Assessment of deep convolutional neural network models for species identification of forensically-important fly maggots based on images of posterior spiracles.

Forensic entomology is the branch of forensic science that is related to using arthropod specimens found in legal issues. Fly maggots are one of crucial pieces of evidence that can be used for estimating post-mortem intervals worldwide. However, the species-level identification of fly maggots is difficult, time consuming, and requires specialized taxonomic training. In this work, a novel method for the identification of different forensically-important fly species is proposed using convolutional neural networks (CNNs). The data used for the experiment were obtained from a digital camera connected to a compound microscope. We compared the performance of four widely used models that vary in complexity of architecture to evaluate tradeoffs in accuracy and speed for species classification including ResNet-101, Densenet161, Vgg19_bn, and AlexNet. In the validation step, all of the studied models provided 100% accuracy for identifying maggots of 4 species including Chrysomya megacephala (Diptera: Calliphoridae), Chrysomya (Achoetandrus) rufifacies (Diptera: Calliphoridae), Lucilia cuprina (Diptera: Calliphoridae), and Musca domestica (Diptera: Muscidae) based on images of posterior spiracles. However, AlexNet showed the fastest speed to process the identification model and presented a good balance between performance and speed. Therefore, the AlexNet model was selected for the testing step. The results of the confusion matrix of AlexNet showed that misclassification was found between C. megacephala and C. (Achoetandrus) rufifacies as well as between C. megacephala and L. cuprina. No misclassification was found for M. domestica. In addition, we created a web-application platform called thefly.ai to help users identify species of fly maggots in their own images using our classification model. The results from this study can be applied to identify further species by using other types of images. This model can also be used in the development of identification features in mobile applications. This study is a crucial step for integrating information from biology and AI-technology to develop a novel platform for use in forensic investigation.

Prevalence of blaCTX-M and Emergence of blaCTX-M-5-Carrying Escherichia coli in Chrysomya megacephala (Diptera: Calliphoridae), Northern Thailand.

This study was undertaken to assess the prevalence of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli (ESBL-EC) among blow fly (Chrysomya megacephala) populations in Northern Thailand. Of 600 blow flies collected from rural (n = 400) and urban (n = 200) areas, 334 blow flies carried ESBL-EC (55.7%). Prevalence of ESBL-EC in blow flies captured from rural areas was significantly higher than that from urban region (72.5% vs. 22.0%, p < 0.001). Susceptibility tests revealed that 68.6% of ESBL-EC possessed multidrug-resistant phenotypes. Coresistance to gentamicin (85%) was common, while resistance to ciprofloxacin was relatively low (18.0%). Of the 334 isolates, 253 isolates (75.7%) harbored blaCTX-M, in which blaCTX-M group 1 was predominant (56.5%), followed by blaCTX-M group 9 (39.1%). Interestingly, a single isolate was found to carry blaCTX-M-5, which resided on the IncA/C conjugative plasmid. This is the first report of blaCTX-M-5 from Thailand and its first identification in blow fly. Pulsed field gel electrophoresis (PFGE) demonstrated high genetic diversity among ESBL-EC isolates. Nevertheless, identical and closely related PFGE profiles were detected among isolates within the same regions and the regions which are several kilometers apart, suggesting that clonal transmission has occurred. Moreover, epidemiologically related isolates were observed between ESBL-EC from blow flies and human intestinal tract. This study provides evidences that blow flies, C. megacephala, are important reservoirs for ESBL-EC and could potentially act as vectors for the spread of ESBL-EC in a Thai community.

Environmental dissemination of mcr-1 positive Enterobacteriaceae by Chrysomya spp. (common blowfly): An increasing public health risk.

Until recently, the role of insects, and particularly flies, in disseminating antimicrobial resistance (AMR) has been poorly studied. In this study, we screened blowflies (Chrysomya spp.) from different areas near the city of Phitsanulok, Northern Thailand, for the presence of AMR genes and in particular, mcr-1, using whole genome sequencing (WGS). In total, 48 mcr-1-positive isolates were recovered, consisting of 17 mcr-1-positive Klebsiella pneumoniae (MCRPKP) and 31 mcr-1-positive Escherichia coli (MCRPEC) strains. The 17 MCRPKP were shown to be clonal (ST43) with few single poly nucleomorphs (SNPs) by WGS analysis. In in-vitro models, the MCRPKP were shown to be highly virulent. In contrast, 31 recovered MCRPEC isolates are varied, belonging to 12 different sequence types shared with those causing human infections. The majority of mcr-1 gene are located on IncX4 plasmids (29/48, 60.42%), sharing an identical plasmid backbone. These findings highlight the contribution of flies to the AMR contagion picture in low- and middle-income countries and the challenges of tackling global AMR.

Larvicidal efficacy of crude and fractionated extracts of Dracaena loureiri Gagnep against Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, and Anopheles minimus mosquito vectors

Objective: To evaluate the larvicidal efficacy of crude and fractionated extracts of Dracaena loureiri endocarp against Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, and Anopheles minimus mosquitos. Methods: Larvicidal activity was tested according to World Health Organization standard protocol.The third-stage larvae of each mosquito species were exposed to various concentrations of Dracaena loureiri crude extract and six groups of Dracaena loureiri fractionated extracts (RC-DT 009–014). Larval mortality rates were observed after 24 h and 48 h of exposure.Then, a computerized probit analysis of the mortality data was performed to determine lethal concentration 50 (LC50) and lethal concentration 90 values. Results: Anopheles minimus larvae (24-h LC5077.88 mg/L) had the highest susceptibility to crude extract, whereas others (Aedes aegypti, 24-h LC50224.73 mg/L; Aedes albopictus, 24-h LC50261.75 mg/L; and Culex quinquefasciatus, 24-h LC50282.86 mg/L) were significantly less susceptible. The most effective groups of fractionated extracts were RC-DT 012 and RC-DT 013. The mosquito species most susceptible to fractionated extracts was Culex quinquefasciatus, with 24-h LC50 values of 0.66 and 0.94 mg/L for RC-DT 012 and RC-DT 013, respectively. Conclusions: The larvicidal activity of fractionated extracts is more effective than that of crude extract against all tested mosquito species. For the most effective alternative larvicide, purification and a phytochemical constituent analysis must be performed.

Variation in the Time of Colonization of Broiler Carcasses by Carrion Flies in Nakhonsawan Province, Thailand.

Carrion flies are the primary insects colonizing vertebrate carrion; however, limited information is available on the variation in the time of colonization (TOC) as related to time of placement (TOP) and time of death (TOD), particularly in Thailand. Three seasonal sets of nine broiler carcasses (euthanized and placed in field within 0.5 h after death) were placed in mesh enclosures within a disturbed deciduous dipterocarp forest at Nakhonsawan Province, upper-central Thailand, for 3 d to determine the colonization time by carrion flies. In total, 21,536 arthropods were collected using traps placed over each carcass. Carrion flies of the family Calliphoridae, Muscidae, and Sarcophagidae predominated (93.42%). Of these, Chrysomya megacephala (F.) (Diptera: Calliphoridae) and Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae) were the dominant species being 36.18% and 35.36%, respectively, across season. These species arrived immediately (5 min) after placement of the carrion in the field during the rainy season, while they were delayed 1-2 h during the dry season. Chrysomya megacephala, C. rufifacies, and Parasarcophaga dux (Thomson) colonized the remains. Time of colonization by C. megacephala and C. rufifacies occurred mostly at ∼1600-1700 hours (10-11 h after placement) for all seasons. In contrast, TOC by P. dux was delayed for 1 d during rainy and dry season. These results mark the first record of carrion fly colonization in this area and also may deserve important information for the further study as they demonstrate time of colonization differs from TOP and most importantly TOD.

Morphological Characteristics of Terminalia of the Wasp-Mimicking Fly, Stomorhina discolor (Fabricius).

Stomorhina discolor (Fabricius), a species of blow fly that mimics wasps, is distributed worldwide, but detailed information about characteristics of its adult terminalia is incomplete. To help fill this gap in the information, the morphology of adult stages of S. discolor was investigated using light microscopy and scanning electron microscopy. Observations using the light microscope revealed unique characteristics of the male genitalia that are markedly different from other blow fly species. More morphological detail, including observation of several sensilla (e.g., sensilla trichoid and sensilla basiconica) along the male terminalia and female ovipositor, was seen under the scanning electron microscope. These details can be taxonomically valuable for identifying males and females of S. discolor and may help address matters concerning copulation in this species.

Prevalence and virulence factors of Candida spp.associated with blow flies

Objective:To investigate the prevalence of Candida spp.and the virulence factors of Candida albicans (C.albicans) isolated from external surfaces of blow flies collected from Mae Sot,Tak Province,Thailand,Methods:The blow flies were collected by sterile sweep nets from three areas in Mae Sot.Yeast isolation was first performed on Sabouraud dextrose agar (SDA) supplemented with chloramphenicol and cycloheximide.The yeast isolates were then identified by using chromogenic agar,a yeast identification test kit,a germ tube formation test and a carbohydrate utilization test.The β-hemolysis was determined on 7％ sheep blood agar,while phospholipase activity was measured on SDA agar supplemented with 10％ egg yolk suspension.Antifungal susceptibility testing was determined by broth micro-dilution testing against ketoconazole and amphotericin B.Results:The prevalence rate of Candida spp.on the external surfaces of the blow flies was 78.1％.All C.albicans isolated from the blow fly demonstrated β-hemolysin and potent phospholipase activities and 47.1％ of C.albicans were resistant to ketoconazole with MIC values 128 μg/mL.Conclusions:The result s indicate that blow flies could play an essential role in the transmission of potentially pathogenic and antifungal resistant C.albicans into the environment.Further investigation on other virulence factors and genetic relatedness among isolates from the blow flies,the environment and clinical specimens is required to confirm this role.

Larvicidal activity of endocarp and seed crude extracts of Dracaena loureiri Gagnep against Aedes aegypti (L.) mosquito

Objective: To evaluate the larvicidal activity of the ethanolic and aqueous extracts of the endocarp and seeds of Dracaena loureiri (D. loureiri) against the dengue mosquito vector, Aedes aegypti. Methods: Bioassays were performed by exposing late third-stage to early fourth-stage larvae of Aedes aegypti to various concentrations of the extracts from D. loureiri. The larval mortality was observed after 24-and 48-h exposure. Results: The larvicidal bioassay in this study demonstrated that the ethanolic endocarp extract was the most effective with the LC50 value of 84.00 mg/L after 24 h exposure and< 50 mg/L after 48 h exposure. Extracts from the other parts of the plant were signifi-cantly less effective as a larvicide. Conclusions: The ethanolic endocarp extract of D. loureiri demonstrated effective lar-vicidal activity. It is an alternative source for developing a novel larvicide for controlling this mosquito species.

Susceptibility to temephos, permethrin and deltamethrin of Aedes aegypti (Diptera: Culicidae) from Muang district, Phitsanulok Province, Thailand.

OBJECTIVE: To investigate the susceptibility to temephos, permethrin and deltamethrin of Aedes aegypti (Ae. aegypti), collected from areas with high incidence of dengue hemorrhagic fever cases in Phitsanulok Province, Thailand. METHODS: The F1 progenies of Ae. aegypti colony, originated from five sub-districts including Aranyik, Hua Ro, Nai Muang, Ban Krang and Tha Pho, were used in the bioassays following the procedures of World Health Organization. For larval bioassay, the late third or early fourth-instar larvae were tested with different concentrations of temephos. For adult bioassay, the females were exposed to 0.75 % permethrin or 0.05% deltamethrin. LC50 value and mortality rate were analyzed to compare the insecticide susceptibility of the larvae and the adults in each area, respectively. RESULTS: The LC50 value of temephos for the larvae from Aranyik, Hua Ro, Nai Muang, Ban Krang and Tha Pho sub-districts was 0.017, 0.017, 0.026, 0.061, and 0.113 ppm, respectively. For permethrin, the highest mortality rate (86.84%) was found in the mosquitoes from Aranyik but the others were more resistant with the lower mortality rates (16.00-42.67%). The adult mortality rates after exposing to deltamethrin were higher (82.34-98.67%) in all areas. CONCLUSIONS: Ae. aegypti larvae were still susceptible to temephos. Conversely, most tested adults tended to resist the permethrin and deltamethrin.

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.

Prevalence of helminthic infections and risk factors in villagers of Nanglae Sub-District, Chiang Rai Province, Thailand.

The purposes of the present study were to survey the prevalence of helminthic infections in people living in Nanglae Sub-District of Chiang Rai Province, Thailand from January to March 2013, and to determine factors that correlated with these infections. Two hundred and sixty-three fecal samples were examined for helminth eggs by the use of Kato's thick smear technique. All data were analyzed by descriptive statistics including frequencies, percentages and correlations (Odds ratio [OR] and 95% confidence interval [CI]). The percentage of overall helminthic infections was 11.8%, comprising 6.1% Taenia spp., 4.5%, Ascaris lumbricoides, 0.8%, Strongyloides stercoralis and 0.4% flukes producing opisthorchiid-like eggs. In addition, the prevalence of helminthic infection correlated significantly with the consumption of raw meat (OR = 2.270, 95% CI = 1.047-4.923) and raising dogs in the house (OR = 2.444, 95% CI = 1.080-5.534).

Two new records of Isomyia paurogonita Fang and Fan, 1986 and Sumatria latifrons Malloch, 1926 (Diptera: Calliphoridae) from northern Thailand, with revised key to the species of Isomyia.

During the annual fly survey at Doi Nang Kaew in Doi Saket District, Chiang Mai Province of Thailand in 2011, Isomyia paurogonita Fang & Fan, 1986 (Diptera: Calliphoridae) and Sumatria latifrons Malloch, 1926 (Diptera: Calliphoridae) were collected for the first time in Thailand. They are the rare species of the subfamily Rhiniinae (tribe Cosminini). Prior to this finding, fifteen species of Isomyia and two species of Sumatria were recorded from Thailand. Therefore, 96 blow fly species have been found in this country. These new locality records of both flies are very important for further research on their biology and ecology in Thailand.

Occurrence of blow fly species (Diptera: calliphoridae) in Phitsanulok Province, Northern Thailand.

Based on the current forensic importance of blow flies (Diptera: Calliphoridae), their biological aspects have been studied increasingly worldwide. The blow fly fauna in Phitsanulok Province, Northern Thailand was studied from May 2009 to April 2010 in the residential, agricultural, mountainous and forested areas of Muang, Wat Bot, Nakhon Thai and Wang Thong districts, respectively, in order to know the occurrence of blow flies in this province. Collections were carried out monthly using commercial funnel fly traps and sweeping methods, with 1-day tainted pork viscera as bait. Identification of adult blow flies exhibited 14 634 specimens, comprising of 5 subfamilies, 14 genera and 36 species. Chrysomya megacephala (Fabricius, 1794) and Achoetandrus rufifacies (Macquart, 1843) were the most and second most abundant species trapped, respectively. These two species of carrion flies prevailed in all the types of land investigated. We calculated and compared the diversity indices, species evenness and richness, and similarity coefficients of the blow fly species in various areas. The data from this study may be used to identify the potential of forensicallyimportant fly species within Phitsanulok Province and fulfill the information on blow fly fauna in Thailand.

Morphology and Developmental Rate of the Blow Fly, Hemipyrellia ligurriens (Diptera: Calliphoridae): Forensic Entomology Applications.

Hemipyrellia ligurriens (Diptera: Calliphoridae) is a forensically important blow fly species presented in many countries. In this study, we determined the morphology of all stages and the developmental rate of H. ligurriens reared under natural ambient conditions in Phitsanulok province, northern Thailand. Morphological features of all stages based on observing under a light microscope were described and demonstrated in order to use for identification purpose. Moreover, development time in each stage was given. The developmental time of H. ligurriens to complete metamorphosis; from egg, larva, pupa to adult, took 270.71 h for 1 cycle of development. The results from this study may be useful not only for application in forensic investigation, but also for study in its biology in the future.

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.

Behavioral responses of Chrysomya megacephala to natural products.

Knowledge of the behavioral responses of medically important flies to natural stimuli is critical for the development of vector control strategies. We designed, constructed, and operated the dual-choice wind tunnel to investigate the behavioral responses of the blowfly, Chrysomya megacephala (Diptera: Calliphoridae), toward two important extrinsic factors (wind speed and olfactory stimuli) that affect the flight behavior of these flies. The dual-choice wind tunnel appeared as a "T-box," consisting of two stimulus partitions and one median release partition. The correlation between wind speed and fly response showed a marked relationship, with the wind speed set at 0.5 m/s being the optimal speed. Of the 72 natural products tested, 24--all having high protein content--attracted flies at the level of greater than 50% in the rearing cages during preliminary tests, thus allowing further investigation in the dual-choice wind tunnel to further assess these responses. The behavioral response between flies exposed to the screening odors in a rearing cage and those exposed in the dual-choice wind tunnel showed a similar trend. One-day-tainted pork viscera yielded the greatest positive response among natural products tested, attracting greater than 90% of the flies included in the test population. One-day-tainted pork viscera exhibited the highest potency (1.578), exceeding 1-day-tainted beef liver, which is commonly used as the standard bait for blowflies. The usefulness and practical applications of the dual-choice wind tunnel for further investigations are also discussed.

Forensic entomology cases in Thailand: a review of cases from 2000 to 2006.

This paper presents and discusses 30 cases of cadavers that had been transferred for forensic entomology investigations to the Department of Forensic Medicine, Faculty of Medicine, Chiang Mai University, northern Thailand, from 2000 to 2006. Variable death scenes were determined, including forested area and suburban and urban outdoor and indoor environments. The fly specimens found in the corpses obtained were the most commonly of the blow fly of family Calliphoridae, and consisted of Chrysomya megacephala (F.), Chrysomya rufifacies (Macquart) Chrysomya villeneuvi Patton, Chrysomya nigripes Aubertin, Chrysomya bezziana Villeneuve, Chrysomya chani Kurahashi, Lucilia cuprina (Wiedemann), Hemipyrellia ligurriens (Wiedemann), and two unknown species. Flies of the family Muscidae [Hydrotaea spinigera Stein, Synthesiomyia nudiseta (Wulp)], Piophilidae [Piophila casei (L.)], Phoridae [Megaselia scalaris (Loew)], Sarcophagidae [Parasarcophaga ruficornis (F.) and three unknown species], and Stratiomyiidae (Sargus sp.) were also collected from these human remains. Larvae and adults of the beetle, Dermestes maculatus DeGeer (Coleoptera: Dermestidae), were also found in some cases. Chrysomya megacephala and C. rufifacies were the most common species found in the ecologically varied death scene habitats associated with both urban and forested areas, while C. nigripes was commonly discovered in forested places. S. nudiseta was collected only from corpses found in an indoor death scene.

A new mucin antibody/enzyme-linked lectin-sandwich assay of serum MUC5AC mucin for the diagnosis of cholangiocarcinoma.

We have previously used agarose gel electrophoresis and immunoblotting to qualitatively measure serum MUC5AC mucin for diagnosing cholangiocarcinoma. In this study, we developed a quantitative determination of serum MUC5AC by sandwich ELISA using MUC5AC mucin monoclonal antibody and soybean agglutinin. A cut-off value of the absorbance 0.074 was obtained from a complete statistical Receiver Operating Characteristic curves with an area under the curve=0.8141. The assay could discriminate cholangiocarcinoma patients from the controls with 71% sensitivity and 90% specificity. The test is simple to perform, reproducible, and probably used for detecting cholangiocarcinoma in a high-risk group or suspected patients.

Fine structure of the eggshell of the blow fly, Lucilia cuprina.

The fine structure of the eggshell of blow fly, Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae), was examined using scanning and transmission electron microscopy. Eggs, 1.09+/-0.07 mm in length and 0.25+/-0.05 mm in width, bore a relatively wide plastron that extending along almost the entire length. The polygonal pattern of chorionic sculpture was indistinct. The ultrathin section indicated a multi-layered eggshell having an exochorion, outer endochorion, pillars, an inner endochorion, innermost chorionic layer, and a wax layer. This study provides new information about the fine morphology of blow flies eggs. A key to differentiate the eggs of forensically important flies in Thailand is given.

Morphological comparison between Chrysomya rufifacies (Macquart) and Chrysomya villeneuvi Patton (Diptera: Calliphoridae) puparia, forensically important blow flies.

In Thailand, the hairy maggots of the blow flies, Chrysomya rufifacies (Macquart) and Chrysomya villeneuvi Patton, are of forensic importance. Both flies are closely related species, not only in the morphological appearance of their larvae and puparia, but also on the aggressive feeding habit of the former. In our continuing studies of forensically important flies, identification of immature ones needs particular attention. In this study, we reported the morphological comparison between the puparia of these two blow fly species using scanning electron microscopy (SEM). Observation revealed that the cuticular sculpture of tubercles along the dorsal and lateral segments had markedly different features: with C. rufifacies having many sharp spines assembling only at the tip, while of C. villeneuvi bore stout spines throughout the tubercle. A larger number of globules at the bubble membrane on the dorsolateral border of the fifth segment was found in C. villeneuvi (average 225) than in C. rufifacies (average 35), and more papillae was observed on the anterior spiracle in C. villeneuvi (13-15) than in C. rufifacies (9-12). However, the morphology of distinct net-like patches of the integument and structure of the posterior spiracle of both species were almost identical. Morphological comparison in this study permitted identification of the puparia of both fly species, particularly in areas where they co-exist.