Effects of Ocean Acidification on Molting, Oxidative Stress, and Gut Microbiota in Juvenile Horseshoe Crab Tachypleus tridentatus

Anthropogenic elevation of atmospheric carbon dioxide (CO2) drives global-scale ocean acidification (OA), which has aroused widespread concern for marine ecosystem health. The tri-spine horseshoe crab (HSC) Tachypleus tridentatus has been facing the threat of population depletion for decades, and the effects of OA on the physiology and microbiology of its early life stage are unclear. In this study, the 1st instar HSC larvae were exposed to acidified seawater (pH 7.3, pH 8.1 as control) for 28 days to determine the effects of OA on their growth, molting, oxidative stress, and gut microbiota. Results showed that there were no significant differences in growth index and molting rate between OA group and control group, but the chitinase activity, β-NAGase activity, and ecdysone content in OA group were significantly lower than those of the control group. Compared to the control group, reactive oxygen species (ROS) and malondialdehyde (MDA) contents in OA group were significantly increased at the end of the experiment. Superoxide dismutase (SOD), catalase (CAT), and alkaline phosphatase (AKP) activities increased first and then decreased, glutathione peroxidase (GPX) decreased first and then increased, and GST activity changed little during the experiment. According to the result of 16S rRNA sequencing of gut microbiota, microbial-mediated functions predicted by PICRUSt showed that “Hematopoietic cell lineage,” “Endocytosis,” “Staphylococcus aureus infection,” and “Shigellosis” pathways significantly increased in OA group. The above results indicate that OA had no significant effect on growth index and molting rate but interfered with the activity of chitinolytic enzymes and ecdysone expression of juvenile horseshoe crabs, and caused oxidative stress. In addition, OA had adverse effects on the immune defense function and intestinal health. The present study reveals the potential threat of OA to T. tridentatus population and lays a foundation for the further study of the physiological adaptation mechanism of juvenile horseshoe crabs to environmental change.

Morphometric variation of the horseshoe crab Tachypleus gigas (Xiphosura: Limulidae) from the Banyuasin estuarine of South Sumatra, Indonesia

Fauziyah, Mustopa AZ, Fatimah, Purwiyanto AIS, Rozirwan, Agustriani F, Putri WAE. 2021. Morphometric variation of the horseshoe crab Tachypleus gigas (Xiphosura: Limulidae) from the Banyuasin estuarine of South Sumatra, Indonesia. Biodiversitas 22: 5061-5070. Morphological studies are essential for fish resource management, ecology, conservation, and stock assessment. This research was conducted to characterize the morphological variation of the coastal horseshoe crab (Tachypleus gigas) from the Banyuasin estuarine, South Sumatra, Indonesia. The body weight (BW), prosoma width (PW), carapace length (CL), telson length (TEL), and total length (TL) were measured for describing the morphometric variability of T. gigas. A total of 70 T. gigas (32 females and 38 males) were found using trammel net fishing and bottom gillnet during the survey. Multivariate and allometric methods analyzed their morphometric characters. Based on Kruskal- Wallis tests, the result showed a significant difference in somebody measurements (TL and CL) between males and females. The principal component analysis (PCA) results showed that all morphometric characters had a strong correlation in both sexes, so the first principal component (PC1) values were 92% and 72%. In comparison, the second principal component (PC2) values were 5% and 20% in females and males, respectively. The results of discriminant function analysis (DFA) suggested that only one (BW) of the five morphometric characters was significant in separating both sexes. Both sexes were also revealed variations in growth patterns based on the allometric analysis results. These results were expected to be used as a basis for managing the horseshoe crab population-based conservation.

Assessing horseshoe crab avoidance response of flicker and continuous red-light LED: Gillnets bycatch mitigation

The horseshoe crab has the important roles as macrobenthos, but it is unfriendly for gillnets fisheries because it can damage the fishing net. The horseshoe crab is an ancient, rare, and protected animal so that the fishermen unable to utilize it, so the effort like mitigations need to be conducted like using red light LED. To avoid the learning behavior of horseshoe crab, so that the technology used is red light LED continuous then the red light led with flicker is used as a comparison. This study aims to determine the right type of red-light LED for horseshoe crab bycatch mitigation by the response pattern. The method used was a laboratory experiment. There are 20 adult horseshoe crabs used. The analysis used was descriptively comparative. The results showed that the red-light LED with flicker was most avoided by horseshoe crabs, which is 75%, while the fastest response of horseshoe crab in avoiding light was found in the light with a flicker effect of 22.97 seconds. Based on the horseshoe crab’s response, it can be concluded that the red LED light flicker is better than the red LED continuous as an alternative technology for the mitigation of horseshoe crab bycatch.

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.

Inter-Subunit Dynamics Controls Tunnel Formation During the Oxygenation Process in Hemocyanin Hexamers

Hemocyanin from horseshoe crab in its active form is a homo-hexameric protein. It exists in open and closed conformations when transitioning between deoxygenated and oxygenated states. Here, we present a detailed dynamic atomistic investigation of the oxygenated and deoxygenated states of the hexameric hemocyanin using explicit solvent molecular dynamics simulations. We focus on the variation in solvent cavities and the formation of tunnels in the two conformational states. By employing principal component analysis and CVAE-based deep learning, we are able to differentiate between the dynamics of the deoxy- and oxygenated states of hemocyanin. Finally, our results identify the deoxygenated open conformation, which adopts a stable, closed conformation after the oxygenation process.