Atchariya Jitpakdi

Larvicidal effect of pepper plants on Aedes aegypti (L.) (Diptera: Culicidae)

ABSTRACT Ethanolic extracts derived from three species of the Piperaceae (pepper) family, Piper longum L., P. ribesoides Wall., and P. sarmentosum Roxb. ex Hunt., were evaluated for efficacy against early 4th instar larvae of Aedes aegypti mosquitoes using larvicidal bioassays. The highest larvicidal efficacy was established from P. longum, followed by P. sarmentosum and P. ribesoides, with LC50 values of 2.23, 4.06, and 8.13 ppm, respectively. Observations of morphological alterations on treated 4th instar larvae revealed that most organs, except anal papillae, had a normal structural appearance that was similar to controls. Under light microscopy, the internal structures of anal papillae in the treated larvae showed shrinkage, while the external features were normal in appearance. Ultrastructural studies, however, clearly demonstrated external destruction, with extensive damage and shrunken cuticle of the anal papillae. The structural deformation of anal papillae probably led to their dysfunction, which may be intrinsically associated with the death of the larvae. This study affords some evidence regarding the action site of the pepper extracts and suggests their potential in developing new types of larvicides used for mosquito control.

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.

Adulticidal activity against Stegomyia aegypti (Diptera: Culicidae) of three Piper spp.

Three Piper species, Piper longum, P. ribesoides and P. sarmentosum, were selected for investigation of adulticidal potential against Stegomyia aegypti, a main vector of dengue and dengue haemorrhagic fever. Successive extraction by maceration with 95% ethanol showed percentage yields of ethanolic extracts, which derived from P. longum, P. ribesoides and P. sarmentosum, of 8.89, 3.21 and 5.30% (w/w), respectively. All Piper extracts illustrated an impressive adulticidal activity when tested against female mosquitoes by topical application. The susceptibility of St. aegypti females to ethanol-extracted Piper was dose dependent and varied among the plant species. The highest adulticidal effect was established from P. sarmentosum, followed by P. ribesoides and P. longum, with LD50 values of 0.14, 0.15 and 0.26 microg/female, respectively. The potential of these Piper species, as possible mosquitocides, established convincing activity for further researches to develop natural substances for combat against adult mosquitoes.

Repellent properties of celery, Apium graveolens L., compared with commercial repellents, against mosquitoes under laboratory and field conditions

In our search for new bioactive products against mosquito vectors, we reported the slightly larvicidal and adulticidal potency, but remarkable repellency of Apium graveolens both in laboratory and field conditions. Repellency of the ethanolic preparation of hexane‐extracted A. graveolens was, therefore, investigated and compared with those of 15 commercial mosquito repellents including the most widely used, DEET. Hexane‐extracted A. graveolens showed a significant degree of repellency in a dose‐dependent manner with vanillin added. Ethanolic A. graveolens formulations (10–25% with and without vanillin) provided 2–5 h protection against female Aedes aegypti. Repellency that derived from the most effective repellent, 25% of hexane‐extracted A. graveolens with the addition of 5% vanillin, was comparable to the value obtained from 25% of DEET with 5% vanillin added. Moreover, commercial repellents, except formulations of DEET, showed lower repellency than that of A. graveolens extract. When applied on human skin under field conditions, the hexane‐extracted A. graveolens plus 5% vanillin showed a strong repellent action against a wide range of mosquito species belonging to various genera. It had a protective effect against Aedes gardnerii, Aedes lineatopennis, Anopheles barbirostris, Armigeres subalbatus, Culex tritaeniorhynchus, Culex gelidus, Culex vishnui group and Mansonia uniformis. The hexane‐extracted A. graveolens did not cause a burning sensation or dermal irritation when applied to human skin. No adverse effects were observed on the skin or other parts of the human volunteers’ body during 6 months of the study period or in the following 3 months, after which time observations ceased. Therefore, A. graveolens can be a potential candidate for use in the development of commercial repellents that may be an alternative to conventional synthetic chemicals, particularly in community vector control applications.

Mosquito repellency of the seeds of celery (Apium graveolens L.).

Abstract When the mosquito repellencies of four fractions of Apium graveolens seeds (one hexane, two dichloromethane and one methanolic) were investigated in the laboratory, all four were found to offer human volunteers some protection against female, adult Aedes aegypti. The hexane fraction, however, was found to exhibit the highest repellency in the laboratory, with median effective doses (ED50) and ED95 of 0.41 and 2.93 mg/cm2 skin, respectively. Only this fraction, which was also found to provide protection against mosquito bites for 3.5 h when applied, in the laboratory, at a concentration of 250 mg/ml, was then investigated for its repellency in the field and its stability. In storage, it was found to retain its repellency for at least 2 months, although significant reductions in its repellency were observed (in terms of shortened complete-protection times) after 3 months, whatever the temperature of storage (-20°C, 4°C, or room temperature). When applied to the skin of volunteers under field conditions, the hexane fraction showed strong repellent activity against a wide range of mosquito species belonging to various genera (Ae. gardnerii, Ae. lineatopennis, Armigeres subalbatus, Culex tritaeniorhynchus, Cx. vishnui group, Cx. quinquefasciatus and Mansonia uniformis). It appeared not to cause dermal irritation or any other adverse effect, either during 6 months of use or in the following 3 months of follow-up. Mosquito repellents based on extracts of Ap. graveolens seeds could be developed commercially, as an effective personal-protection measure against mosquito bites and the diseases caused by mosquito-borne pathogens.

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.

Karyotypic variation and geographic distribution of Anopheles campestris-like (Diptera: Culicidae) in Thailand

Seventy-one isolines of Anopheles campestris-like were established from wild-caught females collected from human-biting and animal-biting traps at 12 locations in Thailand. All isolines had an average branch summation of seta 2-VI pupal skins ranging from 20.3-30.0 branches, which is in the range of An. campestris (17-58 branches). They showed three different karyotypes based on the amount of extra heterochromatin in the sex chromosomes, namely Forms B (X2, Y2), E (X1, X2, X3, Y5) and a new karyotypic Form F (X2, X3, Y6). Form B has been found only in Chaing Mai and Kamphaeng Phet populations, while Forms E and F are widely distributed throughout the species range. Genetic crosses between the 12 isolines, which were arbitrarily selected as representatives of An. campestris-like Forms B, E and F, revealed genetic compatibility that provided viable progeny through F2 generations, suggesting a conspecific nature of these karyotypic forms. These results are supported by the very low intraspecies variation (genetic distance < 0.005) of ITS2, COI and COII from genomic DNA of the three karyotypic forms.

A Glycine- and Glutamate-Rich Protein Is Female Salivary Gland-Specific and Abundant in the Malaria Vector Anopheles dirus B (Diptera: Culicidae)

Abstract Before transmission, malaria parasites reside in the salivary glands of their female mosquito hosts. Saliva proteins assist in blood feeding and also may influence the ability of mosquitoes to transmit malaria. We attempted to identify and isolate cDNAs encoding proteins expressed at a high level in the salivary glands of a malaria vector, Anopheles dirus B Peyton & Harrison (=An. cracens) (Diptera: Culicidae). A major protein with an estimated molecular mass of 35 kDa and an isoelectric point (pI) of ≈4 was detected on a two-dimensional (2D) gel. Internal peptide sequences of the protein spot showed high similarity to sequences present in the conserved C-terminal domain of glycine- and glutamate (GE)-rich proteins. A full-length cDNA encoding this protein was isolated from a salivary gland cDNA library of female An. dirus B. The cDNA encoded a 256-residue protein with a calculated molecular mass of 25.4 kDa and a pI of 3.9. BLAST analysis confirmed that it is a member of the GE-rich family. Compositional and sequence analysis of this and other family members revealed a highly acidic N-terminal region of variable length and low sequence conservation and a well conserved C-terminal domain containing 10 identical residues across the 13 known members of the gene family in mosquitoes. The An. dirus B GE-rich transcript was detected by reverse transcription-polymerase chain reaction (PCR) only in the female salivary glands, indicating that this protein is female saliva-specific. The GE-rich proteins may function as a salivary lubricant to facilitate blood feeding.

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.