Paul Nardon’s research while affiliated with Institut National des Sciences Appliquées de Lyon and other places

7-OV (ouvrages ou chapitres de vulgarisation) 2-1-3 Transfert/animation de la recherche chapitre d'ouvrage 3. Chapitres d'ouvrages En cours d'impression

6-OS (ouvrages ou chapitres scientifiques)

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 γ3-proteobacteria group. It is a pleomorphic rod shaped (1 to 30 μm), 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 α-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 3B) 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 μm and the germinal vesicle 45 μm. 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 μm and 70 μm 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 μm 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 μm. From the oogonia (stage1), 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 autoradiographic 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 great influence on the larval development.

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 γ3-proteobacteria group. It is a pleomorphic rod shaped (1 to 30 µm), 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 α-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 pseudo-syncitium 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 3B) 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 µm and the germinal vesicle 45 µm. 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 µm and 70 µm 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 µm 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 µm. From the oogonia (stage1), 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. Accepté le 1 er novembre 2005 130 P. Nardon 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 autoradiographic 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 great influence on the larval development. Résumé. Ce travail est une revue des recherches effectuées sur l'ovogenèse du charançon Sitophilus oryzae. Ces recherches, originales pour la plupart, ont été réalisées à l'aide de techniques variées, histologiques, histochimiques et ultrastructurales, complétées par des études autoradiographiques destinées à préciser le métabolisme des acides nucléiques (ADN, ARN) et des protéines dans les différents types cellulaires. Les bactériocytes du bactériome apical de l'ovariole sont des cellules géantes polyploïdes abritant de très nombreux endocytobiotes, qui sont des γ3 protéobactéries. Leur métabolisme paraît faible, bien qu'elles se divisent. Elles subissent des phénomènes de lyse. Les ovarioles sont du type télotrophe et les cellules nourricières communiquent entre elles. Elles renferment des symbiotes et de très nombreuses mitochondries. Les noyaux sont plus ou moins polyploïdes, avec des chromosomes au stade pachytène. Le métabolisme de l'ARN et des protéines est très intense. Par contre, les cordons nourriciers sont très peu développés. Les cellules folliculeuses sont également polyploïdes. Les synthèses d'ARN et de protéines y sont très intenses. Elles sécrètent le chorion. L'ovogenèse se déroule en huit stades successifs. Chez la femelle mature on ne trouve que des ovarioles en phase diplotène et fin de pachytène. Un caryonucléole se forme au stade 5. Grâce à une technique de coloration combinant la réaction de Feulgen et le bleu de toluidine, on peut suivre l'évolution des chromosomes pendant toute l'ovogenèse. L'évolution de la vésicule germinative est très complexe. La fragmentation du nucléole s'accompagne de la formation d'un lacis de structures fibreuses et de corps résiduels. Le vitellus est formé de globules lipo-glyco protéiniques et de gouttelettes lipidiques. Sa composition varie au cours de la vitellogenèse. Du glycogène s'accumule dans le cytoplasme des ovocytes âgés. L'ovocyte assure lui-même une synthèse d'ARN et de protéines qui se poursuit pendant la vitellogenèse. Les endocytobiotes se multiplient au cours de l'ovogenèse et colonisent en particulier le pôle postérieur de l'oeuf, dans une zone correspondant au déterminant germinal. Ainsi, dès leur formation, les cellules germinales sont contaminées par les bactéries symbiotiques.

Bacterial intracellular symbiosis in the weevil Sitophilus spp. was discovered in 1927 and since then has been studied by several authors through many approaches. This has led to a deep characterization of variety of symbiotic aspects in relation with morphology, histology, genetic, physiology, as well as host-symbiont molecular and cellular biology. This paper reviews the basic findings from several teams and analyzes them with respect to current knowledge on weevil symbiosis evolution and cell-to-cell molecular interactions. Sitophilus spp. intracellular symbiosis appears as a model appropriate for studying and elucidating molecular and genetic mechanisms and evolutionary features involved in the early steps of insect intracellular symbiosis establishment.

Dryophthoridae weevils are phytophagous insects thriving on a broad host-plant spectrum. Some, such as Sitophilus spp. or Cosmopolites sordidus, are major agricultural pests of cereals and bananas, respectively. Previous studies have concluded that several Dryophthoridae species harbour intracellular bacteria (endosymbionts) in specialized organs named bacteriomes. In this work, we have demonstrated the presence of intracellular symbiosis in seven out of the eight Dryophthoridae species tested, and have assessed the morphological variability of these endosymbionts. Histological analyses of the endosymbionts in either the larval bacteriome or adult female ovaries reveal two fields of variability: physiologic (length variation during host development) and morphologic (both interspecific and intraspecific). These variations are expressed to different degrees in the species studied. Indeed, Sitophilus granarius displays much less pleiomorphism than S. zeamais. Such pleiomorphism may be a consequence of both genetic polymorphism and host/bacteria interaction.

Sitophilus oryzae and S. zeamais are recognized as sibling species, intersterile and difficult to distinguish. We have systematically searched for new morphological and non-destructive characters in order to facilitate their identification. We also revisited two already known characters: pigmentation and pronotum punctures, presenting or not, a smooth median line. In the larvae we discovered a small difference in mandible structure: the apical zone is more depressed in S. oryzae than in S. zeamais. In adults, four new distinctive features are described, i: structure of the metathoracic episternum (two rows of punctures in S. oryzae and three in S. zeamais), ii: the frontal groove (much more depressed in S. zeamais), iii: the complex setae bordering the frontal groove (bigger in S. oryzae than in S. zeamais) and iv: presence of a little structure called « rosette », at the base of the rostrum, easy to see in S. zeamais but less visible in S. oryzae. By the combination of these characters, it is easy to distinguish the insects of the two species. The presence, in several populations, of insects with intermediate features, leads us to consider that the divergence between the two species is still gradually working.