Hard ticks taken from the Japanese serow, Capricornis crispus, in Yamagata Prefecture, Honshu, harboured infective larvae of onchocercid filariae after incubation from the 22nd to the 158th day. Haemaphysalis flava and H. japonica contained one to eight filarial larvae; females, males and a nymph of the ticks were infected. The 44 infective larvae recovered were 612-1,370 μm long, and 11 of them, 930-1,340 μm long, were studied in detail. The larvae possessed the morphologic characteristics of the larvae of the genus Cercopithifilaria, namely an oesophagus with a posterior glandular part, no buccal capsule and a long tail with three terminal lappets. Five types (A to E) of infective larvae were identified based on the morphologic characteristics. While to date five species of Cercopithifilaria have been described from the Japanese serow, a specific identification of the larvae found in this study was generally not possible. Only type E larvae could be tentatively assigned to Cercopithifilaria tumidicervicata, as they had a cervical swelling similar to that of the adults of this species. A key for the identification of the five larval types is presented. The study presents circumstantial evidences indicating that H. flava and H. japonica may transmit Cercopithifilaria spp. to Japanese serows. It also suggests the possibility that such filarial larvae will be found in hard ticks anywhere, because Cercopithifilaria is distributed worldwide, though this genus generally goes unnoticed, as its microfilariae occur in the skin, not in the blood, of host animals.
Human zoonotic onchocercosis is caused by Onchocerca dewittei japonica, parasitic in wild boars (Sus scrofa leucomystax) in Japan. Previously, microfilariae longer than those of Onchocerca dewittei japonica were observed in skin snips from wild boars during the study of O. dewittei japonica. Moreover, the third-stage larvae (L3) of these longer microfilariae were obtained from the blackfly Simulium bidentatum after experimental injections. Based on morphometric and molecular studies, similar L3 were found in blackflies during fieldwork in Oita, Japan. However, except for O. dewittei japonica, adult worms of Onchocerca have not been found in wild boars. In this study, we discovered adult females of a novel Onchocerca species in the skin of a wild boar in Oita, and named it Onchocerca takaokai n. sp. Females of this new species had longer microfilariae and differed from O. dewittei japonica in terms of their morphological characteristics and parasitic location. The molecular characteristics of the cytochrome c oxidase subunit 1 and 12S rRNA genes of the new species were identical to those of the longer microfilariae and L3 previously detected, but they differed from those of O. dewittei japonica at the species level. However, both species indicated a close affinity among their congeners and Onchocerca ramachandrini, parasitic in the warthog in Africa, was basal in the Suidae cluster of the 12S rRNA tree.
The transmission of zoonotic filarial parasites by black flies has so far been reported in the Chiang Mai and Tak provinces, Thailand, and the bites of these infected black flies can cause a rare disease-human zoonotic onchocerciasis. However, species identification of the filarial parasites and their black fly vectors in the Chiang Mai province were previously only based on a morphotaxonomic analysis. In this study, a combined approach of morphotaxonomic and molecular analyses (mitochondrial cox1, 12S rRNA, and nuclear 18S rRNA (SSU HVR-I) genes) was used to clarify the natural filarial infections in female black flies collected by using human and swine baits from two study areas (Ban Lek and Ban Pang Dang) in the Chiang Mai province from March 2018 to January 2019. A total of 805 and 4597 adult females, belonging to seven and nine black fly taxa, were collected from Ban Lek and Ban Pang Dang, respectively. At Ban Lek, four of the 309 adult females of Simulium nigrogilvum were positive for Onchocerca species type I in the hot and rainy seasons. At Ban Pang Dang, five unknown filarial larvae (belonging to the same new species) were detected in Simulium sp. in the S. varicorne species-group and in three species in the S. asakoae species-group in all seasons, and three non-filarial larvae of three different taxa were also found in three females of the S. asakoae species-group. This study is the first to molecularly identify new filarial species and their vector black fly species in Thailand.
Three new species of black flies from Thailand, Simulium wangkwaiense, S. tadtonense and S. maeklongkeense, are described based on their adults, pupal exuviae and cocoons. All three new species are assigned to the Simulium (Simulium) striatum species-group, bringing its total number in Thailand to seven. Simulium wangkwaiense sp. nov. is the species formerly called S. quinquestriatum in Thailand. Certain male and pupal morphological characteristics are shown to separate all seven Thai species of this species-group. All of the three new species have been analyzed genetically for their phylogenetic relationships, with three known related species (except for S. thailandicum), by using the fast-evolving nuclear big zinc finger (BZF) gene.
A new black fly species, Simulium (Gomphostilbia) kiewlomense, is described from females, males, pupae and mature larvae in Thailand. This new species is placed in the S. asakoae species-group and is characterized by having a combination of the elongate female sensory vesicle, widened male hind basitarsus, which is much wider than the hind femur, small pupal terminal hooks, and light greenish larval abdominal segments 1-3. Taxonomic notes are given to separate this new species from other related species. A DNA analysis using the COI gene shows that this new species has two genoforms with 1.21% difference. This is the 28th species of the S. asakoae species-group in Thailand, strengthening the evidence for high species diversity of this species-group.
The female and larva of Simulium (Gomphostilbia) udomi Takaoka & Choochote from Thailand are described for the first time. The female of this species is similar to those of S. (G.) asakoae Takaoka & Davies from Peninsular Malaysia, Thailand, Hong Kong and Vietnam, and S. (G.) chiangdaoense Takaoka & Srisuka from Thailand. The larva of this species is similar to S. (G.) curtatum Jitklang et al. and S. (G.) nr. asakoae 2 from Thailand in having a medium-long postgenal cleft. Taxonomic notes are given to separate this species from these related species. The COI gene sequence of S. (G.) udomi is compared with those of eight species of the S. asakoae species-group and three species of the S. ceylonicum species-group. This species is transferred from the S. ceylonicum species-group to the S. asakoae species-group based on the adult female and male morphological characters, comparisons of the genetic distances and phylogenetic relationships inferred from the COI gene sequences.
Zoonotic onchocerciasis is a human infection caused by Onchocerca species of animal origins and transmitted by black fly vectors. The reported incidence of this disease has increased throughout the world. This study aims to clarify the vectorial roles of black fly species in zoonotic filarial transmission in Tak province, western Thailand. The integrated approach of morphological and DNA sequence-based analyses was used to identify species of both wild-caught female black flies and infective filarial larvae found in the infected black flies. All of 494 female black flies captured were identified as Simulium nigrogilvum, through scanning electron microscopy (SEM) and DNA sequence analyses based on the cytochrome c oxidase subunit I (COI) and subunit II (COII), and the fast-evolving nuclear elongation complex protein 1 (ECP1) genes. Four females of S. nigrogilvum harbored one to three third-stage larvae (infective larvae) in their thoraces, with an infection rate of 0.81% (4/494). All infective larvae were similar in morphology and size to one another, being identified as Onchocerca species type I (= O. sp. type A), a bovine filaria, originally reported from Japan, and also as O. sp. found in S. nodosum in Thailand, based on their body lengths and widths being 1,068-1,346 µm long by 25-28 µm wide, and morphological characters. Comparisons of cytochrome c oxidase subunit I (COI) and 12S rRNA sequences of mitochondrial DNA (mtDNA) and phylogenetic analyses with those of previous reports strongly supported that all larvae were O. sp. type I. This report is the first indicating the presence of O. sp. type I in Thailand and its vector being S. nigrogilvum.
Species of eight species-groups (seven named and one unnamed) of Simulium (Simulium) Latreille are revised chiefly based on the adult female and male genitalia. Four species-groups are each divided into more than two groups. Thus, the S. christophersi species-group is divided into two (S. christophersi and S. nebulicola species-groups), the S. griseifrons species-group into six (S. griseifrons, S. grossifilum, S. chungi, S. crocinum, S. indicum and S. yadongense species-groups), the S. malyschevi species-group into two (S. malyschevi and S. pavlovskii species-groups), and the S. melanopus species-group into three (S. melanopus, S. dumogaense and S. iridescens species-groups). The S. crassifilum species-group is merged with the S. variegatum species-group. Simulium fulvipes (Ono) of the former S. malyschevi species-group and S. dentastylum Yang, Chen & Luo of Simulium s. str. species unplaced to species-group are removed from the subgenus Simulium because their male styles are subequal in length to the coxites. A revised classification of species of the 15 species-groups including eight new species-groups of Simulium (Simulium) is presented. Keys to 20 species-groups of Simulium (Simulium) in the Oriental Region are provided for adult females and males.
Rhipicephalus (Boophilus) microplus serves as an important ectoparasite of livestock and a vector of several pathogens resulting in diseases, subsequently affecting the agricultural field as well as the economy. The extensive use of synthetic acaricides is known to cause resistance over time and therefore a much safer, effective and environmentally friendly alternative to overcome tick infestation should be implemented. Larval immersion tests (LIT) were done to evaluate the effects of Citrus hystrix (Family: Rutaceae) and Cymbopogon citratus (Family: Poaceae) essential oils (EOs) for their individual and combined (1:1) acaricidal activity against the cattle tick. Results showed that LC50 and LC90 values in 24 and 48 hours for Cit. hystrix EO were 11.98% and 24.84%, and 10.95% and 21.71% respectively. LC50 and LC90 values for Cym. citratus EO were 1.21% and 6.28%, and 1.05% and 6.12% respectively. The mixture of EOs from two plants in 1:1 ratio (Cit. hystrix 50%: Cym. citratus 50%) was found to exhibit antagonistic effect (synergistic factor < 1). The 24 hours and 48 hours LC50 and LC90 values for combined EOs were 1.52% and 2.84%, and 1.50% and 2.76% respectively. Individual and combined essential oils were subjected to qualitative analysis using gas chromatography-mass spectrometry (GC-MS) to screen the chemical components present in EOs. Our results showed that the combination of Cit. hystrix and Cym. citratus at 1:1 ratio resulted in an antagonistic effect and the use of Cym. citratus alone is more toxic to R. (B.) microplus, making it a better alternative to chemical based acaricide.
For successful parasitism, parasitoid females must oviposit and the progeny must develop in individual hosts. Here, we investigated the determinants of host acceptance for oviposition and host suitability for larval development of Drosophila parasitoids from Bogor and Kota Kinabalu (≍1,800 km northeast of Bogor), Indonesia, in tropical Asia. Asobara pleuralis (Ashmead) from both localities oviposited frequently (>60%) in all of the drosophilid species tested, except the strain from Kota Kinabalu oviposited rarely (10%) in Drosophila eugracilis Bock & Wheeler. Leptopilina victoriae Nordlander from both localities only oviposited frequently (>77%) in species from the Drosophila melanogaster species group except D. eugracilis (<3.7%), whereas Leptopilina pacifica Novković & Kimura from Bogor oviposited frequently (>85%) only in species from the Drosophila immigrans species group. Thus, host acceptance appeared to be affected by host taxonomy, at least in Leptopilina species. Host suitability varied considerably, even among closely related drosophilid species, which suggests that the host suitability is at least in part independent of host taxonomy and that it has been determined via parasitoid-host coevolutionary interactions (i.e., arms race). Host acceptance did not always coincide with host suitability, i.e., parasitoids sometimes oviposited in unsuitable host species. Geographic origin strongly affected the host acceptance and suitability in the A. pleuralis-D. eugracilis parasitoid-host pair, whereas it only weakly affected the acceptability and suitability in other parasitoid-host combinations.
There are few small animals models for filariasis, even more so for onchocerciasis. Therefore it is difficult to test under drug screening conditions large numbers of potentially macrofilaricidal compounds. One way around this difficulty is to use mice infected with Trichinella spiralis which by reason of anatomical location in the host would show some correlation in antinematode activity between the test and target organisms. This study investigated the activity of 16 compounds against the immature larval stage of T. spiralis. All the nine benzimidazole compounds (albendazole, flubendazole, mebendazole, oxfendazole, oxibendazole 780118, 780120, 790163, and 790392) were active, the most potent being oxfendazole. The benzothiazoles (CGP21306, CGP20376, CGP21833 and CGP24588A) also indicated some anti-nematode activity together with 35vr, an imidazopyridine, but not as marked as the benzimidazole group. However, the organic arsenical compounds (Mel Ga and Mel Ni) showed little activity and this was at a rather highly toxic level. The prospects of using the Trichinella-mouse model as a primary screen to test for potential macrofilaricides are discussed.
OBJECTIVE: Maggot debridement therapy (MDT) has seen a resurgence in recent years in the treatment of hard-to-heal wounds, as a result of rising antibiotic resistance. The sterilised larvae of Lucilia cuprina have been used in MDT in Malaysia since 2003, with encouraging results for the treatment of hard-to-heal diabetic wounds. We report a case series of 30 patients selected from our clinic by convenient sampling with diabetic lower limb ulcers treated with MDT. The average age of patients receiving MDT was >50 years. Of the 30 patients in the study, nine were female and 21 were male. All patients had underlying diabetes, two patients had leg ulcers and 28 patients had diabetic foot ulcers. Sterilised Lucilia cuprina larvae were applied via a standard method of 10 maggots per square centimetre and dressed with sterile gauze. The study endpoint was defined as ≤5% coverage with slough or necrotic tissue following three successive applications of MDT. In this study, maximum debridement of wounds was achieved in 96.6% (29 patients) of our patients, with ≤5% coverage with slough or necrotic tissue, in addition to a reduction in wound-related pain, as assessed by a visual analogue scale. No adverse events were reported. The findings of this study support the use of MDT as a safe, efficacious, and cost-effective method of managing diabetic wounds.
Natural insecticides from plant origin against mosquito vectors have been the main concern for research due to their high level of eco-safety. Control of mosquitoes in their larval stages are an ideal method since Aedes larvae are aquatic, thus it is easier to deal with them in this habitat. The present study was specifically conducted to explore the larvicidal efficacy of different plant parts of Ipomoea cairica (L.) or railway creeper crude extract obtained using two different solvents; methanol and acetone against late third-stage larvae of Aedes albopictus (Skuse) and Aedes aegypti (L.) (Diptera: Culicidae). Plant materials of I. cairica leaf, flower, and stem were segregated, airdried, powdered, and extracted using Soxhlet apparatus. Larvicidal bioassays were performed by using World Health Organization standard larval susceptibility test method for each species which were conducted separately for different concentration ranging from 10 to 450 ppm. Both acetone and methanol extracts showed 100% mortality at highest concentration tested (450 ppm) after 24 h of exposure. Results from factorial ANOVA indicated that there were significant differences in larvicidal effects between mosquito species, solvent used and plant parts (F=5.71, df=2, P<0.05). The acetone extract of I. cairica leaf showed the most effective larvicidal action in Ae. aegypti with LC50 of 101.94 ppm followed by Ae. albopictus with LC50 of 105.59 ppm compared with other fractions of I. cairica extract obtained from flower, stem, and when methanol are used as solvent. The larvae of Ae. aegypti appeared to be more susceptible to I. cairica extract with lower LC50 value compared with Ae. albopictus (F=8.83, df=1, P<0.05). Therefore, this study suggests that the acetone extract of I. cairica leaf can be considered as plant-derived insecticide for the control of Aedes mosquitoes. This study quantified the larvicidal property of I. cairica extract, providing information on lethal concentration that may have potential for a more eco-friendly Aedes mosquito control program.
Cancer still presents enormous challenges in the medical world. Currently, the search for
anticancer compounds has garnered a lot of interest, especially in finding them from the natural
sources. In this study, by using Sulforhodamine B (SRB) colorimetric assay, compounds,
extracted from supermeal worm (Zophobas morio) larvae using two types of acidified organic
solvent (ethanol and isopropanol), were shown to inhibit the growth of a breast cancer line,
MCF-7. A comparative study of the effect was carried out on a normal cell line, Vero. Results
showed that, the two types of extracts inhibits growth of MCF-7 cell at varying degrees, on
the other hand, have much less effect on Vero cell. Extracts analysed by UV-vis spectroscopy,
showed peaks in the range of 260 to 280 nm, inferring the presence of aromatic amino acids,
whereas the highest peak of 3.608 AU at 230 nm indicates the presence of peptide bonds. By
Raman spectroscopy, peaks are observed at 1349 cm-1, 944 cm-1 and 841 cm-1 indicating the
presence of Tyr, Try and Gly, confirming the UV-vis analyses. All results of analyses implied
that the anticancer compounds contain peptides.
Zika virus (ZIKV) has now become a global public health concern. The vectors for ZIKV are Aedes aegypti and A. albopictus. Both these mosquitoes are predominant in Southeast Asia and are also responsible for the spread of other arboviral diseases like dengue virus and chikungunya virus. The incidence of dengue has been increasing over the years and this is of concern to public health workers. Simple laboratory tools for the detection of ZIKV is also lacking. In the absence of drugs and vaccine for these arboviral diseases, vector control is the main option for surveillance and control. Aedes larval surveys have been the hallmark of dengue control along with larviciding and fogging when cases are reported. However, we need new paradigms and options for control of these vectors. The current situation in Southeast Asia clearly proves that effective strategies for vector control need to be proactive and not reactive. This will be the way forward to control epidemics of these diseases inclusive of ZIKV until a vaccine becomes available.
Although myiasis caused by the blow fly, Chrysomya bezziana Villeneuve (Diptera: Calliphoridae), has been reported in Thailand, all of the cases were human. This study described three cutaneous myiasis cases caused by C. bezziana in dogs in Chiang Mai province, northern Thailand. The removal of fly larvae together with specific treatment to cure all cases in this study was represented.
Bacterial polyhydroxyalkanoates (PHA) are expensive partly due to the recovery and purification processes. Thus, many studies have been carried out in order to minimize the cost. Here we report on the use of mealworm, which is the larva of mealworm beetle (Tenebrio molitor) to recover PHA granules from Cupriavidus necator. Mealworms were shown to readily consume the freeze-dried C. necator cells and excrete the PHA granules in the form of whitish feces. Further purification using water, detergent and heat resulted in almost 100% pure PHA granules. Comparison with chloroform extraction showed no signs of reduction in the molecular weight and dispersion of the PHA molecules. Scanning electron microscopy and dynamic light scattering measurements revealed that the biologically recovered PHA granules retained their native spherical morphology. The PHA granules were subjected to a battery of tests to determine their purity and properties in comparison to the chloroform extracted PHA. This study has demonstrated the possibility of using mealworms as a biological agent to partially purify the PHA granules.
Polyhydroxyalkanoates (PHAs) are produced in microbes as a source of carbon and energy storage. They are biodegradable and have properties similar to synthetic plastics, which make them an interesting alternative to petroleum-based plastics. In this study, a refined method of recovering PHA from Cupriavidus necator biomass was proposed by incorporating the use of the yellow mealworm (the larval phase of the mealworm beetle, Tenebrio molitor) as partial purification machinery, followed by washing of the fecal pellets with distilled water and sodium hydroxide. The PHA contents of the cells used in this study were 55wt% (produced from palm olein) and 60 wt% (produced from waste animal fats). The treatment of distilled water and NaOH further increased the purity of PHA to 94%. In parallel, analysis of the 16S rRNA metagenomic sequencing of the mealworm gut microbiome has revealed remarkable changes in the bacterial diversity, especially between the mealworms fed with cells produced from palm olein and waste animal fats. This biological recovery of PHA from cells is an attempt to move towards a green and sustainable process with the aim of reducing the use of harmful solvents and strong chemicals during polymer purification. The results obtained show that - purities of >90%, without a reduction in the molecular weight, can be obtained through this integrative biological recovery approach. In addition, this study has successfully shown that the cells, regardless of their origins, were readily consumed by the mealworms, and there is a correlation between the feed type and the mealworm gut microbiome.
Toxicological studies of four insecticides (malathion, carbaryl, bioresmethrin, and GH 74) against Musca domestica vicinia (Ampang strain) were undertaken with particular reference to age, sex and posttreatment temperature. It was found that bioresmethrin and GH 74, both with a negative temperature coefficient, have great potential for use against houseflies. In vitro inhibitory studies of head and body esterases showed that unlike malathion and carbaryl, bioresmethrin had only negligible effect on these enzymes. The possibilities of using bioresmethrin and GH 74 for controlling the housefly problem in the Cameron Highlands, West Malaysia are discussed.
Toxicity tests carried out on the larvae of A. aegypti and C. quinquefasciatus showed the former to be more tolerant of all insecticides tested, the order of toxicity being temephos greater than DDT greater than DDVP greater than malathion greater than lindane greater than carbaryl; also the second instar larvae of A. aegypti were more susceptible than fourth instar larvae. Enzyme kinetic studies on the total non-specific esterases and CarEs of adults and larvae of both species showed the Km values for total esterases of adult A. aegypti to be 0.333 mM vs 0.233 mM for C. quinquefasciatus; for adult CarEs it was 0.250 mM vs 0.220 mM. For total larval esterases of A. aegypti it was 0.112 mM vs 0.175 mM for C. quinquefasciatus: and for larval CarES it was 0.159 mM vs 0.213 mM respectively. Although some correlation between in vivo toxicity (LD50 values) and in vitro esterase inhibition (I50 values) between species could be discerned, overall correlation could not be established.