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.

Molecular Phylogenetics of Centrocestus formosanus (Digenea: Heterophyidae) Originated from Freshwater Fish from Chiang Mai Province, Thailand.

This study aimed to investigate the morphology and reconstruct the phylogenetic relationships of Centrocestus formosanus originating from 5 species of freshwater fish, i.e., Esomus metallicus, Puntius brevis, Anabas testudineus, Parambassis siamensis, and Carassius auratus, in Chiang Mai province, Thailand. Sequence-related amplified polymorphism (SRAP) and phylogeny based on internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome c oxidase subunit 1 (CO1) were performed. The results showed similar morphologies of adult C. formosanus from day 5 after infection in chicks. C. formosanus originated from 4 species of freshwater fish had the same number of circumoral spines on the oral sucker, except for those from C. auratus which revealed 34 circumoral spines. The phylogenetic tree obtained from SRAP profile and the combination of ITS2 and CO1 sequence showed similar results that were correlated with the number of circumoral spines in adult worms. Genetic variability of C. formosanus also occurred in different species of freshwater fish hosts. However, more details of adult worm morphologies and more sensitive genetic markers are needed to confirm the species validity of C. formosanus with 34 circumoral spines originating from C. auratus in the future.

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.

DISCRIMINATION 28S RIBOSOMAL GENE OF TREMATODE CERCARIAE IN SNAILS FROM CHIANG MAI PROVINCE, THAILAND.

Trematode cercariae are commonly found in many freshwater gastropods. These cercariae can serve to identify the occurrence of such trematodes as Centrocestus formosanus, Haplorchis taichui, Haplorchoides sp, and Stellantchasmus falcatus, which are important parasites in Chiang Mai Province, Thailand. As the species of these cercariae cannot be identified accurately based on morphology, this study employed sequencing of a fragment of 28S ribosomal DNA and phylogenetic analysis to identify the trematode cercariae found in freshwater gastropods in Chiang Mai Province. Eight types of trematode cercariae were identified, namely, distome cercaria (grouped with Philophthalmus spp clade), echinostome cercaria (grouped with Echinostoma spp clade), furcocercous cercaria (grouped with Posthodiplostomum sp/Alaria taxideae/Hysteromorpha triloba clade), monostome cercaria (grouped with Catatropis indicus clade), parapleurolophocercous cercaria (grouped with Haplorchoides sp clade), pleurolophocercous cercaria (grouped with Centrocestusformosanus clade), transversotrema cercaria (grouped with Transversotrema spp clade), and xiphidiocercaria (grouped with Prosthodendrium spp clade). These results provide important information that can be used for identifying these parasites in epidemiological surveys.

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.

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.

Surface ultrastrucure of larva and puparia of blow fly Hypopygiopsis tumrasvini Kurahashi (Diptera: Calliphoridae).

Flies of the genus Hypopygiopsis are forensically important, as their larvae are found to associate with human corpses. In this study, the ultrastructure of larvae and puparia of Hypopygiopsis tumrasvini Kurahashi is presented using scanning electron microscopy (SEM). The larvae are vermiform-shaped, creamy white, and have a smooth integument. The pseudocephalon of larvae bears sensory structures, i.e., antenna, maxillary palpus, and ventral organ. Two tufts of fresh outgrowths (or cerri) were observed along the dorsal margin of the mouth opening of the first instar; whereas a strong mouth hook was apparent in the second and third instars. Keilin's organ, the sensory structure, was noticeable on the ventral surface of the thoracic segments in all instars. In the second and third instars, six conspicuous tubercles were present along the peripheral rims of the last abdominal segment. The puparia were relatively large, measuring 7.77-9.51 mm in length and 3.10-3.97 mm in width. At the latero-dorsal edge of the first abdominal segment, a cluster of ~125 bubble membranes was seen in young puparia; whereas a minute pupal respiratory horn was observed in old one. An SEM image revealed antler-like projections lined within a chamber of a broken pupal respiratory horn. Comparison on number of the bubble membranes of the other blow fly species was shown and the role of pupal respiratory horn compared with other dipterans was discussed.

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.

Morphological descriptions for the identification of Hypopygiopsis tumrasvini Kurahashi (Diptera: Calliphoridae).

This article presents morphological descriptions of larvae and adults of Hypopygiopsis tumrasvini Kurahashi (Diptera: Calliphoridae), a blow fly species that may be of forensic importance. Both second and third instar larvae are illustrated, with emphasis placed on important characteristic features used for larval identification, such as the cephalopharyngeal skeleton, anterior and posterior spiracles, and dorsal spines between the prothorax and mesothorax. Eight to 11 papillae were found on each anterior spiracle arranged in a single row. The dorsal spines between the prothorax and mesothorax were arranged in sets of posteriorly projecting acuminate spines with darkly pigmented tips. The posterior spiracles each bear three prominent and separated long, slender spiracular slits encircled by a dark, thick peritreme that is complete ventromedially around a button. Prominent inner projection of peritreme is seen between the middle and lower spiracular slits. A previously published key for differentiating third instar larvae of flies of possible forensic importance in Thailand is updated to include this additional species. Some characteristic features of males and females of the species are also provided.

Do climatic and physical factors affect populations of the blow fly Chrysomya megacephala and house fly Musca domestica?

The blow fly, Chrysomya megacephala (Fabricius), and house fly, Musca domestica L., are medically and forensically important flies. The population dynamic of these flies is essential for both control and forensical aspects. The aim of this study was to investigate the climatic and physical factors affecting the population trend of both species in Chiang Mai province, northern Thailand, using the Geographic Information System (GIS). Based on systematic random sampling, 18 study sites were selected in three districts (Mueang Chiang Mai, Mae Rim, and Hang Dong). Six land use types were involved in the study sites, i.e., disturbed mixed deciduous, mixed deciduous forest, mixed orchard, lowland village, city, and paddy field. Adult flies were sampled every 2 weeks using an in-house prototype reconstructable funnel trap. Two types of bait were used--one with fresh beef viscera for luring M. domestica and the other with 1-day tainted beef viscera for luring C. megacephala. Collections were conducted from May 2009 to May 2010, and analysis of climatic factors (temperature, relative humidity, and light intensity) was carried out. Correlation bivariate analysis was performed initially to determine the relationship between climatic factors and the number of flies. Consequently, an ordinary co-kriging approach, in ArcGIS 9.2, was performed to predict the spatial distribution of flies with land use and climatic factors as co-variables. A total of 63,158 flies were captured, with C. megacephala being the most common species collected (68.37%), while only 1.3% were M. domestica, thus proving that C. megacephala was the most abundant species in several land use types. A significantly higher number of females than males was found in both species. Fly populations can be collected throughout most of the year with a peak in late summer, which shows a positive relation to temperature but negative correlation with relative humidity. C. megacephala was predicted to be abundant in every land use type, from lowland to forested areas, while the density of house fly was association with altitude and land use types.

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.

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.

Forensically important flesh fly species in Thailand: morphology and developmental rate.

Forensically important flesh fly species in Thailand have been investigated for their larval morphology and developmental rate to increase the forensic entomology database in Thailand and nearby countries. Sarcophaga (Liosarcophaga) dux Thomson, Sarcophaga (Liopygia) ruficornis (Fabricius), and Sarcophaga (Boettcherisca) peregrina (Robineau-Desvoidy) are the species of greatest forensic importance. The third instars of these species are similar in morphological appearance, making it difficult or impossible to differentiate between them based on external morphological characteristics. This study compares these important characteristics and reveals that the most distinctive is the posterior spiracle, followed by the number of papillae on the anterior spiracle. For confirmation of these species, the unique characteristics of adult males are also shown for accurate identification. Both the third instar and adult male characteristics are keys to identifying these species. In addition, the developmental rate of S. dux was examined in northern Thailand during 2002-2003 under natural ambient temperature (approximately 24-28 degrees C) and a natural light/dark photoperiod (approximately 12:12 h), indicating relatively rapid larval development in summer (March-June), pre-pupae (stop moving) initiated at 72 h. In the rainy season (July-October), pre-pupae initiated at 72 or 96 h, whereas pre-pupae initiated at 96 h in winter.

Differentiation between Lucilia cuprina and Hemipyrellia ligurriens (Diptera: Calliphoridae) larvae for use in forensic entomology applications.

Lucilia cuprina and Hemipyrellia ligurriens are forensically important blow flies in many countries. The immature stage, in particular the third instar, of both species are generally similar in morphological appearance. In this study, the use of three main characteristics, the posterior spiracle, the dorsal spines between the first and second thoracic segments, and the cephalopharyngeal skeleton, allows for identification of both fly species, with the number of papillae on anterior spiracle being a supportive characteristic in some cases. Comparison of these characteristics is illustrated in detail.