Copyright @ 2016, Indonesian Aquaculture Journal, p-ISSN 0215-0883, e-ISSN 2502-657 81 Indonesian Aquaculture Journal, 11 (2), 2016, 81-86 # Correspondence: Centre for Aquaculture Research and Development. Gedung Balitbang KP. II, Jl. Pasir Putih II, Ancol Timur, Jakarta Utara 14430, Indonesian. Phone: + (021) 64700928 E-mail: istisugama@yahoo.com Available online at: http://ejournal-balitbang.kkp.go.id/index.php/iaj MOLECULAR DETECTION AND CLONING FOR RICKETTSIA-LIKE BACTERIA OF MILKY HAEMOLYMPH DISEASE OF SPINY LOBSTER Panulirus spp. Isti Koesharyani *)# , Lila Gardenia **) , and Ni Luh Anggra Lasmika ***) *) Centre for Aquaculture Research and Development **) Institute for Freshwater Aquaculture Research and Development ***) Fish Quarantine and Inspection Agency Mataram ABSTRACT Spiny lobster (Panulirus homarus and Panulirus ornatus) are important commodities for Indonesia. The aquaculture of lobster is susceptible for several diseases like parasite, fungi, bacteria, and virus. Among those diseases, milky haemolymph disease (MHD) is often seen as a symptom to mass mortality occurred at lobster farms in Gerupuk Bay of Lombok. The purpose of this study was to determine the lobster diseases on cage culture in Gerupuk Bay of Lombok, West Nusa Tenggara. The study was undertaken from January to March 2015. Diseases status was determined by application of molecular plat-form, polymerase chain reaction (PCR) with designation of specific primer for MHD (254F/R), 254F: 5’-CGA-GGA-CCA-GAG-ATG- GAC-CTT-3’ and 254R: 5’-GCT-CAT-TGT-CAC-CGC-CAT-TGT-3’ with PCR size product of 254 for cloned the pathogen was used TA-cloning Invitrogen for the DNA plasmid as positive control for other analysis. Several tissue samples i.e hepatopancreas, haemolymph, part of muscle hepatopancreas P. homarus and P. ornatus were taken from cage culture farms at Gerupuk Bay then preserved on 90% ethanol for further analysis by PCR and then the amplificated DNA were cloned into pCR ® 2.1 plasmid and transformed into competent E. coli. The result showed almost all lobster samples from Gerupuk Bay were positive infected by MHD, as the results of PCR amplification whereas the band appeared at 254bp. Also MHD plasmid has been successfully cloned and will be used for further examination. Histopathologically in hepatopancreas infection have seen necrosis that contain numerous of rickettsia-like bacteria. KEYWORDS: lobster; diseases; MHD; rickettsia INTRODUCTION Indonesia mainly in Lombok, West Nusa Tenggara (NTB) is a major seed lobster producer and grow-out of wild lobster which have been exported to around the world. The price of lobster is high in both the domestic and export markets. Within a year, NTB pro- duced 78.5 tons of lobster worth IDR 55.25 billion. While the economic value of lobster seed sales reached IDR 16 billion per year (Petersen et al., 2013). There are many species of lobster in Indonesia, such as Panulirus homarus, Panulirus ornatus, Panulirus ver- sicolor, Panulirus longipes, Panulirus polyphagus, and Panulirus penicillatus. Among those species Panulirus homarus were 90% as a grow-out cultured lobster in cage (Jones, 2015; Mustafa, 2013). Intensive capture can lead to a decline in natural population while the lobster can not be cultured. So it needs the proper techniques for handling the seed, density, feed, and disease prevention during grow-out in cage. How- ever, along the grow-out of lobster even with the right technique there always have problem of emerg- ing disease. The disease that commonly attack lob- ster farming include, rickettsia-like bacteria, true bac- teria (Vibrio alginolyticus), protozoa, black gill disease kind of fungi (Fusarium sp.), virus (Panulirusargus V1/ Pa.V1), parasite, and big head syndrome. Those pathogens have had a major impact on the lobster culture (Diggles et al., 2000; Shields & Behringer, 2004; Behringer, 2012; OIE, 2007; Kiryu et al., 2009; Shield, 2011, Lightner & Redman, 2010; Stentiford et al., 2002; Stentiford et al., 2012; Jones, 2015). Among the pathogens, milky haemolymph disease or MHD often infects to several species of cultured spiny lob- ster such as Panulirus homarus, Panulirus ornatus, and Panulirus polyphagus at all life stages and could cause mortality that reached 70%-100% (Jones, 2015). Milky