We have discovered a case of HGT involving adjacent genes in the genomes of Ae. aegypti
and two Wolbachia
strains. The Ae. aegypti
gene AAEL004181 shares around 50% amino acid identity with two genes in the genome of Wolbachia
] from the mosquito Culex quinquefasciatus
, WP1348 and WP1346), which were probably originally a single gene split by insertion of IS element WP1347, and also with WD0513 in strain w
Mel from Drosophila melanogaster
]. The adjacent Ae. aegypti
gene AAEL004188 shows partial similarity to w
Pip WP1349 and to w
Mel WD0514 and is inverted compared to the Wolbachia
genes. The intergenic region between AAEL004181 and AAEL004188 is around 15 Kb (Figure ). The level of sequence identity and the fact that adjacent sets of genes are involved provide a robust case for an HGT event.
A. Percent amino acid identities shared between Aedes aegypti and Wolbachia genes/gene regions.
RT-PCR analysis was conducted and confirmed that Ae. aegypti genes AAEL004181 and AAEL004188 were transcribed in both male and female adult mosquitoes. The Wolbachia wPip genes WP1348 and WP1346 were clearly amplified by RT-PCR in adult Cx. quinquefasciatus of both sexes, indicating that they are also transcribed. Primers AAEL004181 b, c and i were designed to span three introns present in the Aedes aegypti Vectorbase annotation for AAEL004181; based on product size from RT-PCR amplification (Figure and Table ), all of these three introns proved to be mis-annotations and it was concluded that no introns are present. The apparent absence of introns, which would be particularly unusual for a mosquito gene of the size of AAEL004181, is suggestive of a bacterial origin.
Figure 2 Map showing positions of oligonucleotide PCR primers for gene AAEL004181 and the positions of introns (white boxes) present in the Aedes aegypti Vectorbase annotation for this gene (see Table 1 for primer sequences, product sizes and conditions used in (more ...)
Sequences (5'-3'), optimal annealing temperatures (°C) and amplified fragment sizes (base pairs) for primers used in the study.
Full genome microarrays for Aedes aegypti
were hybridized to cDNA from adult females, as shown in Figure . All probes with hybridization signal levels significantly above background (see methods) were ranked in order of signal intensity; 4.9% of these probes (or approximately 15% of all probes) showed lower signal intensity than was seen for either of the two AAEL004181 probes, while 28.2% of probes significantly above background (42% of all probes) showed lower signal intensity than was seen for either of the two AAEL004188 probes. Quantitative RT-PCR data for AAEL004188, AAEL004188 and he act5C
gene used as a control by Hotopp et al
] matched the array results, as shown in figure . The act5C
gene is, as the authors note, highly and constitutively expressed and may in fact be an overly stringent point of comparison to assess whether horizontally transferred genes are likely to be functional. In the microarray data a number of genes of known function showed very similar or lower hybridization intensities relative to the two genes of interest; for example the probes for AAEL012836 (Cytochrome B561
) and AAEL002230 (encoding a chromatin helicase DNA binding protein) showed very similar hybridization intensities as AAEL004181, while AAEL013002 (Cdk9
) and AAEL010226 (Daughterless
) showed very similar hybridization levels as AAEL004188. Based on these data it is considered likely that both AAEL004181 and AAEL004188 are expressed, functional genes, although obviously definitive proof of this will require the raising of antibodies followed by protein studies.
Figure 3 A. Transcript levels of Ae. aegypti genes, AAEL004181 and AAEL004188, relative to whole genome transcription. Microarrays incorporating two distinct 60-mer probes for each annotated Ae. aegypti gene were used to investigate transcript abundance in pools (more ...)
Close homologs of the two Ae. aegypti
genes could not be found in other sequenced mosquito/insect genomes such as Anopheles gambiae
]. Various PCR primer pairs designed for the Aedes aegypti
genes AAEL004181 and AAEL004188 failed to amplify PCR products from several other fellow subgenus Stegomyia
members, but did amplify products for both genes from Ae. mascarensis
(Table ). This species, from Mauritius, is able to produce sterile offspring in laboratory crosses with Ae. aegypti
]. Ae. mascarensis
PCR products from AAEL004181 primers b & f plus g & h (see Figure and Table ), located in diverse regions of the gene, were sequenced and shared a mean 97% nucleotide identity over 1278 base pairs with Ae. aegypti
AAEL004181. Diverged homologs of the two genes may well be present in other more distant Aedes
species, but could not be detected here. Thus, if the direction of the HGT was from Wolbachia
to host it would have occurred at least prior to the species divergence of Ae. aegypti
and Ae. mascarensis
and indeed the accumulation of the 15 Kb of non-coding DNA between the two genes would likely have required a considerable period of time (although it is not possible to make any precise time estimates from the data available).
PCR amplification results from genomic DNA to examine the distribution of Ae. aegypti genes AAEL004181 and AAEL004188, plus presence/absence of Wolbachia, among other species in the Aedes subgenus Stegomyia.
Pip genes are located at the end of a genomic prophage region, providing a putative mechanism for the HGT. Wolbachia
have been shown to contain phage particles by EM in several studies; WO prophage have been shown to be highly variable and rapidly evolving regions in the genomes of mosquito Wolbachia
, and non-congruent with host phylogeny [19
], strongly suggesting that lateral transfer of phage between Wolbachia
strains has occurred. The two w
Mel genes WD0512 and WD0513 are part of an operon that also contains the ankyrin repeat domain (ANK) encoding gene WD0514. This operon is present in mod+ strain variants of w
Mel (able to induce CI in males) but not in the related mod- strain w
Au (unable to induce CI) [25
]. The operon is located in a region of the w
Mel genome that was shown to be missing in w
Au, WD0506-WD0518 in w
Mel, and in fact all these genes have homologs in the prophage regions of the w
Pip genome, except for the ANK gene WD0514. Therefore, although not annotated as prophage [16
], these genes in w
Mel are likely to be remnants of an old prophage region, the rest of which has been deleted or rearranged.
The Ae. aegypti
gene AAEL004181 also shares considerably lower amino acid similarity with a group of genes in the Ae. aegypti
, Anopheles gambiae
and Culex pipiens
genomes. One of these Ae. aegypti
genes showed female salivary gland specific expression and was named aaSGS1 (SGS = Salivary Gland Specific). This gene and homologs in Anopheles
are candidate Plasmodium
sporozoite receptors [26
]. It has already been suggested that the SGS-type mosquito genes might have arisen from an ancient transfer between Wolbachia
and mosquitoes, but with weak support [26
An alternative hypothesis is that the direction of horizontal transfer was in fact from Aedes into Wolbachia, and AAEL004181 is part of a family of SGS-type genes that originated and evolved in mosquitoes. The acquisition of host genes by Wolbachia has not previously been documented. It could be argued that this scenario is more parsimonious since only one inter-domain HGT event would be required, if a subsequent transfer from wPip (or a related strain) to wMel is assumed. In contrast, the hypothesis of Wolbachia to Aedes transfer requires a different origin of the SGS genes compared to AAEL004181. However, phylogenetic reconstruction (Figure ) does not support the hypothesis of a single host-to-Wolbachia HGT, since AAEL004181 clusters with the wPip gene WP1346 with a posterior probability of 1 and a boostrap value of 89. If AAEL004181 had been transferred from mosquito to Wolbachia followed by subsequent transfer between Wolbachia strains, the wPip and wMel sequences would be expected to be more closely related to each other than to AAEL004181 and thus cluster together in the phylogenetic tree, which is not the case. Thus, both phylogenetic evidence and the lack of introns support a Wolbachia-to-host direction of transfer of AAEL004181. The SGS genes may also have had a bacterial origin, as suggested by their apparent lack of introns, but if this is the case then the HGT event or events responsible would be separate from that involving AAEL004181 (and probably pre-date it, given their greater distance from the Wolbachia genes).
Figure 4 Phylogenetic tree of the SGS genes, AAEL004181 in Ae. aegypti and homologous sequences in Wolbachia. The fragments of the AAEL004181 homolog sequenced from Ae. mascarensis are highly similar to AAEL004181, and are not shown here. Boostrap values from (more ...)