Rapid transmission of Bonamia exitiosa by cohabitation causes mortality in Ostrea angasi

The haplosporidian Bonamia was first detected in Australian shellfish in 1991. Australian isolates in Ostrea angasi Sowerby, 1871 were identified as Bonamia exitiosa Hine, Cochennac and Berthe, 2001, which threatens development of an O. angasi aquaculture industry. European field data suggest that Bonamia ostreae Pichot, Comps, Tigé, Grizel and Rabouin, 1980 infections in Ostrea edulis Linnaeus, 1758 build slowly, but infection dynamics of B. exitiosa in O. angasi are unknown. We investigated B. exitiosa infection in O. angasi by cohabiting uninfected juvenile O. angasi with adults infected with B. exitiosa. Oysters were sampled at 10, 21 and 40 days after cohabitation, and B. exitiosa prevalence and intensity were assessed. Bonamia exitiosa rapidly infected and caused disease in O. angasi. Mortalities began at 12 days, with ˜50% mortality by day 21 and >85% mortality by day 40. Mortalities displayed pathology consistent with clinical B. exitiosa infection. Time to first infection is likely influenced by a combination of parasite infectivity, host exposure and host immune capacity. Host death is not required for transmission, but probably facilitates release of parasites from decaying tissue. Understanding B. exitiosa transmission informs design and interpretation of field studies and aids development of management strategies for oyster aquaculture.     

Bonamia in Ostrea angasi: Diagnostic performance,field prevalence and intensity

Bonamia spp. parasites threaten flat oyster (Ostrea spp.) farming worldwide. Understanding test performance is important for designing surveillance and interpreting diagnostic results. Following a pilot survey which found low Bonamia sp. intensity in farmed Ostrea angasi, we tested further oysters (n = 100–150) from each of three farms for Bonamia sp. using heart smear, histology and qPCR. We used a Bayesian Latent Class Model to assess diagnostic sensitivity (DSe) and specificity (DSp) of these tests individually or in combination, and to assess prevalence. Histology was the best individual test (DSe 0.76, DSp 0.93) compared to quantitative polymerase chain reaction (qPCR) (DSe 0.69, DSp 0.93) and heart smear (DSe 0.61, DSp 0.60). Histology combined with qPCR and defining a positive from either test as an infected case maximized test performance (DSe 0.91, DSp 0.88). Prevalence was higher at two farms in a high‐density oyster growing region than at a farm cultivating oysters at lower density. Parasite intensities were lower than in New Zealand and European studies, and this is probably contributed to differences in the performance of test when compared to other studies. Understanding diagnostic test performance in different populations can support the development of improved Bonamia surveillance programs.     

The fertility of the Port Lincoln oyster (Ostrea angasi Sowerby) from West Lakes,South Australia

During 1977–1978 the spawning season of the Port Lincoln oyster (Ostrea angasi Sowerby) extended from about mid-October to early March. Between 5.6% and 21.4% of randomly sampled adult oysters (larger than 68 mm in length) were incubating larvae and between 0.91 and 1.33 broods of larvae were produced per adult oyster. Large oysters (68–94 mm in length) had a mean of 12.1 × 10⁵ larvae per brood. The incubation period under natural and laboratory conditions ranged from 15–22 days and there are similarities in the breeding biology of O. angasi, the European oyster (Ostrea edulis L.) and the Olympia oyster (Ostrea lurida Carpenter).     

Assessment of temperature or salinity effects on larval development by catecholamine‐induced metamorphosis of hatchery‐reared flat oyster,Ostrea angasi (Sowerby 1871) larvae

A need to improve larval rearing techniques led to the development of protocols for catecholamine‐induced settlement of flat oyster, Ostrea angasi, larvae. To further refine these techniques and optimize settlement percentages, the influence of salinity or temperature on development of O. angasi larvae was assessed using epinephrine‐induced metamorphosis. Larvae were reared between salinities of 15–35 and temperatures between 14.5 and 31°C. The greatest percentage survival, growth, development occurred when larvae were reared between 26 and 29°C and between salinities of 30 and 35. Larvae reared outside this salinity and temperature range exhibited reduced growth, survival and/or delayed development. Short‐term (1 h) reduction in larval rearing temperature from 26°C to 23.5°C significantly increased larval metamorphosis without affecting larval survival. Short‐term (1 h) increase in larval rearing temperature from 26°C to 29 and 31°C decreased larval survival and metamorphosis. To ensure repeatability in outcomes, tests showed that larvae sourced from different estuaries did not vary significantly in their metamorphic response to short‐term temperature manipulation and epinephrine‐induced metamorphosis.     

Investigations into the temporal development of epitheliocystis infections in brown trout: a histological study

Epitheliocystis in Swiss brown trout (Salmo trutta) is a chlamydial infection, mainly caused by Candidatus Piscichlamydia salmonis and Candidatus Clavichlamydia salmonicola. To gain a better understanding of the temporal development of infections in wild brown trout, we investigated epitheliocystis infections during the course of the summer and autumn months of a single year (2015), and compared this to sampling points over the span of the years 2012–2014. The survey focused on tributaries (Venoge and Boiron) of the Rhone flowing in to Lake Geneva. When evaluated histologically, epitheliocystis infections were found throughout the period of investigation with the exception of the month of June. Fifty to 86 animals per sampling were investigated. Highest prevalence and infection intensities were seen in September. A correlation between epitheliocystis infection and water temperatures was not evident. Interyear comparison revealed consistent levels of prevalence and infection intensities in late summer. The absence of infections in June, combined with the consistent interyear results, indicates seasonal fluctuation of epitheliocystis infections in brown trout with a reservoir persisting during winter months from which infections can re‐initiate each year. This could either be at levels below detection limits within the brown trout population itself or in an alternative host.     

The effects of stressors on the dynamics of Bonamia exitiosus Hine, Cochennec-Laureau & Berthe, infections in flat oysters Ostrea chilensis (Philippi)

Oysters, Ostrea chilensis, infected with Bonamia exitiosus were held under stressful conditions for 14 days, and then under normal conditions for 3.5 months, to determine the affects of stress on B.exitiosus infection levels. The stressors used were (1) air exposure for 8 h daily; (2) hot water (25–26 °C), or (3) cold water (7 °C) for 1 h daily; (4) hyposaline (15‰), or (5) hypersaline (39–40‰) water; (6) starvation in filtered sea water; and (7) vigorous stirring four times a day. A control tank held oysters in static sea water, which was changed daily. Oysters were also (8) kept in a trough among heavily infected oysters (trough exposure), or (9) in a trough without other oysters (control), for 4 months. Oysters in hyposaline conditions all died within 3 weeks, but this was apparently unrelated to Bonamia infection. Otherwise, cumulative mortalities were highest in trough exposure, cold, hypersaline and hot treatments. There was a significant difference in prevalence between treatments, and the stir, hot, cold, hypersalinity and trough exposure treatments had significantly higher intensities of infection than controls. The mean intensity of Bonamia infection was significantly higher among female and spent oysters than in male and hermaphrodite oysters.     

Gametogenic patterns of the larviparous oyster Ostrea nomades from Karachi, Pakistan (northern Arabian Sea)

The larviparous oyster Ostrea nomades from two different localities was examined histologically and found to be mostly hermaphroditic. However, pure males, pure females and indifferent oysters were also found. The hermaphrodites of O. nomades were classified into three categories: ambisexual, predominantly female and predominantly male. Gonadal development appears to take place throughout the year with increasing activity and ripening in the winter months, leading to immediate spawning and later resorption. The number of brooding larvae of O. nomades was low at both collection sites.     

Dietary addition of the essential oil from Lippia alba to Nile tilapia and its effect after inoculation with Aeromonas spp.

The objective of this study was to evaluate the effect of dietary addition of the essential oil from Lippia alba (EOLA) on growth performance, biochemical and haematological variables and survival after Aeromonas spp. infection of Nile tilapia juveniles (Oreochromis niloticus). Five diets were evaluated with increasing levels of EOLA (0.0 control; 0.25; 0.50; 1.0; and 2.0 ml/kg diet). After 45 days of feeding, the fish were infected with Aeromonas spp. followed by a 14‐day period of observation. There was no mortality during growth assessment. The addition of 2.0 ml EOLA/kg diet improved feed conversion ratio and condition factor and increased lysozyme activity and haematocrit (Hct), and decreased plasma globulin levels compared to the control group. The survival of fish infected with Aeromonas spp. was higher in those fed with 2.0 ml EOLA/kg diet than in fish fed with 0.0–0.5 ml EOLA/kg diet. We concluded that the inclusion of 2.0 ml EOLA/kg diet is recommended for Nile tilapia juveniles, because it improved feed conversion, Hct and immunological activity, did not provoke metabolic changes, and increased survival after Aeromonas spp. infection.     

Prevalence,intensity and pathology of the nasal parasite Nasicola hogansi in Atlantic bluefin tuna (Thunnus thynnus)

Ectoparasitic flatworms of Nasicola (Monogenoidea: Capsalidae), which infect nasal epithelium of true tunas (Thunnus spp.), are not well studied, nor have their impacts on the host's olfactory organ been evaluated. Infections of Nasicola hogansi on Atlantic bluefin tuna, Thunnus thynnus, were investigated with emphasis on the relationship between infection prevalence, abundance and mean intensity with bluefin tuna size, sex, body condition and capture month, as well as histopathological effects. Commercially caught Atlantic bluefin tuna (n = 161, 185–305 cm curved fork length) from the Gulf of Maine were sampled during June through August 2009 for infections by N. hogansi. A total of 247 specimens of N. hogansi were collected, with a prevalence of 45.3%, mean abundance of 1.57 (CI: 1.21–2.03) and mean intensity of 3.45 (CI: 2.91–4.22). Neither fish sex nor landing month had a significant effect on parasite parameters. Larger and better-conditioned Atlantic bluefin tuna had a higher mean intensity of infection. Pathology associated with infection by N. hogansi included extensive necrosis, sloughing of the nasal epithelium and associated inflammation of underlying connective tissues. Further epidemiological and pathological study of this host–parasite system is warranted since impaired olfaction, if present, could adversely affect spawning and migration of this top ocean predator.     

Seasonal dynamics and yearly change in the abundance of metacercariae of <Emphasis Type="Italic">Parabucephalopsis parasiluri</Emphasis> (Trematoda: Bucephalidae) in the second intermediate host in the Uji–Yodo River,central Japan

An introduced pathogenic fish trematode, Parabucephalopsis parasiluri Wang, 1985 (Digenea: Bucephalidae), was studied to determine the seasonal fluctuation in its metacercariae abundance. The timing of the recruitment period of the metacercariae and the relationship between yearly fluctuation in metacercarial abundance and some environmental factors were also examined to develop an effective plan for disease control. Monthly changes in metacercarial abundance in the caudal fin of pale chub (Zacco platypus) and of the prevalence of the first intermediate host (golden mussel Limnoperna fortunei) suggest that P. parasiluri generally has an annual life cycle. The metacercarial abundance in chub increased when the water temperature fell below 7°C in winter, but the rapidity of the increase varied between 2007 and 2008. The fluctuations in the abundance of metacercariae in winter from 2001 to 2008 showed that mean flow rate and mean water level in January were negatively related to the infection level each year, and positively related to the mean water temperature. Based on these results, we propose a control plan for parabucephalopsiosis involving discharge control; that is, more than 50 m³/s (corresponding to 150 m³/s in the Yodo River) of water should be flushed from upstream dams for a period of at least one week starting on the day when the water temperature falls below 7°C.     

Long‐term epidemiological survey of Kudoa thyrsites (Myxozoa) in Atlantic salmon (Salmo salar L.) from commercial aquaculture farms

Kudoa thyrsites (Myxozoa) encysts within myocytes of a variety of fishes. While infected fish appear unharmed, parasite‐derived enzymes degrade the flesh post‐mortem. In regions of British Columbia (BC), Canada, up to 4–7% of fillets can be affected, thus having economic consequences and impacting the competitiveness of BC's farms. K. thyrsites was monitored in two farms having high (HP) or low (LP) historical infection prevalence. At each farm, 30 fish were sampled monthly for blood and muscle during the first year followed by nine samplings during year two. Prevalence and intensity were measured by PCR and histology of muscle samples. In parallel, fillet tests were used to quantify myoliquefaction. Infections were detected by PCR after 355 and 509 degree days at LP and HP farms, respectively. Prevalence reached 100% at the HP farm by 2265 degree days and declined during the second year, whereas it plateaued near 50% at the LP farm. Infection intensities decreased after 1 year at both farms. Blood was PCR‐positive at both farms between 778 and 1113 degree days and again after 2000 degree days. This is the first monitoring project in a production environment and compares data between farms with different prevalence.     

Thelohanellus testudineus n. sp. (Myxosporea: Bivalvulida) infecting the skin of allogynogenetic gibel carp Carassius auratus gibelio (Bloch) in China

A Thelohanellus species was encountered during a survey on Thelohanellus diversity of Carassius auratus gibelio (Bloch) in China. The infection is characterized by the presence of large cysts of 1.4–3.2 cm in diameter in the skin of host. Mature spores were ampullaceous in frontal view and testudinate in lateral view, measuring 19.7 ± 0.7 (18.6–20.8) μm long, 7.6 ± 0.4 (6.6–8.4) μm wide and 7.3 ± 0.5 (6.6–8.8) μm thick. The single polar capsule was elongated pyriform, with 11.1 ± 0.5 (10.0–11.9) μm long and 5.3 ± 0.3 (4.3–5.8) μm wide. Polar filaments coiled with 7–8 turns. Scanning electron microscopy revealed a smooth spore surface with flat side and convex side. The sutural line was straight or ‘S’ like, running near the middle of the valves. Histologically, the large cysts consisting of numerous small plasmodia developed in the dermis of the skin. The BLAST search indicated that the newly obtained ssrRNA gene sequences did not match any available sequences in GenBank and phylogenetic analysis placed it in the Thelohanellus clade. Based on morphology and molecular differences with reported Thelohanellus spp., this parasite was described as a new species of genus Thelohanellus.     

The effect of light intensity on the growth of Brachymystax lenok (Pallas, 1773)

A 35‐day experiment was carried out to investigate the effect of light intensity on growth of Brachymystax lenok under different light intensities: 10, 70, 240 and 1000 lx. Fish(5.5 ± 0.24 g)used in the experiment were fed to satiation twice a day (08:00 hours, 14:00 hours).The photoperiod was 12L:12D (08:00–20:00 hours). The specific growth rate(SGR) of B. lenok under lower intensities(10 lx,70 lx)was significantly higher than the other groups(P < 0.05).No significant difference in feed intake was observed at different light intensities, but feed efficiency (FE) in wet weight at lower intensities (1070 lx) was higher than that at higher intensities(240,1000 lx) (P < 0.05).The final survival rate of juveniles varied from 86.33% to 93.66%,and there was no significant difference between experimental groups. The tested fish under higher light intensities (240 and 1000 lx) spent much more energy in respiration and excretion while depositing less energy for growth than those fish under lower light intensities. It is concluded that light intensity significantly affected growth and optimal light intensity for B. lenok juveniles was about 10–70 lx.     

Effects of temperature and photoperiod on the conditioning of the flat oyster (Ostrea edulis L.) in autumn

The production of the flat oyster Ostrea edulis (L.) natural spat in Europe has decreased almost by 60% in the past ten years. Thus, the importance of the production of oyster spat in hatcheries is evident. One of the critical steps in hatchery production is broodstock conditioning, especially difficult in autumn, when gonadal development is in resting period. Conditioning is influence by temperature, photoperiod and nutrition. In this work, the effects of two temperature and three photoperiod regimes on the conditioning of O. edulis were studied for three years by stereological analyses and registering number and dates of spawning and larval yield. Temperature had a positive effect on the gonadal development of O. edulis during conditioning. The percentages of germinal cells in oysters conditioned with a gradient of temperature (14–18°C) were double compared to oysters conditioned at 15°C. Oysters conditioned with longer photoperiods showed higher percentages of germinal cells. There was no interaction between temperature and photoperiod. Spawning was observed in the oysters treated with daylight (8–16 h) ten weeks from the beginning of conditioning. Flat oysters conditioned with 8 h and 8–12 h of daylight delayed the first spawning for a month. Total larval production was higher in the oysters treated with the longest daylight gradient. Gonadal and gametogenic development was a non‐synchronic process and the spawning extended for around two months. A protocol for flat oyster broodstock conditioning in autumn by using both a gradient of temperature (14–18°C) and daylight (8–16 h) is proposed.     

Incidence and prevalence of Diplectanum aequans and its influence on the fitness of juvenile sea bass (Dicentrarchus labrax) in the Black Sea

High rates of unexplained mortalities (up to 70%) are anticipated in the cultured juvenile sea bass (Dicentrarchus labrax) in farms in the eastern Black Sea at least for the last 8 years. Diplectanum aequans, often blamed for the observed mortalities, is the only prevalent parasite impacting the health of sea bass in the brackish water of the Black Sea. To investigate the seasonal fluctuation of D. aequans prevalence, mean intensities and the potential effects of D. aequans on the fitness of sea bass, eight cages from one farm (Farm A) and four cages at another distinct site (Farm B) were surveyed monthly from May to October of 2008. Six hundred and sixty‐one juvenile sea bass from Farm A and 236 from Farm B were individually examined for the presence and intensity of the parasite. The prevalence of D. aequans in cultured sea bass (96.3 ± 11.11; Mean ± SD) ranged from 66.7% to 100% in June, the beginning of 6‐month growing season. The mean intensity was 6.08 ± 2.19, significantly higher than that (2.74 ± 0.87; P < 0.05) in December (P < 0.05), the end of the growing season. Fish with low fitness had significantly higher number of parasite than the fish with higher fitness (P < 0.005). Although D. aequans negatively influenced host's condition factor, the impact was not enough to lead mortalities in the cultured sea bass in the Black Sea.     

Marteiliosis in mussels: a rare disease?

Among 1280 cultured and wild adult Mediterranean mussels, Mytilus galloprovincialis, collected over a 1 ‐ year surveillance period from the Slovene Adriatic Sea, 0.3% were histologically positive for the presence of Marteilia spp. The infection was concentrated in winter. Employing the molecular methods of PCR, cloning, DNA restriction and sequencing, only Marteilia refringens type M was detected in all the infected mussels. Although all life‐cycle stages of M. refringens severely infected digestive glands, only sporadic disruption of epithelial cells of digestive tubules and focal destruction of digestive tubules were observed in the infected mussels. This was the first detection of M. refringens in M. galloprovincialis from the Slovene Adriatic Sea with the lowest prevalence reported to date. In addition, our results highlight the need for sequencing to complement the established PCR‐RFLP analysis for correct parasite typing.     

Transmission of white spot syndrome virus (WSSV) from Dendronereis spp. (Peters) (Nereididae) to penaeid shrimp

Dendronereis spp. (Peters) (Nereididae) is a common polychaete in shrimp ponds built on intertidal land and is natural food for shrimp in traditionally managed ponds in Indonesia. White spot syndrome virus (WSSV), an important viral pathogen of the shrimp, can replicate in this polychaete (Desrina et al. 2013); therefore, it is a potential propagative vector for virus transmission. The major aim of this study was to determine whether WSSV can be transmitted from naturally infected Dendronereis spp. to specific pathogen‐free (SPF) Pacific white shrimp Litopenaeus vannamei (Boone) through feeding. WSSV was detected in naturally infected Dendronereis spp. and Penaeus monodon Fabricius from a traditional shrimp pond, and the positive animals were used in the current experiment. WSSV‐infected Dendronereis spp. and P. monodon in a pond had a point prevalence of 90% and 80%, respectively, as measured by PCR. WSSV was detected in the head, gills, blood and mid‐body of Dendronereis spp. WSSV from naturally infected Dendronereis spp was transmitted to SPF L. vannamei and subsequently from this shrimp to new naïve‐SPF L. vannamei to cause transient infection. Our findings support the contention that Dendronereis spp, upon feeding, can be a source of WSSV infection of shrimp in ponds.     

Euclinostomum heterostomum and E. ardeolae in tilapia species of Aswan Governorate,Egypt: morphological,molecular, and histopathological characterization

Euclinostomum heterostomum and Euclinostomum ardeolae, both encysted metacercariae (EMC), were found to infect farmed Oreochromis niloticus in the Sahary fish hatchery and wild O. niloticus, Sarotherodon galilaeus, and Tilapia zillii in Lake Nasser, Aswan Governorate, Egypt, at a prevalence of 25.25% and an infection intensity of 1–14 EMC/fish. Macroscopic and microscopic examinations were used to identify them morphologically. PCR amplification, sequencing of the 28S large ribosomal RNA region, and phylogenetic analysis were used for molecular characterization. E. heterostomum and E. ardeolae were isolated from farmed O. niloticus kidneys, peritoneum, abdominal cavity, and under the gills attached to the head bone at prevalence of 13.5% and 6%, respectively, and from wild O. niloticus, S. galilaeus, and T. zillii at a prevalence of 15% and 4.5%, 18% and 10%, and 22.5% and 11.5% respectively. The molecular analyses of rRNA-28S marker revealed that the two Euclinostomum species examined are related yet distinct. This is the first molecular and phylogenetic evidence linking E. ardeolae to Euclinostomum. The present study added two new sequences to the GenBank databases for the genus Eulinostomum from Egypt, with accession numbers MW604803 and MW604806. Euclinostomum spp. have been shown to have detrimental and destructive effects on the kidneys of infected fish; these effects may eventually result in the parasites’ cysts replacing the kidney’s tissues. The kidneys of tilapia spp. infected with Euclinostomum spp. displayed degenerative alterations, with dilated renal tubules associated with migratory tunnels composed primarily of leucocytic infiltrations. Euclinostomum spp. exhibited clear hemophagic characteristics.

     

A simplified method to estimate Diphyllobothrium spp. infection in salmonids

Some fish parasites constitute severe management problems as they may cause mortality of their fish host or are important zoonoses of humans. Parasite assessments are therefore critical to keep track of infections. If conventional sampling techniques can be simplified, parasite assessments might be easier to obtain, less time‐consuming and more extensive. In this study, we compare the assessed number of Diphyllobothrium spp. cysts (CYST) with the counted number of Diphyllobothrium spp. plerocercoid larvae recovered using a conventional digestive technique (LARV). The aim was to determine the potential of using CYST as a simplified methodology for assessing Diphyllobothrium spp. infection in salmonids. In total, 365 brown trout and 424 Arctic charr were sampled from nine lakes in subarctic Norway. Strong correlation, significant linear relationship and large amount of explained variation were found between log₁₀CYST and log₁₀LARV in both fish species. The method had a slight, but not significant tendency to work better in charr compared to trout. In addition, absolute difference between CYST and LARV increased at parasite intensities >100 indicating that the method has reduced functionality when estimating parasite intensity in heavily infected salmonid populations. However, overall, using this simplified and less time‐consuming methodology, a good indication of Diphyllobothrium spp. intensity, abundance and prevalence was obtained. We suggest that this method provides a sound proxy of the Diphyllobothrium spp. burden and have the potential to be used in parasite assessment during fish monitoring and fisheries management surveys, particularly if the time and resources for detailed parasite studies are not available.     

Isolation,characterization and virulence of Mesanophrys sp. (Ciliophora: Orchitophryidae) in farmed swimming crab (Portunus trituberculatus) in eastern China

A disease outbreak occurred in swimming crab (Portunus trituberculatus) farmed in eastern China, with a mortality rate of more than 80%. To further investigate the characteristics and pathogenesis, we reported isolation, characterization and virulence of the causative agent of this disease from 10 sick crabs. Histopathological observation found that multiple tissues, especially haemolymph, contained lots of ciliates. The ciliate was isolated and cultured in vitro, and molecular and morphological studies were done. The results showed that SSU rDNA and LSU rDNA sequences of the ciliate were similar to Mesanophrys ciliates (>96.81%), while ITS1-5.8s-ITS2 sequence was similar to Mesanophrys pugettensis (95.37%) and identical to Orchitophrya stellarum (100%). Furthermore, the results of the morphological study confirmed that the ciliate was similar to Mesanophrys ciliates and O. stellarum cultured in supportive media, but different from O. stellarum cultured in living sperm cells of starfish (Leptasterias spp.). Also, the growth of the ciliate did not interfere with light, which was different from O. stellarum. Accordingly, the ciliate was classified as genus Mesanophrys and temporarily named as Mesanophrys sp. In addition, experimental infection confirmed that Mesanophrys sp. was the pathogen that infected farmed crabs. In summary, Mesanophrys sp. was first isolated and characterized in P. trituberculatus.