Haplosporidium acetes n. sp. infecting the hepatopancreas of jelly prawns Acetes sibogae australis from Moreton Bay, Australia.

Wild Acetes sibogae australis from northern Moreton Bay, Australia displaying opacity of the hepatopancreas were sampled and examined histologically, revealing infection by multinucleate plasmodia of a haplosporidian-like parasite in the epithelial cells of the hepatopancreas. A morphological and phylogenetic investigation identified the parasite as a novel species of the order Haplosporida, and the parasite is described as Haplosporidium acetes n. sp. This is the first report of disease caused by a haplosporidian in wild Australian decapod crustaceans, and the first record of haplosporidiosis in sergestid shrimp. Infections of H. acetes were observed in all cell types (R, B, F and E) within the hepatopancreas. Infected epithelial cells became hypertrophied as they filled with haplosporidian parasites and, in heavy infections, caused almost complete displacement of normal hepatopancreas tissue. Although sporulation was not observed, infected jelly prawns appeared terminally diseased. Infections became grossly evident in around 5% of wild prawns during early autumn at a time of year when jelly prawn populations decline rapidly with decreasing water temperatures, however histopathology indicated at least 13% of apparently normal jelly prawns were also infected. Further studies are required in order to determine if this parasite influences jelly prawn population dynamics. In addition, we report co-infection of a novel microsporidian parasite in the Enterocytozoon Group Microsporidia (EGM) infecting nuclei of hepatopancreatic epithelial cells. The microsporidian was phylogenetically distinct from Enterocytozoon hepatopenaei (EHP) known to infect penaeid shrimp in Asia.

Sea lice Lepeophtheirus spinifer, Tuxophorus sp. and Caligus sp. infections on wild-caught queenfish Scomberoides commersonnianus from northern Australia.

Studies of ectoparasites of wild-caught queenfish Scomberoides commersonnianus from several areas in northern Australia were reviewed to investigate relationships between parasite burdens, environmental conditions and external lesions. A sample of 27 queenfish captured near a dredge spoil disposal site in Gladstone Harbour, Queensland, Australia, in January 2012 was anomalous, with a high percentage of fish (66.6%) exhibiting grossly visible skin lesions including foci of erythema and petechial haemorrhages, particularly on the pectoral girdle and ventrolateral surfaces. Microscopically, lesions comprised acute epidermal erosion, ulceration and/or perivascular dermatitis with dermal oedema and depigmentation. Skin lesions were associated with high prevalence (100%) and intensity (mean = 21.2 copepods fish-1, range 4-46) of infection by sea lice Lepeophtheirus spinifer. Only queenfish infected with >10 L. spinifer presented with skin lesions. This is the first record of L. spinifer from Australia. In contrast, grossly visible skin lesions were not reported from queenfish (n = 152) sampled from other sites in the Northern Territory and Queensland, where the sampled fish had a much lower prevalence (51.3%) and intensity (mean = 3.54, range 0-26) of copepod (L. spinifer, Caligus spp. and Tuxophorus sp.) infections. Copepods from queenfish in studies undertaken outside Gladstone Harbour exhibited an over-dispersed pattern of infection, with the vast majority (n = 137, or 90.1%) of fish infected with <5 copepods. These data demonstrate that heavy L. spinifer infections, combined with poor water quality and/or direct exposure to contaminated dredge spoil and blooms of the cyanobacterium Lyngbya majuscula, can be associated with cutaneous disease in wild-caught queenfish.

Investigation into white spots in the carapace of a moribund mud crab (Scylla serrata) from a white spot syndrome virus (WSSV) positive zone in Moreton Bay, Australia.

BACKGROUND: A freshly deceased mud crab (Scylla serrata) exhibiting multiple white spots under the carapace was found in Pumicestone Passage, northern Moreton Bay in May 2018. This crab was taken from within a biosecurity zone established due to a recent incursion of White Spot Syndrome Virus (WSSV) into populations of wild penaeids (Penaeus spp., Metapenaeus spp.) and crabs (Thalamita crenata) in the area. Because grossly visible white spots have been previously observed under the carapace of moribund S. serrata with white spot disease (WSD) in India, an investigation into the cause of death was undertaken. CASE REPORT: The affected S. serrata was negative for WSSV DNA when gill samples were tested by real-time PCR. Histopathology found no evidence of WSD lesions in the form of basophilic hypertrophied intranuclear inclusions in any tissues of ectodermal or mesodermal origin. Histopathology of the affected carapace showed that the white spots consisted of multiple lighter coloured foci in the exocuticle formed from concentric crystalline-like rings, which extended into the endocuticle. These were interpreted as evidence of mineral mobilisation within the carapace during the pre-moult (D1 or D2) stage of the moult cycle. The cause of death in this case therefore may have been due to moult-related complications. CONCLUSION: These observations confirm that formation of grossly visible white spots under the carapace of S. serrata are not pathognomonic for infection with WSSV. Similar observations in previous studies where WSSV was detected by PCR in this same host may have been incidental findings.

Pathology of finfish and mud crabs Scylla serrata during a mortality event associated with a harbour development project in Port Curtis, Australia.

The objective of this study was to assess the extent and describe the nature of a multi-species marine finfish and crustacean disease event that occurred in Gladstone Harbour, Australia, 2011-2012. Finfish were examined for this study in January to April 2012 from sites where diseased animals were previously observed by the public. Gross abnormalities, including excessive skin and gill mucus, erythema, heavy ecto-parasitism, cutaneous ulceration, corneal opacity, and exophthalmos, were higher (25.5%) in finfish from Gladstone Harbour (n = 435) than in those from an undeveloped reference site, 250 km to the north (5.5%, n = 146, p < 0.0001). Microscopic abnormalities, especially non-infectious erosive to ulcerative dermatitis and internal parasitism, were more prevalent in fish from Gladstone Harbour (n = 34 of 36, prevalence = 94.4%) than in fish from the reference site (3 of 23, prevalence = 13.0% p < 0.0001). The prevalence of shell lesions was higher in mud crabs Scylla serrata sampled from Gladstone Harbour (270 of 718, prevalence = 37.5%) than from the reference site (21 of 153, prevalence = 13.7%; p < 0.0001). The significantly higher prevalence of ulcerative skin disease and parasitism in a range of species suggests affected animals were subjected to influences in Gladstone Harbour that were not present in the control sites. The disease epidemic coincided temporally and spatially with water quality changes caused by a harbour development project. The unique hydrology, geology, and industrial history of the harbour, the scope of the development of the project, and the failure of a bund wall built to retain dredge spoil sediment were important factors contributing to this epidemic.

Effects of temperature on fecundity in vitro, egg hatching and reproductive development of Benedenia seriolae and Zeuxapta seriolae (Monogenea) parasitic on yellowtail kingfish Seriola lalandi.

Globally, aquaculture industries involved with commercial culture of kingfish (Seriola spp.) experience outbreaks of monogenean parasites, which can cause heavy stock losses. In Australia and New Zealand, aquaculturists of kingfish Seriola lalandi incur financial losses caused by two monogenean species: Benedenia seriolae and Zeuxapta seriolae which parasitise the skin and gills, respectively. This study provides information on some basic temperature-dependent life-cycle parameters of these problematic monogeneans on S. lalandi. Hatching times and age at maturity were inversely related to water temperature within the range experienced by wild kingfish in New Zealand (13-21 degrees C). Mature B. seriolae in vitro laid on average 37 eggs/day that hatched over approximately 4 days; peak hatching occurred 9, 11 and 22 days post-deposition at temperatures of 21, 17.5 and 13+/-1.0 degrees C, respectively. Z. seriolae in vitro laid on average 246 eggs/day that hatched over 2 days; peak hatching occurred 7, 9 and 15 days post-deposition at these respective temperatures. B. seriolae matured within 20, 25 and 48 days p.i. at 21, 18 and 13 degrees C. Z. seriolae matured within 25, 37 and >52 days p.i. at the same temperatures. This research describes stages in the reproductive development of B. seriolae and Z. seriolae and discusses the inclusion of basic parasitic life-cycle parameters into management strategies designed to maximise treatment efficacy and limit monogenean epizootics in sea-cage kingfish culture.

Pathology of cultured paua Haliotis iris infected with a novel haplosporidian parasite, with some observations on the course of disease.

Mortalities among juvenile paua Haliotis iris Martyn 1784 in a commercial culture facility were reported in April 2000. Histology of moribund paua showed heavy systemic infections of a uni- to multi-nucleate stage of a novel organism later confirmed by transmission electron microscopy (TEM) and molecular studies to be a haplosporidian. Multinucleate plasmodia up to 25 microm diameter with up to 17 nuclei were detectable in wet preparations of hemolymph from heavily infected paua. The presence of the haplosporidian in the affected facility was associated with mortalities of slow growing 'runt' paua during the summer months. Total mortalities in affected raceways 6 mo after mortalities began were between 82.5 and 90%. Heavily infected paua exhibited behavioural abnormalities including lethargy, loss of righting reflex, and were easily detached from surfaces. Some heavily infected paua exhibited oedema and pale lesions in the foot and mantle, but no reliable gross signs of disease were noted. Light infections of the haplosporidian were also found in apparently healthy paua from the facility. Histology indicated that the early stages of infection were characterised by small numbers of plasmodia in the connective tissue surrounding the gut, amongst glial cells adjacent to nerves in the mantle and foot and within gill lamellae. In heavy infections, large numbers of small plasmodia (mean size 5.5 x 7 microm in histological sections) were present in the hemolymph, gills, heart, kidneys, mantle, foot, epipodium and connective tissue of the digestive gland. Infections were not transferred horizontally at 14 and 19 degrees C after cohabiting heavily infected paua with uninfected paua for 3 mo in aquaria, or 3 mo after injecting healthy paua with hemolymph containing haplosporidian plasmodia. This may indicate that the prepatent period for disease is longer than 3 mo, that disease is not expressed below 20 degrees C, or that an intermediate host is required for transmission. Spore formation was not observed in juvenile paua but sporocyst-like bodies containing putative spores were observed amongst haplosporidian plasmodia in the right kidney of poorly performing adult paua collected from the wild.

Prokaryote infections in the New Zealand scallops Pecten novaezelandiae and Chlamys delicatula.

Four intracellular prokaryotes are reported from the scallops Pecten novaezelandiae Reeve, 1853 and Chlamys delicatula Hutton, 1873. Elongated (1025 x 110 nm), irregular (390 x 200 nm), or toroidal (410 x 200 nm) mollicute-like organisms (M-LOs) occurred free in the cytoplasm in the digestive diverticular epithelial cells of both scallop species. Those in P. novaezelandiae bore osmiophilic blebs that sometimes connected the organisms together, and some had a rod-like protrusion, both of which resemble the blebs and tip structures of pathogenic mycoplasmas. The M-LOs in C. delicatula had a slightly denser core than periphery. Round M-LOs, 335 x 170 nm, occurred free in the cytoplasm of agranular haemocytes in P. novaezelandiae, without apparent harm to the host cell. In P. novaezelandiae, 2 types of highly prevalent (95 to 100%) basophilic inclusions in the branchial epithelium contained Rickettsia-like organisms (R-LOs). Type 1 inclusions occurred in moderately hypertrophied, intensely basophilic cells, 8 to 10 microm in diameter, containing elongate intracellular R-LOs, 2000 x 500 nm. Type 2 inclusions were elongated and moderately basophilic in markedly hypertrophic branchial epithelial cells, 50 x 20 microm in diameter, containing intracellular organisms 500 x 200 nm in diameter. The possible roles of these organisms in pathogenesis is discussed.

Ultrastructure of a haplosporidian containing Rickettsiae, associated with mortalities among cultured paua Haliotis iris.

Uninucleate and multinucleate stages of a protozoan parasite are described from cultured abalone Haliotis iris Martyn, 1784 in New Zealand. The parasite is identified as a haplosporidian by the occurrence of multinucleate plasmodia, mitochondria with tubular cristae, lipid droplets, anastomosing endoplasmic reticulum (aER), multivesicular bodies (MVBs), haplosporogenesis by the production of haplosporosome-like bodies from nuclear membrane-bound Golgi, and their maturation to haplosporosomes. Coated pits occurred in the plasma membrane and coated vesicles were scattered in the cytoplasm, particularly in association with the Golgi face away from the nucleus, and aER. It is concluded that the outward face of the Golgi may be the trans face, and that aER is the trans-Golgi network. Coated pits and bristle-coated vesicles are reported from a haplosporidian for the first time. The vesicles in the MVBs resembled the cores and inner membranes of haplosporosomes, without the outer layer. The possible inter-relationships of these features are discussed. The abalone parasite differs from previously described haplosporidians in the apparent absence of a persistent mitotic spindle, and the presence of intracytoplasmic coccoid to rod-shaped bacteria resembling Rickettsiales-like prokaryotes. Phylogenetic analysis of the 16S rRNA gene sequence of the Rickettsiales-like prokaryotes indicated that these organisms belong to the Rickettsia cluster. The prokaryotes have a high (7%) sequence divergence from known Rickettsieae, with Rickettsia sp. and R. massiliae being the closest relatives. The lack of non-molecular evidence prevents us from proposing a new rickettsial genus at this time.

Luminous vibriosis in rock lobster Jasus verreauxi (Decapoda: Palinuridae) phyllosoma larvae associated with infection by Vibrio harveyi.

Studies were conducted to determine the cause of outbreaks of luminous vibriosis in phyllosoma larvae of the packhorse rock lobster Jasus verreauxi reared in an experimental culture facility. On 2 separate occasions mortalities of up to 75% over a period of 4 wk were observed in 4th to 5th and 8th to 10th instar phyllosomas at water temperatures of 20 and 23 degrees C, respectively. Affected larvae became opaque, exhibited small red spots throughout the body and pereiopods, and were faintly luminous when viewed in the dark. Histopathology showed that the gut and hepatopancreas tubules of moribund phyllosomas contained massive bacterial plaques. The hepatopancreas tubules of moribund larvae were atrophic and some contained necrotic cells sloughed into the lumen. Dense, pure cultures of a bacterium identified as Vibrio harveyi were isolated from moribund larvae. The disease syndrome was reproduced by in vivo challenge and V. harveyi was successfully reisolated from diseased larvae after apparently healthy larvae were exposed by immersion to baths of more than 10(4) V. harveyi ml(-1) at 24 degrees C. Injured larvae were more susceptible to infection than were healthy larvae. Survival of larvae experimentally and naturally exposed to V. harveyi was improved when antibiotics were administered via bath exposures.

Intraspecific variation in Cryptocaryon irritans.

Intraspecific variation in the ciliate Cryptocaryon irritans was examined using sequences of the first internal transcribed spacer region (ITS-1) of ribosomal DNA (rDNA) combined with developmental and morphological characters. Amplified rDNA sequences consisting of 151 bases of the flanking 18 S and 5.8 S regions, and the entire ITS-1 region (169 or 170 bases), were determined and compared for 16 isolates of C. irritans from Australia, Israel and the USA. There was one variable base between isolates in the 18 S region and 11 variable bases in the ITS-1 region. Despite their similar morphology, significant sequence variation (4.1% divergence) and developmental differences indicate that Australian C. irritans isolates from estuarine (Moreton Bay) and coral reef (Heron Island) environments are distinct. The Heron Island isolate was genetically closer to morphologically dissimilar isolates from Israel (1.8% divergence) and the USA (2.3% divergence) than it was to the Moreton Bay isolates. Three isolates maintained in our laboratory since February 1994 differed in sequence from earlier laboratory isolates (2.9% to 3.5% divergence), even though all were similar morphologically and originated from the same source. During this time the sequence of the isolates from wild fish in Moreton Bay remained unchanged. These genetic differences indicate the existence of a founder effect in laboratory populations of C. irritans. The genetic variation found here, combined with known morphological and developmental differences, is used to characterise four strains of C. irritans.

Variation in the development of two isolates of Cryptocaryon irritans.

Two isolates of Cryptocaryon irritans obtained from Acanthopagrus australis from Moreton Bay (isolate C1) and Gymnocranius audleyi from Heron Island (isolate C2) were passaged on Lates calcarifer and Macquaria novemaculeata at 20 and 25 C under identical laboratory conditions. There were significant differences between isolates in the diameter of trophonts and tomonts, the incubation period of tomonts, and the length of theronts. Trophonts of C1 were significantly larger on L. calcarifer than on M. novemaculeata and showed marked size variation with temperature, whereas trophonts of C2 developed equally well on both species and showed little size variation with temperature. Tomonts of C1 were significantly larger than those of C2 when grown on L. calcarifer, whereas on M. novemaculeata tomonts from C1 were significantly smaller than C2 tomonts. The incubation period of tomonts from C1 was significantly shorter than that for tomonts of C2, and theronts of C1 were significantly larger than theronts of C2 under all host/temperature conditions. The differences in the development of these isolates are of biological and epidemiological importance. This indicates that distinct intraspecific variants of C. irritans occur along the coast of southeast Queensland.

Influence of temperature and host species on the development of Cryptocaryon irritans.

The course of infection of the parasitic ciliate Cryptocaryon irritans was followed on Lates calcarifer and Macquaria novemaculeata at 20 and 25 C. The parasite was originally isolated from locally caught Acanthopagrus australis. At 20 C trophonts stayed on the fish longer, tomonts took longer to excyst, and the resulting theronts were larger than at 25 C. On L. calcarifer at 20 C, trophonts grew slowly at first but eventually became significantly larger (mean tomont diameter 466 x 400 microns) than at 25 C (mean diameter 373 x 320 microns). On M. novemaculeata, trophonts never grew as large as on L. calcarifer and at 20 C they grew poorly. The number of theronts produced per tomont was directly related to the size of the tomont but was not influenced by incubation temperature. The tomont incubation period was not related to the diameter of the tomont but was significantly influenced by the host origin of the tomont. Theront size was also significantly affected by the host origin of the tomont but not the diameter of the tomont. These results show that C. irritans exhibits variability in morphometrics on different hosts and under different temperature conditions. This variability needs to be taken into account if utilizing morphometric data for separating strains of C. irritans.

The rate of development of Polylabroides multispinosus (Monogenea: Microcotylidae) parasitic on the gills of Acanthopagrus australis (Pisces: Sparidae).

Experiments were done to determine the rate of development of Polylabroides multispinosus. Eggs hatch after an average 9, 7 and 6 days at 20, 24 and 28 degrees C, respectively. The ciliated oncomiracidium hatched with one pair of clamps (c.p.). Juvenile worms less than 3 days post-infection (p.i.) had 2 c.p. and blood and hematin in the simple, saccular gut. The gut bifurcated at 11 c.p. (6-9 days p.i.) (24 degrees C). The genital atrium, testes, ovary and vitellaria appeared at 16 c.p. (8-11 days p.i.), 22 c.p. (10-14 days p.i.), 30 c.p. (14-18 days p.i.) and 40 c.p. (17-21 days p.i.), respectively. Eggs were produced at 20 days p.i. The period from egg deposition to egg deposition is about one month at 24 degrees C.

The influence of formalin, benzocaine and hyposalinity on the fecundity and viability of Polylabroides multispinosus (Monogenea: Microcotylidae) parasitic on the gills of Acanthopagrus australis (Pisces: Sparidae).

Adult Polylabroides multispinosus exposed in vivo to formalin (200 p.p.m. in sea water, 30 min) deposited a similar number of eggs in 24 h in vitro as did control parasites (sea water, 35 parts per thousand, p.p.t., salinity) and worms exposed in vivo to benzocaine (40 p.p.m. in sea water, 10 min). Worms laid more eggs at 30 p.p.t. salinity in vitro than at 20, 35, 10 and 5 p.p.t. salinity (in decreasing order). Formalin (200 p.p.m., 30 min) decreased the viability of recently laid eggs, the survival of oncomiracidia, removed 70% of juvenile and adult worms from the gills, but decreased only slightly the viability of eggs exposed in utero or after eyespots developed in vitro. Salinities below 30 p.p.t. reduced viability as did an increase in temperature from 24 to 28 degrees C. All adult and juvenile worms were removed by baths in formalin (400 p.p.m., 25 min) or fresh water (1 h).