Potential application of Gustatory Receptor 1 (CmegGr1) gene as a molecular marker for identification of Chrysomya megacephala (Diptera: Calliphoridae).

Chrysomya megacephala larvae can easily be identified using cheap traditional microscopy techniques. Nevertheless, identification using taxonomy keys may be hampered, if the morphological characteristics of the larvae are incomplete, or immature for microscopic identification. To overcome the difficulty of species determination, molecular identification has gained relevance and is applied in forensic investigations. This study aimed to identify a novel target gene, known as the gustatory receptor 1 gene (CmegGr1), which has never been used for identification. The third instar larvae of Ch. megacephala (n = 30) and eight other forensically important fly species were obtained from two sources; rabbit carcasses and the Forensic Entomology Unit collection. Their DNAs were extracted and the CmegGr1 gene was amplified using polymerase chain reaction (PCR). The resulting sequences were subjected to phylogenetic analysis. A 209 bp fragment of the CmegGr1 gene was successfully amplified in 80% (24/30) of Ch. megacephala samples, while all of the non-Ch. megacephala species were not amplified. The phylogenetic analysis revealed that the evolutionary tree of CmegGr1 shares many traits with the 21a gustatory receptors of Calliphora stygia and Lucilia cuprina (Gr21a), which are also classified as necrophagous fly species. The high specificity of species identification was demonstrated in the present study using DNA barcoding, which led to the conclusion that the CmegGr1 gene could serve as an alternative marker for identifying Ch. megacephala.

Potential application of Gustatory Receptor 1 (CmegGr1) gene as a molecular marker for identification of Chrysomya megacephala (Diptera: Calliphoridae)

@#Chrysomya megacephala larvae can easily be identified using cheap traditional microscopy techniques. Nevertheless, identification using taxonomy keys may be hampered, if the morphological characteristics of the larvae are incomplete, or immature for microscopic identification. To overcome the difficulty of species determination, molecular identification has gained relevance and is applied in forensic investigations. This study aimed to identify a novel target gene, known as the gustatory receptor 1 gene (CmegGr1), which has never been used for identification. The third instar larvae of Ch. megacephala (n = 30) and eight other forensically important fly species were obtained from two sources; rabbit carcasses and the Forensic Entomology Unit collection. Their DNAs were extracted and the CmegGr1 gene was amplified using polymerase chain reaction (PCR). The resulting sequences were subjected to phylogenetic analysis. A 209 bp fragment of the CmegGr1 gene was successfully amplified in 80% (24/30) of Ch. megacephala samples, while all of the non-Ch. megacephala species were not amplified. The phylogenetic analysis revealed that the evolutionary tree of CmegGr1 shares many traits with the 21a gustatory receptors of Calliphora stygia and Lucilia cuprina (Gr21a), which are also classified as necrophagous fly species. The high specificity of species identification was demonstrated in the present study using DNA barcoding, which led to the conclusion that the CmegGr1 gene could serve as an alternative marker for identifying Ch. megacephala.

Three repellent gels that contain essential oils from local Malaysian plants against dengue vector.

The essential oils of Litsea elliptica, Piper aduncum, and Piper sarmentosum were prepared as repellents in gel formulation, and their repellent properties against Aedes aegypti were experimentally investigated. The lowest effective doses against adult mosquitoes were 0.8%, 0.5%, and 0.4% for Lit. elliptica, P. sarmentosum and P. aduncum, respectively. In laboratory testing with human subjects, all three gels provided over 90.0% repellency at one hour after application and over 80.0% repellency at four hours, compared with 100% and 95.8% protection after one and four hours, respectively, by DEET. In the field, gels with ED95 concentrations of Lit. elliptica, P. aduncum, and P. sarmentosum essential oils provided 99.3%, 97.5%, and 100% protection, respectively, at two hours. The physical properties and biological stability of the three repellents after storage in hot and cold conditions were also compared. In conclusion, all three gels have the potential for development as repellents against Ae. aegypti.

The formulation of the essential oil of Piper aduncum Linnaeus (Piperales: Piperaceae) increases its efficacy as an insect repellent.

The essential oil (EO) of Piper aduncum Linnaeus, known as 'sireh lada' to locals Malaysian, has the potential to be used as an alternative to synthetic insect repellents such as N,N-diethyl-meta-toluamide. However, the EO's efficacy as a repellent decreases after application due to the high volatility of its active ingredients. A number of studies have showed that optimizing the formulation of plant-based EOs can improve their efficacy as repellents. The present study sought to evaluate the effectiveness of 10% P. aduncum EO in ethanol and in three different semisolid formulations: ointment, cream and gel. These formulations were tested on Aedes aegypti under laboratory conditions. Each formulation was applied to the subject's hands, which were then inserted into a cage containing 25 nulliparous A. aegypti. The number of mosquitoes landing on or biting each subject's hand was recorded, and the repellency percentage, landing/biting percentage and protection time for each of the formulations were compared. There were no statistically significant differences between the semisolid EO formulations with regards to the repellency percentage and the landing/biting percentage at 4 h post-application. All three semisolid EO formulations were able to repel >65% of the A. aegypti at 4 h post-application. The EO ointment formulation provided a protection time (182.5 ± 16.01 min) that was statistically significantly longer than that associated with the EO gel formulation (97.5 ± 14.93 min). Meanwhile, the EO cream formulation provided a protection time of 162.5 ± 6.29 min. As the EO cream and ointment formulations displayed better repellent properties than the EO gel formulation, they appear to be the most promising P. aduncum EO formulations to be developed and commercialized as alternatives to synthetic repellents.

Forensic entomology of high-rise buildings in Malaysia: Three case reports.

The distributions of flies are not only confined to ground level but can also be at higher altitudes. Here, we report three forensic cases involving dipterans in high-rise buildings in Kuala Lumpur, Malaysia. Case 1 involved a corpse of adult female found at the top floor of a fifteen-story apartment. Case 2 dealt with a body of a 75-year-old female discovered in a bedroom on the eleventh floor of an eighteen-story building, while Case 3 was a 52-year-old male found in his fifth floor shop house. Interestingly, entomological analysis revealed that all corpses were infested with similar Dipterans: Megaselia scalaris (Loew) (Diptera: Phoridae), Synthesiomyia nudiseta (Wulp) (Diptera: Muscidae) and sarcophagid (Diptera: Sarcophagidae). The first two species were commonly associated with corpses found indoors at ground level. We noted the additional occurrence of blowflies Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae) and Chrysomya rufifacies Macquart (Diptera: Calliphoridae) larvae in Case 2 and Case 3, respectively. Findings from this study are significant as they demonstrate that certain groups of fly can locate dead bodies even in high-rise buildings. Forensic entomofauna research on corpses found at high elevation is scarce and our study has highlighted the peculiarity of the fly species involved in Malaysia.

Forensic entomology of high-rise buildings in Malaysia: Three case reports

The distributions of flies are not only confined to ground level but can also be at higher altitudes. Here, we report three forensic cases involving dipterans in high-rise buildings in Kuala Lumpur, Malaysia. Case 1 involved a corpse of adult female found at the top floor of a fifteen-story apartment. Case 2 dealt with a body of a 75-year-old female discovered in a bedroom on the eleventh floor of an eighteen-story building, while Case 3 was a 52-year-old male found in his fifth floor shop house. Interestingly, entomological analysis revealed that all corpses were infested with similar Dipterans: Megaselia scalaris (Loew) (Diptera: Phoridae), Synthesiomyia nudiseta (Wulp) (Diptera: Muscidae) and sarcophagid (Diptera: Sarcophagidae). The first two species were commonly associated with corpses found indoors at ground level. We noted the additional occurrence of blowflies Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae) and Chrysomya rufifacies Macquart (Diptera: Calliphoridae) larvae in Case 2 and Case 3, respectively. Findings from this study are significant as they demonstrate that certain groups of fly can locate dead bodies even in high-rise buildings. Forensic entomofauna research on corpses found at high elevation is scarce and our study has highlighted the peculiarity of the fly species involved in Malaysia.

Residual effects of TMOF-Bti formulations against 1(st) instar Aedes aegypti Linnaeus larvae outside laboratory.

OBJECTIVE: To evaluate the effectiveness and residual effects of trypsin modulating oostatic factor-Bacillus thuringiensis israeliensis (TMOF-Bti) formulations against Aedes aegypti (Ae. aegypti) (L.) larvae at UKM Campus Kuala Lumpur. METHODS: Twenty first instar Ae. aegypti larvae were added in each bucket containing 4 L of water supplied with crushed dried leaf powder as their source of food. Combination of TMOF-Bti in rice husk formulation with the following weights viz 10, 25, 50 and 100 mg, respectively in duplicate was distributed in the buckets; while TMOF-Bti in wettable powder formulation each weighing viz 2, 5, 10 and 20 mg, respectively in duplicate was also placed in the buckets. The control buckets run in duplicate with 4 L of water and 20 first instar Ae. aegypti larvae. All buckets were covered with mosquito netting. Larval mortality was recorded after 24 hours and weekly for five weeks. A new batch of 20 1(st) instar larvae Ae. aegypti was introduced into each bucket weekly without additional TMOF-Bti rice husk formulation or wettable powder. The experiment was repeated for four times. RESULTS: The result of the study showed that all formulations were very effective on the first two weeks by giving 100% larval mortality for all concentrations applied. The TMOF (2%) + Bti (2%) had a good residual effect until the end of 3(rd) week, TMOF (4%) + Bti (4%) until 4(th) week, wettable powder TMOF (20%) + Bti (20%) until the third week. CONCLUSIONS: From the results it can be concluded that the TMOF-Bti formulations can be utilized in dengue vector control.

Dengue Infections And Circulating Serotypes In Negeri Sembilan, Malaysia

This study observed the pattern of reported dengue infections, clinical manifestations, and circulating dengue serotypes in Negeri Sembilan, Malaysia. The aim of this study was to determine the co-circulation of the four different dengue virus serotypes in Negeri Sembilan. We analyzed the surveillance data (VEKPRO) from Negeri Sembilan State Health Department and National Public Health Laboratory, Malaysia on reported dengue infections from 1st January 2010 to 31st December 2010. There were 1466 reported dengue infections, 1342 (91.5%) cases were dengue fever (DF) and 124 (8.5%) were dengue hemorrhagic fever (DHF). The mean age was 32.2± 15.8 years old and most were young adults, aged 15 years old and older. Males (p < 0.05), and those residing in Seremban district (p < 0.05) were more likely to get dengue infections. Symptoms presented upon admission were fever (100%), headache (99.9%), myalgia and arthralgia (98.8%), rash(24.2%), petechiae (16.0%),bleeding tendencies (7.0%) and neurological deficits(1.2%). All four dengue serotypes (DEN 1 – 4) were present, the pre-dominant serotype was DEN-3, noted in January, then existed together with DEN-2 until around May. DEN-1 was the most pre-dominant circulating dengue serotype afterwards, reaching a peak in December 2010. Dengue affected all age groups particularly young adults and males. Most cases reported were in urban areas and Seremban district. Most of the dengue infections occurred in the first half of the year, with the DEN-2 and DEN-3 serotypes being the most predominant.

Ovitrap surveillance of the dengue vectors, Aedes (Stegomyia) aegypti (L.) and Aedes (Stegomyia) albopictus Skuse in selected areas in Bentong, Pahang, Malaysia.

Ovitrap surveillance was conducted in methodically selected areas in Bentong, Pahang, Malaysia from June 2008 till December 2009 in order to identify insular sites with stable Aedes aegypti population. Eleven sites were surveyed in Bentong district, Pahang, and one of these locations (N3º33' E101º54') was found to have an ovitrap index of Ae. aegypti and Aedes albopictus ranging from 8%-47% and 37%-78% respectively, indicating that this site could be a high-risk area for dengue outbreak. Ae. aegypti larvae were found in both indoor and outdoor ovitraps (p>0.05) while significant difference between the populations of Ae. albopictus larvae from indoors and outdoors was observed (p<0.01). Data collected in this study could provide important entomological information for designing an effective integrated vector control programme to combat Aedes mosquitoes in this area.

Adulticidal activity of some Malaysian plant extracts against Aedes aegypti Linnaeus.

The adulticidal activity of methanol extracts from three Malaysian plants namely Acorus calamus Linn., Litsea elliptica Blume and Piper aduncum Linn. against adult of Aedes aegypti (L.) were studied. Standard WHO bioassay tests were used to evaluate the effectiveness of these plant extracts. The hexane fraction from methanol extract of Acorus calamus rhizome was the most effective, exhibiting LC50 and LC90 values of 0.04 mgcm(-2) and 0.09 mgcm(-2) respectively. For L. elliptica, the methanol fraction also displayed good adulticidal property with the LC50 and LC90 values of 0.11 mgcm(-2) and 6.08 mgcm(-2) respectively. It is found that hexane fraction of the P. aduncum crude extract was the least effective among the three plants showing LC50 and LC90 values of 0.20 mgcm(-2) and 5.32 mgcm(-2), respectively. However, although A. calamus showed lowest LC values, the LT50 results indicated that the methanol fraction of L. elliptica was most potent extract among the extracts tested.