Spatiotemporal Distribution of Asian Horseshoe Crab Eggs Are Highly Intermingled with Anthropogenic Structures in Northern Beibu Gulf, China

KWAN Kit Yue, FU Yijian, ZHONG Mufeng, 3), KUANG Yang,BAI Haiwei, ZHANG Ce, ZHEN Wenquan, XU Peng,WANG Chun-Chieh, and ZHU Junhua, *

Spatiotemporal Distribution of Asian Horseshoe Crab Eggs Are Highly Intermingled with Anthropogenic Structures in Northern Beibu Gulf, China

KWAN Kit Yue1), 2), FU Yijian1), ZHONG Mufeng1), 3), KUANG Yang1),BAI Haiwei1), ZHANG Ce1), ZHEN Wenquan1), 2), XU Peng1), 2),WANG Chun-Chieh4), and ZHU Junhua1), 2), *

1),,,535011,2),535011,3),535011,4),,530007,

Identification, protection and restoration of spawning habitats are vital for protecting the depleted species. Asian horse- shoe crabs are ecologically important macroinvertebrates in coastal and estuarine ecosystems. However, their spawning habitat stud- ies were limited to several reports in tropical regions, possibly due to the lack of modified survey methods, particularly in habitats with a lower density of spawning adults, and/or intermingled with anthropogenic structures. In this study, the year-round egg distri- bution and spawning habitat baselines ofandwere determined in the northern Beibu Gulf, China. Our findings demonstrated that the peak spawning occurred in June–July and ceased in November–January when the average water temperature dropped below 20℃. Egg aggregations were found <10cm beneath the sediment surface with regular tidal inundation, regardless of seasonal changes, in the vicinity of natural and artificial structures with elevated, mildly sloping sub- stratum within the high tide zones. The nests were characterized by medium-sized sediment grains (0.5–0.9mm), high temperatures (31–34℃), low water contents (0.8%–0.9%), and total organic carbon contents (0.5%–0.7%), which might maximize the hatching success. The identified nesting beaches were close to nursery habitats for juveniles, and tidal creeks were present as the possible cor- ridor connecting these two important habitats through the dominant mangrove forests. The findings provide valuable insights in the scope of spawning behavior and nest-site selection of Asian horseshoe crabs under a mixture of natural and artificial structures, which could benefit future management efforts for the exploited spawning populations.

;; mangrove; tidal creek; sediment texture; slope; elevated substratum

1 Introduction

Coasts and estuaries are thriving but delicate ecosystemsfor a myriad assortment of living species. Horseshoe crabs are typical estuarine denizens inhabiting along the west coast of the North Atlantic and Pacific Oceans (Smith., 2017; John., 2018). Commonly referred to as ‘living fossils’, four extant horseshoe crab species bearing nearly unchanged physical appearance for more than 450 million years (Rudkin and Young, 2009). Horseshoe crabs are eco- logically important in the estuarine food web as both pre- dators and prey (Botton, 2009). Recent studies also point- ed out their potential role as an indicator species in re-flecting the general health of ecosystems (Kwan., 2018,2021). However, the long-term survival of horseshoe crabs does not ensure their future persistence in a changing world. A combination of threats derived from coastal develop- ment and human-induced disturbance have severely affect- ed the remaining horseshoe crab populations (Wang., 2020). Most notably, commercial exploitation of horseshoe crabs fororamoebocyte lysate pro- duction (Gauvry, 2015; Yan, 2018) and human con- sumption (Fu., 2019) pose considerable pressures on their survival. The Atlantic horseshoe craband tri-spine horseshoe crabhave been listed as ‘Vulnerable’ (Smith., 2017) and‘Endangered’ (Laurie., 2019), respectively, based on the recent extensive reviews of the population status and trends across the ranges. The other two Asian species, the coastal horseshoe craband the mangrove horseshoe crabremain ‘Data De- ficient’, but their status are under reassessment due to the

increasing reports depicting their declining trends and the imminent threats to their populations and habitats.

Identification, protection and restoration of critical ha- bitats are the first step for reversing the declining trend of exploited marine organisms (Collins., 2000). Horse- shoe crabs utilize a unique combination of habitats during their entire life cycle: 1) nearshore areas as foraging ground for adults, 2) intertidal flats as nurseries for juveniles, and 3) sandy beaches between the low-tide terrace and thehigh-tide water line for spawning and egg incubation (Smith., 2017). The life history, ecology and habitat ofhave been studied extensively, but the collec- tion of habitat baselines for these three Asian species were disproportional (Wang., 2020). Some Asian countries and territories, including Mainland China (Xie., 2020),Hong Kong region (Kwan., 2016), Taiwan region(Hsieh and Chen, 2015), Singapore (Cartwright-Taylor, 2015) and Indonesia (Meilana., 2021), had gathered rela- tively complete population information from the intertidal nursery habitats. However, the data of spawning ecology and behavior, which can provide particular baseline infor- mation on egg deposition and nest parameters, were large- ly restricted to several studies in tropical countries such as Malaysia (Fairuz-Fozi., 2018; Mohamad., 2019; Zauki., 2019) and Singapore (Cartwright-Taylor, 2015).In China, spawning activities of Asian horseshoe crabs have not been documented since 1984 (Cai., 1984), possi- bly due to the lack of modified spawning and egg survey methods, particularly in areas with a lower density of spawn- ing adults as well as complicated natural and anthropo-genic structures along the shoreline. The nesting pattern of horseshoe crabs that lay clusters of eggs at depths of 10– 20cm beneath the sediment layer also imposes significant challenges for the spawning baseline collection (Botton., 2021).

While the spawning behavior of horseshoe crabs shares many common features, their nesting preference can be site- specific. For the well-studied Atlantic species, spawning beaches in Delaware and New Jersey, USA are typically coarse-grained, while Florida beaches comprised typical- ly fine-grained and poorly drained substratum (Penn and Brockmann, 1994; Smith., 2017). For the Asian spe- cies,and, similar to,seemed to prefer sandy areas between upper- and mid-in- tertidal zones with fine and medium-sized sediment grains (Nelson., 2015; Mohamad., 2019). Itaya. (2019) also observed high spawning activities ofat the moderate slopes near the high tide line.was frequently cited as an excep- tion since their spawning adults are widely observed in themuddy mangrove areas even during low tides (Cartwright- Taylor, 2015; Fairuz-Fozi, 2018; Liao., 2019a). This, therefore, poses an intriguing question on the possi- bly different spawning behavior and nesting preference of

The northern Beibu Gulf is a semi-closed gulf along the coast of southern China and northern Vietnam. The majorestuarine ecosystem is fringed by extensive intertidal beaches (713km2), and include large areas of pristine mangrove forests (74km2) and seagrass beds (0.8km2) (Fan., 2016). On the other hand, the coastline configuration has been largely altered by the construction of aquaculture ponds (344km2), artificial salt pans (23km2), ports (18km2) and seawalls (654km) (Li., 2017). The gulf is frequently claimed to accommodate high densities ofandpopulations in Asia-Paci- fic regions (Brockmann and Smith, 2009; Sekiguchi and Shuster, 2009; Fu, 2019; Liao., 2019a). The in- tertidal habitats for horseshoe crab egg deposition can be influenced by the man-made shoreline features (Botton., 1988; Jackson., 2015; Nelson., 2015). In this study, we examined 1) year-round variation of nest and egg densities, 2) temporal changes in nest distributions, and 3) collected environmental physiochemical parameters of Asian horseshoe crab spawning habitats along the shore- line intermingled with anthropogenic structures. The find- ings can provide valuable baseline data to develop scien- tifically important conservation measures by accounting for critical habitat distribution and peak spawning season of Asian horseshoe crabs in the region.

2 Methods

2.1 Egg Surveys

Preliminary investigation of Asian horseshoe crab spawn- ing habitats was conducted at 18 potential beaches along the northern Beibu Gulf shoreline (Fig.1) during June–August 2019 at day-light low tides around the time of the new and full moons. The sites were chosen based on the identified nursery habitat distribution (Xie., 2020) and fishing community reports (Liao., 2019a). Given that complex natural (., open sandy-mudflats, mangrove forests, tidal creeks and intertidal sand bars) and artificial structures (., seawalls, aquaculture ponds and other coas- tal physical infrastructure) were present along the shore- line, a modified egg survey method was required. Asian horseshoe crab numbers are much low than those in the USA, and egg densities might be too low to be detected using traditional random cores (Botton., 2021). Nest marking method by tracking spawning adults as described in Mohamad. (2019) has been tried. However, none was observed possibly due to the relatively smaller adultpopulation in the gulf. It is also a challenge to closely follow the spawning trail ofduring rising tides when the estuarine water became turbid.

During the preliminary surveys, a team of four people searched for the eggs according to spawning imprints,., shallow depressions left on sediment surface by the females as they deposit their eggs (see John., 2012), within mid- to upper-intertidal areas (<1km from seawalls or road- sides) during low tides. All likely depressions were at- tempted using a handheld shovel to an approximate depth of 20cm from the sediment surface, which can assure that mostandegg clusters can be detected (Fairuz-Fozi., 2018; Mohamad., 2019). All eggs found within the sediment under the same spawn- ing imprint were considered as a ‘nest’. The sampling of Asian horseshoe crab eggs was carried out across a variety of natural habitat types and man-made structures.eggs were distinguished frombytheir discernably smaller size (diameter 1.9–2.2mm3.0–3.3mm for) with greenish-yellow color. Sampling was not conducted during night-time due to safe- ty reasons.

Six shores, including Shanxin (SX), Jiaodong (JD), Yu- zhouping North (YZPN), Yuzhouping South (YZPS), Zhong- sandun (ZSD) and Shajiao (SJ), were identified as the ac- tive spawning habitats for Asian horseshoe crabs (Fig.1). The results of adopted survey method are highly depen- dent on the people who searched for the nests. Therefore, the same team of four people conducted the monthly egg samplings, except during the outbreaks of COVID-19 pan- demic in Mainland of China (February–April 2020), at the designated survey areas with standardized survey efforts (., person-hour per km2survey area; Fig.1, Table 1). Only one nest ofand one nest ofwere found at XGS respectively, therefore XGS was not included in the monthly egg surveys. All identified nests were marked using a colored stick, and all measurements were conducted after the end of the allowed survey time. The GPS locations, nest depth, species, total counts of eggs and their developmental stages (undeveloped eggs, em- bryos at stage 20–21, or hatched trilobite larvae) for each nest were recorded. Only eggs without apparent signs of decay, including those embryos and larvae remained with- in the beach sediments, were included in the counts. The eggs/larvae were returned back to the original pit and co- vered by the sediment once the measurements were com- pleted. Similar approach of egg samplings and measure- ments were conducted in another study ofat Tsuyazaki Cove, Japan (Itaya., under review), and found that the survival and hatching rates of ‘disturbed’eggs (., eggs which were repeatedly sampled, measured and returned back to the original pit) were similar to those ‘control’ eggs in the same region. Since the nest/egg den- sity data derived from the present survey were largely based on the team of people involved in the egg searching, the information was only used to demonstrate the spatiotem- poral patterns, but not for comparing the ‘absolute’ values among studies.

Fig.1 Maps showing the locations of the northern Beibu Gulf, China, and the 18 potential spawning sites of Asian horseshoe crabs. Monthly egg density surveys were conducted at the six study shores (yellow triangles) within the designated survey areas (white contours), except during the outbreaks of COVID-19 pandemic in Mainland China (February–April 2020). The high-density egg areas are indicated in orange rectangles. The scale bar on the map is in 200m. Satellite images were acquired from National Platform for Common Geospatial Information Services (Beijing, China). The site abbreviation is mentioned in Table 1.

Table 1 Asian horseshoe crab egg survey site coordinates, dates and efforts at six study sites in the northern Beibu Gulf, China

Note: The survey time was assigned according to the corresponding total survey area at each site with the consistent number of people in survey team.

2.2 Habitat Physiochemical Parameter Measurements

Measurements of nest temperature, elevation, inclination and oxidation-reduction potential (ORP) were conductedduring low tides. Nest temperature and ORP were measured with a portable ORP meter (Harveson 100P, Su- zhou, China), whereas inclination data were collected us- ing a digital inclinometer (Syntek, Huzhou, China). Ele- vation on the nest surface was determined by a satellite- based real-time kinematic positioning system (Hi-Target, V90, Guangzhou, China).

Sediment samples for determination of grain size, water content, total organic carbon (TOC) and chlorophyll(Chl) were collected in the vicinity of the high-density nest areasat the depth of egg cluster using a 7cm-diameter sediment core. Sediment samples were stored on ice and under dark conditions before they were taken to the laboratory or near- by accommodation within an hour. The grain size was de- termined according to the Blott and Pye (2001) protocol; TOC was analyzed following wet dichromate oxidation method; the water content was calculated from the differ- ence of weight before and after the samples were dried at 60℃ for 72h until achieving a constant weight; and the Chlmeasured using Wisconsin State Laboratory of Hy- giene’s ESS Method 150.1 with modification. All techni- cal details were described in Xie. (2020).

To collect more complete environmental baselines re- garding spawning habitats, surface water physiochemical parameters were also determined at a minimum of three areas per study site, where higher densities of Asian horse- shoe crab nests were recorded. The water samples werecollected during rising tides when the seawater level reach- ed at approximately 40cm, and a high number of matingpairs was observed within the investigation areas. Watertemperature, salinity, dissolved oxygen (DO), pH, water tur-bidity and ORP were measured, whereas suspended particulate matter (SPM), total dissolved nitrogen (TDN) andtotal dissolved phosphorus (TDP) were quantified in the la- boratory. Water temperature and DO were measured by a portable DO meter (YSI ProODO, OH, USA), salinity wasdetermined by an optical refractometer (ATAGO, WA, USA),pH was measured using a handheld pH meter (Thermo Scientific, Orion Star A211, MA, USA), and turbidity was determined with the portable turbidimeter (HACH, 2100Q, CO, USA). SPM concentration was determined by calcu- lating the weight difference between the particulate mat- ter retained on the dried and pre-filtered blank filters, as described by Dan. (2019). TDN was quantified using an auto-analyzer after the decomposition of samples in an autoclave at 120℃ for 0.5h with boric acid-persulfate oxi- dation solution (Lin., 2012). TDP was determined by digesting the samples with acidified 50gL−1K2S2O8solu- tion (pH=1) in an autoclave sterilizer at 120℃ for 90min (Cai and Guo, 2009). The environmental baseline data forat SJ and ZSD were not collected due to the very limited number of nests.

2.3 Data Analysis

All data were tested for normality and homogeneity of variance using Shapiro-Wilk test and Levene’s test, respec-tively. While abundance data of eggs per nest did not meet the normality requirements after all attempts for arithme- tic transformations, the differences among study sites and sampling months were analyzed using non-parametric Schei- rer-Ray-Hare extension of Kruskal-Wallis test. The above calculations were performed in SPSS Statistics (v 26, IBM,NY, USA), and Scheirer-Ray-Hare extension of the Krus- kal-Wallis test was conducted following the modified SPSS protocol described in Shen. (2013).

3 Results

3.1 Temporal Variations of Nest and Egg Densities

Only fournests were found at SJ and one at ZSD during September–November. Monthly changes in densities ofnests and eggs were evident (Fig.2). Nest and egg densities at SX and YZPS peaked in June, whereas those at JD, YZPN and SJ peaked in July (Figs.2A–B). Both the nest and egg densities declined gra- dually after July and entirely ceased in November or De- cember (Figs.2A–B). Spawning ofseem- ed to begin in the coming year in May or earlier in April. The egg and nest densities at ZSD were generally low throughout the year with a peak in August.

Average egg counts (± standard deviation) of eachnest varied monthly between 14 and 30±2 (Fig.2C). However, the egg yields per nest differed neither across sampling month nor among study sites (Month:=3.735,=0.710; Site:=1.079,=0.960; Month×Site:=6.524,=1.000). Egg counts were considerably high- er in eachnest (195–573) compared to those in(13–90). The percentageof undevelop- ed eggs, after excluding those embryos and trilobite larvae, was the lowest in October–November (Fig.2D).eggs were deposited at a depth of 2.4cm (range: 1.4–3.7cm), which was apparently shallower than those of(6.9–7.5cm).

3.2 Spatial Distribution of Nests

Bothandnests were ob- served to be located primarily within the higher intertidal zones limited by the presence of seawalls and/or coastal shrimp ponds (Fig.3). Most nests were found in unshelter- ed areas between mangrove patches with tidal creeks near- by. At SX, spawning also occurred along the artificial ti- dal channels of aquaculture ponds, given that the water con-trol gate was remained open. Temporal changes in egg dis- tributions were indiscernible (Fig.3). Potential nesting po- ints on open sandy mudflats in mid-intertidal zones out- side the mangrove fringes were also searched during the preliminary surveys, however, no nest was detected.

Fig.2 Monthly variations of (A) nest density, (B) egg density, (C) the mean number±standard error of eggs per nest, and (D) the percentage of undeveloped eggs (excluding those embryos and trilobite larvae) of C. rotundicauda at the six study sites. The samplings for Asian horseshoe crab (A) nests and (B) eggs were conducted at the designated survey areas with standardized survey efforts, and their densities were expressed in the amount of sampling work performed by the average person in one hour (person-hour). The data in (C) were analyzed by Scheirer-Ray-Hare extension of Kruskal-Wallis test, but no significant difference among site/month groups was detected. The site abbreviation is mentioned in Table 1.

Fig.3 The monthly nest distribution of C. rotundicauda (red) and T. tridentatus (blue) at the six study sites along the northern Beibu Gulf shoreline (within the orange rectangles in Fig.1). The satellite maps are obtained from the National Platform for Common Geospatial Information Services (https://www.tianditu.gov.cn/). Different sampling months are indicated by the gradients of red/blue. The temporal variation of nest locations was indiscernible. The site abbreviation is mentioned in Table 1.

In terms of beach topography, nests were mostly locatedat elevated substratum beside mangrove tidal creeks, at the bases of mangrovesurface roots or elevated coarse-grained substrate with cobbles, as well as on the sandy slope or narrow band in high tide areas (Fig.3and Figs.4A–F). Nests can also be found not far away from man-made structures, including power transmission faci- lities, mangrove boardwalks, abandoned construction wastes, cemented walkways, water control gates and man-made ti- dal channels of aquaculture ponds (Fig.3 and Figs.4G–I).

Fig.4 Examples for surrounding environmental characteristics of Asian horseshoe crab spawning habitats in the northern Beibu Gulf. A, at the bases of mangrove Excoecaria agallocha surface roots; B, along tidal creeks in mangrove areas; C, at elevated coarser substrates with cobbles; D, at mini sand bars along seawalls; E, at elevated slopes along seawalls; F, elevated slopes outside aquaculture ponds; G, at the bases of power transmission facility; H, near water control gates connected to aquaculture ponds; I, along man-made tidal channels of aquaculture shrimp ponds. Nest distribution points of C. rotundicauda and T. tridentatus are in red and blue, respectively.

3.3 Nest Parameters

The sediment physiochemical conditions ofnests were similar among study sites (Table 2). At low tides, the average nest temperature reached 31–34℃, and the water content was 0.8%–0.9%. The nest sediment was mainly comprised medium sized grains (0.5–0.9mm) with low TOC (0.5%–0.7%), Chl(1–3μgg−1) and ORP (225–252mV). Spawning occurred at substratum with mild and elevated slope (inclination 5˚–14˚, elevation −18m to −20m; Table 2).

The spawning habitats are regularly inundated by estuarine waters with the relatively stable temperature of 31–33℃, salinity 11–21, DO 5–6mgL−1, pH 7.3–7.8 and ORP 217–236mV (Table 3). The water turbidity (36–187NTU) as well as levels of SPM (77–151mgL−1), TDN (61–84μmolL−1) and TDP (2–66μmolL−1) of the spawn- ing grounds were greatly fluctuated during rising tides (Ta- ble 3).

4 Discussion

Asian horseshoe crabs,andcoexist on many intertidal flats in Asia-Pacific re- gions (Mohamad., 2015; Kwan., 2016; Xie., 2020; Meilana., 2021). In this study,eggs were found in the vicinity of those fromat SJ and ZSD (Fig.3), despite the fact that the num- ber of identified nests fromwas limited. In addition to the apparent declines of the localpopulation (Liao., 2019a; Wang., 2020), we also speculate the nesting ofoccurs on sand bars or elevated sand substratum in the lower intertidal zones(Cai, 1984). Extensive stick nets erected along the coastline may also prevent the larger-bodiedadults, compared to other Asian species, from accessing the upper intertidal areas.was observed to spawn throughout the year in tropical regions with a peak in the southwest monsoon period that takes place during May–September (Fairuz-Fozi, 2018; Mat Zauki., 2019). In another report with the con- trasting result, May–August was probably the period with least frequent breeding of the species in Singapore (Cart- wright-Taylor, 2015). In our study which was conductedin the subtropical region, the spawning ofbegins at least before May and peaks in June–July. The nesting activities in most study sites seem to cease in No- vember or not later than December (Fig.2), when the wa- ter temperature typically drops below 20℃ (Gong., 2019). This is consistent with the findings from both field investigations and laboratory experiments showing that Asian horseshoe crabs have restricted tolerance capability to low temperatures (Chiu and Morton, 2004; Liao., 2019b). In October and November, the percentage of un- developed eggs was low, and most had been developed into stage-20 embryos or hatched into trilobite larvae (Fig.2D). In the laboratory,eggs spent approximate- ly 48 days to develop into trilobite larvae (Zadeh, 2009). The hatched larvae would remain in the nests for several more weeks before emerging from the sediment and being transported from the breeding grounds (Rudloe, 1979; Botton., 2010). In this case, most eggs were deposited probably around July, which was identified as the spawning peak in this region. Additionally, 5–8 trilo- bite larvae were found in SJ and SX sediments in May, which suggest that the spawning activities may begin in March–April. The number of eggs per nest of(13–90) and(195–573) in this study was comparable to those noted in Peninsular Malaysia which was 18–107 for(Fairuz-Fozi., 2018; Zauki., 2019), and in Japan which was 200– 500 for(Sekiguchi and Nakumura, 1979); however, it was considerably lower than that in Borneo, Malaysia, which was 967 for(Mohamad., 2019).

Table 2 Nest environmental characteristics during low tides at identified spawning habitats for C. rotundicauda along the northern Beibu Gulf coast

Notes: ORP, oxidation reduction potential; TOC, total organic carbon content; Chl, chlorophyll. The range data are provided in brackets. The data fornests are not shown in table due to the very limited number of nests was found. The site abbreviation is mentioned in Table 1. Data are expressed as mean ± standard deviation;= sample size.

(water level: about 40cm) in the northern Beibu Gulf

Table 3 Surface water physiochemical conditions within the high-density C. rotundicauda nest areas during rising tides

Notes: Data are expressed as mean±standard deviation;=sample size. The range data are also provided. The data fornests are not shown in table because of the very limited number of nests was found. DO, dissolved oxygen; ORP, oxidation reduction potential; SPM, suspended particulate matter; TDN, total dissolved nitrogen; TDP, total dissolved phosphorus. The site abbreviation is mentioned in Table 1.

The nest-site selection by spawning adults can be se- lective, typically in the upper beaches along the high tide line, to increase developmental success by minimizing ex-posure of the developing eggs to hypoxia and hydrogensulfide (Penn and Brockmann, 1994; Vasquez., 2015). Despite the high abundance of spawning adults ofwas noted in the mangrove areas, their nestse- lection characteristics were unclear. Fairuz-Fozi. (2018)found the egg aggregations on a sand bar with pioneer man- grovesandspp. and intertidal areas with domestic discharge points nearby, but not those open flats covered densely with pebbles, shells and pneumato- phore roots of mangroves. In the present study, the eggdistributions ofshowed the following cha- racteristics: 1) in the upper intertidal zones with a tenden- cy of approaching the high tide lines; 2) unsheltered areas on the edges of mangroves with tidal creeks nearby; 3) at the bases of elevated natural substratum (., sand bars, cobbles and mangrove surface roots) or man-made struc- tures (., construction wastes, power transmission faci- lity and mangrove boardwalks); 4) at the sandy slopes alongseawalls or tidal channels of aquaculture ponds (Figs.3 and 4).

The egg distribution pattern can be explained by the li- mited availability of suitable substratum for spawning in low-energy, muddy shores with little slope. Organic matter and hydrogen sulfide concentrations are commonly high in these muddy beaches (Zaldíar-Rae., 2009; Vasquez., 2015). Consequently, Asian horseshoe crabs depo- sited their eggs at high beach elevations, including natural and artificial structures, where the accumulation of coarse- grained (0.5–0.9mm) and well drained sediments occurred (Table 2). Those eggs were also observed to experience high nest temperature (31–34℃) and ORP (225–252mV), but low water content (0.8%–0.9%) and TOC (0.5%–0.7%) during low tides (Table 2). By comparing the present re- sults with previous results from other researchers, relative- ly finer sediment textures were recorded in spawning ha- bitats of(0.2–0.4mm, Zauki., 2019) in Malaysia and thoseofin Japan (0.2–0.3mm, Itaya., 2019).Meanwhile, Mohamad. (2019) observed a wider range of grain sizes (0–2mm)suitable forspawning in the northeastern Borneo.Sediment TOC was consistently low (<3%) in most placeswhereeggs were found (Fairuz-Fozi., 2018; Zauki., 2019). The inclination of nests (5˚–14˚) was also comparable to those recorded in Japan (3˚–8˚, Itaya., 2019). In terms of water physiochemical con- ditions, it seems that spawning adults can tolerate a broad-er range of fluctuations in salinity (2–32), DO (5–7mgL−1) and pH (6–8). The nest depths of(1–4cm) and(7–8cm) were shallow, which can avoid anaerobic conditions of deeper sediments that are known to affect development (Botton., 1988; Penn and Brockmann, 1994; Vasquez., 2015). The depth data were similar to those reported in previous studies (: 2–5cm; Fairuz-Fozi., 2018;: 5–22cm, Botton., 1996; Mohamad., 2019).

Extensive shore protection structures such as seawalls, bulkheads and revetments that extend below mid-foreshore can reduce, fragment and restrict the availability of suit- able spawning habitats for horseshoe crabs (Botton., 1988; Jackson., 2015). Their interaction with wave and hydrology also alters sediment textures and physio- chemical conditions, which reduce the spawning activity of horseshoe crabs (Nelson., 2015). In this study, spawning adults and eggs of Asian horseshoe crabs were noted within the artificial tidal channels when the water control gates remained open (Fig.3 SX). We postulate that the presence of seawalls and other coastal anthropogenic structures have obstructed the mating pairs from access- ing the high tide line. The amplexed pairs were also ag- gregated along the seawalls or at the entrance of water control gates, and such behaviors have previously docu- mented by Itaya. (2019). On the other hand, the clus- tering of horseshoe crab spawning near mangrove board- walks, water control gates and other physical structures may also be attributed to the sheltered areas created by these vertical features extend out onto the intertidal zone. Similar cases were noted in Delaware Bay , USA, where a higher spawning density ofoccurred with- in the enclaves between bulkhead segments with low waveenergy and decreased current velocities (Jackson., 2015). Additionally, the nesting sites were not far away from the identified nursery habitats on sandy mudflats along the outer fridge of mangroves (Xie., 2020). Therefore, tidal creeks that present near the identified nests (Fig.3) were probably the corridor connecting nursery and spawn- ing habitats for Asian horseshoe crabs separated by the do- minant mangrove forests.

Based on the present results, possible management mea- sures for conservation of Asian horseshoe crab spawning populations include: 1) routinely remove ground cages and erected stick nets in high tide areas, including those along tidal creeks, 2) regulate artisanal bivalve aquaculture and beachcombing activities near mangrove areas, 3) close the upper intertidal areas in June–July during the peak spawn- ing period to minimize the detrimental effects from arti- sanal fishing with illegal gears, 4) avoid coastal reclama- tion projects that diminish the beach topography, particu- larly those elevated, coarse-grained substratum and slopes, and 5) restore the spawning habitats by creating mounds with moderate slope and suitable sediment textures along the shoreline. Future studies will explore the nesting behavior of Asian horseshoe crabs, including the influence of lunar tidal rhythmicity and the daily migration to deep- er waters, to further our holistic understanding of their life history and reproduction ecology.

5 Conclusions

Our findings demonstrated apparent temporal variations of nest and egg densities, which peaked in June–July. Ag- gregations of eggs were found embedded in the shallow surface layer (<10cm) of coarse-grained sediment in the upper intertidal zones, and in the vicinity of natural and artificial structures with the elevated, mildly sloping substratum. The nest site selection was to maximize the hat- ching success by incubating the developing eggs with high- er temperature and aerobic conditions. Seawalls and other physical infrastructure seemed to obstruct the access ofspawning pairs to the high tide line. The present study provides valuable insights in the scope of spawning and nest site selection of Asian horseshoe crabs under a mix- ture of natural and artificial structures, which is useful to improve the management efforts of a group of ecological- ly important and endangered species.

Acknowledgements

This study was funded by the National Natural Science Foundation of China (Nos. 32060129, 42067038), the Bei- bu Gulf Ocean Development Research Center under Key Research Base of Humanities and Social Sciences in Guang-xi Universities, the Guangxi Bagui Youth Scholars Pro-gramme, and the Guangxi Recruitment Program of 100 Global Experts.

Blott, S. J., and Pye, K., 2001. GRADISTAT: A grain size distri- bution and statistics package for the analysis of unconsoli- dated sediments., 26: 1237-1248.

Botton, M. L., 2009. The ecological importance of horseshoe crabs in estuarine and coastal communities: A review and specula- tive summary. In:.Tanacredi, J. T.,., eds., Springer, Massachusetts, Boston, 45-63.

Botton, M. L., Colon, C. P., Sclafani, M., Loveland, R. E., Elbin, S., and Parkins, K., 2021. The relationships between spawn- ing horseshoe crabs and egg densities: Recommendations for the assessment of populations and habitat suitability., 31: 1570- 1583.

Botton, M. L., Loveland, R. E., and Jacobsen, T. R., 1988. Beach erosion and geochemical factors: Influence on spawning suc- cess of horseshoe crabs () in Delaware Bay., 99 (3): 325-332.

Botton, M. L., Shuster Jr., C. N., Sekiguchi, K., and Sugita, H., 1996. Amplexus and mating behavior in the Japanese horse- shoe crab,., 13: 151- 159.

Botton, M. L., Tankersley, R. A., and Loveland, R. E., 2010. De- velopmental ecology of the American horseshoe crab.,56: 550-562.

Cai, X., Lin, Q., and Huang, J., 1984. Spawning behavior and early embryonic development of., 6: 663-671 (in Chinese with English ab- stract).

Cai, Y., and Guo, L., 2009. Abundance and variation of colloidal organic phosphorus in riverine, estuarine, and coastal waters in the northern Gulf of Mexico., 54: 1393-1402.

Cartwright-Taylor, L., 2015. Studies of horseshoe crabs around Singapore. In:. Carmichael, R. H.,., eds., Springer, Zug, Cham, 193-211.

Chiu, H. M., and Morton, B., 2004. The behaviour of juvenilehorseshoe crabs,(Xiphosura), on a nur- sery beach at Shui Hau Wan, Hong Kong., 523: 29-35.

Collins, M. R., Rogers, S. G., Smith, T. I., and Moser, M. L., 2000. Primary factors affecting sturgeon populations in the south- eastern United States: Fishing mortality and degradation of es- sential habitats., 66: 917-928.

Dan, S. F., Liu, S. M., Udoh, E. C., and Ding, S., 2019. Nutrient biogeochemistry in the Cross River estuary system and adja- cent Gulf of Guinea, South East Nigeria (West Africa).,179: 1-17.

Fairuz-Fozi, N., Satyanarayana, B., Zauki, N. A. M., Muslim, A. M., Husain, M. L., Ibrahim, S.,., 2018.(Latreille, 1802) population status and spawn- ing behaviour at Pendas coast, Peninsular Malaysia., 15: e00422.

Fan, H. Q., He, B. Y., Mo, C. Z., Wu, B., Yan, B., Qiu, G. L.,., 2016. Marine and coastal wetlands conservation and wise use. In:. Tan, W. F.,., eds., Environmental Science Press, Beijing, 180-213 (in Chinese).

Fu, Y., Huang, S., Wu, Z., Wang, C. C., Su, M., Wang, X.,., 2019. Socio-demographic drivers and public perceptions of consumption and conservation of Asian horseshoe crabs in nor- thern Beibu Gulf, China., 29: 1268-1277.

Gauvry, G., 2015. Current horseshoe crab harvesting practices can- not support global demand for TAL/LAL: The pharmaceutical and medical device industries’ role in the sustainability of horse- shoe crabs. In:. Carmichael, R. H.,., eds., Springer, Zug, Cham, 475-482.

Gong, B., Huang, H., Peng, C., Wang, J., Ma, J., Liu, X.,., 2019. The microbiomic and environmental analysis of sedi- ments in the Indo-Pacific humpback dolphin () habitat in the Northern Beibu Gulf, China., 26 (7): 6957-6970.

Hsieh, H. L., and Chen, C. P., 2015. Current status ofin Taiwan for Red List assessment. In:. Carmichael, R. H.,., eds., Springer, Zug, Cham, 383-396.

Itaya, S., Seino, S., Shuuno, M., Sakurada, A., and Koshiguchi, R., 2019. Spawning site selection of the endangered horse- shoe crabat Tsuyazaki Cove in Fukuo- ka, Japan.. Hanoi, Vietnam, 959-963.

Jackson, N. L., Nordstrom, K. F., Saini, S., and Smith, D. R., 2015. Influence of configuration of bulkheads on use of estuarine beaches by horseshoe crabs and foraging shorebirds., 74 (7): 5749-5758.

John, B. A., 2012. Feeding ecology, molecular phylogeny and TAL production from Malaysian horseshoe crabs (and). PhD thesis. International Islamic University Malaysia, Kuala Lumpur, Malaysia.

John, B. A., Nelson, B. R., Sheikh, H. I., Cheung, S. G., War- diatno, Y., Dash, B. P.,., 2018. A review on fisheries and conservation status of Asian horseshoe crabs., 27: 3573-3598.

Kwan, B. K. Y., Hsieh, H. L., Cheung, S. G., and Shin, P. K. S., 2016. Present population and habitat status of potentially threat-ened Asian horseshoe crabsandin Hong Kong: A proposal for ma- rine protected areas., 25: 673- 692.

Kwan, B. K. Y., Un, V. K. Y., Cheung, S. G., and Shin, P. K. S., 2018. Horseshoe crabs as potential sentinel species for coastal health: Juvenile haemolymph quality and relationship to habi- tat conditions., 69: 894-905.

Kwan, K. Y., Bopp, J., Huang, S., Chen, Q., Wang, C. C., Wang,X.,., 2021. Ontogenetic resource use and trophic dyna-mics of endangered juvenileamong di- versified nursery habitats in the northern Beibu Gulf, China., 16 (6): 908-928, DOI: 10.1111/1749-4877. 12495.

Laurie, K., Chen, C. P., Cheung, S. G., Do, V., Hsieh, H., John, A.,, 2019.(Errata Version Published in 2019). e.T21309A149768986. IUCN, Gland, Switzerland, 60pp.

Li, G. Z., Liang, W., Wang, X., Liu, T., and Nong, H. Q., 2017.. China Ocean Press, Beijing (in Chinese).

Liao, Y., Hsieh, H. L., Xu, S., Zhong, Q., Lei, J., Liang, M.,, 2019a. Wisdom of Crowds reveals decline of Asian horseshoe crabs in Beibu Gulf, China., 53: 222-229.

Liao, Y., Liu, K., Wu, H., Xu, Y., Huang, H., Xu, S.,., 2019b.How survival and food intake of tri-spine horseshoe crabs,respond to thermal variation: Implicationsfor understanding its distribution limit., 53: 1951-1960.

Lin, P., Chen, M., and Guo, L., 2012. Speciation and transforma- tion of phosphorus and its mixing behavior in the Bay of St. Louis estuary in the northern Gulf of Mexico., 87: 283-298.

Meilana, L., Hakim, A. A., and Fang, Q., 2021. Nursery habitat of three species of juvenile Asian horseshoe crabs in Teritip Beach, East Kalimantan, Indonesia: Characterization and im- plication., 26: e01453.

Mohamad, F., Ismail, N., Ahmad, A. B., Manca, A., Rahman, M. Z. F. A., Bahri, M. F. S.,., 2015. The population size and movement of coastal horseshoe crab,(Mül- ler) on the East Coast of Peninsular Malaysia. In:. Carmichael, R. H.,., eds., Springer, Zug, Cham, 213-228.

Mohamad, F., Mohd Sofa, M. F. A., Manca, A., Ismail, N., Che Cob, Z., and Ahmad, A. B., 2019. Nests placements and spawn- ing in the endangered horseshoe crab(Leach, 1819) (Merostomata: Xiphosurida: Limulidae) in Sa- bah, Malaysia., 39: 695-702.

Nelson, B. R., Satyanarayana, B., Zhong, J. M. H., Shaharom, F., Sukumaran, M., and Chatterji, A., 2015. Episodic human ac- tivities and seasonal impacts on the(Müller, 1785) population at Tanjung Selangor in Peninsular Malaysia., 164: 313-323.

Penn, D., and Brockmann, H. J., 1994. Nest-site selection in the horseshoe crab,., 187: 373-384.

Rudkin, D. M., and Young, G. A., 2009. Horseshoe crabs–An ancient ancestry revealed. In:. Tanacredi, J. T.,., eds., Springer, Mas- sachusetts, Boston, 25-44.

Rudloe, A., 1979. Locomotor and light responses of larvae of the horseshoe crab,(L.)., 157: 494-505.

Sekiguchi, K., and Nakamura, K., 1979. Ecology of the extant horseshoe crabs. In:. Cohen, E.,., eds., Alan R. Liss, New York, 37-45.

Shen, X., Qi, H., Liu, X., Ren, X., and Li, J., 2013. Two-way non-parametric ANOVA in SPSS., 30: 913-914 (in Chinese with English abstract).

Smith, D. R., Brockmann, H. J., Beekey, M. A., King, T. L., Mil- lard, M. J., and Zaldivar-Rae, J., 2017. Conservation status of the American horseshoe crab, (): A re- gional assessment., 27: 135-175.

Vasquez, M. C., Johnson, S. L., Brockmann, H. J., and Julian, D.,2015. Nest site selection minimizes environmental stressor ex- posure in the American horseshoe crab,(L.)., 463: 105-114.

Wang, C. C., Kwan, K. Y., Shin, P. K. S., Cheung, S. G., Itaya, S., Iwasaki, Y.,., 2020. Future of Asian horseshoe crab con- servation under explicit baseline gaps: A global perspective., 24: e01373.

Xie, X., Wu, Z., Wang, C. C., Fu, Y., Wang, X., Xu, P.,., 2020. Nursery habitat for Asian horseshoe crabs along the northern Beibu Gulf, China: Implications for conservation management under baseline gaps., 30: 260-272.

Yan, M., Wang, Y., Gu, H., Wang, Y., Li, Q., Fan, Z.,., 2018. Current situation ofamebocyte lysate industry in China and relevant suggestions., 35: 88-91 (in Chinese with English abstract).

Zadeh, S. S., Christianus, A., Saad, C. R., Hajeb, P., and Kama- rudin, M. S., 2009. Comparisons in prosomal width and body weight among early instar stages of Malaysian horseshoe crabs,andin the la- boratory. In:. Tanacredi, J. T.,., eds., Springer, Massachusetts, Boston, 267-274.

Zaldíar-Rae, J., Sapién-Silva, R. E., Rosales-Raya, M., and Brock- mann, H. J., 2009. American horseshoe crabs,, in México: Open possibilities. In:. Tanacredi, J. T.,., eds., Springer, Massachusetts, Boston, 97-113.

Zauki, N. A. M., Satyanarayana, B., Fairuz-Fozi, N., Nelson, B. R., Martin, M. B., Akbar-John, B.,., 2019. Citizen sci- ence frontiers horseshoe crab population regain at their spawn- ing beach in East Peninsular Malaysia., 232: 1012-1020.

J. Ocean Univ. China(Oceanic and Coastal Sea Research)

https://doi.org/10.1007/s11802-022-5164-2

ISSN 1672-5182, 2022 21 (3): 531-540

© Ocean University of China, Science Press and Springer-Verlag GmbH Germany 2022

Corresponding authors. E-mail: zhujunhua2017@bbgu.edu.cn

(August 17, 2021;

September 16, 2021;

November 14, 2021)

(Edited by Qiu Yantao)