**3. Discussion**

To date, only a few instances of individual SHBs bearing bee pathogens have been reported. This is the case for samples from Mexico (positive for *L. passim*, *Apis mellifera* filamentous virus (AmFV), *C. bombi*, *Ascosphera apis*, and *Nosema ceranae* [22]), Florida (positive for *N. ceranae* [20]), and other areas of the USA (positive for DWV, SBV and *P. larvae* [19,20,22,24]).

The present study showed the presence of the honey bee pathogens *L. passim*, *C. mellificae*, DWV, and KBV in SHB adults collected from free-flying colonies in Florida. Furthermore, all SHB samples that were positive for DWV and/or KBV contained replicative viral forms. Although DWV replication in SHB adults is not a new finding [18,23], replication of KBV in SHBs is.

This is not the first time that DWV and KBV have been reported to infect non-Apis hosts. Replicative DWV was found in hornets (Vespa crabro) [67], Asian hornets (V. velutina) [68], and Argentine ants (Linepithema humile) [69]. Replicative KBV has been found in V. velutina [54], Vespula germanica, and Vespula vulgaris [70–72]. However, the current and previous [18,23] detections on coleopterans suggest that DWV and KBV can infect a

wide range of potential hosts, thus envisaging a scenario where wild and managed insect species may act as virus reservoirs that fuel reciprocal spillover. Furthermore, the occurrence of replicative DWV and KBV in the same individuals indicates the possibility of viral co-infections in SHBs, as already reported in A. mellifera and other insect species [59,73,74].

The sequence analysis of DWV and KBV resulted in high identity rates to viral sequences identified in *A. mellifera*. The phylogenetic analysis highlighted that the DWV genome detected in the SHB samples belonged to DWV type A, the less virulent genetic variant of this virus [36]. The KBV genome found in the investigated samples bore a close relationship to other KBV outbreaks reported in the USA, thus excluding the involvement of viruses originating from other countries.

The prevalence of *L. passim*-positive individuals detected in this study (40% of all samples) mirrors that reported in a previous survey in which *C. bombi* was also reported in larval SHBs [22]. Additionally, we report for the first time SHB samples positive for *C. mellificae*. Although *C. mellificae* is generally considered less spread than *L. passim* in honey bees [33–35], the prevalence levels of the two trypanosomatids in our individual samples did not significantly differ.

None of the samples was positive for *P. larvae*, *M. plutonious*, ABPV, IAPV, BQCV, SBV, CBPV, SPV major, or SPV minor. This coincides with the results of previous investigations showing low *P. larvae* [19] and SBV [21] loads in SHB adults. This likely reflects the health of the colonies that were visited by the SHBs prior to sampling.

The finding results highlight the need to clarify pathogen transmission between honey bees and SHB adults better. In the case of DWV, horizontal transmission occurs chiefly by the oral route [18]. In this regard, SHBs are able to trick honey bee adults into feeding them [75,76], possibly acquiring DWV during the exchange of food via trophallaxis. However, the multifaceted host–parasite interaction [77] allows multiple pathways, including oral–oral and fecal–oral transmission. Adult SHBs also may acquire honey bee pathogens by feeding on bee products that are contaminated with multiple microorganism species [41,47,48,78], cannibalizing bee carcasses, or ingesting infected faeces [78–80].

The articulate interactions above and active flying behavior [8] may bring together adult SHBs of different origins that congregate in the same host colony, generating the detected diversity in the pathogen load. On the other hand, the horizontal transmission may occur bi-directionally, as both SHB adults and larvae might defecate inside the hive [77], potentially spreading infected feces that could transmit and perpetuate infective agents within the colony. Infections may also be transmitted vertically. Bee pathogens may be found in SHB larvae [22,23] as consequences of feeding, environmental contamination, and congenital transmission. Nevertheless, the role that SHBs play in the transmission of honey bee pathogens remains unclear.
