A longitudinal study on Anopheles mosquito larval abundance in distinct geographical and environmental settings in western Kenya

Background

Malaria vector control in Africa depends upon effective insecticides in bed nets and indoor residual sprays. This study investigated the extent of insecticide resistance in Anopheles gambiae s.l., Anopheles gambiae s.s. and Anopheles arabiensis in western Kenya where ownership of insecticide-treated bed nets has risen steadily from the late 1990s to 2010. Temporal and spatial variation in the frequency of a knock down resistance (kdr) allele in A. gambiae s.s. was quantified, as was variation in phenotypic resistance among geographic populations of A. gambiae s.l.

Methods

To investigate temporal variation in kdr frequency, individual specimens of A. gambiae s.s. from two sentinel sites were genotyped using RT-PCR from 1996-2010. Spatial variation in kdr frequency, species composition, and resistance status were investigated in additional populations of A. gambiae s.l. sampled in western Kenya in 2009 and 2010. Specimens were genotyped for kdr as above and identified to species via conventional PCR. Field-collected larvae were reared to adulthood and tested for insecticide resistance using WHO bioassays.

Results

Anopheles gambiae s.s. showed a dramatic increase in kdr frequency from 1996 - 2010, coincident with the scale up of insecticide-treated nets. By 2009-2010, the kdr L1014S allele was nearly fixed in the A. gambiae s.s. population, but was absent in A. arabiensis. Near Lake Victoria, A. arabiensis was dominant in samples, while at sites north of the lake A. gambiae s.s was more common but declined relative to A. arabiensis from 2009 to 2010. Bioassays demonstrated that A. gambiae s.s. had moderate phenotypic levels of resistance to DDT, permethrin and deltamethrin while A. arabiensis was susceptible to all insecticides tested.

Conclusions

The kdr L1014S allele has approached fixation in A. gambiae s.s. populations of western Kenya, and these same populations exhibit varying degrees of phenotypic resistance to DDT and pyrethroid insecticides. The near absence of A. gambiae s.s. from populations along the lakeshore and the apparent decline in other populations suggest that insecticide-treated nets remain effective against this mosquito despite the increase in kdr allele frequency. The persistence of A. arabiensis, despite little or no detectable insecticide resistance, is likely due to behavioural traits such as outdoor feeding and/or feeding on non-human hosts by which this species avoids interaction with insecticide-treated nets.     

Distribution and Frequency of kdr Mutations within Anopheles gambiae s.l. Populations and First Report of the Ace.1G119S Mutation in Anopheles arabiensis from Burkina Faso (West Africa)

An entomological survey was carried out at 15 sites dispersed throughout the three eco-climatic regions of Burkina Faso (West Africa) in order to assess the current distribution and frequency of mutations that confer resistance to insecticides in An. gambiae s.l. populations in the country. Both knockdown (kdr) resistance mutation variants (L1014F and L1014S), that confer resistance to pyrethroid insecticides, were identified concomitant with the ace-1 G119S mutation confirming the presence of multiple resistance mechanisms in the An. gambiae complex in Burkina Faso. Compared to the last survey, the frequency of the L1014F kdr mutation appears to have remained largely stable and relatively high in all species. In contrast, the distribution and frequency of the L1014S mutation has increased significantly in An. gambiae s.l. across much of the country. Furthermore we report, for the first time, the identification of the ace.1 G116S mutation in An. arabiensis populations collected at 8 sites. This mutation, which confers resistance to organophosphate and carbamate insecticides, has been reported previously only in the An. gambiae S and M molecular forms. This finding is significant as organophosphates and carbamates are used in indoor residual sprays (IRS) to control malaria vectors as complementary strategies to the use of pyrethroid impregnated bednets. The occurrence of the three target-site resistance mutations in both An. gambiae molecular forms and now An. arabiensis has significant implications for the control of malaria vector populations in Burkina Faso and for resistance management strategies based on the rotation of insecticides with different modes of action.     

Pyrethroid Resistance in Anopheles gambiae,in Bomi County,Liberia, Compromises Malaria Vector Control

Background

Long Lasting Insecticidal Nets (LLIN) and Indoor Residual Spraying (IRS) have both proven to be effective malaria vector control strategies in Africa and the new technology of insecticide treated durable wall lining (DL) is being evaluated. Sustaining these interventions at high coverage levels is logistically challenging and, furthermore, the increase in insecticide resistance in African malaria vectors may reduce the efficacy of these chemical based interventions. Monitoring of vector populations and evaluation of the efficacy of insecticide based control approaches should be integral components of malaria control programmes. This study reports on entomological survey conducted in 2011 in Bomi County, Liberia.

Methods

Anopheles gambiae larvae were collected from four sites in Bomi, Liberia, and reared in a field insectary. Two to five days old female adult An gambiae s.l. were tested using WHO tube (n = 2027) and cone (n = 580) bioassays in houses treated with DL or IRS. A sample of mosquitoes (n = 169) were identified to species/molecular form and screened for the presence of knock down resistance (kdr) alleles associated with pyrethroid resistance.

Results

Anopheles gambiae s.l tested were resistant to deltamethrin but fully susceptible to bendiocarb and fenithrothion. The corrected mortality of local mosquitoes exposed to houses treated with deltamethrin either via IRS or DL was 12% and 59% respectively, suggesting that resistance may affect the efficacy of these interventions. The presence of pyrethroid resistance was associated with a high frequency of the 1014F kdr allele (90.5%) although this mutation alone cannot explain the resistance levels observed.

Conclusion

High prevalence of resistance to deltamethrin in Bomi County may reduce the efficacy of malaria strategies relying on this class of insecticide. The findings highlight the urgent need to expand and sustain monitoring of insecticide resistance in Liberian malaria vectors, evaluate the effectiveness of existing interventions and develop appropriate resistance management strategies.     

ITN Mixtures of Chlorfenapyr (Pyrrole) and Alphacypermethrin (Pyrethroid) for Control of Pyrethroid Resistant Anopheles arabiensis and Culex quinquefasciatus

Pyrethroid resistant Anopheles gambiae malaria vectors are widespread throughout sub-Saharan Africa and continued efficacy of pyrethroid ITNs is under threat. Chlorfenapyr is a promising pyrrole insecticide with a unique mechanism of action conferring no cross-resistance to existing public health insecticides. Mixtures of chlorfenapyr (CFP) and alphacypermethrin (alpha) may provide additional benefits over chlorfenapyr or alphacypermethrin used alone. An ITN mixture of CFP 100 mg/m²+alpha 25 mg/m² was compared with CFP 100 mg/m² and alpha 25 mg/m² in a small-scale experimental hut trial in an area of wild An. arabiensis. The same treatments were evaluated in tunnel tests against insectary-reared pyrethroid susceptible and resistant Culex quinquefasciatus. Performance was measured in terms of insecticide-induced mortality, and blood-feeding inhibition. Tunnel tests showed that mixtures of CFP 100+ alpha 25 were 1.2 and 1.5 times more effective at killing susceptible Cx. quinquefasciatus than either Alpha 25 (P = 0.001) or CFP 100 (P = 0.001) ITNs. Mixtures of CFP100+ alpha 25 were 2.2 and 1.2 times more effective against resistant Cx. quinquefasciatus than either alpha 25 (P = 0.001) or CFP100 (P = 0.003) ITNs. CFP 100+ alpha 25 produced higher levels of blood-feeding inhibition than CFP alone for susceptible (94 vs 46%, P = 0.001) and resistant (84 vs 53%, P = 0.001) strains. In experimental huts the mixture of CFP 100+ Alpha 25 killed 58% of An. arabiensis, compared with 50% for alpha and 49% for CFP, though the differences were not significant. Blood-feeding inhibition was highest in the mixture with a 76% reduction compared to the untreated net (P = 0.001). ITN mixtures of chlorfenapyr and alphacypermethrin should restore effective control of resistant populations of An. gambiae malaria vectors, provide protection from blood-feeding, and may have benefits for resistance management, particularly in areas with low or moderate frequency of pyrethroid resistance. A wash-resistant mixture should be developed urgently.     

Anopheline Larval Habitats Seasonality and Species Distribution: A Prerequisite for Effective Targeted Larval Habitats Control Programmes

Background

Larval control is of paramount importance in the reduction of malaria vector abundance and subsequent disease transmission reduction. Understanding larval habitat succession and its ecology in different land use managements and cropping systems can give an insight for effective larval source management practices. This study investigated larval habitat succession and ecological parameters which influence larval abundance in malaria epidemic prone areas of western Kenya.

Methods and Findings

A total of 51 aquatic habitats positive for anopheline larvae were surveyed and visited once a week for a period of 85 weeks in succession. Habitats were selected and identified. Mosquito larval species, physico-chemical parameters, habitat size, grass cover, crop cycle and distance to nearest house were recorded. Polymerase chain reaction revealed that An. gambiae s.l was the most dominant vector species comprised of An.gambiae s.s (77.60%) and An.arabiensis (18.34%), the remaining 4.06% had no amplification by polymerase chain reaction. Physico-chemical parameters and habitat size significantly influenced abundance of An. gambiae s.s (P = 0.024) and An. arabiensis (P = 0.002) larvae. Further, larval species abundance was influenced by crop cycle (P≤0.001), grass cover (P≤0.001), while distance to nearest houses significantly influenced the abundance of mosquito species larvae (r = 0.920;P≤0.001). The number of predator species influenced mosquito larval abundance in different habitat types. Crop weeding significantly influenced with the abundance of An.gambiae s.l (P≤0.001) when preceded with fertilizer application. Significantly higher anopheline larval abundance was recorded in habitats in pasture compared to farmland (P = 0.002). When habitat stability and habitat types were considered, hoof print were the most productive followed by disused goldmines.

Conclusion

These findings suggest that implementation of effective larval control programme should be targeted with larval habitats succession information when larval habitats are fewer and manageable. Crop cycles and distance from habitats to household should be considered as effective information in planning larval control.     

Pyrethroid resistance in Anopheles gambiae s.s. and Anopheles arabiensis in western Kenya: phenotypic,metabolic and target site characterizations of three populations

Field and laboratory investigations revealed phenotypic, target site and metabolic resistance to permethrin in an Anopheles gambiae s.s. (Diptera: Culicidae) population in Bungoma District, a region in western Kenya in which malaria is endemic and rates of ownership of insecticide‐treated bednets are high. The sensitivity of individual An. gambiae s.l. females as indicated in assays using World Health Organization (WHO) test kits demonstrated reduced mortality in response to permethrin, deltamethrin and bendiocarb. Estimated time to knock‐down of 50% (KDT₅₀) of the test population in Centers for Disease Control (CDC) bottle bioassays was significantly lengthened for the three insecticides compared with that in a susceptible control strain. Anopheles arabiensis from all three sites showed higher mortality to all three insecticides in the WHO susceptibility assays compared with the CDC bottle assays, in which they showed less sensitivity and longer KDT₅₀ than the reference strain for permethrin and deltamethrin. Microplate assays revealed elevated activity of β‐esterases and oxidases, but not glutathione‐S‐transferase, in An. gambiae s.s. survivors exposed to permethrin in bottle bioassays compared with knocked down and unexposed individuals. No An. arabiensis showed elevated enzyme activity. The 1014S kdr allele was fixed in the Bungoma An. gambiae s.s. population and absent from An. arabiensis, whereas the 1014F kdr allele was absent from all samples of both species. Insecticide resistance could compromise vector control in Bungoma and could spread to other areas as coverage with longlasting insecticide‐treated bednets increases.     

Insecticide susceptibility of Anopheles coluzzii and Anopheles gambiae mosquitoes in Ibadan,Southwest Nigeria

The emergence of insecticide resistance in Anopheles (Diptera: Culicidae) mosquitoes has great implications for malaria control in Nigeria. This study aimed to determine the dynamics of insecticide susceptibility levels and the frequency of knock‐down resistance (kdr) mutations (L1014F) in wild Anopheles coluzzii Coetzee & Wilkerson sp. n. and Anopheles gambiae Giles from the Ojoo and Bodija areas of Ibadan, in southwest Nigeria. Insecticide susceptibility to pyrethroids, organophosphates, carbamates and organochlorines was assessed using World Health Organization (WHO) bioassays. A subset of the mosquitoes exposed to pyrethroids and DDT was used for species and molecular form identification; kdr genotyping was determined using the TaqMan real‐time polymerase chain reaction assay. The mosquitoes were resistant to pyrethroids and DDT but completely susceptible to organophosphates and carbamates. Bodija samples (n = 186) consisted of An. gambiae (91.4%) and An. coluzzii (8.1%) and included one An. coluzzii/An. gambiae hybrid specimen. All mosquitoes screened in Ojoo (n = 26) were An. gambiae. The 1014F kdr mutation was detected at frequencies of 24.5 and 5.8% in Bodija and Ojoo, respectively. No correlation was observed between kdr genotypes and resistance phenotypes. The results indicate that metabolic resistance probably plays an important role in the development of resistance and highlight the need to implement insecticide resistance management strategies.     

Genetic Diversity and Population Structure of Sitodiplosis mosellana in Northern China

The wheat midge, Sitodiplosis mosellana, is an important pest in Northern China. We tested the hypothesis that the population structure of this species arises during a range expansion over the past 30 years. This study used microsatellite and mitochondrial loci to conduct population genetic analysis of S. mosellana across its distribution range in China. We found strong genetic structure among the 16 studied populations, including two genetically distinct groups (the eastern and western groups), broadly consistent with the geography and habitat fragmentation. These results underline the importance of natural barriers in impeding dispersal and gene flow of S. mosellana populations. Low to moderate genetic diversity among the populations and moderate genetic differentiation (F _ST = 0.117) between the two groups were also found. The populations in the western group had lower genetic diversity, higher genetic differentiation and lower gene flow (F _ST = 0.116, Nm = 1.89) than those in the eastern group (F _ST = 0.049, Nm = 4.91). Genetic distance between populations was positively and significantly correlated with geographic distance (r = 0.56, P<0.001). The population history of this species provided no evidence for population expansion or bottlenecks in any of these populations. Our data suggest that the distribution of genetic diversity, genetic differentiation and population structure of S. mosellana have resulted from a historical event, reflecting its adaptation to diverse habitats and forming two different gene pools. These results may be the outcome of a combination of restricted gene flow due to geographical and environmental factors, population history, random processes of genetic drift and individual dispersal patterns. Given the current risk status of this species in China, this study can offer useful information for forecasting outbreaks and designing effective pest management programs.     

Vertical and Horizontal Genetic Connectivity in Chromis verater,an Endemic Damselfish Found on Shallow and Mesophotic Reefs in the Hawaiian Archipelago and Adjacent Johnston Atoll

Understanding vertical and horizontal connectivity is a major priority in research on mesophotic coral ecosystems (30–150 m). However, horizontal connectivity has been the focus of few studies, and data on vertical connectivity are limited to sessile benthic mesophotic organisms. Here we present patterns of vertical and horizontal connectivity in the Hawaiian Islands-Johnston Atoll endemic threespot damselfish, Chromis verater, based on 319 shallow specimens and 153 deep specimens. The mtDNA markers cytochrome b and control region were sequenced to analyze genetic structure: 1) between shallow (<30 m) and mesophotic (30–150 m) populations and 2) across the species'' geographic range. Additionally, the nuclear markers rhodopsin and internal transcribed spacer 2 of ribosomal DNA were sequenced to assess connectivity between shallow and mesophotic populations. There was no significant genetic differentiation by depth, indicating high levels of vertical connectivity between shallow and deep aggregates of C. verater. Consequently, shallow and deep samples were combined by location for analyses of horizontal connectivity. We detected low but significant population structure across the Hawaiian Archipelago (overall cytochrome b: Φ_ST = 0.009, P = 0.020; control region: Φ_ST = 0.012, P = 0.009) and a larger break between the archipelago and Johnston Atoll (cytochrome b: Φ_ST = 0.068, P<0.001; control region: Φ_ST = 0.116, P<0.001). The population structure within the archipelago was driven by samples from the island of Hawaii at the southeast end of the chain and Lisianski in the middle of the archipelago. The lack of vertical genetic structure supports the refugia hypothesis that deep reefs may constitute a population reservoir for species depleted in shallow reef habitats. These findings represent the first connectivity study on a mobile organism that spans shallow and mesophotic depths and provide a reference point for future connectivity studies on mesophotic fishes.     

Insecticide resistance monitoring of field‐collected Anopheles gambiae s.l. populations from Jinja,eastern Uganda,identifies high levels of pyrethroid resistance

Insecticide resistance in the malaria vector Anopheles gambiae s.l. (Diptera: Culicidae) threatens insecticide‐based control efforts, necessitating regular monitoring. We assessed resistance in field‐collected An. gambiae s.l. from Jinja, Uganda using World Health Organization (WHO) biosassays. Only An. gambiae s.s. and An. arabiensis (?70%) were present. Female An. gambiae exhibited extremely high pyrethroid resistance (permethrin LT₅₀ > 2 h; deltamethrin LT₅₀ > 5 h). Female An. arabiensis were resistant to permethrin and exhibited reduced susceptibility to deltamethrin. However, while An. gambiae were DDT resistant, An. arabiensis were fully susceptible. Both species were fully susceptible to bendiocarb and fenitrothion. Kdr 1014S has increased rapidly in the Jinja population of An. gambiae s.s. and now approaches fixation (?95%), consistent with insecticide‐mediated selection, but is currently at a low frequency in An. arabiensis (0.07%). Kdr 1014F was also at a low frequency in An. gambiae. These frequencies preclude adequately‐powered tests for an association with phenotypic resistance. PBO synergist bioassays resulted in near complete recovery of pyrethroid susceptibility suggesting involvement of CYP450s in resistance. A small number (0.22%) of An. gambiae s.s. ×An. arabiensis hybrids were found, suggesting the possibility of introgression of resistance alleles between species. The high levels of pyrethroid resistance encountered in Jinja threaten to reduce the efficacy of vector control programmes which rely on pyrethroid‐impregnated bednets or indoor spraying of pyrethroids.     

Population structure of the malaria vector Anopheles sinensis (Diptera: Culicidae) in China: two gene pools inferred by microsatellites

Background

Anopheles sinensis is a competent malaria vector in China. An understanding of vector population structure is important to the vector-based malaria control programs. However, there is no adequate data of A. sinensis population genetics available yet.

Methodology/Principal Findings

This study used 5 microsatellite loci to estimate population genetic diversity, genetic differentiation and demographic history of A. sinensis from 14 representative localities in China. All 5 microsatellite loci were highly polymorphic across populations, with high allelic richness and heterozygosity. Hardy–Weinberg disequilibrium was found in 12 populations associated with heterozygote deficits, which was likely caused by the presence of null allele and the Wahlund effect. Bayesian clustering analysis revealed two gene pools, grouping samples into two population clusters; one includes six and the other includes eight populations. Out of 14 samples, six samples were mixed with individuals from both gene pools, indicating the coexistence of two genetic units in the areas sampled. The overall differentiation between two genetic pools was moderate (F _ST = 0.156). Pairwise differentiation between populations were lower within clusters (F _ST = 0.008–0.028 in cluster I and F _ST = 0.004–0.048 in cluster II) than between clusters (F _ST = 0.120–0.201). A reduced gene flow (Nm = 1–1.7) was detected between clusters. No evidence of isolation by distance was detected among populations neither within nor between the two clusters. There are differences in effective population size (Ne = 14.3-infinite) across sampled populations.

Conclusions/Significance

Two genetic pools with moderate genetic differentiation were identified in the A. sinensis populations in China. The population divergence was not correlated with geographic distance or barrier in the range. Variable effective population size and other demographic effects of historical population perturbations could be the factors affecting the population differentiation. The structured populations may limit the migration of genes under pressures/selections, such as insecticides and immune genes against malaria.     

Impacts of Agricultural Practices on Insecticide Resistance in the Malaria Vector Anopheles arabiensis in Khartoum State,Sudan

Background

Agricultural pesticides may play a profound role in selection of resistance in field populations of mosquito vectors. The objective of this study is to investigate possible links between agricultural pesticide use and development of resistance to insecticides by the major malaria vector Anopheles arabiensis in northern Sudan.

Methodology/Principal Findings

Entomological surveys were conducted during two agricultural seasons in six urban and peri-urban sites in Khartoum state. Agro-sociological data were collected from 240 farmers subjected to semi-structured questionnaires based on knowledge attitude and practice (KAP) surveys. Susceptibility status of An. arabiensis (n=6000) was assessed in all sites and during each season using WHO bioassay tests to DDT, deltamethrin, permethrin, Malathion and bendiocarb. KAP analysis revealed that pesticide application was common practice among both urban and peri-urban farmers, with organophosphates and carbamates most commonly used. Selection for resistance is likely to be greater in peri-urban sites where farmers apply pesticide more frequently and are less likely to dispose of surpluses correctly. Though variable among insecticides and seasons, broad-spectrum mortality was slightly, but significantly higher in urban than peri-urban sites and most marked for bendiocarb, to which susceptibility was lowest. Anopheles arabiensis from all sites showed evidence of resistance or suspected resistance, especially pyrethroids. However, low-moderate frequencies of the L1014F kdr allele in all sites, which was very strongly associated with DDT, permethrin and deltamethrin survivorship (OR=6.14-14.67) suggests that resistance could increase rapidly.

Conclusions

Ubiquitous multiple-resistance coupled with presence of a clear mechanism for DDT and pyrethroids (kdr L1014F) in populations of An. arabiensis from Khartoum-Sudan suggests careful insecticide management is essential to prolong efficacy. Our findings are consistent with agricultural insecticide use as a source of selection for resistance and argue for coordination between the integrated vector control program and the Ministry of Agriculture to permit successful implementation of rational resistance management strategies.     

Pyrethroid Resistance in an Anopheles funestus Population from Uganda

Background

The susceptibility status of Anopheles funestus to insecticides remains largely unknown in most parts of Africa because of the difficulty in rearing field-caught mosquitoes of this malaria vector. Here we report the susceptibility status of the An. funestus population from Tororo district in Uganda and a preliminary characterisation of the putative resistance mechanisms involved.

Methodology/Principal Findings

A new forced egg laying technique used in this study significantly increased the numbers of field-caught females laying eggs and generated more than 4000 F₁ adults. WHO bioassays indicated that An. funestus in Tororo is resistant to pyrethroids (62% mortality after 1 h exposure to 0.75% permethrin and 28% mortality to 0.05% deltamethrin). Suspected DDT resistance was also observed with 82% mortality. However this population is fully susceptible to bendiocarb (carbamate), malathion (organophosphate) and dieldrin with 100% mortality observed after exposure to each of these insecticides. Sequencing of a fragment of the sodium channel gene containing the 1014 codon conferring pyrethroid/DDT resistance in An. gambiae did not detect the L1014F kdr mutation but a correlation between haplotypes and resistance phenotype was observed indicating that mutations in other exons may be conferring the knockdown resistance in this species. Biochemical assays suggest that resistance in this population is mediated by metabolic resistance with elevated level of GSTs, P450s and pNPA compared to a susceptible strain of Anopheles gambiae. RT-PCR further confirmed the involvement of P450s with a 12-fold over-expression of CYP6P9b in the Tororo population compared to the fully susceptible laboratory colony FANG.

Conclusion

This study represents the first report of pyrethroid/DDT resistance in An. funestus from East Africa. With resistance already reported in southern and West Africa, this indicates that resistance in An. funestus may be more widespread than previously assumed and therefore this should be taken into account for the implementation and management of vector control programs in Africa.     

Genetic Diversity in Introduced Golden Mussel Populations Corresponds to Vector Activity

We explored possible links between vector activity and genetic diversity in introduced populations of Limnoperna fortunei by characterizing the genetic structure in native and introduced ranges in Asia and South America. We surveyed 24 populations: ten in Asia and 14 in South America using the mitochondrial cytochrome c oxidase subunit I (COI) gene, as well as eight polymorphic microsatellite markers. We performed population genetics and phylogenetic analyses to investigate population genetic structure across native and introduced regions. Introduced populations in Asia exhibit higher genetic diversity (H _E = 0.667–0.746) than those in South America (H _E = 0.519–0.575), suggesting higher introduction effort for the former populations. We observed pronounced geographical structuring in introduced regions, as indicated by both mitochondrial and nuclear markers based on multiple genetic analyses including pairwise Ф_ST, F _ST, Bayesian clustering method, and three-dimensional factorial correspondence analyses. Pairwise F _ST values within both Asia (F _ST = 0.017–0.126, P = 0.000–0.009) and South America (F _ST = 0.004–0.107, P = 0.000–0.721) were lower than those between continents (F _ST = 0.180–0.319, P = 0.000). Fine-scale genetic structuring was also apparent among introduced populations in both Asia and South America, suggesting either multiple introductions of distinct propagules or strong post-introduction selection and demographic stochasticity. Higher genetic diversity in Asia as compared to South America is likely due to more frequent propagule transfers associated with higher shipping activities between source and donor regions within Asia. This study suggests that the intensity of human-mediated introduction vectors influences patterns of genetic diversity in non-indigenous species.     

Multi-Gene Analysis Reveals a Lack of Genetic Divergence between Calanus agulhensis and C. sinicus (Copepoda; Calanoida)

The discrimination and taxonomic identification of marine species continues to pose a challenge despite the growing number of diagnostic metrics and approaches. This study examined the genetic relationship between two sibling species of the genus Calanus (Crustacea; Copepoda; Calanidae), C. agulhensis and C. sinicus, using a multi-gene analysis. DNA sequences were determined for portions of the mitochondrial cytochrome c oxidase I (mtCOI); nuclear citrate synthase (CS), and large subunit (28S) rRNA genes for specimens collected from the Sea of Japan and North East (NE) Pacific Ocean for C. sinicus and from the Benguela Current and Agulhas Bank, off South Africa, for C. agulhensis. For mtCOI, C. sinicus and C. agulhensis showed similar levels of haplotype diversity (H_d = 0.695 and 0.660, respectively) and nucleotide diversity (π = 0.003 and 0.002, respectively). Pairwise F_ST distances for mtCOI were significant only between C. agulhensis collected from the Agulhas and two C. sinicus populations: the Sea of Japan (F_ST = 0.152, p<0.01) and NE Pacific (F_ST = 0.228, p<0.005). Between the species, F_ST distances were low for both mtCOI (F_ST = 0.083, p = 0.003) and CS (F_ST = 0.050, p = 0.021). Large subunit (28S) rRNA showed no variation between the species. Our results provide evidence of the lack of genetic distinction of C. sinicus and C. agulhensis, raise questions of whether C. agulhensis warrants status as a distinct species, and indicate the clear need for more intensive and extensive ecological and genetic analysis.     

Genome-Based Microsatellite Development in the Culex pipiens Complex and Comparative Microsatellite Frequency with Aedes aegypti and Anopheles gambiae

Background

Mosquitoes in the Culex pipiens complex are among the most medically important vectors for human disease worldwide and include major vectors for lymphatic filariasis and West Nile virus transmission. However, detailed genetic studies in the complex are limited by the number of genetic markers available. Here, we describe methods for the rapid and efficient identification and development of single locus, highly polymorphic microsatellite markers for Cx. pipiens complex mosquitoes via in silico screening of the Cx. quinquefasciatus genome sequence.

Methodology/Principal Findings

Six lab colonies representing four Cx. pipiens and two Cx. quinquefasciatus populations were utilized for preliminary assessment of 38 putative loci identified within 16 Cx. quinquefasciatus supercontig assemblies (CpipJ1) containing previously mapped genetic marker sequences. We identified and validated 12 new microsatellite markers distributed across all three linkage groups that amplify consistently among strains representing the complex. We also developed four groups of 3–5 microsatellite loci each for multiplex-ready PCR. Field collections from three cities in Indiana were used to assess the multiplex groups for their application to natural populations. All were highly polymorphic (Mean  = 13.0 alleles) per locus and reflected high polymorphism information content (PIC) (Mean  = 0.701). Pairwise F_ST indicated population structuring between Terre Haute and Fort Wayne and between Terre Haute and Indianapolis, but not between Fort Wayne and Indianapolis. In addition, we performed whole genome comparisons of microsatellite motifs and abundance between Cx. quinquefasciatus and the primary vectors for dengue virus and malaria parasites, Aedes aegypti and Anopheles gambiae, respectively.

Conclusions/Significance

We demonstrate a systematic approach for isolation and validation of microsatellites for the Cx. pipiens complex by direct screen of the Cx. quinquefasciatus genome supercontig assemblies. The genome density of microsatellites is greater in Cx. quinquefasciatus (0.26%) than in Ae. aegypti (0.14%), but considerably lower than in An. gambiae (0.77%).     

Discriminating between the Effects of Founding Events and Reproductive Mode on the Genetic Structure of Triops Populations (Branchiopoda: Notostraca)

Crustaceans that initially colonize a freshwater temporary pond can strongly bias the subsequent genetic composition of the population, causing nearby populations to be genetically distinct. In addition, these crustaceans have various reproductive modes that can influence genetic differentiation and diversity within and between populations. We report on two species of tadpole shrimp, Triops newberryi and Triops longicaudatus “short”, with different reproductive modes. Reproduction in the tadpole shrimp can occur clonally (parthenogenesis), with self fertilization (hermaphroditism), or through outcrossing of hermaphrodites with males (androdioecy). For all these reproductive modes, population genetic theory predicts decreased genetic diversity and increased population differentiation. Here we use mitochondrial control region (mtCR) sequences and nuclear microsatellite loci to determine if the difference in reproductive mode affects the high genetic structure typical of persistent founder effects. Previous authors indicated that T. newberryi is androdioecious because populations are composed of hermaphrodites and males, and T. longicaudatus “short” is hermaphroditic or parthenogenetic because males are absent. In our data, T. newberryi and T. longicaudatus “short” populations were highly structured genetically over short geographic distances for mtCR sequences and microsatellite loci (T. newberryi: Φ_ST = 0.644, F _ST = 0.252, respectively; T. l. “short”: invariant mtCR sequences, F _ST = 0.600). Differences between the two Triops species in a number of diversity measures were generally consistent with expectations from population genetic theory regarding reproductive mode; however, three of four comparisons were not statistically significant. We conclude the high genetic differentiation between populations is likely due to founder effects and results suggest both species are composed of selfing hermaphrodites with some level of outcrossing; the presence of males in T. newberryi does not appreciably reduce inbreeding. We cannot exclude the possibility that males in T. newberryi are non-reproductive individuals and the two species have the same mating system.     

Population Differentiation and Hybridisation of Australian Snubfin (Orcaella heinsohni) and Indo-Pacific Humpback (Sousa chinensis) Dolphins in North-Western Australia

Little is known about the Australian snubfin (Orcaella heinsohni) and Indo-Pacific humpback (Sousa chinensis) dolphins (‘snubfin’ and ‘humpback dolphins’, hereafter) of north-western Australia. While both species are listed as ‘near threatened’ by the IUCN, data deficiencies are impeding rigorous assessment of their conservation status across Australia. Understanding the genetic structure of populations, including levels of gene flow among populations, is important for the assessment of conservation status and the effective management of a species. Using nuclear and mitochondrial DNA markers, we assessed population genetic diversity and differentiation between snubfin dolphins from Cygnet (n = 32) and Roebuck Bays (n = 25), and humpback dolphins from the Dampier Archipelago (n = 19) and the North West Cape (n = 18). All sampling locations were separated by geographic distances >200 km. For each species, we found significant genetic differentiation between sampling locations based on 12 (for snubfin dolphins) and 13 (for humpback dolphins) microsatellite loci (F _ST = 0.05–0.09; P<0.001) and a 422 bp sequence of the mitochondrial control region (F _ST = 0.50–0.70; P<0.001). The estimated proportion of migrants in a population ranged from 0.01 (95% CI 0.00–0.06) to 0.13 (0.03–0.24). These are the first estimates of genetic diversity and differentiation for snubfin and humpback dolphins in Western Australia, providing valuable information towards the assessment of their conservation status in this rapidly developing region. Our results suggest that north-western Australian snubfin and humpback dolphins may exist as metapopulations of small, largely isolated population fragments, and should be managed accordingly. Management plans should seek to maintain effective population size and gene flow. Additionally, while interactions of a socio-sexual nature between these two species have been observed previously, here we provide strong evidence for the first documented case of hybridisation between a female snubfin dolphin and a male humpback dolphin.     

Malaria vector composition and insecticide susceptibility status in Guinea Conakry,West Africa

This study provides data on malaria vector species composition and insecticide susceptibility status from three localities in Guinea Conakry. A total of 497 mosquitoes were collected resting indoors and morphologically identified as belonging to the Anopheles gambiae complex. The majority of these were An. gambiae s.s. (99.6%), but a small percentage (0.4%) were identified as Anopheles arabiensis. Thirty‐four Anopheles funestus s.s. were also collected. The molecular S form of An. gambiae s.s. was predominant over the M form in Siguiri (95%) and Boffa (97.4%), whereas at Mt Nimba the M form was more abundant (61.4%) than the S form (38.1%). One hybrid M/S specimen was recorded from Mt Nimba. Siguiri populations showed high levels of resistance to DDT, dieldrin and bendiocarb. Anopheles gambiae from Boffa were largely susceptible to the insecticides tested. At Mt Nimba, resistance to DDT and bendicocarb was detected. Biochemical enzyme analysis showed that an altered acetylcholinesterase is operating in the field at low levels. The frequency of the 1014F kdr allele in the An. gambiae S form was 0.24 at Siguiri and 0.14 at Mt Nimba. A single RR specimen was found in the M form. The heterogeneity in species composition and resistance profiles between sites requires vector control interventions to be tailored to each site based on the data collected from ongoing monitoring and surveillance.