Bamboo Shark Research Articles

Single-cell RNA sequencing illuminates the ontogeny, conservation and diversification of cartilaginous and bony fish lymphocytes

Elucidating cellular architecture and cell-type evolution across species is central to understanding immune system function and susceptibility to disease. Adaptive immunity is a shared trait of the common ancestor of cartilaginous and bony fishes. However, evolutionary features of lymphocytes in these two jawed vertebrates remain unclear. Here, we present a single-cell RNA sequencing atlas of immune cells from cartilaginous (white-spotted bamboo shark) and bony (zebrafish and Chinese tongue sole) fishes. Cross-species comparisons show that the same cell types across different species exhibit similar transcriptional profiles. In the bamboo shark, we identify a phagocytic B cell population expressing several pattern recognition receptors, as well as a T cell sub-cluster co-expressing both T and B cell markers. In contrast to a division by function in the bony fishes, we show close linkage and poor functional specialization among lymphocytes in the cartilaginous fish. Our cross-species single-cell comparison presents a resource for uncovering the origin and evolution of the gnathostome immune system.

The Improvement Effects of a Nutritional Fortifier on the Reproductive Performance, Sex Steroid Hormone Production, and Health of the Striped Bamboo Shark Chiloscyllium plagiosum.

To explore a method of improving the reproductive performance of the striped bamboo shark, three groups (D0, D1, and D2) of mature individuals were fed squid with (D1 and D2) or without (D0) a nutritional fortifier during the breeding seasons of 2022 and 2023. Compared with the D0 group, the D1 and D2 groups had an increase of 20.90% and 31.34% in total eggs, increases of 32.73% and 41.82% in the proportion of lecithal eggs, and a total 119.07% increase in hatching rate, respectively, in 2022. In 2023, the corresponding increase was 17.12% and 9.91% in total eggs, 19.63% and 12.15% in the proportion of lecithal eggs, 43.37% and 43.94% in fertilization rate, 23.94% and 22.22% in hatchability rate, and 66.70% and 8.70% in the survival rate of fry. Moreover, the levels of serum estradiol, testosterone, progesterone, albumin, and total antioxidant capacity and the levels of ARA, EPA, DHA, n-3 PUFA, and n-6 PUFA in both serum and lecithal eggs significantly increased, while the levels of serum triglyceride and total cholesterol were the opposite (p < 0.05). The results demonstrate that feeding the sharks with a nutritional fortifier can increase spawn production and the quality of eggs, regulate the production of sex steroids, and improve the nutrition of eggs and the health of broodstocks.

Characterization and humanization of VNARs targeting human serum albumin from the whitespotted bamboo shark (Chiloscyllium plagiosum)

The Shark-derived immunoglobulin new antigen receptors (IgNARs) have gained increasing attention for their high solubility, exceptional thermal stability, and intricate sequence variation. In this study, we immunized whitespotted bamboo shark (Chiloscyllium plagiosum) to create phage display library of variable domains of IgNAR (VNARs) for screening against Human Serum Albumin (HSA), a versatile vehicle in circulation due to its long in vivo half-life. We identified two HSA-binding VNAR clones, 2G5 and 2G6, and enhanced their expression in E. coli with the FKPA chaperone. 2G6 exhibited a strong binding affinity of 13 nM with HSA and an EC50 of 1 nM. In vivo study with a murine model further provided initial validation of 2G6's ability to prolong circulation time by binding to HSA. Additionally, we employed computational molecular docking to predict the binding affinities of both 2G6 and its humanized derivative, H2G6, to HSA. Our analysis unveiled that the complementarity-determining regions (CDR1 and CDR3) are pivotal in the antigen recognition process. Therefore, our study has advanced the understanding of the potential applications of VNARs in biomedical research aimed at extending drug half-life, holding promise for future therapeutic and diagnostic progressions.

Antigen specific VNAR screening in whitespotted bamboo shark (Chiloscyllium plagiosum) with next generation sequencing

IgNAR exhibits significant promise in the fields of cancer and anti-virus biotherapies. Notably, the variable regions of IgNAR (VNAR) possess comparable antigen binding affinity with much smaller molecular weight (∼12 kDa) compared to IgNAR. Antigen specific VNAR screening is a changeling work, which limits its application in medicine and therapy fields. Though phage display is a powerful tool for VNAR screening, it has a lot of drawbacks, such as small library coverage, low expression levels, unstable target protein, complicating and time-consuming procedures. Here we report VANR screening with next generation sequencing (NGS) could effectively overcome the limitations of phage display, and we successfully identified approximately 3000 BAFF-specific VNARs in Chiloscyllium plagiosum vaccinated with the BAFF antigen. The results of modelling and molecular dynamics simulation and ELISA assay demonstrated that one out of the top five abundant specific VNARs exhibited higher binding affinity to the BAFF antigen than those obtained through phage display screening. Our data indicates NGS would be an alternative way for VNAR screening with plenty of advantages.

Biomonitoring of mercury and selenium in commercially important shellfish: Distribution pattern, health benefit assessment and consumption advisories.

This study aims to explore the concentrations of Se and Hg in shellfish along the Gulf of Mannar (GoM) coast (Southeast India) and to estimate related risks and risk-based consumption limits for children, pregnant women, and adults. Se concentrations in shrimp, crab, and cephalopods ranged from 0.256 to 0.275mgkg-1, 0.182 to 0.553mgkg-1, and 0.176 to 0.255mgkg-1, respectively, whereas Hg concentrations differed from 0.009 to 0.014mgkg-1, 0.022 to 0.042mgkg-1 and 0.011 to 0.024mgkg-1, respectively. Se and Hg content in bamboo shark (C. griseum) was 0.242mgkg-1 and 0.082mgkg-1, respectively. The lowest and highest Se concentrations were found in C. indicus (0.176mgkg-1) and C. natator (0.553mgkg-1), while Hg was found high in C. griseum (0.082mgkg-1) and low in P. vannamei (0.009mgkg-1). Se shellfishes were found in the following order: crabs > shrimp > shark > cephalopods, while that of Hg were shark > crabs > cephalopods > shrimp. Se in shellfish was negatively correlated with trophic level (TL) and size (length and weight), whereas Hg was positively correlated with TL and size. Hg concentrations in shellfish were below the maximum residual limits (MRL) of 0.5mgkg-1 for crustaceans and cephalopods set by FSSAI, 0.5mgkg-1 for crustaceans and 1.0mgkg-1 for cephalopods and sharks prescribed by the European Commission (EC/1881/2006). Se risk-benefit analysis, the AI (actual intake):RDI (recommended daily intake) ratio was > 100%, and the AI:UL (upper limit) ratio was < 100%, indicating that all shellfish have sufficient level of Se to meet daily requirements without exceeding the upper limit (UL). The target hazard quotient (THQ < 1) and hazard index (HI < 1) imply that the consumption of shellfish has no non-carcinogenic health impacts for all age groups. However, despite variations among the examined shellfish, it was consistently observed that they all exhibited a Se:Hg molar ratio > 1. This finding implies that the consumption of shellfish is generally safe in terms of Hg content. The health benefit indexes, Se-HBV and HBVse, consistently showed high positive values across all shellfish, further supporting the protective influence of Se against Hg toxicity and reinforcing the overall safety of shellfish consumption. Enhancing comprehension of food safety analysis, it is crucial to recognize that the elevated Se:Hg ratio in shellfish may be attributed to regular selenoprotein synthesis and the mitigation of Hg toxicity by substituting Se bound to Hg.

De novo transcriptome for Chiloscyllium griseum, a long-tail carpet shark of the Indian waters

Sharks have thrived in the oceans for 400 million years, experienced five extinctions and evolved into today’s apex predators. However, enormous genome size, poor karyotyping and limited tissue sampling options are the bottlenecks in shark research. Sharks of the family Orectolobiformes act as model species in transcriptome research with exceptionally high reproductive fecundity, catch prominence and oviparity. The present study illustrates a de novo transcriptome for an adult grey bamboo shark, Chiloscyllium griseum (Chondrichthyes; Hemiscyllidae) using paired-end RNA sequencing. Around 150 million short Illumina reads were obtained from five different tissues and assembled using the Trinity assembler. 70,647 hits on Uniprot by BLASTX was obtained after the transcriptome annotation. The data generated serve as a basis for transcriptome-based population genetic studies and open up new avenues in the field of comparative transcriptomics and conservation biology.

Development of Shark Single Domain Antibodies Specific for Human α-Fetoprotein and the Multimerization Strategy in Serum Detection.

Sensitive detection of cancer biomarkers can contribute to the timely diagnosis and treatment of diseases. In this study, the whitespotted bamboo sharks were immunized with human α-fetoprotein (AFP), and a phage-displayed variable new antigen receptor (VNAR) single domain antibody library was constructed. Then four unique VNARs (VNAR1, VNAR11, VNAR21, and VNAR25) against AFP were isolated from the library by biopanning for the first time. All of the sequences belong to type II of VNAR, and the VNAR11 was much different from the rest of the three sequences. Then VNAR1 and VNAR11 were selected to fuse with the C4-binding protein α chain (C4bpα) sequence and efficiently expressed in the Escherichia coli system. Furthermore, a VNAR-C4bpα-mediated sandwich chemiluminescence immunoassay (VSCLIA) was developed for the detection of AFP in human serum samples. After optimization, the VSCLIA showed a limit of detection of 0.74 ng/mL with good selectivity and accuracy. Moreover, the results of clinical serum samples detected by the VSCLIA were confirmed by an automatic immunoanalyzer in the hospital, indicating its practical application in actual samples. In conclusion, the novel antibody element VNAR exhibits great potential for immunodiagnosis, and this study also provides a new direction and experimental basis for AFP detection.

Loss of noggin1, a classic embryonic inducer gene, in elasmobranchs

Secreted proteins of the Noggin family serve as pivotal regulators of early development and cell differentiation in all multicellular animals, including vertebrates. Noggin1 was identified first among all Noggins. Moreover, it was described as the first known embryonic inducer specifically secreted by the Spemann organizer and capable of inducing a secondary body axis when expressed ectopically. In the classical default model of neural induction, Noggin1 is presented as an antagonist of BMP signalling, playing a role as a neural inducer. Additionally, Noggin1 is involved in the dorsalization of embryonic mesoderm and later controls the differentiation of various tissues, including muscles, bones, and neural crest derivatives. Hitherto, noggin1 was found in all studied vertebrates. Here, we report the loss of noggin1 in elasmobranchs (sharks, rays and skates), which is a unique case among vertebrates. noggin2 and noggin4 retained in this group and studied in the embryos of the grey bamboo shark Chiloscyllium griseum revealed similarities in expression patterns and functional properties with their orthologues described in other vertebrates. The loss of noggin1 in elasmobranchs may be associated with histological features of the formation of their unique internal cartilaginous skeleton, although additional research is required to establish functional connections between these events.

Bacterial abscesses in whitespotted bamboo sharks (Chiloscyllium plagiosum) in managed care.

Sixteen whitespotted bamboo sharks (Chiloscyllium plagiosum) with histologically similar bacterial abscesses were submitted to Northwest ZooPath from nine zoo and aquarium institutions over a 17-year period. These abscesses were characterized by inflammatory cell infiltrates and necrosis with intralesional small, Gram-positive, acid-fast negative, cocci bacteria. The clinical presentation, histologic findings, and culture results indicate that Enterococcus faecalis is a relatively common cause of these lesions in whitespotted bamboo sharks. This organism also provides a treatment challenge due to its inherent antibiotic-resistant properties and ability to form biofilms, confounding the host's immune response. Enterococcus faecalis represents an important cause for abscess formation and cellulitis in captive whitespotted bamboo sharks.

PRIMARY HEPATIC NEOPLASIA IN THREE ADULT BAMBOO SHARKS (CHILOSCYLLIUM PLAGIOSUM AND CHILOSCYLLIUM PUNCTATUM) IN AN AQUARIUM POPULATION.

Neoplasia in elasmobranchs is uncommonly documented. This report describes primary hepatic neoplasia in three adult female bamboo sharks under managed care: biliary adenocarcinoma in a whitespotted bamboo shark (Case 1; Chiloscyllium plagiosum), biliary adenocarcinoma in a brownbanded bamboo shark (Case 2; Chiloscyllium punctatum), and hepatocellular carcinoma in a whitespotted bamboo shark (Case 3). Case 1 presented with extensive cutaneous papillomas and was electively euthanized. At necropsy, a 4-cm-diameter, pale-tan, firm hepatic mass was identified and diagnosed histologically as a biliary adenocarcinoma. Case 2 presented with decreasing body condition despite normal food intake. Coelomic ultrasound and exploratory surgery revealed several large masses in both hepatic lobes, and the patient was euthanized. At necropsy, nine, 1-6-cm-diameter, black to tan, firm hepatic masses were identified and diagnosed histologically as biliary adenocarcinoma with branchial intravascular neoplastic emboli. Case 3 presented for routine health examination and was euthanized for diagnostic purposes after coelomic ultrasound revealed multiple hepatic masses. At necropsy, two 1-3-cm-diameter, brown- and-black mottled, firm hepatic masses were identified and diagnosed histologically as hepatocellular carcinoma. Immunohistochemistry was performed in two of these cases and was noncontributory, likely because of a lack of cross reactivity between antibodies (antipancytokeratin) and elasmobranch tissues.

A singular shark bitter taste receptor provides insights into the evolution of bitter taste perception.

Many animal and plant species synthesize toxic compounds as deterrent; thus, detection of these compounds is of vital importance to avoid their ingestion. Often, such compounds are recognized by taste 2 receptors that mediate bitter taste in humans. Until now, bitter taste receptors have only been found in bony vertebrates, where they occur as a large family already in coelacanth, a "living fossil" and the earliest-diverging extant lobe-finned fish. Here, we have revisited the evolutionary origin of taste 2 receptors (T2Rs) making use of a multitude of recently available cartilaginous fish genomes. We have identified a singular T2R in 12 cartilaginous fish species (9 sharks, 1 sawfish, and 2 skates), which represents a sister clade to all bony fish T2Rs. We have examined its ligands for two shark species, a catshark and a bamboo shark. The ligand repertoire of bamboo shark represents a subset of that of the catshark, with roughly similar thresholds. Amarogentin, one of the most bitter natural substances for humans, also elicited the highest signal amplitudes with both shark receptors. Other subsets of ligands are shared with basal bony fish T2Rs indicating an astonishing degree of functional conservation over nearly 500 mya of separate evolution. Both shark receptors respond to endogenous steroids as well as xenobiotic compounds, whereas separate receptors exist for xenobiotics both in early- and late-derived bony vertebrates (coelacanth, zebrafish, and human), consistent with the shark T2R reflecting the original ligand repertoire of the ancestral bitter taste receptor at the evolutionary origin of this family.

When the penny drops: sharks outsmart cichlids in serial reversal learning

Abstract Two species of fish were tested in a serial reversal learning task using two dimensional objects varying in colour and/or form. After reaching the learning criterion (LC) in the original discrimination task, individuals reversed for up to ten times, i.e., stimulus association was switched and animals rewarded for choosing the stimulus that had not been rewarded in the previous learning phase. This was conducted using the two stimuli green diamond (S+) vs. red square (S−) for the training of eight cichlids, and black star (S+) vs. black circle (S−) for the training of seven bamboo sharks. All fifteen animals completed the initial training phase and reversed successfully at least three times, indicating an ability to forsake a previously learned stimulus association in favour of a new one. Sharks, but not cichlids, also showed a decrease in the number of sessions needed to reach LC with an increase in the number of reversals conducted, showing that some sort of learning strategy, i.e., rule learning, had been applied. Animals that improve their performance within such learning paradigms are considered to possess more behavioural flexibility and advanced cognitive abilities than those who do not. More flexibility could specifically aid species living in complex habitats, social groups and subjected to changing environmental conditions.

DEXMEDETOMIDINE AND MIDAZOLAM INTRAMUSCULAR SEDATION IN BROWNBANDED BAMBOO SHARKS (CHILOSCYLLIUM PUNCTATUM).

Bamboo sharks are some of the most common elasmobranch species in zoos and aquaria and are frequently sedated for medical exams, treatments, and research. This study investigated the use of an IM sedation protocol of a single dose of dexmedetomidine (0.05 mg/kg) and midazolam (2.0 mg/kg) in brownbanded bamboo sharks (Chiloscyllium punctatum). Sharks were serially monitored every 5 min for heart rate, branchial beats, righting reflex, coelomic response, cloacal response, pelvic fin reflex, response to noxious stimulus, voluntary movement, and ability to swim. This sedation dose was effective at rapidly and significantly decreasing responses to tactile and noxious stimuli with minimal respiratory depression and was quickly reversible with atipamezole (0.5 mg/kg) and flumazenil (0.05 mg/kg). Sedated sharks developed a mild metabolic acidosis evidenced by a significant increase in lactic acid (mean < 0.37 mmol/L presedation, 4.2 mmol/L after reversal) and decrease in blood pH (mean 7.464 presedation, 7.277 after reversal); however, clinical intervention was not required. This protocol should be further investigated in different elasmobranch species but is promising for providing sedation for noninvasive procedures in brownbanded bamboo sharks.

Shark and ray genome size estimation: methodological optimization for inclusive and controllable biodiversity genomics

Estimate of nuclear DNA content serves as an independent tool for validating the completeness of whole genome sequences and investigating the among-species variation of genome sizes, but for some species, the requirement of fresh cells makes this tool highly inaccessible. Here we focused on elasmobranch species (sharks and rays), and using flow cytometry or quantitative PCR (qPCR), estimated the nuclear DNA contents of brownbanded bamboo shark, white spotted bamboo shark, zebra shark, small-spotted catshark, sandbar shark, slendertail lanternshark, basking shark, megamouth shark, red stingray, and ocellate spot skate. Our results revealed their genome sizes spanning from 3.18 pg (for ocellate spot skate) to 13.34 pg (for slendertail lanternshark), reflecting the huge variation of genome sizes already documented for elasmobranchs. Our qPCR-based method ‘sQuantGenome’ enabled accurate genome size estimation without using live cells, which has been a severe limitation with elasmobranchs. These findings and our methodology are expected to contribute to better understanding of the diversity of genome sizes in elasmobranchs even including species with limited availability of fresh tissue materials. It will also help validate the completeness of already obtained or anticipated whole genome sequences.

Feeding ecology and reproductive biology of small coastal sharks in Malaysian waters.

Small coastal demersal sharks form a major proportion of the sharks landed in Malaysia. However, little is known about their feeding ecology and reproduction. This study sought to elucidate the dietary patterns, role of ontogeny in prey consumption, and reproductive biology of four dominant small demersal shark species in Malaysian waters: the Hasselt's bamboo shark, Chiloscyllium hasseltii; brownbanded bamboo shark, C. punctatum; spadenose shark, Scoliodon laticaudus; and Pacific spadenose shark, S. macrorhynchos. Dietary analyses revealed a high overlap in prey taxa consumed; clear resource partitioning among co-occurring species based on the percentage Prey-specific Index of Relative Importance (%PSIRI), with higher fish %PSIRI for Chiloscyllium hasseltii, higher cephalopod %PSIRI for C. punctatum, and higher crustacean %PSIRI for both Scoliodon species; and an ontogenetic diet shift, seen through changes in prey size. Based on the examination of reproductive organs, the results showed larger sizes at maturity for males compared to females for all four species; no obvious reproductive cycles, based on hepatosomatic and gonadosomatic indices for all species; female bias in the sex ratio of the embryos of Scoliodon species; and increased reproductive output (number of eggs or embryos and size of eggs) with larger female size for C. hasseltii and Scoliodon species. The partitioning of food resources minimizes direct competition for food and supports coexistence within shared coastal habitats. The reproductive strategies of these small coastal sharks appear to be favorable for supporting short-term population productivity; although a reduction in fishing pressure, especially from bottom trawlers, is essential for the long-term sustainable use of these sharks.

Shark-derived single-domain antibodies for the detection of decapod iridescent virus 1

Decapod iridescent virus 1 (DIV1) has emerged as a novel threat to shrimp farming. The development of a rapid, convenient, and accurate method for diagnosing DIV1 is urgently needed, serving as a key factor in the reduction of the risk of viral outbreaks. This study aimed to develop a new variable antigen receptor (vNAR), derived from shark heavy-chain antibodies, directed against DIV1 for use in detection assays. White-spotted bamboo sharks were immunized with DIV1 virions, and an immune phage-display library was constructed. After three rounds of panning, the lead D02 vNAR was isolated from the library. D02 vNAR was identified to target the viral antigen ORF064L through western blotting, LM-SC/SC, and pull-down analyses. Furthermore, indirect enzyme linked immunosorbent assay (indirect ELISA) and dot-blot assays were developed based on D02 vNAR. The detection limits of the indirect ELISA and dot blot methods were 5 ng/mL and 6.25 ng/spot, respectively. Furthermore, in the indirect ELISA method, a standard curve describing the log concentration of DIV1 versus OD value was established, which was linear in the concentration range of 80–10,240 ng/mL, and the linear regression equation was derived as y = 0.3565x − 0.51601. Furthermore, 300 clinical samples were analyzed by the two methods using quantitative real-time polymerase chain reaction (qRT-PCR) as a reference. Compared to qRT-PCR, the diagnostic sensitivity and specificity of the indirect ELISA and dot blot methods were 96.7 % and 100 % and 93.5 % and 100 %, respectively, demonstrating the practicability and reliability of the vNAR-based indirect ELISA and dot blot methods for the robust detection of DIV1. Overall, this is the first study in which a vNAR has been developed against DIV1. The developed vNAR offers a robust tool with great potential for the diagnosis of DIV1 during shrimp cultivation.

Isolation and Characterization of Targeting-HBsAg VNAR Single Domain Antibodies from Whitespotted Bamboo Sharks (Chiloscyllium plagiosum).

Immunoglobulin new antigen receptor (IgNAR) is a naturally occurring antibody that consists of only two heavy chains with two independent variable domains. The variable binding domain of IgNAR, called variable new antigen receptor (VNAR), is attractive due to its solubility, thermal stability, and small size. Hepatitis B surface antigen (HBsAg) is a viral capsid protein found on the surface of the Hepatitis B virus (HBV). It appears in the blood of an individual infected with HBV and is widely used as a diagnostic marker for HBV infection. In this study, the whitespotted bamboo sharks (Chiloscyllium plagiosum) were immunized with the recombinant HBsAg protein. Peripheral blood leukocytes (PBLs) of immunized bamboo sharks were further isolated and used to construct a VNAR-targeted HBsAg phage display library. The 20 specific VNARs against HBsAg were then isolated by bio-panning and phage ELISA. The 50% of maximal effect (EC50) of three nanobodies, including HB14, HB17, and HB18, were 4.864 nM, 4.260 nM, and 8.979 nM, respectively. The Sandwich ELISA assay further showed that these three nanobodies interacted with different epitopes of HBsAg protein. When taken together, our results provide a new possibility for the application of VNAR in HBV diagnosis and also demonstrate the feasibility of using VNAR for medical testing.

Contrast-enhanced computed tomography and cross-sectional anatomy of the trunk in the brownbanded bamboo shark (Chiloscyllium punctatum).

Computed tomography (CT) is used in veterinary medicine for the diagnosis of bones and soft tissue diseases in various species. In addition, CT has recently been used to diagnose aquatic animals, including Selachimorpha, which are difficult to diagnose out of water. However, because Selachimorpha do not have adipose tissue in the coelomic cavity, the coelomic organs cannot be fully identified using non-contrast CT (NCCT). The aim of this study is to present the anatomical features of the cadaver, NCCT, and contrast-enhanced CT (CECT) as well as the change in CT values of the coelomic organs and musculature of the brownbanded bamboo shark. NCCT scans were performed under anaesthesia in one male and one female shark. CECT was performed 30 min after iopamidol was administered intravenously. The sharks were euthanized, frozen at -20°C, and sliced in the same position in which they were scanned. Using electric band saw, 10-mm transversal sections were obtained. The anatomical structures of both males and females were identified by transversal sections, and CT images homologous to transversal sections were then selected. Sagittal and coronal CECT images were also obtained to facilitate understanding of the location and size of coelomic organs. Although bone structure and air in organs could be sufficiently discriminated on NCCT image, the coelomic organs were almost indistinguishable. On the other hand, CECT images obtained sufficient contrast to identify most coelomic organs in addition to bone and air. The results provide an atlas of a cross-sectional anatomy and CECT images, which is useful information for the medical diagnosis of coelomic organs in live Selachimorpha.

IgNAR characterization and gene loci identification in whitespotted bamboo shark (Chiloscyllium plagiosum) genome

Single domain antibodies (sdAb) are promising candidates in cancer and anti-virus biotherapies for their unique structure characters. Though VHH and IgNAR have been discovered in camelidae and nurse shark (Ginlymostoma cirratum) respectively serval decades ago, expense of these large animals still limits the studies and applications of sdAb. Recently, IgNAR has been found in whitespotted bamboo shark (Chiloscyllium plagiosum), a small-sized sharks, while how to characterize and achieved the IgNAR of whitespotted bamboo shark is still unclear. In our research, we identified four IgNAR coding gene loci in whitespotted bamboo shark chromosome 44 (NC_057753.1), and primers were designed for single domain variable regions of IgNAR (VNAR) libraries preparation. Following sequencing results revealed that all plasmids constructed with our predicted VNAR libraries contained VNAR coding sequences, which confirmed the specificities of our primers in VNAR amplification. To our surprise, ≥90% VNAR sequences were encoded by IgNAR1, which suggests IgNAR1 is the most active IgNAR transcription locus in whitespotted bamboo shark. Interestingly, we found IgNAR(ΔC2-C3) were encoded by IgNAR3. Our findings gave a new sight of whitespotted bamboo shark IgNAR, which would broad the way of IgNAR studies and applications in biotherapies.

Development of a diluent storage media and effect of cryoprotectants on semen collected from white spotted bamboo sharks (Chiloscyllium plagiosum) and cooled to 5 ºC

Three experiments were performed to evaluate the effects of osmolarity, temperature, media and cryoprotectants on white spotted bamboo shark (Chiloscyllium plagiosum) spermatozoa. Semen was collected from 7 different males using a massage technique with the animals held in tonic immobilization with gills submerged. A Computer Assisted Sperm Analysis (CASA) system was used to assess sperm motility. Experiment one evaluated the effects of Hank’s balanced salt solution (HBSS) with different osmolarities (200, 400, 800 and 1000 mOsm Kg1) and incubation temperatures (5 °C and 24 °C [room temperature]) over time (6 h, 12 h and 24 h). Membrane integrity and morphology were degraded (P < 0.05) over time; however, sperm extended with HBSS 1000 and HBSS 800 were least affected. Higher total motility (TM) and average path velocity (VAP, P > 0.05) sperm parameters were observed with HBSS 1000 and HBSS 800, respectively. In addition, TM, VAP, and curvilinear velocity (VCL) were all reduced (P < 0.05) compared to sperm held at 5 °C. Experiment two compared the effects of sperm diluted in crystal reef water (CRW), elasmobranch ringers (ER) or raw held at 5 °C. Results indicated that CRW had improved viability, TM, VAP, VSL, VCL, and straightness (STR) when compared to sperm diluted in ER or Raw at 5 °C. The final experiment compared different cryoprotectants (dimethyl sulfoxide [DMSO] 10%, DMSO 20%, Ethylene-glycol [EG] 10%, EG 20%, and Trehalose [Tre] 10%, Tre 20%) during incubation at 5 °C. Across all time points, sperm incubated in Tre (10 or 20%) had increased (P < 0.05) membrane integrity and normal morphology compared to all other media and raw. Across all time points, sperm exposed to Tre (10% and 20%) and 10% DMSO had significantly improved TM (P < 0.05) compared to EG (10% and 20%) and 20% DMSO which were similar to raw. Although sperm collected from WSBS are robust and appear to tolerate a wide range of osmotic conditions and media, media made at 1000 mOsm Kg1 should provide the best in-vitro osmotic pressure, while storing at cooler temperatures should improve storage survival. For future cryopreservation studies, media using TRE and DMSO as cryoprotectants should be evaluated.