Studies On The Selection Mechanisms Of Oviposition Substrate Of Gasterophilus Pecorum From The Released Przewalski’s Horse(Equus Przewalskii)

After Przewalski’s horse(Equus przewalskii),an endangered species,was reintroduced to its country of origin at Kalamaili Nature Reserve(KNR)for ungulates in Xinjiang,infection by Gasterophilus spp.became an increasingly serious issue.Gasterophilus pecorum is the dominant species in KNR,which differs from the Gasterophilus spp.in the rest of the world among which G.intestinalis is the dominant species.G.pecorum oviposits on shoot tips of Stipa caucasica in KNR,whereas other eight,including G.intestinalis,lay eggs on equine hairs.The selection of the original oviposition substrate by G.pecorum is one of the key factors for the long-term severe infection by G.pecorum of Przewalski’s horse in KNR.The study was based on the special oviposition substrate of G.pecorum,a series of experiments were carried out by means of insect ecology,animal and plant physiology and molecular biology: the correlation between the morphological or biological indexes and the fecundity of G.pecorum was analyzed by traditional biological methods,and the factors influencing the fecundity of G.pecorum were identified;the habitat odour(volatiles from Przewalski’s horse feces)and host odour(S.caucasica volatiles)were collected by dynamic headspace adsorption and determined by automatic thermal desorption gas chromatography-mass spectrometry,and the main and unique volatile components were identified;the olfactory genes of G.pecorum and G.intestinalis were screened by transcriptome technology to clarify the difference and relationship between them.The main results are as follows:1.The weight of the larvae,pupae and adults of G.pecorum was significantly different,and the weight of female was significantly higher than that of the male;the weight loss rate during three stages of G.pecorum was significantly different,the weight loss rate of male was significantly higher than that of female;the larvae weight was positively correlated with the length and width of pupae and adult;the morphological indexes(weight,length and width)of larvae and pupae were significantly positively correlated with empty pupal weight,but there was no correlation between weight in three stages and adult longevity;the morphological indexes in three stages were significantly positively correlated with fecundity of G.pecorum,while pupal duration and adult longevity were not correlation with fecundity.2.The five volatiles with the highest relative contents(top5 volatiles)of fresh feces at the three ovipositing stages of G.pecorum,toluene,1,6-octadiene,3,7-dimethyl-,(S)-,and caprolactam were common to the three stages and acetic acid common to the pre-oviposition and oviposition stages,whereas 1,5,9-undecatriene,2,6,10-trimethyl-,(Z)-,ammonium acetate and butanoic acid,and benzene,2-propenyl-were unique to the pre-oviposition,oviposition,and post-oviposition stages,respectively;and the top5 volatiles of feces with different freshness at the oviposition stage of G.pecorum,caprolactam was common to the three feces and butanoic acid common to the fresh and semi-fresh feces,whereas toluene,1,6-octadiene,3,7-dimethyl-,(S)-,and ammonium acetate were unique to the fresh feces,acetophenone,acetic acid and 1,5,9-undecatriene,2,6,10-trimethyl-,(Z)-unique to the semi-fresh feces,and 3,4-dihydroxybenzyl alcohol,tris(trimethylsilyl)-,nonanal,decanal,and decane,2,2-dimethyl-unique to the dry feces.Particularly,ammonium acetate was the only specific volatile of fresh feces at the oviposition stage,which will require further investigation.The species and relative contents of volatiles varied greatly in semi-fresh and dry feces compared with fresh feces,and changes of dry feces were the largest.3.The top5 volatiles from oviposition and non-oviposition plants of G.pecorum,caprolactam and3-hexen-1-ol,(Z)-were common to all samples.Caprolactam was highest in Ceratoides latens at oviposition stage of G.pecorum and lowest in S.caucasica at postoviposition stage,and that of3-hexen-1-ol,(Z)-was lowest in C.latens and highest in S.caucasica at its oviposition stage.Particularly,in S.caucasica during the three oviposition phenological stages of G.pecorum,3-hexen-1-ol,acetate,(Z)-,2(5H)-furanone,5-ethyl-,and 3-hexen-1-ol,acetate,(E)-were unique,respectively,to the preoviposition,oviposition,and postoviposition stages;in three plant species during the oviposition stage of G.pecorum,3-hexen-1-ol,acetate,(Z)-,3-hexenal,and 1-hexanol were unique to S.orientalis,acetic acid,hexanal,and 2(5H)-furanone,5-ethyl-to S.caucasica,and1,3,6-octatriene,3,7-dimethyl-,cis-3-hexenyl isovalerate,and acetic acid hexyl ester to C.latens;in S.caucasica,2-undecanone,6,10-dimethyl-was unique to the early growth period,acetic acid and2(5H)-furanone,5-ethyl-to the flourishing growth period,and 3-hexen-1-ol,acetate,(Z)-and1,3,6-octatriene,3,7-dimethyl-to the late growth period.Furthermore,substances specific to S.orientalis and C.latens were also present in S.caucasica,except at oviposition stage.4.71 olfactory genes were identified from the transcriptome of G.pecorum,including 26odorant-binding proteins(OBPs),3 chemosensory proteins(CSPs),4 sensory neuron membrane proteins(SNMPs),7 odorant receptors(ORs),20 ionotropic receptors(IRs)and 11 gustatory receptors(GRs);95 olfactory genes were identified from the transcriptome of G.intestinalis,including 23 OBPs,2 CSPs,5 SNMPs,20 ORs,30 IRs and 15 GRs.The specific ORs of Gasterophilus spp.were identified by phylogenetic tree,the homology of 49.2% olfactory genes could not be compared between G.pecorum and G.intestinalis,indicating that olfactory genes were very different between them.The study focused on the special oviposition pattern of G.pecorum in S.caucasica,clarifying the important effects of different weight,length and width on the fecundity of G.pecorum in arid desert steppe,screening out the unique and common components of volatiles from Przewalski’s horse feces and S.caucasica during oviposition location of G.pecorum,indicating the differences in the amounts and homology of olfactory genes between G.pecorum and G.intestinalis.The mechanism that G.pecorum oviposit on S.caucasica were elucidated in the study,providing a reference for predicting the reproductive potential of G.pecorum,providing theoretical basis for the development of high efficiency attractant to trap G.pecorum,providing a molecular basis for behavior control of olfactory sensing mechanism of G.pecorum.