Parasite Inspection in Five Commercially Important Oyster Species (Mollusca: Bivalvia) of Pakistan.
Abstract: A total of five native oyster species namely; Crassostrea gryphoides C. madrasensis C. glomerata Ostrea nomades and Saccostrea cucullata were collected from three sites: Buleji of Sindh coast; Hub and Jiwani of Balochistan coast Pakistan. Parasitic inclusion was present only in histological sections of O. nomades and S. cucullata at Buleji. Individuals of O. nomades found to be infected with protozoan parasite Nematopsis species and Proctoeces sp. trematode parasite. Additionally unknown parasitic cyst was observed at outer epithelium in a male gonadal section of S. cucullata. There is no previous record in literature of pathogens in oyster species at Buleji.
Keywords: Nematopsis Northern Arabian Sea Ostrea Saccostrea Crassostrea Trematode parasite.
Over the past 60 years number of pathogens related diseases and mortalities have been reported in bivalves across the globe [1-7]. The main biological disease-causing agents in marine bivalves are viruses bacteria fungi protists digenean trematodes polychaetes and copepods . Parasitic diseases pose a serious threat to the world's oyster aquaculture industry and to oyster fisheries. Historically some diseases are of regulatory significance [9 10] have been associated with Ostrea spp. and Crassostrea spp. and account for major commercial losses and limit expansion of oyster culture. Pakistan has a sizeable diversity of native species of oysters  which can provide a potential resource for the development of mariculture in Pakistan . However although they are not exploited commercially oyster stocks in Pakistan have become depleted and their natural spat fall is very low . Like elsewhere in the world the study of parasites and disease distressing
oysters is important due to their potential economic impacts on the management of natural stocks and aquaculture [5-6]. Siddiqui et al.  reported the Nematopsis spp. in O. nomades and C. belcheri from Keamari Sea wall area adjacent to Manora Channel along the Sindh coast of Pakistan. The aim of the present study was to broaden this previous study to other species and sites as the diseases of regulatory significance not found in native oyster species which make them exclusively important from the mariculture point of view for commercial exploitation. METHODS
Samples were procured between January 2012 and March 2012 at low tides (Table 1). Five oyster species Crassostrea gryphoides (Schlotheim 1813) C. madrasensis (Preston 1916) C. glomerata (Gould 1850) Ostrea nomads (Iredale 1939) and Saccostrea cucullata (Born 1778) were sampled from the intertidal zones of three sites: Buleji (24 50' N 66 48' E) Hub (24 54' N 66 43' E) and Jiwani (25 07.630' N 61 45.662' E) (Figure 1). Measurements were taken before histological preparations. The animals were brought live to laboratory and the length along their longest axis and shell height of specimens were measured to the nearest millimeter (0.1 mm) by the method described by Galtsoff  and Siddiqui and Ahmed . Wet tissue weight (g) was also recorded prior to further examination. Seven (7) to twenty (20) individuals of each species were histologically examined. A portion (5 mm) of soft tissue was excised with a razor blade along a line extending from the
lower corner of the labial palps across the stomach to the posterior end of the body. This yielded a transverse section through the stomach digestive diverticulum intestine connective tissue demibranchs of both gills and the mantle lobes; tissue samples were then fixed in Davidson's solution  for 48 hrs and preserved in 70% ethanol. Prior to sectioning the tissues were dehydrated in an ethanol series embedded in paraffin wax sectioned (7m) and stained with haematoxylin and eosin. Histologically prepared slides of each species were examined for parasitic inclusion following the World Organization for Animal Health guidelines [9 10]. Gonadal developmental stages were also studies in prepared slides. Gonadal developmental stages
Table 1: Sampling Sites Species Sample Size and Date of Collection
###Site###Species###N###Date of Collection
###"###Crassostrea madransis###7###24/1/2012 and 14/2/2012
###Jiwani and Buleji###Saccostrea cucullata###20###2/2/2012 and 21/2/2012
were determined by assigning the numeric ranks 0-4 for each gametogenic stage (0=spent 1=early developing 2=nearly ripe 3=ripe 4=spawning). Scoring scheme was used to assess gonadal condition of different oyster species at different sites to map gonadal variation among species.
Protozoan Nematopsis sp. Proctoeces sp. (Trematoda) and some unknown parasites were recorded in O. nomades and S. cucullata only at Buleji. Calculated biometrics have shown highest shell length (mm) 86.607.61 for C.madransis followed by 75.603.77 C. gryphoides 55.290.79 O. nomads 41.600.35 C. glomerata and collectively 41.052.35 for S. cucullata obtained from Buleji and Jiwani (Figure 2). Meanwhile highest wet weight (g) 10.63.8 was calculated for C. gryphoides followed by 10.27.6 C. madrasensis and 5.32.3 for S. cucullata communally (Figure 2).
Histological inspection showed specimens of C. gryphoides were set up to be almost in resting state. 95.0% individuals were found spent (0) besides one individual was observed with early developmental (1) stage. Whereas C. madrasensis was found with 42.9% spent (0) 14.3% early developing (1) and 42.9% in ripe (3) condition. C. glomerata showed 10.0% spent (0) 80% nearly ripe (2) and 10% ripe (3) gonads (Figure 2).
In recent studies individuals of O. nomades from Buleji found in ripe (3) state with female gametes and developing (2) male follicles in hermaphroditic individuals. A total of six (6) hermaphroditic and one (1) pure female were encountered during histological investigations. Whereas 15% individuals of S. cucullata found developing or nearly ripe (2) 80% ripe (3) and 5.0% spawning (4) observed.
Histological examination has revealed that both of the candidate species were in the verge of the sexually mature state at Buleji and some of these sexually mature individuals have been found with parasitic burden which shows that mature individuals in the wild population have more chances to acquire parasitic burden as during the present study protozoan Nematopsis sp. Proctoeces sp. Trematode parasite and one unknown parasitic cyst were observed in O. nomades and S. cucullata specimens obtained from Buleji. Seven examined slides of O. nomades (one female and six hermaphrodites) were found with protozoan Nematopsis species; among them one hermaphroditic individual was found heavily parasitized with trematode Proctoeces sp. in addition to Nematopsis sp. (Figure 3a b). Highest prevalence of infestation were recorded in O. nomades at Buleji where Nematopsis sp. and Proctoeces sp. were found respectively in 100% and 14.8% of individuals respectively. Furthermore 8% of S. cucullata at Buleji were infested with an unknown parasite. Parasitic loads differed between different tissues; Nematopsis sp. was found in the gills digestive and connective tissues in 20.4% 38.8% and 40.8% respectively of individuals from Buleji. The parasitic load of Proctoeces sp. was 44.3% in gills 14.8% in gonadal portion and 41.0% in connective tissues from the same site (Figure 2).
A total of thirteen (13) specimens of S. cucullata examined for parasitic inclusion from Buleiji and seven (7) individuals from Jiwani were free of any parasitic infection except for one male specimen that was infected with an unknown parasite was procured from Buleji. A spherical thraustochytrid-like cyst with honeycomb cellular structures and endospores was observed at the outer periphery of gonadal tissue In specimens of C. gryphoides C. madransis and C. glomerata there was no parasitic inclusion in any specimen examined.
Earlier researchers  documented the maximum 241-280 mm size for C. gryphoides at the same site (Hub Delta) the largest size among edible oysters of the world during past decades. whereas same time the populations of C. gryphoides and C. madrasensis were mostly between 41-200 mm size ranges at Hub Delta. Additionally larger sizes (40-70 mm) for C. glomerata from Karachi 32-82 mm for S. cucullata from Paradise Point and 26-97 mm for O. nomads from Buleji and Keamari Sea wall have been recorded formerly . Recent studies have shown the considerable size decrease in existing wild populations when compared to previous data. Although oyster populations at Hub have been considerably depleted during the last decade any contributing reason behind this is not known so far but a possible reason which has never been thoroughly investigated in past was the presence of a nuclear power plant at Hub. Nevertheless these species could be useful potential resource for commercial exploitation being acquiring considerable wet tissue mass. Wet tissue weight data for respective species is not available in previous records. However onset of gametogenesis in C. gryphoides in intermediate and large size classes was the same . Further it was indicated that the percentage of females remains lower in the colder months i. e. from November to January and also stated that in winter months more females remain indifferent and undergo resorption. Siddiqui and Ahmed  examined the larviparous oyster O. nomads from Buleji and Keamari area and histologically individuals were found to be hermaphroditic pure male pure female and indifferent at two sites throughout the year with increasing activity in the winter months followed by immediate ripening and later spawning and resorption around the year.
There is no previous record available pertaining report of trematode parasitic inclusion in Pakistani oyster species. Earlier workers observed 48% to 92% protozoan parasite Nematopsis sp. prevalence with an average of 67% in O. nomads . Moreover parasitophorous vacuoles containing oocysts were investigated in the digestive gland palps mantle gonads gills and kidney of the oysters. According to previous report  inclusion of Nematopsis sp. in O. nomades and C. belcheri was profound from Keamari Sea Wall constructed near Karachi Port. The presence of Nematopsis sp. in Buleji population could be due to the fact that Buleji rocky ledge is rich in fauna and flora and number of arthropod species including brachyuran crabs are quite abundant in the area . Oysters are the intermediate hosts of many species of Nematopsis and life cycle of these parasites is completed in intestine of marine arthropods moreover species of Xanthidae Portunidae Grapsidae Ocypodidae have been designated by authors which could be the possible final host for pathogenic prevalence [5 6 17- 19]. Similarly several species of marine brachyuran crabs from Manora Island have been reported  which lies adjacent to Keamari sea wall and Buleji. Xanthid crab Leptodious exaratus found to be principal species at both sites. Thalamita prymna Charybdis species Eriphia laevimana Pilumnus vespertilio Pseudozius caystrus Grapsus strigosus and Atergatis roseus were also turned out dominant species at both of the sites. These crab species
possibly serve as final host for pathogenic infestation.
Identification of observed parasites to species level was not attempted. Nematopsis sp. oocysts and metacercaria of the trematode Proctoeces sp. were observed in gills digestive and connective tissues of O. nomads from Buleji. Nematopsis has been reported in many bivalve and gastropod mollusks [6 20 21]. Boehs et al.  stated that infestation by sporocysts which are structures of digenetic trematodes could be a threat for host bivalve because of its potential to reduce host fecundity.
Digenean trematodes have complex life cycles in which bivalve mollusks are intermediate hosts harboring larval stages of the digenean. Some finfish species are known ultimate hosts for bucephalid digenean trematodes . By contrast Couch  observed the un-encysted metacercariae of Proctoeces sp. in the gonadal follicles and ducts of oysters from three Gulf of Mexico estuaries and suggested that the presence of this trematode to its oyster host is in fact not harmful. In the present study metacercariae and mature adults of Proctoeces sp. were observed in gills digestive and connective tissues of gonadal sections in O. nomades. Trematode metacercariae usually cause no apparent harm to host bivalve molluscs [24-26] but infection with Proctoeces sp. may cause serious lesions and inhibit gametogenesis .
During the present study protozoan Nematopsis sp. trematode Proctoeces sp. and an unknown (thraustochytrid-like) parasites have been recorded in Parasite Inspection in Five Commercially Important Oyster Species (Mollusca: Bivalvia) Journal of Basic and Applied Sciences 2014 Volume 10 225
O. nomades and S. cucullata at Buleji of Sindh coast. On the contrary Balochistan coast Pakistan was found to be free of pathogenic infestation during the present oyster watch. There is no previous record of trematode parasites in Pakistani oyster species in literature. In this paucity it is recommended that investigative endeavor ought to be expanded in sustainable deployment perspective.
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|Date:||Dec 31, 2014|
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