| Objective:To investigate the parasites of the blood-sucking species S.quinquestriatum in Guiyang,to study the effects of parasitic infections on the development and histopathology of S.quinquestriatum larvae,and to preliminarily explore the changes in gene expression levels of S.quinquestriatum parasitized by mesomermis with RNA-sequencing.Methods:From October to November in 2015,2018,2019,and 2020,S.quinquestriatum larvae and pupae were collected in the channel(26.333424°N,106.671725°E,1036.87 meters)beside the farmland of Qingyan,Guiyang.Black flies larvae and pupae were incubated in the laboratory to obtain adults,which were fixed with 95%ethanol and their species were identified through morphological filming.The parasite-infected larvae were selected from the late-instar larvae and the types of parasites were infected.The post-parasitic juvenile of mesomermis were cultivated in the laboratory,and the morphological characteristics of the adults were observed by scanning electron microscope,where the taxonomic index were measured,the structure of the mesomermis and vegetative bodies was understood with histological filming,and the mesomermis species were identified.The morphology of microsporidia was observed with Giemsa staining and the morphological structure of the xenomas was observed with paraffin slides and HE staining,which helped identify the species of microsporidia.Meanwhile,the histopathological effects of the parasitism on the host were also studied.Infected and healthy larvae were subjected to RNA-sequencing,with three biological replicates.RNA-sequencing was performed on the mesomermis infected S.quinquestriatum larvae and the healthy control group larvae with 3 repeated experiments,and then the bioinformatics analysis was performed on sequencing results.Results:(1)S.(S.)quinquestriatum,S.(S.)bidentatum,S.(S.)qianense,S.(S.)sakishimaense,S.(W.)xingyiense and S.(O.)ornatum were identified in Qingyan,among which S.quinquestriatum occupied about a half of the population and was the dominant local species.(2)A total of 3,343 S.quinquestriatum larvae were screened,357 parasitized by mesomermis,with an infection rate of 5.079%?13.35%;118parasitized by microsporidia,with an infection rate of 1.870%?8.889%;8 larvae were parasitized by Coelomycidium simulii,with an infection rate of 0%?0.496%.The infection rate of mesomermis is the highest.Multiple symbiotes were also discovered.Since the number of C.simulii collected was too small,no follow-up study was conducted.(3)The experiments identified that the mesomermis in S.quinquestriatum collected from Qingyan belongs to a new species,named Mesomermis qingyanese sp.nov.The mesomermis has 6 papillae;its mouth locates at the end of the body;its allelopathys are large and round;the length of its esophageal tube is not as long as the middle part of its body,and the tube end goes deep into its epidermis;it has 2 rows of row cells.The vagina of female mesomermis is short pear-shaped,and the vagina tube is V-type bend.The male mesomermis has 2 sickle-shaped spicules.The post-parasitic mesomermis have sharp cone-shaped appendages.According to the thickness of its epidermis,the length of the male spicules(about 204 ?m),the number and arrangement of reproductive papillae,Mesomermis qingyanese sp.nov.was distinguished from other species of the same genus.(4)The parasitized mesomermis in S.quinquestriatum will interfere with the normal development of the host larvae,making it difficult for the larvae to pupate and leading to significant retardation of adult disc development,massive destruction of adipose tissue,and loss or abnormal development of the gonads.(5)Three species of microsporidia were found in S.quinquestriatum.Based on the characteristics of the spores and cysts of the larvae,Thelohania,Caudospora and Pleistophora were identified and named as Thelohania sp1,Caudospora sp1 and Pleistophora sp1.Thelohania sp1 parasitizes to form syncytial xenomas,while Caudospora sp1 and Pleistophora sp1 form neoplastic xenomas.(6)The process of microsporidia(Pleistophora sp1 as a representative)infecting the host can be divided into three stages: infective phase?proliferative phase and sporogenic phase.The sequence of tissue parasitism is: midgut?fat body,muscle?martensian tube,silk gland,gonad,nerve etc.A larg e number of microsporidian was seen in the parasitized tissues and organs,where the nucleus was hypertrophy,the parasitic vesicles formed,the gap between intestinal epithelial cells in the digestive tract is widened,and the intestinal wall of the digestive tract was irregularly broken.The adipose,glandular,and muscle tissue are damaged.(7)A total of 39.88 Gb transcriptomics data was obtained.After assembly and de-redundancy,65,404 unigene were obtained,with the total length,average length,N50 and GC contents of50,409,279 bp,770 bp,1,208 bp and 43.77%.Respectively,unigene annotated with seven functional databases,and the final results were 37,647(NR: 57.56%)?8,342(NT: 12.75%)?29,840(Swiss Prot: 45.62%)?29,936(KOG: 45.77%)?30,945(KEGG: 47.31%)?27,357(GO: 41.83%)and 28539(Pfam: 43.63%)unigene received functional annotations.A total of 437 genes with significant differences between healthy and parasitic larvae were detected.243 genes were up-regulated and194 genes were down-regulated.Those with significant down-regulation were mainly annotated to genes related to pupal chitin-based cuticle development and endopeptidase inhibitor activity.Conclusions:(1)The investigation found that S.quinquestriatum larvae were parasitized by 1species of nematode(new species: Mesomermis qingyanese sp.nov.),3 species of microsporidia(Thelohania sp1,Caudospora sp1 and Pleistophora sp1)and C.simulii.(2)The parasitized mesomermis and microsporidia in Qingyan has negative impact on the normal growth and development of S.quinquestriatum,where mesomermis mainly cause pupation disorder,fat body destruction and gonads dysplasia,and microsporidia mainly cause pathological changes in the tissues including fat body,digestive tract,muscle,gonads,etc.(3)After the S.quinquestriatum larvae were parasitized by Mesomermis qingyanese sp.nov.,the expression of related unigene such as pupal chitin-based cuticle development proteins and endopeptidase inhibitor activity proteins were down-regulated,suggesting that further research can be performed on the parasitic response of the mesomermis in metamorphosis development and immune response. |
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