Simulated Marine Heat Wave Alters Abundance and Structure of Vibrio Populations Associated with the Pacific Oyster Resulting in a Mass Mortality Event.

Marine heat waves are predicted to become more frequent and intense due to anthropogenically induced climate change, which will impact global production of seafood. Links between rising seawater temperature and disease have been documented for many aquaculture species, including the Pacific oyster Crassostrea gigas. The oyster harbours a diverse microbial community that may act as a source of opportunistic pathogens during temperature stress. We rapidly raised the seawater temperature from 20 °C to 25 °C resulting in an oyster mortality rate of 77.4%. Under the same temperature conditions and with the addition of antibiotics, the mortality rate was only 4.3%, strongly indicating a role for bacteria in temperature-induced mortality. 16S rRNA amplicon sequencing revealed a change in the oyster microbiome when the temperature was increased to 25 °C, with a notable increase in the proportion of Vibrio sequences. This pattern was confirmed by qPCR, which revealed heat stress increased the abundance of Vibrio harveyi and Vibrio fortis by 324-fold and 10-fold, respectively. Our findings indicate that heat stress-induced mortality of C. gigas coincides with an increase in the abundance of putative bacterial pathogens in the oyster microbiome and highlights the negative consequences of marine heat waves on food production from aquaculture.

Contrasting impacts of ocean acidification and warming on the molecular responses of CO2-resilient oysters.

BACKGROUND: This study characterises the molecular processes altered by both elevated CO2 and increasing temperature in oysters. Differences in resilience of marine organisms against the environmental stressors associated with climate change will have significant implications for the sustainability of coastal ecosystems worldwide. Some evidence suggests that climate change resilience can differ between populations within a species. B2 oysters represent a unique genetic resource because of their capacity to better withstand the impacts of elevated CO2 at the physiological level, compared to non-selected oysters from the same species (Saccostrea glomerata). Here, we used proteomic and transcriptomic analysis of gill tissue to evaluate whether the differential response of B2 oysters to elevated CO2 also extends to increased temperature. RESULTS: Substantial and distinctive effects on protein concentrations and gene expression were evident among B2 oysters responding to elevated CO2 or elevated temperature. The combination of both stressors also altered oyster gill proteomes and gene expression. However, the impacts of elevated CO2 and temperature were not additive or synergistic, and may be antagonistic. CONCLUSIONS: The data suggest that the simultaneous exposure of CO2-resilient oysters to near-future projected ocean pH and temperature results in complex changes in molecular processes in order to prevent stress-induced cellular damage. The differential response of B2 oysters to the combined stressors also indicates that the addition of thermal stress may impair the resilience of these oysters to decreased pH. Overall, this study reveals the intracellular mechanisms that might enable marine calcifiers to endure the emergent, adverse seawater conditions resulting from climate change.

Transcriptomic profiling of adaptive responses to ocean acidification.

Some populations of marine organisms appear to have inherent tolerance or the capacity for acclimation to stressful environmental conditions, including those associated with climate change. Sydney rock oysters from the B2 breeding line exhibit resilience to ocean acidification (OA) at the physiological level. To understand the molecular basis of this physiological resilience, we analysed the gill transcriptome of B2 oysters that had been exposed to near-future projected ocean pH over two consecutive generations. Our results suggest that the distinctive performance of B2 oysters in the face of OA is mediated by the selective expression of genes involved in multiple cellular processes. Subsequent high-throughput qPCR revealed that some of these transcriptional changes are exclusive to B2 oysters and so may be associated with their resilience to OA. The intracellular processes mediated by the differentially abundant genes primarily involve control of the cell cycle and maintenance of cellular homeostasis. These changes may enable B2 oysters to prevent apoptosis resulting from oxidative damage or to alleviate the effects of apoptosis through regulation of the cell cycle. Comparative analysis of the OA conditioning effects across sequential generations supported the contention that B2 and wild-type oysters have different trajectories of changing gene expression and responding to OA. Our findings reveal the broad set of molecular processes underlying transgenerational conditioning and potential resilience to OA in a marine calcifier. Identifying the mechanisms of stress resilience can uncover the intracellular basis for these organisms to survive and thrive in a rapidly changing ocean.

Shifting paradigms in restoration of the world's coral reefs.

Many ecosystems around the world are rapidly deteriorating due to both local and global pressures, and perhaps none so precipitously as coral reefs. Management of coral reefs through maintenance (e.g., marine-protected areas, catchment management to improve water quality), restoration, as well as global and national governmental agreements to reduce greenhouse gas emissions (e.g., the 2015 Paris Agreement) is critical for the persistence of coral reefs. Despite these initiatives, the health and abundance of corals reefs are rapidly declining and other solutions will soon be required. We have recently discussed options for using assisted evolution (i.e., selective breeding, assisted gene flow, conditioning or epigenetic programming, and the manipulation of the coral microbiome) as a means to enhance environmental stress tolerance of corals and the success of coral reef restoration efforts. The 2014-2016 global coral bleaching event has sharpened the focus on such interventionist approaches. We highlight the necessity for consideration of alternative (e.g., hybrid) ecosystem states, discuss traits of resilient corals and coral reef ecosystems, and propose a decision tree for incorporating assisted evolution into restoration initiatives to enhance climate resilience of coral reefs.

Two Oyster Species That Show Differential Susceptibility to Virus Infection Also Show Differential Proteomic Responses to Generic dsRNA.

Viral diseases are a significant cause of mortality and morbidity in oysters, resulting in significant economic losses. We investigated the proteomic responses of these two species of oysters to generic double-stranded RNAs (poly I:C and poly A:U). Analysis of proteomic data using isobaric tags for relative and absolute quantitaion (iTRAQ) indicated that there were significant differences in the proteomic responses of the two oyster species resulting from this treatment. Gene ontology analysis showed that several biological processes, cellular components, and molecular function were unique to the different data sets. For example, a number of proteins implicated in the TLR signaling pathway were associated with the Saccostrea glomerata data set but were absent in the Crassostra gigas data set. These results suggest that the differences in the proteomic responses to dsRNA may underpin the biological differences in viral susceptibility. Molecular targets previously shown to be expressed in C. gigas in response to OsHV1 infections were not present in our proteomic data sets, although they were present in the RNA extracted from the very same tissues. Taken together, our data indicate that there are substantial disparities between transcriptomic and proteomic responses to dsRNA challenge, and a comprehensive account of the oysters' biological responses to these treatments must take into account that disparity.

Rapid transcriptional acclimation following transgenerational exposure of oysters to ocean acidification.

Marine organisms need to adapt in order to cope with the adverse effects of ocean acidification and warming. Transgenerational exposure to CO2 stress has been shown to enhance resilience to ocean acidification in offspring from a number of species. However, the molecular basis underlying such adaptive responses is currently unknown. Here, we compared the transcriptional profiles of two genetically distinct oyster breeding lines following transgenerational exposure to elevated CO2 in order to explore the molecular basis of acclimation or adaptation to ocean acidification in these organisms. The expression of key target genes associated with antioxidant defence, metabolism and the cytoskeleton was assessed in oysters exposed to elevated CO2 over three consecutive generations. This set of target genes was chosen specifically to test whether altered responsiveness of intracellular stress mechanisms contributes to the differential acclimation of oyster populations to climate stressors. Transgenerational exposure to elevated CO2 resulted in changes to both basal and inducible expression of those key target genes (e.g. ecSOD, catalase and peroxiredoxin 6), particularly in oysters derived from the disease-resistant, fast-growing B2 line. Exposure to CO2 stress over consecutive generations produced opposite and less evident effects on transcription in a second population that was derived from wild-type (nonselected) oysters. The analysis of key target genes revealed that the acute responses of oysters to CO2 stress appear to be affected by population-specific genetic and/or phenotypic traits and by the CO2 conditions to which their parents had been exposed. This supports the contention that the capacity for heritable change in response to ocean acidification varies between oyster breeding lines and is mediated by parental conditioning.

Proteomic analysis of hemolymph from poly(I:C)-stimulated Crassostrea gigas.

Synthetic double stranded RNA (Poly(I:C)) injection of Crassostrea gigas results in a systemic antiviral response involving many evolutionary conserved antiviral effectors (ISGs). Compared to mammals, the timing of C. gigas ISG expression to viral or poly(I:C) injection is delayed (>12 h p.i.). It could be interpreted that a cytokine is responsible for the systemic, but delayed expression of C. gigas ISGs. We therefore analysed the acellular fraction of C. gigas hemolymph by two-dimensional electrophoresis (2-DE) to identify hemolymph proteins induced by poly(I:C). Poly(I:C) injection increased the relative intensity of four protein spots. These protein spots were identified by tandem mass spectrometry (LC-MS/MS) as a small heat shock protein (sHSP), poly(I:C)-inducible protein 1 (PIP1) and two isoforms of C1q-domain containing protein (C1qDC). RT-qPCR analysis confirmed that the genes encoding these proteins are induced in hemocytes of C. gigas injected with poly(I:C) (p < 0.05). Proteomic data from this experiment corroborates previous microarray and whole transcriptome studies that have reported up-regulation of C1qDC and sHSP during mass mortality events among farmed oysters.

Antiviral immunity in marine molluscs.

Marine molluscs, like all living organisms, are constantly exposed to viruses and have evolved efficient antiviral defences. We review here recent developments in molluscan antiviral immunity against viruses belonging to the order Herpesvirales. Emerging results suggest an interferon-like response and autophagy are involved in the antiviral defence of bivalves to viral infection. Multi-functional plasma proteins from gastropods and bivalves have been identified to have broad-spectrum antiviral activity against mammalian viruses. The antiviral defences present in molluscs can be enhanced by genetic selection, as shown by the presence of oyster strains specifically resistant to ostreid herpesvirus type 1. Whether varying amounts or different isoforms of these antiviral plasma proteins contributes to genetic resistance is worthy of further research. Other evolutionarily conserved antiviral mechanisms, such as RNA interference and apoptosis, still need further characterization.

Oyster viperin retains direct antiviral activity and its transcription occurs via a signalling pathway involving a heat-stable haemolymph protein.

Little is known about the response of non-model invertebrates, such as oysters, to virus infection. The vertebrate innate immune system detects virus-derived nucleic acids to trigger the type I IFN pathway, leading to the transcription of hundreds of IFN-stimulated genes (ISGs) that exert antiviral functions. Invertebrates were thought to lack the IFN pathway based on the absence of IFN or ISGs encoded in model invertebrate genomes. However, the oyster genome encodes many ISGs, including the well-described antiviral protein viperin. In this study, we characterized oyster viperin and showed that it localizes to caveolin-1 and inhibits dengue virus replication in a heterologous model. In a second set of experiments, we have provided evidence that the haemolymph from poly(I : C)-injected oysters contains a heat-stable, protease-susceptible factor that induces haemocyte transcription of viperin mRNA in conjunction with upregulation of IFN regulatory factor. Collectively, these results support the concept that oysters have antiviral systems that are homologous to the vertebrate IFN pathway.

OsHV-1 countermeasures to the Pacific oyster's anti-viral response.

The host-pathogen interactions between the Pacific oyster (Crassostrea gigas) and Ostreid herpesvirus type 1 (OsHV-1) are poorly characterised. Herpesviruses are a group of large, DNA viruses that are known to encode gene products that subvert their host's antiviral response. It is likely that OsHV-1 has also evolved similar strategies as its genome encodes genes with high homology to C. gigas inhibitors of apoptosis (IAPs) and an interferon-stimulated gene (termed CH25H). The first objective of this study was to simultaneously investigate the expression of C. gigas and OsHV-1 genes that share high sequence homology during an acute infection. Comparison of apoptosis-related genes revealed that components of the extrinsic apoptosis pathway (TNF) were induced in response to OsHV-1 infection, but we failed to observe evidence of apoptosis using a combination of biochemical and molecular assays. IAPs encoded by OsHV-1 were highly expressed during the acute stage of infection and may explain why we didn't observe evidence of apoptosis. However, C. gigas must have an alternative mechanism to apoptosis for clearing OsHV-1 from infected gill cells as we observed a reduction in viral DNA between 27 and 54 h post-infection. The reduction of viral DNA in C. gigas gill cells occurred after the up-regulation of interferon-stimulated genes (viperin, PKR, ADAR). In a second objective, we manipulated the host's anti-viral response by injecting C. gigas with a small dose of poly I:C at the time of OsHV-1 infection. This small dose of poly I:C was unable to induce transcription of known antiviral effectors (ISGs), but these oysters were still capable of inhibiting OsHV-1 replication. This result suggests dsRNA induces an anti-viral response that is additional to the IFN-like pathway.

Immune responses to infectious diseases in bivalves.

Many species of bivalve mollusks (phylum Mollusca, class Bivalvia) are important in fisheries and aquaculture, whilst others are critical to ecosystem structure and function. These crucial roles mean that considerable attention has been paid to the immune responses of bivalves such as oysters, clams and mussels against infectious diseases that can threaten the viability of entire populations. As with many invertebrates, bivalves have a comprehensive repertoire of immune cells, genes and proteins. Hemocytes represent the backbone of the bivalve immune system. However, it is clear that mucosal tissues at the interface with the environment also play a critical role in host defense. Bivalve immune cells express a range of pattern recognition receptors and are highly responsive to the recognition of microbe-associated molecular patterns. Their responses to infection include chemotaxis, phagolysosomal activity, encapsulation, complex intracellular signaling and transcriptional activity, apoptosis, and the induction of anti-viral states. Bivalves also express a range of inducible extracellular recognition and effector proteins, such as lectins, peptidoglycan-recognition proteins, thioester bearing proteins, lipopolysaccharide and ß1,3-glucan-binding proteins, fibrinogen-related proteins (FREPs) and antimicrobial proteins. The identification of FREPs and other highly diversified gene families in bivalves leaves open the possibility that some of their responses to infection may involve a high degree of pathogen specificity and immune priming. The current review article provides a comprehensive, but not exhaustive, description of these factors and how they are regulated by infectious agents. It concludes that one of the remaining challenges is to use new "omics" technologies to understand how this diverse array of factors is integrated and controlled during infection.

Dose-dependent effects of metals on gene expression in the sydney rock oyster, Saccostrea glomerata.

In the current study, we tested the effects of common environmental contaminants (the metals zinc and lead) on gene expression in Sydney rock oysters (Saccrostrea glomerata). Oysters were exposed to a range of metal concentrations under controlled laboratory conditions. The expression of 14 putative stress response genes was then measured using quantitative, real-time (q) PCR. The expression of all 14 genes was significantly affected (p < 0.05 vs. nonexposed controls) by at least one of the metals, and by at least one dose of metal. For 5 of the 14 target genes (actin, calmodulin, superoxide dismutase, topoisomerase I, and tubulin) the alteration of expression relative to controls was highest at intermediate (rather than high) doses of metals. Such responses may reflect adaptive (acclimation) reactions in gene expression at low to intermediate doses of contaminants, followed by a decline in expression resulting from exposure at higher doses. The data are discussed in terms of the intracellular pathways affected by metal contamination, and the relevance of such gene expression data to environmental biomonitoring.

Anti-viral gene induction is absent upon secondary challenge with double-stranded RNA in the Pacific oyster, Crassostrea gigas.

Oyster farming is one of the most important aquaculture industries in the world. However, its productivity is increasingly limited by viral disease and we do not yet have management practices, such as protective vaccination, that can control these disease outbreaks. Hence, in the current study we investigated the expression of known anti-viral genes in oysters (Crassostrea gigas) in response to primary and secondary encounter with a virus associated molecular pattern (dsRNA), and tested whether a common form of epigenetic gene regulation (DNA methylation) was associated with the expression of these anti-viral genes. Injection of dsRNA into the adductor muscle resulted in the rapid and transient expression of virus recognition receptors (TLR & MDA5), whereas several anti-viral signalling (IRF & SOC-1) and effector (PKR & viperin) genes were still up-regulated at one week post primary challenge (p < 0.05). This primary encounter with dsRNA appeared to deplete the immune system because anti-viral gene induction was absent in the gills when oysters were given a second injection of dsRNA at 1-week post-primary injection. The expression of DNA methylation genes (DNMT1, DNMT3b, TDG, TET2) and DNA methylation profiles up-stream of specific anti-viral genes (STING, SOC-1 & Viperin) did not change in response to dsRNA injection (p > 0.05). These results collectively suggest that C. gigas does not have an enhanced anti-viral gene response (immune-priming) to secondary dsRNA challenge and that the sustained up-regulation of anti-viral signalling and effector genes following primary challenge is unlikely to be associated with upstream DNA methylation levels.

Quantitative proteomics of heavy metal stress responses in Sydney rock oysters.

Currently, there are few predictive biomarkers in key biomonitoring species, such as oysters, that can detect heavy metal pollution in coastal waterways. Several attributes make oysters superior to other organisms for positive biomonitoring of heavy metal pollution. In particular, they are filter feeders with a high capacity for bioaccumulation. In this study, we used two proteomics approaches, namely label-free shotgun proteomics based on SDS-PAGE gel separation and gas phase fractionation, to investigate the heavy metal stress responses of Sydney rock oysters. Protein samples were prepared from haemolymph of oysters exposed to 100 µg/L of PbCl(2), CuCl(2), or ZnCl(2) for 4 days in closed aquaria. Peptides were identified using a Bivalvia protein sequence database, due to the unavailability of a complete oyster genome sequence. Statistical analysis revealed 56 potential biomarker proteins, as well as several protein biosynthetic pathways to be greatly impacted by metal stress. These have the potential to be incorporated into bioassays for prevention and monitoring of heavy metal pollution in Australian oyster beds. The study confirms that proteomic analysis of biomonitoring species is a promising approach for assessing the effects of environmental pollution, and our experiments have provided insights into the molecular mechanisms underlying oyster stress responses.

Ultrastructural localization of highly variable 185/333 immune response proteins in the coelomocytes of the sea urchin, Heliocidaris erythrogramma.

The 185/333 proteins of sea urchins represent a family of highly variable immune response molecules with unknown functions. In this study, we show that 185/333 proteins are expressed by three cell types: amoebocytes, colourless spherule cells and gut-associated amoebocytes. A sub-population of amoebocytes express 185/333 proteins on the membranes of vesicles emanating from the trans-Golgi and which later fuse with the plasma membranes of the cells. The previously uncharacterized gut-associated amoebocytes also show a high level of 185/333 protein expression on their internal vesicles and plasma membranes. Colourless spherule cells contain 185/333 proteins within large spherules (specialized intracellular vesicles). In the presence of bacteria and yeast, the ultrastucture of colourless spherule cells changes and 185/333 proteins disappear. In contrast, 185/333 proteins were not found in the phagosomes of coelomocytes. The 185/333-positive gut amoebocytes were often associated with anuclear bodies, which appeared to incorporate material of microbial origin that was surrounded by 185/333 proteins. The association between 185/333 proteins on gut amoebocytes and anuclear bodies suggests that these proteins may be involved in the phagocytosis of microbes in the gut epithelium.

In vitro effects of noradrenaline on Akoya pearl oyster (Pinctada imbricata) haemocytes.

Exposure to fluctuating environmental conditions in bivalve molluscs can lead to physiological stress and up-regulated production of stress-associated hormones, such as noradenaline (NA). Since environmental stressors have been found to have an immunosuppressive effect on Pinctada imbricata, we investigated the in vitro affects of NA exposure on their defensive haemocytes, focussing specifically on markers of apoptosis. Terminal dUTP nick-end (TUNEL) labelling was used to detect cells displaying DNA fragmentation within tissue exposed to NA. DNA fragmentation was most significant when haemocytes were exposed to 10.0 ng NA/µg protein relative to non-treated controls. Similarly, Annexin V-FITC staining, a marker of early apoptotic events, was evident in cells exposed to 5.0 and 10.0 ng NA/µg protein after 120 min (p<0.05), and haemocyte adhesion to glass slides declined significantly when cells were exposed to 10.0 ng NA/µg protein (p<0.05). A number of morphological and ultrastructural changes in NA-exposed haemocytes were also identified using transmission and scanning electron microscopy. These alterations included chromatin and cytoplasmic condensation, the formation of apoptotic bodies, vacuolisation and blebbing. In NA-treated cells, polymerisation of F-actin was observed around the periphery of the cytoplasm. All of these data suggest that NA induces apoptosis in P. imbricata haemocytes.

Highly variable immune-response proteins (185/333) from the sea urchin, Strongylocentrotus purpuratus: proteomic analysis identifies diversity within and between individuals.

185/333 genes and transcripts from the purple sea urchin, Strongylocentrotus purpuratus, predict high levels of amino acid diversity within the encoded proteins. Based on their expression patterns, 185/333 proteins appear to be involved in immune responses. In the present study, one- and two-dimensional Western blots show that 185/333 proteins exhibit high levels of molecular diversity within and between individual sea urchins. The molecular masses of 185/333-positive bands or spots range from 30 to 250 kDa with a broad array of isoelectric points. The observed molecular masses are higher than those predicted from mRNAs, suggesting that 185/333 proteins form strong associations with other molecules or with each other. Some sea urchins expressed >200 distinct 185/333 proteins, and each animal had a unique suite of the proteins that differed from all other individuals. When sea urchins were challenged in vivo with pathogen-associated molecular patterns (PAMPs; bacterial LPS and peptidoglycan), the expression of 185/333 proteins increased. More importantly, different suites of 185/333 proteins were expressed in response to different PAMPs. This suggests that the expression of 185/333 proteins can be tailored toward different PAMPs in a form of pathogen-specific immune response.

Comparative proteomic analysis of a sea urchin (Heliocidaris erythrogramma) antibacterial response revealed the involvement of apextrin and calreticulin.

Echinoderms evolved early in the deuterostome lineage, and as such constitute model organisms for comparative physiology and immunology. The sea urchin genome sequence (Strongylocentrotus purpuratus) revealed a complex repertoire of genes with similarities to the immune response genes of other species. To complement these genomic data, we investigated the responses of sea urchins to the injection of bacteria using a comparative proteomics approach on a closely related species. In the sea urchin, Heliocidaris erythrogramma, the relative abundance of many proteins was altered in response to the injection of both bacteria and saline, suggesting their involvement in wounding responses, while others were differentially altered in response to bacteria only. The identities of 15 proteins that differed in relative abundance were determined by mass spectrometry. These proteins revealed a significant modification in energy metabolism in coelomocytes towards the consumption of glutamate and the production of NADPH after injection, as well as an increased concentration of cell signalling molecules, such as heterotrimeric guanine nucleotide-binding protein. The injection of bacteria specifically increased the abundance of apextrin and calreticulin, suggesting that these two proteins are involved in the sequestration or inactivation of bacteria.

Immunosuppressive effects of environmental stressors on immunological function in Pinctada imbricata.

This study assessed the effects of mechanical agitation, hypo-saline conditions, and exposure to the air on the Akoya pearl oyster, Pinctada imbricata, focusing specifically on the immunological activity of haemocytes. Both phagocytosis and phenoloxidase activity decreased significantly when oysters were exposed to all three stressors. Transient decreases were also evident in total haemocyte counts after mechanical stress and exposure to air, while significant increases in total haemocyte counts were evident after exposure to low salinity. Acid phosphatase activity increased significantly when oysters were exposed to air. The frequency of granulocytes in the haemolymph increased significantly when oysters were stressed by hypo-saline conditions, whilst the relative frequency of granulocytes did not differ significantly after mechanical agitation or exposure to air. The total protein content of haemolymph increased significantly when oysters were stressed by mechanical agitation and low salinity. These results suggest that fluctuations in environmental conditions affect circulating haemocytes and their cytochemistry, and that the different immunological parameters tested were influenced uniquely according to the type of stressor.

Haemocyte morphology and function in the Akoya pearl oyster, Pinctada imbricata.

The morphology and cytochemistry of Pinctada imbricata haemocytes were studied in vitro. Three distinct blood cell types were identified; hyalinocytes, granulocytes, and serous cells. Haemocytes were classified based on the presence/absence of granules, and nucleus to cytoplasm ratio. Granulocytes were the most common cell type (62+/-2.81%), followed by hyalinocytes (36+/-2.35%), and serous cells (2+/-0.90%). Granulocytes, and hyalinocytes were found to be immunologically active, with the ability to phagocytose Congo red stained yeast. Of the cells involved in phagocytosis, granulocytes were the most active with 88.8+/-3.9% of these haemocytes engulfing yeast. Cytochemical stains (phenoloxidase, peroxidase, superoxide, melanin, neutral red) showed that enzymes associated with phagocytic activity were localised in granules within granulocytes. Based on their affinities for Giemsa/May-Grünwald stain, haemocytes were also defined as either acidic, basic or neutral. Hyalinocytes and serous cells were found to be eosinophilic, whilst granulocytes were either basophilic (large granulocytes), eosinophilic (small granulocytes) or a combination of the two (combination granulocytes). Light, differential interference contrast and epi-fluorescence microscopy identified three sub-populations of granulocytes based on size and granularity; small (4.00-5.00 microm in diameter, with small granules (0.05-0.5 microm in diameter), large (5.00-9.00 microm in diameter, with large granules (0.50-2.50 microm in diameter) and combination (5.00-9.00 microm in diameter, with both large and small granules). These observations demonstrate that P. imbricata have a variety of morphologically and functionally specialized haemocytes, many of which maybe associated with immunological functions.