2022, Vol. 21
2) School of Marine Sciences, Sun Yat-sen University, Zhuhai 519082, China;
3) College of Marine Sciences, Beibu Gulf University, Qinzhou 535011, China
Horseshoe crabs, known as classic living fossils, have survived on earth for over 450 million years (Rudkin and Young, 2009; Van Roy et al., 2010). In the past few decades, the population of all four extant species of horseshoe crabs, namely, Tachypleus tridentatus, Carcinoscorpius rotundicauda, Tachypleus gigas, and Limulus polyphemus, has declined rapidly (Razali et al., 2020; Wang et al., 2020). In particular, T. tridentatus and C. rotundicauda are found along the coast of southern China, and more than 95% of their populations are distributed in Beibu Gulf surrounded by the provinces of Hainan and Guangdong and the region of Guangxi (Liao and Li, 2001). Current populations of horseshoe crabs in China have drastically declined due to various threats, including overharvest, habitat loss, and anthropogenic pollution. In March 2019, T. tridentatus was formally added to the category of 'Endangered Species' on the International Union for Conservation of Nature's Red List (Laurie et al., 2019), followed by its classification as National Grade Ⅱ protected wild animal in China in February 2021. Horseshoe crab conservation is vital because of its importance to the coastal ecosystem as a major component of the food web and its great contribution to studies on evolutionary biology and local coastal culture and to the biomedical industry. Despite being currently under the strict protection of state laws, these animals are still seriously threatened by habitat loss and destruction, including lack of suitable foods. Understanding the feeding habitats of horseshoe crabs, including their food source, is important in restoring their populations because the variety, distribution, and quality of food sources may determine the survival rate, distribution, and movement patterns of natural populations (Kwan et al., 2015, 2016, 2021).
Tidal flats in estuarine and coastal bays are critical nursery grounds for T. tridentatus and C. rotundicauda juveniles (Shin et al., 2009; Morton and Lee, 2010; Hu et al., 2011; Chen et al., 2015; Xie et al., 2019). These areas are also subjected to severe human disturbance due to their increasing economic development in offshore areas. A diet comprising a combination of algae, seagrass, bivalves, and other plant and animal sources is important in the food chains of Asian horseshoe crabs (Kwan et al., 2015, 2021; Fan et al., 2017). Hence, their omnivorous feeding behavior can help them adapt to harsh intertidal habitats. Feeding ecology, such as the type, quantity, timing, and mode of feeding, is important in understanding the basic survival food requirements of marine organisms and the food web structure of the ecosystem where they live. It is also important to learn their predatory or competitive relationships with other organisms. These data can provide an ecological basis for modeling the flow of materials and energy in the coastal ecosystem. For horseshoe crabs, understanding the contributions of these different potential food sources in their main habitats is important for their conservation (Chen et al., 2015).
Stable isotope analysis (SIA) has been applied to determine the food sources of animals (Estrada et al., 2005; Girard et al., 2012; Lin, 2013), track animal migrations (Ashley et al., 2010), and construct food chains and webs in ecology (Zka et al., 2010). The stable isotope composition of the food consumed by animals corresponds to that of their bodies (Fry, 1988). In addition, the stable isotope ratios of carbon (13C/12C) and nitrogen (15N/14N) provide time-integrated information about food sources assimilated by organisms. SIA is useful in inferring the relative contributions of given primary producers and reconstructing the relationship within the food web in aquatic ecosystems (Pasquaud et al., 2007). Abundant data on nutritional structure and food sources were successfully obtained using stable isotope technology (Lin, 2013; Feng et al., 2015; Jie et al., 2018). Previous diet studies on the Atlantic horseshoe crab L. polyphemus showed that the early-instar juveniles (i.e., 2nd and 3rd instars) derive food from benthic and suspended particulate organic matter, late-instar juveniles (5th to 11th instars) consume polychaetes and crustaceans, and adults prey mainly on bivalves and gastropods (Gaines et al., 2002; Carmichael et al., 2009).
The feeding compositions of T. tridentatus and C. rotundicauda have been previously studied using the stable isotope compositions of muscle samples (Fan et al., 2017; Kwan et al., 2021) and fecal samples (Lee et al., 2021). However, blood samples have never been used for related experiments. The blood (usually called hemolymph) of a horseshoe crab circulates around the organs within the body cavity where it makes direct contact with all internal tissues and organs. Small changes in the internal and external environments of this animal can stimulate its hemolymph and provide necessary feedback to maintain the stability of the internal environment. The hemolymph accepts and responds to changes in environmental signals earlier and more sensitively than muscles and feces. Therefore, hemolymph samples might be useful in reflecting the recent and available food sources (Caut et al., 2009; Therrien et al., 2011). In this study, the hemolymph samples of two Asian horseshoe crab species, T. tridentatus and C. rotundicauda, and their potential food sources were collected from the northern Beibu Gulf and subjected to carbon and nitrogen SIAs to provide information on their food choice at a short time scale and improve their conservation.
2 Materials and Methods 2.1 Study AreaThe study sites were located at Pearl Bay on the west coast of northern Beibu Gulf, Guangxi Region, southwestern China. The bay occupies a total area of 94.2 km2, and the coastline is approximately 46 km long. The bay opens into Beibu Gulf at its southern end (Fig.1). The area is characterized by a subtropical oceanic monsoonal climate with a mean annual temperature of 22.5℃ and 2221 mm of annual precipitation. Tides are regular semidiurnal with an average tidal range of 2.24 m and a spring tidal range of about 5 m (Qiu et al., 2013). The tidal sandand mud-flats in the intertidal zone in the bay are large and generally extend 2 – 3 km when tides recede, thus providing good breeding habitats for horse shoe crabs. Beds of seagrass Zostera japonica and other species are distributed in tidal zones along the seaward edges of the mangrove forests. In this study, samples were obtained from Jintan (mouth of the bay, JT), Jiaodong (middle of the bay, JD), and Fenghuang (top of the bay, FH) in Pearl Bay during low tide in April 2019.
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Fig. 1 Sampling location map in Pearl Bay, northern Beibu Gulf, Guangxi Region, China |
Horseshoe crab distribution was investigated using the systematic quadrat method of Kwan et al. (2016) and Xie et al. (2019) with modifications and aided by the 'Two Step Path' APP to pinpoint the coordinates of the sampling location points. Four horizontal transects were set parallel to the coastline and equally spaced between mean high and low water marks. The lengths of and spacing between the four transects were similar at each study site but differed between study sites with a length range of 0.88 – 2.89 km and a spacing range of 45 – 300 m. Quadrats with 8 m × 8 m dimension (64 m2 area) were placed on each transect at regular intervals of 100 – 140 m in the pre-defined study area. The age classes of horseshoe crabs were classified according to prosomal widths (PWs). C. rotundicauda individuals with a PW range of 50.50 – 63.40 mm are the older juveniles, and those with 90.40 – 145.20 mm are the adults. T. tridentatus individuals with PW of 49.85 – 55.50 mm are the older juveniles, and those with 60.30 – 73.25 mm are the adults. All the older juvenile and adult samples were collected from the survey fields.
2.2.2 Sample preparationThe species of horseshoe crabs were quickly identified within each quadrat, and their sizes were measured. The body size of horseshoe crabs was determined by measuring the PW to the nearest 0.1 mm with vernier calipers. After all the sample sites were preliminarily surveyed, a representative was selected at each location near the inner, middle, and outer bays of the Beilun River Estuary according to the distribution of T. tridentatus and C. rotundicauda.
The horseshoe crab body was divided into three major parts from anterior to posterior: prosoma, opisthosoma, and telson. The heart is located along the dorsal midline, just beneath the carapace of prosoma and opisthosoma (Armstrong and Conrad, 2008). The carapace of horseshoe crabs was cleaned and disinfected before hemolymph was drawn. A 0.5 – 1 mL disposable syringe was used to collect hemolymph samples by cardiac puncture. The needle was not touched with fingers to avoid any potential contamination. The cannula with a needle was safely positioned, and the exposed base of the needle was elevated to avoid contact with any surface to maintain its sterility. Given that horseshoe crab juveniles have a limited amount of hemolymph, only those with PW greater than 50 mm were selected for hemolymph collection. The samples were stored in small 1.5 mL centrifuge tubes and frozen immediately after sampling. Six horseshoe crab hemolymph (three juvenile and three adult samples) were collected for each species from each study site.
Potential food samples were collected from the same areas as the horseshoe crabs. Food samples, including different plant and animal species that the horseshoe crab may consume, were collected and then stored frozen. Gracilaria verrucosa, Ulva lactuca, U. prolifera, Sipunculus nudus, Batillaria zonalis, Perinereis aibuhitensis, Zostera japonica, Azumapecten farrer, Dosinia corrugata, Meretrix lusoria, Crassostrea angulate, and Ostrea cucullate were collected. The plant samples were repeatedly rinsed in distilled water until clean, and were packed, weighed, recorded. Then they were dried in a oven at 70 – 80℃ until the weight was constant. Then they were crushed in a grinder, fully ground to a powder in a mortar, and finally passed through a 150 µm sieve. The animal samples were repeatedly rinsed in distilled water and unshelled. Their muscle tissues were extracted, placed in glass petri dishes, and then freeze-dried in a LABCONCO freeze dryer at −25℃ to −65℃ for 72 h. The muscle samples were then chopped, thoroughly ground to a powder, and passed through a sieve of 250 µm. Freeze-drying was also used for the hemolymph samples.
Sediment and water samples were collected from each study site to determine the environmental conditions, including seawater salinity, pH, and sediment particle. The Malvern 2000 laser particle size analyzer was used for particle size determination. Portable devices were employed for the immediate on-site measurement of the salinity and pH of surficial waters at each study site during low tides.
2.3 Sample Analysis and Isotope Mixing ModelThe carbon isotope ratio (δ13C) and nitrogen isotope ratio (δ15N) of dried hemolymph and food source samples were determined using a Delta V Advantage isotope ratio mass spectrometer coupled with an HT2000-EA elemental analyzer (Thero Fisher Corp., USA). The previsions for δ13C and δ15N analysis were 0.1‰ and 0.2‰, respectively, based on the repeated analysis of a laboratory work standard PRO.
One-way ANOVA was conducted to examine differences in δ13C and δ15N values between different potential food sources. Prior to analysis, raw data were analyzed for variance homogeneity and were log-transformed (log10) to meet the ANOVA assumption. The data were counted and collated using SPSS 22.0 and analyzed for significant differences using a one-way ANOVA; P < 0.05 was considered significant. Significant correlations between data were analyzed using Pearson correlation analysis, and P < 0.05 was considered significant (Feng et al., 2014).
On the basis of the δ13C and δ15N values, the relative importance of different potential food sources to the diet of these investigated horseshoe crabs was calculated using the MixSIR model developed within the Bayesian modeling framework (Moore and Semmens, 2008). Multiple sources of uncertainty, such as food source isotope values and fractionation, were integrated into the estimation by this model. Data on the relative contribution ratios of potential food sources of T. tridentatus and C. rotundicauda from different age groups were calculated separately by MixSIR 1.0.4 software. δ13C and δ15N distribution maps and food source contribution ratio distribution maps were then visualized with Origin 2017 software to study the potential food sources of horseshoe crabs.
3 Results 3.1 δ13C and δ15N Values of Horseshoe Crabs with Different Body SizesSoil and water samples were collected from the research sites. Some related environmental factors were measured, and the representative results are shown in Table 1. Analysis showed a large difference in the soil grain size of the horseshoe crab habitats in the three sample sites from the outer bay to the inner bay. The soils in Jiaodong and Fenghuangtou sites were mostly muddy sediments, and those in the Jintan site were mostly fine sand. Meanwhile, the seawater salinity and pH were similar among the three sample sites of horseshoe crab habitats. However, the food habits of horseshoe crabs in these study areas did not show any significant correlation with their habitat geochemical characteristics.
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Table 1 Geochemical characteristics of horseshoe crab habitats among three study sites of the intertidal zone in Pearl Bay during low tides |
The δ13C and δ15N values of T. tridentatus and C. rotundicauda from different age groups from the three study sites along the northern Beibu Gulf are shown in Table 2. The PW of the investigated C. rotundicauda specimens ranged from 50.50 mm to 142.45 mm, and that of T. tridentatus ranged from 49.85 mm to 73.25 mm. The δ13C and δ15N mean values of C. rotundicauda individuals ranged from −19.01‰ to −16.47‰ and from 10.49‰ to 13.5‰, respectively, and those of T. tridentatus ranged from −19.12‰ to −14.96‰ and from 8.78‰ to 13.48‰, respectively.
For both species, the δ13C value tended to decrease with increasing PW, whereas the δ15N value seemed to increase with increasing body size. However, the Pearson correlation analysis showed that these relationships were not significant (P > 0.10, n = 11 and 18 for T. tridentatus and C. rotundicauda, respectively).
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Table 2 Carbon (δ13C) and nitrogen (δ15N) stable isotope ratios of hemolymph from two Asian horseshoe crab species, T. tridentatus and C. rotundicauda in different size groups sampled along the northern Beibu Gulf shore |
Fig.2 shows that among the 13 possible potential food items for horseshoe crabs, sediment had the lowest mean δ13C value of −22.31‰ ± 3.16‰, and B. zonalis had the highest mean δ13C value of −13.36‰ ± 0.12‰. U. lactuca (−13.49‰) and Z. japonica (−15.16‰) had relatively higher mean δ13C values. The lowest δ15N value was found for U. prolifera at 3.67‰ ± 3.91‰, followed by sediment. The highest mean δ15N value was found for S. nudus at 11.28‰ ± 3.80‰. B. zonalis (10.71‰) and U. lactuca (10.66‰) had relatively higher mean δ13N values. In general, the mean δ15N values of all potential food items were lower than those of horseshoe crabs, except for those of S. nudus, B. zonalis, and U. lactuca that were slightly higher than those for juvenile T. tridentatus. In the horseshoe crab hemolymph samples, the highest and lowest δ13C mean values were obtained from the juvenile T. tridentatus and the adult C. rotundicauda, respectively. Meanwhile, the highest and lowest δ15N average values were found in the adult T. tridentatus and the juvenile T. tridentatus, respectively.
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Fig. 2 Stable isotope biplot for the hemolymph from T. tridentatus and C. rotundicauda and their potential food sources in Pearl Bay (mean ± standard deviation). |
The feasible contributions of each food source to the two species of horseshoe crabs were calculated using the MixSIR model as shown in Fig.3. The contribution of each potential food source in the juvenile C. rotundicauda (PW ranged from 50.50 mm to 63.40 mm) was in the following order: S. nudus > B. zonalis > U. lactuca > P. aibuhitensis > G. verrucosa > Z. japonica > A. farreri > D. corrugata > U. prolifera > M. lusoria > C. angulate > O. cucullate > sedi-ment. The contribution of individual potential food sources to the adults was consistent with those to the juveniles, with only slight differences observed in contribution rates.
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Fig. 3 Relative contribution (%) of potential food sources to the diets of T. tridentatus and C. rotundicauda in Pearl Bay at different growth stages (boxes were interquartile range: low = 25th percentile and upper = 75th percentile). |
B. zonalis, U. lactuca, and S. nudus were the main food sources for T. tridentatus. The most dominant food source for the juveniles was B. zonalis, with an average contribution of 19.80%. For the adults, the most important food source was S. nudus, with an average contribution of 34.20%. O. cucullate and sediment are the food sources of T. tridentatus with the lowest contributions.
S. nudus had a slightly higher median contribution to the juveniles and adults of C. rotundicauda (at 26.90% and 30.50%, respectively) and the juveniles of T. tridentatus (34.20%) than most of the above food sources. The contribution of each potential food source in the small age-class group of T. tridentatus was in the following descending order: B. zonalis > U. lactuca > S. nudus. The lowest-contributing potential food source was sediment, followed by O. cucullate, U. prolifera, and C. angulate. The contribution of potential foods was similar for all groups of horseshoe crabs, except for the potential food items that contributed more to the juvenile T. tridentatus than to the other groups.
4 Discussion 4.1 Comparison of the Potential Food Source Composition of Horseshoe CrabsResults showed that the δ13C and δ15N values of C. rotundicauda and T. tridentatus in the Beilun River Estuary varied over a wide range, indicating their wide feeding array. These obtained value ranges were similar to those previously reported (Hu et al., 2015). The mean δ13C values of both species were within the mean δ13C values of the potential food sources in the present study. In addition, the mean δ15N values of horseshoe crabs were higher than those of the potential food source species, suggesting that all selected food items are potential food sources for C. rotundicauda and T. tridentatus.
According to previous studies on the feeding habits of horseshoe crabs, the juveniles of C. rotundicauda and T. tridentatus feed on a wide range of crustaceans, bivalves, polychaetes, and gastropods (Chatterji et al., 1992; Carmichael et al., 2009). The results presented here also showed the large contribution of B. zonalis and S. nudus, with polychaetes and gastropods being the main sources of the horseshoe crabs, probably due to the convenience of feeding. Another reason is the high abundance and wide distribution of S. nudus in the three sample sites compared with other potential food source species.
This study reported the intermediate contribution ratios of seagrass. By contrast, a previous study using muscle samples indicated that the two Asian horseshoe crab species, C. rotundicauda and T. tridentatus, primarily rely on seagrass-derived nutrients from local vegetated estuarine and coastal habitats in the Beilun Estuary throughout their life stages (Fan et al., 2017). This difference might be attributed to the sample sites being heavily explored for seafood harvesting. Frequent anthropogenic disturbance in the local environment may have a significant effect on the dietary characteristics of C. rotundicauda and T. tridentatus, leading to great changes in food web structure and qualities in the research sites. As a result, the food source compositions of the local horseshoe crabs have been altered. In another light, this finding may also display the difference between hemolymph and muscle samples in the experiments.
The dietary compositions of the juveniles of these two species were assessed using their fecal samples in Hong Kong (Lee et al., 2021), and the results suggested that oligochaetes are the major prey items for C. rotundicauda (41.6%) and T. tridentatus (32.4%), followed by bivalves and crustaceans for C. rotundicauda (8.6% and 8.4%, respectively). The difference in these results might be related to the variation of food resource availability and experimental materials (muscle, feces, and hemolymph).
4.2 Relationship Between the Feeding Habits and Growth of Horseshoe CrabsStudies based on isotope technology by Carmichael et al. (2003) and Gaines et al. (2002) showed that the food chain of horseshoe crab juveniles changes with their age. With the growth of juvenile horseshoe crabs, the nutritional position in the food web structure changes significantly. The food of juvenile horseshoe crabs is mainly organic matter particles on the sediment surface in the environment. Horseshoe crabs of age-class 5 begin to feed on polychaetes and crustaceans, and larger gastropods and bivalves are fed by adult horseshoe crab.
No significant difference was observed between the feeding habits and age classes of horseshoe crabs. One reason might be that the horseshoe crab juveniles are older juveniles from ten instar with PW not less than 50 mm (Liu, 2014; Fan et al., 2017), and they are older than those in previous research. The horseshoe crab juveniles at ten instar already have hard mouthparts and mature organs similar to mature individuals.
4.3 Influence of Environmental FactorsSimilar basic environmental factors revealed similar feeding habitats between C. rotundicauda and T. tridentatus from different sites. Previous studies found a significant correlation between the growth of horseshoe crabs and the environmental conditions of their habitat. C. rotundicauda and T. tridentatus have different habitat preferences: T. tridentatus prefers sandy habitats, and C. rotundicauda prefers a muddy environment (Liu, 2014). In Fenghuang intertidal zone, the sediments are muddy, and the population of horseshoe crabs is small. Meanwhile, a large number of T. tridentatus individuals were found at the fine sandy sediments of the Jintan intertidal zone, and this finding is consistent with previous studies. Horseshoe crabs span their whole life history across diverse habitat types from beaches near the high-tide line through intertidal mudflats and coastal waters within 30 m in bathymetry as their nesting, nursery, and feeding grounds, respectively (Chen et al., 2015). Given that the habitats of horseshoe crabs from different age classes are distributed in different coastal areas, the environmental factors of the habitats might affect the feeding characteristics of these animals. However, no significant correlation was found between the feeding habits of horseshoe crabs and habitat geochemistry. These findings might also be attributed to the complex food sources for horseshoe crabs and the relatively less difference among individuals from different sites. The indiscriminate dietary compositions of C. rotundicauda and T. tridentatus suggested that they might compete vigorously for the food sources that have become limited due to human disturbances in coastal regions.
5 ConclusionsThe δ13C and δ15N values of C. rotundicauda and T. tridentatus of Pearl Bay in southwestern China varied over a wide range. In addition, their food sources vary widely, implying their omnivorousness. No significant correlations were found between the δ13C and δ15N values and individual widths in the two species. S. nudus, B. zonalis, and U. lactuca are the dominant food resources of horseshoe crabs from the inner, middle, and outer bays of the Pearl Bay in northern Beibu Gulf. No significant correlation was observed between the feeding habits of horseshoe crabs and habitat geochemical characteristics. All these results might indicate the highly diverse food sources and indiscriminate dietary compositions of the two Asian horseshoe crabs.
AcknowledgementsThis work was supported by the Scientific Research Project of Huaqiao University (No. 605-50X18005), Guangxi Bagui Youth Scholars Programme, Guangxi Recruitment Program of 100 Global Experts.
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