Molecular phylogeny and evolution of the proteins encoded by coleoid (cuttlefish, octopus, and squid) posterior venom glands.

In this study, we report for the first time a detailed evaluation of the phylogenetic history and molecular evolution of the major coleoid toxins: CAP, carboxypeptidase, chitinase, metalloprotease GON-domain, hyaluronidase, pacifastin, PLA2, SE-cephalotoxin and serine proteases, with the carboxypeptidase and GON-domain documented for the first time in the coleoid venom arsenal. We show that although a majority of sites in these coleoid venom-encoding genes have evolved under the regime of negative selection, a very small proportion of sites are influenced by the transient selection pressures. Moreover, nearly 70 % of these episodically adapted sites are confined to the molecular surface, highlighting the importance of variation of the toxin surface chemistry. Coleoid venoms were revealed to be as complex as other venoms that have traditionally been the recipient of the bulk of research efforts. The presence of multiple peptide/protein types in coleoids similar to those present in other animal venoms identifies a convergent strategy, revealing new information as to what characteristics make a peptide/protein type amenable for recruitment into chemical arsenals. Coleoid venoms have significant potential not only for understanding fundamental aspects of venom evolution but also as an untapped source of novel toxins for use in drug design and discovery.

Pacific ciguatoxins in food web components of coral reef systems in the Republic of Kiribati.

Ciguatera fish poisoning (CFP) is a foodborne illness caused by consumption of coral reef fishes contaminated by ciguatoxins (CTXs); of the known CTX congeners, the Pacific ciguatoxins (P-CTXs) are the most toxic. Little is known about the trophodynamics of P-CTXs in coral reef systems. The present study explores the distribution, transfer, and trophic magnification of P-CTX-1, -2, and -3 in coral reef systems with high (ciguatoxic) and low (reference) ciguatoxicity in a CFP-endemic nation by use of liquid chromatography-tandem mass spectrometry (LC-MS/MS). In ciguatoxic coral reef systems, P-CTXs were detected in 54% of herbivorous fishes [total P-CTXs <0.500-1670 pg/g wet weight (ww)], 72% of omnivorous fishes (<0.500-1810 pg/g ww), and 76% of carnivorous fishes (<0.500-69 500 pg/g ww), as well as a lobster ( Panulirus penicillatus ; 2.36 pg/g ww) and an octopus (Octopodidae; 2.56 pg/g ww). The dominant P-CTXs in grazers and piscivorous fishes were P-CTX-2 and -1, respectively. No significant correlation between P-CTX levels and lipid content in three target predatory fishes indicated that accumulation of P-CTXs does not depend on fat content. A weak but significant positive relationship was observed between δ(15)N and P-CTX-1 levels, but further investigation is required to confirm its biomagnification potential.

Prey decline leads to diet shift in the largest population of Indo-Pacific humpback dolphins?

The Pearl River Delta (PRD) region on the southeast coast of China has long been known as a highly productive fishing ground. Since the late 1980s, fishing pressure in the PRD has been intense, which warrants concerns of potential fishery-related impacts on the food resources and foraging ecology of apex marine predators in this region, such as the Indo-Pacific humpback dolphin (Sousa chinensis). In this study, we examined 54 stomachs with food remains, collected from beached carcasses of humpback dolphins recovered during fifteen years between 2003 and 2017. The 6043 identified prey items represent 62 teleost taxa, primarily small estuarine fish, but also larger reef fish. The dolphins appear to be opportunistic foragers, hunting across the water-column, with preference for shoaling and meaty fishes (e.g. Collichthys lucidus IRI% = 38.6%, Johnius belangerii IRI% = 23.1%, Mugil cephalus IRI% = 14.0%). Our findings suggest a dietary shift in recent years, from primarily demersal (as previously reported) to greater intake of neritic and pelagic fish. Dolphin foraging group size has decreased in recent years, which corresponds with declining size and numbers of prey items retrieved from dolphin stomachs. We suggest that these are indicators of declining food resources. Faced with a shortage of preferred prey, humpback dolphins may have broadened their dietary spectrum to maintain their daily energy intake, while their foraging group size decreased in response to the altered tradeoff between the costs and benefits of group foraging.

Littoral Water in Hong Kong as a Potential Transient Habitat for Juveniles of a Temperate Deepwater Gnomefish, Scombrops boops (Acropomatiformes: Scombropidae).

A total of 40 juveniles belonging to a temperate deepwater gnomefish species, Scombrops boops, were sampled from littoral habitats (2-5 m depth) of eastern Hong Kong waters in April and May 2017 and March 2019. The presence of gnomefish juveniles in subtropical southern China is reported for the first time at a record low latitude of 22°11'-22°21'N. The specimens were identified based on the COI gene sequence. The genetic composition between Japan and Hong Kong gnomefish populations were compared by sequencing the mitochondrial Cytb gene, which showed no genetic differentiation. The juveniles ranged from 3.5-10.1 cm (n = 40) in total length, with 35 individuals caught from Sargassum beds and five from rocky reefs. Our findings highlighted that the littoral habitats in Hong Kong waters, in particular the seasonal Sargassum beds, are important for small juveniles of S. boops.

Long-term variations of phytoplankton community in relations to environmental factors in Deep Bay, China, from 1994 to 2016.

Long-term effects of environmental factors on phytoplankton community in Deep Bay, Hong Kong, were investigated from 1994 to 2016. Phytoplankton community experienced three important shifts: small flagellates and Chroomonas spp. dominated from 1994 to 2006. Heavy blooms of diatoms Skeletonema costatum and Thalassiosira spp. occurred and became the dominant species between 2007 and 2009. Phytoplankton community was dominated by small flagellates and Chroomonas spp. from 2010 to 2015, and their abundance was much higher than earlier periods. Abundance of Chroomonas spp. decreased sharply in 2016, and Thalassiosira spp. became the dominant species. Phytoplankton community changed from consisting mainly of small flagellates and Chroomonas spp. to more diatoms, changing from ~90% to ~45% for other species abundance and from <10% to ~55% for diatoms. Nitrite, 5-day biochemical oxygen demand, salinity, suspended solids and turbidity were more influential on phytoplankton variation (23.65% explained by all the variables).

The effect of temperature on physiology, toxicity and toxin content of the benthic dinoflagellate Coolia malayensis from a seasonal tropical region.

Coolia malayensis is one of the commonly found benthic dinoflagellates in Hong Kong which can produce biotoxins and threaten the early life stages of marine invertebrates. Seawater temperature has been recognized as one of the primary environmental factors that affect the formation of harmful algal blooms. The present study evaluated the responses of C. malayensis, including growth, toxicity and toxin content (putative analogues of okadaic acid and azaspiracids), after exposure to a range of seven different temperatures (i.e., 16°C, 18°C, 20°C, 22°C, 24°C, 26°C, and 28°C). The highest algal density and specific growth rate were recorded at 24°C. Significantly higher Fv/Fm (maximum quantum yield of PSII) and total phaeo-pigment values were observed in the exponential growth phase at 28°C. The toxicity of the algal extract, which was assessed by the lethality rate of Artemia larvae, increased with temperature. The highest toxin content was detected at the second highest temperature treatment, i.e., 26°C. Overall, temperature had significant effects on the physiological activities and toxicity of C. malayensis. This study has raised attention to the potentially increasing risks posed by toxic benthic dinoflagellates during heat waves in coastal waters.

Hemolysis associated toxicities of benthic dinoflagellates from Hong Kong waters.

Benthic dinoflagellates produce a diverse range of phycotoxins, which are responsible for intoxication events in marine fauna. This study assessed the hemolysis associated toxicities of six species of benthic dinoflagellates from the genera Coolia, Fukuyoa, Amphidinium and Prorocentrum. Results demonstrated that Amphidinium carterae, Coolia tropicalis and Fukuyoa ruetzleri were the three most toxic species, while Prorocentrum cf. lima did not have significant hemolytic effect. Grouper samples (Cephalopholis boenak) were more tolerant to the hemolytic algae than the blackhead seabream (Acanthopagrus schlegelii), with decreased heart rate and blood flow being observed in medaka larvae after exposure to toxic algal extracts. LC-MS/MS analysis detected a gambierone analogue called 44-methylgambierone produced by the C. tropicalis isolate. This analogue was also detected in the F. ruetzleri isolate. This study provided new information on the hemolysis associated toxicities of local toxic benthic dinoflagellates, which contributes to better understanding of their emerging threats to marine fauna and reef systems in Hong Kong.

Occurrence and trophic magnification profile of triphenyltin compounds in marine mammals and their corresponding food webs.

The occurrence of triphenyltin (TPT) compounds, a highly toxic antifouling biocide, has been documented in marine environments and organisms all over the world. While some studies showed that marine mammals can be used as sentinel organisms to evaluate the pollution status of emerging contaminants in the environment because of their long lifespans and high trophic levels, information regarding the contamination status of TPT in marine mammal species has been limited over the past decade. More importantly, the primary bioaccumulation pathway of TPT in these long-lived apex predators and the corresponding marine food web is still uncertain. Therefore, this study aimed to evaluate the contamination statuses of TPT in two marine mammal species, namely the finless porpoise and the Indo-Pacific humpback dolphin, and assess the trophic magnification potential of TPT along the food webs of these two species, using stable isotope analysis, and chemical analysis with gas chromatography-mass spectrometry. The results showed that TPT is the predominant residue in majority of the analyzed individuals of two marine mammals, with concentrations ranging from 426.2 to 3476.6 ng/g wet weight in their muscle tissues. Our results also demonstrated an exponential increase in the concentration of TPT along the marine food web, indicating that trophic magnification occurs in the respective food webs of the two marine mammals. The range of trophic magnification factors of TPT in the food webs of finless porpoise and Indo-Pacific humpback dolphin was 2.51-3.47 and 2.45-3.39, respectively. These results suggest that high trophic organisms may be more vulnerable to the exposure of TPT-contaminated environments due to the high trophic magnification potential, and thus ecological risk of these compounds ought to be assessed with the consideration of their bioaccumulation potentials in these marine mammals.

Phylogeny, morphology and toxicity of benthic dinoflagellates of the genus Fukuyoa (Goniodomataceae, Dinophyceae) from a subtropical reef ecosystem in the South China Sea.

Species of Fukuyoa, recently revised from the globular Gambierdiscus, are toxic benthic dinoflagellates associated with ciguatera. In this study, a total of ten strains of Fukuyoa collected from Hong Kong waters were characterized using morphological and phylogenetic analyses. Results from both analyses showed that one of the strains is a putative new species, namely Fukuyoa sp. HK Type 1 (plate formula Po, 3', 7″, 6c, 7s, 5‴, 1p and 2'‴ with a distinctive small and narrow cell shape, narrow Po plate, high Po pore density, large and broad Plate 1' but small and round Po pore size, small and narrow Plate 2', long and narrow Plates 2'‴ and 1p), and the others were F. ruetzleri. This is the first report of these two species of Fukuyoa in the South China Sea and Asia-Pacific region. Phylogenies on 18S, 28S D1/D3 and D8/D10 ribosomal DNA sequences strongly support that Fukuyoa sp. HK Type 1 is currently the most divergent species in the genus Fukuyoa. The diagrammatic plots on the p-distance matrices of 18S, 28S D1/D3 and D8/D10 and ITS regions resolved that the species of Fukuyoa were separated into three main groups, i.e., Fukuyoa sp. HK Type 1, F. paulensis and a group consisting of F. ruetzleri, F. yasumotoi and F. cf. yasumotoi, while Fukuyoa sp. HK Type 1 was always the most distant from the other two groups. Additionally, the pairwise p-distance values calculated based on the ITS region have always been the highest for pairs between Fukuyoa sp. HK Type 1 and other Fukuyoa species, ranging from 0.142 to 0.150. Our molecular results suggested that Fukuyoa sp. HK Type 1 is a putative new species. Both morphological and molecular data of more strains from different localities should be, however, collected to address its intraspecific variability and further evaluate its taxonomic status. A bioassay analysis demonstrated that algal lysates extracted from F. ruetzleri and Fukuyoa sp. HK Type 1 were lethal to brine shrimp larvae, indicating that both species were toxic. Bulk cultures were tested for Pacific ciguatoxins (P-CTXs) and maitotoxins (MTXs) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). All isolates of Fukuyoa produced neither P-CTXs nor MTX-1, but isolates of F. ruetzleri produced a compound putatively assigned as MTX-3. This study has updated the current biodiversity and distribution of the toxic benthic dinoflagellates Fukuyoa, and thus contributes to the understanding of their emerging threats to the sub-tropical reef systems locally and regionally.

Molecular phylogeny and toxicity of harmful benthic dinoflagellates Coolia (Ostreopsidaceae, Dinophyceae) in a sub-tropical marine ecosystem: The first record from Hong Kong.

Coolia are marine benthic dinoflagellates which are globally distributed and potentially toxic. This study provides the first investigation of species diversity and toxicity assessment of Coolia in Hong Kong waters. Fifty-one strains of four Coolia species, including C. malayensis, C. canariensis, C. tropicalis, and C. palmyrensis, were isolated from twelve sub-tidal habitats, and identified phylogenetically using 28S rDNA sequences. Exposure experiments (48-hour) demonstrated that the algal lysates extracted from the four Coolia species exhibited different toxic effects on the lethality and abnormality of two invertebrate larvae, i.e., brine shrimp Artemia franciscana and sea urchin Heliocidaris crassispina. Heliocidaris crassispina was more sensitive to the toxic effects of Coolia species than A. franciscana. Toxicity tests from both larvae revealed that C. malayensis was generally more toxic, and caused higher mortality rates when compared with the other three species. The emerging threat of harmful benthic dinoflagellates to marine environments and sensitive biota is discussed.

Occurrence, distribution and ecological risk assessment of multiple classes of UV filters in surface waters from different countries.

Organic UV filters are common ingredients of personal care products (PCPs), but little is known about their distribution in and potential impacts to the marine environment. This study reports the occurrence and risk assessment of twelve widely used organic UV filters in surface water collected in eight cities in four countries (China, the United States, Japan, and Thailand) and the North American Arctic. The number of compounds detected, Hong Kong (12), Tokyo (9), Bangkok (9), New York (8), Los Angeles (8), Arctic (6), Shantou (5) and Chaozhou (5), generally increased with population density. Median concentrations of all detectable UV filters were <250 ng/L. The presence of these compounds in the Arctic is likely due to a combination of inadequate wastewater treatment and long-range oceanic transport. Principal component analysis (PCA) and two-way analysis of variance (ANOVA) were conducted to explore spatiotemporal patterns and difference in organic UV filter levels in Hong Kong. In general, spatial patterns varied with sampling month and all compounds showed higher concentrations in the wet season except benzophenone-4 (BP-4). Probabilistic risk assessment showed that 4-methylbenzylidene camphor (4-MBC) posed greater risk to algae, while benzophenone-3 (BP-3) and ethylhexyl methoxycinnamate (EHMC) were more likely to pose a risk to fishes and also posed high risk of bleaching in hard corals in aquatic recreational areas in Hong Kong. This study is the first to report the occurrence of organic UV filters in the Arctic and provides a wider assessment of their potential negative impacts in the marine environment.

Changes of accumulation profiles from PBDEs to brominated and chlorinated alternatives in marine mammals from the South China Sea.

The present study investigated the composition profiles and levels of polybrominated diphenyl ethers (PBDEs) and five PBDE alternatives in the blubber of two species of marine mammals, Indo-Pacific humpback dolphins (Sousa chinensis) and finless porpoises (Neophocaena phocaenoides) from the South China Sea. Despite the fact that PBDEs were the most predominant brominated flame retardants in the samples analyzed, decabromodiphenyl ethane (DBDPE), 1,2-bis (2,4,6-tribromophenoxy) ethane (BTBPE), bis- (2-ethylhexyl) -tetrabromophthalate (TBPH), 2-ethylhexyl 2,3,4,5-tetrabromobenzoate (TBB) and Dechlorane Plus (DP) were all detected in both cetacean species. In addition, significantly increasing temporal shifting trends of Deca-BDE to DBDPE, Octa-BDE to BTBPE, and Deca-BDE to DP were observed in porpoise samples between 2003 and 2012 and dolphin samples between 2003 and 2011. These patterns may be attributed to the replacement of PBDEs by alternative halogenated flame retardants (HFRs) and the increasing usage of these alternatives following the restriction/voluntary withdrawal of the production and use of PBDE commercial mixtures. Our findings suggest that the study region may be a source of contamination by PBDE alternative flame retardants due to the high detection frequencies and levels of these compounds in marine mammals.

Stable isotopes as a useful tool for revealing the environmental fate and trophic effect of open-sea-cage fish farm wastes on marine benthic organisms with different feeding guilds.

Environmental fate of fish farm wastes (FFW) released from an open-sea-cage farm at Kat O, Hong Kong was examined by measuring carbon and nitrogen stable isotope (SI) ratios in selected benthic organisms collected along a 2000 m transect from the farm. Our results showed that FFW significantly influenced the energy utilization profile of consumers near the fish farm. Although nitrogen enrichment effect on δ15N was anticipated in biota near the farm, the predicted patterns did not consistently occur in all feeding guilds. Two species of suspension-feeders, which relied on naturally δ15N-depleted sources, were δ15N-enriched near the fish farm. In contrast, both species of benthic grazer and deposit-feeder, which relied on naturally δ15N-enriched algal sources, were δ15N-depleted under the influence of FFW. The SI signatures of biota can, therefore, serve as feasible biomarkers for FFW discharges only when the trophic structure of the receiving environment is fully elucidated.