Repellent effect of local indigenous knowledge-based repellent in Nakhon Si Thammarat, Thailand, against Aedes aegypti mosquito.

Dengue fever has been a significant disease in Thailand for a long time, ranking it as one of the major health problems in the country. Management of the adult stage of mosquito vectors is approached by applying various synthetic chemicals such as adulticides, attractants, deterrents, and repellents. In Thailand, mosquito control and personal protection from mosquito bites are currently the most important measures for preventing and controlling mosquito-borne diseases. Although there are various control strategies for dengue disease, participation from the local community plays a vital role in the success of disease control. At present, a lot of local people have seen the value of local indigenous knowledge and used this to improve their life. The local community in the southern part of Thailand has used mosquito repellent from local knowledge for a long time. The problem regarding mosquito repellent made from local indigenous knowledge is that it has not yet been tested to determine its effectiveness. Therefore, this research aims to assess the effectiveness of mosquito repellent from local learning from Nakhon Si Thammarat provinces in Thailand. From the survey, out of 23 districts, six mosquito repellents were found in 3 communities, including Nabon, Muang, and Thasala. The repellent efficacy against the laboratory strain of Aedes aegypti by using the human-bait technique of the WHO (1996) standard method, with slight modifications. Approximately 0.1 ml of each test sample was applied evenly onto a 30 cm2 test site on one forearm of each human volunteer. Exposure experiments continued at 30 min intervals until at least two bites occurred in a 3-min period, or when a first bite was followed by a confirming bite (second bite) in the subsequent observation period. Each test was duplicated on different days for the two human volunteers. The result shows that three mosquito repellents made from local indigenous knowledge that have protection that lasts for more than 2 h are Ban Ko Sa Child Development Center's citronella spray (Nabon district, Kaew Saen subdistrict), Khun Lang's citronella spray, and Khun Lang's citronella ointment (Muang district, Pak Phun subdistrict). The result of this research was reported back to the local community to re-evaluate their self-reliance on their protection against mosquito biting.

In vitro susceptibility to miltefosine of amphotericin B-resistant Leishmania (Mundinia) martiniquensis.

Leishmania (Mundinia) martiniquensis is a newly described species that causes human visceral, disseminated, and mucocutaneous leishmaniases. Amphotericin B deoxycholate (AmpB) is the first-line drug for the treatment of leishmaniasis in Thailand; however, several relapse cases of leishmaniasis caused by L. martiniquensis have been documented. In this study, in vitro susceptibility to AmpB and miltefosine (MIL) of wild-type (before treatment, LSCM1) and two AmpB-resistant L. martiniquensis strains (an in vitro-induced AmpB-resistant strain, AmpBRP2i, and a relapse strain, LSCM1-6) were determined. Results reveal that the IC50 value and resistance index against both drugs of promastigotes and intracellular amastigotes of the AmpBRP2i and LSCM1-6 strains were statistically significantly higher than those of the LSCM1 strain suggesting that cross-resistance with MIL occurred in both AmpB-resistant strains. The results of this study advocate further investigation into mechanisms that involve the complex nature of AmpB/MIL resistance in L. martiniquensis and development of effective methods for the identification of the AmpB-resistant parasites to help delivery of appropriate treatments for patients and for epidemiological surveys to survey the potential spread of drug-resistant strains.

Natural infection with Leishmania (Mundinia) martiniquensis supports Culicoides peregrinus (Diptera: Ceratopogonidae) as a potential vector of leishmaniasis and characterization of a Crithidia sp. isolated from the midges.

The prevalence of autochthonous leishmaniasis in Thailand is increasing but the natural vectors that are responsible for transmission remain unknown. Experimental in vivo infections in Culicoides spp. with Leishmania (Mundinia) martiniquensis and Leishmania (Mundinia) orientalis, the major causative pathogens in Thailand, have demonstrated that biting midges can act as competent vectors. Therefore, the isolation and detection of Leishmania and other trypanosomatids were performed in biting midges collected at a field site in an endemic area of leishmaniasis in Tha Ruea and a mixed farm of chickens, goats, and cattle in Khuan Phang, Nakhon Si Thammarat province, southern Thailand. Results showed that Culicoides peregrinus was the abundant species (>84%) found in both locations and only cow blood DNA was detected in engorged females. Microscopic examination revealed various forms of Leishmania promastigotes in the foregut of several C. peregrinus in the absence of bloodmeal remnants, indicating established infections. Molecular identification using ITS1 and 3'UTR HSP70 type I markers showed that the Leishmania parasites found in the midges were L. martiniquensis. The infection rate of L. martiniquensis in the collected flies was 2% in Tha Ruea and 6% in Khuan Phang, but no L. orientalis DNA or parasites were found. Additionally, organisms from two different clades of Crithidia, both possibly new species, were identified using SSU rRNA and gGAPDH genes. Choanomastigotes and promastigotes of both Crithidia spp. were observed in the hindgut of the dissected C. peregrinus. Interestingly, midges infected with both L. martiniquensis and Crithidia were found. Moreover, four strains of Crithidia from one of the clades were successfully isolated into culture. These parasites could grow at 37°C in the culture and infect BALB/c mice macrophages but no multiplication was observed, suggesting they are thermotolerant monoxenous trypanosomatids similar to Cr. thermophila. These findings provide the first evidence of natural infection of L. martiniquensis in C. peregrinus supporting it as a potential vector of L. martiniquensis.

Antennal morphology and sensilla ultrastructure of the malaria vectors, Anopheles maculatus and An. sawadwongporni (Diptera: Culicidae).

Mosquitoes rely mainly on the olfactory system to track hosts. Sensilla contain olfactory neuron receptors that perceive different kinds of odorants and transfer crucial information regarding the surrounding environment. Anopheles maculatus and An. sawadwongporni, members of the Maculatus Group, are regarded as vectors of malaria in Thailand. The fine structure of their sensilla has yet to be identified. Herein, scanning electron microscopy is used to examine the sensilla located on the antennae of adults An. maculatus and An. sawadwongporni, collected from the Thai-Myanmar border. Four major types of antennal sensilla are discovered in both species: chaetica, coeloconica, basiconica (grooved pegs) and trichodea. The antennae of female An. maculatus have longer lengths (µm, mean ± SE) in the long sharp-tipped trichodea (40.62 ± 0.35 > 38.20 ± 0.36), blunt-tipped trichodea (20.39 ± 0.62 > 18.62 ± 0.35), and basiconica (7.84 ± 0.15 > 7.41 ± 0.12) than those of An. sawadwongporni. Using light microscopy, it is found that the mean numbers of large sensilla coeloconica (lco) on both flagella in An. maculatus (left: 32.97 ± 0.48; right: 33.27 ± 0.65) are also greater when compared to An. sawadwongporni (left: 30.40 ± 0.62; right: 29.97 ± 0.49). The mean counts of lco located on flagellomeres 1-3, 6, and 9 in An. maculatus are significantly higher than those of An. sawadwongporni. The data in this study indicate that two closely related Anopheles species exhibit similar morphology of sensilla types, but show variations in length, and likewise in the number of large sensilla coeloconica between them, suggesting they might be causative factors that affect their behaviors driven by the sense of smell.

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

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

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.

Diversity of nematodes infecting the human-biting black fly species, Simulium nigrogilvum (Diptera: Simuliidae) in central Thailand.

Black flies (Diptera: Simuliidae) are known as vectors of disease agents in humans and livestock, with some species being vectors of Onchocerca volvulus, the filarial nematode that is the causative agent of human onchocerciasis. Nematode infections in adult female black flies have been reported from some areas in northern and western Thailand, but not from other regions of Thailand. In this study, wild-caught adult female black flies from the central region of Thailand were examined for infections with nematodes. Collections of adult females were carried out at Khlong Lan district, Kamphaeng Phet province, central Thailand. A molecular approach, based on the mitochondrial (cox1, 12S rRNA) and nuclear (18S rRNA) genes, was used to identify the species of nematodes recovered from the specimens collected. A total of 911 wild-caught adult black flies were collected. Simulium nigrogilvum was the most abundant species (n = 708), followed by S. doipuiense complex (n = 179), S. chamlongi (n = 11), S. umphangense (n = 10), S. chumpornense (n = 1), S. multistriatum species-group (n = 1), and S. maewongense (n = 1). Nematode infections were detected in nine specimens of S. nigrogilvum, of which two were positive for filarial worms (one worm each, infection rate 0.28%) and seven were positive for non-filarial nematodes (11 worms in total, infection rate 0.99%). The two filarial nematodes (third-stage larvae) were identified molecularly as Onchocerca species type I, while the 11 non-filarial nematodes were classified into ascaridoid (n = 2), tylenchid (n = 6) and mermithid (n = 3) nematodes. The results of this study demonstrated that adult female S. nigrogilvum were parasitized with diverse nematodes (filarial and non-filarial). Detection of the infective larvae of Onchocerca sp. type I in S. nigrogilvum confirms that occurrence of zoonotic onchocerciasis is highly possible in Thailand. Additional in-depth investigation of the morphology, life cycle and host-parasite relationship of nematodes that parasitized this black fly host is still needed.

Ligusticum sinense Nanoemulsion Gel as Potential Repellent against Aedes aegypti, Anopheles minimus, and Culex quinquefasciatus (Diptera: Culicidae).

Ligusticum sinense Oliv. cv. is a species of Umbelliferae (Apiaceae), a large plant family in the order Apiales. In this study, L. sinense hexane extract nanoemulsion gel (LHE-NEG) was investigated for mosquito repellency and compared to the standard chemical, N,N-diethyl-3-methylbenzamide (DEET), with the goal of developing a natural alternative to synthetic repellents in protecting against mosquito vectors. The results demonstrated that LHE-NEG afforded remarkable repellency against Aedes aegypti, Anopheles minimus, and Culex quinquefasciatus, with median protection times (MPTs) of 5.5 (4.5-6.0), 11.5 (8.5-12.5), and 11.25 (8.5-12.5) h, respectively, which was comparable to those of DEET-nanoemulsion gel (DEET-NEG: 8.5 (7.0-9.0), 12.0 (10.0-12.5), and 12.5 (10.0-13.5) h, respectively). Evaluation of skin irritation in 30 human volunteers revealed no potential irritant from LHE-NEG. The physical and biological stability of LHE-NEG were determined after being kept under heating/cooling cycle conditions. The stored samples of LHE-NEG exhibited some changes in appearance and differing degrees of repellency between those kept for 3 and 6 heating/cooling cycles, thus providing slightly shorter MPTs of 4.25 (4.0-4.5) and 3.25 (2.5-3.5) h, respectively, when compared to those of 5.0 (4.5-6.0) h in fresh preparation. These findings encourage commercially developed LHE-based products as an alternative to conventional synthetic repellents in preventing mosquito bites and helping to interrupt mosquito-borne disease transmission.

Enhancement of Temephos and Deltamethrin Toxicity by Petroselinum crispum Oil and its Main Constituents Against Aedes aegypti (Diptera: Culicidae).

Previous work presented the profound antimosquito potential of Petroselinum crispum essential oil (PEO) against either the pyrethroid-susceptible or resistant strains of Aedes aegypti. This plant oil also inhibited the activity of acetylcholinesterase and mixed-function oxidases significantly, thus suggesting its potential as a synergist for improving mosquitocidal efficacy of insecticidal formulations. This study investigated the chemical composition, larvicidal activity, and potential synergism with synthetic insecticides of PEO and its main compounds for the purpose of interacting with insecticide resistance in mosquito vectors. The chemical profile of PEO, obtained by GC-MS analysis, showed a total of 17 bioactive compounds, accounting for 99.09% of the whole oil, with the most dominant constituents being thymol (74.57%), p-cymene (10.73%), and γ-terpinene (8.34%). All PEO constituents exhibited promising larvicidal effects, with LC50 values ranging from 19.47 to 59.75 ppm against Ae. aegypti, in both the pyrethroid-susceptible and resistant strains. Furthermore, combination-based bioassays revealed that PEO, thymol, p-cymene, and γ-terpinene enhanced the efficacy of temephos and deltamethrin significantly. The most effective synergist with temephos was PEO, which reduced LC50 values to 2.73, 4.94, and 3.28 ppb against MCM-S, PMD-R, and UPK-R, respectively, with synergism ratio (SR) values of 1.33, 1.38, and 2.12, respectively. The best synergist with deltamethrin also was PEO, which reduced LC50 values against MCM-S, PMD-R, and UPK-R to 0.008, 0.18, and 2.49 ppb, respectively, with SR values of 21.25, 9.00, and 4.06, respectively. This research promoted the potential for using essential oil and its principal constituents as not only alternative larvicides, but also attractive synergists for enhancing efficacy of existing conventional insecticides.

Three new species of the Simulium (Simulium) striatum species-group (Diptera: Simuliidae) from Thailand, with their phylogenetic relationships.

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 Multiplex PCR Based on Mitochondrial COI Sequences for Identification of Members of the Anopheles barbirostris Complex (Diptera: Culicidae) in Thailand and Other Countries in the Region.

A multiplex-PCR assay based on mitochondrial cytochrome c oxidase subunit I (COI) sequences was developed for identification of five members of the Barbirostris Complex which occur in Thailand: Anopheles barbirostris s.s., An. dissidens, An. saeungae, An. wejchoochotei and An. barbirostris species A3. Anopheles campestris was not included in the assay due to the lack of unequivocal sequences. Allele-specific primers were designed for specific nucleotide segments of COI sequences of each species. Mismatch method and addition of long GC tail were applied for some primers. The assay provided products of 706 bp for An. barbirostris s.s., 238 bp for An. dissidens, 611 bp for An. saeungae, 502 bp for An. wejchoochotei and 365 bp for An. barbirostris A3. The assay was tested using 111 wild-caught female mosquitoes from Bhutan, Cambodia, Indonesia (Sulawesi) and Thailand. The results of the multiplex PCR were in complete agreement with COI sequencing; however, one of three specimens from Bhutan and all 11 specimens from Indonesia were not amplifiable by the assay due to their distinct COI sequences. This, together with the distinct rDNA sequences of these specimens, suggests the presence of at least two additional new species in the Barbirostris Complex.

Integrated systematics of Anopheles subpictus (Diptera: Culicidae) in the Oriental Region, with emphasis on forms in Thailand and Sulawesi, Indonesia.

The Anopheles subpictus complex consists of four species informally designated, based on fixed inversions of polytene chromosomes and morphology, as species A, B, C and D in India. However, recent studies revealed the presence of only species A and B in Sri Lanka. Little is known about the specific identity of the taxon in other countries in Asia. This paper reports the results of a molecular and morphological study of An. subpictus in Thailand and South Sulawesi, Indonesia. The maxillary palpi of most females from Thailand have the apical pale band longer than the subapical dark band, seta 7-I of pupae branched and short, and eggs with 18-25 float ridges. These characters do not agree with those described for species A, B, C and D in India. The females of An. subpictus from South Sulawesi usually have the subapical dark band of the maxillary palpus equal in length to the apical pale band. Phylogenetic analyses of sequences of the internal transcribed spacer 2 (ITS2) region of rDNA and the cytochrome c oxidase subunit I (COI) gene of mtDNA of specimens from Thailand, and South Sulawesi, and from various localities in GenBank, were conducted. ITS2 sequences of specimens from all localities in Thailand were identical, except for a small divergence in specimen from Phang Nga Province. Three distinct COI clades were detected in specimens from Chiang Mai Province in northern Thailand. However, crossing experiments between the three clades revealed no genetic incompatibility, suggesting that they were conspecific. ITS2 and COI sequences of most specimens from Thailand fell in clades other than those of An. subpictus species A and B and An. subpictus from Indonesia (East Nusa Tenggara, Java, South Sulawesi) and the Philippines. ITS2 sequences from South Sulawesi and East Nusa Tenggara were very similar, and fell in a clade consisting of specimen from Phang Nga in southern Thailand and sequences of some specimens from Cambodia and Vietnam, but their COI sequences were distinct. DNA sequences and morphological differences suggest the presence of two species within An. subpictus in Thailand, and more than one species in Indonesia.

Synergistic Toxicity of Plant Essential Oils Combined with Pyrethroid Insecticides against Blow Flies and the House Fly.

Blow flies (Diptera: Calliphoridae) and the house fly (Diptera: Muscidae) are filth flies of medical importance, and control of their population is needed. As insecticide applications have resulted in fly resistance, and the exploration of plant essential oils (EOs) has increased against filth flies, this study assessed the combination of EOs with pyrethoids to enhance toxic efficacy. The EOs of five effective plants were screened initially against the house fly (Musca domestica L.). Their chemical constituent was performed using gas chromatography-mass spectrometry (GC-MS) analysis. The main components of Boesenbergia rotunda (Zingiberaceae) rhizome, Curcuma longa (Zingiberaceae) rhizome, Citrus hystrix (Rutaceae) fruit peel, Ocimum gratissimum (Lamiaceae) seed, and Zanthoxylum limonella (Rutaceae) fruit were δ-3-caren (35.25%), ß-turmerone (51.68%), ß-pinene (26.56%), p-cumic aldehyde (58.21%), and dipentene (60.22%), respectively. The screening test revealed that the three most effective plant EOs were from B. rotunda, C. longa and O. gratissimum, which were selected for the combination with two pyrethroid insecticides (permethrin and deltamethrin), in order to enhance their synergistic efficacy against the blow flies, Chrysomya megacephala Fabricius, Chrysomya rufifacies Macquart, and Lucilia cuprina Wiedemann, and the house fly. Synergistic action was presented in almost all of the flies tested with permenthrin/deltamethrin/EOs mixtures. It was interesting that the combination of deltamethrin with three EOs showed a synergistic effect on all of the tested flies. However, an antagonistic effect was observed in C. megacephala and M. domestica treated with permethrin-B. rotunda and C. megacephala treated with permethrin-O. gratissimum. The LD50 of insecticides decreased when combined with plant EOs. This alternative strategy will be helpful in developing a formula for effective fly control management.

Biochemical Effects of Petroselinum crispum (Umbellifereae) Essential Oil on the Pyrethroid Resistant Strains of Aedes aegypti (Diptera: Culicidae).

In ongoing screening research for edible plants, Petroselinum crispum essential oil was considered as a potential bioinsecticide with proven antimosquito activity against both the pyrethroid susceptible and resistant strains of Aedes aegypti. Due to the comparative mosquitocidal efficacy on these mosquitoes, this plant essential oil is promoted as an attractive candidate for further study in monitoring resistance of mosquito vectors. Therefore, the aim of this study was to evaluate the impact of P. crispum essential oil on the biochemical characteristics of the target mosquito larvae of Ae. aegypti, by determining quantitative changes of key enzymes responsible for xenobiotic detoxification, including glutathione-S-transferases (GSTs), α- and ß-esterases (α-/ß-ESTs), acetylcholinesterase (AChE), acid and alkaline phosphatases (ACP and ALP) and mixed-function oxidases (MFO). Three populations of Ae. aegypti, comprising the pyrethroid susceptible Muang Chiang Mai-susceptible (MCM-S) strain and the pyrethroid resistant Pang Mai Dang-resistant (PMD-R) and Upakut-resistant (UPK-R) strains, were used as test organisms. Biochemical study of Ae. aegypti larvae prior to treatment with P. crispum essential oil revealed that apart from AChE, the baseline activity of most defensive enzymes, such as GSTs, α-/ß-ESTs, ACP, ALP and MFO, in resistant UPK-R or PMD-R, was higher than that determined in susceptible MCM-S. However, after 24-h exposure to P. crispum essential oil, the pyrethroid susceptible and resistant Ae. aegypti showed similarity in biochemical features, with alterations of enzyme activity in the treated larvae, as compared to the controls. An increase in the activity levels of GSTs, α-/ß-ESTs, ACP and ALP was recorded in all strains of P. crispum oil-treated Ae. aegypti larvae, whereas MFO and AChE activity in these mosquitoes was decreased. The recognizable larvicidal capability on pyrethroid resistant Ae. aegypti, and the inhibitory effect on AChE and MFO, emphasized the potential of P. crispum essential oil as an attractive alternative application for management of mosquito resistance in current and future control programs.

Synergy in the adulticidal efficacy of essential oils for the improvement of permethrin toxicity against Aedes aegypti L. (Diptera: Culicidae).

BACKGROUND: In a previous screening program for mosquitocides from local edible plants in Thailand, essential oils (EOs) of Cyperus rotundus, Alpinia galanga and Cinnamomum verum, were found to possess promising adulticidal activity against Aedes aegypti. With the aim of reducing usage of conventional insecticides and improving the management of resistant mosquito populations, this study was designed to determine the potential synergism in the adulticidal efficacy of EOs on permethrin toxicity against Ae. aegypti, both pyrethroid-resistant and -susceptible strains. METHODS: EOs extracted from rhizomes of C. rotundus and A. galanga as well as C. verum barks were evaluated for chemical compositions and adulticidal activity against Muang Chiang Mai-susceptible (MCM-S) and Pang Mai Dang-resistant (PMD-R) strains of Ae. aegypti. Adulticidal bioassays of EO-permethrin mixtures for synergistic activity were also performed on these Ae. aegypti strains. RESULTS: Chemical characterization by the GC-MS analytical technique demonstrated that 48 compounds were identified from the EOs of C. rotundus, A. galanga and C. verum, representing 80.22%, 86.75% and 97.24%, respectively, of all compositions. Cyperene (14.04%), ß-bisabolene (18.27%) and cinnamaldehyde (64.66%) were the main constituents of C. rotundus, A. galanga and C. verum oils, respectively. In adulticidal bioassays, EOs of C. rotundus, A. galanga and C. verum were effective in killing Ae. aegypti, both MCM-S and PMD-R strains, with LD50 values of 10.05 and 9.57 µg/mg female, 7.97 and 7.94 µg/mg female, and 3.30 and 3.22 µg/mg female, respectively. The adulticidal efficacy against MCM-S and PMD-R Ae. aegypti of these EOs was close to that of piperonyl butoxide (PBO, LD50 values = 6.30 and 4.79 µg/mg female, respectively) but less pronounced than that of permethrin (LD50 values = 0.44 and 3.70 ng/mg female, respectively). Nevertheless, combination-based bioassays discovered the accomplished synergism of EOs together with permethrin. Significant synergistic effects with permethrin against both the strains of Ae. aegypti were recorded in the EOs of C. rotundus and A. galanga. Addition of C. rotundus and A. galanga oils decreased the LD50 values of permethrin against MCM-S dramatically from 0.44 to 0.07 and 0.11 ng/mg female, respectively, with synergism ratio (SR) values of 6.28 and 4.00, respectively. Furthermore, EOs of C. rotundus and A. galanga also reduced the LD50 values of permethrin against PMD-R drastically from 3.70 to 0.42 and 0.003 ng/mg female, respectively, with SR values of 8.81 and 1233.33, respectively. CONCLUSIONS: The synergy of enhanced adulticidal toxicity recorded from EO-permethrin combinations against both strains of Ae. aegypti presents a promising role of EOs as a synergist for improving mosquitocidal efficacy, particularly in situations where conventional compounds are ineffective or inappropriate.

Assessment of Angelica sinensis (Oliv.) Diels as a repellent for personal protection against mosquitoes under laboratory and field conditions in northern Thailand.

BACKGROUND: Angelica sinensis (Oliv.) hexane extract (AHE) has been reported as a proven and impressive repellent against laboratory-reared female Aedes aegypti mosquitoes. With the aim of promoting products of plant origin as a viable alternative to conventional synthetic substances, this study was designed to transform AHE-based repellents for exploitable commercial production by enhancing their efficacy and assessing their physical and biological stability as well as repellency against mosquitoes under laboratory and field conditions. METHODS: The chemical profile of AHE was analyzed by qualitative gas chromatography-mass spectrometry (GC-MS) technique. AHE was supplemented with vanillin, as a fixative, and then investigated for repellency and comparison to the standard synthetic repellent, DEET, under both laboratory and field conditions. Determination of physical and biological stability as a repellent was carried out after keeping AHE samples under varying temperatures and for different storage times. RESULTS: GC-MS analysis revealed that AHE contained at least 21 phytochemical compounds, constituting 95.74 % of the total content, with the major constituent of 3-N-butylphthalide (66.67 %). Ethanolic formulations of AHE and DEET showed improvement of repellency in a dose-dependent manner when vanillin was added in laboratory assessment. While 5-25 % AHE alone provided median complete-protection times of 2.0-6.5 h against Ae. aegypti, these times were increased to 4.0-8.5 h with a combination of AHE and 5 % vanillin (AHEv). Protection times against Ae. aegypti were extended from 2.25 to 7.25 h to 4.25-8.25 h when 5-25 % DEET was combined with 5 % vanillin (DEETv). In determining stability, all stored AHE samples exhibited similar characteristics such as liquid phases with aromatic odor comparable to those of fresh preparations. Furthermore, repellent activity of stored AHE samples lasted for at least six months, with varied efficacy (4.5-10.0 h) against Ae. aegypti. Field trials revealed strong repellency from both 25 % AHEv and 25 % DEETv, with complete protection (100 %) against a wide range of local mosquito populations. A total of 5,718 adult female mosquitoes, with the most predominant being Culex quinquefasciatus (41.47 %), Armigeres subalbatus (41.13 %), and Culex vishnui (10.53 %), was collected during field applications. No local skin reaction or other allergic responses was observed during both laboratory and field study periods. CONCLUSIONS: Angelica sinensis proved to have not only impressive repellency against both laboratory Ae. aegypti and a wide range of natural mosquito populations, but also relative stability in physical and biological performance.

Remarkable repellency of Ligusticum sinense (Umbelliferae), a herbal alternative against laboratory populations of Anopheles minimus and Aedes aegypti (Diptera: Culicidae).

BACKGROUND: For personal protection against mosquito bites, user-friendly natural repellents, particularly from plant origin, are considered as a potential alternative to applications currently based on synthetics such as DEET, the standard chemical repellent. This study was carried out in Thailand to evaluate the repellency of Ligusticum sinense hexane extract (LHE) against laboratory Anopheles minimus and Aedes aegypti, the primary vectors of malaria and dengue fever, respectively. METHODS: Repellent testing of 25% LHE against the two target mosquitoes; An. minimus and Ae. aegypti, was performed and compared to the standard repellent, DEET, with the assistance of six human volunteers of either sex under laboratory conditions. The physical and biological stability of LHE also was determined after keeping it in conditions that varied in temperature and storage time. Finally, LHE was analysed chemically using the qualitative GC/MS technique in order to demonstrate a profile of chemical constituents. RESULTS: Ethanol preparations of LHE, with and without 5% vanillin, demonstrated a remarkably effective performance when compared to DEET in repelling both An. minimus and Ae. aegypti. While 25% LHE alone provided median complete-protection times against An. minimus and Ae. aegypti of 11.5 (9.0-14.0) hours and 6.5 (5.5-9.5) hours, respectively, the addition of 5% vanillin increased those times to 12.5 (9.0-16.0) hours and 11.0 (7.0-13.5) hours, respectively. Correspondingly, vanillin added to 25% DEET also extended the protection times from 11.5 (10.5-15.0) hours to 14.25 (11.0-18.0) hours and 8.0 (5.0-9.5) hours to 8.75 (7.5-11.0) hours against An. minimus and Ae. aegypti, respectively. No local skin reaction such as rash, swelling or irritation was observed during the study period. Although LHE samples kept at ambient temperature (21-35°C), and 45°C for 1, 2 and 3 months, demonstrated similar physical characteristics, such as similar viscosity and a pleasant odour, to those that were fresh and stored at 4°C, their colour changed from light- to dark-brown. Interestingly, repellency against Ae. aegypti of stored LHE was presented for a period of at least 3 months, with insignificantly varied efficacy. Chemical analysis revealed that the main components of LHE were 3-N-butylphthalide (31.46%), 2, 5-dimethylpyridine (21.94%) and linoleic acid (16.41%), constituting 69.81% of all the extract composition. CONCLUSIONS: LHE with proven repellent efficacy, no side effects on the skin, and a rather stable state when kept in varied conditions is considered to be a potential candidate for developing a new natural alternative to DEET, or an additional weapon for integrated vector control when used together with other chemicals/measures.

Proteomic analysis of salivary glands of female Anopheles barbirostris species A2 (Diptera: Culicidae) by two-dimensional gel electrophoresis and mass spectrometry.

Salivary gland proteins of adult female Anopheles barbirostris species A2, a potential vector of Plasmodium vivax in Thailand, were analyzed using a proteomic approach (two-dimensional gel electrophoresis followed by nanoLC-MS). Two-dimensional gel electrophoresis revealed approximately 75 well-resolved spots on the reference gel. Most of the protein spots displayed relative molecular masses from 14 to 85 kDa and isoelectric points ranging from 3.9 to 10. The proteome profiles of A. barbirostris species A2 female salivary glands were affected by aging. The typical electrophoretic pattern of the female salivary glands was reached in 48 h post emergence, suggesting the maturation of salivary glands and saliva contents for blood feeding. Proteins involved in blood feeding, i.e., putative 5' nucleotidase/apyrase, anti-platelet protein, long form D7 salivary protein, D7-related 1 protein, and gSG6 salivary protein, start to accumulate from emergence and gradually increase becoming predominant within 48 h. There are different salivary components expressed within each region of the female glands. The blood-feeding proteins were detected in the distal-lateral lobes and/or medial lobes. Proteins detected and/or identified by this approach could be tested in strategies developed to control pathogen and disease transmission. Moreover, the information of a 2D map of the female salivary gland could be used for comparison with other related species in the A. barbirostris complex to distinguish species members in the complex.

Susceptibility of Anopheles campestris-like and Anopheles barbirostris species complexes to Plasmodium falciparum and Plasmodium vivax in Thailand.

Nine colonies of five sibling species members of Anopheles barbirostris complexes were experimentally infected with Plasmodium falciparum and Plasmodium vivax. They were then dissected eight and 14 days after feeding for oocyst and sporozoite rates, respectively, and compared with Anopheles cracens. The results revealed that Anopheles campestris-like Forms E (Chiang Mai) and F (Udon Thani) as well as An. barbirostris species A3 and A4 were non-potential vectors for P. falciparum because 0% oocyst rates were obtained, in comparison to the 86.67-100% oocyst rates recovered from An. cracens. Likewise, An. campestris-like Forms E (Sa Kaeo) and F (Ayuttaya), as well as An. barbirostris species A4, were non-potential vectors for P. vivax because 0% sporozoite rates were obtained, in comparison to the 85.71-92.31% sporozoite rates recovered from An. cracens. An. barbirostris species A1, A2 and A3 were low potential vectors for P. vivax because 9.09%, 6.67% and 11.76% sporozoite rates were obtained, respectively, in comparison to the 85.71-92.31% sporozoite rates recovered from An. cracens. An. campestris-like Forms B and E (Chiang Mai) were high-potential vectors for P. vivax because 66.67% and 64.29% sporozoite rates were obtained, respectively, in comparison to 90% sporozoite rates recovered from An. cracens.

Susceptibility of Anopheles campestris-like and Anopheles barbirostris species complexes to Plasmodium falciparum and Plasmodium vivax in Thailand

Nine colonies of five sibling species members of Anopheles barbirostris complexes were experimentally infected with Plasmodium falciparum and Plasmodium vivax. They were then dissected eight and 14 days after feeding for oocyst and sporozoite rates, respectively, and compared with Anopheles cracens. The results revealed that Anopheles campestris-like Forms E (Chiang Mai) and F (Udon Thani) as well as An. barbirostris species A3 and A4 were non-potential vectors for P. falciparum because 0 percent oocyst rates were obtained, in comparison to the 86.67-100 percent oocyst rates recovered from An. cracens. Likewise, An. campestris-like Forms E (Sa Kaeo) and F (Ayuttaya), as well as An. barbirostris species A4, were non-potential vectors for P. vivax because 0 percent sporozoite rates were obtained, in comparison to the 85.71-92.31 percent sporozoite rates recovered from An. cracens. An. barbirostris species A1, A2 and A3 were low potential vectors for P. vivax because 9.09 percent, 6.67 percent and 11.76 percent sporozoite rates were obtained, respectively, in comparison to the 85.71-92.31 percent sporozoite rates recovered from An. cracens. An. campestris-like Forms B and E (Chiang Mai) were high-potential vectors for P. vivax because 66.67 percent and 64.29 percent sporozoite rates were obtained, respectively, in comparison to 90 percent sporozoite rates recovered from An. cracens.