Oogenesis and transmission of symbiotic bacteria in the weevil Sitophilus oryzae L. (Coleoptera: Dryophthoridae).

Among Coleoptera, and particularly in numerous Dryophthoridae, the insects share intracellular symbiosis with hereditary Gram-negative bacteria (or endocytobiotes). Sitophilus oryzae Primary Endosymbiote (SOPE) belongs to the gamma 3-proteobacteria group. It is a pleomorphic rod shaped (1 to 30 pm), non-sporulating and non-ciliated bacterium. In the bacteriocyte the bacteria are not included in vacuoles but lie freely in the cytoplasm. SOPE is closely related to Escherichia coli, with 95% homology on the 16S rDNA gene, and also the primary endosymbiotes of Sitophilus zeamais (97,8% identity). In addition to SOPE, several wild strains of S. oryzae harbor a second symbiotic bacterium, that belongs to the B-group of Wolbachia (an (alpha-proteobacterium). They are not considered in this work since studied strains were deprived of Wolbachia. Previous studies have shown that SOPE is transmitted to the offspring strictly through the female. The bacteria are located in ovaries and in the larval bacteriome. The latter organ is at the junction stomodeum-midgut, and does not communicate with the gut lumen. It is dissociated at metamorphosis. This work aims to better describe oogenesis in the weevil Sitophilus oryzae and some other Coleoptera. It was performed with various techniques, some of them being original in histology and histochemistry (BMN fixative, RPH and RPMy). Autoradiographic studies were used to precise the nucleic acid (DNA, RNA) and protein metabolism, in the different cell types of the ovary, and in the symbiotic bacteria. In the adult females, the ovarioles are surrounded with a Tunica propria and a double sheath of flat cells, which present a ribonucleo-proteic metabolism. In the apical bacteriome the bacteriocytes are giant polyploid cells. They contain numerous symbiotic bacteria and exhibit a low cellular activity although they do multiply. They also show a lytic activity with myelinic degeneration figures. Between the bacteriocytes small interstitial cells can be seen. These small cells are also visible between the, trophocytes in the tropharium. The trophocytes, or nurse cells, are polyploid and form a pseudosyncitium like. They harbor numerous symbiotes and mitochondria. The chromosoma are apparently at the end of pachytene or at diplotene stages. In opposition to most other telotrophic insects, the nutritive cords are poorly developped in S. oryzae. Despite an intense metabolism of RNA and proteins, no transfer of these substances to oocytes was seen. The oogenesis is accomplished in 8 serial steps, with the two first stages occurring in the nymph. The stage 1 includes oogonia and the stage 2 begins at the first step of meiosis and ends at pachytene. SOPE are rarely visible. At the stage 3 oocytes are found among the prefollicular tissue, at the basis of tropharium (3A) and are characterized by the presence of phospholipidic inclusions. At the beginning of growth (stage 38) the chromosoma are always in intimate contact with the nuclear membrane. During the stage 4, the oocytes are progressively surrounded by follicular cells. When the chromosoma reach the diplotene stage, they are no longer in contact with the nuclear membrane. The oocytes continue to grow and the nucleolus may begin to fragment. At the stage 5, the follicule is completed. At the beginning of that stage, a karyonucleolus is formed which evolution is very complex. It is characterized by a vacuolisation, followed by a fragmentation and the formation of fibrous structures and endobodies. Oocytes reaches 120 pm and the germinal vesicle 45 pm. The phospholipidic inclusions disappear in the cytoplasm. At this stage the endocytobiotes are still in low amount. The vitellogenesis begins at stage 6, where the vitellus is formed of lipo-glyco proteinic globules and lipid droplets. The oocytes and the germinal vesicles are now about 290 pm and 70 pm long. The endocytobiotes multiply actively and are particularly numerous at the posterior pole and at the central zone of the oocyte, which is not yet occupied by vitellus. The karyonucleolus becomes surrounded by fibrous structures. The stage 7 is characterized by the end of vitellogenesis and the bursting of the germinal vesicle. The oocyte is 350 pm long with a high amount of glycogen in the cytoplasm. The stage 8 is marked by the chorion deposition. The oocyte's size is now 600 to 700 pm. From the oogonia (stage 1), the total volume increases about 100,000 fold. The autoradiographic study shows that the follicle cells are polyploid, and exhibit high activity in DNA, RNA and protein metabolism. The tropharium is actively synthesizing RNA and proteins all along oogenesis. In the ovary, SOPE is present only in the cells of the germ line: apical bacteriocytes, trophocytes and oocytes. We have never seen any symbiotic bacteria in the follicle cells. The autoiadiographic studies show that SOPE has a metabolic activity in the apical bacteriome, in the tropharium and in oocytes. This metabolism results in part in the growth activity of symbiotes, and in other part in a possible physiological activity. Nevertheless, no cytological difference can be seen in the oogenesis of symbiotic and aposymbiotic weevils, despite the fact that SOPE is known to have a groat influence on the larval development.