Hydrophis Cyanocinctus Research Articles

Hydrostatin-TL1, an Anti-Inflammatory Active Peptide from the Venom Gland of Hydrophis cyanocinctus in the South China Sea.

Tumor necrosis factor (TNF)-α is a pleiotropic cytokine with intense pro-inflammatory and immunomodulatory properties, and anti-TNF-α biologics are effective therapies for various inflammatory diseases such as inflammatory bowel disease (IBD) and sepsis. Snake venom, as a traditional Chinese medicine, has been used in the treatment of inflammatory diseases in China for centuries. In this research, we constructed a venom gland T7 phage display library of the sea snake Hydrophis cyanocinctus to screen bioactive compounds that antagonize TNF-α and identified a novel nine-amino-acid peptide, termed hydrostatin-TL1 (H-TL1). In enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) analyses, H-TL1 inhibited the interaction between TNF-α and TNF receptor 1 (TNFR1). Further, H-TL1 attenuated the cytotoxicity of TNF-α in L929 cells as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. H-TL1 also decreased the mRNA expression of TNF-α/TNFR1 downstream targets and suppressed the phosphorylation of well-characterized proteins of downstream signal transduction pathways in HEK-293 cells. In vivo data demonstrated that H-TL1 protects animals against dextran sodium sulfate (DSS)-induced acute colitis and lipopolysaccharide (LPS)-induced acute shock. Given its significant anti-inflammatory activity in vitro and in vivo, H-TL1 is a potential peptide for the development of new agents to treat TNF-α-associated inflammatory diseases.

Corrigendum: Screening of an anti-inflammatory peptide from Hydrophis cyanocinctus and analysis of its activities and mechanism in DSS-induced acute colitis.

Scientific Reports 6: Article number: 25672; published online: 09 May 2016; updated: 25 August 2016 This Article contains errors. In the Results section under subheading ‘H. cyanocinctus venom gland T7 phage display library construction and biopanning’, “DEQHLETELHTHLTSVLTANGFQ” should read: “DEQHLETELHTLTSVLTANGFQ”

Screening of an anti-inflammatory peptide from Hydrophis cyanocinctus and analysis of its activities and mechanism in DSS-induced acute colitis.

Snake has been used for centuries as a traditional Chinese medicine, especially for therapeutic treatment for inflammatory diseases; however, its mechanisms of action and active constituents remain controversial. In our study, a tumor necrosis factor receptor 1 (TNFR1) selective binding peptide, Hydrostatin-SN1 (H-SN1), which was screened from a Hydrophis cyanocinctus venom gland T7 phage display library, was shown to exhibit significant anti-inflammatory activity in vitro and in vivo. As a TNFR1 antagonist, it reduced cytotoxicity mediated by TNF-α in L929 fibroblasts and effectively inhibited the combination between TNF-α with TNFR1 in surface plasmon resonance analysis. H-SN1 was also shown to suppress TNFR1–associated signaling pathways as it minimized TNF-α-induced NF-кB and MAPK activation in HEK293 embryonic kidney and HT29 adenocarcinoma cell lines. We next determined the effect of H-SN1 in vivo using a murine model of acute colitis induced by dextran sodium sulfate, demonstrating that H-SN1 lowered the clinical parameters of acute colitis including the disease activity index and histologic scores. H-SN1 also inhibited TNF/TNFR1 downstream targets at both mRNA and protein levels. These results indicate that H-SN1 might represent a suitable candidate for use in the treatment of TNF-α-associated inflammatory diseases such as inflammatory bowel diseases.

First Record of Laticauda semifasciata (Reptilia: Squamata: Elapidae: Laticaudinae) from Korea

The Chinese sea snake Laticauda semifasciata (Reinwardt in Schlegel, 1837) is newly reported from Korean waters based on three specimens collected from Jeju Island, Korea, in August, September, and November 2015. This is the first time that the genus Laticauda and subfamily Laticaudinae has been reported from Korean waters. The subfamily Laticaudinae has ventrals that are four to five times wider than the adjacent dorsals, which are unlike the ventrals that are similar or up to two times wider than adjacent dorsals in the subfamily Hydrophiinae. Laticauda semifasciata is distinct from other species because it has three prefrontals and its rostrals are horizontally divided into two. As the result of this report, four species (L. semifasciata, Hydrophis (Pelamis) platurus, Hydrophis cyanocinctus, and H. melanocephalus) of sea snakes have been reported in Korean waters.

Identification and Characterization of the First Cathelicidin from Sea Snakes with Potent Antimicrobial and Anti-inflammatory Activity and Special Mechanism

Cathelicidins are a family of gene-encoded peptide effectors of innate immunity found exclusively in vertebrates. They play pivotal roles in host immune defense against microbial invasions. Dozens of cathelicidins have been identified from several vertebrate species. However, no cathelicidin from marine reptiles has been characterized previously. Here we report the identification and characterization of a novel cathelicidin (Hc-CATH) from the sea snake Hydrophis cyanocinctus. Hc-CATH is composed of 30 amino acids, and the sequence is KFFKRLLKSVRRAVKKFRKKPRLIGLSTLL. Circular dichroism spectroscopy and structure modeling analysis indicated that Hc-CATH mainly assumes an amphipathic α-helical conformation in bacterial membrane-mimetic solutions. It possesses potent broad-spectrum and rapid antimicrobial activity. Meanwhile, it is highly stable and shows low cytotoxicity toward mammalian cells. The microbial killing activity of Hc-CATH is executed through the disruption of cell membrane and lysis of bacterial cells. In addition, Hc-CATH exhibited potent anti-inflammatory activity by inhibiting the LPS-induced production of nitric oxide (NO) and pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6. Hc-CATH directly binds with LPS to neutralize its toxicity, and it also binds to Toll-like receptor 4 (TLR4/MD2 complex), which therefore inhibits the binding of LPS to TLR4/MD2 complex and the subsequent activation of LPS-induced inflammatory response pathways. Taken together, our study demonstrates that Hc-CATH, the first cathelicidin from sea snake discovered to have both antimicrobial and anti-inflammatory activity, is a potent candidate for the development of peptide antibiotics.

Sea snake harvest in the gulf of Thailand.

Conservation of sea snakes is virtually nonexistent in Asia, and its role in human-snake interactions in terms of catch, trade, and snakebites as an occupational hazard is mostly unexplored. We collected data on sea snake landings from the Gulf of Thailand, a hotspot for sea snake harvest by squid fishers operating out of the ports of Song Doc and Khanh Hoi, Ca Mau Province, Vietnam. The data were collected during documentation of the steps of the trading process and through interviewers with participants in the trade. Squid vessels return to ports once per lunar synodic cycle and fishers sell snakes to merchants who sort, package, and ship the snakes to various destinations in Vietnam and China for human consumption and as a source of traditional remedies. Annually, 82 t, roughly equal to 225,500 individuals, of live sea snakes are brought to ports. To our knowledge, this rate of harvest constitutes one of the largest venomous snake and marine reptile harvest activities in the world today. Lapemis curtus and Hydrophis cyanocinctus constituted about 85% of the snake biomass, and Acalyptophis peronii, Aipysurus eydouxii, Hydrophis atriceps, H. belcheri, H. lamberti, and H. ornatus made up the remainder. Our results establish a quantitative baseline for characteristics of catch, trade, and uses of sea snakes. Other key observations include the timing of the trade to the lunar cycle, a decline of sea snakes harvested over the study period (approximately 30% decline in mass over 4 years), and the treatment of sea snake bites with rhinoceros horn. Emerging markets in Southeast Asia drive the harvest of venomous sea snakes in the Gulf of Thailand and sea snake bites present a potentially lethal occupational hazard. We call for implementation of monitoring programs to further address the conservation implications of this large-scale marine reptile exploitation.

Distribution patterns of PAHs in different tissues of annulated sea snake (Hydrophis cyanocinctus) and short sea snake (Lapemis curtus) from the Hara Protected Area on the North Coast of the Persian Gulf, Iran

The levels of 22 polycyclic aromatic hydrocarbons (PAHs) were determined in the skin, liver, kidney and muscle tissues of annulated sea snake (Hydrophis cyanocinctus), and short sea snake (Lapemis curtus) collected from the Hara Protected Area, Persian Gulf during October 2010. Low molecular weight (≤3-rings) had the highest levels in skin, while high molecular weight (≥4-rings) was highest in the kidney. The lowest levels of analyzed PAHs were found in muscle in the both species. H. cyanocinctus (59.37ngg−1 dw) revealed to be significantly more contaminated than L. curtus (50.51ngg−1 dw). Naphtalene was the PAH most frequently detected and at the highest level in the different tissues both species. Di- and tri-cyclic PAHs were predominant in both species suggesting petrogenic origin rather than pyrogenic sources of PAHs. The present study represents the first data of contamination by PAHs in sea snake from the Persian Gulf.

Feeding Habits of the Annulated Sea Snake,Hydrophis cyanocinctus, in the Persian Gulf

Abstract We studied the diet of the Annulated Sea Snake (Hydrophis cyanocinctus) in the mangrove habitat of the Iranian Hara Protected Area. Prey items consumed by 34 individual snakes included four species of gobiid fish. Mudskippers (Periophthalmus waltoni, Boleophthalmus dussumieri, and Scartelaos tenuis) comprised the vast majority of the diet. All prey items were ingested head first. There was a positive correlation between snake SVL and total length of prey, but large snakes also ate small prey. Snake neck diameter was correlated positively with maximum prey diameter, and snakes did not consume prey items with a maximum diameter more than 1.2 times their neck diameter, a characteristic that may indicate gape limitation.

Recent rapid speciation and ecomorph divergence inIndo‐Australian sea snakes

The viviparous sea snakes (Hydrophiinae) are a young radiation of at least 62 species that display spectacular morphological diversity and high levels of local sympatry. To shed light on the mechanisms underlying sea snake diversification, we investigated recent speciation and eco-morphological differentiation in a clade of four nominal species with overlapping ranges in Southeast Asia and Australia. Analyses of morphology and stomach contents identified the presence of two distinct ecomorphs: a 'macrocephalic' ecomorph that reaches >2 m in length, has a large head and feeds on crevice-dwelling eels and gobies; and a 'microcephalic' ecomorph that rarely exceeds 1 m in length, has a small head and narrow fore-body and hunts snake eels in burrows. Mitochondrial sequences show a lack of reciprocal monophyly between ecomorphs and among putative species. However, individual assignment based on newly developed microsatellites separated co-distributed specimens into four significantly differentiated clusters corresponding to morphological species designations, indicating limited recent gene flow and progress towards speciation. A coalescent species tree (based on mitochondrial and nuclear sequences) and isolation-migration model (mitochondrial and microsatellite markers) suggest between one and three transitions between ecomorphs within the last approximately 1.2 million to approximately 840,000 years. In particular, the macrocephalic 'eastern' population of Hydrophis cyanocinctus and microcephalic H. melanocephalus appear to have diverged very recently and rapidly, resulting in major phenotypic differences and restriction of gene flow in sympatry. These results highlight the viviparous sea snakes as a promising system for speciation studies in the marine environment.

Snake venomics of two poorly known Hydrophiinae: Comparative proteomics of the venoms of terrestrial Toxicocalamus longissimus and marine Hydrophis cyanocinctus

The venom proteomes of Toxicocalamus longissimus and Hydrophis cyanocinctus, a fossorial and a marine species, respectively, of the Hydrophiinae genus of Elapidae, were investigated by Edman degradation of RP-HPLC isolated proteins, and de novo MS/MS sequencing of in-gel derived tryptic peptide ions. The toxin arsenal of T. longissimus is made up of 1-2 type-I PLA(2) molecules, which account for 6.5% of the venom proteins, a minor PIII-SVMP (1.4% of the venom toxins), and ~20 members of the 3FTx family comprising 92% of the venom proteome. Seventeen proteins (5 type-I PLA(2)s and 12 3FTxs) were found in the venom of H. cyanocinctus. Three-finger toxins and type-I PLA(2) proteins comprise, respectively, 81% and 19% of its venom proteome. The simplicity of the H. cyanocinctus venom proteome is highlighted by the fact that only 6 venom components (3 short-chain neurotoxins, two long-chain neurotoxins, and one PLA(2) molecule) exhibit relative abundances >5%. As expected from its high neurotoxin abundance, the LD(50) for mice of H. cyanocinctus venom was fairly low, 0.132μg/g (intravenous) and 0.172μg/g (intraperitoneal). Our data indicate that specialization towards a lethal cocktail of 3FTx and type-I PLA(2) molecules may represent a widely adopted trophic solution throughout the evolution of Elapidae. Our results also points to a minimization of the molecular diversity of the toxin arsenal of the marine snake Hydrophis cyanocinctus in comparison to the venom proteome of its terrestrial relatives, and highlight that the same evolutionary solution, economy of the toxin arsenal, has been convergently adopted by different taxa in response to opposite selective pressures, loss and gain of neurotoxicity.

Status of the Annulated Sea Snake, Hydrophis cyanocinctus, in the Hara Protected Area of the Persian Gulf

Data on the Annulated Sea Snake (Hydrophis cyanocinctus) were collected during more than one year of field study in the Hara Protected Area, the largest mangrove forest in the Persian Gulf. Mean snoutvent length (SVL) was 117 cm and the largest snake was a female with a SVL of 155 cm and a weight of 1019 g. The Annulated Sea Snake is the dominant sea snake in the area. The species is more active in the warm season (April to November). Local fishing activities are considered the main threat for the species in this area.

Metal concentrations in selected tissues and main prey species of the annulated sea snake (Hydrophis cyanocinctus) in the Hara Protected Area, northeastern coast of the Persian Gulf, Iran

This study is the first detailed ecotoxicological study of the annulated sea snake, Hydrophis cyanocinctus. Concentrations of lead, cadmium, nickel and vanadium were evaluated in muscle, liver, kidney, skin and blood of the annulated sea snake (H. cyanocinctus) and in the whole bodies of its main prey species (Periophthalmus waltoni and Boleophthalmus dussumieri) in the Hara Protected Area, the Persian Gulf. The mean concentrations of lead and vanadium were highest in the kidney, which identified the kidney as a target organ for metals in sea snakes as it is in other reptilian groups. Mean concentrations of cadmium and nickel were highest in the liver and skin, respectively. Mean cadmium concentrations were significantly higher in the liver compared to prey species, which indicated that prey items may be a source of cadmium for the annulated sea snake in the study area. Data presented here may be considered as a baseline for further ecotoxicological studies in sea snakes.

Feeding and reproductive behavior of captive sea snakes Hydrophis cyanocinctus

The annulated sea snake ( Hydrophis cyanocinctus ) is a black and yellow banded neurotoxic hydrophiid, widely distributed along the Indian coast. Observations on the feeding and reproductive behavior of the snake were made for the first time in captivity. It consumed live or chopped fish; prey size depended on snake size. Breeding commenced in October and the matured neonates were observed during the following January. Twenty five gravid snakes gave birth to 87 neonates, i.e. 3-5 neonates per snake. During delivery, they exhibited a different type of movement to release the neonates, and all were delivered only at night. All neonates were measured the morphological traits of weight, snout-vent length, tail length, head length, head width, neck girth and body girth. The relative size of females and males differed significantly. But the relative size of tail length showed an opposite trend compared to other morphological traits. At the end of the experiment, the snakes and neonates were released in the open sea.

Molecular cloning, expression and characterization of three short chain α-neurotoxins from the venom of sea snake—Hydrophiinae Hydrophis cyanocinctus Daudin

Three different genes named sn311, sn316 and sn285 were discovered by large-scale randomly sequencing the high quality cDNA library of the venom glands from Hydrophiinae Hydrophis cyanocinctus Daudin. Sequence analysis showed that these three genes encoded three different short chain α-neurotoxins of 81 amino acids, which contained a signal peptide of 21 amino acids and followed by a mature peptide of 60 amino acids. Amino acid comparison reveals that mature peptides of sn311 and sn316 are highly homologous, with the only variance at position 46, which is Lys 46 and Ser 46, respectively. Whereas the mature peptide of sn285 lacks the most conserved amino acids in short chain α-neurotoxins, Asp 31 and Arg 33. The coding sequences of three neurotoxins were cloned into a thioredoxin (TRX) fusion expression vector (pTRX) and expressed as soluble recombinant fusion proteins in E. coli. After purification, approximately 10 mg/l recombinant proteins with the purity up to 95% were obtained. These three recombinant proteins are designated as rSN311, rSN316 and rSN285, they have a molecular weight of 6.963, 6.920 and 6.756 kDa, respectively, which are similar to those predicted from amino acid sequences. LD 50 values of rSN311, rSN316 and rSN285 are 0.0827, 0.095, and 0.0647 mg/kg to mice, respectively. Studies on effects of these recombinant proteins on neuromuscular transmission were carried out, and results indicate that they all can produce prompt blockade of neuromuscular transmission, but display distinct biological activity characteristic individually. The results from UV-circular dichroism (CD) spectra indicate that they share similar secondary structure compared to other identified α-neurotoxins, and no significant structural differences in these recombinant proteins are observed.

Hematological studies on DIC-like findings observed in patients with snakebite in south China

To clarify the characteristics of the hematological disturbances evoked by snakebite, we measured the antithrombin III (AT-III) activity, α2-plasmin inhibitor (α2-PI) activity, fibrinogen concentration (Fg) and level of fibrin degradation products (FDP) in 21 patients envenomed by several snakes in south China between August 1998 and October 1999. The hematological changes observed were as follows: the mean activities of AT-III were decreased in patients bitten by Ophiophagus hannah (Oh.), Bungarus fasciatus (Bf.), Hydrophis cyanocinctus (Hc.), Rhabdophis subminiatus (Rs.), and Trimeresurus stejnegeri (Ts.), while those of α2-PI were decreased in all patients in the present study; Fg was not detectable in the case of Rs. bite, and the Fg concentration after Ts., Oh., Hc. and Bf. bites also decreased markedly thereby increasing the mean levels of FDP in all patients. It thus appeared that DIC-like syndrome was caused in patients envenomed by snakebite. In the present study, we found that patients who were bitten by Rs., which is still being classified as a non-venomous snake, exhibited complete defibrinogenation and severe hemorrhage without any evidence of severe multiple organ damage. We also found that patients with Ts. bite showed marked hemostatic disturbance without severe multiple organ damage. It is considered that such a discrepancy between the hematological findings and clinical symptoms could be a characteristic phenomenon of the DIC-like syndrome induced by snakebite, especially by Rs. and Ts. bites.

Sea snake Hydrophis cyanocinctus venom. II. Histopathological changes, induced by a myotoxic phospholipase A 2 (PLA 2-H1)

A toxic phospholipase A 2 (PLA 2-H1), isolated from the venom of the sea snake Hydrophis cyanocinctus, was tested for its ability to induce myonecrosis and histopathological changes in albino rats and mice. Induction of myonecrosis was demonstrated by their ability to release creatine kinase (CK) from damaged muscle fibers and direct histopathological examination of the injected muscles (i.m.). PLA 2-H1 exhibits intense myonecrosis characterized by the changes including, necrosis and edematous appearance with cellular infiltrate, vacuolation and degenerated muscle cells with delta lesions and heavy edema in between the cells. No myoglobinuria was noted in any group of animals. The purified PLA 2-H1 was also administered intraperitoneally into the experimental animals and tissue samples were taken at several time intervals. Light microscopic examination of the kidney sections revealed severe damage, evident by focal tubular necrosis, complete disquamation of epithelial lining and epithelial degeneration of tubules in all test animals. Light micrographs of liver sections after 24 h of injection shows fatty infiltration in parenchyma and squashed hepatocytes, while after 48 h, fatty vacuolation of parenchyma in a generalized pattern was observed. Furthermore, sections of the lungs of the same group of animals (48 h) show dilated bronchia and marked infiltration of inflammatory cells within alveoli. Our results suggest that the purified PLA 2-H1 induced moderate myotoxicity in muscles and mild histopathological changes in other vital organs without myoglobinuria.

Sea snake Hydrophis cyanocinctus venom. I. Purification, characterization and N-terminal sequence of two phospholipases A 2

Two phospholipases A 2 (PLA 2, H1 and H2) from sea snake Hydrophis cyanocinctus venom were purified to homogeneity in a single step using reversed-phase high performance liquid chromatography on a Nucleosil 7C 18 column. The molecular weights of H1 and H2, as estimated by MALDI MS, were 13588.1 and 13247.2 Da, respectively. The N-terminal 60 amino acid residues were determined by direct automated Edman degradation analysis. Since both PLA 2s show close sequence homologies to those of PLA 2s from other Elapid snakes (60–84%) they have been tentatively classified as belonging to group-IA and Asp-49 phospholipases A 2. Despite the sequence variation (18%) between H1 and H2, their general structural organization is very similar as shown by their clearly related CD spectra. Furthermore, both enzymes are quite thermostable (60–65°C) as determined by temperature variable CD spectra, indicating that the enzymes contain compact folded structure, mainly based on the core structure of disulfide bridges. However, the major PLA 2 (H1) shows higher toxicity to albino rats (LD 50 i.p. 0.04 mg/kg) and purification resulted in 18-fold increase in toxicity over the crude or whole venom (LD 50 i.p. 0.80 mg/kg). H1 also shows edema-inducing and indirect haemolytic but no haemorrhagic activity. Unlike the toxic PLA 2-H1, enzyme H2 was not toxic to albino rats but showed edema-inducing and indirect haemolytic activities.

Evidence for the presence of adrenocorticotropic and opiate-like hormones in the brains of two sea snakes, Hydrophis cyanocinctus and Lapemis hardwickii

The brain acetone powders of the sea snakes Hydrophis cyanocinctus and Lapemis hardwickii were extracted with a mixture of acetone:water:hydrochloric acid (40:21:1 by volume) and the extracts were then added to a copious volume of acetone, in accordance with the method of C. H. Li (1952, J. Amer. Chem. Soc., 74, 2134) for preparing adrenocorticotropin and β-endorphin from mammalian pituitaries. The resultant precipitate, designated acid acetone powder, possessed adrenocorticotropic activity as evidenced in its ability to stimulate corticosterone production in isolated rat adrenal decapsular cells and lipolysis in isolated hamster adipocytes, and in its cross-reactivity in an ACTH radioimmunoassay. The presence of opioid molecules was indicated by activity in opiate radioreceptor assay using either 3H- d-Ala 2- d-Leu 5 enkephalin or [ 3H]naloxone as ligand and rat brain membranes. The brain acetone powders possessed neither “lactogenic” nor “somatogenic” activity as evidenced by their inability to displace the primary ligand in the rat hepatic prolactin receptor- and growth hormone receptor-binding assays, respectively.

Adaptation to fresh water in the sea snake Hydrophis cyanocinctus: Tissue electrolytes and peripheral corticosteroids

Groups of mature sea snakes, Hydrophis cyanocinctus, were transferred from seawater to tanks of running fresh water and sampled at 10, 16, 21, 27, 31, 35, and 51 days after transfer. The following were measured as parameters of adaptation to fresh water and compared with control (seawater maintained) animals: rate of weight loss; muscle water and ion content; blood hematocrit; plasma osmolarity; plasma Na +, K +, Cl −, and urea concentrations. Peripheral levels of corticosterone and aldosterone were measured using competitive protein binding assay and radioimmunoassay, respectively. Adrenal in vitro 18-oxygenase activity was determined radiometrically using [ 3H]corticosterone. The rate of weight loss was lower in snakes in fresh water, but hematocrit and tissue water composition were unaltered. Plasma ion concentrations and total osmolarity fell significantly within 10 days and stabilized after approximately 16 days. Only after 51 days did muscle ionic composition or plasma Na + K + alter. Urea was below detectable levels in all animals. Peripheral aldosterone concentrations remained at 1 to 4 μg/100 ml plasma whereas mean corticosterone titers fell significantly from 8 to 3 μg/100 ml. Adrenal in vitro 18-oxygenase activity was, however, significantly increased after 51 days of freshwater treatment. It was concluded that H. cyanocinctus is an osmoconforming euryhaline reptile, and possible roles for the adrenal in hydromineral balance are discussed.

Steroid synthesis in the adrenal gland of the sea snake Hydrophis cyanocinctus: the metabolism of exogenous precursors.

Abstract Sea snake ( Hydrophis cyanocinctus ) adrenal glands (whole homogenates, preincubated minces, or mitochondrial preparations) were incubated in vitro with exogenous radioactive precursors. Hydrophis adrenal tissue was capable of synthesizing 17-deoxycorticosteroids from exogenous cholesterol, pregnenolone, progesterone, and DOC, but not from sodium [ 14 C]-acetate. Products identified after incubation were pregnenolone, progesterone, 11β-hydroxyprogesterone, DOC, corticosterone, 18-hydroxycorticosterone, and aldosterone. The major product was corticosterone with lesser quantities also of 18-hydroxycorticosterone and aldosterone. In the case of the mitochondrial preparation 11β-hydroxyprogesterone predominated. No evidence for the biosynthesis of cortisol from cholesterol was found. Two types of kinetic incubation were employed: One sampled the incubation medium alone, while the other sampled both medium plus tissue. It was concluded that sampling the medium only did not allow the identification of the biosynthetic pathways operating in vitro . However, from sampling both the medium and the tissue it was concluded that both the C21-C11 and C11-C21 sequences of hydroxylation operated in the conversion of progesterone to corticosterone. The data contrast with those obtained from previous studies on cobra adrenal tissue, particularly with regard to the ability of sea snake adrenals in vitro to 18-oxygenate exogenous precursors.