Cell physiology in the weevil Sitophilus oryzae is coordinated by three integrated genomes: nuclear, mitochondrial, and the ''S. oryzae principal endosymbiont'' (SOPE). SOPE, a cytoplasmic bacterium (2 ؋ 10 3 bacteria per specialized bacteriocyte cell and 3 ؋ 10 6 bacteria per weevil) that belongs to the proteobacteria ␥3-subgroup, is present in all weevils studied. We discovered a fourth prokaryotic genome in somatic and germ tissues of 57% of weevil strains of three species, S. oryzae, Sitophilus zeamais, and Sitophilus granarius, distributed worldwide. We assigned this Gramnegative prokaryote to the Wolbachia group (␣-proteobacteria), on the basis of 16S rDNA sequence and f luorescence in situ DNA-RNA hybridization (FISH). Both bacteria, SOPE and Wolbachia, were selectively eliminated by combined heat and antibiotic treatments. Study of bacteria involvement in this insect's genetics and physiology revealed that SOPE, which induces the specific differentiation of the bacteriocytes, increases mitochondrial oxidative phosphorylation through the supply of pantothenic acid and ribof lavin. Elimination of this ␥3-proteobacterium impairs many physiological traits. By contrast, neither the presence nor the absence of Wolbachia significantly affects the weevil's physiology. Wolbachia, disseminated throughout the body cells, is in particularly high density in the germ cells, where it causes nucleocytoplasmic incompatibility. The coexistence of two distinct types of intracellular proteobacteria at different levels of symbiont integration in insects illustrates the genetic complexity of animal tissue. Furthermore, evolutionary timing can be inferred: first nucleocytoplasm, then mitochondria, then SOPE, and finally Wolbachia. Symbiogenesis, the genetic integration of long-term associated members of different species, in the weevil appears to be a mechanism of speciation (with Wolbachia) and provides a means for animals to acquire new genes that permit better adaptation to the environment (with SOPE).

The principal intracellular symbiotic bacteria of the cereal weevil Sitophilus oryzae were characterized using the sequence of the 16S rDNA gene (rrs gene) and G + C content analysis. Polymerase chain reaction amplification with universal eubacterial primers of the rrs gene showed a single expected sequence of 1,501 bp. Comparison of this sequence with the available database sequences placed the intracellular bacteria of S. oryzae as members of the Enterobacteriaceae family, closely related to the free-living bacteria, Erwinia herbicola and Escherichia coli, and the endocytobiotic bacteria of the tsetse fly and aphids. Moreover, by high-performance liquid chromatography, we measured the genomic G + C content of the S. oryzae principal endocytobiotes (SOPE) as 54%, while the known genomic G + C content of most intracellular bacteria is about 39.5%. Furthermore, based on the third codon position G + C content and the rrs gene G + C content, we demonstrated that most intracellular bacteria except SOPE are A + T biased irrespective of their phylogenetic position. Finally, using the hsp60 gene sequence, the codon usage of SOPE was compared with that of two phylogenetically closely related bacteria: E. coli, a free-living bacterium, and Buchnera aphidicola, the intracellular symbiotic bacteria of aphids. Taken together, these results show a peculiar and distinctly different DNA composition of SOPE with respect to the other obligate intracellular bacteria, and, combined with biological and biochemical data, they elucidate the evolution of symbiosis in S. oryzae.

The flight activity (take-off) of various strains of Sitophilus oryzae L. and S. zeamais Mots. (Coleoptera, Curculionidae) have been compared on wheat. It was shown that males are more active than females and that S. zeamais, is more active than S. oryzae. Furthermore, in the two species, when reared on wheat kernels, only symbiotic weevils were able to fly, while aposymbiotic insects did not. By supplementation of the diet with vitamins (pantothenic acid, riboflavin) and amino acids (phenylalanine and proline), this activity could be restored in part. The influence of symbiosis on the flight ability of Sitophilus weevil is discussed in relation to the energy metabolism (coenzymes, proline), the role played by symbiosis in this behaviour and its possible effect on the dissemination of the species.