Study On The Effects Of Ciprofloxacin On Development Of Boettcherisca Peregrina Via Gut Microbiota

Background: As an emerging discipline in forensic medicine,forensic entomology is now more widely used in the inference of time of death and cause of death.However,the developmental history of necrophagous flies is often affected by the external environment,or misidentification of the earliest reproducing larvae can lead to biased inferences of the time of death.Another new branch of forensic medicine,forensic microbiology,has also explored a relatively new approach to the inference of time of death: the use of microbial communities associated with corpses to infer the time of death,but also limited by variable environmental factors.Based on the colonisation behaviour of necrophagous flies on carcasses and the feeding and reproduction of larvae,the gut microbiota of necrophagous flies will perhaps be the point of intersection between the two disciplines.At the same time,the presence of exogenous drugs(poisons)has been shown in numerous cases and studies to affect the growth rate of larvae.Exploring whether drugs affect changes in fly development,and whether changes in development are related to fly gut flora,will further improve the accuracy of postmortem interval(PMI)extrapolation.Objectives: To explore the composition of the gut microbiota of Boettcherisca peregrina,to analyze the potential relationship between changes in the gut microbiota and the growth of the fly,to compare the microbiota composition of other fly species and environmental samples(reared minced meat),to identify the effect of ciprofloxacin on the gut microbiota of Boettcherisca peregrina,and to analyze the effect of the gut microbiota on its development,and to improve the accuracy of the postmortem interval(PMI)extrapolated from the developmental patterns of necrophagous flies.Methods:(1)Purified B.peregrina populations were established and reared on blank control,low and high concentrations of ciprofloxacin and their development data were collected.The gut of B.peregrina at three development stages(third instar,pre-pupa instar and adult)and tissue contents(tissues sampled by eliminating puparium)from the pupa were extracted for 16 S r DNA sequencing.(2)Parasarcophaga similis were collected under the same rearing conditions using the same method for comparison.(3)An insect exclusion carcasses model was established under the same rearing conditions and environmental samples(meat)were collected from both the immediate exposure and isolated fly groups during larval development,respectively.(4)Based on the sequencing results,the specific strains were further quantified and verified by quantitative Realtime PCR.Results:(1)Effects on development: under the influence of ciprofloxacin,the developmental rate of B.peregrina was accelerated and both pupa and adult weights were lower;the critical time point of development was shifted forward;(2)Sequencing of the gut microbiota of B.peregrina: The abundance of bacterial communities varied throughout the development of the same group of B.peregrina,and there were large differences in gut microbiota between ciprofloxacin intake group and control group at the same developmental stage due to the influence of ciprofloxacin.The proportion of Ignatzschineria in the gut microbiota of the third instar larvae was much higher in the ciprofloxacin intake group than that in the control group.(3)Even in the same family,there are some structural differences between the bacteria of B.peregrina and P.similis at same life stages.(4)After insect contacts,the structure of the bacterial community in the food resource of insect immediate exposure group changed considerably from that before feeding.Compared to the bacterial community structure of fresh pig lung before fly feeding in the insect isolation group,the main categories of the dominant genera remained unchanged,with Myroides accounting for a significantly higher proportion.The genera of Ignatzschineria and Wohlfahrtiimonas only presented in the meat of insect immediate exposure group.(5)q PCR results for specific strains: Wohlfahrtiimonas chitiniclastica and Ignatzschineria indica showed an overall increasing trend in the gut of the third instar of larvae,the gut of the pre-pupa stage,and the content of the earlier stage of pupa stage.Conclusions: Ciprofloxacin affects the development of B.peregrina and changes the diversity and abundance of the gut microbiota of B.peregrina during the same period of time compared to that of the controlled group;the increase in the content of Ignatzschineria in the gut microbiota of B.peregrina larvae,which is an important genus during their development,may be responsible for the faster growth rate of the larvae in the treated group;B.peregrina is able to select its gut microbiota composition,obtaining and keeping beneficial bacteria from the environment to promote its development.