Relationship between Marteilioides chungmuensis infection and reproduction in the Pacific oyster, Crassostrea gigas

Marteilioides chungmuensis, a protozoan paramyxean parasite, infects the oocytes of the Pacific oyster, Crassostrea gigas. The effects of infection on the reproductive cycle of C. gigas were investigated over two consecutive years at Okayama Prefecture, Japan. In male oysters, gonadal development began during February/March, maturity was achieved in June and spawning activity extended from July to September. In November and December, male oysters were not seen, probably because their gonads regressed to connective tissue and they transformed into undifferentiated oysters. By contrast, female oysters, in which parasite spore formation occurred, were still carrying oocytes until the following March and the spawning process of female oysters took 5 months longer than that of males in epizootic areas. The prevalence of M. chungmuensis infection increased from July to September, when most female oysters had their spawning period, and declined from October to the following April when oysters were at the spent stage. The prevalence of infection increased again in May of the following year and high prevalence was observed in the following July. When prevalence was compared between oysters of different age classes, higher prevalence was detected in older than in younger oysters. Histological examination showed that infected oysters produced oocytes continuously and spawned repeatedly from October to March, during which period healthy oysters were reproductively inactive. Parasites can infect the oocytes of infected oysters throughout the longer spawning period. These observations suggest that M. chungmuensis extends the reproductive period of infected oysters for its own reproductive benefit.     

Effect of acclimatization on hemocyte functional characteristics of the Pacific oyster (Crassostrea gigas) and carpet shell clam (Ruditapes decussatus)

Most experimental procedures on molluscs are done after acclimatization of wild animals to lab conditions. Similarly, short-term acclimation is often unavoidable in a field survey when biological analysis cannot be done within the day of sample collection. However, acclimatization can affect the general physiological condition and particularly the immune cell responses of molluscs. Our aim was to study the changes in the hemocyte characteristics of the Pacific oyster Crassostrea gigas and the carpet shell clam Ruditapes decussatus acclimated 1 or 2 days under emersed conditions at 14 ± 1 °C and for 1, 2, 7, or 10 days to flowing seawater conditions (submerged) at 9 ± 1 °C, when compared to hemolymph withdrawn from organisms sampled in the field and immediately analyzed in the laboratory (unacclimated). The hemocyte characteristics assessed by flow cytometry were the total (THC) and differential hemocyte count, percentage of dead cells, phagocytosis, and reactive oxygen species (ROS) production. Dead hemocytes were lower in oysters acclimated both in emersed and submerged conditions (1%-5%) compared to those sampled in the field (7%). Compared to oysters, the percentage of dead hemocytes was lower in clams (0.4% vs. 1.1%) and showed a tendency to decrease during acclimatization in both emersed and submerged conditions. In comparison to organisms not acclimated, the phagocytosis of hemocytes decreased in both oysters and clams acclimated under submerged conditions, but was similar in those acclimated in emersed conditions. The ROS production remained stable in both oysters and clams acclimated in emersed conditions, whereas in submerged conditions ROS production did not change in both the hyalinocytes and granulocytes of oysters, but increased in clams. In oysters, the THC decreased when they were acclimated 1 and 2 days in submerged conditions and was mainly caused by a decrease in granulocytes, but the decrease in THC in oysters acclimated 2 days in emersed conditions was caused by a decrease in hyalinocytes and small agranular cells. In clams, the THC was significantly lower in comparison to those not acclimated, regardless of the conditions of the acclimatization. These findings demonstrate that hemocyte characteristics were differentially affected in both species by the tested conditions of acclimatization. The phagocytosis and ROS production in clams and phagocytosis in oysters were not different in those acclimated for 1 day under both conditions, i.e. emersed and submerged, and those sampled in the field (unacclimated). The THC was significantly affected by acclimatization conditions, so the differences between clams and oysters should be considered in studies where important concentrations of hemocytes are required. The difference in the immune response between both species could be related to their habitat (epifaunal vs. infaunal) and their ability of resilience to manipulation and adaptation to captivity. Our results suggest that functional characteristics of hemocytes should be analyzed in both oysters and clams during the first 1 or 2 days, preferably acclimated under emersed rather than submerged conditions.     

Pathogenicity of the protozoan parasite Marteilioides chungmuensis in the Pacific oyster Crassostrea gigas

Marteilioides chungmuensis is an ovarian parasite that causes nodule-like structures to appear on the gonads of female Pacific oysters, Crassostrea gigas. It is known that the prevalence of infection increases in summer and decreases from autumn to spring. To investigate the decrease in prevalence of infection and pathogenicity of the parasite, a biopsy method was developed to detect infected oysters, which were then monitored to calculate the mortality rate. Mortality of infected oysters was recorded monthly and changes in reproductive development observed histologically. Compared with control groups, a significant difference in mortality was observed in infected oysters in September and October. Histological observations showed that infected oysters produced oocytes continuously, even in autumn when healthy oysters were reproductively inactive. This prolonged spawning activity of infected oysters resulted in nutritional wasting and mortality. From December onwards, however, almost all infected oysters survived, though the infection persisted. Infection intensity decreased gradually from December. Histological observations revealed that, in winter, infected oysters released infected and uninfected oocytes through the genital canal. The gonad subsequently degenerated and was replaced with connective tissue, as in normal, healthy spent oysters. The results revealed that prevalence of infection decreased from September to May. It is hypothesised that the decline in prevalence within the epizootic area in autumn occurred because infected oysters died and that the winter decrease was due to recovery from infection.     

Early developmental stages of a protozoan parasite, Marteilioides chungmuensis (Paramyxea), the causative agent of the ovary enlargement disease in the Pacific oyster, Crassostrea gigas

A paramyxea, Marteilioides chungmuensis, causes the irregular enlargement of the ovary in the Pacific oyster, Crassostrea gigas in Korea and Japan. The knowledge about the life cycle of the parasite has been limited to the sporulation stages within the oocyte of oysters. In this study, we used the parasite-specific DNA probes and electron microscopy to experimentally infected oysters in a field and successfully clarified early developmental stages of the parasite. The parasite invaded the oysters through the epithelial tissues of the gills, mantle and labial palps. Extrasporogony repeatedly occurred in the connective tissues by binary fusion. The inner cell of the extrasporogonic stage migrated into the gonadal epithelium, invaded the oocyte to start sporulation.     

Phagocytosis of the protozoan parasite, Marteilia sydneyi, by Sydney rock oyster (Saccostrea glomerata) hemocytes

QX disease is a fatal disease in Sydney rock oysters caused by the protozoan parasite Marteilia sydneyi. The current study investigates the phagocytosis of M. sydneyi by Sydney rock oyster hemocytes. It also compares the in vitro phagocytic activities of hemocytes from oysters bred for QX disease resistance (QXR) with those of wild-type oysters. After ingestion of M. sydneyi, hemocyte granules fused with phagosome membranes and the pH of phagosomes decreased. Significantly (p = <0.05) more phagosomes in QXR hemocytes showed obvious changes in pH within 40 min of phagocytosis, when compared with wild-type hemocytes. Phenoloxidase deposition was also evident in phagosomes after in vitro phagocytosis. Most importantly, ingested and melanised M. sydneyi were detected in vivo among hemocytes from infected oysters. Overall, the data suggest that Sydney rock oyster hemocytes can recognise and phagocytose M. sydneyi, and that resistance against QX disease may be associated with enhanced phagolysosomal activity in QXR oysters.     

Hemocyte parameters of the Pacific oyster Crassostrea gigas a year after the Hebei Spirit oil spill off the west coast of Korea

In marine bivalves, hemocytes support various physiological functions, including immune defense, nutrient transport, shell repair, and homeostatic maintenance. Although the effects of marine contaminants on the immunological functions of bivalves have been extensively investigated, the impacts of oil spills are not well understood. Therefore, we investigated hemocyte parameters in the Pacific oyster Crassostrea gigas 13 months after the Hebei Spirit oil spill (December 2007) off the west coast of Korea. The parameters studied included hemocyte concentration and mortality, relative proportion of hemocyte populations, and immunological functions such as phagocytosis and oxidative activity using flow cytometry. These immune-related parameters in oysters damaged by the oil spill were also compared to control oysters that were collected from an area unaffected by the spill. The flow cytometry study indicated that granulocyte population, phagocytic capacity, and reactive oxygen species production in oysters exposed to crude oil 13 months prior were depressed compared to the unexposed control oysters. Our data suggest that immunocompetence in oysters affected by the oil spill had not fully recovered 1 year after the accident, although more detailed studies on the physiology and disease resistance should be performed.     

Isolation and 18S ribosomal DNA gene sequences of Marteilioides chungmuensis (Paramyxea), an ovarian parasite of the Pacific oyster Crassostrea gigas

To develop sensitive detection techniques with the aim of elucidating the life cycle of Marteilioides chungmuensis, an intracellular paramyxean infecting the ovary of the Pacific oyster Crassostrea gigas, we isolated the parasite at the sporont stage from infected oysters using a freeze-thaw procedure at -20 degrees C and differential centrifugations in discontinuous sucrose and Percoll gradients. DNA was extracted from the isolated sporonts, and a PCR amplicon of 18S small subunit ribosomal RNA gene DNA was partially sequenced. In situ hybridization using 3 parasite-specific probes designed from the obtained sequence successfully detected parasite cells in infected oysters, and confirmed that the sequenced DNA was derived from M. chungmuensis.     

Changes in circulating and tissue-infiltrating hemocyte parameters of European flat oysters,Ostrea edulis,naturally infected with Bonamia ostreae

We assayed European flat oyster, Ostrea edulis, hemocyte parameters, circulating and tissue-infiltrating hemocyte densities, circulating hemocyte type distribution and lysosomal enzyme contents, to possibly relate these hematological parameters to Bonamia ostreae infection. Circulating hemocyte densities were not statistically different between infected and uninfected oysters. In contrast, the number of tissue-infiltrating hemocytes increased with infection intensity suggesting a recruitment process at the site of infection and a possibility for cells to migrate from circulatory system to connective tissues. Lysosomal enzymes were localized mainly in granulocytes both infected and uninfected, and mean of alpha-naphtyl butyrate esterase activity decreased with increasing B. ostreae infection level. The main response observed was a change in hemocyte type distribution between uninfected and infected oysters and greater tissue-infiltrating hemocytes with increased infections. These results suggest that the decrease of circulating granulocytes, and, consequently of some cell enzyme activities may be related with B. ostreae infection.     

Changes in biochemical and hemocyte parameters of the Pacific oysters Crassostrea gigas fed T-Iso supplemented with lipid emulsions rich in eicosapentaenoic acid

The aim of this study was to assess the effect of dietary eicosapentaenoic acid (20:5n-3) on hemocyte parameters such as hemocyte concentration, phagocytosis, and non-stimulated reactive oxygen species (ROS) production in Pacific oysters Crassostrea gigas, as well on proximate biochemical and fatty acid compositions. One-year-old oysters (C. gigas) were fed T-Isochrysis aff. galbana (T-Iso), which is low in 20:5n-3, either alone or with supplements of a lipid emulsion rich in 20:5n-3 at 1%, 10% or 50% (dry weight of the algal ration) for up to 7 weeks. Changes in gill fatty acid composition demonstrated that the lipid emulsion was well ingested by oysters during the dietary conditioning. Biochemical analysis indicated that oysters fed supplements of 50% and, to a lesser extent, 10% lipid emulsions had a higher total lipid content compared with oysters fed other diets, suggesting a more advanced reproductive status for the oysters fed high doses of lipid emulsion. Moreover, some oysters in these two treatment groups spawned during the last three weeks of the seven-week feeding experiment. Lipid supplements had a significant influence on hemocyte concentration, phagocytic index and non-stimulated hemocyte ROS production. After 4 weeks, highest hemocyte concentrations were found in oysters fed on a supplement of 50% lipid emulsion compared with those fed on other diets but the hemocytes derived from these oysters had the lowest short-term phagocytic index. After 7 weeks of dietary conditioning, the ROS production in non-stimulated hemocytes of oysters fed 10% and 50% lipid emulsion declined. These results suggested that 20:5n-3, and perhaps its eicosanoid metabolites, affected oyster hemocyte functions; however, the reproductive status of oysters may also have interfered with the 20:5n-3 dietary effect.     

A study of diagnostic methods for Marteilioides chungmuensis infections in the Pacific oyster Crassostrea gigas

The eggs of the Pacific oyster, Crassostraea gigas, become infertile when infected by the parasite Marteilioides chungmuensis. Histologically, M. chungmuensis infects the oyster oocyte cytoplasm, and the ovaries take on a "lumpy" appearance once infected, which lowers commercial value of the oyster. This has a negative economic impact on oyster farms in South Korea and Japan. In this study, we compared traditional diagnostic methods (histology) with two molecular-based methods (polymerase chain reaction [PCR] amplification and in situ hybridization [ISH]) to identify M. chungmuensis-infected oysters. The efficacy of PCR and ISH to identify M. chungmuensis-infected oysters was compared to that of routine histology in 100 oysters. Thirty infections were identified using PCR and 16 using histology, whereas 31 infections were identified using ISH. The ISH and PCR assays were more sensitive compared to using histology with standard epidemiological methods. We strongly recommend that early parasitic invasion should be monitored with PCR/ISH methodologies as a basis for developing effective diagnostic techniques to identify M. chungmuensis-infected oysters.     

Variability in molluscan hemocytes: a flow cytometric study

Reported variability in numbers and relative proportions of hemocytes in marine bivalves may be related to environmental conditions and laboratory method differences. An automated identification assay, flow cytometry, removes much laboratory bias, but its usefulness is limited because the putative cell types in delineated subpopulations have never been confirmed. The present study was designed to: (1) confirm the identity of oyster hemocyte subpopulations described by flow cytometry, and (2) use flow cytometry in an experimental analysis of potential causes of variation. Light-scatter flow cytometry consistently differentiated three subpopulations in oysters from two mid-Atlantic (USA) sites. Cell sorting and microscopy identified them as granular, small granular, and agranular (hyalinocytes and apparently degranulated) hemocytes. Subpopulation proportions estimated by microscopy and by flow cytometry were significantly correlated (r(2) = 0.27 to 0.50). In a 4-week laboratory experiment, neither temperature (12 vs. 22 degrees C) nor food (fed vs. not fed) had a statistically significant effect on total or differential counts, or on hemocyte viability. Most of the variability was attributable to individual differences and was very similar to that reported for vertebrates. We hypothesize that variability in molluscan hemocytes may be more immediately linked to individual metabolic condition than to ambient changes.     

Molecular and histological identification of Marteilioides infection in Suminoe Oyster Crassostrea ariakensis, Manila Clam Ruditapes philippinarum and Pacific Oyster Crassostrea gigas on the south coast of Korea

The oyster ovarian parasite Marteilioides chungmuensis has been reported from Korea and Japan, damaging the oyster industries. Recently, Marteilioides-like organisms have been identified in other commercially important marine bivalves. In this study, we surveyed Marteilioides infection in the Manila clam Ruditapes philippinarum, Suminoe oyster Crassostrea ariakensis, and Pacific oyster Crassostrea gigas, using histology and Marteilioides-specific small subunit (SSU) rDNA PCR. The SSU rDNA sequence of M. chungmuensis (1716 bp) isolated from C. gigas in Tongyoung bay was 99.9% similar to that of M. chungmuensis reported in Japan. Inclusions of multi-nucleated bodies in the oocytes, typical of Marteilioides infection, were identified for the first time in Suminoe oysters. The SSU rDNA sequence of a Marteilioides-like organism isolated from Suminoe oysters was 99.9% similar to that of M. chungmuensis. Marteilioides sp. was also observed from 7 Manila clams of 1840 individuals examined, and the DNA sequences of which were 98.2% similar to the known sequence of M. chungmuensis. Unlike Marteilioides infection of Pacific oysters, no remarkable pathological symptoms, such as large multiple lumps on the mantle, were observed in infected Suminoe oysters or Manila clams. Distribution of the infected Manila clams, Suminoe oysters and Pacific oysters was limited to small bays on the south coast, suggesting that the southern coast is the enzootic area of Marteilioides infection.     

Morphological and quantitative aspects of nodule formation in hemolymph of the blowfly Chrysomya megacephala (Fabricius, 1794)

Insects manifest effective immune responses that include both cellular and humoral components. Morphological and quantitative aspects of cellular and humoral cooperation during nodule formation in Chrysomya megacephala hemolymph against Saccharomyces cerevisae yeast cells were demonstrated for the first time. The analyses were performed in non-injected larvae (NIL), saline-injected larvae (SIL) and yeast-injected larvae (YIL). The hemolymph of injected groups was collected 0.5, 1, 2, 4, 12, 24, 36, or 48-h post-injection. Morphological aspects of YIL nodulation were investigated using transmission electron microscopy (TEM). Quantitative analyses consisted of total (THC) and differential hemocyte counts (DHC) in all the groups and total yeast count (TYC) in YIL, which were performed in an improved Neubauer chamber. Nodule formation was initiated at approximately 2-h post-injection. Twelve hours after the injection, TEM revealed the presence of an amorphous membrane, at the same time that circulating hemocyte number decreased significantly contrasting the increase of yeast number. Our results showed the ability of C. megacephala hemolymph to perform humoral encapsulation when hemocyte population is insufficient to eliminate the microorganisms, warranting consideration in future investigations on the relative roles played by cellular and humoral elements of innate immunity of this calliphorid.     

Infection Status of Estuarine Fish and Oysters with Intestinal Fluke Metacercariae in Muan-gun, Jeollanam-do, Korea

The source of human infection with intestinal flukes was surveyed in estuarine fishes, including the dotted gizzard shad, common sea bass, common blackish goby, redlip mullet, black sea bream, and oyster collected from Muan-gun, Jeollanam-do, Korea during August and September 2007. Collected fishes and oysters were artificially digested in pepsin-HCl solution and examined under a stereomicroscope. In 36 shads (Konosirus punctatus) and 20 basses (Lateolabrax japonicus) examined, Heterophyopsis continua metacercariae were found in 58.3% and 100%, and their average numbers were 12.0 and 6.3 per infected fish, respectively. In 34 gobies (Acanthogobius flavimanus) examined, metacercariae of H. continua were detected in 79.4%, Stictodora lari in 97.1%, and Acanthotrema felis in 92.1%, and their average numbers were 45.8, 189.3, and 235.3 per infected fish, respectively. In 37 redlip mullets (Chelon haematocheilus), Heterophyes nocens metacercariae were found in 56.8%, Pygidiopsis summa in 94.6%, and Stictodora fuscata in 45.9%, and the average metacercarial densities were 17.4, 31.3, and 35.1 per infected fish, respectively. In 30 black sea breams (Acanthopagrus schlegeli) and 45 oysters (Crassostrea gigas) examined, no metacercariae were detected. From the above results, it has been confirmed that the dotted gizzard shad, common sea bass, common blackish goby, and redlip mullet from Muan-gun, Jeollanam-do, Korea are infected with the metacercariae of heterophyid flukes.     

An entomopathogenic bacterium, Xenorhabdus nematophila, inhibits hemocyte phagocytosis of Spodoptera exigua by inhibiting phospholipase A(2)

Phagocytosis is a hemocytic behavior against bacterial infection. An entomopathogenic bacterium, Xenorhabdus nematophila, inhibits immune responses of target insects and causes hemolymph septicemia. This study analyzed how X. nematophila could inhibit phagocytosis to increase its pathogenicity. Granular cells and plasmatocytes were the main phagocytic hemocytes of Spodoptera exigua determined by observing fluorescence-labeled bacteria in the cytosol. X. nematophila significantly inhibited phagocytosis of both hemocytes, while heat-killed X. nematophila lost its inhibitory potency. However, co-injection of X. nematophila with arachidonic acid did not show any significant inhibition of hemocyte phagocytosis. In fact, hemocytes of S. exigua infected with X. nematophila showed significant reduction in phospholipase A(2) (PLA(2)) activity. Dexamethasone, a specific PLA(2) inhibitor, significantly inhibited phagocytosis of both cell types. However, the inhibitory effect of dexamethasone was recovered by addition of arachidonic acid. Incubation of hemocytes with benzylideneacetone, a metabolite of X. nematophila, inhibited phagocytosis in a dose-dependent manner. These results suggest that X. nematophila produces and secretes PLA(2) inhibitor(s), which in turn inhibit the phagocytic response of hemocytes.     

Effects of the mermithid nematode Ovomermis sinensis on the hemocytes of its host Helicoverpa armigera

Little is known about the mechanism by which mermithid nematodes avoid encapsulation responses of insect hosts. In this study, we investigated the influence of the mermithid nematode Ovomermis sinensis on host Helicoverpa armigera hemocyte number, encapsulation activity, spreading behavior and cytoskeleton. Parasitism by O. sinensis caused a significant increase in the total hemocyte counts (THC) and plasmatocyte numbers of H. armigera. However, in vivo encapsulation assays revealed that hemocyte encapsulation abilities of H. armigera were suppressed by O. sinensis. Moreover, parasitism by O. sinensis changed the spreading behavior and cytoskeletons of the host hemocytes. The results suggested that O. sinensis could actively suppress the hemocyte immune response of its host, possibly by destroying the host hemocyte cytoskeleton. This is the first report of a possible mechanism by which mermithid nematodes suppress encapsulation responses of insect hosts.     

Parasitism by Cotesia plutellae alters the hemocyte population and immunological function of the diamondback moth, Plutella xylostella

Cotesia plutellae, a solitary endoparasitoid wasp, parasitizes the diamondback moth, Plutella xylostella, and induces host immunosuppression and lethality in the late larval stage. This study focused on changes of cellular immunity in the parasitized P. xylostella in terms of hemocyte composition and cellular functions. In third and fourth instar larvae of nonparasitized P. xylostella, granular cells represented the main hemocyte type (60-70%) and plasmatocytes were also present at around 15% among the total hemocytes. Following parasitization by C. plutellae, the relative proportions of these two major hemocytes changed very little, but the total hemocyte counts exhibited a significant reduction. Functionally, the granular cells played a significant role in phagocytosis based on a fluorescence assay using fluorecein isothiocyanate-labeled bacteria. The phagocytic activity of the granular cells occurred as early as 5 min after incubation with the bacteria, and increased during the first 40 min of incubation. The parasitism by C. plutellae significantly inhibited phagocytosis of the granular cells. Plasmatocytes also exhibited minor phagocytic activity. Moreover, plasmatocyte phagocytosis was not inhibited by parasitism. On the other hand, hemocyte-spreading behavior in response to pathogen infection was significant only for plasmatocytes, which exhibited a characteristic spindle shape upon infection. A significant spreading of the plasmatocytes was found as early as 5 min after pathogen incubation and their ratio increased during the first 40 min.An insect cytokine, plasmatocyte-spreading peptide 1 (PSP1) from Pseudoplusia includens, was highly active in inducing plasmatocyte-spreading behavior of P. xylostella in a dose-dependent manner. P. xylostella parasitized by C. plutella was significantly inhibited in plasmatocyte-spreading in response to an active dose of PSP1. An in vivo encapsulation assay showed that the parasitized P. xylostella could not effectively form the hemocyte capsules around injected agarose beads. This research demonstrates that the parasitism of C. plutellae adversely affects the total hemocyte populations in number and function, which would contribute to host immunosuppression.     

Discovery of Gymnophalloides seoi metacercariae in oysters from islands of the West Sea known as the habitats of paleartic oystercatchers

An epidemiological survey was performed to know the infection status of oysters with Gymnophalloides seoi metacercariae in 7 islands of the West Sea known as the habitat of paleartic oystercatchers, Haematopus ostralegus osculans, in Korea. The surveyed areas were Aphaedo (Shinan-gun, Chollanam-do), Jangjado, Sonyudo and Munyodo (Okdo-myon, Kunsan-shi, Chollabuk-do), Yubudo (Changhang-up, Sochon-gun Chungchongnam-do), and Polumdo and Chumoondo (Sodo-myon, Kangwha-gun. Inchon-shi). The oysters collected from Aphaedo, the known endemic focus, were examined monthly from August 1995 to October 1996 for observation of any seasonal variation of the metacercarial density. The average metacercarial burden was 761-2,077 by month, but the seasonal variation of the metacercarial density was not obvious. A total of 54 metacercariae was detected in 63 oysters collected from Yubudo. Out of 30 oysters from Sonyudo, 25 (83.3%) were infected with 1-66 metacercariae (12.6 in average). All of 50 oysters (100%) from Munyodo were infected with 3-162 metacercariae (53.5 in average). Only 4 metacercariae were detected in 100 oysters from Chumoondo. However, no metacercariae were found in 55 oysters from Jangjado and 50 oysters from Polumdo. From the above results, it was confirmed that G. seoi is still highly prevalent in oysters from Aphaedo, and several islands of the West Sea known as the habitats of paleartic oystercatchers are new endemic areas of this fluke.     

Commercial-scale sperm cryopreservation of diploid and tetraploid Pacific oysters, Crassostrea gigas

Cryopreservation of sperm from tetraploid organisms (the possession of four chromosome sets) is essentially unexplored. This is the first cryopreservation study to address sperm from tetraploid Pacific oysters, Crassostrea gigas, and addresses the commercial production of triploid oysters (three chromosome sets). Initial motility, refrigerated storage of undiluted sperm, osmolality of extender solutions, sperm concentrations, equilibration time, and cryoprotectants of propylene glycol and dimethyl sulfoxide were evaluated with sperm from diploid and tetraploid oysters. Unlike most teleost fishes, in which the duration of active motility is typically brief, the motility of sperm from oysters lasts for hours. The present study showed that responses to treatment effects by sperm from tetraploids were different from diploids. The majority of tetraploid experiments resulted in less than 10% motility after thawing and less than 5% fertilization. The highest fertilization obtained for thawed sperm was 96% for sperm from diploid oysters and 28% for sperm from tetraploid oysters. Differential responses to treatments by sperm from tetraploid and diploid oysters may be due to differences in gonadal development. However, the use of cryopreserved sperm from tetraploid Pacific oysters produced 100% triploid offspring by fertilization of eggs from diploid females as determined by flow cytometry of larvae. This study demonstrates that sperm from tetraploid oysters can be collected, frozen, and stored for production of triploid offspring.