Mussel Hemolymph Research Articles

Microplastic's Contamination in the Hemolymph and Organs (Gills and Hepatopancreas) of Perna viridis.

The issue of microplastics (MPs) has emerged as a significant concern globally, with discussions surrounding the potential environmental impact of these tiny plastic particles becoming increasingly prevalent. This study aimed to identify the concentration and characteristics of MPs in hemolymph and organs (gills and hepatopancreas) of green mussels (Perna viridis) that are frequently consumed by people in Pangkajene Kepulauan, South Sulawesi Province, Indonesia. Green mussels were collected from two different sampling sites for comparison. Screening was carried out on dispensed hemolymph and dissected organs to identify the characteristics of MPs. Surface seawater sampling was added as information on MP's characteristics from the mussel habitat. Visual observation of MP's characteristics using a stereomicroscope in laminar flow is to prevent contamination. The identification of MP's polymer type is using FTIR-ATR. The results showed that hemolymph, hepatopancreas, gills, and surface water were concentrated with MPs. Small (2-3.9cm) green mussels accumulated more MPs than medium (4-5.9cm) and large (> 6cm). MPs characteristics of fiber shape, transparent color, and size 0.1-0.5mm were dominant in all samples. A total of seven polymers of MPs were identified with polyethylene and polystyrene types most frequently found from all samples. Based on this study, green mussels are good for biomonitoring of MPs.

Polyester Microfibers Exposure Modulates Mytilus galloprovincialis Hemolymph Microbiome.

Microplastic (MP) contamination in the aquatic environment is a cause of concern worldwide since MP can be taken up by different organisms, altering different biological functions. In particular, evidence is accumulating that MP can affect the relationship between the host and its associated microbial communities (the microbiome), with potentially negative health consequences. Synthetic microfibers (MFs) represent one of the main MPs in the marine environment, which can be accumulated by filter-feeding invertebrates, such as bivalves, with consequent negative effects and transfer through the food chain. In the mussel Mytilus galloprovincialis, polyethylene terephthalate (PET) MFs, with a size distribution resembling that of an MF released from textile washing, have been previously shown to induce multiple stress responses. In this work, in the same experimental conditions, the effects of exposure to PET-MF (96 h, 10, and 100 μg/L) on mussel hemolymph microbiome were evaluated by 16S rRNA gene amplification and sequencing. The results show that PET-MF affects the composition of bacterial communities at the phylum, family and genus level, with stronger effects at the lowest concentration tested. The relationship between MF-induced changes in hemolymph microbial communities and responses observed at the whole organism level are discussed.

Nano titanium dioxide alleviates the toxic effects of tris (2-chloropropyl) phosphate on the digestive gland and hemolymph of thick-shell mussel Mytilus coruscus

In the marine environment, nanoparticles play a role in adsorbing and catalytically degrading organic pollutants, thereby mitigating their toxic effects on aquatic organisms. This study aimed to investigate the impact of nano titanium dioxide (nTiO2) and tris (2-chloropropyl) phosphate (TCPP) on the hemolymph and digestive function of the thick-shell mussel Mytilus coruscus. Mussels were divided into a control group, a group exposed to TCPP alone, a group exposed to a combination of TCPP and 0.5 mg/L nTiO2, and a group exposed to a combination of TCPP and 1 mg/L nTiO2. After 14 days of exposure, oxidative stress responses, including superoxide dismutase (SOD) activity and malondialdehyde (MDA) content, immune defense responses, including acid phosphatase (ACP) and alkaline phosphatase (AKP) activities, and gene expression, including HSP70 expression, were measured in the hemolymph and digestive glands of the mussels. Compared to the control group, mussels solely exposed to 100 μg/L TCPP exhibited a significant reduction in SOD activity in the hemolymph. When TCPP was co-exposed with 0.5 mg/L nTiO2, there were significant increases in MDA content and AKP activity in both the digestive gland and hemolymph compared to the control group. Upon co-exposure of TCPP with 1 mg/L nTiO2, MDA content and AKP activity in the digestive gland significantly decreased, while SOD, ACP, and AKP activity in the hemolymph significantly increased and MDA content significantly decreased, returning to the control group levels. Furthermore, in the combined exposure, HSP70 gene expression significantly decreased as the nTiO2 concentration increased from 0.5 mg/L to 1 mg/L. In summary, TCPP impacted the hemolymph and digestive function of mussels, whereas a concentration of 1 mg/L nTiO2 effectively alleviated the toxic effects of TCPP. This study is crucial for assessing the ecological risks of nanoparticles and emerging organic pollutants in marine environments, and provides new insights into the interaction between nTiO2 and TCPP, as well as the influence of nTiO2 concentration on mitigating TCPP toxicity.

Differential effects of ocean warming and BDE-47 on mussels with various personalities

Marine warming and polybrominated diphenyl ethers (PBDEs) pollution are two of the most concerning environmental problems in recent years. However, the impact of their co-occurrence on marine bivalves and the tolerance of bivalves with different traits remain unknown. In this study, thick shell mussels Mytilus coruscus were divided into two personalities according to individual feeding and byssus growth. The reliability of the classification was validated by respiration, self-organization, and post-stress behavior. Then, the survival rate, hemolymph immunity, and digestive glands oxidase activity of classified mussels were evaluated after 21 days of compound exposure to warming and BDE-47. The results showed that mussels could be divided into proactive and reactive types consistently. Compared to reactive mussels, proactive mussels exhibited some traits, such as faster food recovery, more byssus growth, higher metabolic rate, and more efficient clustering. Both single or combined warming and BDE-47 exposure impacted the individual survival, hemolymph, and antioxidase of mussels. Notably, the negative impacts of BDE-47 were exacerbated by warming. Moreover, proactive mussels displayed better adaptability with higher survival rates along with less damage to hemolymph immunity and antioxidant ability compared to reactive ones when facing environmental challenges. This study highlights potential risks associated with the coexistence of marine warming and PBDEs pollution while demonstrating differential fitness among individuals with distinct personalities.

Viruses of Freshwater Mussels during Mass Mortality Events in Oregon and Washington, USA.

Freshwater mussels (Unionida) are globally imperiled, in part due to largely unexplained mass mortality events (MMEs). While recent studies have begun to investigate the possibility that mussel MMEs in the Eastern USA may be caused by infectious diseases, mussels in the Western USA have received relatively little attention in this regard. We conducted a two-year epidemiologic investigation of the role of viruses in ongoing MMEs of the Western pearlshell (Margaritifera falcata) and the Western ridged mussel (Gonidea angulata) in the Chehalis River and Columbia River watersheds in the Western USA. We characterized viromes of mussel hemolymph from 5 locations in 2018 and 2020 using metagenomic methods and identified 557 viruses based on assembled contiguous sequences, most of which are novel. We also characterized the distribution and diversity of a previously identified mussel Gammarhabdovirus related to pathogenic finfish viruses. Overall, we found few consistent associations between viruses and mussel health status. Variation in mussel viromes was most strongly driven by location, with little influence from date, species, or health status, though these variables together only explained ~1/3 of variation in virome composition. Our results demonstrate that Western freshwater mussels host remarkably diverse viromes, but no single virus or combination of viruses appears to be associated with morbidity or mortality during MMEs. Our findings have implications for the conservation of imperiled freshwater mussels, including efforts to enhance natural populations through captive propagation.

Lethal and Sub-lethal Implications of Sodium Chloride Exposure for Adult Unionid Mussel Species: Eurynia dilatata and Lasmigona costata.

The elevated use of salt as a de-icing agent on roads in Canada is causing an increase in the chloride concentration of freshwater ecosystems. Freshwater Unionid mussels are a group of organisms that are sensitive to increases in chloride levels. Unionids have greater diversity in North America than anywhere else on Earth, but they are also one of the most imperiled groups of organisms. This underscores the importance of understanding the effect that increasing salt exposure has on these threatened species. There are more data on the acute toxicity of chloride to Unionids than on chronic toxicity. This study investigated the effect of chronic sodium chloride exposure on the survival and filtering activity of two Unionid species (Eurynia dilatata, and Lasmigona costata) and assessed the effect on the metabolome in L. costata hemolymph. The concentration causing mortality after 28days of exposure was similar for E. dilatata (1893mg Cl-/L) and L. costata (1903mg Cl-/L). Significant changes in the metabolome of the L. costata hemolymph were observed for mussels exposed to non-lethal concentrations. For example, several phosphatidylethanolamines, several hydroxyeicosatetraenoic acids, pyropheophorbide-a, and alpha-linolenic acid were significantly upregulated in the hemolymph of mussels exposed to 1000mg Cl-/L for 28days. While no mortality occurred in the treatment, elevated metabolites in the hemolymph are an indicator of stress.

Age-Related and Seasonal Dynamics of Hemocyte Population in the Mussel <i>Crenomytilus grayanus</i> (Dunker, 1853)

The cellular composition of the hemolymph of the mussel Crenomytilus grayanus is represented by hemoblasts, hyalinocytes, basophilic, neutrophilic and acidophilic granulocytes with a significant predominance of the latter. With the age of the mollusk, the average values of the size and granularity of hemocytes increased due to accumulation of granulocytes in the hemolymph (up to 100% of all cells) and their mature highly differentiated acidophilic forms (up to 99% of all cells); this indicates a decrease in the rate of renewal of the cell population. During the autumn period of sexual inertia, the number of cells in the hemolymph of mussels decreased with increasing age of mollusks but without any correlation with the shell size. However, in summer, during the spawning period, the concentration of hemocytes either did not change or increased with increasing body size, directly correlating with the number of young basophilic granulocytes. This correlation may indicate either a relationship between the number of hemocytes and the ability of mussels to reach large sizes, or a higher potency in aged mussels for emergency mobilization of the immune system in the summer season through rapid replenishment of the pool of circulating cells with subsequent redistribution of young and mature cell forms between hemolymph and the internal organs.

Contaminants of emerging concern in the Maumee River and their effects on freshwater mussel physiology.

Contaminants of emerging concern pose a serious hazard to aquatic wildlife, especially freshwater mussels. The growing number of contaminants in aquatic systems requires scientists and managers to prioritize contaminants that are most likely to elicit a biological response for further monitoring and toxicological testing. The objectives of this study were to identify a sub-category of contaminants most likely to affect Pyganodon grandis and to describe alterations in metabolites and gene expression between various sites. Mussels were deployed in cages for two weeks at four sites along the Maumee River Basin, Ohio, USA. Water samples were analyzed for the presence of 220 contaminants. Hemolymph samples were collected for metabolomics and analyzed using mass spectrometry. Contaminants that significantly covaried with metabolites were identified using partial least-squares (PLS) regression. Tissue samples were collected for transcriptomics, RNA was sequenced using an Illumina HiSeq 2500, and differential expression analysis was performed on assembled transcripts. Of the 220 targeted contaminants, 69 were detected in at least one water sample. Of the 186 metabolites detected in mussel hemolymph, 43 showed significant differences between the four sites. The PLS model identified 44 contaminants that significantly covaried with changes in metabolites. A total of 296 transcripts were differentially expressed between two or more sites, 107 received BLAST hits, and 52 were annotated and assigned to one or more Gene Ontology domains. Our analyses reveal the contaminants that significantly covaried with changes in metabolites and are most likely to negatively impact freshwater mussel health and contribute to ongoing population declines in this group of highly endangered animals. Our integration of "omics" technologies provides a broad and in-depth assessment of the short-term effects of contaminants on organismal physiology. Our findings highlight which contaminants are most likely to be causing these changes and should be prioritized for more extensive toxicological testing.

Applying the concept of liquid biopsy to monitor the microbial biodiversity of marine coastal ecosystems

Liquid biopsy (LB) is a concept that is rapidly gaining ground in the biomedical field. Its concept is largely based on the detection of circulating cell-free DNA (ccfDNA) fragments that are mostly released as small fragments following cell death in various tissues. A small percentage of these fragments are from foreign (nonself) tissues or organisms. In the present work, we applied this concept to mussels, a sentinel species known for its high filtration capacity of seawater. We exploited the capacity of mussels to be used as natural filters to capture environmental DNA fragments of different origins to provide information on the biodiversity of marine coastal ecosystems. Our results showed that hemolymph of mussels contains DNA fragments that varied considerably in size, ranging from 1 to 5 kb. Shotgun sequencing revealed that a significant amount of DNA fragments had a nonself microbial origin. Among these, we found DNA fragments derived from bacteria, archaea, and viruses, including viruses known to infect a variety of hosts that commonly populate coastal marine ecosystems. Taken together, our study shows that the concept of LB applied to mussels provides a rich and yet unexplored source of knowledge regarding the microbial biodiversity of a marine coastal ecosystem.

Assessing the effects of the cytostatic drug 5-Fluorouracil alone and in a mixture of emerging contaminants on the mussel Mytilus galloprovincialis

The assessment of contaminants of emerging concern, alone and in mixtures, and their effects on marine biota requires attention. 5-Fluorouracil is a cytostatic category 3 anti-cancer medication (IARC) that is used to treat a variety of cancers, including colon, pancreatic, and breast cancer. In the presence of other pollutants, this pharmaceutical can interact and form mixtures of contaminants, such as adhering to plastics and interaction with metal nanoparticles. This study aimed to comprehend the effects of 5-Fluorouracil (5FU; 10 ng/L) and a mixture of emerging contaminants (Mix): silver nanoparticles (nAg; 20 nm; 10 μg/L), polystyrene nanoparticles (nPS; 50 nm; 10 μg/L) and 5FU (10 ng/L), in an in vivo (21 days) exposure of the mussel Mytilus galloprovincialis. A multibiomarker approach namely genotoxicity, the antioxidant defence system (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPx), glutathione – S – transferases (GST) activities), and oxidative damage (LPO) was used to assess the effects in gills and digestive gland of mussels. Both treatments cause genotoxicity in mussel's haemolymph, and antagonism between contaminants was observed in the Mix. Genotoxicity observed confirms 5FU's mode of action (MoA) by DNA damage. The antioxidant defence system of mussels exposed to 5FU kicked in and counter balanced ROS generated during the exposure, though the same was not seen in Mix-exposed mussels. Mussels were able to withstand the effects of the single compound but not the effects of the Mix. For oxidative stress and damage, the interactions of the components of the mixture have a synergistic effect.

Impact of environmental microplastics alone and mixed with benzo[a]pyrene on cellular and molecular responses of Mytilus galloprovincialis

The hazard of microplastic (MP) pollution in marine environments is a current concern. However, the effects of environmental microplastics combined with other pollutants are still poorly investigated. Herein, impact of ecologically relevant concentrations of environmental MP alone (50 µg/L) or combined with B[a]P (1 µg/L) was assessed in mussel Mytilus galloprovincialis after a short-term exposure (1 and 3 days) to environmental MP collected from a north-Mediterranean beach. Raman Microspectroscopy (RMS) revealed bioaccumulation in mussel hemolymph of MP, characterized by polyethylene (PE), polyethylene terephthalate (PET), polypropylene (PP), polyethylene vinyl acetate (PEVA) and high-density polyethylene (HDPE), with abundance of MP sized 1.22–0.45 µm. An increase of B[a]P was detected in mussels after 3-day exposure, particularly when mixed with MP. Both contaminants induced cytotoxic and genotoxic effects on hemocytes as determined by lysosomal membrane stability (LMS), micronuclei frequency (FMN), and DNA fragmentation rate by terminal dUTP nick-end labeling (TUNEL). About apoptosis/DNA repair processes, P53 and DNA-ligase increased at 1-day exposure in all conditions, whereas after 3 days increase of bax, Cas-3 and P53 and decrease of Bcl-2 and DNA-ligase were revealed, suggesting a shift towards a cell apoptotic event in exposed mussels. Overall, this study provides new insights on the risk of MP for the marine ecosystem, their ability to accumulate xenobiotics and transfer them to marine biota, with potential adverse repercussion on their health status.

IMPACT OF NAPHTHALENE EXPOSURE ON MARKER ENZYMES OF GREEN MUSSEL Perna viridis

Backgrounds: Environmental contamination has been increased in recent decades as a result of the fast expansion of anthropogenic activities, particularly in the aquatic ecosystem. Under a range of severe environmental circumstances, marine organisms can be susceptible to oxidative stress and results in the changes in the biochemical components which can be assessed to know the health status of organisms.
 Aim of the Works: The aim of this study is to observe the impact of Naphthalene on bivalve mussels and to employ a large number of biomaker to discover distinct and unique patterns. For this the green mussels Perna viridis exposed to naphthalene, in order to understand the changes in marker enzymes in P. viridis haemolymph, gill, and digestive gland.
 Methodology: Green mussels were exposed to naphthalene for 28 days in order to assess changes in biomarker. Acid phosphatase (ACP) and alkaline phosphatase (ALP), alanine transaminase (ALT), and aspartate transaminase activity (AST) were examined in the haemolymph, gill, and digestive gland of mussels P. viridis.
 Results: The haemolymph has considerably higher in all marker enzymes after 28 days of naphthalene exposure. Marker enzymes were inhibited in the gill and digestive gland, and in most cases, all of the marker enzymes in the haemolymph, gill, and digestive gland were concentration dependent. The observed changes in marker enzymes which detected in the haemolymph and the other two tissues of P. viridis were statistically significant.
 Conclusions: The present study showed a significant association between the entire biomarkers tested in mussels exposed to naphthalene. Overall, the results indicate that haemolypmh is the most vulnerable component to naphthalene exposure when compared to tissues, and it may be employed as a bioindicator of organic pollution exposure.

Fecal pollution increases susceptibility to diseases in brown mussel Perna perna from cultured and wild populations

Maricultural systems for food production have expanded worldwide since the 2000s, including the production of brown mussel Perna perna - a key aquaculture species in Venezuela, South Africa, and Brazil. Bivalves grown in sewage-polluted areas can act as sources for human diseases given their filter-feeding behavior. Moreover, sewage can affect mussels' microbiota and fitness, thereby making them more susceptible to infections and diseases, leading to mortalities. In the present study, we investigated the impacts of coastal fecal pollution on P. perna pathological conditions. Fecal indicator bacteria (FIB) were used to assess the microbiological quality in the seawater and mussels' hemolymph from four beaches located at Guanabara Bay, Rio de Janeiro, Brazil: Vermelha (VB), Icaraí (IB), and Urca (UB), where mussels are exploited for consumption and local market sale; and Jurujuba (JB) farming area where mussels are cultured. Additionally, mussels had their organs surveyed for pathological conditions and parasitic infection by histology. UB presented the highest FIB counts in seawater, followed by IB, JB and VB. Moreover, significantly lower values of total coliforms (TC) were found in mussels' hemolymph from VB and IB than JB and UB, which are probably related to differences in the hydrodynamic of these beaches; VB and IB are open beaches, while JB and UB are sheltered. Our findings suggest that mussel's hemolymph could be used as an indicator of the water quality. Mussels from VB and IB presented lower overall incidence of pathological conditions than ones from JB and UB, suggesting that higher fecal contamination increases mussel susceptibility to pathologies. Mussels from VB were associated with an elasmobranch parasite (Tylocephalum sp.) that indicate a conservative ecological dynamic. Our findings highlight that sewage inputs into coastal systems and farming areas raises the risks of parasitosis and diseases in mussels, which may compromise the quantity and quality of shellfish production and lead to economic losses. In response, public investments in urban planning and sanitary regulation close to coastal systems and farming areas are crucial to prevent future problems for the shellfish market and human safety, especially in developing countries such as Brazil.

Traits of a\xa0mussel transmissible cancer are\xa0reminiscent of a parasitic life style

Some cancers have evolved the ability to spread from host to host by transmission of cancerous cells. These rare biological entities can be considered parasites with a host-related genome. Still, we know little about their specific adaptation to a parasitic lifestyle. MtrBTN2 is one of the few lineages of transmissible cancers known in the animal kingdom. Reported worldwide, MtrBTN2 infects marine mussels. We isolated MtrBTN2 cells circulating in the hemolymph of cancerous mussels and investigated their phenotypic traits. We found that MtrBTN2 cells had remarkable survival capacities in seawater, much higher than normal hemocytes. With almost 100% cell survival over three days, they increase significantly their chances to infect neighboring hosts. MtrBTN2 also triggered an aggressive cancerous process: proliferation in mussels was ~ 17 times higher than normal hemocytes (mean doubling time of ~ 3 days), thereby favoring a rapid increase of intra-host population size. MtrBTN2 appears to induce host castration, thereby favoring resources re-allocation to the parasites and increasing the host carrying capacity. Altogether, our results highlight a series of traits of MtrBTN2 consistent with a marine parasitic lifestyle that may have contributed to the success of its persistence and dissemination in different mussel populations across the globe.

Mussel Mass Mortality and the Microbiome: Evidence for Shifts in the Bacterial Microbiome of a Declining Freshwater Bivalve.

Freshwater mussels (Unionida) are suffering mass mortality events worldwide, but the causes remain enigmatic. Here, we describe an analysis of bacterial loads, community structure, and inferred metabolic pathways in the hemolymph of pheasantshells (Actinonaias pectorosa) from the Clinch River, USA, during a multi-year mass mortality event. Bacterial loads were approximately 2 logs higher in moribund mussels (cases) than in apparently healthy mussels (controls). Bacterial communities also differed between cases and controls, with fewer sequence variants (SVs) and higher relative abundances of the proteobacteria Yokenella regensburgei and Aeromonas salmonicida in cases than in controls. Inferred bacterial metabolic pathways demonstrated a predominance of degradation, utilization, and assimilation pathways in cases and a predominance of biosynthesis pathways in controls. Only two SVs correlated with Clinch densovirus 1, a virus previously shown to be strongly associated with mortality in this system: Deinococcota and Actinobacteriota, which were associated with densovirus-positive and densovirus-negative mussels, respectively. Overall, our results suggest that bacterial invasion and shifts in the bacterial microbiome during unionid mass mortality events may result from primary insults such as viral infection or environmental stressors. If so, bacterial communities in mussel hemolymph may be sensitive, if generalized, indicators of declining mussel health.

Evaluation of real-time qPCR-based methods to detect the DNA of the three protozoan parasites Cryptosporidium parvum, Giardia duodenalis and Toxoplasma gondii in the tissue and hemolymph of blue mussels (M. edulis)

The protozoan parasites Cryptosporidium spp., Giardia duodenalis and Toxoplasma gondii can be transmitted to humans through shellfish consumption. No standardized methods are available for their detection in these foods, and the performance of the applied methods are rarely described in occurrence studies. Through spiking experiments, we characterized different performance criteria (e.g. sensitivity, estimated limit of detection (eLD95METH), parasite DNA recovery rates (DNA-RR)) of real-time qPCR based-methods for the detection of the three protozoa in mussel's tissues and hemolymph. Digestion of mussels tissues by trypsin instead of pepsin and the use of large buffer volumes was the most efficient for processing 50g-sample. Trypsin digestion followed by lipids removal and DNA extraction by thermal shocks and a BOOM-based technique performed poorly (e.g. eLD95METH from 30 to >3000 parasites/g). But trypsin digestion and direct DNA extraction by bead-beating and FastPrep homogenizer achieved higher performance (e.g. eLD95METH: 4–400 parasites/g, DNA-RR: 19–80%). Direct DNA recovery from concentrated hemolymph, by thermal shocks and cell lysis products removal was not efficient to sensitively detect the protozoa (e.g. eLD95METH: 10–1000 parasites/ml, DNA-RR ≤ 24%). The bead-beating DNA extraction based method is a rapid and simple approach to sensitively detect the three protozoa in mussels using tissues, that can be standardized to different food matrices. However, quantification in mussels remains an issue.

Toxicity mechanisms of polystyrene microplastics in marine mussels revealed by high-coverage quantitative metabolomics using chemical isotope labeling liquid chromatography mass spectrometry

Marine microplastic has become an important environmental issue of global concern due to its wide distribution and harmful impacts. However, there is still insufficient information on the toxicity mechanism of microplastics to marine organisms. In this study, we developed and applied a high-coverage quantitative metabolomics technique to investigate the toxicity mechanisms of the polystyrene microspheres (micro-PS) on marine mussels (Mytilus coruscus). A total of 3599 metabolites were quantified, including 163 positively identified metabolites, 318 high-confident putatively identified metabolites, and 2602 mass-matched metabolites from the hemolymph of mussels. Metabolomics analysis indicated that micro-PS disrupted the amino acid metabolism, particularly phenylalanine metabolism, which may lead to oxidative stress and neurotoxicity. Micro-PS at environmentally relevant concentrations induced oxidative stress and immunotoxicity in mussels. After 7 days of recovery, along with the significant clearance of micro-PS by mussels, both metabolite levels and biochemical indicators generally returned to the same level as the control group. Overall, the results showed that microplastics at environmentally-relevant concentrations can cause toxic effects on mussels but these influences are reversible. We envisage the usages of high-coverage metabolomics for investigating the toxicity of various types of microplastics under many different conditions, including those relevant to the marine environment.

Multi-residue extraction to determine organic pollutants in mussel hemolymph.

This study assesses the extraction of eleven pharmaceuticals, five pesticides, five perfluoroalkyl substances, and two illicit drugs in hemolymph from (Mytilus Galloprovincialis). Four extraction procedures using Phree™ Phospholipid Removal cartridges were tested using different volumes of methanol (400 and 600μL) and acetonitrile (300 and 450μL). The pollutants were determined by high-performance liquid chromatography-tandem mass spectrometry. The use of methanol gave several problems during the extraction procedure, such as longer times and sample loss. Three methods (acetonitrile 300 and 450μL; and methanol 600μL) were validated. Recoveries at three concentration levels (5, 50, and 100ng/mL) ranged 35.1-129.0 and 29.3-133.0% for acetonitrile 300 and 450μL, respectively, while recoveries for methanol 600μL ranged 52.2-166.0%. Limits of detection were<10ng/mL for most analytes using any of the methods. Methanol 600μL was the only method capable to extract the illicit drug 4-methoxyphencyclidine and provided a better peak shape and higher signal-noise ratio. When applied to non-spiked samples from local markets salicylic acid and diclofenac were detected at 33.50-97.79 and 28.30-30.31ng/mL respectively. To our knowledge, there are no methods to determine organic contaminants in hemolymph and this is the first application of Phree™ cartridges for mussel hemolymph extraction.

Itsas ingurumenerako mikrokutsatzaile organiko hidrofobikoen presentzia eta eraginak aztertzeko estrategien taxutzea

Lan honetan, ingurumen analisirako metodologia planteamendu berri bat proposatu da. Horretarako, kimikariek eta biologoek baldintza kontrolatuetako esposizioa egiteko elkarlanean jardun genuen. Esperimentu honetan, muskuiluak eta lagin-biltze pasiboko tresnak hainbat mikrokutsatzaile hidrofoboren eraginpean jarri ziren. Batetik, kutsatzaileen kontzentrazioak neurtu ziren, lagin-biltze puntualen zein lagin-biltze pasiboen bitartez. Bestetik, muskuiluetan kutsatzaileen kontzentrazioak neurtu ziren. Biologoek, euren aldetik, muskuiluen azterketa histopatologikoa egin zuten. Azkenik, NMR-n oinarritutako metabolomika erabili zen muskuiluek kutsatzaileen aurrean ematen zuten erantzuna aztertzeko. Lau estrategien konbinazioarekin, kausa (kokteleko mikrokutsatzaileen kontzentrazioak eta muskuiluek metatutakoa) eta eragindako efektuak (histologia azterketetan behatutakoak) hobeto uler ditzakegu. kutsatzaileek eragiten dituzten aldaketak ulertzeko eta ondorioak azaltzeko metabolomika baliabide erabilgarria dela ondorioztatu genuen. kutsatzaileen koktelak errute goiztiarra eragin zien muskuiluei; izan ere, NMR metabolomika erabiliz, aldaketa metaboliko nabarienak errute-egunean ikusi ziren.

Metabolomics investigation of summer mortality in New Zealand Greenshell™ mussels (Perna canaliculus)

Increasing water temperatures due to climate change have resulted in more frequent high mortality events of New Zealand Greenshell™ mussels (Perna canaliculus Gmelin 1791). These events have significant impacts within mussel farms which support a major shellfish industry for New Zealand. The present study investigates metabolic responses of farmed mussels during a summer mortality event in order to identify health impacts and elucidate mechanistic effects of external stressors on mussels. A gas chromatography–mass spectrometry (GC–MS)-based metabolomics approach was used to identify metabolic perturbations and flow cytometry assays were used to assess viability, oxidative stress and apoptosis of haemocytes from healthy and unhealthy mussels during a summer mortality event. The results showed significantly higher mortality and apoptosis of haemocytes in unhealthy mussels compared to healthy mussels. Reactive oxygen species (ROS) production, which is an indicator of oxidative stress was very high in both mussel groups, but no differences were observed between the two mussel groups. Metabolomics revealed alterations of many metabolites in both haemolymph and hepatopancreas (digestive gland) of unhealthy mussels compared to healthy mussels, reflecting perturbations in several molecular pathways, including energy metabolism, amino acid metabolism, protein degradation/tissue damage and oxidative stress. An increased level of itaconic acid which is an antimicrobial metabolite and biomarker of pathogen infection was observed in haemolymph, but not in hepatopancreas samples. This investigation provides the first detailed metabolic characterization of mussel immune responses to a summer mortality event and illustrates the benefits of using an integrated metabolomics and flow cytometry workflow for mussel health assessment and biomarker identification for summer mortality early detection.