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%).     

Population Structure and Distribution Patterns of the Sibling Mosquito Species Culex pipiens and Culex torrentium (Diptera: Culicidae) Reveal Different Evolutionary Paths

Nowadays a number of endemic mosquito species are known to possess vector abilities for various diseases, as e.g. the sibling species Culex pipiens and Culex torrentium. Due to their morphological similarity, ecology, distribution and vector abilities, knowledge about these species'' population structure is essential. Culicidae from 25 different sampling sites were collected from March till October 2012. All analyses were performed with aligned cox1 sequences with a total length of 658 bp. Population structure as well as distribution patterns of both species were analysed using molecular methods and different statistical tests like distance based redundancy analysis (dbDRA), analysis of molecular variances (AMOVA) or McDonald & Kreitman test and Tajima''s D. Within both species, we could show a genetic variability among the cox1 fragment. The construction of haplotype networks revealed one dominating haplotype for Cx. pipiens, widely distributed within Germany and a more homogeneous pattern for Cx. torrentium. The low genetic differences within Cx. pipiens could be a result of an infection with Wolbachia which can induce a sweep through populations by passively taking the also maternally inherited mtDNA through the population, thereby reducing the mitochondrial diversity as an outcome of reproductive incompatibility. Pairwise population genetic differentiation (F_ST) ranged significantly from moderate to very great between populations of Cx. pipiens and Cx. torrentium. Analyses of molecular variances revealed for both species that the main genetic variability exists within the populations (Cx. pipiens [88.38%]; Cx. torrentium [66.54%]). Based on a distance based redundancy analysis geographical origin explained a small but significant part of the species'' genetic variation. Overall, the results confirm that Cx. pipiens and Cx. torrentium underlie different factors regarding their mitochondrial differentiation, which could be a result of endosymbiosis, dispersal between nearly located populations or human introduction.     

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.     

Efficacy of leaves extract of Calotropis procera Ait. (Asclepiadaceae) in controlling Anopheles arabiensis and Culex quinquefasciatus mosquitoes

The present study aimed to investigate, the larvicidal, adult emergence inhibition and oviposition deterrent activity of aqueous leaves extract of Calotropis procera against Anopheles arabiensis and Culex quinquefasciatus as natural mosquito larvicide. The larvicidal activity was monitored against 2nd, 3rd and 4th instar larvae of each mosquito species 24 h post-treatment. Adult emergence inhibition activity was tested by exposing 3rd instar larvae of each mosquito species to different concentrations of extracts (200, 400, 600, 800 and 1000 ppm for An. arabiensis and 100, 200, 300, 400, 500 and 600 ppm for Cx. quinquefasciatus). Probit analysis was used to analyze data from bioassay experiments. The oviposition deterrent activity was tested by using three different concentrations of extracts (1000, 500 and 200 for An. arabiensis, and 1000, 500 and 100 for Cx. quinquefasciatus) that caused high, moderate and low larval mortality in the larvicidal experiment against 3rd instar larvae. It was found that, LC50–LC90 values calculated were 273.53–783.43, 366.44–1018.59 and 454.99–1224.62 ppm for 2nd, 3rd and 4th larval instars, respectively, of An. arabiensis and 187.93–433.51, 218.27–538.27 and 264.85–769.13 ppm for 2nd, 3rd and 4th larval instars, respectively, of Cx. quinquefasciatus. Fifty percent of adult emergence inhibition (EI50) was shown at 277.90 and 183.65 ppm for An. arabiensis and Cx. quinquefasciatus, respectively. The pupal stage was not affected till a concentration of 5000 ppm. The extract showed oviposition deterrence and effective repellence against both mosquito species at different concentrations, with the observation on that maximal eggs were laid in low concentration of extract. These results suggest that the leaves extract of C. procera possess remarkable larvicidal, adult emergence inhibitor, repellent and oviposition deterrent effect against both An. arabiensis and Cx. quinquefasciatus, and might be used as natural biocides for mosquito control.     

Modeling Dynamics of Culex pipiens Complex Populations and Assessing Abatement Strategies for West Nile Virus

The primary mosquito species associated with underground stormwater systems in the United States are the Culex pipiens complex species. This group represents important vectors of West Nile virus (WNV) throughout regions of the continental U.S. In this study, we designed a mathematical model and compared it with surveillance data for the Cx. pipiens complex collected in Beaufort County, South Carolina. Based on the best fit of the model to the data, we estimated parameters associated with the effectiveness of public health insecticide (adulticide) treatments (primarily pyrethrin products) as well as the birth, maturation, and death rates of immature and adult Cx. pipiens complex mosquitoes. We used these estimates for modeling the spread of WNV to obtain more reliable disease outbreak predictions and performed numerical simulations to test various mosquito abatement strategies. We demonstrated that insecticide treatments produced significant reductions in the Cx. pipiens complex populations. However, abatement efforts were effective for approximately one day and the vector mosquitoes rebounded until the next treatment. These results suggest that frequent insecticide applications are necessary to control these mosquitoes. We derived the basic reproductive number (ℜ₀) to predict the conditions under which disease outbreaks are likely to occur and to evaluate mosquito abatement strategies. We concluded that enhancing the mosquito death rate results in lower values of ℜ₀, and if ℜ₀<1, then an epidemic will not occur. Our modeling results provide insights about control strategies of the vector populations and, consequently, a potential decrease in the risk of a WNV outbreak.     

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.     

Invasion Pathway of the Ctenophore Mnemiopsis leidyi in the Mediterranean Sea

Gelatinous zooplankton outbreaks have increased globally owing to a number of human-mediated factors, including food web alterations and species introductions. The invasive ctenophore Mnemiopsis leidyi entered the Black Sea in the early 1980s. The invasion was followed by the Azov, Caspian, Baltic and North Seas, and, most recently, the Mediterranean Sea. Previous studies identified two distinct invasion pathways of M. leidyi from its native range in the western Atlantic Ocean to Eurasia. However, the source of newly established populations in the Mediterranean Sea remains unclear. Here we build upon our previous study and investigate sequence variation in both mitochondrial (Cytochrome c Oxidase subunit I) and nuclear (Internal Transcribed Spacer) markers in M. leidyi, encompassing five native and 11 introduced populations, including four from the Mediterranean Sea. Extant genetic diversity in Mediterranean populations (n = 8, N _a = 10) preclude the occurrence of a severe genetic bottleneck or founder effects in the initial colonizing population. Our mitochondrial and nuclear marker surveys revealed two possible pathways of introduction into Mediterranean Sea. In total, 17 haplotypes and 18 alleles were recovered from all surveyed populations. Haplotype and allelic diversity of Mediterranean populations were comparable to populations from which they were likely drawn. The distribution of genetic diversity and pattern of genetic differentiation suggest initial colonization of the Mediterranean from the Black-Azov Seas (pairwise F _ST = 0.001–0.028). However, some haplotypes and alleles from the Mediterranean Sea were not detected from the well-sampled Black Sea, although they were found in Gulf of Mexico populations that were also genetically similar to those in the Mediterranean Sea (pairwise F _ST = 0.010–0.032), raising the possibility of multiple invasion sources. Multiple introductions from a combination of Black Sea and native region sources could be facilitated by intense local and transcontinental shipping activity, respectively.     

Dissecting the Gene Dose-Effects of the APOE ε4 and ε2 Alleles on Hippocampal Volumes in Aging and Alzheimer’s Disease

Objective

To investigate whether there is a specific dose-dependent effect of the Apolipoprotein E (APOE) ε4 and ε2 alleles on hippocampal volume, across the cognitive spectrum, from normal aging to Alzheimer’s Disease (AD).

Materials and Methods

We analyzed MR and genetic data on 662 patients from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) database–198 cognitively normal controls (CN), 321 mild-cognitive impairment (MCI) subjects, and 143 AD subjects–looking for dose-dependent effects of the ε4 and ε2 alleles on hippocampal volumes. Volumes were measured using a fully-automated algorithm applied to high resolution T1-weighted MR images. Statistical analysis consisted of a multivariate regression with repeated-measures model.

Results

There was a dose-dependent effect of the ε4 allele on hippocampal volume in AD (p = 0.04) and MCI (p = 0.02)–in both cases, each allele accounted for loss of >150 mm³ (approximately 4%) of hippocampal volume below the mean volume for AD and MCI subjects with no such alleles (Cohen’s d = −0.16 and −0.19 for AD and MCI, respectively). There was also a dose-dependent, main effect of the ε2 allele (p<0.0001), suggestive of a moderate protective effect on hippocampal volume–an approximately 20% per allele volume increase as compared to CN with no ε2 alleles (Cohen’s d = 0.23).

Conclusion

Though no effect of ε4 was seen in CN subjects, our findings confirm and extend prior data on the opposing effects of the APOE ε4 and ε2 alleles on hippocampal morphology across the spectrum of cognitive aging.     

Avian Host-Selection by Culex pipiens in Experimental Trials

Evidence from field studies suggests that Culex pipiens, the primary mosquito vector of West Nile virus (WNV) in the northeastern and north central United States, feeds preferentially on American robins (Turdus migratorius). To determine the contribution of innate preferences to observed preference patterns in the field, we conducted host preference trials with a known number of adult female C. pipiens in outdoor cages comparing the relative attractiveness of American robins with two common sympatric bird species, European starling, Sternus vulgaris and house sparrow, Passer domesticus. Host seeking C. pipiens were three times more likely to enter robin-baited traps when with the alternate host was a European starling (n = 4 trials; OR = 3.06; CI [1.42–6.46]) and almost twice more likely when the alternative was a house sparrow (n = 8 trials; OR = 1.80; CI = [1.22–2.90]). There was no difference in the probability of trap entry when two robins were offered (n = 8 trials). Logistic regression analysis determined that the age, sex and weight of the birds, the date of the trial, starting-time, temperature, humidity, wind-speed and age of the mosquitoes had no effect on the probability of a choosing a robin over an alternate bird. Findings indicate that preferential feeding by C. pipiens mosquitoes on certain avian hosts is likely to be inherent, and we discuss the implications innate host preferences may have on enzootic WNV transmission.     

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.     

Gender-Specific Effects of Genetic Variants within Th1 and Th17 Cell-Mediated Immune Response Genes on the Risk of Developing Rheumatoid Arthritis

The present study was conducted to explore whether single nucleotide polymorphisms (SNPs) in Th1 and Th17 cell-mediated immune response genes differentially influence the risk of rheumatoid arthritis (RA) in women and men. In phase one, 27 functional/tagging polymorphisms in C-type lectins and MCP-1/CCR2 axis were genotyped in 458 RA patients and 512 controls. Carriers of Dectin-2 _rs4264222T allele had an increased risk of RA (OR = 1.47, 95%CI 1.10–1.96) whereas patients harboring the DC-SIGN _rs4804803G, MCP-1 _rs1024611G, MCP-1 _rs13900T and MCP-1 _rs4586C alleles had a decreased risk of developing the disease (OR = 0.66, 95%CI 0.49–0.88; OR = 0.66, 95%CI 0.50–0.89; OR = 0.73, 95%CI 0.55–0.97 and OR = 0.68, 95%CI 0.51–0.91). Interestingly, significant gender-specific differences were observed for Dectin-2 _rs4264222 and Dectin-2 _rs7134303: women carrying the Dectin-2 _rs4264222T and Dectin-2 _rs7134303G alleles had an increased risk of RA (OR = 1.93, 95%CI 1.34–2.79 and OR = 1.90, 95%CI 1.29–2.80). Also five other SNPs showed significant associations only with one gender: women carrying the MCP-1 _rs1024611G, MCP-1 _rs13900T and MCP-1 _rs4586C alleles had a decreased risk of RA (OR = 0.61, 95%CI 0.43–0.87; OR = 0.67, 95%CI 0.47–0.95 and OR = 0.60, 95%CI 0.42–0.86). In men, carriers of the DC-SIGN _rs2287886A allele had an increased risk of RA (OR = 1.70, 95%CI 1.03–2.78), whereas carriers of the DC-SIGN _rs4804803G had a decreased risk of developing the disease (OR = 0.53, 95%CI 0.32–0.89). In phase 2, we genotyped these SNPs in 754 RA patients and 519 controls, leading to consistent gender-specific associations for Dectin-2 _rs4264222, MCP-1 _rs1024611, MCP-1 _rs13900 and DC-SIGN _rs4804803 polymorphisms in the pooled sample (OR = 1.38, 95%CI 1.08–1.77; OR = 0.74, 95%CI 0.58–0.94; OR = 0.76, 95%CI 0.59–0.97 and OR = 0.56, 95%CI 0.34–0.93). SNP-SNP interaction analysis of significant SNPs also showed a significant two-locus interaction model in women that was not seen in men. This model consisted of Dectin-2 _rs4264222 and Dectin-2 _rs7134303 SNPs and suggested a synergistic effect between the variants. These findings suggest that Dectin-2, MCP-1 and DC-SIGN polymorphisms may, at least in part, account for gender-associated differences in susceptibility to RA.     

Worldwide Distribution of the MYH9 Kidney Disease Susceptibility Alleles and Haplotypes: Evidence of Historical Selection in Africa

MYH9 was recently identified as renal susceptibility gene (OR 3–8, p<10⁻⁸) for major forms of kidney disease disproportionately affecting individuals of African descent. The risk haplotype (E-1) occurs at much higher frequencies in African Americans (≥60%) than in European Americans (<4%), revealing a genetic basis for a major health disparity. The population distributions of MYH9 risk alleles and the E-1 risk haplotype and the demographic and selective forces acting on the MYH9 region are not well explored. We reconstructed MYH9 haplotypes from 4 tagging single nucleotide polymorphisms (SNPs) spanning introns 12–23 using available data from HapMap Phase II, and by genotyping 938 DNAs from the Human Genome Diversity Panel (HGDP). The E-1 risk haplotype followed a cline, being most frequent within sub-Saharan African populations (range 50–80%), less frequent in populations from the Middle East (9–27%) and Europe (0–9%), and rare or absent in Asia, the Americas, and Oceania. The fixation indexes (F_ST) for pairwise comparisons between the risk haplotypes for continental populations were calculated for MYH9 haplotypes; F_ST ranged from 0.27–0.40 for Africa compared to other continental populations, possibly due to selection. Uniquely in Africa, the Yoruba population showed high frequency extended haplotype length around the core risk allele (C) compared to the alternative allele (T) at the same locus (rs4821481, iHs = 2.67), as well as high population differentiation (F_{ST(CEU vs. YRI)} = 0.51) in HapMap Phase II data, also observable only in the Yoruba population from HGDP (F_ST = 0.49), pointing to an instance of recent selection in the genomic region. The population-specific divergence in MYH9 risk allele frequencies among the world''s populations may prove important in risk assessment and public health policies to mitigate the burden of kidney disease in vulnerable populations.     

Type II Transmembrane Serine Protease Gene Variants Associate with Breast Cancer

Type II transmembrane serine proteases (TTSPs) are related to tumor growth, invasion, and metastasis in cancer. Genetic variants in these genes may alter their function, leading to cancer onset and progression, and affect patient outcome. Here, 464 breast cancer cases and 370 controls were genotyped for 82 single-nucleotide polymorphisms covering eight genes. Association of the genotypes was estimated against breast cancer risk, breast cancer–specific survival, and survival in different treatment groups, and clinicopathological variables. SNPs in TMPRSS3 (rs3814903 and rs11203200), TMPRSS7 (rs1844925), and HGF (rs5745752) associated significantly with breast cancer risk (P _trend = 0.008–0.042). SNPs in TMPRSS1 (rs12151195 and rs12461158), TMPRSS2 (rs2276205), TMPRSS3 (rs3814903), and TMPRSS7 (rs2399403) associated with prognosis (P = 0.004–0.046). When estimating the combined effect of the variants, the risk of breast cancer was higher with 4–5 alleles present compared to 0–2 alleles (P = 0.0001; OR, 2.34; 95% CI, 1.39–3.94). Women with 6–8 survival-associating alleles had a 3.3 times higher risk of dying of breast cancer compared to women with 1–3 alleles (P = 0.001; HR, 3.30; 95% CI, 1.58–6.88). The results demonstrate the combined effect of variants in TTSPs and their related genes in breast cancer risk and patient outcome. Functional analysis of these variants will lead to further understanding of this gene family, which may improve individualized risk estimation and development of new strategies for treatment of breast cancer.     

Characterization of DNA Binding Property of the HIV-1 Host Factor and Tumor Suppressor Protein Integrase Interactor 1 (INI1/hSNF5)

Integrase Interactor 1 (INI1/hSNF5) is a component of the hSWI/SNF chromatin remodeling complex. The INI1 gene is either deleted or mutated in rhabdoid cancers like ATRT (Atypical terratoid and rhabdoid tumor). INI1 is also a host factor for HIV-1 replication. INI1 binds DNA non-specifically. However, the mechanism of DNA binding and its biological role are unknown. From agarose gel retardation assay (AGRA), Ni-NTA pull-down and atomic force microscopy (AFM) studies we show that amino acids 105–183 of INI1 comprise the minimal DNA binding domain (DBD). The INI1 DBD is absent in plants and in yeast SNF5. It is present in Caenorhabditis elegans SNF5, Drosophila melanogaster homologue SNR1 and is a highly conserved domain in vertebrates. The DNA binding property of this domain in SNR1, that is only 58% identical to INI1/hSNF5, is conserved. Analytical ultracentrifugation studies of INI1 DBD and INI1 DBD:DNA complexes at different concentrations show that the DBD exists as a monomer at low protein concentration and two molecules of monomer binds one molecule of DNA. At high protein concentration, it exists as a dimer and binds two DNA molecules. Furthermore, isothermal calorimetry (ITC) experiments demonstrate that the DBD monomer binds DNA with a stoichiometry (N) of ∼0.5 and K_d  = 0.94 µM whereas the DBD dimer binds two DNA molecules sequentially with K’_d1 = 222 µM and K’_d2 = 1.16 µM. Monomeric DBD binding to DNA is enthalpy driven (ΔH = –29.9 KJ/mole). Dimeric DBD binding to DNA is sequential with the first binding event driven by positive entropy (ΔH’₁ = 115.7 KJ/mole, TΔS’₁ = 136.8 KJ/mole) and the second binding event driven by negative enthalpy (ΔH’₂ = –106.3 KJ/mole, TΔS’₂ = –75.7 KJ/mole). Our model for INI1 DBD binding to DNA provides new insights into the mechanism of DNA binding by INI1.     

A rural–urban latitudinal study of the distributions of Culex quinquefasciatus and Culex pipiens bioforms in their southernmost sympatric fringe

Mosquitoes grouped in the complex Culex pipiens L. (Diptera: Culicidae) are important vectors of medical and veterinary diseases. In the South American sympatric region, Cx. pipiens and Culex quinquefasciatus Say coexist and potentially hybridize. To identify key drivers of their geographical distribution, mosquito immatures were collected from flower vases of eight urban/rural cemetery pairs within a 5° latitudinal transect along Buenos Aires Province, Argentina. The specimens were identified by molecular methods and their relative proportion modelled as a function of environmental variables. At the beginning of the warm season, northern and southern cemeteries presented exclusively Cx. quinquefasciatus and Cx. pipiens, respectively, with different proportions of both at mid latitudes. By the end of the summer, Cx. quinquefasciatus was present throughout the study area, exclusively in 11 of the 16 cemeteries both rural and urban, whereas Cx. pipiens was predominant only in the southernmost pair. Mean annual temperature, photoperiod variability and time of the season were key drivers of their distributions. All specimens of Cx. pipiens were identified as form molestus and no hybrids were recognized. The reported distribution patterns and the potential absence of Cx. pipiens f. pipiens and hybrids are discussed, along with their implications in disease transmission.     

Enzymatic Characterization of Insecticide Resistance Mechanisms in Field Populations of Malaysian Culex quinquefasciatus Say (Diptera: Culicidae)

Background

There has been no comprehensive study on biochemical characterization of insecticide resistance mechanisms in field populations of Malaysian Culex quinquefasciatus. To fill this void in the literature, a nationwide investigation was performed to quantify the enzyme activities, thereby attempting to characterize the potential resistance mechanisms in Cx. quinquefasciatus in residential areas in Malaysia.

Methodology/Principal Findings

Culex quinquefasciatus from 14 residential areas across 13 states and one federal territory were subjected to esterases, mixed function oxidases, glutathione-S-transferase and insensitive acetylcholinesterase assays. Enzyme assays revealed that α-esterases and β-esterases were elevated in 13 populations and 12 populations, respectively. Nine populations demonstrated elevated levels of mixed function oxidases and glutathione-S-transferase. Acetylcholinesterase was insensitive to propoxur in all 14 populations. Activity of α-esterases associated with malathion resistance was found in the present study. In addition, an association between the activity of α-esterases and β-esterases was also demonstrated.

Conclusions/Significance

The present study has characterized the potential biochemical mechanisms in contributing towards insecticide resistance in Cx. quinquefasciatus field populations in Malaysia. Identification of mechanisms underlying the insecticide resistance will be beneficial in developing effective mosquito control programs in Malaysia.     

Favorable QTL Alleles for Yield and Its Components Identified by Association Mapping in Chinese Upland Cotton Cultivars

Linkage disequilibrium based association mapping is a powerful tool for dissecting the genetic basis underlying complex traits. In this study, an association mapping panel consisting of 356 representative Upland cotton cultivars was constructed, evaluated in three environments and genotyped using 381 SSRs to detect molecular markers associated with lint yield and its components. The results showed that abundant phenotypic and moderate genetic diversities existed within this germplasm panel. The population could be divided into two subpopulations, and weak relatedness was detected between pair-wise accessions. LD decayed to the background (r ² = 0.1182, P≤0.01), r ² = 0.1 and r ² = 0.2 level within 12–13 cM, 17–18 cM and 3–4 cM, respectively, providing the potential for association mapping of agronomically important traits in Chinese Upland cotton. A total of 55 marker-trait associations were detected between 26 SSRs and seven lint yield traits, based on a mixed linear model (MLM) and Bonferroni correction (P≤0.05/145, −log₁₀ P≥3.46). Of which 41 could be detected in more than one environment and 17 markers were simultaneously associated with two or more traits. Many associations were consistent with QTLs identified by linkage mapping in previous reports. Phenotypic values of alleles of each loci in 41 stably detected associations were compared, and 23 favorable alleles were identified. Population frequency of each favorable allele in historically released cultivar groups was also evaluated. The QTLs detected in this study will be helpful in further understanding the genetic basis of lint yield and its components, and the favorable alleles may facilitate future high-yield breeding by genomic selection in Upland cotton.     

Charaterization of mosquito larval habitats in Qatar

Mosquito borne diseases have remained a grave threat to human health and are posing a significant burden on health authorities around the globe. The understanding and insight of mosquito breeding habitats features is crucial for their effective management. Comprehensive larval surveys were carried out at 14 sites in Qatar. A total of 443 aquatic habitats were examined, among these 130 were found positive with Culex pipiens, Cx. quinquefasciatus, Cx. mattinglyi, Ochlerotatus dorsalis, Oc. caspius and Anopheles stephensi. The majority of positive breeding habitats were recorded in urban areas (67.6%), followed by livestock (13.8%), and least were in agriculture (10.7%). An. stephensi larvae were positively correlated with Cx. pipien, Cx. quinquefasciatus, and negatively with water salinity. Large and shaded habitats were the most preferred by An. stephensi. In addition, Cx. pipiens was positively associated with the turbidity and pH, and was negatively associated with vegetation and habitat size. A negative association of Cx. quinquefasciatus with dissolved oxygen, water temperature, and salinity, while positive with habitat surface area was observed. Oc. dorsalis was negatively correlated with pH, water temperature, depth, and habitat surface area, whereas salinity water was more preferable site for females to lay their eggs. These results demonstrate that environmental factors play a significant role in preference of both anopheline and culicine for oviposition, while their effective management must be developed as the most viable tool to minimize mosquito borne diseases.