Parasitic and Fungal Identification in Bamboo Lobster Panulirus versicolour and Ornate Lobster P. ornatus Cultures

Indriyani Nur1)* and Yusnaini2)

1) Department of Aquaculture, Faculty of Fisheries and Marine Science, University of Halu Oleo, Kendari 93232, Indonesia  Corresponding author: [email protected] (Indriyani Nur)

Abstract—Lobster cultures have failed because of mortalities transmissible [5] including Vibrio, Aeromonas, Pseudomonas, associated with parasitic and fungal infections. Monitoring of spawned Cytophaga and Pseudoalteromonas [6,7]. Futher examination eggs and larva of bamboo lobsters, Panulirus versicolour, and ornate of these lobsters revealed four parasite : Porospora lobsters, P. ornatus, in a hatchery was conducted in order to gigantea (: Sporozoea), Polymorphus botulus characterize fungal and parasitic diseases of eggs and larva. One (Acanthocephala: Palaeacanthocephala), Hysterothylacium sp. species of protozoan parasite (Vorticella sp.) was identified from eggs while two species of fungi (Lagenidium sp. and Haliphthoros sp.) were (Nematoda: Secernentea), and Stichocotyle nephropis found on the lobster larvae. Furthermore, adult lobsters cultured in (Platyhelminthes: Trematoda) [8]. Therefore, the aim of this floating net cages had burning-like diseases on their pleopod, uropod, study was to examine and to characterize the infestation of and telson. Histopathological samples were collected for parasite parasites and fungus of potentially cultured lobsters (larval identification and tissue changes. There were two parasites found to stages and adult stages of the spiny lobsters), and also to look infect spiny lobsters on their external body and gill, they are Octolasmis sp. and Oodinium sp..Histopathology showed tissue at the histopathological changes of their tissues. This is changes, necrosis on the kidneys/liver/pancreas, necrosis in the gills regarded as a major cause of low production in aquaculture and around the uropods and telson. systems. Characterization and identification of pathogens is important in disease control and effective treatment. Keywords—Fungal, histopathology, lobster, parasite, infection. II. MATERIALS AND METHODS I. INTRODUCTION A. Broodstock and Larva HE bamboo lobster Panulirus versicolour and ornate Female lobsters that carried eggs under their abdomen were lobster P. ornatus, are common in the ocean. However, the T obtained from South East Sulawesi waters and Banda Sea, lobster has been captured and exploited in over 90 countries [1] Indonesia. They were caught at night and transported to the land which has lead to a loss of spawning stock and overfishing. using air circulation systems. Now, in many area, the population of lobsters has declined After the egg bearing females arrived at the hatchery, the dramatically due to: pollution, loss of habitat and susceptibility lobster were kept in the concrete tank for varying periods of to infection [2]; consequently, the lobsters are now considered time depending on the development stage of their eggs. The rare species. larvae were sampled at 1 – 6 days post hatching (dph). The lobsters remain a significant economic value source for many communities and they are a preferred seafood in the B. Lobster Sampling world. To address the increasing demand for food, lobsters have Adult lobsters were cultured in floating net cages. During the become a major sector of fisheries production through culture period the lobster were fed fresh fish and frozen squid aquaculture. Active research and development programs daily. The live lobsters were kept in aerated containers and worldwide have attempted to grow this sector, but so far none transferred to the laboratory where they were kept in aquariums has been successful. During the culture period, the lobsters for a maximum of two days until they were euthanatized to look often experience many serious problems, particularly those for parasites. Prior to dissection, all lobsters were weighed. caused by parasites and fungus infection. The challenges faced with raising lobsters is great. The most difficult stage to grow C. Examination for Parasites on Larvae and Adult Lobster these species is their larval stage [3]. To date, seed stock cannot yet be produced on demand. Larvae were investigated on outer layers attached to the It has been reported that black gills caused by fungus body. While adult lobsters were killed and then examined Fusarium sp. appeares to have caused mortality in 69.5% of externally and internally for clinical signs of disease and other lobster in cages in Vietnam [4]. Transmission of PaV1 virus abnormalities. Each lobster was necropsied and the following among lobsters also has been a detected [2]. Additionally, organs were checked: external body surface (carapace, gills, several types of have been isolated from lesions of and fins) as well as the kidneys, liver, and pancreas. Lobsters were affected lobsters of shell diseases and were known to be dissected using general methods [9]. Briefly, each lobster was systematically searched for parasites. First the carapace, gills and uropod/telson were examined using a combination of eye Vorticella sp. was observed in outer layers of larvae. and microscope. The outer layer of the carapace was scraped Vorticella sp. are small unicellular often found on with scalpel and then the scrapings were examined using a carapace and skin of many fish species. Vorticella sp. have low microscope. The gills were removed and examined individually host specificity and therefore are widely distributed. Most under microscope, and then scraped into a petri dish filled with families of freshwater fish harbor Vorticella sp. [12, 18]. They clean water to dislodge the parasites and examined under a have also been reported on amphibians, crustaceans, mollusks stereo microscope. The kidneys, liver, and pancreas were dissected and coelenterates. Vorticella sp. inhabit both fresh and salt and examined for parasites using a microscope. Squash water habitats. According to [18], some Vorticella sp. species preparations were prepared and examined under a microscope. are pathogenic. Classification of Vorticella sp. in detail were: All detected parasites were removed and treated with saline to Phylum Ciliophora; Order Peritrichida; Sub Order Sessilina; retain morphological definition. Where possible, photographs Family ; Genus Vorticella [10]. were taken of live specimens and then they were killed, fixed There are various species of Protozoan parasites, which are and preserved in 70% ethanol. Parasites were classed as distinguished by their cilia. Phylum Ciliophora was ectoparasites or endoparasites. characterized with short cilia. Genus Vorticella also consisted of numerous species, all of them are soliter, attach to the D. Identification and Determination of substrate (host) with stalked-like, namely slender, cylindrical, Prevalence and Intensity or contractile stalk [10;19]. External and internal parasites were identified using the Vorticella is bell shaped with big mouth (peristome). following relevant works (e.g. [10-12]). Prevalence and mean color varies, but is usually yellow or green. They live in intensity for selected parasites were determined according to freshwater and sea water, and the adult stage is attach to an [13]. All parasites were identified to at least genus or species object, animal, or aquatic plant. and suctorian ciliates and enumerated. are often found as commensalisms on the external surfaces of E. Fungal Disease Identification on Eggs crustacean embryos, such Corthunia sp, Epistylis sp. and Eggs with fungal infection showed signs of disease. The Vorticella sp. usually infect lobster cultures [20]. They attack symptoms were a cloudy color and cotton-like filaments the body including the: eyestalk, antenna, uropod, and eggs. In attached to the outer layers. Fungi of the lobster eggs were this research, we found that the stalked of parasite Vorticella isolated using PYGS agar (1.25 g peptone; 1.25 g yeast extract; penetrated the inner part of the larvae body. 3.00 g glucose; 12–15 g agar; 1,000 mL sea water). In order to B. Fungus on Lobster Eggs inhibit most bacterial growth antibiotics were added to the Fungus found on the agar media and in the stains was medium. After fungal colonies develop on the agar plates, each identified as two genera of fungi. They are derived from the was isolated onto fresh PYGS agar to obtain a pure culture. For order Lagenidiales, genus Haliphthoros and genus Lagenidium. morphological observations, the isolates were inoculated into Characterization of these fungi can be seen in Figure 1-4. PYGS broth and incubated at 25 ˚C for 3–5 days [14]. Fungal diseases that attack eggs are a serious problem in the Determination and characterization of pure culture colony hatchery because they reduce the success of the eggs. This study isolate based on macroscopic investigation including shape, found several eggs undergoing pathological disorders, size of the colony, and character of the hyphae. While, characterized by a white cloudy egg surface. microscopic morphology included the number of branches, This fungus group was also found infecting eggs and larvae septa, size, and diameter of hyphae. After this the colony was of mangrove crab at Coastal Fisheries Research Center in stained with cotton blue with the guidance of references Gondol, Indonesia [21]. Fungi produce zoospores in the water, [15,16]. and then swim freely to find and attach to the host with the help F. Histological Examination of hyphae. Henceforth, the fungi will take nutrition and certain After examining for parasites, each lobster removed the substances from the host. Infected eggs do not hatch. carapace, gills, kidneys, liver, and pancreas. Tissues were fixed Lagenidium have also been found infecting and penetrating in 10% formalin solution, then processed with paraffin and embryo of berried female crabs, crab larvae, and another lobster stained with haematoxylin and eosin (H & E). Prepared sections species, Homarus americanus [22]. While, an invasive and stains were examined under a light microscope and oomycete identified as Haliphthoros sp. have been observed on histopathological samples were evaluated. Tissues processing [23] infected juvenile spiny lobster when they show signs was done with the procedure in source 17. [17]. lethargy and appetite loss.

III. RESULT A. Parasites Recovered from Larvae Parasites found during this study on larvae 3 – 6 dph was Protozoan Vorticella.

Figure 4. Micro-morphology of Haliphthoros hyphae (400x) with Lactophenol Cotton Blue staining. (A) Wide irregular hyphae, (B) Spore. Figure 1. Morphology of fungi Lagenidium isolated from lobster eggs, the C. Parasites Recovered from Adult Lobster colony is white and thin on PGYA medium. Parasites found from adult lobsters were Octolasmis and Oodinium. Octolasmis is more commonly found attached to the outer body of a lobster and on its gills. These organisms are also commonly found attached to the other crustaceans, but are more numerous on crabs. A possible explanation is that crabs terminally molt. Some of them are opportunistic needing a place to attach into hard surfaces, while others need an obligate symbiote. Some of them are used as an indicator of the host health. Barnacle Octolasmis sp. uses a number of crustaceans as its host, including lobsters Panulirus and crabs Scylla [24]. The barnacle lives on the exoskeletons and gills of its host. They are very adaptable to the host life cycle. The species O. bullata reach 48% in the prevalence of lobster P. polyphagus in Singapore [24]. From our data, it is known at the prevalence of 50% and high intensity (64 individual/lobster). Moreover, lobsters were infected with Oodinium by prevalence of 100% Figure 2. Cell morphology of Lagenidium (400x) isolated from lobster egg staining by Lactophenol Cotton Blue, hyphae looks wide and irregular. and high intensity (69 individual/lobster). Heavy infestation of Octolasmis and Oodinium on our samples may indicate that the organisms in our study were not in good health. The effects of parasites infection in many cases have been observed to reduce growth between molts. Hosts should get priority for treatment so that the infection will not spread. Compare to the larval stage, there are fewer reports about infestations of parasites, diseases, and symbiosis than there is on adult spiny lobster so far. The disease is more frequent and serious in post larva and early juvenile stage.

D. Histopathology Examination

A variety of histopathological lesions were observed in all organs examined. Histopathological changes, including

Figure 3. Morphology of fungi Haliphthoros isolated from lobster eggs, hemorrhage, tubular epithelial cell vacuolization, cell necrosis colony is white and thick on PGYA medium. and mineralization were found in uropod, telson, gill and kidney, liver, and pancreatic tissue. Often, these were generalized reactions, but in many cases they were closely associated with, and likely to be a response to, parasitic infection. This suggestion is not only from histological findings of parasites surrounded but also by pathological changes in host tissue, but also may relation with environment or water quality.

2 1

Figure 7. Telson had necrosis. (1) eosinophil cells and (2) lymphocyte cells. HE 400x.

During this study, gill tissue was the most heavily changed Figure 5. Lobster liver histopathology shows hemorrhagic nephritis. (1) Tubules, (2) hemorrhage, (3) vacuolization tubular epithelial cells, (4) by bacteria and parasite infection. Gill lesions consisted of congestion and (5) tubular necrosis. HE.100x. severe inflammatory reactions with necrosis. It is well known that gills are among the most sensitive organ, which reacts first in changed environment since respiration, osmoregulation and excretion are performed through the gills [25]. Necrosis is an 3 2 irreversible change that occurs following toxicant exposure 5 and/or chronic damage of the gills [26]. Gill necrosis (Figure 6) was detected in this investigation. Necrosis was prominent on the gills of lobsters. These changes were always more severe at the site of parasitic and bacterial infection. Outer layer lesions 4 were principally associated with infections of fungus and parasite, which led to necrosis of the epidermal layer. The massive amount of parasite could occur due to environmental conditions in the cage such as stocking density and/or stress [11]. Since organic matter becomes a substrate for resident microorganisms, once it goes up, the number of parasites will also increase. Elevated organic pollution in lobster culture cage is often a result of excess feed and its decomposition. This affects lobster gills making them more sensitive to pathogens and parasites. On the other hand, liver tissue reaction to organic pollution showed gross signs of Figure 6. Drawing of longitudinal gills. Br: Branchial Stem; Lam: Lamellae; Con: Connective tissues; He (Hemocyte); Parasites (arrows); disease included brown focal necrotic lesions. M: Melanization, is a process characterized by extracellular deposition of melanin in crustacean tissue. IV. CONCLUSIONS

One species of protozoan, an ectoparasite, Vorticella, was

observed to infect the larvae lobster. While, two species of parasites infected adult lobsters, Octolasmis and Oodinium at high levels. The fungus infection Lagenidium sp. and Haliphthoros sp. was observed on bodies of adult lobsters. The main effect of parasite load was that it reduced growth between molts. Necrosis, hemorrhage and vacuolization of liver and pancreas tubule epithelial cells and gill are among the principal histopathological changes due to disease. Environmental management of cage lobster culture is needed to reduce mortality and to increase production.

REFERENCES [1] Philips, B.F. and J. Kittaka. 2000. Spiny Lobsters: Fisheries and Culture. 2nd Edn., Blackwell Publishing, Berlin, Germany. pp 704.

Diagnosis. K. Sugama, H. Ikenoue and K. Hatai (Eds.). 44 p. Gondol [2] Donald C. Behringer, Mark J. Butler IV, Jessica Moss, and Jeffrey D. Research Station for Coastal Fisheries, CRIFI and Japan International Shield . 2012. PaV1 infection in the Florida spiny lobster (Panulirus Cooperation Agency. argus) fishery and its effects on trap function and disease transmission. Can. J. Fish. Aquat. Sci.69: 136–144. [22] Nilson, E. H., Fisher, W. S., and Shleser, R. A. 1975. Filamentous Infestations Observed on Eggs and Larvae of Cultured Crustaceans. [3] Kittaka, J. and Booth, J. D. 2000. Prospectus for aquaculture. In: Phillips, Proceedings of the annual meeting - World Mariculture Society. Volume B.F. and Kittaka, J. (eds.), Spiny Lobsters: Fisheries and Culture. Oxford: 6, Issue 1-4, pages 367–375. Fishing News Books, 465-473. [23] Diggles, B. K. A mycosis of juvenile spiny rock lobster, Jasus edwardsii [4] Nha, V.V., Hoa, D.T. , Khoa, L.V. 2009. Black gill disease of cage- (Hutton, 1875) caused by Haliphthoros sp., and possible methods of cultured ornate rock lobster Panulirus ornatus in central Vietnam caused chemical control. Journal of Fish Diseases. Volume 24, Issue 2, 99–11. by Fusarium species. Aquatic animal health. Volume XIV No. 4. [24] Jeffries, W.B., Voris, H.K., Yang, C.M., 1982. Diversity and distribution [5] National Marine Fisheries Service. 2006. Monitoring of New Hampshire of the pedunculate barnacle octolasmis in the seas adjacent to Singapore. Fishery for Shell Disease in the American Lobster. Final Progress Report. J. Crustac. Biol. 2, 562–569. USA. [25] Mallatt, J. 1985. Fish Gill Structural Changes Induced by Toxicants and [6] Getchel, R. G. 1989. Bacterial shell disease in crustaceans: a review. J. Other Irritants: A Statistical Review. CanadianJournal of Fisheries and Shellfish. Res. 8. 1-6. Aquatic Sciences, , 42(4): 630-648.

[7] Smolowitz, R. 2005. Epizootic shell disease in the American lobster, [26] Takashima and T. Hibiya. 1995. An atlas of fish histologi, Normal Homarus americanus. In: Tlusty, M.F., Halvorson, H.O., Smolowitz, R., and Pathological Feature Second Edition. Kodansha Ltd, Tokyo. 195p. and Sharma, U. (eds.) State of Lobster Science: shell disease workshop. Aquatic Forum Series 05-1. The New England Aquarium, Boston, Massachusetts.

[8] Brattey, J and Campbell, A. 1986. A survey of parasites of the American lobster, Homarus americanus (Crustacea: Decapoda), from the Canadian Maritimes. Canadian Journal of Zoology, 64(9): 1998-2003.

[9] Noga, E. J. 2010. Fish disease : diagnosis and treatment 2nd ed. A John Wiley & Sons, Inc., Publication. p. 497.

[10] Kabata, Z. 1985. Parasites and Diseases of Fish Cultured in the Tropics. Taylor & Francis, London, Philadelphia. 317 pp.

[11] Roberts, R. J. (1989) Fish pathology. Baillier and Tindall, London. 466 pp.

[12] Sminth, S.A. and Noga, E. General Parasitology. In: Stoskopf, M. K.. (ed.). Fish Medicine. W. B. Saunders Company. pp. 132-148.

[13] Margolis, L., Esch, G. W., Holmes, J. C., Kuris, A. M., and Schad, G. A. 1982. The Use of Ecological Terms in Parasitology (Report of an Ad Hoc Committee of the American Society of Parasitologists). The Journal of Parasitology Vol. 68, No. 1 pp. 131-133

[14] Hatai, K. 2012. Diseases of Fish and Shellfish Caused by Marine Fungi. In: C. Raghukumar (ed.), Biology of Marine Fungi, Progress in Molecular and Subcellular Biology 53, Springer-Verlag Berlin Heidelberg.

[15] Alexopoulos, C.J. and C.W. Mims. 1979. Introductory Mycology. Third Edition. John Wiley & Sons, Inc. Canada. 572 p.

[16] Streiblova, E. 1988. Cytological Methods. In: Yeast-a Practical Approach. Campbell, I and J.H. Duffus (Ed.). IRL Press. Oxford-Washington DC. pp. 9-48.

[17] Mumford, S. 2007. Submiting samples and tissue processing. In: Mumford, S., Jerry Heidel, J., Smith, C., Morrison, J., MacConnell, B., Blazer, V. Fish Histology and Histopathology. USFWS-NCTC.

[18] Hoffman, G. L. 1999. Parasites of North American Freshwater Fishes, 2nd Edition. Comstock Publishing Associates, Ithaca, New York.

[19] Lom, J. 1995. Trichodinidae and other Ciliates (Phylum Ciliophora). In: Woo, P.T.K. (Ed.). Fish diseases and disorders Vol. 1. p 229-262. CAB International, Cambridge.

[20] Shields, J. D. 2011. Diseases of spiny lobsters: A review. Journal of Invertebrate Pathology 106:79–91.

[21] Zafran, D. Roza, I. Koesharyani, F. Johnny and K. Yuasa. 1998. Marine Fish and Crustaceans Diseases in Indonesia. In: Manual for Fish Diseases www.waset.org

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Herewith, the international scientific committee is happy to inform you that the peer- reviewed draft paper code 16AE01000031 entitled (Parasitic and Fungal Identification in Culture of Bamboo Lobster Panulirus versicolour and Spiny Lobster P. ornatus by Indriyani Nur, Yusnaini) has been accepted for oral presentation as well as inclusion in the conference proceedings of the ICFAS 2016 : 18th International Conference on Fisheries and Aquatic Sciences to be held in Dubai, UAE during January, 28-29, 2016. The high-impact conference papers will also be considered for publication in the special journal issues at http://waset.org/Publications.

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