Identification, diversity, and evolution analysis of Commd gene family in Haliotis discus hannai and immune response to biotic and abiotic stresses.

The Commd (Copper Metabolism gene MURR1 Domain) family genes play crucial roles in various biological processes, including copper and sodium transport regulation, NF-κB activity, and cell cycle progression. Their function in Haliotis discus hannai, however, remains unclear. This study focused on identifying and analyzing the Commd genes in H. discus hannai, including their gene structure, phylogenetic relationships, expression profiles, sequence diversity, and alternative splicing. The results revealed significant homology between H. discus hannai's Commd genes and those of other mollusks. Both transcriptome quantitative analysis and qRT-PCR demonstrated the responsiveness of these genes to heat stress and Vibrio parahaemolyticus infection. Notably, alternative splicing analysis revealed that COMMD2, COMMD4, COMMD5, and COMMD7 produce multiple alternative splice variants. Furthermore, sequence diversity analysis uncovered numerous missense mutations, specifically 9 in COMMD5 and 14 in COMMD10. These findings contribute to expanding knowledge on the function and evolution of the Commd gene family and underscore the potential role of COMMD in the innate immune response of H. discus hannai. This research, therefore, offers a novel perspective on the molecular mechanisms underpinning the involvement of Commd genes in innate immunity, paving the way for further explorations in this field.

Comparative transcriptome analysis reveals sex bias in expression patterns of genes related to sex steroids and immunity in the skin of spinyhead croaker Collichthys lucidus.

Fish skin is the first barrier against external invasion, and also an important interface for communication between males and females during reproduction. Nonetheless, sexual dimorphism in the physiology of fish skins is still poorly understood. Herein, transcriptomes of skin were comparatively analysed between males and females in spinyhead croaker, Collichthys lucidus. Totally, 170 differentially expressed genes (DEG) were detected, including 79 female-biased genes and 91 male-biased genes. Gene ontology (GO) annotation items of the DEGs were mainly enriched in biological process items (86.2%), including regulation of biological processes, responses to chemical and biological stimuli, transport and secretion, movement, immune response, tissue development, etc. In KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis, the male-biased genes were enriched in pathways including those related to immunity such as the TNF signalling pathway and IL-17 signalling pathway, whereas the female-biased genes were enriched in pathways including those related to female steroids such as ovarian steroidogenesis and oestrogen signalling pathway. In addition, odf3 was found to be a male-specific expression gene, being a candidate marker for phenotypic sex. Thus, the sexual difference in gene expression in fish skin in spawning season was uncovered by transcriptome analysis for the first time, providing new insights into sexual dimorphism in the physiology and functions of fish skin.

Genome-wide identification and transcriptome-based expression profiling of the Sox gene family in the spinyhead croaker (Collichthys lucidus).

Sox genes encode transcription factors with a high-mobility group (HMG) box, playing critical roles in the initiation and maintenance of a variety of developmental processes, such as sex determination and differentiation. In the present study, we identified 26 Sox genes in the genome of spinyhead croaker Collichthys lucidus (Richardson, 1844) with homology-based analysis of the HMG box. The transcriptome-based expression profiles revealed that the expression of the Sox gene in gonads began to differ between sexes when the body length was 2.74 ± 0.24 cm. At that time, three Sox genes (Sox11b, Sox8a and Sox19) were significantly upregulated, accompanied by the downregulation of 12 Sox genes in the ovary, and six Sox genes were temporarily significantly upregulated in the testis. Afterwards, the expression profile of Sox genes changed only with a small amplitude in both the ovary and testis. For adult tissues, huge differences were observed in the expression profiles of Sox genes between ovaries and testes, as well as small differences in somatic tissues between sexes. These results provide clues to further decipher the role of Sox genes in the processes of sex determination and differentiation in spinyhead croaker and other teleosts.

Investigating the Impact of Humic Acid on Copper Accumulation in Sinonovacula constricta Using a Toxicokinetic-Toxicodynamic Model.

In aquatic ecosystems, the interaction between heavy metals and dissolved organic carbon (DOC) plays a pivotal role in modifying the bioavailability of these metals. This study, employing a toxicokinetic-toxicodynamic model, delves into the interactive effects of humic acid (HA), a significant component of DOC, on the bioaccumulation and toxicity of copper (Cu) in the estuarine economic bivalve Sinonovacula constricta. Utilizing the stable isotope 65Cu as a tracer, we evaluated Cu uptake in S. constricta under varied DOC concentrations in a controlled laboratory setting. Our findings reveal that at DOC concentrations below 3.05 mg L-1, the bioavailability of Cu is reduced due to shifts in the speciation distribution of Cu, resulting in decreased bioaccumulation within S. constricta. Conversely, at DOC levels exceeding 3.05 mg L-1, the formation of colloidal Cu-HA complexes allows its entry into the bivalves' digestive system. Moreover, toxicity assays demonstrate an increase in S. constricta survival rates with higher DOC concentrations, suggesting a protective effect of DOC against Cu toxicity. The integration of accumulation and toxicity data infers that Cu-HA complexes, when ingested via the digestive tract, exhibit lower toxicity compared to Cu directly assimilated from the water phase. These findings emphasize the need to consider environmental DOC levels in assessing Cu pollution risks and provide insights for managing heavy metal toxicity in estuarine aquaculture.

Comparative Responses of Orange-Foot and Common-Foot Haliotis gigantea to Carotenoid-Enriched Diets: Survival, Heat Tolerance, and Bacterial Resistance.

Carotenoids, known to enhance survival, heat tolerance, and bacterial resistance, play an essential role in the nutrition of economically important aquatic animals. This study specifically examined their impact as feed additives on the abalone Haliotis gigantea. We prepared 13 compound feeds with varying levels of astaxanthin, zeaxanthin, and ß-carotene, and administered them to both common-footed and orange-footed H. gigantea. The survival rate of H. gigantea was about 70-80%, with no significant differences in survival observed among the various carotenoid-supplemented feeding groups or when compared with the control group, nor between orange-footed and common-footed individuals. In heat attachment duration experiments, orange-foot abalones exhibited longer attachment durations with certain concentrations of astaxanthin and zeaxanthin, whereas common-foot abalones showed extended durations with astaxanthin, zeaxanthin, and ß-carotene, indicating that common-foot abalones might benefit more from these carotenoids. Additionally, our results showed similar patterns and levels of Vibrio harveyi AP37 resistance in both orange-footed and common-footed H. gigantea, suggesting a uniform response to carotenoid supplementation in their bacterial defense mechanisms. This study suggests the potential benefits of carotenoid supplementation in H. gigantea and contributes to the theoretical basis for developing high-quality artificial compound feeds.

Characterization of a RacGTPase up-regulated in the large yellow croaker Pseudosciaena crocea immunity.

The Rac proteins are members of the Rho family of small G proteins and are implicated in the regulation of several pathways, including those leading to cytoskeleton reorganization, gene expression, cell proliferation, cell adhesion and cell migration and survival. In this investigation, a Rac gene (named as LycRac gene) was obtained from the large yellow croaker and it was expressed in Escherichia coli and purified. Subsequently the specific antibody was raised using the purified fusion protein (GST-LycRac). Moreover, the GTP-binding assay showed that the LycRac protein had GTP-binding activity. The LycRac gene was ubiquitously transcribed and expressed in 9 tissues. Quantitative real-time RT-PCR and Western blot analysis revealed the highest expression in gill and the weakest expression in spleen. Time-course analysis revealed that LycRac expression was obviously up-regulated in blood, spleen and liver after immunization with polyinosinic polycytidynic acid (poly I:C), formalin-inactive Gram-negative bacterium Vibrio parahemolyticus and bacterial lipopolysaccharides (LPS). These results suggested that LycRac protein might play an important role in the immune response against microorganisms in large yellow croaker.

Molecular cloning and expression of MyD88 in large yellow croaker, Pseudosciaena crocea.

Myeloid differentiation factor 88 (MyD88) is an adaptor protein involved in the interleukin-1 receptor and Toll-like receptor-induced activation of nuclear factor-kappaB (NF-kappaB). In this report, the full-length cDNA of MyD88 was cloned from the large yellow croaker, Pseudosciaena crocea. It was of 1574 bp, including a 5'-terminal untranslated region (UTR) of 89 bp, a 3'-terminal UTR of 621bp and an open reading frame (ORF) of 864 bp encoding a polypeptide of 287 amino acids. It contained a typical death domain at the N-terminal and a conservative Toll/IL-1R (TIR) domain structure at the C-terminal. The quantitative real-time reverse transcription PCR analysis revealed a broad expression of MyD88 with the highest expression in the spleen and the weakest expression in the muscle. The expression of MyD88 after challenge with formalin-inactivated Gram-negative bacterium Vibrio parahaemolyticus was tested in blood, spleen and liver. It suggested that the highest expression was in the spleen (p<0.05) with 1.9 times (at 48 h) as much as that in the control and the lowest expression of MyD88 was in the liver (p<0.05) with 0.29 times (at 3h) of that in the control. These results indicated that as a universal key adaptor in the Toll-like receptor pathway in mammals, MyD88 might play an important role in large yellow croaker defense against pathogenic infection.

Chromosome assembly of Collichthys lucidus, a fish of Sciaenidae with a multiple sex chromosome system.

Collichthys lucidus (C. lucidus) is a commercially important marine fish species distributed in coastal regions of East Asia with the X1X1X2X2/X1X2Y multiple sex chromosome system. The karyotype for female C. lucidus is 2n = 48, while 2n = 47 for male ones. Therefore, C. lucidus is also an excellent model to investigate teleost sex-determination and sex chromosome evolution. We reported the first chromosome genome assembly of C. lucidus using Illumina short-read, PacBio long-read sequencing and Hi-C technology. An 877 Mb genome was obtained with a contig and scaffold N50 of 1.1 Mb and 35.9 Mb, respectively. More than 97% BUSCOs genes were identified in the C. lucidus genome and 28,602 genes were annotated. We identified potential sex-determination genes along chromosomes and found that the chromosome 1 might be involved in the formation of Y specific metacentric chromosome. The first C. lucidus chromosome-level reference genome lays a solid foundation for the following population genetics study, functional gene mapping of important economic traits, sex-determination and sex chromosome evolution studies for Sciaenidae and teleosts.

Microsatellite-centromere mapping in large yellow croaker (Pseudosciaena crocea) using gynogenetic diploid families.

The large yellow croaker (Pseudosciaena crocea) is an economically important marine fish in China. Inheritance of 22 heterozygous microsatellite loci was examined in normal crossed diploid families and meio-gynogenetic families in P. crocea. Two gynogenetic families were produced via inhibition of the second polar body in eggs fertilized with UV-irradiated sperm. The ratio of gynogenesis was proven to be 100% and 96.9% in the two families, respectively. Of the 22 examined loci, 4 showed a segregation distortion in both control and gynogenetic families. Microsatellite-centromere (M-C) map distances were examined using 18 loci with normal Mendelian segregation. Estimated recombination rates ranged between 0 and 1.0 under the assumption of complete interference. High recombinant frequencies between heterozygous markers and the centromere were found in large yellow croaker, as in other teleosts. The average recombination frequency was 0.586. Ten loci showed high M-C recombination with frequency greater than 0.67. M-C distances provide useful information for gene mapping in large yellow croaker.

{[1-(Arylmethyl)piperidin-4-yl]oxy}-(trifluoromethyl)-pyridines: ketanserin analogues with insect growth regulating activity.

A series of {[1-(arylmethyl)piperidin-4-yl]oxy}-(trifluoromethyl)-pyridine derivatives were designed and synthesized on the basis of the ketanserin (1) framework, a prototypic mammalian 5-HT(2A) receptor antagonist, and the structure-activity relationship (SAR) was also discussed. The result of the bioassay showed that most of the title compounds inhibited the insect growth and exhibited moderate-to-good growth regulating activity against the armyworm Pseudaletia separata Walker. Furthermore, the SAR study revealed that, when the determinant feature, interacting with mammalian 5-HT(2A) receptor, was preserved, a simplified ArCH(2) group greatly contributed to insect growth inhibitory activities. It was also found that the substituted position of the CF(3) group at the pyridine ring played a key role, and that the introduction of 1-[bis(4-fluorophenyl)methyl]piperazine, an equivalent of the benzoylpiperidine moiety of ketanserin, resulted in bioactivities similar to those of the title compounds, which were in agreement with the model of ketanserin analogues binding to mammalian 5-HT(2) receptors.

Nanos3 not nanos1 and nanos2 is a germ cell marker gene in large yellow croaker during embryogenesis.

In this study, three nanos gene subtypes (Lcnanos1, Lcnanos2 and Lcnanos3) from Larimichthys crocea, were cloned and characterized. We determined the spatio-temporal expression patterns of each subtype in tissues as well as the cellular localization of mRNA in embryos. Results showed that deduced Nanos proteins have two main homology domains: N-terminal CCR4/NOT1 deadenylase interaction domain and highly conserved carboxy-terminal region bearing two conserved CCHC zinc-finger motifs. The expression levels of Lcnanos1 in testis were significantly higher than other tissues, followed by heart, brain, eye, and ovary. Nevertheless, both Lcnanos2 and Lcnanos3 were restrictedly expressed in testis and ovary, respectively. No signals of Lcnanos1 and Lcnanos2 expression were detected at any developmental stages during embryogenesis. On the contrary, the signals of Lcnanos3 were detected in all stages examined. Lcnanos3 transcripts were firstly localized to the distal end of cleavage furrow at the 2-cell stage. Subsequently, mounting positive signals started to appear in a small number of cells as the embryo developed to blastula stage and early-gastrula stage. As development proceeded, positive signals were found in the primitive gonadal ridge. These cells of Lcnanos3 positive signals implied the specification of the future PGCs at this stage. It also suggested that PGCs of croaker originate from four clusters of cells which inherit maternal germ plasm at blastula stage. Furthermore, we preliminarily analyzed the migration route of PGCs in embryos of L. crocea. In short, this study laid the foundation for studies on specification and development of germ cell from L. crocea during embryogenesis.

Molecular cloning and expression of Octamer-binding transcription factor (Oct4) in the large yellow croaker, Larimichthys crocea.

Octamer-binding transcription factor 4 (Oct4) is a crucial pluripotent transcription factor in controlling pluripotency of embryonic stem cells (ESCs), formation of primordial germ cells (PGCs), early embryonic development, and gonadal germ cells. To understand the roles of Oct4 in the large yellow croaker (Larimichthys crocea) (Lc-Oct4), the full-length cDNA of Oct4 was cloned and analyzed. Lc-Oct4 includes a 104-bp 5' untranslated region (UTR), a 567-bp 3' UTR and a 1431-bp coding region encoding a protein of 476 amino acids (aa) with a predicted molecular mass (Mm) of 52.40 kDa and an isoelectric point (PI) of 6.34. Quantitative real time PCR (qRT-PCR) in tissues showed that Lc-Oct4 was only expressed in ovary. During ovarian development, the expression level in 635 days post hatching (635-dph) ovary was about 6.3-fold higher than in 270-dph and 1000-dph ovary. The in situ hybridization analysis showed that Lc-Oct4 had a high expression in the different developmental stages of oocytes with the highest level in stages II (later) and III oocytes. Lc-Oct4 was also expressed in spermatogonia and primary spermatocytes. Furthermore, the qRT-PCR analysis of Lc-Oct4 in different developmental embryos revealed that the expression from high to low was multiple-cell stage > mid-blastula stage = mid-gastrula stage, and the expression in multiple-cell stage was at less 1.6-fold higher than in other stages. Accordingly, the whole mount in situ hybridization (WISH) in embryos demonstrated that Lc-Oct4 was expressed only in early embryonic development from 2-cell stage to early-gastrula stage with the peak at 16-cell stage, but not detected in PGCs area. In conclusion, Lc-Oct4 participated in the oogenesis and early embryonic development in large yellow croaker. Overall, this study provided better understanding of Lc-Oct4 gene and lay the foundation for its function research in the large yellow croaker.

Characterization and Spatiotemporal Expression of Klf4 in Large Yellow Croaker Larimichthys crocea.

As one of transcription factors and pluripotency factors, Klf4 plays a crucial role in regulation of cellular processes. In this article, we characterized Klf4 of large yellow croaker (Lc-Klf4), which encodes a 452-amino acid protein (Lc-Klf4) with three highly conserved C2H2 zinc fingers. Lc-Klf4 shares high conservative functions in teleosts with the closest relationship with Stegastes partitus. The spatiotemporal expression showed that Lc-Klf4 was expressed widely in adult tissues with gender difference as follows: brain>gill>eye>heart in female; heart>testis>gill>brain in male; male>female in heart, gill, and testis; and female>male in eye. During growth, the highest expression level of Lc-Klf4 was at 635 dph (days posthatching) in testis and at 270 dph and 635 dph in brain. Besides, Lc-Klf4 was widely and highly distributed in different developmental spermatids especially in spermatocytes. The expression of Lc-Klf4 in embryos was exhibited zygotically beginning from late gastrula stage with high level in closure of blastopore stage and appearance of optic vesicle stage. This expression pattern was supported by whole-mount in situ hybridization with high expression in back and head of late embryos. In conclusion, the spatiotemporal expression patterns of Lc-Klf4 illustrated that Klf4 involves in spermatogenesis, embryogenesis, and adult physiological processes.

Molecular cloning, characterization, and expression analysis of a heat shock protein (HSP) 70 gene from Paphia undulata.

In this study, a full-length HSP70 cDNA from Paphia undulata was cloned using reverse transcriptase polymerase chain reaction (RT-PCR) coupled with rapid amplification of cDNA ends (RACE). The full-length cDNA is 2,351 bp, consisting of a 5'-untranslated region (UTR) of 83 bp, a 3'-UTR of 315 bp, and an open reading frame (ORF) of 1,953 bp. This cDNA encodes 650 amino acids with an estimated molecular weight of 71.3 kDa and an isoelectric point of 5.51. Based on the amino acid sequence analysis and phylogenetic analysis, this HSP70 gene was identified as a member of the cytoplasmic HSP70 family, being the constitutive expression, and it was designated as PuHSC70. The distribution of PuHSC70 mRNA in the mantle, digestive gland, adductor muscle, gonad, gill, heart, and hemocytes suggested that PuHSC70 is ubiquitously expressed. The mRNA levels of PuHSC70 under high temperature and high salinity stresses were analyzed by real-time PCR. Under high temperature stress of 32°C, PuHSC70 mRNA in the mantle, digestive gland, gill, and heart was significantly up-regulated at 1h and 2h, and it was then progressively down-regulated. In the adductor muscle, the level of PuHSC70 mRNA gradually increased throughout the study period; the mRNA levels in the gonad and hemocytes increased significantly at 4h and 8h (P<0.05) and then decreased at 8h and 14 h, respectively, however they increased again afterwards, reaching the highest levels at 50h. Under high salinity (32 ‰) stress, the mRNA levels of PuHSC70 in the mantle and gonad were increased significantly only at 24h and 48 h (P<0.05), and at the rest of the study period they were slightly elevated. Compared with the pretreatment level, the levels of expression in the digestive gland and gill were unchanged or reduced throughout the study period. The levels of PuHSC70 mRNA in the adductor muscle, hemocytes, and heart were significantly increased, reaching a maximum at 24h, and then they gradually decreased; moreover, in the heart, the mRNA expression recovered to the pretreatment level at 50h; while in the adductor muscle and hemocytes, the expression level remained higher than that of the control. The cloning and expression analyses of PuHSC70 provide theoretical basis to further study the mechanism of physiological response to thermal and high salinity stresses.

Design and synthesis of novel insecticides based on the serotonergic ligand 1-[(4-aminophenyl)ethyl]-4-[3-(trifluoromethyl)phenyl]piperazine (PAPP).

1-[(4-Aminophenyl)ethyl]-4-[3-(trifluoromethyl)phenyl]piperazine (PAPP) is a 5-HT(1A) agonist and was reported to display high affinity for serotonin (5-HT) receptor from the parasitic nematode Haemonchus contortus . The present investigation explored the possibility of using PAPP as a lead compound of new insecticides with novel mode of action. On the basis of the PAPP scaffold, a series of 1-arylmethyl-4-[(trifluoromethyl)pyridin-2-yl]piperazine derivatives were designed, synthesized, and evaluated for biological activities against the armyworm Pseudaletia separata (Walker). Bioassays showed that most of the target compounds displayed certain growth-inhibiting activities or larvicidal activities against armyworm. The quantitative structure-activity relationship (QSAR) for growth-inhibiting activities was also analyzed and established.

Cloning and expression analysis of two alternative splicing toll-like receptor 9 isoforms A and B in large yellow croaker, Pseudosciaena crocea.

Toll-like receptors (TLRs) are the archetypal pattern-recognition receptors in sensing foreign pathogens. In this report, two alternative splicing isoforms of TLR9 (named PcTLR9A and PcTLR9B) cDNA were cloned from the large yellow croaker, Pseudosciaena crocea. The full-length cDNA of PcTLR9A was of 3637bp, including a 5'-terminal untranslated region (UTR) of 111bp, 3'-terminal UTR of 355bp and an open reading frame (ORF) of 3171bp encoding a polypeptide of 1056 amino acids. However, the full-length cDNA of PcTLR9B was 119bp longer than that of PcTLR9A from the position of 3079-3197bp, which encoded a polypeptide of 1006 amino acids. Both of the PcTLR9A and PcTLR9B contained 12 typical structures of leucine-rich repeats (LRRs), an LRRTYP and an LRRCT in the extracellular region and a conservative Toll/IL-1R (TIR) domain in the intracellular region. However, compared to PcTLR9A, conservative Box 3 was absent in PcTLR9B TIR domain. Quantitative real-time reverse transcription PCR analysis revealed a broad expression of PcTLR9A and PcTLR9B with the highest expression in spleen and the weakest expression in muscle. Expression of PcTLR9A and PcTLR9B after stimulation with formalin-inactivated Gram-negative bacterium Vibrio parahaemolyticus was tested in blood, spleen and liver. This indicated that the highest expression was 3.3 times (at 24h) as much as that in the control in the spleen (p<0.05) and the lowest expression of PcTLR9A was 1/4 times (at 3h) of that in the control in the liver (p<0.05). PcTLR9B showed a similar expression pattern to PcTLR9A post-injection. These results suggested that both PcTLR9A and PcTLR9B might play an important role in large yellow croaker defense against pathogenic infection.