Mitochondrial pseudogenes in the nuclear genome of Aedes aegypti mosquitoes: implications for past and future population genetic studies

<p>Abstract</p> <p>Background</p> <p>Mitochondrial DNA (mtDNA) is widely used in population genetic and phylogenetic studies in animals. However, such studies can generate misleading results if the species concerned contain nuclear copies of mtDNA (Numts) as these may amplify in addition to, or even instead of, the authentic target mtDNA. The aim of this study was to determine if Numts are present in <it>Aedes aegypti </it>mosquitoes, to characterise any Numts detected, and to assess the utility of using mtDNA for population genetics studies in this species.</p> <p>Results</p> <p>BLAST searches revealed large numbers of Numts in the <it>Ae. aegypti </it>nuclear genome on 146 supercontigs. Although the majority are short (80% < 300 bp), some Numts are almost full length mtDNA copies. These long Numts are not due to misassembly of the nuclear genome sequence as the Numt-nuclear genome junctions could be recovered by amplification and sequencing. Numt evolution appears to be a complex process in <it>Ae. aegypti </it>with ongoing genomic integration, fragmentation and mutation and the secondary movement of Numts within the nuclear genome.</p> <p>The PCR amplification of the putative mtDNA nicotinamide adenine dinucleotide dehydrogenase subunit 4 (<it>ND4</it>) gene from 166 Southeast Asian <it>Ae. aegypti </it>mosquitoes generated a network with two highly divergent lineages (clade 1 and clade 2). Approximately 15% of the <it>ND4 </it>sequences were a composite of those from each clade indicating Numt amplification in addition to, or instead of, mtDNA. Clade 1 was shown to be composed at least partially of Numts by the removal of clade 1-specific bases from composite sequences following enrichment of the mtDNA. It is possible that all the clade 1 sequences in the network were Numts since the clade 2 sequences correspond to the known mitochondrial genome sequence and since all the individuals that produced clade 1 sequences were also found to contain clade 2 mtDNA-like sequences using clade 2-specific primers. However, either or both sets of clade sequences could have Numts since the BLAST searches revealed two long Numts that match clade 2 and one long Numt that matches clade 1. The substantial numbers of mutations in cloned <it>ND4 </it>PCR products also suggest there are both recently-derived clade 1 and clade 2 Numt sequences.</p> <p>Conclusion</p> <p>We conclude that Numts are prevalent in <it>Ae. aegypti </it>and that it is difficult to distinguish mtDNA sequences due to the presence of recently formed Numts. Given this, future population genetic or phylogenetic studies in <it>Ae. aegypti </it>should use nuclear, rather than mtDNA, markers.</p

For the year 2006, mean numbers ± S.E. (per household) of <i>Ae</i>. <i>aegypti</i> pupae and adults from Ou Ruessei, the untreated commune, and Peani, Bti strain AM65-52 treated commune.

<p>The yellow dot indicates that the <i>Ae</i>. <i>aegypti</i> pupae significantly differed between the untreated and Bti commune (p<0.05). The red dot indicates that the <i>Ae</i>. <i>aegypti</i> adults significantly differed between the untreated and Bti commune (p<0.05).</p

For Years 2005 and 2006, the Mean Number ± S.E. of Containers per Household Surveyed in Peani and Ou Ruessei Communes for <i>Ae aegypti</i> Pupae.

<p>Each surveillance covered 50 household per commune.</p

Years 2005 and 2006, <i>Aedes aegypti</i> Pupae Production from Different Types of Larval Habitats in Ou Ruessei Commune.

<p>Years 2005 and 2006, <i>Aedes aegypti</i> Pupae Production from Different Types of Larval Habitats in Ou Ruessei Commune.</p

Number if dengue cases from January-September for the years 2010 and 2011 in each of the 6 districts treated with Bti in 2011.

<p>Number if dengue cases from January-September for the years 2010 and 2011 in each of the 6 districts treated with Bti in 2011.</p

Study site in the years 2005 and 2006, Peani and Ou Ruessei communes separated by National Road 5.

<p>Each purple dot indicates a single house that was surveyed in the year 2006. A total of 12 surveillance was conducted from March-September and it covered 1200 houses from the 2 communes.</p

Year 2006, Percent (%) Household (HH) in Bti Strain AM65-52 Treated Peani Commune and in Untreated Ou Ruessei Commune Categorized according to the Numbers of Indoor <i>Ae</i>. <i>aegypti</i> Adults per Household.

<p>The Bti treatment in Peani Commune was conducted from May 1–5.</p

Number of dengue cases from January-September for the years 2010 and 2011 in each of the 5 districts which were not treated with Bti in 2011.

<p>Number of dengue cases from January-September for the years 2010 and 2011 in each of the 5 districts which were not treated with Bti in 2011.</p

Spatial genetic structure of Aedes aegypti mosquitoes in mainland Southeast Asia

Aedes aegypti mosquitoes originated in Africa and are thought to have spread recently to Southeast Asia, where they are the major vector of dengue. Thirteen microsatellite loci were used to determine the genetic population structure of A. aegypti at a hierarchy of spatial scales encompassing 36 sites in Myanmar, Cambodia and Thailand, and two sites in Sri Lanka and Nigeria. Low, but significant, genetic structuring was found at all spatial scales (from 5 to >2000 km) and significant F(IS) values indicated genetic structuring even within 500 m. Spatially dependent genetic-clustering methods revealed that although spatial distance plays a role in shaping larger-scale population structure, it is not the only factor. Genetic heterogeneity in major port cities and genetic similarity of distant locations connected by major roads, suggest that human transportation routes have resulted in passive long-distance migration of A. aegypti. The restricted dispersal on a small spatial scale will make localized control efforts and sterile insect technology effective for dengue control. Conversely, preventing the establishment of insecticide resistance genes or spreading refractory genes in a genetic modification strategy would be challenging. These effects on vector control will depend on the relative strength of the opposing effects of passive dispersal