Repeat exposure to hypercapnic seawater modifies growth and oxidative status in a tolerant burrowing clam.

Although low levels of thermal stress, irradiance and dietary restriction can have beneficial effects for many taxa, stress acclimation remains little studied in marine invertebrates, even though they are threatened by climate change stressors such as ocean acidification. To test the role of life-stage and stress-intensity dependence in eliciting enhanced tolerance under subsequent stress encounters, we initially conditioned pediveliger Pacific geoduck (Panopea generosa) larvae to ambient and moderately elevated PCO2 (920 µatm and 2800 µatm, respectively) for 110 days. Then, clams were exposed to ambient, moderate or severely elevated PCO2 (750, 2800 or 4900 µatm, respectively) for 7 days and, following 7 days in ambient conditions, a 7-day third exposure to ambient (970 µatm) or moderate PCO2 (3000 µatm). Initial conditioning to moderate PCO2 stress followed by second and third exposure to severe and moderate PCO2 stress increased respiration rate, organic biomass and shell size, suggesting a stress-intensity-dependent effect on energetics. Additionally, stress-acclimated clams had lower antioxidant capacity compared with clams under ambient conditions, supporting the hypothesis that stress over postlarval-to-juvenile development affects oxidative status later in life. Time series and stress intensity-specific approaches can reveal life-stages and magnitudes of exposure, respectively, that may elicit beneficial phenotypic variation.

First record of dark leathery surface of geoduck clams in Panopea globosa.

Dark Leathery Surface of Geoduck Clams (LSGC) is an alteration that affects the periostracum of the mantle and siphon of Panopea generosa from the northwest coast of Canada and Mexico. This alteration affects commercialization and possibly the survival of the clams. The cause of LSGC is unknown but has been correlated with presence of fungi and protozoans. We detected a similar alteration in Panopea globosa from Baja California, Mexico and the histophagous ciliate Uronema marinum was isolated from affected siphon tissue. U. marinum was identified by its morphology and by genetic analysis of the gene 18S rRNA. This is the first record of LSGC in P. globosa and the first identification of a histophagous protozoan associated with it.

Integrating Discovery-driven Proteomics and Selected Reaction Monitoring To Develop a Noninvasive Assay for Geoduck Reproductive Maturation.

Geoduck clams (Panopea generosa) are an increasingly important fishery and aquaculture product along the eastern Pacific coast from Baja California, Mexico, to Alaska. These long-lived clams are highly fecund, although sustainable hatchery production of genetically diverse larvae is hindered by the lack of sexual dimorphism, resulting in asynchronous spawning of broodstock, unequal sex ratios, and low numbers of breeders. The development of assays of gonad physiology could indicate sex and maturation stage as well as be used to assess the status of natural populations. Proteomic profiles were determined for three reproductive maturation stages in both male and female clams using data-dependent acquisition (DDA) of gonad proteins. Gonad proteomes became increasingly divergent between males and females as maturation progressed. The DDA data were used to develop targets analyzed with selected reaction monitoring (SRM) in gonad tissue as well as hemolymph. The SRM assay yielded a suite of indicator peptides that can be used as an efficient assay to determine geoduck gonad maturation status. Application of SRM in hemolymph samples demonstrates that this procedure could effectively be used to assess reproductive status in marine mollusks in a nonlethal manner.

A case of paralytic shellfish poisoning caused by consumption of visceral balls from geoduck Panopea japonica in Japan.

In the end of March 2018, an unprecedented food poisoning incident due to ingestion of the visceral balls of geoduck Panopea japonica occurred in Japan. The patient, presented with symptoms of numbness on the lips and general weakness, was diagnosed as paralytic shellfish poisoning (PSP). The patient immediately treated with the mechanical ventilation recovered and left the hospital after 3 days treatment. Saxitoxins (STXs) in the plasma and urinary samples collected from the patient on the first and second day after hospitalization were analyzed by ultra high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC/MS/MS) and liquid chromatography with post-column fluorescent detection (LC/FLD). The STXs levels of 499.1 and 6.0 µg/L of STX dihydrochloride equivalent (STX·2HCl eq.) were quantitated by LC/FLD in the urinary samples on the first and second day, respectively. In addition, geoducks harvested from the same areas of the PSP causative specimens after the incident were analyzed by LC/FLD, and the results showed the level of STXs in their whole bodies of the geoducks exceeding 0.8 mg STX·2HCl eq./kg which is the maximum levels of STX in CODEX STAN 292-2008. Prominent toxins in STXs that detected in urinary and geoduck samples and identified by UHPLC/MS/MS and LC/FLD were gonyautoxin-1+4 (GTX1+4). These results concluded that the incident was the food poisoning due to STXs accumulated in the geoducks. This is the first PSP case caused by consumption of geoducks in Japan. This is also the first PSP case that causative toxins are detected in urinary samples of patients involved in PSP in Japan.

Physiological responses of juvenile New Zealand geoduck (Panopea zelandica) following emersion and recovery.

The New Zealand geoduck clam is a unique seafood delicacy, with animals selling for up to $US 220-330/kg. Stress accumulated during transport of juveniles to grow-out sites represent a bottleneck in the aquaculture process. In this study, the physiological responses of juvenile geoducks following emersion (3- and 8-h), and recovery (1- and 5-days) were investigated. An integrated approach of flow cytometry, osmolality and metabolomics, along with behavioural assessments was used. Both cellular and chemical haemolymph parameters and metabolite profiles were recorded for P. zelandica juveniles and are reported herein for the first time. An increase in haemolymph osmolality was experienced with an increase in emersion period, with significant differences seen between the 3- and 8-h emersion groups after 5 days of recovery. Viability measures of haemocytes varied insignificantly between experimental groups, creating baseline ranges. The proportion of haemocytes undergoing respiratory burst activity did not appear to be affected by emersion and re-immersion. Haemocyte mitochondrial membrane potential was highest following 1-day of recovery, likely linked to metabolic readjustment, and increased glycolysis, taking place following emersion. Metabolomics analyses suggest that protein, lipid and carbohydrate metabolite classes assist with energy production in geoducks. Activation of anaerobic metabolic pathways, with a high dependence on succinate, were prominent in the 8-h exposure group, with metabolic recovery still taking place following 5-days of immersion, mainly due to proteins restoring energy reserves. Elucidating the physiological responses of juvenile geoduck subjected to transport stress can aid cultivation methods already underway to develop a novel, high value aquaculture industry.

Acidification stress effect on umbonate veliger larval development in Panopea globosa.

Ocean acidification generates a decrease in calcium carbonate availability essential for biomineralization in organisms such as mollusks. This effect was evaluated on Panopea globosa exposing for 7 days umbonate veliger larvae to two pH treatments: experimental (pH 7.5) and control (pH 8.0). Exposure to pH 7.5 affected growth, reducing larval shell length from 5.15-13.34% compared to the control group. This size reduction was confirmed with electron microscopy, also showing shell damage. The physiological response showed an increase in oxygen consumption in larvae exposed to low pH with a maximum difference of 1.57 nmol O2 h-1 larvae-1 at day 7. The gene expression analyses reported high expression values for nicotinamide adenine dinucleotide (NADH) dehydrogenase and Perlucin in larvae at pH 7.5, suggesting a higher energetic cost in this larval group to maintain homeostasis. In conclusion, this study showed that acidification affected development of P. globosa umbonate veliger larvae.

Coupled microbiome analyses highlights relative functional roles of bacteria in a bivalve hatchery.

BACKGROUND: Microbial communities are ubiquitous throughout ecosystems and are commensal with hosts across taxonomic boundaries. Environmental and species-specific microbiomes are instrumental in maintaining ecosystem and host health, respectively. The introduction of pathogenic microbes that shift microbiome community structure can lead to illness and death. Understanding the dynamics of microbiomes across a diversity of environments and hosts will help us to better understand which taxa forecast survival and which forecast mortality events. RESULTS: We characterized the bacterial community microbiome in the water of a commercial shellfish hatchery in Washington state, USA, where the hatchery has been plagued by recurring and unexplained larval mortality events. By applying the complementary methods of metagenomics and metaproteomics we were able to more fully characterize the bacterial taxa in the hatchery at high (pH 8.2) and low (pH 7.1) pH that were metabolically active versus present but not contributing metabolically. There were shifts in the taxonomy and functional profile of the microbiome between pH and over time. Based on detected metagenomic reads and metaproteomic peptide spectral matches, some taxa were more metabolically active than expected based on presence alone (Deltaproteobacteria, Alphaproteobacteria) and some were less metabolically active than expected (e.g., Betaproteobacteria, Cytophagia). There was little correlation between potential and realized metabolic function based on Gene Ontology analysis of detected genes and peptides. CONCLUSION: The complementary methods of metagenomics and metaproteomics contribute to a more full characterization of bacterial taxa that are potentially active versus truly metabolically active and thus impact water quality and inter-trophic relationships.

Metabolic recovery and compensatory shell growth of juvenile Pacific geoduck Panopea generosa following short-term exposure to acidified seawater.

While acute stressors can be detrimental, environmental stress conditioning can improve performance. To test the hypothesis that physiological status is altered by stress conditioning, we subjected juvenile Pacific geoduck, Panopea generosa, to repeated exposures of elevated pCO2 in a commercial hatchery setting followed by a period in ambient common garden. Respiration rate and shell length were measured for juvenile geoduck periodically throughout short-term repeated reciprocal exposure periods in ambient (~550 µatm) or elevated (~2400 µatm) pCO2 treatments and in common, ambient conditions, 5 months after exposure. Short-term exposure periods comprised an initial 10-day exposure followed by 14 days in ambient before a secondary 6-day reciprocal exposure. The initial exposure to elevated pCO2 significantly reduced respiration rate by 25% relative to ambient conditions, but no effect on shell growth was detected. Following 14 days in common garden, ambient conditions, reciprocal exposure to elevated or ambient pCO2 did not alter juvenile respiration rates, indicating ability for metabolic recovery under subsequent conditions. Shell growth was negatively affected during the reciprocal treatment in both exposure histories; however, clams exposed to the initial elevated pCO2 showed compensatory growth with 5.8% greater shell length (on average between the two secondary exposures) after 5 months in ambient conditions. Additionally, clams exposed to the secondary elevated pCO2 showed 52.4% increase in respiration rate after 5 months in ambient conditions. Early exposure to low pH appears to trigger carryover effects suggesting bioenergetic re-allocation facilitates growth compensation. Life stage-specific exposures to stress can determine when it may be especially detrimental, or advantageous, to apply stress conditioning for commercial production of this long-lived burrowing clam.

Dynamic response in the larval geoduck (Panopea generosa) proteome to elevated pCO2.

Pacific geoducks (Panopea generosa) are clams found along the northeast Pacific coast where they are important components of coastal and estuarine ecosystems and a major aquaculture product. The Pacific coastline, however, is also experiencing rapidly changing ocean habitat, including significant reductions in pH. To better understand the physiological impact of ocean acidification on geoduck clams, we characterized for the first time the proteomic profile of this bivalve during larval development and compared it to that of larvae exposed to low pH conditions. Geoduck larvae were reared at pH 7.5 (ambient) or pH 7.1 in a commercial shellfish hatchery from day 6 to day 19 postfertilization and sampled at six time points for an in-depth proteomics analysis using high-resolution data-dependent analysis. Larvae reared at low pH were smaller than those reared at ambient pH, especially in the prodissoconch II phase of development, and displayed a delay in their competency for settlement. Proteomic profiles revealed that metabolic, cell cycle, and protein turnover pathways differed between the two pH and suggested that differing phenotypic outcomes between pH 7.5 and 7.1 are likely due to environmental disruptions to the timing of physiological events. In summary, ocean acidification results in elevated energetic demand on geoduck larvae, resulting in delayed development and disruptions to normal molecular developmental pathways, such as carbohydrate metabolism, cell growth, and protein synthesis.

Establishing the thermal window for aerobic scope in New Zealand geoduck clams (Panopea zelandica).

Geoduck clams (Panopea spp.) are the longest-lived and largest deep burrowing bivalve. Their unique morphology allows them to live buried in the sediment at depths of up to 1 m. The endemic New Zealand geoduck (Panopea zelandica Quoy and Gaimard, 1835) has recently been identified as a potential species for aquaculture. However, very little is known about the biology and physiology of this entirely subtidal geoduck species. Currently, the New Zealand geoduck fishery relies entirely upon wild harvests, but farms are expected to emerge as cultivation protocols are established. A key step in the optimization of cultivation procedures is the identification of optimal temperature and food rations. One method for establishing thermal optima is to identify the temperature window that supports the widest aerobic scope: the degree to which metabolic rate can be increased to support elevated activity demands. Thus, we investigated the aerobic scope for activity at five different temperatures representative of typical environmental conditions (8, 11, 15, 19, and 23 °C) for juvenile and young adult P. zelandica. Clearance rate was also measured at all temperatures. Comparisons of aerobic scope for activity and clearance rates between size classes revealed that juvenile geoducks had a narrower thermal optimum than young adults (15-19 versus 11-19 °C, respectively). Temperatures higher than 19 °C resulted in a reduction of aerobic scope for activity and clearance rate for both juvenile and young adults, which may lead to reduced performance and elevated mortality. These findings provide the first measures of aerobic scope in P. zelandica, a key step towards a meaningful understanding of the ecophysiology of this unusual species.