Animals

Porcine circovirus type 4 (PCV4), first reported in 2019, lacks a stable in vitro culture system, limiting the application of traditional virus neutralization assays. Serological methods are therefore critical for PCV4 surveillance. The capsid (Cap) protein is the primary target for serological diagnosis, but its N-terminal nuclear localization signal (NLS) hinders prokaryotic expression. In this study, the NLS-truncated Cap gene was cloned into pET-22b(+) and expressed in Escherichia coliBL21(DE3). The recombinant NLS-truncated protein (~27 kDa) was purified by nickel-affinity chromatography and specifically recognized by PCV4-positive serum in Western blot. Mice immunized with the protein produced polyclonal antibodies with titers reaching 1:32,000. Using the purified recombinant NLS-truncated protein as coating antigen, an indirect ELISA was established and optimized. The assay showed no cross-reactivity with sera against pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), porcine circovirus type 1(PCV1), porcine circovirus type 2 (PCV2), or porcine circovirus type 3 (PCV3); the detection limit was 1:3200; and both intra- and inter-assay coefficients of variation were <10%. Large-scale surveillance of 1058 sera from four production stages revealed overall seroprevalences of 95.37% for PCV2, 38.85% for PCV3, and 3.50% for PCV4, with PCV2 + PCV3 co-seropositivity as the dominant mixed-exposure pattern (33.6%). These results demonstrate that the NLS-truncated PCV4 Cap protein retains full antigenicity and that the established indirect ELISA is a specific, sensitive, and reproducible tool for PCV4 serosurveillance and epidemiological investigation.

In crustaceans, spermatogenesis is regulated by endocrine and local factors, though many key regulators remain unknown. We identified a previously uncharacterized transcript enriched in male reproductive tissues of the Chinese mitten crab Eriocheir sinensis, encoding a predicted peptide precursor designated MRSS. MRSS encodes a 102-amino acid protein featuring an N-terminal signal peptide and nine C-terminal tandem DEDAA pentapeptide repeats. MRSS is abundantly expressed in the androgenic gland (AG) and accessory sex gland (AsG), with higher expression during the synthesis and secretory stages of AG development. RNAi-mediated knockdown of MRSS altered testicular histology, leading to abnormal vacuolization and abnormal chromatin morphology in spermatocytes. In the AG, MRSS knockdown reduced the expression of IAG and iDMY, while in the testis it downregulated the germ cell maintenance and cell cycle-related genes vasa, PCNA, and cyclin B. MRSS deficiency was also associated with increased apoptosis-related markers, as indicated by an increased Bax/Bcl2 ratio, more prominent TUNEL-positive signals in spermatocytes, and elevated caspase-3-like and caspase-9-like activities. Conversely, in vitro incubation of testes with recombinant MRSS (rMRSS) increased germ cell maintenance and cell cycle-related markers, and reduced apoptosis-related markers, including the Bax/Bcl2 ratio and caspase-3/9-like activities. Our results support a role for MRSS in maintaining testicular homeostasis, potentially in association with IAG expression. This study provides the first evidence of a male reproductive tissue-enriched transcript encoding a predicted peptide precursor involved in testicular structure homeostasis in decapod crustaceans.

Residual feed intake (RFI) measures feed efficiency after accounting for growth and body size. We examined its association with whole-blood gene expression in slow-growing ducks. Of 800 males phenotyped from 21 to 42 d of age, 527 had valid RFI estimates. Eighteen birds with comparable initial body weights were selected for RNA sequencing from low-RFI (LRFI), medium-RFI (MRFI), and high-RFI (HRFI) ranges, with six per group. Screening at |log₂ fold change| ≥ 1 and nominal p < 0.05 identified 707 nonredundant differentially expressed genes (DEGs). The LRFI–MRFI, LRFI–HRFI, and HRFI–MRFI comparisons retained 36, 205, and 6 genes, respectively, after Benjamini–Hochberg correction. Enrichment analysis implicated immune, transport, signaling, and metabolic processes. Weighted gene co-expression network analysis (WGCNA) of the nominal DEG set identified seven modules, five with exploratory associations with intake-related traits. Protein-interaction clustering retained 209 genes, of which 63 correlated strongly with RFI. Elastic Net analysis and repeated subsampling retained 25 genes with high selection stability within the cohort, from which nine named genes were prioritized. These blood transcriptional signals are discovery-stage candidates. These candidates require independent biological and predictive validation.