The Regulation Mechanisms Of Population Density Of Buchnera Aphidicola In Aphis Gossypii Glover

Endosymbiotic bacterium is ubiquitous in aphids. Buchnera aphidicola, an obligate endosymbiont, is an indispensably important component of aphids. Aphids cannot reproduction if their Buchnera are cured. The population density of endosymbiont is closely related to physiological effects and ecological adaptability of its host. However, regulation mechanisms of population density of Buchnera in aphids were still unclear. Therefore,effects of host plant, host genotype, lysozyme and secondary endosymbiont on Buchnera density in cotton-melon aphid Aphis gossypii Glover were studied. The cotton-melon aphid populations exist cotton- and cucurbit-specialized biotypes. The main results are as followings:(1) Net reproductive rate (R0), average life expectancy (T) and intrinsic rate of naturel increase (rm) of cotton-melon aphid population were significantly affected by Buchnera.When Buchnera population were decreased and completely removed by antibiotic, R0 and rm of aphid populations were decreased significantly, and T was obviously prolonged.Under normal conditions, rm of cotton-specialized aphids reared on the zucchini was significantly higher than that on the cowpea and cotton, but removal of Buchnera by high dosage antibiotics, all the rm of aphid populations reared on the zucchini, cowpea and cotton significantly decreased, and there were no significant differences among different host plants.(2) Buchunera population density and average numbers of bacteriocytes in the cucurbit-specialized aphids were significantly higher than that in the cotton-specialized aphids. The population densities of Buchnera were also strongly different in natural populations collected from cotton, cucumber, hibiscus and zucchini, with aphids from hibiscus and zucchini showing higher Buchnera density than those from cotton and cucumber. Host plant significantly impacted Buchnera density in cotton-melon aphids.Genotypes of aphid also significantly affect Bucherna density, although it was dependent strongly on host plants. After the cotton-melon aphids were transferred onto a new host plant, the densities of Buchnera and survival rate of aphids showed remarkable fluctuations in the first several generations. Moreover, when the cotton-specialized aphids were reared on the zucchini, their Buchnera densities were significantly increased and approached to the level in the cucurbit-specialized aphids. In contrast, Buchnera densities in the cucurbit-specialized aphids were declined to the level of the cotton-specialized aphids when they were reared on cowpea.(3) Using diagnostic PCR method, 10 species of facultative symbionts in cotton-melon aphid populations from different host plants in Nanjing, China were detected. Except that Arsenophonus were infected in cotton-melon aphids, others facultative symbionts were not found. Infection rate of Arsenophonus showed a significant difference among aphid populations collected from different host plants. Infection rate of Arsenophonus in aphids on cotton was dramatically higher than that the aphids on cucumber and zucchini. Removal of Arsenophonus significantly improved Buchnera density in cotton-melon aphids(4) Using RT-PCR and RACE techniques, the full-length sequence (680bp) of lysozyme gene in cotton-melon aphids was cloned. Sequence analysis showed that this gene belonged to the i-type lysozyme and it had missed two important active sites of i-type lysozyme in marine animals. SDS-PAGE analysis showed that LYSs were effectively expression in Escherichia coli (BL21) and recombinant protein of LYSs could exist in soluble protein and inclusion bodies forms. qPCR assay showed that lysozyme gene expressed levels in the cucurbit-specialized aphids were highest when aphids were fed on the zucchini, followed on cowpea, and the lowest level on cucumber.(5) Plant extracts added to artificial diets of aphids had a significant impact on Buchnera population density in the cotton- and cucurbit-specialized aphids. Extracts from cucumber, zucchini, pumpkin, and cowpea leaves led to an increase of Buchnera densities in the cotton-specialized aphid populations. In contrast, plant extracts from cotton resulted in a decrease of Buchnera densities in the cucurbit-specialized aphids.The plant secondary metabolites, gossypol and cucurbitacin ingested by aphids from the artificial diets significantly affected the population density of Buchnera in both the cotton-and cucurbit-specialized aphids. Gossypol at a high concentration (50 ppm) suppressed Buchnera titres in aphids, while cucurbitacin led to higher densities.(6) A comparative analysis of differential gene expression profiles among three lineages of the cotton-melon aphid, cotton-specialized (CO), cucurbit-specialized (CU), and CU reared on cowpea (CU-cowpea) was studied using RNA-seq with next-generation sequencing technology. The results showed that there were significant differences at the transcriptional level of CO and CU lineages. 1106 up- and 2835 down-regulated genes in CU, compared to CO, were found, and 812 up- and 14492 down-regulated genes in CU,compared to CU-cowpea, were identified. Differentially expressed genes were enriched in the sugar metabolism, immune system process, pathogen infection or symbiosis, and salivary secretion between CO and CU. Moreover, a majority of genes associated with detoxifying enzymes, digestive enzyme and salivary effectors protein genes were differentially expressed between CO and CU, even between CU and CU-cowpea, and these genes might be related to host specialization of cotton-melon aphids.Four genes, which showed very differentially expressed levels among cotton- and cucurbit-specialized aphid's transcriptome, were selected to perform RNA interference.When a detoxification enzyme gene U4155 was successfully interfered, the Buchenra density in cotton-melon aphids was significantly increased, but the other three genes were interfered, and the density did not altered.In sum, host plants, aphid genotypes, gene expression levels of detoxifying in aphids,and facultative endosymbiont Arsenophonus affected population density of Buchnera, and effect of host plant on Buchnera density was mediated by secondary metabolites of plants.