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.

Ultrastructure of sensilla on the antennae and maxillary palpi of the human-biting black flies, Simulium nigrogilvum and Simulium umphangense, (Diptera: Simuliidae) in Thailand.

Antennae and maxillary palpi are the most important sensory organs involved in the behaviors of black flies. The ultrastructure of sensilla on these sensory appendages of two human-biting black fly species, Similium nigrogilvum and Simulium umphangense, was studied for the first time. Wild adult females of both species were collected in Umphang District, Tak Province, western Thailand. The morphology and distribution of sensilla were examined using scanning electron microscopy. Overall, the morphology of the antennae and maxillary palpi and distribution of sensilla are similar in the two species. Four major types of sensilla were found on the antennae of both species: sensilla basiconica (three subtypes), coeloconica, chaetica (four subtypes), and trichodea. However, sensilla basiconica subtype IV are only present on the antennal surface of S. nigrogilvum. Sensilla trichodea are the most abundant among the four types of sensilla that occur on the antennae of both species. Significant differences in the length of the antennae (scape and flagellomere IX), length of the maxillary palpi (whole and palpal segments I, III, IV and V), and the length and basal width of four sensilla types (trichodea, chaetica, basiconica, and coeloconica) were found. In addition, two types of sensilla were observed on the maxillary palpi: sensilla chaetica (three subtypes) and bulb-shaped sensilla. Differences were observed in the numbers of bulb-shaped sensilla in the sensory vesicles of S. nigrogilvum and S. umphangense. The findings are compared with the sensilla of other insects, and the probable functions of each sensillum type are discussed. The anatomical data on sensory organs derived from this study will help to better understand black fly behavior.

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.

Geometric morphometric wing analysis as a tool to discriminate female mosquitoes from different suburban areas of Chiang Mai province, Thailand.

Mosquitoes are hematophagous insects that transmit parasites and pathogens with devastating effects on humans, particularly in subtropical regions. Different mosquito species display various behaviors, breeding sites, and geographic distribution; however, they can be difficult to distinguish in the field due to morphological similarities between species and damage caused during trapping and transportation. Vector control methods for controlling mosquito-borne disease epidemics require an understanding of which vector species are present in the area as well as the epidemiological patterns of disease transmission. Although molecular techniques can accurately distinguish between mosquito species, they are costly and laborious, making them unsuitable for extensive use in the field. Thus, alternative techniques are required. Geometric morphometrics (GM) is a rapid and inexpensive technique that can be used to analyze the size, shape, and shape variation of individuals based on a range of traits. Here, we used GM to analyze the wings of 1,040 female mosquitoes from 12 different species in Thailand. The right wing of each specimen was removed, imaged microscopically, and digitized using 17 landmarks. Wing shape variation among genera and species was analyzed using canonical variate analysis (CVA), while discriminant function analysis was used to cross-validate classification reliability based on Mahalanobis distances. Phenetic relationships were constructed to illustrate the discrimination patterns for genera and species. CVA of the morphological variation among Aedes, Anopheles, Armigeres, Culex, and Mansonia mosquito genera revealed five clusters. In particular, we demonstrated a high percentage of correctly-distinguished samples among Aedes (97.48%), Armigeres (96.15%), Culex (90.07%), and Mansonia (91.67%), but not Anopheles (64.54%). Together, these findings suggest that wing landmark-based GM analysis is an efficient method for identifying mosquito species, particularly among the Aedes, Armigeres, Culex, and Mansonia genera.

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.

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.

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.

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.

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.

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.

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.