Environment and shipping drive environmental DNA beta-diversity among commercial ports.

The spread of nonindigenous species by shipping is a large and growing global problem that harms coastal ecosystems and economies and may blur coastal biogeographical patterns. This study coupled eukaryotic environmental DNA (eDNA) metabarcoding with dissimilarity regression to test the hypothesis that ship-borne species spread homogenizes port communities. We first collected and metabarcoded water samples from ports in Europe, Asia, Australia and the Americas. We then calculated community dissimilarities between port pairs and tested for effects of environmental dissimilarity, biogeographical region and four alternative measures of ship-borne species transport risk. We predicted that higher shipping between ports would decrease community dissimilarity, that the effect of shipping would be small compared to that of environment dissimilarity and shared biogeography, and that more complex shipping risk metrics (which account for ballast water and stepping-stone spread) would perform better. Consistent with our hypotheses, community dissimilarities increased significantly with environmental dissimilarity and, to a lesser extent, decreased with ship-borne species transport risks, particularly if the ports had similar environments and stepping-stone risks were considered. Unexpectedly, we found no clear effect of shared biogeography, and that risk metrics incorporating estimates of ballast discharge did not offer more explanatory power than simpler traffic-based risks. Overall, we found that shipping homogenizes eukaryotic communities between ports in predictable ways, which could inform improvements in invasive species policy and management. We demonstrated the usefulness of eDNA metabarcoding and dissimilarity regression for disentangling the drivers of large-scale biodiversity patterns. We conclude by outlining logistical considerations and recommendations for future studies using this approach.

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.

Capturing expert uncertainty in spatial cumulative impact assessments.

Understanding the spatial distribution of human impacts on marine environments is necessary for maintaining healthy ecosystems and supporting 'blue economies'. Realistic assessments of impact must consider the cumulative impacts of multiple, coincident threats and the differing vulnerabilities of ecosystems to these threats. Expert knowledge is often used to assess impact in marine ecosystems because empirical data are lacking; however, this introduces uncertainty into the results. As part of a spatial cumulative impact assessment for Spencer Gulf, South Australia, we asked experts to estimate score ranges (best-case, most-likely and worst-case), which accounted for their uncertainty about the effect of 32 threats on eight ecosystems. Expert scores were combined with data on the spatial pattern and intensity of threats to generate cumulative impact maps based on each of the three scoring scenarios, as well as simulations and maps of uncertainty. We compared our method, which explicitly accounts for the experts' knowledge-based uncertainty, with other approaches and found that it provides smaller uncertainty bounds, leading to more constrained assessment results. Collecting these additional data on experts' knowledge-based uncertainty provides transparency and simplifies interpretation of the outputs from spatial cumulative impact assessments, facilitating their application for sustainable resource management and conservation.

Eight habitats, 38 threats and 55 experts: Assessing ecological risk in a multi-use marine region.

Identifying the relative risk human activities pose to a habitat, and the ecosystem services they provide, can guide management prioritisation and resource allocation. Using a combination of expert elicitation to assess the probable effect of a threat and existing data to assess the level of threat exposure, we conducted a risk assessment for 38 human-mediated threats to eight marine habitats (totalling 304 threat-habitat combinations) in Spencer Gulf, Australia. We developed a score-based survey to collate expert opinion and assess the relative effect of each threat to each habitat, as well as a novel and independent measure of knowledge-based uncertainty. Fifty-five experts representing multiple sectors and institutions participated in the study, with 6 to 15 survey responses per habitat (n = 81 surveys). We identified key threats specific to each habitat; overall, climate change threats received the highest risk rankings, with nutrient discharge identified as a key local-scale stressor. Invasive species and most fishing-related threats, which are commonly identified as major threats to the marine environment, were ranked as low-tier threats to Spencer Gulf, emphasising the importance of regionally-relevant assessments. Further, we identified critical knowledge gaps and quantified uncertainty scores for each risk. Our approach will facilitate prioritisation of resource allocation in a region of increasing social, economic and environmental importance, and can be applied to marine regions where empirical data are lacking.

Praziquantel delivery via moist pellets to treat monogenean parasites of yellowtail kingfish Seriola lalandi: efficacy and feed acceptance.

Praziquantel (PZQ) is registered for oral use against Benedenia seriolae infecting Seriola spp. in Japan, but its bitter taste causes poor palatability. Incorporating PZQ in a moist pellet may help mask the flavor to improve intake. Altering delivery, however, may influence efficacy. We assessed the minimum effective concentrations of PZQ in moist pellets delivered by intubation for the monogeneans Zeuxapta seriolae and B. seriolae infecting yellowtail kingfish Seriola lalandi in flow-through tanks. The optimised dose was then evaluated in a sea-cage of S. lalandi to assess feed acceptance and efficacy. During intubation trials, efficacy was assessed as a percent reduction compared to control groups; in the field trial, efficacy was assessed by a percent reduction after treatment. PZQ delivered by intubation at 70 mg kg-1 body weight (BW) for 3 d was 99.7 and 81.7% effective against Z. seriolae and B. seriolae, respectively. Increasing the dose to 120 mg kg-1 BW for 3 d had a similar efficacy against Z. seriolae (98.4%) and increased efficacy against B. seriolae to 89.2%, but partial emesis of the medicated feed was sometimes noted. S. lalandi in a sea-cage at 17°C readily consumed PZQ administered daily in moist pellets at 70 mg kg-1 BW for 3 d (inclusion rate: 5.15 g kg-1), which significantly reduced Z. seriolae and B. seriolae abundance with 99.4 and 81.6% efficacy, respectively. Juvenile B. seriolae were common on the eyes of fish post-treatment which indicates a strategically timed repeat treatment is necessary.

Praziquantel treatment for yellowtail kingfish (Seriola lalandi): dose and duration safety study.

Regulatory approval is being sought to use praziquantel (PZQ) to treat flukes infecting yellowtail kingfish (YTK), but accurate safety data were not available. We investigated the effect of increased doses or prolonged exposure of orally administered PZQ on YTK by assessing changes in haematological and biochemical characteristics, and mortality. Fish were intubated daily for 3 days with 0, 100, 300 and 500 mg PZQ kg(-1) BW day(-1) or once daily for 9 days at 0 and 100 mg PZQ kg(-1) BW day(-1). Blood was taken 24 h after the cessation of treatment. There was no significant difference between any of the haematological or biochemical indices in YTK treated with PZQ and controls, indicating that PZQ is safe for use at 100 mg PZQ kg(-1) BW day(-1) in YTK and that exposure to high doses or prolonged duration does not have negative effects on the YTK haematological or biochemical parameters we measured.

Histological evaluation of sodium percarbonate exposure on the gills of rainbow trout.

Ichthyophthirius multifiliis is a recurring problem in Australian rainbow trout Oncorhynchus mykiss farms and requires strategically timed, repeat treatments for effective management. Sodium percarbonate (SPC) is permitted for use in Australia, with host safety margins based on the toxicity of acute exposures to hydrogen peroxide (HP), the active product released when SPC is added to water. The effects of exposure to HP released by SPC, of repeated doses and of doses exceeding 100 mg l-1 on rainbow trout are unknown. We exposed juvenile rainbow trout (mean weight: 30.5 ± 9 g) to repeated doses of 50, 150 and 250 mg l-1 SPC for 1 h on Days 1, 2, 7 and 8 of a treatment regime. The effect of SPC was assessed by histological evaluation of structural changes in gill tissue. Survival was 100% in all groups, but some fish exposed to 250 mg l-1 SPC displayed impaired swimming performance, and on Day 9 after the final treatment, oedema was present in 9.8% of lamella, which was significantly higher than the mean occurrence of 1.7, 4.2 and 1.3% in fish treated with 0, 50 and 150 mg l-1 SPC, respectively. These changes resolved within 24 h of the cessation of treatment. We conclude that SPC is safe to use on rainbow trout in doses of ≤150 mg l-1 at 17°C, however caution is advised at doses approaching 250 mg l-1. Water temperature, fish age, fish size and maturity, intensity of parasite infection and stocking density could alter the sensitivity of rainbow trout to SPC treatments.

Life cycle and settlement of an Australian isolate of Ichthyophthirius multifiliis Fouquet, 1876 from rainbow trout.

Ichthyophthirius multifiliis Fouquet, 1876, a ciliate parasite, is a cosmopolitan and problematic parasite of cultured freshwater fish. Each geographical isolate of I. multifiliis has variations in life cycle timing under different abiotic water conditions, such as temperature and salinity. We assessed the effects of salinity and temperature on the development and the preferred settlement site of a temperate Australian isolate of I. multifiliis. The time until theront release was significantly different between each temperature; development time was longest at 5 °C with a mean time of 189 h and decreased to a mean time of 11.7 h at 30 °C. At 5 °C our isolate produced a mean of 267 theronts per tomont, which increased to a mean of 493 theronts at 25 °C and reduced to a mean of 288 theronts at 30 °C. Theront length showed an inverse relationship to temperature; mean length was 62 µm at 5 °C and 41 µm at 30 °C. Our isolate reproduced faster at all temperatures and a greater sensitivity to salinity than all reported profiles for temperate isolates. Parasite abundance was highest on the dorsal region of the fish. An accurate understanding of temperature-life cycle information and optimal region to sample for surveillance will aid in the development of specific management plans for the Australian isolate of I. multifiliis, facilitating the strategic timing of treatments.

Minimum effective concentrations of formalin and sodium percarbonate on the free-living stages of an Australian isolate of Ichthyophthirius multifiliis.

Ichthyophthirius multifiliis Fouquet, 1876, a ciliate protozoan, is a common cosmopolitan parasite of freshwater teleosts and is a recurring problem during the summer months on Australian rainbow trout Oncorhynchus mykiss (Walbaum, 1792) farms. Preventative strategies include increasing water flow and filtration, but when an infection is established, chemical intervention is often required. Formalin (FOR) has been traditionally used on Australian trout farms as a treatment for I. multifiliis. Treatment using sodium percarbonate (SPC) that releases hydrogen peroxide when dissolved is being implemented on a number of farms. To assess anecdotal reports of low efficacy we evaluated 1 h exposures of FOR and SPC at 12 °C and 17 °C in both hard and soft water against free-living stages of I. multifiliis. Each free-living stage were exposed to FOR and SPC in vitro; theronts were exposed to 8, 16, 32 or 64 mg/l SPC or FOR every 15 min, for a maximum of 6 h, and the number of live theronts at each time point was recorded. Prototomonts and tomocysts were exposed to 64, 128, 256 and 512 mg/l SPC and 16, 32, 64 and 128 mg/l FOR for 1 h, incubated, with the percentage viability and the number of theronts produced recorded. Theronts were more sensitive to treatment than tomonts, and prototomonts were more sensitive to treatment than tomocysts. FOR and SPC killed all theronts within 15 min at 64 mg/l at both temperatures. FOR was effective against all prototomonts at ≥64 mg/l at both temperatures and was effective against all tomocysts at 128 mg/l at 17 °C but did not achieve complete mortality in any doses tested at 12 °C. SPC was effective against prototomonts and tomocysts at 64 m/l at 17 °C but required ≥256 mg/l at 12 °C. These results can be used to aid development of specific treatment strategies for the management of I. multifiliis on Australian rainbow trout farms.

Validation of a rapid counting method for assessing treatment efficacy against Lepidotrema bidyana infecting silver perch Bidyanus bidyanus.

We used a published sub-sampling method for estimating the abundance of the monogenean Lepidotrema bidyana, a gill parasite of silver perch Bidyanus bidyanus, and determined that it also accurately predicts parasite abundance post-treatment. Post-treatment parasite abundance estimates based on the number of parasites on the first left posterior hemibranch were compared to actual counts on fish after bath and oral treatment trials with praziquantel and fenbendazole. Post-treatment parasite abundance estimates were significantly correlated to real counts of all individual hemibranchs, accurately predicting the parasite abundance on an individual host. There was no significant difference in the post-treatment parasite abundance between individual hemibranchs, however, indicating that the treatment affected L. bidyana abundance on each hemibranch unequally. Use of this method is ineffective at predicting post-treatment abundance; however, it accurately predicts the remaining parasite abundance, aiding evaluation of treatment efficacy, while reducing post-treatment sampling time or facilitating larger sample sizes.

Host impact of monogenean Lepidotrema bidyana infection and intensity estimates for onsite monitoring.

We developed a rapid effective method for accurate estimation of intensity for the monogenean Lepidotrema bidyana, a gill parasite of silver perch Bidyanus bidyanus. This parasite requires monitoring because high-intensity infections reduce host growth and can lead to secondary bacterial and fungal infections. The most accurate method for counting L. bidyana was visual examination of fresh gills. There was a significant relationship between fish size and parasite intensity; however, there was no significant relationship between fish condition and parasite intensity. Parasite intensity estimates were generated by using the mean intensity of worms on the posterior hemibranch on the first left gill arch, compared to the total mean intensity of worms on all hemibranchs. Estimates were validated by predicting L. bidyana intensity from a random sample of silver perch obtained from aquaculture ponds. Parasite intensity estimates correlated strongly to real counts, and this method can be used to accurately predict parasite intensity on an individual host, and thus represents an improvement over previous methods.

New Benedenia species (Monogenea: Capsalidae) from Diagramma labiosum (Perciformes: Haemulidae) on the Great Barrier Reef, Australia, with oncomiracidial descriptions and a report of egg attachment to the host.

The slate sweetlips, Diagramma labiosum Macleay, 1883 (Perciformes: Haemulidae), off Heron Island, Great Barrier Reef, Queensland, Australia, hosts 2 new species of Benedenia Diesing, 1858 (Monogenea: Monopisthocotylea: Capsalidae). Benedenia beverleyburtonae n. sp. infects proximal regions of the primary gill lamellae and gill arches. The adult is characterized by a dorsal vaginal pore anterior to the common genital pore and a voluminous, highly coiled vas deferens. This species also has fine muscle fibrils concentrically arranged in the haptor. Its ciliated oncomiracidium differs little from larvae of other Benedenia species, with the exception of at least 4 gland cells containing a granular secretion on each side of the body at the level of the excretory bladders, with fine ducts opening anterior to the eyes. Benedenia disciliata n. sp. infects gill arches and gill rakers, and the adult is characterized by its small total length, anterior hamuli with a small proximal notch, posterior hamuli with a broad, triangular shape proximally, a conspicuous internal fertilization chamber, and asymmetrical eggs. None of the 5 specimens collected had testes, but their definite absence is undetermined. Their absence and the fact that the vas deferens was discernable only distally in the specimens of B. disciliata examined may represent atrophying of the male organs. Benedenia disciliata is unique among Benedenia species because eggs are attached to the host's gill arches by tight wrapping of the appendages around spines on the gill rakers and the larva is not ciliated. Among Capsalidae species, these features are shared only with species of Dioncus Goto, 1899. Recent molecular evidence has indicated Benedenia is polyphyletic, but with no clear morphological characters available to divide the genus, the 2 new species fit the current concept for the genus more closely than other capsalid genera. Proposal and description of these taxa and accounts of their oncomiracidia and other aspects of their biology indicate potentially useful characters for division of the genus in the future.

Revision of Dioncopseudobenedenia Yamaguti, 1965 including the description of D. ancoralis sp. n. (Monogenea: Capsalidae) from Pacific acanthuroid teleosts.

Dioncopseudobenedenia Yamaguti, 1965 (Monogenea: Capsalidae) is redefined. Dioncopseudobenedenia kala Yamaguti, 1965 (type species) is redescribed from type material from Hawaii and from new specimens from Heron Island, Queensland, Australia and New Caledonia. We made detailed observations on D. macracantha Yamaguti, 1968 from type material from Hawaii, and from new material from Heron Island and New Caledonia. Dioncopseudobenedenia ancoralis sp. n. is described from the gill chamber of Siganus lineatus (Valenciennes) from Green Island and Heron Island, Australia and from New Caledonia. This study confirms that only one pair of large central sclerites is present on the haptor in Dioncopseudobenedenia species. The male copulatory organ in species of Dioncopseudobenedenia is a penis contained in a fluid-filled space (= penis canal) with weakly muscular walls. Dioncopseudobenedenia kala and D. ancoralis bear a sclerite at the tip of the penis. In D. macracantha, the structure of the penis, which has no terminal sclerite, indicates it may combine the functions of a penis and a cirrus. Dioncopseudobenedenia is compared with Calicobenedenia Kritsky et Fennessy, 1999, the other capsalid genus with a single pair of large sclerites on the haptor. The large haptoral sclerites in species of Dioncopseudobenedenia resemble accessory sclerites, whereas those of C. polyprioni Kritsky et Fennessy, 1999 resemble hamuli. Observations of oncomiracidia confirmed that the large haptoral sclerites in D. kala are accessory sclerites. Haptoral morphology suggests that different Dioncopseudobenedenia spp. employ different means of attachment. Mating behaviour was observed twice between two different pairs of D. kala specimens from Heron Island. Two preserved specimens from Nouméa, New Caledonia had structures near the dorsal vaginal pore that we interpret as spermatophores. This is the first report of spermatophores in a capsalid inhabiting the gill chamber. The geographic distribution of Dioncopseudobenedenia spp. is discussed.

Toward routine, DNA-based detection methods for marine pests.

Marine pest incursions can cause significant ongoing damage to aquaculture, biodiversity, fisheries habitat, infrastructure and social amenity. They represent a significant and ongoing economic burden. Marine pests can be introduced by several vectors including aquaculture, aquarium trading, commercial shipping, fishing, floating debris, mining activities and recreational boating. Despite the inherent risks, there is currently relatively little routine surveillance of marine pest species conducted in the majority of countries worldwide. Accurate and rapid identification of marine pest species is central to early detection and management. Traditional techniques (e.g. physical sampling and sorting), have limitations, which has motivated some progress towards the development of molecular diagnostic tools. This review provides a brief account of the techniques traditionally used for detection and describes developments in molecular-based methods for the detection and surveillance of marine pest species. Recent advances provide a platform for the development of practical, specific, sensitive and rapid diagnosis and surveillance tools for marine pests for use in effective prevention and control strategies.