Fourth instars larvae of freshwater midge Chironomus javanus (Diptera, Chironomidae) were exposed for a 4-day period in laboratory conditions to a range of copper (Cu), cadmium (Cd), zinc (Zn), lead (Pb), nickel (Ni), iron (Fe), aluminium (Al) and manganese (Mn) concentrations. Mortality was assessed and median lethal concentrations (LC(50)) were calculated. LC(50) increased with the decrease in mean exposure times, for all metals. LC(50)s for 96 hours for Cu, Cd, Zn, Pb, Ni, Fe, Al and Mn were 0.17, 0.06, 5.57, 0.72, 5.32, 0.62, 1.43 and 5.27 mg/L, respectively. Metals bioconcentration in C. javanus increases with exposure to increasing concentrations and Cd was the most toxic to C. javanus, followed by Cu, Fe, Pb, Al, Mn, Zn and Ni (Cd > Cu > Fe > Pb > Al > Mn > Zn > Ni). Comparison of LC(50) values for metals for this species with those for other freshwater midges reveals that C. javanus is equally or more sensitive to metals than most other tested dipteran.
Biological control using pathogenic microsporidia could be an alternative to chemical control of the diamondback moth (DBM) Plutella xylostella (Lepidoptera: Plutellidae). The microsporidium Nosema bombycis (NB) is one of the numerous pathogens that can be used in the Integrated Pest Management (IPM) of DBM. However, its pathogenicity or effectiveness can be influenced by various factors, particularly temperature. This study was therefore conducted to investigate the effect of temperature on NB infection of DBM larvae. Second-instar larvae at different doses (spore concentration: 0, 1×10²,1×10³,1×10⁴, and 1×10⁵) at 15°, 20°, 25°, 30° and 35°C and a relative humidity(RH) of 65% and light dark cycle (L:D) of 12∶12. Larval mortality was recorded at 24 h intervals until the larvae had either died or pupated. The results showed that the spore concentration had a significant negative effect on larval survival at all temperatures, although this effect was more pronounced (92%) at 35°C compared with that at 20 and 30°C (≃50%) and 25°C (26%). Histological observations showed that Nosema preferentially infected the adipose tissue and epithelial cells of the midgut, resulting in marked vacuolization of the cytoplasm. These findings suggest that Nosema damaged the midgut epithelial cells. Our results suggest that Nosema had a direct adverse effect on DBM, and could be utilized as an important biopesticide alternative to chemical insecticides in IPM.
Myiasis occurs when living tissues of mammals are invaded by eggs or larvae of flies, mainly from the order of Diptera. Most of the previousty reported cases are in the tropics and they were usually associated with inadequate personal hygiene, sometimes with poor manual dexterity. This report describes two cases of oral myiasis in cerebral palsy patients in Seremban General Hospital, Malaysia. This article also discusses the therapeutic property of maggots and highlights the importance of oral health care in the special needs patients.
Many studies have been done on various species of insects to investigate their potential use in industries. This is because insects have high protein content which could be further manipulated. Due to its eating habit, Zophobas morio larvae, also known as super mealworm has been shown to have high amylase activity. In this study, amylase from super mealworm has been immobilized via Cross-Linked Enzyme Aggregates (CLEA) technique and its kinetic performance, evaluated. CLEA is one of the best immobilization method with respect to enzyme stability and reusability. Kinetic performance of both free and CLEA-amylase were evaluated based on the Michaelis-Menten model. Results obtained based on Hanes-Woolf, LineweaverBurk, Eadie-Hofstee and Hyperbolic Regression plots showed that the kinetic parameters, Vmax and KM, changed upon immobilization. For CLEA-amylase, Hanes-Woolf plot showed the bestfitted model based on R2 with Vmax= 1.068 mM/min and KM= 0.182 mM, however, LineweaverBurk plot was used to obtain the kinetic parameters for free amylase, with Vmax and KM of 17.230 mM/min and 2.470 mM, respectively. Thus it is observed that upon immobilization, Vmax for amylase dropped appreciably, however, much lower substrate concentration is needed to saturate the enzymatic sites to reach its maximum catalytic efficiency. The result from this study might open the new path in discovering the potential use of insects in industrial applications, for example, making use of the recovered enzymes in the detergent industry.
We report a case of human intestinal myiasis in a 41-yr-old female patient presented at a clinic in Seri Kembangan, Selangor, Malaysia. Larvae passed out in the patient's feces were sent to the Department of Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia. DNA barcoding confirmed the second case of intestinal myiasis in Malaysia involving the larvae of Clogmia albipunctatus (Duckhouse) (Diptera: Psychodidae). We review reported cases of myiasis and discuss the present case of intestinal myiasis in an urban patient.
We report an unusual cause of gastrointestinal infection occurring in a 1-year-old infant patient who was brought to a public hospital in Kuala Lumpur, Malaysia. Larvae passed out in the patient's feces were confirmed by DNA barcoding as belonging to the species, Lasioderma serricorne (F.), known as the cigarette beetle. We postulate that the larvae were acquired from contaminated food and were responsible for gastrointestinal symptoms in the patient. To our knowledge, this the first report of human canthariasis caused by larvae of L. serricorne.
Mutant lethal giant larvae (lgl) flies (Drosophila melanogaster) are known to develop epithelial tumors with invasive characteristics. The present study has been conducted to investigate the influence of melatonin (0.025 mM) on behavioral responses of lgl mutant flies as well as on biochemical indices (redox homeostasis, carbohydrate and lipid metabolism, transaminases, and minerals) in hemolymph, and head and intestinal tissues. Behavioral abnormalities were quantitatively observed in lgl flies but were found normalized among melatonin-treated lgl flies. Significantly decreased levels of lipid peroxidation products and antioxidants involved in redox homeostasis were observed in hemolymph and tissues of lgl flies, but had restored close to normalcy in melatonin-treated flies. Carbohydrates including glucose, trehalose, and glycogen were decreased and increased in the hemolymph and tissues of lgl and melatonin-treated lgl flies, respectively. Key enzymes of carbohydrate metabolism showed a significant increment in their levels in lgl mutants but had restored close to wild-type baseline levels in melatonin-treated flies. Variables of lipid metabolism showed significantly inverse levels in hemolymph and tissues of lgl flies, while normalization of most of these variables was observed in melatonin-treated mutants. Lipase, chitinase, transaminases, and alkaline phosphatase showed an increment in their activities and minerals exhibited decrement in lgl flies; reversal of changes was observed under melatonin treatment. The impairment of cognition, disturbance of redox homeostasis and metabolic reprogramming in lgl flies, and restoration of normalcy in all these cellular and behavioral processes indicate that melatonin could act as oncostatic and cytoprotective agents in Drosophila.
The sensitivity of larval paralysis assay (LPA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide-formazan (MTT-formazan) assay was compared to evaluate the anthelmintic activity of plant extracts. In this study, the methanolic extract of Azadirachta indica (neem) was evaluated for its activity against the infective-stage larvae (L(3)) of susceptible and resistant Haemonchus contortus strains using the two aforementioned assays. In both in vitro assays, the same serial concentrations of the extract were used, and the median lethal concentrations were determined to compare the sensitivity of both assays. The results revealed a significant difference (P < 0.05) in the sensitivity of the LPA and the MTT-formazan assay. The MTT-formazan assay is more feasible for practical applications because it measured the L(3) mortality more accurately than LPA. This study may help find a suitable assay for investigating the anthelmintic activity of plant extracts against trichostrongylid nematodes.
This paper reports total nematode anthelmintic resistance towards albendazole, fenbendazole, levamisole and ivermectin in a commercial sheep farm located in Terengganu, Malaysia. Faecal Egg Count Reduction Test (FECRT) was conducted on 25 sheep, where five sheep in each group were treated with the respective four anthelmintics based on live bodyweight. The balance of five sheep placed in the control group were not treated with any anthelmintics. At day 13 post-treatment, faecal egg count was conducted and nematode worm egg count reduction percentage was calculated to determine the resistance status towards the respective anthelmintics tested. Results showed that nematodes were resistant to all the anthelmintics tested, namely albendazole, fenbendazole, levamisole and ivermectin with reduction percentage of 87%, 46%, 94% and 68%, respectively. Subsequently, the third stage larvae of Haemonchus contortus and Trichostrongylus colubriformis recovered from post-treatment faecal cultures were subjected to allele-specific polymerase chain reaction (AS-PCR) assay to determine the presence of the benzimidazole resistance gene. This study reports the occurrence of the classical F200Y mutation in the isotype 1 βtubulin gene, for the first time in Malaysia.
Using the cow-baited trap (CBT) method, 1,845 Anopheles mosquitos, comprising 14 species, were caught in malaria-endemic area of Hulu Perak district, Peninsular Malaysia. The two dominant species were An. barbirostris (18.59%) and An. aconitus (18.86%). Anopheles maculatus, the main malaria vector, constituted 9.11% of the total number of mosquitos sampled. Three hundred and seventy-seven Anopheles larvae, comprising 8 species, were sampled using the North Carolina Biological Station dipper. Anopheles barbirostris larvae amounted to 64.69% of the total number of larvae; An. aconitus accounted for 10.65% of larvae. Seven habitats were identified as breeding places of Anopheles. Most species were found to breed in paddies, fishponds, and rivers. Other less popular habitats were temporary pools, mountain streams, and spring wells.
This study was carried out in an oil palm plantation in Bandar Baharu, Kedah using monkey carcasses and focuses in documenting the decomposition and dipteran colonization sequences in 50 days. This is the first study of Diptera associated with the exploitation of carcasses conducted in the north of peninsular Malaysia during the dry and wet seasons thereat. During the process of decomposition in both seasons, five phases of decay were recognized namely fresh, bloated, active decay, advance decay and dry remain. In this decomposition study, biomass loss of carcass occurred rapidly during the fresh to active decay stage due to the colonization and feeding activity of the Diptera larvae. The duration of the fresh and bloated stages of decay were the same in wet and dry seasons but later stages of decay were markedly shorter during the wet season. Twenty one species of adult Diptera were identified colonizing carcasses in the study period. Among the flies from the family Calliphoridae, Chrysomya megacephala Fabricius and Chrysomya nigripes Aubertin were recognized as the earliest arrivals on the first day of exposure. Adult Ch. nigripes was abundant for approximately two weeks after placement of the carcasses. By comparing the percentages of adults collected during the study period, the calliphorids abundance in percentages in wet season was 50.83%, but in dry season, the abundance was only about 35.2%. In contrast, the percentage of Sphaeroceridae in wet season was only 3.33%, but in the dry season, the abundance was 20.8%. Dipteran in family Phoridae, Piophilidae, Sepsidae, Drosophilidae and Dolichopodidae colonized the carcasses for a long period of time and were categorized as long term colonizers.
Despite major insecticide-based vector control programs, dengue continues to be a major threat to public health in urban areas. The reasons for this failure include the emergence of insecticide resistance and the narrowing of the spectrum of efficient products. Cigarette butts (CBs), the most commonly discarded piece of waste, also represent a major health hazard to human and animal life. CBs are impregnated with thousands of chemical compounds, many of which are highly toxic and none of which has history of resistance in mosquitoes. This study was performed to examine whether exposure to CB alters various biological parameters of parents and their progeny. We examined whether the mosquito changes its ovipositional behaviors, egg hatching, reproductive capacity, longevity and fecundity in response to CB exposure at three different concentrations. Females tended to prefer microcosms containing CBs for egg deposition than those with water only. There were equivalent rates of eclosion success among larvae from eggs that matured in CB and water environments. We also observed decreased life span among adults that survived CB exposure. Extracts of CB waste have detrimental effects on the fecundity and longevity of its offspring, while being attractive to its gravid females. These results altogether indicate that CB waste indirectly affect key adult life traits of Aedes aegypti and could conceivably be developed as a novel dengue vector control strategy, referring to previously documented direct toxicity on the larval stage. But this will require further research on CB waste effects on non-target organisms including humans.
Specification on residual action of a possible alternative insecticide derived from plant materials is important to determine minimum interval time between applications and the environmental persistence of the biopesticides. The objective of this study is to evaluate crude acethonilic extract of Ipomoea cairica leaves for its residual and persistence effects against Culex quinquefasciatus larvae. Wild strain of Cx. quinquefasciatus larvae were used for the purpose of the study. Two test designs, replenishment of water and without replenishment of water were carried out. For the first design, a total of 10 ml of test solution containing Ip. cairica extracts was replenished daily and replaced with 10 ml of distilled water. For the second design, treatment water was maintained at 1500 ml and only evaporated water was refilled. Larval mortality was recorded at 24 hours post-treatment after each introduction period and trials were terminated when mortality rate falls below 50%. Adult emergences from survived larvae were observed and number of survivals was recorded. For the non-replenishment design, mortality rate significantly reduced to below 50% after 28 days, meanwhile for replenishment of water declined significantly after 21 days (P < 0.05). There was no adult emergence observed up to seven days for non-replenishment and first two days for replenishment of water design. The short period of residual effectiveness of crude acethonilic extract of Ip. cairica leaves with high percentage of larval mortality on the first few days, endorses fewer concerns of having excess residues in the environment which may carry the risk of insecticide resistance and environmental pollution.
Annually, 4.5 trillion cigarette butts (CBs) are flicked into our environment. Evidence exists that CB waste is deadly to aquatic life, but their lethality to the aquatic life of the main dengue vector is unknown. CBs are full of toxicants that occur naturally, during planting and manufacturing, which may act as larvicidal agents. We assessed Aedes aegypti vulnerability to Marlboro butts during its development. Overall, CBs showed insecticidal activities against larvae. At early phases of development, mortality rates were much higher in two CBs solution (2CBSol) and 3CBSol microcosms (MICRs). Larval survival gradually decreased with development in 1CBSol-MICRs. However, in great presence of CBs, mortality was high even for the late developmental stages. These results suggest that A. aegypti larvae are vulnerable to CB presence in their habitats, but this effect was seen most during the early developmental phases and in the presence of increased amounts of cigarette remnants. CB filters are being used as raw material in many sectors, i.e., brick, art, fashion, plastic industries, as a practical solution to the pollution problem, the observed butt waste toxicity to mosquito larvae open new avenues for the identification of novel insecticide products.
Moisture plays a major role in the dynamics of mosquito populations, especially those breeding in container habitats. Despite this importance, the role of moisture conditions as they affect oviposition and egg development in Aedes vectors remains largely unexplored. We investigated the effect of exposing gravid female Aedes albopictus mosquitoes and their eggs to different moisture levels (MLs) for various periods on oviposition and hatching. Overall, high-moisture substrates (HMSs; 66% and 72%) provided better environments for egg laying. The timing of initial egg laying was far longer at the lowest substrate moisture level (LSML, 25% and 41.2%) than at HMSs. The numbers of eggs laid were much lower in the drier environments. At LSMLs, gravid females retained increasing numbers of mature eggs until death, and egg retention decreased gradually with increasing ML. The HMSs also provided better environments for larval eclosion. The numbers of eggs hatched were lower at the LSML than the HSML environment. No egg hatching occurred after 1 h exposure to moisture. However, egg hatching occurred by installment, with spontaneous hatching (SH) increasing gradually with increasing ML. High-moisture conditions combined with long exposure (30 h and 48 h) favored SH. These results suggest that Ae. albopictus females can respond to better moisture conditions for increased success of embryonation and larval eclosion. This information may be useful in the colonization of floodwater Aedes species.
Discarded cigarette butts (DCB) waste occurs worldwide, pollutes landscapes, is unsightly, and results in added debris removal costs. There is, therefore, a great deal of current interest in making use of DCBs in beneficial ways. Despite evidence that DCBs are harmful to water fleas (Daphnia magna), which breed in aquatic environments as do mosquito larvae, their impact on dengue vectors is unknown. We examined whether Aedes albopictus alters its ovipositional responses, larval eclosion, and development in response to presence of DCBs in its habitats. We found oviposition activity in DCB-treated water similar to that of control water and that ovipositional activity in DCB solutions steadily increased over time as those solutions aged to 10 days. Larval eclosion was initially suppressed on day 1 in DCB solution, but increased thereafter to levels similar to control larval eclosion rates. The DCB-water solutions produced significantly higher mortality in both 1st and 2nd instars over control larvae for several days after initial exposure. Mortality rates decreased sharply 3 to 5 days postexposure as DCBs continued to decompose. We found increased survival rates during late development, but daily input of fresh DCBs prevented most young larvae from completing development. Taken together, these observations suggest that decomposing did not deter gravid Ae. albopictus females from ovipositing in treated containers and that DCB solutions had larvicidal effects on early instars. Our results are discussed in the context of DCB use to control container-breeding Ae. albopictus, a competent dengue vector in Asia and other parts of the world.
Studies were carried out on the bioefficacy and residual activity of Bacillus thuringiensis israelensis H-14 (Bti) (water-dispersible granules of VectoBac ABG 6511 and liquid formulations of VectoBac 12AS) and pyriproxyfen (insect growth regulator, Sumilarv 0.5%) as direct applications for control of larvae of Aedes aegypti and Aedes albopictus. Two dosages of each Bti formulation (285 and 570 international toxic units [ITU]/liter) and the integration of both Bti formulations and pyriproxyfen were used for residual tests with 45-liter earthen jars for a period of 4 wk. In 1 test series, the treated water was replenished daily with 6 liters of seasoned untreated water. In the 2nd test series, the water in the jars was topped up to the 40-liter level during evaluation. Neither Bti formulation remained effective for a full week. Water-dispersible Bti granules provided effective initial control activity against Ae. aegypti and Ae. albopictus for both test designs (with replenishment and without replenishment of water). The higher dosage (570 ITU/liter) for both Bti formulations was only partially effective at the end of 1 wk after being diluted. After 1 wk, water-dispersible Bti granules provided greater larval mortality than did liquid Bti formulation against both mosquito species when integrated with pyriproxyfen. Pyriproxyfen (79.5 and 159 mg/liter) on its own showed low larvicidal activity but provided very effective control of adult emergence. In this study, integration of Bti (285 and 570 ITU/liter) with pyriproxyfen (79.5 mg/liter) extended the duration of partial larval control somewhat, but live larvae persisted throughout the 4-wk test. The integration effect was more obvious when water-dispersible Bti granules were integrated with pyriproxyfen than when liquid Bti was used. Integration of Bti with pyriproxyfen had a negative effect on adult emergence, which was completely inhibited by pyriproxyfen after day 1. Daily replenishment of water increased Bti activity and provided slightly better larval control. Aedes albopictus and Ae. aegypti were both completely susceptible to the higher concentration of Bti and pyriproxyfen in both test designs (with replenishment and without replenishment of water).
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.
Although the microbiome of blood-feeding insects serves an integral role in host physiology, both beneficial and pathogenic, little is known of the microbial community of black flies. An investigation, therefore, was undertaken to identify culturable bacteria from one of Malaysia's most common black flies, Simulium tani Takaoka and Davies, using 16S rDNA sequencing, and then evaluate the isolates for antibiotic resistance and virulence genes. A total of 20 isolates representing 11 bacterial species in four genera were found. Five isolates showed β-hemolysis on Columbia agar, and virulence genes were found in three of these isolates. Some degree of resistance to six of the 12 tested antibiotics was found among the isolates. The baseline data from this study suggest rich opportunities for comparative studies exploring the diversity and roles of the microbiome of S. tani and other Southeast Asian black flies.
A new bufonid amphibian, belonging to a new monotypic genus, is described from the Andaman Islands, in the Bay of Bengal, Republic of India, based on unique external morphological and skeletal characters which are compared with those of known Oriental and other relevant bufonid genera. Blythophryne gen. n. is distinguished from other bufonid genera by its small adult size (mean SVL 24.02 mm), the presence of six presacral vertebrae, an absence of coccygeal expansions, presence of an elongated pair of parotoid glands, expanded discs at digit tips and phytotelmonous tadpoles that lack oral denticles. The taxonomic and phylogenetic position of the new taxon (that we named as Blythophryne beryet gen. et sp. n.) was ascertained by comparing its 12S and 16S partial genes with those of Oriental and other relevant bufonid lineages. Resulting molecular phylogeny supports the erection of a novel monotypic genus for this lineage from the Andaman Islands of India.