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Two members of the species Oryctes rhinoceros nudivirus (OrNV) were detected in O. rhinoceros haplotype G beetles collected from an oil palm plantation in Kluang and a wild coconut tree in Batu Pahat (Johor, Malaysia). OrNV strain Kluang comprised 125,794 bp, encoding 125 open reading frames (ORFs), while OrNV strain Batu Pahat comprised 124,925 bp, encoding 126 ORFs.

Metarhizium anisopliae (Metchnikoff) Sorokin, a pathogenic fungus to insects, infects the subterranean termite, Coptotermes curvignathus Holmgren, a devastating pest of plantation trees in the tropics. Electron microscopy and proteomics were used to investigate the infection and developmental process of M. anisopliae in C. curvignathus. Fungal infection was initiated by germ tube penetration through the host’s cuticle as observed at 6 h post-inoculation (PI), after which it elongated into the host’s integumental tissue. The colonization process continued as seen from dissemination of blastospores in the hemocoel at 96 h PI. At this time point, the emergent mycelia had mummified the host and forty-eight hours later, new conidia were dispersed on the termites’ body surface. Meanwhile, hyphal bodies were observed in abundance in the intercellular space in the host’s body. The proteomes of the pathogen and host were isolated separately using inoculated termite samples withdrawn at each PI-time point and analyzed in two-dimensional electrophoresis (2-DE) gels. Proteins expressed in termites showed evidence of being related to cell regulation and the immune response, while those expressed in M. anisopliae, to transportation and fungal virulence. This study provides new information on the interaction between termites and its entomopathogen, with potential utilization for developing future biopesticide to control the termite population.

Simple Summary The use of Metarhizium anisopliae as a biological control of insect pests has been experimented in the laboratory as well as in field trials. This includes against the termite Coptotermes curvignathus, however the results have varying degrees of success. One reason could be due to the lack of detailed knowledge on the molecular pathogenesis of M. anisopliae. In the current study, the conidial suspension of M. anisopliae isolate PR1 was first inoculated on the C. curvignathus, after which the pathogenesis was examined using two different approaches: electron microscopy and protein expression. At the initiation stage, the progression observed and documented including adhesion, germination, and penetration of the fungus on the cuticle within 24 h after inoculation. Later, this was followed by colonization and spreading of the fungus at the cellular level. Proteomics of C. curvignathus witnessed the expression of proteins related to cell regulation and defense, while in M. anisopliae, protein related to transport and fungal virulence were expressed throughout the infection. These findings offer relevant knowledge for use in the development of M. anisopliae as a prospective biological control agent for termites in the future. Abstract Metarhizium anisopliae (Metchnikoff) Sorokin, a pathogenic fungus to insects, infects the subterranean termite, Coptotermes curvignathus Holmgren, a devastating pest of plantation trees in the tropics. Electron microscopy and proteomics were used to investigate the infection and developmental process of M. anisopliae in C. curvignathus. Fungal infection was initiated by germ tube penetration through the host’s cuticle as observed at 6 h post-inoculation (PI), after which it elongated into the host’s integumental tissue. The colonization process continued as seen from dissemination of blastospores in the hemocoel at 96 h PI. At this time point, the emergent mycelia had mummified the host and forty-eight hours later, new conidia were dispersed on the termites’ body surface. Meanwhile, hyphal bodies were observed in abundance in the intercellular space in the host’s body. The proteomes of the pathogen and host were isolated separately using inoculated termite samples withdrawn at each PI-time point and analyzed in two-dimensional electrophoresis (2-DE) gels. Proteins expressed in termites showed evidence of being related to cell regulation and the immune response, while those expressed in M. anisopliae, to transportation and fungal virulence. This study provides new information on the interaction between termites and its entomopathogen, with potential utilization for developing future biopesticide to control the termite population.

A feeding trial was conducted to investigate the effect of palm kernel cake fermented (FPKC) by Paenibacillus polymyxa ATCC 842 on nutrient digestibility, villi height, and gut microflora. A total of 245 one-day-old broiler chicks (Cobb500) were purchased from a local hatchery and raised in an open house system in the research unit at Universiti Putra Malaysia. The chicks were individually wing-banded, weighed and randomly distributed into seven groups with five replicates (pens) and seven birds per pen. The birds were fed diets containing 0, 5, 10 and 15% palm kernel cake (PKC) and 5, 10, 15% FPKC. All the birds were fed ad libitum during the feeding trial, which lasted 42 days. One bird and two birds per replicate in each dietary treatment were randomly selected at 3 and 6 weeks, respectively. Ileal digesta and small intestinal tissues were collected. The findings showed that feeding of 10% or 15% PKC led to a significant decrease (P0.05) were observed between the dietary treatments in terms of intestinal morphology. However, gut microflora were improved (P

Achroia grisella, is a noxious pest of honey bee hives. In this study the toxicity of honey bee venom and bacterial Cry1C toxin of Bacillus thuringiensis on A. grisella were assessed. In addition, the synergistic interaction between BT Cry1c and crude honey bee venom was determined. The combination of HBV and Cry1C increased the activity for both toxins. Therefore, further analysis was carried out to assess the synergistic interaction of each compound assay separately and in mixture. The results showed LC50 value of 1.105 μg/μl for HBV and LC50 value of 0.201 μg/μl for Cry1C. The combination bioassay result showed an LC50 value of 0.549 μg/μl. The combined calculated LC50 is containing lower concentration of the component toxins, this result proved that the toxicity was enhanced due to the combination. A chi square value of 39.93and relative synergistic factor ratio of 1.14 confirmed that a synergic interaction was achieved in combination.

Lysozyme and prophenoloxidase are two immunity-related enzymes in insect. In our attempt to understand their roles in defence, we profiled expressions of the encoding genes in Spodoptera exigua Hubner and Spodoptera litura Fabricius, during the infection process by an entomopathogenic fungus, Metarhizium anisopliae var. anisopliae. Gene expressions were analysed using quantitative real-time reverse transcription-PCR. Sampling was done at 0, 2, 12 and 24 h after infection, and when the infected larvae reached the moribund stage at 36 and 60 h after infection. The expression profiles of the two genes were similar in both larvae. At 24 h after infection, the lysozyme gene was expressed at a higher level in the infected larvae (fivefold) when compared to the healthy larvae, and reached the highest level (13-fold in S. exigua and 7-fold in S. litura) at the moribund stage. The expression levels of the prophenoloxidase gene in the infected larvae were similar to that of the control, except for a slight increase...

Characterization of pathogenesis genes of Metarhizium anisopliae , will provide better understanding of the role of these genes during pathogenesis. The expression profiles of pathogenesis-related genes encoding for a subtilisin-like protease (PR1), two types of chitinases (CHI2 and CHI3), and a peptide synthetase (PES) were studied during the different stages of M. anisopliae infection in Spodoptera exigua larvae using quantitative real-time RT-PCR. Sampling were at 0, 2, 12, and 24 h after infection, when the infected larvae reached the moribund stage (36 h), when mycelia emerged from the cadavers, when few conidia had formed on the mycelia, and when the cadavers were covered by conidia. For comparison, conidia and mycelial samples harvested from culture media were also included. Among the studied genes, PR1 expression was detected early at 2 h after infection and increased as the infection progressed. CHI2 and CHI3 expressions were detected 12 h after infection and when the mycelia emerged from cadavers, respectively. The expression levels of PR1 , CHI2 and CHI3 genes increased significantly at the beginning of conidiogenesis on cadavers, but decreased at later stages. As expected, their expressions in pure fungal propagules were at very low levels. For PES gene, fold changes were not significant between different samples (less than onefold), indicating it might not have a major role in infecting stages. High expression levels of PR1 , CHI2 , and CHI3 genes during the post-mortem hyphal growth and conidiation stages of M. anisopliae clearly indicate the importance of these genes during the saprophytic phase of this fungus on host insect.

Digestibility trial was conducted to determine the apparent ileal digestibility (AID) of crude protein (CP) and amino acids (AA) in untreated palm kernel cake (PKC) and fermented palm kernel cake (FPKC) on finisher broiler. Paenibacillus polymyxa ATCC 842 and P. curdlanolyticus DSMZ 10248 were used to produce FPKCa and FPKCb, respectively through solid state fermentation (SSF). Broiler male chickens were fed with diets containing 15% PKC from day one until 41 days of age. Birds (36) were selected with uniform body weight, and randomly distributed into 3 groups with 6 replicates in each treatment and 2 birds per replicate. The chickens were deprived from food overnight with free access to drinking water. The birds were fed PKC, FPKCa and FPKCb with indigestible marker. All the chickens were allowed free access to the test ingredients and drinking water for 4 days. The birds were slaughtered; ileal digesta were individually collected, pooled within each replicate in plastic cups; and immediately kept at -20°C for chemical analysis. The findings showed that the process of SSF by cellulolytic bacteria increased the levels of CP from 16.43% in the PKC to 16.68% and 16.80% in FPKCb and FPKCa, respectively. The AID of CP was increased in FPKC compared to the PKC. Additionally, there was an increase in the digestibilities of AA in FPKC compared to untreated PKC. The process of SSF decreased the fibres in FPKC, and there was improvement in the nutrient value of FPKC by cellulolytic bacterial cultures in terms of nutrient content and digestibility.

SummaryApis dorsata is a major pollinator in the rainforests of Peninsular Malaysia, which is threatened by frequent harvesting via the removal of whole comb nests from trees during the harvesting season. There is an urgent need for conservation of this species and the long-term survival of tropical lowland forests. In this paper, the genetic relatedness of A. dorsata aggregations are addressed with a preliminary study on local populations in Malaysia. The analysis of 30 single locus DNA microsatellite markers on three aggregations at intra-aggregation level showed that the queens of different nests within an aggregation were significantly different and not related as mother-daughter (P > 0.063). At inter-aggregation level, the results demonstrated that there was no significant relatedness between the aggregations (P > 0.05) separated by a distance of 0.5 km. The results showed that adjacent aggregations were genetically different and had enough time for establishing the colonies to produce honey.

The oil palm industry of Malaysia has expanded into peat area in Sarawak. Problem statement: The subterranean termite Coptotermes curvignathus was a serious pest of oil palm on peat. Control of this termite has resorted to heavy usage of chemicals which was deemed uneconomical and hostile to the environment. Baiting system has inconsistent success due to the limited knowledge of the behavior of the pest. Approach: This study was to investigate damage and nesting characteristic of the pest. Infested palm in the peat area was dissected using a chainsaw and observation was made on the endoecie and damage. Microclimate inside the palm was recorded using a data logger and acid insoluble lignin from 3 different infested palms was determined. Results: Dissection of infested palm revealed that termite generally attacked the palm from the spear in immature palm or basal region in mature palm due to the energy requirement and level of water table. Spear region infestation was possible because of the moist environment provided by the proximity of the fronds and leaf sheaths. The high lignin content (42-45%) in the thin laminae indicated the concentration of lignin or incorporation of peat in nest construction. Wood stump residues remained in the plantation was one of the main reasons of termite infestation. The stability and protection of the wood stump encouraged termite breeding. A C. curvignathus queen was discovered in the endoecie under the wood stump and was seen mobile. Conclusion: The pest was generally available in area where wood stumps and moisture were available. Manipulation of water table for certain period after spraying of chemical may help reduce infestation by the termite.

Problem Statement: Diamondback moth, Plutella xylostella L. (Lepidoptera, Plutellidae) is the most destructive insect pest of cruciferous plants throughout the world which become resistant to wide range of chemical pesticides. PxGV is known as an effective factor on diamondback moth larval mortality. Approach: Some morphological characteristics, viral DNA isolation and Restriction Endonuclease (REN) analysis of P. xylostella granulovirus and its effects of different concentrations on its larval host Diamondback Moth P. xylostella, were studied. Results: Taiwanian isolate confirmed due to restriction pattern and genome size of PxGV Taiwanian isolate which was compared with PxGV isolates reported earlier. PxGV originally isolated in Taiwan has capsules that are ovocylindrical with a mean size of 272.84±12 by 148.27±19 nm. The virions are 168.44±16±29.57±12 nm. Results from pathogenicity test of the granulovirus to DBM using the leaf disc method shows that first, second and third instars of P. xylostella were significantly susceptible to infection by PxGV. Older larvae were less susceptible to PxGV than younger larvae with the same virus concentration. The LC50 for second instar larvae was 1.39×106 granules mL-1. The LT50 values ranged between 3.813-6.946, 4.965-9.743 and 5145-9.407 days for first to third instars in three different concentrations, respectively. Conclusion: Its high specificity and pathogenicity to its larval host indicate that PxGV is a good candidate as an alternative biopesticide to chemical insecticides in Integrated Pest Management (IPM) of P. xylostella.

A feeding trial was conducted to investigate the effect of palm kernel cake fermented by Paenibacillus polymyxa ATCC 842 (FPKC) on broiler performance. A total of 245 1-day-old broiler chicks (Cobb 500) were raised in the conventional open-sided house. The birds were fed diets containing 0 (Control), 5%, 10% and 15% palm kernel cake (PKC) and 5%, 10%, 15% FPKC. The bodyweight and the feed intake were recorded. The bodyweight gain (BWG) and feed conversion ratio (FCR) were calculated. Carcass characteristics and meat quality were measured at the end of the experiment, whereas blood was collected at 21 (starter) and 42 days (finisher) to determine blood biochemistry. The results showed that the addition of 10% or 15% PKC in broiler diets led to a significant (P < 0.05) decrease in BWG and increase in FCR during the finisher phase or overall performance. However, BWG and FCR were improved (P < 0.05) in chickens fed with 10% or 15% FPKC compared with those fed with 10% or 15% PKC or the Control group. The relative weight of the gizzard was higher (P < 0.05) for the broiler group fed with 15% PKC compared with those birds fed the Control diet or FPKC at 3 weeks of age. No significant differences were observed among the dietary treatments in blood biochemistry, breast meat colour, drip loss, cooking loss and tenderness. In conclusion, the present experiment showed that palm kernel cake fermented by P. polymyxa ATCC 842 could be fed to broiler chickens up to 15% in their rations without any adverse effect on the growth performance and meat quality.

Background Outbreaks of the casuarina moth, Lymantria xylina Swinehoe (Lepidoptera: Lymantriidae), which is a very important forest pest in Taiwan, have occurred every five to 10 years. This moth has expanded its range of host plants to include more than 65 species of broadleaf trees. LyxyMNPV (L. xylina multiple nucleopolyhedrovirus) is highly virulent to the casuarina moth and has been investigated as a possible biopesticide for controlling this moth. LdMNPV-like virus has also been isolated from Lymantria xylin a larvae but LyxyMNPV was more virulent than LdMNPV-like virus both in NTU-LY and IPLB-LD-652Y cell lines. To better understand LyxyMNPV, the nucleotide sequence of the LyxyMNPV DNA genome was determined and analysed. Results The genome of LyxyMNPV consists of 156,344 bases, has a G+C content of 53.4% and contains 157 putative open reading frames (ORFs). The gene content and gene order of LyxyMNPV were similar to those of LdMNPV, with 151 ORFs identified as homologous to those reported in the LdMNPV genome. Two genes (Lyxy49 and Lyxy123) were homologous to other baculoviruses, and four unique LyxyMNPV ORFs (Lyxy11, Lyxy19, Lyxy130 and Lyxy131) were identified in the LyxyMNPV genome, including a gag-like gene that was not reported in baculoviruses. LdMNPV contains 23 ORFs that are absent in LyxyMNPV. Readily identifiable homologues of the gene host range factor-1 (hrf-1), which appears to be involved in the susceptibility of L. dispar to NPV infection, were not present in LyxyMNPV. Additionally, two putative odv-e27 homologues were identified in LyxyMNPV. The LyxyMNPV genome encoded 14 bro genes compared with 16 in LdMNPV, which occupied more than 8% of the LyxyMNPV genome. Thirteen homologous regions (hr s) were identified containing 48 repeated sequences composed of 30-bp imperfect palindromes. However, they differed in the relative positions, number of repeats and orientation in the genome compared to LdMNPV. Conclusion The gene parity plot analysis, percent identity of the gene homologues and a phylogenetic analysis suggested that LyxyMNPV is a Group II NPV that is most closely related to LdMNPV but with a highly distinct genomic organisation.

Knowledge on the population of genetic structure and ecological behaviour of Apis dorsata from Peninsular Malaysia is needed for effective management and conservation of this species since unsustainable whole solitary low nest cutting for product harvesting is the current common practice here. The analysis of 15 single locus DNA microsatellite markers on samples from 20 solitary nests of A. dorsata showed that while these markers were polymorphic, high intracolonial relatedness existed. Furthermore, in general, slightly negative values of intercolony relatedness (R) among the nests of A. dorsata were found. However, positive values of mean intercolony relatedness were observed between 54 pairs of nests out of 190 possible combinations. The R values among nest pairs 3-4 and 3-5 was higher than 0.50 showing that their queens were half siblings, whereas nest pair 19-20 showed relatedness of 0.95 indicating that the same queen was sampled. The results that we obtained could not conclusively support the hypothesis of this study that the honey hunters in Marang district of Malaysia repeatedly harvest the same nest located at a different site and at a different time during the same honey harvesting season. However, our finding of an appreciable level of intercolonial relatedness between several pairs of nests in this pioneer study indicated that a comprehensive study with a larger sample size of solitary nests found throughout the region would be necessary to provide concrete proof for this novel idea.