Sterilization of blow fly eggs, Chrysomya megacephala and Lucilia cuprina, (Diptera: Calliphoridae) for maggot debridement therapy application.

Maggot debridement therapy (MDT) is an application of sterile laboratory-reared blow fly larvae to remove necrotic tissue and disinfect wounds for medical conditions. For effective application, the blow fly larvae used in the wound treatment are required to be in aseptic condition. Here, we report the results of a detailed assessment of bacteria and fungi isolated from the eggs of two blow fly species, Chrysomya megacephala (F.) and Lucilia cuprina (Wiedemann) before and after sterilization by disinfectants Chlorhex-C, povidone-iodine, and sodium hypochlorite. We also assess the survival ability of larvae and their sterility after the cleansing process. The results indicate that the isolated microorganisms from the control group of both the species consisted of 10 species of gram-positive bacteria, 21 species of gram-negative bacteria, and 4 species of yeast. As for sterility testing, the eggs and the larvae of C. megacephala were found to have been completely sterilized after being subjected to thioglycollate medium for 5 days, leading to aseptic larvae. By contrast, some microorganisms from the bacterial culture were still detected in the L. cuprina larvae treated with Chlorhex-C and povidone-iodine. The survival ability of the larvae in both the species was not significantly different between the treated and the control groups. Due to its high disinfection efficacy in destroying microorganisms in both the blow fly eggs, sodium hypochlorite is recommended for preparing sterile larvae before using MDT.

Forensically Important Blow Flies Chrysomya pinguis, C. villeneuvi, and Lucilia porphyrina (Diptera: Calliphoridae) in a Case of Human Remains in Thailand.

This is the first study to report Chrysomya pinguis (Walker) and Lucilia porphyrina (Walker) (Diptera: Calliphoridae) as forensically important blow fly species from human cadavers in Thailand, in addition to Chrysomya villeneuvi (Patton) already known in Thailand. In 2016, a fully decomposed body of an unknown adult male was discovered in a high mountainous forest during winter in Chiang Mai province. The remains were infested heavily with thousands of blow fly larvae feeding simultaneously on them. Morphological identification of adults reared from the larvae, and molecular analysis based on sequencing of 1,247 bp partial mitochondrial cytochrome c oxidase subunit 1 gene (CO1) of the larvae and puparia, confirmed the above mentioned 3 species. The approving forensic fly evidence by molecular approach was described for the first time in Thailand. Moreover, neighbor-joining phylogenetic analysis of the CO1 was performed to compare the relatedness of the species, thereby affirming the accuracy of identification. As species of entomofauna varies among cases in different geographic and climatic circumstances, C. pinguis and L. porphyrina were added to the list of Thai forensic entomology caseworks, including colonizers of human remains in open, high mountainous areas during winter. Further research should focus on these 3 species, for which no developmental data are currently available.

Sarcophaga (Liosarcophaga) dux (Diptera: Sarcophagidae): A flesh fly species of medical importance.

BACKGROUND: Although tropical climate of Thailand is suitably endowed with biodiversity of insects, flies of medical importance is not well investigated. Using information from literature search, fly survey approach and specialist's experience, we review database of Sarcophaga (Liosarcophaga) dux Thomson (Diptera: Sarcophagidae), one of the priorities flesh fly species of medical importance in Thailand. RESULTS: This review deals with morphology, bionomics and medical involvement. Important morphological characteristics of egg, larva, puparia and adult were highlighted with illustration and/or micrographs. Search pertaining to molecular analysis used for fly identification and developmental rate of larvae were included. Medical involvement of larvae was not only myiasis-producing agent in humans and animals, but associated with human death investigations. CONCLUSIONS: This information will enable us to accurate identify this species and to emphasis the increase medically important scene in Thailand.

Impact of abiotic factor changes in blowfly, Achoetandrus rufifacies (Diptera: Calliphoridae), in northern Thailand.

Understanding how medically important flies respond to abiotic factor changes is necessary for predicting their population dynamics. In this study, we investigated the geographical distribution of the medically important blowfly, Achoetandrus rufifacies (Macquart) (Diptera: Calliphoridae), and ascertained the response to climatic and physio-environmental factors in Chiang Mai, northern Thailand. Adult fly surveys were carried out every 2 weeks from May 2009 to May 2010 at 18 systematically randomized study sites in three districts of Chiang Mai province (Mueang Chiang Mai, Mae Rim, and Hang Dong), using reconstructable funnel traps with 1-day tainted beef offal as bait. During the study period, 8,861 adult A. rufifacies were captured, with peak densities being observed at the end of winter (i.e., late February) and throughout most of the summer (May to March). Population density had a weak but significant (α = 0.05) positive correlation with temperature (r = 0.329) and light intensity (r = 0.231), and a weak but significant (α = 0.05) negative correlation with relative humidity (r = -0.236). From the six ecological land use types (disturbed mixed deciduous forest, mixed deciduous forest, mixed orchard, lowland village, city town, and paddy field), greater fly densities were observed generally in the disturbed mixed deciduous forest and lowland village, but not in the paddy fields. In conclusion, A. rufifacies are abundant from the end of winter and throughout most of the summer in northern Thailand, with population density being weakly positively correlated with temperature and light intensity, but weakly negatively correlated with relative humidity. The greatest densities of this fly species were collected in disturbed mixed deciduous forest and lowland village land uses. The prediction of annual and season specific distributions of A. rufifacies were provided in each season and all-year patterns using a co-kriging approach (ArcGIS9.2).

Blow fly maggots (Diptera: Calliphoridae)from a human corpse in a vehicle.

Correct species identification and development data of insects associated with a cadaver can help estimate the time of colonization which could be used to infer a minimal post-mortem interval (minPMI) for forensic investigations. Human remains are found in a variety of locations ranging from open fields to inside automobiles. We report the investigation of blow fly larvae collected from a decomposing body located in the trunk of a car. There were two blow fly (Diptera: Calliphoridae) species: Achoetandrus rufifacies (Macquart) and Chrysomya megacephala (Fabricius). Blow flies can enter the vehicle and colonize human remains. Based on age estimations of third stage larvae of A. rufifacies, the minPMI was estimated to be 4-5 days, which was within the range of 3-5 days estimated by other forensically relevant information.

Isolation of Toxocara eggs from flies in Northeast Thailand.

BACKGROUND: Flies are natural carriers of pathogens and play a considerable role in the mechanical transmission of many pathogens, such as viruses, fungi, bacteria, and parasites, in various regions of the world. OBJECTIVE: To investigate the existence of Toxocara spp. eggs on two types of flies, the Oriental latrine fly, Chrysomya megacephala, and the housefly, Musca domestica, in Ubon Ratchathani, Northeast Thailand. MATERIAL AND METHOD: Flies were collected by the use of sterilized insect sweeping nets at five types of sites, fresh-food markets, garbage piles, restaurants, school cafeterias, and paddy fields from September 2010 to October 2011. After centrifugation, sediments from each fly species were examined for parasites under a light microscope. RESULTS: From the analysis of 555 C. megacephala and 439 M. domestica adult samples, Toxocara eggs were isolated from only the former species in all sites except for the restaurants in the Warinchamrap. No other helminthes and protozoa were found on the body surfaces of both fly species. CONCLUSION: The data suggest that C. megacephala is more likely to be a potential carrier of Toxocara eggs than M. domestica in areas frequented by humans in this region of Thailand.

Geometry of posterior larval spiracles to identify medically and forensically important calliphorids in Thailand.

Fly identification is the primary step of analysis in forensic entomology. Although morphology and molecular techniques are considered satisfactory methods, some constraints may arise from a financial or even human point of view. Over the past decade, the geometric morphometric approach has been increasingly advocated for the classification and identification of arthropods. This study explored the method for species identification of 800 third-instar larvae of eight blow fly species of medical and forensic importance: Chrysomya chani Kurahashi, Chrysomya megacephala (Fabricius), Chrysomya (Ceylonomyia) nigripes Aubertin, Chrysomya pinguis (Walker), Chrysomya (Achoetandrus) rufifacies (Macquart), Hemipyrellia ligurriens (Wiedemann), Lucilia cuprina (Wiedemann), and Lucilia porphyrina (Walker). Based on the posterior spiracles geometry, the cross-validation revealed a relatively high percentage of correct classification in most species, ranking from 86% to 100%. The results of this study confirmed that the geometric morphometric (GM) analysis of posterior spiracles might be utilized as a larva identification tool. Therefore, this GM method represents one way of overcoming difficulties with the identification of blow fly larvae and can support further studies of these flies.

Molecular identification and genetic variation of forensically important fly species (Order: Diptera) in Thailand using DNA barcoding.

Forensic entomology plays a crucial role in criminal investigations by providing vital insights into minimum postmortem interval (PMImin) and corpse relocation by identifying insect species that colonize in decomposing remains. This study aimed to identify and analyze the genetic variation of forensically significant fly species in Thailand, using DNA barcoding of the mitochondrial cytochrome c oxidase subunit I COI gene. A total of 3,220 fly specimens were collected from 18 provinces across six regions of Thailand from October 2017 to September 2022. These specimens were classified by morphological identification into 21 species among three Dipteran families: Calliphoridae, Muscidae, and Sarcophagidae, with Chrysomya megacephala Diptera: Calliphoridae being the most abundant species. DNA barcoding confirmed the morphological identifications with 100 % accuracy, showing low intraspecific K2P distances0.0 to 1.1 %) and significant interspecific K2P distances 2.5 % to 17.2 %. A Neighbour-Joining (NJ) analysis was conducted to assess the molecular identification capabilities of the barcoding region. This analysis successfully recovered nearly all species as distinct monophyletic groups. The species groupings obtained were generally consistent with both morphological and molecular identifications. These findings underscore the effectiveness of DNA barcoding for precise species identification and contribute to a comprehensive database of forensically important flies in Thailand, thus facilitating improved forensic investigations and biodiversity studies.

Evaluation of an I-box wind tunnel model for assessment of behavioral responses of blow flies.

The behavioral response of flies to olfactory cues remains the focus of many investigations, and wind tunnels have sometimes been employed for assessment of this variable in the laboratory. In this study, our aim was to design, construct, and operate a new model of I-box wind tunnel with improved efficacy, highlighting the use of a new wind tunnel model to investigate the behavioral response of the medically important blow fly, Chrysomya megacephala (Fabricius). The I-box dual-choice wind tunnel designed for this study consists of seven conjoined compartments that resulted in a linear apparatus with clear glass tunnel of 30 × 30 × 190 cm ended both sides with wooden "fan compartments" which are equipped with adjustable fans as wind source. The clear glass tunnel consisted of two "stimulus compartments" with either presence or absence (control) of bait; two "trap compartments" where flies were attracted and allowed to reside; and one central "release compartment" where flies were introduced. Wind tunnel experiments were carried out in a temperature-controlled room, with a room light as a light source and a room-ventilated fan as odor-remover from tunnel out. Evaluation of testing parameters revealed that the highest attractive index was achieved with the use of 300 g of 1-day tainted pork scrap (pork meat mixed with offal) as bait in wind tunnel settings wind speed of 0.58 m/s, during 1.00-5.00 PM with light intensity of 341.33 lux from vertical light and 135.93 lux from horizontal light for testing a group of 60 flies. In addition, no significant response of well-fed and 24 h staved flies to this bait under these conditions was found. Results of this study supported this new wind tunnel model as a suitable apparatus for investigation of behavioral response of blow flies to bait chemical cues in the laboratory.

Observations on morphology of immature Lucilia porphyrina (Diptera: Calliphoridae), a fly species of forensic importance.

Lucilia porphyrina (Walker) is a blow fly of forensic importance, and shares its geographical distribution with a related forensically important species, Lucilia cuprina (Wiedemann). The immature stages of both species are similar in general appearance; therefore, correct identification should be given special consideration. This study highlighted the main features of L. porphyrina larvae, as observed under light microscopy and scanning electron microscopy. Particular attention is given to the anterior and posterior spiracles, cephalopharyngeal skeleton, and characteristics of the dorsal spines between the prothorax and mesothorax. In the third instar specifically, morphological information on L. porphyrina showed several features that are shared by L. cuprina, and therefore need certain identification to distinguish between them. Such key features are (1) greater posterior spiracle, (2) apparent inner projection between the middle and lower slits of the posterior spiracle, and (3) strongly sclerotized peritreme. The number of papillae on the anterior spiracle may be a supplement, five to nine and three to six in L. porphyrina and L. cuprina, respectively. The key for identifying third instar of forensically important flies in Thailand has been updated to include L. porphyrina.