Seventeen isolines of Anopheles barbirostris derived from animal-biting female mosquitoes showed three karyotypic forms: Form A (X2, Y1) in five isolines from Phetchaburi province; Form B (X1, X3, Y2) in three and eight isolines from Chiang Mai and Ubon Ratchathani provinces, respectively; Form C (X2, Y3) in one isoline from Phetchaburi province. All 17 isolines exhibited an average branch summation of seta 2-VI pupal skins ranging from 12.1-13.0 branches, which was in the limit of A. barbirostris (6-18 branches). Of the 12 human-biting isolines from Chiang Mai province, five isolines showed Form B (X2, Y2), and seven isolines exhibited a new karyotypic form designated as Form E (X2, Y5). All of 12 isolines had an average branch summation of seta 2-VI pupal skins ranging from 22.4-24.5 branches, which was in the limit of Anopheles campestris (17-58 branches). Thus, they were tentatively designated as A. campestris-like Forms B and E. Hybridization between A. campestris-like Forms B and E showed that they were genetically compatible, yielding viable progeny for several generations suggesting conspecific relationships of these two karyotypic forms. Reproductive isolation among crosses between A. campestris-like Form B and A. barbirostris Forms A, B, and C strongly suggested the existence of these two species. In addition, the very low intraspecific variation (genetic distance<0.005) of the nucleotide sequence of ITS2 of the rDNA and COI and COII of mitochondrial DNA of the seven isolines of A. campestris-like Forms B and E supported their conspecific relationship. The large sequence divergence of ITS2 (0.203-0.268), COI (0.026-0.032), and COII (0.030-0.038) from genomic DNA of A. campestris-like Forms B and E and the A. barbirostris Forms A, B, and C clearly supported cytogenetic and morphological evidence.

Nine isoline colonies of Anopheles barbirostris Form A, derived from individual isofemale lines from Chiang Mai, Phetchaburi, and Kanchanaburi, were established in our insectary at Chiang Mai University. All isolines shared the same mitotic karyotype (X(1), X(2), Y(1)). Molecular analysis of deoxyribonucleic acid (DNA) sequences and polymerase chain reaction (PCR) products of ITS2, COI, and COII regions revealed three distinct groups: A1 (Chiang Mai), A2 (Phetchaburi), and A3 (Kanchanaburi). Crossing experiments among the three groups exhibited strong reproductive isolation, producing low and/or non-hatched eggs, and inviable and/or abnormal development of the reproductive system of F(1)-progenies. Asynaptic regions along the five polytene chromosome arms of F(1)-hybrid larvae clearly supported the existence of three sibling species within A. barbirostris Form A, provisionally named species A1, A2, and A3.

The chemical compositions and larvicidal potential against mosquito vectors of selected essential oils obtained from five edible plants were investigated in this study. Using a GC/MS, 24, 17, 20, 21, and 12 compounds were determined from essential oils of Citrus hystrix, Citrus reticulata, Zingiber zerumbet, Kaempferia galanga, and Syzygium aromaticum, respectively. The principal constituents found in peel oil of C. hystrix were β-pinene (22.54%) and d-limonene (22.03%), followed by terpinene-4-ol (17.37%). Compounds in C. reticulata peel oil consisted mostly of d-limonene (62.39%) and γ-terpinene (14.06%). The oils obtained from Z. zerumbet rhizome had α-humulene (31.93%) and zerumbone (31.67%) as major components. The most abundant compounds in K. galanga rhizome oil were 2-propeonic acid (35.54%), pentadecane (26.08%), and ethyl-p-methoxycinnamate (25.96%). The main component of S. aromaticum bud oil was eugenol (77.37%), with minor amounts of trans-caryophyllene (13.66%). Assessment of larvicidal efficacy demonstrated that all essential oils were toxic against both pyrethroid-susceptible and resistant Ae. aegypti laboratory strains at LC 50 , LC 95 , and LC 99 levels.In conclusion, we have documented the promising larvicidal potential of essential oils from edible herbs, which could be considered as a potentially alternative source for developing novel larvicides to be used in controlling vectors of mosquitoborne disease. Journal of Vector Ecology 35 (1): 106-115. 2010.

Celery-based products were investigated for chemical composition, skin irritation, and mosquito repellency in comparison to commercial repellents and the standard chemical, N,N-diethyl-3-methylbenzamide (DEET), with a goal to develop a natural alternative to synthetic repellents for protection against mosquitoes. Chemical identification by gas chromatography coupled with mass spectrometry discovered that the major constituents of Apium graveolens hexane extract (AHE) were 3-n-butyl-tetrahydrophthalide (92.48%), followed by 5.10% beta-selinene and 0.68% gamma-selinene. Evaluation of skin irritation in 27 human volunteers revealed no irritant potential from 25% ethanolic AHE solution. Laboratory investigated repellent against female Aedes aegypti mosquitoes demonstrated that G10 formula, the best AHE-developed product, provided remarkable repellency with a median protection time of 4.5 h (4.5-5 h), which was greater than that of ethanolic DEET solution (25% DEET, 3.5 h) and comparable to that of the best commercial repellent, Insect Block 28 (28.5% DEET, 4.5 h). According to significantly promising results, including highly effective repellency and no potential skin irritation or other side effects, the G10 formula is a worthwhile product that has the promise of being developed for commercialized registration. This developed AHE product could be an acceptable and affordable alternative to conventional synthetic chemicals in preventing mosquito bites, and in turn, helping to interrupt mosquito-borne disease transmission.

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The increasing and widespread resistance to conventional synthetic insecticides in vector populations has underscored the urgent need to establish alternatives in the mosquito management system. This study was carried out with the aim to investigate the antimosquito property, larvicidal and adulticidal potential, of plant products against both the pyrethroid-susceptible and resistant strains of Aedes aegypti. Seventeen plant products, including essential oils and ethanolic extracts, were obtained by steam distillation and extraction with 95 % ethanol, respectively. Their larvicidal activity was screened, using World Health Organization (WHO) procedures against A. aegypti, Muang Chiang Mai-susceptible (MCM-S) strain. The most effective product was a candidate for investigating larvicidal and adulticidal potential against three laboratory strains of A. aegypti, comprising MCM-S, Pang Mai Dang-resistant (PMD-R), and Upakut-resistant (UPK-R). Potential toxicity of the plant candidate was compared with that of synthetic temephos, permethrin, and deltamethrin. Chemical constituents of the most effective plant product also were analyzed by gas chromatography-mass spectrometry (GC-MS). Results obtained from the preliminary screening revealed the varying larvicidal efficacy of plant-derived products against MCM-S A. aegypti, with mortality ranging from 0 to 100 %. The larvicidal activity of seven effective plant products was found to be dose dependent, with the highest efficacy established from Petroselinum crispum fruit oil, followed by oils of Foeniculum vulgare, Myristica fragrans, Limnophila aromatica, Piper sarmentosum, Curcuma longa, and M. fragrans ethanolic extract (LC values of 43.22, 44.84, 47.42, 47.94, 49.19, 65.51, and 75.45 ppm, respectively). Essential oil of P. crispum was then investigated further and proved to be a promising larvicide and adulticide against all strains of A. aegypti. The pyrethroid-resistant strains of both PMD-R and UPK-R A. aegypti showed significant resistance to temephos, permethrin, and deltamethrin in either the larval or adult stage. Interestingly, high susceptibility to P. crispum oil was observed in the larvae and adults of MCM-S, which are pyrethroid-susceptible A. aegypti, and comparable to those of the pyrethroid-resistant strains, PMD-R and UPK-R. GC-MS analysis of P. crispum oil demonstrated that 19 compounds, accounting for 98.25 % of the whole oil, were identified, with the main constituents being thymol (42.41 %), p-cymene (27.71 %), and γ-terpinene (20.98 %). In conclusion, the profound larvicidal and adulticidal potential of P. crispum oil promises to form a new larvicide and adulticide against either the pyrethroid-susceptible or resistant strain of A. aegypti. Consequently, P. crispum oil and its constituents can be used or incorporated with other chemicals/measures in integrated mosquito management for controlling A. aegypti, particularly in localities with high levels of pyrethroid and organophosphate resistance.

BackgroundIn 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.MethodsEOs 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.ResultsChemical 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%), β-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.ConclusionsThe 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.

BackgroundFor 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 Ligusticumsinense hexane extract (LHE) against laboratory Anopheles minimus and Aedes aegypti, the primary vectors of malaria and dengue fever, respectively.MethodsRepellent 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.ResultsEthanol 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.ConclusionsLHE 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.