DNA Metabarcoding Revealed Interspecific Dietary Difference and Prey Selectivity in Juvenile Horseshoe Crabs Carcinoscorpius rotundicauda and Tachypleus tridentatus From Hong Kong

Horseshoe crabs provide important ecological services including bioturbation and linking of food web in the shallow waters, but their populations are declining globally, leading to major concerns on conservation of these iconic animals. Baseline information of horseshoe crab ecology, such as their trophic role and food source, is pre-requisite for habitat protection plan and captive restocking program. Trophic ecology of Asian horseshoe crabs is relatively poorly understood and previous studies on their juveniles have suggested that they are selective feeders rather than opportunistic generalists. This study demonstrates a non-invasive approach, using DNA metabarcoding analyses of the nuclear 18S rRNA gene on fecal samples to assess the dietary compositions of Carcinoscorpius rotundicauda and Tachypleus tridentatus juveniles to (1) determine their dietary compositions and trophic roles in their ecosystem, (2) determine any prey selectivity, and (3) distinguish the interspecific dietary differences with potential implications on the habitat requirement and ecological partitioning between these two horseshoe crab species. The results based on relative read abundance (RRA) suggested that oligochaetes were the major prey items for both C. rotundicauda (41.6%) and T. tridentatus (32.4%). Bivalves and crustaceans were second major prey groups for C. rotundicauda (8.6 and 8.4%, respectively). Surprisingly, anthozoans contributed a considerable portion of T. tridentatus’s diet (22.8%), which has never been reported. Furthermore, the major prey groups identified in the fecal samples were not the dominant benthic organisms in the studied area as revealed by environmental DNA (eDNA) analyses on the sediment samples, implying that both species are selective feeders rather than dietary generalists. Significant differences observed in the dietary compositions of the two species might be partially due to the difference in habitat preference between the two species. This study provides new insights into the trophic ecology of the two Asian horseshoe crab species in the estuarine habitat and establishes a new framework for future detailed molecular dietary analyses on all developmental stages of horseshoe crabs around the world, which will allow us to evaluate the food sources needed for the survival of horseshoe crabs and facilitate future conservation strategies without killing the animals.

DNA metabarcoding provides insights into the diverse diet of a dominant suspension feeder, the giant plumose anemone Metridium farcimen

ABSTRACTBenthic suspension feeders have significant impacts on plankton communities by depleting plankton or modifying composition of the plankton through prey selectivity. Quantifying diets of planktivorous animals can be difficult because plankton are frequently microscopic, may lack diagnostic characters, and are digested at variable rates. With DNA metabarcoding, the identification of gut contents has become faster and more accurate, and the technique allows for higher taxonomic resolution-n while also identifying rare and highly degraded items that would otherwise not be detected. We used DNA metabarcoding to examine the diet of the giant plumose anemone Metridium farcimen, a large, abundant, competitively-dominant anemone on subtidal rock surfaces and floating docks in the northeast Pacific Ocean. Gut contents of 12 individuals were compared to 80- and 330-μm filtered plankton samples collected one hour prior between 0.02 and 1.5 km from the anemones. The objectives of this study were to determine if M. farcimen has a selective diet and compare our findings with traditional gut content analyses. Metridium farcimen captured a wider range of prey than previously suspected and metabarcoding found many more taxa than traditional sampling techniques. Gut contents were less diverse than 80-μm filtered plankton samples, but more diverse than 330-μm filtered plankton samples. The diet of the anemones was 52% arthropods with a surprisingly high relative abundance of an ant (10%) that has mating flights in August when this study was conducted. The gut contents of M. farcimen likely include all prey that it can detect and that cannot escape. There were no overrepresented taxa in the gut contents compared to the plankton but there were underrepresented taxa. This study highlights the usefulness of the metabarcoding method in identifying prey within the gut of planktivorous animals and the significant terrestrial input into marine food webs.

An Appetite for Destruction: Detecting Prey-Selective Binding of α-Neurotoxins in the Venom of Afro-Asian Elapids

Prey-selective venoms and toxins have been documented across only a few species of snakes. The lack of research in this area has been due to the absence of suitably flexible testing platforms. In order to test more species for prey specificity of their venom, we used an innovative taxonomically flexible, high-throughput biolayer interferometry approach to ascertain the relative binding of 29 α-neurotoxic venoms from African and Asian elapid representatives (26 Naja spp., Aspidelaps scutatus, Elapsoidea boulengeri, and four locales of Ophiophagus hannah) to the alpha-1 nicotinic acetylcholine receptor orthosteric (active) site for amphibian, lizard, snake, bird, and rodent targets. Our results detected prey-selective, intraspecific, and geographical differences of α-neurotoxic binding. The results also suggest that crude venom that shows prey selectivity is likely driven by the proportions of prey-specific α-neurotoxins with differential selectivity within the crude venom. Our results also suggest that since the α-neurotoxic prey targeting does not always account for the full dietary breadth of a species, other toxin classes with a different pathophysiological function likely play an equally important role in prey immobilisation of the crude venom depending on the prey type envenomated. The use of this innovative and taxonomically flexible diverse assay in functional venom testing can be key in attempting to understanding the evolution and ecology of α-neurotoxic snake venoms, as well as opening up biochemical and pharmacological avenues to explore other venom effects.