Toxicological Effects And Mechanisms Of Cadmium On The Wolf Spider Pardosa Pseudoannulata (Araneae: Lycosidae)

The wolf spider Pardosa pseudoannulata?Araneae:Lycosidae?is one of the most common species of wandering spiders in agricultural fields.They act as one of the most important predatory enemies against pest insects and could be a potentially good bioindicator for heavy metal contamination.Cadmium?Cd?is a widespread toxic pollutant in the environment,which presents serious threat to the ecosystem and human health.In the present study,we studied the ecotoxicological effects and mechanisms of Cd on P.pseudoannulata,which would provid theoretical basis for protecting and using the spiders as natural enemies of pests and bioindicator of heavy metals in the fields.Main results were summarized as follows:1.The bioaccumulation of Cd in adult P.pseudoannulata.Adult spiders were provided with 0.2 and 2 mM CdCl₂ solution as their drinking water,the Cd concentration were measured with an inductively coupled plasma optical emission spectrometer after the spiders were exposed to the CdCl₂ solutions for 0,7,14,21 or 28 days.The Cd content in P.pseudoannulata adults increased significantly with the number of days of exposure to a 0.2mM CdCl₂ solution?p<0.05?.When exposed to 2 mM CdCl₂ solution,the Cd content in the spiders increased sharply in the first two?male?or three?female?weeks,and then no significant changes were recorded following with the next three?male?or two?female?weeks exposure?p>0.05?.Cd body burden was significantly lower in females than in males when exposed to 0.2 mM solution for 7 and 28 days,as well as to 2 mM solution for 14,21 and 28days.2.The effects of Cd on the growth,development and reproduction of P.pseudoannulata.Adult spiders were provided with 0.2 and 2 mM CdCl₂ solution as their drinking water,spiderlings were exposured to 0.04 mM?0.2 m M?1 mM and 2 mM CdCl₂ solution in the same way,and all spiders were fed with adults of Drosophila melanogaster.The mortality rates of the spiders and spiderlings,as well as the growth,development and reproduction of spiderlings were recorded.The mortality rate of both adult males and females rose significantly with increased concentration of the CdCl₂ solution?p<0.05?,and the mortality rates of adult males was significantly higher than that of females in 2 mM CdCl₂ solution.Exposure of spiders to Cd contaminated drinking water resulted in reduced body mass,delayed development,fewer eggs and increased mortality of the spiderlings?p<0.05?,but there was no difference between the 0.2 and 1 mM CdCl₂ solutions in their effects on the developmental duration,body mass,eggs deposition of the spiders?p>0.05?.3.The effects of Cd on the activities of antioxidant enzymes of P.pseudoannulata.Adult spiders were provided with 0.2 and 2 mM CdCl₂ solution as their drinking water,the superoxide dismutase?SOD?,catalase?CAT?and glutathione S-transferase?GST?activities in the spiders were measured after the exposure for 0,7,14,21 or 28 days.SOD,CAT and GST activities in P.pseudoannulata adults were increased significantly after a 7 day exposure in both sexes treated with 0.2 mM CdCl₂ solution?p<0.05?and then reverted to the control level.No significant difference in SOD and CAT activities in either sex was found in comparison with the controls when the adults were exposed to 2 mM CdCl₂ solution?p>0.05?.GST activity increased significantly after a 7 day exposure,reached its lowest level after 21 days of exposure,and then reverted to the control level after 28 days of exposure in both sexes when exposed to 2 mM CdCl₂ solution.4.The impacts of Cd on the gene expression at transcriptional level of P.pseudoannulata.Female adult spiders were exposured to 0.2 and 2 mM CdCl₂ solution for 7days.High-throughput transcriptome sequencing was employed to characterize the de novo transcriptome.We obtained 60489 assembled unigenes,18773 of which were annotated in the public databases.A total of 2939 and 2491 differentially expressed genes?DEGs?were detected between the libraries of two Cd-treated groups and the control.When spiders were treated with 0.2 and 2 mM CdCl₂ solution,“metabolic process”and“catalytic activity”were significantly enriched Gene Ontology?GO?and topGO terms?KS<0.05?.“carbohydrate transport and metabolism”,“amino acid transport and metabolism”and“relication,recombination and repair”were significantly enriched in Clusters of Orthologous Groups?COG?classification.“lysosome”,“phagosome”as well as the pathways associated with nutrients and energy metabolism were significantly enriched under Kyoto Encyclopedia of Genes and Genomes?KEGG?pathways type classification.The results indicated the metabolism and digestion processes were seriously affected by Cd,at the same time,cell damage and associated responses were induced at the cellular and molecular level.More DEGs were detected in 0.2 mM Cd-treated spiders compared with 2 mM Cd-treated ones,this indicated that the activities of the enzymes may inhibited with Cd.Certain critical genes involved in defence and detoxification,such as transmembrane transporters,signal transduction molecules,heat shock proteins and antioxidant enzymes may play important roles in response to Cd stress.5.Cloning and sequence analysis of genes encoding detoxifying enzymes GST,gamma-Glutamyltransferase?GGT?and dimethylaniline monooxygenase?FMO?.The open reading frame?ORF?of ppGST is 654 bp and the deduced protein sequence is 217 amino acids with a calculated molecular mass of 24.90 kDa and isoelectric point?PI?of 5.98,and it is belong to delta subtype,with typical N terminal and C terminal domain of protein GST.The ppFMO included an ORF of 1611 bp encoding a putative protein of 536 amino acids.The calculated molecular mass and PI of ppFMO are 61.77 kDa and 9.02.The deduced protein FMO of P.pseudoannulata is belong to FMO5 subtype,with typical NADB_Rossmann domain of protein FMO.Additionally,the coding region of ppGGT is 1038 bp consisting of448 amino acids.Multiple sequence alignments and phylogenetic analysis indicated that the deduced amino acid sequences of P.pseudoannulata GST,GGT and FMO showed high homology to the corresponding sequences of Parasteatoda tepidariorum and Stegodyphus mimosarum.