| With the rapid development of industry,more and more heavy metal,like cadmium,are emitted to the agricultural ecological environment,these cadmium may gradually accumulate in the body of insects,which will eventually produce certain toxic effects.Insects as a key component of global biodiversity,its influence can't be underestimated,therefore,more and more people have begun to pay close attention to the toxic effects of heavy metal cadmium on insects.Excessive intake of cadmium by insects has an effect on their growth and development.The accumulation process of heavy metal cadmium in insects is continuous and dynamic,and its accumulation level is affected by the length of time the insects are stressed,the amount of food intake and the ability of insects to excrete cadmium.Previous research works done on the common and harmful stored-grain pest Tribolium confusum focused mainly on its biology,ecology and control measures by using traditional methods,and studies based on whole genome and transcriptome levels have not been reported.In this study,T.confusum were used as experimental subjects,by feeding of the beetle with medium contaminated with cadmium,to study the effects of cadmium stress with different concentrations of the cadmium on the growth and development of T.confusum,and the content of cadmium in T.confusum body during different development stages.At the same time,transcriptome sequencing was performed using the Illumina Hiseq X Ten platform,and we analyzed the changes of gene expression and related metabolic pathways induced by cadmium stress in T.confusum,so the molecular response mechanisms of cadmium induced T.confusum were described.Finally,qRT-PCR was used to verify the expression levels of the 8 DEGs selected from the transcriptome data.This study can not only provide transcriptome information resources of T.confusum,it also helps to better understand the ecotoxicological effects of heavy metal cadmium.The main conclusions of this study are as follows:1.The third instar T.confusum larvae was placed in medium with different concentrations of cadmium,after feeding for 10 days,the results showed that in the group with higher cadmium content(experimental groups 4 and 5),the effects on the increasing rate of body weight,pupa weight,pupa length and pupation rate of T.confusum were significant(P<0.05).Overall,with the increase of cadmium concentration in medium,the increasing rate of body weight,pupa weight,pupa length and pupation rate of T.confusum were gradually declining,however,there was no significant difference in the increasing rate of body weight,pupa weight,pupa length and pupation rate of T.confusum in the groups with low cadmium(experimental groups 1,2 and 3)(P>0.05).2.The third instar T.confusum larvae was placed in medium with different concentrations of cadmium,after feeding,getting the result of heavy metal cadmium accumulation in the same development period of T.confusum is:with the increase of cadmium concentration in medium,the accumulation of cadmium in T.confusum of each experimental group was significantly higher than that of the control group(P<0.05),and there was a significant difference in the accumulation of cadmium between T.confusum of each experimental group(P<0.05).After the T.confusum were challenged by the same experimental group,the cadmium content in the larvae body increased obviously with the increase of treatment time,there were significant differences in cadmium content in the larvae on the 3rd and 10th day after exposure to cadmium(P<0.05).Compared with larval stage,the content of cadmium in pupae of T.confusum decreased significantly(P<0.05).At the beginning of eclosion,the content of cadmium in adults decreased compared with the pupae(P<0.05).Adults on the 1st day after eclosion to the 7th day after eclosion,the cadmium content in the body decreased significantly with the increase of time(P<0.05).3.According to the transcriptome sequencing of two different treatment groups of T.confusum-six samples(control group and experimental group 4),we obtained 42.77 Gb clean data,the bases percentage of Q30 for more than 87.95%,and the GC content for each sample was approximately 40%.After splicing and de novo assembly,we gained a total of 84,404 All-Unigene with an N50 length of 1,579 bp and a mean length of 927.80 bp.We have annotated the 84,404 All-Unigene,through blasting against various database,totaling 32,013 Unigene can be matched with above databases,accounting for 37.93%of the total All-Unigene.And 11,149?13,775?12,678?19,497?22,410?15,256?26,991?28,137 All-Unigene were annotated in the COG?GO?KEGG?KOG?Pfam?Swissprot?eggNOG?NR database,respectively.Based on NR database queries,All-Unigene's annotation results showed a high proportion of T.confusum sequences had high similarity with sequences of Tribolium castaneum(43.06%).According to the analysis of All-Unigene,we obtained 65,454 CDS,The most CDS were 100-300 bp(21,786,33.28%);the least CDS were over 2000 bp(4,013,6.14%).4.In this study,a total of 4,250 differentially expressed genes were found between two groups of samples,and 1,529 DEGs were annotated by GO functional classification:in the three major categories of biological process,cellular component and molecular function,the number of annotated DEGs were 1,288?594 and 1,319,respectively.KEGG pathway Analysis of DEGs between the two groups of samples showed that:there were 1,198 DEGs involved in 233 pathway.In the KEGG pathway annotation,the Ribosome,Protein processing in endoplasmic reticulum,Glycolysis/Gluconeogenesis,Oxidative phosphorylation,Peroxisome and ABC transporters and so on were significantly enriched.5.Further analysis of several important enrichment pathways found that,in the oxidative phosphorylation pathway,a number of genes related to the electron transport chain complexes were up-regulated,promoted the increase of production capacity suggests that T.confusum incurs energetic expenditures resulting from the necessity to facilitate metal elimination or repair of toxicant-induced damage.In this study,the increasing rate of body weight of T.confusum significantly decreased after stressed caused by cadmium,which may affected by this abnormal energy metabolism.In the protein processing in endoplasmic reticulum pathway,a number of genes like Hsp70,Hsp40,Hsp90 were up-regulated,therefore,it is speculated that HSP genes may be a kind of important cadmium stress response genes of T.confusum,and it plays a crucial role in the path of cadmium stress repair.We also highlighted some genes related ATP-binding cassette transporters including ABCB?ABCC?ABCD and ABCG which were detected simultaneously to be up-regulated in ABC transporters pathway,it has been reported that ABC transporters are associated with the resistance of arthropods,this might be the reason that ABC families were found to be significantly up-regulated in our results.6.The qRT-PCR was used to carry out experimental verification of 8 DEGs.The experimental results were consistent with the results of RNA-Seq,which proved the reliability of the transcriptome data. |
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