Marty R. Deveney

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.

A preliminary phylogenetic analysis of the Capsalidae (Platyhelminthes: Monogenea: Monopisthocotylea) inferred from large subunit rDNA sequences

Phylogenetic relationships within the Capsalidae (Monogenea) were examined using large subunit ribosomal DNA sequences from 17 capsalid species (representing 7 genera, 5 subfamilies), 2 outgroup taxa (Monocotylidae) plus Udonella caligorum (Udonellidae). Trees were constructed using maximum likelihood, minimum evolution and maximum parsimony algorithms. An initial tree, generated from sequences 315 bases long, suggests that Capsalinae, Encotyllabinae, Entobdellinae and Trochopodinae are monophyletic, but that Benedeniinae is paraphyletic. Analyses indicate that Neobenedenia, currently in the Benedeniinae, should perhaps be placed in a separate subfamily. An additional analysis was made which omitted 3 capsalid taxa (for which only short sequences were available) and all outgroup taxa because of alignment difficulties. Sequence length increased to 693 bases and good branch support was achieved. The Benedeniinae was again paraphyletic. Higher-level classification of the Capsalidae, evolution of the Entobdellinae and issues of species identity in Neobenedenia are discussed.

Efficacy of bath and orally administered praziquantel and fenbendazole against Lepidotrema bidyana Murray, a monogenean parasite of silver perch, Bidyanus bidyanus (Mitchell)

We investigated the efficacy of praziquantel (PZQ) and fenbendazole (FBZ), each administered by bath and orally, against the monogenean Lepidotrema bidyana Murray, a gill parasite of the freshwater fish silver perch, Bidyanus bidyanus (Mitchell). PZQ and FBZ were each administered by bath at 10 mg L⁻¹ for 48 h and on surface-coated feed pellets at 75 mg kg⁻¹ per body weight (BW) per day for 6 days. Bath treatments of PZQ and FBZ had an efficacy of 99% and 91%, respectively, against adult L. bidyana. Oral treatments of PZQ and FBZ had an efficacy of 79% and 95%, respectively, against adult L. bidyana. Fish rejected feed pellets surface-coated with PZQ, suggesting that palatability of surface-coated PZQ-medicated feed is poor, which undermined efficacy. In all trials, some juvenile parasites were present on fish after treatment during efficacy assessment, indicating that efficacy may be lower against juvenile parasites or that recruitment occurred post-treatment, demonstrating that repeat treatments are necessary to effectively control L. bidyana in aquaculture.

Gross and histopathological characteristics of two lipomas and a neurofibrosarcoma detected in aquacultured southern bluefin tuna, Thunnus maccoyii (Castelnau), in South Australia.

Three visible lesions were examined from two specimens of southern bluefin tuna. The lesions were examined grossly and two were identified as lipomas, the third bore similarities to a schwannoma. Histopathology confirmed that two consisted of mature adipocytes consistent with a diagnosis of lipoma. The third lesion consisted of spindle cells in Antoni A and B patterns and was tentatively diagnosed as a malignant schwannoma. Immunohistochemistry identified both S100 and glial fibrillary acid protein expression within the lesion, which, together with the histopathological appearance, is consistent with a diagnosis of neurofibrosarcoma.

Uptake, distribution and depuration of paralytic shellfish toxins from Alexandrium minutum in Australian greenlip abalone, Haliotis laevigata

Farmed greenlip abalone Haliotis laevigata were fed commercial seaweed-based food pellets or feed pellets supplemented with 8 × 10⁵ Alexandrium minutum dinoflagellate cells g⁻¹ (containing 12 ± 3.0 μg STX-equivalent 100 g⁻¹, which was mainly GTX-1,4) every second day for 50 days. Exposure of abalone to PST supplemented feed for 50 days did not affect behaviour or survival but saw accumulation of up to 1.6 μg STX-equivalent 100 g⁻¹ in the abalone foot tissue (muscle, mouth without oesophagus and epipodial fringe), which is ∼50 times lower than the maximum permissible limit (80 μg 100 g⁻¹ tissue) for PSTs in molluscan shellfish. The PST levels in the foot were reduced to 0.48 μg STX-equivalent 100 g⁻¹ after scrubbing and removal of the pigment surrounding the epithelium of the epipodial fringe (confirmed by both HPLC and LC-MS/MS). Thus, scrubbing the epipodial fringe, a common procedure during commercial abalone canning, reduced PST levels by ∼70%. Only trace levels of PSTs were detected in the viscera (stomach, gut, heart, gonad, gills and mantle) of the abalone. A toxin reduction of approximately 73% was observed in STX-contaminated abalone held in clean water and fed uncontaminated food over 50 days. The low level of PST uptake when abalone were exposed to high numbers of A. minutum cells over a prolonged period may indicate a low risk of PSP poisoning to humans from the consumption of H. laevigata that has been exposed to a bloom of potentially toxic A. minutum in Australia. Further research is required to establish if non-dietary accumulation can result in significant levels of PSTs in abalone.

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

Abstract 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 hosts 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.

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.

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.

A technique for preserving pigmentation in some capsalid monogeneans for taxonomic purposes

A technique is described to preserve the pigment found in the bodies and the intestine of some brightly coloured and darkly pigmented benedeniine capsalid monogeneans. Previous studies of these pigmented capsalids have proven difficult because the pigmentation usually disappears when the worms are fixed using preservatives containing concentrations of formalin over 5% and/or ethanol, acetic acid, chromic acid, picric acid and mercuric chloride. The technique developed here uses a fixative comprising glycerol, acetone and formalin (GAF). After fixation under light coverslip compression for three minutes, specimens are transferred to absolute acetone for three minutes and cleared in a mixture of nine parts cedar wood oil and one part absolute acetone before mounting in Canada balsam. Processing must be carried out quickly, as these chemicals will cause the pigments to fade if the specimens are exposed to them for too long. Pigmented benedeniines processed using this technique retain the distribution, intensity and colour observed in live worms. The colour and distribution of pigmentation in monogeneans may be of taxonomic importance and this technique aids preparation of whole-mounts suitable for registration as type-material.

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.