Cellular mechanisms underlying extraordinary sulfide tolerance in a crustacean holobiont from hydrothermal vents.

The shallow-water hydrothermal vent system of Kueishan Island has been described as one of the world's most acidic and sulfide-rich marine habitats. The only recorded metazoan species living in the direct vicinity of the vents is Xenograpsus testudinatus, a brachyuran crab endemic to marine sulfide-rich vent systems. Despite the toxicity of hydrogen sulfide, X. testudinatus occupies an ecological niche in a sulfide-rich habitat, with the underlying detoxification mechanism remaining unknown. Using laboratory and field-based experiments, we characterized the gills of X. testudinatus that are the major site of sulfide detoxification. Here sulfide is oxidized to thiosulfate or bound to hypotaurine to generate the less toxic thiotaurine. Biochemical and molecular analyses demonstrated that the accumulation of thiosulfate and hypotaurine is mediated by the sodium-independent sulfate anion transporter (SLC26A11) and taurine transporter (Taut), which are expressed in gill epithelia. Histological and metagenomic analyses of gill tissues demonstrated a distinct bacterial signature dominated by Epsilonproteobacteria. Our results suggest that thiotaurine synthesized in gills is used by sulfide-oxidizing endo-symbiotic bacteria, creating an effective sulfide-buffering system. This work identified physiological mechanisms involving host-microbe interactions that support life of a metazoan in one of the most extreme environments on our planet.

Changes in Red Color Sensitivity over the Spawning Cycle of Female Three-spined Stickleback (Gasterosteus aculeatus).

Male red nuptial coloration is a primary mating signal for three-spined sticklebacks (Gasterosteus aculeatus), and the retinae of both sexes are especially sensitive to red during the breeding season. Red sensitivity is an important aspect of female mate choice in this species, but only when they are ready to spawn and not over the entire breeding period. Here, we aimed to determine if the red sensitivity of female sticklebacks change over their repeat spawning cycle. To this end, we assessed retinal opsin mRNA levels and behavioral red sensitivity in females over this cycle. Both methods indicated that females were more sensitive to red during spawning than in the inter-spawning intervals. Relative expression levels of red color opsin genes (lws) and optical motor sensitivity were high during spawning, decreased after the spawning period, and then increased again 72-96 h later when they were ready to spawn again. Thus, female sticklebacks altered their color sensitivity according to need, but the underlying mechanism remains unclear.

To cope with a changing aquatic soundscape: Neuroendocrine and antioxidant responses to chronic noise stress in fish.

Anthropogenic underwater noises that change aquatic soundscapes represent an important issue in marine conservation. While it is evident that strong underwater acoustic pollutants may cause significant damage to fish at short ranges, the physiological effects of long-term exposure to relatively quiet but continuous noise are less well understood. Here, we present a summary of the known impacts of long-term underwater noise on hypothalamic-pituitary-interrenal (HPI) axis-mediated physiological responses, oxidant/antioxidant balance, and neurotransmitter regulation in fish. Cortisol is known to play a central role in physiological stress response, most often as a mediator of acute response. However, recent research indicates that noise exposure may also induce chronic corticosteroid responses, which involve increased rates of cortisol turnover. Moreover, continuous noise affects oxidative stress and antioxidant systems in vertebrates and fish, suggesting that oxidative species may mediate some noise-induced physiological responses and make these systems valuable noise stress markers. Lastly, noise stress is also known to affect neurotransmitters in the brain that may cause neurophysiological and behavioral changes. The neurochemical mechanisms underlying observed behavioral disorders in fish after exposure to changing acoustic environments are a topic of active research. Overall, a growing body of evidence suggests that chronic noise pollution could be a threat to fish populations. In future work, systematic and comparative investigations into long-term and transgenerational adaptive neuronal and metabolic responses to noise will be important to understand the physiological patterns and dynamics of noise response relevant to fish conservation.

Phylogenetic analysis and ontogenetic changes in the cone opsins of the western mosquitofish (Gambusia affinis).

To convert external light into internal neural signal, vertebrates rely on a special group of proteins, the visual opsins. Four of the five types of visual opsins-short-wavelength sensitive 1 (Sws1), short-wavelength sensitive 2 (Sws2), medium-wavelength sensitive (Rh2), and long-wavelength sensitive (Lws)-are expressed in cone cells for scotopic vision, with the fifth, rhodopsin (Rh1), being expressed in rod cells for photopic vision. Fish often display differing ontogenetic cone opsin expression profiles, which may be related to dietary and/or habitat ontogenetic shift. The western mosquitofish (Gambusia affinis) is an aggressive invader that has successfully colonized every continent except Antarctica. The strong invasiveness of this species may be linked to its visual acuity since it can inhabit turbid waters better than other fishes. By genome screening and transcriptome analysis, we identify seven cone opsin genes in the western mosquitofish, including one sws1, two sws2, one rh2, and three lws. The predicted maximal absorbance wavelength (λmax) values of the respective proteins are 353 nm for Sws1, 449 nm for Sws2a, 408 nm for Sws2b, 516 nm for Rh2-1, 571 nm for Lws-1, and 519 nm for Lws-3. Retention of an intron in the lws-r transcript likely renders this visual opsin gene non-functional. Our real-time quantitative PCR demonstrates that adult male and female western mosquitofish do not differ in their cone opsin expression profiles, but we do reveal an ontogenetic shift in cone opsin expression. Compared to adults, larvae express proportionally more sws1 and less lws-1, suggesting that the western mosquitofish is more sensitive to shorter wavelengths in the larval stage, but becomes more sensitive to longer wavelengths in adulthood.

Largescale mullet (Planiliza macrolepis) can recover from thermal pollution-induced malformations.

It is well known in aquaculture that hyperthermic perturbations may cause skeleton malformations in fish, but this phenomenon has rarely been documented in wild species. One rare location where thermal pollution has increased the proportion of malformed fish in wild population is in the waters near the Kuosheng Nuclear Power Plant in Taiwan. At this site, the threshold temperature and critical exposure time for inducing deformations have not been previously determined. In addition, it was unclear whether juvenile fish with thermal-induced malformations are able to recover when the temperature returns below the threshold. In the present study, juvenile largescale mullet (Planiliza macrolepis) were kept at temperatures ranging from 26°C and 36°C for 1-4 weeks, after which malformed fish were maintained at a preferred temperature of 26°C for another 8 weeks. The vertebrae bending index (VBI) of fish was increased after 2 weeks at 36°C, and deformed vertebral columns were detected by radiography after 4 weeks. However, malformations were not observed in groups kept at or below 34°C. Moreover, at the end of the recovery period, both the VBI and the vertebrae malformations had returned to normal. The results of this study may help to more precisely determine potential environmental impacts of thermal pollution and raise the possibility that the capacity for fish vertebrae to recover from the impacts of chronic thermal exposures may be an important consideration in marine fish conservation.

The Effects of Continuous Acoustic Stress on ROS Levels and Antioxidant-related Gene Expression in the Black Porgy (Acanthopagrus schlegelii).

Hao-Yi Chang, Tzu-Hao Lin, Kazuhiko Anraku, and Yi Ta Shao (2018) Short-term exposure to strong underwater noise is known to seriously impact fish. However, the chronic physiological effects of continuous exposure to weak noise, i.e. the operation noise from offshore wind farms (OWF), remain unclear. Since more and more OWF will be built in the near future, their operation noise is an emerging ecological issue. To investigate the long-term physiological effects of such underwater noise on fish, black porgies (Acanthopagrus schlegelii) were exposed to two types of simulated wind farm noise-quiet (QC: 109 dB re 1 µPa / 125.4 Hz; approx. 100 m away from the wind turbine) and noisy (NC: 138 dB re 1 µPa / 125.4 Hz; near the turbine)-for up to 2 weeks. Measurement of auditory-evoked potentials showed that black porgies can hear sound stimuli under both NC and QC scenarios. Although no significant difference was found in plasma cortisol levels, the fish under NC conditions exhibited higher plasma reactive oxygen species (ROS) levels than the control group at week 2. Moreover, alterations were found in mRNA levels of hepatic antioxidant-related genes (sod1, cat and gpx), with cat downregulated and gpx upregulated after one week of QC exposure. Our results suggest that the black porgy may adapt to QC levels of noise by modulating the antioxidant system to keep ROS levels low. However, such antioxidant response was not observed under NC conditions; instead, ROS accumulated to measurably higher levels. This study suggests that continuous OWF operation noise represents a potential stressor to fish. Furthermore, this is the first study to demonstrate that chronic exposure to noise could induce ROS accumulation in fish plasma.

Hormonal changes over the spawning cycle in the female three-spined stickleback, Gasterosteus aculeatus.

Female three-spined sticklebacks are batch spawners laying eggs in a nest built by the male. We sampled female sticklebacks at different time points, when they were ready to spawn and 6, 24, 48 and 72h post-spawning (hps) with a male. Following spawning, almost all females (15 out of 19) had ovulated eggs again at Day 3 post-spawning (72hps). At sampling, plasma, brain and pituitaries were collected, and the ovary and liver were weighed. Testosterone (T) and estradiol (E2) were measured by radioimmunoassay. Moreover, the mRNA levels of follicle-stimulating hormone (fsh-ß) and luteinizing hormone (lh-ß) in the pituitary, and of the gonadotropin-releasing hormones (GnRHs: gnrh2, gnrh3) and kisspeptin (kiss2) and its G protein-coupled receptor (gpr54) in the brain were measured by real-time qPCR. Ovarian weights peaked in "ready to spawn" females, dropped after spawning, before again progressively increasing from 6 to 72hps. Plasma T levels showed peaks at 24 and 48hps and decreased at 72hps, while E2 levels increased already at 6hps and remained at high levels up to 48hps. There was a strong positive correlation between T and E2 levels over the spawning cycle. Pituitary lh-ß mRNA levels showed a peak at 48hps, while fsh-ß did not change. The neuropeptides and gpr54 did not show any changes. The changes in T and E2 over the stickleback spawning cycle were largely consistent with those found in other multiple-spawning fishes whereas the marked correlation between T and E2 does not support T having other major roles over the cycle than being a precursor for E2.

The effects of continuously acoustical stress on cortisol in milkfish (Chanos chanos).

Strong underwater acoustic noise has been known that may cause hearing loss and actual stress in teleost. However, the long-term physiological effects of relatively quiet but continuously noise on fish were less understood. In present study, milkfish, Chanos chanos, were exposed to the simulated-wind farm noise either quiet (109dB re 1µPa/125.4Hz; approx. 10-100m distant from the wind farm) or noisy (138dB re 1µPa/125.4Hz; nearby the wind farm) conditions for 24h, 3days and 1week. Comparing to the control group (80dB re 1µPa/125.4Hz), the fish exposed to noisy conditions had higher plasma cortisol levels in the first 24h. However, the cortisol levels of 24h spot returned to the resting levels quickly. The fish exposed under noisy condition had significantly higher head kidney star (steroidogenic acute regulatory) and hsd11b2 (11-ß-hydroxysteroid dehydrogenase 2) mRNA levels at the following treatment time points. In addition, noise exposure did not change hypothalamus crh (Corticotropin-releasing hormone) mRNA levels in this experiment. The results implied that the weak but continuously noise was a potential stressor to fish, but the impacts may be various depending on the sound levels and exposure time. Furthermore, this study showed that the continuous noise may up-regulate the genes that are related to cortisol synthesis and possibly make the fish more sensitive to ambient stressors, which may influence the energy allocation appearance in long-term exposures.

Strong Ion Regulatory Abilities Enable the Crab Xenograpsus testudinatus to Inhabit Highly Acidified Marine Vent Systems.

Hydrothermal vent organisms have evolved physiological adaptations to cope with extreme abiotic conditions including temperature and pH. To date, acid-base regulatory abilities of vent organisms are poorly investigated, although this physiological feature is essential for survival in low pH environments. We report the acid-base regulatory mechanisms of a hydrothermal vent crab, Xenograpsus testudinatus, endemic to highly acidic shallow-water vent habitats with average environment pH-values ranging between 5.4 and 6.6. Within a few hours, X. testudinatus restores extracellular pH (pHe) in response to environmental acidification of pH 6.5 (1.78 kPa pCO2) accompanied by an increase in blood [Formula: see text] levels from 8.8 ± 0.3 to 31 ± 6 mM. Branchial Na(+)/K(+)-ATPase (NKA) and V-type H(+)-ATPase (VHA), the major ion pumps involved in branchial acid-base regulation, showed dynamic increases in response to acidified conditions on the mRNA, protein and activity level. Immunohistochemical analyses demonstrate the presence of NKA in basolateral membranes, whereas the VHA is predominantly localized in cytoplasmic vesicles of branchial epithelial- and pillar-cells. X. testudinatus is closely related to other strong osmo-regulating brachyurans, which is also reflected in the phylogeny of the NKA. Accordingly, our results suggest that the evolution of strong ion regulatory abilities in brachyuran crabs that allowed the occupation of ecological niches in euryhaline, freshwater, and terrestrial habitats are probably also linked to substantial acid-base regulatory abilities. This physiological trait allowed X. testudinatus to successfully inhabit one of the world's most acidic marine environments.

GnRH mRNA levels in male three-spined sticklebacks, Gasterosteus aculeatus, under different reproductive conditions.

In vertebrates, reproduction is regulated by the brain-pituitary-gonad (BPG) axis, where the gonadotropin-releasing hormone (GnRH) is one of the key components. However, very little is known about the possible role of GnRH in the environmental and feedback control of fish reproduction. To investigate this, full-length gnrh2 (chicken GnRH II) and gnrh3 (salmon GnRH) sequences of male three-spined sticklebacks (Gasterosteus aculeatus), which are clustered with the taxa of the same GnRH type as other Euteleostei, were cloned and annotated. gnrh1 is absent in this species. The mRNA levels of gnrh2 and gnrh3 in the sticklebacks' brain were measured under breeding and post-breeding conditions as well as in castrated and sham-operated breeding fish and castrated/sham-operated fish kept under long-day (LD 16:8) and short-day (LD 8:16) conditions. Fully breeding males had considerably higher mRNA levels of gnrh2 and gnrh3 in the thalamus (Th) and in the telencephalon and preoptic area (T+POA), respectively, than post-breeding males. Sham-operated breeding males have higher gnrh3 mRNA levels than the corresponding castrated males. Moreover, higher gnrh2 mRNA levels in the Th and higher gnrh3 mRNA levels in the T+POA and hypothalamus (HypTh) were also found in long-day sham-operated males than in sham-operated fish kept under an inhibitory short day photoperiod. Nevertheless, gnrh2 and gnrh3 mRNA levels were not up-regulated in castrated males kept under long-day photoperiod, which suggests that positive feedbacks on the brain-pituitary-gonad axis are necessary for this response.

The complete mitochondrial genome of Hainanese barbel steed Hemibarbus medius Yue (Teleostei: Cyprinidae).

In this study, the complete mitogenome sequence of cyprinid fish, Hainanese barbel steed, Hemibarbus medius Yue (Teleostei: Cyprinidae) has been amplified and sequenced by employing long polymerase chain reaction method. The mitogenome, consisting of 16,614 base pairs (bp), had the typical vertebrate mitochondrial gene arrangement, including 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNAs genes and a non-coding control region (CR). CR of 933 bp length is located between tRNA(Pro) and tRNA(Phe). The overall base composition of Hemibarbus medius is 29.8% for A, 27.2% for C, 25.9% for T and 17.1% for G, with a slight AT bias of 55.7%. The complete mitogenome may provide rather essential and important DNA molecular data for further phylogenetic analysis for not only congeneric species but also higher different taxa of Cyprinid fishes.

The complete mitochondrial genome of Hainan gudgeon Sarcocheilichthys hainanensis Nichols & Pope (Cypriniformes: Cyprinidae).

In this study, the complete mitogenome sequence of cyprinid fish, Hainan gudgeon Sarcocheilichthys hainanensis Nichols & Pope, 1927 (Cypriniformes: Cyprinidae) has been amplified and sequenced by employing long polymerase chain reaction method. The mitogenome, consisting of 16,674 base pairs (bp), had the typical vertebrate mitochondrial gene arrangement, including 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNAs genes and a noncoding control region (CR). CR of 1004 bp length is located between tRNAPro and tRNAPhe. The overall base composition of S. hainanensis is 30.0% for A, 26.7% for C, 26.4% for T and 16.8% for G, with a slight AT bias of 56.4%. The complete mitogenome may provide rather essential and important DNA molecular data for further phylogenetic analysis for not only congeneric species but also higher different taxa of cyprinid fishes.

Differentiation of visual spectra and nuptial colorations of two Paratanakia himantegus subspecies (Cyprinoidea: Acheilognathidae) in response to the distinct photic conditions of their habitats.

BACKGROUND: Vision, an important sensory modality of many animals, exhibits plasticity in that it adapts to environmental conditions to maintain its sensory efficiency. Nuptial coloration is used to attract mates and hence should be tightly coupled to vision. In Taiwan, two closely related bitterlings (Paratanakia himantegus himantegus and Paratanakia himantegus chii) with different male nuptial colorations reside in different habitats. We compared the visual spectral sensitivities of these subspecies with the ambient light spectra of their habitats to determine whether their visual abilities correspond with photic parameters and correlate with nuptial colorations. RESULTS: Theelectroretinogram (ERG) results revealed that the relative spectral sensitivity of P.h. himantegus was higher at 670 nm, but lower at 370 nm, than the sensitivity of P. h. chii. Both bitterlings could perceive and reflect UV light, but the UV reflection patterns differed between genders. Furthermore, the relative irradiance intensity of the light spectra in the habitat of P. h. himantegus was higher at long wavelengths (480-700 nm), but lower at short wavelengths (350-450 nm), than the light spectra in the habitats of P. h.chii. CONCLUSIONS: Two phylogenetically closely related bitterlings, P. h. himantegus and P. h. chii, dwell in different waters and exhibit different nuptial colorations and spectral sensitivities, which may be the results of speciation by sensory drive. Sensory ability and signal diversity accommodating photic environment may promote diversity of bitterling fishes. UV light was demonstrated to be a possible component of bitterling visual communication. The UV cue may assist bitterlings in genderidentification.

Androgens increase lws opsin expression and red sensitivity in male three-spined sticklebacks.

Optomotor studies have shown that three-spined sticklebacks (Gasterosteus aculeatus) are more sensitive to red during summer than winter, which may be related to the need to detect the red breeding colour of males. This study aimed to determine whether this change of red light sensitivity is specifically related to reproductive physiology. The mRNA levels of opsin genes were examined in the retinae of sexually mature and immature fish, as well as in sham-operated males, castrated control males, or castrated males implanted with androgen 11-ketoandrostenedione (11 KA), maintained under stimulatory (L16:D8) or inhibitory (L8:D16) photoperiods. In both sexes, red-sensitive opsin gene (lws) mRNA levels were higher in sexually mature than in immature fish. Under L16:D8, lws mRNA levels were higher in intact than in castrated males, and were up-regulated by 11 KA treatment in castrated males. Moreover, electroretinogram data confirmed that sexual maturation resulted in higher relative red spectral sensitivity. Mature males under L16:D8 were more sensitive to red light than males under L8:D16. Red light sensitivity under L16:D8 was diminished by castration, but increased by 11 KA treatment. Thus, in sexually mature male sticklebacks, androgen is a key factor in enhancing sensitivity to red light via regulation of opsin gene expression. This is the first study to demonstrate that sex hormones can regulate spectral vision sensitivity.

Androgen feedback effects on LH and FSH, and photoperiodic control of reproduction in male three-spined sticklebacks, Gasterosteus aculeatus.

Sexual maturation in the stickleback is controlled by photoperiod. The aim of this study was to find out whether changes in feedback effects exerted by sex steroids could mediate the photoperiodic effect, which is regarded to be of an all-or-nothing character. To that end, males were castrated and treated with different doses of testosterone (T) and in one experiment also with the aromatase inhibitor fadrozole (AI) and kept under different photoperiods. In control fish, long day (LD 16:8) stimulated maturation, associated with more hypertrophied kidneys (a secondary sexual character) and higher levels of pituitary lhb and fshb mRNA than under short day conditions (LD 8:16). Under LD 8:16, low doses of T suppressed both lhb and fshb mRNA levels. However, with the use of high doses of T and/or longer photoperiods the inhibitory effects on lhb and fshb mRNA levels became less clear or instead positive effects were observed. Under intermediate photoperiod conditions, the negative feedback effect of a low dose of T on fshb was more prominent with shorter photoperiods, whereas no such shift was observed for lhb mRNA. The inhibitory effect of the low dose of T on lhb mRNA levels under LD 8:16 was abolished by AI, whereas the stimulatory effect of the high dose of T was not. The negative feedback effects were more marked under short days than under long days, whereas positive feedback effects were more marked under long days. The suppression of both fshb and lhb mRNA levels by low androgen levels, especially under short days, may inhibit maturation completely unless a rise of androgens above threshold levels would allow complete maturation.

Schistocephalus solidus infections increase gonadotropins and gonadotropin releasing hormone (GnRH3) mRNA levels in the three-spined stickleback, Gasterosteus aculeatus.

Parasites often impair the reproduction of their hosts, one well known case being the cestode Schistocephalus solidus which is a common parasite in three-spined sticklebacks, Gasterosteus aculeatus. One of the possible ways that this could be exerted is by suppression on the brain-pituitary-gonadal (BPG) axis. In this study, mRNA levels of FSH-ß and LH-ß and of GnRH2 (cGnRH II) and GnRH3 (sGnRH) were measured via Q-PCR in infected and uninfected fish sampled from the field a few weeks before the onset of breeding. The pituitary mRNA levels of both FSH-ß and LH-ß were higher in infected males than in uninfected males. Also in females, FSH-ß mRNA levels were higher in infected individuals than in others, whereas there was no significant difference found in LH-ß expression. Brain mRNA levels of GnRH3 were higher in infected fish than in uninfected fish in both sexes, but no difference was found in GnRH2 mRNA levels. Thus, infection by S. solidus was able to alter the expressions not only of gonadotropins (GtHs), but also of GnRH which has not been observed previously. However, the effects are opposite to what should be expected if the parasite suppressed reproduction via actions on the brain-pituitary level. The gonads are perhaps more likely to be impaired by the parasites in other ways, and changed feedbacks on the BPG axis could then lead to the increases in GtHs and GnRH.