Navigating the landscape of fear: Fruit flies exhibit distinct antipredator and antiparasite defensive behaviors.

Most organisms are at risk of being consumed by a predator or getting infected by a parasite at some point in their life. Theoretical constructs such as the landscape of fear (perception of risk) and nonconsumptive effects (NCEs, costly responses sans predation or infection) have been proposed to describe and quantify antipredator and antiparasite responses. How prey/host species identify and respond to these risks determines their survival, reproductive success and, ultimately, fitness. Most studies to date have focused on either predator-prey or parasite-host interactions, yet habitats and ecosystems contain both parasitic and/or predatory species that represent a complex and heterogenous mosaic of risk factors. Here, we experimentally investigated the behavioral responses of a cactophilic fruit fly, Drosophila nigrospiracula, exposed to a range of species that include parasites (ectoparasitic mite), predators (jumping spiders), as well as harmless heterospecifics (nonparasitic mites, ants, and weevils). We demonstrate that D. nigrospiracula can differentiate between threat and non-threat species, increase erratic movements and decrease velocity in the presence of parasites, but decrease erratic movements and time spent grooming in the presence of predators. Of particular importance, flies could distinguish between parasitic female mites and nonparasitic male mites of the same species, and respond accordingly. We also show that the direction of these NCEs differs when exposed to parasitic mites (i.e., risk of infection) versus spiders (i.e., risk of predation). Given the opposing effects of predation versus infection risk on fly behavior, we discuss potential trade-offs between parasite and predator avoidance behaviors. Our findings illustrate the complexity of risk assessment in a landscape of fear and the fine-tuned NCEs that arise in response. Moreover, this study is the first to examine these behavioral NCEs in a terrestrial system.

Parasites and the ecology of fear: Nonconsumptive effects of ectoparasites on larvae reduce growth in simulated Drosophila populations.

Predators negatively affect prey outside of direct attack, and these nonconsumptive effects (NCEs) may cause over half the impacts of predators on prey populations. This "ecology of fear" framework has been extended to host-parasite interactions. The NCEs of parasites are thought to be small relative to those of predators. However, recent research shows ectoparasites exert NCEs on multiple life stages of Drosophila. In this study, we apply recent data to a matrix-based model of fly populations experiencing infection/consumption and NCEs from an ectoparasitic mite. We found the NCEs of parasites on larvae, which are not actively parasitized, decreased the size of simulated host populations. By contrast, the NCEs on adult flies increased population size through compensatory egg production. The negative NCEs on larvae outweighed the positive effects on adults to reduce population size. This study suggests that parasitic NCEs can suppress host populations independent of infection.

PHASMARHABDITIS CALIFORNICA (NEMATODA: RHABDITIDAE) HAS REDUCED ESTABLISHMENT SUCCESS AND PROGENY PRODUCTION IN THE PRESENCE OF PRISTIONCHUS ENTOMOPHAGUS (NEMATODA: DIPLOGASTRIDAE).

Phasmarhabditis (syn. Pellioditis) californica is a facultative parasite that has been marketed as a popular biocontrol agent against pestiferous slugs in England, Scotland, and Wales. The necromenic nematode Pristionchus entomophagus has also been recovered from slugs infected with Ph. californica. In this study, we experimentally investigated the outcome of single and mixed applications of Pr. entomophagus and Ph. californica on the slug Deroceras reticulatum (Müller). Host mortality was comparable for single and mixed applications of Ph. californica, with time to death significantly shorter in both treatment groups compared with controls. However, trials with Pr. entomophagus alone did not cause any significant host mortality relative to controls. Compared with the single Ph. californica applications, mixed applications resulted in 67% fewer infective juveniles establishing in the host, and subsequently far fewer infective juveniles were recovered in the next generation. In contrast, the establishment rate and progeny production in Pr. entomophagus were not impacted by the presence of Ph. californica (i.e., mixed applications). Hence, the presence of Pr. entomophagus had a deleterious effect on the establishment success and progeny production of Ph. californica. Our findings reveal an asymmetrical, antagonistic interaction between Ph. californica and Pr. entomophagus and highlight the importance of understanding the ecological relationships between co-occurring species. A decrease in parasite establishment success and progeny production has the potential to directly impact the persistence, sustainability, and efficacy of Ph. californica as a biological control agent.

CHEMOTAXIS RESPONSE OF PHASMARHABDITIS CALIFORNICA (FAMILY: RHABDITIDAE) AND PRISTIONCHUS ENTOMOPHAGUS (FAMILY: NEODIPLOGASTERIDAE) TO THE MUCUS OF FOUR SLUG SPECIES.

The chemotaxis responses of soil nematodes have been well studied in bacteriophagic nematodes, plant-parasitic nematodes, entomopathogenic nematodes, and to a lesser extent malacopathogenic nematodes. Free-living stages of parasitic nematodes often use chemotaxis to locate hosts. In this study, we compared the chemotaxis profile of 2 slug-associated nematodes with overlapping host ranges. Phasmarhabditis californica is a facultative parasite that has been shown to express strain-dependent variation in chemoattraction profile. We tested 4 slug species to determine the attraction index of a Canadian strain of Ph. californica and a sympatric necromenic nematode, Pristionchus entomophagus. When tested against a control (distilled water), Ph. californica showed a clear (positive) attraction towards the mucus of slugs Ambigolimax valentianus, Arion rufus, and Arion fasciatus, but not Deroceras reticulatum. However, when given a choice between the mucus of D. reticulatum and Ar. fasciatus in a pairwise test, Ph. californica was strongly attracted to the former. Other pairwise comparisons did not reveal a clear preference for either slug species in the following pairs: D. reticulatum-Ar. rufus, Am. valentianus-Ar. rufus, D. reticulatum-Am. valentianus. The chemotaxis assay for Pr. entomophagus showed an attraction toward D. reticulatum and Ar. fasciatus (tested against controls); the attraction index for Am. valentianus was positive, but this was not statistically significant. In contrast, the attraction index for Ar. rufus was negative, suggesting possible repulsion to the mucus of this slug species. Given that Pr. entomophagus and Ph. californica occupy overlapping habitats, utilize similar hosts, and exhibit similar chemotaxis profiles, there is a potential for direct interaction between these 2 nematodes. Like other members of the genus Pristionchus, Pr. entomophagus may be able to prey upon the co-occurring Ph. californica, such antagonistic interactions could have important implications for the coexistence of these 2 species and Ph. californica in particular as a biocontrol agent against pestiferous slugs.

A tale of two parasites: Responses of honey bees infected with Nosema ceranae and Lotmaria passim.

Nosema ceranae and Lotmaria passim are two commonly encountered digestive tract parasites of the honey bee that have been associated with colony losses in Canada, the United States, and Europe. Though honey bees can be co-infected with these parasites, we still lack basic information regarding how they impact bee health at the individual and colony level. Using locally-isolated parasite strains, we investigated the effect of single and co-infections of these parasites on individual honey bee survival, and their responsiveness to sucrose. Results showed that a single N. ceranae infection is more virulent than both single L. passim infections and co-infections. Honey bees singly infected with N. ceranae reached < 50% survival eight days earlier than those inoculated with L. passim alone, and four days earlier than those inoculated with both parasites. Honey bees infected with either one, or both, parasites had increased responsiveness to sucrose compared to uninfected bees, which could correspond to higher levels of hunger and increased energetic stress. Together, these findings suggest that N. ceranae and L. passim pose threats to bee health, and that the beekeeping industry should monitor for both parasites in an effort correlate pathogen status with changes in colony-level productivity and survival.

Ectoparasitic mites exert non-consumptive effects on the larvae of a fruit fly host.

The mere presence of predators or parasites can negatively impact the fitness of prey or hosts. Exposure to predators during an organism's development can have deleterious effects on juvenile survival and the subsequent adult stage. Currently, it is unknown if parasites have analogous impacts on host larval stages and whether these effects carry over into other subsequent life stages. However, parasites may be exerting widespread yet underestimated non-consumptive effects (NCEs). We tested if Drosophila nigrospiracula larvae avoid pupating near mite cues (caged Macrocheles subbadius) in arena experiments, and measured the rate of pupation in arenas with mites and arenas without mites. Larvae disproportionately pupated on the side of arenas that lacked mite cues. Furthermore, fewer larvae successfully pupated in arenas containing mites cues compared to arenas without mite cues. We found that ectoparasitic mites exert NCEs on Drosophila larvae, even though the larval stage is not susceptible to infection. We discuss these results in the context of parasite impacts on host population growth in an infectious world.

Parasite preferences for large host body size can drive overdispersion in a fly-mite association.

Body size generally correlates intraspecifically with insect fitness but can also correlate with parasite abundance (number of parasites). Host preferences by parasites, and variation in host immunity, could contribute to this trend. We investigated the effect of host size on mite-fly interactions (Macrocheles subbadius and Drosophila nigrospiracula). Mites strongly preferred to infect larger flies in pair-wise choices, and larger flies were more likely to be infected and acquired more mites in infection microcosms. Preferences of parasites resulted in size-biased infection outcomes. We discuss the implications of this heterogeneity in infection on parasite overdispersion and fly populations.

Relative contributions of parasite consumptive and non-consumptive effects to host population suppression in simulated fly-mite populations.

The "ecology of fear" framework was developed to describe the impacts predators have on potential prey and prey populations, outside of consumption/predation (i.e. non-consumptive effects, NCEs). This framework has recently been extended to symbiotic interactions such as host-parasite associations. Although the NCEs of predators and parasites on their individual victims can be measured experimentally, it is currently not known whether parasites can exert population-level effects on potential hosts through their NCEs. Modelling can be a useful tool for scaling individual-level NCEs to populations to determine impacts on host population growth. In this study, we used previously published data on the consumptive and non-consumptive effects of an ectoparasitic mite (Macrocheles subbadius) on a fruit fly (Drosophila nigrospiracula) to simulate populations experiencing fear (NCEs only), both fear and infection (consumption + NCEs) or neither. Population-level models indicate that NCEs alone were insufficient to reduce population growth. In fact, host populations experiencing NCEs but not infection had slightly larger final populations than unexposed populations (by ~ 550 flies). This result suggests there is compensation (i.e. increased daily reproduction that overcomes shorter lifespans) among exposed flies. By contrast, the consumptive effects of parasites suppressed the growth of simulated host populations, and this deleterious impact grew non-linearly with infection prevalence.

Scared of the dark? Phototaxis as behavioural immunity in a host-parasite system.

Behavioural immunity describes suites of behaviours hosts use to minimize the risks of infection by parasites/pathogens. Research has focused primarily on the evasion and physical removal of infectious stages, as well as behavioural fever. However, other behaviours affect infection risk while carrying ecologically significant trade-offs. Phototaxis, in particular, has host fitness implications (e.g. altering feeding and thermoregulation) that also impact infection outcomes. In this study, we hypothesized that a fly host, Drosophila nigrospiracula, employs phototaxis as a form of behavioural immunity to reduce the risk of infection. First, we determined that the risk of infection is lower for flies exposed in the light relative to the dark using micro-arena experiments. Because Drosophila vary in ectoparasite resistance based on mating status we examined parasite-mediated phototaxis in mated and unmated females. We found that female flies spent more time in the light side of phototaxis chambers when mites were present than in the absence of mites. Mating marginally decreased female photophobia independently of mite exposure. Female flies moved to lighter, i.e. less infectious, environments when threatened with mites, suggesting phototaxis is a mechanism of behavioural immunity. We discuss how parasite-mediated phototaxis potentially trades-off with host nutrition and thermoregulation.

Endosymbiotic Male-Killing Spiroplasma Affects the Physiological and Behavioral Ecology of Macrocheles-Drosophila Interactions.

While many arthropod endosymbionts are vertically transmitted, phylogenetic studies reveal repeated introductions of hemolymph-dwelling Spiroplasma into Drosophila. Introductions are often attributed to horizontal transmission via ectoparasite vectors. Here, we test if mites (Macrocheles subbadius) prefer to infect Spiroplasma poulsonii MSRO (Melanogaster sex ratio organism)-infected flies and if MSRO infection impairs fly resistance against secondary mite attack. First, we tested if mites prefer MSRO+ or MSRO- flies using pairwise choice tests across fly ages. We then tested whether mite preferences are explained by changes in fly physiology, specifically increased metabolic rate (measured as CO2 production). We hypothesize that this preference is due in part to MSRO+ flies expressing higher metabolic rates. However, our results showed mite preference depended on an interaction between fly age and MSRO status: mites avoided 14-day-old MSRO+ flies relative to MSRO- flies (31% infection) but preferred MSRO+ flies (64% infection) among 26-day-old flies. Using flowthrough respirometry, we found 14-day-old MSRO+ flies had higher CO2 emissions than MSRO- flies (32% greater), whereas at 26 days old the CO2 production among MSRO+ flies was 20% lower than that of MSRO- flies. Thus, mite preferences for high-metabolic-rate hosts did not explain the infection biases in this study. To assess changes in susceptibility to infection, we measured fly endurance using geotaxis assays. Older flies had lower endurance consistent with fly senescence, and this effect was magnified among MSRO+ flies. Given the biological importance of male-killing Spiroplasma, potential changes in the interactions of hosts and potential vectors could impact the ecology and evolution of host species. IMPORTANCE Male-killing endosymbionts are transmitted from mother to daughter and kill male offspring. Despite these major ecological effects, how these endosymbionts colonize new host species is not always clear. Mites are sometimes hypothesized to transfer these bacteria between hosts/host species. Here, we test if (i) mites prefer to infect flies that harbor Spiroplasma poulisoni MSRO and (ii) flies infected with MSRO are less able to resist mite infection. Our results show that flies infected with MSRO have weaker anti-mite resistance, but the mite preference/aversion for MSRO+ flies varied with fly age. Given the fitness and population impacts of male-killing Spiroplasma, changes in fly-mite interactions have implications for the ecology and evolution of these symbioses.

Trade-offs between reproduction and behavioural resistance against ectoparasite infection.

Reproduction is a key determinant of organismal fitness, but organisms almost always face the threat of parasite infection. Thus, potential trade-offs between mating and parasite resistance may have substantial impacts on the ecology and evolution of host species. Although trade-offs between microbial resistance and mating in arthropods are well-documented, there is a paucity of evidence that mating compromises host resistance to the ubiquitous threat posed by ectoparasites. Despite the centrality of reproduction to host fitness and the widespread risk of parasites, there is a dearth of experiments showing a trade-off between mating/reproduction and anti-parasite behaviours. In this study, we test if mating increases the susceptibility of female flies to mite infection. We also investigated a potential underlying mechanism for the trade-off: that mating reduces overall endurance and hence anti-parasitic defenses among female flies. We experimentally mated female Drosophila nigrospiracula, with or without a chance to recover from male harassment, and challenged them with a natural ectoparasite, the mite Macrocheles subbadius. Mated females, regardless of time for recovery from male harassment, acquired more infections than unmated females. Furthermore, mated females had lower endurance in negative geotaxis assays, suggesting the increased susceptibility is due to reduced endurance. Our research shows a trade-off between reproduction and parasite resistance in a host-macroparasite system and suggests that trade-off theory is a fruitful direction for understanding these associations.

The influence of infection status and parasitism risk on host dispersal and susceptibility to infection in Drosophila nigrospiracula.

For many organisms, habitat avoidance provides the first line of defence against parasitic infection. Changes in infection status can shift the cost-benefit ratio of remaining in a given habitat vs dispersing. The aim of this study was to test the hypothesis that the propensity to disperse in Drosophila nigrospiracula is mediated by current parasite load and the risk of further infection by an ectoparasitic mite (Macrocheles subbadius). An activity monitor was used to assess dispersal propensity among infected and uninfected flies. The activity level of uninfected females increased threefold upon exposure to a mite, whereas the activity among uninfected males increased by 17-fold in the presence of a questing mite. Among infected flies, the risk of further infection also generated a change in activity, but the magnitude of the response was dependent on host sex. Current infection status influenced the probability of acquiring more parasites due to increased susceptibility to infection with mite load. The probability of acquiring additional mites among males increased more rapidly compared to female flies. Current infection status can potentially determine the risk of further infection, the host propensity and ability to disperse, with consequence for hosts and parasites at the individual, population and species level.

Extending the ecology of fear: Parasite-mediated sexual selection drives host response to parasites.

The 'ecology of fear' describes the negative effects natural enemies have on potential victims even when those victims are not consumed or infected. Although recent work has demonstrated parasites have non-consumptive effects (NCE) on potential hosts, how these effects vary within host populations is not well understood. We investigated how NCE vary based on host risk of infection and relative cost of infection by measuring the metabolic rate (MR) of naive Drosophila nigrospiracula exposed to an ectoparasite, Macrocheles subbadius. We tested two mutually exclusive hypotheses: 1) asymmetrical costs of infection drive adaptions for stronger responses to parasite exposure; or 2) asymmetrical risks of infection drive adaptions for stronger responses to parasite exposure. In this system, male flies have higher costs of infection relative to female flies due to parasite-mediated sexual selection; similarly, virgin females experience higher costs of infection relative to mated females. Risk of infection also varies among flies because mites preferentially infect female flies over males, and mites preferentially infect mated females over virgin females. Our results were compatible with the hypothesis that costs of infection drive the strength of response to mite risk. Female flies responded to parasite exposure with a 15.1% increase in MR, while exposed males showed a stronger response with a 31.3% increase in MR. Mated females increased their MR by 34.8% during mite exposure whereas virgin females experienced an increase of 61.2%. Our findings suggest that NCE of parasites can vary based on state-dependent costs of infection.

Life history of Macrocheles muscaedomesticae (Parasitiformes: Macrochelidae): new insights on life history and evidence of facultative parasitism on Drosophila.

Macrocheles muscaedomesticae is a cosmopolitan macrochelid mite whose populations have likely diverged considering the many locations they inhabit, but most of the work published on this mite species has been on the basis of their association with the house fly, Musca domestica. Here, we studied several aspects of the biology of M. muscaedomesticae associated with drosophilid flies collected in Alberta, Canada. We assessed the degree of divergence of our populations from others, compared their life history to other published populations and experimentally tested whether M. muscaedomesticae feeds on Drosophila hydei hosts by comparing the body mass of mites that attached to hosts to those that did not. There was no strong phylogenetic differentiation among any of the M. muscaedomesticae specimens, suggesting multiple recent introductions of this species to Canada. Compared to other populations, our mites exhibited lower fecundity, which may have been a result of the temperature or nematode-only diet in which they were maintained. Finally, mites that attached to hosts for 4 h weighed significantly more than those that did not. Without direct evidence for host tissue transfer to the mites, it is difficult to determine whether the mites are indeed feeding on their hosts while attached. However, the existing evidence for the costs fly hosts endure at the expense of these mites makes this relationship antagonistic.

Ecology of fear: environment-dependent parasite avoidance among ovipositing Drosophila.

Habitat avoidance is an anti-parasite behaviour exhibited by at-risk hosts that can minimize exposure to parasites. Because environments are often heterogeneous, host decision-making with regards to habitat use may be affected by the presence of parasites and habitat quality simultaneously. In this study we examine how the ovipositing behaviour of a cactiphilic fruit fly, Drosophila nigrospiracula, is affected by the presence of an ectoparasitic mite, Macrocheles subbadius, in conjunction with other environmental factors - specifically the presence or absence of conspecific eggs and host plant tissue. We hypothesized that the trade-off between site quality and parasite avoidance should favour ovipositing at mite-free sites even if it is of inferior quality. We found that although flies avoided mites in homogeneous environments (86% of eggs at mite-free sites), site quality overwhelmed mite avoidance. Both conspecific eggs (65% of eggs at infested sites with other Drosophila eggs) and host plant tissue (78% of eggs at infested sites with cactus) overpowered mite avoidance. Our results elucidate the context-dependent decision-making of hosts in response to the presence of parasites in variable environments, and suggest how the ecology of fear and associated trade-offs may influence the relative investment in anti-parasite behaviour in susceptible hosts.

Phenotypic plasticity more essential to maintaining variation in host-attachment behaviour than evolutionary trade-offs in a facultatively parasitic mite.

A prevailing hypothesis for the evolution of parasitism posits that the fitness benefits gained from parasitic activity results in selection for and fixation of parasitic strategies. Despite the potential fitness advantage of parasitism, facultative parasites continue to exhibit genetic variation in parasitic behaviour in nature. We hypothesized that evolutionary trade-offs associated with parasitic host-attachment behaviour maintain natural variation observed in attachment behaviour. In this study, we used replicate lines of a facultatively parasitic mite, previously selected for increased host-attachment behaviour to test whether increased attachment trades off with mite fecundity and longevity, as well as the phenotypic plasticity of attachment. We also tested for potential correlated changes in mite morphology. To test for context-dependent trade-offs, mite fecundity and longevity were assayed in the presence or absence of a host. Our results show that selected and control mites exhibited similar fecundities, longevities, attachment plasticities and morphologies, which did not provide evidence for life history trade-offs associated with increased attachment. Surprisingly, phenotypic plasticity in attachment was maintained despite directional selection on the trait, which suggests that phenotypic plasticity likely plays an important role in maintaining attachment variation in natural populations of this facultative parasite.

Facultative parasites as evolutionary stepping-stones towards parasitic lifestyles.

Parasites and parasitic lifestyles have evolved from free-living organisms multiple times. How such a key evolutionary transition occurred remains puzzling. Facultative parasites represent potential transitional states between free-living and fully parasitic lifestyles because they can be either free-living or parasitic depending on environmental conditions. We suggest that facultative parasites with phenotypically plastic life-history strategies may serve as evolutionary stepping-stones towards obligate parasitism. Pre-adaptations provide a starting point for the transition towards opportunistic or facultative parasitism, but what evolutionary mechanism underlies the transition from facultative to obligate parasitism? In this Opinion Piece, we outline how facultative parasites could evolve towards obligate parasites via genetic assimilation, either alone or in combination with the Baldwin effect. We further describe the key predictions stemming from each of these evolutionary pathways. The importance of genetic assimilation in evolution has been hotly debated. Studies on facultative parasites may not only provide key insights regarding the evolution of parasitism, but also provide ideal systems in which to test evolutionary theory on genetic accommodation.

Host Respiration Rate and Injury-Derived Cues Drive Host Preference by an Ectoparasite of Fruit Flies.

Host bioenergetics and energy fluxes can be applied to measure the ecological and physiological effects of parasitism. By measuring changes in host metabolic rate, one can estimate the physiological costs of infection. Additionally, metabolic rate dictates the rate of resource conversion within a host and, by extension, the resources available to a parasite. We hypothesize that parasites are selected to respond to cues that indicate high resource availability, that is, host metabolic state. We investigated whether an ectoparasite mite (Macrocheles subbadius) can differentiate between potential hosts (Drosophilia nigrospiracula) on the basis of relative carbon dioxide output as measured by respirometry. In pairwise choice tests, mites were allowed to choose between two size-matched fruit flies with differing metabolic rates or levels of CO2 output. Our results showed that mites preferentially infect flies with relatively higher respiration rates. Accordingly, we investigated whether fly respiratory rate (measured by CO2 production) changes in response to injury, potentially explaining a previously reported preference for injured flies. We also tested whether chemical cues released during injury influence preference for injured hosts. We determined that fly exudate (mostly consisting of hemolymph) applied to an uninjured fly released at the site of injury significantly increased the likelihood of infection, but injury did not significantly change the CO2 output of the flies. Our results suggest that parasites are relying on chemical cues not only for host finding but also to discriminate between hosts on the basis of the rate of respiration, with potentially important implications for the metabolic theory of ecology.

Proximity to parasites reduces host fitness independent of infection in a Drosophila-Macrocheles system.

Parasites are known to have direct negative effects on host fitness; however, the indirect effects of parasitism on host fitness sans infection are less well understood. Hosts undergo behavioural and physiological changes when in proximity to parasites. Yet, there is little experimental evidence showing that these changes lead to long-term decreases in host fitness. We aimed to determine if parasite exposure affects host fitness independent of contact, because current approaches to parasite ecology may underestimate the effect of parasites on host populations. We assayed the longevity and reproductive output of Drosophila nigrospiracula exposed or not exposed to ectoparasitic Macrocheles subbadius. In order to preclude contact and infection, mites and flies were permanently separated with a mesh screen. Exposed flies had shorter lives and lower fecundity relative to unexposed flies. Recent work in parasite ecology has argued that parasite-host systems show similar processes as predator-prey systems. Our findings mirror the non-consumptive effects observed in predator-prey systems, in which prey species suffer reduced fitness even if they never come into direct contact with predators. Our results support the perspective that there are analogous effects in parasite-host systems, and suggest new directions for research in both parasite ecology and the ecology of fear.