Assessment of intestinal parasites in the coexisting Bombus terrestris (Apidae) and Xylocopa augusti (Apidae) in central Chile

Bombus terrestris is a European bumblebee extensively commercialized worldwide for crop pollination. In Chile, this species was introduced in 1997 and after confinement escape, it has spread and established in several localities of central-southern Chile and in the Argentine Patagonia. The South American carpenter bee Xylocopa augusti, in turn, has been recently reported in central Chile, and as B. terrestris, this species has become increasingly common, often found in sympatry with B. terrestris in some localities. While intestinal parasites such as the flagellate trypanosome Crithidia bombi, the microsporidium Nosema bombi, and the neogregarine protozoan Apicystis bombi, show high levels of specialization on the Bombus genus, parasites often increase their host range, especially after invading novel habitats, hence creating new infection disease scenarios. In this work, we used molecular techniques to detect the presence of the intestinal pathogens of B. terrestris in coexisting X. augusti from different localities in the Metropolitan Region of Chile. Our results revealed the presence of the three pathogens in B. terrestris only, with population prevalence broadly similar to that reported in other studies. The carpenter bee X. augusti did not show evidence of any of the three parasites examined, indicating that this invader species is not recipient of any of the parasite species present in B. terrestris.


Introduction
Pathogen spillover, broadly defined as the transfer of one or more pathogens from one reservoir host to a recipient host is one of the major factors influencing disease spread in natural populations. Spillover often occurs when infected host species invade novel habitats and transmit exotic parasites to native hosts, which often experience substantial reduction in reproduction and survival [1,2]. Pathogen spillover occurs commonly in bee colonies transported from one region to another for commercial purposes because commercially-reared colonies often harbor parasites harmful to wild bees, resulting in disease epidemics, and native bee declines in wild populations [3][4][5]. In this regard, Bombus terrestris Linnaeus (Apidae) (Fig. 1a), one of the most extensively used bumblebee species for crop pollination worldwide has been suggested to transmit their parasites to recipient native bees in southern South America [6,7]. One of the parasites commonly harbored by B. terrestris is Crithidia bombi Lipa & Triggiani, a flagellated trypanosome species that infects exclusively species of the Bombus genus. This protozoan is horizontally transmitted by ingestion of infectious cells present in the feces deposited on flowers by infected workers of other colonies or bumblebee species, by contaminated honey stores and nest material, and through vertical transmission from one generation to another within the same colony [8,9]. Like C. bombi, the microsporidium Nosema bombi Fantham & Porter, infects exclusively species of the Bombus genus. The spores (infective stages) locate mostly in the digestive tract of the bumble bees, and after proliferation within the host, are released into the environment [10,11]. As in C. bombi, this parasite transmits vertically across generations in bumble bees [12]. Finally, Apicystis bombi Liu, Macfarlane & Pengelly, is a neogregarine protozoan that in addition to Bombus species, infects Apis mellifera, and Osmia bicornis Linnaeus (Megachilidae). It locates in the host adipose tissue, reducing the colony establishment success [12]. Its transmission is horizontal through shared flower resources, but vertical transmission has also been suggested [13].
In Chile, B. terrestris was imported for tomato pollination in 1997/1998. After greenhouse confinement escape, it has spread and become established in several localities of southern Chile and Argentina (see reviews in [6,14]). In 2009, the presence of C. bombi and A. bombi was reported for the first time in South America [15]. Since then, further reports have confirmed that invasion of B. terrestris to novel habitats in southern Chile and Argentina carried exotic intestinal parasites [6,16], hence increasing the chance of pathogen spillover to other bee species. For example, after B. terrestris invasion, C. bombi was detected in the native bumblebee Bombus dahlbomii Guérin-Méneville in Chile [6], and A. bombi was recorded in the honeybee Apis mellifera in Argentina [17,18], which strongly suggests parasite spillover from B. terrestris to other bee species in southern south America [2].
A recently reported invasive bee species in Chile is the South American carpenter bee Xylocopa augusti Lepeletier ( Fig. 1b and c). Originally distributed in Argentina, Brazil, Uruguay, and Paraguay, this species was recorded for the first time in central Chile in 2013, probably transported through undetected nests in packaging structures from Argentina [19]. After its arrival, new ecological scenarios of coexistence between otherwise allopatric X. augusti and B. terrestris are frequently found in central Chile, providing new opportunities for parasite transmission. It is known that parasite transmission is an expected result when founding populations in novel habitats are genetically depauperate [20,21]. In principle, judging by the pathogens found in B. terrestris and other Xylocopa species, X. augusti has the potential to become infected by some viruses such as DWV (Deformed wing virus), BQCV (Black queen cell virus), and SBPV (Slow bee paralysis virus), all of them detected in B. terrestris also [22,23]. However, as most studies inquiring on the potential transmission of parasites from B. terrestris to other bee species in southern South America have focused almost exclusively on intestinal parasites, and molecular diagnosis techniques for viral testing require a methodology different to that used in this study [24,25], we restricted the analysis to the molecular detection of the protozoans C. bombi and A. bombi, and the microsporidium N. bombi in B. terrestris and X. augusti.

Methods
Overall, 59 specimens of the two species combined were collected from the total 26 campaigns performed to different localities in the Metropolitan Region, Santiago, during October-December 2018 (Fig. 2, Table 1). Individuals were collected in three sites where both species were observed foraging in the same plants and in sites where only one of the species was found, one site per species. Even though campaigns were conceived to check the presence or absence of the focal species, B. terrestris and X. augusti, no native bee species was observed. The only additional bee present in some sites was the honeybee A. mellifera. Specimens of the focal bees were captured using entomological nets, euthanized in killing jars, and stored in 95% alcohol at − 20°C until processing. At the laboratory, separate dissecting tools were

Results and discussion
Among the total 26 bumblebees screened, seven tested positive for the neogregarine A. bombi (26.9%), six for the trypanosome C. bombi (23.07%), and two for the microsporidium N. bombi (7.69%) ( Table 1). Among the seven specimens infected with A. bombi, two of them were also infected with C. bombi and both specimens infected with N. bombi, were also infected with C. bombi.  (Table 3), confirming that the primers used in this work were successful to identify the parasites under study. Unlike B. terrestris, no parasite was detected in the 33 specimens of X. augusti here examined, suggesting that regardless of the status of coexistence with B. terrestris, parasites are not present in X. augusti (Table 1). To confirm this result, primers were designed in order to differentiate between B. terrestris and X. augusti DNA (primer pairs BT-F/BT-R and XA-F/XA-R, respectively) ( Table 2). PCR products of the expected size were obtained from the 26 B. terrestris samples and none from the 33 X. augusti samples when using primer pair BT-F/BT-R. Accordingly, PCR products of the expected size were only obtained from the 33 X. augusti samples and none from the 26 B. terrestris samples when using primer pair XA-F/XA-R. A PCR product from each case was randomly selected and sequenced. The BLAST analysis of the PCR product from a B. terrestris sample (accession no. MT862773) revealed 100% of identity with the partial mitochondrial genome from B. terrestris and the analysis of the PCR product from a X. augusti sample (accession no. MT862774) revealed 90.7% of identity with the partial COI gene sequence from X. violacea. These results indicate that the quality of DNA samples from X. augusti specimens was adequate to perform PCR analysis, which supports the absence of the three parasites in these samples. The prevalence of A. bombi in B. terrestris specimens collected in this work was 26.9%, which is less than the prevalence obtained in a comparable study carried out in Argentina where 14 out of 30 individuals were infected (47%) [16]. Regarding the detection of C. bombi, all the sites where B. terrestris was present had individuals infected with the protozoan, reaching an overall prevalence of 23.1%, which is in the range of values previously reported for populations sampled near Santiago ( [6], 0-41.7%). This result is consistent with the observation that C. bombi is very widespread and abundant wherever B. terrestris is found [6]. Regarding N. bombi, the results obtained in this work indicate a low prevalence of this microsporidium (7.7%), probably because this parasite is more infectious in larvae than in adult bumblebees [26]. Interestingly, Schmid-Hempel et al. [6] did not record the presence of this parasite in B. terrestris populations near Santiago. Likewise, the prevalence of N. bombi in the 12 Chilean populations sampled in 2010 ranged from 0 to 3.7%, with an average prevalence of 1.7% [6], indicating that even if small, our recordings are the highest prevalence recorded for B. terrestris in Chile.
Results from this study indicate that the exotic bee, X. augusti, is not infected by any of the three parasites infecting B. terrestris, suggesting that transmission of pathogens typical of the Bombus genus does not proceed from B. terrestris to sympatric X. augusti. This result is not necessarily unexpected as the pathogens here examined are often considered specific to the Bombus genus. However, as intestinal parasites represent only a fraction of the potential pathogens transmitted (i.e., viruses should also be considered), we cannot rule out completely the idea of parasite transmission from one species to another, especially considering that susceptibility to new parasites is high in novel hosts [27], and new parasites often experience drastic changes in life history traits that permit them to infect novel hosts [28]. While the co-invasion of B. terrestris and X. augusti to Chile has created a new zone of geographical sympatry for the two otherwise allopatric species, the consequence of their coexistence for disease spread is unknown. Although our data suggests absence of intestinal parasite transmission between hosts, works that consider a broader spectrum of intestinal and viral parasites are needed. This information is crucial to understand the extent to which new contexts of host sympatry in species outside their original habitats influence and amplify parasite transmission in novel environments.