*4.6. Statistical Analysis*

The prevalence of the individuals that were positive for *C. mellificae* or *L. passim* and of those showing DWV or KBV infections were statistically compared with a bilateral Fisher's exact test under the null hypothesis of equality. The same test was also used to compare the frequency of multiple vs. single viral infections. Due to the small number of samples, the test for independence χ<sup>2</sup> was not used in this case.

### **5. Conclusions**

This investigation suggests that the honey bee trypanosomatids *L. passim* and *C. mellificae* may colonize, and the viruses DWV and KBV successfully infect *A. tumida* adults. Additional studies are needed to determine whether these pathogens generate clinical evidence and signs of infection in SHBs. The horizontal and vertical transmission routes of these pathogens in/between SHBs should also be clarified, as well as the potential, if any, of these pathogens to limit SHB populations in the wild.

Finally, further research is needed to elucidate the epidemiological role that SHBs play in pathogen transmission to honey bees and other insects as a possible dead-end host or vector.

**Author Contributions:** Conceptualization, G.C.; investigation, G.C.; in-field activity, J.D.E. and I.C.; laboratory activity, G.C.; data curation, G.C. and A.N.; writing—original draft preparation, G.C. and A.N.; writing—review and editing, G.C., J.D.E., I.C. and A.N.; supervision, A.N.; funding acquisition, J.D.E. and A.N. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Italian Ministry of Agricultural, Food and Forestry Policies, grant number AETHINET ("Monitoraggio e tecniche innovative di diagnosi e di controllo del piccolo coleottero dell'alveare, *Aethina tumida*") (A.N.) and by a cooperative agreement provided by the United States Department of Agriculture, Animal and Plant Health Inspection Service (USDA-ARS), AP17PPQS&T00C079 (J.D.E.).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** The authors are grateful to Brandi and Branden Stanford of the Entomology and Nematology Department, University of Florida, for their valuable technical support.

**Conflicts of Interest:** The authors declare no conflict of interest.
