Summer Mortality Events Research Articles

Prediction of heatwave related mortality magnitude, duration and frequency with climate variability and climate change information

Given the link between climatic factors on one hand, such as climate change and low frequency climate oscillation indices, and the occurrence and magnitude of heat waves on the other hand, and given the impact of heat waves on mortality, these climatic factors could provide some predictive skill for mortality. We propose a new model, the Mortality-Duration-Frequency (MDF) relationship, to relate the intensity of an extreme summer mortality event to its duration and frequency. The MDF model takes into account the non-stationarities observed in the mortality data through covariates by integrating information concerning climate change through the time trend and climate variability through climate oscillation indices. The proposed approach was applied to all-cause mortality data from 1983 to 2018 in the metropolitan regions of Quebec and Montreal in eastern Canada. In all cases, models introducing covariates lead to a substantial improvement in the goodness-of-fit in comparison to stationary models without covariates. Climate change signal is more important than climate variability signal in explaining maximum summer mortality. However, climate indices successfully explain a part of the interannual variability in the maximum summer mortality. Overall, the best models are obtained with the time trend and the North Atlantic Oscillation (NAO) used as covariates. No country has yet integrated teleconnection information in their heat-health watch and warning systems or adaptation plans. MDF modeling has the potential to be useful to public health managers for the planning and management of health services. It allows predicting future MDF curves for adaptive management using the values of the covariates.

Characterization of water microbiota and their relationship with resident oysters during an oyster mortality event.

Microorganisms are vital for the health of marine invertebrates, and their assembly is driven by both deterministic and stochastic factors that regulate residents (innate to the host) and transients (from ambient water). However, the role of water microbiota and the significance of deterministic and stochastic processes in aquatic hosts facing mortality threats are largely unknown. This study examines the shifts in water microbiota during an oyster mortality event using amplicon sequencing and compared with those of resident oysters to disentangle the balance of the deterministic and stochastic factors involved. Water temperature and dissolved oxygen significantly shape the microbial community with a distinct monthly pattern, and Cyanobacteria blooms might exacerbate oyster mortality. The comparative analysis of microbial communities in oysters and water revealed that ≤ 21% of the genera were shared between oysters and water, implying that water microbiota cannot easily transfer into oysters. Furthermore, these shared genera had different functions, with oysters more involved in promoting host digestion and nutrient acquisition and water bacteria enriched more in functions promoting their own growth and survival. These findings illustrate that oysters may possess specific selection or barrier mechanisms that permit a small percentage of transients, controlled by stochastic factors and having a minimal effect on oyster mortality, to enter, whereas the majority of oyster microbiota are residents governed by deterministic factors. Consequently, oysters exhibit some plasticity in their symbiotic microbiota, enabling them to maintain microbial homeostasis and adapt to complex microbial surroundings. This may be a shared mechanism among marine invertebrates for survival in complex marine environments.IMPORTANCEPacific oysters are widely cultured and play vital ecological roles. However, the summer mortality hinders sustainable oyster farming. Untangling causative mechanisms of oyster mortality is a complex task due to the intricate "interactome" involving environmental factors, hosts, and pathogens. Interactions between hosts and microorganisms offer an ideal avenue for investigating the truth. We systematically investigated the microbial community in water and resident oysters during a summer mortality event and proposed that the assembly of oyster microbiota is primarily governed by deterministic processes independent of mortality. Pathogens mainly originate from resident members of the oyster microbiota, with a limited influence from the microbial community in the water. Additionally, environmental degraders, such as Cyanobacteria blooms, cannot be overlooked as a contributing factor of oyster mortality. This study evaluated the weight of deterministic and stochastic factors in microbial assembly during an oyster mortality event and greatly broadened our understanding of the "interactome" through the interaction between oysters and water in microbiota.

Histological Examination of Perna canaliculus Mussels during a Summer Mortality Event in New Zealand

The New Zealand Greenshell™ mussel (Perna canaliculus) is endemic to New Zealand and contributes to the success of the country’s aquaculture industry. However, summer mortality and potential disease outbreak events are having an increasing effect on the growth of this industry. The cause of these mortalities remains unknown, and histopathological studies of the pathogen and parasites in mussels are still incomplete. In the present study, a histological approach was used to identify pathogens and parasites, as well as immunological tissue responses in unhealthy- and healthy-looking P. canaliculus during a summer mortality event in 2018. A highly significant association between health conditions and the presence of Perkinsus olseni in mussels was observed. A higher prevalence of P. olseni, Apicomplexan-X (APX), and bacterial (rods and cocci) infections were noted in the unhealthy-looking mussels than in the healthy-looking mussels. In an assessment of stains, hematoxylin and eosin (H&E) staining appeared to be the best method for general pathological and anatomical characterization, while Giemsa provided the clearest visual definition of bacteria. In this aspect, it was comparable to Ziehl–Neelsen (ZN) in apparent sensitivity. Although Gram and ZN staining revealed bacterial cells marginally better than with H&E, their differential staining could not be assessed as no Gram-positive or acid-fast bacteria were seen, and no mussel-positive controls were available for comparison. This study also provides an illustrated guide to some significant mussel health indicators.

Comprehensive Multi-omics Approaches Provide Insights to Summer Mortality in the Clam Meretrix petechialis.

Bivalve mass mortalities have been reported worldwide, which not only can be explained as a result of pathogen infection, but may reflect changes in environments. Although these episodes were often reported, there was limited information concerning the molecular responses to various stressors leading to summer mortality. In the present work, RNA sequencing (RNA-seq), tandem mass tagging (TMT)-based quantitative proteomics, and 16S rRNA sequencing were used to explore the natural outbreak of summer mortality in the clam Meretrix petechialis. We identified a total of 172 differentially expressed genes (DEGs) and 222 differentially expressed proteins (DEPs) in the diseased group compared to the normal group. The inconsistent expression profiles of immune DEGs/DEPs may be due to the immune dysregulation of the diseased clams. Notably, 11 solute carrier family genes were found among the top 20 down-regulated genes in the diseased group, indicating that weakened transmembrane transport ability might occur in the diseased clams. Integration analysis of transcriptomic and proteomic results showed that many metabolic processes such as "arginine and proline metabolism" and "tyrosine metabolism" were inhibited in the diseased group, suggesting metabolic inhibition. Moreover, 16S rRNA sequencing revealed that the microbial composition of clam hepatopancreas was disordered in the diseased group. The comparison of DEGs expression between the natural summer mortality event and an artificial challenge experiment involving both Vibrio infection and heat stress revealed 9/15 genes showing similar expression trends between the two conditions, suggesting that the summer mortality might be caused by a combination of high temperature and Vibrio infection. These results would deepen our understanding of summer mortality and provide candidate resistance markers for clam resistance breeding.

Abundance of Vibrio aestuarianus, water temperature, and stocking density are associated with summer mortality of Pacific oysters in suspended culture

High mortality rates of cultured Pacific oysters (Crassostrea gigas) during the summer months have regularly occurred on oyster farms in British Columbia, Canada over the last 10 years, but little is known about the microbial and environmental conditions that contribute to such mortality events. The objective of the study was to determine correlative factors associated with the onset of a summer mortality event in oysters (mean ± SD shell height: 14.2 ± 0.5 mm) grown in suspended culture at four stocking densities (150, 300, 450, 600 oysters tray−1) from May 11 to September 17, 2018. Variables examined included both biotic (oyster growth, mortality, reproductive development, and microbiome (approximately every week); Vibrio and harmful algal species abundance) and abiotic (temperature, salinity, turbidity, dissolved oxygen, pCO2, pH, and aragonite saturation) ones. Both the absolute abundance of V. aestuarianus and the relative abundance of Vibrio spp. increased with observed oyster mortality and declining health. Mortality was highest on August 12 and associated with a prior period of elevated temperatures (i.e., increasing temperatures from early July to early August) and increased oyster growth/reproductive development. At that time, systemic mixed microbial infections and necrotic gill tissue in histological cross sections were observed in 19% of oysters that appeared healthy macroscopically. Cumulative percent mortalities per tray ranged from 34 to 75%, the highest-density trays having significantly less mortality and smaller shell width, shell length, and gonad length than lower-density trays. This study demonstrates the significant impact of summer mortality on Pacific oysters and highlights the biotic (host growth, reproductive development, and microbiome composition as well as Vibrio spp. abundance) and abiotic (water temperature) factors associated with the observed mortality in this region.

Paternal heat exposure affects progeny larval development in green-lipped mussels Perna canaliculus

The green-lipped mussel Perna canaliculus is critically important to the New Zealand aquaculture industry. However, the rise in marine heatwave (MHW) events poses an emerging threat to this industry through summer mortality events. This study investigated the potential for paternally mediated transgenerational plasticity to improve offspring performance under heat stress. We simulated a week-long MHW event, exposing male P. canaliculus broodstock to elevated (22°C) or ambient (17.5°C) temperatures immediately prior to spawning, and evaluated the effects of paternal heat exposure on successful development, size and acute thermal tolerance of their larvae that were also reared under ambient or elevated (20°C) temperatures through to completion of the lecithotrophic trochophore stage. Elevated paternal and larval temperatures both increased incidence of abnormal development, reducing larval yield, while initial D-veliger shell length was predominantly influenced by developmental temperature, with longer shells formed at 20°C. Veligers from heat-exposed fathers raised under 20°C showed a small, but significant, elevation in lethal tolerance 50 (LT50), the temperature at which 50% of the larvae are predicted to die, when exposed to an additional 1 h heat-shock. These results indicate that paternal heat exposure over a relatively short period can influence offspring performance in this species. The paternal exposure investigated showed limited positive effects on offspring thermal tolerance, which may be outweighed by the negative impact on larval development. As MHWs are forecasted to continue accelerating, understanding transgenerational effects of heat stress will be critical for maintaining high-quality hatchery yields through broodstock selection and may inform wild population forecasting models.

Increased abundance of potentially pathogenic Vibrio and a marine heatwave co-occur with a Pacific Oyster summer mortality event

The Pacific Oyster (Crassostrea gigas) is an important aquaculture species, that has been globally affected by “summer mortality” outbreaks. The causes of summer mortality are often unclear, but several contributing factors have been implicated. To better understand the factors contributing to C. gigas summer mortality, we performed a 24 week experiment in tanks filled by a continuous flow of unfiltered surface seawater during a summer mortality high-risk period. Throughout this period, we measured a suite of environmental parameters, with weekly sampling to characterise the bacterial community composition, the abundance and diversity of the Vibrio genus, and the expression of oyster immune response genes. We observed three clear phases of oyster mortality: Phase 1 (day 0 to 75) was characterised by sporadic and low levels (mean < 1% per week) of oyster mortality; Phase 2 (day 82 to 119) involved constant levels of ongoing mortality (mean < 10% per week); and Phase 3 (day 125 to 161) involved a sharp increase in mortality (mean > 10% per week) reaching up to weekly mortality of 51%. The shift between phase 2 and 3 coincided with heavy rainfall and a marine heatwave (MHW) event at our study site, which lasted 13 days, when the average water temperature was elevated by 0.8 °C when compared to the weeks prior and following the MHW, and a maximum water temperature of 27.7 °C was reached. Oyster mortality was positively correlated with the occurrence of the MHW as well as bacterial and Vibrio abundance, and negatively correlated with seawater salinity that decreased from a salinity of 34.6 to 31.4 psu following rainfall events. In addition, the relative abundance of several Vibrio species, including V. campbellii, V. rotiferianus and V. owensii displayed positive correlations to oyster mortality. Shifts in the oyster-associated bacterial community and oyster immunity gene expression profiles occurred in parallel to increased mortality levels, with genes involved in the regulation of antimicrobial peptides (IkB1 and NF-kB p105) significantly over-expressed during high mortality. Overall, we observed a C. gigas mortality event that coincided with a marine heatwave, rainfall event and a shift in bacterial community composition. Our analyses provides further evidence for the putative role of Vibrio in summer mortality events.

Investigating the effect of bacterial coinfections on juvenile and adult green‐lipped mussels (Perna canaliculus)

AbstractThe New Zealand's Greenshell™ mussel (Perna canaliculus) aquaculture industry is being affected by summer mortality events associated with increasing seawater temperatures and pathogens. In this study, challenge experiments were conducted to investigate, for the first time, the effects of pathogen coinfection on the survivability and haemolymph immune responses of juvenile and adult mussels. Animals were injected with marine broth (control), Vibrio mediterranei, Photobacterium swingsii, or a mixture of V. mediterranei and P. swingsii. Then, mussel survival was monitored for 72 h, and haemolymph was sampled for bacterial quantification and metabolomics analyses at 24‐ and 48‐h post challenge. Coinfected adults and juveniles showed 100% mortality. Bacterial colony counts in haemolymph decreased as infection time continued, especially in juveniles. The haemolymph metabolome of mussels exposed to single bacterial species and coinfection showed response changes largely within energy metabolism. Mussels infected with V. mediterranei exhibited increased metabolites linked to the glutathione pathway, branched‐chain amino acids, and others over time, supporting structural functions. Conversely, mussels infected with P. swingsii showed no metabolic differences over time. The coinfection group exhibited large decreases in important metabolites, such as fatty acids as an alternative energy source and amino acids to support immune functions and protein synthesis.

Comparative chromatin dynamics reveal differential thermal tolerance mechanisms between two congeneric oyster species

Oysters, being globally farmed bivalve of significant economic and ecological value, are increasingly confronted with heat stress induced by global warming, such as the frequent occurrence of mass summer mortality events worldwide. The role of epigenetics in adapting to high-temperature environments has garnered growing attention; however, its application in oysters remains poorly understood. In this study, we conducted a comparative analysis between two closely related oyster species (the relatively thermosensitive Crassostrea gigas and the relatively thermotolerant Crassostrea angulata) using ATAC-Seq and RNA-Seq to reveal the differential thermal tolerance mechanisms at the chromatin dynamics level in response to high-temperature stress. The comparative analysis showed that the inhibitor of apoptosis protein (IAP) family and its associated apoptosis pathways were the major divergent pathway between the two species. The promoter regions of differentially expressed genes in C. gigas under heat stress were found to be enriched with motifs such as Foxp1, Foxk2, Myc, Cebpd, Foxo3, and Hsf1 motifs and expressed genes related to molecular chaperones, including heat shock protein family. And C. angulata activated the genes related to anti-apoptosis, DNA damage repair, and fatty acid synthesis (decreasing fatty acid unsaturation of membrane to regulate membrane fluidity). These findings suggest that C. angulata may have evolved a more centralized and energy-efficient transcriptional regulatory network at the genomic level, thereby facilitating its long-term adaptation to relatively high temperature environments. Furthermore, the key transcription factor Hsf1, involved in heat shock responses, may contribute to the differential heat response patterns in C. gigas and C. angulata through divergent expression levels of its alternatively spliced isoforms (Hsf1a and Hsf1d). Our work will contribute to predicting the adaptive potential of oysters and other marine invertebrates in facing future global warming, and provide a theoretical foundation for genetic improvement of resistance traits and sustainable development of oyster industry.

Changes in Carbohydrates and N-Acetylglucosamine (Glcnac) In Response to an Experimental Increase of Temperature in Tissues of Two Oyster Species, the Temperate Introduced Crassostrea Gigas and the Native Tropical Crassostrea Corteziensis

This information provides valuable insights for further studies into the immunological mechanisms of oyster summer mortality events.

Phylogenomic diversity of Vibrio species and other Gammaproteobacteria isolated from Pacific oysters (Crassostrea gigas) during a summer mortality outbreak.

The Pacific oyster (PO), Crassostrea gigas, is an important commercial marine species but periodically experiences large stock losses due to disease events known as summer mortality. Summer mortality has been linked to environmental perturbations and numerous viral and bacterial agents, indicating this disease is multifactorial in nature. In 2013 and 2014, several summer mortality events occurred within the Port Stephens estuary (NSW, Australia). Extensive culture and molecular-based investigations were undertaken and several potentially pathogenic Vibrio species were identified. To improve species identification and genomically characterise isolates obtained from this outbreak, whole-genome sequencing (WGS) and subsequent genomic analyses were performed on 48 bacterial isolates, as well as a further nine isolates from other summer mortality studies using the same batch of juveniles. Average nucleotide identity (ANI) identified most isolates to the species level and included members of the Photobacterium, Pseudoalteromonas, Shewanella and Vibrio genera, with Vibrio species making up more than two-thirds of all species identified. Construction of a phylogenomic tree, ANI analysis, and pan-genome analysis of the 57 isolates represents the most comprehensive culture-based phylogenomic survey of Vibrios during a PO summer mortality event in Australian waters and revealed large genomic diversity in many of the identified species. Our analysis revealed limited and inconsistent associations between isolate species and their geographical origins, or host health status. Together with ANI and pan-genome results, these inconsistencies suggest that to determine the role that microbes may have in Pacific oyster summer mortality events, isolate identification must be at the taxonomic level of strain. Our WGS data (specifically, the accessory genomes) differentiated bacterial strains, and coupled with associated metadata, highlight the possibility of predicting a strain's environmental niche and level of pathogenicity.

Pathogenicity and virulence of bacterial strains associated with summer mortality in marine mussels (Perna canaliculus).

The occurrence of pathogenic bacteria has emerged as a plausible key component of summer mortalities in mussels. In the current research, four bacterial isolates retrieved from moribund Greenshell࣪ mussels, Perna canaliculus, from a previous summer mortality event, were tentatively identified as Vibrio and Photobacterium species using morpho-biochemical characterization and MALDI-TOF MS and confirmed as V. celticus, P. swingsii, P. rosenbergii, and P. proteolyticum using whole genome sequencing. These isolates were utilized in a laboratory challenge where mussels were injected with cell concentrations ranging from 105 to 109 CFU/mussel. Of the investigated isolates, P. swingsii induced the highest mortality. Additionally, results from quantitative polymerase chain reaction analysis, focusing on known virulence genes were detected in all isolates grown under laboratory conditions. Photobacterium rosenbergii and P. swingsii showed the highest expression levels of these virulence determinants. These results indicate that Photobacterium spp. could be a significant pathogen of P. canaliculus, with possible importance during summer mortality events. By implementing screening methods to detect and monitor Photobacterium concentrations in farmed mussel populations, a better understanding of the host-pathogen relationship can be obtained, aiding the development of a resilient industry in a changing environment.

Impacts of Seawater pH Buffering on the Larval Microbiome and Carry-Over Effects on Later-Life Disease Susceptibility in Pacific Oysters.

Ocean acidification upwelling events and the resulting lowered aragonite saturation state of seawater have been linked to high mortality of marine bivalve larvae in hatcheries. Major oyster seed producers along North America's west coast have mitigated impacts via seawater pH buffering (e.g., addition of soda ash). However, little consideration has been given to whether such practice may impact the larval microbiome, with potential carry-over effects on immune competency and disease susceptibility in later-life stages. To investigate possible impacts, Pacific oysters (Crassostrea gigas) were reared under soda ash pH buffered or ambient pH seawater conditions for the first 24 h of development. Both treatment groups were then reared under ambient pH conditions for the remainder of the developmental period. Larval microbiome, immune status (via gene expression), growth, and survival were assessed throughout the developmental period. Juveniles and adults arising from the larval run were then subjected to laboratory-based disease challenges to investigate carry-over effects. Larvae reared under buffered conditions showed an altered microbiome, which was still evident in juvenile animals. Moreover, reduced survival was observed in both juveniles and adults of the buffered group under a simulated marine heatwave and Vibrio exposure compared with those reared under ambient conditions. Results suggest that soda ash pH buffering during early development may compromise later-life stages under stressor conditions, and illustrate the importance of a long-view approach with regard to hatchery husbandry practices and climate change mitigation. IMPORTANCE Shellfish industries are threatened worldwide by recurrent summer mortality events. Such incidences are often associated with Vibrio disease outbreaks, and thus, it is critical that animals are able to mount sufficient immune responses. The oyster immune system is linked to the microbiome which is laid down during early developmental stages. Consequently, shellfish hatcheries play a key role with regard to shaping the immune competency of later-life stages. This study represents the first in-depth examination of whether the adoption of seawater pH buffering practice by hatcheries for mitigation of ocean acidification may alter the larval microbiome, and thus, have repercussions for adult susceptibility to summer mortality events. Findings demonstrate that even minimal buffering results in a changed microbiome which is paralleled by increased mortality of later-life stages under Vibrio and temperature stressors, highlighting the importance of the hatchery environment with regard to shaping resilience to summer mortality events.

Perkinsus olseni and other parasites and abnormal tissue structures in New Zealand Greenshell™ mussels (Perna canaliculus) across different seasons

The New Zealand Greenshell™ mussel (Perna canaliculus) supports the largest aquaculture industry in the country. However, summer mortality events and potential disease outbreaks may threaten the growth of this industry. As an approach to gauging potential threats through the seasons, a detailed histopathological examination was conducted on 256 adult cultured mussels collected from a farm between April 2018 to September 2019, which covered the austral autumn, winter, spring and summer seasons. Histological sections followed by confirmatory in situ hybridization (ISH) resulted in the identification of Perkinsus olseni at an overall prevalence of 56%. Other parasites and pathogens were identified by histology: apicomplexan parasite X (APX) (78%), copepods (Pseudomyicola spinosus or Lichomolgus uncus) (1%), Microsporidium rapuae (1%), intracellular microcolonies of bacteria (IMCs) (2%) and bacilli and cocci bacteria (4%) in gills, mantle, gonads, digestive epithelium and digestive tubules. There was a significant association between P. olseni and APX infection in mussels. This is the first report on seasonal variations of P. olseni and APX in New Zealand Greenshell™ mussel. There was a significant association between seasons and the presence of P. olseni and APX in mussels. A significant positive association between the brown material accumulation and parasites (P. olseni and APX) and between haemocytosis and P. olseni infections were recorded. A significant association between presence of parasites and health condition (healthy and unhealthy) of mussels was observed. Moreover, a significant association between digestive tubule deterioration (large lumen, with a thin epithelial wall) and P. olseni infection was noted. Therefore, this study provides information regarding the infections of potential parasites and pathogens for the first time in P. canaliculus, their seasonal variations and host-parasite interactions within a commercial farm.

Host-microbiota interactions play a crucial role in oyster adaptation to rising seawater temperature in summer

Climate change, represented by rising and fluctuating temperature, induces systematic changes in marine organisms and in their bacterial symbionts. However, the role of host-microbiota interactions in the host's response to rising temperature and the underlying mechanisms are incompletely understood in marine organisms. Here, the symbiotic intestinal microbiota and transcriptional responses between diploid and triploid oysters that displayed susceptible and resistant performance under the stress of rising temperature during a summer mortality event were compared to investigate the host-microbiota interactions. The rising and fluctuating temperatures triggered an earlier onset and higher mortality in susceptible oysters (46.7%) than in resistant oysters (17.3%). Correlation analysis between microbial properties and environmental factors showed temperature was strongly correlated with indices of α-diversity and the abundance of top 10 phyla, indicating that temperature significantly shaped the intestinal microbiota of oysters. The microbiota structure of resistant oysters exhibited more rapid changes in composition and diversity compared to susceptible oysters before peak mortality, indicating that resistant oysters possessed a stronger ability to regulate their symbiotic microbiota. Meanwhile, linear discriminant analysis effect size (LefSe) analysis found that the probiotics Verrucomicrobiales and Clostridiales were highly enriched in resistant oysters, and that potential pathogens Betaproteobacteriales and Acidobacteriales were enriched in susceptible oysters. These results implied that the symbiotic microbiota played a significant role in the oysters' adaptation to rising temperature. Accompanying the decrease in unfavorable bacteria before peak mortality, genes related to phagocytosis and lysozymes were upregulated and the xenobiotics elimination pathway was exclusively expressed in resistant oysters, demonstrating the validity of these immunological functions in controlling proliferation of pathogens driven by rising temperature. Compromised immunological functions might lead to proliferation of pathogens in susceptible oysters. This study might uncover a conserved mechanism of adaptation to rising temperature in marine invertebrates from the perspective of interactions between host and symbiotic microbiota.

The effects of experimental temperature increase on gametogenesis and heat stress parameters in oysters: Comparison of a temperate-introduced species (Crassostrea gigas) and a native tropical species (Crassostrea corteziensis)

The effect of thermal stress during reproduction was experimentally evaluated in the oyster Crassostrea gigas, a temperate species, and in the tropical oyster Crassostrea corteziensis. The temperature was gradually increased (1 °C day−1) from 20 °C to 34 °C for two weeks. As expected, C. gigas was the species most affected by heat stress, with the highest mortality rate (P < 0.05) starting at 28 °C, while mortality in C. corteziensis was significant only at 34 °C. The reproductive effort at higher temperatures was reflected in C. gigas as the highest index of mature oocytes and the largest rate of atresic and degenerated oocytes. C. corteziensis showed significant increases in the proliferation of early-developing oocytes at maximum temperatures. Lipid peroxidation and lipofuscin accumulation significantly increased in both species at maximum temperatures, with levels in C. gigas being 8-fold higher than in C. corteziensis. A significant loss of biomass and glycogen reserves stored in gonads was found in C. gigas at 34 °C. The mRNA signal of Hsp70 was detected in gonadic tissues from both oysters after thermal stress for in situ hybridization (ISH), with a temperature increase in both species; the cover area of Hsp70 was significantly higher in C. gigas during the experiment. Hemocyte infiltration significantly increased with increasing temperature in both oyster species, and apoptosis was strongly correlated with Hsp70 in both species (r = 0.93; P < 0.05). These results could explain the high tolerance that C. corteziensis has to thermal stress compared to C. gigas and could be used to adapt aquaculture strategies to the use of native species in subtropical climates to reduce summer mortality events.

Phenotypic and molecular characterization of the causative agents of edwardsiellosis causing Nile tilapia (Oreochromis niloticus) summer mortalities

Edwardsiellosis is a serious bacterial disease affecting Nile tilapia (Oreochromis niloticus), causing septicemia and mortalities. Edwardsiella tarda and Edwardsiella anguillarum were isolated from Nile tilapia summer mortality events in Egypt. Diseased fish showed hemorrhagic septicemia, skin erosions, and eye opacity. A total of 24 Edwardsiella spp. isolates were retrieved from the investigated fish specimens. Phenotypic and biochemical characteristics grouped isolates into typical Ed. tarda (n = 14 strains) and atypical Ed. tarda (n = 10 strains). The BLAST analysis of sodB gene sequencing confirmed the conventional identification of typical Ed. tarda strains (n = 14) and reidentified all the atypical strains (n = 10) as Ed. anguillarum. Isolates showed a combination of virulence factors, including biofilm formation (66.6%), hemolysis (100%), chondroitinase (50%), and proteolytic activity (20.8%). The major part of isolates showed high resistance to ampicillin, amoxicillin, gentamycin antibiotics and harbored tetA, blaCTX-M, and aadA1 resistance genes. Pathogenicity testing of isolates in O. niloticus confirmed their virulence. Challenged fish exhibited septicemic signs similar to naturally diseased fish. Infections in naturally infected tilapia triggered acute and chronic histopathological alterations. Degenerative and necrotic changes were noticed in hematopoietic organs. Granulomas were noticed in between the hepatic parenchyma. The data extracted from the study confirm that accurate identification of the causative agents of edwardsiellosis should be reliant on genetic-based approaches. Analysis of the bacterium virulence properties offers insights into establishing novel therapeutics for edwardsiellosis control. The findings refer to the need for antimicrobial sensitivity testing to minimize antimicrobial resistance and increase therapy efficacy.

Characterization of mussel ( Perna canaliculus ) haemocytes and their phagocytic activity across seasons

Perna canaliculus is endemic to New Zealand and is the top shellfish export product. However, the growth of this industry is being adversely affected by summer mortality events. To assess these health threats, differential haemocyte counts (DHC) were performed on haemolymph smears stained with Giemsa, and in vitro phagocytosis activity assays were applied in P. canaliculus for several months covering three seasons (winter, spring and summer). A new optimized classification scheme was developed for P. canaliculus haemocytes where 55% eosinophilic and 27.2% basophilic granulocytes were identified. Eosinophilic granulocytes were classified as dense, semi and small semi, and these new categories were reported herein for the first time in this species. A new haemocyte type (mixed granulocytes), which contains both acidophilic and basophilic granules, is proposed for P. canaliculus. The phagocytosis percentages were significantly affected by incubation time, indicating an increase in zymosan particle uptake from 18.42 ± 1.7% after 30 min, 32.08 ± 3.1% after 60 min and 44.74 ± 3.5% (maximum) after 120 min of incubation. The lowest phagocytosis was observed in the winter season and the highest phagocytosis in summer. This study findings provide a better understanding of the immune function of P. canaliculus haemocytes and serve as a reference for further investigations on the impacts of seasonal variations on haemocyte activity and phagocytosis.

Investigating the biochemical effects of heat stress and sample quenching approach on the metabolic profiling of abalone (Haliotis iris).

Ocean temperatures have been consistently increasing due to climate change, and the frequency of heatwave events on shellfish quality is a growing concern worldwide. Typically, shellfish growing areas are in remote or difficult to access locations which makes in-field sampling and sample preservation of shellfish heat stress difficult. As such, there is a need to investigate in-field sampling approaches that facilitate the study of heat stress in shellfish. This study aims to apply a gas chromatography-mass spectrometry (GC-MS) based metabolomics approach to examine molecular mechanisms of heat stress responses in shellfish using abalone as a model, and compare the effects of different quenching protocols on abalone metabolic profiles. Twenty adult Haliotis iris abalone were exposed to two temperatures (14°C and 24°C) for 24h. Then, haemolymph and muscle tissues of each animal were sampled and quenched with 4 different protocols (liquid nitrogen, dry ice, cold methanol solution and normal ice) which were analyzed via GC-MS for central carbon metabolites. The effects of different quenching protocols were only observed in muscle tissues in which the cold methanol solution and normal ice caused some changes in the observed metabolic profiles, compared to dry ice and liquid nitrogen. Abalone muscle tissues were less affected by thermal stress than haemolymph. There were 10 and 46 compounds significantly influenced by thermal stress in muscle and haemolymph, respectively. The changes of these metabolite signatures indicate oxidative damage, disturbance of amino acid and fatty acid metabolism, and a shift from aerobic metabolism to anaerobic pathways. The study provided insights into the heat response of abalone, which could be useful for understanding the effects of marine heatwaves and summer mortality events on abalone. Dry ice appeared to be a suitable protocol, and safer in-field alternative to liquid nitrogen, for quenching of abalone tissues.

Reciprocal hybrids derived from Crassostrea gigas and C. angulata exhibit high heterosis in growth, survival and thermotolerance in northern China

The Pacific oyster is one of the most economically important species in global aquaculture. However, the production of C. gigas has been compromised by widespread and severe summer mortality events in recent years. To explore the possibility of obtaining a new hybrid line with rapid growth and high summer survival rate traits through hybridization, we performed a systematic comparative study on heterosis for growth and survival of C. gigas (GG), C. angulata (AA) and their reciprocal hybrids of C. gigas × C. angulata (GA) and C. angulata × C. gigas (AG) for 450 days in northern China. The fertilization and D-stage rates of reciprocal hybrids were lower than those of parental species, but obvious heterosis for growth, survival and metamorphosis were observed at larval stages. Meanwhile, the hybrid cohorts showed high heterosis for growth and survival at both juvenile and adult stages. Moreover, compared with growth, heterosis was higher in survival with a mid-parent heterosis ranged from 12.75% to 110.20% throughout the whole period. At day 450, the cumulative survival rate of hybrid GA increased by nearly 34% compared with GG, with a single patent heterosis I(GA/GG) of 191.14%. Notably, the final yield of hybrid crosses was significantly higher than that of parental lines, with a mid-parent heterosis of 182.72% and I(GA/GG) of 239.46%. To investigate the relationship between summer mortality and high temperature tolerance, the survivability of reciprocal hybrids and parental species through exposure to acute increasing temperature stresses for 96 h were compared. The reciprocal hybrids exhibited greater thermotolerance than the parental species, followed the order of GA > AG > AA > GG. Overall, our study confirmed that GA showed the greatest heterosis in terms of growth, survival and thermotolerance, which could be used as the preferred alternative variety in oyster farming in northern China.