Identification and expression pattern of the sex determination gene fruitless-like in Cherax quadricarinatus.

The fruitless (fru) gene has an important function in the courtship behavior and sex determination pathway of Drosophila melanogaster; however, the fru gene has never been reported in shrimps. In this study, the fruitless-like gene was identified in Cherax quadricarinatus (Cqfru) and is reported here for the first time. A sequence analysis revealed a conserved BTB domain in Cqfru which is the same as fru in D. melanogaster. An analysis of the expression level of Cqfru showed that it was highly expressed in the gastrula stage during embryonic development. Furthermore, in situ hybridization and expression distribution in tissues showed that its sexually dimorphic expression may be focused on the hepatopancreas, brains, and gonads. The gonads, brains, and hepatopancreas of males had a higher expression level of Cqfru than those of females; however, the expression level of the abdominal ganglion was found to be higher in females than in males in this study. The results of an RNA interference treatment showed that a knockdown of Cqfru reduced the expression of the insulin-like androgenic gland hormone (IAG) and tumor necrosis factor (TNF). The characteristic fru gene in shrimps is reported here for the first time, with the results providing basic information for research into the sex-determination mechanism in C. quadricarinatus.

Single-Cell Ribonucleic Acid Sequencing Clarifies Cold Tolerance Mechanisms in the Pacific White Shrimp (Litopenaeus Vannamei).

To characterize the cold tolerance mechanism of the Pacific white shrimp (Litopenaeus vannamei), we performed single-cell RNA sequencing (scRNA-seq) of ∼5185 hepatopancreas cells from cold-tolerant (Lv-T) and common (Lv-C) L. vannamei at preferred and low temperatures (28°C and 10°C, respectively). The cells fell into 10 clusters and 4 cell types: embryonic, resorptive, blister-like, and fibrillar. We identified differentially expressed genes between Lv-T and Lv-C, which were mainly associated with the terms "immune system," "cytoskeleton," "antioxidant system," "digestive enzyme," and "detoxification," as well as the pathways "metabolic pathways of oxidative phosphorylation," "metabolism of xenobiotics by cytochrome P450," "chemical carcinogenesis," "drug metabolism-cytochrome P450," and "fatty acid metabolism." Reconstruction of fibrillar cell trajectories showed that, under low temperature stress, hepatopancreas cells had two distinct fates, cell fate 1 and cell fate 2. Cell fate 1 was mainly involved in signal transduction and sensory organ development. Cell fate 2 was mainly involved in metabolic processes. This study preliminarily clarifies the molecular mechanisms underlying cold tolerance in L. vannamei, which will be useful for the breeding of shrimp with greater cold tolerance.

Penaeus stylirostris densovirus proteins CP and NS1 interact with peritrophin of Litopenaeus vannamei.

The Penaeus stylirostris densovirus (PstDNV) is a major virus of shrimps that severely harms the shrimp farming industry. Peritrophin is a peritrophic membrane protein with chitin binding activity. To examine the roles of peritrophin in viral infection, we used yeast two-hybrid to analyze the interaction between the Pacific white shrimp (Litopenaeus vannamei) peritrophin and PstDNV proteins (CP, NS1 and NS2). The yeast two-hybrid results showed that NS1 and peritrophin had an interaction, CP and peritrophin had an interaction as well, and NS2 had no interaction with peritrophin. We validated the interactions with GST pull-down assays. We then conducted RNA interference and qRT-PCR. The results showed that when pre-injection of dsRNA-peritrophin, the quantity of PstDNV in the shrimps injected with viruses was significantly lower than in the control group (P < 0.01), indicating the viral infection was decreased when the peritrophin gene expression was inhibited. The results indicated that peritrophin of L. vannamei participated in the PstDNV infection.

The Penaeus stylirostris densovirus capsid protein interacts with the Litopenaeus vannamei BCCIP protein.

Viral capsid proteins play an important role in the viral infection process. To identify the cellular proteins in shrimp that interact with the Penaeus stylirostris densovirus capsid protein (PstDNV-CP), we constructed a yeast two-hybrid (Y2H) cDNA library of the muscle tissue of Litopenaeus vannamei, and hybridized the bait vector pGBKT7-CP with this library. Cloning and sequencing showed that the shrimp protein interacting with PstDNV-CP was a homolog of BRCA2 and CDKN1A(p21)-interacting protein (BCCIP). We named this protein L. vannamei BCCIP (LvBCCIP). Further analysis showed that LvBCCIP interacted with L. vannamei calmodulin (LvCaM). We validated the interactions between PstDNV-CP and LvBCCIP, and between LvBCCIP and LvCaM, with GST pulldown assays. The gene expression of LvBCCIP increased significantly after PstDNV challenge. In addition, the PstDNV titer of PstDNV-challenged shrimp was significantly reduced after LvBCCIP expression was inhibited using double-stranded RNA (dsRNA) interference. These results indicated that LvBCCIP is critical to PstDNV pathogenesis in L. vannamei. Interestingly, the growth rate of L. vannamei was significantly reduced when LvBCCIP gene expression was silenced, indicating that LvBCCIP may also be associated with growth regulation in L. vannamei. Thus, the interaction between PstDNV-CP and LvBCCIP might explain why PstDNV infection leads to runt-deformity syndrome in shrimp.

The Penaeus stylirostris densovirus capsid interacts with Litopenaeus vannamei troponin I.

The Penaeus stylirostris densovirus (PstDNV) (also known as infectious hypodermal and hematopoietic necrosis virus, IHHNV), a very small DNA virus, is a major shrimp pathogen. The PstDNV genome encodes only two nonstructural proteins and one capsid protein. This virus is thus an ideal, simple model for the investigation of virus-host interactions. To explore the role of the PstDNV capsid in viral infections, a yeast two-hybrid (Y2H) cDNA library was constructed based on Pacific white shrimp, Litopenaeus vannamei mRNA. The Y2H library was then screened, using the PstDNV capsid protein as bait. We identified a host protein that interacted strongly with the PstDNV capsid as L. vannamei troponin I (LvTnI). An in vitro co-immunoprecipitation experiment further supported this interaction. In addition, an in vivo neutralization experiment showed that the vaccination with anti-LvTnI significantly reduced PstDNV copies in PstDNV-challenged shrimp, indicating that the interaction between the PstDNV capsid and cellular LvTnI is essential for PstDNV infection. This result has important implications for our understanding of the mechanisms by which PstDNV infects shrimp.