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.

Chrysomya pinguis (Walker) (Diptera: Calliphoridae), blow fly of forensic importance: A review of bionomics and forensic entomology appraisal.

Role of blow flies as the entomological evidence used in forensic investigations has risen dramatically worldwide. As the diverse habitats of Thailand suitably endowed with biodiversity of insects, blow flies of forensic importance need investigation in their bionomics, which are further applied in forensic investigations. Chrysomya pinguis (Walker, 1858) (Diptera: Calliphoridae) make up one of the most common blow fly species found associated with the human corpses and/or death scenes in several countries of the Asia continent. Given the major species as forensically important, this review is performed by the need for gathering information of C. pinguis from literature search in the future application in the regions where this species exists. This review deals with morphology, current knowledge on bionomics and forensic entomology involvement. Important morphological characteristics of egg, larva, puparium 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. Furthermore, we outline potential issues and challenges of C. pinguis research that necessitate forensic applications in the future.

Wing morphometrics as a tool for the identification of forensic important Lucilia spp. (Diptera: Calliphoridae).

Blow flies of the genera Lucilia Robineau-Desvoidy (Diptera: Calliphoridae) are considered forensically important species across several regions of the world. Due to the similarity of adults, especially females, the usual methods based on morphology or even molecular techniques can experience some limitations; therefore, alternative or supportive tools are required. Recently, the landmark-based geometric morphometric analysis has been applied to discriminate many insects on genus and species level. Herein, we focus on wing morphometric analysis as a tool in classifying five species of Lucilia; three species from Thailand - L. cuprina (Wiedemann, 1830), L. porphyrina (Walker, 1856) and L. sinensis Aubertin, 1933; and two species from Switzerland - L. caesar (Linnaeus, 1758) and L. illustris (Meigen, 1826). Canonical variate analysis of 233 right wings showed four overlapping clusters of L. cuprina, L. sinensis, L. caesar, and L. illustris with one distinct cluster of L. porphyrina. Eighty-eight to 100 percent of correct classification was achieved, with an UPGMA dendrogram analysis revealing clear-cut branch and sub-branch of five species determined. Results from this study suggested that wing morphometric analysis is a useful tool for the identification of adult Lucilia spp.

Ultrastructure of male terminalia of Boettcherisca peregrina and Boettcherisca nathani (Diptera: Sarcophagidae), flesh fly species of forensic importance.

Flesh flies are received greater attention due to their use as evidence in forensic investigations of decomposing human remains. Investigators will often use age of immature insects associated with such remains as the minimal postmortem interval (PMImin) given certain assumptions. Boettcherisca peregrina is a significant flesh fly species of forensic importance in several countries, whereas B. nathani is potentially a species of forensic importance. Distinguishing these two species is difficult and relies on close examination of the male terminalia. For genus Boettcherisca belonging to subfamily Sarcophaginae, identification is primarily based on the morphological structures associated with male terminalia. Using light microscopy and scanning electron microscopy, similarities and differences were determined for these two species. In this study, B. peregrina was collected from an urban area of Mueang District, while B. nathani was collected from the highland forest area of Doi Saket District, Chiang Mai province, northern Thailand. The morphological observations indicated similarities in the morphology of vesica (large, rounded anterior margin, bi-lobed, half upper-outer margins serrated), harpes (well-developed, elongated, broad basally with bifurcated apically) and lateral styli (two parallel tube-like with open ends with microserration apically) in both species. However, differences were found with the cercus and juxta. With regards to the cercus, B. peregrina has bristles or setae only along the posterior surface and the cercal tip is pointed, while B. nathani has bristles or setae on both the anterior and posterior surfaces, and the cercal tip is hook-shaped. Morphometric measurements of three criteria of the cerci (length from top to middle, internal distance at narrow part and distance between apex of cercal prong) revealed that the average length of each criteria of B. nathani cerci (n = 30) were significantly greater than B. peregrina cerci (n = 30). As for the juxta, the juxtal lateral plate was different: primarily bifurcated in B. peregrina and non-bifurcated in B. nathani. The medial part of juxta was different: V-shaped 95.8% (46/48) and, to a lesser extent, W-shaped 4.2% (2/48) in B. peregrina, only W-shaped 100% (52/52) in B. nathani. Such information is useful for entomologists to distinguish between adult males of these two species, which could be valuable when determining colonization patterns in association with decomposing human remains.

Wing morphometrics of medically and forensically important muscid flies (Diptera: Muscidae).

Many muscid flies (Diptera: Muscidae) are well-known as medical, veterinary, and forensically significant insects, thus correct species identification is critically important before applying for fly control and determining a minimal postmortem interval (PMImin) in forensic investigations. Limited in taxonomic keys and taxonomists, as well as scanty in advanced molecular laboratories lead to difficulty in identification of muscids. To date, a landmark-based geometric morphometric analysis of wings has proven to be a promising alternative technique for identifying many insect species. Herein, we assessed wing morphometric analysis for identification of six medically and forensically important muscids, namely Musca domestica Linnaeus, Musca pattoni Austen, Musca ventrosa Wiedemann, Hydrotaea chalcogaster (Wiedemann), Hydrotaea spinigera Stein, and Dichaetomyia quadrata (Wiedemann). A total of 302 right wing images were digitized based on 15 homologous landmarks and wing shape variation among genera and species was analyzed using canonical variate analysis, whereas sexual shape dimorphism of M. domestica, M. ventrosa, and D. quadrata was analyzed using discriminant function analysis. The cross-validation revealed a relatively high percentage of correct classification in most species, ranging from 86.4% to 100%, except for M. pattoni, being 67.5%. Misidentifications were mainly due to cross-pairings of the genus Musca; M. domestica VS M. pattoni VS M. ventrosa. The accuracy of classification using cross-validation test demonstrated that wing shape can be used to evaluate muscid flies at the genus- and species-level, and separate sexes of the three species analyzed, with a high reliability. This study sheds light on genus, species, and sex discrimination of six muscid species that have been approached using wing morphometric analysis.

Daily and seasonal variation of muscid flies (Diptera: Muscidae) in Chiang Mai province, northern Thailand.

Flies of the family Muscidae, or muscids, are of medical and veterinary importance worldwide due to their recognition as nuisance pests and myiasis-producing agents. Effective control of muscids requires biological information on population dynamics daily and across seasons. In this study, such patterns were investigated in three different microhabitats (e.g., forest area, palm plantation and longan orchard) in a suburban area of Chiang Mai Province, northern Thailand. Adult fly samplings were conducted for 24-h intervals using semiautomatic traps and 1-day old beef offal as bait. Samplings were carried out twice per month from July 2013 to June 2014. A total of 3,419 muscids were trapped, comprising nine species, with Musca domestica Linnaeus accounting for the majority (n = 1,329; 38.9%) followed by Hydrotaea spinigera Stein (n = 770; 22.5%) and Musca ventrosa Wiedemann (n = 740; 21.7%). The greatest overall abundance was in the longan orchard location (n = 1,508; 44.1%). Community structure peaked during the rainy season (mid-May to mid-Oct). Peak activity during the day was late morning (9.00 to 12.00 h) for M. domestica, early morning (6.00 to 9.00 h) for H. spinigera, and early afternoon (12.00 to 15.00 h) for M. ventrosa. Temperature had no significant effect on the abundance of M. domestica (rs= -0.030, p = 0.576) or H. spinigera (rs = 0.068, p = 0.200), but had a weak negative correlation with M. ventrosa (rs = -0.238, p = 0.0001). Relative humidity had a weak negative correlation with M. domestica (rs = -0.263, p = 0.0001), H. spinigera (rs = -0.107, p = 0.043) and M. ventrosa (rs = -0.344, p = 0.0001). More females (n = 2,078) were trapped than males (n = 761). These results provide baseline information of daily and seasonal dynamic activity of muscid flies under natural conditions, which is the prerequisite information for effective control measures.

Mitochondrial DNA-Based Identification of Forensically Important Flesh Flies (Diptera: Sarcophagidae) in Thailand.

Flesh flies (Sarcophagidae) are necrophagous insects initially colonizing on a corpse. The species-specific developmental data of the flies collected from a death scene can be used to estimate the minimum postmortem interval (PMImin). Thus, the first crucial step is to correctly identify the fly species. Because of the high similarity among species of flesh flies, DNA-based identification is considered more favorable than morphology-based identification. In this study, we demonstrated the effectiveness of combined sequences (2216 to 2218 bp) of cytochrome c oxidase subunit I and II genes (COI and COII) for identification of the following 14 forensically important flesh fly species in Thailand: Boettcherisca nathani Lopes, Fengia ostindicae (Senior-White), Harpagophalla kempi (Senior-White), Liopygia ruficornis (Fabricius), Lioproctia pattoni (Senior-White), Lioproctia saprianovae (Pape & Bänziger), Parasarcophaga albiceps (Meigen), Parasarcophaga brevicornis (Ho), Parasarcophaga dux (Thomson), Parasarcophaga misera (Walker), Sarcorohdendorfia antilope (Böttcher), Sarcorohdendorfia inextricata (Walker), Sarcorohdendorfia seniorwhitei (Ho) and Seniorwhitea princeps (Wiedemann). Nucleotide variations of Thai flesh flies were evenly distributed throughout the COI-COII genes. Mean intra- and interspecific variations ranged from 0.00 to 0.96% and 5.22% to 12.31%, respectively. Using Best Match (BM) and Best Close Match (BCM) criteria, identification success for the combined genes was 100%, while the All Species Barcodes (ASB) criterion showed 76.74% success. Maximum Likelihood (ML) and Bayesian Inference (BI) phylogenetic analyses yielded similar tree topologies of monophyletic clades between species with very strong support values. The achieved sequences covering 14 forensically important flesh fly species including newly submitted sequences for B. nathani, F. ostindicae and S. seniorwhitei, can serve as a reliable reference database for further forensic entomological research in Thailand and in other areas where those species occur.

Synergistic Toxicity of Plant Essential Oils Combined with Pyrethroid Insecticides against Blow Flies and the House Fly.

Blow flies (Diptera: Calliphoridae) and the house fly (Diptera: Muscidae) are filth flies of medical importance, and control of their population is needed. As insecticide applications have resulted in fly resistance, and the exploration of plant essential oils (EOs) has increased against filth flies, this study assessed the combination of EOs with pyrethoids to enhance toxic efficacy. The EOs of five effective plants were screened initially against the house fly (Musca domestica L.). Their chemical constituent was performed using gas chromatography-mass spectrometry (GC-MS) analysis. The main components of Boesenbergia rotunda (Zingiberaceae) rhizome, Curcuma longa (Zingiberaceae) rhizome, Citrus hystrix (Rutaceae) fruit peel, Ocimum gratissimum (Lamiaceae) seed, and Zanthoxylum limonella (Rutaceae) fruit were δ-3-caren (35.25%), ß-turmerone (51.68%), ß-pinene (26.56%), p-cumic aldehyde (58.21%), and dipentene (60.22%), respectively. The screening test revealed that the three most effective plant EOs were from B. rotunda, C. longa and O. gratissimum, which were selected for the combination with two pyrethroid insecticides (permethrin and deltamethrin), in order to enhance their synergistic efficacy against the blow flies, Chrysomya megacephala Fabricius, Chrysomya rufifacies Macquart, and Lucilia cuprina Wiedemann, and the house fly. Synergistic action was presented in almost all of the flies tested with permenthrin/deltamethrin/EOs mixtures. It was interesting that the combination of deltamethrin with three EOs showed a synergistic effect on all of the tested flies. However, an antagonistic effect was observed in C. megacephala and M. domestica treated with permethrin-B. rotunda and C. megacephala treated with permethrin-O. gratissimum. The LD50 of insecticides decreased when combined with plant EOs. This alternative strategy will be helpful in developing a formula for effective fly control management.

Wing morphometric analysis of forensically important flesh flies (Diptera: Sarcophagidae) in Thailand.

Flesh flies are insects of forensic importance as the larvae associated with human remains can be used to estimate a minimum post-mortem interval (PMImin) in most cases. And, because life-history traits can vary across species, correct identification is a mandatory step before being used as evidence. Adult flesh flies are extremely similar in general appearance, which causes difficulty in species identification as it is largely based on the morphology of the male genitalia; this also makes it difficult to identify females. Currently, landmark-based geometric morphometric analysis of insect wings has proven to be a valuable tool for species identification. Herein, we applied wing morphometric analysis of 524 flesh fly specimens comprising 12 species from Thailand. The right wing of each specimen was removed, mounted on a microscope slide, photographed, and digitized using 18 landmarks. Wing shape variation among genera and species were analyzed using canonical variate analysis, while wing shape variation between sexes of each species was analyzed using discriminant function analysis. A cross-validation test was used to evaluate the reliability of classification. Results of this study demonstrate wing shape can be used to separate genera and species, and distinguish between sexes of the same species, with high reliability. Therefore, the landmark-based geometric morphometric analysis of wings is a useful additional method for species and sex discrimination of flesh flies.

Spatial Distribution of Forensically Significant Blow Flies in Subfamily Luciliinae (Diptera: Calliphoridae), Chiang Mai Province, Northern Thailand: Observations and Modeling Using GIS.

Blow flies of the subfamily Luciliinae (Diptera: Calliphoridae) are one of the main forensically important subfamilies globally. In addition to being used to estimate the minimum post-mortem interval (PMImin), assuming colonization occurred after death, blow fly specimens found infesting a human corpse are used to determine if the corpse was relocated or if the individual ingested narcotics prior to death. The presence of these blow flies in a given area is strongly influenced by abiotic and biotic factors, such as temperature, elevation, and habitat. Having this information, along with geographical distributions and the characteristics of preferred habitats, is necessary to better understand the biology of this group. This study aimed to characterize the spatial distribution of Luciliinae throughout 18 sampling sites within six ecozones (disturbed mixed deciduous forest, mixed deciduous forest, mixed orchard, paddy field, lowland village, and city/town) in central Chiang Mai Province, northern Thailand over one year (May 2009⁻May 2010). The purpose of the study was to elucidate the relationship of blow fly species composition with environmental abiotic factors (e.g., temperature, relative humidity, light intensity), and to predict the distribution of the common species within this subfamily using GIS. Adult collections were performed biweekly, baited with one-day-old beef offal. A total of 2331 Luciliinae flies trapped, comprising eight species, of which the four predominant species were Hemipyrellia ligurriens (Wiedemann) (n = 1428; 61.3%), Lucilia porphyrina (Walker) (n = 381; 16.3%), Hemipyrellia pulchra (Wiedemann) (n = 293; 12.6%), and Lucilia papuensis Macquart (n = 129; 5.5%). Population density across species varied seasonally, peaking in August 2009 coinciding with the rainy season. Predicting population composition was based on a model developed with ArcGIS 9.2, which utilized environmental variables (temperature, relative humidity, and light intensity) in conjunction with abundance data. Models indicated H. ligurriens had the most widespread geographic distribution, while H. pulchra was predicted to occur largely in mixed orchards and lowland villages. Lucilia porphyrina and L. papuensis were less widespread, restricted mainly to mixed deciduous forest. This model, along with knowledge of forensic information, may be useful under certain investigations where the corpse may have been relocated.

A molecular, morphological, and physiological comparison of English and German populations of Calliphora vicina (Diptera: Calliphoridae).

The bluebottle blow fly Calliphora vicina is a common species distributed throughout Europe that can play an important role as forensic evidence in crime investigations. Developmental rates of C. vicina from distinct populations from Germany and England were compared under different temperature regimes to explore the use of growth data from different geographical regions for local case work. Wing morphometrics and molecular analysis between these populations were also studied as indicators for biological differences. One colony each of German and English C. vicina were cultured at the Institute of Legal Medicine in Frankfurt, Germany. Three different temperature regimes were applied, two constant (16°C & 25°C) and one variable (17-26°C, room temperature = RT). At seven time points (600, 850, 1200, 1450, 1800, 2050, and 2400 accumulated degree hours), larval lengths were measured; additionally, the durations of the post feeding stage and intrapuparial metamorphosis were recorded. For the morphometric and molecular study, 184 females and 133 males from each C. vicina population (Germany n = 3, England n = 4) were sampled. Right wings were measured based on 19 landmarks and analyzed using canonical variates analysis and discriminant function analysis. DNA was isolated from three legs per specimen (n = 61) using 5% chelex. A 784 bp long fragment of the mitochondrial cytochrome b gene was sequenced; sequences were aligned and phylogenetically analyzed. Similar larval growth rates of C. vicina were found from different geographic populations at different temperatures during the major part of development. Nevertheless, because minor differences were found a wider range of temperatures and sampling more time points should be analyzed to obtain more information relevant for forensic case work. Wing shape variation showed a difference between the German and English populations (P<0.0001). However, separation between the seven German and English populations at the smaller geographic scale remained ambiguous. Molecular phylogenetic analysis by maximum likelihood method could not unambiguously separate the different geographic populations at a national (Germany vs England) or local level.

Chrysomya chani Kurahashi (Diptera: Calliphoridae), a blow fly species of forensic importance: morphological characters of the third larval instar and a case report from Thailand.

Blow flies are worldwide the most important insects from a forensic point of view. In Thailand, aside from the two most common species, Chrysomya megacephala (F.) and Chrysomya rufifacies (Macquart), Chrysomya chani Kurahashi was also found to be of forensic importance. We present a case of a human female cadaver in its bloated stage of decomposition, discovered at Pachangnoi Subdistrict, northern Thailand. Entomological sampling during the autopsy displayed an assemblage of numerous dipteran larvae. Macroscopic observations showed the coexistence of third instar larvae of the three blow flies C. megacephala, Chrysomya villeneuvi Patton, an unknown blow fly species and one muscid, Hydrotaea sp. The minimum post-mortem interval was estimated to be six days, based on the developmental rate of C. megacephala. The ID of the unknown larva, which is the focus of this report, was revealed later as C. chani by DNA sequencing, using a 1205 bp of cytochrome c oxidase subunit I (COI). The occurrence of C. chani on a human body revealed the need to analyse and describe the morphology of its immature stage, to enable forensic entomologists to identify this fly species in future cases. The morphological examination of the third instar was performed, revealing peculiar characteristics: protuberant tubercles encircling abdominal segments; 9-11 lobes on the anterior spiracle; six prominent pairs of tubercles along the peripheral rim of the eighth abdominal segment; a heavily sclerotized complete peritreme of the posterior spiracles. A key to differentiate the third instar of blow flies of forensic importance in Thailand is provided.

Molecular Analysis of Forensically Important Blow Flies in Thailand.

Blow flies are the first insect group to colonize on a dead body and thus correct species identification is a crucial step in forensic investigations for estimating the minimum postmortem interval, as developmental times are species-specific. Due to the difficulty of traditional morphology-based identification such as the morphological similarity of closely related species and uncovered taxonomic keys for all developmental stages, DNA-based identification has been increasing in interest, especially in high biodiversity areas such as Thailand. In this study, the effectiveness of long mitochondrial cytochrome c oxidase subunit I and II (COI and COII) sequences (1247 and 635 bp, respectively) in identifying 16 species of forensically relevant blow flies in Thailand (Chrysomya bezziana, Chrysomya chani, Chrysomya megacephala, Chrysomya nigripes, Chrysomya pinguis, Chrysomya rufifacies, Chrysomya thanomthini, Chrysomya villeneuvi, Lucilia cuprina, Lucilia papuensis, Lucilia porphyrina, Lucilia sinensis, Hemipyrellia ligurriens, Hemipyrellia pulchra, Hypopygiopsis infumata, and Hypopygiopsis tumrasvini) was assessed using distance-based (Kimura two-parameter distances based on Best Match, Best Close Match, and All Species Barcodes criteria) and tree-based (grouping taxa by sequence similarity in the neighbor-joining tree) methods. Analyses of the obtained sequence data demonstrated that COI and COII genes were effective markers for accurate species identification of the Thai blow flies. This study has not only demonstrated the genetic diversity of Thai blow flies, but also provided a reliable DNA reference database for further use in forensic entomology within the country and other regions where these species exist.

Morphology of immature blow fly Hypopygiopsis infumata (Bigot) (Diptera: Calliphoridae), a potential species of forensic importance.

Blow flies of the genus Hypopygiopsis are forensically-important, as their larvae are commonly associated with human corpses. Within a forensic entomology context, species identification of specimens collected from human corpses is the initial mandatory step in the investigation. Without identification, complete interpretation of entomological evidence is challenged. In this study, the ultrastructures of eggs, all instars, and puparia of Hypopygiopsis infumata (Bigot) are presented based on assessment with scanning electron microscopy (SEM) and light microscopy (LM). Distinctive features used for species identification of all stages are highlighted. Eggs have a slightly widening median area extending almost the entire length. Larvae are vermiform-shaped, creamy white, and have a smooth integument. The pseudocephalon of larvae bears sensory structures (i.e., antennal dome, maxillary palpus and ventral organ). In the first instar, two tufts of cirri are observed along the dorsal margin of the mouth opening. In the second and third instars, six minute tubercles are present along the peripheral rims of the last abdominal segment. The anterior spiracle of the second, third instar, and puparia is fan-shape of single row, comprising 9-11 papillae. The cuticular spines between the 1st and 2nd thoracic segments of the third instar possess many rows of posteriorly-projecting acuminate spines in clusters. In puparia, at the latero-dorsal edge of the 1st abdominal segment, a cluster of ∼92 bubble membranes is present in young puparia (20-24 h). The peristigmatic tufts adjacent to the posterior spiracle of the second instar, third instar, and puparia are heavily branches of long, fine hairs. Our results demonstrate the morphology of eggs, larvae, and puparia of H. infumata are similar to other species in Hypopygiopsis. This study highlighted the main features of cephaloskeleton of H. infumata larvae as observed under LM. Particular attention is given to oral sclerite and rough surface of dorsal cornua which can distinguish between H. infumata and H. tumrasvini.

Predicting Geographic Distribution of Forensically Significant Blow Flies of Subfamily Chrysomyinae (Diptera: Calliphoridae) in Northern Thailand.

Blow flies (Diptera: Calliphoridae) are carrion-breeding flies that are commonly used as evidence in forensic investigation. An adequate knowledge of ecological and geographical data of blow fly has a direct application in forensic science, as far as estimating time of colonization or corpse relocation. The aim of this study was to evaluate the occurrence of four species of Chrysomyinae (Chrysomya pinguis, Chrysomya chani, Chrysomya villeneuvi, and Ceylonomyia nigripes) across six land use types in central Chiang Mai, northern Thailand. Eighteen study sites were selected for sampling across three districts of Chiang Mai province (Mueang Chiang Mai, Mae Rim, and Hang Dong). Adult flies were collected every two weeks using a funnel trap baited with 1-day tainted beef offal. The predicted geographic distributions of forensically important blow fly species were modeled using the computer program ArcGIS, based on selected climatic variables (temperature, relative humidity, and light intensity) recorded at study sites. During the study period, 1298 adult flies were collected, with peak fly occurrence during summer (April⁻May). Seasonal fluctuation patterns varied depending on fly species. Climatic factors displayed diverse impact on associated fly populations. Identified species were restricted mainly to mixed deciduous forests (MDF) especially in the mountainous area. None of these flies were trapped in an urban area.

Bionomics of the oriental latrine fly Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae): temporal fluctuation and reproductive potential.

BACKGROUND: Chrysomya megacephala is a blow fly species of medical and forensic importance worldwide. Understanding its bionomics is essential for both designing effective fly control programs and its use in forensic investigations. METHODS: The daily flight activity, seasonal abundance related to abiotic factors (temperature, relative humidity and rainfall) and reproductive potential of this species was investigated. Adult flies were sampled twice a month for one year from July 2013 to June 2014 in three different ecotypes (forest area, longan orchard and palm plantation) of Chiang Mai Province, northern Thailand, using semi-automatic funnel traps. One-day tainted beef offal was used as bait. RESULTS: A total of 88,273 flies were sampled, of which 82,800 flies (93.8%) were caught during the day (from 06:00 to 18:00 h); while 5473 flies (6.2%) were caught at night (from 18:00 to 06:00 h). Concurrently, the abundance of C. megacephala was higher in the forest area (n = 31,873; 36.1%) and palm plantation (n = 31,347; 35.5%), compared to the longan orchard (n = 25,053; 28.4%). The number of females was significantly higher than that of males, exhibiting a female to male sex ratio of 2.36:1. Seasonal fluctuation revealed the highest abundance of C. megacephala in summer, but low numbers in the rainy season and winter. Fly density was significantly positively correlated with temperature, but negatively correlated with relative humidity. No correlation between numbers of C. megacephala with rainfall was found. Activity occurred throughout the daytime with high numbers from 06:00 to 18:00 h in summer and 12:00 to 18:00 h in the rainy season and winter. As for the nocturnal flight activity, a small number of flies were collected in summer and the rainy season, while none were collected in the winter. Dissection of the females indicated that fecundity was highest during the rainy season, followed by winter and summer. CONCLUSIONS: Since the assessment of daily, seasonal activity and the reproductive potential of C. megacephala remains a crucial point to be elucidated, this extensive study offers insights into bionomics, which may be considered for integrated fly control strategies and forensic entomology issues.

Ultrastructure of male genitalia of blow flies (Diptera: Calliphoridae) of forensic importance.

Male genitalia of blow flies (Diptera: Calliphoridae) are distinctive in their morphological features and are often used for species identification. The aim of this work was to investigate the male genitalia of blow flies of medical and forensic importance from Thailand at the ultrastructural level, using scanning electron microscopy (SEM). Flies in two subfamilies were examined: Chrysomyinae [Chrysomya bezziana Villeneuve, Chrysomya chani Kurahashi, Chrysomya nigripes Aubertin, Chrysomya pinguis (Walker), Chrysomya rufifacies (Macquart), Chrysomya thanomthini Kurahashi & Tumrasvin, and Chrysomya villeneuvi Patton] and Luciliinae [Hemipyrellia ligurriens (Wiedemann), Hypopygiopsis infumata (Bigot), Hypopygiopsis tumrasvini Kurahashi, Lucilia cuprina (Wiedemann), Lucilia papuensis Macquart, Lucilia porphyrina (Walker), and Lucilia sinensis Aubertin]. Particular attention was paid to the main distinguishing features such as the shapes of the cercus and the surstylus, and the complex structure of the distiphallus. The differentiation of the male genitalia of these species at the SEM level is discussed and compared to the conditions in closely related species such as Chrysomya megacephala (Fabricius). A key for the identification of 14 blow fly species based on male genitalia is provided.

Morphology of immature stages of blow fly, Lucilia sinensis Aubertin (Diptera: Calliphoridae), a potential species of forensic importance.

Lucilia sinensis Aubertin (Diptera: Calliphoridae) is a blow fly species of potential forensic importance since adults are attracted to, and colonize, decomposing vertebrate remains. Blow fly larvae associated with human corpses can be useful evidence in forensic investigations; however, their use is dependent in most cases on proper species identification and availability of developmental data. For identification, morphological information on each life stage is traditionally used. We used scanning electron microscopy (SEM) to examine the ultrastructure of eggs, all instars, and puparia, of L. sinensis. The important characteristics used to differentiate L. sinensis from other species are provided. Distinctive features of the eggs are the slight widening median area extending almost the entire length. The last abdominal segment of the first instar bears elongated outer ventral tubercles along the rim of the last abdominal segment. These tubercles, as well as the well developed median and outer dorsal tubercles, are more prominent in the second and third instars. The surface integument of the tubercles is equipped with circular rows of microtrichia. Pairs of inner dorsal tubercle are absent. Each anterior spiracle is comprised of 9-12 papillae arrange in a single row in the second and third instars. As for the third instar, the dorsal spines between the first and second thoracic segments are delicate, narrow, small, and close together (as row or set). The peristigmatic tufts adjacent to the posterior spiracle of the third instar are moderately branches of short, fine hairs, but minute in puparia. In conclusion, the prominent outer ventral tubercle in all instars and puparia is a new diagnostic feature of L. sinensis and helpful in differentiating it from other Lucilia species that are forensically important. The description of immature L. sinensis in this study will be useful for forensic entomologists in countries where this species exists.

Effect of cold argon plasma on eggs of the blow fly, Lucilia cuprina (Diptera: Calliphoridae).

Non-thermal plasma has been used in many medical applications, including treatment of living cells, blood coagulation, wound healing, and sterilization. The process uses an environmentally friendly gas (e.g., argon, helium, oxygen, nitrogen, or hydrogen) to destroy bacteria cells with no serious adverse effect on humans or animals. However, information on the effect of argon plasma on blow fly eggs is lacking. In this study, we explored the ability of cold argon plasma to destroy the eggs of the Australian sheep blow fly, Lucilia cuprina (Wiedemann, 1830); its larvae are a myiasis-producing agent in both human and animals. We tested the effect of cold argon plasma exposure for 1, 2, 3 and 5min on L. cuprina eggs. Since the temperature of cold Ar plasma is around 30°C, to clarify the effect of temperature on the fly eggs, hot air from an electric dryer was tested for comparison. Cold argon plasma exposure in eggs significantly reduced the survival rates of second instar larvae at all exposures tested; the effects were time dependent, with a stronger effect at longer exposure (32% survival rate after a 1-min treatment; 20%, 2min; 20%, 3min; and 6%, 5min), compared to the control (86%). No significant differences were observed in larval survival rates from eggs treated with hot air (80-84%, after 1- to 5-min treatments) versus the control (86%). These results were supported by observing the treated eggshells under a scanning electron microscope (SEM), we found noticeable aberrations only in the plasma treated groups. The emission spectrum of the argon gas discharge revealed emission lines of hydroxyl radicals at 309.1nm; these may cause the deterioration of the treated L. cuprina eggs. Our results have shown the possibility of using cold argon plasma in medical applications, in particular treating myiasis wounds.

Field evaluation of a semi-automatic funnel trap targeted the medically important non-biting flies.

Bait-trapping is a useful approach for monitoring fly population dynamics, and it is an effective tool for physical control of pest species. The aim of this study was to test a newly developed semi-automatic funnel fly trap with some modifications of the former prototype fly trap to study medically important fly population density. The efficacy of the semi-automatic funnel trap was assessed by field sampling during July 2013-June 2014 using 1-day tainted beef offal as bait. The modified semi-automatic funnel traps were able to capture a total of 151,141 adult flies, belonging to the families: Calliphoridae (n=147,248; 97.4%), Muscidae (n=3,124; 2.1%) and Sarcophagidae (n=769; 0.5%), which are the medically important fly species. Among the total of 35 species collected, Chrysomya megacephala (Diptera: Calliphoridae) (n=88,273; 59.95%), Musca domestica (Diptera: Muscidae) (n=1,324; 42.38%) and Boettcherisca peregrina (Diptera: Sarcophagidae) (n=68; 33.01%) were the predominant species of each family. High number of flies was captured in forest area, representing 42.47% (n=64,197) of total specimens. Female flies were trapped more than male with total sex ratio of 0.37 male/female. Flies were trapped throughout the year with peak population in summer. Peak activity was recorded in the afternoon (12.00-18.00h). In summary, the modified semi-automatic funnel fly trap can be used for field collection of the adult fly. By setting the timer, population dynamics, diversity, and periodic activity of adult flies were determined.