| Symbiotic bacteria play a key role in feeding choice of insects.Meantime the feeding choice of herbivorous insects is critical to the co-evolution between insects and plants.Bactrocera minax is one of the main pests in citrus,which is an oligophagous species,only feeding on citrus fruits.How do symbiotic bacteria modulate the feeding choice of adults and affect the food habits in B.minax? To address this question,we focused on the feeding choice of adults of B.minax,clarified the influence of symbiotic bacteria on the foraging behavior and fitness of adults.Further,colonization and transmission of symbiotic bacteria in the gut of B.minax were studied by green fluorescence labeling technology.We also carried out culture-dependent and-independent methods together with metagenomic technology to better understand the symbiotic diversity and function in B.minax.The main results are summarized as follows:1.Effects of symbiotic bacteria on feeding choice of B.minax adultsB.minax feed on a large amount of food to get sexually mature after eclosion.In order to understand the role of bacteria on feeding choice,we set up the different volume(1μl,5μl)and different types of diets,including diets containing all amino acids(Full diet,F)and only Non-Essential amino acids(NE),and then observed the choice behavior of adults treated with antibiotics.The results showed that:(1)Compared with the control,male and female adults treated with antibiotics tended to feed on F diet,and selected diets and feed on them quickly.The food consumption of females and males were 11.77μl and9.53μl respectively,which were significantly higher than the control.(2)When probiotics including Klebsiella oxytoca,Citrobacter freundii and Enterobacter cloacae were added to diets,the feed selectivity and feed intake of adults increased significantly.(3)When the adults were fed with different types of diets,the fecundity of females treated with the antibiotics was significantly reduced.2.Transmission and distribution of gut bacteria in B.minaxThe distribution and transmission of Klebsiella oxytoca,Citrobacter freundii and Enterobacter cloacae in the gut of B.minax were investigated through the green fluorescent protein labeling technique.The results showed that K.oxytoca began to form biofilms after 48 hours of feeding.72 hours later,it colonized in large quantity in the gut and formed thick biofilms.It suggested that K.oxytoca colonized in the gut of adults.In addition,K.oxytoca is observed to distribute in the ovaries and eggs,which gave the clue that it may be transmitted vertically.C.freundii and E.cloacae could also successfully colonized in the gut,and after 48 h they began to form biofilms.But they were not detected in ovaries and eggs.3.The gut bacteria difference of B.minax larvae feeding on varied dietsInsect gut bacteria change a lot upon different diets.We compared the development of eggs and larvae after feeding on artificial diets,ripe navel orange(Citrus sinensis),immature mandarin(Citrus reticulata)and immature navel orange.Then the diversity of gut bacteria of the 2nd instar larvae was analyzed.The results showed that:(1)When rearing in immature mandarin and immature navel orange the hatching time of eggs shorten significantly.The survival rate of the 1st and 2nd instar larvae was significantly improved in the artificial diets.(2)Using culture-dependent method,10 bacteria were identified from the gut of the 2nd instar larvae.Among them,K.oxytoca was always present.(3)The symbiotic diversity of the 2nd instar larvae of B.minax was analyzed by 16S rDNA sequencing,which showed that Providencia was the most abundant(﹥90%)in the gut of the larvae fed on artificial diets.But the proportion was very low when the larvae fed on navel orange,mandarin and ponkan(Citrus reticulata Blanco cv.Ponkan).Enterobacter was the most abundant bacteria for the larvae fed on navel orange and ponkan which achieved 82.56% and 38.58%,respectively.However,Acinetobacter was the highest in the larvae fed on mandarin which accounted for 31.30%.The results indicated that the gut bacteria were varied in the larvae of B.minax and dependent upon food.4.Analysis of gut bacterial function of B.minax by metagenomic sequencingWe carried out metagenomic sequencing analysis to uncover the diversity and function of gut bacteria in the adults,the 2nd instar larvae and pupae of B.minax.The results showed that:(1)The bacteria diversity of adults,pupae and larvae of B.minax remained difference at the genus level.Citrobacter was the most abundant in the gut of adults,accounting for 33.36%,whereas the proportion was low in pupae and larvae.Enterococcus were the most abundant in the gut of 2nd instar larvae,accounting for15.90%,but the proportion is low in adults and pupae.These results suggested that the bacteria were varied with developmental stages of B.minax.(2)The symbionts of B.minax involving in carbohydrate metabolism were mainly Enterobacter,including encoding enzymes of cellulose degradation and pectin degradation.(3)Enterobacteriaceae,Lactobacillus and Pseudomonas engaged in nitrogen fixation and the synthesis of amino acids in B.minax.(4)Enterococcus,Lactococcus,Citrobacter and Enterobacter were the main symbionts,which involved in detoxification and defense against host plants in B.minax.In summary,the present study shows that symbiotic bacteria affect the feeding choice and fitness of B.minax adults.It also suggests that K.oxytoca colonized in the gut and the ovary of adults and may transmtted vertically through eggs.Using metagenomic sequencing analysis,we found that the symbionts of B.minax involved in carbohydrate metabolism and nitrogen fixation to provide nutrition.What’s more,bacteria participate in detoxification defense and enhance immunity of B.minax.These results provide an important clue to understand the bacteria function and the relationship between symbionts and insect food habits. |
