III. MA TERIALS AND METHODS

111.1. Study location

The study location is covered by the tribal belt of the high ranges of

the Western Ghats in Thiruvananthapuram district between north latitude

8O17'50"and 8"53'42" and east longitudes 76"40'24" and 70°17'00", the

Southern most administrative unit of the state with Thiruvananthapuram as

the capital. The district is bounded by the Arabian Sea in the west and the

Districts of the state of Tamil Nadu, namely Kanyakumari in the South and

Tirunelveli in the east and north by Kollam district (Fig.2).

While Thiruvananthapuram district has three physiographic zones

namely the highlands, midlands and low lands (Sreedhara Menon, 1962), it is

only the high land and mid land region is relevant in the present study. This

mountainous zone consists of hills, which form the southern most part of the

Western Ghats. The main Ghats stretch along the eastern border with

elevations varying between 1000 and 1869 m. The top and higher slopes of

the Ghats are very steep and rocky and in many places inaccessible. Such

places are usually barren. The lower slopes of the Ghats on the other hand

are covered with dense vegetation.

The climate of the district varies with the topography, but in general

the typical tropical humid climate with the two monsoons (north west and

north east) exists all over. Temperature and other meteorological conditions

are fairly uniform throughout the district except on the Ghats.

III.l.l. Field survey and collection

A field survey on beekeeping in the district was conducted in order to

identify the zones of the district where the beekeepers depend on for keeping

bees. The potential sites were identified and sample collection was made

from the different apiaries of the district. The tribal settlements of the district

were surveyed for the dependency of the tribes for honey as a Non- wood

forest product. A case study was conducted in selected settlements.

Household level interview was conducted by a questionnaire prepared as per

the format in Annexure 1.

The sample collection at different forest areas was done with

assistance from selected members of the tribal community. Information on

the materials analysed is given in Table 1.

111.2. Methodology

111.2.1. Preparation of acetolysis mixture

9ml of acetic anhydride was mixed with l m l of concentrated Sulphuric

acid, which was added as drops to the acetic anhydride.

111.2.2. Preparation of glycerine jelly

Glycerine Jelly was used as the mounting medium for acetolysed

pollen grains from honey, pollen load and anthers of individual flowers.

Reagents

Gelatin 59

Water 30ml

Glycerine 3511-11

Phenol Crystals 39

Tablel. Materials analysed for nectar and pollen sources

2 . 1 ~ p ~ r a n a indica / Wild / p a l a d e l

SI.

No.

1 YNY -3 I Apis cerana indica rd Pabde 1 HNY -4 -+- -- Trigona irridipennis Palode

/ 5 / HNY -5 1 PL-5 1 Apis cerana indica / Wild I Palode I

Honey I Pollen

Sample Loads

1 6 ~ H N Y - ~ i 1 Trigona irridipennis / Wild I Amboori, Kottur I

-8 - t ~ ~ ~ - r ~ z i r r i d i ~ e n n i s j Wild j palo-1

Bee species

1 12 1 HNY -12 1 1 Trigona inidipennis 1 Wild 1 Njaraneeli I

- b z a n a indica / p i / C h e r a v I

Nature

115 HNY-15 1 1 Apis cerana indica Apiary / Cherappally i

Locality

I I

Apis cerana indica ( Market ( Thiruvananthapuram

118 / HNY-18 1 I Apis cerana indica I Apiary 1 Cherappally,Parandode I I I

HNY -19

HNY -20

HNY -21

HNY -22

HNY -23

HNY -24

HNY -25

HNY -26

HNY -27

HNY -28

HNY -29

HNY -30

HNY -31

HNY -32

HNY -33

HNY -34

HNY -35

HNY -36

HNY -37

HNY -38

HNY -39

I Apis cerana indica Wild

/ Apis cerana indica

I Apis cerana indica

1 Apis cerana indica

PL-24 ) Trigona irridipennis

PL-25 Trigona irridipennis - --t- PL-26 / Trigona irridipennis

I Trigona irridipennis

- 7 A ~ i a n a indica

1 Apis cerana indica

PL-30 Trigona imdipennis ----I-- PL-31 Ap~s cerana indica I

1 Apis dorsata

I Trigona irridipennis

I Apis cerana indica

) Apis cerana indica

( Apis cerana indica

PL-37 Apis cerana indica

PL-38 Apis cerana indica - --I-

I Apis cerana indica

Apiary

Market

Market

Wild

Wild

Wild

Wild

Wild

Apiary

Apiary

Wild

Wild

Wild

Wild

Apiary

Apiary

Apiary

Apiary

Apiary

Apiary

- Amboori, Kottur

Marthandam - Marthandam - Sanghili,Madathara

Peringamala

Peringamala - Cheenikkala, Sanghili

Cheenikkala, Sanghili

Cheenikkala, Sanghili

Parandode

Cherappally ,Parandode

Kesaripuram, Peringamala

Pullivattom, Peringamala

Peringamala

w-40 T -7-erana indica 1 Market (Bt Thiruvananthapuram

142 / HNY -42-t --

I Apis cerana indica Market Thiruvananthapuram i

145 1 HNY -45 1 I Apis cerana indica I Apiary 1 Kesaripuram, Peringamala I

WE7 ~ ~ 4 i b ~ a n a indica / Apiary / C h a l l i m u ~ ~ u j

~ ~ ~ ~ - ~ ~ ~ ~ ~ n a indica A / Arippa. Sanghili

1 1; 1 HNY -50 1 1 Apiscerana indica Apiary 1 Kulathuppuzha 1 HNY -51 Apis cerana indica Apiary Kesarrpuram, Peringamala

5 2 1 HNY -52 cerana indica 1 :;a 'lode 1 - - - - - --

53 HNY -53 Apis cerana indica Elavupalam

1 57 1 HNY -57 I I Apis cerana indica I Wild / Palode 1 58 I HNY -58 Apisceranaindica :I 1 Palode 1 59 HNY -59 PL-59 Yrigona irridipennis Ponmudi

1 60 1 HNY -60 / PL-60 I Trigona irridipennis I Wild I Ponmudi I

-- . . -- - T - 6 1 T PL-61 1 Trigona irridipennis i Wild

62 / HNY -62 I PL-62 Trigona irridipennis 1 Wild 1 Ponmudi I

-4s- t ~ l ? & n a irridipennis Wild 1 ~ o n m u d i l

64 / HNY -64 1 PL-64 1 Trigona irridipennis 1 Wild 1 Ponmudi 1

66 1 HNY -66 / PL-66 / Apis cerana indica I Wild / Kallar, Vithura I

I 68 1 HNY -68 i I Apis cerana indica I Apiary 1 Palode I

i

69 1 HNY -69 1 / Apis dorsata I Wild / Adiparambu, Madathara I

67

70 ) HNY - ~ ~ ~ ~ ~ - / ~ ~ r s ~ t ~ Wild

HNY --67 1

71 1 HNY I 1 1 Apis cerana indica 1 Apiary 1 Palode 1 .-

72 HNY -72 PL-72 Apis cerana indica Wild Palode

73 1 HNY --73 / PL-73 I Trigona irridipennis I Wild Palode I 74 ( HNY -74 1 1 Apis cerana indica 1 Wild 1 Vithura 1

*Y~ i s t -t=rana indica / Market / Peringamala

76 1 HNY -78 1 I Apis cerana indica I Wild / Chonampara, Kottur I

78 / HNY -80 1 I Apis cerana indica / Apiary I Amboori, Kottur I

I

HNY -81 1 PL-81 Trigona irridipennis Apiary

kii-~ \~-~~-jGin*&l

HNY -79 PL-79 Trigona irridipennis

8 1 / ~ ~ y - 8 3 1 1 Trigona irridipennis 1 Wild I K a l t . Vithura I

Wild 17- t--I--

HNY -84

HNY -85

HNY -86

HNY -87

HNY -88

HNY -91

HNY -92

HNY -93

HNY -97

HNY -98

HNY -99

HNY -100

HNY -101

Trigona irridipennis

Apis cerana indica

Apis cerana indica

Jrigona irridipennis

Trigona irridipennis

Trigona irridipennis

Trigona irridipennis

Apis dorsata

Apis cerana indica

Apis cerana indica

Apis cerana indica

Trigona irridipennis

Trigona irridipennis

Trigona irridipennis

Trigona irridipennis .-

Apis cerana indica

Apis florea

Apis cerana indica

Wild

Apiary

Apiary

Wild

Wild

Wild

Wild

Wild

Apiary

Wild

Wild

Wild

Wild

Wild

Wild - Apiary

Wild

Wild -

-

Peringamala

Amboori, Kottur

Chonampara, Kottur

Chonampara, Kottur

Chonampara, Kottur

Chonampara, Kottur

Amboori, Kottur

Peringamala

Agastyamala, Bonnoccord

Bonnoccord

Agastyamala, Bonnoccord

Agastyamala, Bonnoccord - Agastyamala, Bonnoccord

Agastyamala, Bonnoccord - Peringamala

Bonnoccord

Agastyamala, Bonnoccord -

The gelatin is dissolved in warm water with continuous stirring. The

other components are added to it and then filtered hot. It is kept in a closed

bottle. Phenol Crystals are added as a preservative to avoid growth of fungal

elements.

111.2.3. Preparation of pollen: Acetolysis Method (Erdtmann, 1960)

A. From honey

I rnl of honey sample was taken in a test tube and diluted to 10ml by

hot distilled water of 40" C. The diluted honey is sieved through a mesh of

100p rn. The suspension thus obtained was centrifuged at 3000 rpm. for 5

minutes. The supernatant was decanted. The pellet of pollen sediment was

subjected for acetolysis (Erdtman, 1960). Detailed procedure is outlined

below.

Pollen material from honey

& Washed with 51~1170% alcohol Centrifugation

4 Decanted the alcohol Sediment

& Wash with 5ml glacial acetic acid Centrifugation

4 Decanted glacial acetic acid Sediment

4 (9:l acetic anhydride and Con.H2S04) Coloured medium

& Centrifugation Sediment

& Wash and centrifuge in distilled water (3 times)

Sediment

Mounted in Glycerine jelly

B. From pollen load

Individual pollen loads were separated from the beehive and

dispersed directly in 70% alcohol and analysed by acetolysis as above.

C. From anther

For identification of pollen grains isolated from honey and bee pollen

loads, pollen slides from flowering species were prepared as reference

slides. For this, anthers separated from filaments were put in distilled water

and crushed with a glass rod. It is then sieved through a mesh of 100pm

size. The pollen suspension was centrifuged at 3000 rpm for 3 min. and

decanted the supernatant. Pollen pellet was subjected for acetolysis

(Erdtman, 1960) as detailed above.

111.2.4. Field notes on forage preference by Apis cerana indica F.

For the elucidation of the dietary preference shown by the commercial

bee A. cerana indica, three different colonies of this bee were located

between 8"45' and 8"47' north latitudes and 77" 1' and 77" 4' east longitude,

in Thiruvananthapuram district. The colonies were in three different

vegetational patches, one in open cultivated land of mixed vegetation,

second in dry deciduous forest and the third in a transient zone between

introduced exotic vegetation and cultivated land of mixed crops. The bee

colonies were nearly lOOOm apart from each other. The beehive in the forest

patch was a natural one, at the base of a tree trunk of Aporosa lindeliyana L.

(Euphorbiaceae) and the other two were artificial Newton type hives

established for the experiment.

The hives at the three sites were sampled for the corbicular pollen

loads at an interval of 6 days by using pollen traps attached to the entrance

of the hives for a period of 3 months from September-November, which forms

the critical brood rearing season of the honeybee colonies at the area under

investigation. The corbicular pollen loads were acetolysed (Erdtmann, 1960)

and studied by means of Nikon Labophot light microscope-I.

111.2.5. Measurement of pollen volume (Buchman eta/, 1990)

The acetolysed pollen grains (Erdtmann, 1960) were mounted

permanently in glycerine jelly and a Nikon Labophot microscope with

micrometer was used to measure grain diameter in circular grains or length of

long and short axes in elliptical grains. Grain volume was calculated as

nab2/6 where 'a' is grain length and 'b' is grain width. (For circular grains

a=b).

111.2.6. Microphotography

Microphotographs were taken on Nikon-Labophot-l photomicroscope

using lllford NP 55. 35 lnm black and white film.

111.2.7. Nomenclature

Taxonomic revisions and recent local Floras were consulted to bring

the nomenclature of plants up-to-date. Only the legitimate name in

accordance with the International Code of Botanical Nomenclature (1983)

has been followed avoiding synonyms.

111.2.8. Terminology

In general, terms used for describing the pollen grains were that of

Erdtman (1952) incorporating the suggestions made by Reitsma (1970). For

describing the exine stratification, Faegri and lverson (1975) and for amb,

size and shape classes, Walker and Doyle (1975) were followed. Regarding

shapes of apertures, the terminology of simple symmetrical plane shapes

suggested by Systematic Association Committee (1962:) is adopted.

111.2.9. Pollen descriptions

Pollen grains are described in the following sequence; nature of

occurrence, polar outline, equatorial outline, apertures, exine and

sculpturing.

111.2.10. Cluster Analysis

Pollen grains isolated from individual honey samples were acetolysed

and the slides were observed under Nikon Labophot light microscope. The

species identification was done with reference slides. The palynological data

recorded for individual samples were analysed by cluster analysis using

NTSYS-pc package for similarity index between honey collected from

different honeybees and from different floristic locations. The dominant

species represented in all the honey samples were scored as present (1) or

absent (0) across all the honey samples. The two-way data matrix of samples

x species present was subjected for standardization.

The standardized data matrix was used to calculate the similarity

dissimilarity distance using the function 'simint'. This matrix of similarity

coefficients was subjected to unweighted pair group method analysis

(UPGMA) to generate a dendrogram using average linkage procedure. The

similarity matrix coefficients were also analysed by single link and complete

link methods to see the real clustering in the samples based on the

palynological data. All these analysis were conducted using the computer

programme, NTSYS-pc:, version 1.80 (Exter Software, New York). The results

obtained were represented by constructing a dendrogram.

111.2.11. Foraging studies

For the elucidation of foraging behaviour of hive bee A. c. indica, two

species were selected based on the nectar and pollen preference of this bee

species. Bombax ceiba I.. (Bombacaceae) was selected for the behavioural

study of A. c. indica in nectar foraging and Dillenia pentagyna L.

(Dilleniaceae), a major pollen resource for pollen collection.

111.2.11.1. Nectar foraging in Bombux ceiba L.

A sample tree in full bloom was selected and 5 different flowers were

marked. Two of them were bagged using muslin cloth bags and 3 were left

open for bee v~sits. The quantification of nectar productivity of individual

flowers were carried out using micropipette at an interval of I hour.

The frequency of bee visits to individual flowers, time spend by each

bee on individual flower and number of bees visiting marked flowers were

recorded. The diurnal variation in bee visit to this species was also recorded

from 5 AM in the morning when flowers are in bud condition to 6 PM in the

evening till the sunset. Quantification of nectar productivity of individual tree

was assessed by measuring number of branches, branch lets, inflorescences

and individual flowers.

111.2.1 1.2. Pollen foraging in Dillenia pentagyna L.

An individual Willenia tree in full bloom with aggressive foraging by

A. c. indica bees was selected. The quantification of flower production /day

was assessed by counting branchesltree, number of branch letslbranch,

number of inflorescences I branch lets, number of flowerslinflorescences and

number of antherslflower. The data were recorded on the number of bee

visits to individual flower, time spend by each bee in single flower, time taken

by individual bee to load one complete pollen load and diurnal variation in

pollen foraging in this species from dawn to dusk.

111.2.12. Pollen frequency determination (Louveaux's method, 1978)

The honey samples were subjected for pollen frequency determination

by the method of Louveaux (1978). Im l honey was diluted to 10ml by 60' C

water and centrifuged. The pollen sediment was stained by safrannin and

the grains were made into pollen slides. Strip count method was adopted for

counting, as there was not much variation from point counting and total slide

count. Three complete strips were counted across the slide. That sample

where the grain count was very less, total slide count was taken for accurate

frequency of individual species. The results were presented as pollen spectra

of individual samples (Fig. 3 - Fig. 66).

111.2.13. Studies on honey as an NWFP

For the elucidation of the utilization of honey as a non-wood forest

product, five different settlements have been selected at the extreme south

of southern Western Ghats in Thiruvananthapuram district between 8"34

'999 and 8"49'178"N latitude and 77"02'96OU and 7710'083"E longitude.

56

The settlements are potomav in Sanghili forest, Mottamood near

Kallar, Chathancode and Chemmankala at Bonnoccord and Chonampara in

Kottur reserve forest. These settlements were with varying population,

literary level and with different man-day utilization for different jobs. A

questionnaire has been prepared (Annexure 1) and a household level

interview was conducted.

In addition, a seasonal monitoring for traditional apiculture and honey

collection from forests were also made in all these settlements.