While there are persistent calls for developing more marine protected areas (MPAs) for Asian horseshoe crab conservation in response to population declines in Asia–Pacific region, most existing horseshoe crab MPAs were designated without prior comprehensive population assessment and habitat characterization. This study collected biological and habitat information in Hong Kong, in order to identify priority sites for conservation and management of Asian horseshoe crab populations. The territory-wide surveys at 18 spawning/nursery beaches displayed a persistently low mean juvenile density from 0.16 to 2.19 ind/100 m2 in 2012 and 0.08 to 1.41 ind/100 m2 in 2014. These density data were within the same range of that in 2002 and 2007 (0.10–1.97 ind/100 m2), apart from a low survey return of 0.08–0.31 ind/100 m2 in 2004. The current population of juvenile T. tridentatus and C. rotundicauda in Hong Kong is estimated about 2100–4300 and 2400–3000 individuals, respectively. From the 2012–2014 data, no new recruitments of 1st–3rd instar juveniles were found, as the shores were occupied mostly by 6th–9th instar juveniles of prosomal width between 23 and 45 mm, in which considerably high mortality rates were noted. The present findings of existing small and discrete juvenile populations, coupled with relatively few recruits, suggest that the status of juvenile horseshoe crabs in Hong Kong is fragile and vulnerable to local extirpation, especially if no urgent protection measures are implemented. Based on available population data and habitat characteristics, three MPAs, ranging from 5 to 7 km2, are proposed, so as to conserve over 60 and 70 % of the existing juvenile populations of T. tridentatus and C. rotundicauda, respectively. The approach adopted in this study may serve as a case study for proposing other horseshoe crab MPAs in Asia, where increasing human disturbances and over-harvest are imminent.

To enhance our understanding on the foraging biology of juvenile Chinese horseshoe crab on a seagrass-covered intertidal mudflat, we used dual stable isotopes δ13C and δ15N to determine the diet composition and trophic position of 6th–11th instar juveniles in summer and winter. The δ13C and δ15N values in tissues of juveniles and their potential food sources suggested that the juveniles consumed a mixed diet mainly comprised a variety of polychaetes, crustaceans and bivalves, in which these food groups were abundantly available in the study site and largely supported by seagrass biomass in both summer and winter. While δ15N values in juvenile tissues were statistically similar between the two seasons, the δ13C values in tissues of juveniles had a minimal seasonal difference which was probably due to variations in the source values rather than the changes in diet composition of juveniles. There were no differences in δ13C and δ15N values among the size groups of juveniles through 6th–11th instars. Although juvenile Chinese horseshoe crabs may consume the prey in response to the seasonal availability on the mudflat, a healthy condition of habitat with high water quality is essential to support the juveniles and a wide range of intertidal invertebrates they consume.

Responses of growth endpoints and hemolymph constituents in juvenile Chinese horseshoe crab Tachypleus tridentatus under treatments of 0.01 and 0.1 mg/l tributyltin (TBT) and 0.1 and 1 mg/l cadmium (Cd) were examined in a 12-week experiment. A significant decline in final body weight, final prosomal width, percentage of individuals molted and mean molting time was detected under TBT exposures. While morphological abnormalities of the juveniles between TBT treatments and the control were statistically indistinguishable, significantly higher occurrences of carapace erosion and appendage loss were noted under 0.1 and 1 mg/l Cd exposures. Various hemolymph quality indicators, including hemolymph plasma protein level, amebocyte viability and percentage of granular-spherical state of amebocytes of the juveniles exposed to TBT or Cd were significantly lower than the control. Such a decrease in hemolymph quality suggested deleterious effects of metal contaminant-induced stressors on the health status of the juveniles even at low exposure levels (i.e., 0.01 mg/l TBT and 0.1 mg/l Cd). Changes of hemolymph parameters in juvenile horseshoe crabs were more sensitive than growth performance as well as morphological abnormalities in response to metal stressors, and can be used as an indicator to reflect habitat conditions and contaminant levels.

Energy budget is one of the most studied parameters in aquatic animals under environmental challenge. To examine how prolonged starvation would affect their energy budget, respiration rate (RR), ammonia excretion rate (ER), oxygen consumption to ammonia–nitrogen excretion (O:N) ratio and scope for growth (SfG) representing the balance between energy intake and metabolic output, two Asian horseshoe crab species, Tachypleus tridentatus and Carcinoscorpius rotundicauda, were investigated in two feeding regimes (fed and starved) over a period of 7 weeks. No significant effects of species and time course, as well as their interaction, on absorption efficiency were observed in the fed treatments. For both species, RR and ER of the starved treatments significantly decreased, while their O:N ratio significantly increased during the experiment. However, such values for the fed treatments remained relatively stable over the study period. A rapid reduction in SfG was only apparent in the first week of the starved treatments for both species; thereafter, their SfG remained relatively constant. In the fed treatments, SfG of T. tridentatus was significantly lower than that of C. rotundicauda throughout the experiment. In general, C. rotundicauda showed a greater decrease in SfG under starvation than T. tridentatus, suggesting that they may have a more competitive life-history strategy for adjusting to poor nutritional conditions.

The amphioxi Branchiostoma belcheri, Branchiostoma japonicum and Branchiostoma malayanum are recorded from the coast of China Seas. Six amphioxus populations comprising three Branchiostoma species collected from Hong Kong (southern China), Xiamen (south-eastern China) and Qingdao (northern China) were compared at the morphological and molecular levels. Phylogenetic separations among the species and geographic populations were evaluated by the analysis of 12S ribosomal RNA gene sequences and amplified fragment length polymorphism (AFLP) markers. From morphological characters, B. belcheri is more closely related to B. japonicum than to B. malayanum. However, phylogenetic affinities indicated by both 12S rRNA gene sequences and AFLP analysis showed a more recent phylogenetic splitting of B. belcheri and B. malayanum than that of B. belcheri and B. japonicum. In the AFLP similarity tree, geographic populations of B. japonicum were clustered into different clades. The AFLP data also showed that both B. belcheri and B. japonicum populations in Hong Kong have the highest levels of within-population genetic diversity as compared to that in Xiamen and Qingdao, suggesting that genetic diversity of Branchiostoma decreases from low to high latitudes. Results of hierarchical analysis of molecular variance (AMOVA) also revealed a high level of genetic diversity either for the three B. japonicum populations or the two B. belcheri populations in China Seas. However, genetic variation among the three B. japonicum populations was insignificant, indicating that these populations are genetically connected.

•Clam diet showed best growth response in juvenile Chinese horseshoe crabs.•Significant declines in hemolymph parameters at the end of all feed treatments•Hemolymph quality in cultured juveniles significantly poorer than wild counterparts•Hemolymph quality might reflect captivity-induced stress and health status.The growth performance and hemolymph quality of juvenile Chinese horseshoe crabs under four diet treatments: brine shrimp Artemia salina, short-neck clam Ruditapes philippinarum, sandworm Marphysa sanguinea and mixed clam and sandworm were evaluated in a 12-week experiment. The growth responses in terms of final body weight, specific growth rates and percentage of individuals molted under the clam diet were significantly higher than those fed with brine shrimp and sandworm, whereas the mixed clam and sandworm diet was not statistically different from the other three treatments. While the hemolymph properties among these diets were similar, significant declines in hemocyanin concentration, percentage of oxyhemocyanin, and ratio of granular–spherical to granular–flattened states of amebocytes were noted at the end of the study. Such a decrease in hemolymph quality indicated deleterious effects due to prolonged culture on the health status of the juveniles, which might be attributed to captivity-related issues, such as deficiencies of essential diet compositions, movement constraints and absence of tidal rhythms. This was further supported from the finding that the hemolymph quality of the experimental juveniles was significantly poor compared to that of the juveniles collected from the field. Changes of hemolymph constituents could thus be a useful indicator to reflect the health status of juvenile horseshoe crabs under laboratory culture.

•Sediment quality and macrofaunal communities were updated in subtropical Hong Kong.•AMBI and M-AMBI were consistent with sediment characteristics of different benthic environments.•AMBI and M-AMBI were useful in assessing benthic ecological status in subtropics.•Biotic indices were more sensitive than sediment parameters to reflect benthic response.Sediment characteristics and benthic communities on a finer sampling scale in four contrasting environments in subtropical Hong Kong were analyzed in summer and winter 2012. In two harbour habitats which suffered from historic sewage pollution or hypoxic events, organic carbon, nutrient and trace metal content in the sediment were significantly higher than that in an offshore area and a marine reserve. The relatively low organic and nutrient content in the offshore habitat could be resulted from enhanced resuspension of such materials from the seabed owing to intense water mixing and disturbance caused by bottom trawling. The biotic indices AMBI and M-AMBI were shown to be useful in assessing the benthic ecological status of these habitats. Such indices can also be more sensitive than sediment physico-chemical parameters in differentiating the response of macrofauna to seasonal changes in the benthic environment.