Germline transformation of the malaria vector, Anopheles gambiae, with the piggyBac transposable element.

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.

The piggyBac element is capable of precise excision and transposition in cells and embryos of the mosquito, Anopheles gambiae.

The piggyBac transposable element was tested for transposition activity in plasmid-based excision and inter-plasmid transposition assays to determine if this element would function in Anopheles gambiae cells and embryos. In the Mos55 cell line, precise excision of the piggyBac element was observed only in the presence of a helper plasmid. Excision occurred at a rate of 1 event per 1000 donor plasmids screened. Precise excision of the piggyBac element was also observed in injected An. gambiae embryos, but at a lower rate of 1 excision per 5000 donor plasmids. Transposition of the marked piggyBac element into a target plasmid occurred in An. gambiae cells at a rate of 1 transposition event per 24,000 donor plasmids. The piggyBac element transposed in a precise manner, with the TTAA target site being duplicated upon insertion, in 56% of transpositions observed, and only in the presence of the piggyBac helper. The remaining transpositions resulted in a deletion of target sequence, a novel observation for the phenomenon of piggyBac element insertion. 'Hot spots' for insertion into the target plasmid were observed, with 25 of 34 events involving one particular site. These results are the first demonstration of the precise mobility of piggyBac in this malaria vector and suggest that the lepidopteran piggyBac transposon is a candidate element for germline transformation of anopheline mosquitoes.

Tsessebe, Topi and Tiang: three distinct Tc1-like transposable elements in the malaria vector, Anopheles gambiae.

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.

Evidence for two distinct members of the amylase gene family in the yellow fever mosquito, Aedes aegypti.

Genomic DNA fragments encoding a salivary gland-specific alpha-amylase gene, Amylase I (Amy I), and an additional amylase, Amylase II (AmyII) of the yellow fever mosquito, Aedes aegypti, were isolated and characterized. Two independently isolated DNA fragments, G34-F and G34-14A, encode polymorphic alleles of Amy I. A 3.2 kilobase (kb) EcoR I fragment of G34-F, F2, has been sequenced in its entirety and contains 832 base pairs (bp) of the 5'-end, non-coding and putative promoter regions that are adjacent to 2.4 kb of the Amy I coding region. One intron, 59 bp in length, is found towards the 3'-end of the clone. A third genomic clone, 3A, corresponding to Amy II, was sequenced and shown not to contain the primary DNA sequence that encodes the 260 amino acid region that uniquely characterizes the amino terminal end of the Amy I product. Amy I was assigned by restriction fragment length polymorphism (RFLP) mapping to chromosome 2 (23.0 cM) and Amy II to chromosome 1 (44.0 cM). Amy I and Amy II are highly polymorphic and there may be multiple linked copies at each locus. Comparisons between Amy I and Amy II are presented for the putative promoter and conceptual translation products. The identification of two distinct amylase genes and their separate linkage assignments provides evidence for a multigene family of alpha-amylases in Ae. aegypti.

Quetzal: a transposon of the Tc1 family in the mosquito Anopheles albimanus.

A member of the Tc1 family of transposable elements has been identified in the Central and South American mosquito Anopheles albimanus. The full-length Quetzal element is 1680 base pairs (bp) in length, possesses 236 bp inverted terminal repeats (ITRs), and has a single open reading frame (ORF) with the potential of encoding a 341-amino-acid (aa) protein that is similar to the transposases of other members of the Tc1 family, particularly elements described from three different Drosophila species. The approximately 10-12 copies per genome of Quetzal are found in the euchromatin of all three chromosomes of A. albimanus. One full-length clone, Que27, appears capable of encoding a complete transposase and may represent a functional copy of this element.

The Apyrase gene of the vector mosquito, Aedes aegypti, is expressed specifically in the adult female salivary glands.

The yellow fever mosquito, Aedes aegypti, expresses a gene, Apyrase (Apy), that encodes an ATP-diphosphohydrolase. The product of this gene is a secreted enzyme that facilitates hematophagy by preventing platelet aggregation in the host. Apy gene expression is limited to the cells of the distal-lateral and medial lobes of the adult female salivary glands. Apyrase protein levels, detectable by antibodies, peak in the salivary glands about 4 days after adult emergence and remain high after a blood meal. Primary sequence analysis of a genomic clone encoding apyrase reveals a unique TAAATA sequence and seven introns, as well as other conserved features of eukaryotic genes. The temporal, sex- and tissue-specific expression of the Apy gene is consistent with its role as encoding a platelet anti-aggregation factor that functions to facilitate hematophagy and decrease probing time.

The salivary glands of the vector mosquito, Aedes aegypti, express a novel member of the amylase gene family.

Several cDNA clones with similarity to alpha-amylases have been characterized from a library made from adult female salivary gland RNA isolated from the vector mosquito, Aedes aegypti. The corresponding gene, designated Amylase I (Amy I), is expressed specifically in the proximal-lateral lobes of the adult female salivary gland, a pattern overlapping that of another gene, Mal I, involved in carbohydrate metabolism. The deduced amino acid sequence of Amy I indicates that this gene encodes a protein, approximate M(r) = 81,500, that appears to be a novel member of the amylase gene family. The mosquito protein contains a putative signal peptide for secretion and several consensus sites for asparagine-linked glycosylation. The Amy I protein shows significant similarity to invertebrate and vertebrate amylases including the conservation of four reactive and substrate binding sites. However, the amino-terminal region of the Amy-I protein is unique to the mosquito. Similarity with the Drosophila melanogaster protein is evident only after the first 260 amino acids in the mosquito sequence. The identification of this gene and its expression pattern adds to the observed relationship between spatial-specific gene expression in the female salivary glands and the specific feeding mode of the adult mosquito.

Human skin temperature and mosquito (Diptera: Culicidae) blood feeding rate.

The relationship between skin temperature and mosquito blood feeding behavior was examined in nine human subjects. A system implementing computer control of skin temperature was utilized during blood feeding sessions in which feeding behavior (preforaging, foraging, probing, feeding) was timed and compared at five successive skin temperatures (29.0 degrees C-36.2 degrees C). Preforaging, foraging, and probing times were not significantly different at the skin temperatures examined. Average blood meal size (3.3 microliters) also did not differ at these skin temperatures, but the time of engorgement decreased from 249.3 s at 30.8 degrees C to 100.7 s at 36.2 degrees C. The decreased feeding time resulted in an increase in feeding rate from 1.1 microliters/min (29.0 degrees C) to 2.2 microliters/min (36.2 degrees C).