Impact of TEs in Insects
TEs and Genome Size and Organization
As described in the introduction, TEs are integral and significant components of eukaryotic genomes. For example, at least 46% of the human genome (Lander et al., 2001) is TE-derived sequences. It has been proposed that the differing TE abundance may account for the "C-value paradox," which reflects the discrepancy between genome size or DNA content of an organism (as indicated by the C-value) and its biological complexity (Kidwell, 2002). In other words, organisms with similar genetic/biological complexity may have huge variations in genome size due to differences in TE content. Here, some preliminary information gleaned from comparative analysis between different species of mosquitoes is described briefly. The published genomes of Anopheles, Culex, and Aedes mosquitoes vary by five-fold in size, ranging from ~270 Mbp in An. gambiae (Holt et al., 2002) to ~500 Mbp in C. quinquefasciatus (Arensburger et al., 2010) and ~1300 Mbp in Ae. aegypti (Nene et al., 2007). TE contents in these three species are 11-16%, 29%, and 47% of the assembled genomes, respectively, indicating that TEs contributed significantly to the genome size variations among mosquito species. Varied amounts of repetitive elements have also been shown to be a major factor for the nearly three-fold intra-specific differences of genome size in populations of the Asian tiger mosquito, Ae. albopictus (Rai and Black, 1999). Such an intra-specific variation provides a rare opportunity to study the contribution of TEs to genome evolution at the initial stage of the evolutionary process.
As noted before, TEs can induce chromosomal rearrangements through ectopic recombination and other mechanisms (Gray, 2000). Such rearrangements could result in gross reorganization of the chromosomes, which may have a significant evolutionary impact, as suggested by McClintock (McClintock, 1984). A DNA TE named Odysseus was found adjacent to the distal breakpoint of a naturally occurring paracentric chromosomal inversion that is characteristic of An. arabiensis, one of the cryptic species in the An. gambiae complex (Mathiopoulos et al. , 1998). Similar evidence of TE involvement in chromosomal rearrangement has been reported in Drosophila (Lim and Simmons, 1994; Cace-res et al., 2001; Delprat et al., 2009). It is proposed that TEs may indeed be important in generating chromosomal rearrangements in nature, which may result in reproductive isolation and genetic changes that allow An. gambiae to exploit a range of ecological niches (Mathiopoulos et al, 1999).
Evolutionary Impact
The tremendous potential for TEs to generate genetic diversity has long been recognized (McClintock, 1956). They can cause spontaneous mutation, recombination, chromosomal rearrangement, and hybrid dysgenesis (Bregliano et al., 1980; Engels, 1989; Shiroishi et al., 1993; Mathiopoulos et al., 1998). TEs have been generally regarded as "selfish" DNA since the early 1980s (Doolittle and Sapienza, 1980; Orgel and Crick, 1980), as opposed to the original "controlling elements" hypothesis stating that TEs provide the physical basis controlling gene action and mutation (McClintock, 1956). This change of attitude was mainly due to the realization that TEs are somewhat "independent" genetic units, and that their replicative ability allows them to spread even when they are not beneficial to the host organism. The question of whether the "selfish" TEs are just "junk DNA" to the host, or can play important and even adaptive roles in organismal evolution, is at the heart of the debate. Thanks in part to recent studies of insect TEs, it is increasingly clear that there may be a middle ground between the "junk DNA" and the "controlling elements" hypotheses. The host genome can be viewed as an ecological community with complex host— TE and TE-TE interactions (Brookfield, 1995; Kidwell and Lisch, 2000). Given the opportunistic nature of the evolutionary process, "selfish" elements could develop a wide spectrum of relationships with their host. They could be a "junk parasite," a "molecular symbiont," or something in between.
Examples of TEs being co-opted to contribute to organismal biology continue to accumulate. The introduction mentions the early examples of telomere maintenance in Drosophila site-specific insertion ret-rotransposons (Biessmann et al., 1992a, 1992b; Levis et al., 1993). A fascinating genome-wide analysis showed that retrotransposition was responsible for the creation of a significant number of new functional genes in Drosophila (Betran et al., 2002). Some TE copies can be "domesticated" and take on a host function (also see section 3.6.3) – for example, one of the P element repeats appears to have evolved the function of transcription factors (Miller et al., 1995). Other examples of "domesticated" TEs found in non-insect species include the RAG1 and RAG2 genes involved in V(D)J recombination in vertebrate lymphocytes, and the more than 40 new genes derived from TEs in the human genome (Kidwell and Lisch, 2000; Lander et al., 2001; Gellert, 2002). More than 20 adaptive and fixed TE insertions have been found in Drosophila (reviewed in Gonzalez and Petrov, 2009), and a recent genome-wide survey identified 10 TEs associated with adaptations to temperate environments in Drosophila (Gonzalez et al., 2010). TE insertions have been shown to be associated with insecticide resistance in the moth Helicoverpa zea (Chen and Li, 2007), and Culex quinquefasciatus mosquitoes (Itokawa et al, 2010).
In addition to possible insertional mutagenesis associated with TE transposition and imprecise excision, TEs can also serve as substrates for homologus recombination that can result in chromosomal deletion, duplication, and inversion. Polymorphic chromosomal inversions are common in Drosophila and An. gambiae. TEs are often implicated in the generation of these inversions. It is proposed that these natural chromosomal inversions may result in reproductive isolation and perhaps genetic changes that allow An. gambiae to exploit a range of ecological niches (Mathiopoulos et al, 1999).
The mere presence of TEs may represent a powerful genetic force with which the genome has been evolving. The arms race between TEs and the host genomes may drive the evolution of genetic and epigenetic control mechanisms that may be important to the host organisms. For example, recently discovered piRNA and endo-siRNA pathways may have had a profound impact on gene regulation and epigenetic silencing (Aravin et al., 2007; Nishida et al., 2007; Pelisson et al., 2007; Yin and Lin, 2007; Brennecke et al., 2008; Chung et al., 2008; Ghildiyal et al., 2008; Klattenhoff et al., 2009; Lau et al., 2009; Lisch, 2009; Malone and Hannon, 2009; Zeh et al., 2009). On the basis of their broad distribution in bacteria, archea, and eukaryotes (Craig et al., 2002), it is safe to assume that TEs have long been evolving together with the immensely diverse life forms on this planet, and will continue to do so.
Applications of Insect TEs
Endogenous TEs and Genetic Manipulation of Insects
P element-based transgenic and mutagenesis tools in D. melanogaster have played a major role in the tremendous success of this tiny fly as a model organism for genetic analysis. A limited number of DNA transpo-sons, such as hobo, mariner, minos, and piggyBac, which have a broader host range than P, have been developed as tools in insects.Further analysis of TEs in insect genomes may expand the pool of active DNA transposons, which may be used to generate a set of tools with diverse features that can be used collectively for a variety of genetic analyses in different insects. In addition to simply transforming an insect, active TEs mentioned above are used to construct specific vectors to be used in gene trapping, enhancer trapping, and genome-wide insertional mutagenesis studies (Spradling et al., 1999; Klinakis et al., 2000; Horn et al., 2003; Bonin and Mann, 2004). These analyses are powerful ways to investigate gene function and regulation on a genome-scale.
In addition to providing possible new active TEs to be used as tools for genetic manipulation of insects, a better understanding of endogenous TEs will allow better-informed usage of current transposon-based genetic tools. Interactions between exogenous and endogenous transposons that share similar TIRs have been shown to be a potential problem (Sundararajan et al., 1999; Jasin-skiene et al., 2000). Such interactions could be significant in light of the discovery of a diverse range of DNA TEs in a few insects in which genetic manipulation is being actively pursued (see, for example, Nene et al., 2007). Analyses of endogenous insect TEs will lead to better-informed design of transposon-based transformation tools that reduce instability resulting from interactions with endogenous TEs (Ashburner et al., 1998; Atkinson et al., 2001). It is also hoped that the non-Mendelian inheritance of TEs could help beneficial transgenes sweep through insect populations, as the P element did in Dro-sophila (Ribeiro and Kidwell, 1994; Engels, 1997). Such a strategy is being investigated in the context of driving refractory genes into mosquito populations to control mosquito-borne infectious diseases (Ashburner et al., 1998; Alphey et al., 2002), although concerns, including disassociation between transposon and the beneficial gene, will need to be addressed (Marshall, 2008). A better understanding of endogenous TEs may be important to help achieve sustained success of such sophisticated genetic approaches.
SINE Insertion Polymorphism as Polymorphic Genetic Markers
The genetic differences and the pattern of gene flow between insect populations are of fundamental importance to a number of entomological questions, ranging from evolution to practical applications. Single nucleo-tide polymorphisms (SNPs) are powerful markers for population genetic analysis, especially for insects with a large amount of sequence data available. Polymorphic insertion sites of interspersed TEs are potentially rich sources of a different type of genetic markers for population and genetic mapping studies. The discussion here is focused on SINEs, which are especially useful for the reasons described below. In population studies, sequences flanking a SINE at a specific locus are used as primers to amplify genomic DNA isolated from an individual sample. When the PCR products are run on an agarose gel, the genotype of an individual will be revealed on the basis of the number and size of bands. Thus, this locus-specific PCR assay may be used for co-dominant markers that reveal the dimorphism (insertion vs non-insertion) at a specific site. SINEs including the human Alu elements have been shown to be powerful genetic markers (e.g., Batzer et al, 1994; Roy-Engel et al, 2001; Batzer and Deininger, 2002; Salem et al., 2003). The ability of SINE insertion polymorphic markers to differentiate recently separated human populations is a good indication of their power (e.g., de Pancorbo et al., 2001; Nasidze et al., 2001; Watkins et al., 2001). The locus-specific PCR assay of SINE insertions has a few potential advantages over the popular microsatellite markers. The same SINE insertions are identical by descent. The probability that different SINEs of the same size independently insert into the same chromosomal location may be negligible (Stoneking et al., 1997; York et al., 1999; de Pancorbo et al., 2001; Watkins et al., 2001). Moreover, the ancestral state of the SINE insertion polymorphism is likely to be the absence of a SINE because of general lack of excision, although exceptions do exist (Medstrand et al., 2002). The ability to distinguish the ancestral versus derived states provides additional resolving power to address population genetic questions (York et al., 1999). One potential limitation of this approach could be the removal of a SINE insertion by rare recombination or gene conversion events, which may be confused with the non-insertion state. Such events may be revealed during the PCR analysis. Sequence analysis at the insertion site will also allow the investigation of this possibility. Maque and SINE200 have been successfully used to study the incipient speciation between the M and S forms of An. gambiae (Barnes et al., 2005; Santolamazza et al., 2008).
The development of a TE-anchored PCR approach, or TE display, has made it possible to directly screen for TE insertion polymorphism in a few species, including insects (Figure 5; see also Biedler et al., 2003; Yang and Nuzhdin, 2003; Arensburger et al., 2005; Bonin et al, 2008). TE display efficiently scans a large number of loci in the genome, which makes it a very good tool for genotyp-ing. However, as a population genetic tool it has a major limitation common for dominant markers such as AFLP and RAPD; namely, the inability to distinguish between heterozygous and homozygous insertions, rendering the detection of population genetic structure difficult. However, TE display can be used as a direct screen to identify potential polymorphic insertion sites. Therefore, TE display in conjunction with the development of locus-specific PCR markers that are co-dominant will help TE insertion polymorphism markers reach their full potential as population genomic tools for insects.
SINE Insertions as Phylogenetic Markers
TE insertions, more specifically SINE insertions, have been used as molecular systematic tools to trace the evolutionary relationship between whales and Artiodactyla (Shi-mamura et al., 1997; Nikaido et al., 1999), and between Salmonid fishes (Murata et al., 1993). Perhaps the most impressive use of SINE insertions is the resolution of the evolutionary relationship of one of the major tribes of the African cichlid fishes that have evolved through an explosive adaptive radiation (Takahashi et al., 1998; Terai et al., 2003). To obtain TE insertion information for molecular systematics, locus-specific PCR, described above, is used. Here, an RNA-mediated TE such as a SINE is again better suited because its transposition does not involve excision. Therefore, the ancestral state is known to be the absence of a SINE (Shedlock and Okada, 2000; Nishihara and Okada, 2008). The basic concept of this approach is illustrated in Figure 7. The insertion state can be easily determined using an agarose gel or melt-curve analysis. When a fixed insertion is found at a particular site in species 1 and 2 but not in species 3, it can be inferred that 1 and 2 are sister taxa.
Figure 7 A schematic illustration of the principle of using SINE insertions as molecular systematic markers. Monophyletic relationships between species may be inferred on the basis of shared SINE insertions.
One of the requirements of the above approach is that the TE insertion site should be fixed within a species. TE display, a fingerprinting method described earlier (Figure 5), may be used to search for such sites. Potential problems such as non-specific deletions, gene conversions, and sorting of ancestral polymorphisms can either be detected during the PCR and gel electrophoresis analysis, or mitigated by surveying multiple loci (Shedlock and Okada, 2000; Nishihara and Okada, 2008). Sequences of the SINEs themselves in the loci used for the systematic analysis may provide further phylogenetic information. The usefulness of a particular SINE family in molecular systematics studies is dependent on its distribution and lifespan in the taxonomic group of interest. The tremendous diversity of insect species offers interesting challenges to evolutionary biologists. For example, a number of medically and economically important insect organisms exist as cryptic species complexes (Munstermann and Conn, 1997; Krzywinski and Besansky, 2003). New phy-logenetic tools that can be integrated with methods using conventional characters such as morphology and DNA sequences will undoubtedly be of significance. Although SINEs have not been extensively studied in insects, highly repetitive SINEs have already been characterized in different orders, including many medically and economically important species. Therefore, SINEs may provide useful markers for molecular systematic analysis of insects, one of the most diverse groups of life forms on this planet.
In summary, TEs have been successfully used as vectors for genetic manipulation of insects and other organisms. A better understanding of insect TEs will allow better-informed usage of the currently available TE-based tools. The application of TEs as population and phylogenetic markers is at an early stage. Although these markers are promising tools, their scope of application, their resolving power and reliability depend on a better understanding of the population and evolutionary dynamics of the TEs. Therefore, fundamental studies discussed in previous sections also have significant implications for the applications of TE-based molecular tools. As in so many areas of biology, the availability of genome sequences from related species as well as individuals within populations will greatly facilitate the investigation and application of insect TEs.
Summary
The past few years have witnessed an explosive growth both in the development of TE-based genetic and molecular tools and in our fundamental understanding of the diversity, regulation, and impact of TEs in insects. Studies of TEs in insects, especially in D. melanogaster, have more than once led to discoveries that broadly impacted the field of TE research. In addition, studies in D. melanogaster also significantly contributed to the discovery of novel small RNA pathways and the functions of small RNAs in TE suppression, gene regulation, and epigenetic silencing. The availability of new bioin-formatic and experimental tools and the rapidly expanding genome revolution provide an exciting opportunity for the discovery of novel TEs in a wide range of insects, for identification of evidence of TE horizontal transfer, and for in-depth molecular and genomic analysis of these mobile genetic elements. Only through comparative genomic approaches can we come close to a full appreciation of the complex and intricate dynamics governing the evolution of diverse TEs in insect genomes. From an applied perspective, systematic analysis of TEs in many insect species has significant economic and health implications because of the importance of these insects in disease transmission and agriculture.
