- Research article
- Open Access
Transcriptome profiling of a spirodiclofen susceptible and resistant strain of the European red mite Panonychus ulmi using strand-specific RNA-seq
- Sabina Bajda†1,
- Wannes Dermauw†2Email authorView ORCID ID profile,
- Robert Greenhalgh3,
- Ralf Nauen4,
- Luc Tirry2,
- Richard M. Clark3, 5 and
- Thomas Van Leeuwen1, 2Email author
© Bajda et al. 2015
- Received: 13 July 2015
- Accepted: 27 October 2015
- Published: 18 November 2015
The European red mite, Panonychus ulmi, is among the most important mite pests in fruit orchards, where it is controlled primarily by acaricide application. However, the species rapidly develops pesticide resistance, and the elucidation of resistance mechanisms for P. ulmi has not kept pace with insects or with the closely related spider mite Tetranychus urticae. The main reason for this lack of knowledge has been the absence of genomic resources needed to investigate the molecular biology of resistance mechanisms.
Here, we provide a comprehensive strand-specific RNA-seq based transcriptome resource for P. ulmi derived from strains susceptible and resistant to the widely used acaricide spirodiclofen. From a de novo assembly of the P. ulmi transcriptome, we manually annotated detoxification enzyme families, target-sites of commonly used acaricides, and horizontally transferred genes implicated in plant-mite interactions and pesticide resistance. In a comparative analysis that incorporated sequences available for Panonychus citri, T. urticae, and insects, we identified radiations for detoxification gene families following the divergence of Panonychus and Tetranychus genera. Finally, we used the replicated RNA-seq data from the spirodiclofen susceptible and resistant strains to describe gene expression changes associated with resistance. A cytochrome P450 monooxygenase, as well as multiple carboxylcholinesterases, were differentially expressed between the susceptible and resistant strains, and provide a molecular entry point for understanding resistance to spirodiclofen, widely used to control P. ulmi populations.
The new genomic resources and data that we present in this study for P. ulmi will substantially facilitate molecular studies of underlying mechanisms involved in acaricide resistance.
- Cyclic keto-enols
- Target-site mutation
- biRNA virus
- Intradiol ring-cleavage dioxygenases
- Cytochrome P450 mono-oxygenases
The European red mite, Panonychus ulmi, is a member of the family Tetranychidae (Arthropoda: Chelicerata: Arachnida: Acari). Tetranychidae, or spider mites, use their stylet- like chelicerae to puncture the leaf mesophyll-cells to suck out cell contents. This results in chlorotic spots and necrosis and ultimately, leaf abscission . As a result, among mite pests, spider mites are economically important with 80 % of the acaricide market used for their control . Nonetheless, a major problem associated with spider mite control is the rapid development of acaricide resistance. Factors accelerating resistance evolution include not only the frequent use of acaricides, but also high fecundity rates, arrhenotokous reproduction (on haploid males selection is highly effective) and a short life cycle . As a result, the two-spotted spider mite (Tetranychus urticae) and P. ulmi have developed resistance to nearly all acaricide classes  and rank among the top 10 of most resistant arthropod species based on the number of unique active ingredients for which resistance has been reported .
The acaricides spirodiclofen and spiromesifen belong, together with the insecticide spirotetramat, to the family of spirocyclic tetronic acids (keto-enols) and interrupt lipid biosynthesis by direct inhibition of acetyl coenzyme A carboxylase (ACCase) [5, 6]. Introduced relatively recently to the market , spirodiclofen has been shown to be an effective tool in resistance management of tetranychid mites. It shows good to excellent residual control [8, 9] and is effective against spider mites resistant to different acaricide classes [10–16]. Although high levels of spirodiclofen resistance have not yet been reported in field populations, artificial selection studies have shown that spider mites have the potential to develop resistance to spirodiclofen comparatively rapid in the laboratory [17, 18]. Selection for resistance was undertaken with T. urticae, P. ulmi and P. citri and resistance in all species was synergized by piperonylbutoxide (PBO), suggesting the involvement of cytochrome P450s [17–19]. Strikingly, for all three tetranychid species spirodiclofen resistance was age dependent. The concentration of spirodiclofen causing 50 % lethality (LC50) in the resistant strains was much lower in eggs then in mobile stages, a finding Demaeght et al.  suggested could be due to lower expression of detoxification genes in eggs.
In 2011, the high quality genome sequence of the spider mite Tetranychus urticae became available , and afforded new resources and tools to investigate the molecular mechanisms underlying acaricide resistance [20, 22–25]. For example, although biochemical studies had shown that elevated P450 and esterase levels were associated with resistance to spirodiclofen in T. urticae (see above), studies with a genome-wide gene expression microarray allowed the identification of the genes coding for the detoxification enzymes associated with these activities (CYP392E10 and TuCCE-04) . It is clear that our understanding of resistance to acaricides in phytophagous mites improved with the advent of the T. urticae genome, however, such genomic resources are scarce for other key tetranychid mites, particularly P. ulmi. With only 49 nucleotide sequences available in the NCBI public database (accessed May 2015), P. ulmi resistance research at the molecular level has hardly been possible.
For organisms without a sequenced genome, de novo transcriptome assemblies provide an important starting point for genomic analyses, and recent advances in high-throughput transcript sequencing have greatly contributed to our understanding of gene expression and structure [26–29]. This is especially true in agricultural pest management, where the majority of pest species do not have a reference genome and where the elucidation of resistance mechanisms of insecticides is vital [30–34].
In this study we used the Illumina HiSeq2000 technology to generate deep paired-end, strand-specific RNA-seq reads for two strains of P. ulmi. The first strain (HS) is susceptible to most currently used acaricides, while the second strain (PSR-TK) was field collected and shown to be resistant to a number of acaricides, including hexythiazox and clofentezine. The strain also showed decreased susceptibility to spirodiclofen, and was further selected in the laboratory until high levels of spirodiclofen resistance (Resistance ratio of 7000) were reached . Paired-end RNAseq reads were used to assemble a P. ulmi transcriptome, and with the same assembly methodology we constructed two P. citri transcriptomes based on previously published RNA-seq data available as single end reads [33, 34]. We then performed a phylogenetic classification of Panonychus gene families involved in detoxification, such as cytochrome P450 monooxygenases (CYPs), carboxylcholinesterases (CCEs), glutathione-S-transferases (GSTs), UDP-glycosyl transferases (UGTs) and ABC transporters (ABCs), and searched for horizontally transferred genes that were previously uncovered in the genome sequence of T. urticae [21, 35–39]. Moreover, a differential expression analysis between the acaricide susceptible and resistant P. ulmi strain allowed us to identify genes encoding proteins that are candidates for involvement in acaricide resistance. In addition, we identified and compared P. ulmi expressed sequences for known acaricide target sites. The assembled annotated transcriptome of P. ulmi provides an important resource that should substantially facilitate molecular studies in this species, including the elucidation of detoxification mechanisms of xenobiotics.
De novo sequence assembly
We performed high-throughput Illumina sequencing of RNA extracted from adult females from acaricide susceptible (HS) and resistant P. ulmi strains (PSR-TK). With the Illumina HiSeq platform we generated 66,308,047 and 95,412,934 strand-specific paired-end reads for the HS and PSR-TK P. ulmi strains, respectively. Two approaches were used to assemble the P. ulmi transcriptome (see Methods for details). The assembly created with CLC Genomics Workbench (CLC) contains 27,777 unique P. ulmi contigs longer than 200 bp, totalling 27.6 Mb of sequence with an overall GC content of 33.6 %. The average contig size was 993 bp and the N50 was 2087 bp. The Velvet/Oases assembly, on the other hand, consists of 30,044 transcripts totalling 18.6 Mb of sequence with a GC content of 34.5 % and an average contig size/N50 of 619 bp/871 bp. Mapping all RNA-seq reads against both transcriptomes (CLC or Velvet/Oases) revealed that the overall mapping success rate, as a measure for assembly quality, was significantly lower for the Velvet/Oases assembly compared to the CLC assembly (see Additional file 1), with an average overall alignment rate of 91.7 % and 73.4 % for the CLC and Velvet/Oases assembly, respectively. Hence, unless otherwise stated, the CLC assembly was used for all further analyses described in this study. All raw reads and the CLC assembly were submitted to NCBI under BioProject PRJNA271858.
As the transcriptome assemblies of two previously published P. citri transcriptomes [33, 34] were not publicly available, and to generate assemblies with similar methodology, we assembled these transcriptomes in a similar way (CLC) as for P. ulmi (Additional files 2 and 3). P. citri assembly statistics were in line with those of P. ulmi, with 25,529 contigs and an average contig size/N50 of 708 bp/1057 bp for the Liu et al.  P. citri transcriptome and 32,362 contigs and an average contig size/N50 of 646 bp/969 bp for the Niu et al.  P. citri transcriptome.
Homology searches, species distribution and Gene Ontology (GO) analysis
A total of 9190 sequences (33.1 % of all contigs) had a BLAST hit in the NCBI non-redundant (nr) database, with 9109 sequences having a BLASTx hit against the nr protein database and 81 sequences having a BLASTn hit against the nr nucleotide database. As T. urticae gene annotations have not yet been uploaded to the NCBI database, we also performed a local BLASTx search against the T. urticae proteome. We found that 11,250 P. ulmi contigs showed a BLASTx hit with the predicted T. urticae proteome, including 2483 that had no BLASTx-hit in the NCBI nr database. In total, 11,673 P. ulmi contigs (42,0 % of all contigs) had a BLAST-hit in at least one analysis (Additional file 4). Although 42.0 % of contigs with a BLAST-hit may appear to be a relatively small number, we found that this value does not significantly differ from other de novo transcriptome studies with non-model arthropod species (e.g. in [40, 41]). In the Niu et al. transcriptome study  of the related P. citri the percentage of contigs was also of similar magnitude (50.7 %). Such a low percentage can be caused by multiple reasons. The majority of de novo transcriptomes contains a considerable amount of short contigs (<240 bp), containing incomplete (<80 AA) ORFs that in many cases will not BLAST at a given E-value threshold. On the other hand some contigs may be non-coding RNAs which do not BLAST with the non-redundant protein/nucleotide database (or just UTR fragments of otherwise coding genes).
Out of the contigs having a BLAST-hit, 94.1 % (10,989 contigs) of the top BLAST hits belonged to metazoan species, 2.9 % to Bacteria (342 contigs), 1.6 % (181 contigs) to Plants, 0.6 % (75 contigs) to Fungi and 0.7 % (86 contigs) to other organisms. The highest homologies for the majority of the metazoan BLAST hits belonged to the Chelicerata (55.7 %, 6498 contigs) and Arthropoda (20.6 %, 2404 contigs) (Additional file 5). A detailed analysis of all chelicerate BLAST hits revealed a high number of top BLAST hits with T. urticae (21.9 %, 2553 contigs), followed by the social velvet spider Stegodyphus mimosarum (17.8 %, 2080 contigs), the tick Ixodes scapularis (8.5 %, 987 contigs) and the predatory mite Metaseiulus occidentalis (4.7 %, 545 contigs).
Analysis of Panonychus genes involved in xenobiotic resistance
In general, arthropods have developed two types of mechanisms to cope with xenobiotic compounds, both of which can contribute to the development of resistance: mechanisms that decrease exposure due to quantitative or qualitative changes in major detoxification enzymes and transporters (pharmacokinetic mechanisms) and mechanisms that decrease sensitivity due to changes in target site sensitivity caused by point mutations (pharmacodynamic mechanisms) [2, 42, 43]. The pharmacokinetic mechanism can be subdivided into three phases (I-III). In phase I detoxification enzymes such as cytochrome P450 monooxygenases (CYPs) and carboxylcholinesterases (CCEs) incorporate a nucleophilic functional group (a hydroxyl, carboxyl or amine group) into the toxic compound, resulting in a more reactive and water soluble compound. During phase II, enzymes such as UDP-glycosyltransferases (UGTs) and glutathione-S-transferases (GSTs) further increase the water solubility of the phase I metabolite by conjugation with endogenous molecules like sugars and glutathione, respectively. In Phase III, conjugates are transported out of the cell by cellular transporters, e.g., ABC transporters (ABCs).
In this study we mined the P. ulmi and P. citri transcriptomes for genes encoding known target-sites and major detoxification enzymes and transporters (CYPs, CCEs, UGTs, GSTs and ABCs) and compared them with those of T. urticae, for which a high-quality genomic assembly and annotation is available . In addition, we also performed a phylogenetic analysis for all major detoxification gene families. It should be noted, however, that as transcriptomic data for Panonychus sp. is compared to genomic data for T. urticae, care should be taken when comparing numbers and ortho/homologues of Panonychus detoxification genes as recent gene duplications and genes with very low expression levels might be missed. In addition, expression of detoxification genes might also be stage-dependent. Hence, gene number differences between both Panonychus species should also be carefully interpreted as both P. citri transcriptomes were assembled using RNA-seq data from mixed stages [33, 34] while the P. ulmi transcriptome was based solely on RNA-seq data of adult females (see Methods).
Cytochrome P450 monooxygenases
Comparison of the CYP gene number in T. urticae, P. ulmi and P. citri
P. ulmi a
P. citri a
In vertebrates, mitochondrial CYPs (clan M) are associated with steroid or vitamin D metabolism while in insects this clan comprises two groups: one group involved in the ecdysteroid metabolism pathway and another group that metabolize a variety of xenobiotic compounds . We identified 9 P. ulmi and 11 P. citri ORFs in this clan and detected three clear cases of 1:1:1 orthology between P. ulmi, P. citri and T. urticae: CYP314A1, CYP315A1 and CYP302A1 (Additional file 7, Additional file 8, Table 1). Together with T. urticae CYP307A1 of the CYP2 clan (see below), these CYPs are the only T. urticae CYPs that could be assigned as clear orthologs of insect and crustacean CYPs. They are known to be involved in the ecdysteroid pathway in insects and their tetranychid counterparts likely fulfill an analogous role . Similar to T. urticae, orthologs of insect CYP2 clan members CYP306A1 and CYP18A1 could also not be identified for both Panonychus species (Additional file 7, Fig. 2). In insects, CYP306A1s are involved in the synthesis of 20 hydroxyecdysone (20E) by hydroxylating carbon 25, while CYP18A1 is a hydroxylase/oxidase involved in ecdysteroid degradation . Their absence in Panonychus sp. further corroborates that this gene cluster was lost as a whole and is an ancient trait, leading to the use of ponasterone A as the moulting hormone instead of 20E.
In insects, the number of CYP2 genes is relatively well conserved and several insect CYP2 genes are known to be involved in essential physiological functions. Compared to insects the number of CYP2 genes is more diverse in T. urticae and the cladoceran Daphnia sp. [21, 44] and at present the 392 family within the CYP2 clan is best characterized in T. urticae. A whole genome microarray gene expression analysis revealed that several members of the CYP2 clan were upregulated in acaricide resistant T. urticae strains or in a T. urticae strain adapted to a new challenging host plant [20, 36]. Furthermore, functional expression of two T. urticae CYP2 clan enzymes, CYP392A16 and CYP392E10, confirmed that these could metabolise the acaricides spirodiclofen and abamectin, respectively [20, 45]. We identified 16 and 31 CYP2 ORFs in P. ulmi and P. citri respectively. In our phylogenetic analysis, there are two clear cases of 1:1:1 orthology within the CYP2 clan (T. urticae CYP307A1, and CYP392A5). The phylogenetic analysis also suggests that the potential expansion/loss events of certain CYP2 subfamilies, like CYP392A, CYP392D and CYP392E, occurred after the split between the Tetranychus and Panonychus genus. Consequently, it was not surprising that we could not identify a clear P. ulmi/P. citri ortholog of T. urticae CYP392E10 and CYP392A16, suggesting that spirodiclofen or abamectin metabolism must depend on different P450s or other factors in Panonychus species (see differential expression analysis).
Insect CYP3 and CYP4 clans comprise the insect specific families CYP4, CYP6, CYP9 and CYP325 that are well known for their involvement in environmental response/detoxification functions against xenobiotics and phytotoxins . We identified 8 P. ulmi and 26 P. citri ORFs in the CYP3 clan, while 30 P. ulmi and 50 P. citri ORFs were found in the CYP4 clan. Within the CYP3 and CYP4 clan we found 5 (T. urticae CYP382A1, CYP384A1, CYP385A1, CYP385B1, CYP385C1) and 3 (T. urticae CYP407A1, CYP391A1, and CYP4CL1) clear cases of 1:1:1 orthology between T. urticae, P. ulmi and P. citri, respectively. Within the CYP4 clan, the CYP389A subfamily consists of a single gene in T. urticae while 4 paralogs are present in each, P. ulmi and P. citri. In contrast, the CYP389C subfamily appears to be larger in T. urticae compared to the two Panonychus species. Interestingly, Ran et al.  showed that P. citri paralogs of T. urticae CYP389A1 were among the most up- and down regulated CYP genes in an amitraz resistant strain. Ding et al. , on the other hand, showed that a P. citri ortholog of T. urticae CYP4CF2 was highly induced upon exposure to pyridaben while the expression of a P. citri ortholog of T. urticae CYP4CL1 increased after induction by abamectin, azocyclotin, pyridaben, and spirodiclofen. In contrast to the CYP2 clan, none of these tetranychid CYP3 and CYP4 clan members have been functionally characterized.
Carboxylcholinesterases (CCEs) catalyse the hydrolysis of carboxylesters and have a whole gamut of physiological functions including degradation of neurotransmitters, metabolizing hormones and pheromones, regulation of behaviour and detoxification of xenobiotics [48, 49]. CCEs are phase I detoxification enzymes and their role in xenobiotic metabolism and the development of resistance in arthropods has been thoroughly studied (recently reviewed in ). A classification based on the CCE phylogeny, as proposed by  groups insect CCEs into 13 clades spread over 3 classes: the dietary/detoxification enzymes (clades A–C), the generally secreted enzymes (hormone/semiochemical processing class, clades D–G) and the neuro/developmental CCEs (clades I–M, mainly non-catalytic esterases) [48, 49]. While total numbers of CCEs in insects and T. urticae are similar, the neuro/developmental class is expanded in T. urticae with two new subclades J’ and J”. In addition, in contrast to insects no T. urticae CCEs have been identified within the currently recognized classification of insect dietary CCEs .
Comparison of the CCE gene number in T. urticae, P. ulmi, P. citri and D. melanogaster
P. ulmi a
P. citri a
Dietary class (Clade A, B, C)
Undetermined clades (NC)
Glutathione-S-transferases (GSTs) belong to a multifunctional enzyme family, known to play an important role in phase II of xenobiotic detoxification. In addition to pesticide detoxification by conjugation, insect GSTs have also been reported to play a role in attenuation of oxidative stress caused by pesticide exposure and sporadically by metabolism of pesticides directly . Insects cytosolic GSTs can be divided into seven classes [delta (δ), epsilon (ε), omega (ω), sigma (σ), theta (θ) and zeta (ζ)] with delta and epsilon GSTs well known for their role in detoxification of organophosphates and organochlorines [42, 52]. In contrast to insects, Acari also have mu-class GSTs, which were until recently thought to be verterbrate specific [21, 53].
Comparison of the cytosolic GST gene number in T. urticae, P. ulmi, P. citri and I. scapularis
T. urticae a
P. ulmi b
P. citri b
I. scapularis c
Using a whole genome gene-expression microarray, members of the same GST classes were also shown to be highly upregulated in multi-resistant T. urticae strains . Three GST genes (TuGSTd10, TuGSTd14, TuGSTm09) that were upregulated in a strain MAR-AB highly resistant against abamectin and other important acaricides were also functionally expressed. Out of these, TuGSTd14 showed the highest affinity toward abamectin and had a competitive type of inhibition, suggesting the acaricide may bind to the H-site of the enzyme . However, as mentioned earlier no clear Panonychus orthologs of TuGSTd14 could be identified in our study.
The ATP Binding Cassettte (ABC) protein family is a large and ubiquitous family of proteins. Most members of this family use ATP to transport substrates across lipid membranes, and hence are referred to as ABC transporters. Based on the sequence similarity of their nucleotide binding domain (NBD, the domain that binds ATP) the ABC protein family can be divided into eight subfamilies (A to H). Although extensively characterized for their involvement in drug resistance in vertebrates, the role of ABC transporters in arthropod xenobiotic resistance is less well known. Nevertheless, ABC transporters have been associated with resistance to as many as 27 insecticides/acaricides from nine different chemical classes . However, the major source of evidence linking these arthropod ABC transporters to resistance has been ABC transporter expression quantification or synergism studies . A total of 103 ABC genes were identified in the T. urticae genome, the highest number discovered in a metazoan species to date. This high number of ABC genes in T. urticae is due predominantly to lineage-specific expansions of the ABCC, ABCG and ABCH subfamilies .
Comparison of the number of P. ulmi, P. citri and T. urticae ABC protein NBDs
Horizontally transferred genes
Comparison of the UGT gene number in T. urticae, P. ulmi, P. citri and I. scapularis
P. ulmi a
P. citri a
Putative horizontally transferred genes identified in the T. urticae genome and Panonychus spp. transcriptomes
Cysteine synthase A2
P. ulmi a
P. citri a
UDP-glycosyltransferases (UGTs) are common in the majority of living organisms including viruses, bacteria, plants and animals. They catalyze the conjugation of small lipophilic molecules with uridine diphosphate (UDP) sugars, increasing their water solubility. As such, UGTs play an important role in the synthesis, storage and transport of secondary metabolites. In vertebrates, UGTs are also well studied because of their role in phase II drug metabolism [39, 58]. Although a role for UGTs in arthropod xenobiotic resistance was suggested more than twenty years ago , only recently has functional evidence for their role in xenobiotic resistance been presented [60–63]. It has been suggested that the UGT gene family might have been lost early in the Chelicerata lineage and subsequently re-gained in the tetranychid mites by means of the horizontal gene transfer from bacteria. This discovery provides important clues to UGTs functions in relation to detoxification and therefore host adaptation in the phytophagous mites.
Intradiol ring-cleavage dioxygenases (IDRCDs)
Other horizontal gene transfers
Besides UGTs and IDRCDs we also identified Panonychus orthologs of T. urticae carotenoid desaturases, carotenoid cyclases, levanases, cyanate lyase, β-cyanoalanine synthase and methionine synthase (Table 6, Additional file 12). Wybouw et al.  showed that a T. urticae strain overexpresses β-cyanoalanine synthase when adapted to cyanogenic plants. Functional expression also revealed that this enzyme is able to detoxify cyanide which is the main defensive phytochemical of these plants. In contrast to T. urticae, Panonychus sp. almost exclusively feed on Rosaceae , a cyanogenic plant family producing cyanide in various tissues as plant defense . As such, β-cyanoalanine synthase might have been crucial in conferring resistance in Panonychus sp. to this continuous exposure to dietary cyanide.
Target-sites of insecticides/acaricides
Known target sites of acaricides, their T. urticae gene ID and presence in P. ulmi and the IRAC acaricide classes that target these proteins
T. urticae a
P. ulmi b
3 (Sodium channel modulators)
1 (AChE inh.)
23 (Accase inh.)
20 (Mt Complex III inh.)
10 (Mite growth inh.)
6 (Cl channel activators)
Compared to insects T. urticae has a higher number of Rdl and GluCl genes, with the majority of insects having only one Rdl and GluCl gene while T. urticae has 3 and 6, respectively . Similar to T. urticae we identified 5 P. ulmi GluCl and 3 Rdl genes (Additional file 14), suggesting that the Rdl and GluCl gene family diversified before the radiation of the Tetranychidae. Next, we compared all known target-site sequences of the spirodiclofen susceptible (HS) and resistant (PSR-TK) P. ulmi strains to identify non-synonymous single nucleotide polymorphisms (SNPs) that are unique for the resistant strain. For AChE, we identified the F331W substitution (Torpedo californica numbering) that was fixed in the PSR-TK strain but segregating in the HS strain. In the past, it has been shown that a substitution at this position causes resistance to organophosphate (OP) and carbamate (CB) insecticides/acaricides  and the presence of this substitution in both P. ulmi strains emphasizes the scope of selection exerted by OPs and CBs during the second half of the 20th century.
Comparing ACCase sequences of both strains revealed several non-synonymous fixed SNPs in the PSR-TK strain that were not present in the susceptible HS-strain. At present, only one ACCase substitution has been associated with resistance against cyclic keto-enols: an E645K substitution in the whitefly Trialeurodes vaporariorum . However, the residue is not conserved in mites and at the corresponding position in the P. ulmi ACCase protein, the sequence is identical in both strains (a glutamine; at position 566, T. urticae numbering tetur21g02170). Recently, it has been shown that the activated enol derivative of the insecticide spirotetramate interacts with the carboxyltransferase domain of ACCase , suggesting this region as a prime site to expect resistance mutations. Within this domain region, we also found several residues that were different between the ACCase protein sequence of the PSR-TK and the HS-strain (Additional file 14, Additional file 15). However, it is unclear whether these substitutions play a role in resistance. Similarly to ACCase, other target site sequences (VGSC, cytB, GluCl, Rdl and CHS1) contained non-synonymous SNPs in the PSR-TK strain that were not present in the HS strain, but none were located at positions previously reported to be involved in acaricide resistance .
Differential expression analysis between an acaricide resistant (PSR-TK) and susceptible strain (HS) of P. ulmi using RNAseq data
Top 20 of upregulated contigs in the acaricide resistant P. ulmi PSR-TK strain relative to the susceptible HS strain. See Additional file 17 for a full list of differentially expressed contigs
T. urticae BLASTx hit (E-value)b
T. urticae gene name
cytochrome P450 monooxygenase (CYP385C1)
farnesoic acid o-methyl transferase-like protein
farnesoic acid O-methyltransferase-like
collagen alpha-4 chain
adenylyl cyclase associated protein
adenylate cyclase-associated CAP
RNA polymerase 1
RNA-directed RNA polymerase 1
glucosylceramidase isoform 2
intermediate in toll signal transduction
evolutionarily conserved signaling intermediate in Toll pathway
chimeric r1 r2 retrotransposon
similar to gag-like protein
U6 snRNA phosphodiesterase isoform X1 putative
similar to leucine-rich transmembrane protein
RNA polymerase 2-like
RNA-directed RNA polymerase 1
Top 20 of downpregulated contigs in the acaricide resistant P. ulmi PSR-TK strain relative to the susceptible HS strain. See Additional file 17 for a full list of differentially expressed contigs
T. urticae BLASTx-hit (E-value)b
T. urticae gene name
4Fe-4S ferredoxin, iron-sulpur binding domain
krab-a domain-containing protein
zinc finger protein
chemosensory gustatory receptor family
gustatory receptor family
histone-lysine n-methyltransferase setmar-like
transposable element Tc3-related transposase
collagen alpha-4 chain
farnesoic acid o methyltransferase-like protein
farnesoic acid O-methyltransferase-like protein
reverse transcriptase maturase
proteinase inhibitor I19, pacifastin
esterase type b
retrovirus-related pol polyprotein from transposon partial
hypothetical cell surface protein
adenylyl cyclase-associated protein
adenylate cyclase-associated CAP
Acetyl - partial
hypothetical protein TcasGA2_TC016169
similar to gag-like protein
expressed sequence ai451617
zinc finger protein
glycoside hydrolase, catalytic core
paired box and transposase domain
transposable element Tc3-related transposase
Several up- and downregulated contigs showed high homology with sequences of arthropod bisegmented double stranded RNA viruses (biRNA viruses) (Tables 8 and 9, Additional file 17). There are several reports documenting viruses infecting Panonychus species [74–77]. As such, differential expression of biRNA viruses between the two resistant strains might reflect the different virus composition and infection status.
Among the differentially expressed contigs, 29 coded for major detoxification enzyme families or potential new players in detoxification (see above and ). Among the upregulated contigs, we identified 4 CCEs, 3 CYPs, 3 GSTs, 3 ABCs, 4 UGTs, 2 lipocalins and 2 Major Facilitator Superfamily genes while 1 CYP, 2 CCEs, 1 GST, 2 ABCs and 2 IDRCDs were downregulated (Fig. 8, Additional file 17). Such a broad response in detoxifying enzyme families is typically also encountered in the spider mite T. urticae, where numerous gene-expression studies of susceptible and resistant strains have shown an overlap in the gene-families involved [20, 36, 78], but also specificity in term of the members of these families.
Previously, it was shown that the P450 inhibitor PBO and the esterase inhibitor DEF could enhance the toxicity of spirodiclofen in a highly spirodiclofen resistant T. urticae strain  and in a spirodiclofen resistant P. citri strain . Similarly, Kramer and Nauen  showed that PBO enhanced the toxicity of spirodiclofen in PSR-TK P. ulmi strain, suggesting the involvement of CYPs in spirodiclofen resistance in both tetranychid species. The upregulation of a CYP (CYP392E10, tetur27g01030) and a CCE (TuCCE-04, tetur01g10750) was also strongly associated with spirodiclofen resistance in two unrelated T. urticae strains (SR-VP and SR-TK). Furthermore, functional expression confirmed that CYP392E10 metabolizes spirodiclofen . Remarkably, the upregulated P. ulmi CCE and CYP in the PSR-TK strain cluster in different clades in our phylogenetic analyses of tetranychid CCEs and CYPs than those that are upregulated in the spirodiclofen-resistant T. urticae strains (Figs. 2 and 3). Thus, within the Tetranychidae, two different species are able to develop a similar resistance mechanism to spirodiclofen, but the enzymes recruited for this purpose do not need to have followed the same evolutionary path. This independent development of resistance in related species is further reinforced by the fact that only few of the differentially expressed contigs in the PSR-TK P. ulmi strain have a BLASTx hit with amino acid translations of differentially expressed genes in two spirodiclofen resistant T. urticae strains (SR-VP and SR-TK, ) (see Additional file 18). Overall, the analysis has pointed to a number of genes and enzymes that might be causal for the resistant phenotype, and future work including functional expression should confirm that the enzymes can metabolize or sequester spirodiclofen leading to resistance, as was recently undertaken for the model species T. urticae .
Using paired-end strand specific reads from RNA of spirodiclofen susceptible and resistant P. ulmi strains we generated a P. ulmi transcriptome dataset containing 27,777 contigs. As such, our transcriptome data represents a significant increase in the genomic resources that are available for this species. Phylogenetic analyses revealed that in the majority of cases detoxification gene (CCEs, CYPs, UGTs, ABCs) classes/clades/subfamilies are more numerate in T. urticae compared to P. ulmi, suggestive of a link between detoxification gene proliferation and the polyphagous nature of T. urticae. Specific radiations of subfamilies in P. ulmi were also observed. Annotation of all major target-sites in P. ulmi revealed the presence of mutations in AchE that are likely to confer carbamate and organophosphate resistance. Finally, we used the replicated RNAseq data to assess differences in gene expression between a spirodiclofen resistant and susceptible P. ulmi strain and found that a CYP and CCEs are likely to be associated with spirodiclofen resistance in P. ulmi. These results are in line with a previous report on molecular mechanisms of spirodiclofen resistance in T. urticae. However, the upregulated CYP and CCE in the resistant strains from both species seem not to have evolved from the same common ancestor, indicating both species developed spirodiclofen resistance in a similar but nevertheless independent evolutionary manner. To conclude, the new genomic resources and data that we present in this study for P. ulmi will substantially facilitate molecular studies of underlying mechanisms involved in acaricide resistance.
Mites from the three strains (HS, PSR-TK and Ge 16/09) used in this study were all identified morphologically as P. ulmi by Johan Witters (ILVO, Belgium) but also on the basis of cytochrome b (cytB) sequence similarity (cytB sequences of the HS and PSR-TK P. ulmi strain (Additional file 14) had their best BLASTx hit (E-value < 1E−100) with P. ulmi cytochrome b (YP_002808558.1) in the NCBI database. The HS strain was originally collected in 1990 from an apple orchard in Burscheid, Germany and is susceptible to most currently used acaricides, including spirodiclofen . The Ge 16/09 is a field strain, collected in 2009 from an orchard in Heidenfahrt-Heidesheim, Germany, and characterised by high resistance levels to mite growth inhibitors hexythiazox and clofentezine (extrapolated resistance ratios in comparison with the susceptible HS strain were 20,000 and 3900 respectively) and moderate resistant to spirodiclofen with a reported spirodiclofen resistance ratio of 59 compared to the HS strain. The PSR-TK strain is a spirodiclofen-selected laboratory strain derived from Ge 16/09 and with a resistance ratio of more than 7000 compared to the HS strain . All mite strains were reared under identical conditions in climatically controlled chambers at 24 +/− 1 °C, 60 % relative humidity and a 16:8 light:dark period for more than 10 generations on domestic plum trees (Prunus domestica L. var. Brompton). The PSR-TK strain was maintained under constant selection pressure by rearing on plum trees sprayed with 1000 mg a.i./L spirodiclofen until runoff.
RNA extraction, library construction, sequencing and assembly of the P. ulmi transcriptome
Total RNA was extracted from 200 1–3 day old adult female P. ulmi mites from the PSR-TK and HS strains using the RNEasy mini kit (Qiagen, Belgium) with four-fold biological replication (i.e., four replicates each for PSR-TK and HS). Each RNA sample was processed for sequencing by the sequencing service company Fasteris SA (Switzerland) according to the “HiSeq Service Stranded Standard Protocol”. The resulting library was sequenced by Fasteris SA (Switzerland) using the HiSeq 2000 Illumina technology generating strand-specific paired-reads of 2x100 bp. Adapter sequences were removed from the obtained 2x 100 bp reads by Fasteris SA and adapter trimmed reads were used for further analysis. Previously it was shown that a representative transcriptome assembly can be generated from a sub-sample of reads . Hence, to reduce computation time, a subset (the first 12 million paired-reads/sample, 48 million reads/strain, 96 million paired-reads in total) of the total number of reads was used for P. ulmi transcriptome assembly using the CLC Genomics Workbench (CLC) software version 6.5.1 and default settings (mapping mode = fast, automatic bubble size = yes, minimum contig length = 200 nt, automatic word size = yes, perform scaffolding = yes, auto-detect paired distances = yes). Using the same subset of reads we also used an alternative approach to assemble the P. ulmi transcriptome. Reads were first trimmed using Sickle  with quality-cutoff set at 30 and length cutoff set at 90. Trimmed reads were assembled using the Velvet/Oases package  and with the following settings for Oases: −-kmin = 59 --kmax = 61 --kstep = 2 --merge = 61 –d “-shortPaired -strand_specific” -p “ins_length = 200”. Transcripts with maximum sequence length (30,044 transcripts) were filtered from the resulting Velvet/Oases assembly (44,903 transcripts) and used for further analysis. To compare the quality of both assemblies all RNA-seq reads (4 replicates/strain) were mapped using Bowtie version 2.1.0  against either the CLC or Velvet/Oases assembly (see Differential expression analysis section below for mapping procedures).
Raw reads have been submitted to the NCBI Short Read Archive (SRA; experiment accession number SRX833872 (HS) and SRX833917 (PSR-TK)). The CLC assembly was submitted to the NCBI Transcriptome Shotgun Assembly (TSA) Sequence Database and deposited at DDBJ/EMBL/GenBank under the accession GCAC00000000. The version described in this paper is the first version, GCAC01000000. P. ulmi contigs containing more than 10 % of unassigned nucleotides (N) or more than 14 Ns in a row (1630 sequences) and contigs that were shorter than 200 nt after removal of vector contamination sequences (2 contigs) were not uploaded to the TSA Database following database submission procedures. After uploading, an NCBI contamination screen indicated that 227 out of the 26,145 uploaded sequences should be excluded as they showed very high identity with either rRNA of Bacteria, genes of Prunus sp. (host plant of P. ulmi strains) or Wolbachia sp. sequences. These sequences were removed from the TSA assembly and, finally, 25,918 contigs were uploaded. The complete collection of the CLC assembly (27,777 contigs) was added to this manuscript in Additional file 19. The (alternative) Velvet/Oases P. ulmi assembly was added as Additional file 13 to this manuscript.
Blast homology searches and sequence annotation
All P. ulmi contigs (27,777) were used for BLASTx searches against the NCBI non-redundant (nr) protein database (version of September 3rd 2014; E-value cut-off of 1E−5) using Standalone BLAST 2.2.30+ . Sequences that did not yield BLASTx hits were subsequently searched against the non-redundant nucleotide database using BLASTn and with an e-value cut-off of 1E−5. As T. urticae is not yet included in the NCBI nr protein database, P. ulmi contigs were also used as query in BLASTx against the T. urticae proteome (version March 2014, http://bioinformatics.psb.ugent.be/orcae/overview/Tetur). All BLAST results were imported in Blast2GO (version 2.8) to assign gene ontology (GO) terms to sequences retrieved by BLAST search . Further annotation was done with InterPro, where protein motifs were directly queried at the InterProScan web service and consequently merged to the existing annotation . The annotation results were further fine-tuned with (1) the Annex function in order to augment the annotation through the inference of biological process and cellular component terms from molecular function annotations  and with (2) the generic GO slim reduction to summarize the functional information of the transcriptome dataset. Finally, to enable better visualisation of the results, Gene Ontology relationships and enzyme codes (ECs) were highlighted on the Kyoto Encyclopedia of Genes and Genomes (KEGG) maps.
Analysis of genes related to xenobiotic detoxification
Contigs encoding putative P. ulmi CYPs, CCEs, GSTs, UGTs and ABC proteins were retrieved from the P. ulmi transcriptome (CLC assembly) by a tBLASTn search and using protein sequences of T. urticae CYPs, CCEs, GSTs, UGTs and ABCs as query [21, 39, 57]. tBLASTn searches were performed with NCBI Standalone BLAST 2.2.30+  and a cut-off E-value < 1E−5. Open reading frames (ORFs) of P. ulmi contigs encoding detoxification enzymes and ABC proteins were identified using “EMBOSS 6.3.1 getorf” integrated in the Mobyle portal framework (http://mobyle.pasteur.fr/). P. ulmi ORFs were aligned against each other using BLASTn. In cases where two ORFs showed more than 94 % identity at the nucleotide level, they were considered as allelic variants and the longest ORF was retained for phylogenetic analysis. ORFs with identical overlapping sequences were considered to be part of the same gene and merged using BioEdit version , except if the merged ORF misaligned when blasted to the T. urticae protein database.
Two previously published P. citri transcriptomes were also mined for transcripts encoding detoxification enzymes and ABC transporters. For both published P. citri transcriptomes, the cDNA library was constructed using a mixture of RNA extracted from different developmental stages and subsequently sequenced using Illumina HiSeq 2000 technology with a read-length of 90 bp [33, 34]. However, as the P. citri transcriptome sequence assemblies (TSAs) have not been made publicly available, we assembled these transcriptomes de novo using a similar approach as for P. ulmi and using all reads in the sequence read archives (SRAs) ERR044692-ERR044695 and SRR341928 provided by the studies of Liu et al.  and Niu et al. , respectively. P. citri ORFs encoding CYPs, CCEs, GSTs, UGTs, and ABC proteins were identified using a similar approach as for P. ulmi. Likewise, these P. citri ORFs were aligned against each other using BLASTn. In case two P. citri contigs showed at least 94 % identity they were considered as allelic variant and the longest ORF was retained for phylogenetic analysis.
A final selection of Panonychus ORFs encoding CYPs, GSTs UGTs and ABCs was translated into protein sequences and aligned with their homologues in T. urticae using MUSCLE version 3.8.31 . Alignments were inspected by eye and for each alignment only protein sequences showing no misalignment and having a minimum ORF length (CYPs: 450 nt, CCEs: 450 nt, GSTs: 300 nt, UGTs: 450 nt) were retained for the final alignment. Two phylogenetic analyses were performed for the CYP gene family: one with all tetranychid CYPs and one restricted to tetranychid and D. melanogaster mitochondrial CYPs (see ). Panonychus mt CYPs were identified based on the phylogenetic analysis of all tetranychid CYPs. For the phylogenetic analysis of CCEs, a reference set of arthropod CCE protein sequences was also included in the alignment (Additional file 9). As N- and C termini of CCEs are highly variable, the alignment of CCE protein sequences was trimmed as previously described . Only the nucleotide binding domain (NBD) of ABC proteins encoded by Panonychus ORFs was used for phylogenetic analysis of ABC proteins. NBDs were extracted using ScanProsite (http://prosite.expasy.org/scanprosite/) and the PROSITE profile PS5089. Next to T. urticae ABC transporter NBDs, C- and N terminal NBDs of D. melanogaster ABC transporters were also included in phylogenetic analysis of tetranychid ABC transporter NBDs . Model selection was done with ProtTest 2.4 and according to the Akaike information criterion WAG + G + F, LG + I + G + F, LG + I + G + F, LG + I + G, WAG + G + F and LG + G + F were optimal for the phylogenetic reconstruction of CYPs, mitochondrial CYPs, UGTs, GSTs, CCEs and ABC proteins, respectively. Finally, for each alignment a maximum likelihood analysis was performed using Treefinder (version 2011)  bootstrapping with 1000 pseudoreplicates (LR-ELW). The resulting trees were midpoint rooted and edited with MEGA 6.0 software .
Annotation of horizontally transferred genes (excluding UGTs)
Annotation of P. ulmi and P. citri contigs encoding horizontally transferred genes (HTGs) was done in a similar way as for genes involved in xenobiotic detoxification. Model selection and phylogenetic analysis of IDRCD genes was done in a similar way as for genes involved in xenobiotic detoxification. According to the Akaike information criterion WAG + I + G + F was optimal for phylogenetic reconstruction of tetranychid IDRCDs.
Identification of P. ulmi target-site sequences
To obtain P. ulmi target site sequences as full length as possible we mined both P. ulmi transcriptomes (CLC and Velvet/Oases, see above) for contigs encoding target-sites of acaricides using tBLASTn (E-value cutoff < 1E−5) and T. urticae target-site protein sequences as query. P. ulmi contigs encoding target-sites (from both transcriptomes (CLC and Velvet/Oases) were aligned against each other and those contigs having identical overlapping regions were merged. The resulting contigs were manually assembled to obtain P. ulmi target-site sequences as full length as possible. All reads were then mapped against the P. ulmi target site sequences as for the differential expression analysis (see below) with this difference that resulting BAM files were merged for each strain using the SAMtools package . For both the spirodiclofen susceptible and the resistant P. ulmi strain, a consensus sequence was then derived using a perl script (gene_extractor.pl) written by Rutger Vos and available at https://github.com/naturalis/fastq-simple-tools/tree/master/script. The obtained P. ulmi target site sequences were screened for all target site mutations previously described in the literature .
Differential expression analysis
To assess differential gene expression between the HS and PSR-TK strains, we first took the reverse complement of each P.ulmi contig where the majority of its BLASTx hits mapped on the reverse strand. P. ulmi contigs were subsequently concatenated into a single sequence with spacers (“Ns”) of length 100 added between contigs using a custom python script. For each RNA-seq replicate (4 per strain, 8 in total), reads were mapped against this concatenated sequence using Bowtie version 2.1.0  with the preset option “--very-sensitive-local” and with the maximum fragment length for valid paired-end alignments set to 1000 (−X 1000). Mapped reads were counted using htseq-count that is included in the HTSeq package . Forward strand reads were counted using the “--stranded = reverse” option while the “--stranded = yes” option was used to count reverse strand reads. Those P. ulmi contigs of which the proportion between the number of reads mapping to one strand and the total number of reads mapping to either the forward or the reverse strand was more than or equal to 0.95 were considered to have strand-specific reads (20,069 contigs, 72,3 % of all contigs). Read counts of P. ulmi contigs with (1) a BLAST hit, (2) strand-specific reads and (3) not considered contamination (see above) were used for differential expression analysis. Differentially expressed contigs between the resistant (PSR-TK) and susceptible (HS) P. ulmi strain were determined using the DESeq2 (version 1.6.3;) and Bioconductor (http://bioconductor.org/) R-packages. The “unfiltered DESeq2 results” settings (dds < − DESeq(dds, minReplicatesForReplace = Inf) and res < − results(dds, cooksCutoff = FALSE, independent Filtering = FALSE)) were used for differential expression analysis. P. ulmi contigs with a fold change higher than two and a Benjamini-Hochberg adjusted p-value less than or equal to 0.05 were considered differentially expressed.
A set of eight differentially expressed contigs were evaluated by RT-qPCR. This set included 3 down- and 5 upregulated P. ulmi contigs. P. ulmi contig_01010 and contig_00741 showed best BLASTx hits with T. urticae RP49 (tetur18g03590) and ubiquitin (tetur06g00900), E-value of 4E−70 and 5E−59, respectively, and were used as reference genes for RT-qPCR. Primers were designed using Primer3 v.0.4.0  and are listed in Additional file 20. RNA was extracted in triplicate from each strain as described above and 2 μg was reverse transcribed using the Maxima First Strand cDNA synthesis kit for RT-qPCR (Fermentas Life Sciences). All qPCR reactions were conducted with a thermal cycler Mx3005P (Stratagene). Reactions were prepared with Maxima SYBR Green qPCR/Master Mix following the manufacturer’s instructions (Fermentas Life Sciences) and run in two technical and three biological replicates. Non-template-controls were also included to eliminate potential contamination of the samples. The qPCR run protocol was as follows: initial denaturation at 95 °C for 10s, 35 cycles of 95 °C for 15 s, 55 °C for 30s, 72 °C for 30s. To exclude the possibility of nonspecific amplification, each PCR was followed by a melting curve step (ramping from 95 to 55 °C by 1 °C every 2 s) to confirm a single amplicon. Amplification efficiency of each primer pair was calculated from the standard curve, prepared of cDNA of all samples tested, with a dilution range from 0.4 to 50 ng RNA. The Ct values for P. ulmi contig_01010 (RP49) and contig_00741 (ubiquitin) were used for normalization. Significant differences in the relative expression values of the target genes were tested with pairwise fixed reallocation randomization [94, 95].
WD is a postdoctoral fellow of the Fund for Scientific Research Flanders. This work was partially supported by the Research Foundation Flanders (FWO) (grant G.0093.12 N to LT and TVL). RG was supported by National Institutes of Health Genetics Training Grant T32 GM07464. We thank Jan van Arkel for providing a photograph of an adult female of P. ulmi. We thank Johan Witters for morphological identification of P. ulmi species used in this study.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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