A Chromosome-Level Genome Assembly of the Reef Stonefish (Synanceia verrucosa) Provides Novel Insights into Stonustoxin (sntx) Genes.

Reef stonefish (Synanceia verrucosa) is one of the most venomous fishes, but its biomedical study has been restricted to molecular cloning and purification of its toxins, instead of high-throughput genetic research on related toxin genes. In this study, we constructed a chromosome-level haplotypic genome assembly for the reef stonefish. The genome was assembled into 24 pseudo-chromosomes, and the length totaled 689.74 Mb, reaching a contig N50 of 11.97 Mb and containing 97.8% of complete BUSCOs. A total of 24,050 protein-coding genes were annotated, of which metalloproteinases, C-type lectins, and stonustoxins (sntx) were the most abundant putative toxin genes. Multitissue transcriptomic and venom proteomic data showed that sntx genes, especially those clustered within a 50-kb region on the chromosome 2, had higher transcription levels than other types of toxins as well as those sntx genes scatteringly distributed on other chromosomes. Further comparative genomic analysis predicted an expansion of sntx-like genes in the Percomorpha lineage including nonvenomous fishes, but Scorpaenoidei species experienced extra independent sntx duplication events, marking the clear-cut origin of authentic toxic stonustoxins. In summary, this high-quality genome assembly and related comparative analysis of toxin genes highlight valuable genetic differences for potential involvement in the evolution of venoms among Scorpaeniformes fishes.

Marine Envenomation in Okinawa: Overview and Treatment Concept.

Okinawa prefecture is a popular tourist destination due to its beaches and reefs. The reefs host a large variety of animals, including a number of venomous species. Because of the popularity of the reefs and marine activities, people are frequently in close contact with dangerous venomous species and, thus, are exposed to potential envenomation. Commonly encountered venomous animals throughout Okinawa include the invertebrate cone snail, sea urchin, crown-of-thorns starfish, blue-ringed octopus, box jellyfish, and fire coral. The vertebrates include the stonefish, lionfish, sea snake, and moray eel. Treatment for marine envenomation can involve first aid, hot water immersion, antivenom, supportive care, regional anesthesia, and pharmaceutical administration. Information on venomous animals, their toxins, and treatment should be well understood by prehospital care providers and physicians practicing in the prefecture.

The Geographic Distribution, Venom Components, Pathology and Treatments of Stonefish (Synanceia spp.) Venom.

Stonefish are regarded as one of the most venomous fish in the world. Research on stonefish venom has chiefly focused on the in vitro and in vivo neurological, cardiovascular, cytotoxic and nociceptive effects of the venom. The last literature review on stonefish venom was published over a decade ago, and much has changed in the field since. In this review, we have generated a global map of the current distribution of all stonefish (Synanceia) species, presented a table of clinical case reports and provided up-to-date information about the development of polyspecific stonefish antivenom. We have also presented an overview of recent advancements in the biomolecular composition of stonefish venom, including the analysis of transcriptomic and proteomic data from Synanceia horrida venom gland. Moreover, this review highlights the need for further research on the composition and properties of stonefish venom, which may reveal novel molecules for drug discovery, development or other novel physiological uses.

Stonefish toxin defines an ancient branch of the perforin-like superfamily.

The lethal factor in stonefish venom is stonustoxin (SNTX), a heterodimeric cytolytic protein that induces cardiovascular collapse in humans and native predators. Here, using X-ray crystallography, we make the unexpected finding that SNTX is a pore-forming member of an ancient branch of the Membrane Attack Complex-Perforin/Cholesterol-Dependent Cytolysin (MACPF/CDC) superfamily. SNTX comprises two homologous subunits (α and ß), each of which comprises an N-terminal pore-forming MACPF/CDC domain, a central focal adhesion-targeting domain, a thioredoxin domain, and a C-terminal tripartite motif family-like PRY SPla and the RYanodine Receptor immune recognition domain. Crucially, the structure reveals that the two MACPF domains are in complex with one another and arranged into a stable early prepore-like assembly. These data provide long sought after near-atomic resolution insights into how MACPF/CDC proteins assemble into prepores on the surface of membranes. Furthermore, our analyses reveal that SNTX-like MACPF/CDCs are distributed throughout eukaryotic life and play a broader, possibly immune-related function outside venom.

Expression of Recombinant Stonustoxin Alpha Subunit and Preparation of polyclonal antiserum for Stonustoxin Neutralization Studies.

Stonustoxin (SNTX) is a lethal protein found in stonefish venom, responsible for many of the symptoms associated with stonefish envenomation. To counter stonefish venom challenges, antivenom is a well-established and effective solution. In this study, we aimed to produce the recombinant alpha subunit protein of Stonustoxin from Synanceia horrida and prepare antibodies against it The SNTXα gene sequence was optimized for E. coli BL21 (DE3) expression and cloned into the pET17b vector. Following purification, the recombinant protein was subcutaneously injected into rabbits, and antibodies were extracted from rabbit´s serum using a G protein column As a result of codon optimization, the codon adaptation index for the SNTXα cassette increased to 0.94. SDS-PAGE analysis validated the expression of SNTXα, with a band observed at 73.5 kDa with a yield of 60 mg/l. ELISA results demonstrated rabbits antibody titers were detectable up to a 1:256,000 dilution. The isolated antibody from rabbit´s serum exhibited a concentration of 1.5 mg/ml, and its sensitivity allowed the detection of a minimum protein concentration of 9.7 ng. In the neutralization assay the purified antibody against SNTXα protected mice challenged with 2 LD50. In conclusion, our study successfully expressed the alpha subunit of Stonustoxin in a prokaryotic host, enabling the production of antibodies for potential use in developing stonefish antivenom.

Suspected stonefish envenomation in Queensland over eight years: a retrospective series.

INTRODUCTION: Stonefish envenomation results in localized severe pain and swelling and systemic features, including vomiting, arrhythmia, pulmonary oedema, and possibly death. There are limited data regarding the effectiveness of the available antivenom. The aim of this series is to characterize presentations of patients with suspected stonefish envenomation and investigate treatment, including antivenom. METHODS: This is a retrospective observational series of suspected stonefish envenomation as reported to the Queensland Poisons Information Centre or Princess Alexandra Hospital Clinical Toxicology Unit from July 2015 to January 2023. Patients were identified through the databases held by both the Centre and Unit, and data on clinical features and investigations were collected from the patient's electronic medical record. RESULTS: There were 87 suspected stonefish envenomations from July 2015 to January 2023. The median age was 26 (range: 5-69) years, and 69 (79 per cent) patients were male. Pain was reported in 85 (98 per cent) with a median peak pain score of 10 (range 4-12; three rated their pain greater than 10/10). A clear wound was documented in 64 (74 per cent), with local swelling in 63 (72 per cent). A foreign body was retained in eight (9 per cent) presentations. Systemic symptoms were rare, with vomiting in four (5 per cent) and dizziness in two (2 per cent) presentations. There were no instances of hypotension, arrhythmia, or pulmonary oedema. Hot water was administered in 72 (83 per cent) presentations. Oral analgesia was given in 55 (63 per cent). Parenteral analgesia was given in 53 (61 per cent), most commonly opioids. Local anaesthetic block was performed in 19 presentations (22 per cent), with effectiveness documented in 16/19 (84 per cent). Five patients received antivenom for intractable pain, and all received subsequent parenteral analgesia or local anaesthetic block. CONCLUSIONS: Stonefish envenomation is characterized by severe pain. Systemic symptoms were rare and not severe in this series. Local anaesthetic block appeared to be the most effective intervention for severe pain when performed. Antivenom appeared to be ineffective in managing pain.

Stonefish (Synanceia spp.) Ichthyocrinotoxins: An ecological review and prospectus for future research and biodiscovery.

Marine organisms possess a diverse array of unique substances, many with wide ranging potential for applications in medicine, industry, and other sectors. Stonefish (Synanceia spp.), a bottom-dwelling fish that inhabit shallow and intertidal waters throughout the Indo-Pacific, harbour two distinct substances, a venom, and an ichthyocrinotoxin. Stonefish are well-known for the potent venom associated with their dorsal spines as it poses a significant risk to public health. Consequently, much of the research on stonefish focusses on the venom, with the aim of improving outcomes in cases of envenomation. However, there has been a notable lack of research on stonefish ichthyocrinotoxins, a class of toxin that is synthesised within specialised epithelial cells (i.e., tubercles) and exuded onto the skin. This has resulted in a substantial knowledge gap in our understanding of these animals. This review aims to bridge this gap by consolidating literature on the ecological functions and biochemical attributes of ichthyocrinotoxins present in various fish species and juxtaposing it with the current state of knowledge of stonefish ecology. We highlight the roles of ichthyocrinotoxins in predator defence, bolstering innate immunity, and mitigating integumentary interactions with parasites and detrimental fouling organisms. The objective of this review is to identify promising research avenues that could shed light on the ecological functions of stonefish ichthyocrinotoxins and their potential practical applications as therapeutics and/or industrial products.

Inter-species variation in stonefish (Synanceia spp.) ichthyocrinotoxins; an ecological perspective.

Although stonefish (Synanceia spp.) are well-known to harbour a highly noxious defensive venom in their dorsal spines, very little is known about the composition and ecological function of the ichthyocrinotoxins that they secrete onto their epidermis. This study profiled reef (Synanceia verrucosa) and estuarine (Synanceia horrida) stonefish ichthyocrinotoxins via electrophoresis, liquid chromatography, and mass spectrometry to visualise and compare the composition of these toxins between the two species. Stonefish ichthyocrinotoxins were found to be multifarious concoctions that exhibited subtle differences between reef and estuarine species. We speculate that these variations and similarities are driven by the different and similar ecology of these fish species. Further research into the activity of the toxins components is now required to better understand their ecological role.

Getting stoned: Characterisation of the coagulotoxic and neurotoxic effects of reef stonefish (Synanceia verrucosa) venom.

The reef stonefish (Synanceia verrucosa) is a venomous fish which causes excruciatingly painful envenomations. While some research on the pathophysiology and functions of the venom have been conducted, there are still some gaps in the understanding of the venom effects due to the extreme lability of fish venom toxins and the lack of available testing platforms. Here we set out to assess new functions of the venom whilst also attempting to address some unclear pathophysiological effects from previous literature. Utilising a biolayer interferometry assay, our results highlight that the venom binds to the orthosteric site of the α-1 nicotinic acetylcholine receptor as well as the domain IV of voltage-gated Ca2+ (CaV1.2) channel mimotopes. Both these results add some clarity to the previously ambiguous literature. We further assessed the coagulotoxic effects of the venom using thromboelastography and Stago STA-R Max coagulation analyser assays. We reveal that the venom produced anticoagulant activity and significantly delayed time until clot formation of recalcified human plasma which is likely through the degradation of phospholipids. There was a difference between fresh and lyophilised venom activity toward the nicotinic acetylcholine receptor mimotopes and coagulation assays, whilst no difference was observed in the activity toward the domain IV of CaV1.2 mimotopes. This research adds further insights into the neglected area of fish venom whilst also highlighting the extreme labile nature of fish venom toxins.

Is stonefish Synanceia verrucosa envenomation potentially lethal?

Stonefish sting lethality in man has been scarcely documented since the middle of the 20th century. We report three clinical cases, including one fatality, emphasizing the cardiovascular toxicity of the Synanceia verrucosa venom, and its potentially lethal effects. All clinical data have been recently collected in New Caledonia and French Polynesia.

Investigation of the estuarine stonefish (Synanceia horrida) venom composition.

The Estuarine stonefish (Synanceia horrida) is recognised as one of the most venomous fish species in the world but the overall venom composition has yet to be investigated using in-depth transcriptomic and proteomic methods. To date, known venom components are restricted to a hyaluronidase and a large, pore-forming toxin known as Stonustoxin (SNTX). Transcriptomic sequencing of the venom gland resulted in over 170,000 contigs with only 0.4% that were homologous to putative venom proteins. Integration of the transcriptomic data with proteomic data from the S. horrida venom confirmed the hyaluronidase and SNTX to be present, together with several other protein families including major contributions from C-type lectins. Other protein families observed included peroxiredoxin and several minor protein families such as Golgi-associated plant pathogenesis related proteins, tissue pathway factor inhibitors, and Kazal-type serine protease inhibitors that, although not putative venom proteins, may contribute to the venom's adverse effects. BIOLOGICAL SIGNIFICANCE: Proteomic analysis of milked Synanceia horrida venom, paired with transcriptomic analysis of the venom gland tissue revealed for the first time the composition of one of the world's most dangerous fish venoms. The results demonstrate that the venom is relatively less complex compared to other well-studied venomous animals with a number of unique proteins not previously found in animal venoms.

Biochemical and histopathological effects of the stonefish (Synanceia verrucosa) venom in rats.

The Reef Stonefish (Synanceia verrucosa) is one of the most dangerous venomous fish known, and has caused occasional human fatalities. The present study was designed to examine some of the pathological effects of the venom from this fish in Sprague Dawley rats. Crude venom was extracted from venom glands of the dorsal spines of stonefish specimens collected from coral reefs in the Gulf of Aqaba (in the northeastern branch of the Red Sea). The rats were given intramuscular injections of the venom and acute toxicity and effect on selected serum marker enzymes as well as normal architecture of vital organs were evaluated. The rat 24 h LD50 was 38 µg/kg body weight. The serum biochemical markers; alanine transaminase (ALT), lactate dehydrogenase (LDH) and creatine kinase (CK) increased after 6 h of administration of a sub lethal dose of the venom and remained significantly raised at 24 h. Amylase levels also significantly increased after venom injection. The venom caused histological damage manifested as an interstitial hemorrhage, inflammatory cell infiltration, and necrosis. The demonstrated rises in the levels of different critical biochemical parameters in the serum may have led to the observed abnormal morphological changes in these organs. These results may account for some of the clinical manifestations observed in victims of stonefish envenomation. Thus, the presented data provide further in vivo evidence of the stonefish toxic effects that may threaten human life and call for the need for special measures to be considered.

Relationship between food and venom production in the estuarine stonefish Synanceia horrida.

BACKGROUND: The potential costs of venom production may be significant to many marine venomous taxa. In general, the parameters that influence the rate of venom production are poorly understood, but seem to be related to feeding frequency. METHODS: This study examines the effects of starvation on venom profile and venom yield on the estuarine stonefish (Synanceia horrida). In total, the venom of eight stonefishes was tested under two feeding regimes. Over a four week period, one of the two groups underwent an episode of suspended feeding, while the other was fed on a daily basis. The effect of time on venom replacement was determined by a paired T-test. ANOVA was performed to analyze differences in venom weight between fed and unfed treatments. RESULTS: Nutritional suspension was found to have a significant effect on the quantity of venom produced. SDS-PAGE gel and FPLC revealed that the components of the venom collected from both groups were similar, indicating that four weeks is an adequate time to regenerate key venom components but not replenish initial venom quantities. CONCLUSIONS: Venom production was found to be affected by starvation.

Occurrence of a stonefish toxin-like toxin in the venom of the rabbitfish Siganus fuscescens.

Rabbitfish belonging to the order Perciformes are well-known venomous fish that are frequently involved in human accidents. However little research has been done into either the whole venom toxicities or the structures and properties of their venom toxins. In this study, we first examined biological activities of the crude venom extract prepared from dorsal spines of Siganus fuscescens, a rabbitfish most commonly found along the coasts of Japan. As a result, the crude venom extract was shown to have mouse-lethal activity, hemolytic activity against rabbit erythrocytes, edema-forming activity and nociceptive activity, similar to the known scorpaeniform fish toxins (stonefish toxins and their analogues). Then, the primary structure of the S. fuscescens toxin was successfully elucidated by the same cDNA cloning strategy as previously employed for the toxins of some scorpaeniform fish (lionfish, devil stinger and waspfish). The S. fuscescens toxin is obviously an analogue of stonefish toxins, being composed of two kinds of subunits, an α-subunit of 703 amino acid residues and a ß-subunit of 699 amino acid residues. Furthermore, the genes encoding both subunits were cloned from genomic DNA and shown to have an architecture of three exons and two introns, as reported for those of the scorpaeniform fish toxins. This study is the first to demonstrate the occurrence of stonefish toxin-like toxins in perciform fish.

Marine envenomations in returning French travellers seen in a tropical diseases unit, 2008-13.

BACKGROUND: Travel and aquatic activities are increasing in tropical regions. The risk and the spectrum of marine envenomation are unknown in travellers. This work aims to evaluate the prevalence and the characteristics of marine envenomations in returning travellers. METHODS: We retrospectively studied the medical charts of all returning travellers presenting with a health problem in a French tropical disease unit between 2008 and 2013, with focus on travellers complaining of marine envenomation. Characteristics of each type of envenomation are described. RESULTS: Of the 3315 travellers seen during the study period, 43 consulted for a presumed marine envenomation. Six patients were excluded, leaving 37 cases of confirmed marine envenomation. It corresponds to a prevalence of 1.1%. Sex ratio was balanced with 18 men and 19 women. Median age was 42 years (range 25-68 years). Median travel duration was 14 days (range: 6-62 days). The main travel destination was Southeast Asia in 10 cases, followed by islands of East Africa in seven cases. Median elapsed time between envenomation and consultation was 14 days (range: 2-130 days). The purpose of travel was tourism in all cases. The main clinical aspects were oedema, sting marks, cellulitis and flagellations. Eleven cases were presumably caused by corals, 10 by stonefish, 8 by jellyfish, 2 by weever fish, 2 by starfish, 2 by stingray, 1 by lionfish and 1 by sea anemone. CONCLUSION: Prevalence of marine envenomation is low in returning travellers. They are mostly caused by corals, stonefish and jellyfish.

Stonefish envenomation of hand with impending compartment syndrome.

BACKGROUND: Marine stings and envenomation are fairly common in Malaysia. Possible contact to various marine life occurs during diving, fishing and food handling. Even though majority of fish stings are benign, there are several venomous species such as puffer fish, scorpion fish, lionfish, stingray and stonefish that require urgent medical treatment. Stonefish is one of the most venomous fish in the world with potential fatal local and systemic toxicity effects to human. CASE PRESENTATION: We reported a case of stonefish sting complicated with impending compartment syndrome. CONCLUSIONS: Medical staff should be alert about the possibility of this potential emergency in standard management of stonefish stings.

Hot water immersion as a treatment for stonefish sting: A case report.

The North Borneo state of Sabah is known worldwide for its beautiful islands and dive sites. Local hospitals deal with a number of marine-related injuries, including marine fauna envenomation by Scorpaenidae and Synanceiidae families of fish. We report a case of a tourist who presented with excruciating pain on her right foot after stepping on a stonefish. Despite being given parenteral analgesia and regional anaesthesia, the pain persisted. Her pain improved after she soaked her foot in hot water for about 30 minutes. No further treatment was required. We reviewed the literature comparing this inexpensive mode of treatment with other conventional treatments. We also explored the possibility of using hot water immersion for treatment of envenomation by other types of marine animals.