An overview of irritans-mariner transposons in two Mayetiola species ( Diptera : Cecidomyiidae )

Mariner-like elements (MLEs) are widespread Class II transposable elements in insects that are subdivided into several subfamilies. In the current study, we carried out in silico analysis and in vitro experiments to identify MLEs belonging to the irritans subfamily in two cecidomyiid fl ies, Mayetiola destructor and M. hordei. In silico investigation of M. destructor genome allowed the identifi cation of 25 irritans-like elements, which were mostly defective due to several mutations. These defective forms might be the remnants of active elements that ancestrally invaded the host genome. Structural analyses, including signature motifs and transposase-encoding ORFs, revealed structural heterogeneity and the presence of two full length copies. Five consensuses, refl ecting the probable evolutionary groups of these elements, were constructed, based on a similarity matrix. The fi rst consensus (Maymarcons1) belonged to Himar1-like elements reported in other insects, while the remaining four (Maymarcons2 to 5) seemed to be more specifi c to Cecidomyiidae. Moreover, the presence of elements belonging to the Maymarcons4 group was ascertained by PCR amplifi cation, in both Mayetiola species, and was further identifi ed in the Transcriptome Shotgun Assembly (TSA) of the orange fl y, Sitodiplosis mosellana (Cecidomyiidae), suggesting the existence of irritans elements within the Cecidomyiidae, which were derived from an ancestral species by vertical transmission during speciation. On the other hand, consensuses that are specifi c to M. destructor could be derived from a more recent invasion. This study suggests that both M. destructor and M. hordei genomes have been invaded by irritans elements many times with at least two different evolutionary histories. * Corresponding author; e-mail: maha.mezghani@fst.utm.tn INTRODUCTION Transposable elements (TEs) are repeated DNA sequences that are able to move from one site to another in a host genome. These mobile elements are ubiquitous in almost all organisms from different kingdoms and with different proportions depending on species (Chenais et al., 2012). TEs are not simply selfi sh DNA but rather important elements that contribute signifi cantly to genome evolution as well as its shape architecture (Feschotte & Pritham, 2007; Bire & Rouleux-Bonnin, 2012; Hirsch & Springer, 2017). TEs are subdivided into two main classes based on their mechanisms of transposition (Finnegan, 1989; Wicker et al., 2007). Class I elements, also known as retrotransposons, transpose via an RNA intermediate according to the “copy and paste” model. Class II elements, also named transposons move via a DNA intermediate according to the Eur. J. Entomol. 114: 379–390, 2017 doi: 10.14411/eje.2017.049


INTRODUCTION
Transposable elements (TEs) are repeated DNA sequences that are able to move from one site to another in a host genome.These mobile elements are ubiquitous in almost all organisms from different kingdoms and with different proportions depending on species (Chenais et al., 2012).TEs are not simply selfi sh DNA but rather important elements that contribute signifi cantly to genome evolution as well as its shape architecture (Feschotte & Pritham, 2007;Bire & Rouleux-Bonnin, 2012;Hirsch & Springer, 2017).TEs are subdivided into two main classes based on their mechanisms of transposition (Finnegan, 1989;Wicker et al., 2007).Class I elements, also known as retrotransposons, transpose via an RNA intermediate according to the "copy and paste" model.Class II elements, also named transposons move via a DNA intermediate according to the The identifi ed MLEs are mostly inactive owing to mutations affecting different parts of the elements and it has also been shown that many defective copies contain internal deletions that occur non randomly as ascertained by small direct repeats (SDRs) called microhomologies bordering deletion break points (BPs) (Brunet et al., 2002;Kharrat et al., 2015;Ben Lazhar-Ajroud et al., 2016).
The ability of TEs to move enabled them to be used as genetic tools for mutagenesis and transgenesis in several organisms, such as insects (Largaespada, 2003;Ryder & Russell, 2003;Handler & O'Brochta, 2012).The choice of appropriate TEs as transgenetic vectors depends on the TEs present in the target genome since the use of endogenous TEs as genetic tools could result in trans-mobilization and therefore the instability of the host genome (Ashburner et al., 1998).Thus, it is important to study and identify the different TE groups and variants existing in a given genome.
In this study, we focused on two species of Cecidomyiidae; Mayetiola destructor (Say, 1817) and Mayetiola hordei (Kieffer, 1909), which are both major pests of wheat and barley around the world.Previous studies identifi ed a full length MLE copy with intact ORF and perfect TIRs in M. destructor (Russell & Shukle, 1997).This element, named Desmar1, belongs to the mauritiana subfamily and has already been used to study its insertion polymorphisms (Behura et al., 2010).Moreover, an internal region belonging to the irritans subfamily has been characterized and named Des2 (Shukle & Russell, 1995).
Therefore, the aim of this study was to identify and characterize complete irritans elements in M. destructor and its closely related species M. hordei.A combination of in silico and in vitro investigations was carried out and the results used to provide a better overview of the endogenous irritans subfamily in these two cereal pests, which is useful in light of the estimation of these MLEs dynamics and evolutionary history.

Insect sampling
Samples of Mayetiola destructor and M. hordei were collected in the third instar larvae and the fl ax-seed stages of development on wheat and barley.Total DNA was extracted from individual insects using the salting-out protocol (Sunnucks & Hales, 1996).Subsequently, samples from both species of Mayetiola species were identifi ed, based on PCR-RFLP of the cytochrome b gene as reported by Mezghani Khemakhem et al. (2002).

Data sources
The Mdes1.0 release of the Great Plains (GP) M. destructor genome was used for the identifi cation of irritans-like ele-the Himar1-like elements in insects, the third includes the Bytmar1-like elements in marine organisms and the fourth corresponds to the Batmar2-like elements in bats (Sinzelle et al., 2006;Bui et al., 2007).
MLEs are characterized by a typical sequence of 1300 bp in length with terminal inverted repeats (TIRs) of 20-40 bp (Halaimia-Toumi et al., 2004).The MLE TIRs have conserved motifs, such as 5'YYAGRT3' at their extremities, which correspond to the cleavage signal (Bigot et al., 2005).Nevertheless, there are exceptions recorded in at least two irritans transposons, namely Hsmar2 in Homo sapiens (Robertson & Martos, 1997) and Bytmar1 in the hydrothermal crab Bythograea thermydron (Halaimia-Toumi et al., 2004), where the distal motif is modifi ed.The MLE TIRs fl ank one intronless open reading frame (ORF), which encodes a transposase of approximately 350 amino acid residues.This enzyme mediates all transposition steps and allows the integration of the excised MLE in its TA hallmark dinucleotide target site duplication (TSD) (Plasterk et al., 1999;Munoz-Lopez & Garcia-Perez, 2010).The mariner transposase exhibits two signature motifs WVPHEL and YSPDLAP (Robertson, 1993).It is also characterized by an N-terminal domain containing the helix-turn-helix motif (HTH), which serves to bind TIRs during the transposition process (Pietrokovski & Henikoff, 1997), as well as a C-terminal catalytic domain containing a DD34D catalytic triad catalyzing the cleavage of the TE and its integration into the TSD (Brillet et al., 2007;Yuan & Wessler, 2011).The three aspartate residues are generally anchored to three conserved motifs named respectively TGDEKW (TGDETW for the irritans subfamily), HHDNA and YSPDLAPS/CD.The mariner transposase is also characterized by nuclear localization signal (NLS) motifs that transport the transposase through the nuclear envelope (Brillet et al., 2007).
Each TE undergoes different steps during its life cycle.In fact, when a MLE invades a new host genome, it has to increase its copy number by many amplifi cations (Hartl et al., 1997;Le Rouzic & Capy, 2005).The amplifi cation and propagation of such elements may be deleterious for the host genome, which, consequently, develops control strategies to reduce and even inhibit transposon activity.There are two main ways of control.The fi rst is vertical inactivation, which consist of the accumulation of mutations such as frameshifts, nonsense mutations, insertions and deletions (indels) leading to inactive and fossil elements (Lohe et al., 1995;Hartl et al., 1997).The second is the stochastic loss strategy consisting in the autonomous and nonautonomous elimination of MLEs by genetic drift (Lohe et al., 1995;Kidwell & Lisch, 2001).More recently, transposon silencing has proved to be closely related to epigenetic mechanisms including small RNA molecules (siRNA and piRNA) and methylation that control transposon transcription and transposition (Rigal & Mathieu, 2011;Bucher et al., 2012).Thus, in order to escape the host genome selection pressure, MLEs may invade new host genomes by horizontal transfer (HT) as described in several insects (Lampe et al., 2003;Panaud, 2016;Peccoud et al., 2017).
ments.The Mayetiola destructor genome is available in GenBank (NCBI BioProject PRJNA45867).It consists of 26 million reads (34-fold genome coverage) sequenced using the whole genome shotgun (WGS) strategy and assembled in 36,371 contigs with a 14 kb contig N50 length and 24,475 scaffolds with a 756 kb N50 length.The sequenced fraction constitutes 153 Mb with 33 Mb of gaps between contigs, distributed across the M. destructor's four chromosomes.
The transcriptome shotgun assembly (TSA) of the orange wheat blossom midge Sitodiplosis mosellana (Diptera: Cecidomyiidae) was also used to search for irritans-like elements similar to those in M. destructor.The S. mosellana transcriptome is available in GenBank (NCBI Bioproject PRJNA192921) and consists of 24383 complementary DNA (cDNA) contigs.

In silico identifi cation of irritans-like transposable elements
Irritans-like elements were identifi ed in the WGS scaffolds of M. destructor using both the structure-based method with the irritans transposase typical TGDETW motif and the homology-based method using TBLASTN and BLASTN algorithms (https://blast.ncbi.nlm.nih.gov/) with reference to irritans transposases and transposons as queries (Table S1).
Genomic contigs exhibiting similarities with queries (E-value < E -10 ) were identifi ed.In order to extract complete transposon copies, TIRs and TSDs were searched for by extending DNA hits by 1000 bp upstream and downstream of the transposase open reading frame (ORF) and aligning the 5'extension with the reverse complement of the 3' extension.A fi ltration step was then performed by eliminating copies exhibiting an incomplete ORF because of gaps between contigs in the WGS assembly as well as fossil copies whose sizes are less than 300 bp.

Amplifi cation of irritans-like transposable elements
The irritans-like elements from samples of M. destructor and M. hordei were amplifi ed using fi ve TIR primers designed from the alignment of irritans copies previously identifi ed in silico from the M. destructor genome (Table 1).The PCR conditions were programmed as follows: an initial denaturing step at 94°C for 5 min followed by 40 cycles of 3 steps: denaturing at 94°C for 1 min, annealing at 50°C to 56°C for 1 min, extension at 72°C for 1 min 30 s and a fi nal extension step at 72°C for 10 min.PCR amplifi cation products were visualized on 1% agarose gel stained with ethidium bromide.

Cloning of PCR products and sequencing
PCR products were excised from agarose gel and purifi ed using Wizard SV Gel and PCR Clean-Up System kits (Promega, Madison, WI, USA) according to the manufacturer's protocol.Purifi ed DNA was then cloned in a pGEMT-easy Vector System (Promega) and used to transform chimio-competent E. coli DH5α strains.Colonies were then screened as described by Sambrook et al. (2011).Plasmids were extracted from positive colonies (Wiz-ard Minipreps, Promega) and inserts were amplifi ed using T7 and SP6 primers.The same primers were used to sequence, in both directions, the amplifi ed inserts on an automated sequencer (ABI PRISM 3100 Genetic Analyzer, Applied Biosystems, Foster City, CA, USA).Sequences validated as MLEs were then named according to the nomenclature proposed by Robertson & Asplund (1996) and used in further analysis.
Phylogenetic relationships between irritans-like transposable elements identifi ed in M. destructor and M. hordei were inferred using reference elements belonging to the four irritans lineages as well as elements belonging to the mauritiana, cecropia, mellifera, elegans subfamilies.The tree was constructed using the Maximum Likelihood (ML) method with bootstrap analysis of 1000 replicates using the MEGA 6 program (Tamura et al., 2013).Phylogenetic tree management was carried out using the iTOL v3 program (Letunic & Bork, 2007).

In silico identifi cation of irritans-like elements
In silico investigation of the M. destructor genome resulted in the identifi cation of 25 irritans-like elements with sizes ranging from 474 bp to 1590 bp.These elements named Md1 to Md25 were mapped to 22 different scaffolds, among which three were found to contain 2 irritans copies (Table 2).
Most of the copies were defective due to mutations occurring in all parts of the elements and exhibited ORFs encoding truncated transposases lacking or containing some modifi ed signature motifs (Table S2).Only 17 sequences were fl anked by a TA dinucleotide target site duplication (TSD) on one or both sides.Among the 25 irritans elements, two full length copies (Md14 and Md24) exhibited perfect or near perfect TIRs fl anked by the TA dinucleotide TSD and an ORF encoding a transposase.The Md24 transposase is inactive due to two frameshift mutations, while the Md14 transposase bears only a transversion in the start The searches in GenBank database using the BLASTX algorithm revealed that identifi ed elements shared the best amino acid homologies, ranging from 51% to 85%, with irritans-like elements from the tephritid fruit fl y Bactrocera tryoni (APL98287.1)and the green lacewing Chrysoperla plorabunda Cpmar1 (AAC46945.1).
Nucleotide sequence alignment of the identifi ed irritanslike elements allowed the establishment of a similarity matrix from which fi ve consensuses designated by Maymar-cons1-Maymarcons5 were constructed.The comparison of these consensuses with the active Himar1 element of the horn fl y H. irritans (U11642) revealed a nucleic acid similarity ranging from 47% (Maymarcons3) to 58% (Maymar-cons1).A diagram of the consensuses of the irritans-like nucleic acid sequences and their conceptual transposases is shown in Fig. 1a.
For comparative purposes, the transcriptome shotgun assembly (TSA) of the orange wheat blossom midge S. mosellana available in GenBank was investigated using as queries the fi ve built consensuses of Maymarcons.Four cDNA contigs similar to Maymarcons4 were identifi ed with a 79% to 82% nucleotide identity, among which there were two with full length copies of irritans-like elements with an intact ORF.This suggests that these elements are potentially active in the orange cecidomyiid fl y whereas the two others that correspond to incomplete irritans elements have internal deletions spanning the fi rst two motifs of the catalytic triad and occur at positions 684-1071 bp (Fig. 2).

In vitro identifi cation of irritans like transposons in M. destructor and M. hordei
To validate the presence of irritans-like elements in the two species of Mayetiola studied, fi ve primers were designed from the TIRs sequence logos.Results indicate that for M. destructor, PCR products were obtained with primers designed from Maymarcons4 and Maymarcons5, whereas the M. hordei amplifi cations were obtained only with a primer specifi c to Maymarcons4.Cloning and sequencing of these products allowed the identifi cation of 17 irritans-like elements ranging from 802 bp to 929 bp.Alignment of the 11 Maymarcons4-like elements obtained from M. destructor and M. hordei showed nucleotide similarities with Maymarcons4 ranging from 87.03% to 90.27% and 80.66% to 86.02%, respectively.Furthermore, the conceptual translation of Mayamarcons4-like elements was performed and aligned with the putative transposase of Maymarcons4.As shown in Fig. 3a, all elements have a deletion spanning the fi rst signature motif WVPREL and the Moreover, comparison of the six Maymarcons5-like elements identifi ed in M. destructor revealed 84.26% to 96.93% nucleic acid similarity with the consensus of May-marcons5.The 5' and 3' TIRs of these elements differ in their inner region while the 5'CTACTRT3' motif is conserved in its outer region.The alignment of the putative transposases of these elements with the conceptual transposase of Maymarcons5 revealed a deletion spanning the fi rst motif of the catalytic core as shown in Fig. 3b.
Noteworthy, the nucleic acid alignments of several Maymarcons4-like elements identifi ed in the two Mayetiola species and Sitodiplosis transcripts, revealed deletions spanning the same positions.Given that, microhomology analyses have been performed to verify whether these deletions are random or not, two consensuses were established from the identifi ed elements and designated Desmarcons and Hormarcons for M. destructor and M. hordei, respectively.The alignment of both consensuses with Maymar-cons4 sequence revealed a total of six deletions sized from 8 bp to 431 bp (Fig. 4).The Desmarcons has a deletion of 28 bp fl anked by a short direct repeat (SDRs), which occurs near both BPs (Breaking Points Near Near, BPNN) and a 431 bp deletion bordered by SDRs, which are exactly at the BP on one side and near the BP on the other side (BPs Exact Near, BPEN).The Hormarcons has four deletions of 8 bp, 137 bp, 43 bp and 136 bp fl anked by SDRs localized exactly at BPs on both sides (BPs Exact Exact, BPEE) and/or BPNN microhomologies.
The phylogenetic tree (Fig. 5) indicates two major groups; the fi rst belongs to the Himar1-like lineage and contains the Maymarcons1 consensus, while the second diverges from the four known irritans lineages and is divided into three subgroups, one corresponding to the Maymar-cons2 consensus, one to the Maymarcons3 consensus and a third that includes Maymarcons4 and Maymarcons5-like elements.In the latter subgroup, Maymarcons4-like elements of M. destructor diverge from those of M. hordei.

DISCUSSION
In the M. destructor genome, two MLEs named Desmar1 and Des2 were described (Shukle & Russell, 1995;Russell & Shukle, 1997).The Desmar1 is a full length mauritiana-like element with an intact ORF and perfect TIRs, while Des2 is an internal region belonging to irritans-like elements.To date, no complete irritans copies have been identifi ed.
In the current study, complete copies (from TIR to TIR) of irritans-like transposable elements were identifi ed and characterized for the fi rst time in M. destructor and M. hordei using a combination of in silico and in vitro approaches.In silico analysis of the M. destructor genome revealed 25 irritans-like elements from which fi ve con- sensuses were built.This low copy number of elements is congruent with previous studies made by Shukle & Russell (1995).
This study revealed that most of the irritans-like copies were defective and damaged, due to a frameshift, nonsense or indel mutations spanning all the parts of the elements, which indicate an ancient invasion of the genome by these elements, which might be in the senescence stage (Kidwell & Lisch, 2001).
Likewise, the deletions occur mainly in the N-terminal region and the fi rst domain of the catalytic triad, which are crucial for an effi cient MLE mobilization (Lohe & Hartl, 2002).Thus, these deleted elements could act as inhibitors of trans-mobilization by the full-length copies as described for Botmar1-like copies (Rouleux-Bonnin et al., 2005) or as repressors like the KP deleted form, reported in the P element (Black et al., 1987;Andrews & Gloor, 1995).Furthermore, analysis of the M. destructor genome revealed chimerical elements with 3'-3'extremities that might be generated by either an ectopic recombination replacing 5' extremity by 3' extremity or an internal deletion of an initial head-to-tail mariner close copies as proposed by Filée et al. (2015).
The analysis of TIRs revealed specifi c conserved motifs that are different from those described by Bigot et al. (2005) suggesting specifi c interactions between these elements and their protein products.It is noteworthy that such conserved motif modifi cations were previously reported in the two irritans elements, Bytmar1 and Hsmar2 (Bigot et al., 2005).These observations provide evidence of high diversity in the irritans TIRs compared to those of other mariner subfamilies.
The molecular analysis revealed Maymarcons4-like elements in both species, whereas Maymarcons5-like elements were detected only in M. hordei.This could be explained by these elements invading the M. destructor genome following speciation.Conversely, the non amplifi cation of other irritans-like elements detected in the in silico investigation could be related to the high nucleotide variability of mariner TIRs (Bigot et al., 2005) or to the non occurrence of these elements in the Tunisian strains analyzed.Another explanation could be that an eventual ancient invasion of some of these elements (Maymarcons1 and Maymarcons2 like elements) led to the accumulation of mutations in their whole sequences, including ITRs.This would be due to the independent evolution of these copies.
The occurrence of Maymarcons4-like elements in two species of Mayetiola and even in the TSA of the orange blossom midge S. mosellana indicate an ancient invasion of these irritans elements in a common ancestral species of cecidomyiid, which would have been followed by a vertical transmission into derived species, in which it took the form of independently-differentiated, heterologous elements, as is hypothesized for the YSPDLAPCD motif in M. hordei.Likewise, it is also likely that a horizontal transfer between M. destructor and S. mosellana occurred, since they share the same host plant and have full length copies of irritans elements in their genomes.
Strikingly, the deleted regions in the defective forms of Maymarcons4-like elements in M. destructor and M. hordei are the same, suggesting a possible occurrence of these deletions in the ancestor of the two species.Moreover, these gaps are fl anked by microhomologies.The association of microhomology with deletion breakpoints is reported in Mos1 (Brunet et al., 2002), mauritiana (Kharrat et al., 2015) and irritans elements (Ben Lazhar-Ajroud et al., 2016).These deletions do not occur randomly and could result from a host genome control, as well as from additional mechanisms, such as abortive gap repair (Rubin & Levy, 1997) and/or ectopic recombination between homologous short sequences leading to different deletion forms (Negoua et al., 2013;Kharrat et al., 2015).
The phylogenetic analysis grouped the Maymarcons1 consensus within Himar1-like lineage and revealed a novel irritans group, distinct from the four irritans lineages pre-viously reported by Sinzelle et al. (2006).Thus, we recommend that the original classifi cation should be broadened to include the irritans elements characterized in this study, as well as the two irritans elements Tvmar1 (Claudianos et al., 2002) and Pacmmar1 (Bui et al., 2007), which also differ from the four known irritans lineages.
Moreover, the phylogenetic tree revealed a divergence in the Maymarcons4-like elements with respect to Mayetiola species, which favours an independent evolution of these elements after speciation and supports the vertical transfer from an ancestral species.
The high diversity recorded in M. destructor suggests that its genome was invaded many times by different types of irritans elements, as reported in species of Drosophila by Wallau et al. (2014).
In conclusion, the combined results of the in silico and in vitro analyses give an outline of the evolutionary dynamics of the irritans-like elements in the genomes of the two species of Mayetiola.The knowledge of the TE content might be helpful to explore the genome for a better understanding the seeking behavior of these insects with their host and in the case of transposon-based biological pest management for a better vector choice.

Fig. 1 .
Fig. 1.Diagram of the fi ve irritans-like consensuses and the logo of their corresponding ITRs.(a) The fi ve consensuses are compared to the full length irritans element Himar1 (U11642) as a reference.ITRs are indicated by green triangles and UTRs by a continuous black line.HTH and NLS motifs are indicated, respectively, by a blue circle and green rectangle.Motifs of the catalytic triad are boxed in purple rectangles and modifi ed residues are underlined.The aspartate residues are marked in red (with red capital D).The WVPHEL signature motif is indicated by a pink rectangle.The fi rst start residues and last residues are indicated in red.Deletions are represented by dashed lines, whereas insertions (Ins) are indicated by black rectangles.Frameshifts are indicated by empty upside-down triangles.(b) Weblogo representing the ITRs of the fi ve irritans groups identifi ed (Maymarcons-like elements) compared to ITRs of Himar1.The vertical axis is in bits with a maximum of two bits, which is proportional to the nucleotide level conservation at each position.Palindromic and mirror motifs are shown in pink and blue rectangles, respectively.Vertical black lines correspond to symmetry axes.In the Himar1 logo, pink and blue axes correspond to the symmetry of palindromic and mirror motifs, respectively.

Fig. 2 .
Fig. 2. Comparison of the Maymarcons4 consensus and the four cDNA sequences detected in Sitodiplosis mosellana.Deletions are indicated by dashed lines.Accession numbers of cDNA sequences are shown on the left and their identity statistics with Maymarcons4 are shown on the right.

Fig. 3 .
Fig. 3. Alignment of the conceptual translation of Mdemar1 and Mhmar1 elements with (a) the putative transposase of Maymarcons4 consensus (b) the putative transposase of Maymarcons5 consensus.Black and grey blocks correspond to identical and homologous regions.Deletions are represented by discontinuous lines and marked by double pointed arrows.Asterix correspond to stop codons.Binding regions, catalaytic triad domains and signature motifs are boxed in blue.

Fig. 4 .
Fig. 4. Nucleic acidalignment of Maymarcons4 with Desmarcons and Hormarcons generated from in vitro elements in M. destructor and M. hordei, respectively.Short direct repeats (SDRs) microhomologies, fl anking deletions and Breaking Points (BPs) are in bold and underlined by a single or a double line in Desmarcons and Hormarcons, respectively.Microhomologies localized near the BPs (BPNN i.e. breaking point near near) are in red, microhomologies exact near the BPs (BPNE i.e. breaking point near exact) are green and microhomologies localized exactly at BPs (BPEE i.e. breaking point exact exact) are blue.Boxed regions correspond to 5' and 3' TIRs of the 3 consensuses sequences.

Table 2 .
Features of the 25 in silico irritans-like elements identifi ed in Mayetiola destructor.

Table S1 .
Reference sequences used as queries to search for irritans transposable elements in the genomic scaffolds of Mayetiola destructor.

Table S2 .
Features of the 25 transposases conceptually translated from the in silico identifi ed elements in Mayetiola destructor.indicates that the motif is missing.Asterix in motifs designs stop codon occurrence." ins " indicates insertion in the predicted transposase ORF.