The high prevalence (80-100%) of the marine leech Zeylanicobdella arugamensis (De Silva) on cage-cultured Asian sea bass Lates calcarifer (Bloch) led us to investigate the percentage of juvenile leeches hatched from deposited cocoons, survival of juvenile and adult marine leeches at different salinity and temperature. The results showed that the hatching percentage of juvenile leeches was highest at salinity of 30 ppt (32.5 ± 2.8%) followed by 20 ppt (18.0 ± 4.3%) and 10 ppt (12.1 ± 1.4%), respectively. It was found that the adult and juvenile leeches could live up to an average range of 4-7 days at salinity ranging from 10 to 40 ppt. The juvenile leeches were able to hatch at temperature ranging from 25 to 35 °C but unable to hatch at 40 °C. The survival period of adult and juvenile leeches ranged from 11 to 16 days at 25 °C, which was comparatively longer than 5-13 days and 10 h--5 days at 27-30 °C and 35-40 °C, respectively. The study provided the information on the physical parameters of salinity and temperature which are most optimal for the marine leech Z. arugamensis to propagate.
The Asian seabass is euryhaline, therefore it is interesting to describe the infestation and survival of caligids at varying salinity on the host. In this study, two different brackish water culture systems with monoculture and polyculture practices were investigated for the occurrence of Caligus spp. on Lates calcarifer. Polyculture practices mainly consisted of snapper (Lutjanus spp.), grouper (Epinephelus spp.) and seabass (L. calcarifer), while the monoculture was stocked with only seabass. A total of 777 Caligus spp. specimens were isolated from the sampling in 2009, consisting of three species; Caligus chiastos, Caligus epidemicus and Caligus rotundigenitalis. In 2011, the total specimen was increased to 3110 and two additional species were found; Caligus punctatus and one unknown species (Caligus sp.). A 98.6% of the total examination was represented by C. epidemicus. Constant presence of C. epidemicus was observed throughout the study, regardless the differences in between culturing practices and systems. This species was able to survive within wide salinity range, from 5 to 28 ppt. The other isolated species (C. chiastos, C. punctatus, C. rotundigenitalis and Caligus sp.) were only found infesting in polyculture cages with the salinity ranging from 25 to 28 ppt. Despite accounts for less than 2% of the total specimens, these species may able to produce a challenge for L. calcarifer polyculture farming activity due to their capability for host switching. The present study revealed the potential risk for cross-species transmission in polyculture practices.
Two new and two previously described species of diplectanid monogeneans (Heteroplectanum flabelliforme n. sp., Diplectanum sumpit n. sp., D. jaculator Mizelle & Kritsky, 1969 and D. toxotes Mizelle & Kritsky, 1969) were collected from archerfish Toxotes jaculatrix off the Island of Langkawi, Kedah and off Perak, Malaysia. The reproductive systems and squamodiscs of D. jaculator and D. toxotes are described for the first time. D. sumpit n. sp. differs from D. toxotes and D. jaculator in a having a small curved copulatory tube with a distinct accessory piece, compared to the long, tubular copulatory tube of D. jaculator and the slender tube of D. toxotes. D. sumpit n. sp. also differs from D. toxotes in having a larger ventral bar and larger squamodiscs. H. flabelliforme n. sp. differs from all known Heteroplectanum species in the shape and size of the squamodiscs, the arrangement of the sclerites in the squamodiscs, the extremely large ventral bar and the short, curved, non-spinous copulatory tube.
A study of the anterior adhesive apparatus (head organs) of Bravohollisia gussevi Lim, 1995 was carried out using light and electron microscopy. The anterior adhesive apparatus or head organs in B. gussevi comprise 6 circular openings or apertures in the antero-lateral region, associated pits lined with specialized microvillous tegument that differ from the general body tegument, a bundle of ducts, and uninucleate gland cells located lateral to the pharynx. The uninucleate glands of the anterior adhesive apparatus (head organs) comprise 2 types of cells, one kind of cell producing rod-like bodies (S1) and the other oval bodies (S2). The S1 bodies are filled with numerous, less electron-dense vesicles in an electron-dense matrix, while S2 bodies have no vesicles but contain a more homogeneous electron-dense matrix. Interlinking band-like structures were observed between S1 bodies. Similar band-like structures were found between S2 bodies. The formation of S1 bodies was followed by transmission electron microscopy. However, the formation of S2 bodies was unclear and could not be resolved. Uniciliated structures were also observed around the openings of the anterior adhesive apparatus. Each uniciliated structure is usually associated with an opening of a gland cell producing granular, electron-dense, secretory bodies, which differ from the secretions produced by the lateral gland cells of the anterior adhesive apparatus.
A new genus of the Monogenea, Teraplectanum n. g., is proposed for two new species of diplectanids found on the gills of Terapon theraps Cuvier collected off Carey Island, Peninsular Malaysia. The genus is based on a unique arrangement of the male reproductive system. In the new species spermatozoa stored in the seminal vesicle and secretions stored in the prostatic reservoir are transferred into, and mixed to form semen within, a special sclerotized auxiliary piece (SAP), and not within the copulatory tube as occurs in the majority of monogeneans. Teraplectanum species also possess a unique sclerotized vaginal loop through which the vaginal tube passes en route from the vaginal pore to the seminal receptacle. The two new species are Teraplectanum crassitubus n. sp. (type species) and T. angustitubus n. sp. They differ from each other mainly in the morphology of their copulatory tube: in T. crassitubus, the proximal region of this tube is thicker compared to the slender proximal region in T. angustitubus, although in both cases the tube tapers and twists distally. Of the known diplectanid species, only Diplectanum undulicirrosum Zhang et al., 2000 (currently considered incertae sedis) possesses such sclerotized hard parts, which suggests the same type of arrangement of the male reproductive system. Consequently, D. undulicirrosum is re-assigned to this new genus as Teraplectanum undulicirrosum (Zhang et al., 2000) n. comb. The copulatory tube of T. undulicirrosum is similar to the slender, undulating copulatory tube of T. angustitubus but does not taper distally as in the latter species.
Ligophorus belanaki n. sp. and Ligophorus kederai n. sp. are described from Liza subviridis Valenciennes, 1836 and Valamugil buchanani Bleeker, 1854, respectively. Ligophorus kederai n. sp. has fenestrated ventral anchors, while in L. belanaki n. sp. the ventral anchor is not fenestrated. Ligophorus belanaki n. sp. is similar to L. careyensis, one of its coexisting congeners, in the overall shape and size of hard parts, but differs in having a flat median piece in the structure of the AMP (antero-median protuberance of the ventral bar), copulatory organ with non-ornamented initial part and longer vaginal tube, compared to raised median piece in the AMP, ornamented initial part and comparatively shorter vaginal tube in L. careyensis. Ligophorus kederai n. sp. is similar to L. fenestrum, a coexisting congener, in having fenestrated ventral anchors, but differs in having longer points and narrower base. Ligophorus fenestrum, unlike L. kederai n. sp., also possesses fenestrated dorsal anchors. The principal component analysis (PCA) scatterplots indicate that the two new and eight known Ligophorus species from Malaysian mugilids can be differentiated based on the morphometries of their anchors, ventral bars and copulatory organ separately and when combined together. Numerical taxonomy (NT) analyses based on Jaccard's Index of Similarity and neighbour-joining clustering, is used to facilitate comparison of these two new species with the 50 known Ligophorus based on morphological and metric characters. The two new species are different from each other and the other 50 species in the overall shapes and sizes of hard parts, as indicated by the NT analyses.
Cryptocaryoniasis (also known as marine white spot disease) is mediated by Cryptocaryon irritans. This obligate ectoparasitic protozoan infects virtually all marine teleosts, which includes Lates calcarifer, a highly valuable aquaculture species. Little is known about L. calcarifer-C. irritans interactions. This study was undertaken to gain an informative snapshot of the L. calcarifer transcriptomic response over the course of C. irritans infection. An in-house fabricated cDNA microarray slides containing 3872 probes from L. calcarifer liver and spleen cDNA libraries were used as a tool to investigate the response of L. calcarifer to C. irritans infection. Juvenile fish were infected with parasites for four days, and total RNA was extracted from liver tissue, which was harvested daily. We compared the transcriptomes of C. irritans-infected liver to uninfected liver over an infection period of four days; the comparison was used to identify the genes with altered expression levels in response to C. irritans infection. The greatest number of infection-modulated genes was recorded at 2 and 3 days post-infection. These genes were mainly associated with the immune response and were associated in particular with the acute phase response. Acute phase proteins such as hepcidin, C-type lectin and serum amyloid A are among the highly modulated genes. Our results indicate that an induced acute phase response in L. calcarifer toward C. irritans infection is similar to the responses observed in bacterial infections of teleosts. This response demonstrates the importance of first line defenses in teleost innate immune responses against ectoparasite infection.
Culturing fishes in marine cages is a rapidly developing area of marine aquaculture. The Asian seabass Lates calcarifer (Bloch) is a fast growing good quality fish that is readily cultured in intensive systems in the South Asian region and in Malaysia in particular. Although several papers have been published to date on viral, bacterial, parasitic and fungal organisms causing diseases in the Asian seabass, the occurrence of a coccidian infection in this species has only recently been recorded. We collected sporulated and unsporulated oöcysts of a new species of Goussia Labbé, 1986, from the mucus covering the epithelium of the intestine of L. calcarifer. This paper provides a description of Goussia kuehae n. sp. Sporulated oöcysts of this species are ellipsoidal, 37-40 μm in length and 28-30 μm in width. The ellipsoidal sporocysts are relatively small, 15.2-17 × 5.7-8 μm, and located loosely in the oöcyst. There are residual bodies both in the oöcysts and the sporocysts. Goussia kuehae n. sp. differs from all known species of Goussia in the large size of the oöcysts and in having two types of oöcyst residuum.
Tor tambroides, a common and appreciated cyprinid fish of the Tasik Kenyir water reservoir in Malaysia, is one of the species selected for propagation. This fish was first successfully propagated in Malaysia by the Department of Agriculture, Sarawak, Malaysia, and the breeding program continued throughout the country. The gills were frequently infected by a Myxobolus species to be described as Myxobolus tambroides sp. n. The small, 50 to 70 μm, round plasmodia of this species is located intralamellarly. Plasmodia were filled with pyriform myxospores, 9.9 and 7.4 μm wide. In sutural view, the caudal end of the myxospores had a distinctive valvular groove, parallel with the suture. Plasmodia caused deformations on the affected and the neighbouring gill lamellae. The 18S rDNA sequence of M. tambroides sp.n. did not show a close relationship with any other Myxobolus spp., represented in the GenBank. This might be an emerging parasite likely to impact the propagation of this fish.
We describe new myxosporean species from Malaysian fishes cultured in pond farms and net-cages. Myxobolus omari sp. nov. and M. leptobarbi sp. nov. were found in the muscles of Pangasianodon hypophthalmus and Leptobarbus hoevenii, respectively, while plasmodia and spores of Thelohanellus zahrahae sp. nov. and Henneguya daoudi sp. nov. were detected in the gills of Barbonymus gonionotus and Trichogaster trichopterus, respectively. Plasmodia and spores found in these fishes differed from the known myxosporean species in respect of their morphology, tissue tropism and 18S rDNA structure. No major pathological changes were found, but in the future these species might pose a potential threat to more intensified fish culture.
During a survey on fishes of the Tasik Kenyir Reservoir, Malaysia, 5 new Myxobolus spp. and 2 known Henneguya spp. were found. The specific locations for 2 Myxobolus spp. were the host's muscles, while 2 other Myxobolus spp. were found to develop in the host's kidney and gills, respectively. Of the species developing intracellularly in muscle cells, M. terengganuensis sp. nov. was described from Osteochilus hasselti and M. tasikkenyirensis sp. nov. from Osteochilus vittatus. M. csabai sp. nov. and M. osteochili sp. nov. were isolated from the kidney of Osteochilus hasselti, while M. dykovae sp. nov. was found in the gill lamellae of Barbonymus schwanenfeldii. Henneguya shaharini and Henneguya hemibagri plasmodia were found on the gills of Oxyeleotris marmoratus and Hemibagrus nemurus, respectively. Description of the new and known species was based on morphological characterization of spores, histological findings on locations of plasmodia and DNA sequence data.
Cryptocaryon irritans is a parasitic ciliate that causes cryptocaryonosis (white spot disease) in marine fish. Diagnosis of cryptocaryonosis often depends on the appearance of white spots on the surface of the fish, which are usually visible only during later stages of the disease. Identifying suitable biomarkers of this parasite would aid the development of diagnostic tools and control strategies for C. irritans. The C. irritans genome is virtually unexplored; therefore, we generated and analyzed expressed sequence tags (ESTs) of the parasite to identify genes that encode for surface proteins, excretory/secretory proteins and repeat-containing proteins.
Cryptocaryon irritans causes Cyptocaryonosis or white spot disease in a wide range of marine fish including Lates calcarifer (Asian seabass). However, the immune response of this fish to the parasite is still poorly understood. In this study, quantitative polymerase chain reaction (qPCR) was performed to assess the expression profile of immune-related genes in L. calcarifer infected by C. irritans. A total of 21 immune-related genes encoding various functions in the fish immune system were utilized for the qPCR analysis. The experiment was initiated with the infection of juvenile fish by exposure to theronts from 200 C. irritans cysts, and non-infected juvenile fish were used as controls. Spleen, liver, gills and kidney tissues were harvested at three days post-infection from control and infected fish. In addition, organs were also harvested on day-10 post-infection from fish that had been allowed to recover from day-4 up to day-10 post-infection. L. calcarifer exhibited pathological changes on day-3 post-infection with the characteristic presence of white spots on the entire fish body, excessive mucus production and formation of a flap over the fish eye. High quality total RNA was extracted from all tissues and qPCR was performed. The qPCR analysis on the cohort of 21 immune-related genes of the various organs harvested on day-3 post-infection demonstrated that most genes were induced significantly (p fish that were allowed to recover from the C. irritans infection (10 days post-infection), expression of the immune-related genes was down-regulated to levels similar to the control fish. These results provide insights into the interaction between C. irritans and L. calcarifer and suggest that the innate immune system plays an important role in early defence against parasite infection allowing the fish to eventually recover from the infection.
Cryptocaryonosis is a major problem for mariculture, and the absence of suitable sero-surveillance tools for the detection of cryptocaryonosis makes it difficult to screen Cryptocaryon irritans-infected fish, particularly asymptomatic fish. In this study, we proposed a serum-based assay using selected C. irritans proteins to screen infected and asymptomatic fish. Eight highly expressed genes were chosen from an earlier study on C. irritans expressed sequence tags and ciliate glutamine codons were converted to universal glutamine codons. The chemically synthesized C. irritans genes were then expressed in an Escherichia coli expression host under optimized conditions. Five C. irritans proteins were successfully expressed in E. coli and purified by affinity chromatography. These proteins were used as antigens in an enzyme-linked immunosorbent assay (ELISA) to screen sera from experimentally immunized fish and naturally infected fish. Sera from both categories of fish reacted equally well with the expressed C. irritans recombinant proteins as well as with sonicated theronts. This study demonstrated the utility of producing ciliate recombinant proteins in a heterologous expression host. An ELISA was successfully developed to diagnose infected and asymptomatic fish using the recombinant proteins as antigens.
Twenty Asian sea bass Lates calcarifer from a floating cage in Bt. Tambun, Penang were examined for the presence of parasitic gill copepod, Lernanthropus latis. The prevalence of L. latis was 100% with the intensity of infection ranging from 1 to 18 parasites per host or 3.75 of mean intensity. Female parasites having oblong cephalothorax and egg-strings were seen mainly on the entire gill of examined Asian sea bass. The infected gill of Asian sea bass was pale and had eccessive mucus production. Under light and scanning electron microscopies (SEM), L. latis was seen grasping or holding tightly to the gill filament using their antenna, maxilla and maxilliped. These structures are characteristically prehensile and uncinate for the parasite to attach onto the host tissue. The damage was clearly seen under SEM as the hooked end of the antenna was embedded into the gill filament. The parasite also has the mandible which is styliform with eight teeth on the inner margin. The pathological effects such as erosion, haemorrhages, hyperplasia and necrosis along the secondary lamellae of gill filaments were seen and more severe at the attachment site. The combined actions of the antenna, maxilla and maxilliped together with the mandible resulted in extensive damage as L. latis attached and fed on the host tissues.
A total of 17 species of the genus Bifurcohaptor Jain, 1958 have been reported from two fish families namely Bagridae Bleeker, 1858 (Mystus vittatus (Bloch, 1794), M. tengara (Hamilton, 1822), M. keletius (Valenciennes, 1840), Hemibagrus nemurus (Valenciennes, 1840), Rita rita (Hamilton, 1822) and Sperata seenghala (Sykes, 1839)) and Sisoridae Bleeker, 1858 (Bagarius bagarius (Hamilton, 1822)). Out of these, only two species viz. B. indicus and B. giganticus are found valid in India, parasitizing gills of Mystus spp. and Bagarius sp. Taxonomic studies suggest, present specimen of B. indicus and B. giganticus, both are morphologically close to species described by Jain (1958), except morphometric variations and posses 7 pairs of marginal hooks instead of 6 pairs. Present manuscript delves with the characterization of B. indicus and B. giganticus reported from India, using molecular techniques. Partial mt COI nucleotide sequence based insilico protein analysis and partial 28S and ITS-1 rDNA based phylogenetic analysis, estimated by Neighbour-joining (NJ) and Minimum Evolution (ME) methods revealed that the species of the genus Bifurcohaptor are genetically distinct and valid. The grouping of Bifurcohaptor spp. with other representatives of family Dactylogyridae supports morphology based placement into family Dactylogyridae. Present and previous host-parasite information suggests both Bifurcohaptor spp. are species specialist however, the genus Bifurcohaptor is generalist at generic level.
Anisakis spp. (Nematoda: Anisakidae) parasitize a wide range of marine animals, mammals serving as the definitive host and different fish species as intermediate or paratenic hosts. In this study, 18 fish species were investigated for Anisakis infection. Katsuwonus pelamis, Euthynnus affinis, Caranx sp., and Auxis thazard were infected with high prevalence of Anisakis type I, while Cephalopholis cyanostigma and Rastrelliger kanagurta revealed low prevalence. The mean intensity of Anisakis larvae in K. pelamis and A. thazard was 49.7 and 5.6, respectively. A total of 73 Anisakis type I larvae collected from K. pelamis and A. thazard were all identified as Anisakis typica by PCR-RFLP analysis. Five specimens of Anisakis from K. pelamis and 15 specimens from A. thazard were sequenced using ITS1-5.8S-ITS2 region and 6 specimens from A. thazard and 4 specimens from K. pelamis were sequenced in mtDNA cox2 region. Alignments of the samples in the ITS region showed 2 patterns of nucleotides. The first pattern (genotype) of Anisakis from A. thazard had 100% similarity with adult A. typica from dolphins from USA, whereas the second genotype from A. thazard and K. pelamis had 4 base pairs different in ITS1 region with adult A. typica from USA. In the mtDNA cox2 regions, Anisakis type I specimens from A. thazard and K. pelamis showed similarity range from 94% to 99% with A. typica AB517571/DQ116427. The difference of 4 bp nucleotides in ITS1 regions and divergence into 2 subgroups in mtDNA cox2 indicating the existence of A. typica sibling species in the Makassar Strait.
Epidermal pseudotumours from Hippoglossoides dubius and Acanthogobius flavimanus in Japan and gill lesions in Limanda limanda from the UK have been shown to be caused by phylogenetically related protozoan parasites, known collectively as X-cells. However, the phylogenetic position of the X-cell group is not well supported within any of the existing protozoan phyla and they are currently thought to be members of the Alveolata.Ultrastructural features of X-cells in fish pseudotumours are somewhat limited and no typical environmental stages, such as spores or flagellated cells, have been observed. The life cycles for these parasites have not been demonstrated and it remains unknown how transmission to a new host occurs. In the present study, pseudobranchial pseudotumours from Atlantic cod, Gadus morhua, in Iceland and epidermal pseudotumours from the northern black flounder, Pseudopleuronectes obscurus, in Japan were used in experimental transmission studies to establish whether direct transmission of the parasite is achievable. In addition, X-cells from Atlantic cod were sequenced to confirm whether they are phylogenetically related to other X-cells and epidermal pseudotumours from the northern black flounder were analysed to establish whether the same parasite is responsible for infecting different flatfish species in Japan.
Numerous global reports of the species Udonella caligorum, currently thought to be a species complex, suggests that the group may be species-rich. Herein we describe Udonella fugu n. sp., previously described as U. caligorum, found on the parasitic copepod Pseudocaligus fugu infecting Takifugu spp. from Japan. Using morphological data U. fugu can be distinguished from the current valid species by at least one of the traditionally used characters in udonellid taxonomy, and phylogenetic analyses of ssrDNA sequence data for U. fugu and other udonellids confirm that U. fugu forms a distinct clade from other udonellids including U. caligorum. Variable regions in the ssrDNA demonstrated a range of between 2.75 and 5.5% difference between currently recognized species of Udonella. These differences in ssrDNA sequences are phylogenetically useful when distinguishing between morphologically similar udonellids and can be used in conjunction with other data (morphology, phylogeography and fish host) to help clarify udonellid systematics. Udonella fugu was also found to cause significant damage to farmed tiger puffers through their feeding activities. Individual skin lesions were round in shape but merged with adjoining lesions to form more extensive lacerations. In some of the specimens from P. fugu infecting Takifugu niphobles, the protozoan ciliate Trichodina was found on the udonellid body surface and in their intestinal contents. We conclude that the udonellids are a more species-rich group than currently recognized, that early descriptions of new species may have been synonymized with U. caligorum in error and that the frequent global reports of U. caligorum may actually represent new species. This has led to a wide range of morphological descriptions for U. caligorum, blurring the usefulness of morphological data for the group.
Thirty snakehead fish, Channa micropeltes (Cuvier, 1831) were collected at Lake Kenyir, Malaysia. Muscle, liver, intestine and kidney tissues were removed from each fish and the intestine was opened to reveal cestodes. In order to assess the concentration of heavy metal in the environment, samples of water in the surface layer and sediment were also collected. Tissues were digested and the concentrations of manganese (Mn), zinc (Zn), copper (Cu), cadmium (Cd) and lead (Pb) were analysed by using inductively-coupled plasma mass-spectrometry (ICP-MS) equipment. The results demonstrated that the cestode Senga parva (Fernando and Furtado, 1964) from fish hosts accumulated some heavy metals to a greater extent than the water and some fish tissues, but less than the sediment. In three (Pb, Zn and Mn) of the five elements measured, cestodes accumulated the highest metal concentrations, and in remaining two (Cu and Cd), the second highest metal accumulation was recorded in the cestodes when compared to host tissues. Therefore, the present study indicated that Senga parva accumulated metals and might have potential as a bioindicator of heavy-metal pollution.