Transposable elements in mosquitoes - CiteSeerX

Diversity of Retrotransposable Elements Cytogenet Genome Res 110:500–509 (2005) DOI: 10.1159/000084983

Transposable elements in mosquitoes M. Boulesteix, C. Biémont Laboratoire de Biométrie et Biologie Evolutive, UMR C.N.R.S. 5558, Université Lyon 1, Villeurbanne (France)

Manuscript received 10 September 2003; accepted in revised form for publication by J.-N. Volff 27 January 2004.

Abstract. We describe the current state of knowledge about transposable elements (TEs) in different mosquito species. DNA-based elements (class II elements), non-LTR retrotransposons (class I elements), and MITEs (Miniature Inverted Repeat Transposable Elements) are found in the three genera, Anopheles, Aedes and Culex, whereas LTR retrotransposons (class I elements) are found only in Anopheles and Aedes. Mosquitoes were the first insects in which MITEs were reported; they have several LTR retrotransposons belonging to the Pao family, which is distinct from the Gypsy-Ty3 and Copia-Ty1 families. The number of TE copies shows huge variations between classes of TEs within a given species (from 1 to 1000),

in sharp contrast to Drosophila, which shows only relatively minor differences in copy number between elements (from 1 to 100). The genomes of these insects therefore display major differences in the amount of TEs and therefore in their structure and global composition. We emphasize the need for more population genetic data about the activity of TEs, their distribution over chromosomes and their frequencies in natural populations of mosquitoes, to further the current attempts to develop a transgenic mosquito unable to transmit malaria that is intended to replace the natural populations.

Our knowledge about transposable elements (TEs), their structure, sequences, role in the genome, distribution along the chromosomes, and the forces that maintain them in genomes and populations have greatly benefited from the advantages that Drosophila offers as a model species. Its genome has been sequenced (Adams et al., 2000), and the fact that population studies and molecular analyses can be related to developmental processes has furnished invaluable findings that have gradually changed our view about how TEs influence the genome. However, Drosophila melanogaster is only a single species, and it has been shown that other dipteran species, even Drosophila simulans, which is a close sibling of D. melanogaster, present different TE distribution patterns and different copy numbers

(Biémont and Cizeron, 1999; Vieira and Biémont, 2004). Species characteristics, such as effective population size, sensitivity to stressful environmental and genetic conditions, the ability of the genome to limit the TE copy number or even to delete TE copies by various mechanisms, make comparisons between species an important step toward our understanding of the true impact of TEs on genomes. Although the genomes of D. subobscura and D. simulans have not been sequenced, we can already make useful comparisons using the recently sequenced genome of Anopheles gambiae (Holt et al., 2002), a species phylogenetically further from D. melanogaster than D. subobscura and D. simulans. The genome of the mosquito Aedes aegypti, which is currently being sequenced, will give us other opportunities for comparative genomics in the very near future. Here we summarize what is known about the TEs already identified in the genomes of Anopheles and other related mosquitoes, including the as yet scarce data from population analyses.

This work was funded by the Centre National de la Recherche Scientifique (CNRS) (UMR 5558 and GDR 2157 on Transposable elements) and the French Ministry of Research PAL+ program. Request reprints from: Dr. Biémont Christian Laboratoire de Biométrie et Biologie Evolutive, UMR C.N.R.S. 5558 Université Lyon 1, FR–69622 Villeurbanne Cedex (France) telephone: +33-4-72-448198; fax: +33-4-72-431388 e-mail: [email protected]

ABC

Fax + 41 61 306 12 34 E-mail [email protected] www.karger.com

© 2005 S. Karger AG, Basel 1424–8581/05/1104–0500$22.00/0

Copyright © 2005 S. Karger AG, Basel

Mosquito species Mosquitoes belong to the family Culicidae, which includes nearly 3500 species divided into some forty genera (Munstermann and Conn, 1997; Rai and Black, 1999). Most of the litera-

Accessible online at: www.karger.com/cgr

ture about TEs in mosquitoes concerns three genera: Anopheles, Aedes and Culex. Anopheles is part of the subfamily Anophelinae, which has been identified as the basal clade in Culicidae, whereas Aedes and Culex are classified as members of the subfamily Culicinae (Besansky and Fahey, 1997). All the species of the genus Aedes belong to the tribe Aedini, and all the species of the Culex genus belong to the tribe Culicini. A third subfamily that is mentioned below, the Toxorhynchitinae, is basal to the Culicinae. The position of the Armigeres genus, which appears in Table 1, has not been resolved. The relationships between species, complexes of species and subgenera within the Aedes genus and the Anopheles genus are not very clear (Anderson et al., 2001; Krzywinski et al., 2001; Coluzzi et al., 2002; Krzywinski and Besansky, 2003). Reinert (2000) has suggested splitting the composite genus Aedes into two genera: Aedes and Ochlerotatus. In the present paper we use this modification of the taxonomy. Figure 1 summarizes the generally accepted relationships between the taxa within the members of the Culicidae family that are mentioned in this paper. The question of whether the Toxorynchitinae subfamily is a sister

group of Anophelinae or a separately emerging clade is still a matter of debate (Rai and Black, 1999). Within the Anophelinae, the Anopheles gambiae complex is of particular interest. It comprises several closely related and morphologically indistinguishable species: Anopheles gambiae s.s., the major malaria

Anophelinae Sabethini Aedini Culicinae Culicini Toxorhynchitinae Fig. 1. Phylogenetic relationships between the Culicidae subfamilies and between tribes of Culicinae, according to Besansky and Fahey (1997). The Sabethini tribe is not mentioned in the text, but is included in this figure for the sake of clarity.

Table 1. Distribution of transposons (class II elements) among mosquito species Genus

Species

Transposons

ITmD37E 1

MsqTc32 mariner3

crusoe4 Ikirara5 Topi6

Tsessebe6

Quetzal7 Tiang6 P8 Transib9 piggyBac10 Helitrons11

-l

-l

+a

Aedes

+ 100e +f

aegypti albopictus

-g -g

-l

-l

Ochlerotatus atropalpus epactius triseriatus togoi

+ 200/100Mb b + 1000/100Mbb + 2/100Mb(20)b

+f

+f a

Culex

+

-g

pipiens quinquefasciatus restuans

-l h

+ +a

Armigeres

f

subalbatus

+ +a

Toxorhynchites amboinensis Anopheles gambiae arabiensis melas merus quadriannulatus stephensi funestus quadrimaculatus albimanus freeborni dirus

+a 14/100Mb(40)c 12d

+* + 30 g 65i 174 d +g +g +g +g

+ 25j 54d + 50 k 56d + 25 l + 25 l + 25 l + 25 l -l -l

+ 17-31 m 61d +l +l +l +l -l

+ 9-13 m 20d +l +l +l +l -l

-l

-l

+ 20 m 13d +l +l +l +l -l

-l -l -l -l

-l -l -l -l

-l + 10n -l -l

-l -l -l -l

+ +

+

+

+ + + + + + +

+: present (a + in a genus indicates the TE presence in this genus, but gives no information about the species in which the element was detected); +: present in various Asian Anopheles species (see Imwong et al., 2000); -: absent see text for discussion); a: dot blots; b: estimate based on the number of matches in a genomic DNA bank; c: estimate based on the number of matches in an STS database; d: sequenced genome of Anopheles gambiae (Holt et al., 2002); e: estimate based on the number of matches in a genomic DNA bank and confirmed by dot blots; f: preliminary dot blot analysis; g: dot blots; h: degenerate PCR; i: mean number of euchromatic sites in the PEST strain; j: Southern blots; k: slot blots, Southern blots and in situ hybridization; l: Southern blots; m: range of the number of euchromatic insertion sites in different strains; n: Southern blots and in situ hybridization; 1: Shao and Tu (2001); 2: Shao et al. (2001); 3: Imwong et al. (2000); Mukabayire and Besansky (1996); Robertson (1993); Robertson and Lampe (1995); Robertson et al. (1998); 4: Hill et al. (2001); 5: Romans et al. (1998); 6: Grossman et al. (1999); 7: Ke et al. (1996); 8: Sarkar et al. (2003b); 9: Kapitonov and Jurka (2003); 10: Sarkar et al. (2003a); 11: Kapitonov and Jurka (2003); Poulter et al. (2003).

Cytogenet Genome Res 110:500–509 (2005)

501

vector in Africa, An. merus, An. melas, An. arabiensis, An. quadriannulatus. An. bwambae, another species of the An. gambiae complex, is not discussed further in this paper. Throughout the paper, the name An. gambiae sensu stricto, abbreviated as An. gambiae s.s., will be used to identify the species.

DNA-based elements (transposons) Numerous Tc1-mariner family (class II) elements have been found in mosquito genomes. No member of the hAT family has been reported, however, except in the preliminary analysis of the sequenced genome of An. gambiae. Given that hAT-related MITEs exist in Anopheles (Feschotte et al., 2002), it is highly unlikely that this situation will last much longer. Sarkar et al. (2003a) have found 10 families of piggyBac in the sequenced genome of An. gambiae, plus three piggyBac-related sequences apparently domesticated by their host. These elements are present in multiple copies; and one family, AgaPB1, has readily identifiable ITRs. P elements have been reported in various Anopheles species (Sarkar et al., 2003b). The coding sequence of these P elements, which consists of two exons separated by a 61-bp intron, encodes a 776-amino acid transposase. Another family, named Transib, has been reported recently by Kapitonov and Jurka (2003) in D. melanogaster and An. gambiae. It encodes a transposase, which has no resemblance to any known protein. The elements from An. gambiae seem to lack the DDE signature, which has been found in the elements of D. melanogaster. As can be seen in Table 1, the distribution of the various TE families among species is not uniform, with some elements appearing to be restricted to one species, such as Quetzal in An. albimanus, whereas others are very widely distributed, such as ITmD37E, which has been detected in every genus so far examined. MsqTc3 has been found in two genera (Shao et al., 2001), and the elements Ikirara, Tsessebe, Topi, and Tiang in all the species of the An. gambiae complex, but not in the other genera (Romans et al., 1998; Grossman et al., 1999). The apparent absence of TEs in some species must be interpreted with caution. This could be attributable to the fact that the TE probes used were not from the species under study, and thus the apparent distributions observed may in part reflect divergence between the elements in the species analyzed and those in the source species of the probe. This is a likely explanation for elements that no longer transpose, and have therefore have had time to diverge in the absence of selection pressure. This seems to be especially important in species containing large amounts of moderately dispersed repetitive DNA (Marin and Fontdevila, 1996). Moreover, the TEs of a given family could be derived from different ancestral elements in different species, and a highly divergent element could give rise to another new element, which is classified as a new family. A probe derived from another species may not therefore recognize the element, unless the hybridization is carried out under very low stringency conditions. Such considerations could account for the greater number of Ikirara copies in An. gambiae s.s. than in its sibling species, because the probe used for the Southern blot experiments originated from an An. gambiae s.s. fragment (Romans et al., 1998). However, the ITmD37E element shows a

502


highly variable density per megabase among different genomes, indeed even between related species (Shao and Tu, 2001); e.g. its density shows a 100-fold variation within the species of the genus Aedes. Members of this ITmD37E family (see Table 1) can be divided into two groups sharing more than 89 % intragroup similarity. ITmD37E, from An. gambiae, is thus to be grouped with the element of Aedes triseriatus, and ITmD37E, from Aedes epactius, is to be grouped with the element from Aedes atropalpus. The Ae. triseriatus/An. gambiae group is characterized by a low ITmD37E element density per megabase, whereas the Ae. epactius/Ae. atropalpus group is characterized by a high density of this element. Such differences may reflect a difference in permissiveness between genomes for TE mobilization, or a specific mobilization of the elements in some genomes due to a particular evolutionary history or effective population size of the host species. A patchy distribution of a TE is sometimes interpreted as the result of horizontal transfer, a hypothesis that is reinforced if the phylogeny of the TE is not congruent with the phylogeny of its host (Capy et al., 1994). The mariner element is thus thought to transfer horizontally quite easily. This could explain why this element has been found only in the Anopheles species of mosquitoes and in Culex restuans (see Table 1), even though it has been identified in many other insects (Robertson, 1993). The possible loss of this element in some of the lineages leading to Culex and Aedes species cannot, however, be ruled out. Horizontal transfers could also be involved in the distribution of the ITmD37E element, given that the grouping of the families is not congruent with the grouping of host species. Miniature Inverted Repeat Transposable Elements (MITEs) consist of short sequences of DNA, usually less than 500 bp long, with no coding potential, which display conserved terminal inverted repeats (TIRs) (or sometimes subterminal inverted repeats), and a target site preference. They often have a high copy number in the genomes in which they are found. The analogy between the inverted repeats and target site duplications flanking MITEs with those of Class II elements, suggests that the MITEs are DNA-based elements derivatives that could use the machinery of class II elements to transpose, as has been suggested in the nematode (Rezsohazy et al., 1997). Mosquitoes are the first insects in which MITEs were discovered (Tu, 1997). These MITEs are extremely diverse, some being related to PIF/harbinger elements, and others to different subfamilies of Tc1-mariner, even to piggyBac or hAT families. However, some families have no clear relationship with any known TE (Feschotte et al., 2002). In most cases, no attempt has been made to look for the elements of the MITE family discovered in one species of mosquito in other species. Overall, however, it seems as if MITEs in Ae. aegypti have a higher copy number than MITEs in An. gambiae (see Table 2). The Helitrons, which display some similarities to bacterial rolling-circle transposons (Kapitonov and Jurka, 2001), are present in the An. gambiae genome (Kapitonov and Jurka, 2003). The consensus of the first family to be characterized is 8200 bp long and encodes an intronless 1700-amino acid protein containing the canonical Helitron-like domains Rep and SF1. The ends of these elements are 5) TC and CTAG 3), and they have a 12-bp 3) terminal hairpin. There are more than 100

Table 2. Distribution of MITE elements among mosquito species Genus

Species

MITEs

Joey1

Mimo2

Mint12

Nemo12

Pegasus3

Wujin4

Wukong4

Wuneng4

-d -d

+ 2100c

+ 2200-3000c + 2700c

Microuli5

Pony6

+ 3000c

+ 15000c

NOS7

Aedes

aegypti albopictus Culex + 1000c

pipiens quinquefasciatus

+

+ -d

Anopheles gambiae + 1120a 402b arabiensis melas merus quadriannulatus stephensi punctipennis albimanus freeborni dirus

+ 32e 44b +d +d +d +d -d -d -d -d -d

+

8bp-1-Ag8

TA-IE-Ag8 TA-IID-Ag8 TA-ID-Ag8 TA-IIE-Ag8 TA-III-Ag8 TA-IV-Ag8

TA-V-Ag8

TAA-II-Ag8 TAA-I-Ag8

+ 725a 155b

+ 500a

+ 300a

+ 320a 137b

Anopheles

gambiae

+ 630a

+ 1340a

+ 450a 1747b

+ 970a

+ 130a

+ 40a

+: present; -: absent (see text for discussion); a: estimate based on the number of matches in an STS database; b: sequenced genome of Anopheles gambiae (Holt et al., 2002); c: estimate based on the number of matches in a genomic library; 1: Besansky et al. (1996); Tu (2001b); 2: Feschotte and Mouches (2000); 3: Besansky et al. (1996); Mukabayire and Besansky (1996); Tu (2001b); 4: Tu (1997); 5: Tu and Orphanidis (2001); 6: Tu (2000); 7: Luckhart and Rosenberg (1999); 8: Tu (2001b).

copies of various Helitron families in the An. gambiae genome. No attempt has been made to identify Helitrons in other mosquito genomes. Because the coding sequence of these Helitrons seems to be uncorrupted, Poulter et al. (2003) suggest that these elements are active or must have been active recently.

RNA-based elements LTR retrotransposons LTR retrotransposons are the group of TEs that have been the least studied in mosquitoes, although members of the main families, Gypsy, Copia and Pao, have been found in various species of Anopheles (Table 3). Most of the LTR retrotransposons, Aara, Afun, Agam, Amer, and Aste listed in Table 3, have been identified using a degenerate PCR method developed by Cook et al. (2000). This method was designed to amplify fragments of Class I elements from primers homologous to conserved regions of protease and reverse transcriptase. As a consequence, little is known about these TEs. The ozymandias TE was discovered in An. gambiae s.s. by Hill et al. (2001) during an attempt to characterize full-length copies of Ikirara, the class II element mentioned above. Although only the 3) end of ozymandias has been isolated, it seems to be distantly related to 412 of D. melanogaster (40 % nucleotide identity). Moose, found in An. gambiae (Biessmann et al., 1999) is thought to belong to the Pao clade. There is a striking difference between the copy number of Moose estimated by in situ hybridization

and Southern blotting (around 30 copies) by Biessmann et al. (1999), and the number of copies (more than 1000) found in the sequenced genome (Holt et al., 2002) (see Table 3). This difference does not seem to be attributable to the insertion in tandem of different copies or to a high number of copies embedded in the heterochromatin, because 85 copies were reported in the heterochromatin versus 970 in the euchromatin of the sequenced genome of An. gambiae (Holt et al., 2002). This difference could of course reflect specific characteristics of the strains used, PE for Biessmann et al. (1999), PEST for the sequenced genome, or bias due to the techniques used (molecular analyses versus sequenced genome) depending on the percentage of similarity used to define elements of a given family. The case of mtanga/mtanga-Y is puzzling. This Ty1/copia element has been found in the An. gambiae complex. Structurally conserved copies of this element are inserted in cluster on the Y chromosome of An. gambiae s.s.. More divergent copies can be found elsewhere in the genome of this species and in all the species of the complex. This implies that the transposition of mtanga-Y onto the Y chromosome of An. gambiae s.s. probably occurred after the speciation of An. gambiae s.s.. Zebedee, the only LTR retrotransposon described in the Aedes genus, displays a very strange structure, and can be viewed as an unusual retroelement (see below). Non-LTR retrotransposons The non-LTR retrotransposons of mosquitoes are extremely diverse, and members of the different clades defined in Malik


503

Table 3. Distribution of LTR retrotransposons among mosquito species

Genus

Species

LTR retrotransposons

Aara51 Afun11 Agam101 Amer11 Amer31 Amer61 Amer71 Aste111 Aste121 Aste71 Anopheles

gambiae arabiensis merus stephensi funestus

+a +a +a

+a

+a

+a +a

+a

+a

+a Moose2

mtanga3

mtanga-Y3

Ozymandias4

+d +d +d +d +d

+ 12d -d -d -d -d

+ 20e

Anopheles

gambiae + 30 b 1055c arabiensis melas merus quadriannulatus

+: present; -: absent (see text for discussion); a: degenerate PCR approach; b: Southern blots and in situ hybridization; c: sequenced genome of Anopheles gambiae (Holt et al., 2002); d: Southern blots and dot blots; e: Southern blots; 1: Cook et al. (2000); 2: Biessmann et al. (1999); 3: Rohr et al. (2002); 4: Hill et al. (2001).

et al. (1999) have been described. Juan belongs to the Jockey clade, T1, vash and Q to the CR1 clade, Lian to the LOA clade, Guildenstern, RT1 and RT2 to the R1 clade, JAM1 to the RTE clade, and MosquI to the I clade (Tu et al., 1998; Malik et al., 1999; Tu and Hill, 1999). Recently, in a systematic search for non-LTR retrotransposons in the An. gambiae genome, Biedler and Tu (2003) reported 104 families, which belong to all the previously cited clades except LOA, plus some families belonging to the R4 and L1 clades (Malik et al., 1999), the L2 clade (Lovsin et al., 2001), and two new clades, namely Loner and Outcast. There is no detailed information available about the Aara2, Aara8, Agam2, and Amer5 elements. Many of the non-LTR retrotransposon families include several different subfamilies. For example, MosquI displays many truncated copies, which share high sequence similarity and seem to belong to a subfamily distinct from the full-length subfamily of MosquI. The T1Ag family has been split into two subfamilies, T1Ag· and T1Agß, the amount of which depends on the species of the An. gambiae complex concerned. The Juan element is also composed of several subfamilies, each genus having its own subfamily. However, Juan has been found in only one or two isolates of Ae. albopictus and Ae. polynesiensis. Its distribution does not seem to match the phylogeny of the species, suggesting that horizontal transfer has occurred, perhaps as a result of hybridization between species of the Aedes genus (BensaadiMerchermek et al., 1994). The presence of different subfamilies of Juan and T1 is, however, also consistent with a master gene model in which a particular copy is amplified in each lineage (Deininger et al., 1992). The situation is different for RT1 and RT2. These elements are distinct, but belong to closely related families that compete for the same insertion site in the 28S rDNA (Besansky et al., 1992). They probably derive from an ancestral 28S specific transposable element. Their copy number varies considerably between lines and individuals (Besansky et al., 1992), suggesting a high degree of insertion polymor-

504


phism. The relative abundance of RT1 and RT2 differs in the sibling species of the An. gambiae complex: RT1 is present in An. melas, An. gambiae s.s., and An. arabiensis, but not in An. quadriannulatus or An. merus (Table 4). RT2 is present in all the species except An. merus. RT1 seems to be more abundant than RT2 in An. gambiae s.s., and conversely in An. arabiensis. RT1 shows such wide fluctuations in copy number between lines that it is difficult to draw any firm conclusion about its abundance. Nevertheless, RT1 and RT2 occupy significant proportions of the rDNA sequences, and this tends to differ somewhat between populations. In the Drosophila bobbed-8 mutants, that have a long development time, 50 % of the rRNA genes are interrupted by a 5-kb intervening sequence (Jamrich and Miller, 1984). Whether a similar relationship could also apply to RT1 and RT2 of An. gambiae is an open question.

SINEs SINEs are short (usually !500 bp) non-LTR retrotransposons, which do not encode for reverse transcriptase. Most SINEs are composed of three regions: a 5) tRNA-related region, a tRNA-unrelated region, and a 3) AT-rich region (Shedlock and Okada, 2000). Only two SINE families have been reported in mosquitoes: the Feilai family in the Aedes genus and the Twin family in the Culex genus (Table 5). The Feilai element presents a classical SINE structure, with a 5) tRNA-related region (69 % similarity with a Caenorhabditis elegans LeutRNA gene), a highly conserved tRNA unrelated region, and a 3) end with GAA repeats. This element accounts for around 2 % of the genome of Ae. aegypti, and seems to be highly interspersed and frequently associated with genes (Tu, 1999). Twin is approximately 220 bp long, has an unusual structure consisting of two tRNA-Arg related regions separated by a 39-bp spacer, and it is terminated by a short trailer and a poly(A) tract.

Table 4. Distribution of non-LTR retrotransposons among mosquito species Genus

Species

Non-LTR retrotransposons

Juan1

vash2 Guildenstern2 Jam13 Aara24 Aara84 Agam24 Amer54 Lian5

MosquI6 Q7

RT18 RT28 T19

Aedes aegypti albopictus polynesiensis

+ 200a ra ra

triseriatus

-b

pipiens quinquefasciatus tarsalis

+ 2500a +a +a

amboinensis

-b

gambiae arabiensis melas merus quadriannulatus stephensi albimanus

+ 28c

+ 1380d + 14 e 19c

+

Ochlerotatus Culex

Toxorhynchites Anopheles + 20a +

+ 200-300 e 98c 63f + + + + -

+ +

+ +

b

-b

+ + + -

+ + + +

+ 100g 54c 84f + + + + -

+: present; -: absent (see text for discussion); r: element present in a few strains; a: Southern blots; b: Southern blots and PCR; c: sequenced genome of Anopheles gambiae (Holt et al., 2002); d: estimate based on the number of matches in a genomic library and quantitative Southern blotting; e: estimate based on the number of matches in a genomic library; f: mean number of euchromatic insertion sites; g: dot blots; 1: Agarwal et al. (1993); Bensaadi-Merchermek et al. (1994); Hill et al. (2001); Mouches et al. (1991); 2: Hill et al. (2001); 3: Malik and Eickbush (1998); Warren et al. (1997); 4 : Cook et al. (2000); 5: Tu et al. (1998); 6: Tu and Hill (1999); 7: Besansky et al. (1994); Mukabayire and Besansky (1996); 8: Besansky et al. (1992); 9: Besansky (1990a, b); Mukabayire and Besansky (1996).

Table 5. Distribution of SINEs and unclassified elements among mosquito species

Genus

Species

SINEs and unclassified TEs CM-gag1 Odysseus2 Maque3 Feilai4

Twin5 Zebedee6

Aedes

aegypti albopictus

+ 59000c -d -d

-

+e

Ochlerotatus -d

triseriatus Culex + >150a

pipiens

+ 500c

Toxorhynchites -d

amboinensis Anopheles gambiae arabiensis stephensi

+

+ 220b

-

-d

+: present; -: absent (see text for discussion); a: data from dot blots and Southern blots; b: estimate based on the number of matches in an STS database; c: estimate based on the number of matches in a genomic library; d: PCR and Southern hybridization; e: degenerate PCR approach; 1: Bensaadi-Merchermek et al. (1997); 2: Mathiopoulos et al. (1998, 1999); 3: Tu (2001a); 4: Tu (1999); 5: Feschotte et al. (2001); 6: Warren et al. (1997).

This structure is thought to have arisen from the retroposition of a dicystronic tRNA transcript, rather than from the multimerization of two non-LTR retrotransposons (Feschotte et al., 2001). This element has probably been amplified quite recently in the lineage leading to the Culex genus, and it has been found in Culex pipiens and Culex hortensis but not in Aedes or Anopheles species.

Unclassified TEs There are several oddities among the TEs reported in mosquitoes (Table 5). Zebedee is the only family of LTR retroelements isolated from the Aedes genus; two members have been reported, each of which has a single ORF, which can encode a polyprotein with homology to protease, integrase and reverse


505

transcriptase of copia-like elements (Warren et al., 1997). There is no RNAse H domain, and either the gag ORF or the LTRs are lacking. An interesting feature is that the elements Zebedee I and II are both flanked by direct repeats, but there are no similarities between the repeats found in the two elements. CM-gag, which is found in the genome of C. pipiens, contains only a gag gene (Bensaadi-Merchermek et al., 1997), which is most similar to the gag sequence of the non-LTR retrotransposon jockey of D. melanogaster (25.8 % similarity in a 372-amino acid overlap). There are at least 150 copies of CMgag per haploid genome, and the longest members of this family appear to be highly conserved. Most of the mutations responsible for the divergence between copies are found in the third position of codons, suggesting that the ability to encode a gaglike protein was probably necessary for the element to invade the genome. Biedler and Tu (2003) also reported an element named Sponge in An. gambiae, which also contains only a gag gene. This family is related to a CR1 family of non-LTR retrotransposons found also in the An. gambiae genome. Maque is an unusually short repetitive element, only about 60 bp long, of which approximately 220 copies are found in the An. gambiae genome (Table 5). It is thought to be a short product of retrotranscription, as it contains 3) CAA repetitions, and is flanked by target site duplications (Tu, 2001a). Maque can therefore be classified as a SINE element with no internal promoter. Because this kind of short transposable element contains the reverse transcriptase recognition signal, Tu (2001a) suggests that its insertion near a promoter sequence could allow its transcription to take place and generate a SINE. Finally, a TE named Odysseus has been found at the breakpoint of a naturally occurring polymorphic inversion (2Rd)) in An. arabiensis (Mathiopoulos et al., 1998, 1999). This element has not yet been fully characterized.

Population genetic data Although several investigators have pointed out that the insertions of the particular TEs they were describing were polymorphic between strains, the only study in which quantitative information about polymorphism is available concerns the PEST strain of An. gambiae (Mukabayire and Besansky, 1996). The distributions of two non-LTR retrotransposons, Q and T1, a transposon, mariner, and a MITE, Pegasus, were determined by in situ hybridization on polytene chromosomes of several individuals. The mean occupancy frequency per site was 0.91 for mariner, 0.89 for T1, 0.77 for Q, and 0.70 for Pegasus. These values are of the same order as those found for many TEs in laboratory stocks of D. melanogaster (Biémont et al., 1994). The X chromosome is the least polymorphic chromosome; Xlinked sites being fixed for Pegasus and mariner, and no underrepresentation of elements has been detected on this chromosome, suggesting either that these elements do not have any negative recessive effect on fitness (Charlesworth and Charlesworth, 1983) or that they have been mobilized recently (Biémont et al., 1997). In the same strain, Grossman et al. (1999) also performed in situ hybridization with Tsessebe, Topi and Tiang. The most noticeable result is that the 20 euchromatic

506


sites found for Tiang were shared by the four individuals examined, suggesting a strong effect of drift on the PEST strain analyzed.

Impact of TEs on genome size and genome organization In the preliminary analysis of the TE content of the sequenced genome of An. gambiae, Holt et al. (2002) found that TEs constitute 16 % of the euchromatic and more than 50 % of the heterochromatic components of the genome. Consistent with this observation, Mukabayire and Besansky (1996) found that the Q, T1, and mariner elements accumulate in the chromocenter and pericentric regions, giving rise to a diffuse hybridization signal. However this was the case for neither Pegasus (Mukabayire and Besansky, 1996) nor Tsessebe, Topi, or Tiang (Grossman et al., 1999). Moreover, Mukabayire and Besansky (1996) reported a paucity of insertion sites of T1 in the distal part of chromosome arms 2R and 3L, of mariner in the distal part of 3R and 3L, and of Q in the distal part of all the chromosomes except the X chromosome. This contrasts with the high density of repeats described near the telomeres in the sequenced genome of An. gambiae (Holt et al., 2002). This pattern was inferred from a large dataset, which could explain why Mukabayire and Besansky (1996) obtained different results. Mukabayire and Besansky (1996) noted that the number of multiple insertion sites into the same polytene chromosome subdivision was higher than expected for Q, suggesting that the insertion of one copy in a subdivision could favor the insertion of a second element. Furthermore, there was an excess of coincident hybridization sites for the different elements, suggesting the existence of insertion hotspot regions as a result of a particular chromatin conformation. Pegasus showed a strong tendency to be inserted near other TEs, with 40 % of its hybridization signals shared by at least one other TE. This tendency was also observed for other MITEs (Tu, 1997, 2000), suggesting that some chromosomal locations could be a safe haven for some repetitive sequences (Tu, 2000). MITEs appear to be mainly inserted in the vicinity of genes (Tu, 1997), suggesting a possible influence on the expression of these genes, a phenomenon that has been reported for TEs in the human genome (Jordan et al., 2003) and that of the nematode Caenorhabditis elegans (Ganko et al., 2003). This association between MITEs and genes could also play a role in what has been called the short period interspersion pattern. This term has been coined to describe the organization of the genome of some species, including Ae. aegypti (Warren and Crampton, 1991) in which single copy sequences of 1000/ 2000 bp alternate regularly with short (!4000 bp) repetitive sequences. In contrast, a long period interspersion pattern is usually found in organisms with smaller genomes, such as D. melanogaster and An. gambiae (Rai and Black, 1999), and is characterized by the alternation of long repetitive sequences (15600 bp) with very long stretches of single copy sequences. Drosophila seems to contain few MITEs, although two families have recently been identified in D. willistoni (Holyoake et al., 2003).

Genome size variation depends mainly on amplification, deletions, and divergence of various repetitive sequences that are either distributed along the chromosomes or mainly concentrated within heterochromatin (Flavell, 1980; Gregory and Hebert, 1999). The genome size of Ae. albopictus was found to vary between populations (Rao and Rai, 1987; Kumar and Rai, 1990), and this was attributed to variations in the amount of highly repetitive sequences (Black and Rai, 1988). It would be interesting to see whether the MITE elements are implicated in these variations, and whether similar variations also arise in Anopheles and Culex populations, for instance following changes in environmental conditions, as it has been suggested in D. simulans (Vieira et al., 2002). Although MITEs are present in An. gambiae, they have a lower copy number than in Ae. aegypti, which is consistent with the fact that the genome of An. gambiae is smaller than that of Ae. aegypti, and that genome size appears to be correlated with the amount of MITEs (Tu, 1997; Feschotte and Mouchès, 2000; Tu, 2001b). Only a few families of MITEs have been described, however, and none has been checked for simultaneously in both An. gambiae and Ae. aegypti. The An. gambiae complex, which is thought to have split recently and includes species with quite different ecologies, is thus a useful model for testing for possible relationships between TE mobilization, genome size and environmental changes, and the potential influences of TEs in population diversification and adaptation.

Are there any active TEs in mosquitoes? Hypomorphic mutations are rare in An. gambiae (Mukabayire and Besansky, 1996), and nothing resembling the hybrid dysgenesis phenomenon (Kidwell and Lisch, 2001) has been observed in mosquitoes. No direct observation of transposition of an autochthonous TE has been reported to date. Nevertheless, there are some indications that at least some elements could be active. For example, the element mtanga-Y is actively transcribed in males of An. gambiae, and one LTR-LTR circular molecule, probably representing a retrotranscription intermediate, has been isolated (Rohr et al., 2002). Ikirara has been shown to excise in an Anopheles cell line (Leung and Romans 1998), Moose is strongly expressed in male and female germlines, and transcripts have been detected for Zebedee (Warren et al., 1997), Twin (Feschotte et al., 2001), and CM-gag (Bensaadi-Merchermek et al., 1997). Shao et al. (2001) reported that the eight sequenced copies of MsqTc3 were 99 % similar, and Biedler and Tu (2003) showed that several non-LTR retrotransposons families had corresponding ESTs and multiple copies with more than 99 % nucleotide similarity. This suggests that recent transposition has occurred, as has also been concluded from findings from the Drosophila genome (Lerat et al., 2003).

Conclusions Mosquitoes were the first insects in which MITEs were described. They possess several LTR retrotransposons belonging to the Pao family, which is still poorly resolved,

although it has been shown to be distinct from the Gypsy-Ty3 and Copia-Ty1 families (Xiong et al., 1993; Cook et al., 2000; Abe et al., 2001). The transposons (class II elements), non-LTR retrotransposons (class I elements) and MITEs found in the Anopheles, Aedes and Culex genera, display variable copy numbers, from 1 up to 2000 copies. The copy numbers of many TEs must, however, be viewed with caution for at least two reasons: first, some measurements estimate the overall number of copies in a genome, whereas others estimate the euchromatic insertion sites only. Second, the estimates depend on the method employed. Keeping that in mind, there seem to be huge variations in copy number between TE classes within a given species and even between different families within a given class. For example, the class II ITmD37E element has 40 copies or less, whereas the non-LTR retroelement Q has between 63 and 300 copies in An. gambiae. MosquI and Lian, two non-LTR retrotransposons, have around 15 and 1380 copies in Ae. aegypti, respectively. In their recent study, Biedler and Tu (2003) found that the copy number of non-LTR retrotransposons varies from a few copies to 2000, depending on the family. If this variability in the number of copies of different TE families in a given species is confirmed, this will lead to a rather different picture from what is observed in Drosophila, which displays only minor differences in copy number between families (from 1 to 100: Biémont and Cizeron, 1999). This would mean that transposable elements could act in markedly different ways in the genomes of two insects. Mosquitoes offer an opportunity to compare the behavior of TEs between species at different scales: between the distantly related D. melanogaster and An. gambiae genomes (Zdobnov et al., 2002), between different genera, and between very closely related species within the An. gambiae complex. These species diverged very recently (Coluzzi et al., 2002) and this is a unique opportunity to study the impact of TEs on the adaptation to new environments and on the diversification of the complex, or conversely to study the impact that this diversification has had on TE distribution in genomes and populations. In order to understand the evolution and organization of mosquito genomes, a comparative approach is required every time a new element is reported. Moreover, there is a crucial lack of surveys concerning the insertion polymorphism in natural populations. Such surveys are particularly necessary in the light of the current effort to develop a transgenic mosquito unable to transmit malaria (Ito et al., 2002), intended to replace mosquitoes in natural populations. This will not be possible until we can understand the genomic structure of natural populations. The use of TEs as markers, which has led to satisfactory results in several different species of plants (Kalendar et al., 1999; Kumar and Hirochika, 2001) and Drosophila (Lepetit et al., 2002) could help resolve the extremely complex case of An. gambiae populations (Lehmann et al., 2003), eventually by using the transposon display technique (Biedler et al., 2003). Moreover, the transgene will only become fixed within natural populations if there is an efficient genetic drive mechanism to help this to occur (Boete and Koella, 2003; Catteruccia et al., 2003). From this perspective, studying the dynamics of TEs in natural populations is essential if transgenesis is to be made to work. The control of the transgene (Atkinson et al., 2001) is another cru-


507

cial point, because it is known that introduced elements can be mobilized in the absence of endogenous copies (Sundararajan et al., 1999). Accurate knowledge about the TE families present in natural populations of mosquitoes is therefore crucial if we are to understand and avoid the potential mobilization of transgenes.

Acknowledgements We would like to thank Monika Ghosh for reviewing the English text, and Cristina Vieira and the reviewers for comments.

References Abe H, Ohbayashi F, Sugasaki T, Kanehara M, Terada T, Shimada T, Kawai S, Mita K, Kanamori Y, Yamamoto MT, et al.: Two novel Pao-like retrotransposons (Kamikaze and Yamato) from the silkworm species Bombyx mori and B. mandarina: common structural features of Pao-like elements. Mol Genet Genomics 265:375–385 (2001). Adams MD, Celniker SE, Holt RA, Evans CA, Gocayne JD, Amanatides PG, Scherer SE, Li PW, Hoskins RA, Galle RF, et al.: The genome sequence of Drosophila melanogaster. Science 287: 2185–2195 (2000). Agarwal M, Bensaadi N, Salvado JC, Campbell K, Mouches C: Characterization and genetic organization of full-length copies of a LINE retroposon family dispersed in the genome of Culex pipiens mosquitoes. Insect Biochem Mol Biol 23:621–629 (1993). Anderson JR, Grimstad PR, Severson DW: Chromosomal evolution among six mosquito species (Diptera: Culicidae) based on shared restriction fragment length polymorphisms. Mol Phylogenet Evol 20:316–321 (2001). Atkinson PW, Pinkerton AC, O’Brochta DA: Genetic transformation systems in insects. Annu Rev Entomol 46:317–346 (2001). Bensaadi-Merchermek N, Salvado JC, Mouches C: Mosquito transposable elements. Genetica 93: 139–148 (1994). Bensaadi-Merchermek N, Cagnon C, Desmons I, Salvado JC, Karama S, D’Amico F, Mouches C: CMgag, a transposable-like element reiterated in the genome of Culex pipiens mosquitoes, contains only a gag gene. Genetica 100:141–148 (1997). Besansky NJ: A retrotransposable element from the mosquito Anopheles gambiae. Mol Cell Biol 10: 863–871 (1990a). Besansky NJ: Evolution of the T1 retroposon family in the Anopheles gambiae complex. Mol Biol Evol 7:229–246 (1990b). Besansky NJ, Fahey GT: Utility of the white gene in estimating phylogenetic relationships among mosquitoes (Diptera: Culicidae). Mol Biol Evol 14: 442–454 (1997). Besansky NJ, Paskewitz SM, Hamm DM, Collins FH: Distinct families of site-specific retrotransposons occupy identical positions in the rRNA genes of Anopheles gambiae. Mol Cell Biol 12:5102–5110 (1992). Besansky NJ, Bedell JA, Mukabayire O: Q: a new retrotransposon from the mosquito Anopheles gambiae. Insect Mol Biol 3:49–56 (1994). Besansky NJ, Mukabayire O, Bedell JA, Lusz H: Pegasus, a small terminal inverted repeat transposable element found in the white gene of Anopheles gambiae. Genetica 98:119–129 (1996). Biedler J, Tu Z: Non-LTR retrotransposons in the African malaria mosquito, Anopheles gambiae: unprecedented diversity and evidence of recent activity. Mol Biol Evol 20:1811–1825 (2003). Biedler J, Qi Y, Holligan D, Della Torre A, Wessler S, Tu Z: Transposable element (TE) display and rapid detection of TE insertion polymorphism in the Anopheles gambiae species complex. Insect Mol Biol 12:211–216 (2003).

508

Biémont C, Cizeron G: Distribution of transposable elements in Drosophila species. Genetica 105:43– 62 (1999). Biémont C, Lemeunier F, Garcia Guerreiro MP, Brookfield JF, Gautier C, Aulard S, Pasyukova EG: Population dynamics of the copia, mdg1, mdg3, gypsy, and P transposable elements in a natural population of Drosophila melanogaster. Genet Res 63:197–212 (1994). Biémont C, Tsitrone A, Vieira C, Hoogland C: Transposable element distribution in Drosophila. Genetics 147:1997–1999 (1997) Biessmann H, Walter MF, Le D, Chuan S, Yao JG: Moose, a new family of LTR retrotransposons in the mosquito Anopheles gambiae. Insect Mol Biol 8:201–212 (1999). Black WC IV, Rai KS: Genome evolution in mosquitoes: intraspecific and interspecific variation in repetitive DNA amounts and organization. Genet Res 51:185–196 (1988). Boete C, Koella JC: Evolutionary ideas about genetically manipulated mosquitoes and malaria control. Trends Parasitol 19:32–38 (2003). Capy P, Anxolabéhère D, Langin T: The strange phylogenies of transposable elements: are horizontal transfers the only explantation? Trends Genet 10:7–12 (1994). Catteruccia F, Godfray HC, Crisanti A: Impact of genetic manipulation on the fitness of Anopheles stephensi mosquitoes. Science 299:1225–1227 (2003). Charlesworth B, Charlesworth D: The population dynamics of transposable elements. Genet Res 42:1– 27 (1983). Coluzzi M, Sabatini A, della Torre A, Di Deco MA, Petrarca V: A polytene chromosome analysis of the Anopheles gambiae species complex. Science 298: 1415–1418 (2002). Cook JM, Martin J, Lewin A, Sinden RE, Tristem M: Systematic screening of Anopheles mosquito genomes yields evidence for a major clade of Pao-like retrotransposons. Insect Mol Biol 9:109–117 (2000). Deininger PL, Batzer MA, Hutchison CA III, Edgell MH: Master genes in mammalian repetitive DNA amplification. Trends Genet 8:307–311 (1992). Feschotte C, Mouches C: Recent amplification of miniature inverted-repeat transposable elements in the vector mosquito Culex pipiens: characterization of the Mimo family. Gene 250:109–116 (2000). Feschotte C, Fourrier N, Desmons I, Mouches C: Birth of a retroposon: the Twin SINE family from the vector mosquito Culex pipiens may have originated from a dimeric tRNA precursor. Mol Biol Evol 18:74–84 (2001). Feschotte C, Zhang X, Wessler SR: Miniature invertedrepeat transposable elements and their relationship to established DNA transposons, in Craig NL, Craigie R, Gellert M, Lambowitz AM (eds): Mobile DNA II, pp 1147–1158 (ASM Press, Washington, DC 2002). Flavell RB: The molecular characterisation and organization of plant chromosomal DNA sequences. Annu Rev Plant Physiol 31:569–596 (1980).


Ganko EW, Bhattacharjee V, Schliekelman P, McDonald JF: Evidence for the contribution of LTR retrotransposons to C. elegans gene evolution. Mol Biol Evol 20:1925–1931 (2003). Gregory TR, Hebert PD: The modulation of DNA content: proximate causes and ultimate consequences. Genome Res 9:317–324 (1999). Grossman GL, Cornel AJ, Rafferty CS, Robertson HM, Collins FH: Tsessebe, Topi and Tiang: three distinct Tc1-like transposable elements in the malaria vector, Anopheles gambiae. Genetica 105:69–80 (1999). Hill SR, Leung SS, Quercia NL, Vasiliauskas D, Yu J, Pasic I, Leung D, Tran A, Romans P: Ikirara insertions reveal five new Anopheles gambiae transposable elements in islands of repetitious sequence. J Mol Evol 52:215–231 (2001). Holt RA, Subramanian GM, Halpern A, Sutton GG, Charlab R, Nusskern DR, Wincker P, Clark AG, Ribeiro JM, Wides R, et al: The genome sequence of the malaria mosquito Anopheles gambiae. Science 298:129–149 (2002). Holyoake AJ, Kidwell MG: Vege and Mar: two novel hAT MITE families from Drosophila willistoni. Mol Biol Evol 20:163–167 (2003). Imwong M, Sharpe RG, Kittayapong P, Baimai V: Distribution of the transposable element mariner in anopheline mosquitoes. Heredity 85:271–276 (2000). Ito J, Ghosh A, Moreira LA, Wimmer EA, Jacobs-Lorena M: Transgenic anopheline mosquitoes impaired in transmission of a malaria parasite. Nature 417:452–455 (2002). Jamrich M, Miller OL Jr: The rare transcripts of interrupted rRNA genes in Drosophila melanogaster are processed or degraded during synthesis. EMBO J 3:1541–1545 (1984). Jordan IK, Rogozin IB, Glazko GV, Koonin EV: Origin of a substantial fraction of human regulatory sequences from transposable elements. Trends Genet 19:68–72 (2003). Kalendar R, Grob T, Regina M, Suoniemi A, Schulman A: IRAP and REMAP: two new retrotransposonbased DNA fingerprinting techniques. Theor Appl Genet 98:704–711 (1999). Kapitonov VV, Jurka J: Rolling-circle transposons in eukaryotes. Proc Natl Acad Sci USA 98:8714– 8719 (2001). Kapitonov VV, Jurka J: Molecular paleontology of transposable elements in the Drosophila melanogaster genome. Proc Natl Acad Sci USA 100:6569– 6574 (2003). Ke Z, Grossman GL, Cornel AJ, Collins FH: Quetzal: a transposon of the Tc1 family in the mosquito Anopheles albimanus. Genetica 98:141–147 (1996). Kidwell MG, Lisch DR: Perspective: transposable elements, parasitic DNA, and genome evolution. Evolution 55:1–24 (2001). Krzywinski J, Besansky NJ: Molecular systematics of Anopheles: from subgenera to subpopulations. Annu Rev Entomol 48:111–139 (2003). Krzywinski J, Wilkerson RC, Besansky NJ: Evolution of mitochondrial and ribosomal gene sequences in anophelinae (Diptera: Culicidae): implications for phylogeny reconstruction. Mol Phylogenet Evol 18:479–487 (2001).

Kumar A, Hirochika H: Applications of retrotransposons as genetic tools in plant biology. Trends Plant Sci 6:127–134 (2001). Kumar A, Rai KS: Intraspecific variation in nuclear DNA content among world populations of a mosquito, Aedes albopictus (Skuse). Theor Appl Genet 79:748–752 (1990). Lehmann T, Licht M, Elissa N, Maega BT, Chimumbwa JM, Watsenga FT, Wondji CS, Simard F, Hawley WA: Population structure of Anopheles gambiae in Africa. J Hered 94:133–147 (2003). Lepetit D, Brehm A, Fouillet P, Biémont C: Insertion polymorphism of retrotransposable elements in populations of the insular, endemic species Drosophila madeirensis. Mol Ecol 11:347–354 (2002). Lerat E, Rizzon C, Biémont C: Sequence divergence within transposable element families in the Drosophila melanogaster genome. Genome Res 13: 1889–1896 (2003). Leung SS, Romans P: Excisions of the Ikirara1 transposon in an Anopheles gambiae cell line. Insect Mol Biol 7:241–248 (1998). Lovsin N, Gubensek F, Kordis D: Evolutionary dynamics in a novel L2 clade of non-LTR retrotransposons in Deuterostomia. Mol Biol Evol 18:2213–2224 (2001). Luckhart S, Rosenberg R: Gene structure and polymorphism of an invertebrate nitric oxide synthase gene. Gene 232:25–34 (1999). Malik HS, Eickbush TH: The RTE class of non-LTR retrotransposons is widely distributed in animals and is the origin of many SINEs. Mol Biol Evol 15:1123–1134 (1998). Malik HS, Burke WD, Eickbush TH: The age and evolution of non-LTR retrotransposable elements. Mol Biol Evol 16:793–805 (1999). Marin I, Fontdevila A: Evolutionary conservation and molecular characteristics of repetitive sequences of Drosophila koepferae. Heredity 76:355–366 (1996). Mathiopoulos KD, della Torre A, Predazzi V, Petrarca V, Coluzzi M: Cloning of inversion breakpoints in the Anopheles gambiae complex traces a transposable element at the inversion junction. Proc Natl Acad Sci USA 95:12444–12449 (1998). Mathiopoulos KD, della Torre A, Santolamazza F, Predazzi V, Petrarca V, Coluzzi M: Are chromosomal inversions induced by transposable elements? A paradigm from the malaria mosquito Anopheles gambiae. Parassitologia 41:119–123 (1999). Mouches C, Agarwal M, Campbell K, Lemieux L, Abadon M: Sequence of a truncated LINE-like retroposon dispersed in the genome of Culex mosquitoes. Gene 106:279–280 (1991). Mukabayire O, Besansky NJ: Distribution of T1, Q, Pegasus and mariner transposable elements on the polytene chromosomes of PEST, a standard strain of Anopheles gambiae. Chromosoma 104:585–595 (1996). Munstermann LE, Conn JE: Systematics of mosquito disease vectors (Diptera, Culicidae): impact of molecular biology and cladistic analysis. Annu Rev Entomol 42:351–369 (1997).

Poulter RT, Goodwin TJ, Butler MI: Vertebrate helentrons and other novel Helitrons. Gene 313:201– 212 (2003). Rai KS, Black WC IV: Mosquito genomes: structure, organization, and evolution. Adv Genet 41:1–33 (1999). Rao PN, Rai K: Inter and intraspecific variation in nuclear DNA content in Aedes mosquitoes. Heredity 59:253–258 (1987). Reinert JF: New classification for the composite genus Aedes (Diptera: Culicidae: Aedini), elevation of subgenus Ochlerotatus to generic rank, reclassification of the other subgenera, and notes on certain subgenera and species. J Am Mosq Control Assoc 16:175–188 (2000). Rezsohazy R, van Luenen HG, Durbin RM, Plasterk RH: Tc7, a Tc1-hitchhiking transposon in Caenorhabditis elegans. Nucleic Acids Res 25:4048–4054 (1997). Robertson HM: The mariner transposable element is widespread in insects. Nature 362:241–245 (1993). Robertson HM, Lampe DJ: Recent horizontal transfer of a mariner transposable element among and between Diptera and Neuroptera. Mol Biol Evol 12:850–862 (1995). Robertson HM, Soto-Adames FN, Walden KKO, Avancini RMP, Lampe DJ: The mariner transposons of animals: horizontally jumping genes, in Syvanen M, Kado C (eds): Horizontal Gene Transfer, pp 268–284 (Chapman and Hall, London 1998). Rohr CJ, Ranson H, Wang X, Besansky NJ: Structure and evolution of mtanga, a retrotransposon actively expressed on the Y chromosome of the African malaria vector Anopheles gambiae. Mol Biol Evol 19:149–162 (2002). Romans P, Bhattacharyya RK, Colavita A: Ikirara, a novel transposon family from the malaria vector mosquito, Anopheles gambiae. Insect Mol Biol 7:1–10 (1998). Sarkar A, Sim C, Hong YS, Hogan JR, Fraser MJ, Robertson HM, Collins FH: Molecular evolutionary analysis of the widespread piggyBac transposon family and related ‘domesticated’ sequences. Mol Genet Genomics 270:173–180 (2003a). Sarkar A, Sengupta R, Krzywinski J, Wang X, Roth C, Collins FH: P elements are found in the genomes of nematoceran insects of the genus Anopheles. Insect Biochem Mol Biol 33:381–387 (2003b). Shao H, Tu Z: Expanding the diversity of the IS630Tc1-mariner superfamily: discovery of a unique DD37E transposon and reclassification of the DD37D and DD39D transposons. Genetics 159:1103–1115 (2001). Shao H, Qi Y, Tu Z: MsqTc3, a Tc3-like transposon in the yellow fever mosquito Aedes aegypti. Insect Mol Biol 10:421–425 (2001). Shedlock AM, Okada N: SINE insertions: powerful tools for molecular systematics. Bioessays 22:148– 160 (2000). Sundararajan P, Atkinson PW, O’Brochta DA: Transposable element interactions in insects: crossmobilization of hobo and Hermes. Insect Mol Biol 8:359–368 (1999).

Tu Z: Three novel families of miniature invertedrepeat transposable elements are associated with genes of the yellow fever mosquito, Aedes aegypti. Proc Natl Acad Sci USA 94:7475–7480 (1997). Tu Z: Genomic and evolutionary analysis of Feilai, a diverse family of highly reiterated SINEs in the yellow fever mosquito, Aedes aegypti. Mol Biol Evol 16:760–772 (1999). Tu Z: Molecular and evolutionary analysis of two divergent subfamilies of a novel miniature inverted repeat transposable element in the yellow fever mosquito, Aedes aegypti. Mol Biol Evol 17:1313–1325 (2000). Tu Z: Maque, a family of extremely short interspersed repetitive elements: characterization, possible mechanism of transposition, and evolutionary implications. Gene 263:247–253 (2001a). Tu Z: Eight novel families of miniature inverted repeat transposable elements in the African malaria mosquito, Anopheles gambiae. Proc Natl Acad Sci USA 98:1699–1704 (2001b). Tu Z, Hill JJ: MosquI, a novel family of mosquito retrotransposons distantly related to the Drosophila I factors, may consist of elements of more than one origin. Mol Biol Evol 16:1675–1686 (1999). Tu Z, Orphanidis SP: Microuli, a family of miniature subterminal inverted-repeat transposable elements (MSITEs): transposition without terminal inverted repeats. Mol Biol Evol 18:893–895 (2001). Tu Z, Isoe J, Guzova JA: Structural, genomic, and phylogenetic analysis of Lian, a novel family of nonLTR retrotransposons in the yellow fever mosquito, Aedes aegypti. Mol Biol Evol 15:837–853 (1998). Vieira C, Biémont C: Transposable element dynamics in the two sibling species Drosophila melanogaster and Drosophila simulans. Genetica 120:115–123 (2004). Vieira C, Nardon C, Arpin C, Lepetit D, Biémont C: Evolution of genome size in Drosophila. Is the invader’s genome being invaded by transposable elements? Mol Biol Evol 19:1154–1161 (2002). Warren AM, Crampton JM: The Aedes aegypti genome: complexity and organization. Genet Res 58:225–232 (1991). Warren AM, Hughes MA, Crampton JM: Zebedee: a novel copia-Ty1 family of transposable elements in the genome of the medically important mosquito Aedes aegypti. Mol Gen Genet 254:505–513 (1997). Xiong Y, Burke WD, Eickbush TH: Pao, a highly divergent retrotransposable element from Bombyx mori containing long terminal repeats with tandem copies of the putative R region. Nucleic Acids Res 21:2117–2123 (1993). Zdobnov EM, von Mering C, Letunic I, Torrents D, Suyama M, Copley RR, Christophides GK, Thomasova D, Holt RA, Subramanian GM, et al.: Comparative genome and proteome analysis of Anopheles gambiae and Drosophila melanogaster. Science 298:149–159 (2002).


509