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Analysis of virus susceptibility in the invasive insect pest Drosophila suzukii
Accepted Manuscript Short Communication Analysis of virus susceptibility in the invasive insect pest Drosophila suzukii Kwang-Zin Lee, Andreas Vilcinskas PII: DOI: Reference:
S0022-2011(17)30182-9 http://dx.doi.org/10.1016/j.jip.2017.06.010 YJIPA 6964
To appear in:
Journal of Invertebrate Pathology
Received Date: Revised Date: Accepted Date:
6 April 2017 13 June 2017 27 June 2017
Please cite this article as: Lee, K-Z., Vilcinskas, A., Analysis of virus susceptibility in the invasive insect pest Drosophila suzukii, Journal of Invertebrate Pathology (2017), doi: http://dx.doi.org/10.1016/j.jip.2017.06.010
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Short Communication
Analysis of virus susceptibility in the invasive insect pest Drosophila suzukii
Kwang-Zin Leea*, Andreas Vilcinskasa,b
a
Institute for Insect Biotechnology, Justus Liebig University of Giessen, Heinrich Buff Ring 26-32,
D-35392, Germany b
Fraunhofer Institute for Molecular Biology and Applied Ecology, Winchester Strasse 2, D-35394
Giessen, Germany * Corresponding author
ABSTRACT The invasive insect pest Drosophila suzukii infests ripening fruits and causes massive agricultural damage in North America and Europe (Cini, Ioriatti, and Anfora 2012). Environmentally sustainable strategies are urgently needed to control the spread of this species, and entomopathogenic viruses offer one potential solution for global crop protection. Here we report the status of intrinsic and extrinsic factors that influence the susceptibility of D. suzukii to three model insect viruses: Drosophila C virus, Cricket paralysis virus and Flock house virus. Our work provides the basis for further studies using D. suzukii as a host system to develop viruses as biological control agents. KEYWORDS Drosophila suzukii; virus; pastrel; DCV; CrPV; FHV
1. Introduction The spotted wing Drosophila (Drosophila suzukii Matsumura) (Matsumura 1931) has recently emerged as a serious economic pest of soft and stone fruits in the Americas and Europe (Walsh et al. 2011; Calabria et al. 2012). Unlike most other Drosophila species, which feed on rotten fruit, D. suzukii females are attracted to ripe fruit (Lee et al. 2011) and can oviposit into intact fruits using a specialized serrated ovipositor (Atallah et al. 2014). These distinctive attributes, as well as the high reproduction rate and broad host range, have enabled D. suzukii to become one of the most successful invasive species in modern times. The effect of broad-spectrum insecticides is reduced by the inaccessibility of the larvae buried inside fruits and their rapid acquisition of resistance, and the use of such chemicals is also discouraged because they pose a risk to human health and the environment. The development of safe and effective strategies to control D. suzukii could be facilitated by entomopathogenic viruses, which are highly host specific and have negligible effects against beneficial and non-target organisms, making them ideal for integrated pest management programs (Lacey et al. 2015). To establish viruses as biocontrol agents for D. suzukii, we studied factors that influence host susceptibility. One extrinsic factor influencing susceptibility towards viral infection is the presence of the intracellular bacterium Wolbachia, affecting resistance to viruses (Hedges et al. 2008). We also investigated the sequence of the pastrel gene (Magwire et al. 2012), certain alleles of which confer viral susceptibility. Finally, we studied host susceptibility and virus proliferation with three model insect viruses: Drosophila C virus (DCV), Cricket paralysis virus (CrPV) and Flock house virus (FHV). All tested viruses indicated robust virulence towards D. suzukii and might be exploited in future strategies for the reduction of the pest.
2. Materials and methods 2.1. D. suzukii strains and husbandry We used D. suzukii strains from Canada (Ontario), USA, Italy and Germany (Kriftel and Giessen) (Figure 1A, B). Survival assays and quantitative real-time reverse transcriptase polymerase chain
reaction (RT-qPCR) experiments were carried out using Ontario flies at 3–10 days old. Fly stocks were maintained at 26°C and 60% humidity, and were reared on a soybean/cornmeal medium (10.8% (w/v) soybean and cornmeal mix, 0.8% (w/v) agar, 8% (w/v) malt, 2.2% (w/v) molasses, 1% (w/v) nipalgin, 0,625 % propionic acid). 2.2. Wolbachia detection and pastrel SNP determination DNA from 10 flies was extracted using E.Z.N.A. Insect DNA kit (Omega Bio-tek) according to the manufacturer’s instructions. Methods of PCR amplification and the primers listed in Supplementary Table 1. 1.1. Viral isolates and infections Viruses were produced and titrated as previously described (Merkling and van Rij 2015). Insects were infected by the intrathoracic injection of 4.6 nL of a viral suspension in 10 mM Tris-HCl (pH 7.5) using a Nanoject II device (Drummond Scientific). The injections contained 500 focus forming units (FFUs) of DCV or FHV, or 5 FFUs of CrPV. Survival experiments were carried out using three replicate cohorts of 20 females each. Experiments were performed at least three times independently. 1.2. Quantitative real-time RT-PCR DCV, CrPV and FHV titers were measured by quantitative real-time RT-PCR. Total RNA was isolated using TRI Reagent (Zymo Research) according to the manufacturer’s instructions and reverse transcribed using the iScriptcDNA Synthesis Kit (Bio-Rad). The resulting cDNA was amplified using Power SYBR® Green PCR Master Mix (Thermo Fisher Scientific) and the primers listed in Supplementary Table 1. 1.3. Statistical analysis Data were analyzed using an unpaired two-tailed Student’s t-test in GraphPad Prism (GraphPad Software) and survival curves were plotted and analyzed by log-rank analysis (Kaplan-Meier method). P values lower than 0.05 were considered statistically significant: *P < 0.05, **P < 0.01, ***P < 0.0001.
2. Results 2.1. The D. suzukii genome contains the susceptible pastrel allele The presence of Wolbachia was checked by PCR and indicated that all strains except the USA strain were Wolbachia negative. We next investigated the sequence of the pastrel gene. In D. melanogaster, a single nucleotide polymorphism (SNP) in the last exon has the strongest impact on virus susceptibility. All the strains of D. suzukii we tested (Fig. 1C) contained the susceptible allele as described by Magwire et al. (2012). 2.2. D. suzukii is susceptible to DCV infection DCV belonging to the family Dicistroviridae is highly virulent Drosophila (Jousset et al. 1972; Huszar and Imler 2008). Flies infected with DCV succumbed more rapidly than controls injected with buffer alone, and the median survival time (ST 50) was typically achieved around day 6, with most flies dead by approximately day 15 (Fig. 2A). Susceptibility to DCV also correlated with the increasing viral load, as determined by quantitative RT-PCR (Fig. 2B). 2.3. D. suzukii is susceptible to CrPV infection CrPV is closely related to DCV but was initially found in crickets (Reinganum, Oloughli.Gt, and Hogan 1970). Flies infected with CrPV also succumbed more rapidly than controls, but the ST50 was in this case typically achieved on or around day 4, with all flies dead by day 8 (Fig. 2C). As for DCV, susceptibility to CrPV correlated with the viral load, which rose rapidly on day 1, declined slightly on day 2 and then increased further on days 3 and 4 (Fig. 2D). 2.4. D. suzukii is susceptible to FHV infection FHV is an Alphanodavirus in the family Nodaviridae, which was originally isolated from a beetle (Scotti, Dearing, and Mossop 1983). Flies infected with FHV also succumbed more rapidly than controls, and the ST50 was between days 3 and 4, with all flies dead by day 7 (Fig. 2E). As for DCV and CrPV, susceptibility to FHV correlated with the viral load, which rose rapidly on day 1 and then remained steady (Fig. 2F).
3. Discussion Genetic polymorphisms can influence susceptibility to viruses (Magwire et al. 2012). The pastrel gene is associated with resistance against DCV infection, with one SNP in the last exon having the strongest effect. We identified the D. suzukii pastrel locus and found that all the D. suzukii strains we tested contain the sensitive allele. We found that the D. melanogaster model viruses DCV, CrPV and FHV can all kill D. suzukii rapidly. As previously described in D. melanogaster, and in a recent study for D. suzukii (Cattel et al. 2016), infected flies succumbed more rapidly than mock injected controls. All flies died within 15 days after DCV infection, 8 days after CrPV infection and 7 days after FHV infection. Notably, D. suzukii appears to be more susceptible to viral infection than D. melanogaster in previous studies (Barbier 2013; Kemp et al. 2013; Magwire et al. 2012; Martins et al. 2014). Thus far, no natural virus has been described for D. suzukii, suggesting that the three D. melanogaster model viruses are excellent biocontrol candidates for the protection of fruit crops.
Acknowledgements We are grateful to Tobias Richter and Meike Lietzow for technical help, Dr. Marc Schetelig for providing D. suzukii strains and support in fly rearing, and Dr. Jean-Luc Imler for providing the viral strains. This work has been funded by the excellence initiative of the Hessian Ministry of Science, Higher Education and Art (HMWK) supporting the LOEWE Centre for Insect Biotechnology and Bioresources. The authors thank Richard M. Twyman for editing of the manuscript.
References Atallah, J., L. Teixeira, R. Salazar, G. Zaragoza, and A. Kopp. 2014. 'The making of a pest: the evolution of a fruit-penetrating ovipositor in Drosophila suzukii and related species', Proc Biol Sci, 281: 20132840. Barbier, V. 2013. 'Pastrel, a restriction factor for picorna-like viruses in Drosophila melanogaster', University of Strasbourg. Calabria, G., J. Maca, G. Bachli, L. Serra, and M. Pascual. 2012. 'First records of the potential pest species Drosophila suzukii (Diptera: Drosophilidae) in Europe', Journal of Applied Entomology, 136: 139-47.
Cattel, J., J. Martinez, F. Jiggins, L. Mouton, and P. Gibert. 2016. 'Wolbachia-mediated protection against viruses in the invasive pest Drosophila suzukii', Insect Mol Biol, 25: 595-603. Cini, A., C. Ioriatti, and G. Anfora. 2012. 'A review of the invasion of Drosophila suzukii in Europe and a draft research agenda for integrated pest management', Bulletin of Insectology, 65: 149-60. Hedges, L. M., J. C. Brownlie, S. L. O'Neill, and K. N. Johnson. 2008. 'Wolbachia and virus protection in insects', Science, 322: 702. Huszar, T., and J. L. Imler. 2008. 'Drosophila viruses and the study of antiviral host-defense', Adv Virus Res, 72: 227-65. Jousset, F. X., N. Plus, G. Croizier, and M. Thomas. 1972. 'Existence in Drosophila of 2 Groups of Picornavirus of Different Serological and Biological Properties', Comptes Rendus Hebdomadaires Des Seances De L Academie Des Sciences Serie D, 275: 3043-&. Kemp, C., S. Mueller, A. Goto, V. Barbier, S. Paro, F. Bonnay, C. Dostert, L. Troxler, C. Hetru, C. Meignin, S. Pfeffer, J. A. Hoffmann, and J. L. Imler. 2013. 'Broad RNA Interference-Mediated Antiviral Immunity and Virus-Specific Inducible Responses in Drosophila', Journal of Immunology, 190: 650-58. Lacey, L. A., D. Grzywacz, D. I. Shapiro-Ilan, R. Frutos, M. Brownbridge, and M. S. Goettel. 2015. 'Insect pathogens as biological control agents: Back to the future', Journal of Invertebrate Pathology, 132: 1-41. Lee, J. C., D. J. Bruck, H. Curry, D. Edwards, D. R. Haviland, R. A. Van Steenwyk, and B. M. Yorgey. 2011. 'The susceptibility of small fruits and cherries to the spotted-wing drosophila, Drosophila suzukii', Pest Management Science, 67: 1358-67. Magwire, M. M., D. K. Fabian, H. Schweyen, C. Cao, B. Longdon, F. Bayer, and F. M. Jiggins. 2012. 'Genome-wide association studies reveal a simple genetic basis of resistance to naturally coevolving viruses in Drosophila melanogaster', PLoS Genet, 8: e1003057. Martins, N. E., V. G. Faria, V. Nolte, C. Scholtterer, L. Teixeira, E. Sucena, and S. Magalhaes. 2014. 'Host adaptation to viruses relies on few genes with different cross-resistance properties', Proceedings of the National Academy of Sciences of the United States of America, 111: 593843. Matsumura, S. 1931. '6000 illustrated insects of Japan-Empire', Toko Shoin: pp. 1689 [367]. Merkling, S. H., and R. P. van Rij. 2015. 'Analysis of resistance and tolerance to virus infection in Drosophila', Nature Protocols, 10: 1084-97. Reinganum, C., Oloughli.Gt, and T. W. Hogan. 1970. 'A Nonoccluded Virus of Field Crickets Teleogryllus-Oceanicus and T-Commodus (Orthoptera-Gryllidae)', Journal of Invertebrate Pathology, 16: 214-+. Scotti, P. D., S. Dearing, and D. W. Mossop. 1983. 'Flock House virus: a nodavirus isolated from Costelytra zealandica (White) (Coleoptera: Scarabaeidae)', Arch Virol, 75: 181-9. Walsh, Douglas B., Mark P. Bolda, Rachael E. Goodhue, Amy J. Dreves, Jana Lee, Denny J. Bruck, Vaughn M. Walton, Sally D. O'Neal, and Frank G. Zalom. 2011. 'Drosophila suzukii (Diptera: Drosophilidae): Invasive Pest of Ripening Soft Fruit Expanding its Geographic Range and Damage Potential', Journal of Integrated Pest Management, 2: 1-7.
Fig. 1
A
B
C G/A (Ala/Thr)
100 bp
Giessen Italy
Italy Kriftel Ontario Italy
USA
Italy
Fig. 1. Habitat and morphology of D. suzukii and pastrel structure. (A) D. suzukii male on raspberry in Kriftel, Germany. (B) D. suzukii female on the left and male on the right. (C) Structure of the pastrel gene and location of the SNP with the strongest effect on virus susceptibility (sequence variation shown in red), modified from Merkling & van Rij, 2015). Boxes represent exons, lines indicate introns with scale bar indicating 100 base pairs (bp). Sanger sequencing of different D. suzukii strains (left box) revealed the nucleotide conferring the sensitive form of pastrel.
Fig. 2 A
B
C
D
E
F
Fig. 2. Survival and virus titers. Flies (4-6 days old) were infected with (A) DCV (500 FFU), (C) CrPV (5 FFU) and (E) FHV (500 FFU) and the lifespan was monitored daily at 26°C. Data represent the mean ± SE of at least three independent experiments. Log rank test: ***P < 0.0001. Quantitative real-time RT-PCR analysis of the accumulation of (B) DCV, (D) CrPV, (F) FHV RNA at indicated time points after infection in D. suzukii female flies. Data represent the mean ± SD of at least three independent experiments, each involving five flies.
Highlights
Invasive insect pest Drosophila suzukii is susceptible to three insect viruses. Susceptibility links to sensitive allele of pastrel, a gene mediating resistance. Offers model to establish viruses as safe biocontrol agents for Drosophila suzukii.
S0022-2011(17)30182-9 http://dx.doi.org/10.1016/j.jip.2017.06.010 YJIPA 6964
To appear in:
Journal of Invertebrate Pathology
Received Date: Revised Date: Accepted Date:
6 April 2017 13 June 2017 27 June 2017
Please cite this article as: Lee, K-Z., Vilcinskas, A., Analysis of virus susceptibility in the invasive insect pest Drosophila suzukii, Journal of Invertebrate Pathology (2017), doi: http://dx.doi.org/10.1016/j.jip.2017.06.010
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Short Communication
Analysis of virus susceptibility in the invasive insect pest Drosophila suzukii
Kwang-Zin Leea*, Andreas Vilcinskasa,b
a
Institute for Insect Biotechnology, Justus Liebig University of Giessen, Heinrich Buff Ring 26-32,
D-35392, Germany b
Fraunhofer Institute for Molecular Biology and Applied Ecology, Winchester Strasse 2, D-35394
Giessen, Germany * Corresponding author
ABSTRACT The invasive insect pest Drosophila suzukii infests ripening fruits and causes massive agricultural damage in North America and Europe (Cini, Ioriatti, and Anfora 2012). Environmentally sustainable strategies are urgently needed to control the spread of this species, and entomopathogenic viruses offer one potential solution for global crop protection. Here we report the status of intrinsic and extrinsic factors that influence the susceptibility of D. suzukii to three model insect viruses: Drosophila C virus, Cricket paralysis virus and Flock house virus. Our work provides the basis for further studies using D. suzukii as a host system to develop viruses as biological control agents. KEYWORDS Drosophila suzukii; virus; pastrel; DCV; CrPV; FHV
1. Introduction The spotted wing Drosophila (Drosophila suzukii Matsumura) (Matsumura 1931) has recently emerged as a serious economic pest of soft and stone fruits in the Americas and Europe (Walsh et al. 2011; Calabria et al. 2012). Unlike most other Drosophila species, which feed on rotten fruit, D. suzukii females are attracted to ripe fruit (Lee et al. 2011) and can oviposit into intact fruits using a specialized serrated ovipositor (Atallah et al. 2014). These distinctive attributes, as well as the high reproduction rate and broad host range, have enabled D. suzukii to become one of the most successful invasive species in modern times. The effect of broad-spectrum insecticides is reduced by the inaccessibility of the larvae buried inside fruits and their rapid acquisition of resistance, and the use of such chemicals is also discouraged because they pose a risk to human health and the environment. The development of safe and effective strategies to control D. suzukii could be facilitated by entomopathogenic viruses, which are highly host specific and have negligible effects against beneficial and non-target organisms, making them ideal for integrated pest management programs (Lacey et al. 2015). To establish viruses as biocontrol agents for D. suzukii, we studied factors that influence host susceptibility. One extrinsic factor influencing susceptibility towards viral infection is the presence of the intracellular bacterium Wolbachia, affecting resistance to viruses (Hedges et al. 2008). We also investigated the sequence of the pastrel gene (Magwire et al. 2012), certain alleles of which confer viral susceptibility. Finally, we studied host susceptibility and virus proliferation with three model insect viruses: Drosophila C virus (DCV), Cricket paralysis virus (CrPV) and Flock house virus (FHV). All tested viruses indicated robust virulence towards D. suzukii and might be exploited in future strategies for the reduction of the pest.
2. Materials and methods 2.1. D. suzukii strains and husbandry We used D. suzukii strains from Canada (Ontario), USA, Italy and Germany (Kriftel and Giessen) (Figure 1A, B). Survival assays and quantitative real-time reverse transcriptase polymerase chain
reaction (RT-qPCR) experiments were carried out using Ontario flies at 3–10 days old. Fly stocks were maintained at 26°C and 60% humidity, and were reared on a soybean/cornmeal medium (10.8% (w/v) soybean and cornmeal mix, 0.8% (w/v) agar, 8% (w/v) malt, 2.2% (w/v) molasses, 1% (w/v) nipalgin, 0,625 % propionic acid). 2.2. Wolbachia detection and pastrel SNP determination DNA from 10 flies was extracted using E.Z.N.A. Insect DNA kit (Omega Bio-tek) according to the manufacturer’s instructions. Methods of PCR amplification and the primers listed in Supplementary Table 1. 1.1. Viral isolates and infections Viruses were produced and titrated as previously described (Merkling and van Rij 2015). Insects were infected by the intrathoracic injection of 4.6 nL of a viral suspension in 10 mM Tris-HCl (pH 7.5) using a Nanoject II device (Drummond Scientific). The injections contained 500 focus forming units (FFUs) of DCV or FHV, or 5 FFUs of CrPV. Survival experiments were carried out using three replicate cohorts of 20 females each. Experiments were performed at least three times independently. 1.2. Quantitative real-time RT-PCR DCV, CrPV and FHV titers were measured by quantitative real-time RT-PCR. Total RNA was isolated using TRI Reagent (Zymo Research) according to the manufacturer’s instructions and reverse transcribed using the iScriptcDNA Synthesis Kit (Bio-Rad). The resulting cDNA was amplified using Power SYBR® Green PCR Master Mix (Thermo Fisher Scientific) and the primers listed in Supplementary Table 1. 1.3. Statistical analysis Data were analyzed using an unpaired two-tailed Student’s t-test in GraphPad Prism (GraphPad Software) and survival curves were plotted and analyzed by log-rank analysis (Kaplan-Meier method). P values lower than 0.05 were considered statistically significant: *P < 0.05, **P < 0.01, ***P < 0.0001.
2. Results 2.1. The D. suzukii genome contains the susceptible pastrel allele The presence of Wolbachia was checked by PCR and indicated that all strains except the USA strain were Wolbachia negative. We next investigated the sequence of the pastrel gene. In D. melanogaster, a single nucleotide polymorphism (SNP) in the last exon has the strongest impact on virus susceptibility. All the strains of D. suzukii we tested (Fig. 1C) contained the susceptible allele as described by Magwire et al. (2012). 2.2. D. suzukii is susceptible to DCV infection DCV belonging to the family Dicistroviridae is highly virulent Drosophila (Jousset et al. 1972; Huszar and Imler 2008). Flies infected with DCV succumbed more rapidly than controls injected with buffer alone, and the median survival time (ST 50) was typically achieved around day 6, with most flies dead by approximately day 15 (Fig. 2A). Susceptibility to DCV also correlated with the increasing viral load, as determined by quantitative RT-PCR (Fig. 2B). 2.3. D. suzukii is susceptible to CrPV infection CrPV is closely related to DCV but was initially found in crickets (Reinganum, Oloughli.Gt, and Hogan 1970). Flies infected with CrPV also succumbed more rapidly than controls, but the ST50 was in this case typically achieved on or around day 4, with all flies dead by day 8 (Fig. 2C). As for DCV, susceptibility to CrPV correlated with the viral load, which rose rapidly on day 1, declined slightly on day 2 and then increased further on days 3 and 4 (Fig. 2D). 2.4. D. suzukii is susceptible to FHV infection FHV is an Alphanodavirus in the family Nodaviridae, which was originally isolated from a beetle (Scotti, Dearing, and Mossop 1983). Flies infected with FHV also succumbed more rapidly than controls, and the ST50 was between days 3 and 4, with all flies dead by day 7 (Fig. 2E). As for DCV and CrPV, susceptibility to FHV correlated with the viral load, which rose rapidly on day 1 and then remained steady (Fig. 2F).
3. Discussion Genetic polymorphisms can influence susceptibility to viruses (Magwire et al. 2012). The pastrel gene is associated with resistance against DCV infection, with one SNP in the last exon having the strongest effect. We identified the D. suzukii pastrel locus and found that all the D. suzukii strains we tested contain the sensitive allele. We found that the D. melanogaster model viruses DCV, CrPV and FHV can all kill D. suzukii rapidly. As previously described in D. melanogaster, and in a recent study for D. suzukii (Cattel et al. 2016), infected flies succumbed more rapidly than mock injected controls. All flies died within 15 days after DCV infection, 8 days after CrPV infection and 7 days after FHV infection. Notably, D. suzukii appears to be more susceptible to viral infection than D. melanogaster in previous studies (Barbier 2013; Kemp et al. 2013; Magwire et al. 2012; Martins et al. 2014). Thus far, no natural virus has been described for D. suzukii, suggesting that the three D. melanogaster model viruses are excellent biocontrol candidates for the protection of fruit crops.
Acknowledgements We are grateful to Tobias Richter and Meike Lietzow for technical help, Dr. Marc Schetelig for providing D. suzukii strains and support in fly rearing, and Dr. Jean-Luc Imler for providing the viral strains. This work has been funded by the excellence initiative of the Hessian Ministry of Science, Higher Education and Art (HMWK) supporting the LOEWE Centre for Insect Biotechnology and Bioresources. The authors thank Richard M. Twyman for editing of the manuscript.
References Atallah, J., L. Teixeira, R. Salazar, G. Zaragoza, and A. Kopp. 2014. 'The making of a pest: the evolution of a fruit-penetrating ovipositor in Drosophila suzukii and related species', Proc Biol Sci, 281: 20132840. Barbier, V. 2013. 'Pastrel, a restriction factor for picorna-like viruses in Drosophila melanogaster', University of Strasbourg. Calabria, G., J. Maca, G. Bachli, L. Serra, and M. Pascual. 2012. 'First records of the potential pest species Drosophila suzukii (Diptera: Drosophilidae) in Europe', Journal of Applied Entomology, 136: 139-47.
Cattel, J., J. Martinez, F. Jiggins, L. Mouton, and P. Gibert. 2016. 'Wolbachia-mediated protection against viruses in the invasive pest Drosophila suzukii', Insect Mol Biol, 25: 595-603. Cini, A., C. Ioriatti, and G. Anfora. 2012. 'A review of the invasion of Drosophila suzukii in Europe and a draft research agenda for integrated pest management', Bulletin of Insectology, 65: 149-60. Hedges, L. M., J. C. Brownlie, S. L. O'Neill, and K. N. Johnson. 2008. 'Wolbachia and virus protection in insects', Science, 322: 702. Huszar, T., and J. L. Imler. 2008. 'Drosophila viruses and the study of antiviral host-defense', Adv Virus Res, 72: 227-65. Jousset, F. X., N. Plus, G. Croizier, and M. Thomas. 1972. 'Existence in Drosophila of 2 Groups of Picornavirus of Different Serological and Biological Properties', Comptes Rendus Hebdomadaires Des Seances De L Academie Des Sciences Serie D, 275: 3043-&. Kemp, C., S. Mueller, A. Goto, V. Barbier, S. Paro, F. Bonnay, C. Dostert, L. Troxler, C. Hetru, C. Meignin, S. Pfeffer, J. A. Hoffmann, and J. L. Imler. 2013. 'Broad RNA Interference-Mediated Antiviral Immunity and Virus-Specific Inducible Responses in Drosophila', Journal of Immunology, 190: 650-58. Lacey, L. A., D. Grzywacz, D. I. Shapiro-Ilan, R. Frutos, M. Brownbridge, and M. S. Goettel. 2015. 'Insect pathogens as biological control agents: Back to the future', Journal of Invertebrate Pathology, 132: 1-41. Lee, J. C., D. J. Bruck, H. Curry, D. Edwards, D. R. Haviland, R. A. Van Steenwyk, and B. M. Yorgey. 2011. 'The susceptibility of small fruits and cherries to the spotted-wing drosophila, Drosophila suzukii', Pest Management Science, 67: 1358-67. Magwire, M. M., D. K. Fabian, H. Schweyen, C. Cao, B. Longdon, F. Bayer, and F. M. Jiggins. 2012. 'Genome-wide association studies reveal a simple genetic basis of resistance to naturally coevolving viruses in Drosophila melanogaster', PLoS Genet, 8: e1003057. Martins, N. E., V. G. Faria, V. Nolte, C. Scholtterer, L. Teixeira, E. Sucena, and S. Magalhaes. 2014. 'Host adaptation to viruses relies on few genes with different cross-resistance properties', Proceedings of the National Academy of Sciences of the United States of America, 111: 593843. Matsumura, S. 1931. '6000 illustrated insects of Japan-Empire', Toko Shoin: pp. 1689 [367]. Merkling, S. H., and R. P. van Rij. 2015. 'Analysis of resistance and tolerance to virus infection in Drosophila', Nature Protocols, 10: 1084-97. Reinganum, C., Oloughli.Gt, and T. W. Hogan. 1970. 'A Nonoccluded Virus of Field Crickets Teleogryllus-Oceanicus and T-Commodus (Orthoptera-Gryllidae)', Journal of Invertebrate Pathology, 16: 214-+. Scotti, P. D., S. Dearing, and D. W. Mossop. 1983. 'Flock House virus: a nodavirus isolated from Costelytra zealandica (White) (Coleoptera: Scarabaeidae)', Arch Virol, 75: 181-9. Walsh, Douglas B., Mark P. Bolda, Rachael E. Goodhue, Amy J. Dreves, Jana Lee, Denny J. Bruck, Vaughn M. Walton, Sally D. O'Neal, and Frank G. Zalom. 2011. 'Drosophila suzukii (Diptera: Drosophilidae): Invasive Pest of Ripening Soft Fruit Expanding its Geographic Range and Damage Potential', Journal of Integrated Pest Management, 2: 1-7.
Fig. 1
A
B
C G/A (Ala/Thr)
100 bp
Giessen Italy
Italy Kriftel Ontario Italy
USA
Italy
Fig. 1. Habitat and morphology of D. suzukii and pastrel structure. (A) D. suzukii male on raspberry in Kriftel, Germany. (B) D. suzukii female on the left and male on the right. (C) Structure of the pastrel gene and location of the SNP with the strongest effect on virus susceptibility (sequence variation shown in red), modified from Merkling & van Rij, 2015). Boxes represent exons, lines indicate introns with scale bar indicating 100 base pairs (bp). Sanger sequencing of different D. suzukii strains (left box) revealed the nucleotide conferring the sensitive form of pastrel.
Fig. 2 A
B
C
D
E
F
Fig. 2. Survival and virus titers. Flies (4-6 days old) were infected with (A) DCV (500 FFU), (C) CrPV (5 FFU) and (E) FHV (500 FFU) and the lifespan was monitored daily at 26°C. Data represent the mean ± SE of at least three independent experiments. Log rank test: ***P < 0.0001. Quantitative real-time RT-PCR analysis of the accumulation of (B) DCV, (D) CrPV, (F) FHV RNA at indicated time points after infection in D. suzukii female flies. Data represent the mean ± SD of at least three independent experiments, each involving five flies.
Highlights
Invasive insect pest Drosophila suzukii is susceptible to three insect viruses. Susceptibility links to sensitive allele of pastrel, a gene mediating resistance. Offers model to establish viruses as safe biocontrol agents for Drosophila suzukii.