Conservation Program for the Asian Horseshoe Crab Tachypleus Tridentatus in Taiwan: Characterizing the Microhabitat of Nursery Grounds and Restoring Spawning Grounds

Conservation Program for the Asian Horseshoe Crab Tachypleus tridentatus in Taiwan: Characterizing the Microhabitat of Nursery Grounds and Restoring Spawning Grounds

Hwey-Lian Hsieh and Chang-Po Chen

Abstract A study of the physical properties of horseshoe crab (Tachypleus tridentatus) nursery grounds indicated that juveniles preferred sediments consisting of fine sand with median grain size of 0.14–0.27 mm in diameter, 16.9–23.2% water content when tides receded, 0.23–0.41% TOC content, 0.04–0.07% TN content, 2.3–2.8 mg/cm2 chlorophyll a content, and poorly sorted substrates. Juvenile horseshoe crab density increased based on the amount of chlorophyll a content in the sediment and infaunal polychaete density, suggesting that the juveniles prefer nursery grounds containing abundant prey and its supporting food web. An effort to restore horseshoe crab spawning grounds was conducted by covering 20 cm deep mud substrate (0.23 mm in diameter) with coarse sand (1.10 mm in diameter). Although adults transferred to this site succeeded in laying eggs, the hatching rate was only 33.9%. This low rate may be attributed to small tidal amplitude at this restoration site.

1 Introduction 1.1 Significance of the Present Study

The horseshoe crab is a living fossil and has high economic value in the fishing industry. Some of the harvest is used for human consumption and some products are used in the medical industry (Shuster 2001, Swan 2001). It is also a good indicator species for monitoring the health of coastal zones and has local cultural importance in Taiwan (Chen et al. 2004). The Asian horseshoe crab, Tachypleus tridentatus, is one of three horseshoe crabs distributed in the Indo- Pacific region. This species once thrived in the coastal wetlands of Taiwan, but now survives in only a few small areas. A similar decline in abundance and

H.-L. Hsieh (*) Biodiversity Research Center, Academia Sinica, Nankang, Taipei, Taiwan 115, Republic of China e-mail: [email protected]

J.T. Tanacredi et al. (eds.), Biology and Conservation of Horseshoe Crabs, 417 DOI 10.1007/978-0-387-89959-6_26, Ó Springer ScienceþBusiness Media, LLC 2009

[email protected] 418 H.-L. Hsieh and C.-P. Chen endangered status has also been reported by Hong Kong and Japan (Chiu and Morton 1999, Botton 2001). The decline is largely due to loss of suitable habitat caused by anthropogenic impacts such as land reclamation, dike construction, water pollution, and loss due to overfishing for food consumption and biome- dical use. This loss is symptomatic of a wider decline in Taiwan’s natural resources and biodiversity. This decline is in conflict with efforts to sustain Taiwan’s development. In order to replenish what we have lost, the central government administration has recognized restoration of coastal wetlands as an urgent requirement. As a result, implementation of habitat mitigation and restoration has been itemized in the national biodiversity conservation program, which was authorized by Executive Yuan in Taiwan in 2001 (Action Plan in Biodiversity Promotion issued by Executive Yuan August 15, 2001, amended in February 2004). Since conservation of a range of habitats has been acknowledged to be much more effective than the protection of individual species, we have chosen the horseshoe crab as a ‘‘flagship’’ species (Zacharias and Roff 2001). Preserving or conserving their habitat can secure the whole community, including the target flagship species as well as other species dependent on that habitat. More importantly, the recovery of the flagship species populations will indicate the restoration of intertidal ecosystems and the recovery of coastal natural resources, and therefore is beneficial to humans living on the Taiwan islands. In addition, our experiences in conserving this flagship species can be shared with other countries. Since other researchers are likely to encounter situations similar to that in Taiwan, such as harvest pressure and the loss of suitable habitats, our findings can be especially useful in fostering better connections in the international network of marine-protected areas for horseshoe crabs. To implement the conservation plans for the horseshoe crab, we have set up a three-step strategy: (1) Develop an understanding of its life history (2) Characterize its habitat requirements (3) Using the findings of steps 1 and 2, identify methods for repairing and reconstructing a functional ecosystem Throughout its life cycle, the horseshoe crab is highly dependent on environ- mental conditions in all of its coastal habitats. Adults spawn on the coarse sand near the high-tide zone and juveniles inhabit the adjacent intertidal mudflats and then gradually migrate to the deeper subtidal zone for maturation (e.g., Sekiguchi 1988, Chiu and Morton 1999, Anderson and Shuster 2003). Limulus polyphemus nest sites are defined by a set of physical properties including a sandy beach and its hydroclimate. Grain size, sediment moisture content, oxygenation, temperature, and depth of sand over peat have been found to be important (e.g., Botton et al. 1988, Brockmann 2003, Shuster and Sekiguchi 2009). At the macrohabitat level, the geomorphology of the spawning beach as mediated by waves, tides, and currents has also drawn great attention (Anderson and Shuster 2003, Jackson et al. 2005). This data review highlights

[email protected] Conservation Program for the Asian Horseshoe Crab 419 that our knowledge of the horseshoe crab’s habitat requirements is primarily based on observations of the American horseshoe crab L. polyphemus, especially its spawning ground. In contrast, the habitat requirements of the three Asian horseshoe crab species (T. tridentatus, T. gigas, and Carcinoscorpius rotundicauda) are largely unknown.

1.2 Analyses of Habitat Requirements Through the Life History of T. tridentatus and the Purpose of the Study

According to the three-step strategy outlined above, we have made detailed analyses that allow us to understand the habitat requirements of the horseshoe crab at each important life history stage (Fig. 1). Both the aspect of organism

Life history stage

Embryo-first instar Early instars/ Adult Adult Interactions Aspect of Developing Juvenile Feeding/ Spawning Feeding/ Growth/ between fauna organism Growth/ Maturation Dispersion Benthic Water column / Water column / Benthic environment Benthic Benthic environment environment environment Spawning ground Intertidal flat Shallow water High tidal zone Spawning ground

Micro- Temperature, humidity, sediment water salinity, habitat oxygen content, water content, quantity and quality of food, toxic materials, and etc.

support

Aspect of influence influence habitat Macro- Water Sediment Vegetation habitat

estuary particle size seagrass tide topography benthic microalgae flow elevation tidal level influence

Coastal environments

erosion/siltation emerging/sinking

Fig. 1 A scheme showing the analysis of the habitat requirements for a horseshoe crab species throughout its life history

[email protected] 420 H.-L. Hsieh and C.-P. Chen and the aspect of habitat are involved. From the organismal point of view, the horseshoe crab needs different habitats at different life stages. Embryos develop in a benthic environment while embedded in sands on the spawning ground. Newly hatched first instars and early-stage instars are capable of swimming in the water column, and thus are dispersed by currents. Juveniles settle down on tidal flats and commence demersal feeding and growth after a number of molts. As the juveniles grow larger, they gradually leave the tidal flats and migrate to shallow water areas where they become adults. At the start of spawning season, paired mature adults move toward shore and nest on the high tidal zone of sandy beaches. Throughout these stages, they interact with other co-existing fauna. From the habitat point of view, two scales exist for any given habitat: macro and micro. At the macrohabitat scale, the entire coastal environment setting is influenced by the geomorphology and hydrodynamics of that coast, which determines the stability of the coast and whether it is in a state of erosion or deposition. Interactions between geomorphological and hydraulic forces mediate the suitability of beach slope, tidal amplitude, current velocity, and sediment grain size as well as the presence or absence of vegetation for horseshoe crabs to live on. When looking at their microhabitats, components such as sediment grain size, water content, organic content, temperature, salinity, oxygen content, and food availability are critical to the horseshoe crab’s distribution. Due to the lack of records on adults coming to the beach for nesting, mating sites for T. tridentatus have not been reported in recent decades in the Taiwan region. However, spontaneous spawning was induced successfully on a natural, sandy beach, thus suggesting that this kind of beach was a potential and suitable nesting ground (Chen et al. 2004). Since we know this potential nesting ground is sandy and have determined its sand grain size range, we may be able to induce adults to spawn in the field where unfavorable substrate is replaced by a favorable one. In lower mudflats adjacent to a potential spawning ground in higher tidal zones, juveniles from the second instars up to and beyond the sixth instar (carapace length 8–71 mm) are found to be abundant. This kind of mudflat is most likely a nursery ground. Chen et al. (2004) measured several abiotic factors of the potential spawning ground and the nursery mudflat including grain size, water content, total organic carbon, and nitrogen content. Other than this preliminary data, no detailed studies of the microhabitat characteristics of nursery ground have been conducted. The purpose of the present study is to advance our understanding of the habitat requirements of T. tridentatus in the coastal zones of Taiwan so that suitable existing habitat can be preserved and damaged habitats have a chance to be restored. Our approach focuses on the following topics: (1) Characterization of physical properties of nursery grounds (2) Evaluation of potential practices for spawning ground restoration (3) Proposal of future studies regarding habitat restoration and conservation

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2 Nursery Grounds: Relationships Between Juvenile Horseshoe Crab Density, Physical Properties of Sediments, and Infaunal Density 2.1 Materials and Methods

2.1.1 Sampling Sites and Time The physical properties of nursery grounds were examined on Kinmen Island at three sites – Nanshan, Beishan, and Hsiashu – in August of 2005 and July and November of 2006 (Fig. 2). At each sampling site, four 300 m long transect lines, each 50 m apart, were set up parallel to the seashore. On each transect line, ten plots, each 5 m by 5 m, were placed at 30 m intervals and three plots were randomly chosen for the sampling (Fig. 3). A total of 12 plots (3 per transect line) were chosen. Sediment samples were collected, treated, and analyzed as follows.

Fig. 2 Map depicting the present study sites of T. tridentatus distributed in the Taiwan area. On Kinmen Island, samples from three nursery grounds, Hsiashu, Nanshan, and Beishan, were taken from August 2005 to November 2006. At Toungshiao, Miaoli County, sampling was conducted from a restored spawning ground from August to October 2002

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Fig. 3 A scheme showing Low tidal zone L4 the sampling plots for each nursery ground on Kinmen Island where sediment L3 samples were collected to study the sedimentary L2 50 m physical properties and 5 x 5 m2 densities of juvenile L1 horseshoe crabs 30 m T. tridentatus and infauna High tidal zone 1 2 3 ……. 9 10 benthos 300 m

2.1.2 Measurements of Physical Properties Granulometry, water content, total organic carbon and nitrogen contents, chlorophyll a content, pH, and salinity: The top 5 cm of sediment on the mudflat was collected separately using a PVC corer of 2.6 cm diameter. For analyzing chlorophyll a content, the top 0.5 cm of sediment in a 7.07 cm2 surface area was collected. During transportation to the laboratory, all samples were kept cool at approximately 48C while those for chlorophyll a content measurements were also kept in the dark. In the laboratory, samples for the measurement of total organic carbon (TOC) and nitrogen contents (TN) were kept at –708C until cryo-dried. Granulometry was determined by wet sieving the samples through a Wentworth series of screens with mesh openings from 1.0 mm to 63 mm. Silt and clay contents were measured using pipette methods. Median gain sizes and sorting coefficients were also calculated. Detailed procedures were described in Hsieh (1995). Water content of the sediments was measured as the percent weight loss after oven-drying at 608C for 48 h. Water content (% H2O) ¼ [(wet weight – dry weight)/wet weight] 100%. Total organic carbon and nitrogen contents were analyzed using an element analyzer (Perkin-Elmer EA-2400 II). Cryo-dried sediments were sieved through a screen with a mesh opening 0.5 mm in size in order to remove large animal or plant debris. Those that passed through the screen were collected and treated with 1 N HCl to remove all inorganic carbons. Chlorophyll a was extracted by soaking the sample in 90% acetone overnight and analyzed using a fluorometer (Turner Designs, Model: 10-AU). Since no macroalgae and vascular plants were present in the sediment samples, chlorophyll a extracted was considered to be from microalgae. Sediment pH values were measured using a glass electrode pH meter in 1:2 ratio of sediment to deionized water by weight. The sediment was first vigor- ously mixed with water before performing measurements (Chiu et al. 1999). Sediment salinity was measured using a refractometer.

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2.1.3 Estimation of Juvenile Horseshoe Crab Density and Infauna Density In each aforementioned plot (25 m2), the living juvenile horseshoe crabs which were found by sight were counted and infaunal benthos were also sampled. Infauna were collected using a PVC corer with a diameter of 10 cm (0.00785 m2) and treated following the procedures described in Hsieh (1995). The infauna specimens that were retained on a 0.5 mm mesh screen were identified to taxonomic levels higher than species, such as class or order, and the numbers of individuals were counted. The juvenile crab density and infaunal density were expressed as number of individuals m–2.

2.1.4 Statistical Analyses of the Relationships of Juvenile Horseshoe Crab Density with Sedimentary Physical Properties and Infauna Density Differences in the physical properties among the three sites and the properties that contributed to such differences were determined by factor analysis of ordinations (Press 1972). The six physical properties included in the factor analysis were grain size, silt/clay content, sorting coefficient, TOC content, water content, and chlorophyll a content. The relationships between two biotic components, the densities of juvenile horseshoe crab and polychaetes, and five physical properties were determined using Canonical Correlation Analysis (Digby and Kempton 1987). The five physical properties included TOC, grain size, sorting coefficient, water content, and chlorophyll a content. The remaining infauna groups were excluded because they were insignificant based on the Canonical Correlation Analyses. All statistical calculations were produced with the SAS PC software application (SAS Institute 2003).

2.2 Results

2.2.1 Physical Properties of Nursery Grounds Sedimentary physical properties among the three sites exhibited similar pH, sediment salinity, and sediment temperature but dissimilar grain size, silt/clay content, sorting coefficient, total organic content, water content, and chlorophyll a content (Fig. 4). The three sites were all sandy; however, they varied in sediment grain size. Nanshan’s substrate was the finest while Beishan’s was the coarsest and Hsiashu’s was intermediate. Median grain sizes at these sites were 0.08 mm ( SE 0.02), 0.41 mm ( SE 0.08), and 0.16 mm ( SE 0.03) in diameter, respectively. Silt/clay content and grain size were closely inversely correlated (r ¼0.79, p<0.001); thus, Nanshan had the greatest silt/clay content with 45.8% whereas Beishan and Hsiashu had 32.0 and 15.1%, respectively. Nanshan and Beishan had similar total organic carbon content (TOC) of 0.39 and 0.43%, respectively. Both values were greater than 0.24% at Hsiashu. The same trend was seen in total nitrogen content as seen in higher values of 0.06 and 0.07% at Nanshan and Beishan than

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0.05% at Hsiashu. Nanshan possessed 28.6% water content that was greater than those at Beishan (21.6%) and Hsiashu (19.1%). By contrast, Nanshan and Beishan had chlorophyll a concentrations of approximately 1.9–2.0 mg cm–2 that were lower than that of 2.6 mgcm–2 at Hsiashu. Substrates at all three sites were poorly sorted as seen in sorting coefficients that were all greater than 1.0.

0.6 35 8.5 30 0.5 8.0 0.4 25 20 7.5 0.3 15 pH 7.0 0.2 10 6.5 0.1 5 Grain size (mm)

0.0 Water content (%) 0 6.0 60 3.0 35 ) 50 2 2.5 30 40 2.0 25 g/cm

µ 20 30 1.5 15 20 1.0 10

10 Chlorophyll a 0.5 5 Salinity (per mil) content ( content

Silt/clay content (%) content Silt/clay 0 0.0 0 0.5 3.0 30 0.4 2.5 25 2.0 20 0.3 1.5 15 0.2 1.0 10 (degree C) (degree

Total organic 0.1 0.5 Temperature 5 Sorting coefficient carbon content (%) carbon 0.0 0.0 0 0.10 Hsiashu Nanshan Beishan Hsiashu Nanshan Beishan 0.08 Site Site 0.06 0.04

content (%) content 0.02 Total nitrogen Total 0.00 Hsiashu Nanshan Beishan Site

Fig. 4 Measures of benthic physical properties at each nursery ground on Kinmen Island, Taiwan. For each site, data were pooled over all sampling events. Values are means SE

2.2.2 Juvenile Horseshoe Crab Density and Infauna Density Juvenile horseshoe crab density was lowest at the Nanshan site, higher at Beishan, and highest at Hsiashu and averaged 0.006, 0.061, and 0.169 individuals m–2, respectively (Fig. 5). A total of 13 taxa of infauna were collected from the three sites. They were nemerteans, nematodes, bivalves, gastropods, leeches, polychaetes, oligo- chaetes, sipunculans, isopods, insects, amphipods, shrimps, and crabs. Com- pared with the total averaged infauna density of 1783.4 individuals m–2 at Hsiashu, those at Beishan and Nanshan were lower having 1026.2 and 1117.4 individuals m–2, respectively. Among these infauna, the polychaetes were the

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0.25 60 16 Juvenile horseshoe crab Oligochaeta 14 Sipuncula 0.20 50 12 40 0.15 10 30 8 0.10 20 6 0.05 4 10 2 0.00 0 0 1200 50 16 Polychaeta Amphipoda 14 Hirudinea 1000 40 12 800 30 10 600 8 20 400 6 )

2 4 200 10 2 0 0 0 1200 35 10 Bivalvia Gastropoda Macrura 30 1000 8 800 25 20 6 600 15 4 400 10 2 200 5 0 0 0 35 8 140 Insecta Nematoda Isopoda 30 120 6 Density (number of individuals / m 100 25 20 80 4 60 15 40 10 2 20 5 0 0 0 60 25 Hsiashu Nanshan Beishan Brachyura Nemertinea Site 50 20 40 15 30 10 20 10 5 0 0 Hsiashu Nanshan Beishan Hsiashu Nanshan Beishan Site Site

Fig. 5 Density changes of juvenile horseshoe crab T. tridentatus and infauna benthos among nursery grounds on Kinmen Island, Taiwan. For each site, data were pooled over all sampling events. Values are means SE most dominant group at each site and bivalves had the next highest densities (Fig. 5). Similar to the distribution of the juvenile horseshoe crabs, polychaete density was lowest at Nanshan site, greater at Beishan site, and greatest at Hsiashu site and was 513.2, 838.6, and 921.7 individuals m–2, respectively (Fig. 5). The remaining taxa had densities fewer than 50 individuals m–2.

2.2.3 Relationships Among Juvenile Horseshoe Crab Density, Infauna Density, and Physical Properties of the Nursery Ground Microhabitat The first two canonical correlations between two biotic components and five physical components were both significant as seen in Canonical Correlation

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Analysis (cumulative proportions = 0.73 and 1.00, respectively; r1 = 0.54, approx. F(10, 124) = 4.32, p < 0.0001; r2 = 0.37, approx. F(4, 63) = 3.01, p = 0.02). In the first canonical variable, densities of juvenile horseshoe crab and polychaete were positively correlated with chlorophyll a content, but negatively correlated with water and TOC content. In the second canonical variable, densities of juvenile horseshoe crab were negatively correlated with sorting coefficient (Fig. 6).

1.0 Canonical variable 2

0.5

Sorting coefficient Chlorophyll a content TOC Polychaete Water content Grain size

–1.0 –0.5 0 0.5 1.0 Canonical variable 1

Horseshoe crab

–0.5

–1.0

Fig. 6 Ordination and relationships among two biotic (density of horseshoe crab and polychaete) and five sedimentary physical properties by the first two canonical variables using Canonical Correlation Analysis

Among the six physical properties of the sediments, relative importance in differentiating the three sites was demonstrated by Factor Analysis (Figs. 7, 8). The first three principal factors together explained 81% of total variation in the physical properties at the three sites pooled, while the first factor alone explained 48%. On axis factor 1, silt/clay content and chlorophyll a content were important elements, having high positive or negative correlation with the axis (factor loading ¼ 0.88 and 0.52, respectively). On axis factor 2, grain size

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1.0 Factor 2

Grain size

0.5

TOC Water content

–1.0 –0.5 0 0.5 1.0

Silt/clay content Factor 1

–0.5 Sorting coefficient Chlorophyll a content

–1.0

Fig. 7 Ordination of sedimentary physical properties by the first two factor axes, Factors 1 and 2, using Factor Analysis

and chlorophyll a content were important also with high positive or negative correlation with the axis (factor loading ¼ 0.71 and 0.53, respectively) (Fig. 7). When all samples were ordinated by axis factors 1 and 2, the three sites over- lapped with one another; however, Hsiashu and Nanshan were two distinctive sites within a broadly scattered Beishan site (Fig. 8). Hsiashu differed from Nanshan in having greater chlorophyll a content and larger grain size but less silt and clay, water, and TOC content and better-sorted sediments. By contrast, Beishan was characterized by having the largest variation in each of the physical elements that distinguished Hsiashu from Nanshan (Fig. 8).

2.2.4 Microhabitat Requirements of Juvenile Horseshoe Crab To determine which habitat characteristics are most favored by juvenile horseshoe crabs, we analyzed the data by plotting average juvenile density in each site

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5 B

2 B B

B 1 B N Factor 2 B B H B H N N NN B N N H H B BN HH H B H NN 0 H H B NB H H N H H NN H H N BB N B N H N H N H B N N HNB N –1 H H

B B –2 N

–3 –3 –2 –1 0 1 2 3 Factor 1

Fig. 8 Ordinations of the three nursery grounds based on their sedimentary physical properties by the first two factor axes, Factor 1 and 2, using Factor Analysis. H: Hsiashu, N: Nanshan, B: Beishan

at Kinmen against averaged value of each of physical properties at that site. The results showed that juveniles preferred sediments consisting of the following physical traits: grain size ranging from 0.14 to 0.27 mm in diameter, silt/clay content ranging from 13.7 to 36.2%, water content ranging from 16.9 to 23.2%, total organic carbon content ranging from 0.23 to 0.41%, total organic nitrogen ranging from 0.04 to 0.07%, chlorophyll a content ranging from 2.3 to 2.8 mgcm–2, and in poorly sorted condition with sorting coefficients ranging from 1.87 to 2.76 (Fig. 9).

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0.6 0.6 Hsiashu 0.5 Nanshan 0.5 0.4 Beishan 0.4 hoe Crab (ind/m 0.3 0.3

0.2 0.2

0.1 0.1

0.0 0.0

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 102030405060 Density of Juvenile Horses Density of Grain size (mm) of Juvenile Horseshoe Crab (ind/m Density Silt / clay content (%) ) ) 2 2

0.6 0.6

0.5 0.5

0.4 0.4

0.3 0.3

0.2 0.2

0.1 0.1

0.0 0.0

10 15 20 25 30 35 40 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 Density of Juvenile Horseshoe Crab (ind/m Density Density of Juvenile Horseshoe Crab (ind/m Water content (%) Sorting coefficient ) 2 ) 2 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0.0 0.0

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.10 Density of Juvenile Horseshoe Crab (ind/m Density Density of Juvenile Horseshoe Crab (ind/m Density of Juvenile

) TOC (%)

2 TN (%) 0.14

0.12

0.10

0.08

rseshoe Crab (ind/m 0.06

0.04

0.02

0.00

0.5 1.0 1.5 2.0 2.5 3.0 3.5 2 Density of Juvenile Ho Chlorophyll a content (µg/cm )

Fig. 9 Relationships between the densities of juvenile horseshoe crab T. tridentatus and the sedimentary physical properties showing the preferable conditions of the juveniles

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3 Restoration of Spawning Ground 3.1 Preparation of Nesting Substrate and Induction of Spawning

Spawning grounds restoration was conductedina10mby10mnetfencedareaon a small mudflat in the summer of 2002 at Toungshiao Town, Miaoli County, in northern Taiwan. The mesh size of the net was approximately 2 mm which was an adequate size for retaining instars in the fenced area. In July 2002, in the high tidal zone of this experimental area, the original mud substrate (averaging 0.13 mm in diameter) was changed by directly placing a layer of coarse sand (averaging 1.1 mm in diameter, a size suggested by the study of Chen et al. 2004) on top of the existing substrate. After flushing by daily tides for approximately 4 weeks, the coarse sand had been spread slightly down the slope and had formed a zone of approximately 5 m in width by 20 m in length and was 20 cm in depth (Fig. 10). In this restored area, sediment water content in the top 10 cm depth ranged from 3.7 to 9.3% as measured at the time when tides receded. Substrates adjacent to the restored zone were kept intact as mud throughout the study period.

Replaced with 1.1 mm diameter coarse sand Fig. 10 A small net fenced area showing the restored 10 m spawning ground for 5 m wide T. tridentatus at 20 cm original mud flat Toungshiao, Miaoli County, deep 0.13 mm diameter Taiwan, in July 2002. The original mud substrate in the high tidal zone was replaced 10 by 20 cm depth layer of m approximately 1.1 mm diameter coarse sand

3.2 Hatching Rate and Juvenile Survival Rate

In August 2002 on the days when the spring tide arrived, five pairs of gravid adults were placed onto the high tidal zone in order to induce spawning. Locations where ‘‘spawning foams’’ emerged from the substrate were marked with wooden rods (Fig. 11). These air bubbles indicate the presence of nests (Chen et al. 2004). Previous studies showed that hatching takes place 40–50 days after fertilization (Chen et al. 2004). Subsequent observations on hatching rate and survival rates of young instars were made 40 days later and were repeated at 7 day intervals for three more observations until early October. Sand covering the nests was gently scraped away to expose the embedded

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a c

Fig. 11 Induction of spawning on restored spawning ground during August 12–13, 2002. a: gravid adults were put on the beach during tidal floods, b: spawning foams, c: when wooden sticks were placed beneath the spawning foams, the nests could be relocated after tides receded

offspring (Fig. 12). The number of eggs laid and the numbers of the first-stage instars hatched per nest were recorded (Fig. 13). After each recording, all eggs, offspring, and sand were gently placed back in each nest for continuous incuba- tion until the next observation. During each observation time, a search was made on the restored spawning site for crawling instars. A search was also made on the surrounding mud for the first-stage and older instars.

3.3 Results

In total, four nests were found in Area A, eight nests in Area B, and two nests in Area C. In Area B, one nest had no eggs and another nest lost all of its eggs 1 month later; thus, these two nests were not included in further counting. Fifty days after spawning, no nests in Area B and Area C had hatched first instars. Eggs in the nests in Area C turned green and moldy as early as 12 days after spawning (Fig. 13). Only the nests in Area A produced viable offspring. Hatching rates ranged from 0 to 88.5% and averaged 33.9% over a 50-day period (Fig. 14).

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Area A bo un restored da ry Area B sand beach be tw e en m u d/s a nd Original mud flat

Area C

Fig. 12 The nesting localities of the restored spawning ground. Number of nests found in Areas A, B, and C were 4, 6, and 2, respectively

a b

c d

Fig. 13 Developing eggs in the nests. a: nests were discovered by removing covering sand, b: developing embryos, c: the first instars in the nest, d: moldy eggs

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100

Nest 1 Nest 2 Nest 3 80 Nest 4

40 Hatching rate of eggs (%) 20 (Hatched instars / total eggs produced nest) 0 19 Sep 25 Sep 2 Oct Observation date

Fig. 14 Hatching rate changes over the study period of approximately 50 days. Fertilization began on August 12–13, 2002

No juveniles were found on the mudflat within the netted area and the surrounding mudflat during the study period, or over the following autumn, winter, and spring, indicating none of the hatched first instars had survived.

4 Discussion 4.1 Macrohabitat Preferences of T. Tridentatus

In Taiwan, Kinmen Island is known as the best place to find juvenile T. tridentatus. Although spawning swarms on Kinmen Island have not been observed for decades, we realize from local proverbs that the horseshoe crabs once heavily populated the west coast of Kinmen Island. In one proverb, this thriving phenomenon was described as ‘‘horseshoe crabs that died at Shuitou village are smelled at the far distant Gougaun village’’ (Chen et al. 2002). Shuitou is located in a headland bay that has now been partially used to build Shuitou Harbor. The original bay morphology reveals three areas: sandy beach along the upper tidal zone, mudflat stretching across the lower tidal zone, and an offshore trench zone at a depth of 20–35 m. In addition, the bay is subject to faster near-shore currents relative to the surrounding coasts (3.8 vs. 1.8 knots in

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Nanshan and Beishan coast, Taiwan Electronic Navigational Chart Center 2006). Bays with these characteristics have been found to be the most suitable habitat for the horseshoe crabs including T. tridentatus since the three habitats – spawning grounds, nursery grounds, and maturation grounds – required by the horseshoe crab to complete its life history are connected to each other as seen in Delaware Bay for L. polyphemus (Brockmann 2003, Anderson and Shuster 2003) and in T. tridentatus (Sekiguchi 1988, Seino 2009). Compared to Shuitou, the present study sites at Hsiashu, Nanshan, and Beishan are also headland bays, but are smaller. Also, the latter two sites lack an offshore trench and their subtidal zone is much shallower than 20 m. There- fore, integration of the three types of habitats that horseshoe crabs depend on appears to be not as good at Nanshan and Beishan as at Shuitou. Hsiashu has a greater juvenile density than Nanshan and Beishan. This may be attributed to the closer proximity between Hsiashu and Shuitou, making the Hsiashu macrohabitat similar to that of Shuitou as they are co-located in the same bay. Nanshan and Beishan are located in the range of The Horseshoe Crab Con- servation Area, which was designated in order to compensate for the loss of the horseshoe crab’s natural grounds due to the construction of Shuitou Harbor. These two sites represent available and suitable habitat, but probably not the best habitat for the horseshoe crabs on Kinmen Island. The same macrohabitat requirements were shown for the Japanese populations of T. tridentatus distributed in the Seto Inland Sea where the horseshoe crabs prefer semi-enclosed bays having sand bars present at river mouths with tidal mudflats stretching out of these sand bars (Seino 2009).

4.2 Microhabitat Characteristics of Nursery Ground

The juvenile horseshoe crab density at Hsiashu is approximately 2.5 times and 25 times greater than Beishan and Nanshan, respectively. This difference may be explained by both the abundance of the juveniles’ forage base and the food web that supports it as well as by some physical properties. These important biotic determinants include the density of the polychaete prey and the concen- tration of the polychaetes’ food, the microalgae. The abiotic factors include water content and total organic carbon content in the nursery ground sediments. From the viewpoint of food availability and abundance, Hsiashu may represent a better nursery ground than the Beishan and Nanshan sites. Studies on the horseshoe crab’s diets have shown that bivalves, especially thin-shelled and small- sized ones, are the preferable food for the adult L. polyphemus whereas early instars can be raised with polychaetes in aquarium conditions (Botton and Shuster 2003). In addition, polychaetes and crustaceans had also been reported as the food of the adult T. gigas in India (Botton and Shuster 2003). Diet analysis in juvenile T. tridentatus using d13Candd15N measures showed that polychaetes

[email protected] Conservation Program for the Asian Horseshoe Crab 435 were their main prey (Nishida and Koike 2009). According to our study, polychaetes may also be one of the most important food sources for the juvenile T. tridentatus as seen from positively correlated relationships between the densities of the juvenile horseshoe crab and the polychaete (see Fig. 6). Moreover, polychaete density at Hsiashu was found to be greater than at Nanshan (921.7 vs. 513.2 individuals m–2). These results imply that Hsiashu produces more of the favorable food, polychaetes, which nourished the juvenile horseshoe crabs, making Hsiashu capable of producing a larger juvenile population. Regarding the food sources of the polychaetes, deposit-feeding polychaetes assimilate benthic microalgae from sediment surface (Newell et al. 1995, Hentschel 1998, Hsieh et al. 2002). In addition, the chlorophyll a content has been regarded as a measure of microalgal biomass present on the sediment surface. In our study, the polychaete abundance is positively correlated with chlorophyll a content. Chlorophyll a content was greater at Hsiashu than at Nanshan and Beishan, suggesting that Hsiashu possesses higher microalgal biomass (see Figs. 4, 6) and, therefore, more food available for the polychaetes. The positively correlated relationships among the densities of the polychaetes and the juvenile horseshoe crabs and chlorophyll a content lead us to the reason why more juveniles inhabit Hsiashu. From the viewpoint of sedimentary physical properties, Hsiashu may also reflect a better habitat than Beishan and particularly Nanshan. The analyses of relationships between the juvenile horseshoe crab density and the sedimentary physical characteristics show that the juveniles avoid sediments having high water content and high total organic carbon content (see Fig. 6). Waterlogged sediments are known to often lack aeration, resulting in hypoxic or anoxic conditions and even hydrogen sulfide production in the sediments. Oxygen deficiency in sediment has been attributable to organic enrichment (Pearson and Rosenberg 1978). Among the three sites studied, the greater water content and total organic carbon content observed in Nanshan and Beishan sediments may be responsible for the lower densities of the juvenile horseshoe crabs at these two sites as compared to that at the Hsiashu nursery ground.

4.3 The Practice of Spawning Ground Restoration

The present pilot study shows that despite a low average hatching rate, the restoration practice was successful in terms of viable offspring being produced through induction of spawning in a modified substrate. The results from this experiment suggest that restoring spawning ground is feasible and may be a promising approach for enhancing horseshoe crab populations. The low hatching rate observed may be due to various factors. First, the experiment area is located in the inner part of a bay. This bay is enclosed by dikes and has only one tidal outlet. We suspect that tidal amplitude may be too small to aerate the nests, resulting in embryos dying from mold infection.

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Oxygenation of sediment is known to be essential for eggs to develop in the nest. Studies on the characteristics of the nesting site in L. polyphemus showed that mating adults avoided anaerobic peat sediments (Botton et al. 1988). Second, repeated exposure and reburying of nests for observations on embryo survivor- ship may have damaged the fragile embryos. Third, the eggs spawned may be poor in quality because the experiment of spawning induction was conducted at the end of the natural spawning season for the horseshoe crabs.

5 Future Work 5.1 Study on the Geomorphology and Hydrodynamic Regime of the Spawning Ground

Considering the variety of habitats required by the horseshoe crab T. tridentatus throughout its life history, the status of its nursery ground in Taiwan is in much better condition than that of its spawning ground. Mudflats are still present on Kinmen Island, Penghu Island, and the main Taiwan island, but sandy beaches suitable for nesting have largely disappeared due to dike construction, reclamation, or erosion. Although we have initiated a small-scale restoration for spawning and have succeeded in breeding some viable first instars, we failed in our overall goal since we lost many embryos and no hatchlings survived to grow. This may be due to the fact that our knowledge of the physical setting of the spawning ground is still rather limited. In the future, before practicing restoration, the geomorphology and hydrodynamic regime of the natural spawning ground, as perceived as a macrohabitat setting by the mating horseshoe crabs, needs to be studied in detail. Important forces shaping the beach morphology and flow regime are bay shape, tidal amplitude, currents, beach slope, sedimentary composition, sediment transportation, and typhoon- induced wave actions. In addition, on spawning beaches the vertical profiles of physical settings such as water content, temperature, oxygenation, and grain sizes also need further study.

5.2 Restoration Practice of Spawning Ground

In order to restore the ecological integrity of the spawning ground of T. tridentatus, the practice of restoration needs to integrate ecological, engineer- ing, administrative, and community-based conservational disciplines through the entire process of restoration including planning, design, construction, and management. Most sections of the coasts in the Taiwan area have been modified, especially on the Taiwan main island. However, a few places in southwestern Taiwan are undergoing or have used beach nourishment to prevent erosion. These places

[email protected] Conservation Program for the Asian Horseshoe Crab 437 are our target sites for practicing the reformation of spawning grounds. At the present, a small beach at the southern coast of Budai Harbor, Chiayi County, in the southwestern Taiwan seems to be a suitable site. Local NGOs support the recovery of the lost horseshoe crab population that once thrived locally (Chen et al. 2009). Upcoming work for our research team will involve finding and inviting coastal engineers to join the habitat repairs for the horseshoe crabs.

5.3 Evaluation and Conservation of the Existing Nursery Grounds and Potential Spawning Grounds

The status of nursery grounds and potential spawning grounds of T. tridentatus on Kinmen Island need to be continuously monitored. Some of the measurements for evaluating the ecological integrity of these habitats include changes in the number of recruits, the size structure of juvenile populations, and the physical properties of nursery grounds and potential spawning grounds. At the macrohabitat level, beach erosion is a warning sign for the loss of potential spawning grounds. To prevent such loss, the geomorphology of the target bays needs to be monitored and evaluated periodically. The monitoring range should extend from the upper sandy beach area through the tidal flat and down to the offshore trench. The most effective approach to conserve the horseshoe crab T. tridentatus is to designate protected areas for it. On Kinmen Island, 800 hectares of tidal flat has been designated as the horseshoe crab protected area (Chen et al. 2004). This action protects only part of its nursery grounds and not its maturation grounds. Adult populations distributed around the Kinmen area are known to live in the trenches of shallow water surrounding Kinmen, Taiwan, and Xiamen, China. As a result, the designation of protected areas where adult horseshoe crabs harvest- ing is prohibited needs Cross-Strait (Taiwan and China) collaboration.

References

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