Characterisation of multiple Caribbean ciguatoxins and congeners in individual specimens of horse-eye jack (Caranx latus) by high-performance liquid chromatography/mass spectrometry

We studied the variation in toxin profiles of purified extracts of 10 individual specimens and two pools of ciguateric Caranx latus. High-performance liquid chromatography/mass spectrometry (HPLC/MS) identified in all individual samples at least seven Caribbean ciguatoxins (C-CTXs) comprising C-CTX-1 and its epimer C-CTX-2 ([M + H](+) m/z 1141.58), and five new C-CTX congeners with pseudo-molecular ions at m/z 1141.58, 1143.60, 1157.57, 1159.58, and 1127.57. In some samples, additional C-CTX isomers were detected with [M + H](+) ions at m/z 1141.58 (two), 1143.60 (one) and 1157.57 (two). The two low-toxic pools contained only four to six ciguatoxins. The comparison in relative proportions of four different mass classes ([M + H](+) at m/z 1141, 1143, 1157 and 1127) showed that the group at m/z 1157 increased (2-20%) with flesh toxicity. More than 80% of group m/z 1141 comprised C-CTX-1, C-CTX-2 and their isomer C-CTX-1 a whose level in this group correlated with fish toxicity. Contrary to low-toxic fishes, high-risk specimens had C-CTX-1 levels

Lepocreadiidae (Digenea) of Australian coastal fishes: new species of Opechona Looss, 1907, Lepotrema Ozaki, 1932 and Bianium Stunkard, 1930 and comments on other species reported for the first time or poorly known in Australian waters

Opechona austrobacillaris n, sp. is described from Pomatomus saltatrix from marine sites off Western Australia and New South Wales, Australia. It differs from O. bacillaris in its elongate outline, small ventral sucker, longer pseudoesophagus (relative to the oesophagus), relatively shorter ventral sucker to ovary distance and the relatively longer post-testicular region. Lepotrema monile n. sp. is described from Pomacentrus wardi from Heron Island, Queensland. It differs from its congeners in the sphincter around the distal metraterm and the more-or-less oval ovary. Bianium spongiosum n. sp, is described from Ostracion cubicus from Lizard Island, Queensland. It differs from its congeners in lacking lateral flaps in the forebody, but in having large, internal spongiform patches in the lateral forebody. The following species are redescribed from Australian sites: Lepocreadium oyabitcha from Abudefduf whitleyi, Lizard Island; Clavogalea trachinoti from Trachinotus botla, Heron Island and T. coppingeri, New South Wales, Stradbroke Island, Queensland and Heron Island; Myzoxenus insolens from Notolabrus parilus, Western Australia; Bulbocirrus aulostomi from Aulostomus chinensis, Heron Island; Lepocreadioides orientalis [new synonyms: Bicaudum interruptum Bilqees, 1973; Lepocreadioides interruptum (Bilqees, 1973) Madhavi, Narasimhulu & Shameem, 1986; Lepocreadioides discum Wang, 1986; Lepocreadioides sp. of Karyakarte & Yadav (1976)] from Cynoglossus bilineata, Moreton Bay, Queensland; Hypocreadium patellare from Sufflamen chrysopterus, Heron Island; Echeneidocoelium indicum from Echeneis naucrates, Heron Island; Multitestis pyriformis from Epinephelus cyanopodus, Heron Island; Pseudopisthogonoporus vitellosus from Naso brevirostris, Heron Island; and Bianium hispidum from Torquigener whitleyi and T. pleurogramma, southern Queensland. Only M. solens and M. pyriformis have been reported from Australian waters before; both are new host records.

The status of the genera Hysterolecithoides Yamaguti, 1934, Neotheletrum Gibson & Bray, 1979 and Machidatrema Leon-Regagnon, 1998 (Digenea : Hemiuroidea), including a description of M. leonae n. sp from Australian waters

A diagnosis is given for the lecithasterid genus Hysterolecithoides Yamaguti, 1934, which is now found to have two to six (possibly seven) vitelline masses. The species H. frontilatus (Manter, 1969) is returned to the genus, having been considered a member of the bunocotylid genus Neotheletrum by recent authors. It is redescribed from Siganus nebulosus, Moreton Bay, and S. doliatus, Lizard Island, Great Barrier Reef and New Caledonia, with emphasis on the presence of Juel's organ, a uterine seminal receptacle and the blind sac associated with the genital atrium. It differs from its congeners in the trajectory of the pars prostatica which recurves dorsally to the sinus-sac. Oligolecithoides Shen, 1982 is synonymised with Hysterolecithoides and O. trilobatus Shen, 1982 is synomised with H. epinepheli Yamaguti, 1934. Machidatrema Leon-Regagnon, 1998 is diagnosed, and found to be close to Hysterolecithoides, but differs in the lack of a blind-sac projecting from the dorsal genital atrium, by its tandem testes, the coiling of the uterus between the testes and the ovary, and the ventral excretory pore. M. leonae n. sp. is described from Siganus fuscescens, S. lineatus, S. doliatus, S. corallinus, S. vulpinus and Scarus globiceps at Heron Island, Queensland. It differs from its closest congener, M. akeh, in the muscular and tegumental flap over the genital pore and details of the terminal genitalia. M. chilostoma (Machida, 1980) and M. kyphosi (Yamaguti, 1970) are redescribed from Kyphosus vaigiensis from Heron Island. Neotheletrum Gibson & Bray, 1979 is diagnosed: it differs from Hysterolecithoides in its confluent excretory arms, blind seminal receptacle (no Juel's organ) and uniformly tripartite vitellarium. A cladistic analysis suggests that M. chilostoma and M. kyphosi are not best accommodated in Machidatrema, that Machidatrema (sensu stricto) is monophyletic and that Hysterolecithoides is paraphyletic. Hysterolecithoides and Machidatrema are considered hysterolecithine lecithasterids, whilst Neotheletrum is retained as an opisthadenine bunocotylid.

Echinobothrium chisholmae n. sp (Cestoda, Diphyllidea) from the giant shovel-nose ray Rhinobatos typus from Australia, with observations on the ultrastructure of its scolex musculature and peduncular spines

Echinobothrium chisholmae n. sp. is described from Rhinobatos typus Bennett (Rhinobatidae), collected from Heron Island, Great Barrier Reef, Australia. E. chisholmae differs from all congeners in possessing 11 hooks in each dorsal and ventral group on the rostellum and groups of 3-6 hooklets on either side of the hooks. A single metacestode of E. chisholmae was collected from the decapod crustacean Penaeus longistylus Kubo. Yellow pigmentation of the cephalic peduncle in immature adults is caused by the accumulation of large vesicles in the distal cytoplasm of the tegument. The vesicles probably provide materials for spine formation. Ultrastructural examination of the rostellar musculature revealed that the muscles are stratified (striated-like), consisting of a periodic repetition of sarcomeres separated by perforated Z-like lines that are oblique to the long axes of the myofilaments.

Anthropogenic and Natural Organohalogen Compounds in Blubber of Dolphins and Dugongs (Dugong dugon) from Northeastern Australia

A range of organohalogen compounds (10 polychlorinated biphenyl [PCB] congeners, DDT and metabolites, chlordane-related compounds, the potential natural organochlorine compound Q1, toxaphene, hexachlorobenzene, hexachlorocyclohexanes, dieldrin, and several yet unidentified brominated compounds) were detected in the blubber of four bottlenose dolphins (Tursiops truncatus), one common dolphin (Delphinus delphis), and seven dugongs (Dugong dugon), as well as in adipose tissue of a green turtle (Chelonia mydas) and a python (Morelia spilota) from northeast Queensland (Australia). The green turtle and dugongs accumulated lower organohalogen levels than the dolphins. Lower levels in dugongs were expected because this species is exclusively herbivorous. Highest PCB and DDT levels recorded in dugongs were 209 and 173 mug/kg lipids, respectively. Levels of the nonanthropogenic heptachlorinated compound Q1 (highest level in dugongs was 160 mug/kg lipids) were estimated using the ECD response factor of trans-nonachlor. Highest organohalogen levels were found in blubber of dolphins for sumDDT (575-52,500 mug/kg) and PCBs (600-25,500 mug/kg lipids). Furthermore, Q1 was a major organohalogen detected in all samples analyzed, ranging from 450 -9,100 mug/kg lipids. The highest concentration of Q1 determined in this study represents the highest concentration reported to date in an environmental sample. Levels of chlordane-related compounds were also high (280-7,700 mug/kg, mainly derived from trans-nonachlor), but concentrations of hexachlorobenzene, hexachlorocyclohexanes, dieldrin, and toxaphene were relatively low and contributed little to the overall organohalogen contamination. Furthermore, a series of three major (BC-1, BC-2, and BC-3) and six minor (BC-4 through BC-9) unknown brominated compounds were observable by extracting m/z 79 and m/z 81 from the GC/ECNI-MS full scan run. Structural proposals were made for the two major recalcitrant compounds (referred to as BC-1 and BC-2). BC-2 appears to be a tetrabromo-methoxy-diphenylether (512 u) and BC-1 has 14 u (corresponding with an additional CH2 group) more relative to BC-1. In general the organohalogen pattern observed in blubber of dolphins was different compared to similar samples from other locations in the world, which is apparent from the fact that the four major abundant signals in the GC/ECD chromatogram. of D. delphis originated from the four unknown compounds Q1, BC-1, BC-2, and BC-3.

PCDDs in the water/sediment-seagrass-dugong (Dugong dugon) food chain on the Great Barrier Reef (Australia)

Polychlorinated dibenzo-p-dioxin (PCDD) and dibenzofuran (PCDF) concentrations were measured in sediment and seagrass from five locations in or adjacent to the Great Barrier Reef Marine Park. A full spectrum of Cl(5-8)DDs were present in all samples and, in particular, elevated levels of Cl8DD were found. PCDFs could not be quantified in any samples. The PCDD concentrations ranged over two orders of magnitude between sites, and there was a good correlation between sediment and seagrass levels. There were large quantities of sediment present on the seagrass (20-62% on a dry wt. basis), and it was concluded that this was a primary source of the PCDDs in the seagrass samples. The PCDD levels in the seagrass samples were compared with the levels in the tissue of three dugongs stranded in the same region. The relative accumulation of the 2,3,7,8-substituted PCDD congeners in the dugongs decreased by over two orders of magnitude with increasing degree of chlorination. This was attributed to the reduced absorption of the higher chlorinated congeners in the digestive tract, a behaviour that has been observed in other mammals such as domestic cows. (C) 2001 Elsevier Science Ltd. All rights reserved.

Monostephanostomum georgianum n. sp (Digenea : Acanthocolpidae) from Arripis georgianus (Valenciennes) (Perciformes : Arripidae) off Kangaroo Island, South Australia, with comments on Monostephanostomum Kruse, 1979 and Stephanostomum Looss, 1899

Monostephanostomum georgianum n. sp. is described from Arripis georgianus off Kangaroo Island, South Australia. It differs from its congeners by the presence of a short second row of oral spines. M. manteri Kruse, 1979 is reported from A. georgianus off southern Western Australia and Kangaroo Island, South Australia and A. trutta off northern Tasmania. It is considered that the other two species, M. yamagutii Ramadan, 1984 and M. krusei Reimer, 1983, should probably be removed from this genus. Two new combinations are formed, M. gazzae (Shen, 1990) n. comb. (from Stephanostomum) and M. mesospinosum (Madhavi, 1976) n. comb. (from Stephanostomum). A key to the four recognised species of Monostephanostomum is given.

Analysis of toxin profiles in three different fish species causing ciguatera fish poisoning in Guadeloupe, French West Indies

A grey snapper (Lutjanus griseus), a grouper (Serranidae) and a blackjack (Caranx lugubris) were implicated in three different ciguatera poisonings in Guadeloupe, French West Indies. A mouse bioassay indicated toxicity for each specimens: 0.5-1, greater than or equal to 1 and > 1 M Ug g(-1), respectively. After purification by gel filtration chromatography, the samples were analysed by high-performance liquid chromatography coupled to mass spectrometry (LC-MS). The toxin profiles differ from one fish to another. C-CTX-1 was detected at 0.24, 0.90 and 13.8 ng g(-1) flesh in the snapper, grouper and jack, respectively. It contributed only to part of the whole toxicity determined by the mouse bioassay. Other toxins identified were C-CTX-2 (a C-CTX-1 epimer), three additional isomers of C-CTX-1 or -2, and five ciguatoxin congeners (C-CTX-1127, C-CTX-1143 and its isomer C-CTX-1143a, and C-CTX-1157 and its isomer C-CTX-1157b). Putative hydroxy-polyether-like compounds were also detected in the flesh of the grouper with [M+ + H](+) ions at m/z 851.51, 857.50, 875.51, 875.49 and 895.54 Da. Some of these compounds have the same mass range as some known dinoflagellate toxins. In conclusion, this study confirms the usefulness of LC-MS analysis to determine the ciguatoxins levels and the toxin profile in fish flesh hazardous to humans.

A new species of Phyllurus (Lacertilia : Gekkonidae) and a revised phylogeny and key for the Australian leaf-tailed geckos

Phyllurus gulbaru, sp. nov., is a highly distinct species of leaf-tailed gecko restricted to rocky rainforest of Pattersons Gorge, north-west of Townsville. The possession of a cylindrical, non-depressed, tapering original and regenerated tail separates P. gulbaru from all congeners except P. caudiannulatus. From this species P. gulbaru is separated by having a partially divided, as opposed to fully divided, rostral scale. Furthermore, the very small spinose body tubercles of P. gulbaru are in marked contrast to the large spinose body scales of P. caudiannulatus. An analysis of 729 bp of mitochondrial 12S rRNA and cytochrome b genes reveals P. gulbaru to be a deeply divergent lineage with closer affinities to mid-east Queensland congeners than the geographically neighbouring P. amnicola on Mt Elliot. In conservation terms, P. gulbaru is clearly at risk. Field surveys of Pattersons Gorge and the adjacent ranges indicate that this species is restricted to a very small area of highly fragmented habitat, of which only a small proportion receives a degree of protection in State forest. Further, there is ongoing, unchecked destruction of dry rainforest habitat by fire. Under current IUCN criteria, P. gulbaru warrants an Endangered ( B1, 2) listing.

New species of Opechona Looss, 1907 and Cephalolepidapedon Yamaguti, 1970 (Digenea: Lepocreadiidae) from fishes off northern Tasmania

Two new species of lepocreadiid trematodes are described from teleost fishes from off the coast of northern Tasmania. Opechona kahawai sp. nov. from Arripis sp. (Arripidae) differs from congeners by a combination of a longer prepharynx, longer excretory vesicle and the genital pore antero-sinistral to the ventral sucker. Cephalolepidapedon warehou sp. nov. from Seriolella punctata (Centrolophidae) differs from its only congener in the vitellarium reaching into the posterior forebody, a heavy concentration of eye-spot pigment in the forebody, a relatively narrower and more elongate body, a longer prepharynx and a more distinct oesophagus.

Lepocreadiidae (Digenea) from the batfish of the genus Platax Cuvier (Teleostei : Ephippidae) from the southern Great Barrier Reef, Queensland, Australia

The following species are described from Platax spp.: Neomultitestis aspidogastriformis n. sp., from P. teira, off Heron Island, Queensland, which can be distinguished from its congeners by the transversely elongate ventral sucker divided into three loculi and probably by testis number; Multitestis magnacetabulum Mamaev, 1970, from P. teira, off Heron Island, Queensland; Diploproctodaeum rutellum ( Mamaev, 1970), from P. teira, off Heron Island, Queensland; Diploproctodaeum tsubameuo n. sp., from P. batavianus, from the Swain Reefs, off Queensland, which differs from its congeners in its overlapping, posteriorly attenuated testes and 38-55 ovarian lobes; and Diplocreadium sp., from P. batavianus, from the Swain Reefs, off Queensland.

Identification of slow and fast-acting toxins in a highly ciguatoxic barracuda (Sphyraena barracuda) by HPLC/MS and radiolabelled ligand binding

A barracuda implicated in ciguatera fish poisoning in Guadeloupe was estimated to have an overall flesh toxicity of 15 MUg/g using mouse bioassay. A lipid soluble extract was separated into two toxic fractions, FrA and FrB, on a LH20 Sephadex column eluted with dichloromethane/methanol (1:1). When intraperitoneal injected into mice, FrA provoked symptoms characteristic of slow-acting ciguatoxins, whereas FrB produced symptoms indicative of fast-acting toxins (FAT). High performance liquid chromatography/mass spectrometry/radio-ligand binding (HPLC/MS/RLB) analysis confirmed the two fractions were distinct, because only a weak overlap of some compounds was observed. HPLC/MS/RLB analysis revealed C-CTX-1 as the potent toxin present in FrA, and two coeluting active compounds at m/z 809.43 and 857.42 in FrB, all displaying the characteristic pattern of ion formation for hydroxy-polyethers. Other C-CTX congeners and putative hydroxy-polyether-like compounds were detected in both fractions, however, the RLB found them inactive. C-CTX-1 accounted for >90% of total toxicity in this barracuda and was confirmed to be a competitive inhibitor of brevetoxin binding to voltage-sensitive sodium channels (VSSCs) with a potency two-times lower than P-CTX-1. However, FAT active on VSSCs and

A new species of Glaphyromorphus (Reptilia : Scincidae) from Mt Elliot, north-eastern Queensland

Glaphyromorphus clandestinus, sp. nov., is described from granite-slab habitat on Mt Elliot, north-eastern Queensland. This species can be distinguished from its congeners by a combination of the following characters: large size (SVL 72 mm), adpressed limbs of adult separated by noticeably more than the length of the forelimb, 26 mid-body scale rows, and flanks patterned with dark flecks forming a series of longitudinal lines. The distribution, habitat preferences and habits of this species are poorly known. Currently G. clandestinus is known from a single locality where individuals have been found in an exposed area of exfoliating granite, set in a mosaic of rainforest and eucalyptus woodland. The discovery of this species brings to three the number of vertebrate species known to be endemic to Mt Elliot and highlights the evolutionary significance of this southerly outlier to the mountainous rainforest of the Wet Tropics.

Tetra- and tribromopbenoxyanisoles in Marine Samples from Oceania

Some methoxylated polybrominated diphenyl ethers (MeO-BDEs) are known halogenated natural products (HNPs) and are frequently detected in higher organisms of the marine environment. In this study we demonstrate that a prominent MeO-BDE, previously detected in marine mammals from Australia, is identical to 3,5-dibromo-2-(2',4'-dibromo)phenoxyanisole(BC-3,6-MeO-BDE47). Up to 1.9mg/ kg of 6-MeO-BDE 47 was present in cetaceans from Australia, 0.2-0.3 mg/kg in two crocodile eggs from Australia, but concentrations of 1 or 2 orders of magnitude lower were found in shark liver oil from New Zealand and in marine mammals from Africa and the Antarctic. Concentrations of 6-MeO-BDE47 in samples from Australia were in the same range as anthropogenic pollutants such as PCB 153 and p,p'-DDE. Along with 6-MeO-BDE 47 and the known HNP 4,6-dibromo-2-(2',4'-dibromo)phenoxyanisole (BC-2,2'-MeO-BDE 68), several tribromophenoxyanisoles (MeO-triBDE) were present in tissue of Australian cetaceans. To determine their structure, abiotic debromination experiments were performed using 6-MeO-BDE 47 and 2'-MeO-BDE 68 and superreduced di cyanocobalamine. These experiments resulted in formation of eight MeO-triBDEs, all of which were detected in the cetacean samples. Five of these eight MeO-triBDEs could be identified based on two standard compounds as well as gas chromatographic and mass spectrometric features. It was also shown that the first eluting isomer (compound 1), 6-MeO-BDE 17 (compound 2), and 2-MeO-BDE 39 (compound 5) were the most prominent MeO-triBDEs in the Australian cetacean samples. The concentrations of the MeO-triBDEs in two cetacean samples were 0.20 and 0.36 mg/kg, respectively. Although the reductive debromination with dicyanocobalamine resulted in a different congener pattern than was found in the marine mammals, it could not be excluded that the tribromo congeners of 6-MeO-BDE 47 and 2'-MeO-BDE 68 in the samples were metabolites of the latter.

Two new hemiurine species (Digenea : Hemiuridae) from Spratelloides robustus Ogilby (Clupeiformes : Clupeidae) off south-western Australia and records of Parahemiurus merus (Linton, 1910) from Australian and New Caledonian waters

Two new species of hemiurine hemiurid are described from Spratelloides robustus off Woodman Point in southern Western Australia. Hemiurus lignator n. sp. differs from its congeners by a combination of similar-sized suckers, long sinus-sac, tandem testes, relatively elongate shape and unthickened seminal vesicle wall. Parahemiurus xylokopos n. sp. differs from its congeners in a combination of its squat form, its distinctly lobed vitellarium and the proximity of the gonads to the ventral sucker. P. merus (Linton, 1910) is reported from Acanthopagrus australis, Pomatomus saltatrix and Trachinotus coppingeri off northern New South Wales, Caranx sexfasciatus, Scorpis lineolata, Siganus nebulosus, Thunnus tonggol and T. coppingeri off southern Queensland, Cephalopholis boenak and Euthynnus affinis off Heron Island, southern Great Barrier Reef, P. saltatrix off southern Western Australia and Priacanthus hamrur off New Caledonia.