An outbreak of the sessile peritrich Zoothamnium duplicatum in a pilot, commercial-scale Limulus polyphemus hatchery resulted in the loss of ∼96% (40,000) second/third instar larvae over a 61day period. peritrich growth was heavy, leading to mechanical obstruction of the gills and physical damage. The peritrichs were controlled without resultant loss of juvenile crabs by administering 10ppm chlorine in freshwater for 1h and the addition of aquarium grade sand; a medium into which the crabs could burrow and facilitate cleaning of the carapace. Peritrich identity was confirmed from a partial SSU rDNA contiguous sequence of 1343bp (99.7% similarity to Z. duplicatum).
The rhinoceros beetle, Oryctes rhinoceros, has emerged as a serious pest of oil palm since the prohibition of burning as a method for maintaining estate hygiene in the 1990s. The abundance of beetles is surprising given that the Malay peninsula was the site of first discovery of the Oryctes virus, which has been used to effect good as a biological control agent in other regions. A survey of adult beetles was carried out throughout Malaysia using pheromone traps. Captured beetles were examined for presence of virus using both visual/microscopic examination and PCR detection methods. The survey indicated that Oryctes virus was common in Malaysia among the adult beetles. Viral DNA analysis was carried out after restriction with HindIII enzyme and indicated at least three distinct viral genotypes. Bioassays were used to compare the viral strains and demonstrate that one strain (type B) is the most virulent against both larvae and adults of the beetle. Virus type B has been cultured and released into healthy populations where another strain (type A) forms the natural background. Capture and examination of beetles from the release site and surrounding area has shown that the spread and persistence of the applied virus strain is accompanied by a reduction in palm frond damage.
In view of the increasing and devastating damage by rhinoceros beetle (Oryctes rhinoceros) to coconut palms in the middle of last century, many efforts were made to find an efficient natural control factor against this pest, which could not be controlled by pesticides. The basic procedures of these monitoring programmes are outlined together with the final detection of a virus disease in oil palm estates in Malaysia in 1963. In extensive laboratory studies, the virus was isolated and identified as the first non-occluded, rod-shaped insect virus, morphologically resembling the baculoviruses. Infection experiments clarified the pathology, histopathology, and virulence of the virus and demonstrated that the virus was extremely virulent to larvae after peroral application. These findings encouraged the first pilot release of virus in 1967 in coconut plantations of Western Samoa where breeding sites were contaminated with virus. Surprisingly, the virus became established in the Samoan rhinoceros beetle populations and spread autonomously throughout the Western Samoan islands. As a consequence, there was a drastic decline of the beetle populations followed by a conspicuous recovery of the badly damaged coconut stands. This unexpected phenomenon could only be explained after it was shown that the adult beetle itself is a very active virus vector and thus was responsible for the efficient autodissemination of the virus. The functioning of the beetle as a 'flying virus factory' is due to the unique cytopathic process developing in the midgut after peroral virus infection. Pathological details of this process are presented. Because of the long-term persistence of the virus in the populations, rhinoceros beetle control is maintained. Incorporation of virus into integrated control measures and successful virus releases in many other countries are recorded.
The world production of shrimp such as the Malaysian giant freshwater prawn, Macrobrachium rosenbergii is seriously affected by the white spot syndrome virus (WSSV). There is an urgent need to understand the host pathogen interaction between M. rosenbergii and WSSV which will be able to provide a solution in controlling the spread of this infectious disease and lastly save the aquaculture industry. Now, using Next Generation Sequencing (NGS), we will be able to capture the response of the M. rosenbergii to the pathogen and have a better understanding of the host defence mechanism. Two cDNA libraries, one of WSSV-challenged M. rosenbergii and a normal control one, were sequenced using the Illumina HiSeq™ 2000 platform. After de novo assembly and clustering of the unigenes from both libraries, 63,584 standard unigenes were generated with a mean size of 698bp and an N50 of 1137bp. We successfully annotated 35.31% of all unigenes by using BLASTX program (E-value <10-5) against NCBI non-redundant (Nr), Swiss-Prot, Kyoto Encyclopedia of Genes and Genome pathway (KEGG) and Orthologous Groups of proteins (COG) databases. Gene Ontology (GO) assessment was conducted using BLAST2GO software. Differentially expressed genes (DEGs) by using the FPKM method showed 8443 host genes were significantly up-regulated whereas 5973 genes were significantly down-regulated. The differentially expressed immune related genes were grouped into 15 animal immune functions. The present study showed that WSSV infection has a significant impact on the transcriptome profile of M. rosenbergii's hepatopancreas, and further enhanced the knowledge of this host-virus interaction. Furthermore, the high number of transcripts generated in this study will provide a platform for future genomic research on freshwater prawns.
A microsporidian parasite, Enterocytozoon hepatopenaei (abbreviated as EHP), is an emerging pathogen for penaeid shrimp. EHP has been found in several shrimp farming countries in Asia including Vietnam, Thailand, Malaysia, Indonesia and China, and is reported to be associated with growth retardation in farmed shrimp. We examined the histological features from infected shrimp collected from Vietnam and Brunei, these include the presence of basophilic inclusions in the hepatopancreas tubule epithelial cells, in which EHP is found at various developmental stages, ranging from plasmodia to mature spores. By a PCR targeting the 18S rRNA gene, a 1.1kb 18S rRNA gene fragment of EHP was amplified, and this sequence showed a 100% identity to EHP found in Thailand and China. This fragment was cloned and labeled with digoxigenin-11-dUTP, and in situ hybridized to tissue sections of infected Penaeus vannamei (from Vietnam) and P. stylirostris (Brunei). The results of in situ hybridization were specific, the probe only reacted to the EHP within the cytoplasmic inclusions, not to a Pleistophora-like microsporidium that is associated with cotton shrimp disease. Subsequently, we developed a PCR assay from this 18S rRNA gene region, this PCR is shown to be specific to EHP, did not react to 2 other parasitic pathogens, an amoeba and the cotton shrimp disease microsporidium, nor to genomic DNA of various crustaceans including polychaetes, squids, crabs and krill. EHP was detected, through PCR, in hepatopancreatic tissue, feces and water sampled from infected shrimp tanks, and in some samples of Artemia biomass.
Entomopathogenic fungi of the genus Aschersonia are specific for whitefly and scale insects. They can be used as biological control agents against silverleaf whitefly, Bemisia argentifolii and greenhouse whitefly, Trialeurodes vaporariorum. Forty-four isolates of Aschersonia spp. were tested for their ability to sporulate and germinate on semi-artificial media and to infect insect hosts. Seven isolates sporulated poorly (less than 1x10(7) conidia/dry weight) and 10 were not able to infect either of the whitefly species. Several isolates were able to produce capilliconidia. Infection level was not correlated with germination on water agar. After a selection based on spore production and infection, virulence of 31 isolates was evaluated on third instar nymphs of both whitefly species on poinsettia (Euphorbia pulcherrima). Whitefly infection levels varied between 2 and 70%, and infection percentages of B. argentifolii correlated with that of T. vaporariorum. However, mortality was higher for T. vaporariorum than for B. argentifolii, as a result of a higher 'mortality due to unknown causes.' Several isolates, among which unidentified species of Aschersonia originating from Thailand and Malaysia, A. aleyrodis from Colombia, and A. placenta from India showed high spore production on semi-artificial medium and high infection levels of both whitefly species.
The transformation of Lambornella stegomyiae trophonts to theronts, the distribution of invasion cysts on larval Aedes albopictus cuticle, and the virulence of L. stegomyiae to Ae. albopictus and Aedes aegypti were studied in the laboratory. Transformation of trophonts into theronts was induced by a morphogenic agent released from larval Ae. albopictus homogenate. The first transformation was observed 4 hr after exposure to larval mosquito homogenate, but most transformations occurred between 12 and 16 hr. Distribution of invasion cysts on the cuticle of mosquito larvae was not uniform and most cysts were formed on the abdomen and head. L. stegomyiae was highly infective and virulent to Ae. albopictus (mortality rate: 99.53%) and Ae. aegypti (mortality rate: 90.83%) larvae.
The fungal insect pathogen Beauveria bassiana produces a range of insecticidal metabolites and enzymes, including chitinases and proteases, which may assist the disease progression. The enzymes often play a predominant role in the pathogenicity pathway and both chitinases and proteases have previously been shown to be important in host infection. Spray application of supernatants of B. bassiana broth cultures of an isolate from New Zealand caused significant mortality in the green peach aphid, Myzus persicae, within 24 h, demonstrating an apparent contact toxicity. Three-day-old broth cultures were the most effective, with less insect mortality seen using six-day-old broth. However, aphicidal activity increased again when treating aphids with seven-day-old broth. Cultures grew substantially better and produced more potent aphicidal cultures when cultured in media with an initial pH above 5.5. Chitinase was produced a day earlier than the serine protease Pr1, but the peak production periods of these enzymes did not correlate with the aphicidal activities of three- or six-day-old cultures. Cultures treated with EDTA or heated to inactivate the enzymes still showed strong insecticidal activity. Neither beauvericin nor bassianolide, two known insecticidal metabolites, were detected in the supernatants. Therefore the key aphicidal components of B. bassiana cultures were not associated with chitinase nor Pr1 and are yet to be identified.
Global shrimp aquaculture farmers have suffered major economic losses due to disease outbreaks. A notable shrimp disease is Acute Hepatopancreatic Necrosis Disease (AHPND), which is caused by a new strain of Vibrio parahaemolyticus bacteria (VpAHPND) that mainly inhabits the shrimp gut and damages the hepatopancreas. Fewer studies have investigated whether this disease will affect shrimp muscle functioning or cause any muscle damage. We challenged Penaeus monodon shrimp with VpAHPND bacteria using an immersion method. Expression of Dystrophin gene, an important regulatory gene for maintenance of muscle integrity, was quantified from muscle samples using qRT-PCR. Additional verification was conducted by determining calcium concentration and bta-miR-4286 and dre-miR-107b miRNAs expression. P. monodon dystrophin gene demonstrated the highest expression level during AHPND infection when muscle calcium concentration was detected at its lowest level at 6 h post-infection (hpi). The highest muscle calcium concentration, determined at 36 hpi, was supported by higher bta-miR-4286 miRNA expression and lower dre-miR-107b miRNA expression in VpAHPND-infected samples compared to uninfected samples at the same time point. We deduced an interactive relationship between dystrophin gene expression, calcium concentration, and miRNA expression in P. monodon muscle tissues triggered by the invading VpAHPND bacterium.
White feces syndrome (WFS) is an emerging problem for penaeid shrimp farming industries in SE Asia countries, Thailand, Malaysia, Vietnam, Indonesia, China, and in India. This occurrence of this syndrome is usually first evidenced by the appearance of white fecal strings floating on surface of the shrimp ponds. The gross signs of affected shrimp include the appearance of a whitish hindgut and loose carapace, and it is associated with reduced feeding and growth retardation. To investigate the nature of the white feces syndrome, samples of white feces and shrimp hepatopancreas tissue were collected from Penaeus vannamei in affected farms in Indonesia, and these were examined histologically. Within the white feces, we found densely packed spores of the microsporidian Enterocytozoon hepatopenaei (abbreviated as EHP) and relatively fewer numbers of rod-shaped bacteria. From WFS ponds, hepatopancreas samples form 30 individual shrimp were analyzed by histology and in situ hybridization. The results showed that all of the shrimp examined were infected with EHP accompanied by septic hepatopancreatic necrosis (SHPN). Midgut epithelial cells were also infected and this increased the number of tissue types being affected by EHP. By PCR, EHP was detected in all the samples analyzed from WFS-affected ponds, but not in those sampled from healthy shrimp ponds. To determine the modes of transmission for this parasite, we performed feeding and cohabitation bioassays, the results showed that EHP can be transmitted through per os feeding of EHP-infected hepatopancreas tissue to healthy shrimp and through cohabitation ofinfected and healthy shrimp. In addition, we found the use of Fumagillin-B, an antimicrobial agent, was ineffective in either reducing or eliminating EHP in infected shrimp.
Global high demand for Pacific white shrimp Penaeus vannamei has led to intensified cultivation and a wide range of disease problems, including bacterial diseases due to vibrios. Three presumptive luminescent Vibrio harveyi strains (Vh5, Vh8 and Vh10) were isolated from the hepatopancreas (Vh5) and haemolymph (Vh8 and Vh10) of diseased growout Pacific white shrimp from a farm in Setiu, Terengganu, Malaysia, using Vibrio harveyi agar (VHA) differential medium. All three strains were identified as V. harveyi by biochemical characteristics. 16S rRNA gene-based phylogenetic analyses by neighbour-joining, maximum likelihood and maximum parsimony methods showed all three strains in the V. harveyi cluster. All three strains were β-haemolytic and positive for motility, biofilm formation and extracellular products (caseinase, gelatinase, lipase, DNase, amylase and chitinase). Vh10 was subjected to pathogenicity test in Pacific white shrimp by immersion challenge and determined to have a LC50 of 6.0 × 108 CFU mL-1 after 168 h of exposure. Antibiotic susceptibility tests showed that all strains were resistant to oxytetracycline (OXT30), oleandomycin (OL15), amoxicillin (AML25), ampicillin (AMP10) and colistin sulphate (CT25) but sensitive to doxycycline (DO30), flumequine (UB30), oxolinic acid (OA2), chloramphenicol (C30), florfenicol (FFC30), nitrofurantoin (F5) and fosfomycin (FOS50). Each strain was also resistant to a slightly different combination of eight other antibiotics, with an overall multiple antibiotic resistance (MAR) index of 0.40, suggesting prior history of heavy exposure to the antibiotics. Vh10 infection resulted in pale or discoloured hepatopancreas, empty guts, reddening, necrosis and luminescence of uropods, as well as melanised lesions in tail muscle. Histopathological examination showed necrosis of intertubular connective tissue and tubule, sloughing of epithelial cells in hepatopancreatic tubule, haemocytic infiltration, massive vacuolation and loss of hepatopancreatic tubule structure.
The tropical conch, Laevistrombus canarium (Linnaeus, 1758) and Canarium urceus (Linneaus, 1758) are ecologically and economically important shellfish species in Malaysia and neighboring region. Their populations, however are currently declining and this histopathological study investigates the aspect of parasitism and diseases that may affect their well-being. Conch samples were randomly collected from their natural habitat and histological sections (4-5 µm) of various organs and tissues were examined under light microscope. This was followed by ultrastructure analysis on infected tissues using transmission electron microscope (TEM). Based on the histological analysis, large numbers of gamonts, sporocysts and trophozoites of Apicomplexa-like parasites were observed in the vacuolated cells and pyramidal crypt cells of the digestive tubules, and in the digestive ducts. Furthermore, coccidian and oocysts-like Pseudoklossia sp. stages were also observed in the cells of the kidney. Apart from that, spores with cyst-like structure were observed in the digestive gland and kidney. Although the parasites were present in most of the organs analyzed, there was no obvious symptom, inflammatory response or mortality incurred on both species, which implies the possibility of a non-virulent relationship like commensalisms or mutualism. However, more investigations, including molecular studies, are needed to confirm the parasite identification and dynamics, and to further evaluate the nature of relationship between Apicomplexa parasites and their host.
Infection by the microsporidian parasite Enterocytozoon hepatopenaei (EHP) has become a significant problem in the shrimp cultivation industry in Asian countries like Thailand, China, India, Vietnam, Indonesia, and Malaysia. The outbreak of this microsporidian parasite is predominantly related to the existence of macrofauna-carriers of EHP. However, information about potential macrofauna-carriers of EHP in rearing ponds is still limited. In this study, the screening of EHP in potential macrofauna-carriers was conducted in farming ponds of Penaeus vannamei in three states in Malaysia, namely Penang, Kedah, and Johor. A total of 82 macrofauna specimens (phyla: Arthropoda, Mollusca, and Chordata) were amplified through a polymerase chain reaction (PCR) assay targeting genes encoding spore wall proteins (SWP) of EHP. The PCR results showed an average prevalence of EHP (82.93%) from three phyla (Arthropoda, Mollusca and Chordata). The phylogenetic tree generated from the macrofauna sequences was revealed to be identical to the EHP-infected shrimp specimens from Malaysia (MW000458, MW000459, and MW000460), as well as those from India (KY674537), Thailand (MG015710), Vietnam (KY593132), and Indonesia (KY593133). These findings suggest that certain macrofauna species in shrimp ponds of P. vannamei are carriers of EHP spores and could be potential transmission vectors. This study provides preliminary information for the prevention of EHP infections that can be initiated at the pond stage by eradicating macrofauna species identified as potential vectors.