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.

Chemical composition and larvicidal activity of edible plant-derived essential oils against the pyrethroid-susceptible and -resistant strains of Aedes aegypti (Diptera: Culicidae).

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 beta-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 gamma-terpinene (14.06%). The oils obtained from Z. zerumbet rhizome had alpha-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 mosquito-borne disease.

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.

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.

Cytogenetic and molecular evidence for an additional new species within the taxon Anopheles barbirostris (Diptera: Culicidae) in Thailand.

ITS2 DNA sequences of 42 isoline colonies of Anopheles barbirostris species A1 and A2 were analyzed and a new genetic species, temporarily designated as species A4 (Chiang Mai), was revealed. The large sequence divergences of the ITS2 (0.116-0.615), COI (0.023-0.048), and COII (0.030-0.040) genes between A. barbirostris species A4/A1 (Chiang Mai), A4/A2 (Phetchaburi), A4/A3 (Kanchanaburi), and A4/Anopheles campestris-like Form E (Chiang Mai) provided good supporting evidence. Species A1, A2, A3, and A4 share a mitotic karyotype of Form A (X(1), X(2), Y(1)). Crossing experiments between species A4 and the other four species yielded strong reproductive isolation producing few and/or non-hatched eggs and inviable and/or abnormal development of the reproductive system of F(1) progenies. Moreover, available F(1) hybrid larvae showed asynaptic polytene chromosome arms. Hence, molecular and cytogenetic evidence strongly support the existence of A. barbirostris species A4, which is more closely related to A. campestris-like Form E than to species A1, A2, and A3. Additionally, crossing experiments among 12 and seven isolines of different cytological forms of species A1 (A, B, C, D) and A2 (A, B), respectively, yielded fertile and viable F(1) progenies. Thus, different karyotypic forms occurring in natural populations of species A1 and A2 merely represent intraspecies variation of sex chromosomes due to the extra blocks of heterochromatin.

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 (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.

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.

Larvicidal efficacy and biological stability of a botanical natural product, zedoary oil-impregnated sand granules, against Aedes aegypti (Diptera, Culicidae).

Chemical analysis on Curcuma zedoaria rhizome volatile oil, using gas chromatography-mass spectrometer techniques, demonstrated the presence of beta-tumerone (19.88%), 1,8-cineole (8.93%), and 7-zingiberene (7.84%) as major constituents. Larvicidal efficacy against Aedes aegypti mosquitoes of zedoary oil and its formulated preparation, zedoary oil-impregnated sand granules, were investigated and compared with that of Abate(R)sand (temephos). Zedoary oil exhibited pronounced potential against the fourth instar larvae of A. aegypti with an LC(50) and LC(99) of 33.45 and 83.39 ppm, respectively. Application of zedoary oil at a dosage yielding ten times that of LC(99) offered complete larval mortality (100% mortality) for a period of 3 days, and the larval mortality subsequently decreased to lower than 50% after application for more than 5 days. Zedoary oil-impregnated sand granules provided remarkably longer activity, with a larval mortality of 100% for a period of 9 days; and mortality below 50% was obtained in week 3 of application. The complete larval mortality that resulted from applying temephos at dosages of 0.1 and 1 ppm persisted for a period of 6 days and 4 weeks, respectively, and the larval mortality below 50% was reported on day 18 and week 11, respectively. Testing A. aegypti species against stored samples of zedoary oil-impregnated sand granules demonstrated that the product stored at 4 degrees C showed the longest larvicidal activity, followed by those kept at ambient temperature and 45 degrees C, yielding a complete larval mortality for 9, 8, and 6 days, respectively. Most samples of zedoary oil-impregnated sand granules stored at each temperature for 1 month showed slightly higher efficacy than those kept for 2 months. The larvicidal efficacy of samples stored at 4 degrees C seemed to be comparable to that of the fresh sample. The efficacy in killing A. aegypti larvae and good biological stability of zedoary oil-impregnated sand granules make this product promising as an alternative to essential oil in the development of new botanical natural larvicide for use in mosquito control programs.

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

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.

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.

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

Três espécies de Piper, Piper longum, P. ribesoides e P. sarmentosum, foram selecionadas para investigação da potencialidade contra Stegomyia aegypti adultos, principal vetor de dengue e febre do dengue hemorrágico. Sucessivas extrações por maceração com etanol a 95% mostraram uma porcentagem de extratos etanólicos, derivados de P. longum, P. ribesoides e P. sarmentosum, de 8,89, 3,21 e 5,30% (w/w), respectivamente. Todos os extratos de Piper mostraram atividade adulticida expressiva quando testados contra fêmeas de mosquitos através de aplicação tópica. A suscetibilidade das fêmeas do St. aegypt ao extrato de Piper etanólico foi dose dependente e variou entre as espécies de plantas. O mais elevado efeito adulticida foi demonstrado a partir do P. sarmentosum, seguido pelo P. ribesoides e P. longum, valores LD50 de 0,14, 0,15 e 0,26 µg/fêmea, respectivamente. O potencial destas espécies de Piper, como possíveis mosquiticidas, estabeleceu atividade convincente para futuras pesquisas a fim de desenvolver substâncias naturais para o combate a mosquitos adultos.

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.

Chemical composition and anti-mosquito potential of rhizome extract and volatile oil derived from Curcuma aromatica against Aedes aegypti (Diptera: Culicidae).

Crude rhizome extracts and volatile oils of Curcuma aromatica were evaluated for chemical composition and anti-mosquito potential, including larvicidal, adulticidal, and repellent activities against the Aedes aegypti mosquito. Chemical identification achieved by GC/MS analysis revealed that xanthorrhizol, 1H-3a, 7-methanoazulene and curcumene at 35.08 and 13.65%, 21.81 and 30.02%, and 13.75 and 25.71%, were the main constituents in hexane extracts and volatile oils, respectively. Volatile oil of Cu. aromatica possessed a significantly higher larvicidal activity against the 4th instar larvae of Ae. aegypti than that of hexane extracts, with LC50 values of 36.30 and 57.15 ppm, respectively. In testing for adulticidal activity, on the other hand, hexane-extracted Cu. aromatica (LC50: 1.60 microg/mg female) was found to be slightly more effective against female Ae. aegypti than volatile oil (LC50: 2.86 microg/mg female). However, the repellency of these two products against Ae. aegypti adult females differed significantly. The hexane-extracted Cu. aromatica, with a median complete protection time of 1 h (range = 1-1.5 h) when applied at a concentration of 25%, appeared to have significantly higher repellency than that of distillate oil (0.5 h, range = 0-0.5 h). The different results obtained from both products of Cu. aromatica were probably due to variety in quantity and type of active ingredients as well as the biological and physiological characteristics that differed between both developmental stages of mosquitoes, larvae, and adults.

Potential of crude seed extract of celery, Apium graveolens L., against the mosquito Aedes aegypti (L.) (Diptera: Culicidae).

Crude seed extract of celery, Apium graveolens, was investigated for anti-mosquito potential, including larvicidal, adulticidal, and repellent activities against Aedes aegypti, the vector of dengue haemorrhagic fever. The ethanol-extracted A. graveolens possessed larvicidal activity against fourth instar larvae of Ae. aegypti with LD50 and LD95 values of 81.0 and 176.8 mg/L, respectively. The abnormal movement observed in treated larvae indicated that the toxic effect of A. graveolens extract was probably on the nervous system. In testing for adulticidal activity, this plant extract exhibited a slightly adulticidal potency with LD50 and LD95 values of 6.6 and 66.4 mg/cm2, respectively. It showed repellency against Ae. aegypti adult females with ED50 and ED95 values of 2.03 and 28.12 mg/cm2, respectively. It also provided biting protection time of 3 h when applied at a concentration of 25 g%. Topical application of the ethanol-extracted A. graveolens did not induce dermal irritation. No adverse effects on the skin or other parts of the body of human volunteers were observed during 3 mo of the study period or in the following 3 mo, after which time observations ceased. A. graveolens, therefore, can be considered as a probable source of some biologically active compounds used in the development of mosquito control agents, particularly repellent products.

Repellency of aromatic turmeric Curcuma aromatica under laboratory and field conditions.

Three Curcuma species, Curcuma aeruginosa (pink and blue ginger), Cu. aromatica (aromatic turmeric), and Cu. xanthorrhiza (giant curcuma), were selected for investigation of mosquito repellent activity. In a laboratory study, a 95% ethanol extract of each plant was tested for repellent activity of Aedes togoi on human volunteers. Only Cu. aromatica extract showed repellency against Ae. togoi with ED50 and ED95 values of 0.061 and 1.55 mg/cm2, respectively. It also provided biting protection for 3.5 h when applied at a concentration of 25 g%. The ethanolic extract of Cu. aromatica was therefore chosen for further repellent activity under field conditions, where it had a protective effect against Armigeres subalbatus, Culex quinquefasciatus, and Cx. tritaeniorhynchus. The ethanol-extracted Cu. aromatica did not cause dermal irritation when applied to human skin. No adverse effects on human volunteers were observed 2 mo after application. Therefore, Cu. aromatica extract can be applied as an effective personal protection measure against mosquito bites.