A simplified high-throughput method for pyrethroid knock-down resistance (kdr) detection in Anopheles gambiae

Background

A single base pair mutation in the sodium channel confers knock-down resistance to pyrethroids in many insect species. Its occurrence in Anopheles mosquitoes may have important implications for malaria vector control especially considering the current trend for large scale pyrethroid-treated bednet programmes. Screening Anopheles gambiae populations for the kdr mutation has become one of the mainstays of programmes that monitor the development of insecticide resistance. The screening is commonly performed using a multiplex Polymerase Chain Reaction (PCR) which, since it is reliant on a single nucleotide polymorphism, can be unreliable. Here we present a reliable and potentially high throughput method for screening An. gambiae for the kdr mutation.

Methods

A Hot Ligation Oligonucleotide Assay (HOLA) was developed to detect both the East and West African kdr alleles in the homozygous and heterozygous states, and was optimized for use in low-tech developing world laboratories. Results from the HOLA were compared to results from the multiplex PCR for field and laboratory mosquito specimens to provide verification of the robustness and sensitivity of the technique.

Results and Discussion

The HOLA assay, developed for detection of the kdr mutation, gives a bright blue colouration for a positive result whilst negative reactions remain colourless. The results are apparent within a few minutes of adding the final substrate and can be scored by eye. Heterozygotes are scored when a sample gives a positive reaction to the susceptible probe and the kdr probe. The technique uses only basic laboratory equipment and skills and can be carried out by anyone familiar with the Enzyme-linked immunosorbent assay (ELISA) technique. A comparison to the multiplex PCR method showed that the HOLA assay was more reliable, and scoring of the plates was less ambiguous.

Conclusion

The method is capable of detecting both the East and West African kdr alleles in the homozygous and heterozygous states from fresh or dried material using several DNA extraction methods. It is more reliable than the traditional PCR method and may be more sensitive for the detection of heterozygotes. It is inexpensive, simple and relatively safe making it suitable for use in resource-poor countries.     

Proteomic based investigation of rhamnolipid production by Pseudomonas chlororaphis strain NRRL B-30761

We recently reported that a strain of the non-pathogenic bacterial species Pseudomonas chlororaphis was capable of producing the biosurfactant molecule, rhamnolipids. Previous to this report the organisms known to produce rhamnolipids were almost exclusively pathogens. The newly described P. chlororaphis strain produced rhamnolipids at room temperature in static minimal media, as opposed to previous reports of rhamnolipid production which occurred at elevated temperatures with mechanical agitation. The non-pathogenic nature and energy conserving production conditions make the P. chlororaphis strain an attractive candidate for commercial rhamnolipid production. However, little characterization of molecular/biochemical processes in P. chlororaphis have been reported. In order to achieve a greater understanding of the process by which P. chlororaphis produces rhamnolipids, a survey of proteins differentially expressed during rhamnolipid production was performed. Separation and measurement of the bacteria’s proteome was achieved using Beckman Coulter’s Proteome Lab PF2D packed column-based protein fractionation system. Statistical analysis of the data identified differentially expressed proteins and known orthologues of those proteins were identified using an AB 4700 Proteomics Analyzer mass spectrometer system. A list of proteins differentially expressed by P. chlororaphis strain NRRL B-30761 during rhamnolipid production was generated, and confirmed through a repetition of the entire separation process.Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Epilithic and Endolithic Bacterial Communities in Limestone from a Maya Archaeological Site

Biodeterioration of archaeological sites and historic buildings is a major concern for conservators, archaeologists, and scientists involved in preservation of the world's cultural heritage. The Maya archaeological sites in southern Mexico, some of the most important cultural artifacts in the Western Hemisphere, are constructed of limestone. High temperature and humidity have resulted in substantial microbial growth on stone surfaces at many of the sites. Despite the porous natureof limestone and the common occurrence of endolithic microorganisms in many habitats, little is known about the microbial flora living inside the stone. We found a large endolithic bacterial community in limestone from the interior of the Maya archaeological site Ek' Balam. Analysis of 16S rDNA clones demonstrated disparate communities (endolithic: >80% Actinobacteria, Acidobacteria, and Low GC Firmicutes; epilithic: >50% Proteobacteria). The presence of differing epilithic and endolithic bacterial communities may be a significant factor for conservation of stone cultural heritage materials and quantitative prediction of carbonate weathering.     

Effects of AeDNV infection on Aedes aegypti lifespan and reproduction

The effects of Aedes Densovirus (AeDNV) infections on survival, fertility, fecundity and vertical transmission in Aedes aegypti (Diptera: Culicidae) were measured in laboratories in Kiev, Ukraine and Colorado, USA and incorporated into a predictive model of the effects of AeDNV on vector capacity. Adult lifespan and daily survival were reduced in AeDNV infected mosquitoes. This effect was dependent on the dose of the virus. Infected females had decreased fecundity. The oviposition rate was less in infected females and the hatch rate declined in eggs laid by infected females. The amounts of AeDNV in infected females and the infection rate of their offspring were measured with real-time PCR. The average filial transmission rate was 70% and larval infection rates from infected females varied between 42 and 62%. Vertically infected larvae, and individual eggs contained 1 × 10⁵ AeDNV genome equivalents (geq). Modeling the effects of AeDNV infection on Ae. aegypti populations suggested a large decrease in the numbers of eggs, larvae, pupae, and adults arising from infected mothers and suggested that AeDNV treatment of larvae could cause up to a 76% reduction of infectious mosquito days.     

Shaking a leg and hot to trot: the effects of body size and temperature on running speed in ants

Abstract.  1. Data were compiled from the literature and our own studies on 24 ant species to characterise the effects of body size and temperature on forager running speed.
2. Running speed increases with temperature in a manner consistent with the effects of temperature on metabolic rate and the kinetic properties of muscles.
3. The exponent of the body mass-running speed allometry ranged from 0.14 to 0.34 with a central tendency of approximately 0.25. This body mass scaling is consistent with both the model of elastic similarity, and a model combining dynamic similarity with available metabolic power.
4. Even after controlling for body size or temperature, a substantial amount of inter-specific variation in running speed remains. Species with certain lifestyles [e.g. nomadic group predators, species which forage at extreme (>60 °C) temperatures] may have been selected for faster running speeds.
5. Although ants have a similar scaling exponent to mammals for the running speed allometry, they run slower than predicted compared with a hypothetical mammal of similar size. This may in part reflect physiological differences between invertebrates and vertebrates.     

Mosquitoes and West Nile virus along a river corridor from prairie to montane habitats in eastern Colorado

We conducted studies on mosquitoes and West Nile virus (WNV) along a riparian corridor following the South Platte River and Big Thompson River in northeastern Colorado and extending from an elevation of 1,215 m in the prairie landscape of the eastern Colorado plains to 1,840 m in low montane areas at the eastern edge of the Rocky Mountains in the central part of the state. Mosquito collection during June‐September 2007 in 20 sites along this riparian corridor yielded a total of 199,833 identifiable mosquitoes of 17 species. The most commonly collected mosquitoes were, in descending order: Aedes vexans, Culex tarsalis, Ae. dorsalis, Ae. trivittatus, Ae. melanimon, Cx. pipiens, and Culiseta inornata. Species richness was higher in the plains than in foothills‐montane areas, and abundances of several individual species, including the WNV vectors Cx. tarsalis and Cx. pipiens and the nuisance‐biter and potential secondary WNV vector Ae. vexans, decreased dramatically from the plains (1,215‐1,487 m) to foothills‐montane areas (1,524‐1,840 m). Ae. vexans and Cx. tarsalis had a striking pattern of uniformly high abundances between 1,200‐1,450 m followed by a gradual decrease in abundance above 1,450 m to reach very low numbers above 1,550 m. Culex species were commonly infected with WNV in the plains portion of the riparian corridor in 2007, with 14 of 16 sites yielding WNV‐infected Cx. tarsalis and infection rates for Cx. tarsalis females exceeding 2.0 per 1,000 individuals in ten of the sites. The Vector Index for abundance of WNV‐infected Cx. tarsalis females during June‐September exceeded 0.5 in six plains sites along the South Platte River but was uniformly low (0–0.1) in plains, foothills and montane sites above 1,500 m along the Big Thompson River. A population genetic analysis of Cx. tarsalis revealed that all collections from the ≈190 km riparian transect in northeastern Colorado were genetically uniform but that these collections were genetically distinct from collections from Delta County on the western slope of the Continental Divide. This suggests that major waterways in the Great Plains serve as important dispersal corridors for Cx. tarsalis but that the Continental Divide is a formidable barrier to this WNV vector.