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. ;: ;; Gambetta (1928) Lyctidae Lyctus lincarius Old Pine, &c. „ '> ,, 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 1 Buchner (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 bac- terial cell-mass (accessory cell-mass) fuses with the ventral sur- face 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 meta- morphosis. 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 re- ferred 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 any- thing 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.