DNA profiling reveals Neobenedenia girellae as the primary parasitic monogenean in global fisheries and aquaculture.

Accurate identification of parasite species and strains is crucial to mitigate the risk of epidemics and emerging disease. Species of Neobenedenia are harmful monogenean ectoparasites that infect economically important bony fishes in aquaculture worldwide, however, the species boundaries between two of the most notorious taxa, N. melleni and N. girellae, has been a topic of contention for decades. Historically, identifications of Neobenedenia isolates have overwhelmingly been attributed to N. melleni, and it has been proposed that N. girellae is synonymous with N. melleni. We collected 33 Neobenedenia isolates from 22 host species spanning nine countries and amplified three genes including two nuclear (Histone 3 and 28S rDNA) and one mitochondrial (cytochrome b). Four major clades were identified using Maximum Likelihood and Bayesian inference analyses; clades A-D corresponding to N. girellae, N. melleni, N. longiprostata and N. pacifica, respectively. All unidentified isolates and the majority of Neobenedenia sequences from GenBank fell into clade A. The results of this study indicate that N. girellae is a separate species to N. melleni, and that a large proportion of previous samples identified as N. melleni may be erroneous and a revision of identifications is needed. The large diversity of host species that N. girellae is able to infect as determined in this study and the geographic range in which it is present (23.8426°S and 24.1426°N) makes it a globally cosmopolitan species and a threat to aquaculture industries around the world.

Effects of temperature and salinity on the life cycle of Neobenedenia sp. (Monogenea: Capsalidae) infecting farmed barramundi (Lates calcarifer).

Effective parasite management can be achieved through strategically timed treatments that break the life cycle. We examined the effects of temperature (2 °C increments from 22 to 34 °C) and salinity (0, 11, 22, 35, 40‰) on the life cycle (embryonation period, hatching success, oncomiracidia (larvae) longevity, infection success, and time to sexual maturity) of Neobenedenia sp. (Monogenea: Capsalidae), a harmful ectoparasite of farmed marine fishes. Experiments were conducted in controlled conditions in the laboratory. The life cycle was faster in warm, high saline conditions compared to cooler conditions (10-13 days between 26-32 °C, 40‰; 15-16 days between 22-24 °C at 40‰). Warm seawater and high saline conditions (24-32 °C, 35-40‰) improved egg hatching success, reduced time to sexual maturity, and resulted in parasites reaching sexual maturity at a larger size (at 30-32 °C) compared to cooler conditions (22 °C). In contrast, cool, hypersaline conditions (22 °C, 40‰) increased oncomiracidia longevity and infection success. Linear and quantile regression models were used to construct an interactive, online parasite management interface to enable strategic treatment of parasites in aquaculture corresponding to observed temperature and salinity variation on farms in the tropics. It was recommended that farmers treat their stock more frequently during summer (27-31 °C) when parasites can complete their life cycle more quickly. Nevertheless, farmers should be aware of the potential for increased Neobenedenia sp. infections during winter months (21-26 °C) due to increased infection success.

Temperature alters reproduction and maternal provisioning in a fish ectoparasite.

This study quantified the effects of temperature on reproduction and maternal provisioning of the ectoparasite, Neobenedenia girellae (Platyhelminthes: Monogenea), a species known to cause detrimental impacts to aquaculture fishes in tropical and subtropical environments worldwide. At 20 and 25 °C, parasites exhibited relatively slower production of larger eggs that were energy-dense. In contrast, parasites at 30 °C attained sexual maturity faster, were reproductively active over a shorter period, grew to a smaller size and laid smaller, less energy-rich eggs at a faster rate. As such, parasites exhibited two distinct reproductive patterns in response to temperature: parasites at lower temperatures produced larger eggs with higher energy content, while those at the higher temperature had a higher rate of egg production. Larger eggs produced under cooler conditions were better provisioned with energetic reserves and important, membrane-bound lipids that would likely facilitate larval longevity and development success. This is commensurate with previous observations of epizootics of this parasite species in aquaculture systems during winter. Meanwhile, eggs produced at 30 °C contained higher proportions of saturated fatty acids compared with polyunsaturated fatty acids, likely reflecting metabolic regulation of cell membrane fluidity, which is necessary for larvae to survive warm conditions. This study demonstrates that fish ectoparasites have evolved substantial reproductive and metabolic flexibility to maximise infection success under variable environmental conditions.

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.

Biochemical composition of marine monogenean parasite eggs.

This study on the eggs of the tropical monogenean Neobenedenia girellae presents the first detailed quantitative biochemical information of a marine parasite species' eggs. Moisture and protein composed the majority of the contents of freshly laid eggs (79.12±0.82 and 11.51±0.49% respectively) followed by lipid (2.50±0.15%). Lipids were composed of approximately equal amounts of saturated, monounsaturated, and polyunsaturated fatty acids and the predominant lipid class was triacylglycerol (33.82±1.20%). This study represents a fundamental step towards a better understanding of the early life biology of this important species of parasite.

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.