Detection of Pesticides and Viruses in Dead Honey Bees and Other Matrices

Journal of Apicultural Research

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Spring mortality in honey bees in northeastern Italy: detection of pesticides and viruses in dead honey bees and other matrices

Marianna Martinello, Chiara Baratto, Chiara Manzinello, Elena Piva, Alice Borin, Marica Toson, Anna Granato, Maria Beatrice Boniotti, Albino Gallina & Franco Mutinelli

To cite this article: Marianna Martinello, Chiara Baratto, Chiara Manzinello, Elena Piva, Alice Borin, Marica Toson, Anna Granato, Maria Beatrice Boniotti, Albino Gallina & Franco Mutinelli (2017): Spring mortality in honey bees in northeastern Italy: detection of pesticides and viruses in dead honey bees and other matrices, Journal of Apicultural Research To link to this article: http://dx.doi.org/10.1080/00218839.2017.1304878

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Download by: [Istituto Zooprofilatico] Date: 04 April 2017, At: 02:49 Journal of Apicultural Research, 2017 http://dx.doi.org/10.1080/00218839.2017.1304878

ORIGINAL RESEARCH ARTICLE Spring mortality in honey bees in northeastern Italy: detection of pesticides and viruses in dead honey bees and other matrices Marianna Martinelloa , Chiara Barattoa, Chiara Manzinelloa, Elena Pivaa, Alice Borina, Marica Tosonb, Anna Granatoa, Maria Beatrice Boniottic, Albino Gallinaa and Franco Mutinellia* aNational Reference Laboratory for Beekeeping, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Padua, Italy; bVeterinary Epidemiology Department, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Padua, Italy; cIstituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna, Brescia, Italy (Received 28 October 2016; accepted 7 March 2017)

In spring there is often a rise in honey bee mortality incidents. The aim of this study was to investigate the potential correlation, in the reported incidents, between exposure to pesticide treatments and virus infections. Here we summa- rize the situation in northeastern Italy during spring 2014, evaluated by monitoring 150 active ingredients and three honey bee viruses in dead honey bees and other matrices. At least one active ingredient was found in 72.2% of the 79 dead honey bee samples, with the most abundant (59.4%) being insecticides, mainly belonging to the class of neonicotinoids (41.8%), followed by fungicides (40.6%). Imidacloprid, chlorpyrifos, tau-ﬂuvalinate, and cyprodinil were the most frequently detected active ingredients. Multiple virus infections were monitored, revealing a high prevalence of chronic bee paralysis virus (CBPV) and deformed wing virus (DWV), detected in all samples except one. 71 and 37% of the CBPV- and DWV positive samples, respectively, showed a high number of viral copies per bee (>107). This work emphasizes the possible relationship between spring mortality in honey bees and pesticide treatments. Honey bee viruses could synergistically exacerbate the negative impact of pesticides on honey bee health, endangering the survival of colonies.

Mortalidad primaveral en las abejas melı´feras del noreste de Italia: deteccio´n de los plaguicidas y los virus en las abejas melı´feras muertas y en otras matrices

En primavera suele haber un aumento en los incidentes de mortalidad de las abejas. El objetivo de este estudio fue investigar la correlacioń potencial entre los incidentes comunicados y la exposicioń a tratamientos con plaguicidas y las infecciones vı´ricas. Aquı´ resumimos la situacioń en el noreste de Italia durante la primavera de 2014, evaluada mediante la monitorizacioń de 150 ingredientes y tres virus de la abeja melı´fera en las abejas muertas y en otras matrices. Se encontro´ al menos un ingrediente activo en un 72.2% de las 79 muestras de abejas de la miel muertas, siendo los ma´s abundantes los insecticidas (59.4%), principalmente pertenecientes a la clase de los neonicotinoides (41.8%), seguidos por los fungicidas (40.6%). Los ingredientes activos detectados con mayor frecuencia fueron: imidacloprid, clorpirifos, taufluvalinato, y ciprodinil. Se monitorizaron mu´ltiples infecciones vı´ricas, revelando una alta prevalencia del virus de la para´lisis crońica de las abejas (VPCA) y el virus de las alas deformadas (VAD), que se detectaron en todas las muestras excepto en una. El 71% y 37% de las muestras positivas a VPCA y VAD, respectivamente, mostraron un alto nu´mero de copias del virus por abeja (>107). Este trabajo enfatiza la posible relacioń entre la mortalidad primaveral de las abejas de la miel y los tratamientos con plaguicidas. Los virus de las abejas melı´feras podrıán exacerbar sine´rgicamente el impacto negativo de los plaguicidas en la salud de las abejas de la miel, poniendo en peligro la supervivencia de las colmenas. Keywords: honey bees; spring mortality; plant protection products; pesticide; virus

Introduction means, or would produce significantly less food In recent years, the gradual decline in wild or commer- (Kremen et al., 2007). The most recent estimate of the cially managed honey bee colonies (Goulson, Nicholls, global economic benefit of pollination amounts to a Botias, & Rotheray, 2015) is a much debated topic and value of US$ 235–US$ 577 billion, according to data by has attracted increasing interest not only from experts Lautenbach, Seppelt, Liebscher, and Dormann (2012), but also from the general public. Insect-mediated polli- actualized by IPBES in 2016 (IPBES, 2016). nation is critical to the maintenance of biodiversity and Managed honey bees are significant pollinators and global food security. Furthermore, it has been estimated their decline in recent years in North America and that without this “ecosystem service” about one third Europe (Brodschneider et al., 2016; Genersch et al., of the tonnage of the crops we eat (and most of the 2010; Higes et al., 2010; Lee et al., 2015; Potts, Roberts, wild plants) would have to be pollinated by other et al., 2010; Steinhauer et al., 2014; van der Zee et al.,

*Corresponding author. Email: [email protected]

2012) has become a widely studied phenomenon. Differ- 2013a, 2013b, 2013c). In February 2015, EFSA was ent factors are involved in this problem, including honey requested by the European Commission to organize an bee diseases (Dietemann et al., 2012; Higes et al., 2010; open call for new scientific information regarding the Le Conte, Ellis, & Ritter, 2010; Traynor et al., 2016), risk to bees from three neonicotinoids, namely clothian- honey bee management practices and disease control idin, thiamethoxam and imidacloprid (EFSA, 2015b). In treatments (Giacobino et al., 2015), nutrition (Alaux, November 2016, EFSA published its conclusion reports Ducloz, Crauser, & Le Conte, 2010b; Archer, Pirk, on clothianidin and imidacloprid in the light of submitted Wright, & Nicolson, 2014; Brodschneider & Crailsheim, confirmatory data (EFSA, 2016a, 2016b). The evaluation 2010; Odoux et al., 2012), and climatic trends (Le was performed in the peer review setting requested by Conte & Navajas, 2008), but many recent publications the European Commission, following the submission and (Brittain, Vighi, Bommarco, Settele, & Potts, 2010; Gen- evaluation of confirmatory ecotoxicological data con- ersch et al., 2010; Krupke, Hunt, Eitzer, Andino, & cerning the risk assessment for bees. The conclusions Given, 2012; Lee et al., 2015; Mullin et al., 2010; Porrini were based on the evaluation of representative uses of et al., 2016; Potts, Biesmeijer et al., 2010; Potts, clothianidin as an insecticide on winter cereals, beet, Roberts, et al., 2010; vanEngelsdorp et al., 2009) suspect potato, maize/sweet maize, sorghum and forestry nurs- the profound impact of agricultural practices and the ery, and representative uses of imidacloprid as an insec- varied and massive use of chemicals. A fact sheet about ticide on winter cereals, beet, potato, leafy vegetables the European Union’s (EU) agri-environmental indicator and amenity vegetation. It also presented reliable end- on consumption of pesticides is available on the Euro- points deemed appropriate for use in regulatory risk stat website (Eurostat, 2016), providing an overview of assessment, derived from the available studies and litera- recent data, accompanied by exhaustive information on ture in the peer reviewed dossier. Missing information the definitions, measurement methods and context identified as being required to permit a complete risk needed to interpret them correctly. assessment was listed and concerns were identified. New growing seasons bring a rise in the amount and In 2013, EFSA developed a guidance document on number of plant protection product (PPP) applications the risk assessment of PPPs on bees (EFSA, 2013e). The and this may be related to increased honey bee mortal- document is intended to provide guidance for notifiers ity events. In Italy, for example, fairly isolated honey bee and authorities in the context of the review of PPPs and incidents have been described since 2000, but the sever- the active substances they contain under Regulation ity of events increased dramatically in the spring of 2008 (EC) No. 1107/2009. The guidance document suggested (Bortolotti et al., 2009). In 2009, a national monitoring the implementation of a tiered risk assessment scheme network was created (acronym ApeNet), showing that starting with a simple, cost-effective first tier and dust dispersion during sowing with neonicotinoid-coated extending to more complex higher tier studies under maize seeds could lead to honey bee mortality (ApeNet field conditions. Report, 2009; Sgolastra et al., 2012). Systemic pesticides Honey bees are endangered not only by the acute enter the plant’s vascular system and spread throughout toxicity of pesticides but also by their sub-lethal the stems and leaves, guttation water, pollen and nectar (Desneux, Decourtye, & Delpuech, 2007), chronic and during plant growth (Bonmatin et al., 2015). In Italy, delayed lethal effects (Rondeau et al., 2014), depending authorization to use the active ingredients clothianidin, on the specific mode of action of the active ingredient. thiamethoxam, imidacloprid and fipronil for seed dress- A growing number of publications indicate that com- ings was suspended with a decree dated 17/09/2008 bined exposure to individually non-lethal stressors can (Italian Ministry of Health, 2008), and further extended have a harmful effect on honey bees. These encompass to maize seeds until 30/06/2013. A decree dated 25/06/ very diverse types of interaction not only among pesti- 2013 (Italian Ministry of Health, 2013) definitively cides (Biddinger et al., 2013; David et al., 2016; Johnson, ordered the withdrawal of marketing authorizations and Dahlgren, Siegfried, & Ellis, 2013; Rundlo¨f et al., 2015; the use of PPPs containing the active ingredients clothi- Sanchez-Bayo & Goka, 2014; Thompson & Wilkins, anidin, thiamethoxam and imidacloprid for the treatment 2003), but also between pathogens and pesticides (Alaux of seeds and soil, according to Commission Implement- et al., 2010a; Di Prisco et al., 2013; Doublet, Labarussias, ing Regulation No. 485/2013 (EU, 2013a). This regula- de Miranda, Moritz, & Paxton, 2014; Simon-Delso et al., tion banned the use of the three neonicotinoids for 2014; Vidau et al., 2011), and among different pathogens seed treatment, soil application and foliar treatment of (Dainat, Evans, Chen, Gauthier, & Neumann, 2012;Di plants and cereals attractive to bees. This regulation was Prisco et al., 2011; Genersch & Aubert, 2010; Ryabov followed by Commission Implementing Regulation No. et al., 2014). Recent studies have focused on the syner- 781/2013 concerning the approval conditions for the gistic toxicity of several pesticides on honey bee health, active substance fipronil, which prohibits the use and since most pesticides, herbicides, fungicides and acari- sale of seeds treated with PPPs containing this active cides are not highly toxic to honey bees alone, whereas substance (EU, 2013b). Neonicotinoids still remain a in combination there may be heightened toxicity due to subject of intense debate, as clearly also denoted by the interactive effects (Biddinger et al., 2013; Johnson et al., European Food Safety Authority (EFSA) (EFSA, 2013, 2013; Thompson & Wilkins, 2003). It is well known that Honey bee mortality in northeastern Italy 3 low concentrations of pesticides may act as stressors with a total of 79 dead honey bee samples and 15 “re- that make honey bees more susceptible to pathogen lated” matrices, as beeswax (10–50 g), bee bread infections (Alaux et al., 2010a; Di Prisco et al., 2013; (5–15 g), soil, leaves and maize seeds. These non-bee Doublet et al., 2014; Simon-Delso et al., 2014; Thomp- matrices are currently sampled as part of the epidemio- son & Wilkins, 2003). Simon-Delso et al. (2014) showed logical investigation carried out by local veterinary ser- a significant correlation between the presence of fungi- vices after a bee incident is reported, in accordance cide residues and honey bee colony disorders (mainly with Italian national guidelines for the management of caused by deformed wing virus (DWV), black queen cell mortality or depopulation of hives related to the sus- virus (BQCV), and Sac Brood Virus, SBV), while Di pected use of pesticides (Italian Ministry of Health, Prisco et al. (2013) showed that two neonicotinoids, 2014). The samples, individually packed in plastic sam- clothianidin and imidacloprid, but not the organophos- pling bags to avoid cross contamination, were collected phate chlorpyrifos, actively promote DWV replication by both local veterinary services and beekeepers (only due to immunosuppression. Pathogen proliferation four samples, one honey bee sample and three other induced by these neonicotinoid insecticides can thus matrices). The proper storage of all samples was guaran- cause additional mortality, even at sub-lethal doses, and teed by immediate freezing after collection. All samples may contribute to the observed negative influence of delivered to the laboratory were stored at −20 ˚C and some insecticides, or their mixtures, on honey bee long- at −80 ˚C for pesticide and honey bee virus analyses, evity and colony stability. Honey bee viruses have also respectively. Most samples were accompanied by a been reported as a factor contributing to the decline of reporting form completed by the official veterinarian honey bee colonies, usually in association with a foreign with a detailed description of the incident, the number substance (Doublet et al., 2014) or a stressor, as in the of hives concerned, the geographical coordinates, and presence of Varroa destructor infestation (Carreck, Ball, the main, visually assessed nearby crops and clinical & Martin, 2010; Dainat et al., 2012; de Miranda, symptoms of the honey bees (Porrini et al., 2016). The Cordoni, & Budge, 2010; de Miranda & Genersch, 2010; reporting form used to collect information was part of Di Prisco et al., 2011; Francis, Nielsen, & Kryger, 2013; the Italian national guidelines for the management of Genersch & Aubert, 2010; Guzmań-Novoa et al., 2010; mortality or depopulation of hives related to the Le Conte et al., 2010; Ryabov et al., 2014). suspected use of pesticides, and was filled in by the Honey bee viruses persist at low levels in honey bee veterinarians or beekeepers (Italian Ministry of Health, populations without causing overt symptoms, although 2014). The geographical origin of the samples is shown under certain conditions they can become pathogenic, in Figure 1. leading to colony damage and mortality (Traynor et al., 2016). Stress conditions of bees can make covert honey bee virus infections symptomatic (DeGrandi-Hoffman, Pesticide analysis Chen, Huang, & Huang, 2010; Di Prisco et al., 2011, A total of 150 active ingredients (see Table S1) were 2013). Varroa-mediated virus transmission has been clo- studied in this work, using the QuEChERS method sely related to increasing virus transmission and colony followed by high performance liquid chromatography cou- health troubles (vanEngelsdorp, Tarpy, Lengerich, & pled with mass spectrometry (HPLC-MS) or gas chro- Pettis, 2013; Le Conte et al., 2010; Locke, Forsgren, matography coupled with electron capture detection Fries, & de Miranda, 2012). (GC-ECD) analysis. Reference standards with certified Given that incidents involving honey bees have purity were purchased from Sigma Aldrich (St. Louis, MO, recently aroused great interest, this work investigates USA) and a small number from Dr. Ehrenstorfer GmbH the potential causes of mortality events occurring in the (Augsburg, Germany). All solvents were of HPLC grade spring of 2014 in northeastern Italy, evaluating the pres- and purchased from Sigma Aldrich and VWR International ence of pesticides and viruses in honey bees and in PBI s.r.l. (Milan, Italy). Pure water was prepared from a other related matrices submitted to our laboratory. This Milli-Q water purification system (Millipore, Bedford, MA, study is a forensic report that points to possible ‘causes USA). of mortality’, but cannot in any case demonstrate which of the suspected agents are the cause. Sample extraction and clean-up The honey bees or other matrices were treated in the Materials and methods same way and the analytical method was a modification Sampling of that of Anastassiades, Lehotay, Stajnbaher, and The Istituto Zooprofilattico Sperimentale delle Venezie Schenck (2003). The different samples (except bee (IZSVe) is an ISO/IEC 17025 accredited public health bread) were previously pulverised with a grinder (A11 body established in the framework of the National basic IKA-Werke GmbH & Co. KG, Staufen, Germany) Health Service. From April to June 2014 several samples and cooled with liquid nitrogen for proper homogenisa- were received following honey bee mortality incidents, tion. Water (10 ml) was added to the sample (2 g) and 4 M. Martinello et al.

Figure 1. Geographical origin of the samples in the territory of northeastern Italy.

vortexed for 10 min. Acetonitrile with 0.1% acetic acid (at least 5) per detection system were calculated in each (10 ml) was added to the mixture, vortexed for 20 min batch of analyses. A practical default range of 60–140% was and cooled at −20 ˚C for 15 min. QuEChERS salts (EN used for individual recoveries in routine multi-residue analy- method) (sodium citrate 1 g, sodium hydrogen citrate sis. The limit of quantification (LOQ) for analytes detected − sesquihydrate 0.5 g, magnesium sulphate 4 g and sodium with both LC and GC methods was 10 μgkg 1. chloride 1 g) were added and vigorously shaken for 10 min. The mixture was centrifuged (10 min, 3500 xg) and the supernatant (8 ml) transferred to a tube con- LC-MS analysis taining dispersive SPE purification salts for fatty samples The residue was dissolved in reconstitution solution (EN method) (magnesium sulphate 900 mg, PSA 150 mg (0.5 ml) composed of 5 mM ammonium formiate in and C18 150 mg). The solution was vortexed (1 min) water with 0.1% formic acid (mobile phase A), and and centrifuged (10 min, 3500 ×g), and the supernatant 5 mM ammonium formiate in methanol with 0.1% formic (6 ml) transferred to a clean tube and evaporated to acid (mobile phase B) (1 + 1 by volume). LC-MS analysis dryness under a stream of nitrogen at 45 ˚C. was performed on a Shimadzu LCMS-2010 EV (Kyoto, − Fortified samples (50 μgkg 1 for LC-MS and Japan), with a single quadrupole analyzer, using an elec- − 100 μgkg 1 for GC-ECD) were prepared by adding the tron spray ionization source in the positive ion mode. working standard mixture to a blank matrix prior to extrac- Chromatography was performed on a Supelco Analytical tion and, after standing for 30 min, processed as described (Bellafonte, PA, US) Ascentis Express RP-Amide column above. The blank matrices consisted of samples sent to our (10 cm × 2.1 mm, 2.7 μm-particles) with an adequate laboratory or specifically purchased for use as negative con- guard column. The chromatographic eluting condi- trols. They had previously been analysed and had tested tions were optimized as follows: from 5 to 10% negative for the selected active ingredients. Residue con- B (0–1.5 min), from 10 to 48% B (1.5–17 min), from 48 centrations were calculated using solvent-based calibration to 95% B (17–18 min), 95% B maintained for 3 min, curves and the results were recovery corrected (recoveries from 95 to 5% B (21–22 min), and then re-equilibration experimentally determined at each work session). Routine to 5% B for a further 4 min. The flow rate was − recovery checking complied with DG SANCO/12571/2013 0.3 ml min 1 and the injection volume was 10 μl. The (2013) and the recoveries of 10% of representative analytes column was thermostated at 40 ˚C. Honey bee mortality in northeastern Italy 5

GC-ECD analysis (NanoDrop Technologies Inc., Wilmington, DE, USA). − The residue was dissolved in heptane (1 ml) with an RNA was stored at 80 ˚C until use. − internal standard (PCB 209, 200 μgkg 1). GC-MS analysis was performed on a Shimadzu GC 2014 equipped In vitro transcription and quantification of cRNAs with a Restek dual column system (Superchrom S.r.l., The in vitro transcribed RNA (cRNA) of ABPV, CBPV Milan, Italy), an RTX-CLPesticides kit (CLP column and DWV was obtained from a specific PCR 30 m, 0.25 mm ID, 0.25 μm; CLP2 column 30 m, Ni product confirmed by sequencing, cloned into the 0.25 mm ID, 0.20 μm) and a dual 63 electron capture ® pCR™II-TOPO vector (Invitrogen, Carlsbad, CA, detector (ECD). A sample volume of 0.5 μl was injected USA), transcribed to RNA and quantified spectrophoto- in the split mode at an injector temperature of 120 ˚C. metrically. The primers (see Table S2) were designed The oven temperature program was as follows: from viral genome sequences deposited in the GenBank, initial temperature 60 ˚C (held for 2 min), increased by using Primer Express software (Applied Biosystem, Fos- 70 ˚C/min to 200 ˚C; increased by 2 ˚C/min to 300 ˚C ter City, CA, USA). (held for 1 min); increased by 40 ˚C/min to 310 ˚C and Specifically, each PCR product was ligated into the held for 1 min. The detector temperature was held at ® pCR™II-TOPO cloning vector containing the T7 pro- 320 ˚C. Nitrogen served as make-up gas with a flow moter sequence and transformed into One Shot TOP10 rate of 3.05 ml/min. electrocompetent E. coli (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s instructions. Recom- binant plasmid DNA was purified with the GenEluteTM Virus analyses Plasmid Miniprep Kit (Sigma Aldrich Co., St. Louis, MO, Fifty-nine honey bee samples were analyzed using one- USA). The presence and correct orientation of the step quantitative real time RT-PCR (qPCR) to monitor insert was confirmed by M13 PCR, sequencing, and the presence and quantify (absolute quantification) three restriction endonuclease digestion. Plasmid DNA was honey bee viruses: acute bee paralysis virus (ABPV), linearized by restriction enzyme digestion and then sub- chronic bee paralysis virus (CBPV) and DWV. The jected to in vitro transcription using RiboMaxTM Large choice of these viruses was based on the results of pre- Scale RNA Production Systems–T7 (Promega, Madison, vious investigations carried out in Italy, namely: the Ape- WI, USA), according to the manufacturer’s instructions. net (Porrini et al., 2016) and Beenet (http://www.

cRNA concentration was determined by a Nanodrop reterurale.it) nationwide monitoring projects (2009– N1000 spectrophotometer; viral copy numbers were 2014). DWV was the most highly detected (prevalence equivalent to 1.4 × 1013 molecular copies per μl for of 100%) while ABPV and CBPV showed a lower preva- ABPV, 5.6 × 1012 molecular copies per μl for CBPV, lence (approximately 50–60%), but were considered to and 1.0 × 1013 molecular copies per μl for DWV. be particularly relevant in terms of possible effects on honey bees. Under the umbrella of the above-men- tioned monitoring projects, extensive nationwide inves- Absolute quantification using qPCR tigations were carried out on Nosema apis and N. Specific primers and probes for DWV and ABPV were ceranae in 2009–2014. N. apis was only detected on two designed, using Primer Express software and based on all occasions in 2009, while N. ceranae showed a nation- complete viral sequences present in the GenBank database. wide prevalence of about 50%. Accordingly, we did not The specific primers and probe for detecting CBPV, previ- analyze the dead bees found for these pathogens in the ously developed by Blanchard et al. (2007), were slightly present forensic investigation. modified (see Table S2). The specificity of the primers and probes was confirmed by in silico analysis (Blast search) of the amplicon generated by each qPCR and no similarity Sample collection and RNA extraction was found with other sequences in the database. From each sample, five honey bees were individually The qPCR reactions (one for each virus) were per- TM homogenized in Lysis buffer RA1 (700 μl) (Macherey formed on CFX96 Touch Real-Time PCR (BIO-RAD, Nagel GmbH, Du¨ren, Germany) with a bead mill Hercules, CA, USA). For each sample, 250 ng of total homogenizer (Tissue Lyser II; Qiagen GmbH, Hilden, RNA (5 μl) was analysed using a QuantiTect Probe RT- Germany). After centrifugation (5 min, 10,000 ×g), the PCR Kit (Qiagen, Hilden, Germany) in a final volume of supernatants were pooled and an aliquot (350 μl) was 25 μl, containing a final concentration of 0.8 and 0.2 μM used for RNA isolation. Total RNA was extracted using for each primer and probe, respectively. The thermal the Nucleo Spin RNA kit (Macherey Nagel GmbH, cycling conditions consisted of 30 min at 50 ˚C (reverse Du¨ren, Germany). Negative controls (Lysis buffer RA1) transcription), 15 min at 95 ˚C (HotStartTaq DNA Poly- were processed in parallel to detect possible contamina- merase activation) followed by 45 cycles of denaturation tions. The yield and purity of the extracted RNA at 94 ˚C for 15 s and annealing/extension at 60 ˚C for (260/280 and 260/230 nm absorbance ratios) were 1 min. Negative controls (water for molecular biology assessed with a Nanodrop N1000 spectrophotometer applications) were included in each qPCR. 6 M. Martinello et al.

The detection and quantification limits of qPCR for (Mann–Whitney) test for two independent samples was each virus were determined from 10-fold serial dilutions performed with an adjusted alpha (0.05/6 = 0.0083). The of the cRNA, covering the range of 107–102 viral RNA association between categorical variables (i.e., presence/ copies per reaction. Furthermore, to determine the absence of pesticides or region of origin) was evaluated limit of detection (LOD), 50 and 25 viral RNA copies by Fisher’s exact test, when one of the expected values per reaction were also tested. For each virus, two inde- was below 5. The associations between quantitative pendent runs were performed by analysing 5 μl of each variables (i.e., number of viral copies per bee and dilution in triplicate. LOQ was 102 viral copies per bee, concentration of active ingredients) were evaluated by and LOD was 50 viral copies per bee, for each virus. the two-way scatter graph and their strengths measured Ten-fold serial dilution of cRNA transcripts in nucle- by the non-parametric Spearman’s rank correlation ase-free water (107–102 viral RNA copies per reaction) coefficient. was used to generate a standard curve and to quantify RNA in the samples. Triplicates of each dilution were run in each assay. Three independent standard curves, Results one for each virus, were generated by plotting the Determination of pesticide residues threshold cycle (Cq) values against the logarithm of the Table 1 summarizes the results of active ingredient calculated initial copy numbers. The Cq value was determined for each sample run in duplicate. The virus copy determination in the present study and detailed results numbers were calculated using the equation are shown in Tables S3 and S4. In total, 27 different x ¼ 10CqðyinterceptÞ=slope, where x represents the genome active ingredients were found in honey bee samples, with concentrations ranging from 1 to 411.3 ng per bee; copies/bee, to transform the average Cq values of the samples into estimates of genome copies of virus RNA 28 different active ingredients were identified in the μ −1 per individual bee. other matrices analysed, ranging from 10 to 247 gkg μ −1 For those samples that exceeded the standard curve in beeswax and from 12 to 232 gkg in bee bread. by107 viral RNA copies, an appropriate dilution was The main crops reported in the honey bee forage performed and analysed. The resulting standard curves range were maize crops (39.2%), mostly located in the showed high efficiency, ranging from 96.3 to 106.2% for provinces of Udine and Rovigo (northeastern Italy), ABPV, from 98.6 to 103.4% for CBPV, and from 95.9 to apple orchards (33.3%), almost all located in the pro- 2 vince of Bolzano (northern Italy), and vineyards (35.3%), 105.6% for DWV. The R was 0.99 for all viruses (see Figure S1). which had a more varied distribution. Quantitative results were expressed as the number Regarding the identified clinical symptoms, 63.6% of of viral copies per bee and calculated by taking into con- the reports indicated the presence of dying honey bees sideration the amount (μl) of sample used in each step with tremors and 52.3% with uncoordinated move- (sample homogenization, RNA extraction and qPCR). ments. Inability to fly and/or move, increased aggression, and everted proboscis were reported to a lesser extent. Classical honey bee diseases, as European or American Statistical analysis foulbrood, nosemosis or varroosis, were not clinically The data obtained were stored in an ad hoc database confirmed in any incident. and statistical analyses were performed using STATA Residues of many different pesticides were found 12.1 software. The total numbers of fungicide and insec- and most investigated samples were positive for at least ticide residues were calculated by pooling together the one active ingredient (75.8%). Most samples were con- values of the pesticides belonging to these two cate- taminated by more than one residue: 65.3% of the sam- gories. A descriptive analysis was performed: the quanti- ples were contaminated by at least two different tative variables were analyzed by calculating statistical residues, 36.1% by at least 3, with one extreme case, measures, as the median value (p50), and examined from Verano municipality (Bolzano province, northern using vertical box plots. Italy), presenting residues of as many as 11 different The Shapiro-Francia W’ test was used to evaluate data active ingredients. Interestingly, the highest contamina- distribution. The results (p < 0.05) showed that the data tion, as confirmed by the reports attached to the sam- did not follow normal distributions, not even for log- ples, was detected in the province of Bolzano, a transformed virus data, so non-parametric tests were geographical area characterized by intensive apple used. To compare quantitative variables between inde- orchards. pendent samples (i.e., regions), Levene’s robust test Insecticides were the most frequently detected sub- statistic for the equality of variances between the groups stances (59.4%) in honey bees (Tables 1 and S3). Neoni- was preliminarily performed. When the homoscedasticity cotinoids were the most commonly identified (41.8%), hypothesis was satisfied (W50 p-value > 0.05), the and were mainly imidacloprid (19 out of a total of 33 Kruskal–Wallis equality-of-populations rank test was used samples), followed by thiacloprid (8/33) and thiame- for more than two samples. For pairwise comparison toxam (6/33). Acaricides were 24.1% (tau-fluvalinate in between regions, the two-sample Wilcoxon rank-sum most samples, with only 2 testing positive for phosmet Honey bee mortality in northeastern Italy 7

Table 1. Summary of active ingredients detected in the samples analysed.

Honey bees Other matricesa − Active ingredient Prevalence (%) Range (ng per bee) Prevalence (%) Range (μgkg 1) Acrinathrin – – 12 24–41 Azoxystrobin 1 1.3 – – Bromopropylate – – 6 15 Captan – – 6 11 Carbaryl 1 11.7 – – Carbofuran 1 2.9 – – Chlorothalonil 4 3.0–17.0 – – Chlorpyrifos 22 3.5–335.0 12 38–8000 Clothianidin – – 6 10 Coumaphos – – 6 68 Cypermethrin 4 3.2–390.0 – – Cyprodinil 15 1.8–33.0 6 232 Dimethomorph 2 4.7–53.2 6 8534 Dodine 10 1.3–411.3 25 13–247 Fludioxonil 1 1.5 31 12–2415 Fluvalinate 20 2.0–187.4 25 35–154 Folpet 4 1.0–3.0 6 42000 Imidacloprid 24 1.0–205.9 12 33–60 Kresoxim-M 1 4.5 – – Lambda-Cyhalothrin – – 6 2280 Metalaxyl 5 1.0–19.9 12 10–242 Metalaxyl-M – – 12 10–426 Methiocarb – – 12 27–38320 Methiocarb sulfoxide – – 6 3246 Metrofenone 1 8.7 – – Myclobutanil – – 6 36000 Phosmet 2 2.2–3.4 – – Piperonyl Butoxide 6 1.0–398.4 12 17–42 Pirimicarb – – 12 10 Propiconazole – – 6 12 Pyraclostrobin 6 2.9–39.7 6 28 Pyrimethanil 4 1.0–2.8 25 10–1976 S-metolachlor – – 19 17–2511 Tebufenozide – – 6 244 Terbuthylazine 1 2.8 25 15–1026 Tetramethrin 1 n.q.b –– Thiacloprid 10 1–37.1 – – Thiamethoxam 8 1.0–34.6 19 41–4747 Thiophanate methyl 8 1.0–6.5 – – Thiram – – 12 594–10799 Trifloxystrobin 2 3.6–16.7 6 20 Triticonazole 2 4.0–4.5 – – aBeeswax, beebread, soil, leaves and maize seeds. bNot quantifiable. and 1 for carbofuran). Fungicides were also often It is also worth mentioning that some non-autho- detected (40.6%), with a wide variety of active ingredi- rized active ingredients were detected: carbaryl, an ents. Overall, the most frequently found active ingredi- obsolete carbamate pesticide, introduced around 1957 ents were imidacloprid, chlorpyrifos, tau-fluvalinate and and currently not approved for use in the EU (EU, cyprodinil. The highest concentrations in the honey bee 2007a); carbofuran, one of the most toxic carbamate samples were detected for chlorpyrifos and imidaclo- pesticides, banned in the EU since December 2007 (EU, prid, in terms of both mean and absolute values (exclud- 2007b) because the related dietary, worker, and ecologi- ing two samples from Bolzano and Trento provinces, cal risks make all its uses unacceptable; and tetram- northern Italy, contaminated respectively with dodine ethrin, a pyrethroid insecticide registered in 1968, and piperonyl-butoxide, at 411.3 and 398.4 ng per honey which is highly toxic for honey bees and has never been bee). Considering the veterinary medicines currently authorised for use in crop protection (EU, 2016; PPDB, authorized in Italy, tau-fluvalinate and amitraz could 2016). possibly originate from Varroa-control treatments As shown in Table 1 (and Table S4), all seven bees- (Mutinelli, 2016). wax samples were contaminated by at least one active 8 M. Martinello et al. ingredient, mostly insecticides (54.5%), with a wide pres- cantly different (Kruskal–Wallis test: p-value = 0.4696), ence of acaricides (66.7%), and fungicides (45.4%). The except for Bolzano province, which showed the highest four bee bread samples were also positive for at least variability in median values of insecticides (see Figure 2). one residue, equally distributed among fungicides, insecticides and herbicides. Other matrices were collected in accordance with the Italian national monitoring program Virus analysis for bee incidents that may possibly be related to Fifty-nine honey bee samples were investigated by qPCR the use of pesticides (Italian Ministry of Health, 2014). for the detection and absolute quantification of ABPV, The highest number of active ingredients per sample (5, CBPV and DWV. The results are summarised in sup- 6 and 7 respectively), belonging to all the different porting information Table S3. All the samples tested classes except acaricides, were detected in high concen- positive for more than one virus with the exception of trations in maize seeds and soil. Six different pesticides, one sample, which was only positive for CBPV. Of the mainly fungicides, with the highest concentrations were samples, 61% were positive for three viruses, 38% found in vine leaves. The fungicide Folpet displayed the tested positive for CBPV and DWV, and only one was − highest concentration, with 42,000 μgkg 1. positive for DWV and ABPV. At the individual virus Statistical analysis of the data showed no significant level, the samples were 99% positive and quantifiable for association between the presence of pesticides and the CBPV, with values between 3.5 × 104 and 1.0 × 1013 region of origin of the honey bee samples (Fisher’s exact viral copies per bee; 95% for DWV, with values test: p-value = 0.371) (see Table 2). Using Levene’s test between 2.1 × 104 and 8.0 × 1011 viral copies per bee; with a p-value > 0.05, no statistical differences were and 39% for ABPV, with values between 1.4 × 104 found between regions for fungicide residues (Kruskal– and 7.1 × 1010 viral copies per bee. Among the three Wallis test: p-value = 0.9606), or insecticide residues; investigated viruses, ABPV showed the lowest preva- since the p-value of Levene’s test was <0.05, no com- lence. Of the CBPV positive samples, 71% showed a parison was made. The homogeneity of variance test very high number of viral copies per bee (>107); similar showed distributions that were not statistically signifi- results were observed for 37%, and only 12% of positive

Table 2. Comparison between presence of pesticides and relative regions (n = samples with assigned value; p50 = median value) in dead honey bees.

Friuli Venezia Province of Province of Veneto Giulia Trento Bolzano PPP Substance groupa n p50 n p50 n p50 n p50 Azoxystrobin FU 0 1 1.3 0 0 Carbaryl IN 0 1 11.7 0 0 Carbofuran IN 1 2.9 0 0 0 Chlorothalonil FU 0 0 0 3 3.0 Chlorpyrifos IN 3 7.2 0 1 3.5 13 52.8 Cypermethrin IN 1 3.2 0 0 2 198.85 Cyprodinil FU 0 0 1 6.2 11 7.6 Dimethomorph FU 1 53.2 1 4.7 0 0 Dodine FU 2 5.75 0 2 207.3 4 4.0 Fludioxonil FU 0 0 0 1 1.5 Fluvalinate IN 3 5.3 3 4.9 2 95.7 8 5.65 Folpet FU 2 2.0 0 0 1 2.5 Imidacloprid IN 3 1.4 0 0 16 26.55 Kresoxim-M FU 0 0 0 1 4.5 Metalaxyl FU 2 10.55 0 0 2 1.0 Metrofenone FU 0 1 8.7 0 0 Phosmet IN 1 2.2 0 0 1 3.4 Piperonyl Butoxide SY 3 2.5 1 1.0 0 1 398.4 Pyraclostrobin FU 1 4.0 1 39.7 1 3.3 2 4.75 Pyrimethanil FU 0 1 2.4 1 2.8 1 1.0 Terbuthylazine HB 0 1 2.8 0 0 Tetramethrin IN 0 0 0 0 Thiacloprid IN 0 0 1 1.5 7 1.5 Thiamethoxam IN 5 15.3 1 1.9 0 0 Thiophanate methyl FU 4 4.95 2 2.75 0 0 Triﬂoxystrobin FU 1 16.7 0 1 3.6 0 Triticonazole FU 1 4.5 0 1 4.0 0 Total 34 14 11 74 aFU, fungicide; HB, herbicide; IN, insecticide; SY, synergist. Honey bee mortality in northeastern Italy 9

Figure 2. Box plot of number of insecticide (a) and fungicide (b) residues by region. Notes: The white box represents the middle 50% of the observations, with the median at the midline. The whiskers extend to the largest or smallest observations up to 1.5 times the box height above or below the box. The points/dots mark the observations falling outside the box and whiskers (outliers) (BZ, Bolzano; FVG, Friuli Venezia Giulia; TN, Trento). (b) The value of the fungicide residue Dodine, 400 ng per honey bee, has not been shown.

and pairwise comparisons yielded signiﬁcant differences only between Bolzano province and Friuli Venezia Giulia region, and between Bolzano province and Veneto region (two-sample Wilcoxon rank-sum [Mann–Whit- ney] test: p-value = 0.0065 and 0.0019 respectively) (see Figure 3). Comparison of viruses, in the presence and absence of pesticides and assuming homogeneity of variances,

produced no statistically significant differences in ABPV and DWV values (Wilcoxon Mann–Whitney test: p- value = 0.1981 and 0.6227, respectively). No comparison was made for CBPV since Levene’s p-value was <0.05. Spearman’s correlation coefficient, calculated to mea- sure the strength of association between viruses and pesticides, showed a moderately negative correlation Figure 3. Box plot of number of CBPV copies per bee by region. between CBPV and fungicides, and CBPV and insecti- Notes: The white box represents the middle 50% of the cides (Spearman’s rho = −0.3641 and Spearman’s observations, with the median at the midline. The whiskers rho = −0.3991, respectively; both p-values < 0.01). extend to the largest or smallest observations up to 1.5 times the box height above or below the box. The points/dots mark the observations falling outside the box and whiskers (outliers) Discussion (BZ, Bolzano; FVG, Friuli Venezia Giulia; TN, Trento). A significant difference was found in CBPV values between regions Bolzano was the province with the highest number of (Kruskal–Wallis test: p-value = 0.0038), and pairwise compar- honey bee incidents (33% of the total honey bee sam- ison (two-sample Wilcoxon rank-sum [Mann–Whitney] ples submitted to our laboratory). According to the Ital- test) yielded significant values between BZ and FVG ian Institute of Statistics, an average of 53.9 kg of active (p-value = 0.0065) and between BZ and Veneto (p-value = 0.0019) only, but not between FVG and Veneto ingredient per hectare (ha) is used per year in the (p-value = 0.6067), TN and Veneto (p-value = 0.0710), TN and region of Trentino Alto-Adige, against an Italian average FVG (p-value = 0.1052) or TN and BZ (p-value = 0.5050). of 10 kg per ha per year, with peaks of up to 90 kg per ha per year in intensive farming areas as the Val di Non, DWV and ABPV samples, respectively. Six honey bee located between the provinces of Trento and Bolzano, samples showed a very high number of viral copies per which are mainly cultivated with apple orchards (Anony- bee (>107) for all three viruses. mous, 2014). Based on the Usable Agricultural Area Statistical comparison of virus loads among regions, (ISTAT, 2012), 1.37 apiaries per km2 were registered in assuming the homogeneity of variances, showed not sta- the autonomous province of Bolzano, 1.78 in the auton- tistically significant differences in ABPV and DWV values omous province of Trento, 1.25 in the region of Friuli (Kruskal–Wallis test: p-value = 0.9506 and 0.3575, Venezia Giulia and 1.08 in the region of Veneto. The respectively). There was instead a significant difference reported number of bee incidents was 26 (0.77% in CBPV values (Kruskal–Wallis test: p-value = 0.0038), out of 3,352 apiaries), 9 (0.37% out of 2,404 apiaries), 10 M. Martinello et al.

15 (0.55% out of 2,718 apiaries), and 29 (0.33% out of poisoning with lethal effect, and potentially sub-lethal 8,783 apiaries), respectively (Table S3). These incidents harm, to pollinators. These seven priority chemicals do not appear to be linked to the density of apiaries in were: imidacloprid, thiamethoxam, clothianidin, fipronil, the concerned territories, but are more related to the chlorpyrifos, cypermethrin and deltamethrin. In the pre- number of apiaries and to land use, e.g., fruit orchards, sent study, only fipronil and deltamethrin remained sown land and vineyards. undetected, while imidacloprid and chlorpyrifos were The clinical symptoms reported in honey bees, as the most frequently identified substances. Imidacloprid tremors and uncoordinated movements, are among the and chlorpyrifos were also included as high risk com- nervous symptoms caused by neonicotinoid poisoning pounds by Sańchez-Bayo and Goka (2014), correspond- (Blacquie`re, Smagghe, van Gestel, & Mommaerts, 2012), ing to an estimated time of 2 days or less to reach the consistent with the high amounts of imidacloprid LD50 per honey bee, considering topical LD50 to be 60 detected, ranging from 1.0 to 205.9 ng per bee. In its and 70 ng per honey bee (gathering pollen in the field), risk assessment for this active substance in bees, EFSA and oral LD50 to be 13 and 24 ng per honey bee (by concluded that the median lethal dietary doses (LD50) dietary and chronic ingestion of contaminated nectar for acute toxicity was 81 ng per bee for contact expo- and pollen), respectively. Both were the most frequently sure and 3.7 ng per bee for oral exposure (EFSA, found active ingredients in our honey bee samples, and 2015a). According to these LD50 values, the imidaclo- the detected concentrations often exceeded the above prid concentrations detected in this forensic study cited levels. appear to be compatible with a toxic effect on bees. Nevertheless, it is not always possible to determine Tremors can also be caused by certain types of virus, a direct link between a honey bee incident and the including CBPV and ABPV (Genersch & Aubert, 2010). amount or specific molecules of pesticides detected in The high number of honey bee samples contami- the matrices. Honey bees encounter multiple classes of nated with PPPs (72.2%) confirms that the problem pesticides when foraging in the field, potentially exposing arises from intensive agriculture and probably from the them to an array of combinatorial effects. The lethal but improper use of pesticides. The data reported here, and also the sub-lethal effects of pesticides on honey bees, in many of the studies cited in this work, have provided due to the additive and synergistic properties of many further evidence that honey bees are exposed to a wide active ingredients, or in association with pathogens, are spectrum of pesticides. We found 42 different active now well documented (Alaux et al., 2010a; Biddinger ingredients in our samples, and frequently more than et al., 2013; David et al., 2016; Di Prisco et al., 2013; one in the same sample, due to the use of a wide range Doublet et al., 2014; Johnson et al., 2013; Rundlo¨f et al., of chemicals and tank-mixes of insecticides and fungi- 2015; Sanchez-Bayo & Goka, 2014; Simon-Delso et al., cides in agricultural production systems. Furthermore, 2014; Thompson & Wilkins, 2003), but still require fur- repeated treatments even at short time intervals, ther study. Retschnig et al. (2015), for example, showed especially in the presence of specific weather conditions, the effects, but no interactions, of ubiquitous pesticide as in the very rainy spring of 2014, could have con- and parasite stressors on honey bee lifespan and behav- tributed to further worsening the situation. ior in a colony environment. Furthermore, Collison, Honey bees can be contaminated in many different Hird, Cresswell, and Tyler (2016) critically reviewed the ways, both outside and inside the hive, for example by literature and showed that pesticide exposure and direct or indirect contact with crop spray treatments or pathogen infection have not yet been found to interact dust dispersed during coated seed sowing (ApeNet to affect worker survival under field-realistic scenarios. Report, 2009), by ingestion of guttation droplets (Giro- The implications of pesticide effects on Nosema infec- lami et al., 2009), irrigation waters (Porrini, 2010), nec- tions, viral loads and honey bee immunity at the colony tar, pollen and bee bread (Dively & Kamel, 2012; Stoner level remain unclear, as these effects have been & Eitzer, 2012), or by contact with the residues in wax observed in the laboratory setting only. Such potential (Bogdanov, Kilchenmann, & Imdorf, 1997; Wu, Anelli, & impacts should be closely considered and explored to Sheppard, 2011; Zhu, Schmehl, Mullin, & Frazier, 2014). improve investigations into honey bee stressors in natu- Internal contamination can also occur from in-hive use ral conditions. Johnson, Pollock, and Berenbaum (2009) by beekeepers of miticides as tau-fluvalinate and amitraz, stated that honey bee mortality may occur with the which are veterinary medicines currently authorized for application of otherwise sub-lethal doses of miticides use in Italy (Mutinelli, 2016). In our study 72.2% of when tau-fluvalinate and coumaphos are simultaneously honey bee samples were contaminated, but all other present in the hive. Fungicides have long been consid- analyzed matrices also tested positive for a wide variety ered not to be dangerous for honey bees, by virtue of of active ingredients, often at the maximum residue con- their low acute toxicity, but it is now known that there centrations. is an association with poor colony performance and col- Based on the impacts on honey bees and other polli- lapse (Sańchez-Bayo et al., 2016; Simon-Delso et al., nators, Tirado, Simon, and Johnston (2013) drew up a 2014). Fungicides are able to modify the existing micro- list of bee-harming pesticides that should be eliminated flora in bee bread (Joder et al., 2013) and in the honey from the environment in order to avoid any acute bee intestinal tract (Anderson, Sheehan, Eckholm, Mott, Honey bee mortality in northeastern Italy 11

& DeGrandi-Hoffman, 2011), and may therefore have often persist in colonies as latent infections located in both a direct effect on honey bee health, and an indirect honey bees’ fat bodies, with no consequences for colony impact on colony development. Furthermore, many health, and of little concern to beekeepers. Nonetheless, studies have proven the harmful effects of such com- the negative effects of a single viral infection, such as pounds, which diminish honey bees’ detoxification Kashmir virus (Cox-Foster et al., 2007), or of the simulta- mechanisms, thereby exacerbating the negative effects neous presence of viruses and other pathogens are well of other insecticides (Christen, Mittner, & Fent, 2016; known (Alaux et al., 2010a; Di Prisco et al., 2011; Ryabov Iwasa, Motoyama, Ambrose, & Roe, 2004; Johnson et al., 2014). In our study, all three investigated viruses et al., 2013; Pilling & Jepson, 1993; Thompson & Wilkins, were frequently detected, in some cases in the same 2003). According to our results, the number of fungicide honey bee sample, and with a high prevalence of CBPV residues seems to play a role in the appearance of and DWV. This finding is in accordance with the results honey bee colony disorders. Hence the study of the of previous investigations carried out in Italy (Porrini residue load of these pesticides in honey bees opens et al., 2016; http://www.reterurale.it). However, in said new avenues for better understanding of honey bee col- investigations live asymptomatic honey bees were sys- ony disorders. tematically collected from the combs several times a year Regarding other active ingredients found in the pre- (spring-summer-autumn) without any bee incidents being sent study, the synergy between tau-fluvalinate and pyra- reported. This result clearly demonstrates that bee clostrobin could lead to increased mortality related to viruses can be detected even at high concentrations in oxidative stress, considering that pyrethroids can also live asymptomatic bees. The three viruses investigated in cause increased production of reactive oxygen species this study do not seem to be the sole determinant of the (Kale, Rathore, John, & Bhatnagar, 1999). appearance of spring mortality incidents in honey bees, Another condition that poses a high risk for honey because they only become deadly when their virulence is bee health is chronic exposure to certain compounds, as unleashed by an external stressor, as different from what imidacloprid and its main metabolites. Lethal concentra- is observed in winter mortality (Genersch et al., 2010). tions of this insecticide are dependent on exposure time, Our observations suggest that neonicotinoid and with chronic lethality appearing a few days after initial fungicide residues appear to potentially be the main exposure (Rondeau et al., 2014; Suchail, Guez, & stress factor associated with bee disorders. Other stres- Belzunces, 2001). Other researchers also clearly demon- sors could, however, act or interact at the same time, strated an increase in the gut pathogen Nosema ceranae in particularly simultaneous exposure to pathogens and individual bees reared in colonies exposed to imidacloprid insecticides in areas of intensive agriculture. In below the levels considered harmful to bees, thus con- conclusion, the observed mortality events seem to be firming the risk of exposure to chronic sub-lethal doses linked to the high prevalence of two main factors: of this pesticide for honey bee health (Alaux et al., 2010a; viruses and pesticides, with the former being present in Pettis, vanEngelsdorp, Johnson, & Dively, 2012). 100% of dead honey bees and the latter in 75.8% of the The present study confirms that neonicotinoids analyzed samples, albeit below their lethal levels. It is could be implicated in the mortality events explored very likely that the lethality of viruses was due to, pro- here, as their residues in bees are both high and the moted or provoked by the combined stresses of the most prevalent among the observed compounds. Over various pesticides to which the bees were exposed, the past few decades they have become one of the leading to a reduction in the bees’ natural defense most commonly used classes of insecticides (Goulson, mechanisms. 2013) and their widespread use increases the potential Our results emphasise the need for further studies for honey bee exposure to these active ingredients over on the synergism between fungicides, insecticides and longer periods, given that systemic insecticides can be other compounds, and the mechanisms that undermine found in various places throughout the life cycle of a the bees’ defenses. Pesticides are used in agriculture plant. The major knowledge gaps concerning possible according to the concentrations indicated on the labels, impacts of neonicotinoids on pollinators have been use- considered to be sub-lethal doses, but calculated by test- fully summarized in recent reviews by EFSA (2012b, ing individual pesticides in environmental and toxicologi- 2013a, 2013b, 2013c, 2013d, 2016a, 2016b). Neonicoti- cal studies. In agricultural practice, pesticides (as noids (and fipronil) suppress bees’ natural immunity fungicides and insecticides) are often applied in tank mix- (Alaux et al., 2010a; Aufauvre et al., 2012; Brandt, tures to reduce the number of spray applications, but Gorenflo, Siede, Meixner, & Bu¨chler, 2016; Di Prisco the risks of exposure to the simultaneous presence of et al., 2013; Doublet et al., 2014;Sańchez-Bayo et al., several pesticides have currently not been considered 2016), and the mechanisms underlying the synergistic when evaluating the safety of pesticides for honey bees. and/or combined effect of neonicotinoids and fungicides As suggested by EFSA (EFSA, 2012a, 2014), in accor- on viral diseases have recently been described by dance with Regulation (EC) No. 1107/2009 (EU, 2009), Sańchez-Bayo et al. (2016). many knowledge gaps need to be filled, as the assess- Viruses are known to be risk factors causing honey ment of short- and long-term exposure of honey bees to bee mortality and colony collapse. However, viruses pesticides, the toxicity of PPPs in honey bees after 12 M. Martinello et al. repeated exposure to sub-lethal doses, and the evalua- Anderson, K.E., Sheehan, T.H., Eckholm, B.J., Mott, B.M., & tion of cumulative and synergistic effects of active ingre- DeGrandi-Hoffman, G. (2011). An emerging paradigm of dients in honey bees and beehives. The importance of colony health: Microbial balance of the honey bee and hive (Apis mellifera). Insectes Sociaux, 58, 431–444. doi:10.1007/ field studies has also been highlighted by Collison et al. s00040-011-0194-6 (2016) who discuss combining laboratory and field stud- Anonymous. (2014). In val di Non record per uso pesticidi [In ies to better evaluate the impact of pesticides and patho- Non valley, the record for using pesticides]. Retrieved gens on bee populations in a realistic scenario. A direct from http://www.ansa.it/trentino/notizie/2014/05/14/in-val- link between pesticide and virus loads and honey bee di-non-record-per-uso-pesticidi_820f7717-ec15-4af8-a716- 9212d15c1fa7.html mortality events has not, however, been clearly demon- ApeNet Report. (2009). Effects of coated maize seed on honey strated in the course of this study. The data collected bees. Retrieved from http://www.reterurale.it/apenet through our forensic investigation on bee mortality inci- Archer, C.R., Pirk, C.W.W., Wright, G.A., & Nicolson, S.W. dents provides field information supporting current (2014). Nutrition affects survival in African honey bees knowledge on the possible implications of and the syn- exposed to interacting stressors. Functional Ecology, 28, 913–923. doi:10.1111/1365-2435.12226 ergies between pesticides, pathogens and their combina- Aufauvre, J., Biron, D.G., Vidau, C., Fontbonne, R., Roudel, M., tions, which could lead to honey bee mortality incidents. Diogon, M., … Blot, N. (2012). Parasite-insecticide interactions: A case study of Nosema ceranae and fipronil synergy on honey bee. Scientific Reports, 2, 326. doi:10.1038/ Supplementary material srep00326 Biddinger, D.J., Robertson, J.L., Mullin, C., Frazier, J., Ashcraft, Supplemental Tables S1–4 and Figure S1 are available S.A., Rajotte, E.G., … Vaughn, M. (2013). Comparative for this article at: http://dx.doi.org/10.1080/00218839. toxicities and synergism of apple orchard pesticides to Apis 2017.1304878. mellifera (L.) and Osmia cornifrons (Radoszkowski). PLoS ONE, 8, e72587. doi:10.1371/journal.pone.0072587 Blacquie`re, T., Smagghe, G., van Gestel, C.A.M., & Mommaerts, V. (2012). Neonicotinoids in bees: A review on concentra- Acknowledgements tions, side-effects and risk assessment. Ecotoxicology, 21, We acknowledge Mrs. Monica Lorenzetto’s contribution 973–992. doi:10.1007/s10646-012-0863-x to map preparation. Blanchard, P., Ribie`re, M., Celle, O., Lallemand, P., Schurr, F., Olivier, V., … Faucon, J.P. (2007). Evaluation of a real-time two-step RT-PCR assay for quantitation of Chronic bee paralysis virus (CBPV) genome in experimentally-infected Disclosure statement bee tissues and in life stages of a symptomatic colony. Jour- No potential conflict of interest was reported by the authors. nal of Virological Methods, 141, 7–13. doi:10.1016/j.jvi- romet.2006.11.021 Bogdanov, S., Kilchenmann, V., & Imdorf, A. (1997). Acaricide Funding residues in beeswax and honey. Apiacta, 32, 72–80. 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