Efforts to restore the native oyster in the Chesapeake Bay enjoy enormous public support and have consumed and continue to consume vast, some would argue unreasonable and unjustifiable, amounts of funding. Despite this support the stated goals of restoration efforts are poorly defined and consequently provide no realistic measures of success in terms of time, space, or biomass. Quantitative approaches used successfully in management of and rebuilding plans for other marine and estuarine species have not been appropriately applied. Basic information in oyster population dynamics and ecology has been inadequately appreciated in defining the quantitative problem. Given these limitations it is not surprising that little success has been achieved despite the massive investment. We note a lack of ability to predict recruitment, and limit the ingress and impact of disease. Without control of both of these functions, populations cannot be managed in a self-sustaining rebuilding mode within the footprint that they either currently occupy or formerly occupied. Sustained expansion of that footprint through substrate provision is prohibitively expensive, beyond the limits set by availability of substrate material, and futile in the presence of disease and susceptible oysters. Without attaining a substantially increased and rebuilding population, ecological services will be limited. Water quality impacts will, in reality, be modest, local and seasonal, and still subject to being overwhelmed by periodic storm events. Coherent and rational evaluation of biological limitations will lead to more realistic, and indeed very modest goals for ecological restoration. We must accept the fact that efforts to date to restore native oyster populations have failed and the prognosis for improvement of this situation is continued failure. The argument is proffered that stabilizing the present bed footprint with a realistic and sustainable population and the promotion of aquaculture to increase commercial yield is a more predictable and stable economic investment. Each of these options is consistent with the most realistic ecological outcome and should take priority in future efforts.

The eastern oyster, Crassostrea virginica (Gmelin, 1791) has been in decline along the eastern seaboard, and especially in Chesapeake Bay, for decades because of over-harvesting, disease and declines in water quality and suitable habitat. Eutrophication has also been increasing over the past half century, leading to increases in hypoxia and harmful algal blooms (HABs). The effects of two common Chesapeake Bay HAB dinoflagellates, Karlodinium veneficum, and Prorocetnrum minimum were tested on larvae of C. virginica and the Asian oyster being considered for introduction to Chesapeake Bay, C. ariakensis. When embryos from freshly spawned C. virginica and C. ariakensis were exposed immediately to K. veneficum at 10⁴ cells mL⁻¹, virtually all of the developed larvae were deformed within 48 h in one experimental trial, but not in a second trial in which algae were at a different growth stage. No deformities, and mortalities of <45%, were observed in controls to which a standard diet of the haptophyte Isochrysis was added. When 2-wk-old larvae of both species were exposed to the same HAB species, the effect was a severe reduction in motility with K. veneficum, but with P. minimum only C. ariakensis was affected and not C. virginica. Comparisons were made of the frequency of these HABs in Chesapeake Bay from long-term data analysis and the temporal period of spawning. Whereas both blooms are more common during the summer months, the frequency of blooms of K. veneficum and the period of oyster spawning, June to September, coincide more strongly. To compare spatial similarity, results of a larval transport model were compared with observational data for K. veneficum. This comparison demonstrated a significant overlap in July, particularly in the northern reaches of the Bay. These eutrophication-related HABs thus have the potential to reduce survival of early life history stages of oysters and hence to reduce oyster recruitment. Any reduction in recruitment either spatially or temporally, combined with an overall reduction in sheer numbers of larvae that survive, will make restoration or establishment of significant, self-sustaining populations of natural or introduced oyster species much more difficult.

Landings of eastern oysters (Crassostrea virginica) along the United States East Coast (mainly Rhode Island through South Carolina) increased to nearly 27 million bushels by 1890s, but they then declined by nearly 60% to 11.5 million bushels by 1940 and by almost 99% to 0.35 million bushels by 2004. Though overharvesting usually has been cited as the primary factor for the decline in landings from 1890 to 1940, the principal causes were: (1) a fall in demand for oysters, because consumers became aware that oysters could contain pathogens and competition from other foods increased in markets; (2) three economic depressions; and (3) biological and physical damage to the oysters and their beds (predation, siltation, severe storms, channel dredging, and harvesting by dredges). As a consequence of a huge decline in oyster landings in Delaware and Chesapeake Bays during and after the 1960s because of high oyster mortalities caused by the diseases MSX and Dermo, space became available in the United States national oyster market. The oyster industries of Prince Edward Island, Long Island Sound (Connecticut and New York), and the United States Gulf Coast took advantage of this and increased their production.

Recurrent larval and spat mortality (>80%) occurring in most Sydney rock oyster, Saccostrea glomerata, hatchery-runs since 1980 has prevented reliable commercial hatchery supply of spat and ultimately precluded the industry from accessing stock from breeding programs for faster growth and disease resistance. To overcome larval and spat mortality, the interactive effects of temperature and salinity on early life stages (embryos, larvae, and spat) of S. glomerata were investigated to optimize rearing conditions and thereby improve survival and growth. The early ontogenetic stages of S. glomerata were held at temperatures in the range from 16°C to 30°C and salinities in the range 10–35 ppt. Development of embryos to D-veliger larvae was significantly affected by temperature (P < 0.001), salinity (P < 0.001) and the interaction of these factors. Most rapid embryonic development occurred at a salinity and temperature of 35 ppt and 26°C. Growth of D-veliger, umbonate and pediveliger larvae was also significantly affected by salinity (P < 0.001) and temperature, as was the growth of spat. Salinity had a significant effect (P < 0.001) on D-veliger larvae and spat survival, whereas temperature had a significant effect (P < 0.001) on D-veliger and pediveliger survival. Survival and growth of umbonate larvae were not affected by either salinity or temperature within the range tested. Surface-response plots were also used to examine interactions between salinity and temperature. The optimal temperature for growth of D-veliger larvae was 28°C and for umbonate and pediveliger larvae was 30°C. Greatest length increases for D-veliger and umbonate larvae occurred at the maximum salinity level (34 ppt) whereas the salinity at which this occurred for pediveliger larvae was 26 ppt. Survival of larvae at these optima exceeded 95%. Best spat growth was at a salinity of 35 ppt and a temperature of 30°C. Spat survival at this salinity and temperature combination was 82%. Maximum spat survival was 93% and was measured at a temperature and salinity combination of 23°C and 30 ppt.

A tool in the form of a habitat suitability index model (HSI) for the eastern oyster, Crassostrea virginica, was adapted to evaluate and compare the effects of alternative restoration plans in southwest Florida. A component of a large forecasting model, this tool simulates system response by examining the impact of freshwater inputs into the system. The eastern oyster is a good indicator species for modeling because of its sedentary nature and its susceptibility to natural and artificial changes. In addition, oysters form a complex three-dimensional reef structure, which provides habitat and food for numerous species of fish and invertebrates. The model focuses on salinity, temperature, depth, substrate, and high flow frequency as the particular requirements to determine habitat suitability for the eastern oyster. A geographic information system (GIS) incorporates the oyster HSI model, which includes larval and adult components, to determine responses spatially and temporally to facilitate the decision making process. This paper evaluates four hydrologic and land use scenarios for the C-43 West Basin Reservoir Project. Model results indicate that the Preferred Flow scenario and the future conditions with the Comprehensive Everglades Restoration Plan have higher HSI values then either the existing conditions or the future without the Comprehensive Everglades Plan.

Oyster size and morphology affect individual oyster physiology, reproductive biology, and habitat production as well as population ecological services and availability for commercial harvest. Options for oyster restoration and fishery facilitation for eastern oyster (Crassostrea virginica) populations in the Chesapeake Bay include the use of disease resistant diploid eastern oysters (DEBY strain), triploid eastern oysters, and triploid Suminoe oysters (Crassostrea ariakensis) with the objective of providing a marketable product in a reasonable time frame. Shell height-at-age, growth in shell height in relation to environmental conditions, ontogenetic changes in morphology, and changes in biomass for groups of triploid Suminoe, triploid eastern, and diploid DEBY eastern oysters held at identical grow out conditions for the first two years of their lives were evaluated.

Triploid Suminoe oysters reached shell heights of 76 mm (market size in Virginia of 3 in) at 1.1 y with triploid eastern oysters and diploid DEBY oysters attaining the same size at 1.2 y and 1.5 y, respectively. Increases in shell height were positively correlated with water temperature and salinity with the largest increases in shell height typically occurring in warmer months. Holding density significantly affected ratios of shell height (SH) to shell width (SW) and SH to shell inflation (SI) for all three oyster populations. Oysters at lower densities showed a decrease in SH:SI ratio indicative of increased cupping as well as a reduction in SH:SW indicating a trend toward more discoid or rounded form. Tissue dry weight (g) and ash free dry tissue weight (g) increased nonlinearly with size within each population and were statistically different across the three populations examined. Triploid Suminoe oysters had higher tissue weights than either triploid or diploid DEBY eastern oysters of similar ages. Both triploid eastern and Suminoe oysters had higher tissue weights than diploid DEBY oysters of similar age. Observed differences in growth rates and morphology between these groups of oysters affect both the ecological services they provide (filtration rates as well as habitat) as well as their fishery potential (time to market size).

Summer mortalities have been observed in French shellfish areas (including Normandy) since 1994, but origin of this syndrome remains unclear and is suspected to be caused by a combination of several interacting extrinsic (biotic and abiotic) and intrinsic (genetic, physiological, immunological) factors. The French research program, MOREST aimed to identify the origin of oyster summer mortality along the French coast, focusing on the interactions between oysters, their pathogens, and the environment. The present study analyzed spatio-temporal variation in growth, condition and mortality in spat, and half-grown and market-sized oysters reared from February 2000 to October 2003 at six stations within two different areas in the Bay of Veys, Normandy: Grandcamp (GR), and Gefosse (GE) that is more estuarine. These biological parameters were compared between years, age groups, and areas. Results showed that shell growth was significantly lower in the station highest on the shore and similar in the four other sites, whereas tissue growth and condition index were higher in the Gefosse area. Results also showed large interannual, interage, seasonal and spatial variation in oyster mortality. In 2001, mortalities were markedly higher than in other years and all batches and sites were affected by high mortality rates. Moreover oysters suffered much higher mortalities in their second and third years than as spat, and the difference between age classes was accentuated when mortality was high. Increases in mortality occurred when the gonad was most extensively developed and the peak coincided with the spawning and postspawning periods when gonad volume began to decrease. Spatial variability showed that mortality was higher in Gefosse than Grandcamp. Chronology and spatial variations in mortality highlighted the importance of timing and confirmed that mass mortalities may be closely linked to reproduction. The risk seems to be associated with high reproductive effort, partial spawning, and/or slow gonad resorption. Spatial variation also suggests that the study sites experience varying degrees of stress caused by biological changes and probable differences in water quality reflecting the influence of freshwater input in Gefosse where mortality is higher.

The power of sclerochronology as a tool for interpreting the life history of aquatic organisms and the marine environment they inhabited lies in the occurrence of periodic and repeating structures within the skeletal parts of these organisms. Previous work examining the internal growth patterns of northern quahog (=hard clams), Mercenaria mercenaria (L.), along the Atlantic Coast has established their periodicity; however, the two existing descriptions of the annual growth pattern for hard clams from Narragansett Bay, RI are not consistent with one another. One study identified a translucent slow growth band in the summer and the winter, whereas the other study identified a translucent slow growth band only in the winter. To facilitate future sclerochronological analyses using Narragansett Bay hard clams, variations in the existing descriptions of the annual growth pattern were clarified. We found high variability in the annual growth pattern of M. mercenaria collected during the summers of 2005 and 2006. In approximately half the mature individuals observed, the winter growth breaks were separated by three bands, an opaque band of rapid growth in the spring, a translucent slow growth band in the summer, and a second opaque band of rapid growth in the fall. As these individuals approached senility, the pattern became compressed into two seasonal bands, a single opaque band of rapid growth in the spring and a single translucent band of slow growth encompassing both summer and fall. Growth in the remainder of the samples exhibited only two seasonal bands throughout the mature and senile period, an opaque band of rapid spring growth and a translucent band of slow growth throughout the summer and fall. To confirm the patterns observed, we analyzed δ¹⁸O from a subsample of shells. After placing the earlier descriptions and the current description of growth in Narragansett Bay hard clams in chronological order, it appears as though the changing environment of Narragansett Bay was reflected in the growth pattern of these clams. This study emphasizes the importance of validating annual growth patterns even in a region where the periodicity of the species has been confirmed. Changes in the marine environment may alter the patterns in the skeletal structures of aquatic organisms as well as their growth rates.

Increased numbers of benthic predators, especially crabs, have been proposed as a factor contributing to the decline of hard clams (Mercenaria mercenaria Linnaeus, 1758) in Great South Bay, NY. The long-term trend in benthic predators in this system was examined using observations on the distribution and abundance of predators that have been collected by the Town of Islip, NY as part of an annual survey of hard clam populations. The survey began in 1978 and extends to the present and provides concurrent observations of habitat (sediment type, and presence/absence of eelgrass), and hard clam size-frequency distribution and abundance. Predator type and abundance were reported from 1978 to 1981 and 1991 to 2003, which represents one of the most comprehensive benthic predator data sets currently available for any estuarine system. The annual averages of predator abundance in the survey area primarily show interannual fluctuations in abundance. Xanthid crabs (mud crabs, primarily Dispanopeus sayi Smith, 1869) were the numerically dominant predator in the system; blue crabs (Callinectes sapidus Rathbun, 1895) appeared in the late 1990s. Hard clam abundance has declined by 44% since the early 1990s. An Empirical Orthogonal Function (EOF) Analysis of the predator and hard clam data sets showed that fluctuations in predator abundance are: 1) mostly in phase over the survey region and 2) dominated by year-to-year fluctuations in abundance. The EOF results for the hard clams show that hard clam abundance fluctuations are: 1) in phase over the survey region and 2) dominated by a decreasing trend in abundance over the time series. The primary EOF modes essentially were uncoupled, which implies no strong predator-prey interactions between the predators and hard clams. By inference, increasing predator abundance does not appear to be a primary factor producing the long-term decline in hard clam populations. Predation pressure per recruit may still have increased because of declining hard clam population abundance and the concomitant decline in recruitment.

To test the effects of habitat fragmentation per se on the survival and growth of the northern quahog (Mercenaria mercenaria Linné), we stocked juvenile quahogs in 10 seagrass patches (Halodule wrightii Ascherson) ranging in size from 0.5–1,750 m² for two consecutive months during the summer of 2004. As a control, additional quahogs were stocked in cages located in seagrass patches that spanned the same size range. We tested for changes in shell growth, somatic growth, and mortality between caged and non-caged treatments and we estimated the strength of linear relationships between all three measures of growth and mortality with patch area, perimeter, perimeter: area ratio, seagrass aboveground biomass, and epiphyte biomass. Shell and somatic growth were each significantly greater in cages, whereas mortality was significantly less than in uncaged treatments. There were no significant relationships between clam mortality and either patch perimeter, seagrass above ground biomass, or epiphyte biomass, but during July only there was a negative relationship between clam mortality and patch area. Patch area, perimeter, perimeter: area ratio, and seagrass aboveground biomass each had some significant influence on either changes in shell height, length, or somatic growth; but this influence varied between the two trials. Our data suggest that the influence of habitat fragmentation per se declined from June to July, which may the result of increasing predator densities and changes in seagrass patch characteristics.

Along the mid-Atlantic coast of the United States in numerous areas where the water is shallow enough for light to reach the bottom, heavy spring-summer growth of drift seaweed (macroalgae) covers sessile benthic fauna for periods of days to weeks. Coverage for such prolonged periods can exert sublethal effects and eventually lead to death. This study examines the effects of seaweed coverage on body weight, relative abundance, and mortality in an economically important bivalve Mercenaria mercenaria Linnaeus (hard clam) within the Delaware Inland Bays, a shallow estuary representative of systems in which the species occurs and where mortality events have been observed in recent years. Mean body weights (dry weight) were compared between five groups of clams (80–90 mm shell length), taken from two paired sites (each of which had adjacent areas of heavy and light seaweed), and one seaweed-free site during September of 1999. Heavy seaweed density persisted into mid-to-late summer and the groups of clams from these sites had the lowest mean weights. Both heavy seaweed areas also had low abundance of live clams and numerous recently dead clams, identified as such by their shells being still hinged together. Clams at heavy seaweed density sites were consistently out of the bottom, indicating stress. No hinged-dead were found at either of the light seaweed areas and clams were always buried as normal. These results confirm that prolonged coverage by heavy density mats of drift seaweed affects M. mercenaria detrimentally, and document a negative result of human-accelerated eutrophication in coastal waters. The findings emphasize the importance of good management of nutrient loads entering these and other similar shallow estuaries such that the ecological benefits of moderate seaweed growth are retained, whereas extremely dense growth that harms clams and other fauna, creating a public nuisance, is minimized.

Aquacultured hard clams (Mercenaria mercenaria) from Florida were examined for Perkinsus spp. using culture in Rays Fluid Thyioglycollate Medium (RFTM) and PCR. Clams were negative for P. marinus-specific PCR, but low-level infections were observed by RFTM culture and by genus-specific PCR. In contrast, P. marinus was frequently detected in wild oysters, indicating possible host specificity for this parasite.

This paper examines patterns of larval clam settlement on soft-bottom intertidal plots in British Columbia, Canada relative to predator netting (used in beach culture of clams) and adult populations. We studied four sites over two years. Each site had a netted and nonnetted plot where level of clam recruitment was compared. Density (spat/m²) of early recruits was measured for each plot at each site. We found that year (P = 0.0001) was a significant factor, and netting was weakly significant (P = 0.064) in influencing early recruitment of clams. Netted plots also contained high densities of adult clams. Adult filter feeders have been presumed to decrease larval settlement by filtration of recruits. We found that at lower adult density, the relationship between density of spat and density of adults was negative (as predicted). However, at higher densities of adults (450/m²), this relationship no longer held and spat density increased slightly with increase in adult density. Neither presence of netting nor extremely high adult density prevented recruitment of clam larvae, and in plots with nets and high numbers of adults there remained a strong level of recruitment. Our results showed recruitment of bivalve larvae is variable annually and also varies among sites, highlighting the importance of adequate replication of sites and samples to ensure accurate reflection of recruitment patterns. We also found that other factors, netting and adult density, can influence the level of recruitment. Further research is needed to determine whether the magnitude of influence will translate into changes in ultimate population patterns.

A study was conducted in the Îles de la Madeleine (Gulf of St. Lawrence, Canada) to evaluate if it is possible to predict the optimal collector deployment period to reduce the abundance of undesirable species and maintain a high collection of scallop spat. The timing of spat collection was characterized by weekly deployment of spat collectors for one-week periods. These collectors were placed at 2, 5, and 8 m above the bottom to represent the settlement range on commercial line. Other test collectors were deployed weekly between the third and eighth weeks after the onset of spawning at 2 m above the bottom and then were all retrieved in December, June, and October to evaluate cumulative collection for these six different deployment periods. The first peak of Mytilus edulis and Hiatella arctica occurred in September before the Placopecten magellanicus peak. Weekly spat monitoring could be useful to identify when those peaks occur. As a result, growers can delay the collector deployment until after these peaks. The weekly spat collection monitoring appears effective to identify peak settlement of scallop and undesirable species and could be used as a husbandry practice to help growers determine the optimal time to deploy their collectors. Collectors deployed 5–6 wk after spawning was initiated (September 29 and October 6) provided the highest abundance of scallops one year later, at the usual commercial retrieval period. The scallop abundance observed in collectors deployed on October 6, 2003 and retrieved in October 2004 was of 52% of the initial numbers found in December 2003 whereas for mussels, losses were of about 69% over the same period. The abundance of hiatella and anomia increased between December and the following June and decreased afterwards.

The Patagonian scallop Zygochlamys patagonica occurs on the SW Atlantic shelf of South America between 36°S to 55°S in beds at depths around 100 m. Stable oxygen and carbon isotope ratio analysis in scallops from four large beds (Uruguay, 36°17′S, Reclutas, 39°20′S, Tango B, 42°30′S, and Beagle, 55°10′S) in combination with condition indexes and oceanography date strongly suggest that shell growth increments in this species are formed annually. Most translucent growth bands coincide with low values of both δ¹³C and δ¹⁸O (i.e., they are formed at times of high remineralization activity and of higher water temperature). This pattern is consistent throughout the distributional range. Given the specific Argentinean shelf oceanography, higher temperatures at this depth occur during austral autumn—beginning winter in the region of Reclutas and Tango B but during summer–autumn at the northern and southern limit of the distributional range. Year-round investigations at Reclutas beds indicate that translucent shell growth bands coincide with a pause in somatic growth during austral winter, when energy investment is shifted from somatic growth to gonad development.

We examined settlement choices of Pacific calico scallop larvae (Argopecten ventricosus = circularis, Sowerby II, 1842) on eight substrates including live black murex snails (Hexaplex nigritus = Muricanthus nigritus, Philippi, 1845), an endemic neogastropod of the Gulf of California and a common predator of A. ventricosus. We found that A. ventricosus settled on black murex snails with epibiota on their shells in higher densities (6 larvae/cm² ± 1.0) when provided a variety of settlement substrates (P value < 0.0001). In a second experiment, we investigated factors that may contribute to settlement of A. ventricosus on black murex snails such as (1) substrate complexity of murex shells, (2) substrate complexity of murex egg cases, (3) chemical cues from murex snail mucus, and (4) presence or absence of epibiota on black murex snails. Argopecten ventricosus settled in greater densities on black murex snails with epibiota on their shells (8 larvae/cm² ± 1.5) than other substrates (P value < 0.0001). Larval settlement was 4 larvae/cm² (SE = 0.8) on cleaned murex snails where epibiota had been removed, but settlement was only 0.2 larvae/cm² on murex shell fragments, suggesting that the complexity of the shell alone is not what attracts scallop larvae to settle on black murex snails. Settlement was always much higher on living snails than on other substrates which had no more than 1 or 2 larvae/cm². Presence of epibiota on the shell appears to be an important factor for settlement of scallops, and it is probable that scallop larvae do not perceive murex snails as a potential predator but as reef structure.

Large-scale, open, microalgal feed cultures for hatchery and nursery production of marine invertebrates inevitably becomes contaminated with various microbes that can affect productivity and usability of the harvested biomass. In the Greenhouse for Research on Algal Mass Production Systems (GRAMPS) at the NMFS Laboratory in Milford, CT, cultures of Tetraselmis chui (PLY429) often become contaminated with a cyanobacterium; preliminary observations suggested that juvenile bay scallops, Argopecten irradians irradians showed reduced performance when the feed culture became contaminated with this cyanobacterium. We isolated a cyanobacterium from a contaminated culture of PLY429 and conducted a feeding study to determine if this isolate affects survival and growth of juvenile bay scallops, either alone or in combination with PLY429, thereby simulating feeding of a contaminated culture. Bay scallops were given a diet of either 100% PLY429, 50% PLY429 with 50% cyanobacteria, 100% cyanobacteria, or starved. There was 100% mortality of bay scallops by week 3 when they were starved, with a significant difference in survival between diets (P < 0.01). At 6 wk the scallops fed only the cyanobacterium had 63% survival, 93% survived in the mixed diet, and 98% survived when fed 100% PLY429. The net growth of bay scallops on the different diets was also significantly different (P < 0.01) with scallops fed 100% PLY429 having the highest shell-growth rate of 198-μm scallop⁻¹ d⁻¹, and growth rates of 82-μm scallop⁻¹ d⁻¹ on the mixed diet, and 65-μm scallop⁻¹ d⁻¹ for the cyanobacterial diet. These findings suggest that the cyanobacterium will not cause instant mortality, but it will not support sustained survival and growth over time scales of weeks.

The complete ontogeny and postlarval growth of Modiolus capax are described for the first time. Spawning was easily achieved by a gradual increase in water temperature (1°C/h) from 26°C to 31–32°C after exposing the breeders to air for 6 h. The veliger stage (103 ± 8 μm) was reached 23–24 h after fertilization and the low umbo stage (165 ± 9 μm) on the fifth day of culture; growth thereafter was temperature-dependent. At 28.5 ± 0.5°C, competent pediveligers (275 μm) were first observed on the tenth day of culture and on the twelfth day at 26.5 ± 0.5°C, but survival was twice (42%) that obtained at 28.5°C. Most larvae settled between 290 and 310 μm; metamorphosis took 3–4 days. For settlement, several suspended artificial substrata were tested; ropes of twisted anchovy fishing nets captured the larger percentage. However, the use of a downwelling system was the most efficient strategy for the settlement of M. capax over artificial substratum. After metamorphosis, the shell hinge slowly moved toward a more posterior position, and at 440 μm the disconch had adventitious serrated hairs. Seed growth was exponential and temperature-dependent. At 28.0 ± 0.5°C, growth ranged from 26–127 μm day⁻¹, and from 24–63 μm day⁻¹ at 24.8 ± 1.7°C. In both cases, survivorship from pediveliger to seed was approximately 80%.

For the first time since 1990 the Limfjord, which is the main Danish production area for mussels, was partly closed for a period of 14 wk during autumn 2006 because of presence of DSP toxins in blue mussels. The causative organism was Dinophysis acuminata. Only the okadaic acid (OA) and OA esters were observed. The maximum concentration level of approximately 600–700 μg OA equivalents kg⁻¹ was reached at approximately 5,000 cells l⁻¹ of D. acuminata. A further increase in the concentration of D. acuminata did not lead to any further increase in the concentration of OA in the blue mussels. The temporal development in the concentration of D. acuminata cells in the water was in good accordance with the development in the content of OA in blue mussels on a weekly basis. The maximum concentration of D. acuminata during the bloom was 14,000 cells l⁻¹ and the maximum concentration of total OA equivalents in the raw blue mussels was 706 μg/kg. Single cell toxicity measurements showed a mean content of 31 pg OA cell⁻¹ (min 19; max 72 pg OA cell⁻¹). The impact of the concentration procedure was investigated using a range of volumes filtered from 0,2 l–10 l filter⁻¹. Toxins were lost during the filtration process as a response to amount of water concentrated on each filter. The relationship between the volume filtered per filter and the content of OA observed in the cells fitted well the equation y = 32.0·e^−0.12·x, where y = OA cell⁻¹ (pg OA cell⁻¹) and x = volume of water filtered per filter (l). The OA content per cell measured when concentrating only 0.2 L per filter was a good measure of the theoretical content of toxins calculated in a “no handling” case. The influence of heat-treatment on the concentration of DSP toxins in mussels was investigated. Results of OA concentration in paired samples of raw and heat-treated blue mussels showed a 1.8 times higher concentration in heat-treated mussels than in raw blue mussels. In most official monitoring programs raw mussel tissue is used for the analysis of content of DSP toxins. However, most shellfish are eaten cooked or steamed so heat-treated tissue seems to be the most relevant matrix to analyze. This should be recognized in relation to future risk assessments to establish permissible levels of DSP toxins in shellfish and in relation to the control of the permissible levels.

Cultivated mussels may be subjected to heavy predation by several predators including the shore crab Carcinus maenas, and crab predation can result in substantial losses from commercial fisheries. In the present study methods were developed to quantify the effects of crab predation on mussel beds, with the aim of estimating losses of mussels to crabs in the mussel fishery located in the Menai Strait, United Kingdom. There were significant linear relationships between crab carapace width and the number and size of mussels consumed. Increases in the mean length of mussels presented to crabs resulted in an exponential decline in the number of mussels consumed. Consequently, there was a similar decline in the predicted losses of mussels during the cultivation process as mussels increased in body size. It was estimated that C. maenas consumed 10% of seed mussels laid in the Menai Strait during their growth to a marketable size. Although crab abundance and size, and seawater temperature substantially influenced the number of mussels eaten, the size of mussels was by far the dominant factor in determining losses. Therefore, efforts to control crab predation should focus on the early stages of the cultivation process and on areas where the smaller, more vulnerable, mussels are present. The coefficients and formulae presented here could be applied to other areas of Mytilus edulis cultivation subject to predation by C. maenas.

The northern abalone, Haliotis kamtschatkana, fisheries in British Columbia (BC) were closed in 1990 because of substantial declines in the stock biomass. Abalone biomass has remained low since the fishery closure. We used abalone survey data and established growth models to study the stock-recruitment (SR) relationships, estimate mortality rates, and simulate population growth trajectories at various mortality levels for abalone populations in the Queen Charlotte Islands and the Central Coast of BC. The fitted SR curves were flat and near linear, indicating a lack of productivity in the abalone populations. At low spawning stock biomass (<0.05 kg/m²) the SR relationships appeared to be density independent. Annual mortality rates (Z) were estimated to be 0.29–0.36, which included natural mortality and poaching rates. Simulation studies showed that abalone population growth was sensitive to mortality rates. The abalone populations would be sustainable with Z around 0.25, and would increase or decrease with lower or higher Z, respectively. To rebuild the northern abalone populations, measures need to be taken to minimize or eliminate poaching to reduce mortality rates.

The intertidal purple snail Plicopurpura pansa was intensively exploited for “Tyrian Purple,” leading to declining populations and prohibition by the Mexican government of commercial exploitation. A probable cause for the decline is the difficulty of the snails to reattach themselves fast enough before being washed away by the high impact waves during high tides after being removed from the rocks to obtain ‘ink.’ To assess this hypothesis, six experiments were conducted at two rocky beaches in the Baja California Peninsula. Total mortality rates were estimated by a recapture probability model. Mortality rates ranged from 6.34 y⁻¹ to 14.23 y⁻¹ at Playa Cerritos and 5.04 y⁻¹ to 7.79 y⁻¹ at Punta Perico. Male mortality rate values were lower than females. These rates show high vulnerability of purple snail after being removed from rocks. For management and conservation of snail populations, it is not sufficient to handle the snails with care and to “milk” them only periodically. Detachment from the rocks seems to be the main reason for high mortality and should be prohibited.

The 1995–1998 histopathology data from NOAA's Mussel Watch Program were analyzed to: (1) document the occurrence of parasites and pathologies in sentinel bivalves during the 1995–1998 time period, (2) describe and compare the geographic distribution of these parasites and pathologies between different bivalve species and between different geographic regions, and (3) evaluate trends in parasite taxon richness. Parasite taxon richness was higher in oysters than in mytilids and dreissenids. Parasites having higher prevalences in a given host taxon routinely also had higher infection intensities. When different geographic locations were compared, the same trends occurred much more rarely. Oysters were more heavily infected based on total parasite body burden than mytilids, and the frequency of hosts with at least one parasite was higher. Excluding the numerically-dominant gregarines, however, removed the differential between oysters and mytilids, with the exception of East-coast mytilids that were more frequently parasitized than East-coast oysters. Dreissenids had lower prevalence and infection intensity for all parasites than the other host bivalve taxa. Though cestodes offer a discrepancy, most of the more common oyster parasites were unicellular, whereas most of the more common mytilid parasites were multicellular. On the average, parasite distributional patterns along a stretch of coastline were more often clinal in nature in mytilids, in that prevalence and infection intensity tended to change gradually over relatively large distance scales, and more bounded in nature in oysters, in that prevalence and infection intensity tended to change more sharply over shorter distance scales. Latitudinal trends were diametric opposites on the two northern coasts. More parasites occurred in mytilids from northern bays of the East coast, whereas fewer occurred in mytilids from northern bays of the West coast. Mytilids far exceeded oysters in the incidence of pathologies, including digestive gland and gonadal abnormalities and hemocytic infiltration. On the West coast, the vast majority of these pathologies occurred in mussels of the Mytilus edulis complex rather than M. californianus. All pathologies were more common in mytilids from the northeast coast than in West-coast mytilids. Indeed, discounting the gregarines, northeast coast mytilids combined the highest instances of pathologies with among the highest parasite body burdens of any bivalve taxon and coastal area combination in the Mussel Watch program.

Host specificity, extremely high prevalence and infection intensity, and easy sampling make the mantle-cavity ciliate Conchophthirus acuminatus a very convenient model to address numerous fundamental questions relating to symbiosis and commensalism. The acquisition of food by the ciliate as a result of Dreissena filtration activity is probably the basis of the symbiosis, with additional benefits to C. acuminatus being shelter, provision of oxygen, and dispersal. The number of C. acuminatus in a Dreissena population depends on the mussel's size-frequency distribution as there is a direct correlation between Dreissena size and infection intensity. Lack of a correlation between host density and commensal infection intensity may indicate that D. polymorpha and C. acuminatus have a different environmental optimum. Zebra mussels of a given length in each population may have their own carrying capacity of C. acuminatus infection intensity. Upon reaching this carrying capacity, a symbiont population may have density-dependent feedback mechanisms, which slow its reproduction rate within a host and/or increase its emigration from the mussel to maintain an optimal density within its host. Massive emergence of ciliates into open water may be synchronized with a mass occurrence of recently settled Dreissena juveniles to maximize the infection.

An unusual mortality event in the fall of 1999 was observed in Long Island Sound lobsters resulting in the reduction of up to 99% of lobster landings. The die-off corresponded in time with the application of pesticides for the control of mosquitoes that carried West Nile virus, an emerging disease in North America at that time. To determine the possible implications of pesticide application as a direct or contributing factor in the die-off, studies were conducted to determine the effects of experimental exposure to sumithrin on the health of lobsters. Lobsters were exposed in 20-gallon tanks for 5-d or 28-d, and the direct toxicity, as well as sublethal effects on the immune system was determined. The 96-h LC50 for sumithrin upon single exposure, as well as the 28-d LC50 for sumithrin upon repeated exposure, were greater than 1.0 μg/L, the highest concentration tested. No modulation of immune function was detected after sumithrin exposure for either the single or repeated exposures. Water concentrations of sumithrin decreased rapidly over the 5-d single sumithrin exposure, with a half-life of less than 2-h, with similar decay rates for all concentrations tested. Overall, this study showed that sumithrin degrades rapidly in salt water and did not exert significant toxicity in lobsters (it neither killed lobsters nor induced immunotoxicity) at the concentrations tested. It is unlikely that sumithrin significantly contributed to the 1999 lobster die-off in Long Island Sound.

The reproductive season and first-maturity size of the blue lobster Panulirus inflatus were determined at the “Bahía de Loreto” National Park, Gulf of California. Two hundred fifty-six females were analyzed, finding that the onset of gamete development took place in February. The incubation season started in April and ended in November. The highest percentage of ovigerous females was observed in October (88.9%). Histological analysis of gonads revealed two peaks of gonad maturity, in May (75%) and August (75%). Temperature was positively correlated to the percent ovigerous females (r_s = 0.85, P < 0.05) and with the percent ripe females (r_s = 0.73, P < 0.05). The individual first-maturity size was 49.5 mm CL, whereas at a population level it was 80 mm CL, this being proposed as the minimum fishing size. The incubation period was estimated at 2–3 wk, and the histological evidence suggests that each female may spawn more than once and may undergo more than one incubation period during the reproductive season. The reproduction pattern at the Bahía de Loreto National Park in the Gulf of California is discontinuous, likely because the environmental conditions in this zone are more variable than in tropical regions. This feature should be taken into account by the Mexican government when regulating the exploitation of this resource in the study area.

Anti-lipopolysaccharide factors have been described in arthropods, including penaeid shrimp. In this study, we characterize six antilipopolysaccharide-coding clones isolated from a cDNA library from L. vannamei hemocytes. Three different isoforms (named ALFLv1, ALFLv2 and ALFLv3) are observed, based on three nucleotide differences that produce three changes in amino acid sequence (Val11Ala, Val12Ala, Arg83Lys). Other single differences in nucleotide sequences were also noted, but they do not change the translated product. Considering that the signal peptide cleavage site occurs between Ala25 and Gln26, the substitutions Val11Ala and Val12Ala are not present in the mature protein. The mRNA steady state levels of ALFLv3, but not for ALFLv1 or ALFLv2, were up regulated by Vibrio alginolyticus inoculation. Thus, the differences among penaeid ALFs seem to be associated with the Arg83Lys substitution and its expression under bacterial inoculation.

Changes in protein and carbohydrate content during larval development of Lytechinus variegatus were measured under two diet conditions, low concentration (600 algal cells ml⁻¹ day⁻¹) and high concentration (6,000 algal cells ml⁻¹ day⁻¹) to determine the larval stage at which these proximate constituents showed significant changes under different conditions of food availability. In terms of morphology, larvae under the high concentration diet developed fully and metamorphosed after 30 days, whereas under the low concentration diet, some larvae developed up to the 4 arm stage, and some ceased to develop at the 8 arm stage. No significant differences were found in the percentage of larval survival up to day 24 with both treatments (high: 77 ± 18%, low: 66 ± 24%). With the high concentration diet, protein and carbohydrate content per larvae remained relatively constant through day 17 after fertilization, through the 8-arm stage, and then significantly increased by day 20 coinciding with the first appearance of the rudiment stage, remaining high by day 24, coinciding with the first appearance of pedicellariae. Despite the morphological differences between the larvae at both treatments, no significant differences were found in the protein and carbohydrate content per larvae up to day 24. During normal development with a high concentration diet, growth of the pluteus larva seems to be primarily an increase in dimension of the feeding structures, the arms, that requires little production. After full feeding capacity is reached at the 8-arm stage, production increases with development of the rudiment and the pedicellariae.

Pacific whiteleg shrimp (Litopenaeus vannamei, Boone, 1931) was first introduced to Thailand for aquaculture in the late 1990s as an alternative to a native shrimp species, black tiger prawn (Penaeus monodon, Fabricius, 1798). We documented the presence of L. vannamei in the Bangpakong river system, an important watershed in eastern Thailand with a high density of shrimp farms. This paper is a part of a larger study to evaluate potential ecological consequences of introduced L. vannamei. During January–November 2005 (three sampling periods), we sampled wild marine shrimp with commercial shrimp nets (6 m wide × 5 m deep × 25 m long with 2.5 cm mesh) at four sites within the Bangpakong estuary. Results indicated that L. vannamei were present at least once at all sampling sites during the study. Proportion of L. vannamei relative to all Penaeid shrimp per net in the Bangpakong estuary was 0.005 ± 0.0016 (January–March 2005), 0.0005 ± 0.00021 (June 2005), and 0.061 ± 0.0035 (September–November 2005). Litopenaeus vannamei were present in 30%, 16% and 100% of nets used for the three consecutive sampling periods. The mean sizes of L. vannamei captured were 22.4 ± 0.75, 25.1 ± 0.07, and 22.0 ± 0.29 mm postorbital carapace length (PO-CL) and 85.6 ± 2.66, 105.5 ± 6.13, and 85.8 ± 1.04 mm body length. The CL and body sizes were significantly larger in samples collected in June 2005 than the two other periods. Increasing frequencies of occurrence of L. vannamei in the Bangpakong estuary call for the determination of sources (escapes versus a self-sustaining population) and mitigation.

Concentrations of the heavy metals cadmium (Cd), mercury (Hg), nickel (Ni), lead (Pb), chromium (Cr), copper (Cu) and zinc (Zn) in adult banana prawns (Fenneropenaeus merguiensis de Man, 1888) were determined. Banana prawn samples were collected from two provinces of Thailand (Ampur LamSing, Chantaburi, and Ban Huanumkao, Trat) by gill net. Twenty five prawns from each province were randomly chosen from the catch and each prawn sample was divided into two parts: cephalothorax and abdominal muscle. Both parts were digested with concentrated nitric acid and hydrogen peroxide under pressure in a microwave digester. Hg levels were determined using a Cold Vapor Atomic Fluorescence Spectrometer. Cd, Cu, Cr, Ni, Pb, and Zn concentrations were determined using Atomic Absorption Spectrophotometer. Trace concentrations of Cd, Cu, Cr, Ni, Pb, and Zn in cephalothorax of banana prawns from both provinces were significantly higher (P < 0.05) than in muscle, with no significant difference observed for Hg. Concentrations of Hg, Cu, Zn, and Cd in banana prawns from Chantaburi province were significantly higher (P < 0.05) than those from Trat province. There was no significant difference observed between the contents of Pb, Ni, and Cr in banana prawns caught from the two provinces. Heavy metal content in banana prawn, especially in cephalothorax tissue, may be a good indicator (sentinel organism) of heavy metal contamination in the coastal environment. Mean concentrations of all heavy metals in abdominal muscle (edible part) were within the safety limits for human consumption. However, Cd and Cu concentrations in some of the cephalothorax samples from Chantaburi province were higher than the safety limits for human consumption. This suggests that a close monitoring program is needed to ensure the safety of F. merguiensis as a food source.

A large number of polymorphic genetic markers are needed to examine genetic variation in wild and cultured penaeid species, trace pedigrees, and apply marker-assisted selection in breeding programs. The objectives of this study are to (1) isolate and characterize microsatellite genetic markers for the Pacific whiteleg shrimp, Litopenaeus vannamei, (2) demonstrate the usefulness of three randomly selected markers to examine allelic variation in wild and cultured shrimp populations, and trace the pedigree of two families from the breeding program of the US Marine Shrimp Farming Program (USMSFP); and (3) determine the potential usefulness of these microsatellites for linkage mapping. A total of 128 recombinant clones obtained from Sau3 A-digested genomic libraries prepared from ovary of specific pathogen-free L. vannamei were sequenced; 86 of which contained simple sequence repeats (SSRs), or microsatellites, with three or more repeat motifs. The frequency of microsatellites with five or more repeats was estimated at 1/2.74 kb. The most abundant di-, tri, tetra-, penta-, and hexa-nucleotide motifs were (CT)_n, (CCT)_n and (CTT)_n, (CATA)_n, (CTTCT)_n, and (GAGATA)_n. The octa-nucleotide (CCCTCTCT)₃ was also identified. Sixty-two primer sets flanking microsatellites with single or multiple motifs were designed and tested for polymorphism with a small test panel representing individuals of the mapping families being used to develop a linkage map for L. vannamei (ShrimpMap), and 35 of these (56.4%) were polymorphic. Three of these markers (TUGAPv1-3.224, TUGAPv5-7.33, and TUGAPv7-9.17) were used for estimating allele diversity of wild populations of Ecuador and Mexico and tracing the pedigree in two families of the USMSFP breeding program. A large number of alleles (21–31) and allele size range (95–275 bp) was observed in wild shrimp. There was a large allele size range difference at all three loci examined, being smaller in cultured shrimp (32–74 bp) than in wild shrimp (77–180 bp), suggesting null alleles or mutations. The presence of stuttering bands with marker TUGAPv5-7.33 made it difficult to score the wild shrimp from Mexico and suggest the need to first test for inheritance pattern of shrimp microsatellites before using them in population genetics, relatedness/kinship, and traceability studies. Allele segregation in cultured shrimp confirmed codominant inheritance of markers. Observed heterozygosity was 100% for all loci scored. Fourteen randomly selected polymorphic markers were further genotyped with the entire IRMF panel and 8 of these amplified with most of the individuals tested. Linkage analysis using CRIMAP with LOD score of 5.0 placed four of the markers (TUGAPv1-3.132, TUGAPv3-5.213, TUGAPv7-9.179, and TUGAPv7-9.95) in linkage groups LG6, LG5, LG13, and LG14, respectively, and four markers (TUGAPv3-5.271, TUGAPv3-5.391, TUGAPv7-9.94, and TUGAPv7-9.226) remained unlinked. In summary, 35 new microsatellites were developed for L. vannamei, some of which are useful for studies on genetic diversity of wild and cultured stocks, pedigree tracing in breeding programs, and linkage mapping. Moreover, some of the genomic sequences reported here had significant homology to hypothetical proteins of various organisms, known (e.g., reverse transcriptase) or unknown genes, or no homology to any sequence in the GeneBank database, suggesting that sequences from a genomic library can also provide valuable information in identifying functional markers in shrimp.

To explore the possibility of finding interleukin-like gene(s) in Litopenaeus vannamei shrimp, we sought evidence for the presence of interleukin 1 (IL-1)-like genes, because the proteins encoded by these genes are evolutionarily conserved. RT-PCR performed with primers from conserved regions of vertebrate IL-1α genes amplified three bands of 954 bp (ShIL1-21; accession # U94689), 864 bp (ShIL1–28; U94690), and 182 bp (ShIL1-J17; U94691) from various tissues. The cDNA sequences were AT-rich (∼65%) and contained 1–5 copies of the “ATTTA” motif, a possible indicator of mRNA degradation. GenBank search indicated that similar AT-rich elements are present in the 3′-untranslated regions (UTR) of vertebrate cytokines, growth factors and other tissue-specific and developmentally expressed genes. Northern blot hybridization analysis using ShIL1-21 as probe detected mRNA transcripts of ∼1.8 kb and ∼2.9 whereas ShIL1-28 detected a ∼7.5 kb transcript. All transcripts showed tissue and developmental variations in their mRNA expression. The 1.8 kb transcript of ShIL1-21 was expressed at high levels in postlarvae stage 8 (PL8) but not in Zoea stage 2/3 or tail muscle of juvenile and adult shrimp. The 7.5 kb mRNA of SHIL1-28 was present in tail muscle of adult female shrimp but not in male tail muscle. In addition, all transcripts levels increased in juveniles at 24 h and 36 h after TSV challenge indicating that the expression of these genes is modulated during TSV infection. Primer sets flanking simple sequence repeats in ShIL1-21 and ShIL1-28 amplified polymorphic alleles in L. vannamei from various geographic regions, suggesting that these markers will be useful for genetic diversity analyses. ShIL1-21 and ShIL1-28 primers were used for genotyping with the entire international reference mapping family (IRMF) panel developed for construction of a linkage map for shrimp (ShrimpMap). CRIMAP analysis using a limits-of-detection (LOD) score of 5.0 placed ShIL1-21 in linkage group 14 of ShrimpMap whereas ShIL1-28 remained unlinked. The parental alleles segregated in a Mendelian fashion indicating that these genes are useful for pedigree tracing and population analysis.

Cadmium (Cd) is an ubiquitous environmental pollutant of increasing worldwide concern. It has become one of the most hazardous heavy metals in aquatic environments and could threaten aquatic organisms, including marine shrimp. Shrimp are sensitive to Cd and have been found to accumulate it in their bodies in proportion to environmental concentrations. The effects of Cd on the biology and gene expression of the commercially important Litopenaeus vannamei are unknown. The overall hypothesis is that Cd exerts effects on shrimp at both biological and molecular levels. These changes may provide a way to identify genes responsible for toxicity, detoxification and/or tolerance to Cd exposure both acute and chronic. To test the hypothesis, a small-scale pilot project was initiated to obtain baseline information on histological changes associated with Cd treatment and to develop the genomics tools needed to identify genes associated with Cd exposure. The specific objectives of this study were to (1) observe histopathologic changes in control- and Cd-treated postlarvae (PL) during a 48-h period, (2) measure Cd concentrations in Cd-exposed and untreated PLs, and (3) isolate expressed sequence tags (ESTs) from control and Cd-treated PLs for future genomics studies. Specific pathogen-free L. vannamei PLs at stage 42 (PL42, from the Kona Line of the United States Marine Shrimp Farming Program) were used in a waterborne bioassay to determine histological changes in PLs exposed to a range of concentrations of CdCl₂ (0, 0.01, 0.1, 1.0, and 10.0 ppm) for 48 h. Results for objective (1) indicated variable response of individual shrimp to Cd exposure at different concentrations. All but 2 animals from 10-ppm group died by 24 h. Histological lesions were limited to the integument, musculature, gills, hepatopancreas, and midgut-hindgut. For objective (2), results showed that the Cd levels in control PLs at 0 h were low and remained relatively low throughout the study. There was a dose- and time-dependent relationship of waterborne Cd exposure and accumulation. Results from objective (3) suggested differential gene expression in control- and Cd-treated PLs as reflected by the number of ESTs homologous to genes with different molecular function isolated from approximately 1,100 clones each from the control and Cd-exposed (1 ppm) cDNA libraries. These ESTs contributed to the establishment of an EST database for L. vannamei (ShrimpESTBase). Homology searches of the nucleotide and translated amino acid sequences of ESTs isolated from the control and Cd-treated (1 ppm) cDNA libraries identified a significant number of clones similar to (a) known housekeeping genes and genes involved in immune recognition, signal transduction and effector function, (b) other shrimp ESTs of unknown function, (c) ESTs from other species, (d) predicted, unknown or unnamed proteins from other species, and (e) no homology to any sequence in the GenBank database. Some ESTs (∼30%) from the Cd-treated library showed homology to unique sequences representing potential transposable elements. The results provide baseline information on the potential effects of Cd on shrimp health and growth and suggest a complex interaction between environmental conditions, water, feed, stress, and genetic background of PLs and should be further investigated under both laboratory and commercial conditions. Moreover, data suggest L. vannamei may be useful as bioindicators for the condition of their natural environment.

Species and distribution of Penaeoid shrimps in Thailand were studied in specimens of shrimp collected from fish markets and fishing ports of 23 provinces along the Gulf of Thailand and the Andaman Sea coast. Identification of species was based on the morphological characters of rostrum, carapace, branchiae, antennule, antenna, third maxilliped, pereiopods, pleopods, abdomen, telson, petasma, thelycum, and the colored pattern of the whole body based on keys and diagnoses available from the current literature. A total of 50 species from 13 genera and 3 families of Penaeoidea were identified in coastal provinces of the Gulf of Thailand and the Andaman Sea. Additional research is needed to more clearly define the distribution of shrimp species in the Gulf of Thailand and the Andaman Sea coasts.

A large number of polymorphic simple sequence repeats (SSRs) or microsatellites are needed to develop a genetic map for shrimp. However, developing an SSR map is very time-consuming, expensive, and most SSRs are not specifically linked to gene loci of immediate interest. We report here on our strategy to develop polymorphic markers using expressed sequence tags (ESTs) by designing primers flanking single or multiple SSRs with three or more repeats. A subtracted cDNA library was prepared using RNA from specific pathogen-free (SPF) Litopenaeus vannamei juveniles (∼1 g) collected before (0) and after (48 h) inoculation with the China isolate of white spot syndrome virus (WSSV). A total of 224 clones were sequenced, 194 of which were useful for homology comparisons against annotated genes in NCBI nonredundant (nr) and protein databases, providing 179 sequences encoded by nuclear DNA, 4 mitochondrial DNA, and 11 were similar to portions of WSSV genome. The nuclear sequences clustered in 43 groups, 11 of which were homologous to various ESTs of unknown function, 4 had no homology to any sequence, and 28 showed similarities to known genes of invertebrates and vertebrates, representatives of cellular metabolic processes such as calcium ion balance, cytoskeleton mRNAs, and protein synthesis. A few sequences were homologous to immune system-related (allergens) genes and two were similar to motifs of the sex-lethal gene of Drosophila. A large number of EST sequences were similar to domains of the EF-hand superfamily (Ca² binding motif and FRQ protein domain of myosin light chains). Single or multiple SSRs with three or more repeats were found in approximately 61% of the 179 nuclear sequences. Primer sets were designed from 28 sequences representing 19 known or putative genes and tested for polymorphism (EST-SSR marker) in a small test panel containing 16 individuals. Ten (53%) of the 19 putative or unknown function genes were polymorphic, 4 monomorphic, and 3 either failed to satisfactorily amplify genomic DNA or the allele amplification conditions need to be further optimized. Five polymorphic ESTs were genotyped with the entire reference mapping family, two of them (actin, accession #CX535973 and shrimp allergen arginine kinase, accession #CX535999) did not amplify with all offspring of the IRMF panel suggesting presence of null alleles, and three of them amplified in most of the IRMF offspring and were used for linkage analysis. EF-hand motif of myosin light chain (accession #CX535935) was placed in ShrimpMap's linkage group 7, whereas ribosomal protein S5 (accession #CX535957) and troponin I (accession #CX535976) remained unassigned. Results indicate that (a) a large number of ESTs isolated from this cDNA library are similar to cytoskeleton mRNAs and may reflect a normal pathway of the cellular response after im infection with WSSV, and (b) primers flanking single or multiple SSRs with three or more repeats from shrimp ESTs could be an efficient approach to develop polymorphic markers useful for linkage mapping. Work is underway to map additional SSR-containing ESTs from this and other cDNA libraries as a plausible strategy to increase marker density in ShrimpMap.

A preliminary low-density linkage map (ShrimpMap) based solely on simple sequence repeats (SSRs) or microsatellite genetic markers was constructed for specific pathogen-free (SPF) shrimp, Litopenaeus vannamei, from the United States Marine Shrimp Farming Program (USMSFP). Marker loci originated mostly from genomic libraries cloned using DNA from ovaries of adult shrimp or cDNA libraries cloned using RNA of juveniles challenged with either Taura syndrome virus (TSV) or white spot syndrome virus (WSSV). Out of approximately 150 polymorphic markers initially tested with a small test panel consisting of 16 individuals, 100 (83 genomic SSRs and 17 EST-SSRs) were useful for genotyping with the entire mapping panel, a three-generation pedigree comprising two grandparents, two parents and 89 progeny. High frequency of null alleles (approx. 25% of markers) was observed in shrimp microsatellites. Chi-square goodness-of-fit tests revealed that 30 of the 100 markers showed segregation distortion, 17 of which were highly significant distorted (P < 0.001). Linkage analysis using CRIMAP with a LOD score of 3.0 provided a map with coverage for 14 linkage groups consisting of 67 linked markers spaced every ∼22 cM with an observed genome length of 689.1 cM and an estimated genome length for L. vannamei of 2675.7 cM. Variable results were obtained using a LOD score of 5.0. Sex-linked microsatellites were identified in linkage group 4 (LG4) of ShrimpMap, a linkage group that also includes 28 s rRNA, nonLTR retrotransposon reverse transcriptase and other markers with no homology to any sequence in the GenBank database. The map includes the first EST-SSR markers mapped for L. vannamei. Human ESTs also amplified in shrimp DNA and two of them were useful for linkage analysis. Work is underway to place additional EST-SSR markers on to ShrimpMap to serve as a framework map for penaeid species to facilitate integration of linkage and physical maps, study conservation and evolutionary genomics in penaeids, and search for candidate genes associated with fitness traits in wild and cultured shrimp. Moreover, the mapped EST-SSRs provide valuable comparative genomic links not only between L. vannamei and other penaeid species but also between shrimp and other invertebrate and vertebrate genomes.

No Abstract available.