Phuket mar. biol. Cent. Res. Bull 73: 7–14 (2016)
DNA SEQUENCING COMPLEMENTS MORPHOLOGICAL IDENTIFICATION OF CHIROPSOIDES FROM NAM BOR BAY, PHUKET, THAILAND
Phuping Sucharitakul1, Charatsee Aungtonya2 and Siriwadee Chomdej1 *
1 Department of Biology, Faculty of Science. Chiang Mai University, Chiang Mai 50200, Thailand 2 Phuket Marine Biological Center, P.O. Box 60, Phuket 83000, Thailand *Corresponding author: [email protected]
ABSTRACT: This study was conducted by collecting samples of the jellyfishChiropsoides from Nam Bor Bay, Phuket, in southwest Thailand. Specimens were examined and classified using both morphological and molecular methods. Morphological study revealed that all specimens were Chiropsoides buitendijki. Tissues from the gastric saccule, pedalia, exumbrella and rhopalia were extracted and sequenced using nuclear 18s rRNA. The sequences were submitted to the National Center for Biotechnology Information (NCBI). The accession number is KJ135023.
Key words: Molecular analysis, Chiropsoides buitendijki, 18s rRNA
INTRODUCTION because of the arrangement of tentacles and unilat- erally-branching pedalia which differ fromChirop - In the past, jellyfish specimens were studied only salmus with bilaterally branching pedalia. However, by morphological techniques. The morphological the name was unavailable since it was already in study of jellyfish for taxonomic purposes is use for a coleopterid group. In 1956 Southcott challenging because many species of jellyfish exhibit renamed it Chiropsoides (cited by Gershwin, 2006; wide variability in form. In Cubozoa, taxonomy is Williamson et al., 1996). Gershwin (2006) noted still being developed and there have been arguments that the shape of the gastric saccules, pedalial canals, on the validity of nominal species in many genera. and branching of pedalia also supported the However, since 2005, two new families of carybdeids separation of this genus. Therefore, those morpho- were established, i.e., Alatinidae Gershwin, 2005a logical characters were used in order to classify and Carukiidae Bentlage, Cartwright, Yanagihara, Chiropsoides (e.g. gastric saccule, morphology of Lewis, Richards and Collins, 2010. In addition, six pedalia, tentacles, and nematocysts). As a result, new genera were proposed, i.e., Copula Bentlage, Chiropsalmus quadrigatus (Haeckel, 1880) was Cartwright, Yanagihara, Lewis, Richards and Collins, classified into this genus because the arrangement 2010 (Tripedaliidae), Alatina Gershwin, 2005a and of pedalia was similar to Chiropsoides buitendijki. Keesingia Gershwin, 2014 (Alatinidae), Gerongia Chiropsoides buitendijki (Figure 3) was first Gershwin and Alderslade, 2005; Malo Gershwin, described from the harbour of Batavia (now Jakarta), 2005b and Morbakka Gershwin, 2008 (Carukiidae). Java, Indonesia by Horst in 1907 as Chiropsalmus In addition, Chiropsellidae Toshino, Miyake and buitendijki. Later, it was assigned to the genus Shibata, 2015 was newly designated as a chirodropid Chiropsoides by Southcott (1956), as mentioned family and three new genera were assigned, i.e., above. It can easily be distinguished from other Chirodectes Gershwin, 2006 (Chirodropidae), chirodropids by its unilaterally branching pedalia. Chiropsella Gershwin, 2006a and Meteorona C. buitendijki has a cuboid bell with 4–9 tentacles. Toshino, Miyake and Shibata, 2015 (Chiropsellidae). One unilateral branching pedalium arises from The genus Chiropsoides is a member of the each corner of the bell. Eight finger-shaped gastric chirodropid (Multiple-tentacle box jellyfish) group. saccules arise from the perradii and are almost as It was originally described as Chiropsalmus long as the height of the bell cavity. C buitendijki buitendijki (Horst, 1907). However, in 1925 Krumbach has 6 eyes (2 median eyes and 4 lateral eyes) separated it into a new genus called Drepanochirus located in a shallow oval-shaped rhopalial niche 8
Phuket mar. biol. Cent. Res. Bull ostium whereas C. quadrigatus has eight cock’s The present study is focused on the identification comb-shaped gastric saccules and a dome-shaped of the box jellyfish genus Chiropsoides collected rhopalial niche ostium (Horst, 1907; Mayer, 1910; from Nam Bor Bay, Phuket in June 2013. The Kramp, 1961; Gershwin, 2005a; 2006; Gershwin morphology was studied in order to clarify ambiguous and Alderslade, 2006). However, Bentlage (pers. specimens and the 18S gene was amplified using comm.) argued that C. quadrigatus referred by tissues from various parts of the animal. Gershwin (2006) appears to be a species of Chi- ronex, which is characterized by the cock’s comb MATERIALS AND METHODS shaped gastric saccules. In recent years, molecular techniques have been Morphological identification applied to identify specimens. Molecular sequences A total of 14 specimens were collected from from genomes provide more characters that can Nam Bor Bay, Phuket, Thailand. The identification be used to identify and classify specimens (Olsen and classification followed Gershwin (2005 and et al, 1993). rRNA has been extensively used in 2006). The trapped jellyfish were collected from phylogenetic studies since the late 20th century. bamboo stakes. Specimens were observed under The most common rRNA employed as a molecular the stereo-microscope to examine the characters marker is the nuclear encoded small subunit (SSU) defining this genus. The height of the bell was 18S rRNA. Due to its slow evolutionary rate, 18S measured from the top of the exumbrella to the rRNA is appropriate for reconstructing phylogenetic velarium using digital vernier calipers (Fig. 1). relationships. Bentlage et al. (2010) constructed a Internal structures were studied by incising the phylogenetic tree in order to discuss the evolution exumbrella along the body wall from the velarium of venom, life-history and biogeography in Cubozoa. up to the corner of the bell, then exposing the stomach In their study, mitochondrial 16S, nuclear 18S and to observe gastric saccules, phacellae, shape of lips nuclear 28S ribosomal genes were amplified, and manubrium. The bell height (BH), bell width sequenced and analysed. (BW) and other features were recorded. Photographs
Figure 1. Diagrammatic sketch of Chiropsoides showing parameters measured by digital vernier calipers BH= Bell height, BW= bell width. 9
DNA sequencing complements morphological identification were taken under the stereo-microscope using a primer, 0.2 µM reverse primer (Table 1) and 1 µl of Nikon E 8400 camera, while other images were 25 ng DNA. The primer pairs are shown in Table 1. taken with a Nikon SL 1000 camera. PCR was performed in a PCR tube using an initial denaturing step at 95ºC for 5 min followed by 40 DNA analysis cycles of 95ºC for 30 s, 62ºC for 30 s and 72 ºC for DNA extraction (Adapted from Pinto, 2000) 1 min. Finally, a final extension at 72ºC for 5 min Two tissues from 14 specimens were excised was carried out. The PCR products were visualized from tentacles, gastric saccules, eyes and exumbrella using agarose gel electrophoresis. The sequences before being ground into a powder in liquid nitrogen were obtained from DNA sequencing by 1st Base and placed in a microcentrifuge tube in 400 µl of Company, Selangor, Malaysia. lysis buffer. Then, 24 µl of 10 mg/ml RNAase were added, followed by incubation at 37oC for 30 minutes. Phylogenetic tree reconstruction A total of 20 mg/ml Proteinase K was added to a Multiple alignments were performed using final concentration of 1 mg/ml and incubated at SeqMan. The phylogenetic tree was constructed in 60oC for 48 hours. Subsequently, 500 µl of phenol MEGA 6 (Molecular Evolutionary Genetics Analysis) was added and mixed before samples were by adding the 18S sequences from members of centrifuged at 13,500 rpm for 15 minutes. 360 µl Chiropsoides (KJ135023.1), Chironex (AF358104 of the supernatant of the solution were transferred and GQ849076.1), Chirodropidae (GQ849077), to a new microcentrifuge tube and 360 µl of phenol Chiropsalmus (GQ849078), Chysaora (AF358103), added. The total volume was then mixed by vortexing Pelagia (HE591464), Tripedalia (GQ849088) and before samples were centrifuged at 13,500 rpm for 15 Morbakka (GQ849083); Pelagia and Chysaora are minutes. Approximately 340 µl of the supernatant the out-groups for the purpose of this research. were transferred to a new microcentrifuge tube followed by 340 µl of chloroform: isoamylalcohol RESULTS (24:1). The mixture was inverted and centrifuged at Morphological identification. 13,500 rpm for 15 minutes with 300 µl of supernatant Results from the morphological study indicated then being transferred to a new microcentrifuge tube that all of the specimens were Chiropsoides which was filled with 30 µl of 6 M NaCl and mixed buitendijki (Figs. 2–4). by inverting gently. Following this procedure 750 Description. The maximum bell height was µl of extremely cold absolute ethanol was added 74.44 mm and the maximum bell width was 97.98 in order to precipitate the DNA. Samples were mm. The average bell height was 46.87 mm and centrifuged at 13,000 rpm for 10 minutes. The the average bell width is 57.62 mm. supernatant solution was discarded; subsequently Specimens were described carefully using both 1 ml of 70% ethanol was added and the samples external and internal structures. The cubic shape were centrifuged at 13,000 rpm for 5 minutes. of the bell and its smooth texture can be observed The supernatant solution was discarded and directly on first sight (Fig. 2C). Tentacles were flat- air-dried for 24 hours. Finally, 30 µl of TE buffer tened (Fig.4A); furthermore, when they were examined was added and incubated at 37oC for 48 hours. under the stereo-microscope, the wavy-like jagged edge was immediately obvious. The single peda- Polymerase Chain Reaction (PCR) lium in each corner of the bell comprised 5 fingers The 18SAf 5’ CCG AAT TCG TCG ACA that branched unilaterally and bend towards the ACC TGG TTG ATC CTG CCA GT3’ and int6 5’ velarium (Fig.3C). Pedalial canals ran along the GAA TTA CCG CGG CTG CTG 3’ primers used velarium towards the subumbrella lamella, with a in this research amplify about 500 bp according to thorn-shape in the end (Fig. 3D). Each finger had Gershwin (2005c). DNA amplification of partial one tentacle and decreased in size sequentially; the 18S was performed using a thermal cycler. The largest and longest one being inner-most. The reaction mixture for PCR consisted of a total volume rhopaliar niche ostia were covered by a concave of 25 µl which contained 2.5µl of 10X buffer, 0.2 upper scale and a shallow curve on the lower Taq DNA polymerase, 0.2 mMdNTP, 0.2 µM forward covering scale (Fig. 3A); no rhopaliar horns were 10
Phuket mar. biol. Cent. Res. Bull observed (Fig. 3A). There were six eyes per rhopa- specimens from Sri Lanka had 9 fingers and a lium comprised of 2 median lensed eyes and 4 lateral specimen from an unknown locality 7 fingers, eye spots (Figs. 2A and 2B). Eight finger-shape as reported by Gershwin (2006). However, in this gastric saccules pointed down from the stomach study, only 4-5 fingers were found in the 14 specimens to the velarium (Fig. 3B). The v-shaped gastric examined. Specimens varied in size from small phacellae were unbranched; several gastric cirri (22.16 mm.) to large (74.44 mm.). Gershwin were found above the gastric saccules (Figs. 4B (2006) describes preserved material as possessing and 4D). The manubrium was short with triangular hyaline body with pale pinkish tentacles (colour tongue-shape lips (Fig.4C). unknown in living material) whilst specimens in Remarks. Although most of the specimens in the present study had a hyaline body with cloudy this study matched perfectly with those in Gershwin white tentacles and pink internal structures with (2005 and 2006), there were some variations: lavender pink tentacles. In life, specimens had a Chiropsoides buitendijki was recorded from the hyaline body and internal structures with ivory Java Sea with 5-6 fingers (Mayer 1910) whereas white tentacles or with wisteria violet tentacles.
Figure 2. Chiropsoides buitendijki: A, Six eyes, 2 median lensed eyes (red arrow) and 4 lateral eye spots (white arrow); B, Rhopalial stem (arrow); C, Gastric saccules; D, Velarial canals. 11
DNA sequencing complements morphological identification
Figure 3. Chiropsoides buitendijki: A, Rhopaliar niche ostrium; B, Finger-shape gastric saccules; C, Unilateral branching pedalia (arrow); D, Spike pedalial canal (arrow).
Figure 4. Chiropsoides buitendijki: A, Flatted tentacle; B, Numerous gastric cirri; C, Lips; D, V-shape phacellae. 12
Phuket mar. biol. Cent. Res. Bull
Table 1. The oligonucleotide sequence used in this study (Gershwin 2005)
Primer Amplicon Sequence (5’ 3’) size (bp) 18SAf CCG AAT TCG TCG ACA ACC TGG TTG ATC CTG CCA GT 526
Int6 GAA TTA CCG CGG CTG CTG
Molecular analysis for Biotechnology Information (NCBI) (Table 2). DNA was extracted from rhopalia, gastric The accession number is KJ135023. saccule, tentacles and bell. 18S rDNA was then amplified using PCR (Polymerase Chain Reaction) Remarks. In the present study, only tissues from yielding 526 base pair fragments (Fig. 5). Then, eyes (rhopalia) were successfully sequenced while the PCR products were sequenced. A phylogenetic tissues from other organ parts, apart from the eyes tree was constructed in MEGA 6. The phylogeny (rhopalia), failed to produce DNA sequences. In showed that Chiropsoides buitendijki was most contrast to this study, the study by Bentlage et al. closely related to Chiropsalmus quadrumanus (2010) successfully amplified partial mitochondrial (Figure 6). Likewise, the genetic distance between 16S (16S), near-complete nuclear 18S (small Chiropsoides and Chiropsalmus was 0.021 as subunit; SSU) and 28S (large subunit; LSU) calculated using the Kimura 2-parameter model. tentacle tissues. The sequence was submitted to the National Center
Figure 5. PCR products with 526 base pairs. 13
DNA sequencing complements morphological identification
Figure 6. Phylogenetic tree: Chiropsoides buitendijki compared with other cubozoa species and the out group (Chrysaora and Pelagia).
Table 2. Sequence data of Chirodropids available in GenBank
Order Family Genus Species 16S 18S 28S COI
Chirodropida Chiropsalmidae Chiropsoides C. buitendijki +
Chirodropida Chiropsalmidae Chiropsalmus C. quadrumanus + + + +
Chirodropida Chirodropidae Chironex C. fleckeri + + + + Chirodropida Chirodropidae Chironex C. yamaguchii + +
Chirodropida Chiropsellidae Chiropsella C. bronzie + Chirodropida Chiropsellidae Meteorona M. kishinouyei +
CONCLUSION ACKNOWLEDGEMENT
In this study, Chiropsoides buitendijki samples This work was supported by the Graduate have been examined by both morphological and School, Chiang Mai University. We thank Ms. molecular methods. We successfully quantified Krittiya Chanachon and all staff at the Reference DNA extractions from selected tissues and the Collection, Phuket Marine Biological Center for results provide a valuable contribution to the their kind support and assistance. Finally, the first taxonomic study of this species. However, further author gratefully thanks Dr. Somchai Bussarawit investigations concerning the behavior, ecology for his support and for stimulating interest in box and biology of the species remain to be conducted jellyfish, and also first author is beholden to Asst. in the future. Prof. Dr. Sasivimon Swangpol, Mr. Peeranat Bokoranee and Ms. Peeranan Kanthiyah for their advice and illustrative skills. 14
Phuket mar. biol. Cent. Res. Bull
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Manuscript received: 7 August 2015 Accepted: 8 October 2015