| Dietary specialization can occur at different life stages of insects,for example,larvae and adults feed on different food,reducing intraspecific food competition.Gut bacteria are widely involved in the growth,development,immunity and behaviors of host insects.However,whether insect gut bacteria are involved in dietary specialization and the mechanism underlying it have been poorly studied.Plagiodera versicolora(Coleoptera)is an important forestry pest,mainly harmful to willow and poplar,can cause great harm to economic forests and landscape plants.Research has shown that dietary specialization is existed between P.versicolora larvae and adults.At the same time,P.versicolora has stable gut bacterial community that plays important roles in its life history.This study aims to explore the role of gut bacteria in dietary specialization of P.versicolora larvae and its mechanism.Firstly,we established a germ-free leaf beetle system,explored the effects of gut bacteria on the growth and development,and used transcriptome sequencing method to study the influence of gut bacteria on gene expression profile of P.versicolora.Secondly,we verified the dietary specialization of P.versicolora,explored the impact of dietary specialization on larval fitness(including growth and development,immunity),and that indicated that gut bacteria can affect the dietary specialization of P.versicolora larvae.Finally,we used 16 S r RNA sequencing,RT-q PCR,bacteria isolation and culture and bacteria reintroduction to verify the mechanism of gut bacterial influence on dietary specialization of P.versicolora larvae,and analyzed the reasons for the variation of gut bacteria community using metabolomics.The main findings are as follows:1.We established a germ-free beetle system of P.versicolora by sterilizing eggs and feeding germ-free leaves.The germ-free larvae were identified by Culturedependent and Culture-independent methods.The results of medium culture and specific PCR amplification showed that: There was no bacterial growth on the LB medium of germ-free larvae,and there was no obvious light band of 16 S r RNA amplification for total DNA from germ-free larvae,indicating that our germ-free leaf beetle system was successfully established.Subsequently,we compared the differences in the growth and development between germ-free(GF)and conventional-reared(CR)larvae,and the contents of nutrients in the hemolymph.The results showed that GF larval body weight was slightly higher than that of CR larvae on the 6th day,but there was no difference at other times and no difference in the development time of larvae.The glucose content in the hemolymph of GF larvae was higher than that of CR larvae,but the concentrations of protein and trehalose between the two groups have no difference,indicating that the removal of gut bacteria had no adverse effect on the growth and development of P.versicolora larvae.Transcriptome analysis was performed on different tissues of CR and GF larvae.The statistical diagram of differentially expressed genes(DEGs)and volcanic diagrams showed that there were 1,968 up-regulated genes and 2,707 down-regulated genes in body tissues of GF larvae,and 1,368 up-regulated genes and 1,339 down-regulated genes in gut tissues when compared with CR larvae.The results showed that gut bacteria could significantly affect host gene expression.The GO functional enrichment chords showed that these DEGs were enriched in carbohydrate metabolism and digestion.Further RT-q PCR results showed that the expression levels of immune-related genes such as PGRP,defencin and PPO genes were significantly up-regulated in CR larvae.In addition,we also used a mixture of four antibiotics to establish an antibiotic system of P.versicolora.Culturedependent results showed that the number of gut bacteria growing on LB medium of larvae and adults fed antibiotics was less than that of CR groups,suggesting that antibiotics can be used to interfere with P.versicolora gut bacteria.2.We further verified the dietary specialization of P.versicolora,and the results showed that: the adults fed more new willow leaves than mature leaves,but the adults laid more eggs on mature leaves than new leaves,and the larvae fed more mature leaves than new leaves,indicating that the larvae and adults of P.versicolora have dietary specialization.Further experiments showed that when larvae fed mature leaves,the body weight of 2nd instar larvae,pupa weight and adult body weight were significantly higher than that of new leaves,and the development time was significantly shortened.The larvae feeding new and mature leaves were infected with two entomopathogenic fungi,Beauveria bassiana and Aspergillus nomius,and the survival rates of larvae feeding mature leaves was higher,indicating that the dietary specialization of larvae improved their fitness.However,when GF larvae or larvae feeding antibiotics were fed with new and mature leaves,there were no significant differences in the body weight of 2nd instar larvae,pupa weight,adult body weight and development time,suggesting that gut bacteria were involved into and significantly affected dietary specialization of P.versicolora larvae.3.16 S r RNA sequencing results showed that the diversity of gut bacteria decreased when larvae fed new leaves.The PCo A and NMDS analysis showed that gut bacterial communities between larvae feeding new leaves and mature leaves had significant differences and the adults had similar results.The diagrams of Bar and comparative analysis of multiple groups showed that the relative abundance of Pantoea significantly increased after feeding new leaves.Pantoea anthophila,Pantoea dispersa and Pantoea ananatis were isolated from the intestinal tracts of P.versicolora.The bacteria reintroduction experiments showed that P.anthophila and P.dispersa had adverse effects on the growth and development of P.versicolora larvae.These results suggest that new leaves may have adverse effects on the growth and development of larvae by inducing the increase of Pantoea bacteria in the gut.By analyzing the metabolites of new leaves and mature leaves,we found that new leaves and mature leaves of willow and poplar had 136 and 144 differential metabolites,respectively.Based on the large differences of metabolites of willow and poplar,and the similarity of the components of new leaves and mature leaves,29 key metabolites that may cause the variation of gut bacterial communities after feeding new and mature leaves were finally screened.These metabolites belong to at least 19 HMDB categories.By comparing the contents of total protein and total soluble sugar in new leaves and mature leaves,we found that the protein contents in new leaves of willow and poplar were both significantly higher than that in mature leaves,and the protein content in the intestines of larvae feeding new leaves was also significantly higher than that in mature leaves.Therefore,we firstly used two metabolites,Tyrosyl-Valine and Citrulline,which belong to proteins among the 29 key metabolites,to carry out bacterial growth promotion experiments,and found that these two metabolites can promote the growth of P.anthophila in vitro.In conclusion,this study successfully established a germ-free P.versicolora system,and analyzed the effects of gut bacteria on the growth,development and gene expression profile of P.versicolora.Dietary specialization was verified and it was found that dietary specialization significantly improved P.versicolora larval fitness.It was revealed that gut bacteria participated in and enhanced dietary specialization by inhibiting larval growth and weakening immunity.In addition,we preliminarily explored the causes of the bacterial community variation,but the specific factors and mechanisms remain to be further explored.This study provides a novel insight into the ecological function of gut bacteria,provides a new idea for the control of P.versicolora mediated by gut bacteria and a reference for the research of intraspecific and interspecific dietary specialization of other insects. |
PDF Full Text Request