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Horseshoe crab (HSC) is the common name of marine arthropod that belongs to phylum Arthropoda and the order Xiphosura. They are well known unique living fossils that retain their morphology unchanged over millions years. These animals related more to arachnids than crustaceans. There are four species around the world which are Tachypleus gigas (Müller), Tachylpeus tridentatus (Leach), Carcinoscorpius rotundicauda (Latreille), and Limulus polyphemus (Linnaeus).
Three out of four species horseshoe crab, T. gigas, T. tridentatus, and C. rotundicauda inhabit Malaysian coast. T. gigas and C. rotundicauda can be founded in coast of Peninsular Malaysia and East of Malaysia, while T. tridentatus can be founded along the East of Malaysia coast. This study will focus on T. gigas species that has the widest distribution of the three Asian horseshoe crab species (Ismail et al., 2011) and can be founded in Chendor, Pahang. Recently, the number of horseshoe crab reported to be declined worldwide. In Malaysia, the number of horseshoe crab has decline since the horseshoe crab actively consume as delicacy (Tan et al, 2001).
Most studies done on horseshoe crab have focused on their straw-coloured blood. Horseshoe crab blood contains amoebocytes which will turn blue when bacterial toxins present. In United States, they collect the amoebocytes from L. polyphemus and produce Limulus Amoebocytes Lysate (LAL) devices which can be used to detect the endotoxin. It has been commercializes worldwide. Meanwhile in Malaysia, Tachypleus Amoebocytes Lysate (TAL) was produce from T. gigas. This finding can give huge benefits to Malaysia itself. This country no needs to spend massive amount of money for imported LAL. However, before TAL can be produce in a huge amount and support the demand for human and animals' drugs and devices, we need to know the population size of horseshoe crab to be sure that there are enough and continuous supply for the TAL production.
In recent decades, there are just a few numbers of studies on horseshoe crab population size that focus on T. gigas in Malaysia. The status of their population size was not really understand and there are insufficient scientific data. This basic questions and information could solve many problems. In order to address this concern, research on population size of this species should be conducted as an effort to determine the population size of T. gigas. The study will focus on T. gigas in Chendor, Pahang using capture-mark-recapture (CMR) method.
This study is carried out to determine the horseshoe crab (Tachypleus gigas) population size in Chendor, Pahang
2.1 The horseshoe crab
Horseshoe crab (HSC) or also known as king crab is the only primitive marine invertebrate widely distributed on earth (Størmer, 1952). These horseshoe crabs belong to the phylum Arthropoda, which consists of animals having an articulated body and limbs, class Merostomata, and order Xiphosura (Størmer, 1952). These animals phylogenetically more related to arachnids such as spiders and scorpions than crustaceans (Zaleha et al., 2010). Horseshoe crab are not a true crab but the descendent of ancient Eurypterids (Kingsley, 1894) which included sea scorpions and an ancient horseshoe crab, the Aglapsida (Von Roy, 2006).
They are well known unique living fossils which have been continuously withstand for 550 million years of evolution without modified their morphology (Ismail & Sarijan, 2010). Horseshoe crab also known as evolutionary survivors that have remain relatively unchanged (Sekiguchi & Sugita, 1980). Besides, they are most closely related to trilobites that existed 544 million years ago and very similar to a fossil specimen (Mesolimulus walchi) found in the Jurassic deposits (Xia, 2000).
2.2 The anatomy of horseshoe crab
The horseshoe crab body has a 'helmet-shaped'. It has a unique three major parts which are cephalothorax (head), opisthosoma (abdomen), and telson (tail). The cephalothorax means fused head where the head and thorax of horseshoe crab is fused together and also known as prosoma. It has a pair of simple eyes at the front, and a pair of compound eyes positioned laterally. The simple eyes can senses ultraviolet (UV) from the moon while, the compound eyes sensitive to polarized light and can magnifying sunlight ten times (ASMFC).
Besides, the abdomen which also called the opisthosoma, attaches to the cephalothorax by a hinge joint and make these animals flexible to move or fold up. In addition, the hard shell known as carapace covers the whole of the horseshoe crab. The exoskeleton made of chitin, a polysaccharide, and provides an attachment point for muscles. The carapace has a colour range from light grey, greenish-grey to almost black according to the species of horseshoe crab. The glossy carapaces indicating young aged horseshoe crabs and massive scratched carapaces indicating old aged crabs (Walls, 2001).
On the ventral side of the cephalothorax, there are appendages and each have the specific functions. The first pair is the chelicerae, which have small claws at the end that help move food into the mouth. It also has five pairs of walking legs with small claws. But in males, the first pair of claws bigger and modified for grasping the female carapace during mating (Faurby et al., 2011). The last pair of walking legs is the pusher legs with no claws that are used for pushing and for stirring up sediments when the horseshoe crab burrows or lays eggs.
Horseshoe crabs have well-developed external six pairs book gills in the form of appendages which are known as gill lamellae (Suniza et al., 2011).The book gills that located underside the abdomen whish are used for gas exchanged and get the oxygen from the water (Suniza et al., 2011).. To increase the effectiveness the gas exchange, each gill has many folds which increase the surface area. They also bury their self and fold their body to conserve and avoid the loss of water. Besides, if these primitive gills stay moist, horseshoe crab can remain out of water up to four days (U.S Fisheries & Wildlife Service, 2006). It also used the book gills as a paddle while swimming. The first pair is fused and forms the operculum, which protects the other five pairs. Under the operculum are the genital pores, where the eggs and sperm deposited.
These animals also have telson, the spike-like tail. It is not a weapon nor venomous but acts as rudder to help right itself when it is overturned (Jones, 2011). The telson attaches to the abdomen by a ball joint, which allows a wide range of motion. There are several ways to differentiate male and female horseshoe crab. Usually the male carapace is more convex, its edges are more flared, and it is much smaller than the female. The first pair of legs in adult males has claws called claspers that are modified for reproduction. These claspers are larger than the other claws and resemble boxing gloves. Meanwhile, the females and juveniles have the same size and shape of claws on all the walking legs. The genital pores also differ for both sexes. In males they are located at the peaks of hard, conical projections; in females they are softer and appear as elliptical slits.
2.3 The size growth of horseshoe crab
Horseshoe crab is molting by shed their exoskeleton since they are arthropod (Division of Marine Fisheries, 2003). These animals increasing in size by 25-30% with expand their new shells by pumping in water which will harden in about 24 hours and the horseshoe crab will crawl forward from the old exoskeleton (Mid-Atlantic Sea Grant, 2012). Since horseshoe crabs do not moult after they have reached sexual maturity, they are often being a home and colonized by epibionts (Patil & Anil, 2000).
In addition, molting occurs several times during the first three years but decrease in frequent significant as horseshoe crab grow older and stop when they become sexually matured (Division of Marine Fisheries, 2003). They will molt with average 18 time before reach sexually mature and once it matured, these animals can live with additional 8 to 10 years making the total life span as long as 20 years (Misur, 2012).
2.4 Tachypleus gigas
Tachypleus gigas is categorized under subfamily Tachypleinae (Shishikura et al., 2005). It is abundant and can be founded from the Indian Ocean to the Southeast Asia region and also been reported from Malaysia (Chatterji et al., 1992). In Malaysia, T. gigas occur around Peninsular Malaysia and small amount in Borneo Island (Rohizan & Ismail, 2012).
These T. gigas also known as coastal horseshoe crab and commonly found at the sandy zones to muddy zones (Mishra & Mishra, 2011; Cartwright-Taylor et al., 2011) and has the widest distribution of the three Asian horseshoe crab species (Ismail et al., 2011). T. gigas feed on molluscs and decayed organic matter thus; they are called as benthic feeder (Chatterji et al., 1992). This horseshoe crab has a tough greenish-grey colour exoskeleton and the tail is triangular in cross-section (Fortey, 2011).
2.5 Distribution of horseshoe crab
They are only four species of horseshoe crab existing in the world today. There are Tachypleus gigas (Müller), Tachylpeus tridentatus (Leach), Carcinoscorpius rotundicauda (Latreille), and Limulus polyphemus (Linnaeus). Three species from the subfamily Tachypleinae are T. gigas (Shishikura et al., 2005)., T. tridentatus, and C. rotundicauda. These species inhabit the coasts of Indo-Pacific (Ismail & Sarijan, 2010) and has been clustered as Asian horseshoe crabs.
Out of four extant species of horseshoe crabs, three of them, T. tridentatus, T. gigas and C. rotundicauda inhabit Malaysian coast while the distribution of T. tridentatus is restricted to East Malaysia (Kamaruzzaman et al. 2012). The Asian horseshoe crab T. tridentatus and the coastal horseshoe crab T. gigas live in muddy and sandy habitats (Davidson et al., 2008) while mangrove horseshoe crab C. rotundicauda can be founded in muddy area, usually in brackish water (Cartwright-Taylor et al., 2011). Meanwhile, the American horseshoe crab, L. polyphemus can be found along the American coastal of the Northern region from Marine to the Gulf of Mexico and along the Delaware Bay beaches (Carmichael et al., 2003).
According to IUCN (2010) all four species of this marine arthropod classified as either near threated or data insufficient. These due to many factors such as horseshoe crab are destroyed because they feed on commercially important shellfish such as soft shelled clam in Massachusetts (Division of Marine Fisheries, 2003). It also being noticed that anthropogenic activities related to fisheries industry, coastal erosion and construction are the main factor for the shrinking of area for horseshoe crab (Mishra & Mishra, 2011). The habitat degradation by harbour construction also decreases the number of horseshoe crab (Yang et al., 2011).
2.6 Studies on horseshoe crabs
Horseshoe crabs have been used in numerous field of study such as biomedical, genetic, ecology, etc. In recent decades, studies in biomedical field really get the attention (Division of Marine Fisheries, 2003). Today HSC are important to people for their used in medicine. For over 50 years scientists have used HSC in eye research. Scientists easily study the large eye and optic nerve (nerve that send the signal rom eye to brain) of horseshoe crab. Scientist learned a great deal about how human eyes function from the research (Mattei, 2010).
In addition, an important biomedical application to detect bacterial endotoxin using a specialized cell compound from horseshoe crab blood lead to greater exploitation of their natural population worldwide (Tanacredi, 2002). Recently, horseshoe crabs are the source of those compounds technically known as Limulus/Tachypleus Amebocyte Lysate (LAL/TAL) which contributes billion dollar ($) profit annually (Zaleha et al., 2012).
Other than that, chitin also can be founded in exoskeleton of horseshoe crab and it is non-toxic and biodegradable. Chitosan produced from chitin and used as raw materials to manufacture a variety of important product. These substances useful in remove lead from wastewater fight against fat when add to food, hinder the fat from being absorbed by body, boost good cholesterol uptake and inhibit bad cholesterol uptake (OceanQuest). Chitin from horseshoe crab extremely pure and chitin-coated suture reduce healing time in human by 35 - 50% (OceanQuest).
3.1 Study site
The sampling will be carried out along eastern coast of Peninsular Malaysia at Chendor (04°10.642' N and 103°25.261' E), Pahang (Fawwaz Afham, 2012). Fresh specimen of horseshoe crab (Tachylpeus gigas) reported can be founded there.
3.2 Samples collection
Total sampling will be made is three times. The sampling will be conducted on 1/11/2012, 8/11/2012, and 15/11/2012. The minimum day intervals for the next catch are five days after the first catch. Within the time, the marked samples can mingle along with the unmarked Tachypleus gigas population. Samples will be collected with the help of local fishermen using a net. The net measurement is _ metre width and _ metre length.
3.2.1 Capture-mark-recapture (CMR) method
The trapped samples will be taken to the shore for marking. There are three options for the marking which are by using the wood paint, the marking thread and the cable tie. After the marking process, Tachypleus gigas will release back to the sea.
The T. gigas will be marked with wood paint on the carapace (Fawwaz Afham, 2012). Each sample will be marked with different colour according to how many times they get caught. Three different paint's colour will be used which are white, yellow, and blue. For the first time the T. gigas get caught, white colour will be used as identifier colour. The yellow and blue colour will be used for the second and third time. The marking will be done carefully as precaution to avoid the paint from hurting the T. gigas especially the eyes area.
Marking thread will be used as a second option to mark the T. gigas. We need to poke a hole in carapace to tie the marking thread. Each sample will be marked with different colour according to how many times they get caught. Three different marking thread's colours will be used which are white, red, and pink. For the first time the T. gigas get caught, white colour will be used as identifier colour. The red and pink colour will be used for the second and third, and times. The marking will be done carefully to avoid the injury while boring the carapace and hold up their movement.
Cable tie will be used as a third option to mark the T. gigas. We need to bore a hole in carapace to fasten the cable tie. Each sample will be marked with different colour according to how many times they get caught. Three different cable tie's colours will be used which are black, yellow, and blue. For the first time the T. gigas get caught, black colour will be used as identifier colour. The yellow and blue will be used for the second and third, and times. The marking will be done carefully to avoid the injury while boring the carapace and fasten tidily to make sure it will not hold up their movement.
3.2.2 Measurement of the horseshoe crab size
In conducting this study, the size of the horseshoe crab will be measured. The measurement will be taken in centimetre (cm) of total carapace (from the tips of the carapace to the tip of the telson) and prosomal width (Ismail et al., 2011) using a measuring tape (Tan et al., 2011) as morphometric proxy to estimate the approximate age and instar stage of horseshoe crab based on Sekiguchi (1988) in Tan et al., (2011).
The measurement will be taken three times for each horseshoe crab to reduce the measurements error (Faurby et al., 2011). The gender will be determined by observe the present or absent of monodactylus pedipalps (Shuster (1995) and Sekiguchi (1988) in Carmichael et al., 2003) or by checking the shape of the first pedipals (Tan et al., 2011).
The data will be recorded as below:
Sampling site: Chendor, Pahang
Total carapace (TC) (cm)
Prosomal width (PW) (cm)
3.2.3 Analysis of data
Data collected will be analysed using Bailey's triple-catch formula to estimate the Tachypleus gigas population size.
Bailey's triple-catch formula:
Estimated total population, N:
(râ‚‚â‚ + 1) (râ‚ƒâ‚‚ + 1)
aâ‚‚ (nâ‚‚ + 1) râ‚ƒâ‚
aâ‚‚² (nâ‚‚ + 1) (nâ‚‚ + 2) (râ‚ƒâ‚ - 1) râ‚ƒâ‚
(râ‚‚â‚ + 1) (râ‚‚â‚ + 2) (râ‚ƒâ‚‚ + 1) (râ‚ƒâ‚‚ + 2)
Standard error, S.E.:
Population range (95% confidence level):
N + S.E. or ( N - S.E.) to ( N + S.E.)
Total estimated population size, P:
N + Var
To estimate the total abundance of male horseshoe crab, the following equation will be applied:
For female, the following equation will be applied:
Nf = 100% - Nm
aâ‚‚ = Number of individuals that newly mark and release in second sampling
nâ‚‚ = Total number of individuals captured on the second sampling
râ‚‚â‚ = Number of individuals capture on second sampling and marked on first sampling
râ‚ƒâ‚ = Number of individuals captured on third sampling and marked on the first sampling
râ‚ƒâ‚‚ = Number of individuals captured on third sampling and marked on the second sampling
Nm = The total abundance of male
Nf = The total abundance o f female
Rm = Ration of male individuals to the total captured
4.1 Expected outcomes
The horseshoe crab population sizes will be determined. General size of individuals in the studied population will be determined.