Differences In Microbial Diversity And The Effects Of Intracellular Symbiont On The Growth Of Rhyzopertha Dominica

The Rhyzopertha dominica(Fabricius)is an important stored product pest around the world.R.dominica can damge grain quality,which in turn can cause serious economic losses.R.dominica has developed a high resistance to the chemical fumigant phosphine.The grain industry is actively seeking new green and safe control technologies.In this paper,the microbial composition and ensymbiont S.bostrichidophilus RDOM were studied.The Illumina Nova Seq high-throughput sequencing platform was used to sequence the V3-V4 fragment of the bacterial16 S r DNA gene in larvae and adults of R.dominica,and adults of different geographical population R.dominica.In addition,ensymbiont S.bostrichidophilus RDOM was eliminated from the R.dominica by antibiotic feeding,differences in the growth and development of R.dominica before and after elimination were compared.The main findings were as follows:The results of microbial diversity studies in R.dominica showed that Shikimatogenerans was the dominant bacterial genus in R.dominica,comprising for 63.77% of the larval,and88.28% of the adult,microbiome,respectively.Shikimatogenerans was first in relative abundance in the microbiome of the GD,WZ,WH,BJ,k GX,k JX and k FJ R.dominica.S.bostrichidophilus RDOM was the second in relative abundancein in the microbiome of the k ZJG R.dominica,microbiome.Staphylococcus was first in relative abundance in the microbiota of the k ZJG R.dominica,comprising for 35.60%.Shikimatogenerans was second in relative abundance in the microbiota of the k ZJG R.dominica,comprising for 24.38%.Alpha diversity analysis showed that the bacterial diversity index in larvae was significantly higher than that in adults(P<0.05),and the richness index in adults was higher than that in larvae but the difference was not significant(P>0.05).The bacterial diversity index and richness index were significantly higher(P<0.05)in the four granary populations of R.dominica than in the laboratory population.In order to detect the S.bostrichidophilus RDOM populations titer in R.dominica using RT-q PCR relative quantification,the reference genes were screened.Seven genes screened from transcriptome data were used as candidate reference genes.We screened the reference genes under different experimental conditions,including development stages,temperature,hunger and gender.Five algorithms are used to analyze the expression stability,including ΔCt value method,ge Norm,Best Keeper,Norm Finder and Re Finder.The results showed that the most suitable combinations of reference genes were rps3 and rps6 at different developmental stages,18 S r RNA and rps13 under different temperature conditions,rps6 and gadph under starvation stress conditions,and rps6 and actin under different sex conditions.The S.bostrichidophilus RDOM populations in different developmental stages of R.dominica were detected by RT-q PCR using the above-mentioned optimal reference genes at different developmental stages.The results showed that S.bostrichidophilus RDOM populations increased dramatically from the ultimate insect molting and day 6 of adulthood.In R.dominica,S.bostrichidophilus RDOM populations was about 7-fold higher than day 1 at day6 of adulthood.S.bostrichidophilus RDOM populations was close to 0 in males on day 63 of adulthood.S.bostrichidophilus RDOM populations was significantly higher in females than in males at the same age.Screening for suitable antibiotics to eliminate the ensymbiont S.bostrichidophilus RDOM in R.dominica.Four antibiotics include tetracycline hydrochloride,ampicillin,kanamycin sulfate and rifampicin were added to the feed at 30 mg/g.After 7 days of feeding,the S.bostrichidophilus RDOM populations in males decreased significantly(P<0.05).For females,tetracycline hydrochloride,ampicillin and kanamycin sulfate could all significantly reduce S.bostrichidophilus RDOM populations(P<0.05),and rifampicin could reduce the S.bostrichidophilus RDOM populations but not significantly(P>0.05).After one month of feeding,the survival rate of R.dominica was 6.67% in rifampin,43.33% in kanamycin sulfate,97.33% in both tetracycline hydrochloride and ampicillin,and 100% in the control group.The tetracycline was selected as the most suitable antibiotic.Continuous feeding of tetracycline hydrochloride for 3 months resulted in the complete elimination of S.bostrichidophilus RDOM from the R.dominica.Comparing the growth and development history of symbiotic R.dominica and aposymbiotic.The pupal stage of aposymbiotic R.dominica was significantly shorter(P<0.05)at 4.27±0.50 a days compared to 4.57±0.57 b days in the control group.Other growth and development stages were not significantly different(P>0.05).Compared with symbiotic R.dominica,aposymbiotic had lighter outer epidermal color and slower blackening day 1 to 12 of adulthood.Significantly lower body weight at the day 1 to 5 of adulthood(P<0.05),but no significant difference in body size(P>0.05).The above experimental results showed that the obligate symbionts S.bostrichidophilus RDOM had insignificant effects on the growth and developmental stages of the R.dominica,but contributed to the formation of the outer epidermis of the adult moth after feathering.