Domoic acid in Canadian Pacific waters, from 2016 to 2021, and relationships with physical and chemical conditions.

Domoic acid, a phycotoxin produced by species of the marine diatom Pseudo-nitzschia, can cause deleterious impacts to marine food webs and human health. Domoic acid and Pseudo-nitzschia spp. were surveyed from 2016 to 2021 in the Pacific waters of Canada to assess their occurrences, concentrations, and relationships with physical and chemical conditions. Domoic acid was common, occurring in measurable concentrations in 73 % of the 454 samples. It occurred in all regions (west coast of Vancouver Island, Salish Sea, Queen Charlotte Sound / Hecate Strait, deep oceanic NE Pacific), in all years and all seasons. Median concentrations were highest along the west coast of Vancouver Island, and lowest in the oceanic waters of the NE Pacific. Winter had the lowest concentrations; no significant differences occurred between spring, summer, and autumn. High domoic acid concentrations equal to or above 100 ng/L were not common, occurring in about 5 % of samples, but in all seasons and all years except 2019. All six Pseudo-nitzschia taxa identified had similar median concentrations, but different frequencies of occurrence. P. cf. australis appeared to be the major contributor to high concentrations of domoic acid. Physico-chemical conditions were described by ten variables: temperature, salinity, density difference between 30 m and the surface (a proxy for vertical stability), chlorophyll a, nitrate, phosphate, silicate, and the ratios nitrate:phosphate, nitrate:silicate, and silicate:phosphate. Statistical analyses, using general linear models, of their relationships with the absence/presence of Pseudo-nitzschia spp. found silicate (negative) to be the most influential variable common in both the west coast of Vancouver Island and Salish Sea regions. Temperature and chlorophyll a were the most influential variables which determined the log10 abundance of Pseudo-nitzschia spp. in both regions. Analyses of the absence/presence of particulate domoic acid per Pseudo-nitzschia cell (excluding P. americana) found chlorophyll a to be the most influential variable common in both regions, whereas no common influential variable determined the log10 concentration of particulate domoic acid per Pseudo-nitzschia cell (excluding P. americana). These results were generally similar to those of other studies from this area, although this study extends these findings to all seasons and all regions of Canada's Pacific waters. The results provide important background information against which major outbreaks and unusual events can be compared. A domoic acid surveillance program during synoptic oceanographic surveys can help to understand where and when it reaches high concentrations at sea and the potential impacts to the marine ecosystem.

Dissolved Algal Toxins along the Southern Coast of British Columbia Canada.

Harmful algal blooms (HABs) in coastal British Columbia (BC), Canada, negatively impact the salmon aquaculture industry. One disease of interest to salmon aquaculture is Net Pen Liver Disease (NPLD), which induces severe liver damage and is believed to be caused by the exposure to microcystins (MCs). To address the lack of information about algal toxins in BC marine environments and the risk they pose, this study investigated the presence of MCs and other toxins at aquaculture sites. Sampling was carried out using discrete water samples and Solid Phase Adsorption Toxin Tracking (SPATT) samplers from 2017-2019. All 283 SPATT samples and all 81 water samples tested positive for MCs. Testing for okadaic acid (OA) and domoic acid (DA) occurred in 66 and 43 samples, respectively, and all samples were positive for the toxin tested. Testing for dinophysistoxin-1 (DTX-1) (20 samples), pectenotoxin-2 (PTX-2) (20 samples), and yessotoxin (YTX) (17 samples) revealed that all samples were positive for the tested toxins. This study revealed the presence of multiple co-occurring toxins in BC's coastal waters and the levels detected in this study were below the regulatory limits for health and recreational use. This study expands our limited knowledge of algal toxins in coastal BC and shows that further studies are needed to understand the risks they pose to marine fisheries and ecosystems.

The role of salinity in recovery of white sturgeon (Acipenser transmontanus) from stimulated angling stress.

White sturgeon (Acipenser transmontanus) in the Lower Fraser River are the focus of a catch-and-release angling fishery in British Columbia, Canada. However, the lower region of the catch area includes areas where tidal waters invade, and the consequence of salinity levels on recovery from an angling challenge are not characterized in sturgeon, despite theoretical implications of its import. We acclimated white sturgeon to various salinities (0, 10 and 20‰ (parts per thousand)) to investigate the effects of acclimation on recovery from stimulated angling stress that was induced through manual chasing. This challenge elicited the traditional physiological responses such as ion homeostasis disturbance, increases in secondary stress indicators and metabolic acidosis; however, environmental salinity altered the timing of recovery in some of the parameters measured. In addition, the severity of the intracellular pH disturbance in both heart and red blood cell seemed to be mediated in fresh water, yet the recovery pattern of plasma chloride and bicarbonate ions seemed to be facilitated by higher salinity. In general, responses were similar but not identical, leading us to conclude that the role of salinity on recovery from exercise is complex but not insignificant. Salinity may be important to behaviours exhibited by white sturgeon (such as migrations) in their respective saline environments, but less so around the impact of an angling stressor. Further exploration of this response may provide insight on whether the current tidal boundaries for angling white sturgeon are appropriate.

Acute microcystin exposure induces reversible histopathological changes in Chinook Salmon (Oncorhynchus tshawytscha) and Atlantic Salmon (Salmo salar).

Atlantic Salmon (Salmo salar) and Chinook Salmon (Oncorhynchus tshawytscha) develop a severe liver disease called net-pen liver disease (NPLD), which is characterized by hepatic lesions that include megalocytosis and loss of gross liver structure. Based on studies where salmonids have been exposed to microcystin (MC) via intraperitoneal injection, NPLD is believed to be caused by MC exposure, a hepatotoxin produced by cyanobacteria. Despite the link between MC and NPLD, it remains uncertain if environmentally relevant MC exposure is responsible for NPLD. To determine if we could produce histopathology consistent with NPLD, we compared the response of Atlantic and Chinook Salmon sub-lethal MC exposure. Salmon were orally gavaged with saline or MC containing algal paste and sampled over 2 weeks post-exposure. Liver lesions appeared by 6 h but were resolved 2-weeks post-exposure; histopathological changes observed in other tissues were not as widespread, nor was their severity as great as those in the liver. There was no evidence for NPLD due to the absence of hepatic megalocytosis. These results indicate that the development of NPLD is not due to acute MC exposure but may be associated with higher MC concentration occurring in food, long-term exposure through drinking of contaminated seawater and/or interactions with other marine toxins.

Limits and patterns of acid-base regulation during elevated environmental CO2 in fish.

Aquatic CO2 tensions may exceed 30-60 Torr (ca. 30,000-79,000 µatm, respectively; hypercarbia) in some environments inducing severe acid-base challenges in fish. Typically, during exposure to hypercarbia blood pH (pHe) is initially reduced and then compensated in association with an increase in plasma HCO3- in exchange for Cl-. Typically, intracellular pH (pHi) is reduced and recovery is to some degree coupled to pHe recovery (coupled pH regulation). However, during acute hypercarbia, pHe recovery has been proposed to be limited by an "apparent upper bicarbonate threshold", restricting complete pHe recovery to below 15 Torr PCO2. At PCO2 values beyond that which fish can compensate pHe, some fish are able to fully protect pHi despite large sustained reductions in pHe (preferential pHi regulation) and can tolerate PCO2 > 45 Torr. This review discusses pHe and pHi regulation during exposure to hypercarbia starting with modeling the capacity and theoretical limit to pHe compensation in 19 studies. Next, we discuss how fish compensate severe acute hypercarbia exposures beyond the putative limit of pHe compensation using preferential pHi regulation which has recently been observed to be common among fish subjected to severe hypercarbia. Finally, we consider the evolution of pH regulatory strategies in vertebrates, including how the presence of preferential pHi regulation in embryonic reptiles may indicate that it is an embryonic trait that is either lost or retained in adult vertebrates and may have served as an exaptation for key evolutionary transitions during vertebrate evolution.

Water pH limits extracellular but not intracellular pH compensation in the CO2-tolerant freshwater fish Pangasianodon hypophthalmus.

Preferentially regulating intracellular pH (pHi) confers exceptional CO2 tolerance on fish, but is often associated with reductions in extracellular pH (pHe) compensation. It is unknown whether these reductions are due to intrinsically lower capacities for pHe compensation, hypercarbia-induced reductions in water pH or other factors. To test how water pH affects capacities and strategies for pH compensation, we exposed the CO2-tolerant fish Pangasianodon hypophthalmus to 3 kPa PCO2  for 20 h at an ecologically relevant water pH of 4.5 or 5.8. Brain, heart and liver pHi was preferentially regulated in both treatments. However, blood pHe compensation was severely reduced at water pH 4.5 but not 5.8. This suggests that low water pH limits acute pHe but not pHi compensation in fishes preferentially regulating pHi Hypercarbia-induced reductions in water pH might therefore underlie the unexplained reductions to pHe compensation in fishes preferentially regulating pHi, and may increase selection for preferential pHi regulation.

White sturgeon (Acipenser transmontanus) acid-base regulation differs in response to different types of acidoses.

White sturgeon (Acipenser transmontanus) completely protect intracellular tissue pH (pHi) despite large reductions in extracellular (blood) pH (pHe), termed preferential pHi regulation, in response to elevated environmental PCO2 (hypercarbia) and in general appear to be relatively resilient to stressors. Preferential pHi regulation is thought to be associated with hypercarbia tolerance in general, but has also recently been observed to protect pHi against metabolic acidoses induced by exhaustive exercise and anoxia in a tropical air breathing catfish. We hypothesized that preferential pHi regulation may also be a general strategy of acid-base regulation in sturgeon. To address this hypothesis, severe acidoses were imposed to reduce pHe, and the presence or absence of preferential pHi regulation was assessed in red blood cells (RBC), heart, brain, liver and white muscle. A respiratory acidosis was imposed using hyperoxia, while metabolic acidoses were induced by exhaustive exercise, anoxia or air exposure. Reductions in pHe occurred following hyperoxia (0.15 units), exhaustive exercise (0.30 units), anoxia (0.10 units) and air exposure (0.35 units); all acidoses reduced RBC pHi. Following hyperoxia, heart, brain and liver pHi were preferentially regulated against the reduction in pHe, similar to hypercarbia exposure. Following all metabolic acidoses heart pHi was protected and brain pHi remained unchanged following exhaustive exercise and air exposure, however, brain pHi was reduced following anoxia. Liver and white muscle pHi were reduced following all metabolic acidoses. These results suggest preferential pHi regulation may be a general strategy during respiratory acidoses but during metabolic acidoses, the response differs between source of acidoses and tissues.

Characterization of Na+ transport to gain insight into the mechanism of acid-base and ion regulation in white sturgeon (Acipenser transmontanus).

Freshwater fish actively take up ions via specific transporters to counter diffusive losses to their hypotonic environment. While much is known about the specific mechanisms employed by teleosts, almost nothing is known about the basal fishes, such as white sturgeon (Acipenser transmontanus) which may offer insight into the evolution of osmo- and ionoregulation in fishes. We investigated Na+ uptake in juvenile white sturgeon in the presence and absence of transporter inhibitors. We found that sturgeon acclimated to 100µmoll-1 Na+ have Na+ uptake kinetics typical of teleosts and that a Na+/H+ exchanger (NHE) is the predominant transporter for Na+ uptake. White sturgeon are tolerant to hypercarbia-induced respiratory acidoses and recover blood pH (pHe) at 1.5kPa PCO2 but not at higher PCO2 (6kPa PCO2) where they preferentially regulate intracellular pH (pHi). It was hypothesized that during exposure to hypercarbia Na+ uptake would increase at CO2 tensions at which fish were capable of pHe regulation but decrease at higher tensions when they were preferentially regulating pHi. We found that Na+ uptake did not increase at 1.5kPa PCO2, but at 6kPa PCO2 Na+ uptake was reduced by 95% while low water pH equivalent to 6kPa PCO2 reduced Na+ uptake by 71%. Lastly, we measured net acid flux during hypercarbia, which indicates that net acid flux is not associated with Na+ uptake. These findings indicate Na+ uptake in sturgeon is not different from freshwater teleosts but is sensitive to hypercarbia and is not associated with pHe compensation during hypercarbia.

Preferential intracellular pH regulation: hypotheses and perspectives.

The regulation of vertebrate acid-base balance during acute episodes of elevated internal PCO2  is typically characterized by extracellular pH (pHe) regulation. Changes in pHe are associated with qualitatively similar changes in intracellular tissue pH (pHi) as the two are typically coupled, referred to as 'coupled pH regulation'. However, not all vertebrates rely on coupled pH regulation; instead, some preferentially regulate pHi against severe and maintained reductions in pHe Preferential pHi regulation has been identified in several adult fish species and an aquatic amphibian, but never in adult amniotes. Recently, common snapping turtles were observed to preferentially regulate pHi during development; the pattern of acid-base regulation in these species shifts from preferential pHi regulation in embryos to coupled pH regulation in adults. In this Commentary, we discuss the hypothesis that preferential pHi regulation may be a general strategy employed by vertebrate embryos in order to maintain acid-base homeostasis during severe acute acid-base disturbances. In adult vertebrates, the retention or loss of preferential pHi regulation may depend on selection pressures associated with the environment inhabited and/or the severity of acid-base regulatory challenges to which they are exposed. We also consider the idea that the retention of preferential pHi regulation into adulthood may have been a key event in vertebrate evolution, with implications for the invasion of freshwater habitats, the evolution of air breathing and the transition of vertebrates from water to land.

Embryonic common snapping turtles (Chelydra serpentina) preferentially regulate intracellular tissue pH during acid-base challenges.

The nests of embryonic turtles naturally experience elevated CO2 (hypercarbia), which leads to increased blood PCO2  and a respiratory acidosis, resulting in reduced blood pH [extracellular pH (pHe)]. Some fishes preferentially regulate tissue pH [intracellular pH (pHi)] against changes in pHe; this has been proposed to be associated with exceptional CO2 tolerance and has never been identified in amniotes. As embryonic turtles may be CO2 tolerant based on nesting strategy, we hypothesized that they preferentially regulate pHi, conferring tolerance to severe acute acid-base challenges. This hypothesis was tested by investigating pH regulation in common snapping turtles (Chelydra serpentina) reared in normoxia then exposed to hypercarbia (13 kPa PCO2 ) for 1 h at three developmental ages: 70% and 90% of incubation, and yearlings. Hypercarbia reduced pHe but not pHi, at all developmental ages. At 70% of incubation, pHe was depressed by 0.324 pH units while pHi of brain, white muscle and lung increased; heart, liver and kidney pHi remained unchanged. At 90% of incubation, pHe was depressed by 0.352 pH units but heart pHi increased with no change in pHi of other tissues. Yearlings exhibited a pHe reduction of 0.235 pH units but had no changes in pHi of any tissues. The results indicate common snapping turtles preferentially regulate pHi during development, but the degree of response is reduced throughout development. This is the first time preferential pHi regulation has been identified in an amniote. These findings may provide insight into the evolution of acid-base homeostasis during development of amniotes, and vertebrates in general.

Rotational behaviour of encapsulated pond snail embryos in diverse natural environments.

Encapsulated freshwater pond snail embryos display a cilia-driven rotation behaviour that is stimulated by artificially induced hypoxia. Previous studies have suggested that the mixing effect of this behaviour causes enhanced oxygen delivery to embryos within their egg capsules. Despite extensive laboratory-based studies describing this behaviour, it is unclear how this behaviour is used to cope with changes in oxygen concentration and other environmental factors in natural water bodies. We made field measurements of embryo rotation rates in laboratory-reared Helisoma trivolvis embryos placed in ponds of different trophic levels that ranged geographically from the southern Alberta prairie to the Rocky Mountains. Abiotic factors including temperature, pH, conductivity and water oxygen concentration were measured to understand how embryonic rotation is influenced by environmental conditions. Results showed that H. trivolvis embryos exhibit differences in rotational behaviour depending on the environmental conditions. Temperature and oxygen concentration were the primary factors significantly affecting rotation rates. The effect of oxygen concentration on rotation rates was not as widespread as observed under laboratory conditions, probably because the measured oxygen concentrations were above the range that influences embryonic rotation in the laboratory. The rotational behaviour of laboratory-reared Lymnaea stagnalis provided confirmation that embryos of other encapsulated pulmonates exhibit a similar rotational response in natural environments. These results suggest that embryo rotation is influenced by a complex interplay of environmental factors.

Serotonin prolongs survival of encapsulated pond snail embryos exposed to long-term anoxia.

Embryos of the pond snail, Helisoma trivolvis, develop bilateral serotonergic neurons that innervate ciliary bands and stimulate cilia-driven rotation. This behaviour is postulated to increase oxygen availability during hypoxia by mixing the capsular fluid. We hypothesised that the stimulation of ciliary-driven rotation by serotonin (5-HT) enhances the survival of embryos during prolonged hypoxia. Embryo rotation and survival were monitored in different levels of oxygen for 24-48 h while in the presence or absence of 5-HT (100 micromol l(-1)) or a 5-HT antagonist (50 micromol l(-1)). Long-term hypoxia caused delayed embryonic development that appeared morphologically normal. Hypoxia also induced a transient increase in rotation rate in embryos exposed to artificial pond water (APW) or 5-HT that lasted around 3 h. 5-HT-treated embryos had an elevated rotation rate over embryos in APW throughout the long-term exposure to hypoxia. Long-term anoxia also induced a transient increase in rotation rate in embryos exposed to APW or 5-HT. Rotation ceased in embryos exposed to APW by 13 h but persisted in 5-HT-treated embryos for up to 40 h. Fifty percent mortality was reached at 9 h of anoxia in embryos in APW and at 24 h in 5-HT-treated embryos. The 5-HT antagonist mianserin partially inhibited the 5-HT enhancement of rotation but not the prolongation of survival in anoxia. The ability of 5-HT to prolong survival in anoxia reveals a 5-HT-activated metabolic pathway that liberates an alternative energy source.

Integrative biology of an embryonic respiratory behaviour in pond snails: the 'embryo stir-bar hypothesis'.

Embryos of freshwater snails undergo direct development from single cell to juvenile inside egg masses that are deposited on vegetation and other substratum in pond, lake and stream habitats. Helisoma trivolvis, a member of the Planorbidae family of basommatophoran snails, has served as a model for studying the developmental and physiological roles for neurotransmitters during embryogenesis. Early studies revealed that H. trivolvis embryos from stage E15 to E30, the period between gastrulation and the trochophore-juvenile transition, display a cilia-driven behaviour consisting of slow basal rotation and transient periods of rapid rotation. The discovery of a bilateral pair of early serotonergic neurons, named ENC1, which project an apical process to the embryo surface and basal neurites to ciliated cells, prompted the hypothesis that each ENC1 is a dual-function sensory and motor neuron mediating a physiological embryonic response. This article reviews our past and present studies and addresses questions concerning this hypothesis, including the following. (1) What environmental signal regulates ENC1 activity and rotational behaviour? (2) Does ENC1 function as both a primary sensory and motor neuron underlying the rotational behaviour? (3) What are the sensory transduction mechanisms? (4) How does ENC1 regulate ciliary beating? (5) Do other basommatophoran species have similar neural-ciliary pathways and behavioural responses? (6) How is the behaviour manifest in the dynamic natural environment? In this review, we introduce the ;embryo stir-bar hypothesis', which proposes that embryonic rotation is a hypoxia-sensitive respiratory behaviour responsible for mixing the egg capsule fluid, thereby enhancing delivery of environmental oxygen to the embryo.