Mixed infections reveal virulence differences between host-specific bee pathogens
Graphical abstract
Introduction
Pathogens are detrimental to the fitness of their hosts, and changes in pathogen virulence are fueled by various evolutionary pressures (Read, 1994). Theoretical models and empirical studies used to quantify the pressures affecting pathogen virulence show a trade-off between virulence and the ability of the pathogen to be transmitted to future susceptible hosts (Anderson and May, 1982, Anderson and May, 1979, De Roode et al., 2008, Doumayrou et al., 2013, May and Anderson, 1979). Models based on this trade-off predict that, over evolutionary time, host and pathogen populations persist when pathogen virulence is intermediate and pathogen transmission is high (Frank, 1996, Alizon et al., 2009). However, the presence of a second pathogen in the same host can alter a pathogen’s ability to overcome the host’s defenses (Woolhouse et al., 2002). Models that describe the evolution of pathogen virulence based on competition among multiple pathogens infecting the same host generally make predictions based on assumptions of one of two frameworks, superinfection and co-infection (May and Nowak, 1995, Nowak and May, 1994).
In the superinfective framework, one of the pathogens outcompetes the other and the virulence and transmission levels reflect those of only the most competitive pathogen, usually disturbing the balance predicted by the virulence/transmission trade-off theory (Alizon, 2013, Levin and Pimentel, 1981, Mosquera and Adler, 1998, Nowak and May, 1994). While superinfective properties have been observed in some studies (Hughes and Boomsma, 2004, Ben-Ami et al., 2008, Bashey et al., 2011), co-existence of more than one strain of the same pathogen can also occur. In a co-infection, pathogens co-exist until the host dies or recovers, and the resulting virulence and pathogen transmission will reflect a combination of both pathogens (Martcheva and Pilyugin, 2006, May and Nowak, 1995, Van Baalen and Sabelis, 1995). The evolutionary basis for coexistence is more difficult to rationalize than super-infections, as it is not apparently selfish (Alizon et al., 2013). Low genetic diversity among multiple pathogens in a host has been suggested to increase the ability of two pathogens to co-infect the host (Frank, 1996, Buckling and Brockhurst, 2008, Rumbaugh et al., 2012); potentially creating an evolutionarily stable relationship if the pathogens share goods (e.g., digestive enzymes) during the infection process.
Species-specific empirical data on mixed-infections is needed to advance our understanding of pathogen virulence models (Alizon et al., 2013, Brockhurst and Koskella, 2013). In this study, we chose the fungal genus Ascosphaera to quantify parameters associated with within-host, multi-pathogen dynamics in two bee host species. The Ascosphaera are always associated with social and solitary bees, and several species cause a disease known as chalkbrood. Of the 28 described Ascosphaera species, some are commensal pollen saprophytes found in bee nests, some are facultatively pathogenic to bees, and others are obligate pathogens only found in infected bees (Anderson and Gibson, 1998, Wynns, 2012). Chalkbrood is a disease common to megachilid bees (such as the alfalfa leafcutting bee, Megachile rotundata [Megachilidae]), but also afflicts the honey bee, Apis mellifera (Apidae). Chalkbrood only infects bee larvae, infecting per os after the larvae ingest pollen-based food provisions contaminated with fungal spores. After the spores germinate in the larval gut, the hyphae invade the hemocoel, grow throughout the larval body, and eventually emerge through the integument of moribund larvae, or for some Ascosphaera species, the hyphae sporulate just under the larval cuticle (McManus and Youssef, 1984). Only after host death do the hyphae produce spores in vivo, which then get disseminated to future larval food provisions by emerging adult bees, whereby the pathogen gets transmitted.
Virulence studies of Ascosphaera and other fungal infections in honey bees have been mostly limited to describing single pathogen infections (Vandenberg and Goettel, 1995, Goettel et al., 1997, Gilliam, 2000, Vojvodic et al., 2011a), but Vojvodic et al. (2012) found honey bee mortality was significantly increased when Ascosphaera atra was combined with an obligate pathogen, Ascosphaera apis. Thus, mixed infections of Ascosphaera species may be more detrimental to their bee hosts than single infections, but to what extent is that the case throughout the remainder of the Ascosphaera pathogens is hard to predict. We selected three pathogens, A. apis, Ascosphaera aggregata, and Ascosphaera larvis to determine if mixed infections with these pathogens in bees favor the assumptions of the superinfective or co-infective framework. Both A. apis and A. aggregata are the most prevalent obligate pathogens of two economically important bee species, A. mellifera (the European honey bee) and Megachile rotundata (the alfalfa leafcutting bee), respectively (Aronstein and Murray, 2010, James and Pitts-Singer, 2013). A. larvis is pathogenic to the alfalfa leafcutting bee, but it also grows saprophytically on the pollen provisions (Goettel et al., 1997, Bissett, 1988). It is not reported to occur in honey bee hives.
Section snippets
General bioassay methods
To quantify the competition dynamics of mixed Ascosphaera spp. infections in the bees, we compared host survival and fungal production (post host death) in bees fed combinations of pathogens. Bioassays were conducted using three pathogens (A. apis, A. larvis, and A. aggregata) and two hosts (honey bee and alfalfa leafcutting bee). For each treatment replicate, 36–60 bee larvae were given one of seven treatments (252–420 larvae per replicate) with various combinations of one or two pathogens
Honey bees
For honey bees, the survivorship functions significantly differed between treatments (χ26 = 325.11, P < 0.0001). Those treatments with A. aggregata or A. larvis that did not also contain A. apis, had no significant effect on honey bee larval survival, as compared to the control (Fig. 1A, Table A1). The obligate pathogen A. apis was the only factor that significantly decreased survivorship, and reduced the occurrence of cadavers with emerging hyphae (Table 3). In addition, the proportion of larvae
Discussion
We found that the two obligate pathogens in our study (A. aggregata and A. apis) caused greater host mortality and had higher pathogen reproduction in both solo and mixed infections when they infected their common hosts compared to when they infected a novel host. A close host–pathogen relationship in obligate pathogens often arises when a pathogen develops an ability to exploit one host, but this arises to the detriment of being able to exploit other hosts, thus leading to pathogen
Acknowledgments
We thank K. Anderson, M. Alston, M. Chambers, and C. Huntzinger for technical advice and access to resources; E. Babilonia and J. Goolsby for the irradiation of our pollen provisions; C. Marble for access to his farm and leafcutting bees. In addition, we thank S. Alizon, M. Brown, and two anonymous reviewers for helping us improve this manuscript. The authors declare no conflict of interest in the publication of this research. This research was funded by the USDA Agricultural Research Service
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- 1
Present address: Department of Biological Sciences, Rowan University, 201 Mullica Hill Road, Glassboro, NJ 08028, United States.
- 2
Present address: USDA National Programs Office, 5601 Sunnyside Ave, Beltsville, MD 20705, United States.