Kate S. Hutson

Seaweed extracts as a natural control against the monogenean ectoparasite, Neobenedenia sp., infecting farmed barramundi (Lates calcarifer).

Aqueous extracts from common tropical seaweeds were evaluated for their effect on the life cycle of the commercially important ectoparasite, Neobenedenia sp. (Platyhelminthes: Monogenea), through the survival of attached adult parasites, period of embryonic development, hatching success and oncomiracidia (larvae) infection success. There was no significant effect of any extract on the survival of adult parasites attached to fish hosts or infection success by oncomiracidia. However, the extracts of two seaweeds, Ulva sp. and Asparagopsis taxiformis, delayed embryonic development and inhibited egg hatching. The extract of A. taxiformis was most effective, inhibiting embryonic development of Neobenedenia sp. and reducing hatching success to 3% compared with 99% for the seawater control. Furthermore, of the 3% of eggs that hatched, time to first and last hatch was delayed (days 14 and 18) compared with the seawater control (days 5 and 7). Asparagopsis taxiformis shows the most potential for development as a natural treatment to manage monogenean infections in intensive aquaculture with the greatest impact at the embryo stage.

Speciation of the Paradeontacylix spp. (Sanguinicolidae) of Seriola dumerili. Two new species of the genus Paradeontacylix from the Mediterranean

Two new species of teleost blood fluke belonging to the sanguinicolid genus Paradeontacylix are described from the greater amberjack, Seriola dumerili, i.e. Paradeontacylix ibericus n. sp. from the Iberian Peninsula and Paradeontacylix balearicus n. sp. from the Balearic Islands. P. ibericus n. sp. and P. balearicus n. sp. show morphological similarities with Paradeontacylix kampachi and Paradeontacylix grandispinus respectively, which occur in mixed infection in S. dumerili from Japan. Multivariate analysis of morphometrical data provided statistical evidence for the separation of four species. However, component by component analysis did not show statistically significant differences between P. balearicus and P. grandispinus. Molecular data based on rITS2 and mCO1 gene sequences also supported the separation into four species. Morphological and molecular data were used to examine phylogenetic relationships between Paradeontacylix species from S. dumerili and other species in the genus. The results coincided in revealing two main branches with P. kampachi+P. ibericus and (((P. grandispinus+P. balearicus) Paradeontacylix sanguinicoloides) Paradeontacylix godfreyi). Paradeontacylix odhneri, for which little data are available, was located basal in a separate branch. This is the only species of Paradeontacylix which parasitizes a non-carangid host which might probably explain the separation from the other species. Paired similarities between the Japanese and the Mediterranean species, despite the large geographic distance, could be explained by the speciation of parasite geminate lines before host separation by tectonic events. Consequently, geographic and historical isolation support the morphological and genetic differences leading to the evolution of the new species described here.

Efficacy of garlic (Allium sativum) extract applied as a therapeutic immersion treatment for Neobenedenia sp. management in aquaculture

Garlic, Allium sativum L., extract administered as a therapeutic bath was shown to have antiparasitic properties towards Neobenedenia sp. (MacCallum) (Platyhelminthes: Monogenea) infecting farmed barramundi, Lates calcarifer (Bloch). The effect of garlic extract (active component allicin) immersion on Neobenedenia sp. egg development, hatching success, oncomiracidia (larvae) longevity, infection success and juvenile Neobenedenia survival was examined and compared with freshwater and formalin immersion. Garlic extract was found to significantly impede hatching success (5% ± 5%) and oncomiracidia longevity (<2 h) at allicin concentrations of 15.2 μL L(-1) , while eggs in the seawater control had >95% hatching success and mean oncomiracidia longevity of 37 ± 3 h. At much lower allicin concentrations (0.76 and 1.52 μL L(-1)), garlic extract also significantly reduced Neobenedenia infection success of L. calcarifer to 25% ± 4% and 11% ± 4%, respectively, compared with 55% ± 7% in the seawater control. Juvenile Neobenedenia attached to host fish proved to be highly resistant to allicin with 96% surviving 1-h immersion in 10 mL L(-1) (15.2 μL L(-1) allicin) of garlic extract. Allicin-containing garlic extracts show potential for development as a therapy to manage monogenean infections in intensive aquaculture with the greatest impact at the egg and larval stages.

Reproductive strategies of the insidious fish ectoparasite, Neobenedenia sp. (Capsalidae: Monogenea)

Fish monogeneans are lethal parasites in aquaculture. We provide the first experimental evidence that a notorious fish monogenean, Neobenedenia sp., can produce viable eggs in isolation for three consecutive generations. We infected individual, isolated, farmed barramundi, Lates calcarifer (Bloch) with a single oncomiracidium (larva) of the hermaphroditic monogenean Neobenedenia sp. Isolated parasites reached sexual maturity at day 10 post-hatch (24°C, 35‰) and laid ∼3,300 embryonated eggs over 17 days. Egg production rapidly increased following sexually maturity on day 10 (58±15 eggs) and peaked on day 15 (496±68 eggs) before gradually decreasing. Neobenedenia sp. exhibited egg laying and egg hatching rhythms. Parasites laid eggs continuously, but egg production increased in periods of darkness (64.3%), while the majority of oncomiracidia (81%) emerged from eggs in the first three hours of light. Eggs laid by isolated ‘parent’ parasites hatched and individual emerging oncomiracidia were used to infect more individual, isolated fish, with three consecutive, isolated, parasite generations (F1, F2 and F3) raised in the laboratory. Infection success and egg hatching success did not differ between generations. Our data show that one parasite, in the absence of a mate, presents a severe threat to captive fish populations.

Tracking transparent monogenean parasites on fish from infection to maturity

The infection dynamics and distribution of the ectoparasitic fish monogenean Neobenedenia sp. (Monogenea: Capsalidae) throughout its development was examined on barramundi, Lates calcarifer (Bloch) (Latidae), by labelling transparent, ciliated larvae (oncomiracidia) with a fluorescent dye. Replicate fish were each exposed to approximately 50 fluorescent oncomiracidia and then examined for parasites using an epifluorescence stereomicroscope at 10 time intervals post-exposure (15, 30, 60, 120 min, 24, 48 h, four, eight, 12, and 16 days). Fluorescent labelling revealed that parasites attached underneath and on the surface of the scales of host fish. Parasite infection success was 20% within 15 min, and peaked at 93% two days post-exposure, before gradually declining between four and sixteen days. Differences in parasite distribution on L. calcarifer over time provided strong evidence that Neobenedenia sp. larvae settled opportunistically and then migrated to specific microhabitats. Parasites initially attached (<24 h) in greater mean numbers on the body surface (13 ± 1.5) compared to the fins (4 ± 0.42) and head region (2 ± 0.41). Once larvae recruitment had ceased (48 h), there were significantly higher mean post-larvae counts on the head (5 ± 3.4) and fins (12 ± 3) compared to previous time intervals. Neobenedenia sp. aggregated on the eyes, fins, and dorsal and ventral extremities on the main body. As parasites neared sexual maturity, there was a marked aggregation on the fins (22 ± 2.35) compared to the head (4 ± 0.97) and body (9 ± 1.33), indicating that Neobenedenia sp. may form mating aggregations.

Histopathology associated with haptor attachment of the ectoparasitic monogenean Neobenedenia sp. (Capsalidae) to barramundi, Lates calcarifer (Bloch)

[Extract] Capsalid monogeneans are harmful ectoparasites of ornamental and farmed fishes in tropical/subtropical marine environments (Thoney & Hargis 1991; Hirazawa et al. 2011; Whittington 2012). Neobenedenia spp. have low host specificity, a direct life cycle, high fecundity and robust eggs, which contribute to their ability to inflict mass mortalities in aquaculture (Ogawa et al. 1995; Deveney, Chisholm & Whittington 2001; Ruckert, Palm & Klimpel 2008; Whittington 2012). Neobenedenia spp. attach to external surfaces of their host using two attachment organs located anteriorly and one larger posterior attachment organ called the haptor. The haptor is believed to act as the principal anchoring organ of the parasite to the host (Whittington 2012). This organ has chitinous structures that provide mechanical attachment including paired anterior hamuli, accessory sclerites, posterior hamuli and peripheral hooklets (Whittington & Horton 1996). A marginal valve on the haptor allows the organ to create suction on the host. The anterior attachment organs lack accessory chitinous structures and are located directly above the pharynx, which is used to graze on epidermal and mucous cells of the fish (Whittington 2012).

Evidence of multiple species of Chilodonella (Protozoa, Ciliophora) infecting Australian farmed freshwater fishes

Parasitic Chilodonella species, Chilodonella piscicola and Chilodonella hexasticha, cause considerable economic losses globally to freshwater farmed fish production. Some genetic studies of Chilodonella spp. have indicated that many species within the genus may form cryptic species complexes. To understand the diversity of Chilodonella spp. infecting Australian freshwater farmed fish, specimens were isolated from infected barramundi (Lates calcarifer) and Murray cod (Maccullochella peelii) from fish farms in tropical north Queensland (QLD), temperate Victoria (Vic) and New South Wales (NSW) for genetic and morphological analysis. Parasites were stained and measured for morphological description and comparative phylogenetic analyses were performed using the mitochondrial small subunit (mtSSU) rDNA marker. Morphological analyses revealed four distinct morphotypes of Chilodonella infecting farmed barramundi and Murray Cod. Three putative species were isolated from barramundi (Chilodonella hexasticha, C. acuta and C. uncinata) and one from Murray cod (C. piscicola). However, phylogenetic analyses detected only three distinct genotypes, with the putative C. hexasticha and C. piscicola sharing 100% sequence identity. This suggests that Australian isolates of C. hexasticha and C. piscicola could represent the same species and may exhibit phenotypic plasticity. Further molecular analysis, including isolates from the type localities, should be performed to support or refute the synonymy of these species.

Morphological variation in the cosmopolitan fish parasite Neobenedenia girellae (Capsalidae: Monogenea)

Intra-species morphological variation presents a considerable problem for species identification and can result in taxonomic confusion. This is particularly pertinent for species of Neobenedenia which are harmful agents in captive fish populations and have historically been identified almost entirely based on morphological characters. This study aimed to understand how the morphology of Neobenedenia girellae varies with host fish species and the environment. Standard morphological features of genetically indistinct parasites from various host fish species were measured under controlled temperatures and salinities. An initial field-based investigation found that parasite morphology significantly differed between genetically indistinct parasites infecting various host fish species. The majority of the morphological variation observed (60%) was attributed to features that assist in parasite attachment to the host (i.e. the posterior and anterior attachment organs and their accessory hooks) which are important characters in monogenean taxonomy. We then experimentally examined the effects of the interaction between host fish species and environmental factors (temperature and salinity) on the morphology of isogenic parasites derived from a single, isolated hermaphroditic N. girellae infecting barramundi, Lates calcarifer. Experimental infection of L. calcarifer and cobia, Rachycentron canadum, under controlled laboratory conditions did not confer host-mediated phenotypic plasticity in N. girellae, suggesting that measured morphological differences could be adaptive and only occur over multiple parasite generations. Subsequent experimental infection of a single host species, L. calcarifer, at various temperatures (22, 30 and 32 °C) and salinities (35 and 40‰) showed that in the cooler environments (22 °C) N. girellae body proportions were significantly smaller compared with warmer temperatures (30 and 32 °C; P < 0.0001), whereas salinity had no effect. This is evidence that temperature can drive phenotypic plasticity in key taxonomic characters of N. girellae under certain environmental conditions.

Effect of temperature and salinity on egg hatching and description of the life cycle of Lernanthropus latis (Copepoda: Lernanthropidae) infecting barramundi, Lates calcarifer

The parasite Lernanthropus latis (Copepoda: Lernanthropidae) is a major threat to the sustained mariculture of barramundi, Lates calcarifer (Perciformes: Latidae). We investigated the effect of water temperature and salinity on egg hatching success of L. latis and describe the life cycle for the first time. Wild and sea-caged L. calcarifer examined in tropical north Australia exhibited similar parasite prevalence (range: 80-100%) and mean parasite intensity (range: 3-6), whereas land-based maricultured fish were not infected. Hatching success and time to first and last hatch was determined for a range of water temperature (22, 30, 32 and 34°C) and salinity (0, 11, 22, 35 and 40‰) combinations representing current and predicted climate conditions. There was a significant interaction between water temperature and salinity on the hatching success of L. latis nauplii. Eggs hatched in all temperature treatments, with the greatest hatching success at 30°C and 32°C (98 and 92% success, respectively) in 35‰. Hatching did not occur at 0‰ and was severely reduced at 11‰ (1.6% success). Hatching began within 6h at all water temperatures with >95% of eggs hatched within 30h at 30, 32 and 34°C and within 60h at 22°C. Adult parasites differed from the original description by the presence of the parabasal flagellum, small setae on the legs and caudal rami and minor incongruences regarding morphological measurements. The life cycle of L. latis includes three free living stages and five parasitic stages. Although L. latis exhibits broad environmental tolerance, freshwater can be used as an effective management strategy to break the life cycle in aquaculture.

Redescriptions of two species of microcotylid monogeneans from three arripid hosts in southern Australian waters

Microcotyle arripis Sandars, 1945 is redescribed from Arripis georgianus from four localities: Spencer Gulf, Gulf St. Vincent, off Kangaroo Island and Coffin Bay, South Australia, Australia. Kahawaia truttae (Dillon & Hargis, 1965) Lebedev, 1969 is reported from A. trutta off Bermagui, New South Wales and is redescribed from a new host, A. truttaceus, from four localities in South Australian waters: Spencer Gulf, Gulf St. Vincent, off Kangaroo Island and Coffin Bay. Phylogenetic analysis of the partial 28S ribosomal RNA gene (28S rRNA) nucleotide sequences for both microcotylid species and comparison with other available sequence data for microcotylid species across four genera contributes to our understanding of relationships in this monogenean family.