| Feeding nutrition plays a direct role in the growth,development,reproduction and behavior of insects.There are numerous intestinal microbes in insect midgut,which participate in host immunity,reproduction and nutrition acquisition,and are closely related to host nutrition metabolism.The rice leaffolder Cnaphalocrocis medinalis(C.medinalis)is an important migratory pest of rice.The increase in fertilizer uses and the change in feeding nutrition caused by the change of host species are considered to be important reasons to promote its outbreak.In this paper,the effects of different dietary nutrients on feeding behavior,growth and development,nutrient transformation and reproductive parameters of C.medinalis were studied by feeding method,and the diversity of symbiotic bacteria in the gut of C.medinalis was analysised using metagenomic sequencing and traditional isolation and cultivation techniques.The relationship between different feeding nutrients and the intestinal microflora of C.medinalis was discussed.Finally,the effects of relevant nutrients on the growth and development,nutrient metabolism and hormones of C.medinalis larvae were tested by the feedback of intestinal microflora elimination and reinsertion.The main findings are as follows:1.Effects of feeding nutrition on the larvae of C.medinalisThe growth and development,nutritional indicators,the accumulation of nutrients and hormone levels of C.medinalis were studied after feeding on different host nutrients such as rice,maize leaves and artificial diet.The results showed that the eficiency of conversion of digested food(ECD),eficiency of conversion of ingested food(ECI)and relative growth rate(RGR)of C.medinalis fed on rice were the highest(29.19%,8.82%and 0.37%,respectively),which were significantly higher than those fed on maize.The larvae and pupae fed on rice leaves were the heaviest(0.021 g/larva)and the duration of pupae stage was the shortest(7.3 d).The approximate digestibility(AD)of larvae fed with artificial diet was 52.32%,which was significantly higher than that fed with rice and corn.Compared with feeding with rice(17.9 d)and corn(17.7 d),feeding with artificial diet significantly prolonged the duration of larvae stage(21.3 d).The fat content of larvae fed on rice and maize was 27.08%and 25.87%,respectively,which was significantly higher than those fed on artificial diet(17.17%).The contents of soluble sugar and the activities of total protease,amylase and lipase in the midgut of the larvae fed with artificial diet were significantly higher than those of the larvae fed with rice and maize.The lipase activity in midgut of larvae fed with rice was significantly higher than that of larvae fed with maize.When the content of protein and amino acid in maize leaves increased,the content of juvenile hormone increased from 0.05 ?g/g to 0.19?g/g.When the content of protein and amino acid in the leaves fed by the larvae increased,the weight of pupae increased by 15.78%,the content of vitellin increased by 102.17%,and the oviposition of single female increased by 51.11%.2.Effects of feeding nutrition on C.medinalis adultThe C.medinalis adult were fed with various sugar solutions,amino acid components,mixed solution of sugar and ammonia,and water,to analyze the effects of nutrition supplementation during the adult stage on longevity,ovarian development and net population proliferation.The results showed that amino acid supplementation had a negative effect on the longevity of females and males,only 3.7 days and 6.0 days after treatment with distilled water.Moreover,sucrose,fructose,glucose and sugar mixture significantly increased the longevity of females and males.Meanwhile,ovarian development was also rapid:20-40%of the ovaries treated with sugar solution entered Grade ?,and the rest entered Grade ?,while 70-80%of the ovaries treated with distilled water and amino acid entered Grade ?.The egg-laying number and period of adults fed on distilled water were 8.6 eggs per female and 1.5 days,and those fed on glucose and fructose were 164.8 eggs per female and 5.6 days,4.8 eggs per day and 210 eggs per female,respectively.The results of life table analysis showed that the net reproductive rate(R0)of glucose-fed population was 98.33,which was significantly higher than that of distilled water-fed population(24.80).The intrinsic rate of increase Rm and the increment rate were significantly higher than that of distilled water.3.Diversity and function analysis of gut bacteria of C.medinalisThe midgut microbial composition of rice leaf roller,C.medinalis,was determined.The results showed that there were bacteria,fungi,archaea and bacteriophages in the midgut microbiome of C.medinalis.Bacteria was accounting for 99.37%of the total intestinal microbiome.Moreover,10 phyla and 337 species of bacteria were detected.There were 59 species in larvae,274 species in females and 191 species in males,males and females together own 154 species.The highest abundance of Firmicutes was found in the gut of larvae,followed by Proteobacteria,Actinobacteria and Cyanobacteria.More than 99%of the gut bacteria belonged to Enterococcus.Enterococcus(Sp.FDAARGOS-3755),Enterococcus casseliflavus and Enterococcus gallinae were the dominant species in larval intestine.The highest abundance of Proteobacteria was found in the adult,followed by Firmicutes,Bacteroidetes and Actinobacteria.At the genus level,the composition of gut bacteria of male and female adults was similar,only the relative abundance of dominant colonies was different,including Wolbachia(34%and 45%),Pseudomonas(21%and 13%),Morganella(10%and 21%),and Acinetobacter(7%and 5%).There was a major difference in the species of gut bacteria between larval and adult stages,and only 14 species of common bacteria were found.There were significant differences in genus level among Enterococcus in larvae,Arthrobacter and Sphingobacterium in females,and Wolbachia and Morganella in males.The results of isolation and identification of midgut bacteria by traditional culture method showed that five out of 10 strains belonged to Enterococcus casseliflavus,three belonged to Enterococcus Bacillus cereus,and the other two strains were Microbacterium levoglycan and Pseudomonas.Enterococcus was the dominant species of culturable bacteria in the midgut of C.medinalis larvae,which was consistent with the results of metagenomic sequencing.The analysis indicated that the midgut microbial enrichment of C.medinalis larvae had gene functions including metabolism,genetic information processing,etc.Among them,41%of the genes were involved in metabolism,including detoxification of foreign substances,glycan anabolism,carbohydrate metabolism,etc.Carbohydrate and glycan anabolism were the two most abundant KO,accounting for 18%and 11%,respectively.A series of carbohydrate-active enzymes were identified from the metagenomic sequencing of the larvae intestinal flora,there were 37 types of glycosidases including acetyl galactosidases and glucosidase,and 29 types of glycosyltransferases including sucrose synthetase and cellulose synthetase.By comparing the metagenomic pathway of the intestinal bacteria with that of the host's transcriptome,the intestinal bacteria supplemented the functional losses of the larvae of C.medinalis on galactose metabolism,glycosaminoglycan biosynthesis and other metabolic pathways.4.Effect of feeding nutrition on the gut bacteria of C.medinalisThe larvae of C.medinalis were fed with rice,maize leaves and different concentrations of nitrogen-containing solution,and the adults were treated with different sugar solutions,to detect the effect on intestinal flora.The results showed that different feeding nutrition had no significant effect on the predominant intestinal flora,and the relative abundance of Enterococcus was 99.98%(maize)and 99.56%(rice),respectively.However,there were significant changes in the non-dominant microflora in the gut.Microbacterium and Bacillus were the most important microflora in the larvae treated by maize.Compared with rice,Microbacterium pasteuricum,Pasteurella pasteurella and Lactobacillus plantarum were the three bacteria with the maximum changes in abundance at the species level.The relative abundance of the larvae fed on corn increased by 82.37,61.26 and 55.64 times,respectively.The metagenomic genomes of midgut microbes from maize-treated larvae had higher abundance of genes involved in carbohydrate,amino acid,vitamin and glycan metabolism,enrichment of functional genes related to immune system and transport metabolism during rice treatment.Compared with rice,the abundance of genes encoding alpha-amylase,chitinase and glucuronic acid were significantly increased in the gut microbes of larvae feeding on corn.The predominant flora in the gut of larvae fed on the same host with different nutrition levels was the same in all taxonomic levels,and enterococcus remained the predominant flora.With the improvement of nutrition level,the relative abundance of enterobacteriaceae increased from<0.01%to 0.51%.The abundance of three strains of enterobacteriaceae was significantly changed,one was enterobacteriaceae,the other two were enterobacter cloacae.With the improvement of food nutrition level,genes related to lysine and insulin signaling pathways were enriched in the metagenomic genome of intestinal bacteria,and the number of genes involved in lysine synthesis such as DAPD,argD and ASD increased significantly,4.08-5.23 times the number of control genes,and 14 genes involved in insulin signaling pathway were significantly changed.The number of GLGP and FLOT genes increased by 1.46 and 1.57 times,respectively.Gene enrichment associated with the Hedgehog pathway and ubiquitin-mediated proteolysis was significantly reduced.The number of bacteria in the intestine increased obviously after the adults were supplemented with sugar solution.A total of 266 species and 183 species of bacteria were identified in male and female adult worms,31 species and 29 species in the water control.The dominant genera in the intestinal tract of both the male and the female controls were Wolbachia,the relative abundance of which were 98.4%and 93.7%,respectively.The abundance of Wolbachia decreased to 34.4%and 45.5%.The relative abundance of Morganella in males increased from<0.001%to 20.8%,Pseudomonas from 0.07%to 13.4%,Pseudomonas in females from 0.09%to 21.0%,and Morganella from 0.06%to 10.3%.5.Effect of gut bacteria on the growth and development of C.medinalisThe results showed that the relative abundance of predominant Enterococcus decreased from>99%to<0.1%when removal of culturable intestinal colonies by antibiotic treatment.After the elimination of the microbiome,the larval development period was extended from 6.4 days to 9.2 days,the single body weight decreased from 0.025 g to 0.020 g,the relative growth rate of larvae decreased from 33.1%to 12.3%,and the relative feeding amount and other nutritional indexes were also decreased obviously,and there were significant differences between the two groups.The contents of sugar,protein and amino acid decreased by 80.1%,50.3%and 30.8%,respectively.Compared with the control,the contents of juvenile hormone and ecdysone decreased by 81.9%and 27.3%,respectively.The results showed that the larval development period was 6.8 days,the body weight increased to 0.026 g,and the relative growth rate and the relative feeding amount were significantly better after reestablish Enterococcus casseliflavus than those of the antibiotic treatment.The contents of glycogen 8.1 mg/g,protein 1.5 mg/g,juvenile hormone 0.193 ?g/g and ecdysone 0.175 ?g/g were significantly higher than those treated by removing intestinal bacteria. |
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