A Testis-Specific DMRT1 (Double Sex and Mab-3-Related Transcription Factor 1) Plays a Role in Spermatogenesis and Gonadal Development in the Hermaphrodite Boring Giant Clam Tridacna crocea.

The testis-specific double sex and mab-3-related transcription factor 1 (DMRT1) has long been recognized as a crucial player in sex determination across vertebrates, and its essential role in gonadal development and the regulation of spermatogenesis is well established. Here, we report the cloning of the key spermatogenesis-related DMRT1 cDNA, named Tc-DMRT1, from the gonads of Tridacna crocea (T. crocea), with a molecular weight of 41.93 kDa and an isoelectric point of 7.83 (pI). Our hypothesis is that DMRT1 machinery governs spermatogenesis and regulates gonadogenesis. RNAi-mediated Tc-DMRT1 knockdown revealed its critical role in hindering spermatogenesis and reducing expression levels in boring giant clams. A histological analysis showed structural changes, with normal sperm cell counts in the control group (ds-EGFP) but significantly lower concentrations of sperm cells in the experimental group (ds-DMRT1). DMRT1 transcripts during embryogenesis exhibited a significantly high expression pattern (p < 0.05) during the early zygote stage, and whole-embryo in-situ hybridization confirmed its expression pattern throughout embryogenesis. A qRT-PCR analysis of various reproductive stages revealed an abundant expression of Tc-DMRT1 in the gonads during the male reproductive stage. In-situ hybridization showed tissue-specific expression of DMRT1, with a positive signal detected in male-stage gonadal tissues comprising sperm cells, while no signal was detected in other stages. Our study findings provide an initial understanding of the DMRT1 molecular machinery controlling spermatogenesis and its specificity in male-stage gonads of the key bivalve species, Tridacna crocea, and suggest that DMRT1 predominantly functions as a key regulator of spermatogenesis in giant clams.

Identification and involvement of DAX1 gene in spermatogenesis of boring giant clam Tridacna crocea.

DAX1 (dosage-sensitive sex reversal, adrenal hypoplasia congenital critical region on X chromosome gene 1), a key sex determinant in various species, plays a vital role in gonad differentiation and development and controls spermatogenesis. However, the identity and function of DAX1 are still unclear in bivalves. In the present study, we identified a DAX1 (designed as Tc-DAX1) gene from the boring giant clam Tridacna crocea, a tropical marine bivalve. The full length of Tc-DAX1 was 1877 bp, encoding 462 amino acids, with a Molecular weight of 51.81 kDa and a theoretical Isoelectric point of 5.87 (pI). Multiple sequence alignments and phylogenetic analysis indicated a putative ligand binding domain (LBD) conserved regions clustered with molluscans DAX1 homologs. The tissue distributions in different reproductive stages revealed a dimorphic pattern, with the highest expression trend in the male reproductive stage, indicating its role in spermatogenesis. The DAX1 expression data from embryonic stages shows its highest expression profile (P < 0.05) in the zygote stage, followed by decreasing trends in the larvae stages (P > 0.05). The localization of DAX1 transcripts has also been confirmed by whole mount in situ hybridization, showing high positive signals in the fertilized egg, 2, and 4-cell stage, and gastrula. Moreover, RNAi knockdown of the Tc-DAX1 transcripts shows a significantly lower expression profile in the ds-DAX1 group compared to the ds-EGFP group. Subsequent histological analysis of gonads revealed that spermatogenesis was affected in a ds-DAX1 group compared to the ds-EGFP group. All these results indicate that Tc-DAX1 is involved in the spermatogenesis and early embryonic development of T. crocea, providing valuable information for the breeding and aquaculture of giant clams.

Chromosome-level genome assembly and annotation of rare and endangered tropical bivalve, Tridacna crocea.

Tridacna crocea is an ecologically important marine bivalve inhabiting tropical coral reef waters. High quality and available genomic resources will help us understand the population structure and genetic diversity of giant clams. This study reports a high-quality chromosome-scale T. crocea genome sequence of 1.30 Gb, with a scaffold N50 and contig N50 of 56.38 Mb and 1.29 Mb, respectively, which was assembled by combining PacBio long reads and Hi-C sequencing data. Repetitive sequences cover 71.60% of the total length, and a total of 25,440 protein-coding genes were annotated. A total of 1,963 non-coding RNA (ncRNA) were determined in the T. crocea genome, including 62 micro RNA (miRNA), 58 small nuclear RNA (snRNA), 83 ribosomal RNA (rRNA), and 1,760 transfer RNA (tRNA). Phylogenetic analysis revealed that giant clams diverged from oyster about 505.7 Mya during the evolution of bivalves. The genome assembly presented here provides valuable genomic resources to enhance our understanding of the genetic diversity and population structure of giant clams.

Guillain-Barre syndrome: An autoimmune disorder post-COVID-19 vaccination?

SARS-CoV-2 causes Coronavirus Disease 2019 (COVID-19), an infectious condition that can present none or one or more of these symptoms: fever, cough, headache, sore throat, loss of taste and smell, aches, fatigue and musculoskeletal pain. For the prevention of COVID-19, there are vaccines available including those developed by Pfizer, Moderna, Sinovac, Janssen, and AstraZeneca. Recent evidence has shown that some COVID-19-vaccinated individuals can occasionally develop as a potential side effect Guillain-Barre syndrome (GBS), a severe neurological autoimmune condition in which the immune response against the peripheral nerve system (PNS) can result in significant morbidity. GBS had been linked previously to several viral or bacterial infections, and the finding of GBS after vaccination with certain COVID-19, while rare, should alert medical practitioners for an early diagnosis and targeted treatment. Here we review five cases of GBS that developed in different countries after COVID-19 vaccination.

Seasonal variations in biochemical composition and nutritional quality of Crassostrea hongkongensis, in relation to the gametogenic cycle.

Variations in the biochemical composition and nutritional quality with annual changes in gonad development were investigated to identify the optimal harvesting time of C. hongkongensis. The glycogen levels in the mantle, muscle, and gonad-visceral mass were significantly lower in June than in December, associated with changes in the expressions of ChGS and ChGP. Protein content consistently exceeded 52% of dry weight. The only significant change in protein levels was an increase between April and June in the gonad-visceral mass, which was associated with the gonadal transition from proliferation to maturation. Moreover, C. hongkongensis consistently had a well-balanced essential amino acid profile, meeting the essential amino acid requirements of preschool children. The lipid content and fatty acid composition of C. hongkongensis varied with the reproductive cycle, but the omega-3:omega-6 ratio was consistently higher than those of C. gigas and C. virginica. In summary, the optimal harvest time of C. hongkongensis was during the inactive stage of most gonads (from August to February at Beihai).

Dietary supplementations of methionine improve growth performances, innate immunity, digestive enzymes, and antioxidant activities of rohu (Labeo rohita).

The dietary composition of fish constitutes numerous immune and growth-promoting substance; during the current study, methionine was amended in the diet of Labeo rohita (L. rohita), in order to appraise the diverse modulatory effects of methionine supplementation. Fish were grouped into three on the basis of methionine as methionine supplemented group 1 (MSG1), methionine supplemented group 2 (MSG2), and CTRL (control group). We assessed the effects of methionine at the end. Captivating results were obtained from the methionine supplementing groups. The results depict significantly high (p < 0.05) innate immunity parameters, including myeloperoxidase, IgG concentrations, lysozyme, respiratory burst, and total bacteriolytic activity of complement in MSG2. Furthermore, ameliorate growth performances were also noticed such as high weight gain, FCE%, and body length in MSG2, respectively. We also measured high level of antioxidant digestive enzymes, gut length, and survival rate in MSG2 compared to the rest of the groups. The results were further validated by measuring the stress parameters, such as aspartate aminotransferase (AST), glucose, and hematocrit (HCT%), and the concentration remains low in MSG2. Lastly, after 90 days, fish were challenged with Aeromonas hydrophila, the highest survival was observed in the MSG2 and MSG1 compared to CTRL. These results ultimately give the baseline to the credible effects of methionine on L. rohita.

Tetraploid induction of Crassostrea hongkongensis and C. sikamea by inhibiting the polar body 1 release in diploid fertilized eggs.

The production of an all-triploid population by mating tetraploid males with diploid females is the best and most fundamental method for the large-scale production of triploid oysters. Obtaining a stable tetraploid population is essential for guaranteed production in industrialized triploid cultivation. C. hongkongensis and C. sikamea are important oyster breeding species in southern China, and have great economic value. However, there are not any published data on inducing tetraploid C. hongkongensis or C. sikamea. Therefore, we investigated tetraploid induction in these two oyster species by inhibiting the PB1 release in diploid fertilized eggs using Cytochalasin B (CB) under 31 °C, 15 ‰ salinity. The results confirmed that the optimal tetraploid induction conditions for C. hongkongensis were a CB concentration of 0.50 mg/L with induction starting at 9.0 min after fertilization, and stopping at 21.0 min after fertilization; the induction efficiency index reached 0.123 under these conditions. The optimal tetraploid induction conditions for C. sikamea were a CB concentration of 0.50 mg/L, with induction starting at 7.5 min after fertilization and stopping at 18 min after fertilization; the induction efficiency index could be as high as 0.281 under these conditions. However, we confirmed that the tetraploid rate decreased with larval growth, and no tetraploids were detected in the juvenile period of either C. hongkongensis or C. sikamea. This may be attributed to the very low survival of the tetraploid larvae induced by this method, especially as most tetraploid larvae died during the first three days. In summary, it is simple to directly induce tetraploid C. hongkongensis and C. sikamea larvae by inhibiting the PB1 release of diploid zygotes, but the low survival rate makes it challenging to obtain viable juvenile tetraploids.

Autophagy Dually Induced by AMP Surplus and Oxidative Stress Enhances Hemocyte Survival and Bactericidal Capacity via AMPK Pathway in Crassostrea hongkongensis.

Crassostrea hongkongensis (Hong Kong oyster) is an ecologically and economically valuable shellfish endemic to South/Southeast Asia. Due to ocean acidification and warming waters, they have become increasingly vulnerable to invading microbes including Vibrio parahaemolyticus, a significant foodborne human pathogen. In recent years, outbreaks of V. parahaemolyticus have emerged as a perennial phenomenon in parts of the world, necessitating to better understand the biology of host-pathogen interactions in this under-examined marine invertebrate. Although an immunologically relevant autophagy apparatus has been identified in Crassostrea gigas, an evolutionarily close mollusk cousin, the precise mechanistic details of C. hongkongensis autophagy during V. parahaemolyticus infection are still wanting. Here, we compellingly demonstrated that in vivo V. parahaemolyticus challenge robustly triggered autophagic signaling in C. hongkongensis hemocytes peaking at 6 h post-infection, which subsequently promoted bacterial clearance and dampened premature apoptosis. Simultaneously, a large surplus of adenosine monophosphate (AMP) and elevations in reactive oxygen species (ROS, specifically mitochondrial O2 - and cellular H2O2) formation were observed post-infection. Extrinsically applied AMP and ROS could synergistically induce AMP-activated protein kinase (AMPK) phosphorylation to stimulate downstream autophagic events. V. parahaemolyticus infection-induced autophagy was pharmacologically shown to be AMPK-dependent in vivo. Overall, our results establish autophagy as a crucial arm of host defense against Vibrio infections in mollusks, and provide new insights into the underappreciated roles of ROS and AMP as co-regulators of autophagy.

Assessment of cadmium toxicity and its possible effects on goldfish (Carassius auratus), employing microscopy and biochemical techniques.

The present study aimed to evaluate and compare the toxicity level of dietary and waterborne Cadmium (Cd) source on the Goldfish Carassius auratus (C. auratus) using various biochemical and microscopic shreds of evidence. C. auratus were divided into two groups based on Cadmium supplementation methods subjected to the same concentrations in the diet and water; moreover, control (Ctrl) group was set up for comparison. Dietary supplemented group (DSG) group and (Aqueous supplemented groups) ASG were supplied with 0.5% of the Cadmium. Successful completion of the study resulted in the lucrative outcomes, and we noticed significantly high concentrations of Cadmium in the gills of the ASG fish while considerably low levels in the gills of DSG. Histopathological evidence showed clear and distinct changes in the liver cells, dilated sinusoids and unusual occurrence of Kupffer cells were noticed in the AS group fish. At the same time, there were also noticeable changes in the DS group compared to CTRL. Visible changes can be seen on the outer morphology of gills, lamella of ASG and DSG both were shrunk and broken, similar changes were noticed in the muscles myoepithelium. The stress parameters were differentially expressed in both the groups; waterborne Cadmium caused a pronounced release of AST, ALT, cortisol, and glucose concentrations. Comparative mortality showed the highest death rate in the ASG fish compared to DSG fish. The present study depicts the highest toxicity of Cadmium in the soluble Addendum form moreover, administered route and time length are crucial factors.

Microscopic and molecular evidence in support of rodent as a reservoir for dissemination of Leishmaniasis.

Leishmaniasis is a worldwide public health problem and vector-borne disease. It is caused by a diverse group of protozoan parasites that belong to the genus Leishmania and transmitted to humans through a bite of an infected female sand fly. Leishmaniasis has attained epidemic proportion in Khyber Pakhtunkhwa and raises serious concern over its management. The present research work was conducted in cutaneous leishmaniasis (CL) prevalent village named Surgul of district Kohat, Khyber Pakhtunkhwa with a focus to investigate whether rodent can act as a source for dissemination of leishmanial species or not. In this context, rodent samples were analyzed via morphological and molecular approaches to unveil prevalence of CL. It was reported that 12.5% of samples were positive for signs of leishmaniasis through microscopy and 18.75% through polymerase chain reaction (PCR). Supporting the findings further, the color character of rodents was also taken into consideration, which shows that light dark colored rodents were more infected (13.3%) compared to brown colored rodents (11.43%). Based on our findings, we speculate that small rodents are a possible reservoir of various leishmanial parasites and play a significant role in zoonosis and maintenance of their species.

Characterization and functional analysis of a caspase 3 gene: Evidence that ChCas 3 participates in the regulation of apoptosis in Crassostrea hongkongensis.

Caspase 3 plays an important role in apoptotic pathways and contributes to maintaining the homeostasis of the immune system in organisms. The structure, functions, and characteristics of caspase 3 have been extensively investigated in many species, but the research is scarce when it comes to bivalves, particularly oysters. In this study, we identified and cloned a previously unknown caspase 3 gene, named ChCas 3, in C. hongkongensis. The full-length cDNA of ChCas 3 was 1562 bp and included a 175 bp 5'-untranslated region (UTR), a 141 bp 3'-UTR and a 1245 bp open reading frame (ORF) that encoded a polypeptide of 415 amino acids. Similar to caspase 3 in other species, ChCas 3 has a pro-domain, a conserved cysteine active site, a large p20 subunit and a small p10 subunit. Our findings demonstrated the expression of ChCas 3 in all the eight tissues via tissue-specific expression assays with the highest expression in haemocytes. ChCas 3 was confirmed to be expressed throughout the larval development stages, and fluorescence from pEGFP-N1-ChCas 3 was found to be distributed throughout the entire HEK293T cell. In addition, the relative expression of ChCas 3 significantly enhanced in hemocytes post bacterial stimulation, and overexpression of ChCas 3 led to upregulation of the transcriptional activity of NF-κB and p53 reporter genes in HEK293T cells, which indicated that it was involved in innate immune responses. Finally, the apoptosis rate of the haemocytes declined considerably compared with that of the control group after the expression of ChCas 3 was successfully silenced by dsRNA, corroborating its sentinel role in apoptosis. This study provides comprehensive underpinning evidences, affirming caspase 3 crucial role against bacterial infection and apoptosis in C. hongkongensis.

Evaluating the lucrative role of probiotics in the aquaculture using microscopic and biochemical techniques.

The present study focused to elucidate the promising role of probiotics in the fish culture, by employing microscopic and biochemical parameters to rationalize the possible outcomes. After preliminary feeding trial histopathological analysis revealed clear and visible changes in the intestinal and liver cells of CTRL (control group) compared to probiotics supplemented group, the bacterial inoculations resulted in shorten and swelled villi, while liver kupffer cells increased many folds in the CTRL. Moreover, there was clear hypertrophy and lamellar fusion in the gills tissues examined under microscope. Furthermore, intriguing results were obtained, such as a substantial increase in the weight gain, specific growth rate (%BW/day), FCE%, survival%, and improved secretions of amylase, cellulase, and protease activities. Likewise, high crude protein, fats content, while low moisture and total Ash was noticed in EXP I (Probiotics supplemented) compared to CTRL. The microscopic and biochemical analysis disclosed the positive role of probiotics on the fish health, thus we suggest its supplementations in the fish diet.

Molecular characteristics of AMPK and its role in regulating the phagocytosis of oyster hemocytes.

Phagocytosis is one of the fundamental cellular immune defense parameter that helps in the elimination of the invading pathogens in both vertebrates and invertebrates, which require plenty of energy for functioning. In the present study, we identified the critical energy regulator AMP-activated protein kinase (AMPK) in Crassostrea hongkongensis which is composed of three subunits, named ChAMPK-α, ChAMPK-ß, and ChAMPK-γ, and then analyzed the function of AMPK in regulating hemocyte phagocytosis. All the three ChAMPK subunits mRNA were detected to be expressed at various embryological stages, and also constitutively expressed in multiple tissues with high expression in gill and mantle. The phylogenetic tree showed that the three subunits of AMPK were correspondingly clustered with its orthologue branches. Furthermore Western Blot analysis revealed that the AMPK pharmacological inhibitors Compound C could effectively down-regulate the Thr172 phosphorylation level of AMPK-α, and the hemocyte phagocytosis was inhibited by Compound C (CC), which indicate its existence in the oyster. Our results showed that treatment of AMPK inhibitors significantly attenuated the capacity of hemocytes phagocytosis. Moreover, Compound C could also change the organization of actin cytoskeleton in the oyster hemocytes, demonstrating the crucial role of AMPK signaling in control of phagocytosis.