Larvicidal efficacy of crude and fractionated extracts of Dracaena loureiri Gagnep against Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, and Anopheles minimus mosquito vectors

Objective: To evaluate the larvicidal efficacy of crude and fractionated extracts of Dracaena loureiri endocarp against Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, and Anopheles minimus mosquitos. Methods: Larvicidal activity was tested according to World Health Organization standard protocol.The third-stage larvae of each mosquito species were exposed to various concentrations of Dracaena loureiri crude extract and six groups of Dracaena loureiri fractionated extracts (RC-DT 009–014). Larval mortality rates were observed after 24 h and 48 h of exposure.Then, a computerized probit analysis of the mortality data was performed to determine lethal concentration 50 (LC50) and lethal concentration 90 values. Results: Anopheles minimus larvae (24-h LC5077.88 mg/L) had the highest susceptibility to crude extract, whereas others (Aedes aegypti, 24-h LC50224.73 mg/L; Aedes albopictus, 24-h LC50261.75 mg/L; and Culex quinquefasciatus, 24-h LC50282.86 mg/L) were significantly less susceptible. The most effective groups of fractionated extracts were RC-DT 012 and RC-DT 013. The mosquito species most susceptible to fractionated extracts was Culex quinquefasciatus, with 24-h LC50 values of 0.66 and 0.94 mg/L for RC-DT 012 and RC-DT 013, respectively. Conclusions: The larvicidal activity of fractionated extracts is more effective than that of crude extract against all tested mosquito species. For the most effective alternative larvicide, purification and a phytochemical constituent analysis must be performed.

LARVICIDAL AND PUPICIDAL ACTIVITIES OF CRUDE AND FRACTIONATED EXTRACTS OF ACACIA PENNATA (L.) WILLD. SUBSP INSUAVIS SHOOT TIPS AGAINST AEDES AEGYPTI (L.) (DIPTERA: CULICIDAE).

Acacia pennata subsp insuavis, or Cha-om in Thai, is a common vegetable found in Thailand. It has been used as a medicinal herb for a long time. From the literature, antinociceptive, anti-inflammatory, antimicrobial, and anti-helminthic activities were reported. In this study, we investigated two new actions of this plant: larvicide and pupicide. The crude ethanolic and fractionated extracts of A. pennata shoot tips were tested against aquatic stages of the dengue virus vector, Aedes aegypti mosquito. The 1st-4th instar larvae and pupae of Ae. aegypti were subjected for bioassays by following the standard protocol of WHO. The larval and pupal mortalities were observed after 24- and 48-hour exposure times. The bioassays demonstrated that stronger efficacy was found from the fractionated extracts than the crude extracts. The LC50 values against the 3rd instar larvae were 39.45-50.75 mg/l (fractionated extracts) and 244.50 mg/l (crude extracts). It also effects the pupae with the LC50 values of 44.10-53.73 mg/l and 87.27 mg/l for the fractionnated and the crude extracts, respectively. The bioassays demonstrated the effective mosquito larvicide and pupicide of A. pennata extracts. It could be an alternative candidate for the development of phytotoxin for controlling mosquito vectors.

Prevalence and virulence factors of Candida spp.associated with blow flies

Objective:To investigate the prevalence of Candida spp.and the virulence factors of Candida albicans (C.albicans) isolated from external surfaces of blow flies collected from Mae Sot,Tak Province,Thailand,Methods:The blow flies were collected by sterile sweep nets from three areas in Mae Sot.Yeast isolation was first performed on Sabouraud dextrose agar (SDA) supplemented with chloramphenicol and cycloheximide.The yeast isolates were then identified by using chromogenic agar,a yeast identification test kit,a germ tube formation test and a carbohydrate utilization test.The β-hemolysis was determined on 7％ sheep blood agar,while phospholipase activity was measured on SDA agar supplemented with 10％ egg yolk suspension.Antifungal susceptibility testing was determined by broth micro-dilution testing against ketoconazole and amphotericin B.Results:The prevalence rate of Candida spp.on the external surfaces of the blow flies was 78.1％.All C.albicans isolated from the blow fly demonstrated β-hemolysin and potent phospholipase activities and 47.1％ of C.albicans were resistant to ketoconazole with MIC values 128 μg/mL.Conclusions:The result s indicate that blow flies could play an essential role in the transmission of potentially pathogenic and antifungal resistant C.albicans into the environment.Further investigation on other virulence factors and genetic relatedness among isolates from the blow flies,the environment and clinical specimens is required to confirm this role.

Larvicidal activity of endocarp and seed crude extracts of Dracaena loureiri Gagnep against Aedes aegypti (L.) mosquito

Objective: To evaluate the larvicidal activity of the ethanolic and aqueous extracts of the endocarp and seeds of Dracaena loureiri (D. loureiri) against the dengue mosquito vector, Aedes aegypti. Methods: Bioassays were performed by exposing late third-stage to early fourth-stage larvae of Aedes aegypti to various concentrations of the extracts from D. loureiri. The larval mortality was observed after 24-and 48-h exposure. Results: The larvicidal bioassay in this study demonstrated that the ethanolic endocarp extract was the most effective with the LC50 value of 84.00 mg/L after 24 h exposure and< 50 mg/L after 48 h exposure. Extracts from the other parts of the plant were signifi-cantly less effective as a larvicide. Conclusions: The ethanolic endocarp extract of D. loureiri demonstrated effective lar-vicidal activity. It is an alternative source for developing a novel larvicide for controlling this mosquito species.

Susceptibility to temephos, permethrin and deltamethrin of Aedes aegypti (Diptera: Culicidae) from Muang district, Phitsanulok Province, Thailand.

OBJECTIVE: To investigate the susceptibility to temephos, permethrin and deltamethrin of Aedes aegypti (Ae. aegypti), collected from areas with high incidence of dengue hemorrhagic fever cases in Phitsanulok Province, Thailand. METHODS: The F1 progenies of Ae. aegypti colony, originated from five sub-districts including Aranyik, Hua Ro, Nai Muang, Ban Krang and Tha Pho, were used in the bioassays following the procedures of World Health Organization. For larval bioassay, the late third or early fourth-instar larvae were tested with different concentrations of temephos. For adult bioassay, the females were exposed to 0.75 % permethrin or 0.05% deltamethrin. LC50 value and mortality rate were analyzed to compare the insecticide susceptibility of the larvae and the adults in each area, respectively. RESULTS: The LC50 value of temephos for the larvae from Aranyik, Hua Ro, Nai Muang, Ban Krang and Tha Pho sub-districts was 0.017, 0.017, 0.026, 0.061, and 0.113 ppm, respectively. For permethrin, the highest mortality rate (86.84%) was found in the mosquitoes from Aranyik but the others were more resistant with the lower mortality rates (16.00-42.67%). The adult mortality rates after exposing to deltamethrin were higher (82.34-98.67%) in all areas. CONCLUSIONS: Ae. aegypti larvae were still susceptible to temephos. Conversely, most tested adults tended to resist the permethrin and deltamethrin.

SCREENING FOR MOSQUITO LARVICIDAL ACTIVITY OF THAI MUSHROOM EXTRACTS WITH SPECIAL REFERENCE TO STECCHERINUM SP AGAINST AEDES AEGYPTI (L.) (DIPTERA: CULICIDAE).

For over 50 years, biological control of mosquito larvae has depended mainly on plant extracts, fish, bacteria, protozoa, filamentous fungi, viruses or nematodes. In this study, we screened 143 mushroom samples from 44 confirmed species in Thailand for their mosquito larvicidal activity. One g% (w/v) aqueous extracts of dried powdered mushroom samples were tested against 3rd stage Aedes aegypti larvae. Four mushroom species, namely, Thaeogyroporus porentosus, Xylaria nigripes, Chlorophyllum sp and Steccherinum sp, and two unidentified species showed larvicidal mortality ranging from 10%-70% and 18%-90% for 24- and 48-hour exposure time, respectively. Steccherinum sp aqueous crude extract, after 48-hour exposure, did not show any larvicidal activity at 1,000 ppm, whereas that from ethanol, after 24-hour exposure, had 50% and 90% lethal concentration of 203 ppm and 412 ppm, respectively, with higher levels of mortality after 48- hour exposure. This is the first report of mosquito larvicidal properties of Thai mushroom extracts.

LARVICIDAL ACTIVITY OF PERESKIA BLEO (KUNTH) DC. (CACTACEAE) FRUIT ENDOCARP CRUDE AND FRACTIONATED EXTRACTS AGAINST AEDES AEGYPTI (L.) (DIPTERA: CULICIDAE).

The use of insecticides can cause adverse effects in vector control, a plant bio-insecticide is an advantageous substitute. Currently, the promising mosquito larvicidal activity from plant extracts has been reported worldwide, including Thailand. In this study, the endocarp of Pereskia bleo (Kunth) DC. fruit was extracted with distilled water and ethanol. Crudes and fractionated groups of the extracts were evaluated for their larvicidal efficacy against the 3rd instar larvae of Aedes aegypti. At 48 hours of exposure, it was found that the activities of the extracts were higher than 24-hour's. The ethanolic extracts showed stronger activities than the aqueous ones, indicating the lower LC50 values of both crude and fractionated group extracts. The most toxic activity was found in a fractionated group of the ethanolic extract, E-Gr3, with significantly lowest LC50 values of 707.94 and 223.12 ppm for 24- and 48-hour detection times, respectively. The bioassay results indicated the larvicidal property against the Ae. aegypti mosquito of the P. bleo plant extracts. A safety for non-target organisms or an action on other mosquito vectors of this plant, should be further investigated.

Comparative phylogeography reveals a shared impact of pleistocene environmental change in shaping genetic diversity within nine Anopheles mosquito species across the Indo-Burma biodiversity hotspot.

South-East Asia is one of the world's richest regions in terms of biodiversity. An understanding of the distribution of diversity and the factors shaping it is lacking, yet essential for identifying conservation priorities for the region's highly threatened biodiversity. Here, we take a large-scale comparative approach, combining data from nine forest-associated Anopheles mosquito species and using statistical phylogeographical methods to disentangle the effects of environmental history, species-specific ecology and random coalescent effects. Spatially explicit modelling of Pleistocene demographic history supports a common influence of environmental events in shaping the genetic diversity of all species examined, despite differences in species' mtDNA gene trees. Populations were periodically restricted to allopatric northeastern and northwestern refugia, most likely due to Pleistocene forest fragmentation. Subsequent southwards post-glacial recolonization is supported by a north-south gradient of decreasing genetic diversity. Repeated allopatric fragmentation and recolonization have led to the formation of deeply divergent geographical lineages within four species and a suture zone where these intraspecific lineages meet along the Thai-Myanmar border. A common environmental influence for this divergence was further indicated by strong support for simultaneous divergence within the same four species, dating to approximately 900 thousand years ago (kya). Differences in the geographical structuring of genetic diversity between species are probably the result of varying species' biology. The findings have important implications for conservation planning; if the refugial regions and suture zone identified here are shared by other forest taxa, the unique and high levels of genetic diversity they house will make these areas conservation priorities.

Crossing experiments supporting the specific status of Anopheles maculatus chromosomal form K.

There are 3 recognized chromosomal forms (B, E, K) in the taxon of Anopheles maculatus, 1 of the 8 members of the Anopheles maculatus group. Previous studies suggested that forms B and E are cytotypes of the species, but genetic characteristics of form K are unknown. The present study used crossing experiments, and polytene chromosomes of the ovarian nurse cell in F1 hybrids to show that form K is genetically distinct from forms B and E. In addition, postzygotic genetic incompatibility between form K and An. sawadwongporni, An. dravidicus, and An. pseudowillmori are demonstrated. In all crosses, hybrid males were sterile, with atrophied testes and accessory glands, or partially sterile with abnormal spermatozoa. The hybrid females showed varying degrees of atrophied ovaries. The ovarian nurse cell polytene chromosomes of the F1 hybrid females displayed approximately 70% to almost complete asynapsis. The results provide clear evidence that form K should no longer be regarded as a cytotype of An. maculatus. The present study supports previous suggestions that form K represents another species of the Maculatus group.

Crossing experiment of Anopheles maculatus form K and Anopheles willmori (James) (Diptera: Culicidae).

We recently reported crossing experiments between Anopheles maculatus form K and five members of the Maculatus group to support the specific status of form K. In the present study, we performed further crosses between form K and a sixth species of the Maculatus group, Anopheles willmori (James). Low viability was observed in hybrid males and females. All hybrid males were sterile with atrophied testes, or partially sterile with abnormal spermatozoa. The hybrid females showed normal ovaries. The ovarian nurse cell polytene chromosomes of the F1 hybrid females displayed complete asynapsis. Backcrossing showed low viability. All males were sterile with atrophied testes or partially sterile with abnormal spermatozoa, and the females showed varying degrees of atrophied ovaries. The results provide clear evidence that form K is not conspecific to A. willmori supporting previous studies that form K represents another species of the Maculatus group.

The specific status of Anopheles minimus s.l. collected from Taiwan.

At least three members (species A, C, and E) of the Anopheles minimus complex have been described in the Orient. This study investigated the specific status of An. minimus collected in the southern part of Taiwan by crossing experiments with species A from Thailand and species E from Japan. Crosses between Taiwan An. minimus and species A revealed genetic compatibilities. Post-zygotic isolation was observed in crosses between Taiwan An. minimus and species E. Hybrid progeny were only obtained from Taiwan female X species E male. F2 hybrid progeny were not obtained, since the hybrid males were sterile or almost sterile, with atrophied testes or abnormal spermatozoa. The hybrid females backcrossed with either Taiwan F1 progeny and species E males, and laid eggs with lower fertility and viability. This study supports previous published data regarding the analysis of the D3 region of the 28S gene of ribosomal DNA that An. minimus species A is indigenous to Taiwan. Whether other members of the An. minimus complex exist in Taiwan is not conclusive and needs more study.

Crossing experiments of Anopheles minimus species C and putative species E.

In the Anopheles minimus complex, 2 sibling species (A and C) are generally accepted. Recently, a 3rd species, provisionally designated An. minimus species E, has been described from the Ryukyu Archipelago, Japan, based on crossing experiments (A and E), DNA analysis, mitotic karyotypes, and some morphological characteristics. The present study reports the results of crossing experiments between species C and putative species E. Hybridization between the progeny of An. minimus species C from Thailand and putative species E from Japan revealed postzygotic genetic incompatibility. Although F1 hybrid progeny were obtained from both directions of crosses, the hybrid males from C female x E male crosses were completely sterile, with atrophied testes and accessory glands. In addition, the external terminalia of all of these males never completely rotated and the males failed to copulate by artificial mating. In E female x C male crosses, the hybrid males showed partially sterile testes in which most spermatozoa were abnormal (enlarged head) and inactive, and they had very little success in inseminating females. The salivary gland polytene chromosomes of F1 hybrid larvae from species C female x species E male showed a fixed heterozygous inversion on the 3L arm. Those F1 hybrids from species E female x species C male showed partial asynapsis on identified arms (2R and 3L) and a fixed heterozygous inversion on the 3R arm. When the F1 hybrid females from both directions of crosses were backcrossed with either C or E males, they produced male progeny with abnormal spermatozoa. Study of mating behavior in a 30 x 30 x 30-cm cage showed that the C males failed to mate with either C or E females, indicating that species C males cannot breed in confined spaces (lack stenogamy). Putative species E males had little success in inseminating species C females. This study provides strong evidence of genetic incompatibility between An. minimus species C and putative species E, supporting previous data that species E is a distinct species in the An. minimus complex.