Branchial Pathomorphology of Southern Bluefin Tuna Thunnus maccoyii (Castelnau, 1872) Infected by Helminth and Copepodan Parasites.

Three metazoan parasites, a monogenean Hexostoma thynni and two species of copepods Pseudocycnus appendiculatus and Euryphorus brachypterus are known to parasitize the gills of ranched southern bluefin tuna (SBT) and other tuna species. However, there is no detailed information describing the pathological response to infection by these parasites in this species. Wild southern bluefin tuna Thunnus maccoyii (approximately 3 years of age), captured and towed to a grow-out site in the waters immediately south of Port Lincoln, South Australia were subsequently sampled (n = 10) monthly from March until August 2004 during commercial harvest operations. Longitudinal sections of gill hemibranchs with attached parasites were excised and fixed for routine histology and immunohistochemistry. Reference samples were also collected from fish displaying no signs of parasitism or other grossly observable anomalies. Two morphologically distinct granulocytes were observed and putatively identified as eosinophils and mast cells. Pathology was localized to filaments upon and immediately adjacent to parasite attachment sites. Branchial cellular responses, adjunct to the attachment of H. thynni by its opisthaptoral clamps, included hyperplasia and inflammation resulting in structural remodeling of branchial tissues. Inflammatory infiltrates were often dominated by putative eosinophils and lymphocytes when parasitized by H. thynni and P. appendiculatus. Gill associated lymphoid tissue infiltrated the lamellar regions particularly in response to helminth infection. A variable response ranging from hemorrhage with minor hyperplasia or fibroplasia and eosinophilic inflammation to a barely discernible change was seen for gill sections harboring P. appendiculatus and E. brachypterus. The magnitude of the host response to attachment by the latter was congruent with attachment proximity and parasite load. On the basis of the host responses reported here and the low intensity of infection observed in other associated studies these gill ectoparasites are currently considered a low risk for wild and ranched adult SBT.

Three new species of Pseudodactylogyrus (Monogenea: Pseudodactylogyridae) from Australian eels.

Three species of Pseudodactylogyrus Gusev, 1965 (Monogenea: Pseudodactylogyridae) were collected from the gills of Anguilla reinhardtii Steindachner and A. australis Richardson from several localities in Australia and eels imported to Japan from Australia. Pseudodactylogyrus gusevi sp. n. from A. reinhardtii (type host) and A. australis in Queensland, Australia is most similar to P. bini (Kikuchi, 1929), but can be differentiated by the shorter male copulatory tube, heavy sclerotisation of the vaginal tube and the presence of a small projection of the supplementary piece of the hamulus. Pseudodactylogyrus rohdei sp. n. from A. australis (type host) in Queensland, Australia is most similar to P. anguillae (Yin et Sproston, 1948), but differs in the possession of a longer cement gland and the presence of a small projection on the supplementary piece of the hamulus. Pseudodactylogyrus bini sensu Gusev, 1965 and P. anguillae sensu Gusev, 1965 are synonymised with P. gusevi sp. n. and P. rohdei sp. n., respectively. Pseudodactylogyrus mundayi sp. n. from A. australis, originating in Tasmania, Australia and sent alive to Japan, is most similar to P. kamegaii Iwashita, Hirata et Ogawa, 2002, from which it can be discriminated by the shorter male copulatory tube and the shorter vaginal tube. Dactylogyrus bialatus Wu, Wang et Jian, 1988 from Synechogobius ommaturus (Richardson) (Gobiidae) is transferred to Pseudodactylogyrus as P. bialatus comb. n. A phylogenetic tree based on the ITS2 region of six species of Pseudodactylogyrus including P. gusevi and P. mundayi shows that P. haze from a goby diverged first, and that species from eels are monophyletic, forming three lineages differing by their zoogeographical distribution. With the three new species and one new combination proposed in this paper, Pseudodactylogyrus is now comprised of eight species infecting anguillid and gobiid fish, and a key to species is presented.

Factors affecting abundance and prevalence of blood fluke, Cardicola forsteri, infection in commercially ranched southern bluefin tuna, Thunnus maccoyii, in Australia.

A survey of blood fluke, Cardicola forsteri, infection in ranched southern bluefin tuna, Thunnus maccoyii, was undertaken over three farming seasons, from March 2004 to September 2006. Analyses of covariance and logistic regression were used to explore the effects of company, year, season, time in culture, and condition index on intensity, abundance and prevalence of blood fluke infection. Average prevalence of blood fluke infection was 62.64% over the period of the survey. Average intensity was 6.20 (± 0.57) fluke per infected host and the average abundance was 3.70 (± 0.57) fluke per host. Year did not influence mean intensity or abundance although a significant decrease in prevalence in 2005 was evident. Tuna harvested in winter had a significantly greater abundance and prevalence of blood fluke than the tuna harvested in autumn. No effect of intensity or abundance of infection was observed on the condition of the infected tuna. A universal factor in explaining variation in C. forsteri intensity, abundance and prevalence was company. Differences in infection levels between tuna from different companies may be related to differences in husbandry measures employed on each farm, or due to different average sizes of tuna farmed by each of the companies, or due to the location of the operations.

Correlation of humoral immune response in southern bluefin tuna, T. maccoyii, with infection stage of the blood fluke, Cardicola forsteri.

The blood fluke, Cardicola forsteri, is a prevalent infection in ranched southern bluefin tuna. This project aimed to define the timing and intensity of the various developmental stages of C. forsteri within southern bluefin tuna as well as to relate infection to host pathology and immune response. Archival samples from several cohorts of T. maccoyii sampled from 2008 to 2010 were used in this study. The prevalence and intensity of C. forsteri infection was described using heart flushes and histological examination. Humoral immune response, i.e. C. forsteri specific antibody, lysozyme activity, and alternative complement activity, was also described. Based on the validated and detailed C. forsteri infection timeline, relationships between infection events, physiological response, and diagnosis were proposed. Immune response developed concurrently with C. forsteri infection, with the majority of physiological response coinciding with commencing egg production. Further research is needed to confirm the origin of C. forsteri antigen which is responsible for immune response development and how T. maccoyii immune response works against infection. To aide this research, further diagnostic methods for confirmation of infection need to be developed.

The life cycle of Cardicola forsteri (Trematoda: Aporocotylidae), a pathogen of ranched southern bluefin tuna, Thunnus maccoyi.

Aporocotylids (fish blood flukes) are emerging as pathogens of fishes in both marine and freshwater aquaculture. Efforts to control these parasites are hampered by a lack of life cycle information. Here we report on the life cycle of Cardicola forsteri, which is considered a significant pathogen in southern bluefin tuna, Thunnus maccoyi, ranched in South Australia. We surveyed polychaetes, bivalves and gastropods from sites close to tuna pontoons. Infections consistent with the Aporocotylidae were found in terebellid polychaetes, a single Longicarpus modestus and five individuals of Reterebella aloba. All infections were comprised of hundreds of sporocysts in the body cavity of the host, each filled with developing and mature cercariae. Sequences of ITS-2 and lsrDNA from the infection from L. modestus were a perfect match with those of adult C. forsteri from T. maccoyi. This life cycle link is considered confirmed but it is possible that additional terebellid species are infected in South Australia; equally, other species of intermediate host are likely to be involved in other parts of the range of this cosmopolitan trematode. Sequences of the species from R. aloba did not match a known adult but phylogenetic analysis of lsrDNA suggests that it is also a species of Cardicola Short, 1953. These findings show that terebellid polychaetes are a major host group for marine aporocotylids, especially given that Cardicola is the largest marine aporocotylid genus. The two cercarial types are among the smallest known for the family and are unusual, but not unique, in having short, simple tails. We speculate that the form of the tail means that these cercariae are not active swimmers and are thus heavily dependent on currents for dispersal. Control of this parasite might be effected by moving the tuna pontoons appropriate distances to avoid encounter with current-dispersed cercariae, or by increasing the separation of the nets from the sea floor, either by raising the nets or moving to deeper water.

Concurrent epizootic hyperinfections of sea lice (predominantly Caligus chiastos) and blood flukes (Cardicola forsteri) in ranched Southern Bluefin tuna.

Peaks in epizootics of sea lice (mostly Caligus chiastos) and blood flukes (Cardicola forsteri) among Southern Bluefin tuna (Thunnus maccoyii) appear to coincide with the onset of an increased mortality. The mortality event occurs 6-12 weeks after T. maccoyii have been transferred into static ranching pontoons from the wild. However, to date available data on parasite occurrence before commercial harvesting begins, are scant. This research gathered epizootiological data from weeks 4 to 13 post-transfer, for 153 T. maccoyii sampled from two research and four commercial pontoons. Counts of both parasites in the research pontoons reached levels far heavier than previously documented in ranched T. maccoyii. For sea lice, the prevalence in most pontoons was 100%; the highest intensity reached 495 individuals, and mean counts at the peak of the infection exceeded 265 lice per fish. Almost all of the 5407 individual lice counted were identified as adult C. chiastos (89.44% female, 10.14% male); adult females of two other species were also present, C. amblygenitalis (0.13%), in addition to an undescribed species, C. sp. (0.04%). Lice counts were correlated positively with gross eye pathology scores (r(s,151df)=0.3394, p=0.0000), negatively correlated with condition index (r(s,151df)=-0.5396, p=0.0000), and positively correlated with plasma cortisol (r(s,131df)=0.3906, p=0.0000) and glucose (r(s,131df)=0.2240, p=0.0096). For the blood fluke, prevalences were less uniform than those of sea lice, with lower rates of infection at the beginning (ranging from 10% to 40%), reaching 100% mid-study, and declining again (40% in one pontoon). The highest intensity reached 441 individual flukes. Fluke counts were negatively correlated with plasma haemoglobin (r(s,151df)=-0.2436, p=0.0051) and positively with lysozyme (r(s,151df)=0.3013, p=0.0019). Fluke counts were also correlated with sea lice counts (r(s,150df)=0.3143, p=0.0000). Peaks in these epizootics occurred near the onset of elevated mortalities, which started after 7 weeks of ranching.

Parabrachiella jarai sp. nov. (Crustacea: Copepoda: Siphonostomatoida) parasitic on Sillago sihama (Actinopterygii: Perciformes: Sillaginidae).

The presently reported study provides a detailed morphological description of the female and the male of a new species of the genus Parabrachiella-Parabrachiella jarai sp. nov. The parasites were sampled from marine fish, silver sillago, Sillago sihama (Perciformes: Sillaginidae), captured in Malaysia in 1994 and Hong Kong in 1995. The new species bears some resemblance to Parabrachiella lata (Song et Chen, 1976) but differs from it in details of second antenna, mandible, and maxilliped. The genus Parabrachiella currently covers 67 species including those recently transferred from Neobrachiella Kabata, 1979. An amended generic diagnosis is proposed for Parabrachiella and Thysanote. Some members of Parabrachiella are herewith transferred to Thysanote and some Thysanote are now placed in Parabrachiella.

Serological evidence of an antibody response in farmed southern bluefin tuna naturally infected with the blood fluke Cardicola forsteri.

In this study, adaptive immune response was investigated in farmed southern bluefin tuna, Thunnus maccoyii, infected with a sanguinicolid Cardicola forsteri. A cohort (Cohort(2005)) of southern bluefin tuna was sampled between March 2005 and August 2006. Samples were taken at the transfer of wild caught tuna to sea cages and then at regular intervals. Parasite intensity, abundance and prevalence data were recorded. An ELISA was developed to detect and quantify an antibody response against the blood fluke in southern bluefin tuna serum. Intensity and prevalence of the blood fluke were shown to peak in May 2005 at 10.9 flukes per infected fish (SE=1.72) and 97.5% prevalence and then decreased to low prevalence (10%) and intensity (1.0). There were no significant changes in prevalence or intensity in 2006. Antibody titres and seroprevalence increased from 1.37 U microl(-1) and 10% at transfer in March 2005 to reach a peak in December 2005 of 25.86 U microl(-1) (SE=6.26 U microl(-1)) and 66.66%. No significant changes were observed in antibody titres for the same cohort of fish during 2006. Parasitological and serological values from Cohort(2005) were compared to a 2006 cohort (Cohort(2006)) in March 2006 and August 2006 to determine if prior infection in Cohort(2005) elicited any protection against infection in 2006. Although significant differences were not observed in intensities between cohorts it was shown that Cohort(2005) had significantly lower abundances and prevalences of blood fluke infection than Cohort(2006). Although there was no significant difference in mean antibody titres between cohorts in March 2006, the mean antibody titre of Cohort(2006) was significantly greater than that of Cohort(2005) in August 2006. No significant differences were observed in seroprevalence. This is one of the few studies to demonstrate the development of acquired resistance in fish against a parasite in an aquaculture environment under natural infection conditions.

An epizootic of Caligus chiastos on farmed southern bluefin tuna Thunnus maccoyii off South Australia.

In some years, large numbers of Caligus chiastos have been observed on the external surfaces of southern bluefin tuna, particularly on the head and eyes, in some sea cages in Spencer Gulf, Australia. As no epidemiological data were available, we monitored sea lice on tuna (N = 130) in 4 research cages sampled at 6 wk intervals during the 2005 farming season. No lice were observed on a sample of 10 wild-caught tuna when the cohort was transferred to cages in early April. By late May more than half the sampled tuna (22 of 40) were infected, with up to 42 parasites; we also recorded one unidentified Caligus sp. at this time. In early July the number of tuna infected with lice declined to 10%; in the final sample in late August none were detected. Prevalence in May was significantly higher than on other dates (p < or = 0.001), whereas mean abundances did not differ significantly (p > 0.05). The decline in prevalence corresponded with a seasonal fall in temperature, from ca. 17 degrees C in May to 14 degrees C in August. Counts of lice at the peak of infection were associated with the severity of eye damage (Spearman's rank correlation coefficient, r(S,38df) = 0.654, p < 0.001); this may be because lice graze on the cornea or because tuna injure their eyes when flashing (rubbing against objects). Counts at this time were also strongly and inversely correlated with the condition index (r(S,38df) = -0.707, p < 0.001). It appears that tuna become infested with adult sea lice via wild teleosts and elasmobranchs attracted to sea cages.

Epizootics of metazoan gill parasites did not threaten feasibility of farming southern bluefin tuna (Thunnus maccoyii) in a trial extending over summer months.

Tuna farming off Port Lincoln, Australia, involves catching wild 2-4-year-old southern bluefin tuna in summer and then fattening for periods of 2-8 months. As fresh product is not available year-round, the feasibility of maintaining tuna for longer periods was trialled, including over a summer season, when temperatures may exceed 24 degrees C. As the rates of growth and reproduction in ectoparasites of fishes are usually most rapid during warm temperatures, parasite epizootics at this time may adversely affect the health of tuna. We collected epidemiological data on burdens of metazoans on the gills of tuna from the time of stocking in April 2005 through to final harvest in August 2006 (N=220). We document an epizootic of the copepod Pseudocycnus appendiculatus, characterised by a significant increase in both prevalence and mean intensity in the first winter, followed by a decline in these parameters over the next 12 months. This epizootic pattern appears to be independent of seasonal changes in temperature. For two other species, a second copepod (Euryphorus brachypterus) and a polyopisthocotylean flatworm (Hexostoma thynni), there were no clearly discernible trends in infections. As the high water temperatures over the summer period did not lead to increased infections of any species of gill parasites, we conclude that they do not threaten the feasibility of farming of Thunnus maccoyii.

Redescription of the fish parasite Lernanthropus polynemi Richiardi, 1881 (Copepoda: Siphonostomatoida) and relegation of two congeners to synonymy.

Lernanthropus polynemi Richiardi, 1881 is described, for the first time since its original discovery, based on the type-material found in the Museum für Naturkunde in Berlin, Germany. Morphological features of this parasite, which infects the commercially-caught fish Eleutheronema tetradactylum (Shaw), are described and illustrated, including those of the male, which is described for the first time. The types of another species from the same host, L. lappaceus Wilson, 1912, and deposited in the National Museum of Natural History in Washington DC, are also examined based on the type-specimens. The types of a third congener, L. trifoliatus Basset-Smith, 1898, also from the same host, are not available, but the original illustrations are studied. It is considered that the three nominal species described from E. tetradactylum represent a single taxon, and that L. lappaceus and L. trifoliatus be relegated to synonymy with L. polynemi.