Investigation of the feeding ecology of the horseshoe crabs carcinoscorpius rotundicauda and tachypleus tridentatus in Hong Kong using DNA metabarcoding

Abstract

Horseshoe crabs provide important ecological services in estuarine habitats, but their populations are declining globally, leading to major concerns for the conservation of these iconic animals. Baseline information of horseshoe crab ecology, such as their food sources and trophic role, is a pre-requisite for formulating effective habitat protection plans and captive restocking programmes. The feeding ecology of Asian horseshoe crabs is, however, relatively poorly understood. To further investigate feeding preference and temporal variations in food source in horseshoe crabs, this study developed and demonstrated a non-invasive protocol, with DNA metabarcoding analyses of the nuclear 18S rRNA gene of fecal samples to assess the dietary composition of Carcinoscorpius rotundicauda and Tachypleus tridentatus juveniles in Hong Kong to determine 1) their trophic roles and potential prey selectivity; 2) interspecific dietary differences; and 3) any temporal and ontogenetic differences in their diet in response to prey availability.

Firstly, a DNA metabarcoding approach was developed by investigating horseshoe crab fecal samples (46 from C. rotundicauda and 41 from T. tridentatus) and sediment samples collected in 2019. Both species are selective feeders as the major prey items were not the dominant taxa in the corresponding habitats. Oligochaetes were the major prey item for both C. rotundicauda and T. tridentatus. Surprisingly, Anthozoans contributed a considerable portion of the diet of T. tridentatus, which has not been previously reported, suggesting the important contribution of burrowing anemones (in the family Edwardsiidae) as a food source of T. tridentatus. Significant differences were also observed in the dietary compositions of the two species, which was mainly attributed to the high proportion of Anthozoans reads detected in T. tridentatus.

Temporal and ontogenetic dietary assessments were further conducted on fecal samples (135 from C. rotundicauda and 164 from T. tridentatus) and sediment samples collected in 2020 summer (June to October) when was the active foraging period of horseshoe crabs. Findings of this more extensive sampling were largely consistent with the initial survey but also revealed significant temporal variations in the diets of C. rotundicauda and T. tridentatus juveniles. Ontogenetic dietary differences were observed in T. tridentatus but not C. rotundicauda, perhaps as this species exhibit more prominent changes in body size among instar stages. Horseshoe crabs consumed more algae and bivalves when these items were abundant while the selectivity on other food items were not affected by availability, suggesting that the juveniles were able to acclimate to their habitats to certain extend to maximize the foraging efficiency.

This study, therefore. provided new insights into the feeding ecology of the two Asian horseshoe crab species and establishes a novel framework for future detailed molecular dietary analyses on horseshoe crabs around the world.