A TaqMan real-time PCR assay for quantifying type III hepatopancreatic parvovirus infections in wild broodstocks and hatchery-reared postlarvae of Fenneropenaeus chinensis in Korea

A highly sensitive and specific TaqMan real-time PCR was used to quantify hepatopancreatic parvovirus (HPV) type III infections in wild broodstocks and hatchery-reared postlarvae (PL) of Fenneropenaeus chinensis. Totals of 159 and 162 wild brooders from three locations were captured, and 140 and 180 PL were obtained from seven and six commercial hatcheries in 2007 and 2008, respectively. Among the three wild broodstock groups from 2007, only 1 group showed HPV infection and 3.2% of 159 brooders were positive for HPV infection. In 2008, HPV infections were observed from all three wild broodstock groups with 1.93×10(4) copies/mg tissue of pleopods. Of 162 brooders, 26.6% were positive for HPV infection. No PL from the two hatcheries collected in 2007 showed HPV infection, and PL from the rest of the five hatcheries had up to 1.74×10(6) copies/ng of DNA, and PL from three hatcheries showed HPV infections with over 1,000 copies/ng of DNA. The PL from all seven hatcheries collected in 2008 showed up to 2.10×10(5) HPV copies/ng of DNA. PL from two hatcheries showed less than 100 copies/ng of DNA, but PL from the rest of the hatcheries showed HPV infections with over 1,000 copies/ng of DNA. These results show that HPV type III is widely distributed in Korea in addition to previously reported HPV type I, and they can be effectively detected by type-specific realtime PCR.     

Prevalence of white spot syndrome virus (WSSV) in wild shrimp Penaeus monodon in the Philippines

Prevalence of white spot syndrome virus (WSSV) was determined using polymerase chain reaction (PCR) methodology on DNA extracted from the gills of wild black tiger shrimp Penaeus monodon collected from 7 sampling sites in the Philippines. These 7 sampling sites are the primary sources of spawners and broodstock for hatchery use. During the dry season, WSSV was detected in shrimp from all sites except Bohol, but during the wet season it was not detected in any site except Palawan. None of the WSSV-PCR positive shrimp showed signs of white spots in the cuticle. Prevalence of WSSV showed seasonal variations, i.e. prevalence in dry season (April to May) was higher than in the wet season (August to October). These results suggest that WSSV has already become established in the local marine environment and in wild populations of P. monodon. Thus, broodstock collected during the dry season could serve as the main source of WSSV contamination in shrimp farms due to vertical transmission of the virus in hatcheries.     

Various population fluctuation patterns of light-attracted beetles in a tropical lowland dipterocarp forest in Sarawak

The population fluctuation pattern of light-attracted beetles was studied from August 1992 to September 1998 (for 73 months) using ultraviolet light-traps set at three vertical levels in a tropical lowland dipterocarp forest in Sarawak, Malaysia. During our study, a general flowering occurred from April to July in 1996, and flowering on a small scale in 1997 and 1998. We analyzed the data for eight scarabaeid and six meloid species, some of which were anthophilous species. Various fluctuation patterns were observed among the beetle species in aspects of both seasonality and correlation with the supraannual phenological pattern. Three large chafer species (Scarabaeidae, Melolonthini) showed a clear seasonal fluctuation pattern with a peak once from March to May every year, the peak monthly catch greatly fluctuating annually. Other scarabaeid beetles did not show such a clear seasonal population pattern and hardly fluctuated annually. Populations of an anthophilous scarabaeid species, Parastasia bimaculata, a specific pollinator of Homalomena propinqua (Araceae), hardly fluctuated, probably because of its response to the constant flowering of its floral hosts. Monthly catches of an anthophilous scarabaeid, Anomala sp., and meloid beetles showed clear supraannual patterns in response to the general flowering and were significantly correlated with the flowering intensity with or without a lag of a month. The fluctuation pattern of meloids suggests a supraannual population fluctuation pattern of their hosts, i.e., megachilid/anthophorid bees. Received: November 9, 1999 / Accepted: February 8, 2000     

High Occurrence of the Decapod Penstyldensovirus (PstDV1) Detected in Postlarvae of <Emphasis Type="Italic">Penaeus vannamei</Emphasis> Produced in Commercial Hatcheries of Mexico

The decapod penstyldensovirus (PstDV1) is a widely spread shrimp pathogen that causes high mortalities in the shrimp Penaeus stylirostris, while in P. vannamei, it has been associated with induction of the runt deformity syndrome. Using shrimp post-larvae (PL, stages PL13-PL21) collected from 16 commercial hatcheries from Mexico, and a sensitive PCR protocol for its detection, a survey of the PstDV1 prevalence in larvae was undertaken. A high overall prevalence of PstDV1 (49.5 %) in shrimp PL from the studied hatcheries was found. This study reveals that PstDV1 occurs persistently in PL populations, which may have significant implications for its dispersal.     

Polychaete worms--a vector for white spot syndrome virus (WSSV)

The present work provides the first evidence of polychaete worms as passive vectors of white spot syndrome virus (WSSV) in the transmission of white spot disease to Penaeus monodon broodstocks. The study was based on live polychaete worms, Marphysa spp., obtained from worm suppliers/worm fishers as well as samples collected from 8 stations on the northern coast of Tamilnadu (India). Tiger shrimp Penaeus monodon broodstock with undeveloped ovaries were experimentally infected with WSSV by feeding with polychaete worms exposed to WSSV. Fifty percent of polychaete worms obtained from worm suppliers were found to be WSSV positive by 2-step PCR, indicating high prevalence of WSSV in the live polychaetes used as broodstock feed by hatcheries in this area. Of 8 stations surveyed, 5 had WSSV positive worms with prevalence ranging from 16.7 to 75%. Polychaetes collected from areas near shrimp farms showed a higher level of contamination. Laboratory challenge experiments confirmed the field observations, and > 60% of worms exposed to WSSV inoculum were proved to be WSSV positive after a 7 d exposure. It was also confirmed that P. monodon broodstock could be infected with WSSV by feeding on WSSV contaminated polychaete worms. Though the present study indicates only a low level infectivity in wild polychaetes, laboratory experiments clearly indicated the possibility of WSSV transfer from the live feed to shrimp broodstock, suggesting that polychaete worms could play a role in the epizootiology of WSSV.     

Effective size of a wild salmonid population is greatly reduced by hatchery supplementation

Many declining and commercially important populations are supplemented with captive-born individuals that are intentionally released into the wild. These supplementation programs often create large numbers of offspring from relatively few breeding adults, which can have substantial population-level effects. We examined the genetic effects of supplementation on a wild population of steelhead (Oncorhynchus mykiss) from the Hood River, Oregon, by matching 12 run-years of hatchery steelhead back to their broodstock parents. We show that the effective number of breeders producing the hatchery fish (broodstock parents; N(b)) was quite small (harmonic mean N(b)=25 fish per brood-year vs 373 for wild fish), and was exacerbated by a high variance in broodstock reproductive success among individuals within years. The low N(b) caused hatchery fish to have decreased allelic richness, increased average relatedness, more loci in linkage disequilibrium and substantial levels of genetic drift in comparison with their wild-born counterparts. We also documented a substantial Ryman-Laikre effect whereby the additional hatchery fish doubled the total number of adult fish on the spawning grounds each year, but cut the effective population size of the total population (wild and hatchery fish combined) by nearly two-thirds. We further demonstrate that the Ryman-Laikre effect is most severe in this population when (1) >10% of fish allowed onto spawning grounds are from hatcheries and (2) the hatchery fish have high reproductive success in the wild. These results emphasize the trade-offs that arise when supplementation programs attempt to balance disparate goals (increasing production while maintaining genetic diversity and fitness).     

Partitioning kin groups of broodstock in the critically endangered Chinese sturgeon,Acipenser sinensis Gray 1835

Pedigrees of broodstock with unknown relationship of the critically endangered Chinese sturgeon, Acipenser sinensis, was evaluated using microsatellite markers to facilitate genetic management in restocking programs with small broodstock size. We characterized the distributions of relatedness values to reconstruct kin groups in four hatchery families with known pedigrees using microsatellites. The distributions of relatedness values for kin classes were used for partitioning full sibling groups of wild A. sinensis broodstock kept in two hatcheries, resulted in 13 full sibling clusters, four of which containing 62% of all the wild individuals. This indicates high probability of choosing close related breeder pairs in random mating, thus selective breeding is necessary to minimize inbreeding and maintain genetic diversity. This study provides a useful tool for genetic management in conservation programs of A. sinensis in aim of preserving self‐sustained wild populations.     

Changes in Solea senegalensis sperm quality throughout the year

Some of the Senegalese sole (Solea senegalensis) broodstock reproductive constraints are related to sperm quality. Although they present two defined spawning season (spring and autumn), males gave semen during all the year thus an exhaustive annual sperm analysis is important to determine the seasonal changes in semen quality. Sampling was performed monthly during one year, analyzing different cellular parameters to better understand sperm quality limitations obstructing sole mass production. The percentage of progressive motile cells and their linear velocity showed a decrease from March (beginning of the first spawning season) to July (when the highest temperatures were observed), followed by a slight increase in August and October (second spawning season). DNA fragmentation values showed highest values between the two spawning seasons and decreased to the end of the year. The percentage of apoptotic cells was lowest in March (beginning of the first spawning season) and the highest in November. The percentage of cells resistant to seawater exposure presented two peaks related with both spawning seasons. There was a tendency for the semen to attain a quality peak between the beginning and the middle of the first spawning season (March-May), followed by a pronounced decrease, achieving the lowest values during the months with the highest temperature. Also, the different males present in the broodstocks reach their sperm quality peak at different times, which will result in an unequal contribution for the next generation.     

Overview of salmon stock enhancement in southeast Alaska and compatibility with maintenance of hatchery and wild stocks

Modern salmon hatcheries in Southeast Alaska were established in the 1970s when wild runs were at record low levels. Enhancement programs were designed to help rehabilitate depressed fisheries and to protect wild salmon stocks through detailed planning and permitting processes that included focused policies on genetics, pathology, and management. Hatcheries were located away from significant wild stocks, local sources were used to develop hatchery broodstocks, and juveniles are marked so management can target fisheries on hatchery fish. Initially conceived as a state-run system, the Southeast Alaska (SEAK) program has evolved into a private, non-profit concept centered around regional aquaculture associations run by fishermen and other stakeholders that pay for hatchery operations through landing fees and sale of fish. Today there are 15 production hatcheries and 2 research hatcheries in SEAK that between 2005 and 2009 released from 474 to 580 million (average 517 million) juvenile salmon per year. During this same period commercial harvest of salmon in the region ranged from 28 to 71 million salmon per year (average 49 million). Contributions of hatchery-origin fish to this harvest respectively averaged 2%, 9%, 19%, 20%, and 78% for pink, sockeye, Chinook, coho, and chum salmon. Both hatchery and wild salmon stocks throughout much of Alaska have experienced high marine survivals since the 1980s and 1990s resulting in record harvests over the past two decades. Although some interactions between hatchery salmon and wild salmon are unavoidable including increasing concerns over straying of hatchery fish into wild salmon streams, obvious adverse impacts from hatcheries on production of wild salmon populations in this region are not readily evident.     

Results from black tiger shrimp penaeus monodon culture ponds stocked with postlarvae PCR-positive or -negative for white-spot syndrome virus (WSSV).

Commercial, intensive, earthen shrimp ponds (188) in southern Thailand were stocked with postlarvae (PL) of Penaeus monodon that had tested positive or negative for white-spot syndrome virus (WSSV) infection by polymerase chain reaction (PCR) assay. All the PL were grossly healthy. At 2 wk intervals after stocking, shrimp from each pond were examined for gross WSSV lesions and tested for WSSV by PCR. Shrimp from all the ponds stocked with WSSV-PCR-positive PL (Group 0, n = 43) eventually showed gross signs of white-spot disease (WSD) at an average of 40 d after stocking. Of the remaining ponds stocked with WSSV-PCR-negative PL (n = 145), some remained WSSV-PCR-negative throughout the study (Group 5, n = 52), while others (93) became WSSV-PCR-positive after stocking, during the first month (Group 1, n = 23), second month (Group 2, n = 40), third month (Group 3, n = 24), or fourth month (Group 4, n = 6). Crop failure was defined as a pond drain or forced harvest before 14 wk or 98 d of cultivation. For Group 0 the proportion of ponds failing was 0.953, while it was only 0.019 for Group 5. Thus, the relative risk of failure for Group 0 was approximately 50 times that of Group 5. The relative risk of failure for Group 0 was also 3 times that for ponds stocked with WSSV-PCR-negative PL. Obviously, not all WSSV outbreaks resulted in crop failure. Of the 93 ponds stocked with PCR-negative PL that later yielded WSSV-PCR-positive shrimp, 53% reached successful harvest. The study showed that PCR screening of PL and rejection of WSSV-positive batches before stocking could greatly improve the chances of a successful harvest.     

Performance of WSSV-infected and WSSV-negative Penaeus monodon postlarvae in culture ponds.

In a survey of 27 Penaeus monodon culture ponds stocked with postlarvae (approximately PL10) at medium density (approximately 40 shrimp m(-2)), single-step nested white spot syndrome virus (WSSV) PCR was used to measure the WSSV infection rates in the shrimp populations within 1 mo after stocking. Seven ponds were initially WSSV-free, and the shrimp in 5 of these were harvested successfully. In the ponds (n = 6) where detection rates were higher than 50%, mass mortality occurred during the growth period, and none of these ponds was harvested successfully. In a subsequent study, P. monodon brooders were classified into 3 groups according to their WSSV infection status before and after spawning: brooders that were WSSV-positive before spawning were assigned to group A; spawners that became WSSV-positive only after spawning were assigned to group B; and group C consisted of brooders that were still WSSV-negative after spawning. WSSV screening showed that 75, 44 and 14%, respectively, of group A, B and C brooders produced nauplii that were WSSV-positive. Most (57%; 16/28) of the brooders in group A produced nauplii in which the WSSV prevalence was high (>50%).When a pond was stocked with high-prevalence nauplii from 1 of these group A brooders, an outbreak of white spot syndrome occurred within 3 wk and only approximately 20% of the initial population survived through to harvest (after 174 d). By contrast, 2 other ponds stocked with low-prevalence and WSSV-negative nauplii (derived respectively from 2 brooders in group B), both had much higher survival rates (70 to 80%) and yielded much larger (approximately 3x by weight) total harvests. We conclude that testing the nauplii is an effective and practical screening strategy for commercially cultured P. monodon.     

Low impact of infectious hypodermal and hematopoietic necrosis virus (IHHNV) on growth and reproductive performance of Penaeus monodon

No controlled studies on the effect of infectous hypodermal and necrosis virus (IHHNV) on Penaeus monodon have been previously reported. Here we describe domesticated P. monodon that became positive for IHHNV and other viruses at variable levels of prevalence during cultivation in 16 open-air, earthen ponds. These were stocked with domesticated postlarvae (PL) that tested negative for 7 shrimp viruses including IHHNV at 6% prevalence in 3 checks using polymerase chain reaction (PCR) methods. These PL were derived from domesticated female broodstock that individually tested negative for the same viruses. At 4 mo of culture, the shrimp in some ponds without obvious mortality tested positive by PCR methods for IHHNV and 3 other viruses at variable levels of maximum estimated prevalence (MEP). Stained tissue sections showed no lesions typical of IHHNV, but in situ hybridization tests with an IHHNV-specific DNA probe were positive. There was no significant difference in mean body weight (i.e. ca. 25 g) between shrimp groups positive or negative for IHHNV. Similar results were obtained with IHHNV negative and positive adults at 1 yr. Adults that individually tested negative for all 7 viruses and some that tested lightly positive for IHHNV were bred for the next generation. There were no significant differences in the number of eggs (> 600 000) and nauplii (ca. 300,000) produced by females negative and positive for IHHNV. From these females, 11/49 (22%) IHHNV PCR-positive PL batches were obtained from PCR-negative spawners, while 8/11 (73%) were obtained from IHHNV PCR-positive spawners. The results suggested that IHHNV infection can be transmitted vertically but does not seriously retard growth of P. monodon or affect fecundity of lightly infected broodstock.     

Genetic changes in Atlantic salmon stocks since historical times and the effective population size of a long-term captive breeding programme

Human-caused genetic changes in two Atlanticsalmon (Salmo salar L.) stocks, from therivers Iijoki and Oulujoki in Finland, wereassessed by comparing the genetic parameters ofthese stocks before and after the hatcherybreeding of several successive generations,corresponding to 40 and 33 years since the wildstate. The changes were also compared withthose observed in a large wild salmon stock inthe River Teno during 56 years. In all, thevariation at seven microsatellite DNA loci wasexamined in 11 Atlantic salmon samplesoriginating from these three rivers. Theeffective population size, N_e, duringbreeding of the Iijoki broodstock and for theTeno salmon was also estimated by the temporalmethod based on allele frequency changes. Forthe Iijoki broodstock, the changes could betracked generation by generation from thefounding of the stock. Statisticallysignificant changes in allele frequencies werecommon in the hatchery stocks (F = 0.029, forIijoki), but not in the wild Teno stock, whichwas temporally very stable (F = 0.007). Allelicrichness decreased statistically significantly(24.8%) in the Oulujoki broodstock, from 62.1to 46.7 alleles at nine loci. On average, therewere 9.7 fewer alleles (15.7%) in thecontemporary broodstocks than in thecorresponding historical stocks. The meanheterozygosity was 6.6% lower in thecontemporary Oulujoki broodstock, but remainedunchanged in the Iijoki broodstock. Theestimated N_e for the Iijoki broodstock wasunder 80 for 4.5 generations from 1962 to 1995and for the wild Teno salmon over 900 for 56years from 1939 to 1995.     

Comparative analysis of cost factors in sturgeon fingerling production in Iranian hatcheries (2000–2004)

Production cost analysis in aquaculture is an essential exercise to assist farm managers. Economic assessment of a farm operation also provides the basis to formulate governmental aquaculture and enhancement policies in many regions. The present study employed questionnaires and interviewed managers while also using governmental statistics to gain insight into production cost variables in Iranian sturgeon hatcheries. Within a decade, production of sturgeon fingerlings for release and stock enhancement increased in Iran to more than 21 million fingerlings by 2004. Costs and contributions of various production factors were determined using data obtained from a questionnaire involving all hatcheries between 2000 and 2004. A team of experts completed the questionnaire data sets while conducting interviews at all sturgeon centres and other related departments. From 2000 to 2004 the contribution of A. persicus was 79% of the total number of sturgeon fingerlings produced followed by A. nudiventris with 7.5% and Huso huso with 6.6%. Among the various expenditures between 2000 and 2004, the costs for permanent and part‐time employees contributed the greatest share of total costs, averaging 44%, with a noticeable declining trend from 51% in 2000 to 36% in 2004. Obtaining and incubating fertilized eggs averaged 22% of total costs, increasing during the same time period from 6 to 35%, respectively. On average, the 2000–2004 production cost for a single sturgeon fingerling was estimated at Rials 1667 (US$ 0.20), increasing from Rials 992 (US$ 0.12) to Rials 2623 (US$ 0.29) over these 4 years. Permanent staff at a hatchery was determined as being the principal cost, followed by costs for obtaining fertilized eggs (including broodstock handling). Over the 5‐year study period the results indicated that costs for part‐time labour declined yearly and, conversely, the costs of obtaining broodstocks as well as fertilizing and incubating eggs increased. Considering the background of hatchery production and stock enhancement of sturgeon species and the results of fishing data, it is possible to arrive at a first estimate of the potential contribution of Persian sturgeon farming to the total catch in Iranian waters; it is assumed that these increases were most likely through stock enhancement.     

Use of Polymerase Chain Reaction for the Detection of Infectious Hypodermal and Hematopoietic Necrosis Virus in Penaeid Shrimp

A rapid and reliable polymerase chain reaction (PCR) method was developed for the detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in penaeid shrimp. The oligonucleotide primers amplify a 1681-bp fragment of IHHNV, which encompasses the coding sequence for one of the viral coat proteins. The PCR method detects IHHNV in hemolymph and homogenized tissue obtained from the cephalothorax or pleopods of infected shrimp. The technique was also successfully applied to tissue samples preserved in 70% ethanol. The correct size fragment was amplified using IHHNV obtained from six different geographic regions in three different species of penaeid shrimp. No DNA extraction method was necessary for this technique. The use of hemolymph or pleopods provides a nondestructive screening method by which to test juveniles and adult broodstock for the presence of IHHNV. Received September 21, 1999; accepted January 21, 2000     

Sampling and evaluation of white spot syndrome virus in commercially important Atlantic penaeid shrimp stocks

In 1997, white spot syndrome virus (WSSV) was discovered in shrimp culture facilities in South Carolina, USA. This disease was known to cause devastating mortalities in cultured populations in Southeast Asia and prompted concern for the health of wild populations in the USA. Our study surveyed wild shrimp populations for the presence of WSSV by utilizing molecular diagnostics and bioassay techniques. A total of 1150 individuals (586 Litopenaeus setiferus, 477 Farfantepenaeus aztecus and 87 F. dourarum) were examined for the presence of WSSV DNA by PCR. A total of 32 individuals tested positive and were used in a bioassay to examine the transmission of disease to healthy individuals of the culture species L. vannamei. DNA sequencing of PCR products from a positive individual confirmed that the positive individuals carried WSSV DNA. Significant mortalities were seen in test shrimp injected with tissue extracts from heavily infected wild shrimp. These data confirm the existence of WSSV in wild shrimp stocks along the Atlantic Coast and that the virus can cause mortalities in cultured stocks.     

Detection of QTL for growth rate in the blacklip abalone (Haliotis rubra Leach) using selective DNA pooling

The objective of this study was to identify QTL for growth rate in the blacklip abalone Haliotis rubra using selective DNA pooling. Three full-sibling families of H. rubra derived from crosses of wild broodstock were used. DNA was extracted from the largest and smallest 10% of progeny and combined into two pools for each phenotypic tail. The DNA pools were typed with 139 microsatellites, and markers showing significant differences between the peak height ratios of alleles inherited from the parents were individually genotyped and analysed by interval mapping. A strong correlation (r = 0.94, P < 0.001) was found between the t-values from the analysis of pools and the t-values from the analysis of individual genotypes. Based on the interval mapping analysis, QTL were detected on nine linkage groups at a chromosome-wide P < 0.01 and one linkage group at a chromosome-wide P < 0.05. The study demonstrated that selective DNA pooling is efficient and effective as a first-pass screen for the discovery of QTL in an aquaculture species.