Pathogens Spillover from Honey Bees to Other Arthropods

Honey bees, and pollinators in general, play a major role in the health of ecosystems. There is a consensus about the steady decrease in pollinator populations, which raises global ecological concern. Several drivers are implicated in this threat. Among them, honey bee pathogens are transmitted to other arthropods populations, including wild and managed pollinators. The western honey bee, Apis mellifera, is quasi-globally spread. This successful species acted as and, in some cases, became a maintenance host for pathogens. This systematic review collects and summarizes spillover cases having in common Apis mellifera as the mainteinance host and some of its pathogens. The reports are grouped by final host species and condition, year, and geographic area of detection and the co-occurrence in the same host. A total of eighty-one articles in the time frame 1960–2021 were included. The reported spillover cases cover a wide range of hymenopteran host species, generally living in close contact with or sharing the same environmental resources as the honey bees. They also involve non-hymenopteran arthropods, like spiders and roaches, which are either likely or unlikely to live in close proximity to honey bees. Specific studies should consider host-dependent pathogen modifications and effects on involved host species. Both the plasticity of bee pathogens and the ecological consequences of spillover suggest a holistic approach to bee health and the implementation of a One Health approach.


Introduction
Interspecific transmission may occur from a definite maintenance host (aka "reservoir") to an incidental or non-maintenance species (aka "spillover host"). Spillover cases are crucial to pathogen dynamics [1,2].
In a single-host scenario, reservoirs are sufficient and pathogen replication does not need other host species [3]. The basic reproduction number (R0) defines the frequency of new cases originating from each primary event, where R0 = 1 is the threshold between declining infections (R0 < 1) and pathogen persistence within the population by intraspecific transmission (R0 > 1) [4]. When multiple host species are involved, the presence of new maintenance or incidental hosts may result in an increased pathogen transmission [1]. In this case, R0 >> 0 denotes multi-host pathogen scenarios that may be respectively true or apparent, depending on the high or low interspecies transmission. When the R0 is between 0 and 1, the event is called "apparent multi-host pathogen", while "true multi-host pathogen" indicates an event in which there are two different maintenance hosts and the occurrence of interspecies transmission is higher than 1 [5].
Strictly speaking, spillover only occurs when the recipient species is characterized by R0 ≈ 0 [5]. However, in this review, we follow the use of the term sensu lato, commonly indicating a multifaceted range of host shift events [2].
Pollinators are crucial to the generation of crops contributing to the human diet [6]. These agroecosystem service is provided by a range of different species, including honey As shown in Figure 1A, the first article about spillover of honey bee pathogens to other bees was published in 1964, but the number of articles on this topic steadily increased from the year 2020, likely due to the quick development of molecular genetic tools for pathogen detection. Considering other hymenopteran species, the first detection of spillover cases dates back to 2008, with a rapid increase of cases in the following years ( Figure 1B). The first spillover case to other arthropods was assessed in 2009, but later the frequency increased, covering a wide range of species ( Figure 1C).
The geographical distribution of spillover studies present in the literature ( Figure 2) shows a high number of studies in both North and South America, Europe and New Zealand, whereas the reports from other countries were less frequent. The geographical distribution of spillover studies present in the literature ( Figure 2) shows a high number of studies in both North and South America, Europe and New Zealand, whereas the reports from other countries were less frequent. Figures 3 and 4 summarize the spillover cases for each honey bee pathogen in relation to arthropods groups. In events encompassing at least 20 spillover cases, DWV was the most frequently detected (158 cases). BQCV, SBV, IAPV, ABPV, KBV, N. ceranae, SBPV and LSV resulted implicated with progressively decreasing frequency.
The chord graph ( Figure 3) shows all spillover cases described in this review, evaluating the relationship to the investigated arthropod genus. Additionally, Figure 4 highlights the reported frequency of honey bee pathogens in the investigated arthropod communities, to emphazise their plasticity to the host. Figures 3 and 4 summarize the spillover cases for each honey bee pathogen in relation to arthropods groups. In events encompassing at least 20 spillover cases, DWV was the most frequently detected (158 cases). BQCV, SBV, IAPV, ABPV, KBV, N. ceranae, SBPV and LSV resulted implicated with progressively decreasing frequency.
The chord graph ( Figure 3) shows all spillover cases described in this review, evaluating the relationship to the investigated arthropod genus. Additionally, Figure 4 highlights the reported frequency of honey bee pathogens in the investigated arthropod communities, to emphazise their plasticity to the host. Some individuals were found infected with multiple honey bee pathogens ( Figure 5). The highest incidence of coinfections was found in bumblebees, followed by mason bees, mining bees and the honey bee pest Aethina tumida. A high number of co-infections was reported for Eucera nigrescens, Osmia bicornis and Osmia cornuta, for which 6 pathogens were found in the same individuals. Besides, the most abundant coinfecting pathogens able to co-infect the arthropods hosts were DWV, BQCV, SBV, ABPV and N. ceranae. Pathogens 2021, 10, x FOR PEER REVIEW 5 of 24

Discussion
The results of this systematic review highlights that the case history of spillover events involving honey bee pathogens increased over the past six decades. This is consistent with the growing interest of the scientific community in understanding the underlying factors [12,20,22,54]. The higher incidence of spillover cases recorded in Europe, New Zealand, and the Americas may reflect their advances in research and apiculture compared to other regions [60][61][62][63][64][65][66][67].
Bumblebees, mason bees and leafcutter bees were the species in which the spillover was studied more intensely, possibly because of their use in crops and fruit pollination. The fact that some of the surveys were carried out on arthropods ranging freely in the same environment as the managed honey bees is indicative of a pathogen circulation in their common environment. Despite honey bee pathogens were detected in other arthropods, symptoms and other effects on the alternative host populations remain unknown-except for some publications reporting individual bumblebees with crippled wings and scoring positive to DWV [68,69].
The importance of investigating the spillover of honey bee pathogens is also indicated by the discovery of active coinfections in wild hymenopteran individuals. As for the honey bees [30,70,71], multiple infections were found in wild bees, wasps and Aethina tumida individuals, which shows the importance of other arthropods as incidental hosts. The multiple infections that were identified ( Figure 5 and Supplementary Table S1) have both the effect to increase the circulation of pathogens within the arthropod communities, and to recirculate them to the managed honey bee colonies, so generating damage at individual and colony levels.
All of these aspects, including their modifications and effects encompass the implementation of a One Health approach to bee health [72,73]. The health of managed honey bees is dependent on the health of wild bees and other arthropods, and vice versa. This approach is essential to provide suitable ecosystems to pollinators and other arthropods contributing to human livelihoods and environmental health, and for understanding the eco-immunology to prevent the transmission of pathogens and pests, thereby limiting damages in managed and wild insect populations [73][74][75]. Therefore, the circulation/recirculation and the possible impact of honey bee pathogens to the arthropod communities are crucial to build the basis for the One Health approach to the bee health. Here we provide a brief discussion of each of the honey bee pathogens reported in Supplementary Tables S1 and S2, in relation to their spillover hosts.
The DWV is probably the most known, spread, prevalent, and studied honey bee pathogen, often associated to V. destructor [79]. The DWV can be asymptomatically replicated in V. destructor mites [80].
The impact of DWV on honey bees leads to increased interspecific transmission, reaching several species of hymenopterans and other arthropods (Supplementary Tables S1 and S2).
Although the virus is considered endemic in America and New Zealand, it has been rarely reported in other regions, both in honey bees and other arthropods (Supplementary  Tables S1 and S2)

Acute Bee Paralysis Virus (ABPV)
ABPV is a non-enveloped virus and widespread ssRNA (+) virus belonging to Apavirus genus within the Dicistroviridae family [28,96]. As reported above, ABPV is genetically linked to KBV and IAPV [88]. ABPV was detected in V. destructor, where is is reported incapable to replicate [97,98]. ABPV spillover is not recent (Figure 1 and Supplementary  Table S1) as in 1964 various Bombus species were found infected in the United Kingdom [99]. The list of bees in which ABPV was found increases constantly, including many Bombus species as well as a wide range of other bee species [54, 100,101]. In non-bee Hymenoptera, ABPV was detected in Ancistrocerus auctus, Polistes spp., V. germanica, Scolia flavifrons and Linepithema humile [49,54,94].

Israeli Acute Paralysis Virus (IAPV)
Israeli acute paralysis virus (IAPV) is a non-enveloped ssRNA (+) virus, belonging to Apavirus genus within the Dicistroviridae family, whose genome shows high homology to ABPV and KBV [88,102,103]. The virus has been isolated in Israel, but there are several known strains [102]. In honey bees, it induces disorientation, shivering wings, crawling, progressive paralysis and death within or ourside the nest [104].

Slow Bee Paralysis Virus (SBPV)
Slow bee paralysis virus (SBPV) is an icosahedral non-enveloped ssRNA(+) virus from the Iflavirus genus within the Iflaviridae family [105,106]. The infection is responsible for paralysis of the first and second pairs of legs in roughly 12-day old honey bees and their sudden death [107,108].
Recently, it was found in wild Bombus spp., E. nigriscens and O. bicornis in Kyrgyzstan, Germany and Georgia [109], in the United Kingdom [110,111] and Belgium [112]. Furthermore, E. nigriscens and O. bicornis species in Kyrgyzstan, Germany and Georgia scored positive for SBPV infection [109] (Supplementary Table S1). No further spillover events have been reported so far in other arthropods.

Chronic Bee Paralysis Virus (CBPV)
Chronic bee paralysis virus (CBPV) is an unclassified enveloped ssRNA (+) virus carachterized by articulate genome and association to a satellite virus (CBPSV) [113,114]. The infection causes a multifaceted disease encompassing different combinations of symptoms evidencing neurotropism like ataxia, incapability to fly, and trembling, as well as hairlessness and dark colour in the infected bees [114,115].

Lake Sinai Virus (LSV)
LSV is an ssRNA(+) belonging to the Sinhaliviridae family and Sinaivirus genus, of which two strains have been identified so far: LSV-1 and LSV-2 [140]. The virus was discovered in honey bees sampled during a colony transhumance near the Lake Sinai, South Dakota, USA. LSV was reported as involved in the colony collapse disorder, despite both pathogenicity and epidemiology have not been clarified yet [70,141].
Cases of LSV spillover have been reported in Andrena spp. [37,127], Bombus spp., [85,112,127], and species belonging to the families of Halictidae and Megachilidae [127]. LSV has never been detected outside the Apoidea superfamily so far.

Apis mellifera Filamentous Virus (AmFV)
AmFV is an unclassified dsDNA isolated from honey bees, whose relationship with the host and epidemiology are poorly studied. Originally, the pathogen was described as a rickettsia disease, but recently it has been recognized as a virus [142,143]. Severe infections of adult honey bees are associated to milk white hemolymph as a consequence of the high virion concentration. The infected bees show signs of weakness and tend to gather at the hive entrance. Nevertheless, the virus is weakly pathogenic and has low impact on bee lifespan [143][144][145][146].
Few spillover cases have been reported so far. They involved as alternative hosts Andrena spp.

Varroa destructor Macula-like Virus (VdMLV)
VdMLV is an unclassified ssRNA(+) virus of the Tymoviridae family. The mite V. destructor is its primary host and the virus was found in the honey bees as a likely result of the trophic activity of the parasite [149]. Little knowledge is available for this virus. Few spillover cases have been reported so far about VdMLV (Supplementary Table S1), all of them in the wild. Those involved B. lapidarius, B. pascuorum and B. pratorum as host species [112].

Nosema ceranae
Nosema ceranae is a microsporidium that causes nosemosis type C in western honey bees [152,153]. It is an intracellular obligate parasite, infecting the ventricular epithelial cells [154,155]. The effects of N. ceranae infections can be recognized both at individual and colony levels, impacting the bee lifespan, inducing lethargic behaviour, reducing the pollen and honey harvest, and causing colony dwindling [156][157][158][159].
The main known spillover event occurred when the pathogen jumped from the Asian honey bee A. ceranae, which is deemed as the original host, to the western honey bee A. mellifera [152,153].
In addition to A. cerana and A. mellifera, the microsporidium was reported in several other Hymenoptera (Supplementary Table S1 [163]. Besides, it was detected in the small hive beetle as well as in A. tumida [148,164]. Finally, the microsporidium was found in the regurgitated pellets of the European bee-eater Merops apiaster [165].

Nosema apis
Nosema apis is the classic microsporidium infecting A. mellifera, which is responsible for the nosemosis Type A [166]. Like the other microsporidians, it is an intracellular obligate parasite. It causes, in contrast to N. ceranae, severe dysentery that impacts mainly the colony foragers [166][167][168]. Presently, its spread is limited to specific ecological niches as a possible consequence of the competition with the predominant N. ceranae [71,157]. N. apis was detected in commercial B. terrestris colonies [20], but the transmission route remained unclarified.

Ascosphaera apis
The fungus Ascosphaera apis is a honey bee pathogen responsible for the mycosis called chalkbrood disease [169,170]. The infection occurs by spore ingestion in bee larvae, especially in those of the fifth instar, that reduces food consumption and prevents eating [169,170]. The proliferating mycelium invades the larval body, which is transformed into a chalk-like "mummy", so the disease name [169,171,172].
Despite the disease is typical to the honey bees, artificial infections showed the pathogen capability to colonize the intestine of B. terrestris adults and larvae [20].

Melissococcus plutonius
The bacterium M. plutonius is the Gram-negative coccus representing the etiological agent of the European foulbrood disease [173,174].
The pathogen is spread worldwide and infects the brood, which dies by undernutrition [175,176]. The infected larvae become flaccid and yellowish by 5 days after infection [173,175,176].
In the United Kingdom, M. plutonius was found to impair the development of B. terrestris colonies [20].

Spiroplasma apis
Spiroplasma apis is a small, helical and motile Gram-positive Eubacterium deprived of a cell wall [177,178]. The bacterium was isolated in France from colonies showing symptoms of "May disease" [179]. S. apis is lethal to the honey bees when ingested, and the infection may spread by faecal contamination [179].
Strains of S. apis were isolated and detected in wild specimens belonging to B. atratus [125] and O. bicornis [37], with unknown effects.

Spiroplasma melliferum
Spiroplasma melliferum is another Eubacterium isolated from the honey bees [180]. The S. melliferum infection has similar symptoms and transmission route as S. apis, although less virulent [179,180]. As for S. apis, S. melliferum spillover was observed occasionally (Supplementary Table S1). This is the case of O. bicornis individuals, that were found infected in Belgium [37].

Wolbachia spp.
Wolbachia spp. are Gram-negative intracellular bacterial symbionts, which can infect the cells of both female honey bees and drones [181,182]. Wolbachia spp. impacts the host reproduction. The vertical transmission via the eggs represents the main transmission route to persist in honey bee populations [183,184].

Lotmaria passim
Lotmaria passim is a trypanosomatid with a single flagellum, capable to colonize the digestive tract of A. mellifera [185,186]. The parasite spreads within the colony by fecal contact, and the transmission occur via the oro-faecal route [187,188]. The infection impacts the colony by altering behaviour and lifespan of the infected bees [141,189]. L. passim is spread worldwide. The colonization implied the replacing of the other honey bee trypanosomatids Crithidia mellificae [190,191].
L. passim was found also in the small hive beetle, A. tumida, as a possible result of the feeding behaviour of this scavenger [81,148].

Crithidia mellificae
Crithidia mellificae is another trypanosomatid which can replicate in the honey bee intestine to survive [185,186]. Transmission route and impact on bees are very similar to the other parasite L. passim [187,188,192]. C. mellificae was almost completely replaced by L. passim and its infection has been rarely observed [185,193,194].
Despite that, one spillover case was observed in A. tumida, that live in contact with bee colony debris [81].

Crithidia bombi
Crithidia bombi is a trypanosomatid infecting B. terrestris colonies [195,196]. The infection occurs during the external activity of the forager bumblebees [197,198] and, back to the nest, it spreads by fecal contamination to the other workers [199,200]. C. bombi may harm the bumblebee populations as hibernating queens may reduce the success in founding the colonies and remarkably lower their fitness [201]. On queen emergence from the diapause, C. bombi infections grow together with the colony that is being established [200].
C. bombi is transmitted during the foraging activity. The pathogen was detected in the wild on A. vaga and O. bicornis individuals [37] and in small hive beetles collected from the nest of honey bee colonies [148]. Artificial infections showed that C. bombi can replicate in O. lignaria, M. rotundata and H. ligatus [202,203].

Neogregarine Apicystis bombi
Apicystis bombi is a parasite found primarily in bumblebees. It was found to occur also in honey bees from Europe and North America [204][205][206]. Upon the ingestion of the oocytes by the bee, the sporozoites develop and migrate to the fat body, where they develop, multiply and disrupt the adipose tissue. The infection increases the worker mortality rate and, due to the fat body disruption, both queen survival to hibernation and colony foundation success are impaired [84,207,208].

Protocol and Literature Search
This systematic review was carried out according to the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) protocols [209]. The research question to be reviewed was: "Which honey bee pathogens may generate spillover to managed and wild Hymenoptera species and, more in general, to the arthropofauna?" The search intentionally excluded arthropods living in close contact with the honey bees that, like V. destructor, are obligate parasites.
The article search was carried out in PubMed, Web of Science, Science Direct, Google Scholar, and Scopus scientific databases for studies aimed to assess the detection of spillover cases of honey bee pathogens. Filters were used to select articles published from January 1960 to April 2021. The last search date was 31 May 2021.
Studies carried out both in field and laboratory conditions were selected. Besides, studies that did not assess whether the presence of the honey bee pathogens could be related to external contamination were not included. The detected active replication of honey bee viruses was also reported in the Supplementary Materials with an asterisk.
Duplicate studies were excluded. The search and screening for titles, abstracts and results were carried out independently by the authors, including all articles, letters, notes, scientific notes and communications aimed to assess a spillover case of honey bee pathogens and excluding reviews, books, book chapters and theses.
The potentially eligible research articles were read and reviewed independently by the authors and the data were compared to ensure integrity and reliability.
For each article included in this review, relevant information related to the authors, publication year, host species, host conditions, host stage, pathogens and prevalence were extracted. The data from the eligible studies are expressed in the Supplementary Materials and Figures. The authors provided a narrative synthesis of the results for each pathogen capable to generate a spillover case, according to the main characteristics and results related to the topic addressed.

Conclusions
This review shows that, in recent years, the frequency of recorded spillover cases of honey bee pathogens to other arthropods, including wild bees, has dramatically increased. Certainly, human movements and globalization have fostered the inflow of novel pathogenic microorganisms, often with detrimental consequences. However, it should also be considered that the analytical methods currently available give impulse to the research on bee pathology, increasing the chance to identify interspecific transmission events.
The host plasticity shown by some honey bee pathogens raises ecological concern for the potential negative consequences on the pollinating entomofauna and ecosystems in general. Despite the fact that research on these pathogens has significantly improved, we have limited knowledge of their potential impact on other bees, insects, and arthropods in general and the cascade of environmental effects. Laboratory studies are not sufficient to cover this gap, for the intricate interaction of the involved biotic and abiotic factors. For the same reasons, the exploitation of these pathogens in the control of arthropods considered as pests (e.g., A. tumida, G. melonella, V. velutina, L. humile) should be considered with extreme carefulness.
The tight interaction between honey bees and the other environmental components suggests a holistic approach to the study of bee diseases, including their control. Indeed, pathogens may survive in alternate hosts, generating spillback events and possibly jeopardizing the efficacy of the treatments. This emphasizes the beekeeper's responsibility to maintain healthy colonies to benefit both their production and the environment.
Spillover of honey bee pathogens may have undetected yet important repercussions on the health and functioning of an ecosystem. Health management of honey bee colonies is of high importance in this context. Honey bees and the beekeeping industry should, therefore, undertake an essential role in the One Health concept. This requires the adoption of dedicated research actions.