Study On The Response Of Aleuroglyphus Ovatus(Troupeau)to Cadmium Stress And Regulation Of JH-Met Signaling Pathway On Reproduction

Cadmium(Cd)is the food contaminant to be listed by the Food and Agriculture Organization(FAO)and World Health Organization(WHO),which not only pollutes the ecosystem,but also can be transmitted to human beings through the food chain enrichment.Routine environmental monitoring equipment and chemical analysis methods cannot effectively assess the chronic toxicity caused by sustained long-term exposure to heavy metals,and can not more clearly reflect the comprehensive impact of heavy metal environmental pollution on the organisms and ecosystems,so biomonitoring is essential.Aleuroglyphus ovatus is widely found in stored grain,which closely related to environmental heavy metal pollution and changes in the ecosystem structure.The response of mites to long-term cadmium stress was studied for the first time,and the sensitivity of this biological model to cadmium toxicity was evaluated.Reproduction is an important indicator of biological monitoring,and vitellinogen receptor VgR and juvenile hormone receptor Met genes were cloned by RT-PCR and RACE in female adult mites.RT-qPCR,RNAi and juvenile hormone analogue treatment were used to study the role of juvenile hormone via methoprenetolerant in the regulation of reproduction in A.ovatus.At the same time,the molecular mechanism of cadmium effects in A.ovatus was discussed on the reproduction.These studies can provide a basis for evaluating the toxic effects of heavy metal pollution,and also provide a new reference for using Vg,VgR or Met of mites as biomarkers to monitor heavy metal pollution in the future.The main study results are presented as follows:1.Effects of heavy metal cadmium on the growth,development and reproduction of A.ovatus.After short-term or long-term cadmium treatment,A.ovatus developed slowly and its developmental period was prolonged.Long-term cadmium stress would negatively affect the reproduction of female adult mites of A.ovatus,prolonging the preoviposition period,shortening the period after oviposition,and decreasing the total and daily egg laying of each female.And long-term cadmium stress would inhibit the growth rate and reproductive potential of the population,which was reflected in decreasing the net proliferation rate,intrinsic growth rate and weekly growth rate gradually with the increase of Cd2+stress concentration.And the average generation time and population doubling time were significantly prolonged.2.Antioxidant response of Aleuroglyphus ovatus under long-term cadmium stress.The antioxidant enzyme activity of A.ovatus under long-term cadmium stress showed a downward trend.If Cd2+ concentration reached more than 10 mg/kg,the superoxide dismutase(SOD)activity of nymph and adult mites were significantly reduced.The peroxidase(POD)and catalase(CAT)activity in the mites are reduced under long-term cadmium stress during the whole developmental stage.Under different cadmium concentration treatments,the glutathione S-transferase(GST)activity showed no significant difference at the immature stage comparing with the control group,and the GST activity was significantly decreased in adult mites.With the increase of cadmium stress concentration,the malondialdehyde(MDA)content gradually increased at the immature stage.And when Cd2+ concentration reached more than 10 mg/kg,the SOD,POD,CAT,GST activity and MDA content of male adults are all significantly higher than that of female mites.3.AoVgR were obtained and the mRNA expression analysis of AoVgR and AoVg.The 3567 bp sequences of AoVgR were obtained which had the classical functional domain of LDLR family,a transmembrane domain,and an internalization signal NPFY.The protein encoded by AoVgR gene belonged to a secretory protein with the signal peptide sequence“MGCKLKPALLFICCLAAL QTAHLVKS”.It had seven glycosylation sites including NCTS,NNTS,NLSQ,NRTC,NLTG,NHSD,NNSS,and 101 phosphorylation sites.Phylogenetic analysis found the VgRs of all species were branched according to their respective biological classifications,the species belonging to Araneae and Acariformes were also clustered in independent clades,indicating AoVgR had a good evolutionary conservation.The expression of AoVg and AoVgR genes were low in larva and protonymph mites.The expression would gradually increase during the other developmental stages,and the transcript level of both genes would increase rapidly in adult mites.Long-term cadmium stress inhibited the expression of AoVg and AoVgR genes.4.Regulation of reproduction by juvenile hormone receptor.The 1868 bp sequences of AoMet were obtained which had a typical conserved domain of the bHOLH-PAS family.The AoMet amino acid sequences had 79 phosphorylation sites and no glycosylation sites.The phylogenetic tree of AoMet and bHOLH-PAS family proteins(Met,ARNT,Clk)from other species was constructed using NJ method.AoMet was clustered together with the species of Arachnida,and converged on a small branch with Met of Neoseiulus barkeri.The expression of AoMet gene were both higher in the protonymph and tritonymph of A.ovatus,but lower in the egg,larva,and female mites.The expression of AoMet gene was higher in female adults than male adults.Under long-term stress of cadmium,the expression of AoMet gene was significantly reduced,but the expression of AoMet gene gradually decreased with the increase of cadmium concentration,indicating that long-term cadmium stress will significantly inhibit the expression of AoMet gene.RNA interference with AoMet gene was found to inhibit the expression of both AoVg and AoVgR genes,prolong the pre-oviposition period,shorten the oviposition period,and reduce the number of eggs laid.Thus it is suggested that long-term cadmium stress affects the reproduction of A.ovatus by inhibiting the expression of AoMet gene.5.Reproductive regulation of JH-Met signaling in A.ovatus.After the short-term treatment of newly eclosion female mites with juvenile hormone analogue,the expression of AoMet,AoVg and AoVgR genes would be upregulated,and the egg production increased,indicating that JH is involved in regulating AoMet gene and affecting the reproduction of A.ovatus.The titer of JH decreased under long-term cadmium stress in A.ovatus,the total egg production and expression levels of AoMet AoVg and AoVgR genes did not increase after short-term treatment with JHA,indicating that long-term cadmium stress decreased the juvenile hormone titer,shortterm addition of JHA could not change the reproductive situation.It is confirmed that long-term cadmium stress may have serious negative impacts on JH-Met regulation.