Study On Biological Characteristics And Hypodermin Genes Of Parasitic Flies In Gansu Zokor

Zokor belongs to Rodentia,Spalacidae,Myospalacinae,one of the forestry pests in China.Zokor is omnivorous,which harms as many as 95 species of woods,and they mainly feed on the roots of plants,which has a great impact on forestry.However,they live most of their lives in underground tunnels,so it is not easy to be harmed by predator and human,which makes it difficult to control zokors.At present,physical and chemical control have many weaknesses in the control of zokors,in contrast,biological control is environmentally friendly and have less pollution.According to recent surveys,there exist parasitic flies under the subcutaneous tissue of Gansu zokor,and the parasitic rate is about 30%.in the wild.Warble flies belong to Diptera Hypodermatidae,which can be parasitized under the skin of mammals,causing hypodermosis,causing itching of the host animal,and further leading to worse mental health and reduced reproduction rate.In addition,the larvae will drill out of the skin and cause infection through a perforation of skin,the physiological activities will be serious impacted,and it even cause the death of the host.Therefore,from ecological perspective,that is a safe method to control Gansu zokor by using parasitism.The research on hypodermosis disease found that the characteristic antigen hypodermin can be detected by ELISA at the early stage of infection by warble flies.By analyzing the structure and function of Hypodermin,it is found that Hypodermin has three types,Hypodermin A,Hypodermin B and Hypodermin C,they all belongs to serine protease family,and they also have ability to degrade C3 complement and connective tissue,therefore,in the process of parasitism,Hypodermin can help it to escape from host immunity.According to above characteristics,using the effect of warble flies parasitism on zokors or using warble flies as a vector insect to carry the pathogenic pathogens of zokors,it provides a new idea for the biological control of zokors.In this study,the biological characteristics of the warble flies which are subcutaneous parasitism were observed.Based on the investigation of the biological characteristics of warble fly,species of this parasite was identified through morphological and molecular biological measure,and the biological characteristics and occurrence period of parasites were clariffied,these works would provide a basis for further controlling zokors by parasites.Secondly,transcriptome sequencing was used to analyze the Hypodermin gene and determine the key proteins in the parasitic process,select two genes to analyze the expression level in different stage of warble flies were analyzed by RT-q PCR.Finally,the four selected Hypodermin genes were bioinformatically analyzed to clarify their structural characteristics,it provides a theoretical basis for the later research clarification of the molecular mechanism of how warble fly parasiticed in zokor,and the interaction between the two species,provide basic data and theoretical basis for the later application of Hypodermin.The main results are as follows:1.Biological characteristics of parasites in Gansu zokor(1)The spawning period of the adult warble fly is from August to September.The firststage larvae mainly happened in September.The second-stage larvae mainly happened in October.The third-stage larvae often happened in November and lasts until the next year.(2)The larvae are mainly parasitic on the groin and lower back of Gansu zokor,and they also can be parasitic on the forelimb occasionally.The parasite developed directly into thirdstage larvae after parasitism,and did not migrate under the skin of the Gansu zokor.2.Morphology and molecular biology identification(1)Morphological identification: the surface of the spiracular plates of warble fly is flat without raised spines,spiracular opening are arranged on the spiracular plates.There are neatly arranged spines on the edge of the seventh section ventral surface,the edge of the seventh section has irregularly arranged small spines,compared with other warble flies,this warble fly should belong to Oestromyia leporina.(2)Molecular identification: through PCR-RFLP found that the subcutaneous parasites of Gansu zokor were the same species,the length of the highly variable sequence fragment of the COI gene amplified by primers UEA7-UEA10 is about 680 bp.According to the sequence comparison and analysis published in NCBI,the sequence is the closest to that of Oestromyia leporina,with a similarity of 96-97%.3.Transcriptome mining dermosin gene(1)Through RNA-Seq,four sequences with three species of Hypodermin gene were found,the are Hypodermin A,Hypodermin B,Hypodermin C1 and Hypodermin C2.(2)Through RT-q PCR,all Hypodermin genes are expressed in the first-stage larvae.The expression level of Hypodermin B gene are decreases with the development of larvae,the expression level of Hypodermin C gene are decreases in second-stage larvae,then increases after developed into mature third-stage larvae.(3)The Hypodermin gene of Oestromyia leporina belongs to the Tryp_SPc(pancreatic serine protease)superfamily.(4)By the evolutionary analysis based on Hypodermin gene sequence,,the kinship of Oestromyia leporina and Hypoderma diana are more closely related than other warble flies,then gathering with Hypoderma bovis and Hypoderma lineatum.