White spot syndrome virus in decapods from Mississippi Sound, USA, and susceptibility of Palaemonetes pugio and Uca panacea to a Chinese isolate.

The presence and quantity of white spot syndrome virus (WSSV) was surveyed using TaqMan real-time PCR to assess the extent of the virus in Mississippi Sound, USA. A total of 3577 wild decapods comprising 11 species was collected between November 2012 and August 2015: WSSV was present in 10 of the 11 species. Prevalence ranged from 5.1% in Uca rapax to 38.8% in U. spinicarpa. Viral load ranged from 1.8 to 7.3 log10 copies of WSSV µg-1 total DNA. Two Gulf species, Palaemonetes pugio and U. panacea, were injected with a series of doses of a virulent WSSV isolate from China to determine relative susceptibility and virulence because continuing translocation of highly pathogenic isolates of WSSV poses risk to native species. Survival was 0-65% for P. pugio and 5-60% for U. panacea. Median survival time was lower for P. pugio than U. panacea at all doses. Mean (±SD) lethal load was 9.0 ± 8.9 log copies of WSSV µg-1 total DNA in P. pugio and 8.2 ± 8.3 in U. panacea. Mean viral load in survivors was higher in U. panacea than in P. pugio (5.8 ± 6.1 vs. 3.2 ± 3.0 log copies of WSSV µg-1 total DNA); mean viral load was lower in wild individuals of those species (2.9 ± 3.2 for P. pugio and 4.9 ± 5.0 for U. panacea). U. panacea is potentially more tolerant of WSSV than P. pugio and may serve as an important reservoir host in the community.

White spot syndrome virus (WSSV) in cultured juvenile blue crabs Callinectes sapidus: oral versus injection exposure, and feeding frequency effects.

The efficacy of oral versus injection exposure and the effect of feeding frequency on the transmission of white spot syndrome virus (WSSV) in cultured juvenile blue crabs Callinectes sapidus were investigated. Crabs in Group 1 (G-1, n = 48) were exposed once orally to 100 mg of WSSV-infected shrimp tissue mg-1 of body weight (BW). The oral inoculum contained 2.6 × 109 WSSV genome copies mg-1 tissue. Group 2 (G-2, n = 46) received the same dosage once weekly for 5 wk. Group 3 (G-3, n = 12) was injected with 0.01 ml (2.6 × 107 genome copies 0.01 ml-1) WSSV inoculum g-1 BW. Group 4 (G-4, n = 12) was injected with 0.01 ml WSSV-negative shrimp serum and saline mixture g-1 BW. Dead and moribund animals were frozen at -80°C. After 37 d, all remaining crabs were frozen. Genomic DNA from gill tissue was evaluated for the presence and quantity of WSSV using TaqMan real-time PCR. All G-3 animals died and tested positive. No G-4 animals died or tested positive. In the fed groups, WSSV prevalence was approximately 16%, but viral load was higher and survival was lower in G-2 compared to G-1. Injected animals carried a higher viral load than fed animals, and dead animals had higher viral loads than live animals. Blue crab juveniles are susceptible to WSSV, but oral exposure does not efficiently transmit WSSV in juvenile blue crabs. Some animals can die from WSSV if repeatedly exposed.

Development of a modified cortisol extraction procedure for intermediately sized fish not amenable to whole-body or plasma extraction methods.

The corticosteroid hormone cortisol is the central mediator of the teleost stress response. Therefore, the accurate quantification of cortisol in teleost fishes is a vital tool for addressing fundamental questions about an animal's physiological response to environmental stressors. Conventional steroid extraction methods using plasma or whole-body homogenates, however, are inefficient within an intermediate size range of fish that are too small for phlebotomy and too large for whole-body steroid extractions. To assess the potential effects of hatchery-induced stress on survival of fingerling hatchery-reared Spotted Seatrout (Cynoscion nebulosus), we developed a novel extraction procedure for measuring cortisol in intermediately sized fish (50-100 mm in length) that are not amenable to standard cortisol extraction methods. By excising a standardized portion of the caudal peduncle, this tissue extraction procedure allows for a small portion of a larger fish to be sampled for cortisol, while minimizing the potential interference from lipids that may be extracted using whole-body homogenization procedures. Assay precision was comparable to published plasma and whole-body extraction procedures, and cortisol quantification over a wide range of sample dilutions displayed parallelism versus assay standards. Intra-assay %CV was 8.54%, and average recovery of spiked samples was 102%. Also, tissue cortisol levels quantified using this method increase 30 min after handling stress and are significantly correlated with blood values. We conclude that this modified cortisol extraction procedure provides an excellent alternative to plasma and whole-body extraction procedures for intermediately sized fish, and will facilitate the efficient assessment of cortisol in a variety of situations ranging from basic laboratory research to industrial and field-based environmental health applications.

Counter-insurgents of the blue revolution? Parasites and diseases affecting aquaculture and science.

Aquaculture is the fastest-growing segment of food production and is expected to supply a growing portion of animal protein for consumption by humans. Because industrial aquaculture developed only recently compared to industrial agriculture, its development occurred within the context of a growing environmental awareness and acknowledgment of environmental issues associated with industrial farming. As such, parasites and diseases have become central criticisms of commercial aquaculture. This focus on parasites and diseases, however, has created a nexus of opportunities for research that has facilitated considerable scientific advances in the fields of parasitology and aquaculture. This paper reviews Myxobolus cerebralis , Lepeophtheirus salmonis , white spot syndrome virus, and assorted flatworms as select marquee aquaculture pathogens, summarizes the status of the diseases caused by each and their impacts on aquaculture, and highlights some of the significant contributions these pathogens have made to the science of parasitology and aquaculture.

Population model for Amyloodinium ocellatum infecting the spotted seatrout Cynoscion nebulosus and the red snapper Lutjanus campechanus.

The dinoflagellate Amyloodinium ocellatum, a major pathogen in warm water mariculture, has a trophont, a tomont and a dinospore life history stage. This paper presents a population model for A. ocellatum infecting spotted seatrout Cynoscion nebulosus and red snapper Lutjanus campechanus and evaluates the relative effect of each vital rate on the A. ocellatum population growth rate. The vital rates were estimated by incubating trophonts in vitro and tracking their development through the successive life history stages at 25°C and 33 ppt. The A. ocellatum population growth rate was 1.90 d-1 for spotted seatrout and 1.92 d-1 for red snapper. Highest elasticity values (0.24 and 0.23 in spotted seatrout and red snapper, respectively) corresponded to transitions from the dinospore to the trophont stage, the trophont stage to the tomont stage and the tomont stage back to the dinospore stage in both host species (self-loops not included). A 50% change in vital rates showed that the mean number of dinospores produced by a tomont had the largest effect on the A. ocellatum population growth rate (15%), followed by the dinospore infection rate (14%), the tomont sporulation rate (12%) and the dinospore mortality rate (10%) in both host species. A comparison of modeled and experimental vital rate threshold values revealed a 2.5- (spotted seatrout) or a 2.6-fold (red snapper) difference in the values for dinospore mortality, which is the smallest difference among all the modeled and experimental vital rates. Therefore, measures that increase dinospore mortality have a greater likelihood of influencing the outcome of an epidemic.

Development of a rapid assay to detect the dinoflagellate Amyloodinium ocellatum using loop-mediated isothermal amplification (LAMP).

Amyloodinium ocellatum is a highly pathogenic dinoflagellate parasite with global distribution that causes high mortalities in the culture of tropical and sub-tropical marine and estuarine fishes. Diagnosis typically occurs through gross examination following the onset of morbidity, at which point treatment is of limited benefit. In the present study, a new molecular diagnostic tool for the rapid detection of A. ocellatum (AO) was developed using the loop-mediated isothermal amplification method (LAMP). The AO-LAMP assay designed is highly specific using a set of four primers - two outer and two inner primers targeting six different regions on the 5' end of the Small Subunit rDNA region (SSU rDNA) of A. ocellatum. The AO-LAMP assay, optimized for 25-30 min at 62°C, amplified the DNA from A. ocellatum extracted from both water and gill tissue samples and did not amplify DNA from four closely related dinoflagellate sp ecies. The detection limit of the AO-LAMP assay was 10 fg, exceptionally higher than the conventional PCR (1 pg). In addition, the standardized AO-LAMP assay was capable of detecting single tomonts and trophonts; the assay was not affected by the presence of possible inhibitory substances present in environmental water samples or gill samples. The AO-LAMP assay developed in the present study provides a novel useful tool for the simple, rapid and sensitive detection of A. ocellatum in water and gill tissue samples, which could assist in the early detection and improved control of A. ocellatum infections in aquaculture systems.

Susceptibility and tolerance of spotted seatrout, Cynoscion nebulosus , and red snapper, Lutjanus campechanus , to experimental infections with Amyloodinium ocellatum.

Amyloodinium ocellatum is a parasitic dinoflagellate that infects warm-water marine and estuarine fishes and causes mortalities in aquaculture. Its life cycle consists of 3 stages: a feeding trophont that parasitizes the gills and skin where it interferes with gas exchange, osmoregulation, and tissue integrity; a detached reproductive tomont; and a free-swimming infective dinospore. We compared the susceptibility and tolerance of juvenile spotted seatrout, Cynoscion nebulosus, and red snapper, Lutjanus campechanus, to this parasite by individually exposing fish in 3-L aquaria (at 25 C and 33 practical salinity units) to several dinospore doses over different time periods and quantified the size and number of resulting trophonts. We estimated the trophont detachment rate and trophont size at detachment, the 24-hr dinospore infection rate, the dinospore 48-hr median lethal dose (LD(50)), and the trophont lethal load at the 48-hr LD(50). There were no significant differences in dinospore infection rates or dinospore lethal doses between spotted seatrout and red snapper; however, trophonts remained attached longer and attained a larger size in red snapper than in spotted seatrout. The trophont lethal load was significantly higher in spotted seatrout than in red snapper. A proposed model simulating the trophont dynamics reflected our experimental findings and showed that A. ocellatum reproductive success is linked both to the number of dinospores and the size of the trophont, factors that, in turn, are linked to the time the trophont spends on the host and the number of trophonts the host can tolerate.

Kudoa hypoepicardialis n. sp. (Myxozoa: Kudoidae) and associated lesions from the heart of seven perciform fishes in the northern Gulf of Mexico.

Kudoa hypoepicardialis n. sp. infects the space between the epicardium and the compact myocardium and, in intense infections, the pericardial chamber of man-of-war fish (Nomeus gronovii) (Nomeidae) (the type host), blue runner (Caranx crysos) (Carangidae), Warsaw grouper (Epinephelus nigritus) (Serranidae), Atlantic tripletail (Lobotes surinamensis) (Lobotidae), northern red snapper (Lutjanus campechanus) (Lutjanidae), black drum (Pogonias cromis) (Sciaenidae), and bluefish (Pomatomus saltatrix) (Pomatomidae) in the northern Gulf of Mexico. This is the first report of a Kudoa sp. from the heart of a fish in the Gulf of Mexico, and of these hosts, only the bluefish was previously identified as a host for a species of Kudoa. Spores of the new species varied slightly in size among these hosts but were regarded as conspecific based on their nearly identical (99.9%) small-subunit (SSU) ribosomal DNA (rDNA) sequence. The new species differs both from the 4 nominal species of Kudoa reported from fishes in the Gulf of Mexico and from K. pericardialis, an allopatric species that infects the pericardial cavity, by the combination of having a large spore, a small polar capsule, and a polar filament with a single coil. The new species is morphologically and genetically most similar to K. shiomitsui, an allopatric species that infects the heart and pericardial cavity, but is distinguished from it based on a 4.2% difference in the SSU rDNA sequence. Heart lesions primarily were restricted to the vicinity of plasmodia and included a layer of fibrinous inflammation characterized by lymphocytes, macrophages, and granulomas as well as epithelioid encapsulations around plasmodia. Heavily infected hosts had melanin-like deposits and adipose cells beneath the epicardium. and the epicardium was discontinuous and apparently breached by plasmodia in some regions. Cardiac muscle, gill, liver, spleen, intestine, and kidney were normal.