Influence of insular conditions on wing phenotypic variation in two dominant mosquito vectors, Aedes albopictus and Armigeres subalbatus (Diptera: Culicidae), in the border archipelagos of Thailand.

Insects geographically separated into island and mainland populations often exhibit phenotypic variations, a phenomenon known as insular conditions. These conditions can lead to rapid evolutionary changes that affect the morphological characteristics of mosquito vectors. Nevertheless, studies that specifically examine phenotype differences between island and mainland mosquito populations have been limited. In this study, wing variation in size and shape was investigated using the geometric morphometric (GM) technique in two dominant mosquito vectors, Aedes albopictus and Armigeres subalbatus, in the Ranong and Trat archipelagos of Thailand. Significant differences in average wing centroid size (CS) were found in 6 out of 15 population pairs for Ae. albopictus (p < 0.05) and in 5 population pairs for Ar. subalbatus (p < 0.05). After removing the allometric effect, canonical variate analyses (CVA) based on wing shape analysis revealed overlap across all populations for both Ae. albopictus and Ar. subalbatus. However, the statistical analysis indicated that Ar. subalbatus exhibited wing shape differences across all populations (p < 0.05), and most Ae. albopictus populations also displayed distinct wing shapes (p < 0.05), except for the populations from Chang Island and the mainland of Ranong, which showed no significant differences (p > 0.05). These findings enhance our understanding of mosquito adaptability in island regions and provide valuable data for the surveillance and monitoring of vector evolution.

Seroprevalence of melioidosis and its association with blood profiles and pathogens in sheltered dogs in southern Thailand.

Background and Aims: Melioidosis is a notable zoonotic disease in Thailand that can affect both humans and animals. Although dogs are one of the most popular pets worldwide, there is a remarkable lack of information on the prevalence and knowledge of canine melioidosis. This study aimed to estimate the seroprevalence of melioidosis in sheltered dogs and its relationship with the blood profile and blood pathogens. Materials and Methods: Melioidosis in 156 dogs was analyzed using an indirect hemagglutination assay. Hematology and serum biochemistry tests were performed using an automated system. Blood pathogens (e.g., Ehrlichia, Anaplasma, Hepatozoon, and Babesia) were diagnosed using conventional polymerase chain reaction. Results: The seroprevalence rates of canine melioidosis and blood pathogen infection were 5.77% (9/156) and 50.64% (79/156), respectively. Seropositive dogs generally have higher lymphocyte counts and aspartate aminotransferase levels but lower total white blood cell, neutrophil, and platelet (PLT) counts than seronegative dogs. No statistically significant difference (p > 0.05) was observed between the seropositive and seronegative dogs' hematology and serum biochemistry findings. Neither the correlation between melioidosis and blood pathogen infection nor the association between melioidosis and thrombocytopenia was statistically significant (p > 0.05). Remarkably, dogs that had coinfections with both melioidosis and blood pathogens demonstrated a significantly reduced PLTcount (49,167 ± 7,167) compared with dogs that tested positive for melioidosis but negative for blood pathogens (139,333 ± 29,913) (p < 0.01). Conclusion: In southern Thailand, the prevalence of canine melioidosis was low but the prevalence of blood pathogens was high. Coinfection with blood pathogens can significantly reduce PLT counts, which may have a potentially serious impact. Future research should focus on conducting seroprevalence studies in the general dog population.

Preliminary study on prevalence of hemoprotozoan parasites harbored by Stomoxys (Diptera: Muscidae) and tabanid flies (Diptera: Tabanidae) in horse farms in Nakhon Si Thammarat province, Southern Thailand.

Background and Aim: Stomoxys and tabanid flies are of medical and veterinary importance because they play crucial roles in disease transmission as mechanical vectors of various hemopathogens. However, its role as a hemoprotozoan parasite vector in horse farms has not been studied. Therefore, we investigated the occurrence of hemoprotozoan parasites belonging to the genera Babesia, Theileria, and Trypanosoma in Stomoxys and tabanid flies using conventional polymerase chain reaction (PCR) and DNA sequencing. Materials and Methods: All samples (Stomoxys and tabanid flies) were collected using an Nzi trap for three consecutive days each month from November 2022 to March 2023. The flies were morphologically identified to the species level and separated according to sex. Individual (for tabanid flies) or pooled samples (consisting of three specimens of Stomoxys flies of the same species and sex collected from the same site) were used for DNA extraction. Conventional PCR was used to screen for hemoprotozoan parasite DNA, followed by Sanger sequencing to identify the species. Results: In total, 189 biting flies were collected, including four species of Stomoxys (Stomoxys bengalensis, Stomoxys calcitrans, Stomoxys indicus, and Stomoxys sitiens) and five species of tabanids (Atylotus cryptotaxis, Chrysops dispar, Tabanus megalops, Tabanus mesogaeus, and Tabanus rubidus). Stomoxys calcitrans was the most prevalent species, accounting for 58.7% (n = 111) of the collected flies. Ten (12.4%) of the 81 samples (individuals and pools) analyzed by PCR were positive for the 18S rRNA gene of the Theileria/Babesia species. Trypanosoma DNA was not detected in any sample. After performing Basic Local Alignment Search Tool searches and a phylogenetic analysis, only six samples (7.4%), including S. calcitrans (n = 2), S. sitiens (n = 2), T. megalops (n = 1), and A. cryptotaxis (n = 1), were found to be infected with Theileria sinensis. Furthermore, apicomplexan parasites, namely, Mattesia spp. and Colpodella spp., were found on S. indicus, the fungus Meira spp. was found on S. calcitrans, and the pathogenic green alga Helicosporidium spp. was found on A. cryptotaxis. Conclusion: This study is the first to report a variety of Stomoxys and tabanid flies collected from horse farms in Thailand, which were found to be infected with Theileria and Colpodella species that affect mammals, suggesting that Stomoxys and tabanid flies can be used to confirm the presence of hemoprotozoan parasites in the study area. Understanding the presence of hemoprotozoa in flies could help design vector control programs and manage various diseases in the study area.

Hematological analysis of naturally infecting blood parasites in dogs.

Background and Aim: Blood parasite infections such as anaplasmosis, babesiosis, and ehrlichiosis are commonly found in domestic dogs, which adversely influence their health. Many dogs are infected with multiple blood parasites that cause more severe diseases than a single infection. This study aimed to investigate the effect of multiple blood parasite infections on the hematological profiles of dogs at a shelter in Southern Thailand. Materials and Methods: The blood samples from 122 dogs were collected to assess the hematology profiles of uninfected, single-infected, and multiple blood parasite-infected dogs. The results were compared using Kruskal-Wallis test and Dwass-Steel-Critchlow-Fligner pairwise comparisons. The infections were confirmed by polymerase chain reaction. Results: The results showed that all the infected dogs had significantly lower red blood cell (RBC) count, hemoglobin (HB), hematocrit (HCT), and platelet count (PLT) than the uninfected dogs. Although the dogs with triple infection had lower RBC, HB, HCT, and PLT than those with double and single infections, the difference was not statistically significant. Conclusion: We proposed that triple blood parasite infection with Anaplasma platys, Babesia vogeli, and Ehrlichia canis caused more severe disease than double and single infections. Evaluating the hematological profiles of dogs naturally infected with single, double, and multiple blood parasite infections without clinical signs can enhance their health and welfare.

Baseline study of the morphological and genetic characteristics of Haemoproteus parasites in wild pigeons (Columba livia) from paddy fields in Thailand.

Haemoproteus columbae is a common haemosporidian parasite of wild pigeons (Columba livia) reported worldwide. In Thailand, the wild pigeon population is increasing due to paddy field monoculture. However, there are limited reports on the presence of H. columbae in these pigeon populations. The aim of the study was to characterize H. columbae in wild pigeons. A total of 87 wild pigeons were examined using microscopic and molecular methods. Haemoproteus columbae was detected in approximately 27.6% of pigeons and their morphological characteristics were described. The partial cytochrome b (cyt b) gene sequence of H. columbae was then characterized into three common lineages (HAECOL1, COLIV03, and COQUI05). By highlighting the morphologic and genetic characteristics of H. columbae commonly found in this population of pigeons, this study provides essential regional knowledge about haemosporidian parasites that could benefit future taxonomic and phylogeographic studies.

Association of multilocus sequencing types and antimicrobial resistance profiles of methicillin-resistant Mammaliicoccus sciuri in animals in Southern Thailand.

Background and Aim: Mammaliicoccus sciuri, formerly known as Staphylococcus sciuri, is an opportunistic pathogen in the environment, human and animal mucosa, and skin. Although this pathogen is becoming more resistant to drugs and harmful to animals and humans, basic knowledge of this pathogen remains limited. This study aimed to investigate a new multilocus sequencing type (MLST) related to the antibiotic resistance pattern of M. sciuri from animals in southern Thailand. Materials and Methods: We used 11 methicillin-resistant M. sciuri (MRMS) isolates in this study which were obtained from six horses, four cows, and one chicken of the previous study. Antimicrobial resistance (AMR) was re-evaluated based on the minimum inhibitory concentration using the VITEK® 2 automated system. Three AMR genes were examined, namely mecA, mecC, and blaZ. Staphylococcal chromosomal cassette mec (SCCmec) gene detection was performed through the multiplex polymerase chain reaction (PCR). Internal segments of the seven housekeeping genes, ack, aroE, ftsZ, glpK, gmk, pta1, and tpiA, were used for multilocus sequence typing. The population of resistant bacteria and the types of multidrug-resistant, extensively drug-resistant, and pandemic drug-resistant bacteria were classified through descriptive analysis. Results: mecA and blaZ genes were detected in all isolates; however, the mecC gene was not observed in any isolate based on the PCR results. All MRMS isolates revealed a non-typable SCCmec. Seven MLSTs (71, 81, 120, 121, 122, 199, and 200) were identified in this study. Conclusion: The characteristics of MRMS in Southern Thailand were variable, particularly in cattle and horses. The antibiogram and SCCmec types of this pathogen remain concerns with regard to antibiotic-resistant gene transmission among Staphylococcus and Mammaliicoccus species. All MLSTs in Thailand revealed the distribution among clones in Asia, including the virulence of a zoonotic clone in Southern Thailand.

First study on molecular detection of hemopathogens in tabanid flies (Diptera: Tabanidae) and cattle in Southern Thailand.

Background and Aim: Female tabanids play a key role in disease transmission as mechanical vectors for various hemopathogens, but only a limited number of studies have been conducted on them. This study aimed to investigate the occurrence of hemopathogens in tabanid flies compared to those found in nearby cattle hosts. Materials and Methods: Tabanids were collected using a Nzi trap for three consecutive days per month during the dry season (February-May 2021). Furthermore, blood samples were collected from 20 beef cattle (Bos taurus) raised in the same area where the flies were captured. Conventional polymerase chain reaction (PCR) was used to detect hemopathogenic DNA in flies and beef cattle. Results: In total, 279 female tabanids belonging to five species were collected: Tabanus megalops, Tabanus rubidus, Tabanus mesogaeus, Chrysops dispar, and Chrysops fuscomarginalis. Notably, T. megalops was the most abundant species, accounting for 89.2% of the flies collected (n = 249). PCR technique revealed that 76.6% of T. megalops carried at least one pathogen (Anaplasma, Ehrlichia, Babesia, or Theileria). In addition, all beef cattle had multiple hemopathogenic infections (Anaplasma marginale, Ehrlichia spp., Babesia bigemina, Babesia bovis, and Theileria spp.). Conclusion: Although T. megalops could carry many hemopathogens, it might not be an important vector due to the limited number of flies and parasitic load. Furthermore, T. megalops could be utilized to monitor the presence of hemopathogens in the study area, but not the disease occurrence in the individual host species. Knowing the presence of hemopathogens in flies could help manage the disease in this area.

Multiple blood pathogen infections in apparently healthy sheltered dogs in southern Thailand.

In developing countries such as Thailand, free-ranging dogs are frequently involved in road accidents and contribute to the cost of public healthcare. Shelters play a vital role in communities because they help to control the population of unwanted and free-ranging dogs. This study aimed to investigate blood pathogen infection in sheltered dogs, as it is one of the factors contributing to animal welfare. Blood samples were randomly collected from 141 dogs from the largest shelter (approximately 400-500 dogs in total) in southern Thailand. Blood pathogens were detected using both PCR and light microscopy. Four blood pathogens were identified: Anaplasma platys, Ehrlichia canis, Babesia canis vogeli, and Hepatozoon canis. No trypanosomes were detected. The incidence of blood parasite infection was 56.7% (80/141) by PCR, and 28.4% (40/141) by microscopy. E. canis was the most prevalent pathogen, accounting for 46.1% (65/141) of the cases, while multiple infections accounted for 22% (31/141) of the cases. A triple infection with E. canis, A. platys, and B. canis vogeli was observed in 5.7% (8/141) of the cases. Although PCR is far more sensitive than microscopy, it appears to have equivalent specificity. In conclusion, this study reported a high occurrence of blood pathogen infections in clinically healthy sheltered dogs. Many of them were infected with multiple pathogens and may have been infected before entering the shelter. These findings suggest that a blood test is necessary to screen dogs prior to their admission to the shelter to prevent disease transmission and enhance animal welfare.

Antimicrobial resistance in Staphylococcus pseudintermedius on the environmental surfaces of a recently constructed veterinary hospital in Southern Thailand.

Background and Aim: Staphylococcus pseudintermedius is a zoonotic bacterium commonly found in animals, especially dogs. These bacteria can survive on environmental surfaces for several months. The infection of S. pseudintermedius from the environment is possible, but properly cleaning surface objects can prevent it. This study aimed to investigate the prevalence of methicillin-resistant S. pseudintermedius (MRSP) in the environment of a recently constructed veterinary hospital in Southern Thailand, where we hypothesized that the prevalence of MRSP might be very low. Materials and Methods: At three different time points, 150 samples were collected from different environmental surfaces and wastewater across the veterinary hospital. The collection was done after the hospital's cleaning. Bacteria were purified in the culture before being identified as species by biochemical tests and polymerase chain reaction (PCR). Next, the antimicrobial-resistant profile was performed using an automated system (Vitek 2). Finally, the antimicrobial resistance genes were identified using PCR. Results: Fifteen colonies of S. pseudintermedius were isolated from the surfaces of eight floors, four tables, two chairs, and one rebreathing tube. Fourteen colonies (93.3%) were multidrug-resistant (MDR) and carried the blaZ gene (93.3%). The majority of colonies were resistant to benzylpenicillin (93.3%), cefovecin (93.3%), ceftiofur (93.3%), kanamycin (93.3%), and neomycin (93.3%). Notably, only four colonies (26.7%) were methicillin-susceptible S. pseudintermedius, whereas 11 colonies (73.3%) were MRSP and carried both the mecA and blaZ genes. Five MRSP (45.5%) were resistant to at least 14 antimicrobial drugs, represented as extensively drug-resistant (XDR) bacteria. Ten of eleven MRSP (90.9%) were Staphylococcal chromosomal mec type V, while another displayed untypeable. Despite the routine and extensive cleaning with detergent and disinfectant, MRSP isolates were still detectable. Conclusion: Many isolates of MRSP were found in this veterinary hospital. Almost all of them were MDR, and nearly half were XDR, posing a threat to animals and humans. In addition, the current hospital cleaning procedure proved ineffective. Future research should be conducted to determine the bacterial biofilm properties and bacterial sensitivity to certain detergents and disinfectants.

First study on diversity and antimicrobial-resistant profile of staphylococci in sports animals of Southern Thailand.

Background and Aim: Staphylococci are commensal bacteria and opportunistic pathogens found on the skin and mucosa. Sports animals are more prone to injury and illness, and we believe that antimicrobial agents might be extensively used for the treatment and cause the existence of antimicrobial-resistant (AMR) bacteria. This study aimed to investigate the diversity and AMR profile of staphylococci in sports animals (riding horses, fighting bulls, and fighting cocks) in South Thailand. Materials and Methods: Nasal (57 fighting bulls and 33 riding horses) and skin swabs (32 fighting cocks) were taken from 122 animals. Staphylococci were cultured in Mannitol Salt Agar and then identified species by biochemical tests using the VITEK® 2 card for Gram-positive organisms in conjunction with the VITEK® 2 COMPACT machine and genotypic identification by polymerase chain reaction (PCR). Antimicrobial susceptibility tests were performed with VITEK® 2 AST-GN80 test kit cards and VITEK® 2 COMPACT machine. Detection of AMR genes (mecA, mecC, and blaZ) and staphylococcal chromosomal mec (SCCmec) type was evaluated by PCR. Results: Forty-one colonies of staphylococci were isolated, and six species were identified, including Staphylococcus sciuri (61%), Staphylococcus pasteuri (15%), Staphylococcus cohnii (10%), Staphylococcus aureus (7%), Staphylococcus warneri (5%), and Staphylococcus haemolyticus (2%). Staphylococci were highly resistant to two drug classes, penicillin (93%) and cephalosporin (51%). About 56% of the isolates were methicillin-resistant staphylococci (MRS), and the majority was S. sciuri (82%), which is primarily found in horses. Most MRS (82%) were multidrug-resistant. Almost all (96%) of the mecA-positive MRS harbored the blaZ gene. Almost all MRS isolates possessed an unknown type of SCCmec. Interestingly, the AMR rate was notably lower in fighting bulls and cocks than in riding horses, which may be related to the owner's preference for herbal therapy over antimicrobial drugs. Conclusion: This study presented many types of staphylococci displayed on bulls, cocks, and horses. However, we found a high prevalence of MRS in horses that could be transmitted to owners through close contact activities and might be a source of AMR genotype transmission to other staphylococci.

Characterizing the antimicrobial resistance profile of Escherichia coli found in sport animals (fighting cocks, fighting bulls, and sport horses) and soils from their environment.

Background and Aim: Antimicrobial resistance (AMR) is a significant threat to global health and development. Inappropriate antimicrobial drug use in animals cause AMR, and most studies focus on livestock because of the widespread use of antimicrobial medicines. There is a lack of studies on sports animals and AMR issues. This study aimed to characterize the AMR profile of E. coli found in sports animals (fighting cocks, fighting bulls, and sport horses) and soils from their environment. Materials and Methods: Bacterial isolation and identification were conducted to identify E. coli isolates recovered from fresh feces that were obtained from fighting cocks (n = 32), fighting bulls (n = 57), sport horses (n = 33), and soils from those farms (n = 32) at Nakhon Si Thammarat. Antimicrobial resistance was determined using 15 tested antimicrobial agents - ampicillin (AM), amoxicillin-clavulanic acid, cephalexin (CN), cefalotin (CF), cefoperazone, ceftiofur, cefquinome, gentamicin, neomycin, flumequine (UB), enrofloxacin, marbofloaxacin, polymyxin B, tetracycline (TE), and sulfamethoxazole/trimethoprim (SXT). The virulence genes, AMR genes, and phylogenetic groups were also examined. Five virulence genes, iroN, ompT, hlyF, iss, and iutA, are genes determining the phylogenetic groups, chuA, cjaA, and tspE4C2, were identified. The AMR genes selected for detection were blaTEM and blaSHV for the beta-lactamase group; cml-A for phenicol; dhfrV for trimethoprim; sul1 and sul2 for sulfonamides; tetA, tetB, and tetC for TEs; and qnrA, qnrB, and qnrS for quinolones. Results: The E. coli derived from sports animals were resistant at different levels to AM, CF, CN, UB, SXT, and TE. The AMR rate was overall higher in fighting cocks than in other animals, with significantly higher resistance to AM, CF, and TE. The highest AMR was found in fighting cocks, where 62.5% of their isolates were AM resistant. In addition, multidrug resistance was highest in fighting cocks (12.5%). One extended-spectrum beta-lactamase E. coli isolate was found in the soils, but none from animal feces. The phylogenetic analysis showed that most E. coli isolates were in Group B1. The E. coli isolates from fighting cocks had more virulence and AMR genes than other sources. The AMR genes found in 20% or more of the isolates were blaTEM (71.9%), qnrB (25%), qnrS (46.9%), and tetA (56.25%), whereas in the E. coli isolates collected from soils, the only resistance genes found in 20% or more of the isolates were blaTEM (30.8%), and tetA (23.1%). Conclusion: Escherichia coli from fighting cock feces had significantly higher resistance to AM, CF, and TE than isolates from other sporting animals. Hence, fighting cocks may be a reservoir of resistant E. coli that can transfer to the environment and other animals and humans in direct contact with the birds or the birds' habitat. Programs for antimicrobial monitoring should also target sports animals and their environment.

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.

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.

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.

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.

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.