BACKGROUND
transposable elements  are dna sequences that can move within a cell's genome from one chromosomal location to another by a reaction called transposition, augmenting in this way their own representation within genomes.
once having infected the germline of an organism, they can also spread through populations.
these elements are ubiquitous in eukaryotes, accounting in certain species for a high proportion of their genomic dna; for instance, approximately 50% of the human genome  <cit>  and up to 80% of the maize genome  <cit>  are composed of tes.
in anopheles gambiae, the transmitting vector of the malaria parasite, these elements constitute 16% of the euchromatin and up to 60% of the heterochromatin of the genome  <cit> .
these elements have quite simple genetic structures, basically containing genes involved in their transposition--which is generally independent from the cellular replicative machinery--and their flanking recognition sites.
they can be seen as genomic infectious agents and due to this characteristic were originally considered as selfish dna  <cit> ; however, as more information was gathered about te biology, their relevance as important players in evolution and maintenance of eukaryotic genomes has been emphasized  <cit> .
tes can replicate via an rna or a dna intermediate and have been traditionally classified accordingly into two classes   <cit> .
class i elements, also known as retrotransposons, depend on a reverse transcription step.
they are nearly identical to retroviruses in structure and are related phylogenetically  <cit> .
they contain a gag-like gene, a pol-gene  and in certain cases a third open reading frame  coding for an env-like protein  <cit> .
class i is subsequently classified into five orders based on major differences in their insertion mechanism as well as overall genetic organization  <cit> .
class ii elements are genetically and structurally more homogenous and simple.
their basic genetic structure consists of a transposase gene flanked by terminal inverted repeats , which are fundamental for their recognition by transposases and further mobilization.
this class has been classified into two subclasses, which are subsequently grouped into different orders  <cit> .
the classification of class ii tes based in their genetic differences permitted a further classification in superfamilies and families.
the proportion of the different classes, orders, and superfamilies of tes in a given genome varies in different species; some harbor very abundant but few families of tes as mammals, where class i non-long terminal repeats  predominate  <cit> , while others contain many different yet less abundant families.
these differences have been related to the c-value paradox  <cit> , which refers to the huge variations in genome size of eukaryotic organisms despite similar levels of organism complexity  <cit> .
for instance, the large differences observed in genome size between an.
gambiae  and aedes aegypti  is determined by their differential content of tes.
on the other hand, tes can be classified as autonomous and non-autonomous based on their ability to perform their own transposition.
moreover, within these two categories, tes can be also classified as active or inactive based in their actual mobility, which depends on several factors, among which the presence of an active, autonomous element of the same family--or close enough to be able to mobilize it--is fundamental.
an important aspect of the study of tes in a given genome is the understanding of their abundance and diversity as well as the classification and organization of the elements.
currently, repbase is the reference collection of repetitive elements from most eukaryotic genomes that can be consulted online  <cit> .
this database is being used in genome sequencing projects as a collection for masking and annotation of repetitive dna, because it contains a picture of the te landscape in several genomes.
however, most te sequences from repbase are prototypic consensus sequences of families and subfamilies of repeats.
on the other hand, a specific te database called tefam presents te sequences from three mosquito genomes: an.
gambiae, a. aegypti, and culex quinquefasciatus  <cit> .
tefam contains a more restricted number of te families in the mosquito genomes.
previous analyses of tes in an.
gambiae have identified several element families  <cit> ; however, the data are often scattered in the databases or clearly unavailable.
the aim of this work was to describe the diversity of tes within the an.
gambiae genome following the use of the piler tool, which identifies repetitive elements in whole genomes  <cit> --with a particular interest in the detection of new, active elements--as well as to consolidate a database in the form of a hyperlinked spreadsheet containing detailed and readily available information about the te diversity and other repetitive elements found in the genome of an.
gambiae.
the purpose was to present the platform used in the identification and characterization of tes in the genome of an.
gambiae, which, in turn, can be used to screen other sequenced genomes.
RESULTS
an important aspect in the study of the tes in a genome is knowledge of their abundance and diversity, as well as classification and further organization of this information in a clear and comprehensive manner.
aiming at the discovery of unknown, active elements, we performed a search and characterization of tes in the sequenced genome of an.
gambiae.
we used a combined strategy  to identify and characterize repetitive elements present in the genome of the primary vector of malaria, an.
gambiae.
our strategy combines an extensive search of repetitive elements within this genome utilizing the piler-df algorithm as a first screening method, together with a detailed characterization of each of the retrieved sequences by analyzing signature characteristics of tes as well as performing homology-based analysis of the obtained sequences to several databases.
this automated pipeline includes the different steps suggested recently by wicker, et al.,  <cit>  for classifying eukaryotic tes.
the results are compiled in a database of repetitive elements in the mosquito genome, called anotexcel  that provides information for all the te families identified, along with offering the individual sequences found in each family.
database description
anotexcel is organized as an excel spreadsheet with cells containing, in a hyperlinked format, the results obtained after the various analyses performed in the characterization of each family.
each line of the spreadsheet represents a cluster containing a variable number of sequences with a high degree of identity among them, constituting--in most of the cases--te families.
the spreadsheet columns contain the several analyses performed on the sequences and were grouped into four types, colored for easier viewing as follows: blue columns represent the classification of the families; green columns present the general characteristics of the sequences in clusters, such as presence of tirs and their sequences, direct repeats , consensus and centroid sequences, orf sequences, etc.
in orange columns are presented all the results of the blasts performed on several databases , and in yellow columns  are results of new clustering performed by tblastx considering different degrees of identity over at least 50% of the length of the alignments .
this was performed to identify shorter fragments of sequences belonging to the same family that were included in different clusters when more stringent conditions were applied.
clusters that share some degree of identity are colored in column a in anotexcel.
this is not an exhaustive database, as some of the element families already known to be present in an.
gambiae have not been identified by our algorithm; however, it has the important and distinctive characteristic of presenting all the individual sequences within each family  as well as their global alignments in fasta format, the consensus and centroid sequences, and detailed information on their structural characteristics.
in addition, it presents the results obtained after several blasts performed on different databases and includes in hyperlinked format the file with the significant matches to the given database.
this constitutes a rich resource that includes important information on the different families of tes present in the genome of an.
gambiae besides serving as a platform for the analysis of tes present in other sequenced genomes.
anotexcel contains  <dig> repetitive sequences larger than  <dig> nts in length that were grouped into  <dig> clusters .
the repetitive sequences are considered "intact" because they are globally alignable and "isolated" because they are surrounded by unique sequences; therefore, they correspond to insertional events of different repetitive sequences, mainly tes  <cit> .
our strategy in generation of the clusters permitted the grouping of sequences belonging to the same te family with different sizes or corresponding to different regions of the same element.
considering that the sequences identified by piler-df are isolated and unique, it is possible to state that those sequences represent different transposition states of the same te family, allowing for evolutionary or dynamical analysis.
the spreadsheet containing anotexcel can be found at http://exon.niaid.nih.gov/transcriptome/te/a_gambiae/anotexcel-web.zip and the standalone data can be downloaded from http://exon.niaid.nih.gov/transcriptome/te/a_gambiae/anotexcel-sa.zip
the latter file should be decompressed to the user's computer, and the excel file should be opened from within excel.
anotexcel can be downloaded and stored in a personal computer; it occupies approximately  <dig> mb and can be modified by adding or deleting columns according to the user's needs.
we were able to assign most of the retrieved sequences  to a given te class ; the rest were classified as rrna , or repetitive sequences with no te signatures  and possibly expressed pseudogenes .
in order to assess the efficiency of our approach in identifying and characterizing tes from the genome of a. gambiae we compared the number of sequences in anotexcel with those deposited in repbase and tefam.
the results are shown in figure  <dig>  it should be noted that these figures are not 100% comparable since many elements are deposited in repbase and tefam under different names .
also, repbase and anotexcel present data on subfamilies  that were included in figure  <dig>  in addition, some but not all of the elements present in tefam are also deposited in repbase .
we identified approximately half of the ltr elements that have been described so far in the genome of an.
gambiae, few of the nltrs and almost all the known class ii elements.
moreover, we were able to identify and characterize several families of elements  that have not been reported previously, even using similar approaches for identifying tes in the mosquito genome  <cit> .
further characterization of the te families is given below.
te characterization
the te families identified in anotexcel were classified according to their class, subclass, order, superfamily, and family, following the wicker criteria for te classification.
we also classified the families according to the length of sequences in each cluster, i.e., according to the percentage of the cluster consensus sequence representing the full-length canonical element as: full-length , fragments or remnants  and, depending on the case, as solo ltr , mites , or class ii-na .
the methodology used here permitted identification of families presenting both full-length elements and degenerate copies of the same family .
the ltr elements contain several full-length putative active sequences, while nltrs and class ii elements are mainly constituted by fragments or remnant sequences.
for the nltr elements, the percentage of clusters considered as "full" in anotexcel is notably higher than the percentage of full-length sequences that in fact exists, indicating that many of the nltr clusters constitute a mixture of both full-length and fragmented sequences.
notably, although the an.
gambiae genome has been scrutinized in search of tes before using several methodologies  <cit> , we have been able to identify novel te families that have not been previously identified, as detailed further below.
elements class i, order ltr
the ltr elements in the genome of an.
gambiae constitute a numerous order, although there are only three superfamilies: ty1-copia, pao-bel, and t3-gyspsy, the last being the most diverse ltr superfamily within the mosquito genome.
the ltr elements identified in anotexcel were characterized based on the presence of flanking ltrs and/or based on positive matches to peptidase_a <dig>  rvt_ <dig> or  <dig> or rve in pfam or directly due to their significant matches to known ltr elements already deposited in the te databases  or in the genbank non-redundant database.
in anotexcel, they correspond to 19% of all the families identified , and  <dig> % of the total tes identified, totalizing  <dig> different clusters, and are represented by members of the main three superfamilies.
novel elements corresponding to the copia and pao-bel superfamilies were also identified and will be described later.
full-length and fragmented elements are not equally represented in the different ltr superfamilies.
while the majority of the pao-bel elements are represented by full-length copies , the gypsys are represented by solo-ltrs , and the few copia families identified correspond to full-length elements.
the predicted activity of the full-length sequences was also studied .
based on i) the degree of nucleotide identity among the sequences belonging to the same families; ii) the degree of identity among the ltrs of individual elements ; iii) the indication of expression based on positive matches to est databases in anotexcel; and iv) the presence of full-length orfs containing conserved domains for the proteins involved in transposition, we concluded that a substantial fraction of the ltrs described here corresponds to putatively active elements, as indicated by their presence within cdna libraries  .
the ltr families contain elements presenting an average nucleotide identity higher than  <dig> %, and comparison of the ltrs among all the sequences in each of the clusters showed a high degree of identity for the three superfamilies .
in addition, the identity between the two ltrs present in the individual sequences was calculated and appeared to be very high for some of the ltr members  .
the presence of full-length orfs in the sequences reinforces the idea that these families are active or have been very recently inactivated  <cit> .
copia elements
the copia superfamily in the mosquito genome is represented by five different families: copia1-5_ag  <cit>  and the mtanga family  <cit> .
these families have been reported to repbase, and none of them is present in tefam.
in anotexcel, elements belonging to two families, copia3_ag  and copia5_ag  were identified.
the consensus sequence of these two families is identical to the corresponding consensus sequences deposited in repbase.
element copia3_ag is apparently a truncated element with no conserved protein domains.
the copia5_ag sequences present some characteristics of activity, such as the presence of conserved protein domains for rt and rve and identical within sequence ltrs, even if they presented no positive matches to the mrna and the est databases .
novel copia elements
two clusters  were characterized as elements belonging to the copia superfamily, although it was not possible to assign them to any of the already described families within this superfamily since they present high nucleotide distances against all the previous characterized copia elements .
the consensus sequences of these clusters showed significant matches by tblastx with the copia5_ag consensus sequence and copia_dm from drosophila melanogaster  <cit> , respectively .
a phylogenetic analysis including all copia elements previously identified in an.
gambiae and a copia element from d. melanogaster showed no clear clustering of any of these sequences with the previously described copia elements .
additional file  <dig> presents the main characteristics of the sequences belonging to the novel ltr elements described in anotexcel.
the four sequences within cluster  <dig> are  <dig> long with a p-distance at the nucleotide level, considering the full-length alignment of  <dig>  .
the ltrs are  <dig> nts long and present a very high degree of identity .
in two of these sequences, the 3' and 5' ltrs are identical , indicating that they have inserted recently and have not had time to accumulate mutations between the ltrs.
the consensus sequence presents an orf of  <dig> aa containing conserved regions for integrase , reverse transcriptase , and rnaseh, a primer-binding site  for proline, and a polypurine signal at position  <dig>  information that reinforces the idea of activity of this family.
the mean aa distance of the consensus sequence of clusters  <dig> to each of the consensus of the already described copia elements shown in figure  <dig> is  <dig> , indicating that they do not correspond to any of the already described copia families .
the consensus sequence of cluster  <dig> shows two regions spanning  <dig> and  <dig> nts with 65% and 69% identity, respectively, with element copia-5_ag.
the unique orf of cluster  <dig> presents 56% identity along the whole sequence with copia5_ag.
still, the 149-nts-long ltrs of cluster  <dig> do not show any identity with the 108-nts-long ltrs from copia-5_ag, indicating that they are not members of the same te family.
the synonymous substitutions among the sequences in cluster  <dig> were more than six times more frequent than the substitutions at non-synonymous sites, which indicate the presence of purifying selection operating on them.
tajima's test, on the other hand, was not significant for these sequences, although the fact of being four sequences with few segregating sites along the alignment might influence the ability of the test to detect selective pressures operating on them .
the consensus of the sequences in this cluster has been deposited in repbase under the denomination copia-6_ag.
the four sequences in cluster  <dig> are  <dig> nts long with a mean p-distance of  <dig>  and sd =  <dig> , considering the whole alignment.
the ltrs are  <dig> nts long and are identical in all the sequences, suggesting a recent transposition event.
the consensus sequence has an orf of  <dig> aa that contains no conserved regions.
the sequences present two pbss, three sequences for valine, and one for methionine.
the consensus sequence for this cluster shows no regions of identity with previously identified elements.
in the phylogenetic analysis shown in figure  <dig>  the cluster  <dig> sequence groups with a copia element from d. melanogaster, although with a non-significant bootstrap value.
the mean aa distance of the consensus sequence of cluster  <dig> to each consensus of the already described copia elements shown in figure  <dig> was  <dig> .
the blasts performed on an est library as well as on a predicted expressed mrna, both from an.
gambiae, gave positive matches .
so, even if this sequence apparently corresponds to an inactive element, it is being expressed, as demonstrated by its positive match both to the mrna and the est databases, which indicates the activity of this family.
the consensus of the sequences in this cluster has been deposited in repbase under the denomination copia-7_ag.
superfamily pao-bel
according to repbase, there are  <dig> families of pao-bel elements in the mosquito genome .
anotexcel presents sequences of  <dig> of them plus  <dig> novel families.
eleven of these clusters contain full-length sequences that were further analyzed.
they all contain orfs with protein domains for rvt, pep <dig>  and rve , contain very high sequence identity within each cluster and, in all the cases, the within-element ltrs are identical, indicating that they are active at present or were so very recently.
in addition, ten of these families are being expressed, as indicated by their positive matches to the est database .
a phylogenetic analysis of the rt-pepta <dig> conserved domains of the pao-bel elements confirmed the classification of the clusters based on the information present in anotexcel.
in this phylogeny, only those sequences containing conserved domains for the rt were included.
novel pao-bel elements description
two families of elements characterized as pao-bel due to their matches to pao-bel elements deposited in repbase by tblastx  were further characterized.
cluster  <dig> is composed of three sequences presenting a high degree of identity among them   and a total length of  <dig> nts; two of them contain 213-nts-long identical ltrs, indicating that these sequences might have transposed very recently.
the consensus sequence presents an orf of  <dig> aa with conserved domains for rt, pepta <dig>  and rve, also indicating that sequences might belong to an active or recently active family.
the blastn performed on tefam and repbase showed that this family has not been reported before; still, the tblastx on repbase gave positive matches to several pao-bel families, indicating that this family belongs to the pao-bel superfamily.
the consensus sequence of this cluster has three regions  with 63% identity with element bel16-i_ag.
phylogenetic analysis performed with already characterized pao-bel elements showed that the consensus sequence of cluster  <dig> grouped together with the representative sequence of bel16_ag with a significant likelihood branch support value .
nonetheless, the ltrs of both families show no identities and present different sizes, indicating that they do not correspond to the same family.
the sequences in cluster  <dig> present 100% identity with two cdna spanning  <dig> nts from a cdna library, indicating that this elements is being expressed.
on the other hand, we identified more substitutions in synonymous than in non-synonymous positions, with a dn/ds value of  <dig> , indicating a selection pressure on the orf of these sequence.
tajima's test was not executed due the small number of sequences.
the consensus of the sequences in this cluster has been deposited in repbase under the denomination bel20_ag.
the three sequences in cluster  <dig> are  <dig> nts long, with a p-distance of  <dig>  and sd =  <dig> , presenting ltrs of  <dig> nts that are identical within each element.
the consensus sequence presents an orf of  <dig> aa that has no conserved domains for known proteins.
the ltr finder program detected a pbs for asparagine and a ppt signal.
the results obtained after blastn in the te databases, tefam and repbase, indicate no identity with known elements; nevertheless, the tblastx against repbase shows significant e-values with several pao-bel elements, mainly with families bel-18_ag  <cit>  and bel-3_ag  <cit> .
the ltrs of these families are not, however, similar in length or nucleotide composition to the ltrs of cluster  <dig>
the blasts performed on an est library as well as on a predicted expressed mrna, both from an.
gambiae, gave positive matches .
nevertheless, the absence of conserved domains in the orf present in this sequence made it impossible to compare it phylogenetically with other pao-bel sequences.
analysis of ds vs. dn showed no differences in the proportion of substitutions in synonymous vs. non-synonymous positions, indicating a neutral evolution .
the general characteristics of the sequences in this family suggest that it corresponds to a non-autonomous family composed of few elements.
the consensus of the sequences in this cluster has been deposited in repbase under the denomination bel-21_ag.
superfamily gypsy
the gypsy superfamily is the most diverse ltr superfamily in terms of the number of different families that have been previously identified  <cit> .
traditionally, this superfamily had been classified into nine different lineages based on phylogenetic analysis of the rt, rnaseh, and int domains  <cit> .
six of these lineages have been previously reported in insects and five of them  were described in an.
gambiae.
tubio, et al., reported the identification of a huge variety of families within each of these lineages.
in anotexcel, we identified families with full-length sequences belonging to each of the so-called lineages, but we failed to identify the majority of the individual families.
anotexcel presents six clusters with full-length sequences in addition to other clusters presenting fragmented elements or solo ltrs.
the six full-length clusters have signs of activity , presenting functional orfs, and five of them have positive matches to the est library.
most of the gypsys that we identified correspond to solo elements.
in an.
gambiae, tubio et al.
found a high proportion of solo ltrs belonging to this superfamily  <cit>  and interpreted this fact as a slow turnover of ltr elements from the genome of an.
gambiae.
this would mean that each individual copy remains for a longer time in the genome, enhancing the chance of homologous recombination to occur, which is the proposed mechanism to generate this type of deteriorated ltr  <cit> .
the lower proportion of solo ltr elements in the other ltr families might suggest that they are not equally present in all ltr elements.
elements class i, order non-ltr
the non-ltr elements identified previously in the anopheles genome and deposited in tefam and/or repbase constitute a very diverse order of elements composed of  <dig> different superfamilies,  <dig> of which were identified in anotexcel, belonging to the superfamilies rte, outcast, jockey, i, and the majority of them to the cr <dig> superfamily.
some families were considered as remnants of nltr elements for presenting little identity with nltr elements from tefam and/or repbase by tblastx.
the nltrs are usually between  <dig> and  <dig> kb long, and they usually contain two genes: the pol gene, fundamental for their replication, and the gag gene, which it is also present in the ltr retrotransposons and retroviruses.
in anotexcel, the nltr elements were classified based on their positive matches to rvt_ <dig> in pfam and/or their positive matches to polyprotein in the te databases, as well as positive matches to sequences present in the specific te databases or genbank nr database.
in total,  <dig> clusters were classified as nltrs , corresponding to 13% of all the families identified and  <dig> % of the total te families.
the families of nltr elements contain, overall, more numerous sequences than the ltrs ; this is to say, each family is more abundant and more heterogeneous, because most of the clusters are composed of both full-length and fragmented sequences.
most of the full-length sequences present conserved protein domains for exo-endo phosphatase and rt .
nine of these clusters present a second orf but no apparent conserved domains.
the p-distances among all the sequences within each cluster are small and, together with the significant results of tajima's test as well as the significant matches to the est library , indicate that some of these clusters are transcribed.
the majority of the nltr families present in anotexcel correspond to the cr <dig> superfamily.
most correspond to fragmented sequences, and the full-length sequences keep a high nucleotide identity.
anotexcel also presents one cluster with signs of expression belonging to the i superfamily.
both the jockey and outcast superfamilies seem to have active members that have already been reported  <cit> .
three clusters belong to the rte superfamily, two of them present full-length orfs and positive matches to the est library, and one has a positive match to the predicted expressed mrna.
class ii
the actual transposons or class ii elements are characterized by the presence of a gene coding for a transposase enzyme flanked by tirs and have been recently classified into two subclasses  according to the number of dna strands that are cut during the transposition event  <cit> .
elements of class ii belonging to subclass  <dig> and presenting tirs have been subsequently classified into nine superfamilies, six of which have been previously identified in the anopheles genome.
members belonging to all these superfamilies are present in anotexcel, where class ii elements have been classified based according to the presence of tirs and on the positive matches to already characterized elements deposited in any of the databases analyzed.
the majority of all the te families identified in anotexcel belong to this class , , which corresponds to  <dig> % of all the te sequences retrieved by piler.
elements belonging to six different superfamilies  were identified, as well as helitrons that belong to subclass  <dig>  tc1-mariner constitutes the most numerous superfamily, with  <dig> different families, representing 35% of the class ii families .
we were not able to identify copies of full-length piggybac families  <cit>  or the herves element, which correspond to a class ii active element  <cit> .
this element belongs to the hat superfamily, and although we identified other hat elements in the genome, all of them constitute truncated copies  <cit> .
on the other hand,  <dig> clusters with a variable number of sequences harboring tirs with no relationship to previously known elements were also identified.
these elements have been classified as novel class ii mite-like elements and will be later characterized.
the great majority of the class ii elements identified here correspond to highly deteriorated sequences, represented by elements with different degrees of deterioration, including several families already characterized of mites, na , fragments, and a few remnant clusters.
only four clusters harbor full-length sequences, belonging to superfamilies tc1-mariner  and p  .
all contain full-length tirs and, except for cluster  <dig>  they contain conserved domains for transposase and positive matches to the est library, so they probably constitute active or recently active elements .
mites
these elements were originally described in plants  <cit>  and have been found in other eukaryotic organisms, including mosquitoes  <cit> .
they are small, non-autonomous elements  that contain tirs but do not codify for any protein.
they are believed to originate from full-length active elements that lose their coding capacity but maintain the tirs, which allow their mobilization, in a parasitic manner, by active transposases.
there is ample evidence indicating a relationship between active elements and mites, although there is no clear mechanism that explains their generation  <cit> .
they are normally present in high copy number.
in anotexcel,  <dig> clusters were classified as mites of previously characterized families.
some of them had been identified as mites before, belonging to the superfamilies tc1-mariner, p, and harbinger, while others have not been reported as such until now .
the gambol elements belong to the tc1-mariner superfamily; they contain the characteristic dd34e motif  <cit>  and are represented by  <dig> different families that are deposited in tefam.
here we identified ten families related to gambol elements, six of which have tirs with high identity with the tirs of the original elements but smaller size .
this might indicate that just a part of the tir is necessary for element mobilization.
in the six examples shown here, the extreme outer region of the tirs is maintained while the inner region of the tirs is not present.
also, the internal region of all the gambol mite-like elements presented in figure  <dig> have no significant nucleotide similarity with any internal region of the gambol counterpart elements.
they all have quite small sizes, and in the case of the mite-like elements of gambol_ele <dig>  cluster  <dig> and  <dig>  they constitute two subfamilies with almost identical tirs but with different sizes and different internal regions in nucleotide composition, indicating that they probably originated in different events .
it is possible that more than one family of mites evolved from a unique master copy, generating diverse families that keep on transposing and evolving depending on the presence of an active element in the genome, as has been demonstrated for p elements in the an.
gambiae genome  <cit> .
the origin of the internal region of mites is controversial: it might derive from internal regions of the respective master tes followed by nucleotide degeneration or, alternatively, it might originate from an ectopic site  <cit> .
mite-like novel elements
<dig> clusters presenting tirs or palindromic flanking ends, but with no identity to known tes, either in the tir or in the internal region, were identified.
tir lengths vary from  <dig> to  <dig> nts and their total length from  <dig> to  <dig> nts, and six of them are, in fact, palindromic repeats.
palindromic tes have been previously identified in caenorhabditis elegans  <cit>  and d. melanogaster and also in mariner elements identified in an.
gambiae  <cit> .
palindromes are predicted to form secondary structures; in bacteria, repetitive extragenic palindromic elements  have been described as hotspots for transposition, indicating a relationship between reps and tes  <cit> .
little is known about the role of these sequences within genomes, however.
nine of these clusters constitute sequences longer than  <dig> nts, and  <dig> are longer than  <dig> nts .
some of these families present more than  <dig> individual copies, but none showed signs of autonomous activity, as they only present small orfs and no signs of conserved motifs.
a significant proportion presents matches to est databases, suggesting that they are being expressed, and five of them present significant matches to the mrna databases.
tajima's test for the group of sequences within these clusters showed significant values in  <dig> of them.
tajima's d is normally used as a selective neutrality test statistic; however, it has been suggested that sudden population expansions can lead to negative d values, moving the observed d value outside the 95% confidence interval derived for a neutral locus and stationary population  <cit> .
considering this, it is possible that the significantly negative d values obtained for the clusters analyzed here correspond to te families that are in a process of expansion--indeed, they all constitute quite numerous families within the anopheles genome.
we believe that these sequences are mite-like elements, although we cannot rule out the putative master te due to the lack of identity with known elements.
mites are known to be present in several genomes, and they have been associated with master tes  <cit> .
they share their tirs with active elements, and it appears that they manage to survive and spread within genomes by borrowing the transposase from active elements.
what is not known is whether their internal regions derive from internal regions of the master elements or if they are copied from an ectopic site by a conversion process after a double-strand break.
the clustering with less stringent conditions performed by tblastx, shown in the last columns of anotexcel, permitted the identification of sequences with a high degree of identity only among the tir regions, i.e., different subfamilies of mite-like elements.
three groups of clusters among the novel mite-like  were identified by the less stringent clustering.
in all cases, they share high identity in tir regions but low identity in internal regions .
it is possible that the diverging time between these elements is so long that it is impossible to find any detectable similarity.
it is interesting to note that many known mites, such as the stowaway family in rice, have no homologies with other tes, leaving an open question regarding the origin and means of replication of these small, non-autonomous elements  <cit> .
this appears to be the case for the orphan mite-like sequences presented here.
it is apparent that the class ii elements present in the genome of an.
gambiae are composed of a variety of different structurally degenerated sequences that might represent different stages in the process of deterioration of these elements, which in turn might be differentially involved in the regulation of class ii families  <cit> .
CONCLUSIONS
two basic approaches for the task of repetitive sequence identification at the genome scale are commonly used; they are based either on the similarities of a query sequence to already described elements  or on recognition of tes' intrinsic characteristics, such as their repetitive nature within the genome or the presence of structural motifs such as ltrs, tirs, or specific target site duplications  in a query sequence  that are specific of certain families of tes.
here, we used a combined strategy to identify and characterize the repetitive elements present in the genome of the primary vector of malaria, an.
gambiae, and to present all the gathered information in an easy and accessible manner.
we described several families of elements present in the mosquito genome, as well as novel elements that have not been described before.
previous works  <cit>  have shown that use of multiple softwares increases the detection of tes and that the use of piler alone has a low performance for the detection of tes in a genome.
it is worth noting that although our approach missed the detection of some te families, it permitted the identification of novel te families as well as mite-like elements from previously described class ii families that had not been described before.
this is related with a unique feature of the piler program in that it finds and distinguishes different classes of repeats by their characteristic features, implementing different search methods for dispersed families , tandem arrays , pseudo-satellites  and terminal repeats .
the ability of piler to incorporate biologically informed constraints into the repeat discovery process allows the identification of repeats that are more likely to be tes and, in many cases, of unknown te families or subfamilies.
also, it facilitates the identification of fragmented or deteriorated sequences of a given family, which in turn gives a broader idea of the structural diversity within the te landscape of the genome.
it has also been shown previously  that the consensus sequences constructed by piler are longer than those produced by other similar methods, such as repeatscout or repeatfinder and the composition of the repeat libraries generated are similar than the ones obtained by these other programs .
on the other hand, piler suffers from certain limitations including the inability to recognize te families with few members and a relatively low representation of nltrs in its output file which has been previously recognized  with a concomitant higher representation of class ii and ltrs.
this fact has been related to the higher identities that ltr and class ii elements show in their flanking sequences.
anotexcel is a platform for analysis of the repetitive elements present in an.
gambiae that contain tes families with different degrees of deterioration and which include the results of several analyses, allowing its use by researchers interested in several areas of te biology such as variability of terminal regions  and deterioration processes.
this compiled information can serve as a starting point for future investigations of te families in anopheles or in different species.
the platform used to identify and characterize tes in the mosquito genome can be easily used to generate analogue databases in other sequenced genomes, which can be useful for understanding the dynamics of te families evolving in different genomic contexts.
this platform can also serve for analysis of tes in sequenced genomes of other anophelines, allowing for comparative purposes.
this could permit the study of the dynamics of tes in different genomic environments, including the understanding of the process of deterioration of certain te families.

 transposable elements are dna sequences that can move within a cell's genome from one chromosomal location to another by a reaction called transposition, augmenting in this way their own representation within genomes. once having infected the germline of an organism, they can also spread through populations. these elements are ubiquitous in eukaryotes, accounting in certain species for a high proportion of their genomic dna; for instance, approximately 50% of the human genome and up to 80% of the maize genome are composed of tes. in anopheles gambiae, the transmitting vector of the malaria parasite, these elements constitute 16% of the euchromatin and up to 60% of the heterochromatin of the genome. these elements have quite simple genetic structures, basically containing genes involved in their transposition--which is generally independent from the cellular replicative machinery--and their flanking recognition sites. they can be seen as genomic infectious agents and due to this characteristic were originally considered as selfish dna; however, as more information was gathered about te biology, their relevance as important players in evolution and maintenance of eukaryotic genomes has been emphasized. tes can replicate via an rna or a dna intermediate and have been traditionally classified accordingly into two classes. class i elements, also known as retrotransposons, depend on a reverse transcription step. they are nearly identical to retroviruses in structure and are related phylogenetically. they contain a gag-like gene, a pol-gene and in certain cases a third open reading frame coding for an env-like protein. class i is subsequently classified into five orders based on major differences in their insertion mechanism as well as overall genetic organization. class ii elements are genetically and structurally more homogenous and simple. their basic genetic structure consists of a transposase gene flanked by terminal inverted repeats, which are fundamental for their recognition by transposases and further mobilization. this class has been classified into two subclasses, which are subsequently grouped into different orders. the classification of class ii tes based in their genetic differences permitted a further classification in superfamilies and families. the proportion of the different classes, orders, and superfamilies of tes in a given genome varies in different species; some harbor very abundant but few families of tes as mammals, where class i non-long terminal repeats predominate, while others contain many different yet less abundant families. these differences have been related to the c-value paradox, which refers to the huge variations in genome size of eukaryotic organisms despite similar levels of organism complexity. for instance, the large differences observed in genome size between an. gambiae and aedes aegypti is determined by their differential content of tes. on the other hand, tes can be classified as autonomous and non-autonomous based on their ability to perform their own transposition. moreover, within these two categories, tes can be also classified as active or inactive based in their actual mobility, which depends on several factors, among which the presence of an active, autonomous element of the same family--or close enough to be able to mobilize it--is fundamental. an important aspect of the study of tes in a given genome is the understanding of their abundance and diversity as well as the classification and organization of the elements. currently, repbase is the reference collection of repetitive elements from most eukaryotic genomes that can be consulted online. this database is being used in genome sequencing projects as a collection for masking and annotation of repetitive dna, because it contains a picture of the te landscape in several genomes. however, most te sequences from repbase are prototypic consensus sequences of families and subfamilies of repeats. on the other hand, a specific te database called tefam presents te sequences from three mosquito genomes: an. gambiae, a. aegypti, and culex quinquefasciatus. tefam contains a more restricted number of te families in the mosquito genomes. previous analyses of tes in an. gambiae have identified several element families; however, the data are often scattered in the databases or clearly unavailable. the aim of this work was to describe the diversity of tes within the an. gambiae genome following the use of the piler tool, which identifies repetitive elements in whole genomes --with a particular interest in the detection of new, active elements--as well as to consolidate a database in the form of a hyperlinked spreadsheet containing detailed and readily available information about the te diversity and other repetitive elements found in the genome of an. gambiae. the purpose was to present the platform used in the identification and characterization of tes in the genome of an. gambiae, which, in turn, can be used to screen other sequenced genomes.  an important aspect in the study of the tes in a genome is knowledge of their abundance and diversity, as well as classification and further organization of this information in a clear and comprehensive manner. aiming at the discovery of unknown, active elements, we performed a search and characterization of tes in the sequenced genome of an. gambiae. we used a combined strategy to identify and characterize repetitive elements present in the genome of the primary vector of malaria, an. gambiae. our strategy combines an extensive search of repetitive elements within this genome utilizing the piler-df algorithm as a first screening method, together with a detailed characterization of each of the retrieved sequences by analyzing signature characteristics of tes as well as performing homology-based analysis of the obtained sequences to several databases. this automated pipeline includes the different steps suggested recently by wicker, et al., for classifying eukaryotic tes. the results are compiled in a database of repetitive elements in the mosquito genome, called anotexcel that provides information for all the te families identified, along with offering the individual sequences found in each family. database description anotexcel is organized as an excel spreadsheet with cells containing, in a hyperlinked format, the results obtained after the various analyses performed in the characterization of each family. each line of the spreadsheet represents a cluster containing a variable number of sequences with a high degree of identity among them, constituting--in most of the cases--te families. the spreadsheet columns contain the several analyses performed on the sequences and were grouped into four types, colored for easier viewing as follows: blue columns represent the classification of the families; green columns present the general characteristics of the sequences in clusters, such as presence of tirs and their sequences, direct repeats, consensus and centroid sequences, orf sequences, etc. in orange columns are presented all the results of the blasts performed on several databases, and in yellow columns are results of new clustering performed by tblastx considering different degrees of identity over at least 50% of the length of the alignments. this was performed to identify shorter fragments of sequences belonging to the same family that were included in different clusters when more stringent conditions were applied. clusters that share some degree of identity are colored in column a in anotexcel. this is not an exhaustive database, as some of the element families already known to be present in an. gambiae have not been identified by our algorithm; however, it has the important and distinctive characteristic of presenting all the individual sequences within each family as well as their global alignments in fasta format, the consensus and centroid sequences, and detailed information on their structural characteristics. in addition, it presents the results obtained after several blasts performed on different databases and includes in hyperlinked format the file with the significant matches to the given database. this constitutes a rich resource that includes important information on the different families of tes present in the genome of an. gambiae besides serving as a platform for the analysis of tes present in other sequenced genomes. anotexcel contains repetitive sequences larger than nts in length that were grouped into clusters. the repetitive sequences are considered "intact" because they are globally alignable and "isolated" because they are surrounded by unique sequences; therefore, they correspond to insertional events of different repetitive sequences, mainly tes. our strategy in generation of the clusters permitted the grouping of sequences belonging to the same te family with different sizes or corresponding to different regions of the same element. considering that the sequences identified by piler-df are isolated and unique, it is possible to state that those sequences represent different transposition states of the same te family, allowing for evolutionary or dynamical analysis. the spreadsheet containing anotexcel can be found at http://exon.niaid.nih.gov/transcriptome/te/a_gambiae/anotexcel-web.zip and the standalone data can be downloaded from http://exon.niaid.nih.gov/transcriptome/te/a_gambiae/anotexcel-sa.zip the latter file should be decompressed to the user's computer, and the excel file should be opened from within excel. anotexcel can be downloaded and stored in a personal computer; it occupies approximately mb and can be modified by adding or deleting columns according to the user's needs. we were able to assign most of the retrieved sequences to a given te class; the rest were classified as rrna, or repetitive sequences with no te signatures and possibly expressed pseudogenes. in order to assess the efficiency of our approach in identifying and characterizing tes from the genome of a. gambiae we compared the number of sequences in anotexcel with those deposited in repbase and tefam. the results are shown in figure it should be noted that these figures are not 100% comparable since many elements are deposited in repbase and tefam under different names. also, repbase and anotexcel present data on subfamilies that were included in figure in addition, some but not all of the elements present in tefam are also deposited in repbase. we identified approximately half of the ltr elements that have been described so far in the genome of an. gambiae, few of the nltrs and almost all the known class ii elements. moreover, we were able to identify and characterize several families of elements that have not been reported previously, even using similar approaches for identifying tes in the mosquito genome. further characterization of the te families is given below. te characterization the te families identified in anotexcel were classified according to their class, subclass, order, superfamily, and family, following the wicker criteria for te classification. we also classified the families according to the length of sequences in each cluster, i.e., according to the percentage of the cluster consensus sequence representing the full-length canonical element as: full-length, fragments or remnants and, depending on the case, as solo ltr, mites, or class ii-na. the methodology used here permitted identification of families presenting both full-length elements and degenerate copies of the same family. the ltr elements contain several full-length putative active sequences, while nltrs and class ii elements are mainly constituted by fragments or remnant sequences. for the nltr elements, the percentage of clusters considered as "full" in anotexcel is notably higher than the percentage of full-length sequences that in fact exists, indicating that many of the nltr clusters constitute a mixture of both full-length and fragmented sequences. notably, although the an. gambiae genome has been scrutinized in search of tes before using several methodologies, we have been able to identify novel te families that have not been previously identified, as detailed further below. elements class i, order ltr the ltr elements in the genome of an. gambiae constitute a numerous order, although there are only three superfamilies: ty1-copia, pao-bel, and t3-gyspsy, the last being the most diverse ltr superfamily within the mosquito genome. the ltr elements identified in anotexcel were characterized based on the presence of flanking ltrs and/or based on positive matches to peptidase_a rvt_ or or rve in pfam or directly due to their significant matches to known ltr elements already deposited in the te databases or in the genbank non-redundant database. in anotexcel, they correspond to 19% of all the families identified, and % of the total tes identified, totalizing different clusters, and are represented by members of the main three superfamilies. novel elements corresponding to the copia and pao-bel superfamilies were also identified and will be described later. full-length and fragmented elements are not equally represented in the different ltr superfamilies. while the majority of the pao-bel elements are represented by full-length copies, the gypsys are represented by solo-ltrs, and the few copia families identified correspond to full-length elements. the predicted activity of the full-length sequences was also studied. based on i) the degree of nucleotide identity among the sequences belonging to the same families; ii) the degree of identity among the ltrs of individual elements; iii) the indication of expression based on positive matches to est databases in anotexcel; and iv) the presence of full-length orfs containing conserved domains for the proteins involved in transposition, we concluded that a substantial fraction of the ltrs described here corresponds to putatively active elements, as indicated by their presence within cdna libraries. the ltr families contain elements presenting an average nucleotide identity higher than %, and comparison of the ltrs among all the sequences in each of the clusters showed a high degree of identity for the three superfamilies. in addition, the identity between the two ltrs present in the individual sequences was calculated and appeared to be very high for some of the ltr members. the presence of full-length orfs in the sequences reinforces the idea that these families are active or have been very recently inactivated. copia elements the copia superfamily in the mosquito genome is represented by five different families: copia1-5_ag and the mtanga family. these families have been reported to repbase, and none of them is present in tefam. in anotexcel, elements belonging to two families, copia3_ag and copia5_ag were identified. the consensus sequence of these two families is identical to the corresponding consensus sequences deposited in repbase. element copia3_ag is apparently a truncated element with no conserved protein domains. the copia5_ag sequences present some characteristics of activity, such as the presence of conserved protein domains for rt and rve and identical within sequence ltrs, even if they presented no positive matches to the mrna and the est databases. novel copia elements two clusters were characterized as elements belonging to the copia superfamily, although it was not possible to assign them to any of the already described families within this superfamily since they present high nucleotide distances against all the previous characterized copia elements. the consensus sequences of these clusters showed significant matches by tblastx with the copia5_ag consensus sequence and copia_dm from drosophila melanogaster, respectively. a phylogenetic analysis including all copia elements previously identified in an. gambiae and a copia element from d. melanogaster showed no clear clustering of any of these sequences with the previously described copia elements. additional file presents the main characteristics of the sequences belonging to the novel ltr elements described in anotexcel. the four sequences within cluster are long with a p-distance at the nucleotide level, considering the full-length alignment of. the ltrs are nts long and present a very high degree of identity. in two of these sequences, the 3' and 5' ltrs are identical, indicating that they have inserted recently and have not had time to accumulate mutations between the ltrs. the consensus sequence presents an orf of aa containing conserved regions for integrase, reverse transcriptase, and rnaseh, a primer-binding site for proline, and a polypurine signal at position information that reinforces the idea of activity of this family. the mean aa distance of the consensus sequence of clusters to each of the consensus of the already described copia elements shown in figure is, indicating that they do not correspond to any of the already described copia families. the consensus sequence of cluster shows two regions spanning and nts with 65% and 69% identity, respectively, with element copia-5_ag. the unique orf of cluster presents 56% identity along the whole sequence with copia5_ag. still, the 149-nts-long ltrs of cluster do not show any identity with the 108-nts-long ltrs from copia-5_ag, indicating that they are not members of the same te family. the synonymous substitutions among the sequences in cluster were more than six times more frequent than the substitutions at non-synonymous sites, which indicate the presence of purifying selection operating on them. tajima's test, on the other hand, was not significant for these sequences, although the fact of being four sequences with few segregating sites along the alignment might influence the ability of the test to detect selective pressures operating on them. the consensus of the sequences in this cluster has been deposited in repbase under the denomination copia-6_ag. the four sequences in cluster are nts long with a mean p-distance of and sd =, considering the whole alignment. the ltrs are nts long and are identical in all the sequences, suggesting a recent transposition event. the consensus sequence has an orf of aa that contains no conserved regions. the sequences present two pbss, three sequences for valine, and one for methionine. the consensus sequence for this cluster shows no regions of identity with previously identified elements. in the phylogenetic analysis shown in figure the cluster sequence groups with a copia element from d. melanogaster, although with a non-significant bootstrap value. the mean aa distance of the consensus sequence of cluster to each consensus of the already described copia elements shown in figure was. the blasts performed on an est library as well as on a predicted expressed mrna, both from an. gambiae, gave positive matches. so, even if this sequence apparently corresponds to an inactive element, it is being expressed, as demonstrated by its positive match both to the mrna and the est databases, which indicates the activity of this family. the consensus of the sequences in this cluster has been deposited in repbase under the denomination copia-7_ag. superfamily pao-bel according to repbase, there are families of pao-bel elements in the mosquito genome. anotexcel presents sequences of of them plus novel families. eleven of these clusters contain full-length sequences that were further analyzed. they all contain orfs with protein domains for rvt, pep and rve, contain very high sequence identity within each cluster and, in all the cases, the within-element ltrs are identical, indicating that they are active at present or were so very recently. in addition, ten of these families are being expressed, as indicated by their positive matches to the est database. a phylogenetic analysis of the rt-pepta conserved domains of the pao-bel elements confirmed the classification of the clusters based on the information present in anotexcel. in this phylogeny, only those sequences containing conserved domains for the rt were included. novel pao-bel elements description two families of elements characterized as pao-bel due to their matches to pao-bel elements deposited in repbase by tblastx were further characterized. cluster is composed of three sequences presenting a high degree of identity among them and a total length of nts; two of them contain 213-nts-long identical ltrs, indicating that these sequences might have transposed very recently. the consensus sequence presents an orf of aa with conserved domains for rt, pepta and rve, also indicating that sequences might belong to an active or recently active family. the blastn performed on tefam and repbase showed that this family has not been reported before; still, the tblastx on repbase gave positive matches to several pao-bel families, indicating that this family belongs to the pao-bel superfamily. the consensus sequence of this cluster has three regions with 63% identity with element bel16-i_ag. phylogenetic analysis performed with already characterized pao-bel elements showed that the consensus sequence of cluster grouped together with the representative sequence of bel16_ag with a significant likelihood branch support value. nonetheless, the ltrs of both families show no identities and present different sizes, indicating that they do not correspond to the same family. the sequences in cluster present 100% identity with two cdna spanning nts from a cdna library, indicating that this elements is being expressed. on the other hand, we identified more substitutions in synonymous than in non-synonymous positions, with a dn/ds value of, indicating a selection pressure on the orf of these sequence. tajima's test was not executed due the small number of sequences. the consensus of the sequences in this cluster has been deposited in repbase under the denomination bel20_ag. the three sequences in cluster are nts long, with a p-distance of and sd =, presenting ltrs of nts that are identical within each element. the consensus sequence presents an orf of aa that has no conserved domains for known proteins. the ltr finder program detected a pbs for asparagine and a ppt signal. the results obtained after blastn in the te databases, tefam and repbase, indicate no identity with known elements; nevertheless, the tblastx against repbase shows significant e-values with several pao-bel elements, mainly with families bel-18_ag and bel-3_ag. the ltrs of these families are not, however, similar in length or nucleotide composition to the ltrs of cluster the blasts performed on an est library as well as on a predicted expressed mrna, both from an. gambiae, gave positive matches. nevertheless, the absence of conserved domains in the orf present in this sequence made it impossible to compare it phylogenetically with other pao-bel sequences. analysis of ds vs. dn showed no differences in the proportion of substitutions in synonymous vs. non-synonymous positions, indicating a neutral evolution. the general characteristics of the sequences in this family suggest that it corresponds to a non-autonomous family composed of few elements. the consensus of the sequences in this cluster has been deposited in repbase under the denomination bel-21_ag. superfamily gypsy the gypsy superfamily is the most diverse ltr superfamily in terms of the number of different families that have been previously identified. traditionally, this superfamily had been classified into nine different lineages based on phylogenetic analysis of the rt, rnaseh, and int domains. six of these lineages have been previously reported in insects and five of them were described in an. gambiae. tubio, et al., reported the identification of a huge variety of families within each of these lineages. in anotexcel, we identified families with full-length sequences belonging to each of the so-called lineages, but we failed to identify the majority of the individual families. anotexcel presents six clusters with full-length sequences in addition to other clusters presenting fragmented elements or solo ltrs. the six full-length clusters have signs of activity, presenting functional orfs, and five of them have positive matches to the est library. most of the gypsys that we identified correspond to solo elements. in an. gambiae, tubio et al. found a high proportion of solo ltrs belonging to this superfamily and interpreted this fact as a slow turnover of ltr elements from the genome of an. gambiae. this would mean that each individual copy remains for a longer time in the genome, enhancing the chance of homologous recombination to occur, which is the proposed mechanism to generate this type of deteriorated ltr. the lower proportion of solo ltr elements in the other ltr families might suggest that they are not equally present in all ltr elements. elements class i, order non-ltr the non-ltr elements identified previously in the anopheles genome and deposited in tefam and/or repbase constitute a very diverse order of elements composed of different superfamilies, of which were identified in anotexcel, belonging to the superfamilies rte, outcast, jockey, i, and the majority of them to the cr superfamily. some families were considered as remnants of nltr elements for presenting little identity with nltr elements from tefam and/or repbase by tblastx. the nltrs are usually between and kb long, and they usually contain two genes: the pol gene, fundamental for their replication, and the gag gene, which it is also present in the ltr retrotransposons and retroviruses. in anotexcel, the nltr elements were classified based on their positive matches to rvt_ in pfam and/or their positive matches to polyprotein in the te databases, as well as positive matches to sequences present in the specific te databases or genbank nr database. in total, clusters were classified as nltrs, corresponding to 13% of all the families identified and % of the total te families. the families of nltr elements contain, overall, more numerous sequences than the ltrs; this is to say, each family is more abundant and more heterogeneous, because most of the clusters are composed of both full-length and fragmented sequences. most of the full-length sequences present conserved protein domains for exo-endo phosphatase and rt. nine of these clusters present a second orf but no apparent conserved domains. the p-distances among all the sequences within each cluster are small and, together with the significant results of tajima's test as well as the significant matches to the est library, indicate that some of these clusters are transcribed. the majority of the nltr families present in anotexcel correspond to the cr superfamily. most correspond to fragmented sequences, and the full-length sequences keep a high nucleotide identity. anotexcel also presents one cluster with signs of expression belonging to the i superfamily. both the jockey and outcast superfamilies seem to have active members that have already been reported. three clusters belong to the rte superfamily, two of them present full-length orfs and positive matches to the est library, and one has a positive match to the predicted expressed mrna. class ii the actual transposons or class ii elements are characterized by the presence of a gene coding for a transposase enzyme flanked by tirs and have been recently classified into two subclasses according to the number of dna strands that are cut during the transposition event. elements of class ii belonging to subclass and presenting tirs have been subsequently classified into nine superfamilies, six of which have been previously identified in the anopheles genome. members belonging to all these superfamilies are present in anotexcel, where class ii elements have been classified based according to the presence of tirs and on the positive matches to already characterized elements deposited in any of the databases analyzed. the majority of all the te families identified in anotexcel belong to this class,, which corresponds to % of all the te sequences retrieved by piler. elements belonging to six different superfamilies were identified, as well as helitrons that belong to subclass tc1-mariner constitutes the most numerous superfamily, with different families, representing 35% of the class ii families. we were not able to identify copies of full-length piggybac families or the herves element, which correspond to a class ii active element. this element belongs to the hat superfamily, and although we identified other hat elements in the genome, all of them constitute truncated copies. on the other hand, clusters with a variable number of sequences harboring tirs with no relationship to previously known elements were also identified. these elements have been classified as novel class ii mite-like elements and will be later characterized. the great majority of the class ii elements identified here correspond to highly deteriorated sequences, represented by elements with different degrees of deterioration, including several families already characterized of mites, na, fragments, and a few remnant clusters. only four clusters harbor full-length sequences, belonging to superfamilies tc1-mariner and p. all contain full-length tirs and, except for cluster they contain conserved domains for transposase and positive matches to the est library, so they probably constitute active or recently active elements. mites these elements were originally described in plants and have been found in other eukaryotic organisms, including mosquitoes. they are small, non-autonomous elements that contain tirs but do not codify for any protein. they are believed to originate from full-length active elements that lose their coding capacity but maintain the tirs, which allow their mobilization, in a parasitic manner, by active transposases. there is ample evidence indicating a relationship between active elements and mites, although there is no clear mechanism that explains their generation. they are normally present in high copy number. in anotexcel, clusters were classified as mites of previously characterized families. some of them had been identified as mites before, belonging to the superfamilies tc1-mariner, p, and harbinger, while others have not been reported as such until now. the gambol elements belong to the tc1-mariner superfamily; they contain the characteristic dd34e motif and are represented by different families that are deposited in tefam. here we identified ten families related to gambol elements, six of which have tirs with high identity with the tirs of the original elements but smaller size. this might indicate that just a part of the tir is necessary for element mobilization. in the six examples shown here, the extreme outer region of the tirs is maintained while the inner region of the tirs is not present. also, the internal region of all the gambol mite-like elements presented in figure have no significant nucleotide similarity with any internal region of the gambol counterpart elements. they all have quite small sizes, and in the case of the mite-like elements of gambol_ele cluster and they constitute two subfamilies with almost identical tirs but with different sizes and different internal regions in nucleotide composition, indicating that they probably originated in different events. it is possible that more than one family of mites evolved from a unique master copy, generating diverse families that keep on transposing and evolving depending on the presence of an active element in the genome, as has been demonstrated for p elements in the an. gambiae genome. the origin of the internal region of mites is controversial: it might derive from internal regions of the respective master tes followed by nucleotide degeneration or, alternatively, it might originate from an ectopic site. mite-like novel elements clusters presenting tirs or palindromic flanking ends, but with no identity to known tes, either in the tir or in the internal region, were identified. tir lengths vary from to nts and their total length from to nts, and six of them are, in fact, palindromic repeats. palindromic tes have been previously identified in caenorhabditis elegans and d. melanogaster and also in mariner elements identified in an. gambiae. palindromes are predicted to form secondary structures; in bacteria, repetitive extragenic palindromic elements have been described as hotspots for transposition, indicating a relationship between reps and tes. little is known about the role of these sequences within genomes, however. nine of these clusters constitute sequences longer than nts, and are longer than nts. some of these families present more than individual copies, but none showed signs of autonomous activity, as they only present small orfs and no signs of conserved motifs. a significant proportion presents matches to est databases, suggesting that they are being expressed, and five of them present significant matches to the mrna databases. tajima's test for the group of sequences within these clusters showed significant values in of them. tajima's d is normally used as a selective neutrality test statistic; however, it has been suggested that sudden population expansions can lead to negative d values, moving the observed d value outside the 95% confidence interval derived for a neutral locus and stationary population. considering this, it is possible that the significantly negative d values obtained for the clusters analyzed here correspond to te families that are in a process of expansion--indeed, they all constitute quite numerous families within the anopheles genome. we believe that these sequences are mite-like elements, although we cannot rule out the putative master te due to the lack of identity with known elements. mites are known to be present in several genomes, and they have been associated with master tes. they share their tirs with active elements, and it appears that they manage to survive and spread within genomes by borrowing the transposase from active elements. what is not known is whether their internal regions derive from internal regions of the master elements or if they are copied from an ectopic site by a conversion process after a double-strand break. the clustering with less stringent conditions performed by tblastx, shown in the last columns of anotexcel, permitted the identification of sequences with a high degree of identity only among the tir regions, i.e., different subfamilies of mite-like elements. three groups of clusters among the novel mite-like were identified by the less stringent clustering. in all cases, they share high identity in tir regions but low identity in internal regions. it is possible that the diverging time between these elements is so long that it is impossible to find any detectable similarity. it is interesting to note that many known mites, such as the stowaway family in rice, have no homologies with other tes, leaving an open question regarding the origin and means of replication of these small, non-autonomous elements. this appears to be the case for the orphan mite-like sequences presented here. it is apparent that the class ii elements present in the genome of an. gambiae are composed of a variety of different structurally degenerated sequences that might represent different stages in the process of deterioration of these elements, which in turn might be differentially involved in the regulation of class ii families.  two basic approaches for the task of repetitive sequence identification at the genome scale are commonly used; they are based either on the similarities of a query sequence to already described elements or on recognition of tes' intrinsic characteristics, such as their repetitive nature within the genome or the presence of structural motifs such as ltrs, tirs, or specific target site duplications in a query sequence that are specific of certain families of tes. here, we used a combined strategy to identify and characterize the repetitive elements present in the genome of the primary vector of malaria, an. gambiae, and to present all the gathered information in an easy and accessible manner. we described several families of elements present in the mosquito genome, as well as novel elements that have not been described before. previous works have shown that use of multiple softwares increases the detection of tes and that the use of piler alone has a low performance for the detection of tes in a genome. it is worth noting that although our approach missed the detection of some te families, it permitted the identification of novel te families as well as mite-like elements from previously described class ii families that had not been described before. this is related with a unique feature of the piler program in that it finds and distinguishes different classes of repeats by their characteristic features, implementing different search methods for dispersed families, tandem arrays, pseudo-satellites and terminal repeats. the ability of piler to incorporate biologically informed constraints into the repeat discovery process allows the identification of repeats that are more likely to be tes and, in many cases, of unknown te families or subfamilies. also, it facilitates the identification of fragmented or deteriorated sequences of a given family, which in turn gives a broader idea of the structural diversity within the te landscape of the genome. it has also been shown previously that the consensus sequences constructed by piler are longer than those produced by other similar methods, such as repeatscout or repeatfinder and the composition of the repeat libraries generated are similar than the ones obtained by these other programs. on the other hand, piler suffers from certain limitations including the inability to recognize te families with few members and a relatively low representation of nltrs in its output file which has been previously recognized with a concomitant higher representation of class ii and ltrs. this fact has been related to the higher identities that ltr and class ii elements show in their flanking sequences. anotexcel is a platform for analysis of the repetitive elements present in an. gambiae that contain tes families with different degrees of deterioration and which include the results of several analyses, allowing its use by researchers interested in several areas of te biology such as variability of terminal regions and deterioration processes. this compiled information can serve as a starting point for future investigations of te families in anopheles or in different species. the platform used to identify and characterize tes in the mosquito genome can be easily used to generate analogue databases in other sequenced genomes, which can be useful for understanding the dynamics of te families evolving in different genomic contexts. this platform can also serve for analysis of tes in sequenced genomes of other anophelines, allowing for comparative purposes. this could permit the study of the dynamics of tes in different genomic environments, including the understanding of the process of deterioration of certain te families. 
this work contributes to a better understanding of the landscape of tes present in the mosquito genome. transposable elements are dna sequences, present in the genome of most eukaryotic organisms that hold the key characteristic of being able to mobilize and increase their copy number within chromosomes. here we present the spreadsheet named anotexcel that constitutes a database with detailed information on most of the repetitive elements present in the genome of the mosquito. the aim of this work was to catalog the diversity and abundance of tes within the anopheles gambiae genome using the piler tool and to consolidate a database in the form of a hyperlinked spreadsheet containing detailed and readily available information about the tes present in the genome of an.
 transposable elements are dna sequences, present in the genome of most eukaryotic organisms that hold the key characteristic of being able to mobilize and increase their copy number within chromosomes. these elements are important for eukaryotic genome structure and evolution and lately have been considered as potential drivers for introducing transgenes into pathogen-transmitting insects as a means to control vector-borne diseases. the aim of this work was to catalog the diversity and abundance of tes within the anopheles gambiae genome using the piler tool and to consolidate a database in the form of a hyperlinked spreadsheet containing detailed and readily available information about the tes present in the genome of an. gambiae.  here we present the spreadsheet named anotexcel that constitutes a database with detailed information on most of the repetitive elements present in the genome of the mosquito. despite previous work on this topic, our approach permitted the identification and characterization both of previously described and novel tes that are further described in detailed.  identification and characterization of tes in a given genome is important as a way to understand the diversity and evolution of the whole set of tes present in a given species. this work contributes to a better understanding of the landscape of tes present in the mosquito genome. it also presents a novel platform for the identification, analysis, and characterization of tes on sequenced genomes. 