Enzyme-linked immunosorbent assay (ELISA) used in quantification of reproductive output in the pacific oyster,Crassostrea gigas,in Korea

Polyclonal antibody developed from egg proteins of the Pacific oysters, Crassostrea gigas, was used in quantification of the eggs. Quantity of the egg protein was measured using ELISA and a weight-normalized gonadosomatic index (GSI) was then established. Histology indicated that the oysters collected from Goseong Bay, Korea, in 2000 initiated gametogenesis as early as late February and spawned mostly in late June to early July when water temperature reached 23–25 °C. A second spawning peak was also observed in late August and the spawning activity continued to late October. Oyster egg proteins could be detected by ELISA in all months except March and November; a few oysters collected during December and January contained a measurable amount of eggs. The highest monthly mean GSI, 0.423, was observed in mid-June when the oysters were ready to spawn. The maximum GSI recorded in this study was 0.667. A positive correlation was found between oyster size and fecundity; the number of eggs increased as oyster biomass increased (r²=0.7497). Our data suggest that oysters in Goseong Bay produce 3–196 million eggs during the spawning season. The immunological method developed in this study was sensitive enough to measure a small quantity of eggs and considered to be method of choice for studying bivalve reproduction.     

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.     

Seasonality in the infection and invasion of Marteilioides chungmuensis in the Pacific oyster Crassostrea gigas

The protozoan parasite Marteilioides chungmuensis causes irregular enlargement of the ovary in the Pacific oyster Crassostrea gigas. The parasite invades the oyster through the epithelial tissue of the labial palp, replicates in the connective tissue, and then moves to the gonad, producing spores inside the oocytes. In this study the seasonality and invasion period of the parasite into the host was investigated over a 1 yr cycle. Uninfected 1 and 0 yr old (spat) oysters were placed in an epizootic area every month from July 2004 to July 2005 and September 2005 to March 2006, respectively, and left for 1 mo. Labial palps and gonad were sampled monthly and examined for infection by nested PCR and histological observations. Prevalence of infection detected by PCR was 70% or higher from August to October, but declined sharply in November and reached 7% or lower from February to April. To explain the low detection rate in winter, 1 yr old uninfected oysters were placed in an epizootic area in winter (water temperature: 8 to 10 degrees C) for 2 wk and then transferred to M. chungmuensis-free seawater at 24 degrees C. Although prevalence of infection was ca. 7% before transfer to heated seawater, levels of 87% were detected after 1 wk. After a 3 wk exposure to heated seawater, parasites were found in host oocytes by histological observation. It was concluded that the low prevalence in winter was due to insufficient replication of M. chungmuensis at low seawater temperatures, resulting in levels not detectable by nested PCR, and not to the absence of invasion.     

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.     

REPRODUCTIVE PERIODICITY OF THE SEA URCHIN,TRIPNEUSTES GRATILLA (L.) IN TAIWAN COMPARED WITH OTHER REGIONS

An annual reproductive periodicity of Tripneustes gratilla in northern Taiwan is revealed by both the gonad development stages and the gonad index. The gonads of T. gratilla recovered from spawning in February—March, propagated gametocytes in April—June, came to prematurity in July—August, and matured in September—November. Spawning occurred in October—December. The breeding pattern of T. gratilla over a broad geographical area appears to be related to seasonal changes of sea temperatures.     

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.     

Quantitative assessment of reproductive effort of the Manila clam Ruditapes philippinarum in a lagoon on Jeju Island (Korea) using enzyme-linked immunosorbent assay

We investigated gonad development and reproductive effort (RE) of the Manila clam Ruditapes philippinarum at Jeju Island, Korea. Gonad maturation and RE were determined using histology and an indirect enzyme-linked immunosorbent assay (ELISA). In June 2006, most of the clams (80%) in the lagoon were in the resting stage. Spawning clams first appeared in late July, and most clams spawned from early August to mid-September. The condition index increased gradually from early July to late August, then declined from early to mid-September, suggesting that spawning occurred during this period. The gonadosomatic index assessed by ELISA also increased dramatically from June (0.9), peaked in early August (19.7) then declined from late August to mid-September, indicating that clams at the study site had only one spawning pulse during the spawning period. Spawning at Jeju Island was one month later than Manila clams on the west coast of Korea. The delayed spawning and low RE of the clams could be in part, be explained by lower food availability, as the level of chlorophyll-a recorded in this study was much lower than that found in water from the west and south coast.     

Reproductive cycle of the winged pearl oyster,Pteria penguin (Röding 1793) (Pteriidae) in north-eastern Australia

The spawning cycle of the winged pearl oyster, Pteria penguin, was studied at Orpheus Island, north-eastern Queensland to observe seasonal changes in gonad development, mean oocyte diameter and sex ratio over time. A total of 201 oysters were collected over 10 sampling periods, approximately every 5–6 weeks (mean?=?20 oysters/sample). Histological analysis of gonads showed that spawning activity occurred mainly through the austral mid-spring to late summer (November to March), when mean monthly water temperature ranged between 27.5°C and 29.4°C. There was a primary spawning peak at the beginning of the spawning season in December, followed by a secondary peak in March. Mean oocyte diameter (µm) was highest during the warmer months, ranging from 31.2?±?2.4 in November to 36.2?±?3.2 in March, and sex ratio analysis showed the percentage of females in the population also peaked between 30% and 50% during this period.     

Spawning periodicity of three species of Nodilittorina in Hong Kong

The spawning periodicity of Nodilittorina trochoides, N. radiata and N. vidua from two rocky shores of different wave exposure, Big Wave Bay (more exposed site) and Cape d'Aguilar, in Hong Kong was monitored for one year (November 1994–November 1995). The breeding season was similar for the three species, mainly during the summer (June–September). The spawning duration was 9 months for N. vidua and 7 months for the other two littorinids. Spawning was initiated at different times for the two sites. At Big Wave Bay, spawning occurred between May–November for N. trochoides; March–September for N. radiata and March–November for N. vidua. At Cape d'Aguilar, the spawning period of N. trochoides and N. radiata was similar (April–October), whilst for N. vidua, spawning extended up to December. A decrease in monthly fecundity was recorded during July and August at Big Wave Bay and Cape d'Aguilar respectively. A weak relationship was found between the size of reproductive females and egg production. The seasonal fecundity of N. trochoides and N. vidua was similar at both sites and estimated as 10 000 and 7500 eggs per female respectively. Fecundity of N. radiata varied between sites, with 15 000 eggs and 9000 eggs per female being recorded from Cape d'Aguilar and Big Wave Bay respectively.     

Assessment of Maturity,Reproduction and Reproductive Potentials of Torpedo Scad, <Emphasis Type="Italic">Megalaspis cordyla</Emphasis> (Linnaeus, 1758) from Northern Arabian Sea Coast of Pakistan

Port landing specimens of torpedo scad, Megalaspis cordyla were collected at monthly intervals from August 2013 to June 2014 from the northern Arabian Sea coast of Pakistan. The life history characteristics revealed that torpedo scad has a prolonged breeding season which occurred with two peaks: from May to June and from November to February. The spawning seasonality was also validated with microscopic gonad examination.The highest GSI value was noted in May and January whereas the lowest in November was recorded for both sexes. The age at which individuals of both sexes become 50% mature (t₅₀) was estimated as 2.44 year. Life span (t_max) calculated as 3.96 years for male and females. The size of 270 and 265 mm at which 50% individuals attained sexual maturity (L₅₀) was determined for males and females respectively. The reproductive potential (fecundity)was made from ten ripe females ranged from 29–43.8 cm TL and weighing 220–540 g of IV–V developmental stage was enumerated as 32306–236677 eggs (CV = 2.135) with an average number of eggs 86544 counted per ripe female/season. This study contains fundamental data of an important fish species in the northern Arabian Sea coast of Pakistan. It provides vital information to the fisheries managers for implementation on sustainable use of the resource during spawning season.     

Gonadic cycle of the American oyster Crassostrea virginica (Lamellibranchia: Ostreidae) in Mecoacán, Tabasco, México

The American oyster Crassostrea virginica is exploited along Gulf of Mexico. This resource represents a job source and incomes for fishermen. In Mexico the production is supported by Tabasco state, the first producer. However, the mexican landings of this bivalve had been dropped about 40% last ten years. By 1999, Tabasco presents a unique ban season fishery of oysters. This season was based in evaluation of gonadal development by visual observations of color and texture, larvae and seeds abundance. In 2000, the government set up two ban seasons in terms of evaluation of populations every year, without gonadal analysis. In general, exists different spawning seasons for American oyster, in accordance with the environmental conditions. It is necessary to establish a ban season according to reproductive cycle. This study presents the gonadic cycle for an oyster population of Mecoacin lagoon along a year. It was defined five phases of the gonad development: resting, gametogenesis, mature or ripe, spawn and post spawn. Gametogenesis is present all the year, except December. The spawning activity was detected all year, except July and August. The ripe phase presented maximum values in August and December. It was proposed a modification of the ban seasons from April 15 - May 30, and September 15 - October 30 to March 15 - May 15 and September 1 - October 30, respectively, according to gonadic cycle obtained in this work. This modification would to avoid capture mature organisms (ready to spawn).     

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.     

Spawning frequency, fecundity, egg weight and spawning type of silver pomfret, Pampus argenteus (Euphrasen) (Stromateidae), in Kuwait waters

Silver pomfret, Pampus argenteus, were collected by fishing with drift gillnets on one spawning ground in Kuwait waters during 1998–2000. Fish size frequency, sex ratio, maturation cycle, spawning frequency, fecundity and egg weight were assessed. The length–weight relationship differed between sexes whereby females were significantly bigger than males. Spawning started in mid‐May and continued until early October. During this time the water temperature ranged from 26.0 to 32.8°C, salinity was ? 39.0‰ and water depth ranged between 5 and 12 m. Large females spawned earlier than young spawners and the overall percentage of males during the spawning period was 70.3%. Spawning occurred after 13.00 h, with peak spawning between 15.00 and 18.00 hours during outgoing tide. Mean daily spawning frequency amounted to 63.2%. Spawning activity was found to be associated with the lunar cycle and spawnings were concentrated during the first and third quarters of the moon period, indicating a semilunar reproduction cycle. It was concluded that a female would spawn at least six times during the season. No change was observed in relative fecundity during the peak spawning season (June–August). Average relative batch fecundity was 176.3 eggs g^?1 somatic weight (SW), corresponding to a relative total fecundity of 1058 eggs g^?1 SW, which is 1.5 times higher than estimates obtained from counting the standing stock of oocytes. Bigger fish produced heavier eggs and the egg weight decreased as the spawning season progressed. Based on gonadal cycles, oocyte size frequency distribution and total fecundity, we concluded that silver pomfret is a multiple batch spawner with indeterminate fecundity.     

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.     

Effect of water level fluctuation on shore spawning of Mirogrex terraesanctae (Steinitz), (Cyprinidae) in Lake Kinneret, Israel

Mirogrex terraesanctae (Steinitz), is an endemic, open water, zooplanktivorous fish. It spawns in the shallow littoral (0–50 cm) of Lake Kinneret, from November to May with a peak in mid winter (January-February). Spawning begins shortly after nightfall, when schools of the fish move along the shoreline in rocky regions, and release their milt and eggs. The adhesive eggs are attached to the surface of recently inundated, algae-free stones. A negative relationship exists between epilithic growth and egg density and survival. Algal growth may play a key role in the determination of the spawning depth. Breeding success of M. terraesanctae depends on the rate and extent of rise in lake level which, in turn, determines the availability of a suitable spawning substratum.     

Reproduction of the Arctic copepodCalanus hyperboreus in the Greenland Sea-field and laboratory observations

The seasonal vertical migration of adultCalanus hyperboreus Krøyer and their female reproductive biology have been studied in the central Greenland Sea, based on 7-month coverage during one annual cycle. Females dwelled in surface waters only between April and July. Gonad maturation began after the summer descent into deeper waters between September and October in the absence of food. Breeding was assessed from gonad development and egg production experiments. It started around the beginning of November and was mostly completed by March/April, before the spring ascent. The overwintering depth of females ranged from close to the surface to 3000 m, and mature females had a preference for shallower depths. Males were mostly found during the breeding period, where they inhabited the 500- to 1000-m layer. Female dry weight was highest in August and November and lowest in February. Field observations were supplemented by observations on gonad development and egg production of females, which were collected in June and August and kept in the laboratory for up to 6 months. Clutch size, spawning interval and fecundity were determined at 0°C. Females collected in June were mature earlier. Due to their low dry weight, they developed only small gonads and very few of them spawned. Females from August were much fatter and produced up to 1000 eggs in several clutches deposited at regular intervals.     

Sex-, gametogenesis, and tidal height-related differences in levels of HSP70 and metallothioneins in the Pacific oyster Crassostrea gigas

Pacific oysters, Crassostrea gigas, living at a range of tidal heights, routinely encounter large fluctuations in temperature. We demonstrate that levels of heat shock proteins (HSP) and other stress proteins (metallothioneins, MTs) quantified by ELISA, remained similar in gills, mantle and digestive gland between oysters inhabiting low and high tidal heights. In contrast, endogenous HSPs and MTs levels in gonad changed significantly during gametogenesis. In female gonads, the constitutive form of HSP70 and the MTs increased from immature (or resting) to mature stages (about more than 3-fold) and decreased after spawning. In male gonads, the same expression patterns were observed, whereas proteins levels decreased once fully mature. Females presented higher concentration of HSP70 and MTs than males during the spawning period. No significant difference in HSPs and MTs patterns was found among oysters sampled at low and high tidal heights. We hypothesize that the high level of stress proteins in eggs may increase survival of oyster progeny.     

Mass spawning of Caesio teres (Pisces: Caesionidae) at Enewetak Atoll,Marshall Islands

Synopsis Mass spawning of Caesio teres occurred between March and August, 1983 on a reef emerging from deep water just inside the East Channel of Enewetak Atoll, Marshall Islands. Aggregations and spawning were observed from one day before until three days after the full moon. Spawning aggregations were not observed during the new or quarter moons. Spawning commenced after high tide, when the current began to flow out the pass from lagoon to ocean. The spawning aggregation of close to 1000 individuals migrated to the spawning site. Spawning occurred when the aggregate ascended to near the water's surface. Subgroups dashed horizontally within the aggregate, releasing a highly visible gamete cloud. Predation on spawning adults was not observed. Predation on spawned eggs was noted. The eggs of C. teres are described. Mid-Pacific Research Laboratory, Enewetak Atoll, Republic of the Marshall Islands Present address: Motupore Island Research Station, P.O. Box 320, University of Papua New Guinea, Papua New Guinea     

The reproductive ecology of the invasive Asian paddle crab,Charybdis japonica (Brachyura: Portunidae), in northeastern New Zealand

The Asian paddle crab, Charybdis japonica, native to the northwest Pacific, is an invasive species that has established populations within northeastern New Zealand. Here, we provide a detailed examination of C. japonica's reproductive seasonality and gametogenesis, sizes at physiological maturity, and sex ratios outside its native range. Trapping in the Weiti River Estuary, Whangaparoa (36°38.4′S, 174°43.6′E) from February 2010 to May 2012 indicated a male‐biased population. Reproductive seasonality largely depended on female condition, since males contained ripe spermatozoa year‐round. Female gametogenesis began during late autumn, with oocyte development occurring throughout winter to early spring. Based on the presence of mature gametes during late winter and the appearance of gravid females in early November, mating began during early spring, with spawning in mid‐spring. Spawning coincided with a large drop in the gonad index, and occurred between sea surface temperatures of 17–22°C and 11.75–13.5 h day length. Females remained reproductive for up to 5 months of the year. Histological observation suggested that CW50 (the carapace width at which 50% of the population is mature) for females was 46.39 mm, while CW50 for males could not be determined due to a paucity of immature males. Histological analysis also suggested that females of C. japonica could produce store sperm and multiple broods annually.     

Lemon sole Microstomus kitt in the northern North Sea: a multidisciplinary approach to the early life-history dynamics

Lemon sole Microstomus kitt is a commercially valuable flatfish species that occurs in shelf waters around the northeast Atlantic. Only the most basic life-history information is available for the North Sea. Spawning is generally assumed to occur between early May and October, with a peak between May and August. Lemon sole larvae have been found in the water column in the northern North Sea in winter during standard surveys. Larvae captured in November/December 2016 and January/February 2017 using the International Council for the Exploration of the Seas standard 2 m Midwater Ring trawls (MIK) were analysed to gain a better understanding of the pelagic early life-history stages of lemon sole, especially in relation to the timing of spawning and the dispersal of overwintering larvae. Larval age was estimated from sagittal otolith primary increment counts. The larvae caught in November/December ranged in nominal age from 4 to 45 days post-hatching which suggests that spawning continues into late October and November. Most, but not all, of the larvae caught in January/February were post metamorphosis, and the difference in age between the two sampling dates was consistent with the elapsed time between samplings. The estimated hatching dates confirm that lemon sole spawning extends into late autumn in the northern North Sea, with overwintering larvae in all developmental stages. Drift modelling of eggs and larvae released at historically documented spawning grounds in the northern North Sea suggests that these grounds are also the source for all of the larvae sampled during the 2016–2017 surveys.