Appendix C. Results of generalized mixed-effect models.

Results of generalized mixed-effect models

Appendix D. Results of parasitoid host choice experiment.

Results of parasitoid host choice experiment

Appendix A. Photographs of experimental vegetation islands.

Photographs of experimental vegetation islands

Appendix B. Parasitoid diversity data showing percentage of parasitoid species reared from Liriomyza helianthi and Calcomyza platyptera.

Parasitoid diversity data showing percentage of parasitoid species reared from Liriomyza helianthi and Calcomyza platyptera

Introduction of Non-Native Pollinators Can Lead to Trans-Continental Movement of Bee-Associated Fungi

<div><p>Bees are essential pollinators for many flowering plants, including agriculturally important crops such as apple. As geographic ranges of bees or their host plants change as a result of human activities, we need to identify pathogens that could be transmitted among newly sympatric species to evaluate and anticipate their effects on bee communities. We used PCR screening and DNA sequencing to evaluate exposure to potentially disease-causing microorganisms in a pollinator of apple, the horned mason bee (<i>Osmia cornifrons</i>). We did not detect microsporidia, <i>Wolbachia</i>, or trypanosomes, which are common pathogens of bees, in any of the hundreds of mason bees screened. We did detect both pathogenic and apathogenic (saprophytic) fungal species in the genus <i>Ascosphaera</i> (chalkbrood), an unidentified species of <i>Aspergillus</i> fungus, and a strain of bacteria in the genus <i>Paenibacillus</i> that is probably apathogenic. We detected pathogenic fungal strains in asymptomatic adult bees that therefore may be carriers of disease. We demonstrate that fungi from the genus <i>Ascosphaera</i> have been transported to North America along with the bee from its native range in Japan, and that <i>O</i>. <i>cornifrons</i> is exposed to fungi previously only identified from nests of other related bee species. Further study will be required to quantify pathogenicity and health effects of these different microbial species on <i>O</i>. <i>cornifrons</i> and on closely-related native North American mason bees that may now be exposed to novel pathogens. A global perspective is required for pathogen research as geographic ranges of insects and microorganisms shift due to intentional or accidental introductions.</p></div

Summary statistics on diapaused adult bees from cells of <i>Osmia cornifrons</i> nests collected at one residence (R) and five orchards with organic (O) or conventional (C) management practices.

<p>* Some nests were destroyed when a farm vehicle hit the nesting box.</p><p>Summary statistics on diapaused adult bees from cells of <i>Osmia cornifrons</i> nests collected at one residence (R) and five orchards with organic (O) or conventional (C) management practices.</p

<i>Osmia cornifrons</i>.

<p>A) Mating pair of <i>O</i>. <i>cornifrons</i>; male above female. Photo credit: L. Russo, used with permission. B) Nest box used to provide shelter for mason bee trap nests. C) <i>O</i>. <i>cornifrons</i> nest opened shortly after nest closure; pollen provision masses with small larvae and eggs are visible; note mud partitions separating pollen provision masses. D) <i>O</i>. <i>cornifrons</i> nest opened after all larvae have completed feeding, defecated and spun cocoons (early fall); each cocoon contains one adult, diapausing bee. Nest entrances in figures C and D are to the left.</p

Maximum-likelihood estimate of <i>Ascosphaera</i> species based on ITS-1, 5.8S, and ITS-2 DNA sequences.

<p>Numbers above nodes represent bootstrap proportions; numbers below nodes represent posterior probabilities. Values below 50% have been removed to enhance readability and interpretation. Sequences from this study are indicated by sample location code and nest number; sequences from dead larvae have an asterisk (*). Colors indicate fungal life history (and possible pathogenicity); red: pathogenic, blue: saprophytic, black: unknown. Circles indicate host family; black circles: found in nests of Megachilidae, gray circles: found in nests of Colletidae, open circles: found in colonies of Apidae. Host and pathogenicity from references [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130560#pone.0130560.ref024" target="_blank">24</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130560#pone.0130560.ref028" target="_blank">28</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130560#pone.0130560.ref031" target="_blank">31</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130560#pone.0130560.ref065" target="_blank">65</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130560#pone.0130560.ref067" target="_blank">67</a>]. <i>Ascosphaera naganensis</i>, a species whose holotype was collected from <i>Osmia cornifrons</i> in its native range in Japan, is indicated by an arrow.</p

Maximum-likelihood estimate of <i>Aspergillus</i> species based on 28S DNA sequence data.

<p>Numbers above nodes represent bootstrap proportions; numbers below nodes represent posterior probabilities. Values below 50% have been removed to enhance readability and interpretation. Sequences from this study are indicated by sample location code and nest number. Colors indicate pathogenicity as tested in honey bees [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130560#pone.0130560.ref032" target="_blank">32</a>]; red: pathogenic, blue: apathogenic, black: not tested.</p

Counts and mean weight per site of <i>Osmia cornifrons</i> that tested positive (+) or negative (-) based on PCR screens for <i>Ascosphaera</i> (Asc), <i>Aspergillus</i> (Asp), <i>Paenibacillus</i> (Pae), and multiple microbes (mul).

<p>^a Mean values of the weight in milligrams (mg) of live bees that were then screened for the presence of microbes (mean weight), of only those bees testing negative (mean weight -), and of only those bees testing positive for one or more microorganism(s) (mean weight +).</p><p>* Mean weight of bees testing positive versus negative were significantly different based on a Welch's t-test at p < 0.05.</p><p>Counts and mean weight per site of <i>Osmia cornifrons</i> that tested positive (+) or negative (-) based on PCR screens for <i>Ascosphaera</i> (Asc), <i>Aspergillus</i> (Asp), <i>Paenibacillus</i> (Pae), and multiple microbes (mul).</p