The potential use of mitochondrial ribosomal genes (12S and 16S) in DNA barcoding and phylogenetic analysis of trematodes.

BACKGROUND: Genetic markers like the nuclear ribosomal RNA (rRNA) genes, internal transcribed spacer regions, mitochondrial protein-coding genes, and genomes have been utilized for molecular identification of parasitic trematodes. However, challenges such as the design of broadly applicable primers for the vast number of species within Digenea and the genetic markers' ability to provide sufficient species-level resolution limited their utility. This study presented novel and broadly applicable primers using the mitochondrial 12S and 16S rRNA genes for Digenea and aimed to show their suitability as alternative genetic markers for molecular identification of orders Plagiorchiida, Echinostomida, and Strigeida. RESULTS: Our results revealed that the mitochondrial 12S and 16S rRNA genes are suitable for trematode molecular identification, with sufficient resolution to discriminate closely related species and achieve accurate species identification through phylogenetic placements. Moreover, the robustness of our newly designed primers to amplify medically important parasitic trematodes encompassing three orders was demonstrated through successful amplification. The convenience and applicability of the newly designed primers and adequate genetic variation of the mitochondrial rRNA genes can be useful as complementary markers for trematode molecular-based studies. CONCLUSIONS: We demonstrated that the mitochondrial rRNA genes could be alternative genetic markers robust for trematode molecular identification and potentially helpful for DNA barcoding where our primers can be widely applied across the major Digenea orders. Furthermore, the potential of the mitochondrial rRNA genes for molecular systematics can be explored, enhancing their appeal for trematode molecular-based studies. The novelty of utilizing the mitochondrial rRNA genes and the designed primers in this study can potentially open avenues for species identification, discovery, and systematics in the future.

A molecular phylogeny of Asian species of the genus Metagonimus (Digenea)--small intestinal flukes--based on representative Japanese populations.

Metagonimus Katsurada, 1912 is a genus of small intestinal parasites. The genus comprises eight species, primarily from far-eastern Asia, with two exceptions reported from Europe. Metagonimus yokogawai, the most widespread species, is the main agent responsible for the intestinal disease, metagonimiasis, in Japan and some other East Asian countries. On the basis of the ratio of the size of the ventral and oral suckers, Metagonimus has traditionally been morphologically divided into two groups; however, the genus has not been extensively studied using molecular data. To reveal phylogenetic relationships within Metagonimus based on molecular data, we analyzed six of the seven species present in Asia using samples collected in central Japan. Maximum likelihood and Bayesian analyses of a combined 28S ribosomal DNA (rDNA), internal transcribed spacer 2 (ITS2), and mitochondrial cox1 gene sequence dataset separated the six species into two well-supported clades. One clade comprised M. yokogawai, M. takahashii, M. miyatai, and M. hakubaensis, whereas the other consisted of M. otsurui and M. katsuradai. Genetic distances calculated from 28S rDNA and ITS2 nucleotide sequences and a comparison of the predicted amino acid sequences of cox1 gene suggested that M. otsurui and M. katsuradai may have diverged recently. None of the four main morphological characters used to delimit species of Metagonimus (i.e., sucker ratio, positions of the uterus and testes, and distribution of vitelline follicles) was consistent with the distribution of species in the molecular tree.

Landmark and outline-based geometric morphometrics analysis of three Stomoxys flies (Diptera: Muscidae).

Adult flies of the genus Stomoxys Geoffroy, 1762 (Diptera: Muscidae), especially S. pullus Austen, 1909, S. uruma Shinonaga et Kano, 1966 and S. indicus Picard, 1908, are morphologically similar and sometimes difficult to distinguish when using external morphological characteristics. These species may act as vectors and/or potential vectors of many pathogens (virus, bacteria and protozoa). Their correct identification is important to target the vectors involved in the transmission of the pathogens and also helps in the fly control program.The aim of the present study was to distinguish three species which are difficult to separate using traditional diagnostic characters for species of Stomoxys such as colour patterns and body proportions. Modern morphometrics, both landmark and outline-based, was used to access wing geometry of S. pullus, S. uruma and S. indicus. A total of 198 and 190 wing pictures were analysed for landmark- and outline-based approaches, respectively. Wing shape was able to separate species and sexes of the three Stomoxys flies with highly significant difference of Mahalanobis distances. The cross-validated classification scores ranged from 76% to 100% for landmark and 77% to 96% for outline-based morphometrics. The geometry of wing features appears to be a very useful, low-cost tool to distinguish among the vectors S. pullus, S. uruma and S. indicus.

CORRELATION OF HOST SPECIFICITY, ENVIRONMENTAL FACTORS AND ORIENTAL RAT FLEA ABUNDANCE.

Fleas are the vectors of many communicable diseases that are normally found in oriental rats. Climate and environmental changes influence the habitat and migration patterns of vectors. In this study, the oriental rat flea abundance, represented as total flea index, was determined in correlation to host specificity and various environmental factors. The number of hosts and fleas calculated from 3 specific habitats (shipping area, decayed area, and market area) from July 2010 to June 2011. The results showed that the common hosts in the shipping area and decayed area were Rattus rattus and R. exulans, with the total flea indexes of 3.36 and 1.58. R. norvegicus was the most common host identified in the market area. Fleas were virtually absent in rat hosts collected from the market area. Both the density of reservoir hosts and the total flea index were positively correlated with the mean annual rainfall and temperature. These data could be useful for control of rat populations in each specific habitat.

Subtype distribution of Blastocystis in Thai-Myanmar border, Thailand.

Blastocystis sp. is a common zoonotic intestinal protozoa which has been classified into 17 subtypes (STs). A cross-sectional study was conducted to determine the prevalence and subtype distribution of Blastocystis in villagers living on the Thai-Myanmar border, where the risk of parasitic infection is high. A total of 207 stool samples were collected and DNA was extracted. PCR and sequencing using primers targeting small-subunit ribosomal RNA (SSU rRNA) gene were performed. The prevalence of Blastocystis infection was 37.2% (77/207). ST3 (19.8%; 41/207) was the predominant subtype, followed by ST1 (11.6%; 24/207), ST2 (5.3%; 11/207), and ST4 (0.5%; 1/207). A phylogenetic tree was reconstructed using the maximum likelihood (ML) method based on the Hasegawa-Kishino-Yano + G + I model. The percentage of bootstrapped trees in which the associated taxa clustered together was relatively high. Some sequences of Blastocystis positive samples (TK18, 39, 46, 71, and 90) were closely related to animals (pig and cattle) indicating zoonotic risks. Therefore, proper health education in parasitic prevention for the villagers should be promoted to improve their personal hygiene. Further longitudinal studies are required to monitor the prevalence of parasitic infections after providing health education and to investigate Blastocystis ST in animals living in these villages.

Molecular insights versus morphological traits: rethinking identification of the closely related Angiostrongylus cantonensis and Angiostrongylus malaysiensis.

BACKGROUND: The closely related Angiostrongylus cantonensis and Angiostrongylus malaysiensis have been reported to coexist in Thailand and share similar hosts and life cycles. Recently, in an angiostrongyliasis outbreak in Thailand, both A. cantonensis and A. malaysiensis were found in the cerebrospinal fluid of affected patients. Morphological similarities, overlapping distribution, shared hosts and habitats, and the close genetics of the two Angiostrongylus species can complicate accurate species identification. Addressing these challenges, this study aims to evaluate whether a correlation between the morphological and genetic identities of A. cantonensis and A. malaysiensis can improve species identification accuracy. METHODS: Angiostrongylus spp. specimens from five zoogeographical regions in Thailand were subjected to morphological and molecular identification using the mitochondrial cytochrome b gene and the nuclear internal transcribed spacer 2 region (ITS2). The morphological characters for males and females were then validated using the species identity obtained from the nuclear ITS2 region. RESULTS: The results revealed that morphological misidentifications between these two closely related species are common due to overlapping morphological characters. Although certain male traits such as body length and width aided species differentiation, female traits were found to be less reliable. Furthermore, hybrid forms (8.2%) were revealed through the ITS2 results, which can further complicate morphological identification. Mito-nuclear discordance was also present in 1.9% of the Angiostrongylus specimens from Thailand, suggesting a complex historical interbreeding between the species. CONCLUSIONS: Based on our findings, we suggest that nuclear ITS2 is a reliable marker for species identification of A. cantonensis and A. malaysiensis, especially in regions where both species coexist. Additionally, the scope and consequences of hybridization between the two closely related Angiostrongylus species should be further investigated in Thailand.

Hydroxyapatite-binding Silver/Titanium Dioxide as a Potential Control Compound Against Mosquito Vectors, Aedes aegypti (Diptera: Culicidae) and Anopheles dirus (Diptera: Culicidae).

Controlling mosquitoes is vital for counteracting the rising number of mosquito-borne illnesses. Vector control requires the implementation of various measures; however, current methods lack complete effectiveness, and new control agents or substances are urgently needed. Therefore, this study developed a nonwoven fabric sheet coated with hydroxyapatite-binding silver/titanium dioxide compound (hydroxyapatite-binding silver/titanium dioxide sheet [HATS])and evaluated its effectiveness on all stages of laboratory Aedes aegypti (Linnaeus); Diptera: Culicidae and Anopheles dirus (Peyton & Harrison); Diptera: Culicidae. We reared larvae with HATS and control sheets and assessed their mortality, emergence, and hatching rates. The submersion rates of engorged female mosquitoes in submerged HATS and control sheets were also compared. The HATS strongly affected mosquito development, resulting in high mortality rates (mean ± SE) of 99.66 ± 0.58% (L1-L2) and 91.11 ± 9.20% (L3-L4) for Ae. aegypti and 100% of both stages for An. dirus. In contrast, mosquitoes raised in the control sheet showed relatively high survival rates of 92.33 ± 3.21% (L1-L2) and 95.67 ± 0.58% (L3-L4) for Ae. aegypti and 86.07 ± 3.53% (L1-L2) and 92.01 ± 8.67% (L3-L4) for An. dirus. Submersion of engorged females was found in the HATS oviposition cup, leading to a decreased number of eggs and a low hatching rate compared to that of the control. Overall, HATS may be a useful new control method for Ae. aegypti and An. dirus.

Distribution and abundance of Stomoxyini flies (Diptera: Muscidae) in Thailand.

Stomoxyini flies (Diptera: Muscidae) include species of parasitic flies of medical and veterinary importance. The adult flies feed on the blood of mammals and may transmit several parasites and pathogens. We conducted an entomological survey of Stomoxyini flies from different sites in Thailand. Stomoxyini flies were collected at four major types of sites: zoos, livestock farms, wildlife conservation areas and a national park using vavoua traps between November 2010 and April 2011. A total of 3,314 Stomoxyini flies belonging to the genera Stomoxys, Haematobosca, Haematostoma and Haematobia were collected. Eight species were identified: S. calcitrans (46.6%), S. uruma (26.8%), S. pulla (4.3%), S. indicus (0.7%), S. sitiens (0.1%), H. sanguinolenta (11.2 %), H. austeni (0.5%) and H. irritans exigua (9.8%). The diversity of Stomoxyini flies in the livestock farms was higher than the other sites. Altitude correlated with the number of flies. This study provides information that may be useful for Stomoxyini flies control.

Effectiveness of Herbal Essential Oils as Single and Combined Repellents against Aedes aegypti, Anopheles dirus and Culex quinquefasciatus (Diptera: Culicidae).

Mosquito repellents reduce human-vector contact of vector-borne diseases. We compared the repellent activity of 10 undiluted essential oils (anise, basil, bergamot, coriander, patchouli, peppermint, petitgrain, rosemary, sage and vetiver) against A. aegypti, A. dirus and C. quinquefasciatus using the arm-in-cage method. Petitgrain oil was the most effective against A. aegypti (270 min). Peppermint oil was the most effective against A. dirus (180 min). Interestingly, all single oils had attributes of repellency against C. quinquefasciatus (ranged, 120−360 min). Moreover, we integrated their binary combinations of highly effective essential oils against A. aegypti and A. dirus to potentially increase the protection time. A 1:1 combination of petitgrain/basil, petitgrain/coriander, basil/coriander and basil/sage reduced the median complete-protection time of 150 min for A. aegypti; a combination of sage and patchouli oils prolonged the median complete-protection time of 270 min for A. dirus. Combining essential oils effect protection time from these two mosquito species.

Mitochondrial DNA barcoding of mosquito species (Diptera: Culicidae) in Thailand.

The correct identification of mosquito species is important for effective mosquito vector control. However, the standard morphological identification of mosquito species based on the available keys is not easy with specimens in the field due to missing or damaged morphological features during mosquito collections, often leading to the misidentification of morphologically indistinguishable. To resolve this problem, we collected mosquito species across Thailand to gather genetic information, and evaluated the DNA barcoding efficacy for mosquito species identification in Thailand. A total of 310 mosquito samples, representing 73 mosquito species, were amplified using mitochondrial cytochrome c oxidase subunit I (COI) primers. The average maximum intraspecific genetic variation of the 73 mosquito species was 1% ranged from 0-5.7%. While, average minimum interspecific genetic variation (the distance to the nearest neighbour) of the 73 mosquito species was 7% ranged from 0.3-12.9%. The identification of success rates based on the "Best Match," "Best Close Match," and "All Species Barcodes" methods were 97.7%, 91.6%, and 81%, respectively. Phylogenetic analyses of Anopheles COI sequences demonstrated a clear separation between almost all species (except for those between An. baimaii and An. dirus), with high bootstrap support values (97%-99%). Furthermore, phylogenetic analyses revealed potential sibling species of An. annularis, An. tessellatus, and An. subpictus in Thailand. Our results indicated that DNA barcoding is an effective molecular approach for the accurate identification of mosquitoes in Thailand.

Insecticidal Activity of Plectranthus amboinicus Essential Oil against the Stable Fly Stomoxys calcitrans (Diptera: Muscidae) and the Horse Fly Tabanus megalops (Diptera: Tabanidae).

The stable fly, Stomoxys calcitrans (Diptera: Muscidae), and the horse fly, Tabanus megalops (Diptera: Tabanidae), are important ectoparasites of livestock in Thailand. These species affect animal health and cause economic losses. This study investigated the insecticidal activity of Plectranthus amboinicus essential oil against S. calcitrans and T. megalops through contact and fumigant toxicity tests and evaluated the effects of the essential oil on these flies through histopathological and scanning electron microscopic (SEM) studies. The results of the contact toxicity test indicated that the median lethal dose against S. calcitrans and T. megalops was 12.05 and 131.41 µg/fly, and the 90% lethal dose was 45.53 and 200.62 µg/fly, respectively. The results of the fumigant toxicity test showed that the median lethal concentration against S. calcitrans and T. megalops was 1.34 and 7.12 mg/L air, and the 90% lethal concentration was 4.39 and 30.37 mg/L air, respectively. Histopathology revealed neuronal degeneration in the brain of S. calcitrans and interstitial neuronal edema of the brain and ovarian necrosis in T. megalops. No external morphological changes were observed via SEM. Given its insecticidal properties against S. calcitrans and T. megalops, P. amboinicus essential oil could be developed into a natural insecticide to control these fly species.

Species Discrimination of Three Odontomachus (Formicidae: Ponerinae) Species in Thailand Using Outline Morphometrics.

All members of the ant genus Odontomachus Latreille, 1804 are venomous ants. Four species in this genus have been identified from Thailand: Odontomachus latidens Mayr, 1867; O. monticola Emery, 1892; O. rixosus Smith, 1757; and O. simillimus Smith, 1758. The three latter species are available and have been used for an outline morphometric study. They display similar morphology, which makes their distinction very difficult except for highly qualified individuals. A total of 80 worker specimens were studied, exploring the contour shapes of their head and pronotum as possible taxonomic characters. The size of each body part was estimated determining the contour perimeter, the values for which were largely overlapping between O. rixosus and O. simillimus; most O. monticola specimens exhibited a significantly larger size. In contrast to the size, each contour shape of the head or pronotum established O. rixosus as the most distinct species. An exploratory data analysis disclosed the higher taxonomic signal of the head contour relative to the pronotum one. The scores obtained for validated reclassification were much better for the head (99%) than for the pronotum (82%). This study supports outline morphometrics of the head as a promising approach to contribute to the morphological identification of ant species, at least for monomorphic workers.

Sensitive and accurate DNA metabarcoding of parasitic helminth mock communities using the mitochondrial rRNA genes.

Next-generation sequencing technologies have accelerated the pace of helminth DNA metabarcoding research, enabling species detection in bulk community samples. However, finding suitable genetic markers with robust species-level resolution and primers targeting a broad species range among parasitic helminths are some of the challenges faced. This study aimed to demonstrate the potential use of the mitochondrial 12S and 16S rRNA genes for parasitic helminth (nematodes, trematodes, cestodes) DNA metabarcoding. To demonstrate the robustness of the 12S and 16S rRNA genes for DNA metabarcoding, we determined the proportion of species successfully recovered using mock helminth communities without environment matrix and mock helminth communities artificially spiked with environmental matrices. The environmental matrices are human fecal material, garden soil, tissue, and pond water. Our results revealed the robustness of the mitochondrial rRNA genes, through the high sensitivity of the 12S rRNA gene, and the effectiveness of the 12S and 16S primers targeting platyhelminths. With the mitochondrial rRNA genes, a broad range of parasitc helminths were successfully detected to the species level. The potential of the mitochondrial rRNA genes for helminth DNA metabarcoding was demonstrated, providing a valuable gateway for future helminth DNA metabarcoding applications like helminth detection and biodiversity studies.

Utility of a set of conserved mitochondrial cytochrome oxidase subunit I gene primers for Mansonia annulata identification.

DNA-based identification system using the mitochondrial cytochrome oxidase subunit I (COI) gene has enabled validation of many species in certain taxonomic groups. These primer combinations were able to work universally across Insecta. Here, a set of three primer pairs were successful in amplifying COI of Mansonia annulata mosquito, a potential vector of Brugia malayi. By merging all three amplicons the whole COI was obtained. Primer pair TY-J-1460/C1N2087 amplified 5' region of COI, LepF1/LepR1 the central and C1J2090/TL2N3014 the 3' region, generating COI amplicons of 650, 700 and 950 base pairs, respectively. When Ma. annulata sequences were compared with those from online sources, they formed a cluster group that is clearly distinct from other allied species.

Bionomic status of Anopheles epiroticus Linton & Harbach, a coastal malaria vector, in Rayong Province, Thailand.

A longitudinal entomological survey was conducted to provide in-depth information on An. epiroticus and determine whether ecological and entomological factors could influence malaria transmission in Rayong Province, Thailand. The mosquitoes were collected monthly from May 2007 to April 2008 by human landing catch technique from 6:00-12:00 PM for 2 consecutive nights, at 3 collection sites. A total of 3,048 mosquitoes within 5 species were captured: An. epiroticus, Culex quinquefasciatus Say, Cx. sitiens Wiedemann, Aedes aegypti (L.) and Ae. albopictus Skuse. PCR was used for molecular identification of An. sundaicus complex, by determination of COI, ITS2, and D3 genes. The target mosquitoes were An. epiroticus, which was the predominant species, accounting for 43.8% of specimens collected. The biting cycle pattern increased during 6:00-8:00 PM and reached a maximum of 6.6 bites/person/hour by 12:00 PM. The mosquitoes varied in population density throughout the year. The highest biting rate was 37.6 bites/person/ half night in September and the lowest (10.2 bites/person/half night) in January. Nested PCR and real-time PCR techniques were used to detect the malaria parasite in An. epiroticus adult females. Nine of 926 (0.97%) mosquitoes tested were malaria parasite positive: 6 P. falciparum and 3 P. vivax. The infective mosquitoes were found in the dry and early rainy seasons. The overall annual entomological inoculation rate (EIR) in the village was 76.6. The overall parity rate was 74%. A total of 38 cement tanks were used to characterize the nature of the breeding places of An. epiroticus. An. epiroticus larvae coexisted with Aedes and Culex larvae; the maximum larval density was more than 140 larvae per dip in May. Breeding places included fresh, brackish and salt water, typically with full sunlight and mats of green algae on the water surface. The salinity of the water ranged from 0.5 to 119.4 g/l, with a narrow pH range of 8.2-8.7. Dissolved oxygen was highest in November (6.27 mg/l) and lowest in March (3.46 mg/l). The water temperature varied between 24.6 and 32.8 degrees C.

Application of recombinant SMR-domain containing protein of angiostrongylus cantonensis in immunoblot diagnosis of human angiostrongyliasis.

The aim of this study was to find novel proteins expressed from an Angiostrongylus cantonensis adult female worm cDNA library for serodiagnosis of angiostrongyliasis. An immuno-dominant clone, fAC22, was identified by immunoscreening with pooled positive sera from proven angiostrongyliasis patients. The clone contained an open reading frame of 2,136 bp encoding a 80.5 kDa protein with a predicted isoelectric point of 5.8. The deduced amino acid sequence (712 amino acids) contained the conserved domain of Small mutS related (Smr) superfamily protein, with similarity with the Smr domain protein of Brugia malayi. The fusion His-tagged 81 kDa recombinant protein expressed as inclusion body in Escherichia coli was solubilized and purified by Ni-affinity chromatography for use in immunoblot analysis. Its sensitivity, specificity, positive and negative predictive values in immunodiagnostic test was 93.5, 91.5, 79.0 and 97.5%, respectively. Although some cross-reactivity of the antigen was observed among gnathostomiasis, bancroftian filariasis, ascariasis, echinococcosis, paragonimiasis and opisthorchiasis, sera from 14 other infections were all negative. These data indicate its possible application in immunodiagnosis of clinically suspected angiostrongyliasis. Key words: Angiostrongylus cantonensis,eosinophilic meningitis, recombinant fusion protein, immunodiagnosis

Molecular Identification and Geometric Morphometric Analysis of Haematobosca aberrans (Diptera: Muscidae).

The genus Haematobosca Bezzi, 1907 (Diptera: Muscidae) contains haematophagous flies of veterinary importance. A new fly species of this genus was recognised from northern Thailand based on morphological characters and described as Haematobosca aberrans Pont, Duvallet & Changbunjong, 2020. In the present study, the mitochondrial cytochrome c oxidase I (COI) gene was used to confirm the morphological identification of H. aberrans. In addition, landmark-based geometric morphometrics was used to determine sexual dimorphism. The molecular analysis was conducted with 10 COI sequences. The results showed that all sequences were 100% identical. The sequence was not highly similar to reference sequences from GenBank and did not match any identified species from Barcode of Life Data Systems (BOLD). Phylogenetic analysis clearly differentiated this species from other species within the subfamily Stomoxyinae. For geometric morphometric analysis, a total of 16 wing pictures were analysed using the landmark-based approach. The results showed significant differences in wing shape between males and females, with a cross-validated classification score of 100%. The allometric analysis showed that wing shape has no correlation with size. Therefore, the COI gene is effective in species identification of H. aberrans, and geometric morphometrics is also effective in determining sexual dimorphism.

Insecticidal and Histopathological Effects of Ageratum conyzoides Weed Extracts against Dengue Vector, Aedes aegypti.

Crude extracts and essential oils of A. conyzoides were tested with larva and adult stages of Ae. aegypti mosquitoes to determine their insecticidal properties. The crude extracts and essential oils came from three varieties of A. conyzoides (with white flowers, purple flowers, or white-purple flowers) and from two places on each plant (leaves and flowers), giving six types overall: leaf-white (LW); leaf-purple (LP); leaf white-purple (LW-P); flower-white (FW); flower-purple (FP); and flower white-purple (FW-P). Chemical constituents and components of the essential oils were identified using gas chromatography-mass spectrometry (GC-MS). Electron microscopic and histopathological studies were performed to determine the toxicological effects on mosquitoes in terms of morphological alterations. The six types of crude extracts exhibited no activity against individuals in the larval stages. However, six types of essential oils were effective against adult Ae. aegypti females. The mortality of adult Ae. aegypti females was higher from leaf extracts, particularly LP (median lethal dose, LD50 = 0.84%). The number of chemical constituents identified by GC-MS was high in flowers, especially W-P. Precocene I was the most abundant chemical component among the five types of essential oils, except in LP, in which precocene II was the most abundant. Histopathological alterations in adult Ae. aegypti females included compound eye degeneration, muscular damage with cellular infiltration, gut epithelial degeneration and necrosis, pyknotic nuclei in the malpighian epithelium and ovarian cell degeneration. FW and FP plant types exhibited the highest severity of histopathological alterations in mosquitoes compared with other plants, probably owing to the presence of monoterpene compounds in their tissues. The present study demonstrated LP plant extracts from A. conyzoides could be effective adulticides against adult Ae. aegypti. As natural products are biodegradable and exhibit low toxicity to mammalian and non-target organisms, they are suitable candidates for use in vector control programmes.

New records and DNA barcoding of deer flies, Chrysops (Diptera: Tabanidae) in Thailand.

Chrysops spp. or deer flies (Diptera: Tabanidae) are hematophagous flies of medical and veterinary importance and some species are important vectors of Trypanosoma evansi, the causative agent of surra in Thailand. However, data regarding deer fly species and their molecular identification are limited. Accurate species identification will indicate the appropriate control measures. In this study, an entomological survey of deer flies from different sites in Thailand between May 2018 and June 2019 were conducted. In addition, mitochondrial cytochrome oxidase subunit I (COI) barcoding region was used for species identification. A total of 82 females were collected and 6 species were identified. Of these, three species are new records for Thailand: C. designatus, C. fuscomarginalis and C. vanderwulpi bringing the species total found in Thailand to nine. The COI sequences revealed an intraspecific divergence of 0.0%-2.65% and an interspecific divergence of 7.03%-13.47%. Phylogenetic analysis showed that all deer fly species were clearly separated into distinct clusters according to morphologically identified species. These results indicated that COI barcodes were capable in discriminating between deer fly species on the basis of the barcoding gap and phylogenetic analysis. Therefore, DNA barcoding is a valuable tool for species identification of deer flies in Thailand.

Efficacy of Ageratum conyzoides extracts against Giardia duodenalis trophozoites: an experimental study.

BACKGROUND: Giardia duodenalis causes giardiasis in humans, particularly in developing countries. Despite the availability of treatments, resistance to some of the commercial anti-Giardia drugs has been reported in addition to their harmful side effects. Therefore, novel treatments for giardiasis are required. In this study, we aimed to assess the in vitro activity of crude extracts of Ageratum conyzoides against G. duodenalis trophozoites. METHODS: Plants were classified into three groups based on their flower colors: white (W), purple (P), and white-purple (W-P). Plants were separately cut into leaf (L) and flower (F) parts. Changes in internal organelle morphology of trophozoites following exposure to crude extracts were assessed using transmission electron microscopy (TEM). In subsequent experiments, efficacy of the most active essential oils from crude extracts [half maximal inhibitory concentrations (IC50) ≤ 100 µg/mL] against G. duodenalis trophozoites was tested. In vitro anti-Giardia assays using essential oils were performed in the same way as those performed using crude extracts. RESULTS: LW-P and FP extracts showed high activity (IC50 ≤ 100 µg/mL) against G. duodenalis trophozoites, with IC50 ± SD values of 45.67 ± 0.51 and 96.00 ± 0.46 µg/mL, respectively. In subsequent experiments, IC50 ± SD values of LW-P and FP essential oils were 35.00 ± 0.50 and 89.33 ± 0.41 µg/mL, respectively. TEM revealed the degeneration of flagella and ventral discs of G. duodenalis trophozoites following exposure to crude extracts. CONCLUSION: Crude LW-P and FP extracts of A. conyzoides showed the highest activity against G. duodenalis. Exposure to crude extract induced changes in the flagella and ventral discs of G. duodenalis trophozoites, which play important roles in attachment to the surface of mucosal cells. Our results suggest that the tested extracts warrant further research in terms of their efficacy and safety as giardiasis treatment.