The mosquito Aedes aegypti is the world's most important vector of yellow fever and dengue viruses. Work is currently in progress to control the transmission of these viruses by genetically altering the capacity of wild Ae. aegypti populations to support virus replication. The germline transformation system reported here constitutes a major advance toward the implementation of this control strategy. A modified Hermes transposon carrying a 4.7-kb fragment of genomic DNA that includes a wild-type allele of the Drosophila melanogaster cinnabar (cn) gene was used to transform a white-eyed recipient strain of Ae. aegypti. Microinjection of preblastoderm mosquito embryos with this construct resulted in 50% of the emergent G 0 adults showing some color in their eyes. Three transformed families were recovered, each resulting from an independent insertion event of the cn ؉ -carrying transposon. The cn ؉ gene functioned as a semidominant transgene and segregated in Mendelian ratios. Hermes shows great promise as a vector for efficient, heritable, and stable transformation of this important mosquito vector species.
Germline transformation of the major African malaria vector, Anopheles gambiae, was achieved using the piggyBac transposable element marked with the enhanced green fluorescent protein (EGFP) injected into mosquito embryos. Two G1 generation male mosquitoes expressing EGFP were identified among 34 143 larvae screened. Genomic Southern data and sequencing of the piggyBac insertion boundaries showed that these two males arose from one piggyBac insertion event in the injected G0 embryos. Genetic cross data suggest that the insertion site of the element either resulted in, or is tightly linked to, a recessive lethal. This was demonstrated by a deficiency in the number of EGFP-expressing offspring from inbred crosses but expected ratios in outcrosses to non-transformed individuals and failure to establish a pure-breeding line. The insertion was weakly linked to the collarless locus on chromosome 2 and was shown by in situ hybridization to be located in division 28D of that chromosome. Particularly high levels of expression were observed uniformly in salivary glands and, in most individuals, in the anterior stomach. An improvement in the injection technique at the end of the studies resulted in increased G0 hatching, transient expression and EGFP-expression rates among G1 progeny.
Anopheles stephensi is an invasive malaria vector that is endemic to south Asia and the Arabian Peninsula. It was recently reported in the Horn of Africa countries including Djibouti (2012), Ethiopia, Sudan (2019), Somalia (2019) and most recently Nigeria (2020). This mosquito is a competent vector for both Plasmodium falciparum and P. vivax. It is characterized by a high degree of behavioral plasticity and the ability to reproduce in various types of breeding sites including containers and therefore has the potential to propagate malaria transmission in rapidly urbanizing settings with poor drainage and disposal of waste containers. The World Health Organization (WHO) has called on all countries to scale up surveillance efforts to detect and report invasion by this vector and institute appropriate and effective control mechanisms. In Kenya, the Division for National Malaria Program (DNMP) and its partners have been conducting entomological surveillance in all coastal and northern counties that are suspected to be at risk of An. stephensi invasion as well as in all counties at risk of malaria. These efforts were supported by molecular surveillance of all unidentified Anopheles mosquitoes from other studies conducted by the Kenya Medical Research Institute (KEMRI) to try and identify An. stephensi. In this article, we report the first detection of An. stephensi in two sub counties of Marsabit County, Kenya in December 2022. We used Polymerase Chain Reaction (PCR) as the primary method of identification and confirmed results using morphological keys and sequencing of the ITS2 region. With the detection of this vector in Kenya, there is an urgent need for intensified surveillance to determine its occurrence and distribution and develop tailored approaches towards control to prevent further spread.
Abstract. A simple method for rapid identification of large numbers of Anopheles mosquitoes was developed based on polymerase chain reaction (PCR) amplification of the rDNA intergenic spacer and internal transcribed spacer 2. By means of previously described primers for the Anopheles gambiae and An. quadrimaculatus species complexes, rDNA was amplified simultaneously from 96 whole mosquitoes or parts. No homogenization or individual DNA preparation was necessary, and transfer of 96 samples to PCR reactions was performed simultaneously with a bacterial replicator. Control reactions indicate that the level of cross-contamination is negligible, and false-negative findings are rare. The method was tested on larvae, pupae, adult heads, whole adult males and females, and single tarsi. All parts except tarsi provided satisfactory template. Fresh, ethanol-preserved, dried, and frozen adults were also tested with similar results. The method was also tested for amplification of a single-copy gene, white. Results were generally positive, although some false-negative findings were observed. This method allows rapid analysis of large numbers of mosquitoes without robotic equipment and should enable rapid and extensive PCR analysis of field-collected samples and laboratory specimens.
Three distinct types of Tc1-family transposable elements have been identified in the malaria vector, Anopheles gambiae. These three elements, named Tsessebe, Topi and Tiang, have the potential to encode transposases that retain most of the conserved amino acids that are characteristic of this transposon family. However, all three are diverged from each other by more than 50% at the nucleotide level. Full-length genomic clones of two types, Topi and Tsessebe, have been isolated and fully sequenced. The third, Tiang, is represented only by a 270 bp, PCR-amplified fragment of the transposase coding region. The Topi and Tsessebe elements are 1.4 kb and 2.0 kb in length, respectively, and differ in the length of their inverted terminal repeats (ITRs). The Topi elements have 26 bp ITRs, whereas the Tsessebe clones have long ITRs ranging in length from 105 to 209 bp, with the consensus being about 180 bp. This difference is due primarily to variation in the length of an internal stretch of GT repeats. The copy number and location of these elements in ovarian nurse cell polytene chromosomes varies greatly between element subtypes: Topi elements are found at between 17-31 sites, Tsessebe at 9-13 and Tiang at 20 euchromatic sites, in addition to several copies of these elements in heterochromatic DNA. The copy number and genomic insertion sites of these transposons varies between A. gambiae strains and between member species of the A. gambiae complex. This may be indicative of transpositionally active Tc1-like elements within the genome.
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