Research Article Algae 2011, 26(4): 317-325 http://dx.doi.org/10.4490/algae.2011.26.4.317 Open Access The occurrence of the ciguatera fish poisoning producing dinoflagellate genus Gambierdiscus in Pakistan waters Sonia Munir1,*, P. J. A. Siddiqui1 and Steve L. Morton2 1Centre of Excellence in Marine Biology, University of Karachi, Karachi 75270, Pakistan 2National Oceanic Atmospheric and Administration, Marine Biotoxins Program, 219 Fort Johnson Road, Charleston, SC 29412, USA Five benthic species of the genus Gambierdiscus (Dinophyceae) were observed for the first time in the coastal waters of Pakistan, Northern Indian Ocean. The morphology of the epiphytic, ciguatera-related toxic species G. toxicus, G. belizea- nus, G. polynesiensis, G. australes and G. cf. yasumotoi are presented here, described by the Kofoid system of thecal plates Po, 3΄, 7˝, 6c, 8s, 5˝΄, 1p, 2˝˝ with differences in cell shape, cell size, plates, pores around the apical pore plate by using light and scanning electron microscopy. The occurrence of these potentially toxic dinoflagellate species in Pakistani coastal areas of Manora Channel and Balochistan during high temperatures of 28-32ºC is cause of concern for human health impacts from ciguatera fish poisoning. Key Words: ciguatera fish poisoning; Dinophyceae;Gambierdiscus ; Northern Indian Ocean; Pakistan INTRODUCTION The primary causative agent of ciguatera fish poison- Buenconsejo-Lum 2001). Worldwide an estimated 50,000 ing (CFP) are nonplanktonic dinoflagellates of the genus people suffer every year from cigutera toxicity (Ragelis Gambierdiscus (Dinophyceae). The type species is G. toxi- 1984, Anderson et al. 2000). cus Adachi et Fukuyo 1979, an armored, unicellular, pho- Species level description are very difficult for anteri- tosynthetic and toxic dinoflagellate species distributed posterior compressed dinoflagellate cells such as G. toxi- circumtropically. This benthic dinoflagellate’s preferred cus (Adachi et Fukuyo 1979), G. belizeanus (Faust 1995), habitat is attached to macroalgae surface, algal turf, de- G. australes, G. polynesiensis, G. pacificus(Chinain et al. tritus, and sand (Bomber et al. 1988, Bagnis et al. 1990, 1999), G. carolinianus, G. caribaeus, G. carpenteri (Lita- Morton and Faust 1997, Masó et al. 2003, Aligizaki and ker et al. 2009) as well as globular shaped cells such as G. Nikolaidis 2008). Gambierdiscus is an important species yasumotoi (Holmes 1998) and G. ruetzelri (Litaker et al. complex that produce ciguatoxins which cause of the 2009). Distingushing these two groups depend upon the CFP in the coral reef areas. CFP had been troublesome in ornamentation and shape and size of plates. island countries where human populations rely on reef The first case of CFP in Indian Ocean was described in fish as a primary protein source. Ciguatera is a syndrome Mauritius, an island in the western Indian Ocean (Quod that causes gastrointestinal, neurological and cardiovas- and Turquet 1996). This is first observation of the Cigutara cular disorders in humans (Lewis et al. 1988, Palafox and species from the northern part of the Indian Ocean, Paki- This is an Open Access article distributed under the terms of the Received September 21, 2011, Accepted November 22, 2011 Creative Commons Attribution Non-Commercial License (http://cre- Corresponding Author ativecommons.org/licenses/by-nc/3.0/) which permits unrestricted * non-commercial use, distribution, and reproduction in any medium, E-mail: [email protected] provided the original work is properly cited. Tel: +92-21-9261397, Fax: +92-21-9261398 Copyright © The Korean Society of Phycology 317 http://e-algae.kr pISSN: 1226-2617 eISSN: 2093-0860 Algae 2011, 26(4): 317-325 water temperatures from both locations were 32ºC and 28ºC, respectively. Microscopy techniques Samples were preserved in Lugols solution and brought to the Marine Biotoxin lab, USA and examined under 5600LV Scanning electron microscopy (JOEL, Tokyo, Ja- pan) and under light microscopy / Fluorescence micros- copy (BX51; Olympus, Tokyo, Japan) with a DP71 camera. Cells were stained with calcoflour white, examined under the 200-400× and images were captured with camera. For scanning electron micrograph, Lugol’s fixed sample were desalted in seawater gradient (100, 70, 50, and 25%) and cleaned with 100% freshwater, dehydrated in an acetone series (10, 30, 60, 80, and 100%), mounted and sputter Fig. 1. Coastal map of Pakistan. The sampling points along coated with gold-platinum (Desk IV; Denton Vacuum, the Manora Channel coast of Karachi and Churna Island coast of Moorestown, NJ, USA). The morphometric analysis was Balochistan are marked by arrows. performed using Microsuite 5 (Olympus). stan is described according to modern taxonomical clas- sification following Faust (1995), Holmes (1998), Chinain RESULTS et al. (1999), Litaker et al. (2009). This study provides basic information about the toxic benthic dinoflagellates from Five species of Gambierdiscus were present in the the non-coral area of Manora Channel with high man- coastal waters of Pakistan, including. G. toxicus, G. beliz- grove vegetation along the Karachi coast and the Coral eanus, G. polynesiensis, G. australes and G. cf. yasumotoi. reef area of the Balochistan coast Pakistan. Identification was based on Kofoidean thecal plate tabu- lation and structure (Yasumoto et al. 1977, Faust 1995, Holmes 1998, Chinain et al. 1999, Litaker et al. 2009). MATERIALS AND METHODS Gambierdiscus toxicus Adachi and Fukuyo 1979 Sampling collection (Figs 2A-H, 3A-C, 4A & B) Samples sites were the Manora Channel Sindh coast of Cells of G. toxicus are round, flattened and dorso- Karachi and Churna Island Balochistan coast. The Sindh ventrally compressed in apical and antapical view. Cell coastline is about 250 km long and the continental shelf size was 71.0-96.4 µm dorsoventrally and 61.9-90.1 µm deep stretches into the ocean and adjacent to souther- in transdiameter. Both epitheca and hypotheca valves eastern part to Indian border along Sir Creek on the east. are smooth, porose and similar in height. The epitheca The Balochistan coast is 800 km long with a steep and is ellipsoidal, compressed in apical view. Epithecal plates narrow continental shelf to the Iranian border near Jiwani are comprised of 11 plates from pore plate to precingular in the west. These coastal areas are commercially impor- plates as apical pore plate (Po), 3΄, 7˝ are variable in shape tant and provide a transit trade port via Karachi harbor and size (Fig. 2D & F). The Po is fish hooked shaped and and Gawader port of Balochistan. is 7.7-8.0 µm long and 4.4-6.1 µm wide. The oval shaped Samples were collected from Manora Channel Po covered with dense pores openings (n = 28-30) is po- (24º47.93′ N, 66º58.87′ E) Sindh coast and Churna Island sitioned at the ventral side of the apical plate (Figs 2F, 3A (24º46.70′ N, 66º26.66′40˝ E) Balochistan coast of Pakistan & B). The first apical plate, 1΄, is small, pentagonal and is (Fig. 1). The samples from the Manora channel were col- seven sided at the left corner of epitheca and edge of 1˝ lected with a 55 µm mesh size net during July 2007 and a and 7˝ plates of precingular plates, connected with Po, 2΄, Niskin bottle (1.7 L) sampler at 1 m depth from the Chur- 3΄, 6˝, 7˝, 1˝, and 2˝. The 2΄ is a sub rectangular plate, much na Island, Balochistan coast during November 2007. The broader and larger that is 41.1-45.8 µm long and 13.3-27.2 http://dx.doi.org/10.4490/algae.2011.26.4.317 318 Munir et al. Gambierdiscus in Pakistan Waters A B C D E F G H Fig. 2. Gambierdiscus toxicus. (A-C) Light microscopy and fluorescence stained cells. (D-H) Scanning electron micrograph of the anterior- posterior compressed surface, the flattened shaped, and the smooth thecal surface. (F) Epithecal plates and fish hook shape (Po) (black arrow) in apical view. (G) The 1˝, 7˝ small precingular plates (black arrows). (H) Postcingular plates and posterior intercalary plate (1p) in antapical view. A B C D E F G H Fig. 3. Scanning electron microscopy of apical pore plates. (A-C) Tear shaped apical plate with apical pores of Gambierdiscus toxicus. (D & E) Apical plate of rugose G. belizeanus. (F) Apical plate of G. australes. (G & H) Triangle shaped apical plate of G. polynesiensis. 319 http://e-algae.kr Algae 2011, 26(4): 317-325 A B C D E F G H Fig. 4. Scanning electron micrograph of posterior intercalary plates (1p). (A & B) Gambierdiscus toxicus. (C & D) G. belizeanus. (E & F) G. australes. (G & H) G. polynesiensis. µm wide, comes in contact with 2˝, 3˝, and 4˝ plates. Of fish hooked shaped and is 7.7-8.0 µm long and 4.6-6.1 µm the precingular plates, 1˝ and 7˝ are smallest, and 3˝ much wide and covered with dense pores (n = 30) positioned larger 51.3-68.8 µm and 8.0-14.5 µm in wide (Fig. 2F & G). ventrally with the 1΄, 2΄, and 3΄ plates (Figs 3D, E, 5B & C). The hypotheca is more excavated with eight plates (5˝΄, The first apical plate ΄1 is small , pentagonal, seven sided, 1p, and 2˝˝). The postcingular plate 4˝΄ is large 32.5-54.0 located in the left corner of epitheca, and connected with µm and 13.8-23.5 µm wide. The 1˝˝ and 2˝˝ plates are small Po, 2΄, 3΄, 6˝, 7˝, 1˝ and 2˝. The 2΄ is sub rectangular that is and connect with 1˝΄, 1p and 5˝΄ plates (Fig. 2H). The larg- much broader and larger 39.6-40.0 µm long and 17.0-18.8 est plate is a posterior intercalary plate (1p) that is 45.3- µm wide and has wide contact with 2˝, 3˝ and 4˝ plates.