Cadmium (Cd) is one of the dominant metal pollutants present in the aquatic environment that affects ion homeostasis, oxidative stress (OS) and immune responses of aquatic organisms. Given the physicochemical similarities between Cd²⁺ and calcium (Ca²⁺) ions, their antagonism may facilitate the mitigation of Cd-induced toxicity. To better understand the role of Ca in protecting against Cd-induced toxicity in teleosts, juvenile grass carp were exposed to Cd (measured concentration 3 μg/L) and a gradient of Ca concentrations (measured concentration 1.5 mg/L, 2.5 mg/L, 3.0 mg/L, and 3.5 mg/L in the control (CTL) group, low calcium (LCA) group, medium calcium (MCA) group, and high calcium (HCA) group, respectively) for 30 days. Inductively coupled plasma mass spectrometry (ICP-MS) data analyses showed that simultaneous exposure to Ca impaired the accumulation of Cd in all tested tissues. Besides, Ca addition maintained the plasma ion (Na⁺, K⁺, Cl^-) homeostasis, alleviated Cd-induced oxidative stress (OS), and regulated the activities and transcriptional levels of ATPase. Furthermore, transcriptional heatmap analysis demonstrated that several indicator genes for OS and calcium signaling pathway were found to be significantly modulated by Ca addition. This work delineates a protective effect of Ca against Cd-induced toxicity in grass carp, providing new insight into the possible solutions to Cd pollution issues in aquaculture industry.

SUMMARY: Actin depolymerizing factors regulate actin dynamics involved in cellular processes such as morphogenesis, motility, development and infection. Here, a novel actin depolymerizing factor gene (CiADF₂) was cloned from the cDNA library ofCryptocaryon irritans, a parasitic ciliate causing cryptocaryonosis. The full-length cDNA ofCiADF₂was 531 bp. Its open reading frame (ORF) was 417 bp, encoding a polypeptide of 138 aa with typical features of the ADF/cofilin family. Reverse transcription-PCR suggested thatCiADF₂is expressed in all stages of the life cycle. After site-directed mutagenesis of a non-universal genetic code, the ORF was subcloned inEscherichia coli. The bacteria were induced with the addition of isopropylthio-β-D-galactoside to express a fusion protein of recombinant CiADF₂(rCiADF₂) with glutathioneStransferase. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot confirmed the predicted molecular mass of rCiADF₂of 16·2 kDa. A mouse antibody against rCiADF₂recognized native CiADF₂, and rCiADF₂reacted with mouse antisera againstC. irritanstrophonts. CiADF₂was abundant in the plasma around cytostomes, suggesting that CiADF₂is involved in ciliate movement. Moreover, rCiADF₂showed F-actin binding and depolymerizing activity. This study will help to clarify the pathogenic biology of the parasite and develop effective control measures for cryptocaryonosis.

The hybrid offspring of barbel chub Squaliobarbus curriculus and grass carp Ctenopharyngodon idella exhibit stronger resistance to the grass carp reovirus (GCRV) infection than grass carp. Toll-like receptors (TLRs) play indispensable roles in the antiviral immunity of fish. In this study, the structures and antiviral immune functions of barbel chub TLR19 (ScTLR19) and grass carp TLR19 (CiTLR19) were compared. The amino acid sequence of ScTLR19 shared high similarity (97.4%) and identity (94.0%) with that of CiTLR19, and a phylogenetic tree revealed the close evolutionary relationship between ScTLR19 and CiTLR19. Protein domain composition analyses showed that ScTLR19 possessed an additional leucine-rich repeat (designated as LRR9) located at amino acid positions 654–677 in the extracellular region, which was absent in CiTLR19. Multiple sequence alignment and three-dimensional structure comparison also indicated that the extracellular regions of ScTLR19 and CiTLR19 exhibited greater differences compared to their intracellular regions. Molecular docking revealed that the extracellular region of ScTLR19 (docking score = −512.31) showed a stronger tendency for binding with polyI:C, compared to the extracellular region of CiTLR19 (docking score = −474.90). Replacing LRR9 in ScTLR19 with the corresponding amino acid sequence from CiTLR19 reduced the binding activity of ScTLR19 to polyI:C, as confirmed by an ELISA. Moreover, overexpression experiments suggested that ScTLR19 could regulate both the IRF3–TRIF and IRF3–MyD88 signaling pathways during GCRV infection, while CiTLR19 only regulated the IRF3–MyD88 signaling pathway. Importantly, replacing LRR9 in ScTLR19 with the corresponding amino acid sequence from CiTLR19 altered the expression regulation on IRF3, MyD88, and TRIF during GCRV infection. These findings collectively reveal the structural and functional differences between ScTLR19 and CiTLR19, and they may provide data to support a deeper understanding of the molecular mechanisms underlying the differences in GCRV resistance between barbel chub and grass carp, as well as the genetic basis for the heterosis of GCRV resistance in their hybrid offspring.