Reproductive Strategies of the Insidious Fish Ectoparasite, Neobenedenia sp. (Capsalidae: Monogenea) Truong Dinh Hoai1,2, Kate S. Hutson1* 1 Marine Parasitology Laboratory, Centre for Sustainable Tropical Fisheries and Aquaculture and the School of Marine and Tropical Biology, James Cook University, Queensland, Australia, 2 Aquatic Environment and Fish Pathology Department, Faculty of Animal Science and Aquaculture, Vietnam National University of Agriculture, Hanoi, Vietnam Abstract 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 (24uC, 35%) and laid ,3,300 embryonated eggs over 17 days. Egg production rapidly increased following sexually maturity on day 10 (58615 eggs) and peaked on day 15 (496668 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. Citation: Dinh Hoai T, Hutson KS (2014) Reproductive Strategies of the Insidious Fish Ectoparasite, Neobenedenia sp. (Capsalidae: Monogenea). PLoS ONE 9(9): e108801. doi:10.1371/journal.pone.0108801 Editor: La´szlo´ Orba´n, Temasek Life Sciences Laboratory, Singapore Received March 18, 2014; Accepted August 31, 2014; Published September 29, 2014 Copyright: ß 2014 Dinh Hoai, Hutson. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. Data are available from the James Cook University Research Database: https://research.jcu.edu.au/researchdata/published/detail/d9eda2a2d49ef0b1ab4de74a2c77c826/. Funding: This work was funded by an AusAID Scholarship (www.ausaid.gov.au) awarded to TDH. The Australian Society for Parasitology (http://parasite.org.au/) provided a Student Travel Award for TDH to present this research at the annual conference in Launceston, Tasmania, Australia in 2012. The funders had norolein study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: [email protected] Introduction reproductive potential when a parasite finds itself alone on a host. Monogeneans also exhibit egg laying and egg hatching rhythms, Monogeneans exhibit sophisticated life history strategies in which can reduce the risk of predation and coincide with host order to ensure their survival in contrasting and unpredictable behaviours to ensure infection success. Monogeneans maximise environments. Evolutionary strategies include multiple reproduc- their chances of finding a host by releasing eggs into the tive mechanisms, predator avoidance and behavioural responses to environment during certain times of the day or night [7], host and environmental cues that favour enhanced infection extending the hatching period [11], responding to hatching cues success. In wild populations these strategies ensure some parasites such as shadows [12], chemicals [11,13], mechanical disturbance survive to the next generation, whereas in captive populations, [14–16] and osmotic changes [17], most of which are generated by where host organisms are confined in high densities, it can lead to the host. parasite epizootics. Neobenedenia are marine capsalid monogeneans of critical Various reproductive mechanisms have been observed in concern to aquaculture because they exhibit several life history monogeneans including oviparity [1], viviparity [2] and self- traits that aid their survival. Neobenedenia spp. have direct life fertilisation [3]. Most oviparous monogeneans deposit fewer than cycles with short generation times [18,19] and low host specificity 100 eggs/parasite/day [4–7], although some species can produce [20,21] which has resulted in major stock losses in several more than 550 eggs/parasite/day [8]. In viviparous monogeneans, aquaculture fish species (see [18,22–24]). Furthermore, attached up to three consecutive generations can develop inside the mother parasite stages are transparent, which can reduce predation by parasite. For example, a single individual Gyrodactylus salaris cleaner organisms [21]. Eggs are encapsulated by a proteinaceous bears the first daughter within 24 hours of the birth of the parent shell which confers protection to the developing embryo from [9] and thus has the capacity to produce six million offspring in digestion [25], most chemicals [26–29] and bacteria [1]. Capsalid four weeks [1]. Self-fertilisation is known to occur in monogenean monogeneans are hermaphrodites, displaying several types of species that infect the bladder of amphibians [3,10] and ensures reproduction including mutual cross-insemination [30], attach- PLOS ONE | www.plosone.org 1 September 2014 | Volume 9 | Issue 9 | e108801 Reproductive Strategies of Neobenedenia ment of spermatophores to other individuals [31] and self- monitored daily under a stereomicroscope using both transmitted insemination (as observed by the copulatory organ lodged in the and incident light. When eye spots were observed in the eggs, parasite’s own uterus; see [30,32]). However, no studies have monitoring was increased to every 2 h in order to obtain newly experimentally examined whether fish monogeneans can success- hatched oncomiracidia. Individual oncomiracidia ,4 h old were fully reproduce in isolation and produce viable eggs and larvae. gently aspirated using a fine-tip glass pipette under a stereomi- The aim of this research was to experimentally examine the croscope and introduced to a 10 L aquarium containing an reproductive strategies of Neobenedenia. Specifically we sought to individual L. calcarifer in 6 L of sea water. Each oncomiracidium determine: 1) whether Neobenedenia can reproduce in isolation; 2) was released at the bottom of the aquarium to avoid the effects of Neobenedenia fecundity over time; 3) whether Neobenedenia surface tension and currents which can trap and kill oncomiracidia exhibit egg laying rhythms and/or hatching rhythms. We used a [1]. When the oncomiracidia were introduced, air supply to the barramundi, or Asian seabass, Lates calcarifer – Neobenedenia sp. fish was stopped for 1 h in order to reduce water currents and model system for our experiments. Barramundi (Perciformes: thereby increase infection success [19]. Thirty replicates were Latidae) are distributed in estuaries and coastal seas from south- made at room temperature (24.360.2uC). Salinity in each western India to north-eastern Australia, with approximate aquarium was checked using a refractometer every 24 h and latitudes of 625u [33]. This species is among the most important adjusted by adding distilled water to maintain 3561%. food fishes in tropical Australasia and is farmed throughout eastern To determine the onset of egg production, a piece of 5 cm2 fine and western Asia (China, India, Israel, Indonesia, Malaysia, gauge (0.560.5 mm) netting was immersed in each aquarium and Philippines, Singapore, Tahiti, Taiwan, Thailand) and Australia checked daily under a stereomicroscope. Eggs have filamentous [34]. High intensities of Neobenedenia on farmed fish damage host strings which easily entangle on netting [40]. The day that eggs epidermis through attachment and feeding [19,23,35] and were observed on the netting was recorded as time to sexual increase secondary infections [36,37]. maturity. Eggs always entangle on netting on the day of sexual maturity (AK Brazenor, unpublished data). Netting was renewed Materials and Methods daily and any suspended eggs remaining in the sea water were collected daily by filtering the solution through a 60 mm mesh. Ethics Statement In order to confirm isolated fish were infected by a single, This work was conducted using a barramundi, Lates calcarifer – individual parasite, fish were immersed in fresh water at the end of Neobenedenia sp. model system with all procedures approved by the experiment, which kills Neobenedenia [see 35]. Fish were the James Cook University Animal Ethics Committee (A1579). bathed in 1 L of fresh water containing a mild sedative (Aqui-S Neobenedenia sp. used in experiments were collected from private 1:1000) for 5 min. The fresh water solution and the body surface land in north Queensland, Australia. Future permissions should be of the fish were examined twice under a stereomicroscope to directed to Coral Coast Barramundi Pty Ltd. collect detached and attached parasites, respectively. We sought to determine