e Rese tur arc ul h c & a u D q e A v e f l o o Ceesay et al., J Aquac Res Development 2012, 3:2 l p a m n Journal of Aquaculture r e u n o t DOI: 10.4172/2155-9546.1000123 J ISSN: 2155-9546 Research & Development Research Article OpenOpen Access Access Biodiversity and Adaptability of Holothuria leucospilota and Stichopus japonicus Sea Cucumber Species in Artificial Environment Abdoulie Ceesay1, Mariana Nor Shamsudin1*, Norfarrah Mohammed Alipiah1 and Intan Safinar Ismail2 1Laboratory of Marine Biotechnology, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 2Chemistry Department, Faculty of Science, Universiti Putra Malaysia Abstract Sea cucumber species respond to challenging environmental conditions differently thus, to culture them in artificial environment requires a better understanding of their environmental needs. Two species of sea cucumber Stichopus japonicus and Holothuria leucospilota in Teluk Kamang Port Dickson (PD) (2° 27’ 46.61″N 101° 50’ 42.98″E), Malaysia, were investigated to determine their biodiversity. H. leucospilota was separately investigated to establish its abundance in three (3) separate periods of the year- March, July and November. Global Positioning System (GPS) and Yellow Springs Instruments (YSI) were used to measure the physical parameters. Stichopus japonicus and Holothuria leucospilota were found in temperature ranges of 31 to 32°C and 30 to 31°C, respectively. Salinity was 30.00 to 31.00 part per thousand (ppt) for S. japonicus and 30.00 to 31.00 ppt for H. leucospilota. The pH ranged from 7.80 to 8.40 and 8.00 to 8.60 for S. japonicus and H. leucospilota respectively. H. leucospilota species were more abundant in March than in July, and more in July than in November. Amplification of their genomic DNA using the 16S primer, showed H. leucospilota to be approximately 550 bp and S. japonicus 600 bp in size. The sequenced PCR products analyzed using the Basic Local Alignment Search Tool (BLAST) Version 2.01G identifiedHolothuria leucospilota as another species in Malaysia. These findings provide an insight into Holothurians environment needs and established a nucleotide sequence for H. leucospilota in nucleotide database for future use. Keywords: Biodiversity; Stichopus japonicas; Sea cucumber; 16S 30 cm to 100 cm in two sites: a coral reef site at Teluk Kemang beach for primer; BLAST; Holothuria leucospilota H. leucospilota, and sandy or muddy areas behind Costa Rica Hotel for S. japonicus. The species were collected within three different quadrats, Introduction each measuring 5 m x 20 m. YSI was used to take salinity, temperature, Sea cucumbers are organisms belonging to the phylum Echinoder- and pH measurements, and the GPS was used to determine the location mata from the class Holothuroidea composed of five genera. Even thou- of the species at the sites. The sea cucumber species collected from each gh some are found in shallow waters, they are typically ocean dwellers quadrat was counted and then stored in buckets containing sea water and are found living in the deep ocean. They live on or near the ocean with battery aerators to provide oxygen [5]. They were then transported floor and are often buried beneath it [1]. They are widely distributed in to the lab in this condition before being transferred to the aquarium. all regions of the oceans worldwide. To date, as many as 1500 species and new species identified each year have been recorded among the Abundance of H. leucospilota at Port Dickson six valid orders of Apodida, Aspidochirotida, Elasipodida, Molpadiida, To determine the abundance of H. leucospilota, a survey was first Dendrochirotida and Dactylochirotida [2]. Holothurians have a long history of being consumed by the oriental people, mostly the Chinese conducted from March to July 2010, and then in November of the and Japanese. Being a source of food and medicine to many has resul- same year. The number of species per survey was determined randomly ted in their being harvested heavily in many communities with tonnes due to the vastness of the area and it was conducted in three quadrats leaving the ocean annually [3]. This situation led some species to the measuring 100 m-2 (5 m x 20 m) along a 100 m transect line. The sea point of extinction. However, some institutions and communities have cucumber species were collected and counted before transferring them embarked on aquaculture and breeding projects, in addition to other in improvised aquariums. Upon completion of the work the species conservation efforts geared towards protecting the endangered species were returned to their respective locations [5]. The physical parameters from complete disappearance. In Malaysia, approximately 50 species were also determined during each sampling period. from three orders and seven genera have been recorded while 34 spe- cies still require further identification [2,4]. Two sea cucumber species, Stichopus japonicus and Holothuria leucospilota, found around the coa- *Corresponding author: Mariana Nor Shamsudin, Laboratory of Marine Biotech- stline in Port Dickson are among the types recorded in Malaysia, with nology, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, H. leucospilota being the commonest ones [2,4]. Studies on the identi- Selangor, Malaysia, Tel. 03-8947 2192; Fax: 03-8947 2191, E-mail: mariana@ fication of local sea cucumbers at the genomic level are limited. Thus, medic.upm.edu.my this study was conducted to identify these two species of sea cucumber Received January 24, 2012; Accepted March 16, 2012; Published March 26, using DNA sequencing and also to highlight some of the environmen- 2012 tal factors that support the survival of one species over the other in an Citation: Ceesay A, Shamsudin MN, Alipiah NM, Ismail IS (2012) Biodiver- artificial environment. sity and Adaptability of Holothuria leucospilota and Stichopus japonicus Sea Cucumber Species in Artificial Environment. J Aquac Res Development 3:123 Materials and Methods doi:10.4172/2155-9546.1000123 Copyright: © 2012 Ceesay A, et al. This is an open-access article distributed un- Sampling of sea cucumbers der the terms of the Creative Commons Attribution License, which permits unre- stricted use, distribution, and reproduction in any medium, provided the original The sea cucumber samples were collected at low tide at a depth of author and source are credited. J Aquac Res Development ISSN: 2155-9546 JARD, an open access journal Volume 3 • Issue 2 • 1000123 Citation: Ceesay A, Shamsudin MN, Alipiah NM, Ismail IS (2012) Biodiversity and Adaptability of Holothuria leucospilota and Stichopus japonicus Sea Cucumber Species in Artificial Environment. J Aquac Res Development 3:123 doi:10.4172/2155-9546.1000123 Page 2 of 6 Estimation of population size conditions. S. japonicus was cultured in artificial sea water at 27.00 ppt below its optimum salinity level of approximately 30.00 ppt and left in To determine the estimated population size of H. leucospilota sea that condition for two days. H. leucospilota was also cultured in artificial cucumber we collected data such as quadrat size (100 m-2), the size -2 sea water at salinity level of 27.00 ppt, below its optimum salinity level of the survey area (8000 m ) and the number of species per quadrat. of 30.00 ppt, and left in such a condition for two days. The salinity was ℜ The mean ( ) value of all the quadrat counts generates the population adjusted to 28.00 ppt on the third day when organisms showed signs of density expressed in numbers of individuals per quadrat area. To stress. Temperature on the other hand was control at 31 and 32°C for estimate the total population size of H. leucospilota at the survey site, S. japonicus and H. leucospilota respectively using heaters for the first we calculated N for the quadrat data using the formula: N = (A/a) × n, week but when the salinity was normalized, the temperature was left where: N = the estimated total population size, A = the total study area, ambient and was normally between 24 and 30°C. Their conditions were a = the area of the quadrat and n = the mean number of organisms per monitored both in the morning and in the afternoon. Observations quadrat. To also determine the distribution of the species at the site we made on each species were recorded on a daily basis. calculated the variance (s 2) and divided the variance by the mean (ℜ) to compute the variance-to-mean ratio (s 2 /ℜ) which tells us whether the Total genomic DNA Extraction species were aggregated, random or uniformly dispersed. To calculate DNA extraction was carried out using Epicenter’s DNA and the variance and variance-to-mean ratio, we used the following set of RNA extraction kit (Epicenter Biotechnologies) according to the formulas: (i) = quadrat number, Σ (x ) = total number of organisms, Σ i manufacturer’s instructions. Five to ten milligram (5-10 mg) of H. (x ) / n = mean, Σ (d2) = sum of squared deviations, s 2 = Σ (d2) / (n-1) i i i leucospilota and S. japonicus body tissue and intestine samples collected = sample variance and s 2 / ℜ = variance-to-mean ratio. from one individual sacrificed from each species were mixed with 300 The values are given below: µL of the tissue and cell lysis buffer containing 1 µL proteinase K in a microcentifuge tube and incubated at 65°C for 15 mins, vortexing A = 8000 m-2, a = 100 m-2, n = 19, 61, 38, 13, 10, 14, 4, 3, 3 every 5 mins. Samples were then placed on ice for 3-5 minutes before N=? addition of the MPC protein, and centrifuged for 10 mins at 4°C at 8,500 x g. The supernatant was collected and the pellet discarded.