PesTrapp mobile app: A trap setting application for real-time entomological field and laboratory study.

Diseases such as malaria, dengue, Zika and chikungunya remain endemic in many countries. Setting and deploying traps to capture the host/vector species are fundamental to understand their density and distributions. Human effort to manage the trap data accurately and timely is an exhaustive endeavour when the study area expands and period prolongs. One stop mobile app to manage and monitor the process of targeted species trapping, from field to laboratory level is still scarce. Toward this end, we developed a new mobile app named "PesTrapp" to acquire the vector density index based on the mobile updates of ovitraps and species information in field and laboratory. This study aimed to highlight the mobile app's development and design, elucidate the practical user experiences of using the app and evaluate the preliminary user assessment of the mobile app. The mobile app was developed using mobile framework and database. User evaluation of the mobile app was based on the adjusted Mobile App Rating Scale and Standardized User Experience Percentile Rank Questionnaire. The process flows of system design and detailed screen layouts were described. The user experiences with and without the app in a project to study Aedes surveillance in six study sites in Selangor, Malaysia were elucidated. The overall mean user evaluation score of the mobile app was 4.0 out of 5 (SD=0.6), reflects its acceptability of the users. The PesTrapp, a one-stop solution, is anticipated to improve the entomological surveillance work processes. This new mobile app can contribute as a tool in the vector control countermeasure strategies.

PesTrapp mobile app: A trap setting application for real-time entomological field and laboratory study

@#Diseases such as malaria, dengue, Zika and chikungunya remain endemic in many countries. Setting and deploying traps to capture the host/vector species are fundamental to understand their density and distributions. Human effort to manage the trap data accurately and timely is an exhaustive endeavour when the study area expands and period prolongs. One stop mobile app to manage and monitor the process of targeted species trapping, from field to laboratory level is still scarce. Toward this end, we developed a new mobile app named “PesTrapp” to acquire the vector density index based on the mobile updates of ovitraps and species information in field and laboratory. This study aimed to highlight the mobile app’s development and design, elucidate the practical user experiences of using the app and evaluate the preliminary user assessment of the mobile app. The mobile app was developed using mobile framework and database. User evaluation of the mobile app was based on the adjusted Mobile App Rating Scale and Standardized User Experience Percentile Rank Questionnaire. The process flows of system design and detailed screen layouts were described. The user experiences with and without the app in a project to study Aedes surveillance in six study sites in Selangor, Malaysia were elucidated. The overall mean user evaluation score of the mobile app was 4.0 out of 5 (SD=0.6), reflects its acceptability of the users. The PesTrapp, a one-stop solution, is anticipated to improve the entomological surveillance work processes. This new mobile app can contribute as a tool in the vector control countermeasure strategies.

Status of pyrethroid resistance in Aedes (Stegomyia) aegypti (Linneaus) from dengue hotspots in Klang Valley, Malaysia.

The continued absence of an effective and safe tetravalent dengue vaccine and the lack of specific anti-viral treatment have made mosquito vector control using chemical insecticides as the mainstream for dengue prevention and control. However, the long-term use of chemical insecticides may induce resistance. Hence detection of insecticide resistance in dengue vectors is crucially important in ensuring the insecticide-based intervention in dengue control program is still effective and reliable. In this study, the susceptibility status of Aedes aegypti from five selected dengue hotspots in Klang Valley, Malaysia against pyrethroids was determined by employing the World Health Organization (WHO) protocol of adult bioassay. Four types of pyrethroids were tested against adult female Aedes aegypti to determine the knockdown rate, post 24-h adult mortality and resistance ratio. All field-collected Aedes aegypti strains were resistant to the four pyrethroids tested, except for the Taman Sungai Jelok (TSJ) strain. Permethrin exhibited the lowest knockdown rate against Aedes aegypti, followed by deltamethrin, cyfluthrin and lambda-cyhalothrin. This present study indicated the widespread of pyrethroid resistance in Aedes aegypti in Klang Valley, indicating the needs of implementing alternative measures in vector control program. The data in this study can be utilised as an input for insecticide resistance management of Aedes aegypti in Malaysia.

Status of pyrethroid resistance in Aedes (Stegomyia) aegypti (Linneaus) from dengue hotspots in Klang Valley, Malaysia

@#The continued absence of an effective and safe tetravalent dengue vaccine and the lack of specific anti-viral treatment have made mosquito vector control using chemical insecticides as the mainstream for dengue prevention and control. However, the long-term use of chemical insecticides may induce resistance. Hence detection of insecticide resistance in dengue vectors is crucially important in ensuring the insecticide-based intervention in dengue control program is still effective and reliable. In this study, the susceptibility status of Aedes aegypti from five selected dengue hotspots in Klang Valley, Malaysia against pyrethroids was determined by employing the World Health Organization (WHO) protocol of adult bioassay. Four types of pyrethroids were tested against adult female Aedes aegypti to determine the knockdown rate, post 24-h adult mortality and resistance ratio. All field-collected Aedes aegypti strains were resistant to the four pyrethroids tested, except for the Taman Sungai Jelok (TSJ) strain. Permethrin exhibited the lowest knockdown rate against Aedes aegypti, followed by deltamethrin, cyfluthrin and lambda-cyhalothrin. This present study indicated the widespread of pyrethroid resistance in Aedes aegypti in Klang Valley, indicating the needs of implementing alternative measures in vector control program. The data in this study can be utilised as an input for insecticide resistance management of Aedes aegypti in Malaysia.

Insight into the origin of chikungunya virus in Malaysian non-human primates via sequence analysis.

Chikungunya virus (CHIKV) is maintained in the sylvatic cycle in West Africa and is transmitted by Aedes mosquito species to monkeys. In 2006, four verified CHIKV isolates were obtained during a survey of arboviruses in monkeys (Macaca fascicularis) in Pahang state, Peninsular Malaysia. RNA was extracted from the CHIKV isolates and used in reverse transcription polymerase chain reactions (RT-PCR) to amplify PCR fragments for sequencing. Nucleic acid primers were designed to generate overlapping PCR fragments that covered the whole viral sequence. A total of 11,238 base pairs (bp) corresponding to open reading frames (ORFs) from our isolates and 47 other registered isolates in the National Center for Biotechnology Information (NCBI) were used to elucidate sequences, amino acids, and phylogenetic relationships and to estimate divergence times by using MEGA 7.0 and the Bayesian Markov chain Monte Carlo method. Phylogenetic analysis revealed that all CHIKV isolates could be classified into the Asian genotype and clustered with Bagan Panchor clades, which are associated with the chikungunya outbreak reported in 2006, with sequence and amino acid similarities of 99.9% and 99.7%, respectively. Minor amino acid differences were found between human and non-human primate isolates. Amino acid analysis showed a unique amino acid at position 221 in the nsP1region, at which a glycine (G) was found only in monkey isolates, whereas arginine (R) was found at the same position only in human isolates. The time to the most recent common ancestor (MRCA) estimation indicated that CHIKV probably started to diverge from human to non-human primates in approximately 2004 in Malaysia. The results suggested that CHIKV in non-human primates probably resulted from the spillover of the virus from humans. The study will be helpful in understanding the movement and evolution of CHIKV in Malaysia and globally.

Modelling the Effect of a Novel Autodissemination Trap on the Spread of Dengue in Shah Alam and Malaysia.

In this paper, we will start off by introducing the classical Ross-Macdonald model for vector-borne diseases which we use to describe the transmission of dengue between humans and Aedes mosquitoes in Shah Alam, which is a city and the state capital of Selangor, Malaysia. We will focus on analysing the effect of using the Mosquito Home System (MHS), which is an example of an autodissemination trap, in reducing the number of dengue cases by changing the Ross-Macdonald model. By using the national dengue data from Malaysia, we are able to estimate λ, which represents the initial growth rate of the dengue epidemic, and this allows us to estimate the number of mosquitoes in Malaysia. A mathematical expression is also constructed which allows us to estimate the potential number of breeding sites of Aedes mosquitoes. By using the data available from the MHS trial carried out in Section 15 of Shah Alam, we included the potential effect of the MHS into the dengue model and thus modelled the impact MHS has on the spread of dengue within the trial area. We then extended our results to analyse the effect of the MHSs on reducing the number of dengue cases in the whole of Malaysia. A new model was constructed with a basic reproduction number, R 0,Mala MHS, which allows us to identify the required MHSs coverage needed to achieve extinction in Malaysia. Numerical simulations and tables of results were also produced to illustrate our results.

Pteridine fluorescence in age-determination of immature Chrysomya megacephala (Fabricius) and Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae).

In the practice of forensic entomology, the chronological age of the maggots retrieved from the cadaver is used to determine the minimum post-mortem interval (mPMI) i.e. minimum time of death. The conventional method of aging the maggots is based on measuring the growth rate of these maggots. Although effective, the constraint associated with conventional method necessitates the development of new age determination method, such as pteridine determination. Pteridine, a by-product of protein metabolism in insects is known to correlate with the age of a variety of dipterans. A number of studies were conducted on aging the adults of forensically important flies. In this study, pteridine was extracted from Chrysomya megacephala and Chrysomya rufifacies maggots of known age using established methods and determined by measuring the fluorescence at excitation of 330nm and the emissions between 350nm and 600nm. Results exhibited significant positive linear relationships between the pteridine accumulations and age of the fly immature. Pteridine determination is a potential new age determination tool that can be used to determine mPMI.

A preliminary proteomic study of permethrin resistant and susceptible Aedes aegypti (L.).

The mechanism of insecticide resistance is traditionally attributed to detoxification enzymes, target site alteration, decreased penetration of insecticides and behavioural resistance. Other form of mechanisms, such as the role of protein(s) in resistance is unknown. In the present study, the protein profiling of both IMR-PSS strain (permethrin-selected) and IMR-LS strain (laboratory-susceptible) 24 hours post exposure period to permethrin was carried out via 1D-gel electrophoresis and liquid chromatography mass spectrometry (LC-MS/ MS). The bands which appeared in the gel of 1D-electrophoresis revealed an abundance of proteins. The band pattern of both strains looked macroscopically alike and differed only in band intensity. However, LC-MS/MS analysis revealed that the IMR-PSS strain produced extra 388 peptides that were not found in the IMR-LS strain, indicating that IMR-PSS strain reacted differently from IMR-LS strain as a result of persistent exposure to permethrin. Since the complex banding patterns of 1D-gel electrophoresis were difficult to interpret the significance of the protein difference between IMR-PSS and IMR-LS strain, hence LC-MS/MS analysis is ideally suited for better protein resolution and thus will allow more in-depth comparison of the complex pattern. The findings here provide the first preliminary evidence that insecticide resistance in mosquito induces up regulation of proteins that may be protective to mosquitoes against insecticide and proteins could be another mechanism that contributes to development of resistance.

Pteridine fluorescence in age-determination of immature Chrysomya megacephala (Fabricius) and Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae)

@#In the practice of forensic entomology, the chronological age of the maggots retrieved from the cadaver is used to determine the minimum post-mortem interval (mPMI) i.e. minimum time of death. The conventional method of aging the maggots is based on measuring the growth rate of these maggots. Although effective, the constraint associated with conventional method necessitates the development of new age determination method, such as pteridine determination. Pteridine, a by-product of protein metabolism in insects is known to correlate with the age of a variety of dipterans. A number of studies were conducted on aging the adults of forensically important flies. In this study, pteridine was extracted from Chrysomya megacephala and Chrysomya rufifacies maggots of known age using established methods and determined by measuring the fluorescence at excitation of 330nm and the emissions between 350nm and 600nm. Results exhibited significant positive linear relationships between the pteridine accumulations and age of the fly immature. Pteridine determination is a potential new age determination tool that can be used to determine mPMI.

A preliminary proteomic study of permethrin resistant and susceptible Aedes aegypti (L.)

@#The mechanism of insecticide resistance is traditionally attributed to detoxification enzymes, target site alteration, decreased penetration of insecticides and behavioural resistance. Other form of mechanisms, such as the role of protein(s) in resistance is unknown. In the present study, the protein profiling of both IMR-PSS strain (permethrin-selected) and IMR-LS strain (laboratory-susceptible) 24 hours post exposure period to permethrin was carried out via 1D-gel electrophoresis and liquid chromatography mass spectrometry (LC-MS/ MS). The bands which appeared in the gel of 1D-electrophoresis revealed an abundance of proteins. The band pattern of both strains looked macroscopically alike and differed only in band intensity. However, LC-MS/MS analysis revealed that the IMR-PSS strain produced extra 388 peptides that were not found in the IMR-LS strain, indicating that IMR-PSS strain reacted differently from IMR-LS strain as a result of persistent exposure to permethrin. Since the complex banding patterns of 1D-gel electrophoresis were difficult to interpret the significance of the protein difference between IMR-PSS and IMR-LS strain, hence LC-MS/MS analysis is ideally suited for better protein resolution and thus will allow more in-depth comparison of the complex pattern. The findings here provide the first preliminary evidence that insecticide resistance in mosquito induces up regulation of proteins that may be protective to mosquitoes against insecticide and proteins could be another mechanism that contributes to development of resistance.

Single and co-breeding of different mosquito species in fogging-free and dengue risk areas in West Malaysia

@#Ovitrap surveillance was carried out in fifteen localities encompassing foggingfree and dengue risk areas in West Malaysia to determine on the dispersal and prevalence of single and co-breeding of mosquito larvae particularly Aedes. Ovitraps were sited randomly indoors and outdoors within human settlements in all study areas. All the localities exhibited positive ovitraps with single breeding of Ae. albopictus that ranged between 64.29% and 100.00%. These findings indicated Ae. albopictus as the predominant container-breeding species in all study areas. The co-breeding of Ae. aegypti with Ae. albopictus larvae (34 ovitraps), Ae. albopictus with Culex quinquefasciatus larvae (32 ovitraps) as well as Cx. quinquefasciatus with Armigeres subalbatus larvae (1 ovitrap) were also detected in certain study localities. Interestingly, co-breeding of Ae. albopictus with Ar. subalbatus larvae as well as Ae. albopictus with Uranotaenia sp. larvae in Malaysia is reported for the first time in the present study. Better understanding of the co-breeding scenario involving different species of mosquito larvae is needed to ensure the efficacy of vector control actions to be conducted.

Similar vertical transmission rates of dengue and chikungunya viruses in a transgenic and a non-transformed Aedes aegypti (L.) laboratory strain.

The increase of the burden of dengue and chikungunya and the relative failure of traditional vector control strategies have highlighted the need to develop new control methods. RIDL-SIT, a vector control method based on the release of engineered male mosquitoes, has shown promising results from field trials conducted in the Cayman Islands and Brazil. In large scale use, a small proportion of females might be released along with the males. Such females are potential virus vectors; here we investigate the vertical transmission of dengue and chikungunya of homozygous OX513A females.We provided females of OX513A-My1 and a wild type comparator strain with blood meals artificially infected with dengue serotype 1, 2, 3, 4 or chikungunya viruses. For 14 days post-feeding, eggs laid by females were collected. Larvae and their mothers were first tested by qRT-PCR, then by inoculation on cell cultures to search for infectious viral particles. We found no significant difference between the minimum infection rate of OX513A-My1 and wild type females. We also discussed the potential number of females being released, a fraction of the female wild population. Consequently, we conclude that there are no evidence that OX513A-My females, if released into the environment, would cause more harm than their wild counterparts.

Effects of vitamin B fortified sucrose solution on the longevity and reproductive potentials of laboratory-bred Culex quinquefasciatus Say adult.

Laboratory colonised mosquitoes are usually maintained on vitamin B complex fortified sucrose solution, however only few studies were conducted to evaluate the effects of such practice. This study aimed to determine the effects of different concentrations of sucrose solution fortified with and without 1% vitamin B complex on the longevity and reproductive potential of adult female of a local strain of Culex quinquefasciatus Say. Two arms of studies were carried out separately and each arm was triplicated. In one arm, concentrations of sucrose solution at 0%, 1%, 3%, 5% and 10% fortified with 1% Vitamin B complex were fed to the mosquitoes, while in the other arm, similar sucrose concentrations were used but without 1% vitamin B complex. Adult Cx. quinquefasciatus maintained on 5% sucrose solution fortified with 1% vitamin B complex exhibited significant extended vitality and longevity in stimulating ovarian development, compared with other vitamin fortified sucrose concentrations (p<0.05). The vitality and longevity of F0 and F1 males were 76.67±2.19 days and 57.67±8.19 days respectively. The F0 females survived the longest duration of 107.67±5.61 days and the F1 females survived 90.67±12.47 days with higher number of eggs laid, i.e. 1427.67±62.89 eggs at a higher hatchability rate of 57.05±8.39% or 814.49 eggs hatched. Thus, 5% sucrose solution fortified with 1% Vitamin B complex should be used to produce colonies of homogenous mosquitoes as this exerts positive biological effects on laboratory-bred Cx. quinquefasciatus.

Effects of vitamin B fortified sucrose solution on the longevity and reproductive potentials of laboratory-bred Culex quinquefasciatus Say adult

Laboratory colonised mosquitoes are usually maintained on vitamin B complex fortified sucrose solution, however only few studies were conducted to evaluate the effects of such practice. This study aimed to determine the effects of different concentrations of sucrose solution fortified with and without 1% vitamin B complex on the longevity and reproductive potential of adult female of a local strain of Culex quinquefasciatus Say. Two arms of studies were carried out separately and each arm was triplicated. In one arm, concentrations of sucrose solution at 0%, 1%, 3%, 5% and 10% fortified with 1% Vitamin B complex were fed to the mosquitoes, while in the other arm, similar sucrose concentrations were used but without 1% vitamin B complex. Adult Cx. quinquefasciatus maintained on 5% sucrose solution fortified with 1% vitamin B complex exhibited significant extended vitality and longevity in stimulating ovarian development, compared with other vitamin fortified sucrose concentrations (p<0.05). The vitality and longevity of F0 and F1 males were 76.67±2.19 days and 57.67±8.19 days respectively. The F0 females survived the longest duration of 107.67±5.61 days and the F1 females survived 90.67±12.47 days with higher number of eggs laid, i.e. 1427.67±62.89 eggs at a higher hatchability rate of 57.05±8.39% or 814.49 eggs hatched. Thus, 5% sucrose solution fortified with 1% Vitamin B complex should be used to produce colonies of homogenous mosquitoes as this exerts positive biological effects on laboratory-bred Cx. quinquefasciatus.

Similar vertical transmission rates of dengue and chikungunya viruses in a transgenic and a non-transformed Aedes aegypti (L.) laboratory strain

The increase of the burden of dengue and chikungunya and the relative failure of traditional vector control strategies have highlighted the need to develop new control methods. RIDL-SIT, a vector control method based on the release of engineered male mosquitoes, has shown promising results from field trials conducted in the Cayman Islands and Brazil. In large scale use, a small proportion of females might be released along with the males. Such females are potential virus vectors; here we investigate the vertical transmission of dengue and chikungunya of homozygous OX513A females.We provided females of OX513A-My1 and a wild type comparator strain with blood meals artificially infected with dengue serotype 1, 2, 3, 4 or chikungunya viruses. For 14 days post-feeding, eggs laid by females were collected. Larvae and their mothers were first tested by qRT-PCR, then by inoculation on cell cultures to search for infectious viral particles. We found no significant difference between the minimum infection rate of OX513A-My1 and wild type females. We also discussed the potential number of females being released, a fraction of the female wild population. Consequently, we conclude that there are no evidence that OX513A-My females, if released into the environment, would cause more harm than their wild counterparts.

Detection of Wolbachia from field collected Aedes albopictus Skuse in Malaysia.

BACKGROUND & OBJECTIVES: Wolbachia-based vector control strategies have been proposed as a mean to augment the existing measures for controlling dengue vector. Prior to utilizing Wolbachia in novel vector control strategies, it is crucial to understand the Wolbachia-mosquito interactions. Many studies have only focused on the prevalence of Wolbachia in female Aedes albopictus with lack of attention on Wolbachia infection on the male Ae. albopictus which also affects the effective expression of Wolbachia induced- cytoplasmic incompatibility (CI). In this study, field surveys were conducted to screen for the infection status of Wolbachia in female and male Ae. albopictus from various habitats including housing areas, islands and seashore. METHODS: Adult Ae. albopictus (n=104) were collected using human landing catches and hand aspirator. Standard ovitraps were also set in the selected areas for five days and the larvae were identified to species level. All the collected Ae. albopictus were screened for the presence of Wolbachia using multiplex polymerase chain reaction (PCR) and gene sequencing of Wolbachia surface protein (wsp) gene. RESULTS: A 100 per cent positivity of Wolbachia infection was observed for individual Ae. albopictus screened. For pooled mosquitoes, 73 of the 76 pools (female) and 83 of the 87 pools (male) were positive with Wolbachia infection. The wsp gene sequence of the Wolbachia strain isolated from individual and pooled mosquitoes showed a 100 per cent homology with Wolbachia sp. of Ae. albopictus isolated from various geographical regions. Phylogenetic analysis based on wsp gene fragments showed that the isolates were clustered into groups A and B, respectively. INTERPRETATION & CONCLUSIONS: The results indicated that Wolbachia infection was widespread in Ae. albopictus population both in female and male Ae. albopictus. All the infected females were superinfected with both A and B strains while the infected males showed a combination of superinfection of A and B strains and single infection of B strain.

Surveillance of Aedes albopictus Skuse breeding preference in selected dengue outbreak localities, peninsular Malaysia.

Entomological surveillance was conducted in order to determine the abundance and to evaluate any changes of biological vectors or ecology, especially in the dengue outbreak areas. The abundance and breeding preference of Aedes albopictus and Aedes aegypti were conducted in selected dengue outbreak localities in three states of peninsular Malaysia namely Selangor, Federal Territory of Kuala Lumpur, and Penang Island using ovitraps and larval survey method. It was determined that Ae. albopictus was predominant in most of the localities and found to breed more outdoor than indoor. A wide range of breeding foci were recorded in this study. It was also determined that ovitrap method was more effective to detect the presence of Aedes mosquitoes when the larval survey was at low rate of infestation. The abundance of Ae. albopictus in dengue outbreak localities emphasis that the vector control programme should also target this species together with the primary dengue vector, Ae. aegypti.

Morphological descriptions on the larvae of Hypopygiopsis fumipennis (Walker, 1856) (Diptera: Calliphoridae).

The study on biodiversity of forensically important Diptera in the tropical rain forest in Malaysia is scarce. Thus, a preliminary survey was conducted at a jungle fringe near Kampung Bahagia Bukit Lagong, Sungai Buloh, Selangor. A rat carcass was offered to attract carrion flies and we collected an adult female calliphorid, Hypopygiopsis fumipennis (Walker, 1856) during the fresh stage of carcass decomposition. The female fly was allowed to oviposit on chicken liver in a container and the resulting larvae were reared to the adult stage. Along the developmental process, several individuals from each instar were collected and preserved in 70% ethanol and then processed on the slides. We recorded the duration of development for each instar and described its larval features for the first time. The third instar larvae of H. fumipennis showed accessory oral sclerite present, anterior spiracle with 13-15 papillae, intersegmental spines mostly unicuspid with pointed end, and posterior spiracles heavily sclerotized with inter-slit projections. Some larval differences between H. fumipennis and Hypopygiopsis violacea were noted.

ABSENCE OF AEDES AEGYPTI (L.) ON AN ECOLOGICAL ISLAND: COMPETITIVE EXCLUSION?

Ovitrap surveillance was conducted in 2012 and 2006 in Malay and Aboriginal Villages on Carey Island. In each village, standard ovitraps were placed indoors and outdoors at randomly selected houses/locations. All L3 larvae recovered were identified up to species level. Results demonstrated that only larvae of Aedes albopictus were found in all the positive ovitraps placed indoors and outdoors. In 2012, a high ovitrap index (OI) of 66.7% indoor and 84.0% outdoor in the Malay Village; and 62.5% indoor and 88.0% outdoor in Aboriginal Village with an apparent absence of Aedes aegypti. In 2006, a 100% OI was recorded in all ovitraps set indoors and outdoors in both villages.

Morphological descriptions on the larvae of Hypopygiopsis fumipennis (Walker, 1856) (Diptera: Calliphoridae)

The study on biodiversity of forensically important Diptera in the tropical rain forest in Malaysia is scarce. Thus, a preliminary survey was conducted at a jungle fringe near Kampung Bahagia Bukit Lagong, Sungai Buloh, Selangor. A rat carcass was offered to attract carrion flies and we collected an adult female calliphorid, Hypopygiopsis fumipennis (Walker, 1856) during the fresh stage of carcass decomposition. The female fly was allowed to oviposit on chicken liver in a container and the resulting larvae were reared to the adult stage. Along the developmental process, several individuals from each instar were collected and preserved in 70% ethanol and then processed on the slides. We recorded the duration of development for each instar and described its larval features for the first time. The third instar larvae of H. fumipennis showed accessory oral sclerite present, anterior spiracle with 13-15 papillae, intersegmental spines mostly unicuspid with pointed end, and posterior spiracles heavily sclerotized with inter-slit projections. Some larval differences between H. fumipennis and Hypopygiopsis violacea were noted.