growth pattern, never reaching a final adult size, and never forming a permanent lip. Since it is not possible to number the whorls in any conventional way, therefore, it was decided that 2 views of each shell should be obtained:-

1. From the whorl-orientated view (WOV)(Fig 7). The W O V enables direct comparison of shells with different numbers of whorls since they are all drawn from a standard view, and are all orientated so that the same stages in the spiral progression of the shell are represented. The number of coils to the pre-body whorl was assessed by positioning the shell in the watchglass so that it was balanced on its lower apertural lip, as shown in fig 6.1, giving a planar view of the spire. Using the graticule, the number of whorls was then counted off, to the nearest sixteenth, and the shell lowered perpendicularly into the horizontal position (Fig 6.3), in the direction shown by the arrow, pivoting on the lower lip.

2. From the traditional view of the shell, which is drawn to give maximum exposure to the aperture (Fig 8). Most shell characters used in the past have been constructed from this view. Oberholzer et al (1970) published extensive details showing differences in measurement obtained by drawing shells orientated at different degrees of tilt and roll. Significant differences in measurements are obtained only on short-spired shells, where the degree of tilt affects the apparent spire height. All shells were therefore orientated so that the tip of the apex and the outer edge of the lower lip of the aperture were in focus at the same time (after Oberholzer, 1970)(Fig 6.4). This is the aperture-orientated view (AOV). However, the A O V does not take into account the fact that different sized snails were at different stages in the development of their coils, and that measurement of apertural dimensions, spire height and other variables may be subject to ailometric growth during the development of the shell. Hence, measurements of the pre-body whorl dimensions were taken from the WOV.

From the drawings obtained, measurements were made on all variables using a 300mm ruler for linear measurements, and a 360 degree protractor for the angles. All measurements were then converted into the equivalent values at a magnification of X 6. These were then recorded on computer program sheets, before being keyed onto disc in the Biometric Unit computer system at the BM(NH).

41

FIG 6.3 - POSITION OF WHORL ORIENTATED SHELL FOR DRAWING

FIG 6.4 - POINTS OF FOCUS (P & Q) FOR STANDARDISING SHELL ORIENTATION

42

LONGITUDINALAXIS

FIG 7.1 - WOV: SHELL MEASUREMENTS USED IN PI AND P2

1. HW2 — height of second whorl2. HW3 — height of third whorl3. HW4 — height of fourth whorl4. WW2 — width of second whorl5. WW3 — width of third whorl6. WW4 — width of fourth whorl7. B4 — distance from apex to widest point

on fourth whorl on right side8. 65 — distance from apex to widest point

on fifth whorl on right side

9. H4 — distance from apex to widest point on fourth whorl on left side

10. H5 — distance from apex to widest pointon fifth whorl on left side

11. W 4— total width of fourth whorl12. W5 — total width of fifth whorl13. A — apex angle

FIG 7.2 - WOV: FURTHER SHELL MEASUREMENTS USED IN PI AND P2

43LONGITUDINAL

AXIS

r

3

IK

14-

<=---13---J6

2

■5F15XL

1. L — shell length Z W — shell width3. S — spire height4. BWL — height of body whorl5. AL1 — aperture height = perpendicular

measurement6. AW1 — aperture width: perpendicular

measurement7. AL2 — aperture height = oblique

measurement8. AW2 — aperture width : oblique

measurement9. d — apertural protrusion

FIG 8.1 AOV: LINEAR SHELL MEASUREMENTS USED IN PI AND P2

10. Apertural protrusion angle (APA)

11. Body whorl shoulder angle (BWSA)

12. Apertural axis angle (AAA)

13. C14-. Cl

IS. usu - u p p e r , spire h e ight

BWSA is drawn at a tangent to the extrapolated curve ot the apertural lip, where it aeets the body whorl.

AAA is drawn between the points of maximum separation on the apertural lip. These two points invariably lie near the junction of the apertural lip with the body whorl and near the apertural lip near the lowest point on the columella.

FIG 8.2 - AOV: ANGULAR SHELL MEASUREMENTS USED IN PI AND P2

OVERLEAF:-FIG 8.3 - C O L L U M E L L A R VARIABLES USED IN PI A N D P2

1 - w

2 - x

3 - y

4 - z

45

FIG 8.3 AOV: C O L L U M E L L A R M E A S U R E M E N T S USED IN PI A N D P2

3.2 N U M E R I C A L ANALYSIS

(a) INTRODUCTION

Two separate analyses were carried out. In the preliminary study PI, 4 samples were looked at, taken from collections made in Western Kenya. No assumption was made regarding the taxonomic status of the samples, only that distinct shell forms were apparent in each. A brief examination showed clear uniformity within each sample, with strong similarities to the typical shell form of one of the 4 main species believed to be present in Kenya (Brown, 1980). Once the variables used in PI had been assessed, a more comprehensive analysis - P2, was undertaken, incorporating the most useful variables found in PI. This involved 10 larger samples, taken from further collections made in Western Kenya and the Machakos District, Nairobi. It also included the 4 samples used in PI and undamaged shells from snails used in the isoelectrophoretic study.

(b) Preliminary Analysis, PI

0 ) Choice of Variables

Firstly comparisons between samples were carried out by eye to obtain a visual impression of the variables likely to express the most obvious inter-sample differences. When examining any taxa, the best characters should be reasonably accessible, especially if they need to be used by field workers. They should also be stable - that is consistently found on most individuals in the taxa concerned, at defined stages in their life cycles, regardless of environmental influences or nutritional state. The most valuable characters are likely to maximise interspecific differences whilst minimising conspecific variation as well. With bulinids, however, characters providing such clear cut differences have not been found using morphological or anatomical sources.

The final variables chosen (Table 4) represent a mixture of traditional ones, such as shell length, shell width and body whorl height, and some new ones, such as the angles. The W O V measurements represent a departure from previous methods of measurements, taken from the A O V only, in an attempt to standardise potentially useful variables, such as BWSA, on shells which have a significantly different number of whorls*. In addition, the collumellar ridge was also standardised to allow direct measurement of 4 linear dimensions, in preference to the coded multistate data used by other workers in the past, such as Brown et al (1971a), since continuous data is usually more informative.

25 variables were chosen (Figs 7 <5c 8), all of which were linear measurements, except for the 2 angles. Linear measurements were made to the nearest { m m and angles to the nearest j degree.

(2.) Number of individuals measured

This was limited by the number of individuals having undamaged shell apices. 74- shells were measured - the details of each sample are given in table 7. Unless every snail in each of the sampled populations had been collected, it is never possible to assess how accurately the range of variation for any given variable in a sample represents that of the original population.

* The character B W S A is used in the field to separate the Rough Periwinkle (Littorina saxatilis) from the Small Periwinkle (L neritoides) and the Flat Periwinkle (L littoralis). In L saxatilis the outer lip of the aperture meets the body whorl at a right angle, whereas this angle is more acute in the Other tWO species (Barret and Yonge, 1958) .

TABLE » SUMMARY OF VARIABLES USED IN THE PI AND P2 ANALYSES

VARIABLE NAME PI P2L / /W / /Wo / /AL1 / -AW I / -AL2 - /AW2 - /BWL / /S - /BWSA / -a / -c / -USH - /APA - /AAA - /w / /X / / !y / ' |3 / ' id / / jHW2 / /WW2 / /HW3 / /WW3 / /HW4 / /WW*f / /B4 / /B5 / /A / /

- fW - lH 5 / /W5 / 1 1

/ Used in t h i s a n a ly s is - N o t used in t h i s a n a ly s is

In addition the stability of any multidimensional scaling technique or hierarchical cluster also depends on the number of individuals measured and

the number of variables employed, tending to be more stable as the number of both increase.

(3) Assessment of Allometry

Gould (1984) suggests that all shelled molluscs exhibit allometric growth at some stage during their ontogenetic development. Since extensive allometry can affect the relationships between different variables, a brief analysis of the four PI samples was undertaken to assess its effect on certain shell variables. Ten specimens were chosen from each sample covering a wide shell range, from 47.5 to 124.5mm. The relationships between the ratios W/L with L, AW/AL with L, S/L with L and AL/L with L were assessed. Since there are insufficient measurable stages on any one snail shell for any assessment of individual allometry, bivariate plots were produced for each sample with a fitted regression line where applicable.

(4) Multivariate analysis

In multivariate analysis, each individual is known as an Operational Taxonomic Unit or OTU. With 74 snail shells measured on 25 different variables, a large primary data matrix was produced to summarise the information (Appendix). The data was then analysed using 2 main numerical techniques:

i. Ordination (multidimensional scaling)

The relationships between the OTUs can be represented as Euclidean Distances in multidimensional space. Since many of the variables may be correlated with each other, the original data may be transformed in such a way that the OTUs may be represented in a much lower dimensional space (or A-space, Sneath and Sokal, 1973), without a significant loss of information. When sufficient information about the ordinal structure is captured in 2 or 3 dimensions, relations between the OTUs can be shown diagramatically. A-space is constructed using new axes in such a way that they capture the greatest amount of variation possible between the individual OTUs. 2 main techniques are used in multidimensional scaling : Principal Components Analysisand Principal Co-ordinates Analysis. Both are very useful in the detection of taxonomic groupings and in the allocation of OTUs of unknown or doubtful origin to groups. They require no a priori knowledge of the taxa involved, and only need the insertion of raw data to function. The 2 techniques are described and compared below. However, neither technique is fundamentally discriminative - a third technique, canonical variate analysis, is more suitable for this.

ii. Clustering

Although ordination may lead to the identification of phena, cluster analysis is required when a classification is attempted (Sneath & Sokal, 1973). This technique arranges OTUs into an heirarchy of groups, based on the similarity levels between them, and represents their relationships visually as a dendrogram.

In addition to these techniques, a minimum spanning tree was constructed and superimposed on the Principal Coordinates Analysis plots (Sucai* & Sokau, m 3 ).

Principal Components Analysis (PCMA)

This technique is described clearly by Davis (1971). It has the major advantage over Principal Coordinates Analysis (PCOA), in that the most important variables can be identified by their eigenvector values on the principal component axes. Unfortunately, the Fortran 77 program written to run P C M A requires a complete data set to work unlike PCOA. Because data was missing from PI, 3 OTUs and 5 variables were omitted. All 4 sample data sets were pooled giving a primary data matrix of 71 OTUs and 20 variates.

In PCMA, a correlation half-matrix is first computed, in which each element represents a product-moment correlation coefficient between a pair of variates. These values range from +1, where variates are identical to -1, where variates represent the extremes of co-variation.

The program then summarises the relationships implied by the variables by giving scores, principal components, for each O T U on a set of new axes. Each axis has the property of being uncorrelated with any other, since they are arranged orthogonally. Each is associated with a latent root, equal to the variance accounted for by that particular axis. The first principal axis is arranged so that it accounts for the maximum amount of variation possible, whilst the second principal axis accounts for the maximum amount of variation between OTUs in a direction orthogonal to the first and so on.

50To transform the original variables into their principal component scores, the correlation eigenvectors are calculated, then normalised. Each vectordetermines the relative contribution of a single variable to one particular principal axis, once the primary data has been standardised. Since variables used in the PI analysis differed widely in order of magnitude (by an order of X2 in some cases), standardisation is necessary to diminish the influence of the larger variables, which would otherwise tend to dominate the principal axes with their large eigenvectors. Standardisation involved subtracting from each observed value the mean value of the variable and then dividing each by its SD, to equalise the spread of data along each axis. Each variate then has a SD=1. The biggest positive and negative eigenvectors correspond to the variables which contribute most to the position of an O T U on that particular principal axis. Each eigenvector must be normalised so that the sum of squares of the correlation matrix elements are equal to one.

By calculating the latent vectors of normalised eigenvectors of the original correlation matrix, the principal component matrices can then be computed, giving a set of k orthogonal axes in A-space, where k is much less than t, the original number of variables. The new O T U coordinates were computed by the equation (davis,iqn) :

P=VXwhere V= the principal component matrix and X= the standardised data matrix

P= the r x t matrix of coordinates of rOTUs on the k principal axes

Canonical variate analysis (CVA)

C V A is similar to P C M A in that it represents multivariate data on a number of transformed axes. Unlike PCMA, however, it is primarily a discriminating technique, used to resolve phenological groups too similar for P C M A (Sneath & Sokal, 1973). C V A works on the assumption that O T U ’s can be separated into a number of pre-ordained groups, with each O T U showing a central tendency in the value of its characters towards the group mean for each variate. Hence, C V A attempts to maximise separation between each group and minimise it within each one. In effect, it works like a principal components analysis on group means. It also assumes that the relationship between the variables is the same in all groups.

This analysis was run as a Minitab program in the BM(NH) . A primary data matrix of 4 groups, representing the 4 homogenous samples was constructed, containing 71 OTU's and 20 variates.

In the first step of C V A group means, maximum and minimum values, SD and SE for each variate is calculated as well as a correlation matrix. Similarly a pooled between group summary is produced, and a table of reduced means for each group for ^ach variate (ie, group mean minus overall mean), giving an indication of group differences. Instead of simply calculating the latent roots and vectors from the correlation matrix, as in PCMA, between-group and within-group variation must be taken into account.

51

If the between-group sums of squares and products matrix is A, and the pooled within-group sums of squares and products matrix is Z, the latent roots and vectors of matrix Z“*A are found. The latent roots measure the discriminatory power associated with each canonical variate.

Since 3 groups may be shown in 2 dimensions, only 3 canonical variate axes were produced for the 4 samples. Since the data was not standardised, normal eigenvector values were obtained by multiplying each value by the pooled SD value for each variate. In addition to the CV plots, the program also calculates the Mahalanobis Distance between the multivariate means for all 4 groups (MD is in fact the squared distances between groups). Tight groups are considered to be further apart than less cohesive groups whose centres are the same distance apart but which have many outlying points.

Sijk = 1 when Xij and Xik are identicalSijk = 0 when Xij and Xik span the extremes of the sample

In the second step of the program, the Principal Axes and Principal Coordinates are calculated. The distance between any 2 OTUs on the multidimensional plot roughly correspond to the similarities between the OTUs, with the most similar ones being closer together and the dissimilar ones being more widely widespread.

Principal Coordinates Analysis (PCOA)

This method was developed by Gower (1966); a recent account is given by Mardia et al (1979). It is not so restrictive in its choice of data as PCMA, particularly since it does not appear to be as disturbed by missing data (Rohlf, 1972). In addition, it can be used on data regardless of whether the relationships between the OTUs are Euclidean or not. It is primarily a technique for using on binary and multistate data, where OTUs are represented in multidimensional space on the basis of their overall similarities (or dissimilarities). Distances between OTUs in multidimensional space have no linear relationship, unlike those in PCMA, which approximate to Euclidean Distances between OTUs in the original character space. The main disadvantage with P C O A is that the importance of individual variables cannot be assessed. However, unlike PCMA, which cannot cope with missing values for a variable, it is possible in P C O A to include variables for which measurement is restricted to a limited number of OTUs. In the present study, many of the smaller snails had not completed their 4th or 5th whorls (particularly in the sample showing similarities with the B ugandae type). These snails were included in P C O A even though variables measured on the 4th and 5th whorls were absent.

With 74 OTUs and 25 variables, a large primary data matrix was constructed (Appendix 1). This data was keyed onto disc via terminals in the Biometric Unit in the BM(NH), and run through a compiled P C O A Fortran program on a PDP11/24 mini computer.

In the first step of the program, the % similarity between each pairwise combination of OTUs was calculated, using Gower's Similarity Index, Sg (after Gower 1966):-

52

Where

s3 =-I

jk = i =

t

Wijk =

Sijk =

first OT U

second OT Ufirst variable compared

t^variable compared

weight for variable i

measure of similarity between i. and i

Wijk is set to 1 if a valid comparison is possible between i. and i . Wijk is set to 0 if no valid comparison is possible. *Sijk is always 0 and < 1

Sijk = 1 - ( Xij-Xik /Ri)

Where Xij = Value of variable i for O T U j.Ri = Range of variable i in the sample.

Minimum Spanning Tree

This technique was first developed by Prim (1957) and improved by various other authors since, such as Rohlf (1973). Although not a multidimensional technique, it is a useful way of highlighting the inadequacies which can occur in multidimensional scaling, as first used by Rohlf (1970). When representing a large number of OTUs in A-space, a certain amount of variation is 'lost to view' in dimensions perpendicular to the principal axes. In a 2-dimensional view in particular, 2 OTUs may appear to be closely associated, whereas in another dimension they may in fact diverge greatly. This may be picked up by referring to the similarity matrix in the case of a PCOA, where it was used in this study. However, it is not easily seen. The links of the minimum spanning tree, calculated from the similarity matrix, can be plotted on the principal coordinates to highlight these distortions, since they link each O T U with its nearest neighbour, regardless of their principal coordinates.

Hierarchical Clustering Technique

This technique is useful since it leads to the separation of OTUs into homogenous subsets and attempts to produce a classification of them. The scaling techniques previously described do not do this. It should be noted however, that it is most useful if the data is strongly clustered and hierarchical (Everitt, 1974), and can be very misleading if none is present, since it will produce an hierarchical cluster anyway. The multivariate analyses were therefore carried out first in order to see whether such a technique was justified. Hierarchical clustering is represented diagrammatically by a dendrogram. It has the disadvantage, like PCOA, in that it gives no information about the relative importance of the different variables. Although hierarchical clustering may represent the relationships between OTUs with a high level of similarity, it leaves doubt about the relationships between larger clusters at lower levels of similarity. Conversely, P C M A is less reliable in representing relationships between closer OTUs, but more accurate in summarising them between groups of OTUs.

All hierarchical techniques involve the construction of a similarity or dissimilarity matrix. In agglomerative methods, a similarity matrix is constructed (in this analysis Gower's Similarity Index was used), where the % similarity between 2 OTUs is calculated.

53Dendrogram construction involves this agglomerative hierarchical technique. Initially, each O T U is considered as a single 'cluster*. There follows a series of successive fusions between OTUs with the highest level of similarity. Fusion then continues with an O T U being added to either another O T U or a new cluster, at each stage, until finally, all OTUs are in the same group. The level at which each O T U joins another cluster defines the point at which each dendrogram link is constructed.

The linkage procedure varies according to the method used. In this analysis, the single-linkage technique was used (Sneath and Sokal, 1973). An O T U may be joined to another cluster if it is more similar to any individual in that cluster, than to any individual in any other cluster. The main disadvantage with singlelinkage is the problem of 'chaining', where distinct clusters of OTUs may be linked by a 'chain' of OTUs lying between them. Hence, the technique may fail to resolve distinct clusters if a number of intermediates are present. Conversely, 2 clusters may be merged with little justification that a member of one cluster is necessarily similar to a member of the other.

(C) MAIN ANALYSIS, P2

Following the P C M A in PI, it was necessary to revise the list of variables. Details of the changes are given in the results section. The number of variables went up to 2 1, all of which were either continuous linear measurements (mm), or angles. 10 main samples were analysed from Western and Central Kenya, as well as one miscellaneous sample of mixed shells from the biochemical analysis. In all, 271 shells were measured, producing a very large data matrix (appendix1) containing over 5600 elements. The number of OTUs was eventually reduced to 230, after removing all OTUs with missing values. The 4 samples used in PI were incorporated in P2, having been remeasured according to the modified method, as described earlier on. With such a large sample it was not possible to carry out a PCOA. Instead, a P C M A was carried out separately, firstly using data with the size effect removed and then with it present. The method used was identical to that used in PI.

Because of the nature of the results, it was decided not to construct either the Minimum Spanning Tree or a dendrogram.

Finally in P2 a C V A was carried out on 11 of the samples, for 20 of the variables.

3 .3 BIOCHEMICAL ANALYSIS

(a ) ISOELECTRIC FOCUSING (IE F)(1 ) INTRODUCTION

Because of the acute morphological plasticity shown by the genus Bulinus, the inclusion of enzyme patterns provides useful additional information not normally available to the traditional taxonomist (Rollinson, 1980).

At present, IEF is the most widely used technique for separating proteins. Polyacrylamide IEF was carried out in the ETU at the BM(NH) using the LKB Multiphor System. This consists of a basic unit of a covered glass plate with electrode connections to a power unit and underlain by a coolant system connected to a multitemp thermostatic circulator. The procedure adopted in this analysis follows that outlined by Karlsson et al (1973) with the additional modifications recommended by Ross (1976). With the advent of polyacrylamide gels, pore size can now be determined very precisely, giving better resolution than either cellulose acetate or starch gel electrophoresis. It is also very stable, both chemically and mechanically, providing a good stabilising medium for the ampholines in IEF.

Wild caught snails were first screened to ensure none were shedding schistosome cercariae, since parasitised snails give enzyme patterns complicated by additional bands which are often due to the prescence of the schistosome in the hosts tissue. Digestive gland extracts were then prepared from 68 specimens, representing 12 different samples. Some snails, particularly from the coastal sites, did not respond well to laboratory conditions, so were prepared 3 weeks before the main bulk of material; these were stored at -20 degrees centigrade to prevent denaturation of the digestive gland enzymes. All snails were extracted whole from the shell wherever possible, allowing the unbroken shells to be used in the morphological analysis. Digestive gland extracts were then transferred to microtubes and frozen at -20 degrees centrigrade for 12 hours to disrupt the cell membranes and facilitate enzyme extraction. After thawing, extracts were homogenised in 20 pi. of distilled water per tube using glass rods. At first this was done manually, then using an automatic stirrer. Extracts were then spun in a centrifuge at 10000 rpm for j hour to give a clear supernatent ready for application. Care was taken to centrifuge at 4 degrees centigrade in order to minimise enzyme activity.

(2 ) GEL PREPARATION AND RUNNING A SYSTEMThis followed the procedure recommended by Ross (1976). The recipe below gives the amounts required for the preparation of 2 gels:-

Acrylamide (29.1%) 20 mlN N Bis-acrylamide 20 mlSucrose 15gAmpholine 8mlRiboflavin 0.8 mlWater 72ml

Sucrose and water was added to a conical flask and stirred continuously while the acrylamide and bis-acrylamide were added. The ampholine was then pipetted in and the flask stoppered. A vacuum pump was connected to the flask and run for 10 minutes to remove air from the solution, then was disconnected. In a separate beaker 5mg of riboflavin (0.005%) was added to 100 ml of distilled water and then mixed in the conical flask. This gave a gel solution with a pH range 4-9.5, as determined by the ampholine concentration, suitable for all the enzyme systems run.

The 2 glass plates forming the mould for the gel were bound together using metal clips and sealed with a rubber gasket, opening at one corner to allow the gel to be poured in. The thinner lower plate was scored across 5 m m from one end. The mould was then stood upright on its sealed side and the gel then poured in through the opening using a 25cc pipette. Once the air bubbles were removed and the gasket sealed, the plates were left to set overnight. When ready, the plates were cooled in the fridge for 20 minutes before the clips and gasket were removed and the thicker top plate prised off. Prepared plates were then covered with a sheet of clean plastic film to protect them from condensation, labelled and stored in the fridge for use. When needed, plates were removed from the fridge and left to reach room temperature before the sheets were removed.

A test run on 5 enzyme systems was carried out initially, using extracts from 22 snails representing 7 different samples. The phosphoglucomutase system (PGM) failed to give clear enough zymograms, so only 4 systems were continued : Acid phosphatase (AcP), Hydroxybutyrate dehydrogenase (HBDH), Glucose phosphate isomerase (GPI) and Malate dehydrogenase (MDH).

ttumnNOn the main run, 29 extracts were applied to each plate, as well as 2\Hb extracts, acting as markers, since the lower alpha-band in the main fraction has an isoelectric point (pi) at pH 7.25. All snail extracts were applied at the cathodal end of the gel, except in the case of the M D H system. A micropipette was used to apply 15-20 pi of each extract of each plate, spaced out according to the template placed underneath the gel plate. Electrode strips were soaked in the electrolyte solutions (5.6% 1M H 3P O 4 for the anode and 1M Na OH for the cathode), and then layed parallel to the longest edges of the gel to give a uniform field strength. Each plate was then loaded on to the horizontal cooling plate of the basic multiphor unit, which was kept at a constant 2 degrees centigrade. Gels were run two at a time for 1 \ hours with an applied voltage of 1200V. Because the gel was cooled, separation of extract components was more efficient and faster, giving better resolution of the different fractions. In addition, at lower temperatures, lateral diffusion of the extracts is minimised. During each run the current fell to about 20mA.

When a current is applied, the ampholines in the gel form a linear pH gradient between the 2 electrodes. Each extract component carries a different overall charge according to the carrier proteins in their molecular structure, and may have either positive or negative polarity. Ail components move towards the electrode of opposite charge, but their speed of movement through the polyacrylamide gel depends on their net charge - the greater the pd across the electrodes, the faster each component separates out - although in IEF this speed differential is not so important (although faster movement does minimise lateral diffusion and therefore gives sharper banding). Each component continues to move across the pH gradient until it reaches the point where it has no net charge. This is its isoelectric point (pi), where it is concentrated or focused into a narrow zone. Unlike other typesof electrophoresis, the components will not continue to move through the gelonce they have reached their pi.

Since each protein has a particular pi, it was necessary to measure the ampholine pH gradient in order to identify the different components and to allow comparison with other plates. The scored portion of each plate was carefully broken off and the pH gradient across it taken at 2 degrees centigrade, using a Phillips electrode, measuring at 10mm intervals along the gel, starting from the anode end. The isoelectric point for each component was then found by correlating its position in the gel to the pH gradient.

Zymograms for each enzyme system were obtained using specific stains which are precipitated by the catalytic action of the prosthetic group of specific enzymes. The enzyme assay details are given in table 5. The reagents were previously mixed with an agar solution before pouring onto the gels. These were left to set for 10 minutes before leaving on a hot plate for 1-2 hours at 50 degrees centigrade to develop. Enzymes were then fixed in the gels by immersing in several changes of a solution of 4% glycerine in 5% acetic acid, over 2k hours. Finally, a solution of 7% gelatin in 3% glycerin at 50 degrees centigrade was added to each plate, covering the gel, and left to harden at room temperature for 2 days.

TABLE 5 ENZYME ASSAY SOLUTIONS

ENZYME SUBSTRATE CO-ENZYM ES/ OTHER ADDITIVES

BU FFER(Substrate: coenzyme mix)

MDH 20 ml1M Na malate pH 7.0

70 mg NAD 30 mg MTT 10 mg PMS

130 ml 0.2 M tris - HC1pH 7.0(30:100)

GPI 50 mgFructose-6-Phosphate

25 mg NADP 25 mg MTT 4 ml 10% Mg C l 25 Units G6 PDF?

150 ml 0.2 M tris - HC1 pH 8.0 pH 8.0 ( IO0 : SO )

A CP 200 mg- Naphthyl Phospate

100 mg Fast Blue 4 mg 10% Mn C l,

150 ml 0.1 M Na acetate pH 5.0 (100:50)

HBDH 400 mgDL-B-Hydroxybutyrate

60 mg NAG 30 mg MTT 10 mg PMS 3 ml 10% Mg Cl

150 ml 0.2 M tris-HCl pH 8.0 (too: IS)

1.5 g AGAR TO SOLUTIONS

NAD = NICOTINAMIDE ADENINE DINUCLEOTIDE

MTT = MTT TETRAZOLIUM

PMS = PHENAZINE METHOSULPHATE

NADP = NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE

For a given zymogram, the major bands which develop are thought to represent alloenzymes, that is, genetic variants of an enzyme which have the same activity, but which differ slightly in amino acid sequence, produced by different alleles at a single locus. Support for this idea comes from the fact that they appear to conform to the Hardy-Weinberg law (7£«.nes,

Band distances were measured on a m m grid and recorded on graphpaper. Each band was quantified visually, according to its density as either faint, 1+, 2+, 3+ or 4+, all measurements being, made from the anodal end of the gel. In addition, each plate was photographed, then stored in a metal, dust-free box.

Cb) Cellulose acetate electrophoresis of egg proteins - method

This was carried out according to the method followed by Wright and Ross (1966), with some minor modifications to voltage and amperage levels.Egg capsules less than 48 hours old were taken from trays and inverted under a light microscope. A micropipette was used to draw off the perivitelline fluid from the eggs, ensuring that the embryos remain untouched. Cellulose acetate strips 50 m m wide and 100 m m long were soaked in a saturated solution of 0.073 M glycine/sodium hydroxide buffer (pH 11.78 at 25 degrees centigrade). The electrophoresis tank was filled with 0.058 M solution of the same buffer, obtained by diluting it by 20%. Each cellulose acetate strip was then partitioned into 2 x 50 m m strips and 2 samples from each egg capsule was applied with a micropipette, one to each strip, at a point 18 m m from the cathodal end of the strip. Separation was carried out with a constant voltage of 130V and 0.4mA for 2 hours. The tank was surrounded by a jacket of iced water to maintain a constant 18 degrees centigrade in the buffer. Up to 4 separate egg capsule extractions were carried out at a time, shielded by a wet absorbent cover to maintain a constant level of humidity. On completion of the runs, the separated proteins were fixed in 5% trichloroacetic acid for 15 minutes and then stained in 0.002% nigrosin in 2% acetic acid. Finally, the excess stain was washed out in 5% acetic acid. On drying each plate was photographed. The Vitatron TLD 100 universal densitometer was used to plot the strip densities in a graphical form. Hamilton-Atwell (1976) discusses the quantification of acetate strips more precisely.

TABLE 6 . Details of the samples analysed by C A E

1249 Tiengre

1250 Asembo Bay

1251 Dunga Beach

1252 Migosi

1260 Shamba, Kisumu

1261 Paponditi

1262 Tiengre - 4

1254 Kinyui

1347 Marigiza Dam1348 Marizani Dam

3. H- Scanning electron microscopy - method (SEM)

Two undamaged shells from each of the samples analysed in the PI numerical analysis were first cleaned to remove deposits, according to the method described previously. These were then mounted on metal studs and coated with gold, to give enhanced resolution of the surface detail under SEM.

The SEM work was carried out in the Electron Microscopy Unit at the BM(NH), using the ISI 60A and Cambridge SI80 Stereoscan SEMs. These gave highly detailed images, and magnification of up to X900 were obtained. A camera built into both microscopes enabled photographs to be taken.

TABLE 7 SUMMARY OF SAMPLE SIZE FOR EACH ANALYSIS

SAMPLE10 LO C A LIT Y

NUM ERICALANALYSIS(MAX)

IEF EGGPROTEINS

PARASITESCREENING

M ICROSCULPTURESEM

LR1249-1 Kisian Stream 5 4 10-2 Kisian Stream - 10 5 12 --3 Kisian Stream - 2 2 6 --4 Kisian Stream 25 6 4 25 -

LR1250 Asembo Bay 24 4 3 2 -LR1251 Dunga Beach - 10 5 18 -LR1252 Migosi 25 2 2 13 -LR1254 Kinyui 25 8 5 22 -LR1260 Shamba 25 5 5 25 -LR1261 Paponditi 25 3 3 8 -LR1263 Marigiza Dam - 5 3 14 -LR1264 Marizani Dam - 6 4 21 -LR1265 Kiziamkala Dam - 2 2 17 -LR1266 Nimbodze Dam - 8 - 12 -- Bovo Dam - 1 - - -PC3746 Kapsimotwa Dam 14 - - - 3

PC2739 Miwani 21 - - - 3PC2872. Aram Market 20 - - - 3PC2842 Kisumu Abattoir

1

19

1f< I

3

i

CHAPTER 4 - RESULTS4.1 M O R P H O M E T R I C ANALYSIS

A) P R O G R A M M E PI

Table 3 gives a summary of the samples discussed below. The details of collection localities are given in Section 3.1.

a. Visual inspection of samples

The Miwani sample (2739) was visually most distinct (Fig 9.3 and 9.4). The shell was very slender, with a relatively high spire and relatively low aperture. Generally, the whorls were very weakly shouldered. The aperture was very slender compared to the other three samples, also having a moderately developed columellar ridge. The angle of attachment of the upper aperture lip to the body whorl (BWSA in Fig 8.2) was distinctly acute. Also very noticeable was the spout-like columellar margin of the aperture below the ridge, combined with a distinctive sharp angle to the lower aperture lip (where it joins the columella ). Well developed nodules were apparent on the body whorl. This sample corresponds to the preliminary identification as B.nasutus (henceforth called the B.nasutus form or sample).

The Kapsimotwa D a m sample (3746) was distinguishable from the B.nasutus form by a combination of a more squat shell Relatively lower spire and more developed columellar ridge (Fig 9.5 and 9.6). It had a more obtuse B W S A and had a relatively higher, less slender aperture. In addition, the base of the aperture was not spout-like. Nodules appeared to be less extensive, rarely found on the body whorl. This sample conforms to a preliminary identification of B.africanus.

The Kisumu abattoir (2842) and Aram Market (2812.) samples appeared closest together on the graphs, although some differences were apparent between samples for certain features. In general, shells from the Kisumu abattoir sample reached a greater size, with narrower apertures and more pointed spires. However, both samples were separable from the B.africanus and B.nasutus forms on a number of features. Both samples were generally more globose (Figures 9.1 and 9.2; 9.7 - 9.9) with relatively wider apertures and much lower spires (the Aram Market sample had an even lower spire than Miwani snails), with strongly convex shoulders (particularly in the Aram Market sample). In addition, both samples had very weak columellar ridges, (often absent in the Aram Market sample) and a distinctive kink in the parietal margin above the ridge (smoothly rounded in the B.africanus and B.nasutus forms). Neither sample showed evidence of nodular microsculpture.

62

FIG 9 - REPRESENTATIVE SHELLS FROM THE FOUR PI SAMPLES9.1 & 9.2 9.3 & 9.4 9.5 & 9.6 9.7 - 9.9

ARAM MARKET MIWANIKAPSIMOTWA DAM KISUMU ABATTOIR

X6 MAGNIFICATION.

Each of the four samples appeared to be morphologically uniform. In addition Shell Colour and texture were uniform throughout each sample.

b. Assessment of Allometry

Neither the B.ugandae nor B.africanus forms showed any clear allometric relationship between the variables W, L, AW, AL and spire height. A significant negative allometric relationship was found for both B.globosus and B.nasutus forms for AL/L with L, ie, aperture length became relatively smaller as the shell grew. These two forms also exhibited positive allometric relationships between S/L and L and a negative allometric relationships between W/L and L. As these shells grow, the spire height becomes relatively higher and the overall shell shape becomes relatively more slender compared to overall length. No allometric change occurred in aperture shape with increasing shell size.

c. Univariate and Bivariate Analysis

Univariate analysis failed to separate clearly any of the four samples because there was generally too much overlap between the sample ranges for the different variables.

Bivariate analysis was used to compare individuals within the B.nasutus sample using certain variables, including L against W. Variable ratios were not incorporated into the analysis, since any ratio differences between samples should have been adequately represented on separate bivariate plots. In addition, many ratios have no actual physical analogue - it is important, in so far as it is possible, to describe differences between samples in simple visual terms since it is desirable that the non-specialist should be able to make reliable identifications. For these reasons ratios were not employed in multivariate analysis. Bivariate analysis of the individual samples failed to show any consistent separation of the OTU's into sub-samples, supporting the visual observation of sample uniformity. This was also born out by the standard deviation values for each sample variable. It was thought unnecessary to use contingency tables to confirm this.

d. Principal component analysis

Fig 10 shows the correlation maxtrix representing 20 standardised variables, measured from 71 OTUs. Only coefficients greater than + or -0.5 have been coded.

Most of the large variables showed high positive correlations with L and W. These are c, AL, AW, BWL, and whorl number (Wo). BWSA showed a medium negative correlation with both L and Wo. None of the variables taken from the whorl-orientated view (WOV) were correlated with the size- related variables. However, angle A showed a medium negative correlation with HW3, and a high correlation was found between W W 2 and WW3, and H W 2 and HW3. None of the columella variables correlated highly, although x showed a medium correlation with W and AL, and z the same with L and AL1.

d. (Cont'd)

(1) Full data set

The % cumulative variation accounted for by the first 5 principal axes was 38.6, 54.0, 66.0, 76.0 and 81.0. This suggested that the data has been summarised only moderately on the first 3 axes shown in Fig 11.1 - 11 .4. 33% of the variation is therefore Tost from view'. The cause of these figures may be due to 2 factors: the lack of any significant correlation between many of the smaller variables, or because the data itself is inherently very variable.

The first 3 plots show that there is no clear separation between the samples nor does any other pattern of separation emerge. Each of the samples is, however, generally restricted to a particular region of each of the plots. This is most noticeable in fig 11.1 for samples A, U and N, for G and N in fig 11.4 and A in 11.3. Fig 11.2 represents the distributions of the different samples shown in fig 11.1. This suggests no overlap occurs between A, U and N on the first 2 principal axes, although G overlaps with all three. However, sample G appeared distinct in Fig 11.4 (showing the plot of the second principal axis (PC2) against the third (PC3).

Table 8, lists the Eigenvector elements for the first 3 principal axes. These have been normalised to remove the effects of certain variables being up to 2 orders of magnitude greater than some others. These large variables would have dominated the ordination without normalisation. The first axis was largely influenced by 7 variables - BWL^W, AL, AW, and Wo, all of which showed high positive correlations with\L. The importance of size-effect on distribution along the first principal axis (PCI) can also be seen by looking at the signs of their various eigenvector values, all of which are negative. Sneath and Sokal (1973) suggested that the presence of so many eigenvectors with the same sign indicates size dominance. The greatest spread of individual shells occurred along the PCI, with smaller specimens occurring towards the right hand side of the plots in Fig 11.1 and 11.3. Size effects may well explain some of the separation of sample A along PCI, especially since univariate analysis revealed no overlap in the length ranges of samples A and U. PC2 was influenced by 6 variables -A, HW3, BWSA, HW2, z and Wo, in descending order of importance. Only the latter shows any correlation with L. Three of these 6 variables have positive eigenvectors, indicating that shape differences are more important along this axis. It was noted that the N sample OTUs all had larger PC2 components than those in the A sample. The 4N specimens in the top left hand corner of the plot of PCs 1 against 2 could all be distinguished visually, having slender shells, acute apices and small apertures. All the A OTUs were similar however, apart from size differences. This confirms the impression that the N sample is seperable from the other samples on shape differences, whereas the A sample separates to a large extent because of differences in size. Pc3 is influenced mostly by characters W W 3 and WW2. The outlying N O T U seen in figs 11.3 and 11.4 is due to an error in the calculation of WW2, WW3, H W 2 and HW3, the values of which w*re all accidentally doubled. It illustrates the importance of the variables in determining the position of the OTU's on the second and third principal axes.

OVERLEAFs-

F1G 10 CORRELATION MATRIX OF PI VARIABLES

Variable 1 - Wo

2 - H W 2

3 - W W 2

4 - HW 3

5 - W W 3

6 - B4

7 - A

8 - L

9 - W

10 - BW L

11 - AL112 - AW1

13 - a14 - c

15 - d

16 - BWSA

17 - W

18 - X

19 - y20 _ z

-K-See f ig s 6,7 and 8 for f u l l descriptions

1920

-1-2

8 4 59 2 4 510 3 5 4 511 2 4 3 4 512 2 4 3 2 513 1 2 1 4 514 2 4 5 4 4 4 1 515 2 4 216 -2 -1 -11718 1 1 1 1

2 3 4 10 11 12 13 14

The original correlation coefficients were converted to integer values for ease of reference.Total correlation equates to a value of 5, with the value decreasing as the correlation coefficient falls.

5

15

55

5

16 17 18

5____ 519 20

FIG 10 C O R R E L A T I O N M A T R I X O F PI VARIABLES0*3

67

KEY TO PI SAMPLES:

A 37*6 KAPSIMOTWA DAM I s &*AFR(CANUS F 0R m )N 2739 MIWANI ( - &.NASUTUS fORm }

G 28*2 KISUMU ABATTOIR ( = 6 . OLoaoSUS FoRm )

U 2812. ARAM MARKET (= R FoRm )

68

PCI AGAINST PC2

FIG 11.2 PCI AGAINST PC2 - AREA OF DISTRIBUTION OF THE FOUR SAMPLES

FIG 11.3 PC 1 AGAINST PC3

PC 3

3.86 -

0.00 -

G GG G

G G G GG G G N

G G G G G NG A G GG G U G NU A u G NN

NN N NNAA AA UU N

u u N A NA N ..A A N NAU UU

U NUU

u U

N

-5.79 ---1—-5.79

T

FIG 11.4 PC2 AGAINST PC3o.oo 5.79 PC 2

TABLE 8 P I - NORMALISED EIGENVECTOR ELEMENTS ON THE FIRST tHREE AXES FROM THE PRINCIPAL COMPONENT ANALYSIS SF 228 oTUs AND 20 VARIABLES--------------------------------------

VARIABLE NORMALISED EIGENVECTOR ELEMENTS

AXIS 1 AXIS 2 AXIS 31. - Wo -0.281 0.28* 0.132. - HW2 0.09 0.31* 0.213. - WW2 0.02 0.18 -0.43*4. - HW3 0.12 0.43* 0.165. - WW3 0.05 0.05 -0.53*6. - B4 0.15 0.00 -0.36*7. - A -0.05 -0.43* -0.258. - L -0.331 0.16 -0.059. - W -0.341 -0.10 -0.0410.- BWL -0.341 0.06 -0.0811.- ALj -0.321 0.04 -0.0812.- AWj -0.29 -0.22 -0.1013.- a -0.24 0.12 -0.1714.- c -0.341 -0.01 -0.0415.- d -0.20 -0.27 0.1316.- BWSA 0.16 -0.33* 0.2017.- W -0.07 -0.02 0.2518.- X -0.22 -0.16 -0.0919.- y -0.17 0.10 0.2320.- z -0.18 0.29* -0.14

1. - Most Important Variables on PCI2. - Most Important Variables on PC23. - Most Important Variables on PC3

d. (2) Size-affect removed

The taxonomic significance of size differences between OTUs which are truly genetic in origin is obscured by the enormous range of variation which occurs due to age differences, and various environmental and biotic factors, such as parasite burden.The plots for the modified principal component analysis, with isometric size effects removed is shown in figs 12.1 - 12.3. Table 9 lists the eigenvector values of the first three axes. The transformed data resulted in a general agglomeration of OTU's in all plots, since all variables had to be logged. This also meant that a greater amount of variation was captured on the first 3 principal axes - 56.9, 79.6 and 88.1% cumulative variation respectively. Apart from the outlying OTUs, some separation was apparent. In fig 12.1, the plot of PCI against PC2, there were no separations, although sample A appeared most distinct, being well away from sample G. PCI is dominated by columella characters y and w. Most separation within the main group occurs along PC2, which is dominated by columella variable x. Plots of PC3 against PCI and PC2 against PC3 revealed that the N sample seemed to form a separate group. PC2 against PC3 also suggested that sample A is separate. None of the plots separate either samples G nor U. PC3 is dominated by variable z.

The 5 outlying OTUs - four from sample U and one from G, have no visible columellar ridge. These were scored as zero for variables w, x, y and z. However, this appears to have given undue weighting to these variables, having led to a profound increase in the standard deviations of each variable. This would then explain their distribution. A further principal component analysis was carried out with the size effect removed, and with these outliers also absent. No significant change in the overall ordination was found. In addition, PCI was still heavily influenced by the x variable, PC2 by z and PC3 by w. It would therefore appear that the outliers had no significant eigenvector distortion either.

d. (3) Reduction in number of variables

Variable reduction was then carried out in an attempt to reduce the effect of random variation between samples and to minimise the effects of individual variation within each sample. Variables with consistently low eigenvector values on all 3 principal axes were excluded from the next principal component analysis. Davis and Boratynski (1979) have shown that this is a useful technique for reducing the number of variables. 8 variables were removed by this method.

PC 22.98

U U0.00 -

-1.49 -

A AA A A A A A

A AA N A N

N U G N N U U N N G N N N N G N U U U

U U U U G G G U G N G

N U G G N G G N G

G G G G G G G

•4.48 0.00 4.48 PC1

FIG 12.1 PI - PRINCIPAL COMPONENT PLOT FOR FULL DATA SET: PCI AGAINST PC2WITH SIZE-EFFECT REMOVED

PC 34.48 - G U

OjOO -

U u u

-4.48 -

-4.48

U GU G G U G

U G G G U U G G U U G A A G

A A A A A G G A G G A A A U G A G

AN N N

N N NN N N N N N N

N N N N

NN

0.00 4.48 PC1

FIG 12.2 - PCI AGAINST PC3WITH SIZE-EFFECT REMOVED.

PC32.98-

G

G

G G G U U UG U G G

G U G U UG G G U U U A A A

0 . 0 0 - G G G U G U A A A A AG G G U A A A

G AN A

r N N UN N U U N A

N N N N NN N N N

-208 -

NN

N

N N N

— i---------------------------- !------------------------------1---------------4.48 000 4.48 PC 2

FIG 12.3 - PC2 AGAINST PC3WITH SIZE-EFFECT REMOVED.

TABLE 9 PI - NORMALISED EIGENVECTOR ELEMENTS ON THE FIRST THREE AXES FROM THE PRINCIPAL COMPONENT ANALYSISOF 228 OTUs AND 20 VARIABLES WITH ISOMETRIC SIZE-------EFFECT REMOVED

VARIABLE NORMALISED EIGENVECTOR ELEMENTS

AXIS 1 AXIS 2 AXIS 31. - Wo -0.09 -0.03 -0.022. - HW2 -0.13 -0.14 0.023. - WW2 -0.13 -0.04 -0.024. - HW3 -0.11 -0.16 -0.015. - WW3 -0.14 -0.02 0.006. - B4 -0.14 -0.04 0.057. - A -0.12 0.01 0.098. - L -0.07 0.01 -0.059. - W -0.08 0.01 0.0410.- BWL -0.07 0.02 -0.0311.- ALj -0.08 0.02 -0.0212.- AW j -0.08 0.00 0.1213.- a -0.08 0.02 -0.0614.- c -0.09 0.01 0.0215.- d -0.09 -0.04 0.1616.- BWSA -0.13 -0.08 0.2017.- W 0.511 -0.21 0.2818.- X 0.26 0.91* 0.1119.- y 0.69' -0.27 0.0320.- z 0.15 0.01 -0.90s

1.2.3.

Most Important Variables on PCI Most Important Variables on PC2 Most Important Variables on PC3

75

The % variation accounted for by the first 5 axes following this principal component analysis was 49.3, 65.9, SI.5, 87.6 and 92.2. This is higher than in the corresponding analysis involving all the variables, suggesting that much of the background variation was indeed removed by this method.

PCI was again dominated by size related variables, particularly BWL, L , c, W, AL, Wo and AW. PC2 was mainly influenced by HW3, A and HW2, and PC3 by W W 3 and WW2. These results are similar to the first analysis.

The ordination plots are shown in figs 13.1 - 13.3. They are similar to the first analysis, except that in figs 13.1 and 13.2, smaller specimens occur towards the left hand side. Some separation of sample A is apparent along PCI. Samples A and U are most cohesive in the plot of PCI against PC2, with sample A again so in the plot of PCI against PC3. Sample N appears most distinctive in the plot of PC2 against PC3. Overall, the reduction in variables does not appear to have improved resolution of the OTUs. Consequently, no size-reduction analysis was carried out.

e. Canonical Variate Analysis

In a further attempt to resolve the relationship between the 4 samples, a Canonical Variate Analysis was carried out.

The method has been described previously.

Figs 14.1 - 14.3 show the plots of the first 3 canonical variate axes. The percentage cumulative variation accounted for by each was 48.07, 88.06 and 100.00.

These 3 plots show that there is a clear distinction between all 4 samples. The greatest separation occurs between samples N and G, with a Mahalanobis Distance of 67.6 (Table 10). N Sample is the most isolated of the samples overall, although A is also very distinct. Samples U and G both separate out on the plot of CV1 against CV3, although they are the closest samples by far, with a Mahalonobis Distance of 21.3.

Size effect was not removed from the analysis. However, on CV1, AL1, A and L have the highest eigenvector values. On CV2, L and BW SA are highest, while on CV3, L, W and A dominate. This indicates that separation of samples U and G is largely based on size differences. Samples A and N separate best on CV2, suggesting that L and BW SA are responsible. However L and W are important in influencing O T U distribution along CV3, where A and N do not separate out. This suggests that BW SA is largely responsible for the separation of A and N on CV2.

f. Principal co-ordinate analysis

This technique produced results which were very similar to those in Pi, with the full data set, since all the data was continuous, either as linear measurements or angles. Because of the similarity in results, it appears that the missing values had no substantial effect on the overall ordination, nor does it suggest that the additional information provided by these additional variables were of much use in aiding resolution of the OTUs.

76

PC 23 2 3 -

0.00 _

-4.85 -

-435 0.00 4.85 PC1

FIG 13.1 PI - PRINCIPAL COMPONENT PLOT FOR FULL DATA SET WITH 6 VARIABLES REMOVED: PI AGAINST P2

77

FIG 13.2 - PCI AGAINST PC3

FIG 13.3 - PC2 AGAINST PC3

78

KEY TO PI SAMPLES:

1 3746 KAPSIMOTWA DAM ( - A USED PR E V IO U SL Y )

4 2739 MIWANI ( . N USED PR EVIOUSLY )3 2812. ARAM MARKET ( - & USED PR EVIOUSLY )2 2842 KISUMU ABATTOIR ( = u USED PR EV IO U SLY )

79

FIG 14.1 PI - CANONICAL VARIATE ANALYSIS FOR FULL DATA SET: CV1 AGAINST CV2

80

FIG 14.2 - CV2 AGAINST CV3

FIG 14.3 - CV1 AGAINST CV3

TABLE 10

SAMPLENO:

P I - MAHALANOB1S DISTANCES BETWEEN MULTIVARIATE SAMPLE MEANS FROM CANONICAL VARIATE ANALYSIS

SAMPLE NO:1 2 3

1. 0.02. 62.6 0.03. 55.2 21.3 0.04.

4

67.6 71.2 55.7 0.0

82Fig 15 shows the plot of the first 2 principal co-ordinate axes. The first 5 axes account for 24.5, 34.25, 41.50, 47.30 and 51.65% of the total variation. This is quite poor, and may be due to the large number of missing values. Over the plot has been superimposed a Minimum Spanning Tree, which allows the relationships between the OTUs in 5 dimensions to be conveniently summarised in 2.

Given the low amount of variation captured by the 5 principal axes, any apparent groupings seen in the plot in fig 15 should be interpreted with caution. From this plot, the A sample in particular, seems quite distinct, apart from one outlier, among sample G. The N sample also appears quite distinct, as does most of the U sample. G falls mainly in the middle of the plot, and overlaps with the other samples. This ordination is very similar to that see in Fig 11.2, the Principal component analysis for the full data set.

g. Hierarchical Clustering Analysis

This was carried out in an attempt to further resolve the tentative separation implied by the Principal co-ordinate results. Fig 16 shows the dendrogram from this analysis. Overall it gave similar results again to the Principal co-ordinate analysis, with an almost complete separation of sample A, and a division of G, giving one separated sub-group, and another overlapping with U. The N group also resolves into two sub-groups, although the relationships between OTUs seems more tentative than the other samples considering the low level of similarity between the connecting groups.

F IG 1 5 P I - P R IN C IP A L COORDINATE PLOT AND M IN IM UM SPANN ING TREE P C I A G A IN S T PC2

FIG 16 PI - HIERARCHICAL CLUSTERING DENDROGRAM (SINGLE LINKAGE)

B. P R O G R A M M E P2

a. Visual inspection of samples

The Kisian stream sample (1249) (fig 17.1 - 8) contained two visually distinct shell forms among the larger specimens. This was apparent even in the natural habitat. One form corresponds to the B.nasutus form in PI, having a slender shell with a relatively high spire and a relatively low, slender aperture (fig 17.5 - 8). The other form corresponds to the B.africanus form in PI, having a more globose shell shape, a medium height spire and a medium height aperture, which is relatively wider (fig 17.1 -4). Smaller specimens (less than 80 m m at x 6)'were not separable.

Among the other samples, the Shamba snails (1260) (Fig 17.13 and 17.14) and Asembo Bay snails (1250) (Fig 17.15 and 17.16) were visually similar to the PI B.globosus/ugandae forms; these had very globose shell shapes, with relatively low spires and very weak columellar ridges. In addition they had strongly convex shoulders (especially to the body whorl) and distinctly angled parietal margins.

The Migosi sample (1252) (Fig 17.11 and 17.12) and the Paponditi sample (1261) (fig 17.9 and 17.10) were very similar to the PI B.africanus form, being generally less globose than the Shamba/Asembo Bay forms. They also had relatively higher spires, narrower apertures and more strongly developed ridges. The Migosi snails in particular had weakly convex shoulders to the whorls. Generally, smaller specimens were less distinguishable from small Shamba/Asembo Bay specimens having relatively strongly convex shoulders and low spires.

The Kinyui sample from Central Kenya (1254) (fig 17.17 and 17.18) appeared more like PI B.nasutus, having a relatively high spire, with an acute BWSA. However, overall shell shape was more globose, like PI B.africanus, with a medium height aperture.

Snails from the coastal localities at Msambweni (fig 2 for the map) were not included in the morphometric analysis due to excessive shell damage particularly on the upper whorls. Snails from the four main localities - Margiza Dam, Maridzani Dam, Kiziamkala D a m and Nimbodze D a m were visually very similar. All were very slender, having a medium-height spire, with a medium height aperture which was rather narrow. The columella ridge was relatively weak, with a distinctive spout-like columella below it. In addition, the shouldering was weakly convex on all whorls, whilst the nodular microsculpture extended onto the body whorl in all specimens examined. These samples correspond in their shell morphology most closely to the B.nasutus nasutus type.

b. Assessment of allometry

The effects of allometry were not investigated in °2, since it was clear from PI that it did not affect clustering of the samples to any significant degree.

iA pP R o X im A T E L y I 3 m m .

X6 MAGNIFICATION.

F IG 1 71 7 . 1 - 1 7 . 4 1 7 . 5 - 1 7 . 8

R EPR ESEN TAT IVE SHELLS OF A D D IT IO N A L P2 SAM PLES : B AFR IC AN U S FORM (C V A SAMPLE A ) K IS IA N STREAM B NASUTUS FORM (C VA SAMPLE S ) K IS IA N STREAM

17.6

17

.7

17.8

86

1 7 . 9 1 7 . 1 0

1 7 .1 1 1 7 . 1 2

X6 MAGNIFICATION.

F IG 1 7 - R EP R ES EN TA T IVE SHELLS OF A D D IT IO N A L P2 SAM PLES :1 7 . 9 a n d 1 7 . 1 0 B AFR IC AN U S FORM, P A P O N D IT I 1 7 . 1 1 a n d 1 7 . 1 2 B A FR IC A N U S /N A SU TU S FORM, M IG O S I

X6 MAGNIFICATION.

F IG 1 7 - R EPR ESEN TAT IVE SHELLS OF A D D IT IO N A L P 2 SAM PLES :1 7 . 1 3 & 1 7 . 1 4 B (? )G LO B O S U S FORM, SHAMBA1 7 . 1 5 & 1 7 . 1 6 B (? )U G A N D A E FORM, ASEMBO BAY1 7 . 1 7 & 1 7 . 1 8 B A FR IC A N U S /N A SU TU S FORM, K IN Y U I

88c. Univariate and Bivariate Analysis

As expected, univariate analysis gave no separation of any samples because of great overlap in their ranges for all variables (overlap was even more extensive than in PI for many characters). Bivariate analysis failed to separate any of the samples with the exception of snails from the Kisian stream locality, where two shell forms were separable. A bivariate plot of upper spire height against shell length gave consistent separation of certain shells (Fig 18). The shells in the upper half of Fig 18, correspond most closely to the B.nasutus productus type, described already in part (a) (these specimens were separated into sample S for the Canonical Variates Analysis). Shells in the lower half of the same figure correspond to the B.africanus type (sample R in the Canonical Variates Analysis). Shells from the Paponditi and Migosi localities were found to lie well below the dividing line in Fig 18, ie, they also correspond to the B.africanus type using these variables. Similar separations of these shells were obtained from bivariate plots of whorl number against shell length and aperture height against shell length.

O - kisian stream snails used in the electrophoretic work as well

FIG 18 BIVARIATE PLOTS SEPARATING TH E T W O KISIAN S T R E A M MORPHOTYPES:

UP P E R SPIRE HEIGHT AGAINST SHELL LE N G T H GOCO

90

d. Principal Component Analysis

The Principal Coordinate and Hierarchical Clustering techniques used in PI, were omitted from P2 since they gave very similar results to the Principal Component Analysis.

-*Variables ALi and AWJ were replaced by AL2 and A W 2 in P2. It was felt that these new variables give a more meaningful measure of the maximum physical dimensions of the aperture. In addition, variables 13 and 14 (Fig 10) were removed, since they are almost identical to ALj and AWj, correlating highly with them. Variables AAA, S and upper spire height (USH) were added (USH is the same as spire height, S, but excludes body whorl measurement).

1) Full data set

Fig 19 shows the correlation matrix, representing 21 standardised variables measured from 22S shells. Only correlation coefficients larger than 0.5 are shown. Overall, the matrix is very similar to the corresponding one in PI, with the largest variables being highly correlated with each other. The additional variable S is very highly correlated with L. The columella variables w and z also show little correlation with other variables, whereas x and y show moderate correlation with some of the size-related variables, particularly W and AW2. Variables taken from the W O V show no significant correlation with any of the size-related variables. As in PI, however, H W 2 and H W 3 are highly correlated, whereas W W 2 and W W 3 are only moderately so. AP A is quite highly inversely correlated with S and AAA.

A large percentage cumulative variation was captured on the first five principal axes - 37.7, 52.8, 66.8, 77.7 and 80.0 respectively. These values were significantly higher than the values in PI.

As in PI, the first principal axis (PCI) was dominated by size-related variables, mainly L, BWL, AL2, W, S, Wo and AW2. PC2 was dominated by a number of variables, most notably d, AAA, A and AP A (AAA and AP A were not included in PI). Also like PI, PC3 was dominated by W W 3 and WW2, as well as B4. On PCI, larger O T U ’s were found towards the right hand side.

ALl - Perpendicular apertural length AWl - Perpendicular apertural widthAL2 - Oblique apertural lenghth AW2 - Oblique apertural widthSee fig 8.1

OVERLEAF:-FIG 19 - CORRELATION MATRIX OF P2 VARIABLES

Variable 1 - L

2 - W

3 - Wo

4 - AL2

5 - AW2

6 - BW L

7 - S

8 - USH

9 - APA

10 - A A A

11 - W

12 - X

13 - y14 - z15 - d16 - HW 2

17 - W W 2

18 - HW 3

19 - W W 3

20 - B421 _ A

1 52 4 53 4 2 54 5 5 3 55 3 5 1 46 5 5 3 57 5 2 4 28 3 4 19 -2 -2

101112 1 113 1 2 11415 1161718

1 12 1

3

20

2 1 ____________________________________________1 2 3 4 5 6

FIG 19

55

-3

7

The original correlation coefficients were converted to integer values for ease of reference.Total correlation equates to a value of 5, with the value decreasing as the correlation coefficient falls.

52 5

-2 55

55

55

3 52 5

1 5_______________________________ 5_

8 9 10 11 12 13 14 15 16 17 18 19 20 21

C O R R E L A T I O N M A T R I X O F P2 VARIABLES

Z Oro

93

Fig 20 shows the plot of PCI against PC2.

With a much larger number of OTU's represented in this analysis than in the equivalent PI ordination, even less sample differentiation was seen. Sample 273*1 is most distinguishable, although 28 land 3746 are quite localised. 12.61 is widely scattered. Overall, it is immediately apparent that one would be far less likely to be able to accurately identify a randomly chosen shell according to its sample, than in the corresponding.Pl plots. The other two plots of PCI v PC3 and PC2 v PC3 are not shown since they provide no additional information.

2) Size-effect removed

Fig 21 shows a plot of the first two principal axes following transformation of the data. For the first five principal axes, the percentage cumulative variation was: 56.7, 74.9. 85.8, 91.8 and 95.2PCI was dominated by the columella variables x, w and z, as was PC3. PC2 was dominated by x and z. The size-related variables had very small eigenvector values. Fig 21, shows outlying OTU's, present for the same reason as the ones in PI, ie, all columella variables were scored zero. However, a further Principal Component Analysis with these OTU's omitted failed to change the overall ordination to any significant degree. In addition, the columella variables were still found to dominate the eigenvector valves on the first three principal axes. From the plot of PCI against PC2, there is greater clarity of certain samples, most notably 273S (separating into two groups), 1250 and 3746. However, the coded letters attest to an enormous amount of overlap between OTUs from different samples. The other two plots gave no additional information and are not shown.

3) Reduction in number of variables

Using the technique described in section 4.1.A, seven variables were removed - USH, w, x, y, z, HW2, WW3. Fig 22 shows the plot for the first two principal axes, which account for 65.9% of the variation between them. No improvement in resolution of the OTUs was obtained. The other two plots are not shown. The plot for size- effect removed gave no further resolution either.

4) Inclusion of variables from 4th and 5th Whorls

In order to assess the importance of variables HW4, WW4, b5, H4, H5, W4 and W5, all OTU's with values for these characters were subjected to Principal Component Analysis. 108 OTU's were analysed, using 28 variables.

94

K E Y T O P2 SAMPLES:

V 1250 AS EM BO BA Y

□ 1252 MIGOSI

0 125* KINYUI

T 1260 S H A M B A

A 1261 PAPONDITI

O 1262 TIENGRE

□ 37*6 KAPSIMOTWA D A M

O 28*2 KISUMU ABATTOIR

■ 2739 MIWANI

▲ 287Z A R A M M A R K E T

A _ MISCELLANEOUS

PC 2■

A ■

3.04-

0.00

-3.04

aA □ ■ ■ □ ■ □

□ ■ ■ A A □ • oA T A A oA

• oA O ^ • • AA A A • • □ • □ oA A ▼ A A A • • o o • • • o AA • O A □ OA O A A o ▼ o □ □ a • • • •

▼ A A □ OQ o o A □ □ A □ D AV A ▼ D □ ▼ A a A A o O A

V V ▼ V A ▼ A ▼ o A O O B • •A A A A T O a a O A A A A oV A V V A A o AA o ▼ O • OA □ V V T D ▼ A A

O A • ▼ O ▼ ▼ □V o o ▼ A

▼A□

□□

V o o o □o TV ▼ A □ □ □

▲ A T

-1---3.04 “ I------------------------------------- 1--------------------------------------

0.00 3.04 PC1

FIG 20 P2 - PRINCIPAL COMPONENT PLOT FOR FULL DATA SET - PCI AGAINST PC2 ONLY

PC 2

3.15 - ■

■ ■

0.00 - © o o ©▼ A o

AA *

▼

A

o o ■▼ o ■ ■* A U ■© ■ ■ ■ ■ ■ ■ Ao O ■ A V V V

▼ A O A B C A O OG ▼ • 0 t F O O A• G H I j K l A *▼ • ▼ M N O P A a0 ▼ R A S A» W ▼ V O V V O O▼ T v O O

▼ V V OV vV

AO o-3.15 -

-------------- ,---------------|---------------1—- 3.15 0.00 3.15

S ym b o ls A - S r e p r e s e n t c o o r d in a t e s w h e re m o re t h a n o ne 0TU o c c u p ie s t h e same m o rp h o s p a c e w i t h i n t h e tw o a x e s s h o w n .

PC1

A O A ▼ A □ □B O 0 A AC • A A □D © • • A DE © • • • A A T A D O D D F © • • A A A O D D DG © • •H • • ▼ AI 0 * A A A A D DJ O O • • □ □

K © © • • ▲ ▲ A D DL © © A A A AM O ▼ ▼ AN A A □O © A AP O V A DQ v a tR • t aS A A A

FIG 21 P2 - PRINCIPAL COMPONENT PLOT FOR FULL DATA SET, WITH ISOMETRIC SIZE-EFFECT REMOVED - PCI AGAINST PC2 ONLY

OVERLEAF:-

FIG 22 P2 - PRINCIPAL C O M P O N E N T PLOT F O R FULL D A T A SET WITH 7 VARIABLES R E M O V E D PCI AGAINST PC2 O N L Y

FIG 23 P2 - PRINCIPAL C O M P O N E N T PLOT F O R OTU'SWITH FULL D A T A SET A N D COMPLETE F O U R T H A N D FIFTH W H O R L MEASUREMENTS PCI AGAINST PC2 O N L Y

PC 2 982.75-

0 .00 -

AA A A

V D□ D OA D O V O V V A • ▼ VA O A ▼ A T

▼ A O V O T AO t O t • A A O A A A A A T A A q A

G A T O O ▼

V A O▼ v oT To ▼A

A

A□ □

A□O □

A □

A O T• AO

A □

D o D □ •O □ •

G

G G

-2.75-

2.75 0.00 2.75 PC1

FIG 2 2

FIG 23

None of these additional variables correlate highly with any other variable. The only reasonbly high correlations exist between H4 and B4 (0.76), W4 and W W 4 (0.64), H 5 and W W 4 (0.7), H 5 and b4 (0.7) and W 5 and (0.7).

Analysis of the eigenvector scores on the first 3 principal axes shows that the first is size related, with B WL, AL, W, L, A W all having high values. Only W 5 contributes significantly to the second axes, whereas H 5 and H 4 are important to the third. HW% is the largest contributor to the second, with W o to the third.

Fig 23 shows the plot of the first 2 principal components from this analysis, accounting for 47.1% of variance.

There is no distinct separation of any sample or group of shells. The other 2 plots produced similar ordinations and are not shown.

Principal Component Analysis in P2 gives far less clear results than in PI. It included more samples, each with a greater number of shells. The expression of individual variation is much greater, exacerbated by a greater size range overall, which further increases intrasample variation due to ontogenetic differences between specimens of different sizes. By eye, it was far less easy to correctly identify shells according to their sample.

e. Canonical Variate Analysis

Since the Principal Component Analysis in P2 gave far less sample resolution than Pi, a Canonical Variate Analysis was performed.

Fig 24 shows the plot for the first two canonical variate axes (CV1 and CV2). The percentage cumulative variation accounted for by the first three axes was 41.50, 69.72 and 80.50. The amount of variation in P2 was greater than in PI since seven more samples were included. The amount of variation accounted for on the first three axes was therefore less.

From Fig 24, it is clear that there is far less sample separation than in PI, although samples R and S are distinct. The other two plots are not shown since they are similar. However, in CV2 against CV3, S is again distinct, whereas sample O shows greatest resolution in the plot of CV1 against CV3. Within the main cluster shown in Fig 24, certain samples are clearly confined to small areas of the ordination. Sample N appears the most cohesive of these, and is very distinct from C, D and H. These three samples are most similar to A. In the plot of CV2 against CV3, sample R is most cohesive within the main cluster, along with sample O. In the plot of CV1 against CV3, samples N and A are most cohesive within the main cluster.

100

KEY TO P2 SAMPLES:E 1250 A S E M B O B A Y

C 1252 MIGOSI

D 1254 KINYUI

I 1260 S H A M B A

H 1261 PAPONDITI

A 1262 KISIAN-A

L 3746 K A PS IM OT WA D A M

N 2842 KISUMU ABATTOIR

R 2739 MIWANI

O 2872 A R A M M A R K E T

S 1262 KISIAN-N

OVERLEAFs-

FIG 24

FIG 25

P2 - CANONICAL VARIATE ANALYSIS F O R FULL D A T A SET. CV1 AGAINST CV2 O N L Y

P2 - MAHALANOBIS DISTANCE D E N D R O G R A M (SINGLE LINKAGE) FO R 11 SAMPLES

102

CV 25 70 -

R r

0 00 -

s S Ns N N

N NN N N N

N N NN

N N NRR N E 0

S IE NR R ON 1 N Et

SC 01 ItR H OH t o 1 E TER R R 1 0 01 OE ir e i

RR H H 10 E 0 II El 1RRR n H l H t o 01 E E

R d h o i i I I u OE 1 1C H 0 A H 01 10 0 E

C H H A D A D 0 l A ED c c C O A D L 1

HD d H D D l C DD D H H A A

C C C C C A D C C AD D C D A H A

D CC D H H H D HHC

DC

- 5 .70 -

— I—- 5 7 0

~i—0 00 5 70

FIG

CV1

FIG 25

103e. (Cont'd)

The Mahalanobis Distances between each sample are given in Table 11. For ease of analysis, the relationships are summarised in the single-linkage dendrogram shown in Fig 25. This shows that two main sample groups are apparent: one contains the B.africanus shell forms from Paponditi, Migosi, Kinyui, Kisian and Kapsimotwa D a m (samples H, C, D, A and L); the other contains the B.globosus/ugandae shell forms from Aram Market, Asembo Bay, Shamba and Kisumu abattoir (O, E, I and N). The Miwani’and Kisian Stream (sample S) snails are most distinct, even from each other. Visually, both correspond to B.nasutus productus shell forms. The Aram Market*and Asembo Bay3sites are actually synonymous, so similarity was to be expected.

1 S aitvl£O

3S ample £

104

TABLE 11 P2 - MAHALANOBIS DISTANCES BETWEEN MULTIVARIATE SAMPLE MEANS FROM CANONICAL VARIATE ANALYSIS

SAMPLEA C D E H I L N O R S

A 0.00C 8.84 0.00D 12.00 7.25 0.00E 20.14 31.89 28.40 0.00H 9.48 3.01 6.78 24.52 0.00I 20.30 26.06 22.76 8.93 17.12 0.00L 14.01 19.00 12.76 14.66 15.88 17.39 0.00N 35.93 44.63 40.26 14.30 30.92 10.27 24.30 0.00O 16.14 21.04 21.67 6.43 15.86 12.49 15.64 17.10 0.00R 37.71 25.27 29.45 54.89 25.04 55.77 39.14 52.06 33.70 0.00S 46.55 41.72 40.17 54.90 36.49 50.76 40.72 40.15 46.88 26.23 0.00

4.2 ISOELECTROPHORESIS RESULTS

The AcP separations (plate 1) produced the clearest separations, whereas the GPI separations were generally less distinct (plate 3). The M D H and H B D H systems both gave reasonably good separations (plates 2 and 4 respectively).

No comparison was made of differences in band intensities between extracts, apart from those where parasites were indicated. The significance of differences between banding intensities has yet to be assessed (Ross, pers comm). However, figs 26.1-4 only show bands with relative intensities of 2 or above, in addition to fainter ones where they appeared significant. Wright and Rollinson (1979) found no age or size -related enzyme differences in the B africanus group for enzymes MDH, GPI or AcP. This aids interpretation to a certain extent, although most snails involved in this analysis were of a comparable size (85-100mm at x 6 mag). The results for the four enzyme systems run are given below.

a. AcP

Bands range from pH 4.7-7.1 (similar to the results of Rollinson and Southgate, 1979). The main variation occurs between pH 6.2-7.1 (corresponding to zone c of Rollinson and Southgate, 1979, where they also found most variation).

Seven main banding patterns are apparent (represented diagramatically in fig 26.1). Pattern 2 is essentially the same as pattern 3, with the additional bands between pH 6.3-6,7 due to parasite infection. The presence of additional bands in the M D H pattern (fig 26.2, pattern 1, between at pH 7.3 and 7.9) and GPI (fig 26.3, patterns 1 and 2, between pH6.5 and 7.6), confirms this. These patterns were produced from only two snails, both from the Kisian stream, which contained other snails showing a high infection rate of Schistosoma bovis. Although all wild caught snails were regularly screened for evidence of cercarial shedding, a very recent parasitic infection may have gone undetected since the schistosome may have been present in the snail's digestive gland as a sporocyst.

It is difficult to match up the bands in fig 26.1 with those identified by Rollinson and Southgate (1979). The three bands in pattern 2 attributed to parasite infection, correspond to certain AcP isozymes. However, if these are omitted, the following interpretation can be given:-

Band at pi 6.3 (pattern 5)6.5 (patterns 4 and 5)6.6 (pattern 4)7.0 (pattern 3)

In addition, three extra bands occur:-

Band at pi 6.75 (patterns 1 and 6) 6.85 (patterns 1 and 6)7.10 (patterns 2 and 3)

= A C P - cl = A C P - c2 = A C P - c3 = A C P - c4

OVERLEAFs-REPRESENTATTYEJEF PATTERNS FOR THE FOUR ENZYME SYSTEMS:R U N NO: SAMPLE

Hb Haemoglobin Control

29 Kinyui (1254)

30-33 Aram Market (1250)

34-43 Dunga (1251)

44-46 Shamba (1260)

47 Maridzani Da m (1264)

48<3c 49 Kiziamkala Dam (1265)

50-52 Nimbodze Da m (1266)

53-55 Kinyui (1254)

107

AcP. Bulinus MFRICANUS 6POUP. 36.1.85.

Iimiiuii lillilll!'!!!A l W h i - . :

H R Jt RR.ll R H t t H t t M V. 51. «( * »• 15. If. 50. M.5*. 55. 59. SO. 51.55.

PLATE 1 - AcP

PLATE 2 - MDH

P L A T E 3 - GPI

H6DH l u l l .

4 ^ * i ♦ •

I H i ' ! ' 111 il!| I il

« . » . n ,Hfc. H .

, W . n H * • « • **.

P L A T E <f - H B D H

Consistent differences occur also towards the acid end of the plate (around pH 5.0). Table 12 shows the overall banding patterns for the samples analysed. It is clear that the Kisian stream, Migosi and Paponditi snails all have similar AcP patterns as do four of the seven Nimbodze D a m snails from the coast (pattern 3 in fig 26.1). The Dunga Beach, Asembo Bay and Shamba snails also show the same overall banding pattern (pattern 6 in fig 26.1), consistently different from the other main group. The Kinyui snails shown an AcP pattern intermediate between these two main groups, although the main bands (at pi 4.7 and 5.0 in fig 26.1, pattern 1) separate out at the same pi as the Kisian group. The coastal snails show a variety of AcP patterns (4, 5 and 7 in fig 26.1). Most Maridzani D a m and Margiza D a m snails show pattern 4, whereas the two Kiziamkala D a m snails and two of the Nimbodze D a m snails show pattern 7.

b. M D H

Fig 26.2 shows the main banding patterns obtained. Not much variation is shown in this pattern, the main differences occurring between pH 6.3 - 6.8 (corresponding to MDH-3 (Rollinson and Southgate, 1979)) and pH 8.1 - 8.5. The two mid-bands in pattern 1 (with pi values of 7.3 and 8.0) are most probably due to to parasite infections, since these refer to the two Kisian stream snails which give the additional bands in pattern 1 of the AcP plates and pattern 1 of the GPI plates. There are two main patterns, then: the Margiza Da m snails all show pattern 3 (table 12); all other snails analysed show pattern 2.

c. GPI

Fig 26.3, shows the main banding patterns obtained for GPI. Bands range from pH 5.3 to 7.6, although most variation occurs between pH 5.3 and 6.0. Patterns 1 and 2 refer to the parasitised Kisian Stream snails. Bands between pH 6.5 and 7.6 are attributable to the parasite. Four other banding patterns emerge (table 12). Again, the Kisian Stream, Migosi and Paponditi snails have similar GPI patterns, as do the Kinyui snails and the same 4/7 Nimbodze Da m specimens (pattern 3). The Dunga Beach, Asembo Bay and Shamba snails show the same overall banding pattern (pattern 4) as they do for AcP patterns. This is consistently different from the Kisian group, having a single main band with a slightly more alkaline pi. The coastal snails again show a variety of patterns, with only the Maridzani D a m and the single Bovo D a m snails showing the same AcP and GPI patterns. All the Margiza Da m snails show pattern 6 (table 12), whereas the Maridzani, Bovo and Kiziamkala D a m snails shown pattern 5 (which is basically very similar, except for the band at pi 6.75). The Nimbodze Da m snails are most varied: one shows pattern 5, three show pattern 6, and four show pattern 3 (as previously mentioned).

d. H B D H

Interpretation of these plates proved most difficult, since the main region of variation between pH 4J-5.2 is very narrow (plate 4). Future separaiions using this enzyme system would be best carried out using a gel with a narrower pH range, perhaps between pH 4-7 (rather than pH 4-9.5 as used here). The more alkali bands which separated between pH 6.0-7.6 are not shown since they were impossible to interpret . Fig 26.4 shows that 4 main banding patterns were present. Comparison with the work done by

Rollinson and Southgate (1979) on the Bulinus africanus group, suggests that at least three isoenzymes can be identified:-

H B D H - 11 = pattern 1- 10 = pattern 2- 2 = pattern 3

Similar to the results of the AcP and GPI systems, snails from the Kisian Stream, Migosi and Paponditi share the same overall pattern (pattern 1). The four coastal snails from Nimbodze D a m (85/8, 9, 10 and 11) also share this. Similarly, all snails from the Dunga Beach, Asembo Bay and Shamba localities share pattern 2. None of the Kinyui snails gave any bands for this enzyme (denoted as pattern 5). Apart from the Nimbodze D a m snails already mentioned, all the coastal snails showed banding pattern 3, except a single snail from Maridzani D a m (85/17). This showed pattern shared with snail 86/17 from the Kisian Stream.

OVERLEflF;-

FIG 26 - D I AG RA MM AT IC REPRESENTATION OF THE MAIN IEF BANDING PATTERNS:

FIG 26.1 AcP

FIG 26.2 M D H

FIG 26.3 GPI

FIG 26.4 H B D H

112

1 2 3 4 5 6 7

pH

1 2 3

FIG 26.1 - AcPFIG 26.2 - M D H

1 2 3 4 5 6

p H 5.0 J

FIG 26A - H B D H

FIG 26.3 - GPI

113TABLE 12 : ENZYME BANDING PATTERNS OF THE Bulinus africanus GROUP SAMPLES

SNAIL ENZYME PATTERNLOCALITY AcP GPI MDH HBDH

KISIAN - 1 3 3 2 13 3 2 13 3 2 13 3 2 13 3 2 1

- 2 3 3 2 13 3 2 13 3 2 13 3 2 13 3 2 13 3 2 13 3 2 13 3 2 13 3 2 _

3 3 2 1- 3 3 3 2 _

3 3 2 1- 4 2 2 1 1

2 1 1 13 3 2 1- 5 2 43 3 2 13 3 2 1

MIGOSI 3 3 2 13 3 2 1

PAPONDITI 3 3 2 13 3 2 13 3 2 1

KINYUI 1 3 2 51 3 2 51 3 2 51 3 2 -

1 3 2 51 3 2 51 3 2 51 3 2 5

SHAMBA 6 4 2 26 - - _

6 it 2 26 it 2 26 it 2 2

ASEMBO BAY 6 4 2 26 it 2 26 4 2 26 it 2 2

Each number represents one of the banding patterns shown in figs 26.1 - 26.4 on page 112.

114

TABLE 12 ; ENZYME BANDING PATTERNS OF THE Bulinus africanus GROUP SAMPLES

SNAIL ENZYME PATTERNLOCALITY AcP GPI MDH HBDHDUNGA BEACH 6 U 2 2

6 It 2 26 it 2 26 It 2 26 If 2 26 It 2 26 It 2 26 it 2 26 It 2 26 It 2 2MARI Z AN I DAM 5 5 2 3k 5 2 itif 5 2 3it 5 2 3it 5 2 37 5 2 3KIZIAMKALA DAM 7 5 2 37 5 2 3NIMBODZE DAM 5 5 2 _

3 3 2 13 3 2 13 3 2 17 6 2 _

- 6 _ _

7 6 2 -MARIGIZA DAM 5 6 3 3

it 6 3 _

it 6 3 3it 6 3 3

BOVO DAM tt 5 2 3

115

4.3 E G G PROTEIN RESULTS

For technical reasons the two scales on the densitometer graphical recorders were set at different levels for each acetate strip, so only the relative differences between peaks can be compared for each sample. Each trace was set to measure a relative density of about 50 units for each darkest band, so that traces were more comparable.

The results for the different samples are shown in figs 27.1 - 10.

The coastal snails from the Margiza and Maridzani Dams (figs 27.1 & 2) showed the clearest overall pattern, with 3 distinct peaks.1 The Kinyui sample showed a similar pattern. Although the Shamba sample showed similarities with these 3 samples, the right hand peak is much less conspicuous.

The other samples show more complicated patterns, with a number of intermediate peaks being present, culminating in the very rounded appearance of the Dunga sample, where a number of peaks occur together in a very close succession. The Migosi, Paponditi and Kisian -1 and 4 samples share a prominent peak*(27.3-6), corresponding to a dark band at the anodal end of the strip. This is not shared by other Western Kenyan snails (27.7-9).

The variation between samples was considered too great to warrant any quantitative assessment of the different bands.

peaks 1, 2 and 3

The far righthand peak, 4

OVERLEAF;-

FIG 27 - RELATIVE E G G PROTEIN B A N D INTENSITY PROFILESA N D CELLULOSE ACETATE STRIPS FO R WILD C A U G H T SNAILS

FIG 27.1 Margiza Da m

FIG 27.2 Maridzani D a m

FIG 27.3 Kisian Stream (Pool 4)

FIG 27.4 Kisian Stream (Pool 1)

FIG 27.5 Migosi

FIG 27.6 Paponditi

FIG 27.7 Asembo Bay

FIG 27.8 Shamba

FIG 27.9 Dunga Beach

FIG 27.10 Kinyui

d'2U-

S ‘TD

CvAil

S^Q

3AU-

Vn"Jf

» 3A

U.*n

3-tf

RELATIVE BAND DENSITYRELATIVE BAND DENSITY RELATIVE BAND DENSITY

©

FIG 27.5 ^ 5°1

FIG 27.6 ditLi

s ONc

riy g

oNda

siQ S

ftmna

y Jiy

xs 'S

Ncnv

97M

w.ss»c

r SAu

-tngy

RELATIVE BAND DENSITY RELATIVE BAND DENSITY

These are summarised in table 13. Only the Kisian-3 and 4 pools and the Migosi site were found to contain snails shedding cercariae. These were subsequently identified as S bovis by egg morphology and IEF of homogenates. Kisian-4 in particular shows a high prevalence of infection at 80%. No infected snails were found in pools 1 and 2. Prevalence at Migosi was 69%. There was no evidence of S haematobium infection in any of the living snails. Certain coastal samples shed unidentified cercariae of fish heterophyids. These can be separated from schistosome cercariae since they have forked tails and eyespots.

4 A PARASITOLOGICAL RESULTS

119

TABLE 13 PARASITE INFECTIONS OF WILD CAUGHT SNAILS

SAMPLE N*: SCREENED N°: SCHISTO PARASITE IDENTIFICATION

Kisian Stream - 1 - 2 - 3 - 4

Asembo bay Dunga Beach Migosi Kinyui Shamba Paponditi Margiza Dam Maridzani Dam Kiziamkala Dam Nimbodze Dam Tiwi Dam Mazeras (Bot Gdns)

101 2

6

252

IS13 22 25 814 21 17 12 4 7

003

20

0090000

0

0

0

0

0

Schistosoma bovis Schistosoma bovis

1 with ech in ostom e s p e c ie s Schistosoma bovis

4 with fish h e tero p h y id s p e c ie s 1 with fish h e tero p h y id s p e c ie s

7 with fish h e tero p h y id s p e c ie s

4.5 EXAMINATION O F SHELL MICROSCULPTURE

a. Assessment of the Protoconch Margin

Gould (1969) suggests that small differences between individuals in the early stages of ontogeny, may eventually give rise to a wide variety of adult forms, through ontogenetic divergence.

In order to test this idea with regard to the different shell forms in the Bulinus africanus group, it was first necessary to define the protoconch. This would then enable comparison between the protoconchs of different shell forms, such as B nasutus and B ugandae, with a view to explaining differences in shell variables in the mature forms.

The exact definition of the protoconch in Bulinus has always been rather vague. Connolly (1939) referred simply to the first whorl, whereas Hubendick (195S) mentioned a nuclear whorl, followed by post-nuclear whorls, as did Walter (1962). Walter (1962) also referred to the embryonic whorl, suggesting the simplest definition of the protoconch, ie that portion of the shell laid down while the snail is still within the egg. Gould (1969) described the protoconch as that portion of the hatched shell which has been laid down before accretional growth occurs. This assumes no accretional growth has occurred in the egg. From my observations, striae (which are transverse ridges strongly suggestive of accretional growth) appear even in the egg.

This problem in defining the limit of the protoconch (whether it represents the point at which accretional growth occurs, or whether it represents the emergence stage of the snail from the egg, with a rapid increase in its growth rate) is also important for another reason, apart from the ontogenetic comparison of shell forms. The protoconch margin would provide the only recognisable standardised point on the whole shell, regardless of its size at the snails death, since Bulinus shows indeterminate growth, like Peocilozonites (Gould 1969).

Newly-hatched snails, less than 30 minutes old were observed from laboratory stocks of the Kisian and Kinyui collections. In addition, high- resolution SEM photographs were taken of specimens from the four PI samples involved in the numerical analysis.

(1) Newly-hatched Snails

Hatchling examination failed to provide any evidence of consistent delimitation of the new shell within the first 7/8 whorls (using a light microscope at X50 magnification). On hatching, a snail is born with a shell having completed approximately 6/8 to 7/8 of a whorl. It is possible to recognise a region where the embryonic punctae give way to striae, lying approximately 5/8 of the way along the shell. There appears to be no difference between the Kisian and Kinyui snails, of which five from each locality were examined.

2. SEM Photographs

Even using these more detailed images (plates 5-15) the protoconch margin proved impossible to delimit. There are no visible interruptions in the sculpturing pattern, nor any prominent growth ridge. However, the transition zone between the punctated region and the accretional region can be seen in much greater detail (Plate 6). From the six shells studied, this transition occurs consistently at about 5/8 whorl (confirming observations using the light microscope). It was too difficult to find any transition in this region when studying the Aram Market shell photographs (Plate 14) because of the apparent lack of striation on the first two whorls. This transition zone appears most obvious in the Kisumu Abattoir sample (B nasutus shell form). However, it is not as clear cut as shown in plate 12.

From this brief study, there appears to be no justification for suggesting the existence of a protoconch margin in the Kenyan B africanus group. Consequently, the study to compare protoconch differences between samples was abandoned.

b. Examination of Shell Microsculpture

Since there are a number of problems associated with regular surveillance of shell microsculpture using SEM, particularly with measurement of size and density, a detailed descriptive account of the sequential arrangement and development of the different elements is presented below.

Plates 5-15 show the SEM photographs representing each sample. Three shells from each of the PI samples were examined.

Three basic types of microsculptural elements are present:

i. Punctae - generally confined to the apical whorl; these are thelinearly-arranged pin-prick depressions seen in plate 5 for example.

ii. Transverse ribs (or striae) - confined mainly to the second whorl;

iii. Nodules - on the third whorl and beyond, depending on the sample. These appear to be unique to the B africanus group (Brown 1980).

1. Punctae

Plate 8 taken from a Kapsimotwa D a m specimen (B africanus form), shows details of the punctal arrangement on the apical whorl prior to striae formation. Punctae are arranged in a series of transverse rows which often bifurcate. In addition, they form a wider-spaced spiral progression (Plate 15) up to 22 rows wide. No observable shape or distributional differences were apparent, except in the Aram Market specimens (B ugandae form). Plate 15 shows that punctae appear to be more extensively distributed, especially on the second whorl, due to the lack of other microsculptural elements). Walter’s (1962) observations on differences in 'the relative coarseness of punctae' were not noticed.

122

2. Transverse Ribs

Transverse ribs begin to appear at approximately 5/8 whorls in most specimens Plate 5. Punctae become incorporated into the rib as a pit Plate 7, only on the face directed down the spiral. This does not occur in the Aram Market sample (Plate 15). When the rib is viewed from an angle looking down the spiral (Plate 10 these may give a delicate corrugated appearance, especially on the second whorl. The absence of ribs on Aram Market specimens (B ugandae forms) gives the shell an almost smoothe appearance under SEM, so that the punctae continue onto the second and third whorls as well spaced pits.

3. Nodules

Just as the relatively weak striae gradually strengthen into large ribs, so there is a further progression as the ribs appear to broaden and disrupt into smaller transverse segments (Plate 6) and the pits disappear. These transverse segments form nodules, characteristically rounded at the growing edge with a flattened trailing edge. Their asymmetry may be explained by the position of pits on the ribs, which are also asymmetrically distributed, as mentioned previously (Plate 11). Nodule development in the

Kisumu Abattoi r sample (B globosus shell form) is extremely weak (Plate 13), and under a light microscope may be missed. This is due in part to the relatively weak transverse ribbing found in the sample. Both the Kapsimotwa D a m (B africanus form) and Mi wan i

(B nasutus form) samples have stronger post-punctae microsculpture than the other two samples, with relatively strong transverse ribs (Plate 7 ) in particular. However, the nodular sculpture is rather weaker in the Kapsimotwa D a m sample (Plate 9) than in the Miwani sample (Plate 11) on all whorls.Nodular microsculpture is most developed on the end of the second and start of the the third whorls, with up to 17 nodules occurring in a transverse row on the second whorl, and up to 28 on the fourth whorl (B nasutus form). Like punctae, these transverse rows of nodules are not always uniform and may bifurcate in places (Plate 11). No shape differences are apparent between the samples, although nodules appear fainter and more elongate as they progress down the spiral. In the Miwani sample,nodules are present even on the fourth and fifth whorls, but in the Kapsimotwa Da m sample, they peter out by the middle of the fourth whorl, and the shell is smooth from then on. In the Ki sumu sample, no nodules were apparent (when they did occur) beyond the third whorl.

Overlap between the different microsculptural elements is very much in evidence, particularly between the ribs and nodules. Rib pits appear to be gradually restricted to the edges of the whorl, near the suture, as the nodules developed (Plate 7). Walter (1962) considered punctae to be well differentiated from the other elements. However, he was impressed with the idea that post- embryonic sculpture 'evolves' from punctae, as this present study suggests.

Abattoir

There are a number of fundamental problems when analysing shell microsculpture using SEM. Since specimens are fixed to the mounting stub and then gold plated, they are unusable for further morphometric study. In addition, a great amount of time (and expense) is necessary to carry out a statistically valid quantitative sampling of shells. Since shells are immobilised on the stub, they cannot be rotated sufficiently to compare sculptural progression over more than about 5/8ths of any given whorl. To ensure that ontogentically equivalent sections of shell are compared between all shells, each specimen would need to be orientated in a similar manner to that described for obtaining the W O V in the numerical analysis (section 3.1). At such high magnifications as the Scanning Electron Microscope affords, problems of scale and curvature deviations complicate any quantitative assessment of element size or density.

Using a light microscope, a maximum magnification of X50 was obtained, so that it was exceedingly difficult to assess accurately size and density of the various elements, particularly the punctae. In addition, sculpturing varied greatly in shape and distribution according to the light conditions.

124

PLATE 5 - B.africanus FORM, KAPSIMOTWA DAM x 190

PLATE 6 - B.africanus FORM, KAPSIMOTWA DAM x i

125

PLATE 7 - B.africanus FORM, KAPSIMOTWA DAM x 123

P L A T E 8 B .africanus FO R M . KA P SIM O TW A DAM x 342

126

PLATE 9 - B.africanus FORM, KAPSIMOTWA DAM

127

PLATE 10- B.nasutus FORM. M1WAN1 x 133

PLATE 11 B.nasutus FORM, MIWANI x H 3

PLATE 12- B.nasutus FORM, MIWANI x 39

PLATE 13- B.Rlobosus FORM, KITSUMU ABATTOIR x 72

129

PLATE 14- B.ugandae FORM, ARAM MARKET x 140

PLATE 15- B.ugandae FORM, ARAM MARKET x 17

CHAPTER 5 - DISCUSSION5.1 NUMERICAL DISCUSSIONBrown et al (1986) stressed the importance of improving standardisation of variables in the study of Bulinus. Morphometric studies of the bulinid shell present unique problems in this respect since all species exhibit indeterminate growth so size-related variables cannot be standardised simply by selecting shells of mature forms, such has been carried out by Woodruff and Gould (1980) on the land snaiiCerion. Shells of Bulinus also lack other ontogenetic markers - it has been shown already that a definable protoconch margin is absent, for example. To overcome this problem, Gould (1969), working on fossilised shells of the bermudan land snail Poecilozonites, recommended that all shell measurements are taken at standard whorl widths. In this way the effects of differences between shells of different sizes might be overcome. However, apertural and columellar measurements could only be obtained by either structural damage to the shell or by means of X-Ray photography (expensive and time consuming). An alternative approach to shell standardisation, would be to obtain mass curves from each sample of each variable plotted against shell length. A standard variable measurement could be obtained for a standard shell length, within the region of isometric shell growth. However, this technique severely restricts the expression of sample variation for a given variable, so that the range of sample variation cannot be accurately assessed. A final approach to standardisation of variables in relation to shell length, would be to divide O T U ’s into size classes small enough so that the effects of allometric growth are minimalised. Morphometric analysis would then be carrried out separately on each class.

The present study involved standardisation of about half the shell variables according to whorl number, so that measurements taken from the W O V are comparable between specimens of different size. The two correlation matrices, for PI (Fig 10) and P2 (Fig 19), show that this has been effective, since none of the W O V variables correlate with L (nor any of the L - related variables). Ontogenetic differences were confined to variables taken from the AOV. Three main factors influenced sample distribution in PI and P2:

1) Individual O T U variation - the most important factor. In P2, the range of individual variation has been such that sample overlap is continuous in most cases.

2. Isometry - separation of Kapsimotwa Da m and Asembo Bay snails in PI was enhanced by the non-overlap in shell length between the two samples. Since a number of variables correlate highly with L, the isometric loading effect on PCI is significant. In P2 however, where sample overlap in shell length is extensive, isometric differences would have served to reduce sample cohesion on PCI.

3. Allometry - this acts to reduce sample cohesion. However, its effects are difficult to determine, and also difficult to remove. To a large extent, its effects are masked by the influence of individual variation, as shown by the Kapsimotwa Da m and Asembo Bay samples in PI. In PI, it appears that significant allometry exists among certain size-related variables, particularly in the B.nasutus group, although this appears to have had little affect on sample cohesion.

Allometric growth occurs when differential rates of accretional growth occur at the apertural lip in snails of different shell length. This results in a continual change in shell shape with shell length; in a morphometric analysis involving shell variables, this reduces the overall similarity between a sample of snails taken from the same population, especially between the older, usually larger specimens and the younger, usually smaller specimens.

131

3. (Cont’d)

Allometric effects may be partly responsible for the poor resolution of the P2 samples, although undoubtedly most of this is due to individual variation. The bivariate plots of snails from the Kisian stream (Figs 18.1-3) show that smaller specimens of the S and A subsamples are far less clearly distinguished from each other than are the large snails. This suggests that allometric effects gain significance in snails greater than about 13 mm.

PI Principal Component Analysis gives incomplete separation of the samples, although 3 clusters appear to be present. Removal of low-scoring eigenvector variables produced no significant change in the ordination probably because the effects of individual variation are too large for it to make any difference. Canonical Variate Analysis gave the clearest resolution of the PI samples since it enhances intersample variation whilst minimising it within each one. These PI analyses suggest that:

(1) The B.nasutus form is most clearly separated from the other three forms using variables A, HW2, H W 3 in Principal Component Analysis. In Canonical Variate Analysis, variables L and BWSA separate it clearly from the B.africanus form. Variables a, A and L also separate it from the B.globosus and B.ugandae forms.

(2) The B.africanus form is separated in Principal Component Analysis from the B.ugandae form by a number of size-related variables -BWL, c, W, L, AL, AW and WO. In Canonical Variate Analysis, variables L and BWSA in particular separate the B.africanus form from the other three samples.(3) The B.globosus form separates from the B.ugandae form using variables W W 2 and W W 3 and Principal Components Analysis. In Canonical Variate Analysis, separation is achieved using L, W and A.

These PI results give support to the theory that the B.ugandae form is a localised (perhaps ecophenotypically modified) form of the more widespread species, B.globosus. (Mandahl-Barth (195*0 originally described the ugandae taxon as B.globosus ugandae). From Principal Component Analysis, there is extensive overlap between the Kisumu Abattoir and Asembo Bay samples. (In addition, in the Principal Component Analysis with isometric - size effects removed, they overlap extensively). In the Canonical Variate Analysis they are adjacent in multidimensional space, with a very small Mahalanobis Distance between them. This short separation is accounted for mainly by differences in L and W values, along with A.

The Principal Component Analyses of P2 illustrate a common problem in resolving populations of Bulinus. With an increased number of samples and shells, the range of character overlap is too great for any resolution. However, samples of the B.nasutus and B.africanus forms from PI are well separated using variables d, a , a /\A ana a f a , emphasising the importance oi shape differences between these two samples. P2 Canonical Variate Analysis shows just how extensive variable overlap is between the samples. However, it also confirms the morphological separation of the Kisian Stream S sample and A sample, using variables z, x, WH2, d and L. This separation supports Jelnes claim (1979b) for the existence of two types of snail in the stream, based on the discovery of two electromorphs, having different enzyme mobilities for PGI. The existence of two sympatric morphotypes lends further support for the existence of two separate species in the stream. The P2 Canonical Variate Analysis also clearly distinguishes within the main group, between the B.globosus/ugandae forms of Asembo Bay/Aram Market, Shamba and Kisumu Abattoir from the B.africanus forms. The Mahalanobis Distance dendrogram gives support to the idea of 3 morphotypes: (H-L) B.africanus form, (O-N)B.globosus/ugandae form and (S - R), the distinct B.nasutus forms. P2 analysis thus also gives evidence to support the conspecifity of B.globosus and B.ugandae forms in Western Kenya.

5.2 VARIABLE ASSESSMENTAmong the most useful variables used in the multivariate analyses have been the angles A, BWSA, A A A and APA. A is particularly useful since it does not correlate with L, whereas the other three do at a low level. Generally, the W O V variables were insignificant in the resolution of samples, including those from the fourth and fifth whorls. However, variables W W 3 and W W 2 were useful in resolving the PI B.globosus and B.ugandae forms. H W 2 and H W 3 were useful in separating the B.nasutus form from these two samples. The importance of the columella variables should be treated with caution. The Principal Component Analysis in P2 with isometric size-effect removed is dominated by variables x, z and w. However, the Asembo Bay and PI B.africanus forms are plotted close together, yet the Asembo Bay sample has almost no columellar ridge, whereas it is very strongly developed in the B.africanus sample (The same ordination was obtained when the outliers were removed). These three columellar variables are small to draw and measure. Fig 6.3 shows that they all lie in the same plane of measurement, ie, perpendicular to the longitudinal shell axis. Their accurate standardisation depends heavily on the exact orientation of the shell as it is swivelled around the longitudinal axis (like a top). The margin of error, and hence the standard deviation of these variables is large. Their contribution to eigenvetor scores is disproportionate, and they would appear unsuitable for morphometric quantification. The other columella variable, y, appears to contribute little to the resolution of different samples. Future assessment of the columellar ridge should perhaps be carried out visually, giving scores for the degree of development. It is difficult to assess the importance of size-related variables in resolving the different samples, once the isometric effect has been removed, due to the predominance of the columellar variables. In addition, many of them are much less important in Canonical Variate Analysis than in Principal Component Analysis. The bivariate separation of the two Kisian Stream morphotypes suggests that certain ratios of these variables with shell length are important. The incorporation of ratios in future studies would preclude further isometric transformations amongst variables showing a high correlation with shell length.

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5.3 DISCUSSION O F 1SOELETROPHORESIS RESULTS

There are four important points to bear in mind when interperating enzyme electrophoretic patterns:-

1. Enzyme polymorphism.

2. Parasitism - extra bands may be separated using infected snails, since parasite homogenate is present in the extract. Wright, Rollinson and Goll (1979) were able to identify different parasites in Bulinus senegalensis from the pi of these additional bands, when compared to the banding pattern obtained from uninfected snails.

3. Post-transcriptional enzyme changes - bands are not necessarily the direct expression of an allele.

4 Gel differences between plates stained for the same enzyme - since 16 plates were run, only the most representative samples from each system are presented with this study (plates 1-4). The clear AcP separations (plate 1) were produced from large amounts of extract. The GPI separations were generally less successful (plate 3). Trails in the«'koitn«. end of the plate

(towards the top of plate 3) indicated slight enzyme degradation in some extracts, although this has not affected the main pattern. Since this was the last enzyme system run, alternate freezing and thawing may have caused this. Wave patterns were also seen across certain plates, towards the acid end of the gel; however, there are as yet no satisfactory explanations to account for this (Ross, pers comm).

Separation of the various samples proved far easier than in the morphometric analysis. This was facilitated by the general lack of heterozygosity, and the low level of parasite infection. However, with only four enzyme systems analysed and only 75 snails characterised, no general conclusions can be drawn, nor can any adequate assessment of either individual or sample variation be made. Until breeding experiments are carried out, the degree of influence of post-transcriptional change on the banding patterns remains unknown.

With these factors borne in mind, the results suggest that:-

1. Kisian Stream snails are very similar enzymatically. Fig 18.1, showing the bivariate plot of USH against L for these snails, shows that all the specimens chosen for IEF (white circles) correspond to the africanus form. Thus, the nasutus form is not represented in IEF, so the apparent uniformity of enzyme patterns does not contradict Jelnes (1979b) observation of two PGI electromorphs in the stream. The Kisian Stream snails represented in IEF are also close to those of the Migosi and Paponditi localities, especially on AcP, GPI and H B D H patterns.

2. Asembo Bay, Dunga Beach and Shamba snails share the same distinct basic patterns. These are different from (1). Thus, these IEF results support the existence of these two separate morphotypes. Since the Shamba IEF banding patterns are similar to those of Asembo Bay and Dunga Beach, further support is given to the conspecificity of these two shell forms.

3. The Kinyui sample, from Central Kenya, has AcP and GPI banding patterns similar to (1), supporting the morphological similarity of these populations, seen in P2 Canonical Variate Analysis. This confirms Jelnes (1979b) who reported snails from this locality with the B africanus PGI pattern.

4. The coastal snails appear enzymatically more variable, but nevertheless, consistently different from the other inland samples, with the exception of certain snails from the Nimbodze Dam. 4/6 of these snails gave enzyme patterns very similar to (1). Visual inspection of the coastal snails however, suggests the presence of only a single morphotype, corresponding to B nasutus nasutus. This includes snails from Tiwi Da m from where Jelnes (1979b) reported snails giving the B nasutus PGI banding pattern.

A greater difference was noted in the present work between the enzyme banding patterns corresponding to the B africanus and B globosus/ugandae forms around Lake Victoria than Rollinson and Southgate (1979) found along the south-eastern shore of the lake in north-east Tanzania.

In general, therefore, IEF results clearly confirm the morphometric observations. It is not possible to characterise individual populations from these results since minor banding differences between samples were not consistent. However, the results do reflect differences at a higher taxonomic level, between population groups. In order to characterise adequately individual populations, more extensive sampling, involving a greater number of enzyme systems is required. The identification of allozymes would then be followed up by an assessment of allelelic frequencies for each enzyme system. This could give some indication of genetic distances between sample populations. However, to establish the genetic basis for phenotypic banding patterns, breeding experiments are required (Johnson, 1975). The concept of a ’biotype* has been put forward (Bullini, 1982) to describe populations having distinct allele frequencies (as, for example, of allozymes). However the taxonomic status of such a concept is as yet unresolved (similar to the Variety* argument raised in section 2-3, to describe lichen morphotypes having distinct lichen acids).

5.4 EGG PROTEINS DISCUSSIONWright and Rollinson (1979) stated their view that egg protein electrophoretic patterns have contributed little to the resolution of similar taxa and that their taxonomic use is primarily supportive.

The results from the present study show great variability, even within the small number of snails examined. It would be rash to attempt to extrapolate these results into any sort of conclusion. However, with an a priori knowledge of the relationships between the different samples, based on the morphometric and IEF results, it would aPPear' judged by the anodal peak, that the Migosi, Paponditand Kisian Stream samples are distinct to the Western Kenyan B africanus forms. The B globosus/ugandae forms from Western Kenya lack this peak, i Asembo Bay, Suamba and Dunga Beach snails. The Kinyui snails also appear to show this peak, although overall it is more akin to the distinctive coastal snails, with their three large peaks. Overall, these results accord with the morphometric and IEF results, especially in separating the Western Kenyan africanus and globosus/ugandae forms. In addition, the distinct character of the coastal snails, represented by the Margiza and Maridzani specimens is supported.

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Rollinson and Southgate (1979) observed that snails identified as B nasutus from Tanzania had distinctive egg protein profiles (compared to africanus, ugandae and globosus forms). This would match the Kenyan coastal snails if their preliminary identification as B nasutus nasutus is proved correct.

5.5 PARASITOLOGICAL DISCUSSIONIt is very difficult to generalise about the parasitological characteristics for a given population of Bulinus. Even within susceptible populations, there may be many individuals which prove refractive to miracidial infection. The taxonomic significance of parasitological results is therefore strictly limited. When examining wild caught snails, this situation may be exacerbated by differential survival of non-infected specimens in transit. In addition, rearing laboratory stocks usually involves breeding from non-parasitised snails since most of the infected individuals fail to lay eggs.

These results are interesting therefore from a parasitological point of view rather than a taxonomic one. Apart from the above considerations, the sample sizes are too small anyway to give any firm conclusions.

The presence of S bovis in Kisian - 3 and 4 confirms the data presented by Wright and Rollinson (1979) and by Southgate and Knowles (1975). The latter authors found a prevalence of 76% here, suggesting a consistently high level of snail infection. Both pools are used daily by cattle for drinking, unlike pools 1 and 2 nearby, which were much smaller, with steep sides. This might explain the lack of S bovis in these pools. Migosi was also easily accessible to cattle, although none were seen. Members of the B forskalii and B truncatus groups also carry S bovis in Western Kenya (Southgate and Knowles, 1975), although B ugandae does not appear to.

Sturrock (1985) has reported the presence of S haematobium at Msambweni on the Kenyan coast. The identity of the host snail is unclear at present, although B globosus has been implicated.* Prevalence of infection is extremely low here 8/5000 snails examined = 0.19%), so it is not surprising that none of the coastal material was found to be shedding cercariae. The fish heterophyid infections may have been responsible for the poor egg-laying success among coastal samples.

IEF shows the 2 additional Kisian-*f snails carried infections. This is strongly implied by the additional bands obtained from 3 of the enzyme systems :

AcP (pattern 2, 3 bands between pH 6.3-6.7 and 2 bands between pH 7.0 and 7.1), GPI (pattern 1, 6 bands between pH 6.5 and 7.6, and pattern 2, between pH6.5 and 7.5), and M D H (pattern 1, 2 bands at pH 7.3 and 8.0).

elsewhere on the coast.

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5.6 S U M M A R Y

Since this is only a preliminary investigation of the Bulinus africanus group in Kenya, it would be unwise to draw any firm taxonomic conclusions from the results obtained. The study involved only a limited number of samples, and particularly in the IEF, far fewer variables were analysed than would be desirable to be sure of a representative analysis. Although the numerical analysis is, by and large, objective in data manipulation, there are a number of different algorithms which could have been chosen to resolve data. Each may give different results, depending on how they are applied. Since results may depend on the subjective selection of the numerical technique, the argument of subjectivity can be aimed not only at the traditional malacologist, but on occasions to the numercial taxonomist as well. This present study does have the advantage over more ambitious projects in that most samples came from the same geographical area, so that the effects of geographical variation were minimised.

With these factors in mind, the following main points summarise the findings of this study:-

1. Morphometric analysis suggests the existence of three distinct shellmorphotypes in Western Kenya, corresponding to the B africanus, B nasutus productus and B globosus/ugandae forms. Separation of the B africanus and B nasutus forms as biological species is further supported by their sympatric occurrence in the Kisian Stream. Support is also given to the conspecificity of B globosus and B ugandae forms, which showed extensive morphological overlap. Where clear separation was apparent, size-differences were important. However, since this is based on the preliminary identification of one B globosus sample and the B ugandae samples, the general significance of this is open to question.

2. The most useful variables found for separating Western and Central Kenyan samples were the angles A, APA, A A A and BWSA, as well as WH2, WH3, W W 2 and WW3. Undoubtedly, a number of size-related variables are also useful. However, their importance is obscured by isometric and allometric effects.

3. Principal Component Analysis gives limited resolution of more distinctive morphosamples in PI, but is not sensitive enough to separate less distinct samples in P2. Canonical Variate Analysis proves more useful since it maximises intersample variation at the expense of intrasample variation.

4. Microsculptural analysis confirms the lack of a distinct protoconch margin in the Bulinus africanus group in Kenya. Microsculptural elements and their ontogenetic sequencing was described. The absence of well- developed ribbing and the absence of nodulation is seen in the B ugandae form. The reduced rib development and poor nodulation of the B globosus form is noted. The presence of extensive nodulation in the B nasutus form is seen, and its weaker, less extensive development observed in the B africanus form. However, the unsuitability of microsculptural differences as taxonomic criteria is discussed.

5. IEF gives evidence of consistent differences in the banding patterns of snails corresponding to B africanus and B globosus/ugandae forms, especially in enzymes AcP, GP1 and HBDH. Evidence is also given of two coastal forms - certain snails from Nimbodze D a m gave enzyme patterns similar to the B africanus forms, whereas the other coastal snails appeared enzymologically distinct.

137

6. Egg protein patterns highlighted the distinction of coastal snails. Differences between Western Kenyan B africanus and B globosus/ugandae forms were also observed.

7. These results reinforce the current approach in bulinid taxonomy that the shell cannot be used on its own as a source of taxonomic variables.

S. Future research on the Bulinus africanus group in Kenya involves many areas of study. Morphological re-assessment of the coastal snails in a similar numerical analysis is important. However, more extensive characterisation of further samples is paramount. Specifically, the characterisation of enzymes of the Kisian B nasutus form is important, although this applies to many other Kenyan populations as well. Until there is more extensive analysis of enzymes and an assessment of allele frequency, a comprehensive characterisation of the Bulinus africanus group using shell variables will be slow.

138

Acknowledgements.

It is a pleasure to thank Dr. David Brown, my supervisor at the BM(NH) for all his help and advice and Professor Roy Anderson, my Imperial College supervisor, for his valuable support.

From the BM(NH) I am particularly grateful to Mr. Graham Ross and Ms. Jill Lines for their help with the IEF work.

Thanks are also due to Professor G. Kinoti of the University of Nairobi and to Mr. Crispin Apat for his help and assistance in the field.

Finally I should like to thank my parents, Mr. Peter Archer and Mrs. Margaret Archer and my wife Mandy, for the tremendous patience and support they have given me during the writing of this thesis.

This study was supported by a Medical Research Council 3grant sibo.

grant from the

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67. L O W E R , E S, MOYO, H G and GARDNER, S L (1981). Trematode - Gastropod Associations in nine non-Lacustrine Habitats in the Mwanza Region of Tanzania. Parasitology, 83: 381-399.

68. MACDONALD, G (1965). The Dynamics of Helminth infections, with special reference to Schistosomes. Trans Roy Soc Trop Med Hyg, 59: 489-506.

69. MA ND A H L - B A R T H , G (1954). The Freshwater Molluski of Uganda and adjacent territories. Annls Mus R Congo Beige, 8°, Zool, 32: 1-206.

70. M A ND AH L- BA RT H, G (1957). Intermediate hosts of Schistosoma African Biomphalaria and Bulinus: ii. Bull Wld Health Org., 17: 1-65.

71. M A N D A H L - B A R T H , G (1958). Intermediate Hosts of Schistosoma African Biomphalaria and Bulinus. Wld Health Org Monogr Ser., 37: 132 S; Geneva.

72. M A ND AH L- BA RT H, G (1960). Intermediate Hosts of Schistosoma in Africa. Some recent information. Bull Wld Health Org., 22: 565-573.

73. MA ND A H L - B A R T H , G (1965). The species of the Genus Bulinus, intermediate hosts of Schistosoma. Bull Wld Health Org., 33: 33-44.

74. MA ND A H L - B A R T H , G (1968). Revision of the African Bithyniidae (Gastropoda:Prosobranchia). Review De Zoologie Et Botanique Africaines, 78: 129-160.

75. M A ND AH L- BA RT H, G, FRANDSEN, F and 3ELNES, J E (1976). Bulinus sp (2n = 26) (from Salisbury, Rhodesia, a close relative of B.truncatus (Audouin), being a potential intermediate host for Schistosoma haematobium in South EastAfrica. Trans Roy Soc Trop Med Hyq, 70 s 88.

76. MARDIA, K V, KENT, J T and BIBBY, 3 M (1979). Multivariate Analysis. London: Chapman and Hall.

77. MARTENS, E (1897). Beschalte Weichtiere Deutzche-Ost-Afrika (Berlin).

78. MA YR , E, LINSLEY, E G and USINGER, R L (1953). Methods and Principles of Systematic Zoology. McGraw-Hill, New York and London.

79. Mc CU LL OU GH , F S (1957). The seasonal density of populations of Bulinus (Physopsis) globosus in natural habitats in Ghana. Ann Trop Med Parasit, 51: 235.

80. MORELET, A (1866). Coquilles Nouvelles Recueillis Par Le Dr Fr Welwitch Dans L'Afrique Equatoriale. 3 Conch Paris, 6: 153-163.

81. MORELET, A (1868). Mollusques Terrestres Et Fluviatiles Voyage Du Dr Friedrich Welwitsch. Paris:Bailiere.

82. M O R G A N , E and LAST, V K (1982). The behaviour of B.africanus: A circadian profile. Animal Behav, 30 (2): 557-567.

83. MOZELEY, A (1939). The freshwater Mollusca of the Tanganyika Territory and Zanzibar Protectorate and their relation to human schistosomiasis. Trans R. Soc. Edinburgh, 59: 687-744.

84. MULLER, O F (1781). New name for 'Le Bulin' Adanson, 1757, Histoire Naturelle de Senegal; Histoire Des Coquilles Per Naturforscher, Halle, 15: 6.

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145

85. OBERHOLZER, G (1970). Contributions to the Morphology, Distribution and Taxonomy of some African Species of the Subgenus Bulinus (Muller) (MolluscatBasomatophora). Um»u6usrtet> thesis presented ppg -the oe&Ree t*>c-T&R ScteN-rAE.S outh Africa • W & stetrn 6wpg Potchefstoom Umiv 8.86. OBERHOLZER, G, BROW N, D S and V A N EEDEN, 0 A (1970). Taxonomic characters of the Radula in the Bulinus natalensis/tropicus Complex in Eastern Southern Africa. Western Bydr Potchefstroom Univ B., 10: 1-41.

87. O ’KEEFE, J H (1985a). Population biology of the freshwater snail Bulinus globosus on the Kenya coast. I. Population Fluctuations in relation to climate. Journal of Applied Ecology 22: 73-84.

88. O ’KEEFE, J H (1985b). Population biology of the freshwater snail Bulinus 9lobosus on the Kenya coast. II. Feeding and Density Effects on Population Parameters. Journal of Applied Ecology, 22: 85-90.

89. PAGGI, L ORECCHIA, P, BULLINI, L, NASCETTI, G and BIOCCA, E (1978). Studi Morfologici, Biologici E Biochemici Su Una Nuova Specie Di Bulinus (Gastropoda:Planorbidae). Parassitologia, 20: 1-6.

90. PATTERSON, C M and BURCH, J B (1978). Chromosomes of Pulmonate Molluscs. Pulmonates Systematics, Evolution and Ecology. Academic Press, New York, 2A: 171-217.

91. PORTER, A (1938). The Larval Trematoda found in certain South African Mollusca with special reference to Schistosomiasis. Pubis SmAfr Inst Med Res 42: 1-492.

92. PRETORIUS, S J (1979). The population dynamics of the pulmonate snail Bulinus (P) africanus (Krauss). The influence of temperature on mass increase and survival. Malacologia, 18 (1-2): 237-243.

93. PRETORIUS, S J (1982). Mark -Recapture Studies on Bulinus (Physopsis) africanus (Krauss) (Mollusca, Pulmonata), Malacologia, 22 (1-2) : 93-102.

94. PRIM, R C (1957). Shortest connection networks and somegeneralisations. Bell System Tech J, 36: 1389-1401.

95. PRINSLOO, J F and V A N EEDEN, J A (1973). The distribution of thefreshwater molluscs of Lesotho with particular reference to the intermediate hosts of Fasciola hepatica. \*/e.sT£Tzn Bhor f>oT H£FS'ffi.op<w U h w & jS"”! * •


96. ROHLF, F J (1970). Adaptive Hierarchical clustering schemes. Syst Zool. 19 : 58-82.

97. ROHLF, F J (1972). A empirical comparison of three ordination techniques in numerical taxonomy. Syst Zool 21 : 271-280.

98. ROHLF, F J (1973). Hierarchical clustering using the minimum spanning tree. Computer J 16.

146

99. ROLLINSON, D (1980). Enzymes as a Taxonomic Tool: A Zoologist’s View. Systematics Association Special Volume Number 16, "Chemosystematics: Principles and Practise", edited by F A Bisby, 3 G Vaughan and C A Wright, pp 123 - 146. Academic Press, London and New York.

100. ROLLINSON, D and SOUTHGATE, V R (1979). Enzyme Analysis of Bulinus africanus Group Snails (Moll Planorbidae) from Tanzania. Trans R Soc Trop Med Hyg, 73: 667-672.

101. ROSS, G C (1976). Isoenzymes in Schistosoma spp. : LDH, M D H and Acid Phosphatases separated by Isoelectric Focusing in Polyacrylamide Gel. Comp Biochem Physiol. B, 55: 343-346.

102. RU DOLPH, P H and WHITE, J K (1979). Egg laying behaviour of B.octoploidius Burch (Basommatophora: Planorbidae). Journal Moll Stud., 45(3): 355-363.

REFERENCES (Cont'd)

103. SALADIN, B, DE GR EM ON T, A and WEISS, N (1976). Isoelectric Focusing in the Taxonomy of Bulinid Snails. Acta Trop., 33: 376-379.

104. SHIFF, C J (1964 b). Studies on Bulinus (P) globosus in Rhodesia III. Bionomics of a natural population existing in a temporary habitat. Ann Trop Med Parasit, 58, 240.

ShiFFjIAfcM-R.SeeA d w t «o»j a l t o rEeeNce s p.m-9.

105. SHIFF, C J and COUTTS, W C C (1979). Seasonal patterns in the transmission of Schistosoma haematobium in Rhodesia and its control by winter application of molluscicide. Trans R Soc Trop Med Hyd, 73 (4): 375-380.

106. SMITH, V G F (1982). Distribution of snails of medical and veterinary importance in an originally polluted watercourse in Nigeria. Ann Trop Med Parasit, 76 (5): 539-546.

107. SMITHERS, O (1955). Zone electrophoresis in starch gels: Group variations in the Serum Proteins of normal human adults. Biochemical Journal, 61: 629- 641.

108. SMITHERS, S R (1956). On the ecology of schistosome vectors in the Gambia, with evidence of their role in transmission. Trans R Soc Trop Med Hyg., 50, 354.

109. SNEATH, P H A and SOKAL, R R (1973). Numerical Taxonomy, W H Freeman and Co, San Francisco.

110. SOUTHGATE, V R and KNOWLES, R J (1975). The intermediate hosts of Schistosoma bovis in Western Kenya. Transactions of the Society of Tropical Medicine and Hygiene, 69: 356-357.

111. SOUTHGATE, V R and KNOWLES, R J (1977). On Schistosomamargrebowiei Le Roux 1933: The morphology of the egg, miracidium andcercaria, the compatibility with species of Bulinus and development in MeSOCricetUS auratUS. z Parasitenkunde, S4-: 133 - zso .

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113. STURROCK, R F (1973). Field studies on the population dynamics of Biomphalaria glabrata, intermediate host of Schistosoma mansoni on the West Indian Island of St Lucia, International Journal of Parasitology, 3 : 165-17**.

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115. STURROCK, R F. Report on visit to Msambweni Schistosomahaematobium project, July 1985. Unpublished report.

116. TAGGART, J., FERGUSON, A and MASON, F M (1980). Genetic variation in Irish populations of Brown Trout (Salmo trutta L): Electrophoretic analysis of allozymes. Comp Biochem Physiol, 69 (B): 393-** 12.

117. TEESDALE, C (1962). Ecological observations on the Molluscs of significance in the transmission of Bilhagiasis in Kenya. Bull Wld Health Org, 27: 759.

118. V A N AARDT, W J and V A N EEDEN, J A (1969). Bydraes Tot Die Morfologie Van Bulinus (Physopsis) africanus (Krauss). Wetensk Bydr Potchefstroom Univ, 5: 1-72.

119. V A N AARDT, W J and FREY, B J (1981a). Oxygen-binding characteristics of the Haemolymph of the freshwater snail Bulinus (P) globosus. South Afr J Zool, 16 (1): 1-**.

120. V A N AARDT, W J and FREY, B J (1981b). Evidence for non-assimilation of Chlorella by the African freshwater snail Bulinus (P) globosus. South Africa J Sci, 77 (7): 319-320.

121. WALTER, H J (1962). Punctuation of the embryonic shell of Bulininae (Planorbidae) and some other Basommatophora and its possible Taxonomic- Phylogenetic implications. Malacologia, 1 (1): 115-137.

122. WEBBE, G (1962 a). Population studies of intermediate hosts in relation totransmission of Bilharziasis in East Africa. Ciba Foundation Symposium on Bilharziasis, pp 7-22. ____

123. WEBBE, G (1965). Natural trends in snail populations in relation to control of Bilharziasis in East Africa. East African Med J., 42: 605-613.

12**. WEBBE, G and MSANGI, A S (1958). Observations on three species of Bulinus on the east coast of Africa. Ann Trop Med Parasit, 52: 302.

\Alebs£J \W2b .Aoditicmal.KeTEfccwCES

*1.

125. WIUM-ANDERSEN, G (1973). Electrophoretic studies on esterases of some African Biomphalaria Sdp. (Planorbidae). Malacologia, 12 (1): 115-122.

126. WIUM-ANDERSEN, G (1974). A systematic examination of a Biomphalaria species from Namibia by means of esterase electrophoresis. Steenstrupia, 3: 183-186.

127. W O O D R U F F , D S and GOULD, S 3 (1980). Geographic Differentiation and Speciation in Cerion - a preliminary discussion of patterns and processes. Biol 3 Lin Soc., 14 : 389-416.

128. W O R L D H E A L T H ORGANISATION (1985). The Control of Schistosomiasis Technical Report Series, 728. World Health Organisation, Geneva.

129. WRIGHT, C A (1957). Studies on the structure and taxonomy of Bulinus jousseaumei (Dautzenberg). Bulletin of the British Museum (Natural History) Zoology, 5 (l): 1-28.

130. WRIGHT, C A (1959). The application of Paper Chromatography to a Taxonomic Study in the Molluscan Genus Lymnaea. 3 Linn Soc., XLIV (296) : 222-237.

131. WRIGHT, C A (1960 a) The crowding phenomenon in Laboratory Colonies of freshwater snails. Annals of Tropical Medicine and Parasitology, 54 (2): 224- 232.

132. WRIGHT, C A (1960 b) Relationships between Trematodes and Molluscs. Ann Trop Med Parasit.,54: 1-7.

133. WRIGHT, C A (1963a). The freshwater Gastropod Mollusca of Angola. Bull Br Mus Nat Hist., Zool., 10: 449-528.

134. WRIGHT, C A (1963b). Schistosomiasis in the Western Aden Protectorate: A preliminary survey. Trans Roy Soc Trop Med Hyg., 57.

135. WRIGHT, C A (1964). Biochemical variation in Lymnaea peregra (Mollusca:Basommatophora). Proc Zool Soc Lond,, 40: 277-286.

136. WRIGHT, C A (1971). Bulinus on Aldabra and the subfamily Bulininae in the Indian Ocean Area. Philosophical Transactions of the Royal Society, London, B 260: 299-313.

137. WRIGHT, C A and FILE, S K (1968). Digestive gland esterases in the genus Bulinus (Mollusca: Planorbidae). Comp Biochem Physiol., 27: 871-874.

138. WRIGHT, C A and KLEIN, 3 (1967). Serological studies on the taxonomy of Planorbid snails. 3 Zool, London, 151: 489-495.

139. WRIGHT, C A and ROLLINSON, D (1979). Analysis of enzymes in the Bulinus africanus Group (Mollusca: Planorbidae) by Isoelectric focusing. 3.Nat Hist 13: 263-273.


149REFERENCES (Cont'd)140. WRIGHT, C A & ROLLINSON, D (1981). Analysis of enzymes in the Bulinus tropicus/truncatus complex (Mollusca:PIanorMdae). Journal of Natural History, 13: 263-273.

142o f

141. WRIGHT, C A, ROLLINSON, D and GOLL, P H (1979). Parasites in Bulinus sene^alensis (MolluscasPlanorbidae) and their detection. Parasitology, 79: 95- 105. *—

. WU, S K (1 9 7 2 ) . C o m p a ra t iv e s tu d ie s on a p o ly p lo id s e r ie s th e A f r i c a n Genus B u l in u s t r u n c a t u s . Ann t r o p Med P a ra s i

WRI6HT 8.Ross ,1^ 5- WflMbKT 8.Ross.inu*.See

Additional ReFBeerjtes • / fie tow.12 : 51 - 5 8 .

ADDITIONAL REFERENCES :

BARRET, J and YONGE, C M (1958). Collins Pocket Guide to the Sea Shore : 134. London and Glasgow : Collins.

BROWN, D S and ROLLINSON, D (1982). The southern distribution of the freshwater snail Bulinus africanus. S. Afr. J. Sci, 78 : 290-293.

BULLINI, L (1982). Genetic, Ecological and Ethological Aspects of the Speciation Process. In C Barigozzi (Ed), Mechanisms of Speciation, : 241-264. New York : Liss.

SHIFF, C J (1964a). Studies on Bulinus (Ph .) globosus in Rhodesia.I. The influence of temperature on the intrinsic rate of natural increase. Annals of Tropical Medicine and Parasitology, 58 : 94-105.

WEBBE, G (1962b). The transmission of Schistosoma haematobium in an area of Lake Province, Tanganyika. Bull. Wld Hlth Org, 27 : 59-85.

WRIGHT, C A and ROSS, G C (1965). Electrophoretic studies of some planorbid egg proteins. Bull. Wld Hlth Org, 32 : 709-712.

WRIGHT, C A and ROSS, G C (1966). Electrophoretic studies on planorbid egg proteins. The Bulinus africanus and B. forskalii species groups. Bull. Wld Hlth Orq, 32 : 727-731.

150

APPENDIX 1

1) D A T A F O R SNAILS F R O M PRESERVED

2) D A T A F O R FIELD CO LL EC TE D SNAILS.

COLLECTION.

TABLE 1 - P I PROGRAM - PRIMARY DATA MATRIX FOR PRESERVED KENYAN SNAILS 151(ALL M E AS UR EM EN TS GIVEN AT X6 MAGNIFICATION)

PC/LRNO:

L(mm)

W(mm)

Wo(l/16 of a whorl)

AL1(mm)

AW1(mm)

B W L(mm)

BWSA(Deg)

a(mm)

c(mm)

w(mm)

X

(mm)y(mm!

3(mm)

d(mm)

H W 2(mm)

W W 2(mm)

H W 3(mm)

W W 3(mm)

H W 4(mm!

W W 4(mm!

B4(mm!

B5(mm!

A(Deg)

H5(mm)

W 5 (mm)

3746-20

TI8J) 80.4 84.0 82.0 39.0 T 5 S 3 52.0 80.0 35.5 "OcT 3.50 T 7 X 4.50 19.0 T o o " 2.50 4.00 5.50 11.0 11.0 20.5 47.5 119.5 33.5 56.0

3746-18

108.0 71.5 77.0 70.0 37.5 98.5 39.5 70.0 32.0 7.25 3.25 17.0 2.25 16.0 1.00 3.50 3.50 7.00 13.5 17.0 25.5 27.5 124.0 40.0 59.0

3746-17

102.5 70.0 76.0 69.5 34.5 93.0 51.0 69.0 30.5 5.75 3.00 15.5 1.75 16.5 1.00 3.00 3.00 7.50 11.5 17.0 26.0 27.5 115.0 38.5 58.0

3746-16

89.5 64.0 75.0 61.5 32.0 82.5 39.0 60.0 26.5 3.75 3.00 11.5 0.75 13.0 0.50 3.00 2.00 8.00 10.0 17.0 21.0 49.5 128.0 36.0 56.0

3746-15

99.5 69.0 78.0 70.0 35.0 90.0 54.0 66.0 29.0 5.00 3.75 15.0 1.25 15.0 1.00 3.00 4.00 6.50 13.0 13.5 22.0 55.0 114.5 34.0 55.0

3746-14

105.5 69.5 76.0 73.0 36.5 97.0 44.5 73.5 31.0 6.00 2.25 19.3 1.75 16.0 1.00 3.00 3.50 7.50 12.0 16.5 25.5 53.0 118.0 39.0 58.0

3746-12

107.5 74.5 79.0 78.0 37.0 99.0 51.5 75.5 34.5 6.25 2.50 18.3 1.75 19.0 1.50 3.00 4.00 6.50 12.5 15.0 24.0 56.0 115.0 36.0 57.5

3746-10

92.0 60.0 75.0 57.5 29.5 83.0 42.5 59.5 24.5 4.25 3.75 14.0 1.25 11.5 1.50 3.00 3.00 7.00 11.0 15.0 24.0 56.5 113.5 38.0 53.5

3746-09

92.0 61.0 76.0 63.0 29.5 84.0 51.0 60.0 25.0 5.50 3.00 14.3 1.25 11.5 1.50 3.00 3.50 6.50 9.5 15.0 20.5 50.0 115.5 34.0 53.5

3746-OS

96.0 68.0 73.0 69.0 32.0 89.0 55.0 65.5 28.5 4.00 3.25 14.0 2.50 14.0 0.50 2.50 1.50 6.00 7.0 16.0 13.5 42.5 134.0 38.0 59.0

3746-06

95.0 65.5 76.0 61.0 31.5 86.0 44.5 57.5 24.0 4.50 3.00 12.8 1.50 12.5 1.50 2.50 4.00 7.00 10.5 15.5 23.0 53.5 109.5 38.0 57.0

3746-05

100.0 70.5 81.0 70.0 36.0 90.5 51.0 69.5 31.0 5.00 3.25 16.5 1.50 16.0 1.50 2.50 3.50 5.50 8.5 10.0 19.0 50.5 104.0 31.0 50.5

3746-01

109.5 73.5 83.0 75.0 36.5 100.0 47.5 69.5 31.0 4.00 1.25 17.0 5.25 13.0 1.50 3.50 4.50 7.50 11.5 13.0 17.0 48.5 113.5 28.0 53.0

TABLE 1 - PI PROGRAM - PRIMARY DATA MATRIX FOR PRESERVED KENYAN SNAILS 132(ALL MEASUREMENTS GIVEN AT X6 MAGNIFICATION)

PC/LRNO:

L(mm)

W(mm)

Wo(l/16 of a whorl)

ALt(mm) AW1(mm)B W L(mm)

BWSA(Deg)

a(mm)

c(mm)

w(mm)

X(mm]

y(mm)

3(mm)

d(mm!

H W 2(mm)

W W 2(mm)

HW 3(mm)

W W 3(mm)

H W 4(mm!

W W 4(mm)

B4(mm!

B5(mm!

A(Deg!

H5(mm) W 5(mm)

3746-21

82.0 54.5 74.0 57.0 25.5 76.0 59.5 55.0 26.0 3.50 2.00 13.5 3.00 12.0 2.00 2.50 4.00 6.50 - - 21.5 42.5 - - -2842-25

72.5 53.0 70.0 28.5 26.5 66.0 56.5 48.0 21.5 5.25 2.00 14.3 0.75 15.0 2.00 2.50 6.00 6.50 16.5 15.5 25.5 - - 37.0 -2842-24

83.5 57.5 74.0 29.5 29.5 73.5 55.5 56.0 25.0 9.00 0.25 22.0 2.25 14.0 1.00 2.50 4.50 7.00 13.5 16.0 23.5 51.5 38.0 50.5

2842-23

87.0 57.0 79.0 28.5 34.0 74.5 61.0 54.0 25.0 7.00 0.75 20.5 1.50 13.0 2.00 2.00 5.50 6.00 14.5 14.0 21.0 49.5 33.5 45.0

2842-21

69.0 48.0 73.0 25.8 24.5 60.0 62.0 45.0 22.3 3.50 - 16.5 1.00 12.5 2.00 3.00 6.00 6.00 14.5 12.0 21.0 39.0 33.0 43.0

2842-20

69.5 48.0 65.0 24.8 15.5 63.0 61.5 52.3 23.3 3.25 0.75 15.5 - 13.0 2.00 2.00 6.00 6.00 15.5 12.0 29.0 - - -2842-19

86.5 58.5 80.0 30.0 36.0 73.5 53.0 52.0 22.5 5.50 0.25 20.0 1.25 14.5 3.50 3.00 7.00 5.00 16.0 8.5 20.0 49.0 29.0 43.0

2842-18

90.0 64.5 76.0 33.5 35.0 79.0 59.0 57.0 26.0 4.00 - 17.0 - 16.0 2.50 3.00 5.50 7.00 15.0 15.0 25.0 53.0 37.5 51.5

2842-17

59.0 45.0 66.0 42.3 23.5 54.0 59.0 59.0 20.3 3.75 0.50 14.5 1.50 12.5 2.00 2.50 6.50 6.00 18.0 10.0 27.0 - - -

2842-16

72.0 57.0 72.0 50.0 29.8 63.5 69.0 47.8 24.5 4.50 0.75 14.5 0.50 16.0 1.00 3.00 4.00 8.00 11.0 15.5 18.5 46.0 33.0 51.0

2842-15

92.0 60.0 80.0 57.0 29.0 77.5 61.5 55.5 26.0 6.25 1.00 16.5 1.00 13.5 2.00 2.00 6.00 5.00 15.5 9.0 20.5 47.0 30.0 44.5

2842-14

47.0 37.0 62.0 37.8 18.0 44.0 78.0 37.0 17.3 1.75 0.75 10.0 0.50 10.5 1.50 2.00 4.50 5.00 - - 20.0 - 33.0 -

2842-13

66.0 46.5 71.0 45.0 23.5 59.0 60.5 43.8 18.8 4.50 0.75 14.5 0.50 13.0 2.00 3.00 6.00 6.00 14.0 12.0 21.0 - 36.0 51.5

TABLE 1 - P I PROGRAM - PRIMARY DATA MATRIX FOR PRESERVED KENYAN SNAILS 153(ALL M E A S U R E M E N T S GIVEN AT X6 MAGNIFICATION)

PC/LRNO:

L(mm)

W(mm)

Wo(l/16 of a whorl)

ALl(mm)

AW1(mm)

BW L(mm)

BWSA(Deg)

a(mm)

c(mm!

w(mm)

X

(mm)y(mm)

3(mm)

d(mm)

H W 2(mm)

W W 2(mm!

H W 3(mm)

W W 3(mm!

H W 4(mm!

W W 4(mm!

B4(mm)

B5(mm)

A(Deg)

H5(mm)

W 5 (mm)

2842-12

78.5 56.0 73.0 54.5 29.5 72.0 56.5 53.0 25.5 3.25 0.25 19.0 1.75 13.0 1.50 3.00 5.00 6.00 12.0 13.0 21.0 57.0 96.5 36.0 51.5

2842-11

72.5 52.0 72.0 48.5 27.5 64.0 65.0 47.0 22.5 4.00 0.50 19.00 1.00 13.5 2.50 3.00 7.00 7.00 16.0 14.0 22.5 38.5 88.5 42.0 48.0

2842-10

80.5 55.5 79.0 49.0 27.0 68.0 58.0 47.0 22.5 5.50 1.00 17.5 1.00 12.0 1.50 3.00 6.00 5.50 13.5 9.5 19.0 41.0 92.5 27.0 42.0

2842-09

87.0 61.0 76.0 56.0 30.0 76.0 65.0 54.5 27.0 5.50 - 17.8 2.00 14.00 1.00 2.50 3.50 6.00 12.5 12.5 21.0 52.5 109.5 33.0 48.5

2842-OS

67.0 52.0 70.0 47.5 27.5 60.0 62.0 46.0 22.8 5.50 0.25 16.0 0.50 15.5 1.50 3.00 5.00 6.00 11.5 13.0 22.0 - 99.5 34.5 35.0

2842-07

62.0 46.5 66.0 45.5 24.0 57.5 70.0 44.5 21.0 4.00 0.50 15.5 0.50 12.5 2.00 2.50 5.50 6.00 15.5 12.0 20.0 - 106.5 35.0 -

2842- 06

89.5 65.5 75.0 58.0 34.0 79.0 63.0 57.0 27.5 4.50 1.00 20.5 15.5 1.00 3.00 3.50 7.00 13.5 16.5 21.0 51.0 106.5 37.5 58.0

2842-05

79.5 59.5 75.0 55.5 31.0 72.0 57.5 54.5 26.0 5.50 - 18.0 2.00 17.0 1.00 3.00 4.00 7.00 10.0 13.0 19.0 44.5 103.0 27.0 47.0

2842-04

82.5 59.0 75.0 52.0 32.0 72.0 57.5 52.0 23.5 7.50 1.00 18.3 0.50 16.5 1.00 3.00 4.50 7.00 12.5 14.0 22.0 50.0 96.5 33.5 47.0

TABLE 1 - PI PROGRAM - PRIMARY DATA MATRIX FOR PRESERVED KENYAN SNAILS(ALL M E A S U R E M E N T S GIVEN AT X6 MAGNIFICATION)

PC/LRNO:

L(mm)

W(mm)

Wo(l/16 of a whorl)

A H(mm)

AW1(mm)

B W L(mm)

BWSA(Deg)

a(mm)

c(mm)

w(mm)

X

(mm)y(mm)

3(mm)

d(mm)

H W 2(mm)

W W 2(mm)

H W 3(mm!

W W 3(mm)

H W 4(mm)

W W 4(mm!

B4(mm!

B5(mm!

A(Deg)

H5(mm)

W5(mm)

i m r16

7T75" 3 0 ” s o — 3 0 ” 3C5- (>7.0 51.5 51.0 22.5 1 W 0.75 13.5 - 12.0 2.50 3.00 £Too" 7.00 31.5 111.0 41.0

2872-15

59.0 40.5 61.0 43.3 18.5 56.0 61.5 42.3 19.3 2.50 0.50 14.0 1.25 8.5 1.50 3.50 5.00 8.00 - - 32.0 - 105.5 - -

2872-13

62.0 46.0 66.0 46.8 23.5 58.0 62.5 46.5 21.3 3.00 0.75 12.0 0.50 12.5 1.00 2.50 5.00 6.50 - - 23.5 - 120.5 34.5 -

2872-12

69.0 50.0 61.0 55.5 25.0 67.0 63.0 53.0 24.0 2.00 0.50 16.5 1.00 11.5 1.00 3.50 3.00 9.00 - - 32.0 - 125.5 - -

2872-10

76.0 51.5 67.0 52.3 26.3 69.0 56.0 53.0 22.8 5.25 0.50 18.0 0.50 13.5 2.00 3.00 6.00 7.00 - - 26.0 - 111.0 - -

2872-09

69.0 50.0 66.0 49.5 23.3 65.0 53.0 49.0 24.8 3.50 0.25 14.0 1.50 11.5 2.00 3.00 6.00 7.00 - - 29.5 - 112.5 41.0 -

2872-08

58.5 42.5 59.0 47.8 21.0 56.0 61.0 47.5 20.8 1.75 1.00 10.0 1.75 10.5 1.00 3.00 2.50 8.00 - - 29.0 - 128.0 - -

2872-06

55.0 39.0 60.0 41.3 17.5 51.0 56.0 40.3 18.8 1.75 0.50 9.0 0.75 8.0 2.00 4.00 5.00 10.0 - - 29.0 - 105.0 - -

2872-05

62.0 46.0 63.0 48.5 22.0 58.5 72.5 47.5 22.0 3.00 1.50 11.5 0.50 11.5 2.00 3.00 5.50 7.00 - - 26.0 - 110.5 - -

2872-04

63.0 43.0 66.0 46.5 21.0 58.5 65.5 45.3 19.5 3.00 15.0 0.50 9.5 2.00 2.50 5.50 6.00 18.0 12.0 28.0 - 113.5 33.0 -

2872-03

66.0 47.5 62.0 47.0 23.0 63.0 61.0 46.0 22.8 2.25 12.5 - 11.0 1.00 3.00 4.00 9.00 - - 29.0 - 118.0 - -

2872-02

65.0 47.0 67.0 47.5 23.8 61.5 54.0 46.5 20.8 2.75 1.00 13.3 1.00 11.0 1.00 2.50 4.00 7.00 - - 27.0 - 122.5 - -

2872-01

67.5 48.0 62.0 ' 53.3

1

24.5 64.0 65.5 52.5 25.0 3.00 1.50 15.0 2.50 11.5 2.00 3.50 5.00 8.00 - - 31.0 - 115.0 - -


PC/LRNO:

L(mm)

W(mm)

Wo(l/16 of a whorl)

ALl(mm)

AW1(mm)

BW L(mm)

BWSA(Deg)

a(mm)

C(mm)

w(mm)

X(mm)

y(mm'

3(mm]

d(mm!

H W 2(mm]

W W 2(mm)

HW 3(mm]

W W 3(mm)

H W 4(mm)

W W 4(mm]

B4(mm)

B5(mm]

A(Deg)

H5(mm)

W 5 (mm)

"2739-08

90.5 &2.6 9i.5 22.0 73.0 40.0 51.0 2l0 2.66 T>33" T57T 3.66 7J 2.66 T W 6.00 w T O " Ii.3 26.6 44.5 90.6 30.0 38.0

2739-05

75.0 47.0 74.0 52.0 20.5 65.5 35.5 21.0 21.5 2.00 1.75 12.0 3.25 7.5 1.50 2.50 4.00 6.50 11.0 12.0 18.0 42.0 98.5 31.0 24.5

2739-03

92.5 52.0 82.0 56.0 24.0 76.0 39.0 55.0 24.5 2.25 0.75 13.5 3.25 8.5 2.50 4.00 8.00 7.00 19.0 11.0 27.0 46.5 80.5 46.5 42.5

2739-02

95.0 54.0 82.0 56.5 23.0 77.5 43.5 55.0 24.0 1.75 0.75 13.0 2.75 8.0 2.00 3.00 7.00 6.50 19.0 11.0 23.0 44.0 91.0 32.5 44.0

2739-18

76.0 43.0 77.0 49.5 18.0 65.5 40.0 48.5 20.0 2.50 1.25 14.0 2.75 7.0 1.00 3.00 5.00 6.50 13.5 12.0 20.5 44.5 91.5 33.5 37.5

2739-17

78.5 47.0 69.0 53.0 21.5 69.5 43.0 51.5 23.5 1.75 0.75 12.8 3.75 8.0 1.50 2.50 5.00 6.50 15.5 13.5 23.0 49.5 94.5 35.0 42.5

2739-16

73.0 43.5 69.0 52.3 20.5 64.0 48.5 49.8 21.5 2.00 0.50 12.5 4.50 8.0 1.00 2.00 4.00 6.00 11.0 12.0 18.0 40.0 94.0 33.5 39.5

2739-15

85.0 47.0 76.0 55.0 21.0 72.5 36.5 52.5 22.5 2.50 - 13.3 4.00 7.0 1.50 3.00 5.00 6.50 15.5 13.5 23.0 48.0 91.0 35.0 42.0

2739-13

119.0 58.0 73.0 66.5 23.5 94.5 33.0 66.0 29.5 2.00 0.50 14.0 3.50 9.0 2.00 3.00 6.00 6.00 17.0 11.5 23.5 42.5 89.0 38.5 27.5

2739-12

84.0 49.0 78.0 52.0 21.0 70.0 36.0 50.5 22.0 2.00 0.50 12.3 2.25 7.5 1.00 2.50 4.50 6.50 14.0 11.5 21.5 40.5 11.0 31.0 43.0

2739-11

122.0 59.5 86.0 67.5 24.5 91.5 40.0 67.5 26.5 2.50 - 20.0 6.50 11.0 2.50 3.00 7.50 7.00 19.5 20.5 29.0 57.5 86.5 40.5 41.0

2739-10

70.0 38.0 68.0 47.5 16.8 61.0 52.5 91.5 20.0 2.00 - 12.8 5.25 7.5 1.50 3.00 6.00 7.50 20.5 12.0 33.0 - 94.0 43.0 -

2739-09

59.5 37.0 70.0 40.8 15.8 51.5 41.0 79.9 16.8 1.50 0.50 10.8 1.75 5.0 2.00 3.50 6.00 7.50 15.0 11.5 22.0 - 90.0 32.0 -


PC/LRNO:

L(mm)

W(mm)

Wo(l/16 of a whorl)

AL1(mm)

AW1(mm)

B W L(mm)

BWSA(Deg)

a(mm)

c(mm)

w(mm)

X

(mm)y(mm)

3(mm!

d(mm)

H W 2(mm)

W W 2(mm)

HW 3(mm)

W W 3(mm)

H W 4(mm)

W W 4(mm)

B4(mm)

B5(mm)

A(Deg)

H5(mm)

W5(mm)

2739-08

123.0 59.5 92.0 67.0 23.0 97.5 29.0 65.5 27.5 1.00 - 18.0 6.00 7.5 O o - 2.50 6.00 7.00 15.5 12.5 18.5 40.0 88.0 3J.5 37.0

2739-07

91.0 50.0 78.0 57.0 22.5 71.5 45.5 54.5 24.0 2.00 0.75 13.5 2.00 8.0 2.00 2.50 6.00 6.00 16.5 12.5 25.0 49.5 92.0 34.5 43.0

2739-OS

68.5 40.0 70.0 47.8 18.5 61.0 39.5 91.0 20.5 1.50 0.50 10.0 3.00 7.0 1.50 3.00 5.00 7.00 16.0 14.0 25.0 - 101.5 34.0 -

2739-05

89.0 48.0 76.0 57.0 21.5 75.0 38.0 55.5 24.5 2.00 - 13.0 4.00 17.5 1.50 3.00 5.00 7.00 17.5 14.5 25.0 54.0 97.5 41.5 45.0

2739-04

80.5 43.0 75.0 52.0 19.5 68.0 51.0 50.0 22.0 2.00 1.00 12.0 4.25 7.0 1.50 2.50 6.00 7.00 15.0 12.0 23.5 49.5 87.5 38.0 39.0

2739-03

82.0 47.5 77.0 52.0 20.0 71.0 41.0 49.0 21.0 2.00 - 14.0 2.50 6.0 2.00 2.50 5.00 5.00 13.5 10.5 19.5 40.5 97.5 29.0 40.5

2733-01

116.5 58.0 91.0 67.0 27.5 92.5 31.5 65.0 29.0 4.00 0.75 24.0 6.25 9.0 1.00 3.00 4.50 7.50 13.0 12.5 22.5 41.5 96.5 31.0 35.5

157

(ALL MEASUREMENTS GIVEN AT X6 MAGNIFICATION)

TABLE 2 - P2 PROGRAM - PRIMARY DATA MATRIX FORFIELD COLLECTED KENYAN SNAILS

PC/LRNO:

126203

126222

126201

126206

126229

126234

126211

126216

126223

126212

126236

126215

126239

L(mm) 101.0 123.0 108.0 127.0 116.0 89.0 70.5 134.0 106.5 88.0 70.5 112.0 115.0W(mm) 68.0 66.5 68.0 67.0 75.0 51.0 49.0 65.5 69.5 63.0 50.5 72.0 75.0W o(l/16 o f a Whorl) 74.0 94.0 75.0 90.0 72.0 76.0 63.0 95.0 78.0 72.0 68.0 75.0 82.0AL2(mm) 72.0 68.0 70.0 72.0 78.0 55.5 54.5 70.5 71.5 60.5 48.5 71.0 75.0AW2(mm) 33.5 33.0 33.5 33.0 37.5 24.0 25.0 29.5 35.5 31.0 25.0 36.5 37.5BWL(mm) 94.0 99.0 94.5 103.5 106.0 75.5 66.0 101.0 97.0 81.0 48.5 103.0 101.5S(mm) 29.0 55.0 40.0 57.0 38.5 34.0 17.5 65.5 35.0 26.5 22.0 40.0 41.5USH (mm) 7.0 24.0 14.0 23.5 10.5 13.5 4.5 33.0 9.5 7.0 7.0 9.0 13.5

APA(Dogs) 31.0 17.0 26.0 16.0 24.0 24.0 34.0 14.0 22.0 31.5 32.5 23.0 22.0AAA(Degs) 14.0 15.0 17.0 20.0 14.0 14.0 17.0 21.0 16.0 16.0 17.0 14.5 16.0w(mm) 3.50 - 3.25 3.0 6.0 3.75 3.0 - 6.0 6.25 2.75 5.75 3.75x(mm) 2.25 - 1.00 1.00 2.00 1.00 1.50 - 2.75 1.50 1.25 3.00 2.00y(mm) 18.0 14.8 15.8 16.8 21.0 13.5 13.5 14.8 17.8 14.0 10.8 19.5 18.03(mm) 4.00 - 2.50 1.75 6.00 2.50 4.50 - 5.25 4.00 3.25 4.00 5.75d(mm) 15.5 13.0 14.5 12.5 14.5 10.0 11.5 10.0 15.0 14.0 12.0 15.5 15.5HW2(mm) 2.00 2.00 1.50 1.50 2.00 1.50 1.50 1.50 1.00 2.00 2.00 1.00 1.50WW2(mm) 3.50 3.00 4.00 3.00 4.50 3.00 3.50 2.50 3.00 3.00 3.00 3.00 2.50HW3(mm) 5.50 4.50 7.00 5.00 9.00 6.50 5.50 6.00 5.00 5.00 7.50 4.00 4.50WW3(mm) 8.00 5.50 9.50 7.00 11.00 8.00 10.0 6.50 7.50 7.50 7.50 9.50 7.50HVMmm) 16.0 12.5 21.0 18.0 30.0 18.0 - 16.5 11.5 16.0 22.0 17.5 16.0WW(mm) 17.0 13.0 19.0 13.0 20.5 15.0 - 13.0 16.5 15.5 15.0 20.0 16.0B4(mm) 29.5 19.0 36.0 23.5 43.5 28.0 33.5 24.0 23.5 28.0 34.5 33.5 25.0B5(mm) - 41.0 70.0 49.0 - 52.0 - 46.0 52.5 - - - 62.0A(Degs) 109.9 88.0 90.0 95.0 107.0 84.0 108.0 86.0 112.0 119.0 98.0 114.0 111.0H4(mm) 19.0 15.5 27.0 20.5 26.5 19.0 24.0 18.0 19.0 19.5 22.0 22.0 20.0fMnun) 39.5 19.0 39.0 28.0 50.0 28.0 45.0 27.0 36.0 40.0 39.0 41.0 35.5H5(mm) - 29.5 54.0 34.5 - 40.0 - 33.0 44.0 - - - 40.0W5(mm) - 39.0 64.0 42.5 - 46.0 - 40.0 61.5 - - - 59.0

158



PC/LRNO:

12522*

125229

125216

125203

125211

125227 1252

38126227

126219

126217 1262

1*126226

126232

L(mm) 6 *.5 110.0 69.0 98.0 62.5 100.0 68.0 125.0 121.0 83.0 67.0 80.5 90.0W(mm) *5.5 71.0 *8.0 61.0 *1.0 65.5 *8.0 67.0 6*.0 55.0 *7.0 53.5 61.0W o(l/16 of a Whorl) 62.0 81.0 62.0 82.0 66.0 78.0 6*.0 9*.0 90.0 69.0 66.0 79.0 69.0AL2(mm) 51.0 75.5 57.0 66.0 *8.0 68.0 55.0 67.5 66.0 6*.0 5*.0 56.5 66.0AW2(mm) 20.0 33.5 21.0 30.0 19.0 30.5 23.0 32.5 31.0 25.5 22.5 26.0 26.5BWL(mm) 59.0 98.0 6*.5 87.0 58.0 92.0 63.5 99.5 95.0 77.5 63.0 71.0 83.0S(mm) 1*.5 35.5 12.5 21.0 16.5 3*.0 1*.0 57.5 5*.0 19.5 15.5 25.0 26.0USH (mm) 6.0 12.0 *.5 11.5 5.0 8.0 *.5 26.0 26.0 5.5 *.5 9.5 7.0APA(Degs) *2.0 27.0 *5.0 22.0 31.0 20.0 **.0 17.0 20.0 37.0 38.0 32.0 27.0AAA(Degs) 17.0 18.0 15.0 16.0 19.0 17.0 15.0 13.0 15.0 15.0 15.0 16.0 1*.0w(mm) 1.0 6.50 1.50 5.75 2.50 2.25 2.50 - - 1.50 3.50 *.50 3.25x(mm) 1.0 2.00 0.75 1.25 1.00 2.00 1.25 - - 0.25 1.00 1.50 2.00y(mm) 11.5 17.3 13.8 16.8 12.0 15.3 16.0 15.8 15.3 17.0 13.0 1*.0 16.33(mm) *.0 *.25 *.50 3.75 *.00 *.00 *.00 - - 5.50 2.00 *.50 5.50d(mm) 10.5 1*.5 12.0 11.5 9.5 12.0 12.5 1*.5 13.0 13.5 11.5 11.5 10.5HW2(mm) 2.00 2.00 2.00 2.00 2.00 1.50 1.50 2.00 2.00 1.50 1.50 1.00 2.00WW2(mm) 3.50 3.00 3.50 3.00 3.50 3.50 3.50 2.00 2.50 2.50 *.00 3.00 3.00HW3(mm) 6.00 5.50 *.50 5.50 6.00 *.00 5.00 5.50 6.50 5.50 6.00 3.50 7.50WW3(mm) 8.50 6.50 9.00 7.00 7.50 8.00 8.00 6.00 7.00 6.50 10.0 5.00 8.00HV4(mm) - 1*.0 - 15.0 - 13.5 - 1*.0 17.5 20.0 - 10.0 -

WW(mm) - 1*.5 - 13.5 - 18.0 - 10.5 11.5 15.0 - 12.0 -B*(mm) 27.0 26.0 27.0 21.5 26.0 28.0 27.0 21.0 25.5 29.5 35.5 23.0 26.5B5(mm) - 59.5 - 52.0 - 62.0 - *0.0 50.0 - - 53.0 -A(Degs) 100.0 99.9 113.0 109.0 95.0 11*.0 110.0 98.0 86.0 111.0 10*.0 96.0 110.0H*(mm) 19.0 16.0 19.0 18.5 17.0 20.0 17.0 1*.5 18.0 18.0 26.5 1*.0 21.5V4(mm) 38.0 32.0 *1.0 31.0 - 37.0 37.0 2*.0 2*.5 39.0 *2.0 25.0 *5.5H5(mm) - *1.5 - 33.0 - *6.5 - 30.0 39.5 - - 31.0 -W5(mm) - 58.5 *8.0 - 59.0

«38.0 *1.5 - - *6.0

,

159(ALL MEASUREMENTS GIVEN AT X6 MAGNIFICATION)

TABLE 2 - P2 PROGRAM - PRIMARY DATA MATr ix FORHELD COLLECTED KENYAN SNAILS

PC/LRNO:

1252 0 4

125217

125232 1252

30125234 1252

35125213

125237

125206

125236

125209 1252

01 125223

L(mm) 86.0 106.5 106.0 66.0 73.0 87.5 106.5 93.5 81.5 89.0 115.0 100.0 113.0W(mm) 58.0 66.5 64.0 43.5 51.5 52.0 69.5 63.0 52.5 62.0 69.0 65.5 68.0W o(l/16 of a Whorl) 73.0 82.0 76.0 71.0 67.0 74.0 82.0 78.0 69.0 74.0 83.0 76.0 83.0AL2(mm) 63.0 66.0 73.0 50.5 54.0 58.0 73.0 69.0 60.5 62.0 78.0 72.5 78.5AW2(mm) 28.0 32.0 30.5 20.0 24.0 25.5 30.5 30.0 24.0 27.0 33.5 31.0 33.5BWL(mm) 79.0 87.5 95.0 59.5 67.0 75.0 95.5 83.5 72.0 79.5 101.0 90.0 99.0S(mm) 24.0 40.0 34.0 16.5 15.0 30.0 34.5 26.0 20.0 29.0 38.0 28.5 36.5USH (mm) 7.0 19.0 11.0 7.0 6.0 12.0 11.0 10.0 9.5 10.0 14.0 10.0 14.0APA(Degs) 25.0 24.0 23.0 33.0 46.0 27.0 22.0 29.0 36.0 24.0 24.0 28.0 26.0AAA(Degs) 18.0 12.0 18.0 17.0 17.0 16.0 19.0 18.0 14.0 19.0 17.0 17.0 19.0w(mm) 2.50 4.25 3.50 2.50 2.25 4.25 2.75 4.50 4.00 3.25 1.75 4.75 3.25x(mm) 1.00 3.00 2.00 2.00 0.75 2.00 2.25 2.00 2.50 1.00 4.00 2.00 1.50y(mm) 14.5 15.3 16.5 12.5 14.3 16.5 12.0 18.0 14.5 14.5 13.0 16.5 14.53(mm) 4.00 1.75 5.00 5.50 4.00 3.00 6.00 4.50 5.50 4.75 6.00 6.75 6.00d(mm) 11.5 8.0 12.0 9.5 12.0 11.0 12.0 12.0 12.0 12.0 12.5 12.5 13.0HW2(mm) 2.00 3.00 1.00 3.00 2.00 3.00 2.00 1.50 4.00 2.50 2.00 1.50 2.00WW2(mm) 3.50 4.00 2.50 3.50 3.00 4.00 3.00 3.50 5.00 5.50 3.50 3.50 3.00HW3(mm) 6.00 8.50 4.00 6.50 65.0 8.00 4.50 5.50 10.0 8.50 5.50 4.50 5.50WW3(mm) 8.50 6.50 8.00 7.00 8.00 8.00 7.00 8.50 9.00 10.0 7.50 8.50 6.50HWMmm) 9.50 9.50 16.0 16.0 - 23.0 11.5 14.5 - 20.5 16.0 13.0 15.5WW(mm) 16.5 10.0 18.0 12.0 - 15.0 13.5 15.5 - 18.0 14.0 18.0 18.5B4(mm) 31.0 28.0 29.0 24.5 27.5 30.0 19.5 24.0 34.5 30.5 30.5 27.0 27.5B5(mm) - 51.5 61.0 - - - 59.5 50.5 - - 61.0 54.0 56.5A(Degs) 116.0 92.0 106.0 94.0 109.0 85.0 110.0 111.0 90.0 101.0 107.0 111.0 105.0H4(mm) 21.0 18.5 19.0 16.5 16.0 22.5 14.0 20.0 23.0 21.0 21.0 21.5 16.5fM m m ) 41.0 31.5 36.0 30.0 39.0 33.0 31.0 36.5 39.0 40.0 36.5 38.0 31.5H5(mm) - 36.5 55.0 - - 43.5 32.5 38.5 39.5 48.5 40.5W5(mm) - 50.0 60.0 - - 46.0 54.5 60.0

l56.0 63.5 55.5

160



PC/LRNO:

125433

125428

125414

125418

125417

125423

125436

125412

125424

125411

125435 125202 1252

10L(mm) 113.0 105.0 78.0 95.5 96.0 87.0 108.0 105.0 86.5 101.5 73.0 84.0 103.0W(mm) 75.0 70.5 55.0 64.0 70.0 59.5 73.5 66.0 58.0 65.0 53.0 53.0 61.5WoO/16 of a Whorl) 80.0 82.0 69.0 80.0 74.0 76.0 82.0 77.0 77.0 78.0 68.0 72.0 80.0AL2Cmm) 70.5 70.0 57.0 66.5 70.5 60.0 70.0 69.5 63.5 67.5 57.5 60.5 70.0AW2(mm) 38.5 34.0 26.0 29.5 32.0 29.0 34.0 29.0 23.5 31.0 25.0 25.0 32.0BWL(mm) 99.0 92.5 70.0 86.5 90.0 79.0 93.5 94.5 78.5 90.0 67.0 76.0 91.0S(mm) 41.0 37.0 22.5 29.0 26.0 28.0 42.0 37.5 25.5 34.5 16.0 24.0 34.0USH (mm) 14.0 13.0 8.9 9.5 6.0 8.0 14.5 10.5 8.0 11.0 6.0 8.0 12.0

APA(Degs) 22.0 22.0 26.0 20.0 29.0 21.0 21.0 15.0 26.0 22.0 39.0 30.0 22.0AAA(Degs) 16.0 18.0 16.0 18.0 19.0 19.0 20.0 20.0 18.0 16.0 12.0 15.0 17.0w(mm) 5.75 7.00 3.25 5.50 4.25 4.75 5.25 6.75 3.75 6.50 5.00 3.50 6.00x(mm) 2.25 2.50 1.50 1.75 0.75 1.50 2.25 3.00 1.00 2.00 1.50 1.50 2.75y(mm) 18.8 16.3 14.0 15.5 19.5 14.5 16.0 18.5 12.5 16.5 14.5 14.5 17.83(mm) 3.00 3.8 2.00 3.75 2.00 2.75 4.00 4.25 1.75 4.25 2.25 5.00 2.00d(mm) 16.0 14.0 11.5 12.0 13.0 11.5 14.5 8.5 10.0 11.5 14.0 11.0 11.0HW2(mm) 3.50 2.50 3.00 2.00 1.50 2.00 2.50 2.00 1.50 2.00 3.00 1.50 1.50WW2(mm) 4.50 3.50 3.50 3.00 4.50 3.00 3.50 4.00 3.00 4.00 4.00 3.50 2.00HW3(mm) 7.00 7.00 6.50 5.00 4.00 5.50 7.50 5.50 4.00 5.50 7.50 6.00 6.00WW3(mm) 7.50 9.00 7.50 6.50 9.00 9.00 8.50 8.50 6.50 8.00 8.50 8.00 7.50HW4(mm) 16.5 14.5 16.0 10.5 13.0 12.5 16.0 12.0 9.5 12.0 18.0 - 16.5WW(mm) 14.5 16.0 15.0 12.5 19.0 16.0 15.0 17.0 13.0 16.0 14.5 - 16.0B4(mm) 27.5 24.5 25.5 16.5 26.0 23.0 25.5 24.0 23.5 24.0 30.0 27.5 25.0B5(mm) 58.0 54.0 - 43.0 - 52.5 54.5 58.5 46.0 55.5 - - 49.0A(Degs) 100.0 94.0 104.0 102.0 129.0 104.0 99.0 102.0 108.0 100.0 111.0 111.0 98.0H4(mm) 21.0 19.5 25.0 13.0 17.0 19.5 21.0 21.5 17.0 20.5 19.0 19.0 18.0V4(mm) 37.5 35.0 40.0 27.0 - 35.0 36.5 39.0 31.0 35.0 38.5 35.5 33.5H5(mm) 40.5 37.0 - 30.5 - 37.5 37.0 40.0 36.0 34.0 - - 42.5W5(mm) 60.0 56.5 - 55.0

______- 51.5 57.5 63.0 52.5 54.0 - - 54.0

161



PC/LRNO:

125005

125406

125415 1254

08125405

125426

125404

125401

125402

125437

125438

125421 1254

13L(mm) 69.5 103.5 80.5 82.0 113.5 98.0 108.0 107.0 105.5 125.5 120.5 72.5 97.0W(mm) 50.5 67.5 56.0 56.0 77.0 65.5 70.0 70.5 71.0 83.0 80.0 53.5 65.5V oU /16 of a Whorl) SO.O 80.0 74.0 76.0 79.0 77.0 76.0 83.0 81.0 82.0 84.0 65.0 77.0AL2(mm) 53.0 65.0 57.0 52.0 73.0 67.0 73.0 73.0 70.0 80.0 76.5 54.0 65.0AW2(mm) 25.5 32.0 25.5 27.5 36.5 33.5 34.5 35.5 35.0 41.0 41.0 25.5 31.0BWL(mm) 65.5 91.0 73.0 73.0 99.0 88.0 97.0 96.0 93.0 110.0 106.5 64.5 86.5S(mm) 15.5 39.5 24.0 26.0 40.0 31.5 35.0 34.0 36.0 48.0 45.0 20.0 32.0

USH (mm) 4.0 12.0 7.5 8.5 15.0 10.0 11.5 11.0 11.5 15.5 14.0 8.0 10.5APA(Degs) 40.0 18.0 27.0 29.0 23.0 23.0 22.0 18.0 21.0 22.0 22.0 35.0 18.0AAA(Degs) 9.0 19.0 19.0 19.0 16.0 16.0 16.0 19.0 18.0 16.0 15.0 17.0 18.0w(mm) 5.75 5.50 3.75 3.00 6.25 5.50 5.50 3.75 5.50 5.25 7.00 2.25 7.00x(mm) 0.50 2.50 1.50 1.50 3.00 2.00 2.75 1.75 2.50 2.00 2.25 1.00 2.00y(mm) 14.5 14.0 16.0 13.0 18.5 17.8 17.0 16.8 17.0 21.8 20.5 12.3 16.33(mm) 0.75 4.00 2.25 2.00 3.25 2.75 3.25 3.25 2.50 3.25 3.25 1.00 2.75d(mm) 13.5 10.0 11.0 14.5 13.0 13.0 12.5 13.5 16.0 15.5 11.5 11.5HW2(mm) 1.50 3.00 2.50 2.00 2.50 2.00 2.50 1.50 3.00 4.00 2.25 2.00 2.00WW2(mm) 3.00 4.00 3.00 3.00 4.00 3.00 4.00 3.50 4.50 5.50 3.50 3.50 3.50HW3(mm) 5.00 6.50 6.50 4.00 7.50 4.50 6.00 4.50 6.50 9.99 7.00 5.50 6.00WW3(mm) 6.50 7.50 8.00 7.00 8.00 6.50 8.00 7.50 8.00 10.0 9.00 8.00 7.00HW4(mm) - 14.0 14.5 10.5 15.5 13.5 13.0 12.0 13.0 19.0 16.5 12.5 11.5WW(mm) - 15.5 14.5 14.5 15.0 14.0 15.0 15.0 15.5 18.0 15.5 14.5 13.5B4(mm) 23.5 27.0 22.5 23.0 26.0 28.5 26.0 21.0 23.0 31.0 23.5 23.0 24.0B5(mm) - 56.0 - 44.0 56.5 51.0 53.5 52.5 47.5 71.0 60.0 - 47.0A(Dcgs) 122.0 98.0 103.0 106.0 100.0 109.0 103.0 112.0 99.0 98.0 104.0 103.0 98.0H4(mm) 15.5 19.0 19.0 13.0 18.0 15.5 17.0 16.5 16.5 25.0 17.0 22.0 17.5W4(mm) 35.5 34.0 35.5 27.5 35.0 32.5 33.0 33.5 33.0 44.0 36.4 35.0 31.0H5(mm) - 36.0 - 35.0 38.5 36.0 39.0 32.0 35.0 45.0 41.0 31.5 34.0WMmm) - 57.5

i

52.0 60.5 55.5 59.5 58.0 58.5 65.0 63.5 56.0

162



PC/LR 1250 1250 1250 1250 1250 1250 1250 1250 1250 1250 1250 1250 1250NO: 11 25 15 02 30 03 27 21 32 20 19 36 09

L(mm) 81.0 76.0 58.0 73.0 50.0 56.0 55.5 85.0 77.5 77.0 69.0 72.0 56.0W(mm) 59.0 56.0 01.5 52.0 37.5 01.0 02.5 61.0 56.5 58.0 51.5 57.0 00.0W o(l/16 of a Whorl) 73.0 68.0 63.0 65.0 58.0 62.0 59.0 70.0 69.0 60.0 60.0 67.0 59.0AL2(mm) 56.0 58.0 03.0 50.0 03.0 01.0 03.0 60.0 50.0 58.5 53.0 58.0 05.5AW2(mm) 31.0 30.0 21.5 26.5 20.0 21.5 21.0 32.0 28.0 32.0 26.5 28.5 23.5BWL(mm) 75.0 72.5 55.0 69.0 51.0 52.5 53.0 79.5 73.5 73.5 60.5 69.5 53.5S(mm) 22.0 18.0 10.0 22.5 10.0 15.0 13.0 29.5 23.0 16.5 15.0 13.0 10.0USH (mm) 5.0 0.0 3.0 0.0 3.0 3.5 2.5 6.0 0.0 3.0 0.5 2.5 2.5APA(Degs) 31.0 36.0 39.0 26.0 03.0 36.0 37.0 31.0 28.0 35.0 02.0 00.0 08.0AAA(Degs) 9.0 8.0 9.0 10.0 8.0 12.0 10.0 13.0 10.0 10.0 8.0 10.0 11.0w(mm) 3.50 0.25 2.00 2.50 3.00 3.00 2.00 3.75 1.50 2.00 0.00 5.00 -x(mm) 2.00 0.50 0.50 1.00 0.75 0.75 1.00 1.00 0.50 1.00 1.25 0.50 -y(mm) 10.0 18.3 10.5 12.3 13.0 12.0 11.8 16.3 10.0 15.0 10.3 15.5 10.33(mm) 0.50 0.75 0.25 0.25 0.75 0.75 0.25 1.25 0.25 0.25 0.75 1.00 -d(mm) 15.5 15.5 12.0 12.5 10.0 11.0 10.5 15.5 13.0 16.0 10.5 15.5 12.0HW2(mm) 1.00 1.50 1.00 1.00 2.00 1.00 1.00 2.00 1.50 1.00 1.50 1.50 1.00WW2(mm) 3.00 0.00 3.00 3.00 3.50 3.00 5.00 3.00 2.50 3.50 0.00 2.50 3.50HW3(mm) 3.00 5.00 0.00 5.00 6.00 0.00 0.50 6.00 5.00 3.50 5.50 3.50 3.00WW3(mm) 7.00 7.50 8.00 9.00 9.50 8.50 10.0 7.50 7.00 9.50 9.00 6.50 8.50HWO(mm) 12.5 - - - - - - - 20.0 - - - -WW(mm) 16.0 - - - - - - - 13.5 - - 16.0 -BO(mm) 21.0 30.0 25.5 31.0 20.5 27.0 28.0 35.0 32.5 31.5 30.0 31.5 20.5B5(mm) - - - - - - - - - - 01.0 - -A(Degs) 121.0 126.0 106.0 110.0 101.0 101.0 112.0 113.0 118.0 126.0 108.0 131.0 120.0H4(mm) 10.0 19.0 18.5 21.0 19.0 22.5 20.0 19.5 10.0 21.5 22.0 17.0 19.5WO (mm) 33.5 00.0 33.0 01.0 33.0 37.0 00.0 01.5 32.5 - - - 39.0H5(mm) - - - - - - - - - - - - -V5(mm) - - - - - - - - - - -

163



PC/LRNO:

126112

126120

126121

126116

126132

126109

126119

126135

126126

126137

125029

125013

125016

L(mm) 85.0 68.0 94.0 110.5 116.5 115.0 88.5 88.0 85.0 72.0 77.0 82.0 63.5W(mm) 59.5 49.5 67.0 74.5 83.0 78.0 62.5 58.0 57.0 51.0 59.0 61.0 46.0WoO/16 of a Whorl) 75.0 70.0 75.0 82.0 82.0 84.0 73.0 72.0 75.0 69.0 65.0 64.0 64.0AL2(mm) 58.0 53.0 69.0 77.5 79.5 77.0 65.0 62.0 61.5 54.0 60.5 58.0 47.0AW2(mm) 28.0 23.0 32.5 34.5 40.0 37.0 30.0 28.0 27.0 23.5 32.5 31.0 23.0BWL(mm) 76.0 62.0 87.5 100.0 103.0 100.5 79.0 79.0 76.0 66.0 74.0 75.5 60.0S(mm) 26.5 16.0 25.0 34.5 36.5 37.5 24.0 25.5 24.0 18.5 16.5 24.0 15.5

MSH (mm) 9.0 6.0 7.0 11.0 13.5 14.5 9.5 9.5 9.0 6.0 3.0 6.0 3.5APA(Degs) 30.0 38.0 32.0 30.0 28.0 29.0 34.0 27.0 31.0 35.0 38.0 32.0 34.0AAA(Degs) 15.0 18.0 15.0 18.0 18.0 18.0 15.0 18.0 18.0 18.0 10.0 9.0 15.0w(mm) 4.00 2.25 5.25 5.00 4.25 4.25 3.50 3.00 4.50 2.25 5.00 5.00 1.50x(mm) 1.50 0.75 1.50 2.25 3.50 1.25 1.25 1.25 1.25 0.50 1.25 2.00 0.50y(mm) 13.5 11.0 18.0 18.0 16.25 17.0 14.5 13.5 15.0 13.0 16.5 15.0 13.03(mm) 3.00 4.50 5.00 4.50 3.50 5.00 3.00 4.00 4.00 4.25 0.50 1.50 1.50d(mm) 13.0 11.5 16.0 16.0 17.0 16.5 15.0 12.0 13.5 11.0 16.0 15.5 10.5HW2(mm) 1.00 1.50 1.50 2.00 1.50 2.50 3.00 3.00 1.50 2.00 1.50 2.00 1.00WW2(mm) 3.00 3.00 4.00 3.50 3.00 3.00 4.00 4.00 3.50 3.50 4.00 3.50 3.00HW3(mm) 4.00 5.50 5.50 5.50 5.00 6.00 7.50 8.00 4.50 6.00 3.50 7.50 3.00WW3(mm) 7.50 6.50 9.00 7.50 8.00 7.00 7.50 8.50 7.50 8.00 9.50 8.50 7.00HW4(mm) 10.5 - 12.5 12.0 15.0 15.5 16.0 18.0 14.5 - - - 12.5WW(mm) 16.0 - 15.5 14.5 16.5 14.5 15.5 15.5 15.0 - - 16.0 16.0B4(mm) 22.5 23.0 24.0 23.0 25.5 22.5 28.5 27.0 24.5 27.0 34.0 35.5 21.0B5(mm) 47.0 - 50.0 51.0 53.5 52.5 - - 53.0 - - _ -A(Degs) 100.0 109.0 111.0 109.0 118.0 103.0 91.0 94.0 113.0 105.0 122.0 110.0 121.0H4(mm) 14.0 15.5 16.5 17.0 17.0 19.0 22.0 22.0 17.0 17.0 18.0 19.5 14.0W4(mm) 28.0 - 35.5 34.0 39.0 35.0 39.0 37.5 32.0 35.0 44.5 45.0 33.5H5(mm) 33.0 - 36.0 33.5 38.0 39.5 - - 34.5 - - - -W5(mm) 51.5 - 62.0 57.5 67.5 60.0 - - 48.0 - - : -



PC/LRNO:126006 1261

11126107 1261

34126124 1261

23126127

126138

126117

126105

126104 1261

10 126113L(mm) 65.0 57.0 76.0 65.5 55.5 71.0 82.5 62.0 71.0 80.0 63.5 79.0 76.0W(mm) 47.0 39.0 50.0 44.0 39.0 48.0 54.0 42.0 48.0 57.0 46.0 54.0 53.0W o(l/16 of a Whorl) 66.0 68.0 70.0 65.0 64.0 69.0 74.0 65.0 72.0 71.0 69.0 73.0 71.0AL2(mm) 48.0 39.5 55.0 51.0 40.0 58.5 60.5 48.0 50.0 58.0 45.0 56.0 55.0AW2(mm) 23.0 19.5 23.0 21.0 18.5 22.0 25.0 19.0 22.0 25.5 22.0 29.5 25.0BWL(mm) 60.0 51.0 68.5 60.5 50.5 64.5 71.0 56.5 63.5 71.5 58.0 69.5 69.0S(mm) 16.0 18.0 22.0 15.5 15.5 17.0 22.5 14.0 21.0 23.0 18.5 23.0 22.0

L\SH (mm) 5.0 6.0 8.0 6.0 5.0 6.5 9.0 5.0 7.5 9.0 6.0 9.0 7.5APA(Degs) 36.0 29.0 31.0 38.0 32.0 34.0 32.0 34.0 28.0 34.0 31.0 31.0 30.0AAA(Degs) 14.0 18.0 17.0 15.0 15.0 14.0 18.0 14.0 16.0 19.0 16.0 16.0 20.0w(mm) 2.75 2.25 2.50 3.00 2.00 2.50 2.50 1.00 2.25 3.50 2.75 2.50 3.00x(mm) 0.50 1.25 1.00 1.00 1.00 1.50 0.75 0.50 1.00 1.00 0.50 1.00 0.75y(mm) 12.3 10.0 12.0 12.0 8.50 13.3 13.3 10.5 12.5 14.0 12.0 12.8 14.53(mm) 0.25 1.50 4.00 2.75 0.75 3.50 4.75 3.25 1.75 3.50 2.50 2.50 5.00d(mm) ‘ 11.0 8.50 10.5 11.0 10.0 10.5 12.0 10.0 9.5 12.5 10.5 11.0 11.0HW2(mm) 3.00 2.00 3.00 2.50 1.50 2.00 2.50 2.50 3.00 2.00 2.00 2.00 2.00WW2(mm) 3.50 3.00 4.00 3.00 3.50 3.00 3.00 3.00 3.00 3.50 3.00 3.50 3.00HW3(mm) 5.50 6.50 8.50 6.00 5.50 6.50 8.00 5.50 6.50 6.50 6.00 6.50 6.50WW3(mm) 7.50 7.00 8.00 6.50 7.50 7.50 7.00 6.50 6.50 8.50 7.00 7.00 8.00HW4(mm) 19.5 18.5 - - - - 14.5 - 17.0 18.5 19.0 15.0 -WW(mm) 15.5 12.5 - - - - 13.0 - 12.5 15.5 12.5 14.5 -B4(mm) 28.0 25.0 27.5 25.0 27.5 27.5 20.0 21.0 25.0 25.5 27.5 22.5 27.0B5(mm) - - - - - - 42.5 - - - - - -A(Degs) 103.0 96.0 93.0 96.0 95.0 101.0 90.0 100.0 99.0 109.0 105.0 95.0 104.0H4(mm) 21.0 15.5 17.5 19.0 18.0 16.5 16.5 16.5 18.0 19.0 16.5 22.0 17.0W4(mm) 36.0 30.0 34.0 35.5 32.0 35.0 31.0 34.5 33.0 38.5 33.0 35.0 35.0H5(mm) - - - - - - 34.0 - - 35.5 - - -W5(mm) _ - - - - 52.0 - - - - - -

165



PC/LRNO:

126032

126027

126004

126024

126031

126028

126010

126012

126022

126021

126011

126023

126015

L(mm) S7.0 54.0 82.0 81.0 82.0 70.0 97.5 80.5 96.0 69.0 83.0 34.0 83.0W(mm) 62.0 37.0 58.0 55.0 60.0 51.0 67.5 56.5 69.0 48.5 60.5 60.0 61.5WoU/16 of a Whorl) 72.0 65.0 69.0 70.0 71.0 65.0 71.0 71.0 73.0 68.0 70.0 69.0 72.0AL2(mm) 58.0 39.5 57.0 56.5 58.0 51.0 64.0 55.5 69.0 51.0 60.5 59.5 57.0AW2(mm) 33.0 18.5 29.5 28.5 32.5 25.5 36.0 30.5 37.5 25.5 32.5 32.5 32.5BWL(mm) 80.0 49.0 75.0 74.0 75.0 65.0 89.0 70.5 88.0 62.5 78.0 78.0 74.0S(mm) 26.5 13.5 24.0 23.0 22.0 18.5 31.0 23.0 25.0 16.0 21.0 23.0 25.0USK (mm) 7.5 5.5 7.5 7.0 7.0 5.0 9.0 10.0 7.0 6.0 5.0 6.0 12.0APA(Degs) 28.0 36.0 33.0 31.0 32.0 34.0 31.0 38.0 36.0 41.0 35.0 35.0 39.0AAA(Degs) 10.0 11.0 12.0 9.0 8.0 15.0 11.0 13.0 9.0 8.0 10.0 8.0 12.0w(mm) 5.00 3.00 3.75 4.00 5.00 4.75 7.50 6.00 5.25 4.50 4.50 4.00 5.00x(mm) 2.00 0.25 0.75 0.25 0.50 0.25 0.25 - 1.00 0.50 1.00 0.50 0.50y(mm) 18.0 13.5 19.0 20.5 21.5 19.0 25.0 17.3 25.0 15.3 19.5 19.8 19.53(mm) 0.50 1.00 1.75 1.50 1.25 2.00 2.25 0.50 1.00 0.50 2.00 0.50 1.00d(mm) 16.5 10.5 15.0 13.5 16.0 13.5 18.0 15.0 18.0 13.5 15.5 15.5 17.5HW2(mm) 2.00 2.50 2.50 2.00 2.50 2.50 2.50 2.50 2.00 4.00 2.00 3.00 2.00WW2(mm) 3.50 6.00 3.50 3.00 3.00 3.00 3.50 4.00 3.00 3.50 3.00 3.50 3.50HW3(mm) 6.00 7.50 8.00 8.50 5.50 5.50 8.50 6.50 5.00 7.50 6.00 8.00 7.50WW3(mm) 7.50 6.50 7.50 7.00 6.50 7.50 8.00 8.00 8.00 7.00 7.00 7.50 8.00HW^(mm) 22.0 - 26.5 24.0 18.0 - 27.0 21.5 19.0 19.0 20.5 - 24.0WW(mm) 15.0 - 13.5 12.0 13.5 - 15.0 15.0 18.5 13.5 14.0 - 15.0BMmm) 31.0 24.0 39.0 31.5 30.5 30.5 36.0 32.0 29.5 28.5 34.0 35.0 31.5B5(mm) - - - - - - - - - - - *A(Degs) 105.0 89.0 100.0 102.0 108.0 103.0 98.0 91.0 118.0 93.0 119.0 106.0 95.0H4(mm) 21.0 15.0 20.0 19.0 17.5 20.0 21.0 18.0 21.5 18.5 17.0 20.5 23.0W4(mm) 39.0 27.0 40.0 39.0 35.0 39.0 40.0 33.0 42.0 35.0 40.0 42.0 37.5H5(mm) - - - - - - - - - - - -W5(mm) - - - - - - - - - - - - -

166



PC/LRNO:

B10C21 B10C

08126003

126017

126016

126019

126034

126020

126033

126036

126009

126013

126005

L(mm) 83.0 91.0 87.5 90.5 72.5 77.0 86.5 84.0 78.0 68.0 97.5 59.0 62.0W(mm) 56.5 66.5 59.5 67.0 53.5 55.0 62.5 59.0 60.0 47.0 68.0 42.0 44.5W o(l/I6 of a Whorl) 70.0 70.0 81.0 83.0 70.0 70.0 70.0 72.0 70.0 70.0 75.0 64.0 66.0AL2(mm) 53.5 61.5 54.0 60.0 54.5 52.0 58.5 58.5 56.5 46.5 66.5 44.0 46.0AW2(mm) 27.0 31.0 30.5 35.5 27.5 28.0 34.5 31.0 31.5 23.5 36.5 21.5 22.0BWL(mm) 77.0 83.5 71.0 81.0 67.0 68.0 78.0 76.0 73.0 60.0 88.0 54.0 57.0S(mm) 29.0 28.5 33.0 28.0 16.0 23.5 25.5 23.0 20.5 19.5 29.5 14.0 15.0

USH (mm) 6.0 7.5 16.5 10.0 6.0 9.0 8.5 8.0 5.0 8.5 9.5 4.5 5.0APA(Degs) 20.0 31.0 34.0 34.0 38.0 34.0 35.0 36.0 36.0 37.0 32.0 39.0 39.0AAA(Degs) 15.0 13.0 12.0 8.0 13.0 9.0 9.0 9.0 13.0 10.0 8.0 9.0 12.0w(mm) 3.00 5.50 - 6.75 3.00 3.00 6.25 7.25 3.50 4.75 6.50 3.25 3.00x(mm) 1.50 1.00 - 1.00 - 0.75 1.00 0.25 1.25 0.50 1.25 0.25 -

y(mm) 15.5 20.5 15.3 19.8 14.8 18.0 22.0 23.0 16.5 14.0 17.8 13.5 16.33(mm) 1.50 1.00 - 1.75 1.00 0.25 1.50 2.00 1.25 0.50 0.75 0.50 0.75d(mm) 10.5 16.0 15.0 17.5 15.5 14.5 18.0 16.0 16.5 12.5 9.5 . 10.5 12.5HW2(mm) 1.00 1.00 2.50 1.50 3.00 3.50 3.00 2.00 2.00 4.00 1.50 2.00 2.00WW2(mm) 2.50 2.50 3.00 2.50 3.00 3.00 2.50 3.50 3.00 3.00 3.00 4.00 3.00HW3(mm) 3.00 2.50 7.00 3.50 7.00 9.00 9.00 7.50 5.50 8.50 4.50 5.50 5.50WW3(mm) 7.00 7.00 6.50 8.00 6.00 6.00 6.50 7.00 6.50 5.50 7.00 8.50 7.50HW^(mm) 12.0 13.0 20.0 17.5 - 23.0 25.5 18.0 20.0 20.5 18.0 - -

WW(mm) 16.0 17.5 12.0 12.0 - 11.0 13.0 14.5 13.5 10.0 17.5 - -

B4(mm) 24.5 23.5 26.0 21.0 29.5 33.0 36.5 27.0 28.5 29.0 23.5 28.5 27.5B5(mm) - - 51.5 54.5 - - - - - - 64.5 - -

A(Degs) 117.0 114.0 87.0 101.0 97.0 88.0 93.0 92.0 118.0 86.0 96.0 97.0 100.0H4(mm) 16.5 17.0 20.0 20.0 16.5 18.5 21.5 15.5 16.5 18.0 13.5 20.5 17.0W4(mm) 32.5 35.0 29.0 33.0 35.5 35.0 40.5 34.0 38.5 33.0 33.0 35.5 33.0H5(mm) 40.0 39.5 35.0 41.0 - - - 39.0 - - 44.5 - -

W5(mm) - - 44.0 55.5 - - _______________ _____________

60.0a

- -

167



PC/LRNO:

B10C41

B10C23 B10C

05 B10C12 B10C45 B10C28 B10C34 B10C17 B10C24 B10C47 B10C22 B10C16 B10C36L(mm) 83.0 87.5 117.0 85.0 81.0 90.0 90.5 100.0 80.0 84.0 82.0 90.0 83.5T(mm) 62.5 58.0 80.5 64.0 54.0 62.0 66.5 69.0 56.0 60.0 57.0 65.5 57.0W o(l/16 of a Whorl) 67.0 74.0 80.0 68.0 69.0 68.0 71.0 75.0 71.0 68.0 73.0 74.0 72.0AL2(mm) 55.0 61.0 79.0 65.0 58.0 52.0 63.5 66.0 52.0 55.0 57.0 58.0 59.0AW2(mm) 31.5 29.0 36.0 34.0 24.5 29.0 32.0 33.5 28.0 30.0 27.0 36.0 27.0BWL(mm) 79.0 78.0 116.0 80.0 71.5 83.5 83.0 91.0 70.0 80.0 75.0 80.5 75.5S(mm) 24.5 25.0 38.5 18.5 19.5 34.0 26.0 32.0 26.5 27.0 24.0 30.5 24.0USH (mm) 4.0 9.5 11.0 5.0 9.5 6.5 7.0 9.0 10.0 4.0 7.0 9.5 8.0APA(Dcgs) 31.0 30.0 23.0 41.0 36.0 19.0 29.0 24.0 25.0 25.0 28.0 37.0 27.0AAA(Degs) 14.0 13.0 18.0 10.0 15.0 17.0 15.0 14.0 15.0 16.0 15.0 11.0 18.0w(mm) 5.75 3.00 4.25 - 4.00 - 4.75 3.75 3.50 - 3.25 7.50 2.25x(mm) 1.00 1.50 1.50 - 2.00 - 1.75 2.00 2.50 - 2.00 2.00 1.00y(mm) 17.8 16.0 18.8 14.5 14.5 15.5 16.5 16.5 14.0 15.3 14.5 20.8 14.83(mm) 1.00 1.00 6.00 - 1.50 - 3.00 1.25 1.50 - 1.50 2.75 1.50d(mm) 17.0 12.0 16.5 18.5 11.0 13.5 16.0 16.0 10.5 14.5 11.5 19.5 11.5HW2(mm) 2.00 1.50 1.50 1.00 1.00 1.00 1.50 1.00 1.00 1.00 1.00 1.00 1.00WW2(mm) 4.00 2.50 3.00 2.50 3.00 3.00 2.50 2.00 2.50 2.50 2.50. 2.50 2.50HW3(mm) 4.50 4.00 4.50 3.00 4.50 6.00 4.50 3.50 3.50 3.00 3.50 2.50 3.00WW3(mm) 5.00 6.50 6.00 5.50 8.00 8.50 7.00 7.00 7.50 6.00 6.50 6.50 7.00HW4(mm) - 13.5 12.0 17.5 15.0 - 10.5 13.0 13.0 - 10.0 13.0 11.0WW(mm) - 15.5 13.5 15.0 17.0 - 15.0 16.0 18.0 - 14.0 16.5 16.0B4(mm) 33.0 25.0 22.5 24.5 32.5 33.5 25.0 27.0 25.0 26.5 20.5 21.5 20.5B5(mm) - 38.5 54.0 - - - 49.0 58.0 - - 40.5 55.5 42.5A(Degs) 26.0 102.0 116.0 130.0 107.0 114.0 110.0 115.0 106.0 133.0 111.0 125.0 ’ 113.0H4(mm) 19.0 16.5 16.0 19.0 19.5 19.0 16.0 18.5 19.0 15.5 14.0 16.5 | 16.0WMmm) 40.0 30.0 36.5 38.0 34.5 41.5 17.0 34.5 33.0 37.5 29.5 33.5 ' 31.5H5(mm) 45.5 38.5 36.5 45.5 - - 39.0 40.0 38.0 44.0 35.5 42.0 37.0W5(mm) - 52.0 64.0 - -

t

61.0 58.0 52.5 - 51.0 55.5 52.5

168



PC/LRNO:

374621 B10C

27 B10C26 B10C04 B10C42 B10C11 B10C14 B10C37 B10C33 B10C25 B10C51 B10C43 B10C38L(mm) 82.0 121.5 104.5 97.0 93.0 95.0 96.5 95.0 95.0 114.0 100.0 103.5 118.0W(mm) 54.5 80.0 71.0 63.0 59.5 61.5 65.0 63.0 64.5 73.0 65.5 66.0 77.5W o(l/16 of a Whorl) 74.0 86.0 82.0 69.0 76.0 73.0 76.0 78.0 85.0 84.0 74.0 76.0 84.0AL2(mm) 57.0 77.0 69.0 61.0 56.5 62.5 62.5 60.0 66.0 69.0 65.0 68.0 72.5AW2(mm) 2 5.5 40.0 36.0 28.0 28.0 29.0 32.0 29.5 34.0 33.5 33.0 31.5 38.0BWL(mm) 76.0 108.5 91.0 84.0 84.5 86.0 85.5 83.0 89.0 98.0 90.0 94.5 103.5S(mm) 26.5 43.5 38.0 32.5 37.0 33.0 33.5 33.0 28.0 46.0 34.0 35.5 46.5USH (mm) 6.0 12.5 14.0 12.5 8.5 8.5 11.0 12.0 6.0 16.0 10.0 |9.0 14.5

APA(Degs) 26.0 23.0 26.0 24.0 22.0 19.0 23.0 22.0 30.0 17.0 22.0 20.0 21.0AAA(Degs) 16.0 17.0 17.0 20.0 14.0 16.0 14.0 14.0 13.0 23.0 16.0 17.0 21.0w(mm) 3.50 5.50 3.00 5.75 4.00 3.75 4.00 4.75 2.50 6.25 5.75 5.25 5.75x(mm) 2.00 2.75 3.25 1.75 0.50 1.00 3.00 0.75 0.75 2.00 1.25 1.25 2.00y(mm) 13.5 19.0 14.8 15.0 16.3 15.8 16.0 19.5 18.0 19.0 19.3 20.8 17.03(mm) 3.00 3.75 2.75 1.75 1.00 1.75 2.00 2.00 1.50 5.00 4.00 4.75 2.00d(mm) 12.0 18.0 2.50 11.5 13.0 9.50 13.0 13.0 15.5 11.0 13.0 10.5 12.5HW2(mm) 2.00 1.50 3.00 2.00 1.00 1.00 1.50 1.00 1.00 1.50 1.00 1.50 1.50WW2(mm) 2.50 3.00 6.50 4.50 2.50 2.00 3.00 2.00 2.50 2.50 2.50 3.00 2.50HW3(mm) 4.00 4.00 6.00 7.50 3.50 3.50 3.50 4.50 3.00 5.50 4.00 3.50 5.50WW3(mm) 6.50 6.50 14.0 11.0 6.50 7.00 8.00 6.50 6.00 6.00 6.50 6.50 6.50HW4(mm) - 8.0 10.5 21.0 - 12.0 15.0 12.5 - 13.0 13.0 9.5 10.5WW(mm) - 10.0 17.0 19.5 16.5 19.0 11.0 - 11.0 15.0 14.5 11.5B4(mm) 21.5 17.0 44.0 31.5 19.5 25.0 25.0 19.0 25.5 22.5 31.0 21.5 22.5B5(mm) 42.5 41.0 99.0 53.5 52.5 53.5 57.0 45.0 - 49.0 54.0 54.5 47.5A(Degs) 99.0 108.0 140.0 102.0 118.0 111.0 110.0 117.0 145.C 108.0 104.0 110.0 112.0H4(mm) 15.5 11.5 28.5 24.0 15.0 17.0 21.5 12.5 19.0 13.0 20.0 15.5 11.5W4(mm) 28.0 24.0 29.0 40.0 30.0 34.0 37.5 29.0 38.0 28.5 34.0 33.0 29.0H5(mm) - 31.0 29.0 46.0 39.5 35.0 43.0 26.0 45.5 32.0 34.0 37.0 ,3 1 .5V5(mm) - 51.5 50.5 61.5 49.0 52.0 56.5

_____________

48.0 59.0 52.5 56.0 57.5 i 56.5l

169



PC/LR 37^6 3746 3746 3746 3746 3746 3746 3746 3746 3746 3746 3746 3746NO: 01 05 06 08 09 10 12 14 15 16 17 18 20L(mm) 109.5 100.0 95.0 96.0 92.0 92.0 107.5 105.5 99.5 89.5 102.5 108.0 118.0W(mm) 73.5 70.5 65.5 68.0 61.0 60.0 74.5 69.5 69.0 64.0 70.0 71.5 80.5W o(l/16 of a Whorl) 83.0 81.0 76.0 73.0 76.0 75.0 79.0 76.0 78.0 75.0 76.0 77.0 84.0AL2(mm) 74.0 69.0 '60.5 66.5 62.0 57.0 77.0 72.5 69.0 60.5 69.0 68.5 81.5AW2(mm) 36.0 35.0 32.0 32.5 29.0 30.0 37.0 36.5 35.0 32.0 34.5 37.5 38.5BWL(nmi) 100.0 90.5 86.0 89.0 84.0 83.0 99.0 97.0 90.0 82.5 93.0 98.5 106.5S(mm) 37.0 29.5 35.0 29.5 30.5 31.5 30.0 30.5 31.5 28.0 32.0 37.0 36.5USH (mm) 9.5 10.0 9.0 7.5 8.0 8.5 8.5 9.0 9.5 7.0 9.5 9.5 12.0APA(Degs) 24.0 32.0 21.0 28.0 22.0 20.0 32.0 27.0 28.0 25.0 31.0 |23.0 31.0AAA(Degs) 20.0 15.0 17.0 15.0 16.0 15.0 13.0 9.0 15.0 18.0 16.0 U5.0

112.0

w(mm) 4.00 5.00 4.50 4.00 5.50 4.25 6.25 6.00 5.00 3.75 5.75 7 .2 5 6.50x(mm) 1.25 3.25 3.00 3.25 3.00 3.75 2.50 2.25 3.75 3.00 3.00 3.25 3.50y(mm) 17.0 16.5 12.8 14.0 14.3 14.0 18.3 19.3 15.0 11.5 15.5 17.0 17.03(mm) 5.25 1.50 1.50 2.50 1.25 1.25 1.75 1.75 1.25 0.75 1.75 2.25 4.50d(mm) 13.0 16.0 12.5 14.0 11.5 11.5 19.9 16.0 15.0 13.0 16.5 16.0 19.0HW2(mm) 1.50 1.50 1.50 0.50 1.50 1.50 1.50 1.00 1.00 0.50 1.00 1.00 1.00WW2(mm) 3.50 2.50 2.50 2.50 3.00 3.00 3.00 3.00 3.00 3.00 3.00 3.50 2.50HW3(mm) 4.50 3.50 4.00 1.50 3.50 3.00 4.00 3.50 4.00 2.00 3.00 3.50 4.00WW3(mm) 7.50 5.50 7.00 6.00 6.50 7.00 6.50 7.50 6.50 8.00 7.50 7.00 5.50HV4(mm) 11.5 8.5 10.5 7.0 9.5 11.0 12.5 12.0 13.0 10.0 11.5 13.5 11.0WW(mm) 13.0 10.0 15.5 16.0 15.0 15.0 15.0 16.5 13.5 17.0 17.0 17.0 11.0B4(mm) 17.0 19.0 23.0 13.5 20.5 24.0 24.0 25.5 22.0 21.0 26.0 25.5 20.5B5(mm) 48.5 50.5 53.5 42.5 50.0 56.5 56.0 53.0 55.0 49.5 57.5 57.5 47.5A(Degs) 113.5 104.0 109.5 134.0 115.5 113.5 115.0 118.0 114.5 128.0 115.0 124.0 119.5H4(mm) 14.0 14.0 13.5 17.5 15.5 16.5 16.0 17.5 15.0 14.5 16.5 19.5 15.5W4(mm) 30.0 29.0 31.0 35.5 31.5 30.5 33.5 34.5 32.5 32.5 33.5 38.0 30.5H5(mm) 28.0 31.0 38.0 38.0 34.0 38.0 36.0 39.0 34.0 36.0 38.5 40.0 33.5W5(mm) 53.0 50.5 57.0 59.0 53.5 53.5 57.5 58.0 55.0 56.0 58.0 59.0 56.0

170



PC/LRNO:

284212

284213

284214

284215

284216

284217

284218

284219

284220

284221 2842

23 284224 284225L(mm) 78.5 66.0 47.0 92.0 72.0 59.0 90.0 86.5 69.5 69.0 87.0 83.5 72.5W(mm) 56.0 46.5 37.0 60.0 57.0 45.0 64.5 58.5 48.0 48.0 57.0 57.5 53.0V o (l/1 6 of a Whorl) 73.0 71.0 62.0 80.0 72.0 66.0 76.0 80.0 65.0 73.0 79.0 74.0 70.0AL2(mm) 54.5 45.0 38.0 57.0 50.0 42.0 58.5 52.5 53.0 46.0 55.0 56.5 48.0AW2(mm) 29.5 24.0 18.0 29.5 30.0 24.0 33.5 30.0 25.0 26.0 28.5 29.5 29.0BWL(mm) 72.0 59.0 44.0 77.5 63.5 54.0 79.0 73.5 63.0 60.0 74.5 73.5 66.0S(mm) 23.5 21.0 9.5 34.5 22.0 17.0 31.0 33.0 13.5 27.5 31.5 26.0 23.0MSH (mm) 6.5 7.0 3.5 14.5 8.0 5.0 11.0 13.0 4.0 9.0 12.5 10.0 7.0

APA(Degs) 32.0 32.0 48.0 29.0 39.0 34.0 32.0 28.0 41.0 35.0 28.0 32.0 32.0AAA(Degs) 14.0 10.0 9.0 9.0 10.0 9.0 10.0 10.0 8.0 10.0 10.0 10.0 6.0w(mm) 3.25 4.50 1.75 6.25 4.50 3.75 4.00 5.50 3.25 3.50 7.00 9.00 5.25x(mm) 0.25 0.75 0.75 1.00 0.75 0.50 - 0.25 0.75 - 0.75 0.25 2.00y(mm) 19.0 14.5 10.0 16.5 14.5 14.5 17.0 20.0 15.5 16.5 20.5 22.0 14.33(mm) 1.75 0.50 0.50 1.00 0.50 1.50 - 1.25 - 1.00 1.50 2.25 0.75cKmm) 13.0 13.0 10.5 13.5 16.0 12.5 16.0 14.5 13.0 12.5 13.0 14.0 15.0HW2(mm) 1.50 2.00 1.50 2.00 1.00 2.00 2.50 3.50 2.00 2.00 2.00 1.00 2.00WW2(mm) 3.00 3.00 2.00 2.00 3.00 2.50 3.00 3.00 2.00 3.00 N

>O O 2.50 2.50

HW3(mm) 5.00 6.00 4.50 6.00 4.00 6.50 5.50 7.00 6.00 6.00 5.50 4.50 6.00WW3(mm) 6.00 6.00 5.00 5.00 8.00 6.00 7.00 5.00 6.00 6.00 6.00 7.00 6.50HW4(mm) 12.0 14.0 - 15.5 11.0 18.0 15.0 16.0 15.5 14.5 14.5 ■ .3.3 16.5WW(mm) 13.0 12.0 - 9.0 15.5 10.0 15.0 8.5 12.0 12.0 14.0 16.0 15.5B4(mm) 21.0 21.0 20.0 20.5 18.5 27.0 25.0 20.0 29.0 21.0 21.0 23.5 25.5B5(mm) 57.0 - - 47.0 46.0 - 53.0 49.0 - 39.0 49.5 51.5A(Dcgs) 96.5 88.5 106.5 97.0 101.5 102.5 94.0 85.5 109.5 90.5 93.5 98.5 95.0H4(mm) 14.5 15.0 14.0 15.5 14.0 15.0 17.0 15.5 15.5 15.5 16.0 16.0 16.5W4(mm) 26.5 25.5 25.5 27.0 27.5 30.0 29.0 24.5 32.0 25.0 26.0 27.0 29.5H5(mm) 36.0 33.0 - 30.0 33.0 - 37.5 29.0 - 33.0 ( 33.5 38.0 37.0W5(mm) 51.5 - .44.5 52.0 - 51.5 43.0 - 43.0 45.0 50.5

171



PC/LRNO:

287217

287221

287223

287214 2872

25 284204 284205

284206

284207

284208

284209 284210 284211

L(mm) 61.0 60.0 58.0 51.5 63.5 82.5 79.5 89.5 62.0 67.0 87.0 80.5 72.5^(inm ) 43.0 41.0 41.5 37.0 47.5 59.0 59.5 65.5 46.5 52.0 61.0 55.5 52.0W o(l/16 of a Whorl) 66.0 60.0 62.0 60.0 62.0 75.0 75.0 75.0 66.0 70.0 76.0 79.0 72.0AL2(mm) 47.0 47.0 43.5 40.5 43.0 52.0 55.0 58.0 45.5 47.0 56.0 49.0 48.0AW2(mm) 22.0 18.5 20.0 18.0 23.0 32.5 32.0 34.5 24.0 28.0 30.0 27.0 27.5BWL(mm) 57.0 56.5 53.0 48.5 59.5 72.0 72.0 79.0 57.5 60.0 76.0 68.0 64.0S(mm) 14.0 13.0 15.0 11.0 20.5 29.0 23.0 30.5 16.0 19.0 31.5 32.0 24.0USH (mm) 4.5 3.0 5.0 3.0 4.0 11.0 8.0 10.5 4.0 6.5 11.5 12.5 8.5

APA(Degs) 37.0 33.0 35.0 37.0 25.0 33.0 36.0 33.0 38.0 40.0 31.0 26.0 31.0AAACDegs) 12.0 13.0 14.0 14.0 12.0 5.0 8.0 9.0 9.0 7.0 14.0 13.0 11.0w(mm) 2.75 2.25 1.50 0.75 3.25 7.50 5.50 4.50 4.00 5.50 5.50 5.50 4.00x(mm) 0.50 0.50 0.50 0.75 1.00 1.00 - 1.00 0.50 0.25 - 1.00 0.50y(mm) 13.5 12.0 9.5 7.5 12.5 18.3 18.0 20.5 15.5 16.0 17.8 17.5 19.03(mm) 1.75 1.50 1.00 1.50 1.00 0.50 2.00 - 0.50 0.50 2.00 1.00 1.00d(mm) 10.5 9.00 10.0 9.0 10.0 16.5 17.0 15.5 12.5 15.5 14.0 12.0 13.5HW2(mm) 2.00 1.00 1.50 1.00 1.00 1.00 1.00 1.00 2.00 1.50 1.00 1.50 2.50WW2(mm) 3.00 4.00 4.00 3.00 3.00 3.00 3.00 3.00 2.50 3.00 2.50 3.00 3.00HW3(mm) 4.00 4.00 6.00 4.50 5.00 4.50 4.00 3.50 5.50 5.00 3.50 6.00 7.00WW3(mm) 7.00 9.00 8.50 8.00 8.00 7.00 7.00 7.00 6.00 6.00 6.00 5.50 7.00HV4(mm) - - - - - 12.5 10.0 13.5 15.5 11.5 12.5 13.5 16.0WW(mm) - - - - - 14.0 13.0 16.5 12.0 13.0 12.5 9.5 14.0B4(mm) 23.0 25.5 26.5 22.0 33.5 22.0 19.0 21.0 20.0 22.0 21.0 19.0 22.5B5(mm) - - - - - 50.0 44.5 51.0 - - 52.5 41.0 38.5A(Degs) 115.5 108.3 102.0 101.5 111.0 96.5 103.0 106.5 106.5 99.5 109.5 92.5 88.5HMmm) 14.0 19.0 16.5 - 19.5 14.0 14.0 15.0 16.0 13.5 14.0 13.5 18.5WMmm) 30.0 35.5 32.5 - 38.0 25.5 26.5 30.5 31.0 27.5 27.0 24.5 29.0H5(mm) 32.5 - - - - 33.5 27.0 37.5 35.0 34.5 33.0 27.0 42.0W5(mm)

i____________- - - - - 47.0 47.0 58.0 - 35.0 48.5 42.0 48.0

172



PC/LR 2872 2872 2872 2872 2872 2872 2872 2872 2872 2872 2872 2872 2872NO: 01 02 03 04 05 06 08 09 10 12 13 15 16L(mm) 67.5 65.0 66.0 63.0 62.0 55.0 58.5 69.0 76.0 69.0 62.0 59.0 71.5W(mm) 48.0 47.0 47.5 43.0 46.0 39.0 42.5 50.0 51.5 50.0 46.0 40.5 50.0W o(l/16 of a Whorl) 62.0 67.0 62.0 66.0 63.0 60.0 59.0 66.0 67.0 61.0 66.0 61.0 65.0AL2(mm) 52.5 47.0 46.5 46.0 48.0 40.5 47.0 49.0 52.0 55.0 46.0 41.5 41.5AW2(mm) 24.5 23.5 23.0 21.0 22.0 17.5 20.5 23.0 26.0 25.0 23.5 18.5 25.0BWL(mm) 64.0 61.5 63.0 58.5 58.5 51:0 56.0 65.0 69.0 67.0 58.0 56.0 67.0S(mm) 15.0 18.0 19.0 16.5 13.0 14.0 10.5 19.5 22.0 15.0 14.5 16.5 20.0USH (mm) 3.5 4.0 3.0 4.5 3.0 4.0 2.0 4.0 7.0 2.0 4.0 3.5 5.0

APA(Degs) 36.0 30.0 29.0 31.0 40.0 29.0 38.0 28.0 31.0 35.0 37.0 27.0 30.0AAA(Degs) 14.0 11.0 13.0 13.0 11.0 16.0 12.0 15.0 12.0 12.0 10.0 13.0 9.0w(mm) 3.00 2.75 2.25 3.00 3.00 1.75 1.75 3.50 5.25 2.00 3.00 2.50 3.00x(mm) 1.50 1.00 - - 1.50 0.50 1.00 0.25 0.50 0.50 0.75 0.50 0.75y(mm) 15.0 13.3 12.5 15.0 11.5 9.0 10.0 14.0 18.0 16.5 12.0 14.0 13.53(mm) 2.50 1.00 - 0.50 0.50 0.75 1.75 1.50 0.50 1.00 0.50 1.25 -d(mm) 11.5 11.0 11.0 9.5 11.5 8.0 10.5 11.5 13.5 11.5 12.5 8.5 12.0HW2(mm) 2.00 1.00 1.00 2.00 2.00 2.00 1.00 2.00 2.00 1.00 1.00 1.50 2.50WW2(mm) 3.50 2.50 3.00 2.50 3.00 4.00 3.00 3.00 3.00 3.50 2.50 3.50 3.00HW3(mm) 5.00 4.00 4.0 5.50 5.50 5.00 2.50 6.00 6.00 3.00 5.00 5.00 6.00WW3(mm) 8.00 7.00 9.00 6.00 7.00 10.00 8.00 7.00 7.00 9.00 6.50 8.00 7.00HV4(mm) - _ - 18.0 - - - - - - -WW(mm) - - 12.0 - - - - - - - - ~B4(mm) 31.0 27.0 29.0 28.0 26.0 29.0 29.0 29.5 26.0 32.0 23.5 32.0 31.5B5(mm) - - - - - - - - - - -A(Degs) 115.0 122.5 118.0 113.5 110.5 105.0 128.0 112.5 101.0 125.5 120.5 105.5 111.0IW(mm) 20.0 14.0 18.5 16.5 17.0 18.5 23.0 19.0 17.5 21.5 14.0 17.5 18.5WMmm) 39.5 31.0 37.5 32.0 35.5 34.5 37.5 37.5 32.5 42.5 32.0 33.5 37.0H5(mm) - - - 33.0 - - - 41.0 - - 34.5 0 41.0W5(mm) - - ! - - - - - - - 1 - - ~



PC/LRNO:

273909 273910 273911 273912 273913 273915 273916 273 9 17 273918 273902 273903 273905 273908

L(mm) 59.5 70.0 122.0 84.0 119.0 88.0 73.0 78.5 76.0 95.0 92.5 75.0 90.5W(mm) 37.0 38.0 59.5 49.0 58.0 47.0 43.5 47.0 43.0 54.0 52.0 47.0 49.5W o(l/16 of a Whorl) 70.0 68.0 86.0 78.0 73.0 76.0 69.0 69.0 77.0 82.0 82.0 74.0 82.0AL2(mm) 40.0 47.0 66.5 52.0 64.0 53.5 51.0 52.5 49.0 55.5 55.0 51.5 52.5AW2(mm) 16.0 17.0 24.5 21.0 23.5 21.0 20.5 21.5 18.5 23.0 24.0 19.5 21.5BWL(mm) 51.5 61.0 91.5 70.0 94.5 72.5 64.0 69.5 65.5 77.5 76.0 65.5 73.0S(mm) 20.5 25.0 56.0 33.5 56.0 32.5 24.0 27.5 27.5 40.5 38.5 24.0 39.5USH (mm) 8.0 9.0 30.0 13.5 24.0 12.5 9.0 9.0 10.5 17.5 16.5 9.0 17.5

APA(Degs) 19.0 18.0 16.0 15.0 10.0 19.0 23.0 20.0 20.0 16.0 16.0 23.0 16.0AAA(Degs) 18.0 20.0 15.0 17.0 20.0 20.0 18.0 18.0 15.0 17.0 21.0 18.0 20.0w(mm) 1.50 2.00 2.50 2.00 2.00 2.50 2.00 1.75 2.50 1.75 2.25 2.00 2.00x(mm) 0.50 - - 0.50 0.50 - 0.50 0.75 1.25 0.75 0.75 1.75 0.25y(mm) 10.8 12.8 20.0 12.3 14.0 13.3 12.5 12.8 14.0 13.0 13.5 12.0 12.03(mm) 1.75 5.25 6.50 2.25 3.50 4.00 4.50 3.75 2.75 2.75 3.25 3.25 3.00d(mm) 5.0 7.5 11.0 7.5 9.0 7.0 8.0 8.0 7.0 8.0 8.5 7.5 7.5HW2(mm) 2.00 1.50 2.50 1.00 2.00 1.50 1.00 1.50 1.00 2.00 2.50 1.50 2.00WW2(mm) 3.50 3.00 3.00 2.50 3.00 3.00 2.00 2.50 3.00 3.00 4.00 2.50 3.50HW3(mm) 6.00 6.00 7.50 4.50 6.00 5.00 4.00 5.00 5.00 7.00 8.00 4.00 6.00WW3(mm) 7.50 7.50 7.00 6.50 6.00 6.50 6.00 6.50 6.50 6.50 7.00 6.50 6.00HW4(mm) 15.0 20.5 19.5 14.0 17.0 15.5 11.0 15.5 13.5 19.0 19.0 11.0 16.0WW(mm) 11.5 12.0 20.5 11.5 11.5 13.5 12.0 13.5 12.0 11.0 11.0 12.0 11.5B4(mm) 22.0 33.0 29.0 21.5 23.5 23.0 18.0 23.0 20.5 23.0 27.0 18.0 20.0B5(mm) - - 57.5 40.5 42.5 48.0 40.0 49.5 44.5 44.0 46.5 42.0 44.5A(Degs) 90.0 94.0 86.5 110.0 89.0 91.0 94.0 94.5 91.5 91.0 80.5 98.5 90.0H4(mm) 16.5 20.0 21.5 113.5 18.5 19.0 13.0 16.5 15.5 21.0 20.5 15.5 -WMmm) 25.5 28.0 28.0 25.5 26.5 27.0 22.5 26.0 24.5 29.0 27.5 25.0 -H5(mm) 32.0 43.0 40.5 31.0 38.5 35.0 33.5 35.0 33.5 32.5 36.5 31.0 30.0WMmm)

•____________________________

- 41.0 43.0 27.5 42.0 39.5 42.5 37.5 44.0 42.5 24.5 38.0

174(ALL MEASUREMENTS GIVEN AT X6 MAGNIFICATION)


PC/LR 273*1 2739 2739 2739 2739 2739 2739NO: 01 03 04 05 06 07 08L(mm) 116.5 82.0 80.5 89.0 68.5 91.0 123.0W(mm) 58.0 47.5 43.0 48.0 40.0 50.0 59.5W o(l/16 of a Whorl) 91.0 77.0 75.0 76.0 70.0 78.0 92.0AL2(mm) 65.5 51.0 51.5 56.0 47.0 56.0 63.0AW2(mm) 27.5 19.5 19.5 21.5 18.5 23.0 23.0 1

BWL(mm) 92.5 71.0 68.0 75.0 61.0 71.5 97.5S(mm) 53.5 33.5 31.0 34.0 23.5 37.0 59.0USH (mm) 23.5 10.5 12.5 14.0 7.5 14.5 26.0

APA(Degs) 10.0 13.0 16.0 16.0 20.0 15.0 10.0AAA(Degs) 22.0 19.0 18.0 18.0 19.0 20.0 20.0w(mm) 4.00 2.00 2.00 2.00 1.50 2.00 1.00x(mm) 0.75 - 1.00 - 0.50 0.75 -

y(mm) 24.0 14.0 12.0 13.0 10.0 13.5 18.03(mm) 6.25 2.50 4.25 4.00 3.00 2.00 6.00d(mm) 9.0 6.0 7.0 17.5 7.0 8.0 7.5 *HW2(mm) 1.00 2.00 1.50 1.50 1.50 2.00 1.50WW2(mm) 3.00 2.50 2.50 3.00 3.00 2.50 2.50HW3(mm) 4.50 5.00 6.00 5.00 5.00 6.00 6.00WW3(mm) 7.50 5.00 7.00 7.00 7.00 6.00 7.00HWMmm) 13.0 13.5 15.0 17.5 16.0 16.5 15.5WW(mm) 12.5 10.5 12.0 14.5 14.0 12.5 12.5B4(mm) 22.5 19.5 23.5 25.0 25.0 25.0 18.5B5(mm) 41.5 40.5 49.5 54.0 - 49.5 40.0A(Degs) 96.5 97.5 87.5 97.5 101.5 92.0 88.0H4(mm) 15.0 14.0 16.0 19.5 15.5 18.0 17.0W4(mm) 23.0 75.0 24.0 29.0 26.5 26.5 24.5H5(mm) 31.0 29.0 38.0 41.5 34.0 34.5 33.5W5(mm) 35.5 40.5 39.0 45.0

<43.0 37.0

taxonomic study of the bulinus africanus … · 2 abstract certain species of the bulinus africanus...

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