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Gene silencing by RNA interference in the house dust mite, Dermatophagoides pteronyssinus
Molecular and Cellular Probes xxx (2015) 1e5
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Short communication
Gene silencing by RNA interference in the house dust mite, Dermatophagoides pteronyssinus Edward J. Marr a, b, Neil D. Sargison b, Alasdair J. Nisbet a, Stewart T.G. Burgess a, * a b
Division of Vaccines and Diagnostics, Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Edinburgh, Scotland, United Kingdom Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Roslin, Midlothian, Scotland, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 July 2015 Received in revised form 22 July 2015 Accepted 22 July 2015 Available online xxx
This is the first report of gene silencing by RNA interference (RNAi) in the European house dust mite, Dermatophagoides pteronyssinus, Trouessart, 1897. Using a non-invasive immersion method first developed for the honey bee mite, Varroa destructor, a significant reduction in the expression of D. pteronyssinus glutathione-S-transferase mu-class 1 enzyme (DpGST-mu1) was achieved following overnight immersion in double stranded RNA encoding DpGST-mu1. Although no detrimental phenotypic changes were observed following silencing, this technique can now be used to address fundamental physiological questions and assess the potential therapeutic benefit in silencing D. pteronyssinus target genes in selected domestic situations of high human-mite interface. © 2015 Elsevier Ltd. All rights reserved.
Keywords: House dust mites Dermatophagoides pteronyssinus RNA interference Glutathione-S-transferase Astigmatid Atopy
1. Introduction Dermatophagoides pteronyssinus, the model European house dust mite (HDM), is a free-living Psoroptidean of medical importance due to the potent allergenicity of a number of its faecal antigens and the impacts that these allergens have in relation to asthma and other allergic conditions in humans, including chronic allergic rhinitis [1e3]. In total, 24 allergens have been identified thus far from HDM species [1]. The inhalant allergens produced by HDMs have been estimated to account for 50% of allergic disease cases [1], and sensitisation to D. pteronyssinus allergens in asthmatic or allergic individuals has been estimated to be as high as 80e90% [4,5]. The experimental reduction of the presence of D. pteronyssinus in the homes of asthma sufferers by chemical means has been shown to result in a considerable decrease in symptoms associated with asthma over a trial period of 8 weeks [6]. Current clinical treatment for alleviating or minimising the impact of HDM allergy involves medication with preventative and alleviating chemotherapies as well as allergen-specific immunotherapy, although novel treatments are currently being sought. The group one allergen, Der p 1, has been the subject of considerable study,
* Corresponding author. E-mail address: [email protected] (S.T.G. Burgess).
given its role in triggering allergic asthma. Recently, this antigen has been considered for development of a DNA based vaccine for both asthma and allergic rhinitis, which might represent a promising new therapeutic alternative [7e10]. Der p 1 has also been assessed as the basis for a small-molecule-based therapy using reversible peptidase inhibitors, revealing another alternative means of treatment [11]. Currently, the control of the mites themselves involves a modification of the environment to reduce the available HDMsuitable habitat, or application of acaricides (e.g. synthetic pyrethroids or organophosphates) to the habitat. However, due to the high reproductive potential and large population numbers of mites involved, repeated treatment with acaricides has the potential to lead to the development of acaricide resistance; such resistance to the widely used pyrethroids has already been demonstrated in vitro, confirming this concern [12]. An additional, important consideration of applying regular prophylactic doses of pesticides is the potential emergence or exacerbation of existing resistance to pyrethroids in other domestic arthropod pests, such as the German cockroach, Blattella germanica [13]. Alternative strategies, such as regulating the ambient relative humidity, so that it is maintained at 35% for a minimum of 22 h daily [14], has been shown to impact on the population growth of Dermatophagoides farinae, although the practicalities and equipment required for implementing this
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Please cite this article in press as: E.J. Marr, et al., Gene silencing by RNA interference in the house dust mite, Dermatophagoides pteronyssinus, Molecular and Cellular Probes (2015), http://dx.doi.org/10.1016/j.mcp.2015.07.008
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E.J. Marr et al. / Molecular and Cellular Probes xxx (2015) 1e5
particular solution might be prohibitively expensive. RNA inference (RNAi) is a relatively conserved, ancient mechanism of cell defence against viruses, now widely used experimentally as a means of achieving specific gene silencing to elucidate or alter function [15,16]. A novel application of RNAi developed for combating colony collapse disorder in the honey bee, Apis mellifera, has conferred protection against Israeli acute paralysis virus (IAPV) using Remembee-IAPV, a food supplement containing IAPVspecific dsRNA [17], an approach with commercial potential as a means of controlling pathogens within the hive. This approach could pave the way for the development of RNAi-based control in other controlled environments against various pathogens, parasites or pests, such as HDMs. In addition, RNAi silencing has been demonstrated in a range of Acari, including the honey bee mite, Varroa destructor, and the two-spotted spider mite, Tetranychus urticae [18,19]. RNAi can be triggered by the introduction of double-stranded RNA (dsRNA) or small interfering RNA (siRNA) into an organism. The subsequent processing of the ds/siRNA and incorporation within the RNA-induced silencing complex (RISC) leads to the cleavage of gene-specific messenger RNA (mRNA) sharing homologous sequence to the ds/siRNA [20e25]. The phenotype resulting from reduced transcript levels following RNAi can suggest or indicate the function(s) of the target gene and its importance to the host organism and furthermore, might inform about its potential as a novel intervention target [26e28]. Until now, there has been no published account of RNAi in HDMs, possibly due to limitations imposed by their diminutive size (440 mm in length) [29]. However, the demonstration in V. destructor of a non-invasive immersion-based methodology for achieving RNAi has recently facilitated the ongoing development of this method for application to smaller mite species. The RNAi methodology developed in V. destructor focussed on dsRNA encoding VdGST-mu1, a mu class glutathione-Stransferase (GST) with orthology to the HDM Der p 8 panallergen [4,18,30]. GSTs are also known to play critical roles in detoxification and are widely implicated in the development of drug resistance [4]. Here we demonstrate successful silencing of DpGST-mu1 in D. pteronyssinus following development of a non-invasive RNAi methodology. 2. Material and methods 2.1. D. pteronyssinus culture and harvesting Dermatophagoides pteronyssinus mites, originally acquired from a culture maintained at the University of Vienna, Austria, were housed within vented 75 cm2 Corning Flasks (Corning, UK), fed on a mixture of high quality goldfish flake food (Aquarian, UK) and wheat germ (Neal's Yard, UK) (equal ratio w/w). The flasks were kept at room temperature (22e24 C) within a box containing saturated salt solution to maintain constant humidity. Mites were harvested from the cultures by flotation in saturated saline solution.
photographic images were captured using an Axiovert 25 CFL inverted fluorescent microscope (Zeiss, UK) with 10 Achrostigmat magnification lens (Zeiss, UK) using a D90 AF-5 DX NIKKOR digital camera with 18e105 mm f/3.5e5.6 G ED VR lens kit (Nikon, Japan). 2.3. dsRNA/heterogeneous siRNA preparation Double-stranded RNA (dsRNA) encoding 500 bp of D. pteronyssinus gluthathione-S-transferase mu class 1 (DpPoGST-mu1) (AY825939.1) or 319 bp Escherichia coli strain K-12 sub-strain MG1655 lacZ (NC_000913.3) was produced using the dsRNA production vector pL4440 from the Fire Lab C. elegans Vector Kit 1999(Addgene plasmid #1654); pL4440 containing the target gene sequence was produced by cloning the target gene into pL4440 using Sac1 and Sma1 restriction sites (Roche, UK); PCR cycling times were 95 C for 5 min, followed by 30 cycles of 95 C for 15 s, 57 C for 30 s, and 70 C for 60 s with final extension of 70 C for 60 s (primer sequences are available from the authors upon request). The vector pL4440 was dephosphorylated following restriction digestion with Thermo-Stable Alkaline Phosphatase (Promega, UK) prior to T4 DNA ligation (Promega, UK) to maximise ligation efficiency. Cloning was confirmed by sequencing (Eurofins Genomics, Germany) using the M13 uni (21) standard primer. The dsRNA was synthesised from linearised plasmid using T7 RiboMAX™ Express RNAi System (Promega, UK) according to the manufacturer's protocol. The quality of dsRNA was assessed using gel electrophoresis and the concentration determined using an ND-1000 Nanodrop spectrophotometer (Thermo Scientific, UK). Heterogenous siRNA (het_siRNA) was prepared for DpGST-mu1 and lacZ using the ShortCut® RNase III enzyme (New England Biolabs, UK) according to the manufacturer's protocol, resulting in a conversion of the 319e500 bp dsRNA constructs into a heterogeneous mixture of 18e25 bp siRNAs. The het_siRNAs were diluted to a working concentration of 2.5 mg/ml in physiological saline. 2.4. Liposome enclosure of dsRNA The effect of enclosing the dsRNA within liposomes was assessed on the basis that enhanced RNAi was demonstrated in the nematode Haemonchus contortus using this technique [31]; dsRNA was incubated with Lipofectamine® 2000 Reagent (Invitrogen, UK) for 10 min at room temperature at ration of 1 ml Lipofectamine to 10 ml dsRNA. 2.5. Non-invasive RNAi Twenty adult male D. pteronyssinus mites were immersed in the detached cap of a 1.5 ml micro-centrifuge tube (Axygen, USA) in 15 ml dsRNA encoding either DpGST-mu1 or lacZ, diluted in saline to a final concentration of 2.5 mg/ml, with 5 biological replicates per treatment group. The mites were then incubated at 4 C overnight for 24 h. Experiments were replicated also to assess both the dsRNA encoding DpGST-mu1 and lacZ encapsulated in liposomes and the het_siRNAs encoding DpGST-mu1 and lacZ.
2.2. Uptake of fluorescently-labelled siRNA by D. pteronyssinus 2.6. RNA extraction The protocol developed for V. destructor [18] was adapted to accommodate the challenges presented in handling these tiny mites. Twenty adult female D. pteronyssinus mites were immersed in the detached cap of a 1.5 ml micro-centrifuge tube in 15 ml solution containing either a fluorescently labelled AllStars Negative Control siRNA (Fluoro-siRNA) (QIAGEN, UK) in sterile normal saline (final Fluoro-siRNA concentration 0.05 mg/ml) or 15 ml saline only. The mites were incubated at 4 C overnight and then washed three times in molecular grade water (Sigma Aldrich, UK) before
Following the 24 h immersion, mites were washed three times with molecular grade water before being transferred to a ZR BashingBead™ lysis tube containing 800 ml of RNA Lysis Buffer (Zymo Research, USA). Mites were then homogenised using a Precellys 24 homogeniser (Bertin Technologies, France) with 3 cycles of 23 s at 6000 rpm. Samples were then centrifuged at 12,000 g for 1 min at 4 C, and the supernatant transferred to a sterile microcentrifuge tube. Thereafter, RNA was extracted using ZR Tissue
Please cite this article in press as: E.J. Marr, et al., Gene silencing by RNA interference in the house dust mite, Dermatophagoides pteronyssinus, Molecular and Cellular Probes (2015), http://dx.doi.org/10.1016/j.mcp.2015.07.008
E.J. Marr et al. / Molecular and Cellular Probes xxx (2015) 1e5
and Insect RNA Microprep kits (Zymo Research, USA) according to the manufacturer's protocol, incorporating an on-column DNase I step. RNA was eluted in 14 ml molecular grade water (Sigma Aldrich, UK), and RNA quality assessed using Agilent Bioanalyser Pico Chips (Agilent, UK). RNA integrity numbers (RINs) were disregarded in favour of visual appraisal of the 18S ribosomal RNA (rRNA) electropherogram peak on the basis that the 21S rRNA fragment is either degraded or co-migrates with 18S, as observed for P. ovis and other arthropods [32,33]. 2.7. Quantitative PCR (qPCR) Gene-specific primers were designed for DpGST-mu1(Accession number AY825939.1) and E. coli lacZ (Accession number NC_000913.3) using Primer3 [34] (primer sequences are available from the authors upon request). Primers for D. pteronyssinus elongation factor 1-alpha (DpEF1a) were designed as described by Mounsey et al. [35]. First strand cDNA synthesis was performed using Superscript II (Invitrogen, UK) and oligo(dT)23 anchored primers (SigmaeAldrich, UK) using 10 ml of total RNA of D. pteronyssinus. The qPCRs were performed in triplicate on cDNA samples (diluted 1:3 with molecular grade water) using an ABI Prism 7500 real-time thermal cycler (Applied Biosystems, UK); qPCR cycling times were 50 C for 2 min then 95 C for 5 min, followed by 40 cycles of 95 C for 30 s, 57 C for 40 s, and 72 C for 40 s. Melt curve analysis cycle times were 95 C for 15 s, 60 C for 60 s and 95 C for 15 s. A single 25 ml PCR reaction consisted of 1 ml cDNA, 12.5 ml SYBR® GreenER™ qPCR SuperMix for ABI PRISM® (Life Technologies, UK), 0.5 ml of each primer (2 mM) and 10.5 ml of molecular grade water. Standard curves (108e102 copies/ml) were prepared from 109 copies/ml stock of plasmid DNA and amplified in triplicate. Correlation co-efficients of the standard curves were used to calculate PCR efficiencies, which were consistently >90%. The average number of copies/ml of cDNA was calculated, and the results were normalised to those of the reference gene DpEF1a. 2.8. Statistical analysis Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software Inc, USA). A Student's t-test was used to determine the statistical difference in transcription between the means of the two treatment groups in each experiment. No transformations were required, and p-values of <0.05 were considered significant. 3. Results 3.1. Uptake of Fluoro-siRNA by D. pteronyssinus Following
overnight
immersion
in
Fluoro-siRNA
at
a
3
concentration of 0.05 mg/ml in saline, concentrated fluorescent siRNA was visible in the foreguts of D. pteronyssinus (Fig. 1, Panel 1). Control mites immersed overnight in saline demonstrated some degree of autofluorescence, a feature common in invertebrates (Fig. 1, Panel 2). 3.2. qPCR validation of gene silencing by RNAi in D. pteronyssinus mites Overnight immersion of adult male D. pteronyssinus in DpGSTmu1 dsRNA resulted in significantly reduced mRNA transcript for this gene (p ¼ 0.0073), compared with those immersed overnight in lacZ dsRNA, with an average reduction in normalised DpGSTmu1 gene transcription relative to the lacZ control of 59% (Fig. 2, Panel 1). The administration of dsRNA with lipofectamine did not result in an enhanced gene-silencing phenotype (Fig. 2, Panel 2) and, although the trend of reduced DpGST-mu1 gene transcription was evident, the 40% average reduction compared with the mites immersed in lacZ dsRNA was not significant (p ¼ 0.1850). Immersion of mites in het-siRNA encoding DpGST-mu1 did not show a reduction of DpGST-mu1 transcription (p ¼ 0.3996) (Fig. 2, Panel 3). 4. Discussion Here we have demonstrated that D. pteronyssinus gene-specific silencing of the DpGST-mu1 gene can be achieved using a noninvasive immersion method. Incubation overnight at 4 C was chosen on the basis that 100% survival rates were recorded using this technique, and mites remained under complete immersion throughout the duration of the incubation. This finding contrasts with those immersed at room temperature or in a humidity incubator at 25 C; here, the mites were not only more mobile and able to escape the immersion, but displayed increased mortality over 24 h (data not shown). In spite of achieving a significant reduction in DpGST-mu1 gene transcription, averaging 59%, the silencing did not result in any detrimental phenotypic changes to the mites. This result was not unexpected, given that observations were limited to the 24 h experimental period, and no abiotic stressors that might result in a need for GST activity were introduced to compare survival between the treatment groups. There are multiple classes of GSTs, of which at least one other class has been identified in D. pteronyssinus, showing similarity to the delta and epsilon class in insects. This redundancy could also explain the lack of a non-wild-type phenotype, with the remaining GST belonging to one or more of the different classes left intact following successful RNAi specific for the mu class GST alone [30]. Further experiments would be required to ascertain longevity of the gene silencing, whether or not the silencing was carried over to the translational level and
Fig. 1. Fluoro-siRNA uptake in Dermatophagoides pteronyssinus. Mites were immersed overnight in Fluoro-siRNA solution (1) or sterile saline (2) then viewed by inverted fluorescence microscopy (Axiovert 25 CFL; Zeiss, UK) at 10 magnification. Photographic images taken on a Nikon digital SLR camera (D90 AF-5 DX NIKKOR with 18e105 mm f/ 3.5e5.6 G ED VR lens kit) (Nikon, Japan). Scale bar: 225 mm.
Please cite this article in press as: E.J. Marr, et al., Gene silencing by RNA interference in the house dust mite, Dermatophagoides pteronyssinus, Molecular and Cellular Probes (2015), http://dx.doi.org/10.1016/j.mcp.2015.07.008
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Fig. 2. Gene silencing of DpGST-mu1 in Dermatophagoides pteronyssinus by RNAi. Normalised mean DpGST-mu1 expression normalised to EF1a (copies/ml) determined by qPCR are shown for mites immersed in; (1) DpGST-mu1 dsRNA compared to lacZ dsRNA; (2) DpGST-mu1 dsRNA compared to lacZ dsRNA with lipofectamine (LPF); and (3) DpGST-mu1 het_siRNA compared to lacZ het_siRNA. n ¼ 5 for each treatment group, error bars indicate mean ± SEM.
whether or not phenotypic changes do occur over a prolonged experimental period. Such experiments might involve assessing mite survival when exposed to acaricides following RNAi, given the role that GSTs are known to play in acaricide resistance within the Acari [36e38]. In addition, translation level silencing of DpGST-mu1 could be ascertained by means of a fluorescence-based GST enzyme assay, whereby the fluorescence emitted as a result of the conjugation of reduced glutathione to a substrate such as monochlorobimane indicates GST activity [18,36]. However, the caveat would be that the remaining GSTs of different classes could account for residual GST activity through functional redundancy in mites following successful RNAi-mediated silencing of the DpGST-mu1 gene. In addition to physiological similarities, D. pteronyssinus shares extensive antigen and allergen homology with Psoroptes ovis and other important mite species, which indicates that useful comparative studies could be conducted. For example, the important P. ovis mite allergens Pso o 2, Pso o 8, Pso o 10, Pso o 11, Pso o 14 and many more antigens, each have ontological equivalents in D. pteronyssinus termed Der p 2, Der p 8, Der p 10, Der p 11 and Der p 14, respectively [39,40]. Given the similarities observed, the development of RNAi in D. pteronyssinus could be considered as a method of developing protocols for RNAi in other astigmatid mites of medical and veterinary importance, including P. ovis and Sarcoptes scabiei that cannot be cultured in vitro, are difficult to reliably harvest in sufficient numbers, and for which there is currently a paucity of genomic information [26,41]. This demonstration of RNAi in D. pteronyssinus is particularly timely, as it intersects with the publications of the complete mitochondrial genome of D. pteronyssinus and the draft genome, transcriptome and microbiome of D. farinae, which provide a wealth of new information that might enable high throughput genome-wide RNAi studies [1,42]. Genome-wide RNAi screens have proved informative in a wide range of organisms and would be of great value, in terms of better understanding biology of HDMs and the mechanisms by which they interact in a synanthropic
environment, providing insights into how they might be more effectively and sustainably controlled. One intriguing possibility could be the silencing of allergen-specific gene transcription in HDMs by RNAi through the use of dsRNA applied to HDM habitats in a domestic environment (e.g., pillows, mattresses and carpets) to alleviate allergic disease. This approach warrants future investigation.
5. Conclusions This demonstration of a significant reduction in the transcription of the DpGST-mu1 gene represents the first published account of RNAi in any HDM and paves the way for future RNAi studies in these synanthropic mites. HDMs are implicated in asthma and other allergy-related illnesses in humans and are therefore of considerable medical relevance. With current chemotherapeutic control being inadequate, the present RNAi approach has the potential to facilitate future research towards developing novel intervention methods. Furthermore, D. pteronyssinus might represent a model for RNAi development in astigmatid mites of both veterinary and medical importance such as species of Sarcoptes and Psoroptes, for which obtaining live mites for experimentation remains a major challenge.
Acknowledgements EJM was funded by the Perry Foundation and the Scottish Government. AJN and STGB received funding from the Scottish Government and DEFRA. EJM drafted the manuscript and performed the experiments; NDS, AJN and STGB contributed to the drafting of the manuscript. All authors read and approved the final manuscript. The authors would also like to express their gratitude to Drs Alan Bowman and Ewan Campbell (University of Aberdeen) for their advice and helpful discussions.
Please cite this article in press as: E.J. Marr, et al., Gene silencing by RNA interference in the house dust mite, Dermatophagoides pteronyssinus, Molecular and Cellular Probes (2015), http://dx.doi.org/10.1016/j.mcp.2015.07.008
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Short communication
Gene silencing by RNA interference in the house dust mite, Dermatophagoides pteronyssinus Edward J. Marr a, b, Neil D. Sargison b, Alasdair J. Nisbet a, Stewart T.G. Burgess a, * a b
Division of Vaccines and Diagnostics, Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Edinburgh, Scotland, United Kingdom Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Roslin, Midlothian, Scotland, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 July 2015 Received in revised form 22 July 2015 Accepted 22 July 2015 Available online xxx
This is the first report of gene silencing by RNA interference (RNAi) in the European house dust mite, Dermatophagoides pteronyssinus, Trouessart, 1897. Using a non-invasive immersion method first developed for the honey bee mite, Varroa destructor, a significant reduction in the expression of D. pteronyssinus glutathione-S-transferase mu-class 1 enzyme (DpGST-mu1) was achieved following overnight immersion in double stranded RNA encoding DpGST-mu1. Although no detrimental phenotypic changes were observed following silencing, this technique can now be used to address fundamental physiological questions and assess the potential therapeutic benefit in silencing D. pteronyssinus target genes in selected domestic situations of high human-mite interface. © 2015 Elsevier Ltd. All rights reserved.
Keywords: House dust mites Dermatophagoides pteronyssinus RNA interference Glutathione-S-transferase Astigmatid Atopy
1. Introduction Dermatophagoides pteronyssinus, the model European house dust mite (HDM), is a free-living Psoroptidean of medical importance due to the potent allergenicity of a number of its faecal antigens and the impacts that these allergens have in relation to asthma and other allergic conditions in humans, including chronic allergic rhinitis [1e3]. In total, 24 allergens have been identified thus far from HDM species [1]. The inhalant allergens produced by HDMs have been estimated to account for 50% of allergic disease cases [1], and sensitisation to D. pteronyssinus allergens in asthmatic or allergic individuals has been estimated to be as high as 80e90% [4,5]. The experimental reduction of the presence of D. pteronyssinus in the homes of asthma sufferers by chemical means has been shown to result in a considerable decrease in symptoms associated with asthma over a trial period of 8 weeks [6]. Current clinical treatment for alleviating or minimising the impact of HDM allergy involves medication with preventative and alleviating chemotherapies as well as allergen-specific immunotherapy, although novel treatments are currently being sought. The group one allergen, Der p 1, has been the subject of considerable study,
* Corresponding author. E-mail address: [email protected] (S.T.G. Burgess).
given its role in triggering allergic asthma. Recently, this antigen has been considered for development of a DNA based vaccine for both asthma and allergic rhinitis, which might represent a promising new therapeutic alternative [7e10]. Der p 1 has also been assessed as the basis for a small-molecule-based therapy using reversible peptidase inhibitors, revealing another alternative means of treatment [11]. Currently, the control of the mites themselves involves a modification of the environment to reduce the available HDMsuitable habitat, or application of acaricides (e.g. synthetic pyrethroids or organophosphates) to the habitat. However, due to the high reproductive potential and large population numbers of mites involved, repeated treatment with acaricides has the potential to lead to the development of acaricide resistance; such resistance to the widely used pyrethroids has already been demonstrated in vitro, confirming this concern [12]. An additional, important consideration of applying regular prophylactic doses of pesticides is the potential emergence or exacerbation of existing resistance to pyrethroids in other domestic arthropod pests, such as the German cockroach, Blattella germanica [13]. Alternative strategies, such as regulating the ambient relative humidity, so that it is maintained at 35% for a minimum of 22 h daily [14], has been shown to impact on the population growth of Dermatophagoides farinae, although the practicalities and equipment required for implementing this
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Please cite this article in press as: E.J. Marr, et al., Gene silencing by RNA interference in the house dust mite, Dermatophagoides pteronyssinus, Molecular and Cellular Probes (2015), http://dx.doi.org/10.1016/j.mcp.2015.07.008
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particular solution might be prohibitively expensive. RNA inference (RNAi) is a relatively conserved, ancient mechanism of cell defence against viruses, now widely used experimentally as a means of achieving specific gene silencing to elucidate or alter function [15,16]. A novel application of RNAi developed for combating colony collapse disorder in the honey bee, Apis mellifera, has conferred protection against Israeli acute paralysis virus (IAPV) using Remembee-IAPV, a food supplement containing IAPVspecific dsRNA [17], an approach with commercial potential as a means of controlling pathogens within the hive. This approach could pave the way for the development of RNAi-based control in other controlled environments against various pathogens, parasites or pests, such as HDMs. In addition, RNAi silencing has been demonstrated in a range of Acari, including the honey bee mite, Varroa destructor, and the two-spotted spider mite, Tetranychus urticae [18,19]. RNAi can be triggered by the introduction of double-stranded RNA (dsRNA) or small interfering RNA (siRNA) into an organism. The subsequent processing of the ds/siRNA and incorporation within the RNA-induced silencing complex (RISC) leads to the cleavage of gene-specific messenger RNA (mRNA) sharing homologous sequence to the ds/siRNA [20e25]. The phenotype resulting from reduced transcript levels following RNAi can suggest or indicate the function(s) of the target gene and its importance to the host organism and furthermore, might inform about its potential as a novel intervention target [26e28]. Until now, there has been no published account of RNAi in HDMs, possibly due to limitations imposed by their diminutive size (440 mm in length) [29]. However, the demonstration in V. destructor of a non-invasive immersion-based methodology for achieving RNAi has recently facilitated the ongoing development of this method for application to smaller mite species. The RNAi methodology developed in V. destructor focussed on dsRNA encoding VdGST-mu1, a mu class glutathione-Stransferase (GST) with orthology to the HDM Der p 8 panallergen [4,18,30]. GSTs are also known to play critical roles in detoxification and are widely implicated in the development of drug resistance [4]. Here we demonstrate successful silencing of DpGST-mu1 in D. pteronyssinus following development of a non-invasive RNAi methodology. 2. Material and methods 2.1. D. pteronyssinus culture and harvesting Dermatophagoides pteronyssinus mites, originally acquired from a culture maintained at the University of Vienna, Austria, were housed within vented 75 cm2 Corning Flasks (Corning, UK), fed on a mixture of high quality goldfish flake food (Aquarian, UK) and wheat germ (Neal's Yard, UK) (equal ratio w/w). The flasks were kept at room temperature (22e24 C) within a box containing saturated salt solution to maintain constant humidity. Mites were harvested from the cultures by flotation in saturated saline solution.
photographic images were captured using an Axiovert 25 CFL inverted fluorescent microscope (Zeiss, UK) with 10 Achrostigmat magnification lens (Zeiss, UK) using a D90 AF-5 DX NIKKOR digital camera with 18e105 mm f/3.5e5.6 G ED VR lens kit (Nikon, Japan). 2.3. dsRNA/heterogeneous siRNA preparation Double-stranded RNA (dsRNA) encoding 500 bp of D. pteronyssinus gluthathione-S-transferase mu class 1 (DpPoGST-mu1) (AY825939.1) or 319 bp Escherichia coli strain K-12 sub-strain MG1655 lacZ (NC_000913.3) was produced using the dsRNA production vector pL4440 from the Fire Lab C. elegans Vector Kit 1999(Addgene plasmid #1654); pL4440 containing the target gene sequence was produced by cloning the target gene into pL4440 using Sac1 and Sma1 restriction sites (Roche, UK); PCR cycling times were 95 C for 5 min, followed by 30 cycles of 95 C for 15 s, 57 C for 30 s, and 70 C for 60 s with final extension of 70 C for 60 s (primer sequences are available from the authors upon request). The vector pL4440 was dephosphorylated following restriction digestion with Thermo-Stable Alkaline Phosphatase (Promega, UK) prior to T4 DNA ligation (Promega, UK) to maximise ligation efficiency. Cloning was confirmed by sequencing (Eurofins Genomics, Germany) using the M13 uni (21) standard primer. The dsRNA was synthesised from linearised plasmid using T7 RiboMAX™ Express RNAi System (Promega, UK) according to the manufacturer's protocol. The quality of dsRNA was assessed using gel electrophoresis and the concentration determined using an ND-1000 Nanodrop spectrophotometer (Thermo Scientific, UK). Heterogenous siRNA (het_siRNA) was prepared for DpGST-mu1 and lacZ using the ShortCut® RNase III enzyme (New England Biolabs, UK) according to the manufacturer's protocol, resulting in a conversion of the 319e500 bp dsRNA constructs into a heterogeneous mixture of 18e25 bp siRNAs. The het_siRNAs were diluted to a working concentration of 2.5 mg/ml in physiological saline. 2.4. Liposome enclosure of dsRNA The effect of enclosing the dsRNA within liposomes was assessed on the basis that enhanced RNAi was demonstrated in the nematode Haemonchus contortus using this technique [31]; dsRNA was incubated with Lipofectamine® 2000 Reagent (Invitrogen, UK) for 10 min at room temperature at ration of 1 ml Lipofectamine to 10 ml dsRNA. 2.5. Non-invasive RNAi Twenty adult male D. pteronyssinus mites were immersed in the detached cap of a 1.5 ml micro-centrifuge tube (Axygen, USA) in 15 ml dsRNA encoding either DpGST-mu1 or lacZ, diluted in saline to a final concentration of 2.5 mg/ml, with 5 biological replicates per treatment group. The mites were then incubated at 4 C overnight for 24 h. Experiments were replicated also to assess both the dsRNA encoding DpGST-mu1 and lacZ encapsulated in liposomes and the het_siRNAs encoding DpGST-mu1 and lacZ.
2.2. Uptake of fluorescently-labelled siRNA by D. pteronyssinus 2.6. RNA extraction The protocol developed for V. destructor [18] was adapted to accommodate the challenges presented in handling these tiny mites. Twenty adult female D. pteronyssinus mites were immersed in the detached cap of a 1.5 ml micro-centrifuge tube in 15 ml solution containing either a fluorescently labelled AllStars Negative Control siRNA (Fluoro-siRNA) (QIAGEN, UK) in sterile normal saline (final Fluoro-siRNA concentration 0.05 mg/ml) or 15 ml saline only. The mites were incubated at 4 C overnight and then washed three times in molecular grade water (Sigma Aldrich, UK) before
Following the 24 h immersion, mites were washed three times with molecular grade water before being transferred to a ZR BashingBead™ lysis tube containing 800 ml of RNA Lysis Buffer (Zymo Research, USA). Mites were then homogenised using a Precellys 24 homogeniser (Bertin Technologies, France) with 3 cycles of 23 s at 6000 rpm. Samples were then centrifuged at 12,000 g for 1 min at 4 C, and the supernatant transferred to a sterile microcentrifuge tube. Thereafter, RNA was extracted using ZR Tissue
Please cite this article in press as: E.J. Marr, et al., Gene silencing by RNA interference in the house dust mite, Dermatophagoides pteronyssinus, Molecular and Cellular Probes (2015), http://dx.doi.org/10.1016/j.mcp.2015.07.008
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and Insect RNA Microprep kits (Zymo Research, USA) according to the manufacturer's protocol, incorporating an on-column DNase I step. RNA was eluted in 14 ml molecular grade water (Sigma Aldrich, UK), and RNA quality assessed using Agilent Bioanalyser Pico Chips (Agilent, UK). RNA integrity numbers (RINs) were disregarded in favour of visual appraisal of the 18S ribosomal RNA (rRNA) electropherogram peak on the basis that the 21S rRNA fragment is either degraded or co-migrates with 18S, as observed for P. ovis and other arthropods [32,33]. 2.7. Quantitative PCR (qPCR) Gene-specific primers were designed for DpGST-mu1(Accession number AY825939.1) and E. coli lacZ (Accession number NC_000913.3) using Primer3 [34] (primer sequences are available from the authors upon request). Primers for D. pteronyssinus elongation factor 1-alpha (DpEF1a) were designed as described by Mounsey et al. [35]. First strand cDNA synthesis was performed using Superscript II (Invitrogen, UK) and oligo(dT)23 anchored primers (SigmaeAldrich, UK) using 10 ml of total RNA of D. pteronyssinus. The qPCRs were performed in triplicate on cDNA samples (diluted 1:3 with molecular grade water) using an ABI Prism 7500 real-time thermal cycler (Applied Biosystems, UK); qPCR cycling times were 50 C for 2 min then 95 C for 5 min, followed by 40 cycles of 95 C for 30 s, 57 C for 40 s, and 72 C for 40 s. Melt curve analysis cycle times were 95 C for 15 s, 60 C for 60 s and 95 C for 15 s. A single 25 ml PCR reaction consisted of 1 ml cDNA, 12.5 ml SYBR® GreenER™ qPCR SuperMix for ABI PRISM® (Life Technologies, UK), 0.5 ml of each primer (2 mM) and 10.5 ml of molecular grade water. Standard curves (108e102 copies/ml) were prepared from 109 copies/ml stock of plasmid DNA and amplified in triplicate. Correlation co-efficients of the standard curves were used to calculate PCR efficiencies, which were consistently >90%. The average number of copies/ml of cDNA was calculated, and the results were normalised to those of the reference gene DpEF1a. 2.8. Statistical analysis Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software Inc, USA). A Student's t-test was used to determine the statistical difference in transcription between the means of the two treatment groups in each experiment. No transformations were required, and p-values of <0.05 were considered significant. 3. Results 3.1. Uptake of Fluoro-siRNA by D. pteronyssinus Following
overnight
immersion
in
Fluoro-siRNA
at
a
3
concentration of 0.05 mg/ml in saline, concentrated fluorescent siRNA was visible in the foreguts of D. pteronyssinus (Fig. 1, Panel 1). Control mites immersed overnight in saline demonstrated some degree of autofluorescence, a feature common in invertebrates (Fig. 1, Panel 2). 3.2. qPCR validation of gene silencing by RNAi in D. pteronyssinus mites Overnight immersion of adult male D. pteronyssinus in DpGSTmu1 dsRNA resulted in significantly reduced mRNA transcript for this gene (p ¼ 0.0073), compared with those immersed overnight in lacZ dsRNA, with an average reduction in normalised DpGSTmu1 gene transcription relative to the lacZ control of 59% (Fig. 2, Panel 1). The administration of dsRNA with lipofectamine did not result in an enhanced gene-silencing phenotype (Fig. 2, Panel 2) and, although the trend of reduced DpGST-mu1 gene transcription was evident, the 40% average reduction compared with the mites immersed in lacZ dsRNA was not significant (p ¼ 0.1850). Immersion of mites in het-siRNA encoding DpGST-mu1 did not show a reduction of DpGST-mu1 transcription (p ¼ 0.3996) (Fig. 2, Panel 3). 4. Discussion Here we have demonstrated that D. pteronyssinus gene-specific silencing of the DpGST-mu1 gene can be achieved using a noninvasive immersion method. Incubation overnight at 4 C was chosen on the basis that 100% survival rates were recorded using this technique, and mites remained under complete immersion throughout the duration of the incubation. This finding contrasts with those immersed at room temperature or in a humidity incubator at 25 C; here, the mites were not only more mobile and able to escape the immersion, but displayed increased mortality over 24 h (data not shown). In spite of achieving a significant reduction in DpGST-mu1 gene transcription, averaging 59%, the silencing did not result in any detrimental phenotypic changes to the mites. This result was not unexpected, given that observations were limited to the 24 h experimental period, and no abiotic stressors that might result in a need for GST activity were introduced to compare survival between the treatment groups. There are multiple classes of GSTs, of which at least one other class has been identified in D. pteronyssinus, showing similarity to the delta and epsilon class in insects. This redundancy could also explain the lack of a non-wild-type phenotype, with the remaining GST belonging to one or more of the different classes left intact following successful RNAi specific for the mu class GST alone [30]. Further experiments would be required to ascertain longevity of the gene silencing, whether or not the silencing was carried over to the translational level and
Fig. 1. Fluoro-siRNA uptake in Dermatophagoides pteronyssinus. Mites were immersed overnight in Fluoro-siRNA solution (1) or sterile saline (2) then viewed by inverted fluorescence microscopy (Axiovert 25 CFL; Zeiss, UK) at 10 magnification. Photographic images taken on a Nikon digital SLR camera (D90 AF-5 DX NIKKOR with 18e105 mm f/ 3.5e5.6 G ED VR lens kit) (Nikon, Japan). Scale bar: 225 mm.
Please cite this article in press as: E.J. Marr, et al., Gene silencing by RNA interference in the house dust mite, Dermatophagoides pteronyssinus, Molecular and Cellular Probes (2015), http://dx.doi.org/10.1016/j.mcp.2015.07.008
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Fig. 2. Gene silencing of DpGST-mu1 in Dermatophagoides pteronyssinus by RNAi. Normalised mean DpGST-mu1 expression normalised to EF1a (copies/ml) determined by qPCR are shown for mites immersed in; (1) DpGST-mu1 dsRNA compared to lacZ dsRNA; (2) DpGST-mu1 dsRNA compared to lacZ dsRNA with lipofectamine (LPF); and (3) DpGST-mu1 het_siRNA compared to lacZ het_siRNA. n ¼ 5 for each treatment group, error bars indicate mean ± SEM.
whether or not phenotypic changes do occur over a prolonged experimental period. Such experiments might involve assessing mite survival when exposed to acaricides following RNAi, given the role that GSTs are known to play in acaricide resistance within the Acari [36e38]. In addition, translation level silencing of DpGST-mu1 could be ascertained by means of a fluorescence-based GST enzyme assay, whereby the fluorescence emitted as a result of the conjugation of reduced glutathione to a substrate such as monochlorobimane indicates GST activity [18,36]. However, the caveat would be that the remaining GSTs of different classes could account for residual GST activity through functional redundancy in mites following successful RNAi-mediated silencing of the DpGST-mu1 gene. In addition to physiological similarities, D. pteronyssinus shares extensive antigen and allergen homology with Psoroptes ovis and other important mite species, which indicates that useful comparative studies could be conducted. For example, the important P. ovis mite allergens Pso o 2, Pso o 8, Pso o 10, Pso o 11, Pso o 14 and many more antigens, each have ontological equivalents in D. pteronyssinus termed Der p 2, Der p 8, Der p 10, Der p 11 and Der p 14, respectively [39,40]. Given the similarities observed, the development of RNAi in D. pteronyssinus could be considered as a method of developing protocols for RNAi in other astigmatid mites of medical and veterinary importance, including P. ovis and Sarcoptes scabiei that cannot be cultured in vitro, are difficult to reliably harvest in sufficient numbers, and for which there is currently a paucity of genomic information [26,41]. This demonstration of RNAi in D. pteronyssinus is particularly timely, as it intersects with the publications of the complete mitochondrial genome of D. pteronyssinus and the draft genome, transcriptome and microbiome of D. farinae, which provide a wealth of new information that might enable high throughput genome-wide RNAi studies [1,42]. Genome-wide RNAi screens have proved informative in a wide range of organisms and would be of great value, in terms of better understanding biology of HDMs and the mechanisms by which they interact in a synanthropic
environment, providing insights into how they might be more effectively and sustainably controlled. One intriguing possibility could be the silencing of allergen-specific gene transcription in HDMs by RNAi through the use of dsRNA applied to HDM habitats in a domestic environment (e.g., pillows, mattresses and carpets) to alleviate allergic disease. This approach warrants future investigation.
5. Conclusions This demonstration of a significant reduction in the transcription of the DpGST-mu1 gene represents the first published account of RNAi in any HDM and paves the way for future RNAi studies in these synanthropic mites. HDMs are implicated in asthma and other allergy-related illnesses in humans and are therefore of considerable medical relevance. With current chemotherapeutic control being inadequate, the present RNAi approach has the potential to facilitate future research towards developing novel intervention methods. Furthermore, D. pteronyssinus might represent a model for RNAi development in astigmatid mites of both veterinary and medical importance such as species of Sarcoptes and Psoroptes, for which obtaining live mites for experimentation remains a major challenge.
Acknowledgements EJM was funded by the Perry Foundation and the Scottish Government. AJN and STGB received funding from the Scottish Government and DEFRA. EJM drafted the manuscript and performed the experiments; NDS, AJN and STGB contributed to the drafting of the manuscript. All authors read and approved the final manuscript. The authors would also like to express their gratitude to Drs Alan Bowman and Ewan Campbell (University of Aberdeen) for their advice and helpful discussions.
Please cite this article in press as: E.J. Marr, et al., Gene silencing by RNA interference in the house dust mite, Dermatophagoides pteronyssinus, Molecular and Cellular Probes (2015), http://dx.doi.org/10.1016/j.mcp.2015.07.008
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Please cite this article in press as: E.J. Marr, et al., Gene silencing by RNA interference in the house dust mite, Dermatophagoides pteronyssinus, Molecular and Cellular Probes (2015), http://dx.doi.org/10.1016/j.mcp.2015.07.008