| Marsupenaeus,a member of Crustacea,Decapoda,and Penaeidae,is an important commercial species.Traditionally,M.japonicus was regarded as the only species of Marsupenaeus,and now there is no distinction in production.Work to date suggests that Marsupenaeus has two morphologically similar varieties,which exhibit wide variation in many aspects,including their phenotypes,hepatopancreas index,seminal vesicles,growth tare,temperature tolerance,and molecular sequences(mitochondria and nuclear genes).A comprehensive understanding of the genetic differences between the two forms can provide scientific basis and genomic data for the identification of effective species,germplasm resources and genetic breeding of M.japonicus.This study focused on major industrial and scientific issues,including germplasm resources and evolutionary adaptation mechanism of M.japonicus along the coast of China.By using genotyping by sequencing and comparative transcriptomics technology,the fine population genetic structure of two closely related Marsupenaeus species was analyzed from the aspects of population structure,gene character,physiology and biochemistry,and the evolutionary adaptation of immune defense and hypoxia tolerance.Finally,the comprehensive response mechanism of M.japonicus under air exposure stress was investigated.The main results of the study are as follows:1.The population genetic structure based on a single copy orthologous geneIn the study,the length of caspase gene was 1050 bp,and 24 variable sites were detected,including 20 parsimony informative sites.There were 57 haplotypes.The haplotype diversity was 0.837?0.947,and the nucleotide diversity was between 0.0086?0.0129,which showed up the high haplotype diversity and low nucleotide diversity.The haplotype network and SAMOVA analysis showed that the wild populations of M.japonicus were significantly divided into two groups.The pairwise genetic distance and Fst values between varieties was higher than that within the variety.The gene flow values were less than 1,which showed that two marsupenaeus species have weak exchange.The values of Tajima's and Fu'Fs test were not significant.The nucleotide mismatch distribution was distinct multi-peak distribution,which showed that the wild populations of M.japonicus experienced population expansion.2.Fine-scale population genetic structure based on genotyping-by-sequencing and interbreeding experimentsThe kuruma shrimp(Marsupenaeus japonicus)includes two cryptic species,which are distributed mostly allopatrically but co-occur in the northern South China Sea(from Huilai to Beihai).To obtain a better understanding of the fine-scale genetic structure and parapatric diversification of these two varieties in the northwestern Pacific region,we used a genotyping-by-sequencing(GBS)approach to establish their phylogenetic relationships.Using the GBS technique,we genotyped 28891 SNPs in 160 individuals in the Northwest Pacific.The results supported two highly diverged evolutionary lineages of kuruma shrimp(var.? and ?).The ND and XM populations showed complex genetic patterns,which might be affected by the complex environment of the Taiwan Strait.In addition,the migration rates and inbreeding coefficients of XM and BH were much lower than those of the other populations,which might be related to the land-sea changes and complex ocean currents in the Taiwan Strait and Qiongzhou Strait.Choice and no-choice interbreeding experiments provided support for the biological species concept,by showing the existence of reproductive isolation or incompatibility.In view of these differences between the two Marsupenaeus species,we believe that it is essential and urgent to establish a genetic database for each and reevaluate their ecological suitable conditions in order to improve species-specific culturing techniques.3.Evolutionary adaptation analysis of immune defense and hypoxia tolerance in two closely related Marsupenaeus species based on comparative transcriptomicsKuruma shrimp,a major farmed shrimp species in the world,includes two cryptic or sibling species,form I and form II.Due to the lack of genomic resources,little is known about the molecular mechanisms associated with immune defense and hypoxia tolerance.Here,we sequenced the transcriptomes of two closely related Marsupenaeus species and compared genomic divergence.This study obtained 77049 and 84561 unigenes with N50 values of 1281 bp and 1244 bp for form ? and form ?,respectively,and 5036 pairs of putative orthologs were identified between two Marsupenaeus species.Estimation of Ka/Ks ratios indicated that 165 orthologous genes may be under positive selection(Ka/Ks>0.5),including 49 pairs with a Ka/Ks ratio>1.According to the peak of synonymous rates,the divergence time between form ? and form ? was about 0.26-0.69 Mya.These positively selected orthologous genes related to the immune process mainly comprised single VWC domain protein,legumain,ras-related C3 botulinum,caspase,C-type lectin and were enriched in functions related to immune(Toll-like receptor and PI3K-Akt signaling)and hypoxia signaling(HIF-1 signaling and VEGF signaling).In this study,dozens of caspase-like unigenes were screened from two Marsupenaeus transcriptomes.Among these,the PjCaspase orthologous gene was subjected to positive selection(Ka/Ks=1.22),which had different secondary and threedimensional structure prediction.We characterized the transcriptomes of the two Marsupenaeus species and obtained several key orthologs associated with immune defense and hypoxia tolerance,which provides new insights into the immunity and genetic divergence of the two varieties.Moreover,this study will facilitate further,comparative genomic studies of the two varieties.4.Comparative analysis of the codon usage patterns in two closely related Marsupenaeus species based on comparative transcriptomicsUsing the CodonW 1.4.2 software,we performed codon bias analysis of two marsupenaeus species transcriptomes.The average contents of GC were 51.61%and 51.54%for form ? and form ?,respectively.Parity Rule 2(PR2)plot analysis showed that purines(A and G)were used more frequently than pyrimidines(C and T)in two marsupenaeus species.The average ENc value were 52.1 and 52.22 for form ? and form?,respectively.Overall,orthologous genes that underwent positive selection(?>1)had a higher correlation coefficient than that experienced purifying selection(?<1).In the form ?,the relationships were highly significant positive between Axis 1 and A3,T3 and ENc(p<0.01).However,all relationships in the form ? were completely opposite.We determined 12 and 14 optimal codons for form I and form ?,respectively.Two marsupenaeus species had 31 different codon pairs.The results of multi-species clustering based on codon preference are consistent with those of traditional classification.5.Air exposure affects physiological responses,innate immunity,apoptosis and DNA methylation of Kuruma shrimp,Marsupenaeus japonicusAir exposure stress is a common phenomenon for commercial crustacean species in aquaculture and during waterless transportation.However,the antioxidant responses to air exposure discussed in previous studies may be insufficient to present the complexities involved in this process.The comprehensive immune responses,especially considering the immune genes,cell apoptosis,and epigenetic changes,are still unknown.Accordingly,we investigated the multifaceted responses of M.japonicus to air exposure.The results showed that the expression profiles of the apoptosis genes(e.g.,IAP,TXNIP,caspase,and caspase-3)and the hypoxia-related genes(e.g.,hsp70,hif-1?,and HcY)were all dramatically induced in the hepatopancreas and gills of M.japonicus.Heart rates,mortality rate,T-AOC(Total antioxidant capacity)and lactate contents showed time-dependent changes upon air exposure.Air exposure significantly induced apoptosis in hepatopancreas and gills tissues.The apoptosis index(AI)of the 12.5 h experimental group increased significantly(p<0.05)in the hepatopancreas and gills.In the experimental group(EG,12.5 h),most individuals had lower methylation ratios than the control group(CG).Air exposure markedly reduced the full-methylation and total-methylation ratios(31.39%for the CG and 26.46%for the EG).This study provided a comprehensive understanding of the antioxidant responses of M japonicus considering its physiology,innate immunity,apoptosis,and DNA methylation levels,and provided theoretical guidance for waterless transportation. |
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