A genotyping array for the globally invasive vector mosquito, Aedes albopictus.

BACKGROUND: Although whole-genome sequencing (WGS) is the preferred genotyping method for most genomic analyses, limitations are often experienced when studying genomes characterized by a high percentage of repetitive elements, high linkage, and recombination deserts. The Asian tiger mosquito (Aedes albopictus), for example, has a genome comprising up to 72% repetitive elements, and therefore we set out to develop a single-nucleotide polymorphism (SNP) chip to be more cost-effective. Aedes albopictus is an invasive species originating from Southeast Asia that has recently spread around the world and is a vector for many human diseases. Developing an accessible genotyping platform is essential in advancing biological control methods and understanding the population dynamics of this pest species, with significant implications for public health. METHODS: We designed a SNP chip for Ae. albopictus (Aealbo chip) based on approximately 2.7 million SNPs identified using WGS data from 819 worldwide samples. We validated the chip using laboratory single-pair crosses, comparing technical replicates, and comparing genotypes of samples genotyped by WGS and the SNP chip. We then used the chip for a population genomic analysis of 237 samples from 28 sites in the native range to evaluate its usefulness in describing patterns of genomic variation and tracing the origins of invasions. RESULTS: Probes on the Aealbo chip targeted 175,396 SNPs in coding and non-coding regions across all three chromosomes, with a density of 102 SNPs per 1 Mb window, and at least one SNP in each of the 17,461 protein-coding genes. Overall, 70% of the probes captured the genetic variation. Segregation analysis found that 98% of the SNPs followed expectations of single-copy Mendelian genes. Comparisons with WGS indicated that sites with genotype disagreements were mostly heterozygotes at loci with WGS read depth < 20, while there was near complete agreement with WGS read depths > 20, indicating that the chip more accurately detects heterozygotes than low-coverage WGS. Sample sizes did not affect the accuracy of the SNP chip genotype calls. Ancestry analyses identified four to five genetic clusters in the native range with various levels of admixture. CONCLUSIONS: The Aealbo chip is highly accurate, is concordant with genotypes from WGS with high sequence coverage, and may be more accurate than low-coverage WGS.

Detection of viruses from feces of wild endangered Macaca maura: a potential threat to moor macaque survival and for zoonotic infection.

BACKGROUND: To date, there is a scarcity of information and literature on Macaca maura health status relative to viral diseases. The objectives of the present study were to investigate on the potential spread of enteric and non-enteric viruses shed in the environment through a wild macaque feces and to understand the possible interrelation in the spread of zoonotic viruses in a poorly studied geographical area, the Sulawesi Island. This study will also contribute providing useful information on potential threats to the health of this endangered species. METHODS: The sampling was conducted between 2014 and 2016 in the Bantimurung Bulusaraung National Park, in the south of the Sulawesi Island and non-invasive sampling methods were used to collect fresh stools of the M. maura, one of the seven macaque species endemic to the island of Sulawesi, Indonesia. The population under study consisted in two wild, neighboring social macaque groups with partially overlapping home ranges; twenty-four samples were collected and examined using negative staining electron microscopy and a panel of PCR protocols for the detection of ten RNA and two DNA viruses. RESULTS: Viral particles resembling parvovirus (5 samples), picornavirus (13 samples) and calicivirus (13 samples) were detected by electron microscopy whereas the PCR panel was negative for the 12 viruses investigated, except for one sample positive for a mosquito flavivirus. The results did not correlate with animal sex; furthermore, because all of the animals were clinically healthy, it was not possible to correlate feces consistency with viral presence. CONCLUSIONS: As information on viral infections in wild moor macaques remains limited, further studies are yet required to identify the fecal-oral and blood transmitted potentially zoonotic viruses, which may infect the moor macaque and other macaque species endemic to the South Sulawesi Island.

Characterization of vector communities and biting behavior in South Sulawesi with host decoy traps and human landing catches.

BACKGROUND: Indonesia has high mosquito diversity, with circulating malaria and arboviruses. Human landing catches (HLC) are ethically questionable where arboviral transmission occurs. The host decoy trap (HDT) is an exposure-free alternative outdoor sampling device. To determine HDT efficacy for local culicids, and to characterize local mosquito fauna, the trapping efficacy of the HDT was compared to that of HLCs in one peri-urban (Lakkang) and one rural (Pucak) village in Sulawesi, Indonesia. RESULTS: In Lakkang the outdoor HLCs collected significantly more Anopheles per night (n = 22 ± 9) than the HDT (n = 3 ± 1), while the HDT collected a significantly greater nightly average of Culex mosquitoes (n = 110 ± 42), than the outdoor HLC (n = 15.1 ± 6.0). In Pucak, there was no significant difference in Anopheles collected between trap types; however, the HDT collected significantly more Culex mosquitoes than the outdoor HLC nightly average (n = 53 ± 11 vs 14 ± 3). Significantly higher proportions of blood-fed mosquitoes were found in outdoor HLC (n = 15 ± 2%) compared to HDT (n = 2 ± 0%). More blood-fed culicines were collected with outdoor HLC compared to the HDT, while Anopheles blood-fed proportions did not differ. For the HDT, 52.6%, 36.8% and 10.5% of identified blood meals were on cow, human, and dog, respectively. Identified blood meals for outdoor HLCs were 91.9% human, 6.3% cow, and 0.9% each dog and cat. Mosquitoes from Pucak were tested for arboviruses, with one Culex pool and one Armigeres pool positive for flavivirus, and one Anopheles pool positive for alphavirus. CONCLUSIONS: The HDT collected the highest abundance of culicine specimens. Outdoor HLCs collected the highest abundance of Anopheles specimens. Although the HDT can attract a range of different Asian mosquito genera and species, it remains to be optimized for Anopheles in Asia. The high proportion of human blood meals in mosquitoes collected by outdoor HLCs raises concerns on the potential exposure risk to collectors using this methodology and highlights the importance of continuing to optimize a host-mimic trap such as the HDT.

Molecular analysis reveals a high diversity of Anopheles species in Karama, West Sulawesi, Indonesia.

BACKGROUND: Understanding local Anopheles species compositions and bionomic traits are vital for an effective malaria vector intervention strategy. Though eight malaria vectors, including species complexes, have been documented across the island of Sulawesi, Indonesia, a comprehensive survey linking morphological and molecular species identification has not been conducted in this global hotspot of biodiversity. RESULTS: Eighteen distinct species of Anopheles were molecularly identified in a 1 km2 area in Karama village, West Mamuju Province, Sulawesi. Known species included An. aconitus, An. karwari, An. peditaeniatus, An. vagus, An. barbirostris, An. tessellatus, An. nigerrimus, An. crawfordi, An. maculatus, An. flavirostris and An. kochi. Of the 18 distinct sequence groups identified through both ribosomal DNA internal transcribed spacer region 2, and mitochondrial DNA cytochrome c oxidase subunit 1 loci, 8 could not be identified to species through comparison to published sequences. The comparison of morphological and molecular identities determined that interpretations of local species compositions for primary and expected species in Karama (An. barbirostris and An. vagus) had the highest rate of accuracy (92.1% and 87.6%, respectively) when compared to molecular analysis. However, the remaining distinct sequences molecularly identified to species were identified correctly by morphological methods less frequently, from 0 to 83%. CONCLUSIONS: Karama, Indonesia has a high diversity of Anopheles spp. The unexpected high number of Anopheles species in a small area points to possible complex transmission dynamics and limitations with vector control based on possible varying behaviors and interactions with both humans and interventions. Morphological identification of Anopheles spp. in this study was more accurate for primary and expected species than secondary or unexpected species. Finally, the inability to identify seven sequence groups to species with consensus sequences implies that future studies employing sequencing are required to clarify species compositions in the Nigerrimus Subgroup, among others, as well as their distribution and vector status. Use of molecular methods in conjunction with morphological investigations for analysis of species composition, population dynamics and bionomic characteristics is directly implicated in understanding drivers of malaria transmission, intervention effectiveness, and the pursuit of malaria elimination.

Integrated vector management with additional pre-transmission season thermal fogging is associated with a reduction in dengue incidence in Makassar, Indonesia: Results of an 8-year observational study.

Dengue virus transmission is endemic in Makassar, Indonesia, with the majority of cases reported soon after the start of the annual rainy season. Before 2006, larval source reduction, larvaciding, and reactive routine, outdoor, insecticide fogging campaigns did not result in a reduction in seasonal dengue incidence. Beginning in 2006, village volunteers conducted comprehensive surveys for immature Aedes during the dry season, when vector populations were at their lowest. Based on this pre-season vector data, a single additional pre-emptive outdoor fogging with Malathion was conducted once annually before the rains began in villages with a pre-defined proportion of sampled houses positive for Aedes immatures. This additional procedure was associated with reduced temporal larval indices as well as an 83% reduction in reported cases during the transmission season over the 8-year period of implementation. Two cities adjacent to Makassar experienced substantial but smaller reductions in dengue incidence; while other cities further from the intervention area did not. This represents the first time an integrated intervention strategy has been coupled with substantially reduced dengue transmission in Indonesia.

Molecular identification of mosquitoes of the Anopheles maculatus group of subgenus Cellia (Diptera: Culicidae) in the Indonesian Archipelago.

This study reports the molecular differentiation of females of Anopheles maculatus s.l. collected in eight localities on five islands in the Indonesian Archipelago: Hargowilis and Hargotirto villages of Central Java Province, North Kalimantan Province, Sabang off the northern tip of Sumatra Province, Sumba Island of East Nusa Tenggara Province and Sulawesi Province. Analyses based on rDNA (ITS2 and D3) and mtDNA (COII) sequences revealed the presence of An. greeni for the first time in North Kalimantan, and at least one novel (previously unrecognized) species of the Maculatus Group in Central Java (Hargowilis). Despite the similarity of rDNA markers of specimens of An. maculatus s.l. from Central Java and Sulawesi, their COII sequences are highly divergent (3.3%), which might indicate the presence of a further new species. Specimens of An. maculatus s.l. from the other localities had identical rDNA sequences to most An. maculatus s.s. from mainland Southeast Asia, but moderate divergence in their COII sequences (1.2-2.1%). The latter might indicate there are further novel species within the Maculatus Complex. However, as the divergence at COII may be the result of geographical structuring within species related to the historical biogeography of the region, further studies are needed to shed light on this possibility.

Comparative field evaluation of kelambu traps, barrier screens and barrier screens with eaves for longitudinal surveillance of adult Anopheles mosquitoes in Sulawesi, Indonesia.

BACKGROUND: Sampling methodologies for mosquitoes that are capable of transmitting vector-borne infectious diseases provide critical information on entomological endpoints. Reliable and meaningful field data is vital to the understanding of basic vector biology as well as disease transmission. Various traps take advantage of different vector behaviors and are inevitably subject to sampling biases. This study represents the first comparison of kelambu traps (KT) to barrier screens (BS), barrier screens with eaves (BSE) and indoor and outdoor human landing catches (HLCs). METHODS: Two trap comparison studies were undertaken. In the first study, mosquitoes were collected in Karama over 26 trapping nights to evaluate the kelambu trap relative to indoor and outdoor HLCs. In the second study, mosquitoes were collected in Karama over 12 trapping nights to compare the kelambu trap, barrier screen, barrier screen with eaves and outdoor HLCs. The kelambu trap, barrier screen and barrier screen with eaves obstruct the flight of mosquitos. HLCs target host-seeking behaviors. RESULTS: There was no significant difference between indoor and outdoor HLCs for overall Anopheles mosquito abundance. All five of the molecularly identified Anopheles species collected by HLCs, An. aconitus, An. barbirostris, An. peditaeniatus, An. vagus and An. tessellatus, are reported as vectors of malaria in Indonesia. The kelambu trap (n = 2736) collected significantly more Anopheles mosquitoes than indoor HLCs (n = 1286; Z = 3.193, P = 0.004), but not the outdoor HLCs (n = 1580; Z = 2.325, P = 0.053). All traps collected statistically similar abundances for the primary species, An. barbirostris. However, both comparison studies found significantly higher abundances for the kelambu trap for several secondary species compared to all other traps: An. nigerriumus, An. parangensis, An. tessellatus and An. vagus. The kelambu trap retained the highest species richness and Gini-Simpson's diversity index for both comparison studies. CONCLUSIONS: This study demonstrates that the kelambu trap collects overall Anopheles abundance and species-specific abundances at statistically similar or higher rates than HLCs in Sulawesi, Indonesia. Therefore, the kelambu trap should be considered as an exposure-free alternative to HLCs for research questions regarding Anopheles species in this malaria endemic region.

Mark-release-recapture studies reveal preferred spatial and temporal behaviors of Anopheles barbirostris in West Sulawesi, Indonesia.

BACKGROUND: Population density, dispersion patterns, flight distances, and survival rate of vector mosquitoes are all contributors to vectorial capacity that may be estimated in a single experimental method: mark-release-recapture (MRR). In this study, these key parameters were measured for mosquito populations in Karama, West Sulawesi, Indonesia. METHODS: Two mark-release-recapture (MRR) experiments were carried out in Karama village to characterize seasonality differences, if any: wet season (December 2013, MRR1) and dry season (May 2014, MRR2). For both experiments, mosquitoes were marked according to release site/date and were released on four consecutive nights. Four sampling methodologies were utilized to enable recapture: human landing catches (HLCs), kelambu traps and barrier screens. RESULTS: 98.7% of all catches were molecularly confirmed as Anopheles barbirostris. During the wet season, An. barbirostris demonstrated no preference toward endophagy. In the dry season, An. barbirostris demonstrated an endophagic preference. The duration of the feeding cycle for An. barbirostris was determined to be 5 days during the wet season and 3.7 days during the dry season, though an anomaly likely caused the wet season feeding cycle to be overestimated. The largest percentages of recaptured mosquitoes were collected in a single site during both seasons. The only significant relationship with mosquito dispersal was site of release and recapture. Finally, dispersal rates of An. barbirostris frequently ranged up to 800 m (the maximum measurable distance in this study) within a single day of release. CONCLUSIONS: This study estimated key vector parameters for An. barbirostris an understudied species complex, in Karama, West Sulawesi, Indonesia. Despite the length of the feeding cycle, the high indoor biting rates demonstrated by An. barbirostris in Karama suggest that the use of IRSs and LLINs, especially during the dry season, would have a substantial impact on the panmictic An. barbirostris population.

Genetic and morphological evidence for a new species of the Maculatus Group of Anopheles subgenus Cellia (Diptera: Culicidae) in Java, Indonesia.

BACKGROUND: Anopheles maculatus, a species of the Maculatus Group of subgenus Cellia (Diptera: Culicidae), is an important vector of human malarial protozoa in Java, Indonesia. However, the identity of this species in Indonesia has been questionable because published reports and records are based mainly on morphological identification, which is unreliable for distinguishing members of the Maculatus Group due to overlapping characters. METHODS: We performed morphological assessments, metaphase karyotype preparations, phylogenetic analyses of ITS2 and cox2 sequence data and cross-mating experiments to determine whether the Javanese form and An. maculatus (s.s.) from Thailand were conspecific. RESULTS: The adults of the Java strain are similar to those of An. maculatus (s.s.), but the larvae and pupae exhibit significant differences. The metaphase karyotype of Javanese specimens includes a long acrocentric X chromosome and a small telocentric Y chromosome, which are distinct from other members of the Maculatus Group. Cross-mating of the Java strain with An. maculatus (s.s.) revealed genetic incompatibility. Phylogenetic analysis of ITS2 and cox2 sequences revealed that the Java strain forms a single clade that is distinct from clades of other members of the group (Kimura 2-parameter, K2P, genetic distances 3.1-19.2% and 1.6-9.6%, respectively). CONCLUSIONS: This study provides evidence that the Javanese form of An. maculatus is not conspecific with An. maculatus (s.s.) and constitutes a previously unrecognized species of the Maculatus Group.

Negligible Impact of Mass Screening and Treatment on Mesoendemic Malaria Transmission at West Timor in Eastern Indonesia: A Cluster-Randomized Trial.

Background: Mass screening and treatment (MST) aims to reduce malaria risk in communities by identifying and treating infected persons without regard to illness. Methods: A cluster-randomized trial evaluated malaria incidence with and without MST. Clusters were randomized to 3, 2, or no MST interventions: MST3, 6 clusters (156 households/670 individuals); MST2, 5 clusters (89 households/423 individuals); and MST0, 5 clusters (174 households/777 individuals). All clusters completed the study with 14 residents withdrawing. In a cohort of 324 schoolchildren (MST3, n = 124; MST2, n = 57; MST0, n = 143) negative by microscopy at enrollment, we evaluated the incidence density of malaria during 3 months of MST and 3 months following. The MST intervention involved community-wide expert malaria microscopic screening and standard therapy with dihydroartemisinin-piperaquine and primaquine for glucose-6 phosphate dehydrogenase-normal subjects. All blood examinations included polymerase chain reaction assays, which did not guide on-site treatment. Results: The risk ratios for incidence density of microscopically patent malaria in MST3 or MST2 relative to that in MST0 clusters were 1.00 (95% confidence interval [CI], .53-1.91) and 1.22 (95% CI, .42-3.55), respectively. Similar results were obtained with molecular analysis and species-specific (P. falciparum and P. vivax) infections. Microscopically subpatent, untreated infections accounted for 72% of those infected. Conclusions: Two or 3 rounds of MST within 3 months did not impact the force of anopheline mosquito-borne infection in these communities. The high rate of untreated microscopically subpatent infections likely explains the observed poor impact. Clinical Trials Registration: NCT01878357.

Genomic analysis and growth characteristic of dengue viruses from Makassar, Indonesia.

Dengue fever is currently the most important mosquito-borne viral disease in Indonesia. In South Sulawesi province, most regions report dengue cases including the capital city, Makassar. Currently, no information is available on the serotypes and genotypes of the viruses circulating in the area. To understand the dynamic of dengue disease in Makassar, we carried out dengue fever surveillance study during 2007-2010. A total of 455 patients were recruited, in which antigen and serological detection revealed the confirmed dengue cases in 43.3% of patients. Molecular detection confirmed the dengue cases in 27.7% of patients, demonstrating that dengue places a significant disease burden on the community. Serotyping revealed that dengue virus serotype 1 (DENV-1) was the most predominant serotype, followed by DENV-2, -3, and -4. To determine the molecular evolution of the viruses, we conducted whole-genome sequencing of 80 isolates. Phylogenetic analysis grouped DENV-2, -3 and -4 to the Cosmopolitan genotype, Genotype I and Genotype II, respectively. Intriguingly, each serotype paints a different picture of evolution and transmission. DENV-1 appears to be undergoing a clade replacement with Genotype IV being supplanted by Genotype I. The Cosmopolitan DENV-2 isolates were found to be regionally endemic and is frequently being exchanged between countries in the region. By contrast, DENV-3 and DENV-4 isolates were related to strains with a long history in Indonesia although the DENV-3 strains appear to have been following a distinct evolutionary path since approximately 1998. To assess whether the various DENV serotypes/genotypes possess different growth characteristics, we performed growth kinetic assays on selected viruses. We observed the relatively higher rate of replication for DENV-1 and -2 compared to DENV-3 and -4. Within the DENV-1, viruses from Genotype I grow faster than that of Genotype IV. This higher replication rate may underlie their ability to replace the circulation of Genotype IV in the community.

Distance to Anopheles sundaicus larval habitats dominant among risk factors for parasitemia in meso-endemic Southwest Sumba, Indonesia.

BACKGROUND: The decline in intensity of malaria transmission in many areas now emphasizes greater importance of understanding the epidemiology of low to moderate transmission settings. Marked heterogeneity in infection risk within these populations creates opportunities to understand transmission and guide resource allocation to greater impact. METHODS: In this study, we examined spatial patterns of malaria transmission in a hypo- to meso-endemic area of eastern Indonesia using malaria prevalence data collected from a cross-sectional socio-demographic and parasitological survey conducted from August to November 2010. An entomological survey performed in parallel, identified, mapped, and monitored local anopheline larval habitats. RESULTS: A single spatial cluster of higher malaria prevalence was detected during the study period (relative risk=2.13; log likelihood ratio=20.7; P<0.001). In hierarchical multivariate regression models, risk of parasitemia was inversely correlated with distance to five Anopheles sundaicus known larval habitats [odds ratio (OR)=0.21; 95% confidence interval (CI)=0.14-0.32; P<0.001], which were located in a geographically restricted band adjacent to the coastline. Increasing distance from these sites predicted increased hemoglobin level across age strata after adjusting for confounders (OR=1.6; 95% CI=1.30-1.98; P<0.001). CONCLUSION: Significant clustering of malaria parasitemia in close proximity to very specific and relatively few An. sundaicus larval habitats has direct implications for local control strategy, policy, and practice. These findings suggest that larval source management could achieve profound if not complete impact in this region.

Impact of a spatial repellent on malaria incidence in two villages in Sumba, Indonesia.

A randomized, double-blinded, placebo-controlled study was conducted to examine the effect of spatial repellent (SR) in households at risk of malaria in Indonesia. Following presumptive radical cure for malaria in 180 adult men representing sentinels of new infection in four clusters within two villages, all households were given either metofluthrin or placebo mosquito coils. Weekly blood smear screening and human-landing mosquito catches were done throughout the 6 months intervention. Malaria infections occurred in 61 subjects living in placebo households and 31 subjects living in SR coil households, suggesting a 52% protective effect of SR. Likewise, anopheles indoor human landing rates were 32% lower in homes receiving SR coils. Differences in the malaria attack rate between SR- and placebo-treated homes was significant when not accounting for the effects of clustering. When the analysis was adjusted for intra-cluster correlation, the differences between SR- and placebo-treated homes were not statistically significant. The findings provide evidence of SR public health benefit and support a larger trial statistically powered to detect those effects.

Towards eradication: three years after the tsunami of 2004, has malaria transmission been eliminated from the island of Simeulue?

The island of Simeulue was the first landfall of the tsunami of December 2004. The tsunami destroyed many villages on the island, leaving one third of the population homeless. Malaria is endemic in Simeulue and an epidemic was reported to have occurred three months prior to the tsunami. Information concerning malaria was, however, not easily available. The earthquakes related to the tsunami may have created extensive potential breeding sites of Anopheles sundaicus, the probable vector, and increased vulnerability of the human population; a possibility of increased transmission made a further outbreak possible. Consequently, subsequent to the tsunami, considerable amounts of aid, including anti-malarial measures such as insecticide treated mosquito-nets, were deployed on the island. A series of island-wide cross-sectional surveys were conducted in 2005-2007 to determine whether these had had any effect on malaria prevalence. Larval sampling, and CDC light-trap and landing collections of hungry mosquitoes were also undertaken. The results indicate that despite the continuing presence of potential vectors in some places the anti-malaria measures introduced following the tsunami have controlled, and may be close to eliminating, malaria from the island.

Malaria prevalence in Nias District, North Sumatra Province, Indonesia.

BACKGROUND: The Nias district of the North Sumatra Province of Indonesia has long been known to be endemic for malaria. Following the economic crisis at the end of 1998 and the subsequent tsunami and earthquake, in December 2004 and March 2005, respectively, the malaria control programme in the area deteriorated. The present study aims to provide baseline data for the establishment of a suitable malaria control programme in the area and to analyse the frequency distribution of drug resistance alleles associated with resistance to chloroquine and sulphadoxine-pyrimethamine. METHODS: Malariometric and entomology surveys were performed in three subdistricts. Thin and thick blood smears were stained with Giemsa and examined under binocular light microscopy. Blood blots on filter paper were also prepared for isolation of parasite and host DNA to be used for molecular analysis of band 3 (SAO), pfcrt, pfmdr1, dhfr, and dhps. In addition, haemoglobin measurement was performed in the second and third surveys for the subjects less than 10 years old. RESULTS: Results of the three surveys revealed an average slide positivity rate of 8.13%, with a relatively higher rate in certain foci. Host genetic analysis, to identify the Band 3 deletion associated with Southeast Asian Ovalocytosis (SAO), revealed an overall frequency of 1.0% among the 1,484 samples examined. One hundred six Plasmodium falciparum isolates from three sub-districts were successfully analysed. Alleles of the dhfr and dhps genes associated with resistance to sulphadoxine-pyrimethamine, dhfr C59R and S108N, and dhps A437G and K540E, were present at frequencies of 52.2%, 82.5%, 1.18% and 1.18%, respectively. The pfmdr1 alleles N86Y and N1042D, putatively associated with mefloquine resistance, were present at 31.4% and 2%, respectively. All but one sample carried the pfcrt 76T allele associated with chloroquine resistance. Entomologic surveys identified three potential anopheline vectors in the area, Anopheles barbirostris, Anopheles kochi and Anopheles sundaicus. CONCLUSION: The cross sectional surveys in three different sub-districts of Nias District clearly demonstrated the presence of relatively stable endemic foci of malaria in Nias District, North Sumatra Province, Indonesia. Molecular analysis of the malaria parasite isolates collected from this area strongly indicates resistance to chloroquine and a growing threat of resistance to sulphadoxine-pyrimethamine. This situation highlights the need to develop sustainable malaria control measures through regular surveillance and proper antimalarial drug deployment.

Maxillae and mandibles of male mosquitoes and female autogenous mosquitoes (Diptera: Culicidae).

Maxillae and mandibles of males of 44 species of 12 mosquito genera and females of three autogenous genera and two partially autogenous species were examined under light microscopy and scanning electron microscopy. The maxillae and mandibles of male mosquitoes are delicate, tape-like structures with lengths characterizing genera or higher level classification units. Five patterns are recognized: (A) long maxillae and mandibles with mandibles longer than maxillae in Anopheles; (B) long maxillae and mandibles with maxillae longer than mandibles in Toxorhynchites; (C) short or intermediate lengths of maxillae with short mandibles in Aedes, Armigeres, Culex, Ochlerotatus, Orthopodomyia, and Uranotaenia; (D) short or intermediate length of maxillae with no mandibles in Mimomyia and Tripteroides; and (E) no maxillae and mandibles in Malaya and Topomyia. Maxillary and mandibular lengths of male mosquitoes show a positive correlation. Length of maxillae and mandibles of autogenous females are reduced to the same level as conspecific males. In contrast, females of partially autogenous species have complete maxillae and mandibles as in females of anautogenous species.

Mouthparts of male Aedes (Stegomyia) mosquitoes.

Mouthparts of adult males of 17 strains of 8 species from the subgenus Stegomyia of the genus Aedes, including 5 strains of Aedes aegypti and 6 strains of Aedes albopictus, were examined. Lengths of maxillae, mandibles, maxillary palpi, and proboscises were measured under light microscopy and their detailed structures were examined by scanning electron microscopy. Lengths were presented as ratios to proboscis lengths. In contrast to previous reports, mandibles were found in all 5 strains of male Ae. aegypti examined. Variations in maxillary and mandibular lengths were significant among strains, even within Ae. aegypti and Ae. albopictus. High variation of these structures among and within species indicates that the average length of these structures in only 1 species may not be a reliable representative of a subgenus, and those of 1 strain may not be reliable for a species. However, their range in length (maxillae 0.13-0.50, mandibles 0.04-0.17 length of the proboscis) may be regarded as a subgeneric attribute. Maxillae and mandibles distinctly shorter than the proboscis, together with their delicate structures and the large coefficient of variation, suggest that they exist only as vestigial structures. A positive correlation was found between lengths of maxillae and those of mandibles, but mandibles are usually shorter than maxillae. The hypopharynx is discernible from the labium wall by its texture and border, and this suggests that it was a free stylet in the past.