Assessing the Toxicity of Lagocephalus sceleratus Pufferfish from the Southeastern Aegean Sea and the Relationship of Tetrodotoxin with Gonadal Hormones.

Given the dramatic increase in the L. sceleratus population in the southeastern Aegean Sea, there is growing interest in assessing the toxicity of this pufferfish and the factors controlling its tetrodotoxin (TTX) content. In the present study, liver, gonads, muscle and skin of 37 L. sceleratus specimens collected during May and June 2021 from the island of Rhodes, Greece, were subjected to multi-analyte profiling using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in order to quantitate TTX and evaluate whether this biotoxin interrelates with hormones. TTX and its analogues 4-epiTTX, 11-deoxyTTX, 11-norTTX-6-ol, 4,9-anhydroTTX and 5,11/6,11-dideoxyTTX were detected in all tissue types. Liver and gonads were the most toxic tissues, with the highest TTX concentrations being observed in the ovaries of female specimens. Only 22% of the analyzed muscle samples were non-toxic according to the Japanese toxicity threshold (2.2 µg TTX eq g-1), confirming the high poisoning risk from the inadvertent consumption of this species. Four steroid hormones (i.e., cortisol, testosterone, androstenedione and ß-estradiol) and the gonadotropin-releasing hormone (GnRH) were detected in the gonads. Androstenedione dominated in female specimens, while GnRH was more abundant in males. A positive correlation of TTX and its analogues with ß-estradiol was observed. However, a model incorporating sex rather than ß-estradiol as the independent variable proven to be more efficient in predicting TTX concentration, implying that other sex-related characteristics are more important than specific hormone-regulated processes.

Monthly Variation of Tetrodotoxin Levels in Pufferfish (Lagocephalus sceleratus) Caught from Antalya Bay, Mediterranean Sea.

The silver-cheeked toadfish (Lagocephalus sceleratus), an invasive alien pufferfish species that has rapidly settled throughout the Mediterranean region, poses significant threats not only to native marine species and fisheries but also to public health due to the tetrodotoxin (TTX) they harbor. In this study, TTX concentrations in L. sceleratus from Antalya Bay in the Northeastern Mediterranean Sea were investigated using Q-TOF-LC-MS on a monthly basis over a one-year period. Pufferfish were caught by angling from May 2018 to April 2019. The TTX levels in three different tissues (gonads, liver, and muscle) of 110 pufferfish in total were determined in both male and female individuals caught for 11 months. The highest TTX mean levels generally occurred in the gonads and the lowest in the muscle samples. As regards the maximum TTX contents, the highest concentrations determined were 68.2, 34.2, and 7.8 µg/g in the gonad, liver, and muscle tissues, respectively. The highest levels were generally observed in late autumn to winter (especially in November and December) in all tissues from both genders. Female individuals were generally found to be more toxic than male individuals. The TTX levels found confirm that the consumption of L. sceleratus from Antalya Bay remains dangerous throughout the year, and thus L. sceleratus constantly constitutes an important risk source for public health.

Envenomations caused by fish in Brazil: an evolutionary, morphological, and clinical vision of a neglected problem

ABSTRACT Venomous fish are commonly found in Brazilian waters. The most important marine venomous fish species are stingrays (Dasyatidae, Gimnuridae, Myliobatidae, and Rhinopteridae families), catfish (Ariidae family), scorpionfish and lionfish (both Scorpaenidae family), and toadfish (Batrachoididae family). Meanwhile, Potamotrygonidae stingrays and Pimelodidae catfish are the most important venomous freshwater fish. The mechanisms of envenomation vary and involve various venomous apparatuses and glands. Despite not being highly developed, these venomous apparatuses in fish appear rudimentary, using structures such as fins and rays to inoculate toxins and rarely presenting with specialized structures. Toxins are produced by glandular tissue made up of proteinaceous cells, lacking true glands, and are positioned along the inoculation structures. However, systemic manifestations are rare. No antivenom serum has been developed for any species of American venomous fish. Brazilian venomous fish and their venoms have only recently attracted attention, leading to new studies not only addressing clinical issues in humans, but also exploring the discovery of new active substances with immense pharmacological potential.

Envenomations caused by fish in Brazil: an evolutionary, morphological, and clinical vision of a neglected problem

Venomous fish are commonly found in Brazilian waters. The most important marine venomous fish species are stingrays (Dasyatidae, Gimnuridae, Myliobatidae, and Rhinopteridae families), catfish (Ariidae family), scorpionfish and lionfish (both Scorpaenidae family), and toadfish (Batrachoididae family). Meanwhile, Potamotrygonidae stingrays and Pimelodidae catfish are the most important venomous freshwater fish. The mechanisms of envenomation vary and involve various venomous apparatuses and glands. Despite not being highly developed, these venomous apparatuses in fish appear rudimentary, using structures such as fins and rays to inoculate toxins and rarely presenting with specialized structures. Toxins are produced by glandular tissue made up of proteinaceous cells, lacking true glands, and are positioned along the inoculation structures. However, systemic manifestations are rare. No antivenom serum has been developed for any species of American venomous fish. Brazilian venomous fish and their venoms have only recently attracted attention, leading to new studies not only addressing clinical issues in humans, but also exploring the discovery of new active substances with immense pharmacological potential.

An update on the evolutionary origin of aglomerular kidney with structural and ultrastructural descriptions of the kidney in three fish species.

The kidney of fish contains numerous nephrons, each of which is divided into the renal corpuscle and renal tubules. This glomerular structure is the filtration unit of the nephron and is important for the kidney function, but it has been reported that the renal corpuscle was lost in at least four independent linages of fish (i.e., aglomerular kidney). In this study, the authors newly described renal structures for three species by histological and ultrastructural observations: two aglomerular kidneys from a seahorse Hippocampus barbouri and a toadfish Allenbatrachus grunniens and a glomerular kidney from a snake eel Pisodonophis boro. The renal development of H. barbouri was also described during 1-35 days after birth. In all species tested, the anterior kidney was comprised of haematopoietic tissues and a few renal tubules, whereas the posterior kidney contained more renal tubules. Although the glomerular structure was present in P. boro, light microscopic observations identified no glomeruli in the kidney of H. barbouri and A. grunniens. Ultrastructurally, abundant deep basal infoldings with mitochondria in the renal tubules were observed in A. grunniens compared to H. barbouri and P. boro, suggesting the possible role of basal infoldings in maintaining the osmotic balance. By integrating the results from the three species and comprehensive literature search, the authors further showed that 56 species have been reported to be aglomerular, and that the aglomerular kidney has evolved at least eight times in bony fishes.

Long duration advertisement calls of nesting male plainfin midshipman fish are honest indicators of size and condition.

The plainfin midshipman fish (Porichthys notatus) has long served as a model organism for neuroethology research on acoustic communication and related social behaviors. Type I or 'singing' males produce highly stereotyped, periodic advertisement calls that are the longest known uninterrupted vertebrate vocalizations. Despite the extensive literature on the acoustic behaviour of this species, it remains unclear whether reproductive males signal their quality via their highly energetic, multiharmonic advertisement calls. Here, we recorded the advertisement calls of 22 reproductive type I males at night in a controlled laboratory setting in which males were housed in aquaria maintained at a constant temperature (13.9±0.3°C). The duration of the advertisement calls from type I males was observed to increase from the first call of the night to the middle call after which call duration remained steady until the early morning hours and first light. A strong positive correlation was observed between loudness (sound pressure level and maximum sound pressure level) of the advertisement call and body size (mass and standard length; rs>0.8). In addition, an asymptotic relationship was observed between the harmonic frequencies (f0-f10) of the advertisement calls and male body condition, with harmonic frequencies initially increasing with body condition indices, but then plateauing when body condition measures were high. Taken together, our results suggest that type I male advertisement calls provide reliable honest information about male quality regarding size and body condition. Such condition-dependent information of calling males could potentially be used by receptive females to help facilitate mate choice decisions.

Genome Analysis of Lagocephalus sceleratus: Unraveling the Genomic Landscape of a Successful Invader.

The Tetraodontidae family encompasses several species which attract scientific interest in terms of their ecology and evolution. The silver-cheeked toadfish (Lagocephalus sceleratus) is a well-known "invasive sprinter" that has invaded and spread, in less than a decade, throughout the Eastern and part of the Western Mediterranean Sea from the Red Sea through the Suez Canal. In this study, we built and analysed the first near-chromosome level genome assembly of L. sceleratus and explored its evolutionary landscape. Through a phylogenomic analysis, we positioned L. sceleratus closer to T. nigroviridis, compared to other members of the family, while gene family evolution analysis revealed that genes associated with the immune response have experienced rapid expansion, providing a genetic basis for studying how L. sceleratus is able to achieve highly successful colonisation. Moreover, we found that voltage-gated sodium channel (NaV 1.4) mutations previously connected to tetrodotoxin resistance in other pufferfishes are not found in L. sceleratus, highlighting the complex evolution of this trait. The high-quality genome assembly built here is expected to set the ground for future studies on the species biology.

Fish sounds and boat noise are prominent soundscape contributors in an urban European estuary.

Passive acoustic monitoring is a valuable tool for non-intrusive monitoring of marine environments, also allowing the assessment of underwater noise that can negatively affect marine organisms. Here we provide for the first time, an assessment of noise levels and temporal soundscape patterns for a European estuary. We used several eco-acoustics methodologies to characterize the data collected over six weeks within May 2016 - July 2017 from Tagus estuary. Biophony was the major contributor dominated by fish vocalizations and the main driver for seasonal patterns. Maritime traffic was the major source of anthropogenic noise, with daily patterns monitored using 1584 Hz third-octave band level. This indicator avoided biophony and geophony, unlike other indicators proposed for the EU Marine Strategy Framework Directive. Furthermore, the frequency overlap between anthropophony and biophony demands precautionary actions and calls for further research. This study provides an assessment that will be useful for future monitoring and management strategies.

Gap junction-mediated glycinergic inhibition ensures precise temporal patterning in vocal behavior.

Precise neuronal firing is especially important for behaviors highly dependent on the correct sequencing and timing of muscle activity patterns, such as acoustic signaling. Acoustic signaling is an important communication modality for vertebrates, including many teleost fishes. Toadfishes are well known to exhibit high temporal fidelity in synchronous motoneuron firing within a hindbrain network directly determining the temporal structure of natural calls. Here, we investigated how these motoneurons maintain synchronous activation. We show that pronounced temporal precision in population-level motoneuronal firing depends on gap junction-mediated, glycinergic inhibition that generates a period of reduced probability of motoneuron activation. Super-resolution microscopy confirms glycinergic release sites formed by a subset of adjacent premotoneurons contacting motoneuron somata and dendrites. In aggregate, the evidence supports the hypothesis that gap junction-mediated, glycinergic inhibition provides a timing mechanism for achieving synchrony and temporal precision in the millisecond range for rapid modulation of acoustic waveforms.

Exposure and Recovery of the Gulf Toadfish (Opsanus beta) to Weathered Deepwater Horizon Slick Oil: Impacts on Liver and Blood Endpoints.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants that can be responsible for a variety of deleterious effects on organisms. These adverse outcomes are relatively well studied, but at concentrations rarely found in the environment. Among the documented effects of sublethal acute PAH exposure are reductions in osmoregulatory capacity and immune function, and changes in the function of critical metabolic organs such as the liver. Gulf toadfish (Opsanus beta) were exposed to control seawater (0.006 µg tPAH50 /L) or water accommodated fractions of Deepwater Horizon spill oil diluted to 3 flow-through exposure regimes (0.009, 0.059, and 2.82 µg tPAH50 /L) for 7 d, with a recovery period of equal duration. We hypothesized that these chronic exposures would induce the aryl hydrocarbon receptor (AhR)-mediated pathways and result in significant impacts on markers of osmoregulatory, immune, and metabolic function. We further hypothesized that measurable reversal of these impacts would be observed during the recovery period. Our results indicate that activation of cytochrome P 450 (CYP)1A1 was achieved during exposure and reversed during the recovery phase. The only significant deviations from controls measured were a reduction in plasma glucose in fish exposed to medium and high levels of PAH after 7 d of exposure and a reduction in plasma osmolality fish exposed to high levels of PAHs after 7 d of recovery, when CYP1A1 messenger (m)RNA levels had returned to control levels. Our study illustrates a disconnect between the activation of CYP1A1 in response to environmentally realistic PAHs concentrations and several physiological endpoints and supports the idea that the AhR might not be associated with mediating osmoregulatory, immune, and metabolic changes in Gulf toadfish. Environ Toxicol Chem 2021;40:1075-1086. © 2020 SETAC.

Impacts of a local music festival on fish stress hormone levels and the adjacent underwater soundscape.

An understudied consequence of coastal urbanization on marine environments is sound pollution. While underwater anthropogenic sounds are recognized as a threat to aquatic organisms, little is known about the effects of above-surface coastal sound pollution on adjacent underwater soundscapes and the organisms inhabiting them. Here, the impact of noise from the 2019 Ultra Music Festival® in Miami, FL, USA was assessed at the University of Miami Experimental Hatchery (UMEH) located directly adjacent to the music festival and on underwater sound levels in Bear Cut, a nearby water channel. In addition, stress hormone levels in fish held at UMEH were measured before and during the festival. Air sound levels recorded at UMEH during the Ultra Music Festival did not exceed 72 dBA and 98 dBC. The subsurface sound intensity levels in the low frequency band increased by 2-3 dB re 1 µPa in the adjacent waterway, Bear Cut, and by 7-9 dB re 1 µPa in the fish tanks at UMEH. Gulf toadfish (Opsanus beta) housed in the UMEH tanks experienced a 4-5 fold increase in plasma cortisol, their main stress hormone, during the first night of the Ultra Music Festival compared to two baseline samples taken 3 weeks and 4 days before Ultra. While this study offers preliminary insights into this type of sound pollution, more research is needed to conclude if Ultra caused a stress response in wild organisms and to fully understand the implications of this type of sound pollution.

Boat noise in an estuarine soundscape - A potential risk on the acoustic communication and reproduction of soniferous fish in the May River, South Carolina.

The impact of boat related noise on marine life is a subject of concern, particularly for fish species that utilize acoustic communication for spawning purposes. The goal of this study was to quantify and examine the risk of boat noise on fish acoustic communication by performing acoustic monitoring of the May River, South Carolina (USA) from February to November 2013 using DSG-Ocean recorders. The number of boats detected increased from the source to the mouth with the highest detections near the Intracoastal Waterway (ICW). Boat noise frequency ranges overlapped with courtship sounds of silver perch (Bairdiella chrysoura), black drum (Pogonias cromis), oyster toadfish (Opsanus tau), spotted seatrout (Cynoscion nebulosus), and red drum (Sciaenops ocellatus). In the May River estuary, red drum may experience the greatest risk of auditory masking because of late afternoon choruses (21% time overlap with boat noise) and only one spawning location near the noisy ICW.

The complete mitochondrial genome of Lagocephalus sceleratus (Tetraodontiformes; Tetraodontidae) and phylogenetic studies of Tetraodontidae.

The complete mitochondrial genome sequence of Lagocephalus sceleratus was determined. The complete mitochondrial genome was 16,444 bp in length and contained 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and two non-coding region (the control region and the origin of light strand replication). The overall base composition was A 27.59%, C 31.43%, G 17.12%, T 23.85%. All protein-coding genes started with an ATG initiation codon, except COI used GTG. With the exception of ND6 and eight tRNA genes, all other genes were encoded on the heavy strand. Additionally, the phylogenetic relationship of 37 Tetraodontidae species based on the complete genome was analyzed, and the result showed that L. sceleratus was clustered with other Lagocephalus species. These results would be useful for the investigation of phylogenetic relationship, taxonomic classification and phylogeography of the Tetraodontidae.

Fundamental constraints in synchronous muscle limit superfast motor control in vertebrates.

Superfast muscles (SFMs) are extremely fast synchronous muscles capable of contraction rates up to 250 Hz, enabling precise motor execution at the millisecond time scale. SFM phenotypes have been discovered in most major vertebrate lineages, but it remains unknown whether all SFMs share excitation-contraction coupling pathway adaptations for speed, and if SFMs arose once, or from independent evolutionary events. Here, we demonstrate that to achieve rapid actomyosin crossbridge kinetics bat and songbird SFM express myosin heavy chain genes that are evolutionarily and ontologically distinct. Furthermore, we show that all known SFMs share multiple functional adaptations that minimize excitation-contraction coupling transduction times. Our results suggest that SFM evolved independently in sound-producing organs in ray-finned fish, birds, and mammals, and that SFM phenotypes operate at a maximum operational speed set by fundamental constraints in synchronous muscle. Consequentially, these constraints set a fundamental limit to the maximum speed of fine motor control.

The impact of acute PAH exposure on the toadfish glucocorticoid stress response.

The objective of the present study was to determine whether the polycyclic aromatic hydrocarbons (PAHs) associated with the Deepwater Horizon (DWH) oil spill impacted the stress response of teleost fish. The hypothesis was that intraperitoneal (IP) treatment with PAHs associated with the DWH oil spill or waterborne exposure to DWH oil high energy water-accommodated fraction (HEWAF) would result in the downregulation of the stress response of Gulf toadfish, Opsanus beta, a benthic marine teleost fish that resides in the Gulf of Mexico. In vivo plasma cortisol levels and adrenocorticotropic hormone (ACTH)-mediated cortisol secretion by in vitro isolated kidney tissue were measured. Toadfish at rest IP-treated with naphthalene had higher plasma cortisol compared to fluorene-treated and control fish; phenanthrene-treated fish tended to have higher plasma cortisol levels that fluorene-treated and controls. When subjected to an additional crowding stress, naphthalene and phenanthrene-treated fish were no longer able to mount a stress response compared to fluorene-treated and control fish, suggesting exhaustion of the stress response. Supporting this in vivo data, there tended to be less cortisol released by the kidney in vitro from naphthalene and phenanthrene-treated fish in response to ACTH compared to controls. In contrast, toadfish at rest exposed to 3% Slick A HEWAF did not have significantly different plasma cortisol levels compared to controls. But, exposed fish did have significantly less cortisol released by the kidney in vitro in response to ACTH. When toadfish were subjected to an additional stress, there were no significant differences in plasma cortisol or ACTH, suggesting the action of a secondary secretagogue to maintain plasma cortisol in vivo. Combined, these data suggest that in response to acute PAH exposure, there may be internalization or downregulation of the melanocortin 2 receptor (MC2R) that mediates the action of ACTH.

An implantable two axis micromanipulator made with a 3D printer for recording neural activity in free-swimming fish.

BACKGROUND: Chronically implanted electrodes allow monitoring neural activity from free moving animals. While a wide variety of implanted headstages, microdrives and electrodes exist for terrestrial animals, few have been developed for use with aquatic animals. NEW METHOD: A two axis micromanipulator was fabricated with a Formlabs 3D printer for implanting electrodes into free-swimming oyster toadfish (Opsanus tau). The five piece manipulator consisted of a base, body, electrode holder, manual screw drive and locking nut. The manipulator measured approximately 25×20×30mm (l×w×h) and weighed 5.28g after hand assembly. RESULTS: Microwire electrodes were inserted successfully with the manipulator to record high fidelity signals from the anterior lateral line nerve of the toadfish. COMPARISON WITH EXISTING METHOD(S): The micromanipulator allowed the chronically implanted electrodes to be repositioned numerous times to record from multiple sites and extended successful recording time in the toadfish by several days. CONCLUSIONS: Three dimensional printing allowed an inexpensive (<$US 5 material), two axis micromanipulator to be printed relatively rapidly (<2h) to successfully record from multiple sites in the anterior lateral line nerve of free-swimming toadfish.

Optical micro-tomography "OPenT" allows the study of large toadfish Halobatrachus didactylus embryos and larvae.

Batrachoidids, which include midshipman and toadfish are less known among embryologists, but are common in other fields. They are characteristic for their acoustic communication, and develop hearing and sound production while young juveniles. They lay large benthic eggs (>5mm) with a thick chorion and adhesive disk and slow development, which are particularly challenging for studying embryology. Here we took advantage of a classical tissue clearing technique and the OPenT open-source platform for optical tomography imaging, to image a series of embryos and larvae from 3 to 30mm in length, which allowed detailed 3D anatomical reconstructions non-destructively. We documented some of the developmental stages (early and late in development) and the anatomy of the delicate stato-acoustic organs, swimming bladder and associated sonic muscles. Compared to other techniques accessible to developmental biology labs, OPenT provided advantages in terms of image quality, cost of operation and data throughput, allowing identification and quantitative morphometrics of organs in larvae, earlier and with higher accuracy than is possible with other imaging techniques.

Analysis of the intersexual variation in Thalassophryne maculosa fish venoms.

Gender related variation in the molecular composition of venoms and secretions have been described for some animal species, and there are some evidences that the difference in the toxin (s) profile among males and females may be related to different physiopathological effects caused by the envenomation by either gender. In order to investigate whether this same phenomenon occurs to the toadfish Thalassophryne maculosa, we have compared some biological and biochemical properties of female and male venoms. Twenty females and males were collected in deep waters of the La Restinga lagoon (Venezuela) and, after protein concentration assessed, the induction of toxic activities in mice and the biochemical properties were analyzed. Protein content is higher in males than in females, which may be associated to a higher size and weight of the male body. In vivo studies showed that mice injected with male venoms presented higher nociception when compared to those injected with female venoms, and both venoms induced migration of macrophages into the paw of mice. On the other hand, mice injected with female venoms had more paw edema and extravasation of Evans blue in peritoneal cavity than mice injected with male venoms. We observed that the female venoms had more capacity for necrosis induction when compared with male venoms. The female samples present a higher proteolytic activity then the male venom when gelatin, casein and FRETs were used as substrates. Evaluation of the venoms of females and males by SDS-PAGE and chromatographic profile showed that, at least three components (present in two peaks) are only present in males. Although the severity of the lesion, characterized by necrosis development, is related with the poisoning by female specimens, the presence of exclusive toxins in the male venoms could be associated with the largest capacity of nociception induction by this sample.

Acoustic Communication in Fishes and Potential Effects of Noise.

Many soniferous fishes such as cods and groupers are commercially important. Sounds are produced during courtship and spawning, and there is the potential for aquatic noise to interfere with critical behaviors and affect populations. There are few data on the response of wild populations of sound-producing fishes to acoustic noise. New motion and sound exposure fish tags could be used to assess the behavioral responses of large numbers of fish to noise exposure. Many factors, such as fishing mortality and environmental variability in prey supply, could also affect populations and potentially interact with the behavioral responses to noise.

Steroidogenesis by testis and accessory glands of the Lusitanian toadfish, Halobatrachus didactylus, during reproductive season.

In teleost fish sex steroids are essential for gonadal function and have marked effects in reproductive and agonistic behavior and in the expression of secondary sexual characteristics. The Lusitanian toadfish, Halobatrachus didactylus, has two male morphotypes: type I males are territorial nest-holders and have large accessory glands while type II males are smaller, have a relatively large testis and small accessory glands. In the present study, the steroidogenic activity of the testis and accessory testicular glands of the Lusitanian toadfish were examined in vitro as well as their presence in urine. The testis of type I males produced 11-ketotestosterone (11KT) and 11ß-hydroxy-4-androstene-3,17-dione (11ßA) from tritiated 17-hydroxyprogesterone, while those of type II males produced testosterone (T) and 11ß,17ß-dihydroxy-4-andosten-3-one (11ßT), but not 11KT. Additionally, the testis and accessory glands of both morphs produced mostly 5ß,3α-reduced and 17,20α-hydroxylated metabolites. Type I, but not of type II, males synthesised 5ß-reduced androgens in their accessory glands. The presence of 11ßA exclusively in the urine of type I males during reproductive season suggests an association with maintenance of secondary sexual characteristics and behavior in this morph. The urine of both types of males contained two 5α-androstane and 5ß-pregnane glucuronides. Among the latter steroids, those that are 17,21-dihydroxylated are potentially metabolites from cortisol and were found only in type I males during the spawning season. The diversity of metabolites produced by the testis and accessory glands and the presence of some in urine is suggestive of a potential role in chemical communication and reproductive behavior.