Anuluck Junkum

Chemical Composition and Larvicidal Activity of Edible Plant-Derived Essential Oils Against the Pyrethroid-Susceptible and -Resistant Strains of Aedes aegypti (Diptera: Culicidae)

ABSTRACT: 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 &bgr;-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 y-terpinene (14.06%). The oils obtained from Z. zerumbet rhizome had &agr;-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 LC50, LC95, and LC99 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.

Molecular and cytogenetic evidence of three sibling species of the Anopheles barbirostris Form A (Diptera:Culicidae) in Thailand

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 (X1, X2, Y1). 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 F1-progenies. Asynaptic regions along the five polytene chromosome arms of F1-hybrid larvae clearly supported the existence of three sibling species within A. barbirostris Form A, provisionally named species A1, A2, and A3.

Cytogenetic and molecular evidence for two species in the Anopheles barbirostris complex (Diptera: Culicidae) in Thailand

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.

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.

Establishment of a Self-Mating Mosquito Colony of Anopheles sinensis From Korea

ABSTRACT Many Korean entomologists have attempted to colonize Anopheles sinensis Korean strain, a primary malaria vector in Korea, previous efforts, however, were unsuccessful. Only a study that a successful colonization of An. sinensis Korean strain over 5 generations was recently reported. Recently we obtained a self‐mating colony from this mosquito strain. The original colony was established from progenies obtained from females collected from So‐Rae District, Incheon, Republic of Korea (ROK), and was maintained for 23 successive generations. While the adult females of first five generations were fed on humans as a source of blood, those of the subsequent 18 generations were fed on white rats as a source of blood. A self‐mating colony was initiated from the 9th generation by rearing 200 and 300 newly emerged females and males, respectively, in a 30x30x30 cm cage. The insemination rates increased from approximately 40% to 81% by the 20th generation. Comparisons of biological aspects between artificial mating and self‐mating colonies are reported.

Chemical Constituents and Combined Larvicidal Effects of Selected Essential Oils against Anopheles cracens (Diptera: Culicidae)

A preliminary study on larvicidal activity against laboratory-colonized Anopheles cracens mosquitos revealed that five of ten plant oils at concentration of 100 ppm showed 95–100% larval mortality. The essential oils of five plants, including Piper sarmentosum, Foeniculum vulgare, Curcuma longa, Myristica fragrans, and Zanthoxylum piperitum, were then selected for chemical analysis, dose-response larvicidal experiments, and combination-based bioassays. Chemical compositions analyzed by gas chromatography coupled to mass spectrometry demonstrated that the main component in the oil derived from P. sarmentosum, F. vulgare, C. longa, M. fragrans, and Z. piperitum was croweacin (71.01%), anethole (63.00%), ar-turmerone (30.19%), safrole (46.60%), and 1,8-cineole (21.27%), respectively. For larvicidal bioassay, all five essential oils exerted promising efficacy in a dose-dependent manner and different performances on A. cracens after 24 hours of exposure. The strongest larvicidal potential was established from P. sarmentosum, followed by F. vulgare, C. longa, M. fragrans, and Z. piperitum, with LC50 values of 16.03, 32.77, 33.61, 40.00, and 63.17 ppm, respectively. Binary mixtures between P. sarmentosum, the most effective oil, and the others at the highest ratio were proved to be highly efficacious with a cotoxicity coefficient value greater than 100, indicating synergistic activity. Results of mixed formulations of different essential oils generating synergistic effects may prove helpful in developing effective, economical, and ecofriendly larvicides, as favorable alternatives for mosquito management.

Salivary gland proteins of the human malaria vector, Anopheles dirus B (Diptera: Culicidae)

Salivary gland proteins of the human malaria vector, Anopheles dirus B were determined and analyzed. The amount of salivary gland proteins in mosquitoes aged between 3--10 days was approximately 1.08 +/- 0.04 microg/female and 0.1 +/- 0.05 microg/male. The salivary glands of both sexes displayed the same morphological organization as that of other anopheline mosquitoes. In females, apyrase accumulated in the distal regions, whereas alpha-glucosidase was found in the proximal region of the lateral lobes. This differential distribution of the analyzed enzymes reflects specialization of different regions for sugar and blood feeding. SDS-PAGE analysis revealed that at least seven major proteins were found in the female salivary glands, of which each morphological region contained different major proteins. Similar electrophoretic protein profiles were detected comparing unfed and blood-fed mosquitoes, suggesting that there is no specific protein induced by blood. Two-dimensional polyacrylamide gel analysis showed the most abundant salivary gland protein, with a molecular mass of approximately 35 kilodaltons and an isoelectric point of approximately 4.0. These results provide basic information that would lead to further study on the role of salivary proteins of An. dirus B in disease transmission and hematophagy.

Comparative morphometry and morphology of Anopheles aconitus Form B and C eggs under scanning electron microscope

Comparative morphometric and morphological studies of eggs under scanning electron microscope (SEM) were undertaken in the three strains of two karyotypic forms of Anopheles aconitus, i.e., Form B (Chiang Mai and Phet Buri strains) and Form C (Chiang Mai and Mae Hong Son strains). Morphometric examination revealed the intraspecific variation with respect to the float width [36.77 +/- 2.30 microm (Form C: Chiang Mai strain) = 38.49 +/- 2.78 microm (Form B: Chiang Mai strain) = 39.06 +/- 2.37 microm (Form B: Phet Buri strain) > 32.40 +/- 3.52 microm (Form C: Mae Hong Son strain)] and number of posterior tubercles on deck [2.40 +/- 0.52 (Form B: Phet Buri strain) = 2.70 +/- 0.82 (Form B: Chiang Mai strain) < 3.10 +/- 0.32 (Form C: Chiang Mai strain) = 3.20 +/- 0.42 (Form C: Mae Hong Son strain)], whereas the surface topography of eggs among the three strains of two karyotypic forms were morphologically similar.

Field evaluation of G10, a celery ( Apium graveolens )-based topical repellent, against mosquitoes (Diptera: Culicidae) in Chiang Mai province, northern Thailand

The potential of G10, a celery (Apium graveolens)-based topical product, as a repellent against natural mosquito populations was evaluated in comparison to commercial (Insect Block 28) and standard (25% DEET) repellents in Chiang Mai province, northern Thailand. These repellent products afforded encouragingly excellent personal protection against a broad range of mosquito species belonging to various genera, including Aedes, Anopheles, Armigeres, Culex, and Mansonia. No mosquito bite was observed on the volunteers treated with G10 and Insect Block 28 throughout the field study, whereas two species, i.e., six A. barbirostris and two A. subalbatus, came to bite or land on 25% DEET-treated volunteers. Thus, it can be concluded that while G10 and Insect Block 28 exhibited similarly powerful repellent activities with complete (100%) protection, 25% DEET was effective in minimizing bites with 99.68% protection. G10 formula was also studied for physical properties and biological stability after being kept under two conditions; a heating and cooling cycle, and varying temperature and time storage. Most samples of stored G10 not only demonstrated a similarity in appearance and physical properties, but also provided comparable repellency to that of the fresh preparation. These findings encourage commercial development of G10 formula as an alternative to conventional synthetic repellents.

Evaluation of the colchicine-like activity of Gloriosa superba-extracted fractions for mosquito (Diptera: Culicidae) cytogenetic study

Abstract Four fractions of Gloriosa superba L., i.e., hexane fraction, dichloromethane fraction 1, dichloromethane fraction 2, and methanol fraction, were investigated for colchicine-like activity using a mosquito cytogenetic assay. The results revealed that the latter three fractions yielded promisingly high colchicine-like activity, whereas the hexane fraction yielded very low activity compared with 1% colchicine in a 0.85% sodium chloride solution. The metaphase rates and average number of metaphase chromosomes per positive brain ganglion (range) of Aedes aegypti L. larvae after incubation with 0.25–2% solutions of dichloromethane fraction 1, dichloromethane fraction 2, 0.5–2% solutions of methanol fraction, and 1% colchicine solution were 90–100% and 7 (2–19) to 22 (7–47); 90–100% and 4 (1–11) to 30 (4–73); 95–100% and 11 (1–28) to 17 (2–62); and 100% and 6 (2–11), respectively. The temperature stability tests of the three promising fractions were performed by heating 0.5% working solution at 121°C for 15 min and preparing 0.5% working solution from stock frozen at −20°C for 10 mo. These fractions also yielded satisfactory outcomes of metaphase rates and an average number of metaphase chromosomes per positive brain ganglia compared with 1% colchicine solution.