The Morphology and Physiology of the SalivaryGlands of Hemiptera-Heteroptera.

By

B. A. Baptist, Ph.D.(From the Department of Entomology, Zoological Laboratory,

Cambridge.)

With 46 Text-figures.

CONTENTS.

PAGEINTBODUCTION . . . . . . . . . . 91MATERIAL AND TECHNIQUE . . . . . . . 93MOBPHOLGGY AND HLSTOPHYSIOLOGY OF GLANDULAR SYSTEMS . 97

Morphology, p. 97; Histology, p. 106; Secretion, p. 119;Physiological Activity, p. 121.

DISCUSSIONS AND CONCLUSIONS 122Morphology, p. 122; Histology, p. 126; Secretion, p. 132;

pH and Enzymes, p. 134.SUMMARY 136

INTRODUCTION.

GLANDS associated with the intake of food are generallypresent among Insects although they are wanting in many ofthe Coleoptera. In most insects they are morphologically thelabial glands, but in those where they have taken on some otherrole the mandibular and sometimes also the maxillary glandsperform a salivary function. The special role of the labialglands in such cases is generally that of silk secretion, as isfound in larval Lepidoptera, Trichoptera, and Psocoptera; orotherwise they may be greatly reduced or absent as in the adultLepidoptera.

The size and shape of the glands are highly variable indifferent insects, but within an order a certain degree of uni-formity becomes apparent. The glands are usually situatedin the thorax lying on either side of the gut, but may lie inthe head and may extend into the abdomen. They may berelatively simple or complexly branched and convoluted. A

92 B. A. BAPTIST

reservoir is frequently developed from the conducting part ofthe system, which in its purely conductive part frequentlypossesses taenidia in its internal lining similar to that found intracheae. The common opening of the salivary duct alwayslies in a salivary pocket or 'salivarium' at the base of thehypopharynx. In the higher orders this becomes modified foractively expelling the secretion of the glands. It becomes thesalivary syringe of Hemiptera, Hymenoptera, and Diptera, andthe silk press of larval Lepidoptera.

In the Hemiptera the labial glands exhibit a very wide rangein form and structure and the object of the present investigationwas to study these glands by modern microchemical methods,and attempt to find the biological and physiological explana-tion of their striking diversity. In spite of this diversity,however, the system conforms to a common plan, that of aprincipal gland with an accessory gland on each side, the latteropening through its duct into the hilus of the principal gland.The principal salivary duct leaves the hilus of the principalgland and passes forward into the head, where it joins its fellowto form a very short common duct which immediately passesinto the characteristic salivary pump and opens into its distal end.

The form and disposition of the salivary glands of theHeteroptera were first studied by Dufour (1833) in manydifferent species in an investigation of the alimentary system.He did not, however, follow out the course of the ducts com-pletely except in N o t o n e c t a , and made the mistake ofsupposing that the ducts both of the principal glands and theaccessory glands opened into the mouth cavity. More recentlyBugnion (1908) and Bugnion and Popoff (1910) published amore accurate account working on various species. Since thework of Bugnion and Popoff other authors who have describedthe glands in the same or similar Heteroptera are Faure-Fremiet (1910) those of the aquatic Heteroptera, Cornwall andPatton (1914) and Puri (1924) those of Cimex, Poisson (1924)the aquatic Heteroptera, Hamilton (1931) Nepa c ine r ea ,Hamner (1936) Solubea p u g n a x , Breakey (1936) Anasat r i s t i s , Hood (1937) Oncope l tus fasc ia tus , andEawat(1939) Naucor i s c imicoides . No mention has been made,

SAIJIVAEY GiANDS OF HEMIPTERA 93

however, of the nervous and tracheal supply of these glands,except for a few observations by Faure-Fremiet of the com-paratively rich tracheal supply of the accessory glands in theCryptocerata, and the tracheal supply of the glands of Nepacinerea by Hamilton, and Naucor i s c imicoides byBawat. With reference to the histology, a few observationshave been made by Bugnion and Popoff and Faure-Fremiet.No observations have been made, however, on the cytologicalstages of the glandular cycle. Faure-Fremiet has also worked onthe chemical nature of the salivary secretion of the Gryptocerata.

With regard to physiological action there has been very littleinvestigation in Heteroptera, there being only some observa-tions by Smith (1920,1926) on certain Capsids, and by Cornwalland Patton of an anticoagulin in the glands of T r i a t o m a ,and Puri (1924) in the glands of Cimex. Among the Hemi-ptera-Homoptera, which have been studied much more exten-sively, Davidson (1923) has found an amylase in the salivaryglands of Aphis r u m i c i s , Weber (1928a) has found anamylase and an invertase in the salivary glands of Empo-asca so lanum (Jassidae), and Butler (1938) has found anamylase and an invertase in Aleyrodes b r a s s i c a e .

MATEKIAL AND TECHNIQUE.

The material used constituted representatives of all thesuperfamilies of Heteroptera except the Aradoideae.

The species used were: N o t o n e c t a g lauca L. (Noto-nectidae), Naucor i s c imicoides L. (Naueoridae), Nepacinerea L. (Nepidae), Gerr is l a c u s t r i s L. (Gerridae),Salda l i t t o r a l i s L. (Saldidae), Corixa geoffroyiLeach (Corixidae), Nabis a p t e r u s L. (Nabidae), Tr ia -toma in f e s t ans Klug (Eeduviidae), E h o d n i u s p ro-l ixus Stal. (Eeduviidae), Cimex l e c t u l a r i u s L. (Cimi-cidae), Lygus p r a t e n s i s L. (Capsidae), Monan th iacardu i L. (Tingitidae), An thocor i s nemorum L.(Anthocoridae), P e n t a t o m a ruf ipes L. (Pentatomidae),Gas t rodes f e r rug ineus L. (Lygaeidae), Chilacist y p h a e Perr (Lygaeidae), M e t a c a n t h u s e legans L.(Berytidae), Corizus p a r u m p u n c t a t u s Schill. (Coreidae),

94 B. A. BAPTIST

Dysdercus howard i de Geer (Pyrrhoeoridae), P y r r h o -coris a p t e r u s L. (Pyrrhoeoridae).

Of these the aquatic forms N o t o n e c t a , l . a u c o r i s ,Nepa, Corixa, and G e r r i s were obtained in the immediateneighbourhood of Cambridge, as also were the terrestrial formsLygus , A n t h o c o r i s , M o n a n t h i a , and Chi lac i s .P e n t a t o m a , Gas t rodes , Corizus , and M e t a c a n t h u swere obtained in the Mildenhall district of Suffolk. S a 1 d a wasobtained from the coast in the neighbourhood of Bournemouth;P y r r h o c o r i s from Germany; Dysde rcus from Jealott'sHill Eesearch Station; C i m e x from London; while E h o d -nius and T r i a toma were kindly supplied by the LondonSchool of Hygiene and Tropical Medicine from their cultures.

It was sometimes necessary to employ momentary chloro-form anaesthesia before dissection. Although avoided wherepossible, it was found that this did not have any deleteriouseffects on the subsequent fixation; thus confirming the ex-perience of du Boissezon (1930) with mosquitoes. The glandswere dissected in the insect's own blood, or if this was insuffi-cient in quantity, in a drop of Einger's solution, and trans-ferred immediately to the fixative.

For vital staining methylene blue 1 :1,000, Janus green1 :10,000, and neutral red 1 :10,000 were used.

For histological purposes the fixatives used were aqueousBouin, Zenker's fluid, corrosive sublimate in Saline, andFlemming without Acetic. In some cases certain other fixa-tives, alcoholic Bouin, Carnoy, Petrunkevitch, &c, were alsoused but gave no specially noteworthy results. For cytologicalwork the most successful of the fixatives used were Kolatchev(Nassanov's modification) for Golgi bodies and Altmann (Baker'smodification) for mitochondria. For staining iron haemo-toxylin and orange G, safranin and light green, Delafield'shaemotoxylin and eosin, and Mallory's triple stain, were thestain combinations generally employed. Other staining tech-niques used were those of Champy-Kull and Benda (as modifiedby Baker) for staining mitochondria. To obtain secretorystages the glands were fixed at varying intervals of time afterthe commencement of feeding.

SALIVABY GLANDS OF HEMIPTBRA 95

For histochemical analysis proteids were tested for by thexanthoproteic and Millon's reactions as modified for Mstologicalsections: xanthoproteic reaction: the sections are treated withfuming nitric acid in the cold. After some minutes the slide isremoved and rinsed; proteids are coloured yellow; subsequentexposure to ammonia vapour changes the colour to orange.Millon's reaction: the sections are placed in the reagent for3 or 4 hours, then washed in 1 per cent, nitric acid and examined;the proteids are coloured red. The reagent was prepared asrecommended by Bensley and Gersh (1933) thus: dilute 400 c.c.of cone. HN03 to 1 litre of distilled water; allow to remain48 hours; then dilute anew ten times; to the solution of 4 percent, nitric acid so obtained crystals of mercuric nitrate areadded in great excess and left for several days until completesaturation; filter; to 400 c.c. of the solution so obtained add3 c.c. of the initial solution of 40 per cent. HN03 and 1-4 gm.of sodium nitrite.

Nucleic acid derivatives were tested for by Feulgen's tech-nique for chromatin. The chromaffine reaction was employedto test for possible phenolic derivatives of protein metabolismthus: tissue is fixed in (1) Miiller-formol and (2) potassiumiodate-formol (equal parts of 5 per cent, potassium iodate and10 per cent, formol), and sections prepared in the usual way.A positive reaction is recognized by the yellow or brownish tintwhich is taken up by the chromaffine substances.

Examination for substances of a fatty nature was carriedout with the fresh gland and after fixation with 10 per cent,formol. Examination for fat was carried out by staining withSudan 111 and Sudan black after formol fixation. The glandswere also tested with osmium peroxide.

In the determination of pH the capillator method was used.The glands were dissected out in normal saline and placed ina drop of the same rn a clean watch-glass until a sufficientnumber had been accumulated. The saline was then drainedoff with a capillary pipette as completely as possible, and theglands then broken open with fine needles. The salivary fluidthus obtained was taken up by a capillary pipette and mixedwith an equal quantity of the indicator on a clean waxed slide.

96 B. A. BAPTIST

This fluid was then drawn up in a clean capillary tube similar tothose containing the standards for comparison. The indicatorsused were bromo-cresol purple and phenol red. Tue standardswere those supplied by the B.D.H. capillator set. In those caseswhere the quantity of salivary fluid available was very minute,Wigglesworth's modification (1927 a) for determination of thepH of very minute quantities of fluid was used.

Preparation of the salivary fluid for enzyme investigationwas carried out as follows. The glands were dissected out insaline and placed in a clean watch-glass in a drop of saline.After a sufficient number had been accumulated the saline wasdrawn off and the glands quickly washed in a few drops ofdistilled water. This was then drained away as completely aspossible and the glands broken up with fine needles. In thosecases in which the glands did not yield sufficient salivary fluidby this treatment, a brei of the glands was made by crushingthem up with a little clean sand in a few drops of distilled water.In all cases a minute quantity of toluol was added as an anti-septic. The substrates used were gelatin (slide method of Pick-ford and Doris, 1932), and blood fibrin stained with carmine(Wigglesworth, 1927) for protease; starch-agar (slide method ofPickford and Doris, 1932) and 2 per cent, starch solution foramylase; pure recrystallized cane sugar, 5 per cent, aqueoussolution, for invertase; maltose, 5 per cent, aqueous solution,for glycogenase; olive oil for lipase; filter paper and cottonwool for cellulase. In the slide method the slides were placedin a closed glass vessel which contained filter paper saturatedwith buffer solution, and kept at a temperature of 25° C. Inthe other cases the experiments were carried out in micro-tubes4 cm. x 0-5 cm. at 25° C. from 3-12 hours. The buffer solutionsemployed were phosphate mixtures (Clark and Lubs). In allcases controls were carried out with boiled salivary extractprovided by heating in boiling water for 5 minutes. For testingfor reducing sugars Fehling's solution was used. Lipase diges-tion was recognized by liberation of fatty acid indicated byphenol red.

In testing for* anticoagulant enzymes human blood drawndirectly from a vein was used. About 0-5 c.c. of the blood was

SALIVARY GLANDS OF HEMIPTEEA 97

added to the enzyme extract contained in a micro-tube andthoroughly mixed up with it. The tubes were then closed andconstantly agitated until clotting had occurred in the controls.The end point was taken as the first indication of the formationof a clot, whether it involved the whole mass of blood or onlya part of it. The tests were carried out at 25° C. The controlswere provided by heating the gland extract in boiling water for5 minutes. Tests were also carried out with smaller quantitiesof blood in capillary tubes.

MORPHOLOGY AND HISTOPHYSIOLOGY OF GLANDULAR SYSTEMS.

Morphology.

The salivary system in all Hemiptera-Heteroptera can besaid to consist of a pair of principal and accessory glands. Theprincipal glands are fundamentally bilobed, comprising anteriorand posterior lobes. They are situated in the thorax on eitherside of the gut except in the Notonectidae where they lie in thehead under the fronto-occipital region of the cranium above thebrain and between the compound eyes. The principal gland isfound to be simply bilobed in the families Notonectidae (Text-fig. 1), Naucoridae (Text-fig. 2), Nabidae (Text-fig. 13), someEeduviidae (Text-fig. 7), Capsidae (Text-fig. 10 A), Corixidae(Text-fig. 5), Pentatomidae (Text-fig. 14), as also shown byprevious workers in various species of these families; and alsoin the Saldidae (Text-fig. 6), Anthocoridae (Text-fig. 12), andTingidae (Text-fig. 11). The relative size of the lobes variesin the different forms but the anterior lobe is always the smaller.A modified bilobed gland is found in the Nepidae (Text-fig. 3),and Gerridae (Text-fig. 4), where the anterior lobe is separatedfrom the posterior lobe by an anterior lobe duct (aid.). Further,in these cases both anterior lobe and posterior lobe are sub-divided into numerous lobules. In N e p a the two most anteriorlobules of the posterior lobe are larger in size and different instructure and in the nature of their contents from the otherlobules. A trilobed principal gland has been found in the familyLygaeidae (Text-figs. 15, 16) and also in the Berytidae (Text-fig. 17) which belongs to the same super-family. The Pyrrho-coridae which also belong to the same superfamily have, how-

NO. 329 H

B. A. BAPTIST

•5mm.

TEXT-FIG. 1.

Salivary glands of N o t o n e c t a g l a u o a .

K E Y TO LETTEBMG OF TEXT-FIGS. 1^16.

a.d., accessory duct; a.g., accessory gland; a.l., anterior lobe ofprincipal gland; a.l.d., anterior lobe duct; 6.6., striated margin;b.m., basement membrane; c , cuticular lining; c.c, central canal;c.v., collecting vacuoles; g., granules; I., large anterior lobule ofposterior lobe; l.l., lateral lobe; m., muscle; m.l., median lobe;m.p., membrana propria; n., nerve; n.p., nerve plexus; nu.,nucleus; nu.', nucleolus; p.', posterior process; p.d., principalduct; p.I., posterior lobe of principal gland; r., replacement cell;r.m., reserve material; s.g., secretion granules; s.d., secondaryduct; s., secretion; *., trachea; t,', tracheole; t.p., tubular processof accessory gland; v., vacuole.

ever, a quadrilobed principal gland (Text-figs. 18, 20), whichcondition is also present in the Coreidae (Text-fig. 19). Byfollowing the openings of the lobes in these quadrilobed glands

SALIVARY GLANDS OF HEMIPTERA 99

ag -

TEXT-FIGS. 2-3.

Fig. 2. Salivary glands of Naucoris eimicoides.Fig. 3. Salivary glands of Nepa einerea.

it appears that the antero-median lobe comprises the typicalanterior lobe "while the remaining lobes comprise the typicalposterior lobe. This division is also borne out by the stainingreactions of the secretion contained in these lobes. Finally, inRhodn ius (Text-fig. 8) and Cimex (Text-fig. 9) a unilobed

100 B. A. BAPTIST

•5mm....

•Smm.TnxT-mas. 4-6.

Fig. 4. Salivary glands of Gerris laoust r is .Fig. S. Salivary glands of Oorisa gooff royi.Fig. 0. Salivary glands of Salda l i t to ra l i s .

SALIVARY GLANDS OF HEMIPTEBA 101

pd—

pi— -

TBXT-ITGS. 7-8.Fig. 7. Salivary glands of Triatoma infestans.Fig. 8. Salivary glands of Bhodnius prol ixus .

principal gland is present. In the former there is a faint traceof a division into anterior and posterior parts, and the principalduct emerges from the gland in this region. In Cimex,

102 B. A. BAPTIST

10 b

TEXT-FIGS. 9-11.Kg. 9. Salivary glands of Cimex lec tu la r ius .Fig. 10a. Salivary glands of Lygus pra tens i s .Fig. 10b. Nervous plexus on principal gland of Lygus pra tensia .Kg. 11. Salivary glands of Monanthia cardui . .

SALIVARY GLANDS OF HEMIPTERA 103

however, there is no trace of a division into lobes, and theprincipal duct leaves the gland at its anterior extremity, thegland apparently being primitively unilobed.

The accessory gland (ag.) varies from a tubular to a vesicularform. In the Pyrrhocoridae (Text-figs. 18, 20) it takes the formof a duct-like tubule which cannot be distinguished into acces-sory gland and duct. In the Lygaeidae (Text-fig. 15), Berytidae(Text-fig. 17), and Coreidae (Text-fig. 19) a duct-like part canbe distinguished from a slightly thicker terminal glandularpart. In all the other families the accessory gland has the formof a vesicle generally somewhat ovoid in shape. In the Gerridae(Text-fig. 4) it is subdivided to some extent into three lobuleswhich taper off into narrow processes. In all cases exceptE h o d n i u s the accessory duct passes forwards from theaccessory gland into the posterior region of the head whereit then turns back and returns to the thorax to join the principalduct as it emerges from the hilus of the principal gland. InE h o d n i u s (Text-fig. 8) the accessory duct opens at one endinto the principal duct while at the other end it tapers into a finethread to end blindly in the head region.

Both principal and accessory glands and their ducts generallyreceive a tracheal supply. This is derived from a trachealtrunk of the< first spiracular trachea as in N o t o n e c t a ,Nauco r i s , Nepa,and Nabis or from the visceral tracheaesupplying the thoracic part of the gut. In No tonec t a wherethe principal gland is situated in the head, the anterior lobederives its tracheal supply from a branch derived from theventral cephalic trachea. The tracheae supplying the posteriorlobe and accessory glands are, however, derived from the firstspiracular trunk. The glands of Corixa , Cimex, An tho-cor i s , M o n a n t h i a , M e t a c a n t h u s , and Chilacis pos-sess no tracheal supply.

A distinct nervous plexus (np.) which is stained by methyleneblue (used supravitally) is found on the principal gland of allthe insects used. The nerve (n.) which supplies this plexus wastraced in N o t o n e c t a , Nauco r i s , T r i a t o m a , Ehod-n ius , Cor ixa , and D y s d e r c u s , and is derived from thehypocerebral ganglion of the stomatogastric nervous system.

13

TKXT-JTOS. 12-15.Fig. 12. Salivary glands of Anthocoris nemorum.Fig. 18. Salivary glands of Nabia ap te rus .Fig, 14. Salivary glands of Pentatoma rufipes.Fig. 15. Salivary glands of Gastrodes ferrugineua.

SALIVARY GLANDS OF HEMIPTEBA 105

•25M.

-ag

TEXT-EIGS. 16-17.Fig. 16. Salivary glands of Chilacis typhae .Fig. 17. Salivary glands of Metacanthus elegans.

The accessory glands and salivary ducts in no case possessedany trace of a nervous plexus.

106 E. A. BAPTIST

TEXT-FIG. 18.

Salivary glands of Dysdercus howardii .

His to logy .The glandular epithelium proper is of various types -which,

however, form a fairly connected series in their general form

SAMVAKY GLANDS OP HEMIPTEEA 107

and cytology. The type as represented by the principal glandof N o t o n e c t a (Text-figs. 21-5) resembles that of typical

pi-

ll

TEXT-FIGS. 19-20.Fig. 19. Salivary glands of Corizus pa rumpunc ta tus .Kg. 20. Salivary glands of Pyrrhocoris ap te rus .

vertebrate serous glandular tissue. As in other Heteropterathere is only a single layer of glandular cells in each lobe, butthese are columnar in form, their narrower inner ends abuttingon a very narrow central canal (cc.) which is itself lined by

108 B. A. BAPTIST

a separate layer of flattened cells. Each glandular cell possessesusually two nuclei more or less centrally placed. The nucleusis large and irregular in shape with fine extensions into thecytoplasm. It possesses one or two large and irregular nucleoli

—CC

CV

•5mm

TEXT-BIG. 21.

Longitudinal section of principal gland ofNotonecta glauca.

(nu.1), while the chromatin is in the form of minute microsomesdistributed more or less uniformly in the nucleus. The cyto-plasm is traversed by large collecting vacuoles (cv.) containingsecretion. These increase in size towards the inner parts of thecells. Eegular rounded secretion granules (sg.) are concentratedround the borders of the collecting vacuoles and more sparselydistributed in the other parts of the cytoplasm. The cytoplasm

SAUVAKY GLANDS OF HEMIPTEKA 109

23 b

TEXT-FIGS. 22-23 b.Fig. 22. Longitudinal section of resting glandular cell of posterior lobe

of Notonecta glauca.Fig. 23a. Longitudinal section of posterior lobe cell of Notonecta

glauca during secretory activity.Fig. 23b. Longitudinal section of anterior lobe cell of Notonecta

glauca during secretory activity.

110 B. A. BAPTIST'

of the anterior- and posterior-lobe cells differ slightly in texture.In the former (Text-fig. 23 b) it is of a rather frothy nature, beingpermeated by minute vacuoles, whereas in the latter (Text-fig.23a) this uniform vacuolization is absent and the cytoplasm isof a dense nature. In the posterior process (Text-fig. 21, p.) ofthe posterior lobe the cells consist of a laige central vacuole,the protoplasm being confined to a thin parietal layer containinga rounded nucleus. Besides secretion granules, the cytoplasmalso contains fine granular and rod-shaped mitochondria, andcircular and semi-circular Golgi bodies, both of which are uni-formly distributed and bear no special relation to the nucleusor secretion granules.

The gland of N o t o n e c t a when examined at varying inter-vals of time after feeding showed fairly distinct secretory stages.In the gland of an insect which has not fed for several hours(Text-fig. 22) the cytoplasm is densely packed with largesecretion granules and the collecting vacuoles are moderate insize and full of densely staining secretion. In the nucleus thechromatin is rather uniformly distributed and the nucleolusis large and prominent. In the glands of insects taken immedi-ately after the prey has been killed there is no marked differencein the gland cells. In the gland of an insect taken after about15 minutes of feeding (Text-fig. 23 a) the secretion granulesare very much less numerous but the collecting vacuoles arestill full of somewhat dense secretion and have increased some-what in size. In the gland of an insect taken after prolongedfeeding of about \ to 1 hour (Text-fig. 24) the secretiongranules are practically absent, and the cytoplasm is greatlyreduced in quantity at the expense of the collecting vacuoleswhich have increased in size and number but contain only avery thin and lightly staining secretion. In an insect takenabout 2 hours after the cessation of a prolonged feed (Text-fig.25) the cytoplasm has assumed normal proportions and a fewsecretion granules have appeared. In still later stages thegranules increase in number and a heavier secretion appearsin the collecting vacuoles. In the examination of the gland inits various stages of secretory activity it is found that all thecells do not act with the same rapidity on the stimulus of feeding.

SALIVARY GiAHBS OF HBMIPTBEA

24

-nu

Ill

25

5

•05 mm. •05 mm.

•05 mm. •05 mm.

TEXT-EIGS. 24-27.Fig. 24. Longitudinal section of posterior lobe cell of Notonecta

g 1 a u c a after prolonged secretory activity.Fig. 25. Longitudinal section of posterior lobe cell of Notonecta

glauca 2 hours after cessation of secretory activity.Fig. 26. Longitudinal section of wall of accessory gland of Noto-

necta glauca.Fig. 27. Longitudinal section of principal duct of Notonecta

glauca.

In those glands taken in the midst of feeding activity the secre-tion granules of some cells were found to be almost entirelydischarged, while in others they were yet quite numerous.

nu

0 5 mm.

-#- cc

nu•05 mm.

TEXT-PIGS. 28-29.Fig. 28. Longitudinal section of posterior lobe cells of principal

gland of Naucoris cimicoides.Fig. 29. Longitudinal section of posterior lobe lobule of principal

gland of Nepa cinerea.

SALIVARY GLANDS OF HEMIPIEBA 118

Further, this non-synchronous activity is not orderly, since insome glands it is the cells of the anterior part which havedischarged their granules, while in others it is the cells of themiddle or posterior parts.

In Naucor i s (Text-fig. 28) the glandular epithelium is stillmade up of large columnar cells abutting on a very narrowcentral canal which is lined as in N o t o n e e t a by a flattenedepithelium. Each cell, however, contains a single large centralvacuole and only a parietal layer of protoplasm. The nucleusis generally small, oval in shape, and contains one or twonucleoli and chromatin in the form of microsomes. The struc-ture of the gland cell may be said to resemble somewhat thatof the posterior process cells of the posterior lobe of the gland ofNotonec ta . The cytoplasm of the cells is uniform in con-sistency, contain? no trace of secretion granules or of any othermaterial in definite form. In the posterior lobe of Nauco r i s ,which in the male is slightly marked off from the rest of thelobe as a posterior lobule, the central vacuole is extremely largeand the layer of parietal protoplasm very narrow indeed.

In Nepa the glandular epithelium is modified a stagefarther in that each cell becomes rounded off into a relativelyfree lobule opening into a central canal (Text-fig. 29), each lobeof the gland consisting of a series of small rounded lobules. Themost anterior pair of lobules of the posterior lobe, which aresomewhat larger than the others, differ also in having a syncitiallayer of protoplasm containing several nuclei (Text-fig. 30). Inthese as also in other lobules the nuclei are oval or flattened withone or two large nucleoli and chromatinal microsomes, and thecytoplasm contains no secretion granules but frequently hassmall dense masses (rm.) in it.

In Gerr is (Text-fig. 31) each lobe as in Nepa consistsof a number of lobules which, however, open together at ahilus and not into a longitudinal central canal. The walls of allthe lobules are made up of a syncitial layer of protoplasmsimilar to the syncitial lobules of Nepa. The cytoplasm,moreover, contains irregular granular masses disposed in themiddle and outer parts of the epithelium.

In the other Heteroptera the glandular epithelium consistsNO. 329 I

114

nu

•05 mm.

TEXT-FIGS. 30-31.Fig. 30. Longitudinal section of anteriormost lobule of posterior lobe of

Nepa einera.Fig. 31. Longitudinal section of principal gland ofGerr i s lacus t r i s .

of a single layer of narrow glandular cells surrounding a singlelarge central cavity in each lobe, thus giving the latter a vesi-cular form. The glandular cells are simple in structure and arerectangular or cubical in shape. The nucleus is centrally placed

SALIVARY GI/ANBS OF HEMIPTERA 115

and is rounded or oval in shape and always contains chromatinin the form of minute granules or microsomes. The nucleus alsocontains several relatively small more or less regular nucleoli.

•05mm.

•fill

33

-nu

l l

•05 mm.

TEXT-EIGS. 32-33.

Fig. 32. Longitudinal section of cells of principal gland of N a b i sa p t e r u s during secretory activity.

Fig. 33. Longitudinal section of cells of principal gland of N a b i sa p t e r u s after prolonged secretory activity.

In the gland of L y gu s (Text-fig. 34) thenucleoli are remarkablein being exceedingly numerous and having a regular roundedform. The cytoplasm of the glandular epithelium in all theseHeteroptera is of a finely granular nature but does not containsecretion granules as found in Notonec ta . Nevertheless,except in Nabis and the blood-sucking forms, the cytoplasmcontains generally rather larger accumulations (rm.) stainingmore deeply than the surrounding cytoplasm, and distributedchiefly in the basal parts of the cells. These accumulations are

116 B. A. BAPTIST

blackened by prolonged osmification, and are also brought outsharply by postchroming. The internal border of the epitheliumis always of a simple granular nature. Typical mitochondriaare present in the form of irregular rod-like and granular bodiesscattered throughout the cytoplasm.

38Jill

: - • • * & • ' mrWg • ;•!• W •'••-•w •'•'.' • / '•••S'^E " '

TEXT-ETGS. 34-38.Fig. 34. Longitudinal section of cells of principal gland of Lygus pra-

tensis .Fig. 35. Longitudinal section of wall of accessory gland of Lygus pra-

tensis .Fig. 36. Transverse section of principal duct of Lygus p ra tens i s .Fig. 37. Transverse section of accessory duct of Lygus p ra tens i s .Fig. 38. Longitudinal section of cells of principal gland of Corixa geof-

froyi.

In the glands of those insects taken after feeding activity(Text-fig. 42) the dense patches (rm.) of the cytoplasm are nolonger evident and the cytoplasm frequently contains vacuolesinstead. The nuclei are generally found closer to the internalborder with indistinct nuclear membrane. The microsomes are

SALIVARY GLANDS OF HEMIPTEBA 117

more or less clumped together, and the nucleoli less numerousand with reduced staining capacity. In N a b i s during intensesecretory activity (Text-fig. 33), that is after prolonged feeding,large masses of cytoplasm on the internal aspect of the epithe-lium are budded off and apparently transformed into secretion

TEXT-FIGS. 39-40.Fig. 39. Longitudinal section of cells of principal gland of Penta toma

rufipes.Fig. 40. Transverse section of accessory gland of Penta toma rufipes.

in a manner resembling a holocrine method of secretion forma-tion. On the other hand, in R h o d n i u s , as also in T r i a tomaand Cimex, -which takes a few large meals at intervals ofseveral days, there is no difference in the structural appearanceof the glandular cell before and immediately after feeding.

The accessory gland whether tubular or vesicular in formalways consists of a single layer of glandular cells surroundedexternally by a basement membrane. In the vesicular type ofaccessory gland as seen in N o t o n e c t a (Text-fig. 26) as wellas in many others, the glandular epithelium is thin and flattened,consisting of flat polygonal cells with small rounded nuclei andclear uniform cytoplasm, devoid of any secretion granules or

118 B. A. BAPTIST

43

•05mm.

TEXT-FIGS. 41-43.Fig. 41. Longitudinal section of cells of principal gland of

Dysdercushowardi i before secretory activity.Fig. 42. Longitudinal section of cells of principal gland of

Dysdercus howardii after secretory activity.Fig. 43. Transverse section of accessory gland of Dys-

dercus howardii .

•nu

mp

•05 mm. '05 mm.

TEXT-FIGS. 44 A-46.

Fig. 44a. Longitudinal section of cells of anterior lobe ofprincipal gland of Tr ia toma infes tans .

Fig. 44b. Longitudinal section of cells of posterior lobe ofprincipal gland of Triatoma infestans.

Fig. 45. Longitudinal section of accessory gland of Tria-toma infestans.

Fig. 46. Longitudinal section of accessory gland and wallof principal gland ofRhodnius prol ixus .

SALIVARY GLANDS OF HEMIPTERA 119

any special inclusions. The inner margins (&&.) of the cellsexhibit a striated appearance and in some cases, as in theaquatic forms, separate filaments can be distinguished in thestriated margin. In the tubular type of accessory gland asfound in P e n t a t o m a (Text-fig. 40), Dysde rcus (Text-fig.43), and others the cells are cubical or columnar and have a cyto-plasm permeated with large clear vacuoles. The inner borderis strongly thickened but does not present a striated appear-ance.

The walls of the salivary ducts are also made up of flattenedcells varying from cubical and much vacuolated epithelium toa thin flattened epithelium. In Lygus (Text-figs. 86, 37)both types are present in that the principal duct is of a simpleflattened epithelium, while the accessory duct has a thickcubical epithelium ^ith numerous vacuoles. In the principalducts, and frequently also in the accessory ducts, the internalborder is marked by a thickened cuticular lining which is raisedinto spiral taenidia just as in tracheae. The principal duct ofN o t o n e c t a (Text-fig. 27) is peculiar in having instead ofspiral thickenings radially arranged pocket-like bulgings of thelumen into the epithelium giving the duct a cord-like appearancesuperficially.

Sec re t ion .

In all the glands studied the staining reactions of the secretionwere noted. The secretion was always precipitated by theordinary fixatives such as Bouin and Zenker showing the pro-tein nature of the secreted product. The type of precipitateproduced, however, on fixation differed in some cases in theanterior and posterior lobes. In No tonec t a the precipitateof the anterior lobe was thinner and finer than that of theposterior. In Naucor i s no appreciable difference was noted.In Nepa again the secretion of the lobules of the anteriorlobe was frequently precipitated as a reticulum, while that ofthe posterior lobe was precipitated as fine granules. Thereticular form of precipitate was also obtained in the lobulesof the anterior lobe of Ger r i s , and in the anterior lobe of

120 B. A. BAPTIST

P e n t a t o m a . With these exceptions the precipitated secre-tion was always granular and more or less similar in all the lobes.The accessory gland contained no precipitated secretion in anycase with the single exception of a starved specimen of N o t o -n e c t a .

With regard to the chemical nature of the secretion m nocase was it found that the secretion was soluble in fat solvents,or that it was stainable in Sudan III, the specific stain for fattysubstances. In some cases, however, it was found that thesecretion of certain lobes blackened on treatment with osmiumperoxide. This was found in the glands of Nepa where thecontents of the two large anterior lobules were blackened; inGerris where the contents of all the lobules of the anteriorgroup were blackened; the anterior lobe of Gas t rodes andthe anterior lobe of Chi lac is . With vital dyes the secretionwas not specifically stained in any case. The glands of N o t o -nec ta and Naucor i s were also tested after formol fixationwith the xanthoproteic and Millon's tests for proteid substancesas modified for histological work. The secretion in all parts ofthe gland gave a positive and similar reaction.

With respect to staining reactions the secretion generallytook the acid dye in typical acid-basic dye combinations suchas safranin and light green. In a few cases, however, theanterior and posterior lobes stained differently. Thus inP e n t a t o m a , Gas t rodes (anterior and lateral lobes),Dysde rcus , P y r r h o c o r i s , and Cor izus , the anteriorlobe secretion took the basic dye, while that of the posterior lobetook the acid dye. With haemotoxylins, both Delafield's andiron-alum-haemotoxylin the secretion in both anterior andposterior lobes took the acid dye used, namely the eosin andorange G. With Mallory's triple stain no constant stainingreactions were obtained in most cases, the stains, however,reacting generally according to the heaviness of the precipitatedsecretion. Thus a certain amount of differentiation was some-times obtained between anterior and posterior lobes. In a few-cases, No tonec t a , Nepa, the posterior lobe always tookthe fuchsin of the triple stain, while the anterior lobe took theaniline blue.

SALIVARY GLANDS OF HBMIPTEBA 121

Physiological Activi ty.The pH of the principal glands varied from 5-6 to 6-8. In

the cases of Notonecta, Naucoris, Nepa, Corixa,Gerris, Nabis, Triatoma, Lygus, and Pentatoma,the pH of the lobes, anterior and posterior, were tested separ-ately. In all cases the pH was the same in both lobes. The pHof the accessory gland was in all cases found to be 6-8. In somecases the pH of the first part of the midgut was also determined.

With regard to the digestive enzymes the lobes were testedseparately in all the cases where they could be easilyseparated, namely in Notonecta, Nepa, Naucoris,Gerris, Corixa, Salda, Lygus, Triatoma, Penta-

Enzymes of Principal Gland.

II ! :

Insect. -INotonecta glauca .Naucoris cimicoidesNepa cineraGerris lacustrisSalda littoralisCorixa geoffroyiNabis apterusTriatoma infestansRhodnius prolixusCimex lectularius .Lygus pratensisMonanthia cardui .Anthocoris nemorumPentatoma rufipesGastrodes ferrugineus .Chilacis typhaeMetancanthus elegans .Corizus parumpunctatusDysdercus howardiiPyrrhocoris apterus

6-26-26-2S-66-46-46-46-26-26-46-86-66-46-66-86-86-46-46-06-4

+

No glycogenase, maltase, or cellulase.The pH of the anterior part of the gut was in Notonecta 6-6, Nabis

6-6, Lygus 6*4, Penta toma 6-4, Dysdercus 6-4.

122 B. A. BAPTIST

to ma, and D y s d e r c u s . Similar results, however, werealways obtained for both lobes. The enzymes tested for weregenerally all the enzymes concerned with the diet of the parti-cular insect, namely protease, lipase, glycogenase, and invertasein the carnivorous forms and protease, lipase, invertase,maltase, amylase, and cellulase in the vegetarian and mixed-diet species. Either one or two enzymes were always present,except in the case of the vertebrate blood-sucking forms whichcontained no digestive enzymes but an anticoagulating enzymeinstead. Not more than two enzymes were found in anysalivary gland. The following table shows the results of theenzyme analysis and pH of the principal gland. The accessorygland was always tested separately for enzymes but in no casewas any trace of an enzyme found.

Insect.

Triatoma,,ff

Rhodnius,,,,

Cimex

Coagulation

No. of Glands.

2 pr. glands10 pr.10 ace. ,2pr. ,

10 pr.10 pr. ,

(heated to '30 pr. ,30 ace. ,

10° C.)

Tests.

Vol. ofBlood.

0-1 c.c.0-5 ,0-1 ,0-1 ,0-5 ,0-5 ,

0 1 ,0-1 ,

Time forCoagulation.

2 hr. 45 min.50 „

8 „1 „ 45 „

55 „7 „

2 „ 15 „12 „

Time forControl.

15 min.6 ,

11 ,18 ,4 ,5 ,

17 ,11 ,

DISCUSSIONS AND CONCLUSIONS.

Morphology.

The underlying form of the salivary glands of Hemiptera isobviously that of a two-group system consisting of a principalpart and an accessory part, the latter in many cases playinga very subsidiary role. The principal part is fundamentallybilobed, and where it is further subdivided it is usually possibleto assign the subdivisions to anterior and posterior groups withreference to the issuing salivary duct. This has been done byfollowing the openings of the lobules which usually fall into twogroups, but where this has not been possible staining reactions

SALIVARY GLANDS OF HBMIPTEBA 123

of the secretions have suggested the affinities of the lobes. Theaccessory part in the system may be said to be of two maintypes, namely that of a tubular or a vesicular type. The formershows various shades of differentiation from the salivary ducts.In some eases there is no difference at all, and they are identicaleven in size. The essential difference, when there is one, is in thethickness of the epithelium. In the case of the vesicularaccessory gland a striated border to the epithelium is invariablypresent, and the cells remain rather flattened and enclose a largecavity which gives to this part its characteristic vesicular form,whereas in the tubular form the central cavity is small, but thecells are large and contain large collecting vacuoles. The ductof the accessory part into which the tubular accessory glandoften gradually merges is generally characterized as a purelyconducting part, its epithelium being narrow and its cuticularlining being raised into taenidia just as in the duct of theprincipal gland. But, on the other hand, both accessory andprincipal ducts may be thick and glandular, a feature generallyassociated with the tubular type of accessory gland. Theglandular nature of these ducts was first pointed out byBugnion and Popoff (1910) who concluded that the ducts ofthe terrestrial Hemiptera possessed a 'glandular mantle' ascontrasted to a simple pavement-like epithelium of the ductsof aquatic Hemiptera. Now, although this does not apply toall terrestrial forms as generalized by Bugnion and Popoff, itdoes in a large number of cases. The case of Lygus presentsan interesting intermediate in that the principal duct is of asimple flattened epithelium, while the accessory duet has athick glandular form although it leads to a vesicular accessorygland. Bugnion and Popoff also make another erroneousgeneralization in that they conclude that the accessory glandof the terrestrial Hemiptera is of a tubular nature; whereas,according to the species studied in this work and all otherspecies hitherto described, it occurs among the terrestrialHemiptera in fewer families than the vesicular type. Anothercharacteristic and constant feature is the course of the accessoryduct. It always originates at the proximal end of the principalduct; that is, where the latter emerges from the principal gland.

124 B. A. BAPTIST

It then follows a variable course, but always eventually passesanteriorly into the back of the head lying dorsally to the princi-pal duct and laterally to the oesophagus. Here it turns roundand returns to the thorax where it terminates in the accessorygland. Until, however, the development of the glands has beencarefully studied it will not be possible to give a conclusivereason for the course of the accessory duct. It seems fairlyclear, however, that whatever its nature may be, the accessorygland is purely a development of the primary conducting partof the glandular system, and is homologous with the reservoirof the salivary glands of other orders.

The cases of Cimex and E h o d n i u s call for specialmention. The former provides the single exception in theHeteroptera in having a unilobed gland. This condition,occurring as it does in a parasitic group, is possibly a degenerateone produced probably by complete suppression of the anteriorlobe. In E h o d n i u s where there is only a very faint trace ofdivision of the principal gland into anterior and posterior parts,the position of the issuing principal duct indicates that thiscorresponds to the normal division into anterior and posteriorlobes. The accessory gland in E h o d n i u s is, however, peculiarin consisting of a minute vesicle which opens directly into theprincipal gland. The principal duct, a little distance fartheralso gives off another secondary duct (Text-fig. 8, sd.) whichsoon tapers into a blind thread. If the minute vesicle is regardedas homologous with the accessory gland of other Hemiptera, asits position indicates, the morphological value of this secondaryduct in E h o d n i u s cannot be ascertained, unless its develop-ment can throw some light upon it. Mellanby (1936) gives ashort account of the development of the gland in E h o d n i u s ,but does not mention anything about the accessory gland orsecondary duct.

The tracheal supply of the salivary glands in most casescomes from the first visceral tracheae which are generally thelargest of the tracheae supplying the gut. It runs forwardfrom its origin, alongside the gut, and at the level of the salivaryglands supplies them with branches. As a general rule a commontrunk is given off to supply both principal and accessory glands,

SALIVARY GLANDS OF HEMIPTEEA 125

but this is certainly not always the case, even the principalgland being sometimes supplied by several different branchesfrom the gut trachea. In N o t o n e c t a , Nauco r i s , andNepa , however, the supply comes from the first spiraculartrunk, and is a large branch which breaks up to supply bothprincipal and accessory glands. It may also give small branchesto prothoracic structures. Why this is so, apart from the reasonthat the glands in these insects are placed rather more forwards,is difficult to say. Those cases in which no tracheal supply wasnoticed are with the exception of C or ixa minute glands whichapparently receive enough oxygen from the blood to satisfytheir needs without a special tracheal supply. The very richtracheal supply of the accessory vesicles, first pointed out byFaure-Fremiet, is remarkable and led him to suggest that theseaccessory glands have a glandular function. That this is un-doubtedly true is shown also by the full condition of the salivaryvesicles when dissected, although the secretion appears to bealways of a watery nature, enzymes never having beenfound.

A well developed nervous plexus is always present on thesurface of the principal gland. Though in many cases the nerveswhich supply this plexus were not traced owing to the minutesize of the whole system, presumably they must be present.In those cases where they have been followed out they are seento be derived from the hypo-cerebral ganglion of the stomato-gastric nervous system. In their course to the gland they areoften associated with a strand of fatty tissue extending from theregion of the head to the salivary glands which makes themrather difficult to detect. Perhaps the nerve plexus is respon-sible for an acceleration of cellular activity, which seems toresult in the production of a somewhat thinner secretion thanthat ordinarily found stored up in the lumen of the gland. Thisin indicated by the histological changes which are well shownin the gland of N o t o n e c t a , where glands of animals afterprolonged feeding showed bulky collecting vacuoles contain-ing a very slight and sparsely distributed precipitate. It isremarkable that the accessory gland in no case possesses anytrace of a nervous plexus, resembling in this way the salivary

126 B. A. BAPTIST

duets, thus further indicating its origin from the primary con-ducting portion of the salivary system.

Hi s to logy .A membrana propria is very well marked on the principal

gland in some cases, while in others scarcely any trace of itremains. It has not been possible to correlate this with thestructure or function of the glands themselves. When presentit contains nuclei and in some cases muscle-fibres, the latterbeing especially developed at the distal ends of the lobes. Thepresence of muscle-fibres seems to be correlated with the pre-datory habit, being best developed in N o t o n e c t a (Text-fig. 21) and Nab is (Text-fig. 38). These forms may requirea large quantity of saliva at the beginning of a feed for paralys-ing or killing the prey, so that the presence of muscle-fibres inthe sheath of the gland will no doubt help to some extent in theexpelling of secretion from the gland. The absence of a sheathseems also to be correlated in certain cases with the absence orpoor development of a tracheal supply.

This is in line with the view of Dreher (1936) and others thatmembranous envelopes of insect organs are often developed byanastomosis of tracheal end cells.

Keplacement cells of the glandular epithelium may be saidto be of no great significance. There is no trace of them in anyof the aquatic forms, and in the majority of the others theyare very poorly represented. Further, no dividing nuclei havebeen observed.

The glandular epithelium always consists of a single layerof glandular cells disposed round a central lumen, the characterof which marks out certain morphological types of gland.

The type found in N o t o n e c t a represents a typical glan-dular tissue, and is directly comparable to typical vertebrateserous glands. There is, as in other Hemiptera, only a singlelayer of glandular cells, but there they consist of very large cellsfilling up practically the whole gland. Another characteristicfeature is that the central lumen of the gland is itself lined bya special flattened epithelium, and forms a longitudinal collect-ing canal in the middle of the gland. The nucleus is peculiar

SALIVARY GLANDS OF HEMIPTBRA 127

in having a very irregular form with radiating processes extend-ing into the cytoplasm. In the active gland it becomes somewhatreduced in size but does not show any other remarkable change.The cytoplasm possesses typical secretion granules and alsocontains large collecting vacuoles round which the secretiongranules are aggregated. These characteristic collectingvacuoles are no doubt developed as an adaptation to theexceptionally large size of the cell, and also serve the purposeof storing up quite an appreciable quantity of secretion. Itmust be assumed that normally secretion granules are builtup in cytoplasm to the zymogen-granule stage, transformedinto secretion, and transferred to the collecting vacuoles. Whenthe collecting vacuoles are filled, the glandular synthesis pro-ceeds as far as the zymogen-granule stage, and is stored up assuch in the cytoplasm. Mitochondria and Golgi bodies typicalof insect tissue are present but show no relation to the secretiongranules, and thus do not appear to contribute to secretionsynthesis. It might be mentioned here that Parat and Painleve(1924) and Beams and Goldsmith (1930) do not find any evidencethat these bodies play any part in secretion synthesis in thesalivary glands of the larva of Chi ronomus , or in thesalivary glands of the grasshoppers (Beams and King, 1932).Lesperon (1937) comes to the same conclusion in a cytologicalstudy of secretion in the silk glands of the silk-worm and otherinsects.

Viewing the gland of N o t o n e c t a as a whole it appears toshow a very high degree of development as regards its glandularmechanism. Not only has it developed the mechanism of form-ing zymogen-granules and thus producing a large quantity ofsecretion at short notice; but, retaining this, has also acquireda device for storing a supply of ready-formed secretion for im-mediate use by the development of collecting vacuoles.

The glands of the related aquatic forms represent a serieswhich take their origin from that of N o t o n e c t a . What maybe considered the next stage in this series is found in N a u -cor i s . In this case the cytoplasm has become somewhatreduced and the collecting vacuoles have run together so as toform a large central vacuole. The nucleus is relatively small

128 B. A. BAPTIST

and regular in outline, and the cytoplasm has lost the pro-perty of storing up secretion granules, the glandular synthesisproceeding normally to the production of secretion which istransferred to the large central vacuole. The arrangement ofthe cells in the gland is exactly the same as in N o t o n e c t a,except that the cells tend to bulge out and consequently becomevery marked on the surface of the gland.

In the next type found in N e p a there is a transformation ofthe type of cell found in Naucor i s into rounded lobules,which are in the process separated from each other, but stillopen into an axial central canal which is lined by its ownepithelium. The two most anterior lobules of the posteriorlobe, however, are different from the others. Each is a vesiclewhose wall is made up of a syncitial layer of cytoplasm withseveral nuclei, which may be regarded as derived by the fusionof several of the simple lobules. Another characteristic featurein Nepa is the development of an anterior lobe duct whichseems to be an extension of the central canal of the lobe outsidethe gland.

The type found in G e r r i s can be directly derived from thatof Nepa. Here there are two sets of lobules, each meetingat a hilus and not opening into a longitudinal central canal asin the previous types. Furthermore, these lobules have asyncitial epithelium resembling the two anterior vesicles ofNepa, and must have been derived in a manner similar tothose. There is also present an anterior lobe duct just as inNepa.

The remaining type of gland which is found in Corixa andin the terrestrial Heteroptera is the vesicular type, which isfundamentally different from those already described, in that inthis case the gland consists of a narrow glandular epitheliummade up of a single layer of small cells surrounding a largecentral cavity which is kept filled with secretion. In the cellsof these glands the nucleus is normally regular and rounded inform, with a large number of nucleoli in most cases. In certaineases, notably Lygus and Corixa , the nucleoli are regularand rounded in form and strongly reminiscent of secretiongranules of the cytoplasm. The nuclei are also seen, particularly

SALIVARY GLANDS OF HBMIPTBEA 129

in those insects taken soon after feeding, to lie towards or onthe inner border of the cell, and the nuclear contents may oftenbe seen in the process of extrusion into the gland lumen,features which suggest that in these cases there is a directparticipation of the nucleus in the production of the secretionby nucleolar emission in addition to its normal indirect con-trolling influence on the activities of the cytoplasm. Withreference to nucleolar activity in glandular tissue in general, thebulk of evidence from other sources both in Invertebrates andVertebrates is not in its favour. In the silk gland of the silk-worm which is particularly favourable for study of nucleolaractivity, as the nuclei are extremely large and nucleoli exceed-ingly numerous, and also specially relevant for comparison withthe salivary glands, Nakahara (1917) produced convincingevidence for the emission of nucleoli into the cytoplasm, whichthus presumably formed a contribution to the secreted product.Lesperon (1937), however, in an extensive work on the cytologyof the gland was not able to confirm this or find any evidence fornucleolar participation in glandular activity. In the light ofthis evidence it is not reasonable to exclude altogether thepossibility of the features mentioned above in the nuclei of thevesicular salivary glands of Hemiptera being degenerativechanges consequent on glandular activity. With regard to otherchanges of the nucleus Buchmann (1929, 1930) in the salivaryglands of the larvae of Musca and S tomoxys , and Oka(1930) in the salivary glands of Odonata have observed changesin the appearance and position of chromatin in the nucleusduring secretory activity, there being a general tendency ofthe chromatin to clump together in active glandular tissue.From this, however, it is not possible to suggest what part thenucleus plays in the activity of the cell. Similar changes havebeen observed in the hemipterous salivary glands, but not asregular and constant features.

With regard to the cytoplasm a very prominent feature foundin most of the vesicular hemipterous glands is the presence ofsmall dense masses (rm.), distinct from secretion or zymogengranules (sg.), distributed towards the bases or outer parts ofthe cells. They are strongly impregnated by osmium peroxide

NO. 329 K

130 B. A. BAPTIST

and are well preserved and prominent in tissue treated speciallyfor preserving protein-lipoidal elements. They are also generallyabsent in the glands of insects taken after feeding, in whichcases their place is taken by vacuoles. They seem, therefore, torepresent a reserve material stored up in the cytoplasm foruse in glandular synthesis. Typical mitochondria and Golgibodies are present, but do not suggest by their form or distribu-tion any particular activity in glandular synthesis. A striatedborder characteristic of the midgut epithelium, and also foundby Buchmann (1929, 1930) in the salivary glands of Muse aand S t o m o x y s , is never present in the epithelium of theprincipal gland of Hemiptera. The internal border is generallyregular and distinct but is of a granular nature. In active glands,however, it is often broken up. No secretion granules (sg.) arepresent in the cytoplasm. The secretion, on the whole, seems tobe formed by the entire cytoplasm and is not distinguishable inthe intermediate stages preceding its transference into thelumen of the gland. In this respect it resembles the mode ofsecretion of the silk gland as described by Lesperon (1937). Insome cases, however, a breaking up of the cytoplasm and eventhe nucleus in the inner (towards the lumen) part of the cellhas been observed. This is particularly prominent in the glandsof N a b i s. Here even in the normal gland the cytoplasm canbe seen to be transformed into the secretion, and in activeglands parts of the cytoplasm are found to be budded off intothe lumen, a characteristic feature of the holocrine mode ofsecretion.1 In N a b i s, however, the replacement cells are veryfew in number and consequently the glandular epithelium mustpossess a remarkable power of regeneration, being able toreconstruct itself after having lost quite an appreciable portionof its cytoplasm. The glandular mechanism in this as in othercases is therefore strictly speaking of the merocrine type.

Taking the vesicular gland as a whole, it is clear that itsessential characteristic is in the formation and storage of arelatively large quantity of secretion for immediate use, in

1 The controversy over this method of secretion has been summarizedby Wigglesworth (1939), p. 263. I t seems probable that, while in somecases an artefact, holocrine secretion is in others a normal process.

SALIVARY GLANDS OF HEMIPIEEA 1.31

contrast to the typical serous gland where the secretory productis stored in the cytoplasm as zymogen-granules. This featurecauses a great difference in the appearance of the cytoplasm, onwhich feature the glandular epithelium may be separated intodistinct types, one with secretory granules and the other with-out. From the cytological point of view, therefore, the glandularepithelium of the principal salivary glands of Motonectabelong to the first type, while the other Heteropteran principalsalivary glands belong to the second type.

With regard to the accessory glands, it is obvious from therange of forms studied that the accessory vesicle has beenderived from the tubular accessory gland which, as described,gradually merges into the duct-like part of the system. Thechange is brought about by an enlargement of the cells whichacquire a water-secretory function. The cytoplasm remainsclear, and a striated border is developed to the epithelium inthe vesicular type. In some cases mitochondria are prominent.The accessory vesicles, when freshly dissected out, are alwaysfull of a transparent fluid neutral in reaction, which is in allcases devoid of enzymes, and precipitated secretion is neverfound in its lumen. It is generally richly supplied with tracheae.All these features point to the conclusion that it is an activestructure secreting a watery fluid. This, however, does not ruleout the possibility of its acting as a reservoir for the secretionof the principal gland, under special circumstances as shown instarved specimens of N o t o n e c t a . Here, however, the acces-sory vesicle is not separated from the principal gland by anygreat length of duct as it is in the great majority of Heteroptera.In these cases it is clear that it does not and cannot serve asa reservoir as is often supposed. Poisson (1924) suggested thatit may have an excretory function, as he affirmed that in theaquatic forms the accessory gland took up and secreted methy-lene blue which was injected into the body. In this work,however, it has been found that the accessory glands do notreact in this way, and they present no evidence whatever ofperforming an excretory function.

From an evolutionary point of view it seems probable that thevarious types of glandular system as found in the Heteroptera must

132 B. A. BAPTIST

have been derived from a system such as is found in the Aphids,as illustrated by Aphis fabae (Weber, 1928). Here theprincipal gland is composed of massive glandular cells clusteredround the hilus. The cells contain secretion granules and smalland large vacuoles. From this the type found in N o t o n e c t awould be derived by a prolongation of the duct iuto the bodyof the gland forming a central canal into which the glandularcells can open, and a specialization of the cells in size and in theirmethod of glandular synthesis by the production of well-developed secretion granules and well-developed collectingvacuoles. The other principal type, the vesicular, could bederived by a great reduction in the size of the cells, with theconsequent formation of a large space at the hilus round whichthey would come to lie, in the case of each lobe. In the case ofthe multi-lobed gland it could be assumed that the primary lobehad further constricted itself into lobules and thus given riseto several vesicles.

Secre t ion .All the information obtained in this work is strongly against

the popular idea that the various lobes of all Hemipteroussalivary glands produced widely different chemical substances,each with a special function. This has largely been due toFaure-Fremiet's work (1910) on the salivary glands of theaquatic Heteroptera and the remarkable conclusions he drawsfrom his results. He finds that in the Notonectidae, Naucoridae,Nepidae, and Belostomatidiae, the principal gland can bedivided into three parts according to the chemical nature of thesecretion: (1) a rhagiocrine erythrophil gland where the productof secretion is precipitated by alcohol and coloured by acidfuchsin and orange G and picric acid; (2) a rhagiocrine cyanophilgland whose product of secretion is precipitated by alcohol andcoloured by aniline blue and picric acid; (3) a lipocrine glandwhose product is an oily substance of variable nature blackenedby osmium peroxide and soluble in alcohol and fat solvents.Apart from not being able to agree with his conclusions, theresults he claims have been shown to be erroneous. He wasparticularly able to distinguish a lipocrine part in the glandular

SALIVARY GLANDS OF HEMIPTBEA 133

system which produced an oily secretion. In the entire rangeof glands studied no evidence of such a secretion or such asecretory part in the gland was obtained, using the specifictechnique for lipide detection, namely that of Sudan III andSudan black, and preparing sections by the freezing microtome.However, in one of the cases (Xepa) where he claims a lipo-crine substance, it has been found that the substance concerneddoes blacken with osmium peroxide but does not stain withSudan III. It is, moreover, preserved even with such fixativesas alcoholic Bouin, so that undoubtedly it cannot be of lipidenature. Eawat (1939) has repeated Faure-Fremiet's work withrespect to Nauco r i s , and also fails to get any of the resultswhich the latter claims. There are a few cases in the insectsstudied where a blackening has been observed with osmiumperoxide, but in no case did these glands also stain withSudan III. From the blackening by osmium peroxide aloneno conclusions can be drawn except that it probably containssome reducing substance.

Further, the staining reactions which Faure-Fremiet hastaken as a basis for histochemical differentiation, namelyMallory's triple stain, is entirely unsatisfactory for this purpose.The stain itself originally used by Mallory for the differentialstaining of connective tissue consists of a combination techniqueusing acid fuchsin and aniline blue and orange G, three acidstains which are not even capable of differentiating acid andbasic substances, but which in various heterogeneous tissues arecapable of being taken up differently by the various componentsof the material, and has been used successfully for this purposeby numerous workers. In the salivary glands of Hemiptera,however, although the secretion is in some cases precipitated indifferent physical forms, their nature is not sufficiently anddistinctly different in most of these cases to produce a differ-ential staining with Mallory as a constant and distinct feature.In the few cases where such a difference has been noted it isnot possible to make any conclusion from it, except that thephysical nature of the precipitate is different, which is quiteobvious even without staining with Mallory.

The secretions, however, have in all cases also been tested

134 B. A. BAPTIST

with a specific acid-basic combination, namely that of safraninand light green with no mordanting. By this method it hasbeen possible to differentiate the secretion in some cases; butthis difference is, where present, not very strongly marked.Feulgen's chromatin test which is specific for nucleic acid failedto stain the secretion in every case, showing thau nucleic acidderivatives do not enter into the composition of the secretion.The precipitation and preservation of the secretion in all casesby the various fixatives used proves its proteid nature, and thiswas further confirmed by the histochemical application of thexanthoproteic and Millon's reaction in the case of Noto-n e c t a . Also the chromaffine reaction for the detection ofcertain phenolic derivatives of protein metabolism such asadrenalin was applied to N o t o n e c t a and P e n t a t o m a ,but with negative results. From what is known, therefore, ofhistochemical technique it is not possible to differentiate thesecretions produced, apart from demonstrating their proteidnature.

pH and E n z y m e s .The pH of the salivary fluid of the principal gland was found

to be either slightly acid or neutral. In this it agrees with thepH of the salivary fluid as found in insects of other orders.The pH of the fluid in the accessory gland was always found tobe neutral and is further proof of the production of only awatery fluid in the accessory gland.

Enzymes were found without exception in the glands, twoenzymes being the maximum number found in any particulargland. The enzymes were found to be always related to the typeof food consumed, and were those concerned with the digestionof that particular component of the food which was presentin the greatest proportion. In those species with a relativelymixed diet such as P y r r h o c o r i s , D y s d e r c u s , P e n t a -toma , only an invertase was found, indicating perhaps thatthese species are primarily and chiefly vegetable feeders. Inthose cases where two enzymes were found, there was nocorresponding differentiation of the slightest kind in the struc-ture of the glandular epithelium, nor was there any differentia-

SALIVARY GLANDS OF HEMIPTBEA 185

tion in the lobes on this account, both enzymes being found inthe same lobe. This is, of course, in agreement with enzymesproduced by the gut. It is remarkable that the enzyme cellulasewas not found to be present in any case. Both Smith (1920) andDavidson (1923), however, have postulated a cellulase in thesaliva of Capsids and Aphids. According to Smith the salivaof the Capsid bug Lygus p r a t e n s i s produced, as shown inthe sections of the host plant tissue, cell-wall disintegration atthe place of puncture. Such disintegration, however, may notbe necessarily due to the effect of cellulase present in the saliva,but could just as probably follow on disintegration of the sur-rounding tissue caused by the mechanical destruction of adjacentcells, or a breakdown of the cells as a result of some toxicprinciple in the saliva. The same type of evidence has been putforward by Davidson to show the presence of a cellulase in thesaliva of Aphis r u m i c i s . The enzyme tests used in thepresent work were of a qualitative nature, and so it was notpossible to estimate accurately how their activity was affectedby the pH. In testing for the rapidity of action of the enzymesit was generally found that at least half an hour was necessarybefore the first traces of digestion appeared. Comparing thiswith the feeding habits of the animals concerned it seems thatexternal digestion by the saliva cannot be utilized to any greatextent, as the feeding time at any particular spot is generallytoo short for this purpose. It seems, however, certain that quitean appreciable quantity of the injected saliva is imbibed againand salivary digestion continues in the first stomach where thefood taken in is first stored. In those cases where the pH of thisregion of the gut was tested it was found to be similar to that ofthe salivary glands or very near it. Moreover, the range ofaction of the enzymes themselves is quite wide, as far as can bestated from qualitative tests. There is undoubtedly, therefore,a continuation of the salivary enzyme activity in the gut, andit must be of appreciable value in the digestive economy of theinsect. It is not strange that no digestive enzymes are presentin the blood-sucking forms, as a single meal is retained for avery long period in the first part of the gut and only transferredin minute quantities to the second part of the midgut where

136 B. A. BAPTIST

digestion occurs. The presence of anticoagulant enzymes foundin these forms serves the purpose not only of preventingcoagulation from occurring while the blood is being sucked up,but also in keeping it uncoagulated in the first part of the gutwhere it is stored, so that it can be easily transferred in minutequantities to the posterior part of the gut where digestion occurs.

From the physiological point of view the salivary glands ofHemiptera may be said to produce a secretion which is primarilydigestive in function. The mechanical facilitation it affords inthe sucking up of the food, however, must be an equally im-portant function. The toxic nature of the saliva of the predaceousforms seems to be incidental to their being of the nature ofdigestive juices.

SUMMAEY.

The salivary glands of the Heteroptera consist of a pair ofprimarily bilobed principal glands and accessory glands whichvary very greatly in form and structure in different families.

The glands are usually supplied with tracheae, and theprincipal glands are invested by a nervous plexus which issupplied by a glandular nerve from the hypocerebral ganglionof the stomatogastric system.

The principal salivary gland of Notonecta is characterizedby the presence of large cells having zymogen granules andby the storage of fluid secretion in vacuoles. In contrast, mostof the remaining Heteropteran salivary glands belong to thevesicular type, having a one-layered glandular epithelium madeup of small cells which discharge their secretion into a largecentral storage cavity or axial canal. This type of gland lackszymogen granules but has small dense masses of reservematerial in the basal or outer parts of the cells. There isnormally no difference in the structure of the glandularepithelium in the different lobes. The accessory glands areeither in the form of a thin-walled bladder-like vesicle,or are tubular or duct-like; they seem to be purely a develop-ment of the primary conducting glandular system, and arethus homologous with the salivary reservoir of other orders.All the information obtained in this work is strongly againstthe idea that the various lobes of Hemipterous salivary glands

SALIVARY GLANDS OP HEMIPXEBA 137

produce widely different chemical substances, each with aspecial function. The results obtained by Faure-Premiet havenot been confirmed.

Except with blood-sucking forms digestive enzymes werealways found in the glands, two enzymes being the maximumnumber found in any particular gland. The enzymes were foundto be always related to the type of food consumed, and werethose concerned with the digestion of that particular componentof the food which was present in the greatest proportion. Inno case was a cellulase found. An anti-coagulant principle wasfound to be present in the glands of blood-sucking forms.

The accessory glands appear to produce only a waterysecretion, enzymes being absent.

The pH of the principal gland is generally slightly acid, whilethat of the accessory gland is neutral.

Mitochondria and Golgi bodies typical of insect tissue arepresent in certain glands, but show no relation to the secretiongranules, and thus do not appear to contribute to secretionsynthesis.

From a number of experiments it appears that the action ofthe digestive enzymes is not sufficiently rapid for externaldigestion to take place to any great extent. It seems, however,certain that quite an appreciable quantity of the injectedsaliva is imbibed again, and that the salivary digestion con-tinues in the stomach, where the food taken in is first stored.The pH activity range of the enzymes is in general wide.

ACKNOWLEDGEMENTS.

This problem was undertaken at the suggestion of Dr. W. H.Thorpe to whom I am greatly indebted for guidance andadvice. I am also indebted to Dr. A. D. Imms, F.E.S., for hiskind advice and criticism. I am very grateful to Professor J.Stanley Gardiner and Professor James Gray for accommodatingme in the Zoological Laboratory, Cambridge, and to theCeylon Government for a grant during this period. My thanksare also due to Miss L. Poom, Mrs. H. H. Brindley, Dr. G. H.Mansbridge, and Mr. P. C. Hawker for help in procuringmaterial.

138 B. A. BAPTIST

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