The Development of the larval and adult mid-gutof Calandra oryzae (Linn.): The Rice "Weevil.

By

K. Mansour, B.Sc, D.I.C.,F r o m t h e D e p a r t m e n t of E n t o m o l o g y , I m p e r i a l C o l l e g e of S c i e n c e a n d

T e c h n o l o g y , L o n d o n , S . W . 7 .

W i t h P l a t e s 2 9 - 3 3 a n d 1 T e x t - f i g u r e .

C O N T E N T S .

P A G E

I . I N T R O D U C T I O N . . . . . . . . . 3 1 4

I I . HlSTOEICAL . . . . . . . . . 3 1 4

(a) E m b r y o n i c . . . . . . . . 3 1 4

(6) P o s t - e m b r y o n i c . . . . . . . 3 1 8

(c) P r e v i o u s W o r k o n C a l a n d r a . . . . . 3 1 8

I I I . MATERIAL AND TECHNIQUE . . . . . . 3 1 8

(a) C a l a n d r a o r y z a e : Breeding Habits and the variousStages in the Life-history . . . . . 3 1 8

(6) Other Material 319(c) Technique . . . .' . . . . 3 2 0

IV. EMBRYONIC STUDIES ON C A L A N D R A O R Y Z A E . . . 321

(a) The Blastoderm Stage and the Formation of theGerminal Layers . . . . . . . 3 2 1

(6) Growth of the Embryonic Band 326(c) Appearance of the Stomodaeum and Proctodaeum . 328(d) Development of the Rudiments of the Mid-gut Epithe-

lium of the Larva . . . . . . . 329(e) Formation of the Larval Mid-gut and the Appearance

of the'Accessory Cell-mass' . . . . . 333( / ) The Accessory Alimentary Mass and the Sub-oesophageal

Body 336

V. OCCURRENCE OF THE ACCESSORY CELL-MASS IN OTHER INSECTS 336

VI. POST-EMBRYONIC STUDIES ON C A L A N D R A O R Y Z A E . . 337

(a) Degeneration of the Larval Mid-gut and the Initiationof that of the Adult 337

(6) Behaviour of the Accessory Alimentary Cell-mass . 338

314 K. MANSOUE

VI.— COIltd. PAGE(c) The Yellow Body . . . 338(d) A p p e a r a n c e of t h e Mesen t e r i c Caeca a n d t h e F a t e of t h e

A c c e s s o r y M a s s . . . . . . . . 3 3 9(e) F o r m a t i o n of t h e Def in i t ive M i d - g u t E p i t h e l i u m of t h e

A d u l t 339

V I I . F O R M A T I O N o r T H E A D U L T M I D - G U T I N O T H E R R H Y N C H O -

F H O R A . . . . . . . . . 3 4 0

V I I I . T H E A C C E S S O R Y C E L L S I N T H E A D U L T . . . . 3 4 1

I X . G E N E R A L C O N S I D E R A T I O N O F T H E K N O W N T Y P E S O F A D U L T

M I D - G U T F O R M A T I O N I N T H E C O L E O P T E R A . . . 3 4 1

X . S U M M A R Y . . . . . . . . . 3 4 4

X I . A C K N O W L E D G M E N T S . . . . . . . 3 4 5

B I B L I O G R A P H Y . . . . . . . . . 346

E X P L A N A T I O N O F P L A T E S . . . . . . . 349

I. INTRODUCTION.

IN November 1925, while examining a series of sectionsthrough the larva of the rice weevil (0alandra oryzae,Linn.), I was puzzled by the presence of a comparatively largemass of cells between the nervous system and the alimentarycanal in the region where the fore-gut passes into the mid-gut.Later on, by examining sections through metamorphosinglarvae, it was noticed that this mass takes part in the formationof the adult mid-gut. This suggested that a detailed study ofthe origin and behaviour of this mass, together with a study ofthe development of the larval mid-gut and that of the adult,might be of some interest to the embryologist and probably tothe systematist also.

II. HISTORICAL.

(a) Embryonic .The development of the mid-gut epithelium of insects has

for the last fifty years been the subject of numerous investiga-tions, not only from the point of view of comparative insectembryology, but also from the point of view of its relation tothe germ-layer theory. According to this theory the material

MID-GUT OF CALANDRA 315

from which the mid-gut epithelium is built should be derivedfrom the innermost of the germinal layers, i. e. the endoderm.

It is not the intention here to enumerate the results obtainedby the various investigators, nor to describe in detail the en-deavours made to apply the germ-layer theory to the case ofinsects, since the whole problem has often been reviewed atlength in the text-books of embryology, but only to give a briefaccount of the main views which still divide the more recentinsect embryologists into two opposing groups.

1. Mid-gut epithelium derived from an anterior and a pos-terior endodermal rudiment and therefore of an endo-dermal origin.

Grassi (1884) showed that in A p i s the mid-gut epitheliumwas derived from the anterior and posterior ends of the innerlayer which he considered as a combination of the mesodermaland endodermal layers.

Kowalevsky (1886) observed the same phenomenon inM u s c a, and was led to regard the insect egg at the time of theformation of the germinal layers as comparable to a gastrulawith an extremely elongated furrow-like invagination. Throughthis lengthening the endodermal layer was cut into halves,which were dragged apart to the anterior and posterior ends ofthe inner layer. Prom these two halves the larval mid-gutdeveloped.

The view of Grassi as elaborated by Kowalevsky was acceptedby Heider (1889), Wheeler (1889), Escherich (1900), Nussbaumand Fulinski (1906 and 1909), and in a modified form by Strind-berg (1913).

Hirschler (1909) confirmed Grassi's observations, and in 1912gave an interpretation which is totally different from that ofKowalevsky. According to Hirschler there are two gastrula-tion phases in the development of pterygote insects. The firstphase involves the segregation of the primary yolk-cells—which this investigator called primary endoderm—from theblastodermal skin which was referred to as the ectoderm. Thesecond phase lies in the differentiation of the mid-gut epithelium

316 K. MANSOUR

rudiments from the inner layer. This observer also maintainedthese views in Schroder's ' Handbuch der Bntomologie ' (1924).

While all the above-mentioned investigators agreed that therudiments of the mid-gut epithelium are differentiated from theinner layer, Carriere (1890), Carriere and Burger (1897), andNoak (1901) claim that, at least in the species they investigated,the mid-gut epithelium rudiments arise quite independently ofthe inner layer, from proliferating areas of the blastoderm, oneat each end of the germ-band corresponding to the future loca-tion of the stomo- and proctodaeum respectively. According tothese investigators the proliferated cells represent the endodermwhile the inner layer gives rise exclusively to the mesoderm. Thisview received partial support from the work of Nelson (1915).

2. Mid-gut epithelium derived from proliferations of the innerends of the stomo- and proctodaeum and therefore ofan ectodermal origin.

Ganin (1874) observed that in certain insects the mid-gutepithelium was derived from the inner ends of the stomo- andproctodaeum. He concluded that owing to the fact that theseorgans are developed from the ectodermal layer, the mid-gutepithelium which is derived from them is of an ectodermalorigin.

Voeltzkow (1888, 1889, and 1889a), working on Muscaand M e l o l o n t h a , while confirming Ganin's observations,came to an entirely different conclusion. According to thisobserver, the stomodaeal and proctodaeal invaginations origin-ated from the base of the gastrula-furrow and therefore be-longed to the inner layer, a conclusion which meant that thestomodaeum and proctodaeum of insects were not of ecto-dermal origin. To the adherents of the germ-layer theoryneither Ganin's nor Voeltzkow's conclusions were acceptable.

Heymons (1895), who devoted special attention to the develop-ment of the mid-gut in Orthoptera and Dermaptera, fully con-firmed Ganin's and Voeltzkow's observations, and concludedthat all the larval and imaginal structures of insects were de-rived from two germinal layers only, viz. the ectoderm and the

MID-GUT OF CALANDRA 317

mesoderm. This conclusion led Heymons to believe that thegerm-layer theory had been shaken, and he endeavoured toexplain the case of insects by assuming that the yolk-cellsoriginally gave rise to the mid-gut epithelium. He also sug-gested that, owing to the gradual displacement of the originalmid-gut epithelium by epithelial growths from the ends of thefore- and hind-guts, the assimilating action of the yolk-cellshad become restricted to the embryonic period. This hypothesisreceived great support from Heymon's discovery (1897) that inL e p i s m a , an Apterygote insect, the mid-gut epithelium isactually derived from the yolk-cells.

In 1903 Heymon's hypothesis received further support byMadame Tschuproff-Heymons's discovery that in some repre-sentatives of the O d o n a t a (Libellulidae) the middle portionof the mid-gut epithelium is derived from the yolk-cells whilethe anterior and posterior portions are formed from the stomo-and proctodaeum respectively. This worker considered this con-dition as constituting a transitional stage between the L e p i s -ma type and that of the more highly developed pterygote in-sects as described by Heymons.

Lecaillon (1898), stxidying the embryology of certain Chryso-melid beetles, again confirmed Ganin's and Heymons's observa-tions. This investigator concluded that, owing to the fact thatinsects are the most highly developed group among Arthropods,and also owing to the presence of a large mass of yolk in thedeveloping egg, insects follow an atypical form of development,and their case can in no way affect the germ-layer theory whichholds good for all the animals whose developmental processeshave not been interfered with.

In considering the theoretical problems involved, Lecaillonaccepted Heymons's theory and proceeded to homologize thevery early developmental stages of insects with those of otheranimals. According to this investigator there is no blastulastage in the more highly developed pterygote insects, and theperiod when the primary yolk-cells are segregated from theother cleavage cells corresponds to the gastrulation period ofother animals.

NO. 282 Y

318 K. MANSOUR

The observations of the supporters of the ectodermal originhave been confirmed by Deegener (1900), Czerski (1904),Friederichs (1906), and Saling (1907) in the Coleoptera;Schwartze (1899) and Toyama (1902) in the Lepidoptera;Eabito (1898) in the Orthoptera ; and Pratt (1900) in theDiptera.

(b) P o s t - e m b r y o n i c .

Kowalevsky (1887) described the adult mid-gut epithelium ofMusca as derived from scattered groups of cells between thelarval digestive epithelium cells. During metamorphosis thesegroups of cells, through active proliferation, form a continuouslayer which eventually becomes the adult digestive epithelium,while the larval one is sloughed off into the lumen, awaitingrejection to the exterior when the adult emerges. With onlyone exception, such has been found to be the case in all holo-metabolic insects that have been studied. The exception weowe to Poyarkoff (1910), who found that in Ga le ruce l l au lmi , a Chrysomelid beetle, the adult raid-gut epithelium isderived from some of the cells proliferated from the posteriorside of the oesophageal valvule.

(c) P r e v i o u s Work on Oa landra .To my knowledge no account of the embryonic or post-

embryonic development of the^mid-gut epithelium of Calan-dra o ryzae has been published, but Tschomiroff (1890) gavean extremely short account of the embryology of Ca land rag r a n a r i a , a closely-related species to the weevil under con-sideration. The larval mid-gut here was described as formedfrom the yolk-cells.

III. MATERIAL AND TECHNIQUE.

(a) Ca landra o r y z a e : Breed ing H a b i t s and thev a r i o u s S tages in the L i f e - h i s t o r y .

This weevil breeds without any obvious difficulty in an incu-bator at about 30° C.

The eggs are laid singly inside the grain.

MID-GUT OF CALANDRA 319

The fertilized female when ready to oviposit eats out a holelarge enough to receive its egg. Turning round, it brings theapex of the abdomen just above the hole, inserts its ovipositor,and lays the egg. The egg is elliptical in form, about 0-7 mm.in length and 0-3 mm. in breadth. It occupies about the innerfour-fifths of the hole, which is filled up with a mucilaginoussecretion flowing from the ovipositor. This secretion on dryingup acquires the colour of the testa of the grain, thus making it ex-tremely difficult to determine the spot where the egg has been laid.

Hatching occurs in about 4^5 days.The larva is apodous and lives inside the grain.There are four larval instars. The first and second require

1-2 days each, the third 5-6 days, and the last 7-9 days.About 3 days before pupation the larva ceases to feed and,

by sticking together the loose debris which has accumulatedinside the grain, it constructs a neat cell, inside which it under-goes the whole process of metamorphosis. In this cell the meta-morphosing larva contracts rhythmically, peristaltic move-ments being recognizable from the thorax backwards for abouta day and in the meantime undergoes marked change in form.After being nearly hemispherical with the head capsule partiallysunk in the thoracic region, and with no marked difference be-tween the latter and the abdomen, the larva becomes very muchreduced in girth with the head capsule well marked from therest of the body. This stage, which is only a transitional one, isthe ' prepupal' stage.

After about a day the pupal moult sets in, and in the cellappears a pupa libera and the old larval skin.

The pupal stage lasts 6-7 days. Towards the end of thisperiod the pigmentation of the adult begins.

When the adult emerges it has a brownish colour whichgradually gets darker. After about 3 days inside, the adultbores its way to the open and the cycle starts again.

(b) Other Mater ia l .Besides following the development of the mid-gut in all the

above-mentioned stages of Calandra oryzae, stages in theY2

320 K. MANSOUR

life-cycle of the following insects were examined for checkingthe results and also for comparative purposes.

Curcul ionids .Calandra granaria, L. . . larvae pupaeHylobius abietis, L. „ „Odoiporus glabricollis, Gyll. . „Anthonomus grandis, Boh. . ,, prepupaeAnthonomus pomorum, L. . „ pupaeBalaninus nucum, L. . . „Bhyncolus lingnarius, Marsh. . ,,Cionus sp. . . . „

Scoly t ids .Dendroctonus ponderosae, Hop. „Scolytus destructor, 01. . . ,, prepupaeXyleborus saxesinae, Eatz. . „

Chrysomelids.Melasoma populi, L. . ,, ,, pupaeGalerucella sp. . . ,.

Anobiids.Anobium paniceum, L. . ,, „Ptinus tectus, Boield. . „ ,, ,,

L amel l icorns .Dorcus paralellopipedus, L. . ,, ,, ,,

(c) T e c h n i q u e .The fixative that proved most suitable for this work was

Carl's, which is a mixture of 15 pts. absolute alcohol, 6 pts.formalin, 2 pts. glacial acetic acid, and 30 pts. distilled water.This gave the best results when used hot at about 70° 0. forfrom 3 to 8 minutes, according to the size of the object. It wasthen followed by 70 per cent, alcohol for at least 24 hours.After dehydrating, the material was then cleared in cedar-woodoil and embedded in paraffin wax in the usual way. Sectionswere cut 8/x thick and stained in Delafield's haematoxylin.

MID-GUT OF CALANDRA 321

Prom what has been mentioned about the life-history ofCalandra oryzae, it is to be gathered that the durationof the embryonic, larval and pupal stages is not fixed. For thisreason it has been thought advisable not to consider the ageas a definite criterion of the progress of development, but torefer to the various stages according to certain developmentalprocesses.

For the purpose of this work the earliest embryonic stageshave been omitted, and the fully formed blastoderm is thestarting-point of the developmental processes dealt with.

IV. EMBRYONIC STUDIES ON CALANDRA ORYZAE.

(a) The Blas toderm Stage and the Format ion ofthe Germinal Layers .

In the developing egg at the fully formed blastoderm stage,the following can be easily recognized :

1. A peripheral unicellular layer.2. Yolk material.3. Cells in the yolk.4. A mass of cells at one end, between the peripheral cellular

layer and the yolk.

The peripheral unicellular layer consists of uniform cubicalcells closely packed together round the yolk.

The yolk in fixed preparations is in the form of globules and,scattered among these globules, are the few ' cleavage cells'that did not take part in the formation of the peripheralcellular layer. Similar cells in other insects have been describedby various authors and are the ' primary yolk-cells '.

The mass of cells at one end between the yolk and the peri-pheral unicellular layer is the genital rudiment, which nowmarks the posterior end of the developing egg.

In the advanced blastoderm stage the peripheral unicellularlayer is no longer of a uniform thickness, there being a longitu-dinal band of columnar cells along one side and a similar bandof distinctly flattened cells along the other, the two bands pass-

322 K. MANSOUB

ing insensibly one into the other. The columnar band indicatesthe ventral side of the future embryo.

The appearance of a mid-ventral groove in the columnar banddenotes the beginning of the formation of the germinal layers(fig. 1, PI. 29). This groove first appears in the posterior region,but later on extends the whole length of the egg. During theformation of this groove two lateral furrows appear along theedges of the columnar cell-layer, and these mark off betweenthem the definitive embryonic band of the developing egg(fig. 1, PI. 29). This band is divided into three regions, viz. onemedian, which constitutes the walls of the mid-ventral groove,and two laterals.

The cells of the walls of the ventral groove multiply rapidly,and on account of this activity and also owing to the approachof the edges, the cavity of the groove is gradually reduced andfinally disappears (fig. 2, PL 29).

The reduction and disappearance of the ventral groove causesthe two lateral bands to unite, and this broad band of tissuehas been regarded by all previous workers as constituting theectoderm.

Some of the cells proliferated from the walls of the ventralgroove retain their extra-vitelline situation while others wanderinto the yolk (fig. 2, PI. 29). These two sets of cells are totallydifferent from one another. The cells entering the yolk are verypoorly defined and even form a syncytium with deeply stainingconstituents. The extra-vitelline cells, on the other hand, areirregular in form but with clearly marked cell walls (fig. 2,PI. 29), and possess very feebly staining cytoplasm.

Soon after the disappearance of the ventral groove, the extra-vitelline products of its walls, probably through amoeboidmovements, spread themselves between the yolk and the newlyformed ventral columnar layer, i. e. the ectoderm (fig. 3, PI. 29).In their new positions these cells are quite separate from oneanother and form a non-continuous layer.

By this time the entrance of the other cells into the yolk iscompleted. No evidence of them can be seen anywhere nearthe non-continuous layer. Thus we are now left with only two

MID-GUT OF CALANDRA 323

layers outside the yolk : the ectoderm, the origin of which hasalready been explained, and the non-continuous layer within,so that there are no anterior and posterior rudiments such ashave been assumed to exist and have been described as endo-derm.

In most of the details given the mode of formation of thegerminal layers of C a l a n d r a o r y z a e is similar to that de-scribed in other insects, and the chief points of importanceseem to be :

1. The absence of the so-called endodermal rudiments, and2. The determination of the origin of the cells which disappear

into the yolk, a point apparently missed by most otherobservers (cf. Graber, 1888 ; Voeltzkow, 1888 ; Wheeler,1889 and 1893 ; Heymons, 1895 ; Schwartze, 1899 ;IN oak, 1901 ; Petrunkewitsch, 1901 ; Schwangart,1904; Hammerschmidt, 1910; Nelson, 1915 ; andLeuzinger, 1926.

Students of embryology recognize the ' gastrula ' as the stageproduced by an invagination of part of the ' blastula ' wall, theinvaginated cells forming the lining of the primary gut, thislayer being the ' endoderm '.

In order to trace the origin of the endoderm in the pterygoteinsects, where the details of development are not so simple asjust described, the first point to be decided is whether theinsect blastoderm corresponds to the ' blastula' of other types,and various views have been expressed upon this point.

Kowalevsky (1887) considered the blastoderm stage of theinsect embryo as corresponding to the blastula stage of otheranimals. To this worker the blastoderm of insects was ablastula full of yolk. Kowalevsky also assumed that the pre-paration of this yolk for assimilation by the developing em-bryo was carried out by cleavage cells left in the yolk duringthe formation of the blastoderm, i. e. the ' primary yolk-cells ',and to this observer these cells had no phylogenetic significance.

As to the ventral groove, Kowalevsky homologized it withan elongated shallow gastrula invagination, and derived the

324 K. MANSOUB

endodermal and mesodermal layers from its walls. In the dif-ferentiation of these germinal layers, he assumed that the endo-dermal layer, through the lengthening of the gastrula invagina-tion, was dragged apart to opposite ends of the developingembryo.

Strindberg (1913) differed from Kowalevsky in regarding theendodermal layer as being formed along the whole stretch ofthe ventral groove, and not at the anterior and posterior endsonly. This observer also took into consideration the cells enter-ing the yolk after the formation of the blastoderm, and con-cluded that they were undifferentiated endodermal cells, i. e.mesoendodermal cells.

On the other hand Lecaillon (1S98) considered that in thedevelopment of the more highly organized pterygote insectsthere is no true blastula stage, and that the period towards theend of the cleavage, when some of the cells rise to the surfaceof the developing egg to form the blastoderm, corresponds tothe gastrulation period. According to this worker the primaryyolk-cells represent the endodermal layer, while the blasto-dermal wall which he called ' primitive ectoderm ' contains therudiments of the mesodermal and ectodermal layers only. Thegroove itself Lecaillon considered as a coenogenetic feature ofthe developing insect egg, and referred to it as the ' mesodermalgroove '.

The third and last view to be considered here we owe toHirschler (1912). According to this investigator there are twogastrulation phases in the development of the pterygote insects.The first phase occurs towards the end of the segmentationperiod when the wall of the blastoderm is being formed and afew cells are left behind in the yolk. The peripheral layerHirschler called ectoderm, while the cells left in the yolk, i. e.the primary yolk-cells, he called the ' primary endoderm '.The second gastrulation phase is marked by the appearance ofthe ventral groove, whose cells later on differentiate into meso-dermal and secondary endodermal layers, both of which remainoutside the yolk. The cells entering the yolk after the blasto-derm stage Hirschler considered of secondary endodermal origin

MID-GUT OF CALANDRA 325

and as representing a secondary phenomenon. Neither theblastoderm stage nor the ventral groove, according to this view,has a phylogenetic significance.

Now it seems to me that whether or not the blastoderm stagecorresponds to the ' blastula ' stage depends entirely on thesignificance of the ventral groove and whether or not its wallscontain the endodermal elements.

A consideration of the behaviour of the endodermal layeramong Arthropods other than pterygote insects, and possessingeggs rich in yolk, will assist us in the solution of this problem.Among Crustacea (vide MacBride's ' Text-book of Embryo-logy'), as shown in the case of Homarus , Palaemon,M y s i s, and representatives of Amphipods and Isopods ;among Arachnids, e.g. Agelena, Euscorpius andLimu 1 us ; and in Myriapods as described for Scolopendra,the endodermal layer is proliferated either from the walls ofan invagination or from a ridge or thickening along the blasto-derm. Soon after proliferation the constituent cells enter theyolk and spread themselves there.

Again, among apterygote insects it is definitely establishedthat the endodermal cells reside in the yolk. In Anuridamar i t ima , which possesses an egg comparatively poor inyolk, Claypole (1898) observed that the endodermal cells aredifferentiated at an early stage of development, probably bya process of gastrulation, and that these cells remain in theyolk quite distinct from the vitellophags. According to Hey-mons (1897) and Uzel (1897), in Lepisma and other aptery-gote insects, the endodermal cells are budded off into the yolkfrom a thickened area of the blastoderm. The budded-off cellssoon become unrecognizable from the cells left in the yolkduring the formation of the blastoderm.

Similarity between the cells entering the yolk in the above-mentioned Crustacea, Arachnida, Myriapoda and apterygoteinsects, and those budded off into the yolk in the case ofCalandra oryzae and the other pterygote insects, is at oncesuggested.

It is therefore not unjustifiable to conclude that in the

326 K. MANSOUR

pterygote insects the cells budded off into the yolk from thewall of the ventral groove are probably homologous with theendodermal cells of other Arthropods and apterygote insects,and thus represent the true endodermal cells : hence the wallof the ventral groove here contains endodermal elements, andin accordance with the embryological definition of the ' gas-trula ' the ventral groove will represent an elongated gastrulainvagination and the stage prior to the appearance of theventral groove must be the ' blastula ' and not a post-gastrulastage, as Lecaillon and Hirschler had concluded.

Kowalevsky's view, so far as it concerns the significanceof the blastoderm, the meaning of the primary yolk-cells andthe significance of the ventral groove, is fully supported by thewriter's observations onCa land ra oryzae, but as to themode of origin and the behaviour of the endodermal cells it isunsound, as will be shown now and later on.

Kowalevsky assumed that originally the endodermal layerwas dragged apart to opposite ends of the embryo, and thusit was represented by an anterior and a posterior rudiment.My observations on Calandra oryzae fail to show thepresence of such rudiments, but demonstrate very clearly theentrance of the endodermal cells into the yolk ; a fact which isin full harmony with what is known to occur in other Arthro-pods including the apterygote insects.

A table, showing the views expressed by different authors, ishere inserted (p. 327) as a summary of the first part of this paper.

(b) Growth of the Embryonic Band.During the formation of the germinal layers, the anterior

and posterior ends of the embryonic band grow very actively.As a result of this growth, which is more marked posteriorly,two folds appear one at either end of the egg. These folds arethe anterior and posterior amniotic folds.

The growth of the anterior end is more marked laterally whereit forms the cephalic lobes.

On the other hand, the growth of the posterior end is in alongitudinal direction only, and the germ-band extends round

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328 K. MANSOUR

the posterior end of the egg to the dorsal side where it con-tinues to grow until it nearly reaches the anterior extremity.At this stage, therefore, the embryonic band is nearly twice thelength of the egg.

The growth in length involves the three longitudinal stripsof the embryonic band, i. e. the median or walls of the ventralgroove and the two laterals. The newly formed material is beingrapidly differentiated into endodermal cells which wander intothe yolk, mesodermal and ectodermal cells which behave asdescribed above.

As this growth proceeds the genital rudiment correspondinglyshifts so that it is all the time situated in a morphologicallyfixed position, i. e. between the posterior end of the embryonicband and the yolk.

(c) Appearance of the Stomo- and P r o c t o d a e u m .After the formation of the germinal layers the stornodaeum

appears as an ectodermal invagination in the anterior part ofthe embryonic band, in a mid-ventral position (fig. 4, PI. 29).

It is to be emphasized here again, that at this stage ofdevelopment the germinal layers represented outside the yolkare the columnar ectodermal layer and the non-continuousmesodermal one together with the genital rudiment which issituated at the posterior end of the embryonic band.

The stomodaeum pushes its way dorsally between the scat-tered mesodermal cells, and for some time remains as a well-defined pit with uniform walls (fig. 4, PI. 29 ; fig. 6, PL 30).

While the stomodaeum is appearing, peculiar structures,which probably are disintegrating cells, are to be seen justinside the yolk in the stomodaeal region (deg.c, fig. 4, PI. 29 ;fig. 6, PI. 30). Probably these structures are of mesodermal origin.

The proctodaeum originates in a similar manner from amedian ectodermal invagination at the posterior extremity ofthe embryo. The pit soon becomes deep and at first is at rightangles to the embryonic band (fig. 5, PI. 29), but later on be-comes parallel to it and extends forwards along it (fig. 11,PI. 30). The wall of the proctodaeal tube at this stage is not

MID-GUT OF CALANDRA 329

of uniform thickness, since the morphologically dorsal side thinsout gradually as it passes into the amnion (fig. 11, PI. 30).

(d) Development of the Eud imen t so f the LarvalMid-gut Ep i the l ium.

The posterior wall of the stomodaeal pit, which is roundedevenly (fig. 6, PI. 30), soon becomes the seat- of active cellulardivision which leads to the formation of two processes one oneither side of the pit (fig. 7, PI. 30). In fig. 8, PL 30, one ofthese processes is shown in an oblique section.

At first the component cells of these processes are packedtogether and thus exhibit a polygonal form. Their cytoplasmis dense, and in all respects is exactly similar to that of the cellsof the stomodaeal wall. Gradually these cells become irregularin form and show light and vacuolated cytoplasm, but never-theless they continue to divide actively and, insinuating them-selves posteriorly between the yolk and the mesodermal cells,they form two lateral discontinuous bands one on either side ofthe mid-ventral line (fig. 9, PI. 30). These bands.meet similarbut less evident structures advancing anteriorly in a similarmanner from the wall of the proctodaeum after the latter hasgiven rise to the rudiments of the Malpighian tubules (fig. 11,PI. 30).

The lateral bands, which are definitely derived from theposterior and anterior walls of the stomodaeum and procto-daeum respectively, contain the rudiments of the future larvalmid-gut epithelium.

Towards the end of the proliferation of these bands numerouscells are to be seen migrating into the yolk from the blind endof the stomodaeum (fig. 10, PI. 30). At first these cells are recog-nizable by the deep stain of their nuclei, but soon they lose thischaracter and become indistinguishable from the ones alreadypresent in some of the spaces in the yolk. Nothing definite canbe said about the fate of these cells. It is only certain that theydo not take part in the formation of any definite structure. Itis possible that they are destined to help in the preparation ofthe yolk for assimilation.

330 K. MANSOUE

The derivation of the larval mid-gut epithelium from pro-liferations of the blind ends of the stomodaeum and procto-daeum was first claimed by Ganin (1874). Since then this claimhas been supported by Heymons (1895), Korotneff (1894), andEabito (1898) in the Orthoptera ; Witlaczil (1884) and Hey-mons (1897) in the Ehynchota ; Schwartze (1899) and Toyama(1902) in the Lepidoptera ; Lecaillon (1898), Deegener (1900),Czerski (1904), Friederichs (1906) and Saling (1907) in theColeoptera ; Pratt (1900) in the Diptera ; and Heymons (1895)in the Dermaptera.

All the above-mentioned investigators concluded that owingto the fact that the stomo- and proctodaeum are ectodermalstructures, the mid-gut epithelium derived from them is ecto-dermal in origin. This conclusion is obviously at variance withthe germ-layer theory and, in spite of the numerous confirma-tions Ganin's view had received, the derivation of the mid-gutepithelium from ectodermal structures remains unaccepted bypractically all the embryologists.

The majority of those who contested Ganin's view originallyaccepted Kowalevsky's theory (see p. 323), and derived themid-gut epithelium from anterior and posterior endodermalrudiments.

It has been mentioned (see p. 326) that the representation ofthe endodermal layer by two cellular masses one at either endof the embryonic band is quite contrary to what occurs amongother Arthropods with eggs rich in yolk and also in C a l a n d r ao r y z a e .

Now, if the mid-gut epithelium of insects has an endodermalorigin, one would expect it to be formed from the endodermalcells spread in the yolk. That this should be the case is evidentfrom the study of the development of the apterygote insects.According to Heymons (1897), Uzel (1898), and Claypole (1898),the mid-gut epithelium of various representatives of this groupis formed from the endodermal cells which were spread in theyolk in a way similar to that described for various Arthropods.That such probably was the case among the ancestors of thepterygote insects is an hypothesis which was originally suggested

MID-GUT OF CALANDRA 831

by Heymons (1895) and has since received support from variousauthors. Carriere and Burger (1897) recorded that ' at the timeof the appearance of the endoderm bands (i. e. the rudimentsof the mid-gut epithelium which these observers derive froman anterior and a posterior endodermal mass), the yolk-cells atthe periphery, regularly distributed and connected together,form a complete sac, or if you will a primary mid-intestinalepithelium ' (p. 358). A similar phenomenon has been describedby Wheeler (1889), Heymons (1895), Hammerschmidt (1910),and Leuzinger (1926). One is therefore led to conclude that theanterior and posterior endodermal rudiments of the variousauthors are not homologous with the true endoderm of theapterygote insects, and if such rudiments do really exist theywill be merely analogous in as far as they contribute to theformation of the mid-gut epithelium. This conclusion receivesgreat support from a close examination of the works of Wheeler,Nusbaum and Fulinski, and Hirschler, all of whom endeavouredto demonstrate the presence of the anterior and posterior endo-dermal rudiments.

Wheeler (1889) described an anterior and a posterior thicken-ing in the inner layer of D o r y p h o r a at the moment thestomodaeal invagination is formed. He claimed that fromthese thickenings the endodermal rudiments are differentiated.Wheeler's fig. 88 which is intended to illustrate this differentia-tion in the posterior mass fails to do so. The line of demarcationbetween what this author believed to be endoderm and the restof the mass looks very artificial and probably was due to anartifact. As to the anterior mass this writer did not say much.Prom a comparison between Wheeler's fig. 82 and a correspond-ing stage in C a 1 a n d r a it is perhaps not unreasonable to con-clude that the posterior undifferentiated mass of this author isprobably the genital rudiment. A clear idea of this can begained by comparing the posterior part of Wheeler's fig. 82with the writer's fig. 5, PI. 29.

Pigs. 3 and 4 of Nusbaum and Fulinski (1906) show at oncethat the supposed anterior endodermal rudiment is an out-growth from the posterior wall of the stomodaeum. These

332 K. MANSOUE

workers (1909), while admitting that at certain developmentalstages the rudiments of the mid-gut epithelium look like out-growths of the stomodaeum and proctodaeum, assumed thatin such cases the differentiation of the anterior and posteriorendodermal rudiments is slightly retarded while the appearanceof the stomodaeal and proctodaeal invaginations is somewhatearlier, so that each of the uridifferentiated regions of the germ-band at the most anterior and posterior ends of the gastrulafurrow correspond to the stomodaeal and proctodaeal invagina-tions respectively.

Nusbaum and Fulinski's assumption is based on Kowalevsky'stheory, which is at variance with what occurs in the develop-ment of the apterygote insects and the other Arthropods withyolky eggs, and therefore it cannot be accepted as an explana-tion of the origin of the mid-gut epithelium.

Hirschler (1909), on the other hand, described the appearanceof blade-like processes from the posterior stomodaeal wall ofD o n a c i a, and stated that similar structures occur in certainother insects. His figs. 33 and 36 are intended to illustratethese processes. He, however, believed that these outgrowthsgradually pass inwards into the stomodaeal wall, and probablyform the separating lamellae between the stomodaeum and themid-gut. In deriving this latter organ, Hirschler assumed thatthe median cells of the inner layer at the anterior end of theembryonic band are endodermal and derived the mid-gut epithe-lium from caps of flattened cells between the stomodaeal andproctodaeal invaginations and the yolk. However, Hirschlerdid not explain why these cells were to be regarded as endo-dermal.

Three years before Hirschler's work the development ofD o n a c i a was studied by Friederichs (1906), who agreed withHirschler in the description of events but described the processesas ectodermal, and said that they give rise to the mid-gutepithelium.

One is therefore greatly tempted to believe that the capswhich Hirschler claimed to be the rudiments of the mid-gutepithelium probably correspond to the few mesodermal cells

MID-GUT OF CALANDRA 333

dorsal to the stomo- and proctodaeal imaginations, while theblade-like ectodermal outgrowths to which this author referredcorrespond to the stomodaeal outgrowths which give rise tothe greater bulk of the mid-gut epithelium rudiments inC a l a n d r a .

It is now obvious that Wheeler, Nussbaum and Fulinski, andHirschler have not been referring to identical structures in theirdescription of ' endodermal rudiments ', and that in no case dothe structures they refer to exhibit any similarity of composi-tion or origin and thus cannot be called ' endodermal' in theproper embryological sense of the word. In fact no clear exposi-tion of the differentiation of the so-called ' endodermal rudi-ments ' has yet been given by any author, and my observationsin the case of C a l a n d r a o r y z a e confirm the view that nosuch rudiments do differentiate in the regions where the mid-gutepithelium originates and that therefore the larval mid-gut is

' indeed ectodermal in origin.Therefore we may conclude that the origin of the larval

mid-gut epithelium in C a l a n d r a oryzae , and probably allpterygote insects with the exception of the Libellulidae (videHeymons, 1896, and Tsehuproff-Heymons, 1903), differs en-tirely from that in the apterygote insects and other Arthropodswith yolk-bearing eggs where the true endodermal cells whichat first wander into the yolk become the mid-gut epithelium.In most of the pterygote insects these cells disappear, and theirfunction of producing the larval mid-gut epithelium is taken onby ectodermal cells proliferated from the stomodaeum andproctodaeum.

(e) F o r m a t i o n of the L a r v a l Mid-gu t and t h eA p p e a r a n c e of t h e ' A c c e s s o r y C e l l - m a s s ' .

The rudiments of the larval mid-gut epithelium which nowlie beneath the yolk become closely associated with the innerwalls of some of the abdominal mesodermal somites (fig. 12,PI. 31). These portions of the somites are destined to form themuscular wall of the mid-gut.

The cells of the rudiments of the mid-gut epithelium divideNO. 282 Z

334 K. MANSOUR

actively and, together with their mesodermal associates, spreadround the yolk in a dorso-ventral direction (fig. 13, PL 31).

During the spreading a very curious phenomenon occurs.Some of the cells proliferated from the mid-gut rudiments enterthe yolk instead of spreading round it (fig. 13, PL 31). Duringtheir passage into the interior these cells undergo remarkablecytological changes. Their nuclei become much lighter thanthe original ones, while their cytoplasm becomes denser andacquires a very fine reticulate texture. These transformed cellsagglomerate in the middle of the yolk as a comparatively largemass (figs. 13 and 14, PL 31). This group of cells has a peculiarhistory, as will be seen during the course of this paper, but asat present it is only certain that its cells are accessory alimen-tary cells with an unknown function, they will be referredto merely as ' accessory cells '.

Meanwhile the stomodaeal tube has been growing pos-teriorly into the yolk, and eventually it abuts on the anteriorend of the accessory cell-mass (fig. 14, PL 31).

After the epithelial rudiments have budded off the accessorymass they continue to spread themselves round the yolktogether with their mesodermal associates, so that by the timethe formation of the dorsal wall of the embryo has been com-pleted, the yolk is to be seen surrounded by a rather loose layerof irregular cells derived from the rudiments of the mid-gutepithelium, and a layer of elongated cells with long nuclei,derived from the inner walls of the mesodermal somites. Theformer layer will form the future larval mid-gut epithelium,while the latter will give rise to the mid-intestinal musculature.

During the growth period of the epithelial and muscularrudiments, the accessory cell-mass has been undergoing slightchanges in position. Probably partly due to the activities of itsown cells, and partly on account of the elongation of the stomo-daeal tube, this mass is to be seen in a more anterior situation.Again, probably because of the development of the supra-oesophageal ganglion, it is also pushed downwards to a moreventral position. As a result of these processes a portion of thismass now separates the ventral stomodaeal wall from the ventral

MID-GUT OF CALANDRA 335

rudimentary layers of the mid-gut. As development proceedsthe accessory cell-mass slowly moves from the yolk into a sub-stomodaeal situation (fig. 15, PI. 31). Eventually it entirelyleaves the yolk (fig. 16, PI. 31), and is to be found dorsal to thenervous chain and closely applied to the wall of the alimentarycanal at the point where the stomodaeum opens into the mid-gut.

In its sub-stomodaeal position the accessory mass remainsduring the latter part of the embryonic life, and the wholeperiod of larval growth apparently undergoing no visible changeapart from increasing in size. Its fate and behaviour duringmetamorphosis will be discussed later on.

As soon as the accessory mass is extruded from the yolk, thecells of the rudimentary epithelial layer of the larval mid-gutbecome closely packed together to form a sac whose ends jointhe stomodaeal and proctodaeal tubes. The wall of this newlyformed sac is the mid-gut epithelium of the larva.

At this stage the epithelial cells of the mid-gut are large withweakly staining cytoplasm in contrast with those of the stomo-daeum and proctodaeum, which are much smaller in size andpossess very dense cytoplasm (fig. 16, PI. 31).

The rudimentary muscular layer gives rise to the circular andlongitudinal muscles of the mid-gut which may be very looselyjoined to those of the stomodaeum, but in all sections examinedthere is a gap between the two.

By the time the mid-gut is fully formed the other embryonicprocesses are also completed and hatching soon follows.

In the feeding larva the cells of the mid-gut epithelium,although retaining their large size, are quite different incharacter from the ones described for the fully formed embryo.The most marked change lies in the cytoplasm, which is nowvacuolated and stains very deeply. Dispersed between thesecells and situated at their bases are the replacement cells (figs. 17and 18, PI. 32).

Apart from these cytological differences, the presence of astriated fringe and a chitinous intima on the inner side ofthe mid-gut, and stomodaeum and proctodaeum respectively,marks the three regions of the alimentary canal.

z2

336 K. MANSOUR

The anterior portion of the mid-gut is dilated and endsposteriorly in a narrow long tube which is coiled twice andpasses into the proctodaeum. Mesenteric caeca are only presenton this narrow portion. They are papillae-form, consisting ofabout ten cells each.

(/) The Accessory Al imentary Mass and the Sub-oesophageal Body.

The accessory mass must not be confused with the sub-oesophageal body described by Wheeler (1893), Nussbaum andFulinski (1906 and 1909), and Hirschler (1909). Such a bodyis present in Calandra oryzae (fig. 14, PI. 31) but it takesno part in the formation of the mid-gut as Nussbaum and Fulin-ski and Hirschler have suggested, and in the insects theseauthors investigated no accessory mass has been described.

In Calandra the cells of the sub-oesophageal body aremostly binueleate, suggesting that this body is nephrocytic.(Compare the ventral nephrocytes of some Dipterous larvae(Keilin, 1917), the peri-oesophagealnephrocytes of Pediculushum an us (Nuttall and Keilin, 1921), and similar structures ofCimex lec tu la r ius (Puri, 1924).) This body is to be seenin Calandra before the migration thither of the accessorymass (fig. 14, PI. 31), and is actually pushed away from the wallof the gut by the advancing accessory cells (fig. 16, PI. 31).

V. OCCURRENCE OF THE ACCESSORY CELL-MASS IN OTHER

INSECTS.

Examination of eight Curculionid species, three Scolytids, twoAnobiids, two Chrysomelids, and one Lamellicorn (see p. 320),revealed the fact that an organ similar in all respects to theaccessory mass is present in Calandra granar ia , Odoi-porus glabr icol l i s , and Hylobius ab ie t i s , while asimilar organ but in a totally different situation is present inRhyncolus l ignar ius . In this last-mentioned species itoccupies a dorsal situation somewhere near the junction of themid-gut and the proctodaeum.

While one can perhaps assume that in Calandra granaria,

MID-GUT OF CALANDRA 337

Hylobius abiet is and Odoiporus glabr icol l is ,tbe mode Of origin and behaviour of the accessory mass issimilar to that described in Calandra oryzae, one is hardlyjustified in drawing a similar conclusion for Rhyncolusl ignar ius , and it is hoped to carry out further research uponthis species as soon as the necessary material has been pro-cured.

VI. POST-EMBRYONIC STUDIES ON CALANDRA ORYZAE.

(a) Degenerat ion of the Larval Mid-gut and theIn i t i a t i on of t ha t of the Adul t .

The processes involved in the metamorphosis of £he mid-gutbegin towards the end of the last larval instar about three daysbefore the pupal moult sets in. The habits of the larva andthe external changes in form during this period have beendescribed on p. 319.

The internal changes are most peculiar and are begun bythe collapse of the ventricular portion of the gut to a narrowtube with an irregular wall. The posterior portion also undergoesdiminution in lumen. Not only does the larval mid-gut diminishconsiderably in width, but also in the same time it is markedlyreduced in length. Its coils become less evident, and it is nowtransformed to a narrow and almost straight tube which isblocked anteriorly with numerous degenerating cells and istotally cut off from the posterior end of the fore-gut (fig. 19,PI. 32).

During the collapse, the replacement cells between the basesof those of the epithelium grow to form a more or less con-tinuous flattened layer, which envelops the degenerating epi-thelial wall and separates it from the muscular one (fig. 19,PI. 32).

As a result of these processes the larval mid-gut epitheliumwith its replacement cells, except that it remains loosely joinedto the proctodaeum, now lies loose within the muscular wall(fig. 19, PI. 32). The latter is undergoing regeneration, and willbecome the muscular wall of the adult mid-gut.-

At the same time the cells at the posterior end of the fore-gut

338 K. MANSOUR

become elongate, multiply rapidly, and obliterate the openinginto the larval mid-gut. The stomodaeum then becomes cutoff from the latter and grows backwards into a blind tube insidethe transforming muscular layer of the mid-gut (fig. 20, PI. 32),pushing before it the degenerating larval epithelium until thenew tube reaches the proctodaeum. By the time this happensonly a few irregular cells and scattered pieces of chromatinrepresent the last of the larval mid-gut (fig. 21, PI. 32). Thewalls of this newly formed tube will later on yield the definitiveepithelium of the adult mid-gut.

(b) Behaviour of the Accessory Al imentary Cell-mass .

As soon as the collapse of the larval mid-gut begins, theaccessory mass awakens from its larval quiescence. After beingclosely applied to the ventral side of the alimentary canal(fig. 17, PI. 32), it fuses •with the wall of the metamorphosingfore-gut (fig. 19, PI. 32). This fusion takes place in the regionbetween the muscular walls of the fore- and mid-guts of thelarva (fig. 18, PI. 32). The anterior margin of the fusion denotesthe anterior boundary of the mid-gut tube.

The fusion now extends posteriorly (fig. 20, PI. 32), probablypartly due to the intercalary growth of the portion of the mid-gut tube fused with the accessory mass and partly to the activi-ties of this mass itself. Extension also occurs laterally, andeventually the mid-gut tube is surrounded by the accessory cells(fig. 20, PL 32). Posterior extension continues, and by the timethe mid-gut tube reaches the proctodaeum only about one-thirdof it remains free from these cells (fig. 21, PI. 32).

(c) The Yel low B o d y .Up to the stage when the mid-gut tube reaches the procto-

daeum its lumen is extremely narrow (fig. 21, PI. 32), but it thenbecomes enlarged anteriorly (fig. 22, PI. 33) owing to the appear-ance of a homogeneous substance which is probably secreted bythe cells of the walls of this tube. This substance is the yellowbody; a term which was originally given by Weismann (1864) to

MID-GUT OF CALANDRA 839

the contents of the gut of M u s c a at a similar stage of develop-ment.

During the secretion of the yellow body the pupal moult setsin and the development of the adult mid-gut continues withoutany break during the pupal stage.

(d) A p p e a r a n c e of the Mesen te r i c Caeca and theF a t e of the Accessory Mass.

The development of the mesenteric caeca, which begin toappear all along the mid-gut, is more easily seen in the posteriorregion where the accessory cells are lacking. Here they appearas groups of cells derived from the wall of the mid-gut tube.These groups of cells are numerous and are scattered all overthe outer surface of the wall.

The individual groups, through active division of their cells,form small tubes closed at their outer ends and opening into thelumen of the gut (fig. 25, PI. 33).

In the anterior region, although development commences, thecaeca do not grow into tubes, probably because of the pressureof the accessory cells which collect round the budding caeca,squeezing them, so that the lumens are obliterated (fig. 24, PI. 33).

By the time the formation of the mesenteric caeca is com-pleted, the accessory cells, which once formed a more or lessregular layer round most of the mid-gut tube, have becomesegregated into small groups, forming the outer walls of mostof the caeca.

(e) F o r m a t i o n of the Def in i t i ve Mid-gut E p i t h e -l ium of t he A d u l t .

During the formation of the mesenteric caeca the remainderof the cells of the mid-gut tube have been undergoing activedivision and reorganization. After being extremely irregularand distinctly flattened, where the accessory cells exist (fig. 22,PL 33) they become more or less cubical, and by the time theaccessory cells have become incorporated in the walls of thenumerous mesenteric caeca these cubical cells form a columnarlayer which is the definitive mid-gut epithelium of the adult.

340 K. MANSOUR

From the beginning of the development of the adult mid-gutits lumen has been in direct communication with that of themetamorphosing fore-gut. The hind-gut communication, how-ever, is not effected until after the definitive mid-gut epitheliumhas been fully formed.

By this time the other metamorphosis processes have beencompleted and the adult is almost ready to emerge.

The post-embryonic processes detailed above can be summedup as follows :

1. The degeneration of the larval mid-gut and the formationof that of the adult start about three days before thepupal moult sets in.

2. The larval mid-gut epithelium with the replacement cellscollapses, degenerates and totally disappears before theemergence of the imago.

3. The adult mid-gut epithelium is derived entirely from theposterior end of the metamorphosing fore-gut.

4. The accessory alimentary cells envelop the developingmid-gut epithelium and later on become localized in thewalls of most of the mesenteric caeca.

VII. FORMATION OF THE ADULT MID-GUT IN OTHEREHYNCHOPHORA.

It was pointed out on p. 336 that in certain other Ehyncho-phora the accessory mass had been observed, and in the formsstudied the type of adult mid-gut formation is similar to thatdescribed for Calandra oryzae.

InSco ly tus des t ruc to r , Anthonomuspomorum,and Antbonomus grandis no accessory cell-mass ispresent, but in all these cases examined the adult mid-gutepithelium arises exactly as in Calandra oryzae.

This shows that the accessory mass has nothing to do withthe type of development of the adult mid-gut epithelium de-scribed above. To the embryologist the significance of thistype lies in the fact that the adult mid-gut epithelium is de-rived from a structure which has a definite ectodermal origin.

MID-GUT OF CALANDRA 341

This recalls and supports what has been stated earlier in thispaper about the origin of the larval mid-gut epithelium of mostpterygote insects.

VIII. THE ACCESSORY CELLS IN THE ADULT.

The activities of the accessory cells during imaginal life havenot been so far studied. From a brief examination it can be saidthat the number of these cells becomes markedly reduced.After being densely aggregated in the walls of most of the mesen-teric caeca (fig. 24, PI. 33), they apparently disappear graduallyduring the imaginal life of the individual until in aged adultsonly a few cells are to be found near the distal ends of thesecaeca (fig. 26, PI. 33).

In considering the possible function of these cells a digestiveone is at once suggested on account of their close intimacy withthe digestive tract, but there is at present no experimentalevidence in support of this. If this should prove to be correct,it suggests that probably the adult extracts something fromthe food material which was unobtainable by the larva.

IX. GENERAL CONSIDERATION OF THE KNOWN TYPES OF

ADULT MID-GUT FORMATION IN THE COLEOPTERA.

The type of formation of the adult mid-gut epithelium de-scribed here for C a l a n d r a o r y z a e and other Ehyncho-phorous species is totally different from that described by Eegnel(1897) in T e n e b r i o m o l i t o r , Karawaiew (1898) in A n o -b i u m p a n i c e u m , Deegener (1904) in C y b i s t e r r o s e l i ,and Korschelt (1924)in Dy t i s c u s m a r g i n a l i s . In all thesevarious Coleopterous insects the adult mid-gut epithelium wasfound to develop from islands of replacement cells scatteredat the bases of the larval mid-gut epithelium cells, and my ownobservations on P t i n u s ' t e c t u s agree with those of theauthors mentioned. This ' P t i n u s ' type can easily be dif-ferentiated from the ' C a l a n d r a ' during the pupal stage. Atthis stage in insects with the ' P t i n u s ' type the ' yellow body'is to be found surrounding a large mass of cellular debris which

342 K. MANSOUR

is the sloughed-off larval mid-gut epithelium (Text-fig, 1, B,relep). In those insects of the ' C a 1 a n d r a ' type the ' yellowbody' contains no remains of the larval mid-gut epithelium

TEXT-FIG. 1.

A, longitudinal section through developing adult mid-gut ofAnthonomus pomorum. B, longitudinal section throughdeveloping adult mid-gut of Ptinus teotus. Camera drawingsemi-diagrammatic. For lettering see p. 349.

(Text-fig. 1, A, and fig. 22, PI. 33), since such remains have beencontinually pushed backwards in front of the advancing mid-gut tube (Text-fig. 1, A, relep, and figs. 20 and 21, PI. 32).

MID-GUT OF CALANDRA 343

On the question as to which of these two types is to be re-garded as the more primitive, we might agree with Perez (1902)that the formation of the adult mid-gut epithelium from scat-tered replacement cells is comparable to the ordinary processesof regeneration occurring in the functional larval and nymphalmid-gut of the various insects.

Now the replacement cells are represented in the larva ofCalandra oryzae (figs.l7andl8, P1.32). During metamor-phosis they form a more or less continuous layer (fig. 19, PI. 32),but this layer undergoes the same fate as the cast-off larval mid-gut epithelium. Possibly, therefore, similar replacement cells ori-ginally gave rise to the mid-gut epithelium of the ancestral adult.

The failure of the replacement cells to form the epithelium ofthe adult mid-gut in the Ehynchophorus species studied recallsthe reference earlier in this paper to the failure of the endo-dermal cells to form the larval mid-gut—two points, the signi-ficance of which must for the present remain obscure.

The type of development of the adult mid-gut epithelium de-scribed for Calandra oryzae, on the other hand, exhibitsa distinct relationship to that described by Poyarkoff (1910) inGalerucella ulmi, a phytophagous beetle. In this insectPoyarkoff found that during metamorphosis the larval epithe-lium is stripped off with its replacement cells from the muscularwall and is passed into the hind-gut. According to this observer,the cells of the posterior side of the oesophageal valvule multiplyrapidly and extend backwards in the form of a solid plug,totally blocking the space once occupied by the mid-gut epi-thelial sac. These proliferated cells, which are fusiform, are ofvarious sizes. Some of the smallest become fixed to the meta-morphosing muscular wall and are destined to form the defini-tive adult mid-gut epithelium, while few larger ones form a pro-visional epithelium which lasts only during the pupal stage.The remaining fusiform cells perish and take no part in theformation of the adult organs.

The important point in Poyarkoff's account is the formationof the adult mid-gut epithelium from some of the cells pro-liferated from the posterior end of the fore-gut.

344 K. MANSOUB

An incomplete examination of Melasoma populi ,another phytophagous beetle, suggests that in this species thedevelopment is similar to that described by Poyarkoff.

This resemblance between examples of Phytophaga andRhynchophora, if it turns out to be characteristic of the groups,is interesting because of the view of recent systematists as tothe close relationship between these two series which Lameere(1903), on certain anatomical characters, actually united intoone group, Phytophaga.

With reference to the assumed relations of the Phytophagaand Rhynchophora, it is worth mentioning that the Lamelli-cornia agree with these two series in the possession of compoundpedicellate testis which are only present in these three groupsamong the Coleoptera. An examination of Dorcus para l -le lopipedus , however, revealed the fact that the mode ofadult mid-gut formation is of the ' P t inus ' type. If the sameis true of other Lamellicorns, it may indicate either that thistest is of no value or that the Lamellicornia are not very closelyrelated to these series.

With regard to the question of origin of the Phytophaga andEhynchophora, Kolbe (1908) thought that they have been de-rived from primitive Glavicornia. Ganglbauer (1903), on theother hand, suggested that these series might have had a com-mon descent with the Malacodermata. Possibly the systematicstudy of the mode of formation of the adult mid-gut, which I amundertaking, will throw light on this point.

X. SUMMARY.

(i) Embryonic .1. The blastodermal stage of insects corresponds to the

blastula stage of other animals.2. The primary yolk-cells are a secondary feature of the

developing insect egg.3. The ventral groove corresponds to the invagination of

the gastrula.4. The endodermal cells of insects are budded off into the

MID-GUT OF CALANDRA 345

yolk from the walls of the ventral groove or from thickenings inthe blastodermal wall.

5. The endodermal cells inC. oryzae and most Pterygotado not give rise to any larval or imaginal structures.

6. The larval mid-gut epithelium in C. oryzae and mostpterygote insects is derived from the inner ends of the stomo-daeum and proctodaeum and is therefore of ectodermal origin.

7. The accessory alimentary cell-mass present in the larva ofCalandra oryzae is developed from the rudiments of thelarval mid-gut epithelium.

(ii) Pos t -embryonic .8. The metamorphosis of the mid-gut in C. oryzae begins

about three days before the pupal moult occurs.9. The larval mid-gut epithelium collapses and degenerates.10. The replacement cells take no part in the formation of

the mid-gut of the adult.11. The adult mid-gut epithelium in Calandra oryzae

and five other Ehynchophorus species is derived from theposterior end of the metamorphosing fore-gut, and is therefore ofdefinite ectodermal origin.

12. The accessory alimentary cells are incorporated in thewalls of most of the mesenteric caeca.

13. The presence of the accessory cell-mass has nothing to dowith the ' Ca landra ' type of formation of adult mid-gutepithelium.

14. The number of the accessory cells is reduced duringimaginal life.

15. In insects with the ' C a l a n d r a ' type the ' yellow body'contains no remains of the larval mid-gut epithelium. Suchremains characterize the ' P t i n u s ' type, and differentiate itfrom that of Calandra .

16. The ' P t inus ' type probably is the more primitive.

XI. ACKNOWLEDGMENTS.

This work has been carried out under the supervision ofProfessor F. Balfour-Browne, without whose constant en-couragement and stimulating advice it would not have been

346 K. MANSOUR

accomplished. The writer also has to thank Professor E. W.MacBride, Professor H. Graham Cannon, and Dr. J. W. Munrofor the interest they have taken in this work and the help theygave.

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EXPLANATION OF PLATES 29-33.

All figures are from Camera lucida drawings.

LIST OF COMMON REFERENCE LETTERS.

ace c, accessory cell; ace m, accessory cell-mass ; am, amnion; ap, appen-dage ; be me ca, beginning of a mesenteric caecum; be mg r, beginning of mid-gut rudiment; be mg t, beginning of mid-gut tube ; br, brain ; ch i, chitinousintima ; ei mus, circular muscles ; co me ca, core of mesenteric caecum ;degc, degenerating cell; deglep, degenerating larval mid-gut epithelium;

350 K. MANSOUR

ect, ectoderm ; end, endoderm; fg, fore-gut; ge r, genital rudiment;i w so, inner wall of mesodermal somite; If, lateral furrow; Ig mus, longi-tudinal muscles ; Ip, lateral plate ; Mal.t, Malpighian tubule ; mdp, middleplate; mes, mesoderm; rng ep, mid-gut epithelium; tng ep c, mid-gutepithelium cell; mg r, mid-gut rudiment; mg t, mid-gut tube; mig c,migrating cells; mus c, muscle cell; mus w, muscular wall; neph, nephro-cyte ; nrb, neuroblast; phag, phagocyte; prod, proctodaeum; pr yc,primary yolk-cell; p w stom, posterior wall of stomodaeum; re I mg, remainsof larval mid-gut epithelium ; re y, remains of yolk; r Mai, rudiment ofMalpighian tubule ; ry mg w, rudimentary wall of mid-gut; ry mus w,rudimentary muscular wall ; rp c, replacement cells ; so 6, sub-oesophagealbody; stom, stomodaeum; siom raws, stomodaealmuscles; stomw, stomodaealwall; strfr, striated fringe ; vg, ventral groove or gastrula furrow ; vnch,ventral nerve-chain ; y, yolk ; y b and yeb, yellow body.

PLATE 29.

All Figures are from Sections of Eggs.Fig. 1.—Ventral portion of a transverse section through an advanced

blastoderm showing the gastrula furrow (vg) and the lateral furrows (If).X380.

Fig. 2.—Ventral portion of a transverse section to illustrate the entranceof the endodermal cells (end) into the yolk. X 380.

Fig. 3.—Portion of transverse section showing the mesoderm (mes) andthe ectoderm (ect) after the entrance of the endodermal elements into theyolk, x 380.

Fig. 4.—Portion of transverse section through the early stomodaeal pit.X380.

Fig. 5.—Portion of a longitudinal section through the early proctodaealpit. X 400.

PLATE 30.

All Figures are from Sections of Eggs.Fig. 6.—Portion of a transverse section through the posterior wall of the

early stomodaeal pit. X 380.Fig. 7.—Portion of a transverse section through the posterior wall of the

stomodaeum to show the beginning of the formation of the rudiments ofthe mid-gut epithelium of the larva, x 380.

Fig. 8.—Portion of an oblique section through the posterior wall of thestomodaeum and one of the rudiments of the mid-gut epithelium of thelarva (mgr). X 380.

Fig. 9.—Portion of a transverse section through the thoracic region ofthe embryo to show the presence of the rudiments of the mid-gut epithe-lium (mgr) on either side of the mid-ventral line, x 380.

MID-GUT OF CALANDHA 851

Fig. 10.—Portion of sagittal section illustrating the entrance of cellsfrom the blind end of the stomodaeum into the yolk. X 400.

Fig. 11.—The posterior portion of a sagittal section showing the begin-ning of one of the rudiments of the larval mid-gut epithelium derived fromthe proctodaeum (mg r). x 400.

PLATE 31.

All Figures are from Sections of Eggs.Fig. 12.—Lateral portion of a transverse section showing the association

of one of the rudiments of the larval mid-gut epithelium (mg r) with theinner wall of an abdominal mesodermal somite (i w so). X 380.

Fig. 13.—The inner portion of a transverse section showing the buddingoff of the cells of the accessory mass (ace m) from the rudiments of the larvalmid-gut epithelium into the yolk. X 300.

Fig. 14.—The anterior portion of*a sagittal section showing the stomo-daeum abutting on the accessory mass (ace m). This figure also shows thesub-oesophageal body (so 6). x 300.

Fig. 15.—Portion of a sagittal section showing the accessory mass migrat-ing from the yolk into a sub-stomodaeal situation. X 400.

Fig. 16.—Portion of a sagittal section through a fully formed embryoshowing the accessory mass (ace tn) outside the lumen of the alimentarycanal. This figure also shows the binucleate cells (neph) derived from thesub-oesophageal body. X 400.

PLATE 32.

Figs. 17 and 18 are from Sections of a third Instar Larva.Figs. 19 and 20 are from Sections of Prepupae.

Fig. 17.—Portion of a sagittal section showing relative position of acces-sory mass in the growing larva. X 80.

Fig. 18.—Portion of a sagittal section illustrating the difference betweenthe cells of the stomodaeum and those of the mid-gut. This figure alsoshows the gap between the muscles of the stomodaeum and those of themid-gut. X 600.

Fig. 19.—Portion of a sagittal section through a metamorphosing larva,showing the collapse and the degeneration of the larval mid-gut and thefusion of the accessory mass (ace m) to the ventral side of the stomodaeum.X80.

Fig. 20.—The same as the previous one but more advanced, showing theincreased fusion of the accessory mass and the growth of the stomodaeumto form the mid-gut tube (mg t). X 80.

Fig. 21.—The same as the previous sections but more advanced, showingthe remains of the larval mid-gut epithelium (relmg) and the mid-gut tubenearly reached the proctodaeum. X 80.

3 5 2 « • " " . . K. MANSOUK

PLATE 33.

Figs. 22-5 are from Sections of Pupae.Fig. 26 is from a Section of the Gut of an aged Adult.

Fig. 22.—Portion of a longitudinal section showing the lumen of the mid-gut tube much enlarged. X 80.

Fig. 23.—Portion of a longitudinal section of the wall of the developingmid-gut showing the beginning of the formation of a mesenteric caecumwhere the accessory cells are present. X 380.

Fig. 24.—Section through one of the anterior mesenteric caeca in anadvanced pupa showing the accessory cells {ace c) arranged round a coreof cells continuous with the cells of the mid-gut epithelium, x 220.

Fig. 25.—Section through one of the posterior mesenteric caeca. X 220.Fig. 26.—Section through one of the most anterior mesenteric caeca in

an aged adult showing the reduction of the number of the accessory cells.x220.