Ecobiology of horseshoe crab in Brebes, Northern Coast of Java: Preliminary study

Horseshoe crabs are known as living fossils and three species are found in Indonesia where all of them are being protected. The research aimed to assess ecology, species composition, and biological aspects in Brebes estuary. The research was conducted in August and October 2017 in Brebes Estuary. Samples were caught using local fishermen and experimental gears. Parameters of water quality were observed insitu and substrate was analyzed in the laboratory. The horseshoe crabs were measured morphologically, their stomach and eggs were taken to be analyzed in the laboratory. Total specimens of horseshoe crabs were 33 individuals (Tachypleusgigas=8; Carsinoscorpius rotundicauda=25). T. gigas has 16.3-22.6 cm of prosomal width and 216.1-717.0 gram of weight. C. rotundicauda has 11.2-14.5 cm of prosomal width and 96.0-275.5 gram of weight. Stations with a muddy sand substrate (silt >50%) have the highest number of captured horseshoe crabs. Food habits of T. gigas are macrophytes and detritus while C. rotundicauda are macrophytes, molluscs, annelids, and coral. Niche breadth of C. rotundicauda were wider than T. gigas. High niche overlap value between T. gigas and C. rotundicauda indicates high competition in utilizing food sources. The fecundity of C. rotundicauda was higher than T. gigas but the egg diameter of C. rotundicauda smaller than T. gigas.


Introduction
Horseshoe crabs are called ancient creatures where they have changed from 200 million years and the lineage of evolution is at least ~ 510 million years [1,2]. Rudkin et al. [3] also reported that these creatures had an age longer than 445 million years. The horseshoe crab currently consists of only four species. Limulus polyphemus (Linnaeus, 1758) occurs in the coastal waters of the eastern continental shelf of North America and in the Gulf of Mexico and three other species (Tachypleus gigas (Müller, 1785), Carcinoscorpius rotundicauda (Latreille, 1802) and Tachypleus tridentatus (Leach, 1819)) are found in the coastal waters of India, Southeast Asia, China, and Japan [4]. Indonesian coastal waters are reported to have three species of Asian horseshoe crabs, namely T. gigas, C. rotundicauda and T. tridentatus, which are distributed in several regions [5][6][7].
The horseshoe crab's role in ecology are food source for other species, a voracious eater, and can disrupt extensive benthic and coastal habitats [4]. The blood of L. polyphemus and T. tridentatus has a substance known as Limulus Amebocyte Lysate (LAL) [8]. The other two species T. gigas and C. 2 rotundicauda can also produce LAL. LAL is used by the US Food and Drug Administration (FDA) as a test for the presence of endotoxins in biology, pharmaceutical drugs, and medical devices and is used worldwide [9]. Tachypleus Amebocyte Lysate (TAL) is an alternative to LAL [10].
Despite its important role in ecology, the horseshoe crab poses several threats to its survival. Significant changes in the natural habitat of horseshoe crabs for nesting are caused by continuous anthropogenic activities [1,10)]. Development in coastal areas is one of the contributors to the loss of suitable habitat for horseshoe crab nesting, especially in the Eastern Asian region [11][12][13][14]. In the Asian region, the population of horseshoe crabs has some pressures in terms of medical use and human consumption [15].
The IUCN Red List of Threatened Species releases the status that Limulus polyphemus is vulnerable [16], T. tridentatus is endangered [17], T. gigas is data deficient [18] and C. rotundicauda is data deficient [19]. Three species of horseshoe crabs (T. gigas, C. rotundicauda and T. tridentatus) in Indonesia have a protected status through Regulation of the Minister of Environment and Forestry of the Republic of Indonesia No. P.20/MENLHK/SETJEN/KUM.1/6/2018 concerning protected flora and fauna species [20].
In Indonesia, studies on the ecology and biology (food habit, reproductive aspect) of T. gigas and C. rotundicauda are still scant. Ecological and biological studies of T. gigas and C. rotundicauda are needed in Indonesia to fill in the lack of data on horseshoe crab in Indonesia. This research aimed to assess ecology, species composition, and biological aspects of horseshoe crab in Brebes estuary.

Study site
This study was carried out on August and October 2017 in Brebes, Northern Coast of Java. The research stations cover the entire delta Pemali estuary area which is located in seven locations determined according to the typology of the waters. The seven stations are Mangrove creek (station 1), Polang Canal (station 2), Mouth of Pemali (station 3), Mouth of West Pemuda Canal (station 4), East Pemuda Canal (station 5), River embankment (station 6), and Kaliwlingi (station 7) (figure 1).

Data collection
Sampling was carried out using local fisherman's fishing gear (single-layer nets/snapper nets/three-inch gillnet monofilament nets, trammel nets, and trash nets), and also experimental gear (centripede nets, and fyke nets). The horseshoe crab was recorded for gender, measurements of prosoma (length and width), and weight. The horseshoe crabs were dissected and their gonads and stomachs were preserved in 5% formaldehyde for further analysis. Gonads and stomach content observation were conducted in Research Institute for Fish Resources Enhancement. In the laboratory, ovaries were weighted to the nearest 0.001 g, then some eggs of each ovary were counted as subsample and its diameter was measured under a microscope equipped with an ocular micrometer.

Data analysis
Species diversity is a comparative analysis of the horseshoe crab species composition caught at each station that represents habitat typology. The comparison is displayed in histogram form so we can find out which species are mostly caught in the estuary of the Pemali Delta.
Index of preponderance is used to determine the feeding habits of horseshoe crab [42]: The niche breadth (Ba) values vary from 0 to 1. The niche breadth (Ba) values 0 mean species consume a single item and 1 mean species exploits available items in equal proportion. Values of Ba are considered low when below 0.4, moderate when between 0.4-0.6, and high when the value is higher than 0.6 [45].
Niche overlap between the most common species was analyzed using Pianka's index [59]: . The value of niche overlap (Ojk) is classified as low (<0.33), medium (0.33-0.67) and high (> 0.67) [46]. The assumption here is that all species can access different food sources equally [47,48], because it did not collect data on the availability of resources in the study area [47].
The Gonadosomatic Index (GSI) was calculated using Effendi's formula [49], representing the percentage of the gonad weight with total horseshoe crab weight. Fecundity was determined using a combination of the gravimetric method [49,50] against the ovaries of mature female organisms [51] determined by the formula: T. gigas has characteristics: brownish, has a size of up to 40 cm, has a convex body shape with a horseshoe-like carapace, a triangular telson-shaped cross section, and has special characteristics in the form of a protoreceptor in the caudal spin [52], while C. rotundicauda is the smallest species, with the largest sizes ranging from 25-30 cm, the body shape is like a steel shell, brownish, and the cross-section of the telson is round [53]. A total of 33 individuals (Eight T. gigas; 25 C. rotundicauda) of horseshoe crab were caught during the study. The composition of horseshoe crab species caught at each research station is presented in figures 2 and 3. Based on figures, we discovered that horseshoe crab caught in five out of seven stations, Mangrove Creek, Polang Canal, Mouth of West Pemuda Canal, East Pemuda Canal, and Upstream Estuary. In this study, horseshoe crab were not found in the Muara Pemali and the river embankments, but based on information from local fishermen, horseshoe crabs were generally caught there.   C. rotundicauda and T. gigas can be found at Polang Canal and East Pemuda Canal, while at the other three stations (Mangrove Creek, Mouth of West Pemuda Canal, and Kaliwlingi) only found C. rotundicauda. C. rotundicauda is a species that dominates the catch compared to T. gigas and almost found in all research stations with a percentage ranged from 67.67 -100%. The Northern Coast of Java generally has shallow waters with soft, sandy bottoms or broad mudflats or mangrove-mudflat ecosystems and these characteristics are suitable as habitat for C. rotundicauda. Horseshoe crab usually spends most of its life near or at the bottom of brackish swamp waters [5,7,54,55,56]. T. gigas is found in much lesser numbers. This condition is presumably because this species prefers a slightly different or more specific habitat than C. rotundicauda. All sampling locations were thought to be less suitable or ideal as a habitat for genus Tachypleus, so that this species was found in a smaller percentage. These findings similar to previous studies where the genus Tachypleus, C. rotundicauda is almost always found in greater numbers than T. tridentatus and T. gigas [57].
The distribution of carapace length frequencies of the two horseshoe crabs caught in the Pemali Delta Estuary is showed in figure 4. The horseshoe crab species that dominates Pemali River estuary were C. rotundicauda with carapace lengths ranged from 11.4-13.3 cm which is divided into three interval classes (10-11, 12-13, and 14-15 cm). The highest number of horseshoe crabs was found in the 12- T. gigas species found in the research locations had a carapace length ranged between 16.1-24.5 cm and were in class intervals of 16-17, 22-23, and 24-25 cm. Overall T. gigas had the largest size and wide class intervals. Mashar et al. [7] in their research along the north coast of Java, found this type of crab in the size range between 15-16 cm to 25-26 cm. The variation in the size of horseshoe crabs is most likely influenced by geographical conditions, population density, food availability, and environmental conditions [60,61]. Malaysian mangrove horseshoe crab has the carapace length longer than India and Thailand [62]. The results showed that the T. gigas species were only found limited to the estuary areas which had the lowest salinity range up to 14‰. Another specific characteristic that is quite light is the typology of the substrate as its habitat. This species found in locations that have a muddy sand substrate with a silt percentage more than 50%. However, salinity is thought to be a limiting factor compared to substrate types for the distribution of this species, as explained in previous studies that the Asian Limulidae T. tridentatus and T. gigas live in shallow and calm seas, to river estuaries with muddy sand bottoms [63]. C. rotundicauda was found in all study sites. This condition is similar to the results from previous studies where C. rotundicauda have a wider range habitat preference, that were brackish, muddy waters in mangrove ecosystems, and shallow beaches with soft sandy bottoms or large mud flats [54,56,63]. In this study, C. rotundicauda species were found from the end of estuary to the upstream estuary (salinity 0.5‰), and a wider range of muddy substrate type, between 8.46% to more than 50%, even more, frequent found in sandy mud type habitat with a balanced composition of sand and mud.

Biology of horseshoe crab 3.2.1. Food habit.
The food habits of horseshoe crabs were analyzed from the stomach contents of the horseshoe crabs which have food inside. The number of stomach content analyzed were 4 individuals of T. gigas and 21 individuals of C. rotundicauda. T. gigas index of preponderance consisted of macrophyte (95.45%) and detritus (4.55%), while C. rotundicauda consisted of macrophyte (68.99%), molluscs (29.07%), annelids (1.54%), and corals (0.41%) (figure 5). In this study, the food composition of C. rotundicauda was more varied compare to T. gigas. These results are similar to research conducted by Nuraisah et al. [27]. The index of preponderance is a useful tool to study the food preferences of organisms [42]. Nikolsky [51] distinguishes the food preferences of organisms into three categories, main foods with an Index of Preponderance of more than 25%, complementary foods between 5-25%, and supplementary foods less than 5%. The index of preponderance of T. gigas shows that its main food was macrophyte, and its supplementary food was detritus. C. rotundicauda has the main foods of macrophytes and molluscs, and supplementary foods of annelids and corals. Both of them have commonly used macrophytes as their main food. Recent studies among horseshoe crabs suggest a variety of foods, including particulate organic matter such as algae/macrophyte, may play a significant role in the diet of horseshoe crabs [64,65,66].
The index of preponderance of T. gigas in Brebes shows that its main food is macrophyte and supplementary food is detritus. Food habit of T. gigas in Balikpapan (Indonesia) consists of gastropods, bivalves, scaphopods, litter, and others with the main food being gastropods and bivalves [27]. The food composition of T. gigas in Cherok Paloh (Pahang, Malaysia) showed that echinoderms dominated food item during the open sea migration phase and the dominations were substituted by macrophyte as the T. gigas migrated inshore [25]. Hajisamae et al. [26] found that T. gigas in the east coast of Peninsular  [24] research showed that T. gigas was a selective feeder, and analysis of its stomach content in different months showed that molluscs and decayed organic matter were the main constituents of its diet. The major portion of the mollusca was contributed by bivalves. From July to October, the percentage of decayed organic matter, sand particles, and pieces of plant material was high in the stomach of T. gigas. Unidentified organic matter (mostly plant material) in decayed condition constituted about 28.0% of the total diet. It occurred regularly and throughout the year with a maximum contribution in October (50%). The index of preponderance of C. rotundicauda in Brebes shows that its main foods are macrophyte and molluscs, supplementary foods are annelids and corals. The food habit of C. rotundicauda in Balikpapan (Indonesia) is consists of gastropods, scaphopods, bivalves, crustaceans, echinoderms, Polychaeta, litter, and others with the main food being gastropods and leaf litter [27]. In Sunderban Estuarine, C. rotundicauda feeds mainly on gastropods, bivalves, polychaetes, and decayed organic matter. Decayed organic matter consisting mainly of plant material [28]. Seasonal variations of the food composition of C. rotundicauda in Pahang (Malaysia) showed that mollusks formed the main item especially gastropods. Unidentified organic matters in the stomach content analysis of C. rotundicauda showed a high preference for plant materials [12]. Zhou & Morton [29] found that the food composition of C. rotundicauda juveniles from Pak Nai beach (Hongkong) consisted mainly of insect larvae, followed by polychaetes and amphipods.
The food compositions of horseshoe crabs were closely related by the abundance and availability of food in the environment [25]. The Malaysian peninsular land barrier effect has been shown in macrophyte [67,68] and marine animals [69,70], which depends on the water currents for their dispersal. In several studies, macrophytes were found to be the main food in the intestines of male horseshoe crabs [12,24,25,28,71,72]. Sediment analysis encountered that the composition of macrophyte was higher in the beach area [25], so it is suspected that this affects the stomach contents of horseshoe crabs in coastal areas. Though horseshoe crabs are selective feeders, their adaptation to prefer a variety of macrobenthic communities (adaptive feeding) could have helped them in maintaining their population over time [73].

Niche breadth.
Nice breadth describes the amount of food resources that can be utilized by a group of organisms and the selectivity of a type of organism between species and between individuals within the same species to food resources in certain habitats [44]. The niche breadth (Ba) between the C. rotundicauda and T. gigas two types of horseshoe crabs is different.  Table 3. Niche breadth of horseshoe crabs T. gigas and C. rotunicauda in Brebes.

Species
Nice Breadth (Ba) Tachypleus gigas 0.10 Carcinoscorpius rotundicauda 0.78 T. gigas has a niche breadth (Ba) value of 0.10, lower than C. rotundicauda which has a value of 0.78 (table 3). Nice breadth of T. gigas in Brebes is low (Ba = 0.10), similar to the nice breadth of T. gigas in Balikpapan (Ba = 0.15) [27]. The low value of nice breadth indicates T. gigas is selective in nature's food preferences. This can be seen from the number of food types on T. gigas less than C. rotundicauda. T. gigas has only two types of food (macrophyte and detritus). Low nice breadth characterizes that the species is selective towards the food resources available in the waters [44,74]. The low value of nice breadth indicates that the species has a lower adaptability to the new environment [75].
The nice breadth of C. rotundicauda in Brebes is high (Ba = 0.78), higher than the nice breadth of C. rotundicauda in Balikpapan (Ba = 0.53). A high value of nice breadth indicates C. rotundicauda is a generalist species or not selective in choosing food resources in nature. C. rotundicauda has more food types than T. gigas in Brebes. C. rotundicauda has four types of food (macrophyte, molluscs, annelids and corals). A high value of nice breadth characterizes that the species is not selective towards the food resources available in the waters [44,74]. The dietary pattern of an organism that is a generalist (highvalue nice breadth) indicates that the species has high adaptability to new environments [75].

Niche overlap.
The niche overlap value between T. gigas and C. rotundicauda is high (Ojk = 0.97) (table 4). This niche overlap value illustrates the high competition opportunities between T. gigas and C. rotundicauda according to the classification by Moyle & Senanayake [46]. The niche overlap value between T. gigas and C. rotundicauda in Brebes is different from Balikpapan. In Balikpapan, the niche overlap value between T. gigas and C. rotundicauda has a small niche overlap value (Ojk = 0.28) which indicates low competition in food utilization [27]. Niche overlap of food is an area of space-niches inhabited by two or more niche inhabitants [59]. If the value of the niche overlap approaches 1, it indicates that two species being compared have almost the same type of food [74]. Niche overlap occurs when two or more organisms use the same food resource so that it can describe the competition between organisms in an ecosystem. Sa et al. [76] has explained that niche overlaps can occur in similar organisms that utilize the same food resource. T. gigas has high competition against C. rotundicauda due to their high similarity in using macrophyte as the main food.  [30] found that GSI from T. gigas ranged from 18.03 to 29.11%. with carapace length were 121-170 mm.  The fecundity of T. gigas ranged from 330 to 4,109 eggs, while C. rotundicauda's fecundity was 10,236 eggs. It means the fecundity of C. rotundicauda is higher than T. gigas. The size of oocytes from both species showed different sizes, where the oocytes of C. rotundicauda is smaller than T. gigas. This result was similar to a study that has been conducted by Chatterji [30], that stated fecundity of T. gigas (maximum fecundity was 6565 eggs) is lower than C. rotundicauda (maximum fecundity was 10,982 eggs) and eggs diameter of T. gigas (ranged between 1.54 to 2.09 mm) is smaller than C. rotundicauda (1.59 to 2.35 mm). In a study conducted by Khan [28] states that C. rotundicauda has fecundity between 3,540 to 13,490 eggs. The fecundity of both C. rotundicauda and T. gigas increasing with body size and weight [28,30,31].
The mature females from both species found in estuarine area, with shallow water (Polang Canal and Mouth of west pemuda canal). Due to limited samples, we cannot conclude the spawning period and its location of the two species of horseshoe crab in Brebes waters. However, Ehlinger & Tankersley, 2009 [77] stated that the timing of spawning of horseshoe crabs varies among areas with different tidal regimes. According to Brockman& Smith [78], all four species of horseshoe crabs migrate to shallow water for spawning.

Conclusion
A total of 33 individuals (Eight T. gigas; 25 C. rotundicauda) of horseshoe crab were caught during the study. The condition of aquatic environment based on six parameters in the research locations shows that these waters still support aquatic fauna. Stations with a muddy sand substrate with a silt percentage of more than 50% have the highest number of captured horseshoe crabs. Food habits of T. gigas are macrophytes and detritus while C. rotundicauda are macrophytes, molluscs, annelids, and coral. Niche breadth of C. rotundicauda's was wider than T. gigas. Niche overlap between T. gigas and C. rotundicauda was high that indicates high competition between them in utilizing food sources. The fecundity of C. rotundicauda was higher than T. gigas but the egg diameter of C. rotundicauda smaller than T. gigas.