Due to the great advantages in selection accuracy and efficiency, genomic selection (GS) has been widely studied in livestock, crop and aquatic animals. Our previous study based on one full-sib family of Litopenaeus vannamei (L. vannamei) showed that GS was feasible in penaeid shrimp. However, the applicability of GS might be influenced by many factors including heritability, marker density and population structure etc. Therefore it is necessary to evaluate the major factors affecting the prediction ability of GS in shrimp. The aim of this study was to evaluate the factors influencing the GS accuracy for growth traits in L. vannamei. Genotype and phenotype data of 200 individuals from 13 full-sib families were used for this analysis. In the present study, the heritability of growth traits in L. vannamei was estimated firstly based on the full set of markers (23 K). It was 0.321 for body weight and 0.452 for body length. The estimated heritability increased rapidly with the increase of the marker density from 0.05 K to 3.2 K, and then it tended to be stable for both traits. For genomic prediction on the growth traits in L. vannamei, three statistic models (RR-BLUP, BayesA and Bayesian LASSO) showed similar performance for the prediction accuracy of genomic estimated breeding value (GEBV). The prediction accuracy was improved with the increasing of marker density. However, the marker density would bring a weak effect on the prediction accuracy after the marker number reached 3.2 K. In addition, the genetic relationship between reference and validation population could influence the GS accuracy significantly. A distant genetic relationship between reference and validation population resulted in a poor performance of genomic prediction for growth traits in L. vannamei. For the growth traits with moderate or high heritability, such as body weight and body length, the number of about 3.2 K SNPs distributed evenly along the genome was able to satisfy the need for accurate GS prediction in the investigated L.vannamei population. The genetic relationship between the reference population and the validation population showed significant effects on the accuracy for genomic prediction. Therefore it is very important to optimize the design of the reference population when applying GS to shrimp breeding.

Crustins are crustacean cationic cysteine-rich antimicrobial peptides that contain one or two whey acidic protein (WAP) domain(s) at the carboxyl terminus and mainly show antimicrobial and/or proteinase inhibitory activities. Here, we performed genome and transcriptome screening and identified 34 full-length crustin-like encoding genes in Litopenaeus vannamei. Multiple sequence analysis of the deduced mature peptides revealed that these putative crustins included 10 type Ia, two type Ib, one type Ic, 11 type IIa, three type IIb, four type III, one type IV, one type VI, and one type VII. These putative crustins were clustered into different groups. Phylogenetic analysis, considering their domain composition, showed that different types of crustin-like genes in crustaceans might be originated from the WAP core region, along with sequence insertion, duplication, deletion, and amino acid substitution. Tissue distribution analysis suggested that most crustin-like genes were mainly detected in immune-related tissues while several crustin-like genes exhibited tissue-specific expression patterns. Quantitative PCR analysis on 15 selected crustin-like genes showed that most of them were apparently upregulated after Vibrio parahaemolyticus or white spot syndrome virus (WSSV) infection. One type Ib crustin-like gene, mainly expressed in the ovary, showed the highest expression levels before the gastrula stage and was hardly detected after the limb bud stage, suggesting that it was a maternal immune effector. Collectively, the present data revealed the molecular and functional diversity of crustins and their potential evolutionary routes in crustaceans.

Acute hepatopancreatic necrosis disease (AHPND) has caused a heavy loss to shrimp aquaculture since its outbreak. Vibrio parahaemolyticus (VP_AHPND) is regarded as one of the main pathogens that caused AHPND in the Pacific white shrimp Litopenaeus vannamei. In order to learn more about the mechanism of resistance to AHPND, the resistant and susceptible shrimp families were obtained through genetic breeding, and comparative transcriptome approach was used to analyze the gene expression patterns between resistant and susceptible families. A total of 95 families were subjected to VP_AHPND challenge test, and significant variations in the resistance of these families were observed. Three pairs of resistant and susceptible families were selected for transcriptome sequencing. A total of 489 differentially expressed genes (DEGs) that presented in at least two pairwise comparisons were screened, including 196 DEGs highly expressed in the susceptible families and 293 DEGs in the resistant families. Among these DEGs, 16 genes demonstrated significant difference in all three pairwise comparisons. Gene set enrichment analysis (GSEA) of all 27,331 expressed genes indicated that some energy metabolism processes were enriched in the resistant families, while signal transduction and immune system were enriched in the susceptible families. A total of 32 DEGs were further confirmed in the offspring of the detected families, among which 19 genes were successfully verified. The identified genes in this study will be useful for clarifying the genetic mechanism of shrimp resistance against Vibrio and will further provide molecular markers for evaluating the disease resistance of shrimp in the breeding program.

A mutant of the ridgetail white prawn, which exhibited rare orange-red body color with a higher level of free astaxanthin (ASTX) concentration than that in the wild-type prawn, was obtained in our lab. In order to understand the underlying mechanism for the existence of a high level of free astaxanthin, transcriptome analysis was performed to identify the differentially expressed genes (DEGs) between the mutant and wild-type prawns. A total of 78,224 unigenes were obtained, and 1863 were identified as DEGs, in which 902 unigenes showed higher expression levels, while 961 unigenes presented lower expression levels in the mutant in comparison with the wild-type prawns. Based on Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes analysis, as well as further investigation of annotated DEGs, we found that the biological processes related to astaxanthin binding, transport, and metabolism presented significant differences between the mutant and the wild-type prawns. Some genes related to these processes, including crustacyanin, apolipoprotein D (ApoD), cathepsin, and cuticle proteins, were identified as DEGs between the two types of prawns. These data may provide important information for us to understand the molecular mechanism of the existence of a high level of free astaxanthin in the prawn.

Crustins are a kind of antimicrobial peptide (AMP) that exist in crustaceans. Some crustins do not have direct antimicrobial activity but exhibit in vivo defense functions against Vibrio. However, the underlying molecular mechanism is not clear. Here, the regulatory mechanism was partially revealed along with the characterization of the immune function of a type I crustin, LvCrustin I-2, from Litopenaeus vannamei. LvCrustin I-2 was mainly detected in hemocytes, intestines and gills and was apparently up-regulated after Vibrio parahaemolyticus infection. Although the recombinant LvCrustin I-2 protein possessed neither antibacterial activity nor agglutinating activity, the knockdown of LvCrustin I-2 accelerated the in vivo proliferation of V. parahaemolyticus. Microbiome analysis showed that the balance of intestinal microbiota was impaired after LvCrustin I-2 knockdown. Further transcriptome analysis showed that the intestinal epithelial barrier and immune function were impaired in shrimp after LvCrustin I-2 knockdown. After removing the intestinal bacteria via antibiotic treatment, the phenomenon of impaired intestinal epithelial barrier and immune function disappeared in shrimp after LvCrustin I-2 knockdown. This indicated that the impairment of the shrimp intestine after LvCrustin I-2 knockdown was caused by the dysbiosis of the intestinal microbiota. The present data suggest that crustins could resist pathogen infection through regulating the intestinal microbiota balance, which provides new insights into the functional mechanisms of antimicrobial peptides during pathogen infection.