Study On The Intestinal Microbial Community Structure Of The Rainbow Stag Beetle

Lucanidae is a common saprophytic insect in Scarabaeoidae of Coleoptera.Larvae live in rotten wood or soil,decompose rotten wood and humus to obtain growth and development nutrients,while adults use sap,nectar or rotten fruit as food.They are very strict with the environment around them,and very sensitive to climate change and environmental change of forest ecosystem.It also benefit to keep the ecombine homeostasis of ecosystem,as forest ecosystem health assessment indicators.Phalaclorgnathus muelleri,the rainbow stag beetle,is popular among people because of its colorful body and interesting male upper jaw.At present,it has established a mature artificial breeding system.So far,the research on stag beetle mainly focuses on Systematics and Taxonomy,the research on intestinal microorganisms is rarely involved.This study represents the first comprehensive analysis of the gut bacterial community of the rainbow stag beetle Phalacrognathusmuelleri during different life stages using highthroughput sequencing(Illumina Miseq).We investigated 1st,2nd,and 3rd instar larvae and adults gut microbial diversity and community composition that were raised under standard rearing conditions.Our aim was to compare the differences of gut microbiota structure in different developmental stages and different genders.The results indicate that:1.The dominant intestinal bacterial phyla across all samples were Firmicutes(74.72%),Proteobacteria(12.67%),Bacteroides(10.87%)and Tenericutes(1.80%)across all stages and Firmicutes was the highest,Tenerictes was the lowest in the relative abundance.From the first instar larvae to the adult stage,the relative abundance of Firmicutes showed a decreasing trend of larval abundance than that of adults.However,the larvae abundance reveled an increasing trend than that of adults.These three phyla were significantly different in larval stages and adults stages(one way ANOVA: P < 0.05).Tenericutes was no ups and downs in the whole life stage,and there is no obvious difference in each stage.Gut fungi predominant phyla were Ascomycota(80.86%)and Basidiomycota(12.36%),Ascomycota was significantly higher than Basidiomycota in the relative abundance.There were also significantly different between adults and the first instar larvae(one-way ANOVA: P < 0.05).However,there were no significant differences in dominant phyla between adults and the second,third instar larvae(one-way ANOVA: P > 0.05).The relative abundance of Basidiomycota shows significantly different between adults and larval(one-way ANOVA: P < 0.05).2.The results of alpha diversity analysis suggested that: from the first instar larvae to the adults,the bacterial alpha diversity the 1st instar to the 3rd instar larvae was increased.Adults showed the lowest gut bacterial alpha diversity.The fungal alpha diversity showed the trend of larvae was higher than adults.The beta diversity analysis demonstrated dramatic shifts in gut microbial community structure between larvae and adults(bacteria ANOSIM: P = 0.001;fungi ANOSIM: P = 0.001),the first and second instar larvae(bacteria ANOSIM: P = 0.007;fungi ANOSIM: P = 0.039)had significant differences in the bacterial community,little differences in the fungal community.In the second and third instar larvae,the difference of the microbial community composition was not significant(bacteria ANOSIM: P=0.059;fungi ANOSIM:P=0.037).Notably,there was a barely significantly different between females and males.(Bacteria ANOSIM: P = 0.047;Fungi ANOSIM: P = 0.431)and were little difference between female and male in adult stage was also very small(Bacteria ANOSIM: P = 0.578;Fungi ANOSIM: P = 0.258).In general,the microbial community composition have great differences between larvae and adults,and between first instar and third instar of larvae,and has no significant difference in the different genders.3.Our study demonstrated dramatic shifts in gut bacterial community structure between larvae and adults,reflected in the microbial community compostions,the relative abundance and the diversity.Becasuse,larvae fed on decaying wood and adults fed on beetle jelly,illustrated that diet is a crucial factor shaping the gut bacterial community structure.In addition,the rain bown stag beetle belongs to complete metamorphosis insect,and the intestinal structure has changed greatly in pupae stage.It has a great impact on beetle gut microbes,which is also a key factor leading to significant differences in the community structure of larvae and adults.There were significantly different in microbial community structure between early instar and final instars larvae,suggesting that certain life stages are associated with a defined gut microbial community,and different development stages will affect the composition of gut microbe.It should be noted that gender has no significant effect on the composition and abundance of intestinal microorganisms.We infer that this is due to the high standard artificial feeding condition,uniform diet and the same living environment.Moreover,our rain stag beetle has not go through the dormant phase and the sexual organs are not mature,the number of endogenous and exogenous microorganisms has not increased or changed,thus the gut microbial community is highly conserved.In summary,the gut microbial community composition have no great divergence between male and female.The aim of study was to investigate the structure of intestinal microbial community in different developmental stages and genders under artificial rearing conditions.Understanding the effects of diet,developmental stages and genders on the structure of intestinal microbial community.It provides a basis for the conservation of spade beetles under the condition of artificial breeding,helps to better understand the symbiotic relationship between insects and their colonizing microorganisms,and is of great significance to the utilization of biomass energy and the recycling of waste.