On the So-Called Symbiotic Relationship Between Coleopterous Insects and Intracellular Micro-Organisms

On the so-called Symbiotic Relationship between Coleopterous Insects and Intracellular Micro-Organisms. By K. Mansour, Ph.D. (Lond.) (Department of Zoology, The Egyptian. University, Abbassiah, Cairo).

With Plates 17-18.

CONTENTS. PAOK I. INTRODUCTION ...... 255 II. CALANDRA GRANARIA AND CALANDBA ORYZAE . . . 257 III. BABIS GRANXJLIPENNIS ...... 261 IV. ORYZAEPHILUS SUBINAMENSIS . . . . . ' . 262 V. SlTODBEPA PANICBA ...... 262 VI. WOOD-EATING INSECTS ...... 263 1. With Intracellular Micro-organisms in connexion with the Alimentary Canal ...... 264 (a) Some Anobiidae and Cerambycidae . . . 264 (6) Some Curculionidae ...... 265 2. With Intracellular Micro-organisms away from the Ali- mentary Canal ...... 265 (c) Some Bostrychidae and Lyctidae .... 265 VII. DISCUSSION AND CONCLUSION ...... 266 BIBLIOGRAPHY ...... 269

I. INTEODUCTION. RECENTLY a number of investigators have paid a great deal of attention to the study of the intracellular micro-organisms occurring in insects. The coleopterous species so far known to harbour such micro-organisms are given in table I. In all the cases where intracellular micro-organisms occur, the mode of transmission from one generation of the host to the next ensures the infection of all the eggs. This infection takes place at different developmental stages of the egg in the different families. In the Curculionidae it takes place in the oocyte stage (Mansour, 1930), in the Cucujidae it occurs just TABLE I.

Food Material. Intracellular Micro- Author. Family. Species. Larva. Adult. organisms. Breitsprecher (1928) Anobiidae Anobium stria turn, 01. Old fir wood Similar to larva Yeast-like Emobius abietis, F. Felled wood Xestobium rufovillosum ,De. G. Old wood Tripopitys carpini Pine wood Lasioderma Redtenbacheri. Cured tobacco Fungus-like Buchner(1921) Sitodrepa panicea, Thorns. Bread, biscuits, &c. Yeast-like ;; Ernobius mollis, L. Felled wood ;; :; Mansom (1934) Bostrychidao Bostrychoplitcs Zickeli, Mars. Wood rich in starch and sugar Bacteria-like Sinoxylon ceratoniae, L. Ehizopertha dominica, F. Grain, biscuits, &c. ;: ;; Old Pine, &c. Gambetta (1928) Lyctidae Lyctus lincarius „ '> ,, ITeitz (1927) Cerambycidae Rhagium spp. Old stumps of fir, birch, &c. Phytophagous Yeast-like Leptura rubra Similar to Rhagium Fungus-like Spondylis buprestoides Koch (1931) Cucujidae Oryzaephilus surinamensis, L. Stored grain „ Bacteria-like Mansour (1930) Curculionidae Calandra oryzae, h. Similar to larva Hylohius abietis, L. Old pine stumps Young shoots and needles Bans granulipennis Fruit of Citrullus Atrophic Odioporus glabricollis Plantin Same as larva Not studied Khyncolus hgnarius, March. Willow and elm wood

Buchner1 (1928,1930, » Numerous spp. and 1933) 1 This author records the occurrence of mycetomes similar to those of Calandra and Hylobius in 74 spp. of weevils. For the full list, see his contribution of 1933. COLEOPTERA AND MICRO-ORGANISMS 257 before the formation of the shell (Koch, 1931), and in the Bostrychidae the infecting micro-organisms pass into the egg through the micropyle while the egg is in the uterus (Mansour, 1934). In the Anobiidae, on the other hand, the outer cover of the egg during oviposition is smeared with a fluid containing a culture of the micro-organisms, and the actual infection occurs during hatching through the ingestion of a piece of the shell by the issuing larva (Buchner, 1921). All such methods of transmission reveal a close intimacy between the micro-organisms and their respective hosts, and the relation therefore has been referred to as symbiotic by several authors, especially Buchner. In a previous paper (Mansour, 1930) dealing mainly with Calandra oryzae and Hylobius abietis, the supposed symbiotic relation between the intracellular micro-organisms and their respective hosts has been doubted. Since then new facts have come to our knowledge, and in this work it is proposed to deal with these facts with the object of denning the biological meaning of this particular association.

II. CALANDRA GRANARIA AND CALANDRA ORYZAE. These two species are pests of stored grain. They are almost identical in their life-cycle and bionomics. Systematically also they are very closely related. In Calandra oryzae (Mansour, 1927) a big accessory cell- mass has been described in detail. A similar cell-mass has also been referred to in Calandra granaria. In a more recent study (Mansour, 1930) the cells of the accessory mass of Calandra oryzae have been found to harbour numerous bacillus-like micro-organisms. During metamorphosis the bacterial cell-mass (accessory cell-mass) fuses with the ventral surface of the posterior end of the stomodaeum and ultimately surrounds it. This end, together with the bacterial cell-mass which surrounds it, grows backwards to form the definitive mesenteron of the adult, and thus the bacterial cells are spread around the developing epithelium of the mid-gut. Later on they become mainly localized around the epithelium of the mesenteric caeca. During the adult life the micro-organisms 258 K. MANSOUR grow and multiply very actively and pass from their harbouring cells into the lumen of the gut, where they mix with food, and later on pass to the outside. During this period of activity the micro-organisms invade the originally non-harbouring epithelial cells and multiply in a similar fashion. Calandra granaria, as mentioned above, also possesses an accessory cell-mass which behaves similarly during metamorphosis. This similarity in behaviour of the mass, and the close relation of the two species, led the author to infer that the cells of the accessory mass of Calandra granaria also harbour bacteria (Mansour, 1930). Buchner (1930 and 1983) and Scheinert (1933) also come to a similar conclusion. Thorough investigation of Calandra granaria, however, has shown definitely that the accessory cell-mass, though similar in origin and behaviour during metamorphosis to the corresponding mass of Calandra oryzae, is quite free from any micro-organisms. A comparative study of the two species of Calandra referred to above revealed the following facts: 1. No intracellular micro-organisms are present in Calan- dra granaria. Thorough search of smears and sections always gave negative results. 2. The accessory cell-mass of granaria is paired and is much smaller in size than that of oryzae. This is easily seen by comparing figs. 1 and 3, PL 17, of granaria with figs. 2 and 4, PI. 17, of almost corresponding stages of oryzae. 8. The few accessory cells at the anterior end of the developing mesenteron of granaria (fig. 1, ace. c, PI. 17) later on become localized around the epithelium of the first circle of mesenteric caeca. All the other caeca are quite free from such cells (compare fig. 5, PI. 17, of granaria with fig. 6, PI. 17, of a corresponding caecum of Calandra oryzae). 4. Unlike the tips of the ovarioles of Calandra oryzae (fig. 7, PI. 18), those of granaria (fig. 8, PI. 18) are quite free from micro-organisms. Cells similar to those of the accessory mass are present at the tips of the ovarioles but do not harbour any micro-organisms. This last observation receives confirma- tion from the work of Krautwig (1930) on the genital organs of Calandra granaria. This author describes thoroughly the COLEOPTERA AND MICRO-ORGANISMS 259 $ genitalia of this species and gives an illustration of a section through the tip of an ovariole without mentioning anything about the occurrence of micro-organisms. Also the work of Inkmann (1988) on the early embryonic development of Calandra granaria lends support to this observation. This author, like Krautwig, does not mention anything about the presence of micro-organisms in any of the cells of the embryo or in between the yolk globules, as would be the case had the tips of the ovarioles been serving as an infection centre as in Calandra oryzae. Masses similar to that of Calandra have been described in Hylobius abietis, Odioporus glabricollus, and Baris granulipennis (Mansour, 1927 and 1930), Otior- rhynchus inflatus, Pissodes notatus, Cryptor- rhynchus lapathi, Cionus sp., Sibina pellucens, Protapion aeneum, and many other weevils (Buchner, 1928, 1930, and 1933, and Scheinert, 1933). A study of An- thonomues pomorum, Anthonomues grandis, Hy- pera variabilis, Cionus sp., and Balaninus nucum, has proved the absence of any masses homologous with the one under consideration. It follows, therefore, that the accessory cell-mass is a specific feature of some Curculionidae. Probably it has been induced to appear through the presence of a micro- organism infection. In the case of Calandra granaria, however, presumably through agencies quite unknown to us (but having nothing to do with the eating habits) the micro- organisms have totally disappeared. The formerly micro- organism-harbouring cells, on the other hand, were left behind quite free from any infection. Compared with those of Calan- dra oryzae they are smaller in size and much less in number. Consequently the mass in the full-grown larva is much smaller. Biologically and systematically the two species of Calandra are very closely related. The fact that one harbours micro- organisms and the other not, in spite of the presence of the accessory cell-mass, leads to the conclusion that such micro- organisms are of no importance in the Life of their host. The absence of bacteria in the case of Calandra granaria without any apparent hindrance to the weevil lends support 260 K. MANSOUR to the view that the micro-organisms of Calandra oryzae are not symbiotic in the strict meaning of the term. In this connexion also, the recent work of Margaret Lilien- stern (1932) on the intracellular micro-organisms of ants is very illuminating. This author studied the mycetomes of Formica f u s c a, and followed the transmission from one generation to the next. In the embryo the harbouring cells lie ventrally at the posterior end of the egg and are continuous with the blastoderm. During gastrulation the infected cells become better differen- tiated from the rest and sink inwards independently of the sink- ing in of the walls of the gastral groove. They then form a separate mass whose cells are full of bacteria-like organisms. In the larval and imaginal stages of all castes this mass lies ventrally to the junction of the mesenteron and the proctodaeum. During the egg-laying period of the queen the micro- organisms break loose from their harbouring cells and invade the ovarioles, and hence the growing oocytes. In the queenless colonies, however, this author found that the workers generally lay eggs which develop normally if spared the voracity of the other members of the colony. In spite of the presence of mycetomes full of micro-organisms in these parthenogenetic workers, their progeny is always free from the bacteria-like intracellular micro-organisms or very poorly infected. In the development of these bacteria-free individuals a cell-mass, similar in all respects to the harbouring mycetome, appears and follows a similar course. This author also records the remarkable fact that in Formica fusca var. glebaria, cells, corresponding to those of the mycetome of the ordinary fusca and following a similar course, appear in the embryo, but have never been found to harbour micro-organisms. In the related species, Formica rufa and Formica san- guine a, similar mycetomes also appear, and remain throughout life quite distinct but free from micro-organisms. This last point has been overlooked by Buchner (1921) in his description of these species. The occurrence of these evanescent and empty mycetomes can only give support to the view that the intracellular micro- organisms are not as vital to their host as many authors assume. COLEOPTERA AND MICRO-ORGANISMS 261

III. BARIS GRANULIPENNIS. This is a desert Curculionid which breeds in the fruits of Citrullus colocynthis. It possesses an accessory cell- mass which harbours intracellular micro-organisms. The behaviour of the mass in the larval period and during metamorphosis is similar to what has been described in the case of Calandra oryzae. The mode of transmission is also pre- cisely similar. Both in the larvae and pupae it is comparatively easy to demonstrate the presence of the micro-organisms either in smears (fig. 9, PL 18) or sections. In the adult the matter is somewhat different. The micro-organisms could only be detected in the very young adults, i.e. fairly soon after emer- gence (fig. 10, PI. 18). During this period they pass in great quantities from their harbouring cells into the lumen of the alimentary canal in a way exactly similar to that described in Calandra oryzae. In the more advanced adults, however, the alimentary tract is quite free from any infection (fig. 11, PI. 18). The cells which formerly harboured the micro- organisms remain distinguishable from the rest (fig. 11, defl. ace. c, PI. 18) but have no traces of the micro-organisms whatsoever. During this bacteria-free period (save for the ovarioles), which lasts till the end of the life of the imago (a few months), the processes of copulation and oviposition take place. It has been assumed by several authors that intracellular micro-organisms, when present in such close intimacy to the alimentary canal, render digestive services to their host and help greatly in the preparation of its food for assimilation. The micro-organisms in the young adults of the insect under consideration pass in great quantities into the lumen of the alimentary canal. This insect, however, is known not to take any food at all during its imaginal life. This fact at once ex- cludes that assumed role for these intestinal intracellular micro-organisms. 262 K. MANSOUR

IV. OEYZAEPHILUS SUEINAMENSIS. Pierantoni (1930) and Koch (1931) have described the mycetomes of this species. Also the mode of transmission has been studied thoroughly by the latter author. The mycetomes are four in number and are situated two on each side at the posterior end near the junction of the mesenteron and the proctodaeum. Throughout life the mycetomes remain quite free from the alimentary canal, and seem to have no direct connexion with any other system. The micro-organisms here have been referred to as symbionts though without any known service to the host. Koch, however, mentions that during his work on this species he has been able to get beetles with mycetomes quite free from micro-organisms simply by a change of the food material. This result, if confirmed, is quite sufficient to preclude the possibility of any symbiotic relationship between Oryzaephilus and its intracellular micro-organisms.

V. SlTODEBPA PANICEA. It has been long known that Sitodrepa panicea has mycetocytes scattered between the epithelial cells of the four mesenteric pouches at the anterior end of the mid-gut in larval and adult stages (Karawaiew, 1899; Escherich, 1900; and Buchner, 1921). The mode of transmission of the yeast-like micro-organisms in these mycetocytes has been elucidated by the latter author and confirmed by Breitsprecher (1928). The importance of these micro-organisms to the host has been quite obscure right from the date of their discovery. Karawaiew originally referred to them as parasitic but, soon after, this view has given way to another postulating a symbiotic relation between these yeast-cells and their host. This latter view is based on the intimacy between these micro-organisms and their host, and also on their relation to the alimentary canal. The benefit the host is supposed to draw from the micro-organisms is therefore believed to be digestive. Becently Koch (1933) has procured experimental evidence which in his opinion proves a definite symbiotic relation between the beetle and the yeast. The yeast-cells are supposed by this author to be utilized by COLEOPTERA AND MICRO-ORGANISMS 263 the host as a source of vitamins. He claims to have succeeded in procuring yeast-free larvae by treating the eggs with a 0-5- 2'0 per cent, chloramin solution and extracting the embryo from the shell when it is about fully developed. The yeast-free larvae were much smaller in size; the comparison here being made between an operated-upon larva and a perfectly normal one. Probably the difference in size might have been due mainly to the sterilizing operation and the untimely extraction from the egg. In another experiment Koch kept a yeast-free larva on pea-cake and dried yeast. This latter substance was in- tended to replace the checked intracellular yeast. As a control he used two larvae, one yeast-free and the other quite normal, both being kept on pea-cake only. The compensated yeast-free larva was found to be almost of normal size. He therefore concludes from this experiment that the vital substance con- tributed by the intracellular yeast must be equal in its effect to the vital substance in the dry yeast. In view of the experiments of Abderhalden (1919) it would have been more con- vincing if Koch had added to the controls a normal larva fed on pea-cake and dry yeast. This would have given us perhaps an idea of the effect of the dried yeast irrespective of the presence of the intracellular micro-organisms. Abderhalden fed the larvae of Deilephila euphorbiae on leaves sprayed with yeast extract. This had a marked effect on the size of the larvae and the issuing moths were very big in size. To arrive at a definite conclusion from the experiments of Koch, therefore, seems to us very early, and we must wait for more data and a more thorough investigation of the problem.

VI. WOOD-EATING INSECTS. Intracellular micro-organisms occurring in wood-eating insects have been assigned an important role in the nutrition of their hosts. Buchner (1928) assumes that in return for what they receive the micro-organisms break down cellulose and thus make it possible for the host to obtain the carbohydrates necessary for its growth. The cases known so far among these insects can be grouped as follows: NO. 306 T 264 K. MANSOUR 1. With intracellular micro-organisms in connexion with the alimentary canal. (a) Some Anobiidae and some Cerambycidae. (b) Some Curculionidae. 2. With intracellular micro-organisms away from the alimentary canal. (c) Some Lyctidae and Bostrychidae. (a) Anobiidae and Cerambycidae. Breitsprecher (1928) described yeast-cells similar to those of Sitodrepa in Ernobius mollis, Anobium striatum, Xestobium rufovillosum, Oligomerus brunneus, and Tripopitys carpini. Buchner also described similar micro-organisms from Ernobius abietis. Heitz (1927) succeeded in cultivating the yeast-like organisms of Ernobius abietis on artificial media, but failed to show any splitting of the cellulose through the activity of these micro-organisms. Among the Cerambycidae, intracellular yeast-like and fungus-like micro-organisms are known to exist in E h a g i u m inquisitor, Eh. bifaciatum and Leptura rubra respectively (Heitz, 1927). As in the case of the Anobiidae, this author has cultivated these micro-organisms on artificial media but failed again to demonstrate any splitting of the cellulose. Notwithstanding the results of Heitz, Buchner (1928) cites the occurrence of these fungus- and yeast-like organisms in Ceram- bycidae and Anobiidae in support of his hypothesis of symbiosis. Uvarov (1929), in his summary of the literature on 'Insect Nutrition and Metabolism', also favours the view of Buchner and stresses the importance of such organisms in the nutrition of wood-eating insects. Eipper (1930), from an investigation on the fate of cellulose in some Anobiid and Cerambycid wood-eating insects, comes to a totally different conclusion. This author has succeeded in demonstrating clearly the presence of cellulase in micro- organism-free Cerambycid larvae closely related in all respects to those species with intracellular micro-organisms which also possess a similar cellulase. Moreover, Eipper points out that COLEOPTERA AND MICRO-ORGANISMS 265 no yeast is known yet to cause a breaking down of cellulose. In a very recent work by Mansour and Mansour-Bek (1934), some aspects of the digestive processes in two Cerambycid species (Xystrocera globosa and Macrotoma palmata) have been studied. These two species are quite free from intracellular micro-organisms. Also the examination of the lumen of the gut for extra-cellular micro-organisms gave negative results. Xystrocera proved to possess no cellulase, while Macro- t o m a showed the presence of a very strong one. A comparison between food materials of the two species helped a great deal in comprehending this result. Macrotoma lives on the dry wood of Morus, which by analysis was found to contain only 0-48 per cent, of soluble sugars and starch. Xystrocera, on the other hand, lives on the sapwood of Albizzialebbek, which contains 10-5 per cent, of these substances. In the first case the presence of a cellulase enables the larva to feed on wood quite poor in ordinary nutritive substances, while in the second the insect larva can derive enough carbo-hydrates without the utilization of cellulose. In view of the results of Heitz, Eipper, and Mansour and Mansour-Bek, it must be concluded that the role assigned to the intracellular micro-organisms of the Anobiids and Cerambycids in breaking down cellulose is unjustifiable. (b) Curculionidae. Intracellular micro-organisms are known to occur in a number of Curculionids with wood-eating habits (see Table I). The case of Hylobius abietis has been previously considered by the author (Mansour, 1930). It was then concluded that the supposed symbiotic relation between Hylobius and its bacteria is quite unjustified. The recent study on some Curculionidae (p. 257), though without wood-eating habits, augments this doubt and indicates that it is highly improbable that wood- feeding Curculionids derive any benefit whatsoever from their intracellular micro-organisms. (c) BostrychidaeandLyctidae. In a recent work on the Bostrychidae mycetomes have been described in Bostrychoplites Zickeli, Sinoxylon 266 K. MANSOUR ceratoniae, and Rhizopertha dominica Mansour (1934). The two first species have similar wood-eating habits, the third is a household pest living in grain, biscuits, &c. The mycetomes of these species remain quite separate from the alimentary canal throughout life. Gambetta (1928) also referred to similar mycetomes in some Lyctidae. In these two wood-eating families, the presence of mycetomes without direct communication with the alimentary tract indicates the lack of relation between the wood-eating habit and the occurrence of intracellular micro-organisms.

VII. DISCUSSION AND CONCLUSION. Intracellular micro-organisms have been always referred to as symbiotic by Buchner and his school. The host is supposed to derive vital help from the small organisms it harbours. Such help, especially in the case of insects with wood-eating habits, has been supposed to be digestive. (The micro-organisms in return for what they receive are thought to break down cellulose into simpler carbohydrates which are then available to the host.) This view is based on the following assumptions: 1. The close intimacy between the micro-organisms and the host as revealed by the elaborate and sure methods of transmission. 2. The relation of the intracellular micro-organisms to the alimentary canal. 3. The assumption that wood is exceedingly low in nutritive content. 4. The assumption that wood-eating insects are unable to secrete cellulase. With regard to the first two points it must be admitted that the association of wood-eating insects and intracellular micro- organisms is certainly remarkable. On the other hand, it must be pointed out that such association is far from being regular or confined to wood-eating insects. Similar associations are known to occur in insects with totally different feeding habits, COLEOPTERA AND MICRO-ORGANISMS 267 e.g. Calandra oryzae, where the food is the contents of grain. Moreover, it has been pointed out previously that among the wood-eating Curculionids, like Hylobius abietis, the micro-organisms pass into the alimentary canal only during the adult stage when the insect is not feeding on wood. In the cases among the Bostrychidae and Lyctidae, the micro-organisms are away from the alimentary tract throughout life. In the Ano- biidae and Cerambycidae where yeast- and fungus-like intracellular micro-organisms occur, the work of Heitz (1927) gives us direct evidence as to the disability of these micro-organisms to break down cellulose. The third and fourth points are much easier to decide. Analysis of a number of kinds of wood showed that some have a comparatively high content of soluble sugars and starch (Mansour and Mansour-Bek, 1934). The available substances in the ingested wood in this case are certainly much more abundant than in the material from which some Holothurians and earth- worms derive their nutrition. In Holothurians the available food in the ingested material is only about 0-25 per cent. (Oomen, 1926). On the other hand, other kinds of wood were found to be really quite poor in such substances. The presence of cellulase in some wood-feeding insects without any micro-organisms is now well established. Among Ceram- bycids it has been demonstrated in Cerambyx cerdo (Ripper, 1930), Hylotrupes bajulus (Falck, 1930), and Macrotoma palmata (Mansour and Mansour-Bek, 1934). It is also worth mentioning that our search of Xystrocera g 1 o b o s a, another Cerambycid without micro-organisms, gave negative results. Therefore, contrary to the views of Buchner, we must em- phasize the facts that there are kinds of wood with a high nutritive content, and also that there are wood-eating insects (without micro-organisms) which are able to secrete a cellulose splitting enzyme. The two last assumptions therefore can no longer be quoted in support of the hypothesis of symbiosis between wood-eating insects and intracellular micro-organisms. Also our study of the physiology of Xystrocera globosa and Macrotoma 268 K. MANSOUR palmata lends no corroboration to this view of symbiosis. The larvae of these two species live on sound wood, and are quite free from intracellular micro-organisms or any organisms living freely in the lumen of the alimentary tract. Therefore superficially these two larvae are quite similar in their feeding habits. The larva of Xystrocera, however, is devoid of cellulase, and apparently derives its necessary carbohydrates from the soluble sugars and starch content of the sap-wood of Albizzia lebbek on which it feeds (10-5 percent.). The larva of Macrotoma, on the other hand, secretes a cellulase. The wood of Morus on which it lives is very poor in soluble sugars and starch (0-48 per cent.). It is not unreasonable therefore to infer that this larva, in contrast with that of Xystrocera, derives its necessary carbohydrate nutriment from the cellulose of the wood through the activity of its enzyme secretion. In view of this comparative physiological study it must be concluded that the intracellular micro-organisms are of no importance in the nutrition of insects with cellulose-feeding habits. Insects not feeding on wood and harbouring intracellular micro-organisms have been referred to above, and the idea of symbiosis between the micro-organisms and their host in such cases is even less tenable. It has been often surmised that the intracellular micro- organisms of insects are able to assimilate atmospheric nitrogen (Heitz, 1927, and Cleveland, 1925), or represent a source of vitamins (Uvarov, 1929, and Koch, 1933), thus supplementing the deficiency of these substances in the food. Such conjectures come mainly from our incomplete knowledge of the nutrition of insects. They cannot at this stage, however, be accepted as a vindication of the idea of symbiosis in its strict meaning. The case of Calandra granaria as compared with that of Calandra oryzae points clearly to the non-importance of the intracellular micro-organisms in the life of their insect hosts. Also the case of Baris granulipennis points to a similar conclusion. The intracellular micro-organisms in insects are probably more correctly referred to as commensals, or parasites causing COLEOPTERCOLEOPTERA AAN AND DMICRO-ORGANISM MICRO-ORGANISMS S 26269 9 COLEOPTERA AND MICRO-ORGANISMS 269 non oconceivabl conceivable hare harm mto tothei their hostr host. .Probably Probably, a,s aEippes Eipper (1930r (1930) ) hanhas opointe s conceivablpointed outd out,e the ,har theym ary earto analagoue thei analagour hosts wits. withProbably thh eth gall-formine gall-formin, as Eippeg insectgr insect(1930s )s anhaand sdthei pointetheir hosr hosdt outplantst plants, the. y. are analagous with the gall-forming insects and their host plants. I desirI desire teo toexpres express ms ym besy best thankt thanks tso tProfessoo Professor Jr. JW. W. Munr. Munro o forfo kindlrI kindldesiry eyallowin tallowino expresg mg sem mteo y t ofinisbes finisth thankhthi this wors tworok Professo ka t atht eth Laboratoriere JLaboratorie. W. Munrs os of foothfr ethkindl eDepartmen Departmeny allowint got fm oEntomologyfe Entomologyto finish thi, Eoya,s Eoyaworl kCollegl aCollegt thee oeLaboratorief oSciencef Science, s, LondonoLondonf the, S.WDepartmen, S.W. 7. . 7. t of Entomology, Eoyal College of Science, London, S.W. 7. BIBLIOGRAPHYBIBLIOGRAPHY. . AbderhaldenAbderhalden, E, . E(1919).—"Studie. (1919).—"StudieBIBLIOGRAPHYn iin. iid. dvo. nvo einzelnen . einzelnen Organen Organen hervorgen hervorge- - brachteAbderhaldenbrachten Substanzen Substanze, E. (1919).—"Studien min mit spezifischet spezifischen rii .Wirkun rd .Wirkun von geinzelne (IIg (II. 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Morphn VII. u.. . uDiOekol. eOekol symbion. de. rde-r Tiere'tischeTiere', 26n, .26Einricht. . der Rtisselkafer", 'Zeitsohr. f. Morph. u. Oekol. der ClevelandClevelandTiere', , L26, . L.R. . R(1925).—"Th. (1925).—"The abilite ability oyf otermitef termites tso tlivo live perhape perhaps indes inde- - finitelyClevelandfinitely on , oaLn die. a Rdiet. o(1925).—"Thtf opurf pure cellulose"e cellulose"e abilit, 'Biol, y'Biol o. fBull.' .termite Bull.', 48,s .48 to. live perhaps inde- CrozierCrozierfinitely, W, .W oJn. . aJ(1918).—"Th . die(1918).—"Tht of pure ecellulose" eamoun amount, 'Biolot f obottof . bottoBull.'m m,materia 48materia. l ingestel ingested db yby Holothurians"CrozierHolothurians", W. J., ' (1918).—"ThJourn, ' Journ. Experimenta. Experimentae amounl Zool.'tl Zool.'of ,botto 26, .26m. material ingested by EscherichEscherichHolothurians", R, . R(1900).—"U. (1900).—"U, ' Journ.. Experimentad.. dregelmassig. regelmassigl eZool.' Vorkommee Vorkomme, 26. n von nvo Sprosspilzen Sprosspilzen n iEscherichn iden mde mDarmepithe , DarmepitheR. (1900).—"Ul einel eines Kafers".s dKafers". regelmassig, 'Biol, 'Biol. eZentralbl.' . VorkommeZentralbl.', 20n, .20vo.n Sprosspilzen GambettaGambettain dem, ,LDarmepithe . L(1928).—"Ricerch. (1928).—"Ricerchl eines Kafers"e esull ,sull a'Biol asimbios .simbios Zentralbl.'i ereditarii ereditari, 20.a ad i dialcun alcuni i coleotterGambettacoleotteri ,silofagi" i Lsilofagi". (1928).—"Ricerch, 'Ric, 'Ric. Morf. Morf. Biol. eBiol . Animsull. Anima . simbiosNapoli'. Napoli'i, volereditari, vol. 1. 1.a di alcuni HeitzHeitzcoleotter, ,E . E.(1927).—"Ube i (1927).—"Ubesilofagi", 'Ricr .rintrazellular Morfintrazellular. Biol. eAnim eSymbios Symbios. Napoli'e ebe , volibe i.holzfressende 1holzfressende. n n HeitzKaferlarvenKaferlarven, E. ,(1927).—"Ube I", ,I" 'Zeitschr, 'Zeitschr. rf .. intrazellularMorphf. Morph. u.. uOekol.e OekolSymbios. d. dTiere'. Tiere'e ,be Bdi, Bd. holzfressende7.. 7. n InkmannInkmannKaferlarven, Ferd, Ferd. ,(1933).—'Beitr .I" (1933).—'Beitr, 'Zeitschr. .f .z .Morph Entwicklungagz. Entwicklungag. u. Oekol.. dde. sTiere'de Kornkafers.s Kornkafers., Bd. 7.' ' KarawaiewInkmannKarawaiew,, FerdW, .W .(1899).—"Anatomi. (1933).—'Beitr(1899).—"Anatomi. z.e Entwicklungageu . uMetamorphos. Metamorphos. dees Kornkafers.ede sde Darmkanals Darmkanal' s s deKarawaiewrde Larvr Larve vo,e Wnvo .Anobiun (1899).—"AnatomiAnobium mpaniceum" paniceum"e, 'Biolu, . 'BiolMetamorphos. Zentralbl.'. Zentralbl.',e 19,de .19s .Darmkanals KochKochde, rA , Larv. A(1931).—"Symbios. (1931).—"Symbiose von Anobium epaniceum" voe nvo nOryzaephilu Oryzaephilu, 'Biol. s Zentralbl.'surinamensis surinamensi, 19s .Ls . L(Cucu. (Cucu- - jidaeKochjidae,, Coleoptera)"A, Coleoptera)". (1931).—"Symbios, 'Zeitschr, 'Zeitschr.e f..vo Morphf.n Morph Oryzaephilu. un. und Oekold Oekols surinamensi. d. dTiere'. 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Mansour, K. (1927).—"Development of the larval and adult mid-gut of Calandra oryzae L.", 'Quart. Journ. Micr. Sci.', 71. (1930).—"Prelim. Studies on the Bacterial cell-mass of Calandra oryzae (Linn.). The Rice-Weevil", ibid., vol. 73, part. 3. Pierantoni, U. (1930).—"Origine e sviluppo degli organi symbiotici di Oryzaephilus surinamensis L.", 'Atti della R. Accademia delle Science fisiche e matematiche di Napoli', vol. 18, Serie 2 a, no. 4. Ripper, Walter (1930).—"Zur Frage des Celluloseabbaus bei der Holz- verdauung xylophager Insektenlarven", 'Zeitschr. f. vergl. Physio- logie', Bd. 13. Scheinert, Willi (1933).—"Symbiose u. Embryonalentwicklung bei Riissel- kafern", 'Zeitschr. f. Morph. u. Oekol. der Tiere', Bd. 27. Uvarov, B. P. (1929).—"Insect Nutrition and Metabolism", 'Trans. Ento. Soc. London', vol. 76.

EXPLANATION OF PLATES 17 AND 18. All figures are from camera lucida drawings.

LIST OF REFERENCE LETTERS. ace. c, accessory cell; ace. cm,., accessory cell-mass; b., bacteria; 6. ace. c, bacterial accessory cell; co. me. ca., core of mesenteric caecum; defl. ace. c, deflorated accessory cell; d. mg. ep., developing mid-gut epithelium; fg., fore-gut; mg. ep., mid-gut epithelium; mus. w., muscle-wall; n. b. c, nucleus of bacterial cell. PLATE 17. All figures are from preparations fixed with Carl's fluid and stained with Delafield's haematoxylin. Fig. 1.—Portion of a sagittal section through an advanced pupa of Calandra granaria showing the few accessory cells (ace. c.) free from micro-organisms and situated at the ventral surface of the anterior region of the developing mesenteron. x 150. Fig. 2.—Portion of a sagittal section through an early pupa of Calan- dra oryzae showing the accessory cells full of micro-organisms and surrounding the developing mid-gut of the adult. X 150. Fig. 3.—Portion of a transverse section through an early pupa of Calandra oryzae at the junction of the stomodaeum with the mesenteron showing the accessory cells full of micro-organisms and surrounding the developing mid-gut of the adult. X 150. Fig. 4.—Portion of a transverse section through a prepupa of Calandra granaria showing the fusion of the paired accessory mass with the tip of the stomodaeum. x 150. Fig. 5.—Section through a mesenteric caecum of an adult Calandra granaria (corresponding in level to that of the caecum of Calandra oryzae shown in fig. 6) free altogether from accessory cells. X 210. COLEOPTERA AND MICRO-ORGANISMS 271

Fig. 6.—Section through a mesenteric caecum from the middle region of the mesenteron of an adult Calandra oryzae showing the presence of a large number of bacterial accessory cells (b. ace. m.). X 210.

PLATE 18. All figures are from preparations fixed with Schaudinn's fluid and stained with Giemsa. Fig. 7.—Section through the anterior tip of an ovarioleof an egg-laying Calandra oryzae showing the presence of numerous micro-organisms set free from the bacterial cells in this region, x 650. Fig. 8.—Section through the anterior tip of an ovariole of an egg-laying Calandra granaria showing cells similar in nature to those of the accessory mass of this species and similarly free from micro-organisms. X 650. Fig. 9.—Portion of a smear from the mesenteric region of an advanced pupa of Baris granulipennis showing the thread-like micro-organisms. X 650. Fig. 10.—Portion of a section through the mesenteron of a young adult of Baris showing the intracellular micro-organisms in the mycetocytes. X 320. Fig. 11.—Portion of a section through the mesenteron of an aged adult of Baris showing the absence of intracellular micro-organisms and a deflorated mycetocyte {defl ace c). x 320. Quart. Journ. Micr. Sci. Vol. 77, N. S., PI, 17

d mg ep

d mg ep Quart. Journ. Micr. Sci. Vol. 77, N. S., PI. 18

n b c

b a cc c

K. Mansour, del.