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.

Acaricidal activity of the essential oils from Citrus hystrix (Rutaceae) and Cymbopogon citratus (Poaceae) on the cattle tick Rhipicephalus (Boophilus) microplus larvae (Acari: Ixodidae).

Rhipicephalus (Boophilus) microplus serves as an important ectoparasite of livestock and a vector of several pathogens resulting in diseases, subsequently affecting the agricultural field as well as the economy. The extensive use of synthetic acaricides is known to cause resistance over time and therefore a much safer, effective and environmentally friendly alternative to overcome tick infestation should be implemented. Larval immersion tests (LIT) were done to evaluate the effects of Citrus hystrix (Family: Rutaceae) and Cymbopogon citratus (Family: Poaceae) essential oils (EOs) for their individual and combined (1:1) acaricidal activity against the cattle tick. Results showed that LC50 and LC90 values in 24 and 48 hours for Cit. hystrix EO were 11.98% and 24.84%, and 10.95% and 21.71% respectively. LC50 and LC90 values for Cym. citratus EO were 1.21% and 6.28%, and 1.05% and 6.12% respectively. The mixture of EOs from two plants in 1:1 ratio (Cit. hystrix 50%: Cym. citratus 50%) was found to exhibit antagonistic effect (synergistic factor < 1). The 24 hours and 48 hours LC50 and LC90 values for combined EOs were 1.52% and 2.84%, and 1.50% and 2.76% respectively. Individual and combined essential oils were subjected to qualitative analysis using gas chromatography-mass spectrometry (GC-MS) to screen the chemical components present in EOs. Our results showed that the combination of Cit. hystrix and Cym. citratus at 1:1 ratio resulted in an antagonistic effect and the use of Cym. citratus alone is more toxic to R. (B.) microplus, making it a better alternative to chemical based acaricide.

Acaricidal activity of the essential oils from Citrus hystrix (Rutaceae) and Cymbopogon citratus (Poaceae) on the cattle tick Rhipicephalus (Boophilus) microplus larvae (Acari: Ixodidae)

@# Rhipicephalus (Boophilus) microplus serves as an important ectoparasite of livestock and a vector of several pathogens resulting in diseases, subsequently affecting the agricultural field as well as the economy. The extensive use of synthetic acaricides is known to cause resistance over time and therefore a much safer, effective and environmentally friendly alternative to overcome tick infestation should be implemented. Larval immersion tests (LIT) were done to evaluate the effects of Citrus hystrix (Family: Rutaceae) and Cymbopogon citratus (Family: Poaceae) essential oils (EOs) for their individual and combined (1:1) acaricidal activity against the cattle tick. Results showed that LC50 and LC90 values in 24 and 48 hours for Cit. hystrix EO were 11.98% and 24.84%, and 10.95% and 21.71% respectively. LC50 and LC90 values for Cym. citratus EO were 1.21% and 6.28%, and 1.05% and 6.12% respectively. The mixture of EOs from two plants in 1:1 ratio (Cit. hystrix 50%: Cym. citratus 50%) was found to exhibit antagonistic effect (synergistic factor < 1). The 24 hours and 48 hours LC50 and LC90 values for combined EOs were 1.52% and 2.84%, and 1.50% and 2.76% respectively. Individual and combined essential oils were subjected to qualitative analysis using gas chromatography-mass spectrometry (GC-MS) to screen the chemical components present in EOs. Our results showed that the combination of Cit. hystrix and Cym. citratus at 1:1 ratio resulted in an antagonistic effect and the use of Cym. citratus alone is more toxic to R. (B.) microplus, making it a better alternative to chemical based acaricide.

A comparison of different types of ovitraps for outdoor monitoring of Aedes mosquitoes in Kuala Lumpur.

Dengue is a significant public health problem in Malaysia and vector surveillance is one of the important components in a vector control program. Routinely vector surveillance in Malaysia is performed through larval surveys. However, larval surveys have several limitations. Thus ovitraps are used as an alternative method for monitoring dengue vectors. The aim of this study was to determine the effectiveness of Standard Ovitrap (SO), Mosquito larvae Trapping Device (MLTD), Double Sticky Ovitrap (DST) and NPK Fertiliser Trap to monitor the abundance of Aedes mosquitoes. Each ovitrap was placed at four different sampling points and rotated to the next position every one week. Larvae and adult mosquitoes were collected and identified in the laboratory. All four trapping methods successfully collected larvae of Aedes mosquitoes. The mean number of larvae per ovitrap in DST was significantly higher (p < 0.05) compared to SO and MLTD. DST and NPK Fertiliser Trap were capable of capturing adult mosquitoes. Ovitrap Index and the mean number of adults per ovitrap in NPK Fertiliser trap were significantly higher (p < 0.05) as compared to DST. Another set of experiments were conducted to compare NPK Fertiliser traps containing fresh NPK fertiliser solution with those containing stock NPK Fertiliser solution. The fresh solution was prepared fresh while the stock solution was stored for a month before being used in the field. Result shows no significant differences (p > 0.05) between these solutions. Findings from this study conclude that DST is the most effective ovitrap to monitor Aedes larvae, while NPK Fertiliser trap is the most effective ovitrap to trap Aedes adult mosquitoes in the study area.

A comparison of different types of ovitraps for outdoor monitoring of Aedes mosquitoes in Kuala Lumpur

@#Dengue is a significant public health problem in Malaysia and vector surveillance is one of the important components in a vector control program. Routinely vector surveillance in Malaysia is performed through larval surveys. However, larval surveys have several limitations. Thus ovitraps are used as an alternative method for monitoring dengue vectors. The aim of this study was to determine the effectiveness of Standard Ovitrap (SO), Mosquito larvae Trapping Device (MLTD), Double Sticky Ovitrap (DST) and NPK Fertiliser Trap to monitor the abundance of Aedes mosquitoes. Each ovitrap was placed at four different sampling points and rotated to the next position every one week. Larvae and adult mosquitoes were collected and identified in the laboratory. All four trapping methods successfully collected larvae of Aedes mosquitoes. The mean number of larvae per ovitrap in DST was significantly higher (p < 0.05) compared to SO and MLTD. DST and NPK Fertiliser Trap were capable of capturing adult mosquitoes. Ovitrap Index and the mean number of adults per ovitrap in NPK Fertiliser trap were significantly higher (p < 0.05) as compared to DST. Another set of experiments were conducted to compare NPK Fertiliser traps containing fresh NPK fertiliser solution with those containing stock NPK Fertiliser solution. The fresh solution was prepared fresh while the stock solution was stored for a month before being used in the field. Result shows no significant differences (p > 0.05) between these solutions. Findings from this study conclude that DST is the most effective ovitrap to monitor Aedes larvae, while NPK Fertiliser trap is the most effective ovitrap to trap Aedes adult mosquitoes in the study area.

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.

Effects of preservatives and killing methods on morphological features of a forensic fly, Chrysomya megacephala (Fabricius, 1794) larva.

Preservation of larvae retrieved from cadavers is important in ensuring the quality and integrity of entomological specimens used for the estimation of post-mortem interval (PMI). The process of killing and preserving larvae could distort the larvae leading to inaccurate estimation of PMI. In this study, the effects of killing Chrysomya megacephala larvae with hot water at different temperatures and subsequent maintenance in various preservatives were determined. Larvae not killed by hot water but preserved directly were used as control. The types of preservative used were 10% formalin, 70% ethanol and Kahle's solution. The morphological features examined were length, turgidity, curvature and coloration of larvae. Larvae killed in 80ºC hot water have shorter mean length (12.47 ± 2.86 mm) compared to those in 60ºC hot water (12.95 ± 2.69 mm). Increasing the duration of preservation in all types of preservative caused elongations of larvae treated or untreated with hot water. There were no significant changes in larval turgidity preserved in Kahle's solution compared to other two preservatives and were unaffected by the duration of storage. Larvae preserved in Kahle's solution experienced the least changes in coloration and shape compared to other preserved larvae in 70% ethanol or 10% formalin. Larvae directly immersed alive in 70% ethanol experienced the most changes in curvature, coloration and turgidity. This study suggested that killing larvae with hot water at 80ºC and preservation in Kahle's solution is the optimum method resulting in least changes in morphological features of Ch. megacephala larvae.

An occurrence of Synthesiomyia nudiseta (Wulp) (Diptera: Muscidae) from a human corpse in a high-rise building in Malaysia: a case report.

This is the first report of Synthesiomyia nudiseta (Wulp) (Diptera: Muscidae) on a human corpse discovered in a high-rise building in Malaysia. On 5 March 2008, a decomposing body of an adult female was found on the top floor of a thirteen-story building in Kuala Lumpur, Malaysia. Her body was colonized by S. nudiseta larvae, which were normally associated with corpses found indoors at ground level. The post-mortem interval (PMI) was estimated at approximately 5 to 9 days. This case is significant as it demonstrates that this species can locate a dead body even in a high-rise building. Further findings of fly distribution especially in high-rise buildings should be reported to assist entomologists in PMI analysis.

An occurrence of Synthesiomyia nudiseta (Wulp) (Diptera: Muscidae) from a human corpse in a high-rise building in Malaysia: A case report

Abstract. This is the first report of Synthesiomyia nudiseta (Wulp) (Diptera: Muscidae) on a human corpse discovered in a high-rise building in Malaysia. On 5 March 2008, a decomposing body of an adult female was found on the top floor of a thirteen-story building in Kuala Lumpur, Malaysia. Her body was colonized by S. nudiseta larvae, which were normally associated with corpses found indoors at ground level. The post-mortem interval (PMI) was estimated at approximately 5 to 9 days. This case is significant as it demonstrates that this species can locate a dead body even in a high-rise building. Further findings of fly distribution especially in high-rise buildings should be reported to assist entomologists in PMI analysis.

Morphological descriptions of second and third instar larvae of Hypopygiopsis violacea Macquart (Diptera:Calliphoridae), a forensically important fly in Malaysia.

Hypopygiopsis violacea, a species of fly of forensic importance, was recovered from a corpse and described for the first time. The morphological structures of the second and third instar larvae of four specimens were examined using light microscope. Observations were focused on three main morphological characters: cephalopharyngeal skeleton, anterior and posterior spiracles. Cephalopharyngeal skeleton of second instar larva is darkly pigmented and without accessory sclerite below the mouth hook. The anterior spiracles of second and third instar larvae have 8-9 papillae each, arranged in a single row. The posterior spiracle of second instar larva has two spiracular slits with no thickening of peritreme. This differentiates it from the third instar, whereby the latter has three slits for each posterior spiracle. Cephalopharyngeal skeleton of third instar larva is heavily pigmented. An accessory sclerite is found below the hook part of third instar larva but is absent in second instar. Peritreme of the posterior spiracle of third instar larva is thick almost complete encircling a button. The intersegmental spines of the cuticular surface are dome-shaped and unicuspid. Third instar larva of this species is large with size approximately 15 mm long. These findings provide important identification features of immature stages of Hy. violacea which could be useful in forensic entomology.

An analysis of forensic entomological specimens by Universiti Kebangsaan Malaysia.

This study reviews forensic entomological specimens analysed by the Department of Parasitology & Medical Entomology, Universiti Kebangsaan Malaysia for the year 2004. A total of 10 cases (6 males and 4 females) were observed for the entomological specimens. Various types of death scenes were obtained including indoor and outdoor area such as bushes field, rubbish dumping site, and aquatic areas. Identified fly species collected from the death sites were blow flies, Chrysomya megacephala, Chrysomya rufifacies and Lucilia cuprina and unknown sarcophagid larvae, with Ch. megacephala being the most common species found in the ecologically varied death scene habitats. The post-mortem interval (PMI) estimation ranged from one to five days, based on the entomological specimens collected.

Morphological descriptions of second and third instar larvae of Hypopygiopsis violacea Macquart (Diptera:Calliphoridae), a forensically important fly in Malaysia

Hypopygiopsis violacea, a species of fly of forensic importance, was recovered from a corpse and described for the first time. The morphological structures of the second and third instar larvae of four specimens were examined using light microscope. Observations were focused on three main morphological characters: cephalopharyngeal skeleton, anterior and posterior spiracles. Cephalopharyngeal skeleton of second instar larva is darkly pigmented and without accessory sclerite below the mouth hook. The anterior spiracles of second and third instar larvae have 8-9 papillae each, arranged in a single row. The posterior spiracle of second instar larva has two spiracular slits with no thickening of peritreme. This differentiates it from the third instar, whereby the latter has three slits for each posterior spiracle. Cephalopharyngeal skeleton of third instar larva is heavily pigmented. An accessory sclerite is found below the hook part of third instar larva but is absent in second instar. Peritreme of the posterior spiracle of third instar larva is thick almost complete encircling a button. The intersegmental spines of the cuticular surface are dome-shaped and unicuspid. Third instar larva of this species is large with size approximately 15 mm long. These findings provide important identification features of immature stages of Hy. violacea which could be useful in forensic entomology.