Accelerating the Morphogenetic Cycle of the Viral Vector Aedes aegypti Larvae for Faster Larvicidal Bioassays.

Any bioassay to test new chemically synthesized larvicides or phytolarvicides against Culicidae and more harmful mosquito species, such as Aedes aegypti and Aedes albopictus, which specifically transmit dengue, yellow fever, chikungunya viral fevers as well as Zika virus, or Anopheles gambiae, a vector for malaria and philariasis, requires thousands of well-developed larvae, preferably at the fourth instar stage. The natural morphogenetic cycle of Aedes spp., in the field or in the laboratory, may extend to 19 days at room temperature (e.g., 25°C) from the first permanent contact between viable eggs and water and the last stage of larval growth or metamorphosis into flying adults. Thus, accelerated sequential molting is desirable for swifter bioassays of larvicides. We achieved this goal in Aedes aegypti with very limited strategic and low-cost additions to food, such as coconut water, milk or its casein, yeast extract, and to a lesser extent, glycerol. The naturally rich coconut water was excellent for quickly attaining the population of instar IV larvae, the most advanced one before pupation, saving about a week, for subsequent larvicidal bioassays. Diluted milk, as another food source, allowed an even faster final ecdysis and adults are useful for mosquito taxonomical purpose.

Detection of diarrheagenic Escherichia coli using a two-system multiplex-PCR protocol.

BACKGROUND: Diarrheagenic Escherichia coli (DEC) strains are important causes of diarrhea. However, they cannot be distinguished from E. coli of the intestinal microbiota by conventional microbiological tests. METHODS: This work presents a two-system multiplex PCR for detection of DEC. Primers for 16S rRNA gene were added as internal amplification control to validate negative reactions. The multiplex-PCR system 1 contains primers for detection of Shiga toxin producing E. coli (STEC; stx1, stx2), enteropathogenic E. coli (EPEC; eae, bfpA), atypical enteropathogenic E. coli (aEPEc; eae), enteroinvasive E. coli (ETEC; lt, st), enteroinvasive E. coli (EIEC; ial), and the internal amplification control 16S rRNA. The system 2 contains primers for EIEC (ipaH), enteroaggregative E. coli (CVD432), diffusely adherent E. coli (daaE), and 16S rRNA. The protocol was tested with E. coli reference strains, and also with cultures of fecal specimens of people with diarrhea and healthy controls. RESULTS: The protocol correctly identified the DEC reference strains. No DEC marker was amplified for negative controls; these results were validated by the amplification of a fragment of the 16S rRNA gene. The frequency of DEC was 7.6% for both patients and healthy controls; two Shigella sonnei strains were detected in the group with diarrhea. The identity of the amplicons was confirmed by DNA sequencing. CONCLUSION: The protocol is specific for DEC Shigella and is suitable for clinical laboratories.