Damrongpan Thongwat

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 of Anopheles minimus species C and putative species E

ABSTRACT 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 × 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 × 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 × species E male showed a fixed heterozygous inversion on the 3L arm. Those hybrids F1 from species E female × species C male showed partial asynapsis on identified arms (2R and 3L) and a fixed heterozygous inversion on the 3R arm. When the hybrid females from both directions of crosses were F1 backcrossed with either C or E males, they produced male progeny with abnormal spermatozoa. Study of mating behavior in a 30 × 30 × 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.

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

ABSTRACT 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 ∼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.

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.

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 LC50 77.88 mg/L) had the highest susceptibility to crude extract, whereas others (Aedes aegypti, 24-h LC50 224.73 mg/L; Aedes albopictus, 24-h LC50 261.75 mg/L; and Culex quinquefasciatus, 24-h LC50 282.86 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.