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Evidence of Varroa-Mediated Deformed Wing Virus Spillover in Hawaii Authors Title: Evidence of Varroa-mediated Deformed Wing virus spillover in Hawaii Authors: Jessika Santamaria Corresponding Author: [email protected] University of Hawaii at Mānoa, United States 3050 Maile Way, Honolulu, HI 96822 Ethel M. Villalobos University of Hawaii at Mānoa, United States 3050 Maile Way, Honolulu, HI 96822 Laura E. Brettell Hawkesbury Institute for the Environment at the West Sydney University, Science Rd, Richmond, NSW 2753, Australia ScottUniversity Nikaido of Hawaii at Mānoa, United States 3050 Maile Way, Honolulu, HI 96822 Jason R. Graham University of Hawaii at Mānoa, United States 3050 Maile Way, Honolulu, HI 96822 Stephen Martin The University of Salford Manchester, UK M5 4WT Abstract: Varroa destructor, a parasitic mite of honey bees is also a vector for viral diseases. The mite displays high host specificity and requires access to colonies of Apis spp. to , viruses transmitted by V. destructor Previouscomplete studiesits lifecycle. have Indetected contrast, DWV the inDeformed a variety Wing of insect Virus groups (DWV), that one are of not the many , appears to have a much broader ehost discrete range. distribution of the Varroa mite in the Hawaiian archipelago to compare DWV prevalencedirectly parasitized on non-Apis by the flo mite. In this study, we take advantageVarroa presenceof th is linked where V. destructor wer visitors, and test whether absent.to a “viral We spillover”. sampled individualsWe selected of two Apis islands mellifera withCeratina different smaragdula viral landscapes:Polistes Oahu, aurifer Polistes hasexclamens been present since 2007, and Maui, where the mite is Hymenoptera community of the two isla , , honey bee, and colonies on Oahu have, to muchassess higher and compare incidence the of DWV DWV prevalence compared in to the Maui. nds. The- Apisresults Hymenoptera indicated that, collected as expected, from The study sites selected Correspondingly, DWV was detected on the Non ence of theOahu, Varroa but was absent in the species examined on Maui. inshared the Hymenoptera a similar geography, community climate, on mite and- insectinfected fauna, islands. but differed in the pres mite, suggesting an indirect, but significant, increase on DWV prevalence Keywords: Varroa Apis mellifera mite, deformed wing virus, pollinator health, viral transmission, viral Abbreviations:spillover, 1.Deformed Introduction Wing Virus (DWV) crops In the last two decades, emerging diseases have caused extensive damage to and livestock (Morens and Fauci, 2013; Voyles et al., 2014,). Pathogens have been repeatedly shown to jump between species (Levitt et al., 2013; Li et al., 2011; affectingMalmstrom honey and Alexander, 2016) and the Deformed Wing Virus (Iflaviridae; DWV) have shown thatbees the DWVis no exceptionmay have co(Villalobos,-evolved with2016). the Recent European molecular honey studiesbee Apis mellifera the original virus may have been a low prevalence pathogen with( many variants), and and low virulence . (Martin et al , 2012; Wilfert et al., 2016). Upon contact with the Asian honey Apis cerana Varroa destructor beeA. ( cerana to A.), mellifera a new mite and vector, with this new transmission, jumped route speciesthe prevalence from and virulence of DWV in A. mellifera was . amplified (Martin et al.,colonies 2012; ofWilfert the Asian et al., honey 2016 ). RecentApis studiescerana byand Yanez A. mellifera et al indicated(2015) on that sympatric there are a large number of sharedbee, strains of DWV ,circulating in the Asian and the European honey bee populations in the European honey , however the virus is more prevalent via the mite and/or greaterbee colonies,susceptibility suggesting of A. mellifera a more efficient to infection transmission to the virus route or the vector. A similar situation has been reported for the native Japanese honey bee Apis cerana japonica A. mellifera but at a much , which shares DWV infections with sympatric lower prevalence (Kojima et al., 2011). While DWV evolved in close association with Apis capable of infecting a broad range of non-Apis bees, it also appears hosts (Genersch et al., 2006; insect Li etgenera al., 2011; Melathopoulos et al., 2017). So far, DWVGuzman has- been detected in 23 across Europe, North and South America ( Novoa et al., 2015; Levitt- et al., 2013; Reynaldi et al., 2013; Singh et al., 2010), including social and non socialut the bees, wasps, ants, and a myriad of other insect groups. Not much is known abo impact of the virus in these host species (Tehel et al., 2016). Negative strands of nonDWV,-Apis suggestive of viral replication in the host, have been However found only in 5 generaery of of DWV amonginsects a wide (Levitt range et ofal., species 2013; Tehel has created et al., 2016 concerns). about ,a the possible discov “viral spillover” from honey specially economically bee colonies to other insect species, e important pollinators such as bumble bees (Graystock et al., 2016regionsa, Graystock where V. et al., destructor2016b). Work is present on viral and spillover DWV is has prevalent been conducted, in the honey so far, in Budge et bee population (V. destructoral., 2015; Tehel in honey et al., 2016; Traynor et al., 2016). In fact, the presence of and prevalence of DWVbee incolonies honey has been linked to increased viral loads, virulence, Varroa has also been associated withbee populationsother viral diseases (Martin inet honeybeesal., 2012). Additionally, including AKI, KBV, BQCV, and SBV (Francis et al., 2013; Martin, 2001; Shen et al., 2005). The fragmented distribution of the Varroa mite on the Hawaiian archipelago withoutmakes for Varroa ideal studymites sitesin the in ecosystem. which to examine Honey bees pollinator first arrived communities to the Hawaiian with or archipelago in 1857 and became establishedwe sampled across local the honey eight islandsbees and by non 1909-Apis Hymenoptera(Szalanski et al., species 2016). on In the this Varro study,a- positive island of Oahu and the Varroa-negative island of Maui. The selected study sites shared similar bees havegeography, been in floral contact resources, with V. destructorand insect communities; however, Oahu’s honey since 2007, and have high DWV prevalence and increased bee populations viral loads. on that In contrast, Maui remains mite free to this date, and the honey -Apis Hymenopteraisland have a much species lower selected incidence as representatives of DWV (Martin of tethe al., community 2012). The were: non Ceratina smaragdula, Polistes aurifer Polistes exclamens. C. smaragdula , and , commonly known as the small carpenter bee, is a mostly solitary bee abundant in garden bees . environments in Hawaii, sharing nectar and pollen resources with honey Polistes spp. are common social wasps that visits flowers occasionally to feed on nectar. hunt caterpillar prey, and Re-emerging viral diseases such as DWV represent one of the major threats to honey ld bees and other insects bee health, and the “spillover” of pathogens to wi may also contribute to the current global pollinator decline (Fürst et al., 2014; Genersch et al., 2006; Graystock et al., 2013a; Graystock et al., 2013b; Manley et al., incidence2015; Tehel of etDWV al., 2016).on non -HereApis weinsects carry in out areas a preliminary with and without comparison V. destructor of the . 2. Methods 2.1 Specimen collection We selected three species within two different Hymenoptera genera as representatives of the local community of flower visitors: the introduced small carpenter bee Ceratina smaragdula (Apidae) which was first recordedPolistes in aurifer Hawaii and in Polistes1999 (Magnacca exclaman ands King, 2013), and introduced paper wasps th century and in (Vespidae) first recorded in Hawaii were in collected the 19 from five sites on 1952 respectivelyVarroa-positive (Beggs et al., 2011). All samples Varroa-negative Oahu ( island), and four sites on Maui, ( island). Collection sites on both islands consisted onThe a mix selected of agricultural insect species fields, are parks, all gardens, and beach edge vegetation strips. whererelatively they abundant overlap inand resource can be founduse with in urban A. mellifera. and agricultural Our selection environments, of diverse habitats provided us with a preliminary bird’s eye view of the viral distribution on each island the micro-climate diversity that characterizes the Hawaiian archipelag, and representso. Polistes wasps collected on Oahu are P. aurifer and the specimens from Maui are P. exclamans. Consequently the comparisons between the paper wasps were at the genus level. Samples were collected from August 2014 to November 2015. handInsects-held were net. collected Paper wasp whiles samples they were were foraging also collected in fields fromor flower around patches, their nestsvia a . Each insect was stored individually and kept on ice in the field until transferred to a - °C freezer for long term storage. 80 2.2 RNA Extraction & Reverse Transcription PCR centrifuge which wasEach submerged individual inwas liquid transferred nitrogen to before a nuclease the sample free 1.5ml was crushed usingtube, a sterile mini pestle. Total RNA was then extracted froms theconditions resulting and powder resuspended using the RNeasy Mini -Kit (Qiagen) following manufacturer’ in 30 µl of RNase free water. RNA concentration was determined using a Nanodrop Transcription2000c (Thermo- Scientific)- and samples were diluted to 25ng/µl. Reverse carried out to determinPCR (RT ePCR) whether protocols samples adapted contained from DWV.Martin E etndogenous al (2012)
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