In Malaysia, maggot debridement therapy (MDT) utilizes maggots of Lucilia cuprina (Wiedemann) to debride necrotic tissue from wound surface, reduce bacterial infection and therefore, enhance wound healing process. To evaluate the sterility of the sterile maggots produced after sterilization process before delivering onto patient wounds. Sterility of sterile maggots is crucial in ensuring the safe usage of MDT and patient's health. Eggs of L. cuprina collected from a laboratory colony were divided into treated group (sterilized) and control group (non-sterilized). Treated group underwent sterilization while eggs from control group were allowed to hatch without sterilization. Sodium hypochlorite and formaldehyde were the main disinfectants used in this sterilization process. Scanning electron microscope (SEM) was used to examine and ascertain the sterility of sterile maggots. SEM results showed that all sterilized L. cuprina eggs and maggots achieved sterility and all were cleared from bacterial contamination. In contrast, all non-sterilized eggs and maggots were found to be colonized by microorganisms. Sterilization method employed to sterilize eggs and maggots used in Malaysia MDT was proven successful and MDT is safe to be used as wound management tools.
The toxic effects of Abate (temephos) on mosquito larvae and non-target organisms were studied in the rice-field and in the laboratory. In the laboratory tests, Culex tritaeniorhychus larvae and cladoceran zooplanktons (predominantly Diaphanosoma and Moinodaphnia species) were found to be highly susceptible to Abate with LC50 values of 0.27 and less than 0.10 parts per billion respectively. Other non-target species in decreasing degree of susceptibility to Abate were copepods (Tropodiaptomus spp.), Aplocheilus panchax and Tubifex worms. In field study, Abate at concentrations 60, 100 and 200 gm hectare-1 is effective in maintaining the rice-fields free of Anopheles and Culex mosquitoes for at least 2 days. No mortality was observed for Aplocheilus panchax and Tubifex worms at the above concentrations; nevertheless, populations of cladoceran zooplanktons and copepods were reduced up to seven days posttreatment.
Insecticides Abate, DDT, Dowco-214, Dursban, fenitrothion, fenthion, gamma-HCH, and malathion were tested against the field collected fourth instars larvae of Mansonia from Penang Island, Malaysia. The larvae appeared to be highly susceptible to Dursban and Abate with LC50 values of 1.54 and 1.92 parts per billion respectively. Other chemicals, in decreasing degree of effectiveness, were gamma-HCH, fenthion, P,P'-DDT, Dowco-214, fenithrothion and malathion. The potential use of these chemicals in Mansonia control was discussed. A simple method for collecting and testing Mansonia larvae was also described.
Bioefficacy of thermal fogging application of 2 Bacillus thuringiensis israelensis formulations, Vectobac ABG 6511 water-dispersible granules (3,000 international toxic unit [ITU]/mg) and Vectobac 12AS liquid (1,200 ITU/mg), was assessed for larvicidal activities against Aedes aegypti, Aedes albopictus, Anopheles dirus, and Culex quinquefasciatus. Portable Agrofog AF35 sprayers were used to apply the 2 formulations indoors in residential premises on Penang Island, Malaysia. Vectobac ABG 6511 showed good larvicidal effect against all 4 mosquito species at 3 of the higher doses tested (2.91 x 10(9), 1.45 x 10(9), and 0.71 x 10(9) ITU/ha), with more than 96% mortality at 48 h after spraying. As a comparative formulation, Vectobac 12AS also showed good larvicidal activity against all 4 mosquito species at 2 of the higher doses tested (2.87 x 10(9) and 1.46 x 10(9) ITU/ha), with more than 92.5% mortality at 48 h after spraying. Larvae of An. dirus were significantly more susceptible to both water-based Vectobac formulations when compared to the other 3 mosquito species. Both microbial formulations showed better efficacy at higher doses. However, even at the lowest dose tested, Vectobac ABG 6511 and Vectobac 12AS (both at 0.36 x 10(9) ITU/ha) showed larvicidal properties, with more than 66% mortality at 48 h after spraying. Overall, for this bacterial agent, the water-dispersible granule formulation has better prospects than the liquid formulation for the control of larvae of vector mosquitoes.
Five formulations of Bacillus sphaericus (strain 2362) including aqueous suspension BSP 1, BSP 2, technical powder ABG 6184, corncob granules ABG 6185 (potencies 2 x 10(10), 2 x 10(7), 9.5 x 10(10), 5 x 10(10), 5 x 10(10) spore/g, respectively) and wettable powder ABG 6232 (1,000 BS ITU/mg) were tested against laboratory-cultured late third/early fourth instar larvae of Mansonia uniformis in floating screened cages in small plots at swampy ditches on Penang Island, Malaysia. Mean dosage/response values at 90% mortality levels were 6.93, 95.32, 1.45, 11.92 and 2.86 liters or kg per ha, respectively, for the formulations tested. There were practically no residual effects for the formulations tested with larvae introduced at 48, 96, and 168 h post-treatment. In trials of BSP 1, ABG 6184 and ABG 6185 (1 liter or 1 kg per ha) against immature Mansonia spp. in impounded paddy field ditches, improved efficacy and residual effects were obtained with mean reductions of 93.1, 91.9 and 80.4% at days 3, 7 and 14 posttreatment, respectively.
Emulsifiable concentrates of DursbanR (chlorpyrifos) and Dowco 214 (chlorpyrifos-methyl) were tested as mosquito larvicides using Hudson knapsack sprayers on small plots of rice-fields on Penang Island. The mosquitoes found in these rice-fields are predominantly Anopheles campestris and Culex tritaeniorhynchus. At dosages of 14, 28 and 56 gm hectare-1, Dursban is effective in maintaining the rice-fields free of Anopheles larvae for at least 2, 3 and 7 days respectively. Dowco 214 at 56 gm hectare-1 is able to keep the fields free of all mosquito larvae for at least 2 days.
Detailed studies of larval development of Octolasmis angulata and Octolasmis cor are pivotal in understanding the larval morphological evolution as well as enhancing the functional ecology. Six planktotrophic naupliar stages and one non-feeding cyprid stage are documented in details for the first time for the two species of Octolasmis. Morphologically, the larvae of O. angulata and O. cor are similar in body size, setation patterns on the naupliar appendages, labrum, dorsal setae-pores, frontal horns, cyprid carapace, fronto-lateral gland pores, and lattice organs. Numbers of peculiarities were observed on the gnathobases of the antennae and mandible throughout the naupliar life-cycle. The setation pattern on the naupliar appendages are classified based on the segmentation on the naupliar appendages. The nauplius VI of both species undergoes a conspicuous change before metamorphosis into cyprid stage. The cyprid structures begin to form and modify beneath the naupliar body towards the end of stage VI. This study emphasises the importance of the pedunculate barnacle larval developmental studies not only to comprehend the larval morphological evolution but also to fill in the gaps in understanding the modification of the naupliar structures to adapt into the cyprid life-style.
Coral-associated organisms often exhibit a continuum of host specificities. We do not know whether the variation in host specificity is related to the settlement organs or preferential settlement behaviours of the larvae. We examined the morphology of attachment discs, the settlement and metamorphosis of coral barnacles-Pyrgoma cancellatum (lives in a single coral species), Nobia grandis (two families of corals), and Armatobalanus allium (six families of corals). Our results revealed that the attachment organ of all three species are spear-shaped with sparse villi, indicating that the morphology of the attachment organs does not vary among species with different host specificities. Larvae of P. cancellatum and N. grandis only settle on their specific hosts, suggesting that chemical cues are involved in the settlement. Cyprids of N. grandis display close searching behaviour before settlement. Cyprids of P. cancellatum settle immediately on their specific host corals, without any exploratory behaviour. The host specificity and exploratory behaviours of coral barnacle cyprids are results of adaptive evolution. We argue that there is a trade-off between exploration and energy conservation for metamorphosis processes. Coral barnacle metamorphosis is longer when compared to free-living species, likely because it involves the development of a tube-shaped base on the coral surface.
A retrospective study was undertaken to determine the clinical features of cutaneous larva migrans (CLM) seen in the Department of Dermatology, Hospital Kuala Lumpur (Kuala Lumpur, Malaysia) and to assess the rate of correct diagnosis made by the referring primary care doctors. Clinical records of all 31 patients with CLM seen between January 2006 and June 2010 were retrieved. The majority of patients were male. The mean age was 32.2 years. Pruritus was reported in 83.9% of cases and serpiginous tracts in 100%. The mean lesion count was 4.4 and the mean duration of disease before presentation was 3.1 weeks. The majority of skin lesions were on the buttock and lower extremities. Only 45.2% of patients had the correct diagnosis made by the referring primary care doctors. Older age of patients and lower number of lesions were associated with a higher rate of correct diagnosis. The low rate of correct diagnosis made by the referring primary care doctors to the dermatologists in this study warrants the need for education of not only primary care doctors but also future primary care providers, consisting of medical students, house officers and junior medical officers.
Study site: Department of Dermatology, Hospital Kuala Lumpur
A T. spiralis serine protease 1.2 (TsSP1.2) was identified in the muscle larvae (ML) and intestinal larvae surface/excretory-secretory (ES) proteins by immunoproteomics. The aim of this study was to determine the TsSP1.2 function in the process of T. spiralis intrusion, growth and reproduction by using RNA interference (RNAi). RNAi was used to silence the expression of TsSP1.2 mRNA and protein in the nematode. On 2 days after the ML were electroporated with 2 µM of TsSP1.2-specific siRNA 534, TsSP1.2 mRNA and protein expression declined in 56.44 and 84.48%, respectively, compared with untreated ML. Although TsSP1.2 silencing did not impair worm viability, larval intrusion of intestinal epithelium cells (IEC) was suppressed by 57.18% (P < 0.01) and the suppression was siRNA-dose dependent (r = 0.976). Infection of mice with siRNA 534 transfected ML produced a 57.16% reduction of enteral adult burden and 71.46% reduction of muscle larva burden (P < 0.05). Moreover, silencing of TsSP1.2 gene in ML resulted in worm development impediment and reduction of female fertility. The results showed that silencing of TsSP1.2 by RNAi inhibited larval intrusion and development, and reduced female fecundity. TsSP1.2 plays a crucial role for worm invasion and development in T. spiralis life cycle, and is a potential vaccine/drug target against Trichinella infection.
In previous studies, a Trichinella spiralis serine protease (TsSP) was identified in excretion/secretion (ES) products from intestinal infective L1 larvae (IIL1) using immunoproteomics. The complete cDNA sequence of TsSP gene was 1372 bp, which encoded 429 amino acids with 47.55 kDa. The TsSP was transcribed and expressed at all T. spiralis life cycle phases, as well as mainly located at the cuticle and stichosome of the parasitic nematode. Recombinant TsSP bind to intestinal epithelial cells (IEC) and promoted larva invasion, however, its exact function in invasion, development and reproduction are still unknown. The aim of this study was to confirm the biological function of TsSP during T. spiralis invasion and growth using RNA interference (RNAi) technology. The results showed that on 1 day after electroporation using 2.5 µM siRNA156, TsSP mRNA and protein expression of muscle larvae (ML) was suppressed by 48.35 and 59.98%, respectively. Meanwhile, silencing of TsSP gene by RNAi resulted in a 61.38% decrease of serine protease activity of ML ES proteins, and a significant reduction of the in vitro and in vivo invasive capacity of IIL1 to intrude into the IEC monolayer and intestinal mucosa. When mice were infected with siRNA 156-transfected larvae, adult worm and muscle larva burdens were decreased by 58.85 and 60.48%, respectively. Moreover, intestinal worm growth and female fecundity were evidently inhibited after TsSP gene was knockdown, it was demonstrated that intestinal adults became smaller and the in vitro newborn larval yield of females obviously declined compared with the control siRNA group. The results indicated that knockdown of TsSP gene by RNAi significantly reduced the TsSP expression and enzymatic activity, impaired larvae intrusion and growth, and lowered the female reproductive capacity, further verified that TsSP might participate in diverse processes of T. spiralis life cycle, it will be a new prospective candidate molecular target of anti-Trichinella vaccines.
Rhynchophorus ferrugineus or red palm weevil (RPW) is a destructive insect pest of major cultivated palms such as coconut, date and oil palm. One of the control management of RPW is trunk injection using monocrotophos or methamidophos, but these chemicals are found to affect ecosystems and human health. Thus, we aimed to determine a bio-pesticide to replace these synthetic chemicals. We tested the antifeedant activity of three eugenol-based compounds as potential control agent against RPW larvae in vitro condition for two weeks. All these compounds show significant effect as feeding deterrent agent on 4th instar larvae, while WN16 (4-allyl-2-methoxy-1-(4-trifluoromethyl-benzyloxy)-benzene) shows the highest feeding deterrent index (FDI = 64.42%). Here we present the data regarding the biological aspect on treated RPW larvae as well as antifeedant activity index of these eugenol derived compounds.
Background: Dirofilaria ursi is a filarial nematode that parasitizes the subcutaneous tissues of the American black bear (Ursus americanus) and Japanese black bear (Ursus thiabetanus japonicus). D. ursi that has parasitized black bears has the potential to subsequently infect humans. In addition, extra-gastrointestinal anisakiasis is less common in Japan.
Case presentation: We report a case of ventral subcutaneous anisakiasis and dorsal subcutaneous dirofilariasis that was acquired in Fukushima, in the northern part of Japan. The patient was an 83-year-old Japanese female, and subcutaneous parasitic granulomas were present on her left abdomen (near the navel) and left scapula. A pathological examination of the surgically dissected tissue sections from each region demonstrated eosinophilic granulomas containing different species of parasites. To enable the morphological and molecular identification of these parasites, DNA was extracted from paraffin-embedded sections using DEXPAT reagent, and the cytochrome oxidase 2 (COX2), internal transcribed spacer 1 (ITS1), 5.8S and ITS2 regions of the Anisakis larvae, and the 5S rRNA region of the male Dirofilaria were sequenced. The PCR products were examined and compared with DNA databases. Molecular analysis of the COX2 and 5S rRNA sequences of each worm revealed that the nematode found in the ventral region belonged to Anisakis simplex sensu stricto (s.s.) and the male Dirofilaria found in the dorsal region was classified as D. ursi.
Conclusion: The present case showed a combined human case of D. ursi and A. simplex s.s. infections in subcutaneous tissues. The results of this study will contribute to the identification of unknown parasites in histological sections.
Aquaculture is the fastest growing animal production sector. However, the production of marine fish is still hampered by the high mortality rate in the first few weeks after hatching. Mortality in larvae is often caused by microbial infections. Today, the incorporation of immunostimulants into microparticles provides us new tools to enhance disease resistance in marine larviculture. In this study, we prepared alginate microparticles loaded with the model antigen fluorescein isothiocyanate conjugated-bovine serum albumin. Optimum concentrations of alginate and CaCl2, the correct alginate viscosity and the appropriate preparatory conditions led to the creation of desirable microparticles with the correct size for oral feeding in gnotobiotic European sea bass larvae. The prepared alginate microparticles were stable in sea water and were successfully ingested by gnotobiotic sea bass larvae at day after hatching 7 without causing any negative effects. Results suggest the suitability of this drug delivery system for targeting the innate immune system of fish larvae in order to enhance disease resistance and thus reduce mortality in larviculture.
Vibrio anguillarum causes high mortality in European sea bass (Dicentrarchus labrax) larviculture. In this study, we evaluated if the recombinant sea bass ferritin-H could stimulate the innate immune system of gnotobiotic European sea bass larvae resulting in protection against a V. anguillarum challenge. We also evaluated the effect of a V. anguillarum infection on the transcription of immune-related genes in gnotobiotic European sea bass larvae. Recombinant sea bass ferritin-H was produced, encapsulated in calcium alginate microparticles and orally delivered to sea bass larvae at seven days after hatching. Our results showed V. anguillarum caused an acute infection, resulting in high mortality. The infection significantly upregulated the expression of tlr3, tlr5, cas1, il1β, tnfα, mif, il10, cc1, cxcl8 at 18, 24 and 36 h post infection, but not of the chemokine receptor genes cxcr4 and ccr9. There was no protective effect of ferritin-H. Remarkably, ferritin-H caused significantly higher transcript levels for cxcr4 and ccr9. Sea bass ferritin-H was more likely involved in immune-suppression and results point in the direction of a negative regulation of CXCR4 resulting in inhibition of cell proliferation, differentiation and migration which is detrimental to innate immunity and might explain the non-protective effect of ferritin-H in fish larvae.
Simulium (Nevermannia) ledangense sp. nov. is described from females, males, pupae and mature larvae from Peninsular Malaysia. This new species is assigned to the Simulium feuerborni species-group of the subgenus Nevermannia, and is characterized by the pupa having a very long stalk of the ventral paired gill filaments, which is almost five times longer than the interspiracular trunk and female tergites of segments 2 and 5 to 7 shiny. Taxonomic notes are given to distinguish this new species from three known species of the S. feuerborni species-group from Malaysia.
Simulium (Gomphostilbia) hiroyukii is described based on females, males, pupae and larvae collected in Mount Murud, Sarawak, Malaysia. This new species is assigned to the Simulium darjeelingense species-group of the subgenus Gomphostilbia, and is characterized by the darkened fore coxae and the pupal gill with eight long filaments, of which middle and dorsal triplets have elongated primary and secondary stalks, respectively.
To investigate the breeding habitat preference of black flies, a comprehensive black fly survey was conducted for the first time in Peninsular Malaysia. Preimaginal black flies (pupae and larvae) were collected manually from 180 stream points encompassing northern, southern, central and east coast of the Peninsular Malaysia. A total of 47 black fly species were recorded in this study. The predominant species were Simulium trangense (36.7%) and Simulium angulistylum (33.3%). Relatively common species were Simulium cheongi (29.4%), Simulium tani (25.6%), Simulium nobile (16.2%), Simulium sheilae (14.5%) and Simulium bishopi (10.6%). Principal Component Analysis (PCA) of all stream variables revealed four PCs that accounted for 69.3% of the total intersite variance. Regression analysis revealed that high species richness is associated with larger, deeper, faster and higher discharge streams with larger streambed particles, more riparian vegetation and low pH (F=22.7, d.f.=1, 173; P<0.001). Relationship between species occurrence of seven common species (present in >10% of the sampling sites) was assessed. Forward logistic regression analysis indicated that four species were significantly related to the stream variables. S. nobile and S. tani prefer large, fast flowing streams with higher pH, large streambed particles and riparian trees. S. bishopi was commonly found at high elevation with cooler stream, low conductivity, higher conductivity and more riparian trees. In contrast, S. sheilae was negatively correlated with PC-2, thus, this species commonly found at low elevation, warmer stream with low conductivity and less riparian trees. The results of this study are consistent with previous studies from other geographic regions, which indicated that both physical and chemical stream conditions are the key factors for black fly ecology.
Sexually anomalous individuals, typically intersexes or gynandromorphs, bear a mixture of male and female traits. Twelve sexually anomalous individuals of the black fly Simulium (Gomphostilbia) trangense Jitklang, Kuvangkadilok, Baimai, Takaoka & Adler were discovered among 49 adults reared from pupae. All 12 sexually anomalous adults were parasitized by mermithid nematodes, although five additional parasitized adults had no overt external anomalies. Sequence analysis of the 18S rRNA gene revealed that the mermithids, possibly representing a new species, are related to Mesomermis spp., with genetic distances of 5.09-6.87%. All 12 anomalous individuals had female phenotypical traits on the head, thorax, forelegs, midlegs, and claws, but male features on the left and right hind basitarsi. One individual had mixed male and female genitalia. The findings are in accord with the trend that mermithid infections are associated with sexually anomalous adult black flies.