Authentication scheme for routine verification of genetically similar laboratory colonies: a trial with Anopheles gambiae.

BACKGROUND: When rearing morphologically indistinguishable laboratory strains concurrently, the threat of unintentional genetic contamination is constant. Avoidance of accidental mixing of strains is difficult due to the use of common equipment, technician error, or the possibility of self relocation by adult mosquitoes ("free fliers"). In many cases, laboratory strains are difficult to distinguish because of morphological and genetic similarity, especially when laboratory colonies are isolates of certain traits from the same parental strain, such as eye color mutants, individuals with certain chromosomal arrangements or high levels of insecticide resistance. Thus, proving genetic integrity could seem incredibly time-consuming or impossible. On the other hand, lacking proof of genetically isolated laboratory strains could question the validity of research results. RESULTS: We present a method for establishing authentication matrices to routinely distinguish and confirm that laboratory strains have not become physically or genetically mixed through contamination events in the laboratory. We show a specific example with application to Anopheles gambiae sensu stricto strains at the Malaria Research and Reference Reagent Resource Center. This authentication matrix is essentially a series of tests yielding a strain-specific combination of results. CONCLUSION: These matrix-based methodologies are useful for several mosquito and insect populations but must be specifically tailored and altered for each laboratory based on the potential contaminants available at any given time. The desired resulting authentication plan would utilize the least amount of routine effort possible while ensuring the integrity of the strains.

IMP PCR primers detect single nucleotide polymorphisms for Anopheles gambiae species identification, Mopti and Savanna rDNA types, and resistance to dieldrin in Anopheles arabiensis.

BACKGROUND: Polymerase chain reactions to distinguish single-nucleotide polymorphisms are commonly used for mosquito identification and identifying insecticide resistance alleles. However, the existing methods used for primer design often result in analyses that are not robust or require additional steps. METHODS: Utilizing oligonucleotides that are unique in having an intentional mismatch to both templates three bases from the SNP at the 3-prime end, three new PCR assays that distinguish SNP targets using standard gel electrophoresis of undigested DNA fragments were developed and tested. These were applied to: (1) an alternative ribosomal DNA PCR assay to distinguish five members of the Anopheles gambiae complex; (2) detection of the Mopti and Savanna rDNA types; and (3) an assay to distinguish resistance to dieldrin (Rdl) alleles in Anopheles arabiensis. RESULTS: Reproducible specific amplification of the target alleles was observed in all three assays. The results were consistent with existing analyses but proved simpler and the results more distinct in our hands. CONCLUSION: The simplicity and effectiveness of the method should be utilized in these and other PCR analyses to increase their specificity and simplicity. These results have the potential to be extended not only to mosquito analyses but also to parasite and human polymorphisms.

Methylparaben in Anopheles gambiae s.l. sugar meals increases longevity and malaria oocyst abundance but is not a preferred diet.

The antimicrobial and antifungal chemical methylparaben (methyl-4-hydroxybenzoate) was added to the adult sucrose diet of Anopheles gambiae and Anopheles arabiensis, and its effect on longevity was determined. In all cases, significant increases in longevity were observed when 0.2% (w/v) methylparaben was added to meals that were refreshed weekly. When fresh sugar diet was refreshed daily, no increase in longevity was observed due to methylparaben suggesting that the effect of methylparaben is to preserve the quality of the sugar diet. No longevity effect of providing pure water in addition to sugar- or methylparaben-supplemented meals was observed. Feeding preference tests were performed to determine whether meals containing methylparaben were preferred, and whether, when given no choice but the less-preferred diet, mosquitoes would consume less sugar. Using the stable carbon isotope (13)C in paired tests, we show that the sugar diet containing methylparaben was clearly avoided by A. gambiae but not A. arabiensis. Little effect of methylparaben on the total amount of sugar consumed was observed when mosquitoes were given no diet choice. Methylparaben effects on Plasmodium cynomolgi B oocyst formation and encapsulation were observed in a normal A. gambiae stock and one which encapsulates at a high frequency. Nearly two-fold increases in the number of both normal and encapsulated oocysts were observed as a result of methylparaben in the diet. Because of its longevity effects, we have implemented methylparaben use for all mosquitoes in our holdings and recommend it as a routine sugar meal supplement.