Bacillus Popilliae Research Articles

Dynamic pathology and pathogen distribution of the yeast Metschnikowia bicuspidata in the Chinese mitten crab (Eriocheir sinensis).

In this study, the "milky disease" model of Eriocheir sinhensis was constructed via intramuscular injection with the pathogenic yeast Metschnikowia bicuspidata. The dynamic pathological changes of E. sinensis after injection were elucidated with two staining methods (haemotoxylin-eosin and alcian blue periodic acid-Schiff) and fluorescence insitu hybridization technology. Anatomical observation revealed three stages of the "milky disease": no clinical signs (1-4 days after infection), the appearance of signs of disease (5-7 days), and significant liquefaction (10 days). Histological observation also revealed three stages of the disease: yeast diffusion (1-2 days after infection), yeast slow development (3-4 days), and yeast rapid proliferation (5 days). And FISH technique was suitable for the early detection of infection with M. bicuspidata in E. sinensis. We found that M. bicuspidata spread to the whole body of the crab through the haemolymph and developed into fungal septicaemia. These results elucidate the systemic pathological characteristics of "milky disease" in E. sinensis and suggest the pathogenic mechanism of M. bicuspidata.

Effect of temperature on the occurrence of Metschnikowia bicuspidata disease in Eriocheir sinensis

Eriocheir sinensis aquaculture has been significantly impeded by “milky disease,” which is caused by Metschnikowia bicuspidata. This disease exhibits distinct seasonality and is closely associated with temperature fluctuations. The present study investigated the impact of temperature on the proliferation and pathogenicity of M. bicuspidata and the immune capacity of E. sinensis. We observed a significant temperature-dependent effect on the proliferation rate of M. bicuspidata in vitro and in vivo. Although low temperatures (4 °C) suppressed M. bicuspidata proliferation, different temperatures did not have a considerable influence on pathogenicity (P > 0.05). Cultivation at lower temperatures significantly reduced the proliferation rate of pathogens in infected E. sinensis and the time to death. Further, the bacteriostatic capacity of E. sinensis serum was significantly enhanced under high temperatures compared with that under low-temperature conditions (P < 0.01). Transcriptome analysis of hemocytes from E. sinensis at different breeding temperatures indicated that the host's metabolism and immunity were differentially affected at 4 °C compared to 32 °C, with pathways associated with growth and development primarily affected under low-temperature conditions and pathways related to energy supply predominantly influenced under high-temperature conditions. Notably, temperature also led to changes in the expression of genes closely linked to immunity and apoptosis, such as acid phosphatase, C-type lectin, glutathione peroxidase, caspase, and initiator caspase-8. The current findings have significant implications for understanding the effect of temperature on M. bicuspidata pathogenicity in E. sinensis and preventing milky disease infection during aquaculture.

The Pathogenic Yeast Metschnikowia bicuspidata var. bicuspidata in the Aquacultured Ecosystem and Its Biocontrol.

M. bicuspidata var. bicuspidata is a pathogenic yeast which can affect aquacultured and marine-cultured animals such as brine shrimp, ridgetail white prawn, chinook salmon, giant freshwater prawn, the Chinese mitten crab, marine crab, the mud crab, the mangrove land crab, the Chinese grass shrimp, sea urchins, sea urchins, Daphnia dentifera and even snails, causing a milky disease, and it has caused big economic losses in aquacultural and marine-cultural industries in the past. However, the detailed mechanisms and the reasons for the milky disease in the diseased aquatic animals are still completely unknown. So far, only some antimycotics, killer toxins and Massoia lactone haven been found to be able to actively control and kill its growth. The ecofriendly, green and renewable killer toxins and Massoia lactone have high potential for application in controlling the milky disease.

Milky Disease: A Review of Discovery, Pathogens, and Detection Methods in Crabs

As an important economic crustacean in China, the crab has been a popular focus in aquaculture in recent years due to its high production and economic value. The epidemic of milky disease has severely damaged the crab farming industry, resulting in heavy economic losses and hindering the sustainable development of high-quality crab farming. The disease is characterized by severe emulsification of all tissues and the presence of large amounts of opaque, milky fluid in the body, with a high infection rate and high mortality. Determining the cause of the disease, its life cycle, transmission routes, and detection methods will help to provide direction for the prevention and control of crab milky disease. This article aims to review the discovery, main pathogens, and pathologies of milky disease, along with the currently established detection methods. We have also attempt to classify the control measures for the prevention and control of milky disease in crabs.

Establishment and Application of Real-Time Fluorescence Quantitative PCR Detection Technology for Metschnikowia bicuspidata Disease in Eriocheir sinensis.

Metschnikowia bicuspidata causes a "milky disease" in Chinese mitten crab, Eriocheir sinensis, which inflicts significant damage on the breeding industry, but there are no effective drugs for this disease. Precise detection technologies and clarification of transmission routes are now essential to prevent its occurrence. A real-time fluorescent quantitative PCR (qPCR) detection method targeting the mitochondrial cytochrome c oxidase subunit VIA (COX6A) of M. bicuspidata was developed and its sensitivity, specificity, repeatability, and application effectiveness evaluated. There was a robust linear relationship between the qPCR threshold cycle value (Ct) and copy number of the standard with a wide dynamic range. The standard curve had a correlation coefficient (R2) of 0.996, amplification efficiency of 103.092%, and a lower limit of detection sensitivity of 7.6 × 101 copies/µL. The COX6A-qPCR method exhibited high specificity for the detection of M. bicuspidata, with no cross-reactivity. The intra- and inter-group variation coefficients were <1% and 2%, respectively. The qPCR exhibited superior sensitivity compared to existing detection methods, with a positivity rate of 76.67%. The M. bicuspidata content ranged from 1.0 × 101-2.7 × 106 copies/µL. The COX6A-qPCR detection technology exhibited high sensitivity, strong specificity, and excellent repeatability, enabling the accurate quantification of M. bicuspidata.

Virome Analysis Provides an Insight into the Viral Community of Chinese Mitten Crab Eriocheir sinensis.

Recent advances in viromics have led to the discovery of a great diversity of RNA viruses and the identification of a large number of viral pathogens. A systematic exploration of viruses in Chinese mitten crab (Eriocheir sinensis), one of the most important aquatic commercial species, is still lacking. Here, we characterized the RNA viromes of asymptomatic, milky disease (MD)-affected, and hepatopancreatic necrosis syndrome (HPNS)-affected Chinese mitten crabs collected from 3 regions in China. In total, we identified 31 RNA viruses belonging to 11 orders, 22 of which were first reported here. By comparing viral composition between samples, we observed high variation in viral communities across regions, with most of the viral species being region-specific. We proposed to establish several novel viral families or genera based on the phylogenetic relationships and genome structures of viruses discovered in this study, expanding our knowledge of viral diversity in brachyuran crustaceans. IMPORTANCE High-throughput sequencing and meta-transcriptomic analysis provide us with an efficient tool to discover unknown viruses and explore the composition of viral communities in specific species. In this study, we investigated viromes in asymptomatic and diseased Chinese mitten crabs collected from three distant locations. We observed high regional variation in the composition of viral species, highlighting the importance of multi-location sampling. In addition, we classified several novel and ICTV-unclassified viruses based on their genome structures and phylogenetic relationships, providing a new perspective on current viral taxa.

Metabolomic and metagenomic analyses of the Chinese mitten crab Eriocheir sinensis after challenge with Metschnikowia bicuspidata.

Milky disease caused by Metschnikowia bicuspidata fungus has significantly harmed the Chinese mitten crab Eriocheir sinensis aquaculture industry. However, the effect of M. bicuspidata infection on the metabolism and intestinal flora of the crab remains unclear. In this study, we aimed to explore the changes in the metabolism and intestinal flora E. sinensis after 48 h of infection with M. bicuspidata, using metabolomic and metagenomic analyses. Metabolomic analysis results revealed 420 significantly different metabolites between the infected and control groups, and these metabolites were enriched in 58 metabolic pathways. M. bicuspidata infection decreased the levels of metabolites related to amino acid biosynthesis, the tricarboxylic acid cycle, as well as lysine, histidine, linolenic, arachidonic, and linoleic acid metabolism. These results indicated that M. bicuspidata infection significantly affected the energy metabolism, growth, and immunity of E. sinensis. The results of metagenomic analysis showed that the anaerobes and ascomycetes populations significantly increased and decreased, respectively, after M. bicuspidata infection. These changes in intestinal flora significantly upregulated metabolic and synthetic pathways while downregulating immunity-related pathways. The results of integrated metabolomic and metagenomic analyses showed that 55 differentially expressed genes and 28 operational taxonomic units were correlated with 420 differential metabolites. Thus, the intestinal flora changes caused by M. bicuspidata infection also affected the metabolites. This study provides novel insights into the metabolic-and intestinal microflora-based effects of M. bicuspidata infection in E. sinensis, as well as a theoretical basis for the interaction between fungi and crustaceans.

Development of a nested PCR assay for specific detection of Metschnikowia bicuspidata infecting Eriocheir sinensis.

In recent years, the “milky disease” caused by Metschnikowia bicuspidata has seriously affected the Eriocheir sinensis culture industry. Discovering and blocking the transmission route has become the key to controlling this disease. The existing polymerase chain reaction (PCR) detection technology for M. bicuspidata uses the ribosomal DNA (rDNA) sequence, but low sensitivity and specificity lead to frequent false detections. We developed a highly specific and sensitive nested PCR method to detect M. bicuspidata, by targeting the hyphally regulated cell wall protein (HYR) gene. This nested HYR-PCR produced a single clear band, showed no cross-reaction with other pathogens, and was superior to rDNA-PCR in specificity and sensitivity. The sensitivity of nested HYR-PCR (6.10 × 101 copies/μL) was greater than those of the large subunit ribosomal RNA gene (LSU rRNA; 6.03 × 104 copies/μL) and internal transcribed spacer (ITS; 6.74 × 105 copies/μL) PCRs. The nested HYR-PCR also showed a higher positivity rate (71.1%) than those obtained with LSU rRNA (16.7%) and ITS rDNA (24.4%). In conclusion, we developed a new nested HYR-PCR method for the specific and sensitive detection of M. bicuspidata infection. This will help to elucidate the transmission route of M. bicuspidata and to design effective management and control measures for M. bicuspidata disease.

The complete mitochondrial genome sequence of the Metschnikowia bicuspidata (Metschnikoff, 1884), an emerging pathogen of ‘milky disease’ in Chinese mitten crab

The outbreak of milky disease of Chinese mitten crab caused by M. bicuspidata seriously restricted the development of the crab industry. In this study, the mitochondrial genome sequence of M. bicuspidata was assembled, annotated, and further analyzed. The results indicated that the complete mitochondrial genome of M. bicuspidata was 75,095 bp, which contained two rRNAs, 23 tRNAs, and 13 protein-coding genes. The phylogenetic tree of 13 yeasts based on the complete mitochondrial genome was constructed which showed that M. bicuspidata (accession number OK514652) and M. bicuspidata (accession number MW147605.1) were clustered in a clade. To sum up, our research results would further provide essential data for the systematics and evolution study of M. bicuspidata.

Making of Massoia Lactone-Loaded and Food-Grade Nanoemulsions and Their Bioactivities against a Pathogenic Yeast

Nanoemulsions (NEs) have been made for improving the delivery and disperse of bioactive compounds. In this study, it was found that the best ingredients for the stable Massoia lactone-loaded and food-grade NEs making were 560.0 µL of Tween-80, 240.0 µL of Span-80 and 200.0 µL of Massoia lactone. Then, 9.0 mL of distilled water was titrated into the mixture under continuous magnetic stirring (750 rotations min−1) with about 2 drops per second for 20 min. Finally, the system was treated by ultrasonication using an ultrasonic generator (180 W and 22 KHz) for 5 min. All the prepared particles with a mean droplet diameter of 43 nm were spherical, had uniform size distribution and were equally distributed in the Massoia lactone-loaded NEs. The obtained Massoia lactone-loaded nanoemulsions (NEs) were very stable without changes of the mean droplet diameter and polydispersity indexes (PDI) for over two months under different conditions. As with free Massoia lactone, Massoia lactone loaded in the NEs had high anti-fungal activity against Metschnikowia bicuspidate LIAO, a pathogenic yeast causing milky disease in the Chinese mitten crab by damaging its cell membrane and causing cellular necrosis. Massoia lactone loaded in the NEs also had the DPPH radical scavenging activity and the hydroxyl radical scavenging activity.

Experimental Transmission of the Yeast, Metschnikowia bicuspidata, in the Chinese Mitten Crab, Eriocheir sinensis.

The Chinese mitten crab, Eriocheir sinensis, is an important farmed crustacean species in China, outranking other farmed crabs in yield and economic importance. An infection called “milky disease”, caused by the yeast, Metschnikowia bicuspidata, has emerged in E. sinensis farms in northeast China and has caused progressive economic losses. The diseased crabs present with opaque, whitish muscles and milky hemolymph. Currently, there are no effective drugs to treat the infection. Clarifying the transmission route of M. bicuspidata would help to treat and prevent the disease. We investigated the effects of three different M. bicuspidata infection methods (feeding, immersion, and cohabitation) on E. sinensis. All three infection methods led to a high infection rate in healthy crabs. After 35 d, the infection rate was 76.7%, 66.7%, and 53.3% in the feeding, immersion, and cohabitation groups, respectively. Diseased crabs exhibited the typical symptom of hemolymph emulsification, with a high pathogen load of M. bicuspidata. The yeast was not detected in the oocytes of infected crabs. Fertilized embryos, zoea larvae, and megalopae of infected ovigerous crabs tested negative for yeast, indicating that direct transmission from mother to offspring does not occur. Our results highlight avenues for the prevention and control of this yeast.

First report of Metschnikowia bicuspidata infection in Chinese grass shrimp (Palaemonetes sinensis) in China.

The Chinese grass shrimp (Palaemonetes sinensis) was found with a white turbidity appearance in the Panjin area. After dissection, typical symptoms of milky disease with hemolymph emulsification and noncoagulation were observed; however, the pathogen was unknown. In this study, we aimed to isolate the pathogen of the diseased P. sinensis. We found that the pathogen could grow on the fungal medium Bengal red, and microscopic examination showed that it reproduced by budding. Molecular identification of the isolated and purified yeast strain LNMB2021 based on 26S rDNA sequence showed that the pathogenic pathogen was Metschnikowia bicuspidata (GenBank OK094821), with 98.74% homology with M. bicuspidata strain LNES0119 (GenBank OK073903) and 98.56% with M. bicuspidata strain Liao (GenBank MT856369). The results of an artificial infection test showed that M. bicuspidata caused the same clinical symptoms in P. sinensis, and the isolated pathogen was still the same, which proved that P. sinensis was a new host of M. bicuspidata. Histopathological analysis showed that there were obvious pathological changes in the hepatopancreas and muscle tissue of the diseased P. sinensis. Identification of the pathogen is essential for the prevention and control of the disease and the healthy culture of P. sinensis. Furthermore, considering the transmissibility and cross-host transmission of M. bicuspidata, its risk of infecting other aquatic animals deserves high attention.

Paenibacillus popilliae (milky disease of insects)

Paenibacillus popilliae (milky disease of insects)

Development of a TaqMan real-time quantitative PCR assay to detect Metschnikowia bicuspidata in Chinese mitten crab Eriocheir sinensis.

Milky disease of Chinese mitten crab Eriocheir sinensis caused by Metschnikowia bicuspidata is a novel disease with high mortality. No effective treatment is currently available, but a rapid, accurate detection method is required for the prevention and control of the disease. In this study, the genome-sequencing results of M. bicuspidata and similar species were used for comparative genomic analysis for genes specific to M. bicuspidata. A quantitative PCR (qPCR) detection method for M. bicuspidata was then established using the specific primers and probes designed according to the sequence of a hypothetical protein gene specific to M. bicuspidata. The assay was found to have a high degree of repeatability and reproducibility, with a linear dynamic range (R2 = 0.998) extending over 9 log10 dilutions and a high efficiency (100.7%). Furthermore, the method showed high sensitivity, being able to detect at least 11.3 copies μl-1 of recombinant plasmid, and strong specificity, without any cross-reaction with any of the 9 species of yeast that are closely related to M. bicuspidata or any of 16 species of pathogenic bacteria commonly observed in aquatic animals. The established method was used to examine 138 apparently healthy crabs collected from 22 farms, with 21 samples (15.2%) found to be M. bicuspidata-positive. Thus, the developed qPCR assay is a specific, sensitive, stable, and rapid diagnostic method for the detection and quantification of M. bicuspidata DNA from E. sinensis tissues.

Metschnikowia bicuspidata isolated from milky diseased Eriocheir sinensis: Phenotypic and genetic characterization, antifungal susceptibility and challenge models.

A severe disease occurred in farmed Eriocheir sinensis characterized by milky liquid accumulation in the pectoral cavity, in the province of Liaoning, China, during October 2018-April 2019. Diseased crabs moved sluggishly, exhibited appetite loss and readily lost legs. Under the microscopic analysis, it was observed that the milky liquid contained a large number of yeastlike microorganisms (0.8-1.2μm × 1.5-1.9μm), which were also present in the muscle, hepatopancreas and gills. A dominant strain was isolated from the milky liquid and other tissues of diseased crabs. It grew on nutrient agar and formed 1- to 3-mm white opaque colonies, each with a protuberance in the centre. Besides, the results of TEM and SEM also demonstrate a typical multilateral budding model of the yeast clearly. We identified the strain, which we named 2EJM001, as Metschnikowia bicuspidata based on 18S rDNA, ITS and 26S rDNA sequence analyses and on its morphological, physiological and biochemical characteristics. Phylogenetic analysis revealed that 26S rDNA of 2EJM001 was clustered with M. bicuspidata (LNES0119) as reported by Bao et al. In addition, unlike Bao et al., two challenge experiments (injection and immersion) were used in this study. The results of challenge experiments show that 2EJM001 was pathogenic to E. sinensis and caused signs similar to those found in the naturally infected crabs. At the same time, the minimum inhibitory concentrations (MIC80 and MIC90 ) were determined. This study further confirms that M. bicuspidata 2EJM001 was the pathogen responsible for 'milky disease' in E. sinensis from Liaoning Province.

Proteomic Analysis of the Hemolymph After Metschnikowia bicuspidata Infection in the Chinese Mitten Crab Eriocheir sinensis.

The “milky disease” of the Chinese mitten crab, Eriocheir sinensis, is a highly lethal fungal disease caused by Metschnikowia bicuspidata infection. To elucidate the immune responses of the hemolymph of E. sinensis to M. bicuspidata infection, a comparative analysis of the hemolymph of E. sinensis infected with M. bicuspidata and that treated with phosphate buffered saline was performed using label-free quantitative proteomics. A total of 429 proteins were identified. Using a 1.5-fold change in expression as a physiologically significant benchmark, 62 differentially expressed proteins were identified, of which 38 were significantly upregulated and 24 were significantly downregulated. The upregulated proteins mainly included cytoskeleton-related proteins (myosin regulatory light chain 2, myosin light chain alkali, tubulin α-2 chain, and tubulin β-1 chain), serine protease and serine protease inhibitor (clip domain-containing serine protease, leukocyte elastase inhibitor, serine protein inhibitor 42Dd), catalase, transferrin, and heat shock protein 70. Upregulation of these proteins indicated that phenoloxidase system, phagocytosis and the ROS systems were induced by M. bicuspidata. The downregulated proteins were mainly organ and tissue regeneration proteins (PDGF/VEGF-related factor protein, integrin-linked protein kinase homing pat-4 gene) and hemagglutination-associated proteins (hemolymph clottable protein, hemocyte protein-glutamine gamma-glutamyltransferase). Downregulation of these proteins indicated that M. bicuspidata inhibited hemocyte regeneration and hemolymph agglutination. Fifteen differentially expressed proteins related to immunity were verified using a parallel reaction monitoring method. The expression trend of these proteins was similar to that of the proteome. To the best of our knowledge, this is the first report on the proteome of E. sinensis in response to M. bicuspidata infection. These results not only provide new and important information on the immune response of crustaceans to yeast infection but also provide a basis for further understanding the molecular mechanism of complex host pathogen interactions between crustaceans and fungi.

Metschnikowia bicuspidate associated with a milky disease in Eriocheir sinensis and its effectitve treatment by Massoia lactone

The pathogenic yeast strain LIAO causing the milky disease in the Chinese mitten crab belonged to one member of Metschnikowia bicuspidate which could grow well at different temperatures from 28 to 4 °C. It was also found that the pathogenic yeast strain LIAO could grow in the extracts of the muscle, gill, heart tissues, intestinal tracts of the healthy Chinese mitten crabs by using the reducing sugars, amino acids and other nutrients in them. Massoia lactone released from liamocins produced by Aureobasidium melanogenum had high anti-fungal activity against the pathogenic yeast strain LIAO and M. bicuspidate WCY isolated from the diseased marine crabs. The minimal inhibitory concentrations (MIC) and the minimal fungicidal concentration (MFC) in the liquid culture against the pathogenic yeast strain LIAO were 0.15 mg/mL and 0.34 mg/mL, respectively. Massoia lactone as a bio-surfactant could damage the cell membrane, even break the whole cells of the pathogenic yeast strain LIAO and cause cellular necrosis of the pathogenic yeast LIAO. Therefore, Massoia lactone could be used to effectively kill the pathogenic yeast strains and as an effectitve treatment for milky disease in the Chinese mitten crab.

First description of milky disease in the Chinese mitten crab Eriocheir sinensis caused by the yeast Metschnikowia bicuspidata

The Chinese mitten crab, Eriocheir sinensis, is an economically important crustacean species in China. A recent epidemic of “milky disease” in Panjin, China resulted in a large loss of cultured E. sinensis. Clinical signs of the disease include weak response, swollen pereopod and claw joints, opaque or whitish muscles, and milky hemolymph, culminating in tissue or organ failure and death. Histological analysis of the crabs showed a marked disorder of fibers, discrete necrotic lesions, and high yeast infiltration in the muscles, heart, gills, and other organs. The yeast cells (LNES0119) isolated from the diseased crab tissues were subjected to polymerase chain reaction analysis. The resulting product, the D1/D2 domain of 26S rDNA of the LNES0119 yeast isolate, was used to identify the pathogen for milky disease, which was found to have 98% sequence identity with the sequence of Metschnikowia bicuspidata (GenBank accession nos. U44822 and U94944), a pathogenic yeast found in aquatic invertebrates. A laboratory challenge experiment showed that the pathogen was infectious and caused the same pathological alterations in crab hosts. The present findings demonstrated that the pathogen isolated from the Chinese mitten crab with milky disease was the yeast M. bicuspidata. This study is the first description of M. bicuspidata infection in the Chinese mitten crab and thus warrants further research on the effects of M. bicuspidata in crustaceans

Effects of biological and mineral fertilization on the growth, nutrition, and yield of Capsicum chinense under greenhouse conditions

Habanero peppers (Capsicum chinense Jacq.) are cultivated in the American continent and consumed around the world for their flavor, aroma, and biofunctional compounds in addition to being used as a food colorant. Increasing the production and quality of peppers using biological and mineral fertilization represents a challenge for producers. The present study aimed to evaluate the effects of the application of microorganism consortia on the plant growth, nutrition, and fruit yield of C. chinense under greenhouse conditions. Bonasol® (Azotobacter sp., Azospirillum brasilense, Pseudomonas fluorescens, Bacillus subtilis, and Rhizophagus intraradices) and Lilasol® (Bacillus popilliae, Bacillus thuringiensis, and Purpureocillium lilacinum) consortia were added at two mineral fertilization levels (75% and 100%). The Bonasol® consortium increased the plant height and stem diameter of C. chinense plants by 16.7–18.7% and 20.1–25.2%, respectively, and the number of flowers per plant by 84.7%. Consequently, fruit yield expressed as t ha−1 was enhanced by 32.4% and 46.1%, regarding to the mineral fertilization at 75% and 100%, respectively. Additionally, fruit quality was improved, as evidenced by heavier (17.8%) and longer (5.19%) fruits. Aerial (65.6%) and root (116.5%) biomass were also higher compared to non-inoculated plants. The microorganism consortia did not increase the N and P content in C. chinense leaves, but the Lilasol® consortium did increase the foliar K content. In conclusion, the mixture of the plant growth-promoting and mycorrhizal fungus in the Bonasol® consortium increased the plant growth and production of C. chinense.

THE OCCURRENCE AND SEQUENCE ANALYSIS OF MILKY HEMOLYMPH DISEASE IN LOBSTER PANULIRUS HOMARUS FROM DIFFERENT LOCATIONS IN INDONESIA

Among the high significance diseases in spiny lobster farming, Milky Hemolymph Disease of Spiny Lobster (MHD-SL) is considered as the most devastating disease which caused in mortality up to 80 % and morbidity to 100%. Investigations of this disease were mostly performed for farmed lobster and no observation was conducted for wild lobster. This study was carried out first, to investigate milky hemolymph disease occurrence both in wild and farmed spiny lobster Panulirus homarus from different locations by PCR analysis. Second, to perform nucleotide sequence analysis in order to identify, and to describe relationship of the milky disease agent obtained in this study with the disease agent from several geographical regions by phylogenetic analysis. Adult farmed lobsters were collected from Lombok, Pangandaran and Pegametan, while wild lobsters were obtained from Jembrana, Lombok, and Banyuwangi coastal areas. One farmed lobster from Pegametan and two farmed lobsters from Lombok were infected with the milky disease. Nucleotide sequence analysis demonstrated that the causative agent of milky disease obtained in this study (MHD-1, 2, and 3) exhibited 99% homologous nucleotide sequence with milky disease agent from Vietnam that was uncultured Rickettsia-like Bacteria (RLB). As a consequence, further work is needed most importantly on how to cultivate milky disease agent in order to find proper methods to alleviate milky disease problems. This paper is the first report on phylogenetic analysis of milky disease from Indonesia that pointed out a closed relationship between milky disease in the present study and from Vietnam, Madagascar, Mozambique and Tanzania.