Investigation on the presence of known and emerging pathogens of Apis mellifera in Apis florea samples accidentally introduced into Malta and Italy

Apis florea Fabricius, 1787, the red dwarf honey bee, is a wild bee species native to Asia. Its adaptability to various climates, nesting behaviour and ease of colonization has recently attracted attention due to its possible ecological impact as an invasive species to compete with Apis mellifera during foraging and the risk to introduce novel pathogens in the local apoidean population. In 2024,after the discovery for the first time in Europe of a fully established colony of A. florea in Malta, a small nest was found in the same area, near the Malta Freeport Terminals, a very busy cargo and transhipment port in the southern part of the island. In May of the same year, a small colony of A. florea was reported in the port of Gioia Tauro (Reggio Calabria) on the wall of a container coming from India. Immediately before colonies eradication, samples of A. florea collected from both sampling site were subjected to visual inspection to verify the presence of parasites or morphological alterations and subsequently to molecular investigations to: 1) confirm the species, by amplification and Sanger sequencing of a region of the subunit I of cytochrome c oxidase (COI) of mitochondrial DNA; 2) detect the presence of known and emerging pathogens of A. mellifera, such as Nosema apis, Nosema ceranae, Crithidia mellificae and Lotmaria passim and several bee viruses (acute paralysis virus - ABPV; chronic bee paralysis virus - CBPV; deformed wing virus - DWV; royal black cell virus - BQCV; brood sac virus - SBV; Kashmir virus - KBV; Israel virus acute paralysis virus - IAPV; bee filamentous virus - AmFV; rhabdovirus-1 - ARV-1; rhabdovirus-2 - ARV-2; aphid lethal paralysis virus - ALPV; Bee Macula-like virus - BMLV; Lake Sinai Virus - LSV). Parasites or morphological alterations were not detected on the A. florea specimens collected from both sampling sites. The morphological identification of A. florea was confirmed by comparing the COI sequences obtained with data available in the GenBank database. Among all the pathogens investigated, the bees collected in Malta resulted positive only for ABPV, AmFV, BQCV, DWV, and SBV (presence of viral co-infections), while only SBV was detected in the sample from the port of Gioia Tauro. The presence of known and emerging pathogens in A. florea have contributed to increase the available knowledge on this species. Furthermore, the data demonstrated that, due to the increase in global trade, the spread of this alien species in the Mediterranean region could pose not only a potential ecological issue for local biodiversity but also a possible risk of pathogen transmission to local bee populations. This highlights the need to monitor the potential presence of A. florea nests through an early warning system and to take prompt action for their eradication.