Small RNA biosynthetic genes
The proteins such as Drosha, Pasha, Exportin1, 2 and 5, Loquacious, R2D2 and Dicer are the first in the biosynthesis of miRNAs and siRNAs in eukaryotes [17, 19, 37, 39]. In miRNA pathway, the RNase III enzyme Drosha associates with a dsRNA binding protein, Pasha to generate pre-miRNAs in the nucleus [17, 19]. Subsequently, the pre-miRNAs are exported into the cytoplasm by nuclear export receptors, Exportin1, 2 and/or 5 for further processing by Dicer [37, 39]. Also, Dicer is the first protein that is required in the siRNA biogenesis to cleave long dsRNA precursors into short siRNAs capable of regulating anti-viral responses and transposable elements [17]. In D. melanogaster two Dicer proteins are known: Dicer1 partners with a dsRNA binding protein, Loquacious to generate the miRNAs and Dicer2 partners with another dsRNA binding protein, R2D2 to generate siRNAs [40].
Using the orthology detection tool called Proteinortho5 [41] and/or Blastp against the NCBI protein database, we found orthologs of Drosha, Pasha, Exportin1 and Exportin2 in the genomes of ecto-parasitic mites: Varroa destructor, V. jacobsoni and Tropilaelaps mercedesae, the predatory mite: Metaseiulus occidentalis and the black-legged tick: Ixodes scapularis. All of these species belong to Parasitiformes. Genes similar to Exportin5 were also found in the genomes of these species, except T. mercedesae. Using the same search approaches, we also found homologs of all these genes in Acariformes species that include the dust mites: Dermatophagoides pteronyssinus, Euroglyphus maynei, the two-spotted spider mite: Tetranychus urticae and the scabies mite: Sarcoptes scabiei. As presented in Table 1, multiple copies of orthologs of Drosha, Pasha, Exportin1 and 5 were found only in the genomes of V. destructor, V. jacobsoni, T. urticae, D. pteronyssinus, E. maynei and I. scapularis. Using both InterProScan [42] and HmmScan [43], we further confirmed the presence or absence of known conserved domains in orthologs of these proteins identified in all these Acari species (Table 1).
Table 1
Number of gene orthologs involved in the biosynthesis of miRNAs and their characteristic conserved domains. Asterick (*) following species name abbreviations indicates that its genome is fully sequenced whereas no asterisk indicates that its genome is partially sequenced. (#) Indicates that the gene contains no conserved domains.
Genes | | Acari Super-order | Parasitiformes | Acariformes |
| Acari orders | Ixodida | Mesostigmata | Trombidiformes | Sarcoptiformes |
Domain description | Domain ID (IPR/Pfam) | Is* | Mo* | TM | Vd | Vj | Tu* | Dp | Em | Ss |
Drosha | Ribonuclease domain | IPR000999/PF14622 | 1 | 1 | 1 | 6 | 3 | 1 | 1 | 1 | 1 |
One DsRNA binding domain | IPR014720/PF00035 |
Pasha | DsRNA binding domain | IPR014720/PF00035 | 2 | 1 | 1 | 3 | 3 | 2 | 2 | 1 | 1 |
Exportin1 | Importin-beta_N | IPR001494/PF03810 | 1 | 1 | 1 | 2 | 2 | 3 | 1 | 1 | 1 |
Exportin-1/Importin-b-like | IPR013598/PF08389 |
Exportin-1, C-domain | IPR014877/PF08767 |
Exportin2 | Importin-beta_N | IPR001494/PF03810 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
Exportin-2_C | IPR005043/PF03378 |
Exportin-2_Central | IPR013713/PF08506 |
Exportin5 | Exportin-1/Importin-b-like | IPR013598/PF08389 | 1 | 1 | 0 | 1 | 1 | 2 | 1# | 2# | 1# |
Proteins shown are from Ixodes scapularis (Is), Metaseiulus occidentalis (Mo), Tropilaelaps mercedesae (Tm), Tetranychus urticae (Tu), Varroa destructor (Vd), Varroa jacobsoni (Vj), Dermatophagoides pteronyssinus (Dp) Euroglyphus maynei (Em) and Sarcoptes scabiei (Ss).
With the same search approaches mentioned above, we identified homologs of Dicer1 (Dcr1) and Dicer2 (Dcr2) proteins in the genome assemblies of all the studied Acari species. Phylogenetic analysis based on the alignment of the two catalytic conserved Ribonuclease domains of all Dicer orthologs identified in these species along with orthologs of this protein in C. elegans (Ce), D. melanogaster (Dm) and an Acari species Dermatophagoides farina (Df) was carried out. Additionally, we searched for the presence of known conserved domains of Dicer proteins in each identified ortholog as outlined in the method's section. The analysis revealed that orthologs of Dicer1 and Dicer2 in Acariformes species clustered separately from those found in Parasitiformes species (Fig. 1A). Moreover, we found that Dicer1 orthologs identified in V. destructor (Vd), V. jacobsoni (Vj), T. urticae (Tu), I. scapularis (Is) and D. pteronyssinus (Dp), clustered together with significant bootstrap support (Fig. 1A) and have similar domain architecture to Dicer1 of both D. melanogaster (Dm-Dcr1) and C. elegans (Ce-Dcr1) (Fig. 1B). In the same vein, Dicer2 orthologs in these species clustered together and have similar domain architecture to Dm-Dcr2 with the exception of the ortholog identified in T. urticae, which clustered with Ce-Dcr1 (Fig. 1A, B). A single Dicer1 ortholog was found in all these species while several copies of Dicer2 were detected in the genome assemblies of V. destructor (2), V. jacobsoni (2) and D. pteronyssinus (2). As shown in Fig. 1A, duplicates of Dicer2 in V. destructor and V. jacobsoni clustered together while those identified in D. pteronyssinus did not. It is worthwhile to note that duplicates of Dicer2 in V. destructor, V. jacobsoni and D. pteronyssinus have similar domain architecture to Dm-Dcr2 (Fig. 1B).
Dicer1 ortholog found in T. mercedesae (Tm) clustered with Vj-Dcr1 and Vd-Dcr1 with significant bootstrap values (Fig. 1A) though it did not have similar domain architecture with Dm-Dcr1 and Ce-Dcr1 (Fig. 1B). Homolog of Dicer2 in this species has a similar domain structure with Dm-Dcr2 and clustered closely with Vj-Dcr2 and Vd-Dcr2 (Fig. 1A, B). On the other hand, orthologs of Dicer1 and Dicer2 identified in E. maynei and S. scabiei clustered closely with Dp-Dcr1, Df-Dcr1, Dp-Dcr2 and Df-Dcr2 (Fig. 1A) though they differed significantly in domain architecture from Dm-Dcr1/Ce-Dcr1 and Dm-Dcr2, respectively (Fig. 1B). As shown in Fig. 1A and B, one ortholog of Dicer1 found in M. occidentalis (Mo-Dcr1) clustered with Tm-Dcr1, Vj-Dcr1 and Vd-Dcr1 with significant bootstrap values and clearly shared similarities in domain architecture with Dm-Dcr1 and Ce-Dcr1. However, the three Dicer2 orthologs identified in this species (Mo-Dcr2a,b,c) grouped significantly with orthologs of this protein identified in I. scapularis, T. mercedesae, V. destructor and V. jacobsoni though they did not have similar domain structure with Dm-Dcr2 (Fig. 1A, B).
Figure 1 Phylogenetic analysis and schematic domain architecture of Dicer proteins. The tree in (A) was built based on the alignment of the two catalytic conserved Ribonuclease domains (IPR000999/PF00636) of Dicer orthologs identified in the studied Acari species using MAFFT [44]. The studied species include: Varroa destructor (Vd), Varroa jacobsoni (Vj), Tetranychus urticae (Tu), Ixodes scapularis (Is), Dermatophagoides pteronyssinus (Dp), Tropilaelaps mercedesae (Tm), Euroglyphus maynei (Em), Metaseiulus occidentalis (Mo) and Sarcoptes scabiei (Ss). Orthologs of Dicer proteins in Caenorhabditis elegans (Ce), Drosophila melanogaster (Dm) and Dermatophagoides farinae (Dp) were also included in this tree. The species names were abbreviated for convenience and the letters (a, b and c) following the names of some species indicate the number of gene copies found in their genomes. Names in “Red” and “green” colors on the tree are Parasitiformes and Acariformes species, respectively while those in “Blue” and “Purple” are D. melanogaster and C. elegans, respectively. The tree was constructed using PhyML [45], with the model recommended by Lefort et al. [46] under the Akaike information criterion (LG+G) with 500 bootstrap replicates. The domain architecture of Dicer proteins in (B) was generated by searching for known conserved domains in the Pfam database using both InterProScan [42] and HmmScan [43] and the letters (a, b and c) indicate the number of gene copies found in their genomes.
We identified homologs of Loquacious in the genomes of all Acari species investigated herein and several copies of this gene were found only in V. jacobsoni (6) and T. urticae (2) genomes (see Additional file). All orthologs of this protein found in these Acari species have the characteristic conserved dsRNA binding domain (IPR014720/PF00035). However, we did not find either R2D2 protein nor its ortholog C3PO protein previously identified in the genome of the red flour beetle Tribolium castaneum [33], in the genomes of any of the studied Acari species (see Additional file).
Argonautes and RISC components
Argonautes are the core effector proteins of the RNA-induced silencing complexes (RISCs) in the cytoplasm. They belong to the Argonaute superfamily, which is functionally divided into three families based on their interactions with specific RNA substrates [19]. Members of the Ago family interact with miRNAs and siRNAs, those of the Piwi family interact with piRNAs and those of the WAGO family, which are restricted to nematodes, interact with secondary siRNAs produced by RdRPs [19, 36]. Members of the Ago family are further divided into two distinct groups based on their involvement in either the miRNA- or siRNA-directed gene silencing. For instance, in D. melanogaster, Argonaute1 (Ago1) is required for miRNA-directed gene silencing while Argonaute2 (Ago2) is required for siRNA-directed gene silencing alone [47]. Meanwhile, in C. elegans, Alg1 and Alg2 are required for miRNA-directed gene silencing and Rde1 and Ergo1 are required for siRNA-directed gene silencing [21, 36]. A typical Argonaute protein is comprised of four domains: The N-terminal, PAZ, Mid and PIWI domain and the latter domain is the slicing domain that degrades the target mRNA transcript [19].
In this study, we found homologs of Ago family Argonautes in Parasitiformes and Acariformes species using the same search approaches mentioned above. A maximum likelihood tree based on the alignment of the PIWI domain showed that orthologs of the Ago family identified in Acari belong to two distinct groups that appear to be specialized in either mi- or siRNA- directed gene silencing (Fig. 2). It was intriguing to find that some of the Acari orthologs clustered together with Dm-Ago1, Ce-Alg1 and Ce-Alg2, whereas others clustered closely with Dm-Ago2 and Ce-Rde1. Therefore, we referred to these orthologs as Ago1 and Ago2, respectively. Except in E. maynei whose genome encodes a single copy of Ago1 and Ago2, we found more copies of Ago2 than Ago1 in the genomes of the remaining Acari species. The tree further showed that orthologs of Ago1 and Ago2 in Acariformes species clustered separately from those identified in Parasitiformes species, with significant bootstrap values (Fig. 2).
Figure 2 Phylogenetic analysis of Agonaute proteins. The tree was constructed based on the alignments of the conserved PIWI domain of Argonaute orthologs identified in the studied Acari species: Varroa destructor (Vd), Varroa jacobsoni (Vj), Tetranychus urticae (Tu), Ixodes scapularis (Is), Dermatophagoides pteronyssinus (Dp), Tropilaelaps mercedesae (Tm), Euroglyphus maynei (Em), Metaseiulus occidentalis (Mo) and Sarcoptes scabiei (Ss) using MAFFT [44]. Orthologs of these proteins in Caenorhabditis elegans (Ce), Drosophila melanogaster (Dm), Tribolium castaneum (Tc), Psoroptes ovis (Po) and Dermatophagoides farinae (Dp) were also included in this tree. The species names were abbreviated for convenience and the letters (a, b, c, d, e, f, g and h) following the names of some species indicate the number of gene copies found in their genomes. Names with “Red” and “green” colors on the tree are Parasitiformes and Acariformes species, respectively while those in “Blue” and “Purple” are D. melanogaster/T. castaneum and C. elegans, respectively. The tree was constructed using PhyML [45], with the model recommended by Lefort et al. [46] under the Akaike information criterion (LG + G + I + F) with 500 bootstrap replicates.
The search for the presence of known conserved domains revealed that almost all the orthologs of Ago1 in Acari have the same domain architecture with Dm-Ago1 and Ce-Alg1 and 2, with the exception of the single ortholog identified in E. maynei, which lacks the N-terminal domain (Fig. 3). Contrary to Ago1 homologs identified in Acari, which mostly shared the same domain architecture with orthologs of this protein in C. elegans and D. melanogaster, most of the Ago2 homologs in Acari had a domain architecture that was quiet distinct from those of both C. elegans and D. melanogaster (Fig. 3). The majority of them lack the Mid domain or both the N-terminal and the Mid domains. Only all the homologs of Ago-2 identified in I. scapularis (Ago-2a, b, c and d) and one homolog of this protein identified in M. occidentalis (Ago-2c) and T. urticae (Ago-2 h) have similar domain structure with Dm-Ago2.
Figure 3 Schematic domain architecture of Argonaute proteins of the Ago family in Acari. The domain architecture of the proteins was generated by searching for known conserved domains in the Pfam database using both InterProScan [42] and HmmScan [43]. The letters (a, b, c, d, e, f, g and h) following the names of the gene (Ago1 and Ago2) indicate the number of gene copies found in in the individual Acari species.
Argonautes of the Piwi family such as Argonaute-3 (Ago3), Aubergine (Aub) and Piwi were found in the genomes of I. scapularis, T. urticae, V. destructor, V. jacobsoni, M. occidentalis and T. mercedesae, but not in genomes of D. pteronyssinus, E. maynei and Sarcoptes scabiei. We identified homologs of Ago3 in the genomes of I. scapularis, T. urticae, V. destructor, V. jacobsoni, M. occidentalis and T. mercedesae (see Additional file). Notably, several copies of this protein were found only in the genomes of I. scapularis (2) and V. jacobsoni (2). We further identified putative homologs of D. melanogaster-Aub and Tribolium castaneum (Tc)-Aub/Piwi only in the genome assemblies of I. scapularis and T. urticae (see Additional file). It is important to note that one homolog of Drosophila Aub/Piwi and Ago-3 were previously detected in the genome of the red flour beetle, Tribolium castaneum (Tc) [33]. In our study, we identified one and two copies of Dm-Aub and Tc-Aub/Piwi, respectively, in I. scapularis and one and four copies of Tc-Aub/Piwi and Dm-Aub, respectively in T. urticae (see Additional file). All homologs of the Piwi family Argonautes identified in T. urticae and I. scapularis had the PIWI and PAZ domains, while those identified in V. destructor, V. jacobsoni, M. occidentalis and T. mercedesae had only the PIWI domain. Phylogenetic analysis placed homologs of Ago3, Aub and Piwi in Acari with orthologs of these genes in D. melanogaster and T. castaneum (Fig. 2).
We found homologs of the WAGO family Argonautes (Ce-PPW1, -PPW2, -SAGO1 and -SAGO2) in some Acari species studied herein using Proteinortho5 [41] (see Additional file). One and two homologs of Sago2 and Sago1, respectively, were identified in I. scapularis's genome. Also, one homolog of PPW2 was found in T. mercedesae while one and three homologs of Sago1 were identified in V. jacobsoni and V. destructor, respectively. In T. urticae, homologs of Sago1 (1) and PPW2 (1) were also found. Interestingly, most of the orthologs of the WaGO family Argonautes were the same homologs, which were highly similar to Ago family Argonautes (Ago2) identified in these species (represented as Is-Ago2a and Ago2b, Tm- Ago2c, Vd-Ago2b, Vj-Ago2b and Tu-Ago2c) (see Additional file). Since all the orthologs of the WAGO family Argonautes clustered with orthologs of the Ago family Argonautes, with significant bootstrap support (Fig. 2), we tentatively referred to them as members of the Ago family Argonautes that is Ago2 homologs. It is worth mentioning that all C. elegans WAGO family Argonautes used as query sequences in our study have both the PAZ and the PIWI domains.
Orthologs of the Ago and Piwi families Argonautes identified in all these Acari species were further examined for the presence of the conserved residues, Aspartate-Aspartate-Histidine/Aspartate/Lysine (DDH/DDD/DDK). These residues that are present within the catalytic PIWI domain presumably enable some Argonaute proteins to cleave the target mRNAs (reviewed in [48, 49]). As shown on Table 2, the DDH catalytic residue was found in all the Argonautes identified in I. scapularis. Similarly, Argonautes of the Ago family identified in E. maynei’s genome contained this motif. The only member of the Piwi family that is Ago3 identified in the genomes of M. occidentalis, T. mercedesae and V. jacobsoni did not have any of these residues in the sequences of their PIWI domains, whereas all the Argonautes of the Ago family identified in these species have the DDH residue. Also, Ago3 and one homolog of Ago-2 (that is Ago2d) identified in V. destructor lacked these conserved residues, though the remaining proteins of the Ago family had the conserved DDH residue. Also, Ago1 of S. scabiei did not have these residues, whereas Ago2 homologs did have. In T. urticae, all the Argonautes of the Ago family had the DDH motif, while only four out of the six Argonautes of the Piwi family had the DDH motif. In D. pteronyssinus, Ago1 and Ago2a have the DDH motif, while the remaining Argonautes that were Ago2b to 2 g had the DDD motif.
Table 2
Catalytic residues of Argonautes of the Ago, Piwi and Wago groups in Acari species, Caenorhabditis elegans, Drosophila melanogaster and Tribolium castaneum.
Ce-Alg-1 | V | I | F | F | G | C | D | I | | V | V | Y | R | D | G | V | S | | I | P | A | P | A | Y | Y | A | H | L | V | A | F | Ago |
Ce-Alg-2 | V | I | F | L | G | C | D | I | | V | V | Y | R | D | G | V | S | | I | P | A | P | A | Y | Y | A | H | L | V | A | F | Ago |
Ce-Rde-1 | T | M | Y | V | G | I | D | V | | V | V | Y | R | D | G | V | S | | L | P | V | P | V | H | Y | A | H | L | S | C | E | Ago |
Ce-PPW-1 | R | L | I | V | G | F | V | T | | L | L | Y | F | N | G | V | S | | L | P | V | P | L | Y | I | A | D | R | Y | S | G | Wago |
Ce-PPW-2 | H | L | I | I | G | V | G | I | | T | I | Y | F | S | G | V | S | | I | P | T | P | L | Y | V | A | N | E | Y | A | K | Wago |
Ce-Sago-1 | R | L | I | I | G | F | E | T | | L | I | Y | F | S | G | V | S | | L | P | I | P | L | H | I | A | G | T | Y | S | E | Wago |
Ce-Sago-2 | R | L | I | V | G | F | V | T | | L | L | Y | F | N | G | V | S | | L | P | V | P | L | Y | I | A | D | R | Y | S | G | Wago |
Dm-Ago-1 | V | I | F | L | G | A | D | V | | I | L | Y | R | D | G | V | S | | I | P | A | P | A | Y | Y | A | H | L | V | A | F | Ago |
Dm-Ago-2 | T | M | Y | I | G | A | D | V | | I | Y | Y | R | D | G | V | S | | Y | P | A | P | A | Y | L | A | H | L | V | A | A | Ago |
Dm-Ago-3 | V | M | I | C | G | I | D | S | | I | I | Y | R | D | G | I | G | | I | P | A | C | C | M | Y | A | H | K | L | A | Y | Piwi |
Dm-Aub | L | M | T | V | G | F | D | V | | L | F | F | R | D | G | V | G | | V | P | A | V | C | H | Y | A | H | K | L | A | F | Piwi |
Dm-Piwi | L | M | T | I | G | F | D | I | | V | F | Y | R | D | G | V | S | | V | P | A | V | C | Q | Y | A | K | K | L | A | T | Piwi |
Tc-Ago-1 | V | I | F | L | G | A | D | V | | I | L | Y | R | D | G | V | S | | I | P | A | P | A | Y | Y | A | H | L | V | A | F | Ago |
Tc-Ago-2 | C | I | I | M | G | A | D | V | | V | F | F | R | D | G | V | S | | Y | P | A | P | T | Y | Y | A | H | L | A | A | A | Ago |
Tc-Ago-3 | W | M | V | C | G | I | D | V | | I | V | F | R | D | G | V | G | | V | P | A | P | C | L | Y | A | H | K | L | A | A | Piwi |
Tc-Aub | L | M | V | V | G | Y | D | V | | L | I | Y | R | D | G | V | G | | V | P | A | P | C | Q | Y | A | H | K | L | A | F | Piwi |
Tc-Piwi | L | M | V | V | G | Y | D | V | | L | I | Y | R | D | G | V | G | | V | P | A | P | C | Q | Y | A | H | K | L | A | F | Piwi |
Is-Ago-1 | V | I | F | L | G | A | D | V | | I | F | Y | R | D | G | V | S | | I | P | A | P | A | Y | Y | A | H | L | V | A | F | Ago |
Is-Ago-2a | V | I | I | I | G | A | D | V | | I | F | Y | R | D | G | V | S | | I | P | A | P | V | Y | Y | A | H | L | A | A | Y | Ago |
Is-Ago-2b | V | I | V | M | G | A | D | V | | I | F | Y | R | D | G | V | S | | I | P | V | P | V | Y | Y | A | H | H | A | T | Q | Ago |
Is-Ago-2c | V | I | I | I | G | A | D | V | | I | F | Y | R | D | G | V | S | | I | P | A | P | V | Y | Y | A | H | L | A | A | F | Ago |
Is-Ago-2d | V | I | I | L | G | A | D | V | | I | F | Y | R | D | G | V | S | | I | P | A | P | A | Y | Y | A | H | W | V | A | F | Ago |
Is-Ago-3a | V | M | V | I | G | I | D | V | | F | V | F | R | D | G | V | G | | V | P | A | P | C | Q | Y | A | H | K | L | A | N | Piwi |
Is-Ago-3b | V | M | V | I | G | I | D | V | | F | V | F | R | D | G | V | G | | V | P | A | P | C | Q | Y | A | H | K | L | A | N | Piwi |
Is-Aub | T | M | V | I | G | Y | D | T | | L | F | F | R | D | G | V | S | | V | P | A | P | C | Q | Y | A | H | K | L | A | F | Piwi |
Is-Aub/Piwia | M | M | C | V | G | Y | D | T | | L | F | F | R | D | G | V | S | | V | P | A | P | C | Q | Y | A | H | K | L | A | F | Piwi |
Is-Aub/Piwib | M | M | C | V | G | Y | D | T | | L | F | F | R | D | G | V | S | | V | P | A | P | C | Q | Y | A | H | K | L | A | F | Piwi |
Mo-Ago-1 | V | I | F | F | G | C | D | V | | I | F | Y | R | D | G | V | S | | I | P | A | P | A | Y | Y | A | H | L | V | A | F | Ago |
Mo-Ago-2a | Y | M | A | I | G | V | D | V | | F | I | F | R | D | G | V | A | | I | P | A | P | A | Y | Y | A | H | L | V | A | F | Ago |
Mo-Ago-2b | F | M | I | L | G | A | D | V | | Y | Y | L | R | D | G | V | S | | I | P | A | P | V | Y | Y | A | H | L | A | A | F | Ago |
Mo-Ago-2c | F | I | V | F | G | A | D | V | | L | F | Y | R | D | G | V | S | | I | P | A | P | V | Y | Y | A | H | L | A | A | A | Ago |
Mo-Ago-2d | F | M | I | L | G | A | D | V | | Y | Y | L | R | D | G | V | S | | I | P | A | P | V | Y | Y | A | H | L | A | A | F | Ago |
Mo-Ago-2e | T | L | I | I | G | M | D | V | | I | V | Y | R | D | G | V | S | | L | P | A | P | V | Y | Y | A | H | L | V | A | F | Ago |
Mo-Ago-3 | T | L | V | I | G | L | S | L | | - | - | - | - | - | - | - | - | | - | - | - | - | - | - | - | - | - | - | - | - | - | Piwi |
Tm-Ago-1 | V | I | F | F | G | C | D | V | | I | F | Y | R | D | G | V | S | | I | P | A | P | A | Y | Y | A | H | L | V | A | F | Ago |
Tm-Ago-2a | F | I | V | F | G | G | D | V | | F | F | F | R | D | G | V | S | | I | P | A | P | I | Y | Y | A | H | L | A | A | A | Ago |
Tm-Ago-2b | Y | M | V | V | G | A | D | V | | Y | F | Y | R | D | G | V | S | | I | P | A | P | V | F | Y | A | H | L | A | A | K | Ago |
Tm-Ago-2c | F | M | I | V | G | A | D | V | | Y | I | Y | R | D | G | V | A | | I | P | A | P | A | Y | Y | A | H | L | A | A | T | Ago |
Tm-Ago-2d | Y | M | I | L | G | A | D | V | | Y | Y | F | R | D | G | V | S | | I | P | S | P | I | Y | Y | A | H | L | A | A | F | Ago |
Tm-Ago-3 | T | L | V | I | G | M | S | G | | V | V | F | R | R | - | - | - | | L | P | A | P | L | L | Y | A | G | K | C | V | E | Piwi |
Vd-Ago-1 | V | I | F | F | G | C | D | V | | I | F | Y | R | D | G | V | S | | I | P | A | P | A | Y | Y | A | H | L | V | A | F | Ago |
Vd-Ago-2a | Y | I | V | F | G | A | D | V | | F | F | F | R | D | G | I | S | | I | P | A | P | V | Y | Y | A | H | L | A | A | S | Ago |
Vd-Ago-2b | F | I | V | F | G | A | D | V | | F | F | F | R | D | G | V | S | | I | P | A | P | V | Y | Y | A | H | L | A | A | A | Ago |
Vd-Ago-2c | Y | M | I | V | G | A | D | V | | Y | Y | Y | R | D | G | V | A | | I | P | A | P | V | Y | Y | A | H | W | A | A | Q | Ago |
Vd-Ago-2d | F | M | V | V | G | I | D | I | | I | I | F | R | A | S | V | P | | I | P | A | P | L | E | Y | A | N | L | A | L | R | Ago |
Vd-Ago-2e | Y | I | V | L | G | A | D | V | | Y | Y | L | R | D | G | V | S | | I | P | A | P | I | Y | Y | A | H | L | A | A | F | Ago |
Vd-Ago-2f | T | I | I | I | G | L | D | V | | I | I | Y | R | D | G | V | S | | I | P | A | P | I | Y | Y | A | H | L | V | A | F | Ago |
Vd-Ago-2 g | F | I | V | F | G | A | D | V | | F | F | F | R | D | G | V | S | | I | P | A | P | V | Y | Y | A | H | L | A | A | A | Ago |
Vd-Ago-2 h | F | I | V | F | G | A | D | V | | F | F | F | R | D | G | V | S | | I | P | A | P | V | Y | Y | A | H | L | A | A | A | Ago |
Vd-Ago-3 | I | M | V | I | G | M | S | A | | F | V | F | R | N | - | - | - | | L | P | A | P | L | L | Y | A | S | K | C | A | E | Piwi |
Vj-Ago-1a | V | I | F | F | G | C | D | V | | I | F | Y | R | D | G | V | S | | I | P | A | P | A | Y | Y | A | H | L | V | A | F | Ago |
Vj-Ago-1b | V | I | F | F | G | C | D | V | | I | F | Y | R | D | G | V | S | | I | P | A | P | A | Y | Y | A | H | L | V | A | F | Ago |
Vj-Ago-2a | Y | M | I | V | G | A | D | V | | Y | Y | Y | R | D | G | V | A | | I | P | A | P | V | Y | Y | A | H | W | A | A | Q | Ago |
Vj-Ago-2b | F | I | V | F | G | A | D | V | | F | F | F | R | D | G | V | S | | I | P | A | P | V | Y | Y | A | H | L | A | A | A | Ago |
Vj-Ago-2c | Y | I | V | L | G | A | D | V | | Y | Y | L | R | D | G | V | S | | I | P | A | P | I | Y | Y | A | H | L | A | A | F | Ago |
Vj-Ago-2d | T | I | I | I | G | L | D | V | | I | I | Y | R | D | G | V | S | | I | P | A | P | I | Y | Y | A | H | L | V | A | F | Ago |
Vj-Ago-2e | T | I | I | I | G | L | D | V | | I | I | Y | R | D | G | V | S | | I | P | A | P | I | Y | Y | A | H | L | V | A | F | Ago |
Vj-Ago-3a | I | M | V | I | G | M | S | A | | F | V | F | R | N | - | - | - | | L | P | A | P | L | L | Y | A | S | K | C | A | E | Piwi |
Vj-Ago-3b | I | M | V | I | G | M | S | A | | F | V | F | R | N | - | - | - | | L | P | A | P | L | L | Y | A | S | K | C | A | E | Piwi |
Tu-Ago-1a | V | I | F | L | G | A | D | V | | I | F | Y | R | D | G | V | S | | I | P | A | P | A | Y | Y | A | H | L | V | A | F | Ago |
Tu-Ago-1b | V | I | F | L | G | A | D | V | | I | F | Y | R | D | G | V | S | | I | P | A | P | A | Y | Y | A | H | L | V | A | F | Ago |
Tu-Ago-1c | V | I | F | L | G | A | D | V | | I | F | Y | R | D | G | V | S | | I | P | A | P | A | Y | Y | A | H | L | V | A | F | Ago |
Tu-Ago-2a | T | I | I | M | G | A | D | V | | I | F | Y | R | D | G | V | S | | I | P | A | P | V | Q | Y | A | H | L | A | A | F | Ago |
Tu-Ago-2b | T | I | I | M | G | A | D | V | | I | F | Y | R | D | G | V | S | | I | P | A | P | V | Q | Y | A | H | L | A | A | F | Ago |
Tu-Ago-2c | I | M | I | I | G | A | D | V | | I | F | L | R | D | G | V | S | | A | P | A | P | V | M | H | A | H | N | L | A | Y | Ago |
Tu-Ago-2d | I | M | I | I | G | A | D | V | | I | F | L | R | D | G | V | S | | A | P | A | P | V | M | H | A | H | N | L | A | Y | Ago |
Tu-Ago-2e | I | M | I | I | G | A | D | V | | I | F | L | R | D | G | V | S | | A | P | A | P | V | M | H | A | H | N | L | A | Y | Ago |
Tu-Ago-2f | I | M | I | I | G | A | D | V | | I | F | L | R | D | G | V | S | | A | P | A | P | V | M | H | A | H | N | L | A | Y | Ago |
Tu-Ago-2 g | T | M | V | V | G | V | D | V | | V | V | Y | R | D | G | V | S | | D | P | A | P | A | R | Y | A | H | H | A | A | A | Ago |
Tu-Ago-3 | A | M | I | V | G | L | D | S | | F | I | Y | R | D | G | V | G | | V | P | A | P | C | Q | Y | A | H | K | L | A | F | Piwi |
Tu-Auba | C | M | I | V | G | Y | D | T | | I | I | Y | R | D | G | V | S | | V | P | A | P | C | Q | Y | A | H | K | L | A | L | Piwi |
Tu-Aubb | W | M | I | V | G | Y | N | T | | I | I | Y | R | D | G | V | S | | V | P | A | P | C | Q | Y | A | H | K | L | A | F | Piwi |
Tu-Aubc | T | M | F | V | G | Y | N | T | | I | I | Y | R | D | G | V | S | | V | P | A | P | C | H | Y | A | Y | K | L | A | F | Piwi |
Tu-Aubd | C | M | I | V | G | Y | D | T | | I | I | Y | R | D | G | V | S | | V | P | A | P | C | Q | Y | A | H | K | L | A | L | Piwi |
Tu-Aub/Piwi | C | M | I | V | G | Y | D | T | | V | I | Y | R | D | G | V | S | | V | P | A | P | C | Q | Y | A | H | K | L | A | F | Piwi |
Df-Ago-1 | V | I | F | L | G | A | D | V | | I | L | Y | R | D | G | V | S | | I | P | A | P | A | Y | Y | A | H | L | V | A | F | Ago |
Df-Ago-2a | T | M | A | I | G | V | D | V | | I | L | Y | R | D | G | V | S | | I | P | T | P | V | F | Y | A | H | L | A | A | F | Ago |
Df-Ago-2b | T | M | I | I | G | L | D | V | | I | I | F | R | D | G | V | S | | I | P | T | Q | V | M | Y | A | D | L | C | A | Y | Ago |
Df-Ago-2c | T | M | A | I | G | V | D | V | | V | I | F | R | D | G | V | S | | M | P | V | P | V | R | Y | A | D | L | C | A | Y | Ago |
Df-Ago-2d | T | M | I | I | G | I | D | V | | L | I | F | R | D | G | V | S | | I | P | T | P | I | R | Y | A | D | L | C | A | Y | Ago |
Df-Ago-2e | T | M | A | I | G | I | D | V | | I | V | F | R | D | G | I | G | | M | P | I | P | V | R | Y | A | D | L | C | A | Y | Ago |
Df-Ago-2f | T | M | I | I | G | I | D | V | | I | I | F | R | D | G | V | S | | I | P | T | P | I | K | Y | A | D | l | C | A | y | Ago |
Df-Ago-2 g | T | M | V | C | G | I | D | V | | A | V | F | R | D | G | V | S | | L | P | T | P | V | R | Y | A | D | L | C | A | Y | Ago |
Po-Ago-1 | V | I | F | L | G | A | D | V | | I | L | Y | R | D | G | V | S | | I | P | A | P | A | Y | Y | A | H | L | V | A | F | Ago |
Po-Ago-2 | T | M | V | I | G | I | D | V | | L | V | F | R | D | G | V | S | | I | P | T | P | I | R | Y | A | D | L | C | A | Y | Ago |
Em-Ago-1 | V | I | F | L | G | A | D | V | | I | L | Y | R | D | G | V | S | | I | P | A | P | A | Y | Y | A | H | L | V | A | F | Ago |
Em-Ago-2a | T | M | A | I | G | V | D | V | | I | F | Y | R | D | G | V | S | | I | P | T | P | V | F | Y | A | H | L | A | A | F | Ago |
Dp-Ago-1c | V | I | F | L | G | A | D | V | | I | M | Y | R | D | G | V | S | | I | P | A | P | A | Y | Y | A | H | L | V | A | F | Ago |
Dp-Ago-2a | T | M | A | I | G | V | D | V | | I | F | Y | R | D | G | V | S | | I | P | T | P | V | F | Y | A | H | L | A | A | F | Ago |
Dp-Ago-2b | T | M | V | V | G | I | D | V | | I | I | F | R | D | G | V | S | | I | P | T | P | I | R | Y | A | D | L | C | A | Y | Ago |
Dp-Ago-2c | T | M | V | V | G | I | D | V | | I | I | F | R | D | G | V | S | | I | P | T | P | I | R | Y | A | D | L | C | A | Y | Ago |
Dp-Ago-2d | T | M | V | L | G | V | D | V | | A | V | F | R | D | G | V | S | | L | P | T | P | V | R | Y | A | D | L | C | A | Y | Ago |
Dp-Ago-2e | T | M | A | I | G | I | D | V | | I | I | F | R | D | G | I | G | | M | P | V | P | V | R | Y | A | D | L | C | A | Y | Ago |
Dp-Ago-2f | T | M | V | I | G | L | D | V | | I | I | F | R | D | G | V | S | | I | P | T | P | V | M | Y | A | D | L | C | A | Y | Ago |
Dp-Ago-2 g | T | M | A | I | G | V | D | V | | I | V | F | R | D | G | V | S | | M | P | I | P | V | R | Y | A | D | L | C | A | Y | Ago |
Ss-Ago-1c | V | I | F | L | G | A | D | V | | I | F | Y | R | D | G | V | S | | - | - | - | - | - | - | - | - | - | - | - | - | - | Ago |
Ss-Ago-2a | T | I | V | F | G | V | D | V | | V | I | F | R | D | G | V | S | | I | P | T | A | I | R | Y | A | D | L | C | A | Y | Ago |
Ss-Ago-2b | T | M | I | V | G | I | D | V | | I | V | Y | R | D | G | I | D | | M | P | V | P | L | R | Y | A | D | L | C | A | Y | Ago |
Argonautes shown are from Caenorhabditis elegans (Ce), Drosophila melanogaster (Dm), Tribolium Castaneum (Tc), Ixodes Scapularis (Is), Metaseiulus occidentalis (Mo), Tropilaelaps mercedesae (Tm), Varroa destructor (Vd), Varroa jacobsoni (Vj), Tetranychus urticae (Tu), Dermatophagoides farinae (Dp), Psoroptes ovis (Po), Euroglyphus maynei (Em), Dermatophagoides pteronyssinus (Dp), and Sarcoptes scabiei (Ss). The amino acid sequences of the PIWI domains were aligned with MAFFT [44]. Substitutions for the Aspartate-Aspartate-Histidine (Asp-Asp-His) motif such as Aspartate or Histidine to either Serine (S), Arginine (R), Alanine (A), Asparagine (N), Glycine (G) or Tyrosine (Y), are colored in pink. Substitutions of the Histidine to Aspartate (D) or Lysine (K) are colored in light blue. (-) indicate the absence of amino acid residues from the sequence. |
Other components of the RISC complex notably Vasa intronic gene (Vig-1) and Tudor-staphylococcal nuclease (Tsn-1), that also contribute to the degradation of the target mRNAs [50, 51], were also identified in the genome of the Acari species in this study, with the exception of E. maynei, which had Vig-1 but not Tsn-1 (see Additional file).
DsRNA uptake and spread
We searched for Sid-1, Rsd-2, Rsd-3, Rsd-6, Eater and SR-CI genes that are involved in cellular dsRNA uptake and systemic spread of the silencing dsRNA molecules as mentioned above. We were unable to identify genes similar to Sid-1 nor its orthologs SilA, SilB and SilC in the studied Acari species, previously found in the T. castaneum genome [33] in the studied Acari species. Similarly, orthologs of Rsd-2 and Rsd-6 were not found in any of the studied Acari species, though orthologs of Rsd-3 protein were present in all of them (see Additional file). Duplicates of this protein were only found in the genomes of I. scapularis (2), V. jacobsoni (5) and V. destructor (9) respectively. Interestingly, all the identified orthologs had a single conserved domain, the epsin N-terminal homology (ENTH) domain that has been shown to be sufficient to mediate the transport of dsRNA molecules into both somatic and germ cells of C. elegans [29]. We also identified orthologs of Dm-Eater in the genomes of some Acari species except in those of D. pteronyssinus, E. maynei and S. scabiei. Again, duplicates of this protein were found in T. urticae (3), V. destructor (5) and V. jacobsoni (2). Orthologs of this protein in the Acari species have several epidermal growth factor (EGF)-like modules. Furthermore, one homolog of Dm-SR-CI gene was identified only in the genome of T. urticae. The Drosophila SR-CI receptor and its homolog had a characteristic conserved Meprin, A5 protein, and protein tyrosine phosphatase Mu (MAM) domain.
siRNA secondary amplification
Systemic and trans-generational gene interference rely on the amplification of the initial siRNA trigger for efficient gene knockdown throughout the treated organism. This mechanism for enhancing RNAi potency necessitates the action of a cellular RNA-dependent RNA polymerase (RdRP) protein, which has been extensively studied in C. elegans [52, 53]. Interestingly, we found that all Acari species from the two sister lineages, Acariformes and Parasitiformes studied here, poses such RdRP proteins. Duplicates of this protein were found in the genomes of all studied species. Moreover, all the Acari RdRP proteins identified in this study including those previously identified in C. elegans (Ego-1 and Rrf-1) [37] and other Acari species e. g. D. farinae [54] and Psoroptes ovis [55] had the characteristic conserved RdRP domain [34]. In order to infer the evolutionary history of Acari RdRP proteins, we conducted a phylogenetic analysis based on the amino acid alignment of RdRP domain using C. elegans as outgroup, as described in the methods section. The phylogeny revealed that proteins of Acariformes (indicated with “green” color on Fig. 4) clustered strongly together (494/500 bootstraps) and separately from those of Parasitiformes. The Parasitormes proteins (indicated with “red” color on Fig. 4) also clustered strongly together (408/500 bootstraps). However, three out of the total four proteins identified in I. scapularis were found to be evolutionary closer to Acariformes proteins than to those of Parasitiformes (479/500 bootstraps) (Fig. 4).
Figure 4. Phylogenetic distribution of RNA-dependent RNA polymerase (RdRP) genes in Acari species: Varroa destructor (Vd), V. jacobsoni (Vj), Tropilaelaps mercedesae (Tm), Metaseiulus occidentalis (Mo), Ixodes scapularis (Is), Dermatophagoides pteronyssinus (Dp), Euroglyphus maynei (Em), Tetranychus urticae (Tu), Sarcoptes scabiei (Sc) and Psoroptes ovis (Po). The nematode species, Caenorhabditis elegans (Ce) was used as outgroup. The species names were abbreviated for convenience and the numbers (1, 2, 3, 4 and 5) following their names indicate the gene copies found in each species. Names with “Red” and “green” colors on the tree are Parasitiformes and Acariformes species, respectively while those in “Purple” are C. elegans. The tree was constructed using PhyML [45], with the model recommended by Lefort et al. [46] under the Akaike information criterion (LG + G + I + F) with 500 bootstrap replicates and is based on the amino acid alignment of the conserved RdRP domain (IPR007855/PF05183) using MAFFT [44].