Gut microbiomes of mobile predators vary with landscape context and species identity

Abstract Landscape context affects predator–prey interactions and predator diet composition, yet little is known about landscape effects on insect gut microbiomes, a determinant of physiology and condition. Here, we combine laboratory and field experiments to examine the effects of landscape context on the gut bacterial community and body condition of predatory insects. Under laboratory conditions, we found that prey diversity increased bacterial richness in insect guts. In the field, we studied the performance and gut microbiota of six predatory insect species along a landscape complexity gradient in two local habitat types (soybean fields vs. prairie). Insects from soy fields had richer gut bacteria and lower fat content than those from prairies, suggesting better feeding conditions in prairies. Species origin mediated landscape context effects, suggesting differences in foraging of exotic and native predators on a landscape scale. Overall, our study highlights complex interactions among gut microbiota, predator identity, and landscape context.

Currie, Gillette, & Raffa, 2010;Coon, Brown, & Strand, 2016;Toju & Fukatsu, 2011;Yun et al., 2014). The gut microbiome of wild insect populations likely represents a sample of microbiota from local food and other sources in their surrounding environment (Borer, Kinkel, May, & Seabloom, 2013). On a local scale (small quadrats of 0.025 m²), correlations among gut microbial richness of two ground-dwelling cricket species and prey richness in the habitat have been reported (Schmid, Lehman, Brözel, & Lundgren, 2015); yet, the landscape-level consequences for mobile organisms such as flying predators have remained largely unexplored.
Yet, systematic studies on the effects of landscape context on predator gut microbiota are lacking.
Ideally, studies investigating landscape configuration and composition are performed in experimental landscapes, where landscape attributes are controlled by the experimenter (Hadley & Betts, 2016, p. 59). However, such studies are often performed within only a single habitat type and cover often cover less than 1 km² (Haddad et al., 2015); such scales are considerably smaller than the foraging range of many insects, including pollinators or predatory beetles. Here, we report results from a mensurative experiment, in which study sites are selected a priori on a meaningful biological scale. We present evidence for landscape-level effects on insect gut microbiota on a scale of several thousand km².
Predator fitness may be affected by landscape context directly through variability in food quality and quantity. Prior work has shown that landscape context is associated with fitness-related measures of body condition, such as body size or fat content, in grounddwelling predators (Bommarco, 1998;Öberg, 2009;Östman, Ekbom, Bengtsson, & Weibull, 2001), but this relationship has not been examined in mobile arthropod predators and the role of gut microbes has remained elusive. As the microbiome can directly affect the nutritional state and health of an organism (Bahrndorff et al., 2016;Borer et al., 2013;Gibson & Hunter, 2010;Ruokolainen et al., 2016), changes in the microbiome associated with the landscape could also have indirect microbe-mediated effects on body condition.
In this study, we examined the effects of landscape context on the gut bacterial community and body condition of predatory insects.
We used aphidophagous lady beetles as our study system, as they are locally widespread and important natural enemies of aphids in agricultural crops (Obrycki, Harwood, Kring, & O'Neil, 2009;Snyder, 2009) and seminatural habitats (Bianchi et al., 2013). Although aphids are their preferred prey, the lady beetles' food spectrum includes a broad range of other soft-bodied arthropods, as well as fungal or plant resources (Dixon, 2000;Evans, 2009;Hodek & Honěk, 1996;Trilitsch, 1999;Weber & Lundgren, 2009). In a proof-of-concept laboratory experiment, we first show that even a single meal can increase the richness and alter the community composition of gut bacteria in individual beetles, indicating that diet diversity can affect gut communities. In a mensurative field experiment (Hadley & Betts, 2016), we sampled six lady beetle species that differ in their phylogenetic relatedness (including three in the same genus), origin (native and exotic), and body size to explore the contribution of host-specific factors to differences in the gut microbiome and physiological response to landscape context. We tested the effects of landscape context at two spatial scales by sampling beetles in two field types with contrasting plant diversity: (1) species-rich prairies and soybean monocultures that (2) were systematically selected to be surrounded by landscapes ranging from low to high proportion of land covered by annual crops in southern Wisconsin, USA. We expected that mobile predators that forage in prairies have access to a broader range of prey types compared to beetles foraging in soybean and therefore would have a richer gut community. Because mobile predators may forage on a landscape scale, we further predicted that lady beetles would have a relatively simpler gut community when the collection sites are surrounded by crop-dominated landscape compared to sites surrounded by more natural habitats. In addition, we examined whether landscape-mediated changes in predator gut microbiota were associated with differences in body condition, assessed using estimates of beetle fat content.
Fat content reflects the available energy reserves for survival and reproduction and resistance to nutritional stress (Arrese & Soulages, 2010;Roma, Bueno, & Camargo-Mathias, 2010). We predicted that prairies and landscapes with low proportions of arable land would foster greater body condition. We show that changes at the field and landscape scale affected the gut bacterial community and physiological response of predators, but the direction of the effect differed significantly between exotic and native species, raising the possibility of inherent differences in habitat use and foraging preferences among these groups.

| Feeding experiment
In a laboratory feeding experiment, we tested whether a single meal has the potential to alter the gut bacterial community of lady beetles.
Adult Coleomegilla maculata De Greer (pink spotted lady beetle) were collected in April 2012 in Arlington, Wisconsin (USA), from dandelion flowers where they commonly aggregate in the spring (Harmon, Ives, Losey, Olson, & Rauwald, 2000;Figure 1d). Beetles were maintained in the laboratory on dandelion flowers and moistened cotton balls for 7 days to allow their gut bacteria to equilibrate to similar diet environments. Prior to testing, beetles were starved for 48 hr. The beetles were randomly allocated to three treatments: (1) no food (control), Beetles that finished their meal completely within 1 hr (n = 19 beetles) were transferred into 1.5-ml microtubes containing 70% ethanol and frozen at −20 °C (n = 7 for the control, n = 5 for the 1-species diet, and n = 7 for the 5-species diet).

| Field study
We sampled wild populations of lady beetles in southern Wisconsin, USA, in 2012. The region is dominated by agricultural row crops (mainly corn [Zea mays L.] and soybean [Glycine max L.]) with remaining patches of seminatural habitat (i.e., forest, grasslands, wetlands). We initially selected 10 prairies and 10 conventionally managed soy fields as two field types with contrasting diversity of plants and likely associated prey species. The fields were at least 2.6 km apart ( Fig. S1 in Appendix S1). We analyzed the landscape composition within a 2 km radius of each field, which is an ecological meaningful distance for foraging flights in lady beetles (Woltz & Landis, 2014). The proportions of land cover types within each sector were analyzed with ArcGIS (10.0, ESRI, Redlands, CA, USA) and the Geospatial Modeling Environment software (Beyer, 2012) with the Cropland Data Layer (CDL, USDA, NASS 2012). As a metric for landscape complexity, we used the proportion of annual crop monocultures (0.16-0.77; cropland hereafter) as it represents a habitat that is frequented by lady beetles but is intrinsically species poor and, in contrast to seminatural habitat, is easy to unambiguously categorize. The proportion of cropland and seminatural habitat were negatively correlated (Pearson's r = −.88, p < .001) and the later produced essentially the same results when used in the analysis instead.
We sampled each field multiple times by sweep netting or hand collection from July through mid-August. During this time, soybean aphid (A. glycines) populations usually reach high densities, but in 2012, they remained exceptionally low likely due to the severe drought in the Midwest (Liere et al., 2015). It was also difficult to find lady beetles (compared to our previous experience), and we succeeded in only eight soy fields and nine prairies. In total, we collected 243 beetles (n = 139 in prairie, n = 104 in soy) belonging to six aphidophagous species (Coccinellidae: Coccinellinae: Coccinellini) including the exotic Collected beetles were immediately placed separately into microtubes containing 70% ethanol, transported to the laboratory on ice and preserved at −20°C until later analysis.

| Analysis of gut bacteria
We characterized the total gut bacterial community of lady beetles with Automated Ribosomal Intergenic Spacer Analysis (ARISA), a costand time-efficient fingerprinting technique. ARISA detects bacterial phylotypes based on the length heterogeneity of the intergenic spacer region between the 16S and 23S rRNA genes (Fisher & Triplett, 1999).
We analyzed up to four technical PCR replicates for each sample of the feeding experiment due to the low number of biological replications. No technical replications were used for wild populations.
Reagent-only controls were included from the PCR step onwards.
The PCR fragments were separated with a capillary sequencer (ABI

| Estimation of body fat content
We visually estimated the fat content in individual beetles during gut dissections. Beetles were assigned to the categories low, medium, and high fat content (Anderson, 1981): "Low": little visual fat, mainly accumulated in the parietal layer; "Medium", clearly visible fat accumulations also in regions of the gut or reproductive organs; "High": fat filling and expanding the abdomen. Compared to whole body fat extraction, visual estimates of body fat do not provide quantitative data but allowed us to distinguish between storage fat and accumulated lipids in reproductive organs. Considering the fluctuations in total body fat in females during egg laying, estimates of storage fat provide a suitable assessment of the nutritional state.

| Feeding experiment
For the feeding experiment, technical replications existed for all but three samples and were averaged prior to the analysis. The relationship between bacterial richness and the number of prey species in the meal (zero in the control, 1-species diet, 5-species diet) was analyzed with linear regression. The number of bacterial taxa in a sample was log-transformed, and the model included number of technical replicates per sample as known prior weights, giving more weight to samples with more replications.
We analyzed the gut bacterial community assemblage using

Bacterial richness
We tested the effects of host-specific factors, sex, field type, and proportion of annual cropland in the surrounding 2 km on the logtransformed gut bacterial richness using linear mixed-effects mod-

Bacterial community structure
Bacterial community composition in wild collected species was visualized as in the laboratory experiment with NMDS based on Bray-Curtis distances and by mean relative abundance of bacterial taxa per beetle species and habitat type ( Fig. S2 in Appendix S1). We tested the effect of species, and species grouped by genus, origin, and body size on bacterial composition using separate (one-way) perMANOVA (adonis; Oksanen et al., 2017). Species, as the best predictor, was included in a model testing the interactions between species and field type, and species and proportion cropland. Additionally, we tested the interaction between species and sex. All models included sex within species within collection site as random effect. Homogeneity of sample dispersion was tested (vegan: betadisper, permutest; Oksanen et al., 2017).

Body fat content
We analyzed the proportion of beetles in three ordinal categories (low, medium, and high fat content) using cumulative link mixedeffects models (ordinal: CLMM; Christensen, 2015) as a function of beetle species contrasts, field type, proportion cropland, and bacterial richness as fixed effects and beetle species within collection site as random effects. The full models included all two-way interactions, and models were simplified as described above. To assess the effect of sex, three-way interactions with sex were included in the best fit model and deleted from maximal models based on AICc.

| Feeding experiment
In guts of the 19 beetles from the feeding experiment, we found 313 bacterial phylotypes (OTUs). The bacterial richness in individual beetle guts increased with the number of prey species in the meal (Table 2; Figure 2a) from 28 ± 7 (mean ± SD) in the beetles in the unfed (control) diet, to 31 ± 5 in the 1-species diet, and 39 ± 11 in the 5-species diet. Overall, we detected a significant but weak effect of the meal type on the bacterial community (perMANOVA; Table 2A; Figure 2b).
In pairwise tests (Table 2b-d), the gut communities between beetles from the 1-species diet and the 5-species diet differed from the control but not from each other. Nonsignificant differences in sample dispersion (

| Bacterial richness
In total, we found 551 bacterial taxa (OTUs) in the guts of 243 field collected beetles; the mean bacterial richness was 80 ± 20. Most of the variance in richness was explained by the differences between beetle species, which was higher in the three exotic species than in the three native species (Table 3 and Figure 3a; Table S3 in Appendix S1). Moreover, exotic and native species responded differently to landscape context: the bacterial richness in native species guts increased with increasing proportion of cropland surrounding the collection side, but decreased for exotic species (Tables 3 and Figure 3a; Table S3 in Appendix S1). Further, there was an effect of field type with higher bacterial richness in beetles collected in soy than in prairies (Table 3; Table S3 in Appendix S1). Sex had no effect.

| Bacterial community structure
The bacterial assemblages were largely associated with beetle species identity (perMANOVA ; Table 3a and Figure 3b). Origin, genus, and body size, also, had significant effects on the community structure, but the fit of the models was weaker (Table 3b-d). Sex, field type (corn vs. soy), and proportion cropland did not explain additional variability (Table 3e,f). The detected effects on the bacterial community might be partly driven by variances in sample dispersion between species (Table 3), but species also had distinct sets of abundant bacteria indicating compositional differences among species (Fig. S2 in Appendix S1).

| Body fat content
The relative fat content of beetles was associated with species identity (Tables 3 and Table S4 in Appendix S1). Most beetles of the genus  Figure 4a; Table S4 in Appendix S1). Gut bacterial richness also interacted with the proportion of cropland to affect variation in beetle fat content. Bacterial richness had a negative effect on fat content when the proportion of cropland was low and a positive effect when the beetles were collected in crop-dominated areas (Tables 3 and Figure 4b; Table S4 in Appendix S1). Further, beetles collected in prairie had a higher fat content compared to soy (Table 3; Table S4 in Appendix S1) and tended to be fatter when the prairie was surrounded by cropland, but this interaction was only marginally significant (Table 3 and Figure 4c; Table S4 in Appendix S1). When sex was included as a fixed effect in the analysis, the interaction between prairie and the proportion of cropland also became significant.
Additionally, we found an interaction between crop and sex with only females responding positive to increasing proportions of cropland.
Further, there was an interaction between species and sex (Table S5 and S6 in Appendix S1).

| DISCUSSION
We hypothesized that the diversity and composition of gut microbes in mobile arthropod predators would be affected by landscape context, both at the local (field) and at broader (among field, landscape) scale. Consistent with this prediction, we found that changes in landscape composition were associated with changes in richness of bacterial OTUs in the guts of beetles, but this effect was strongly species-dependent. In fact, one of the strongest patterns observed in this study was the distinct difference in abundance and composition of gut bacteria across species of lady beetles. Moreover, a significant amount of bacterial community variation, and the response of microbes to landscape composition, was related to whether species were native or exotic, an unexpected finding. Native lady beetles had a richer gut bacterial community, and this richness increased as the landscape became more crop-dominated; in contrast, the gut bacterial richness of exotic beetles was generally lower than that of natives and decreased as the amount of cropland increased in the landscape.

| Species effects on bacterial richness and composition
The significant effect of lady beetle species on the gut bacterial community composition raises three nonmutually exclusive hypotheses about T A B L E 3 Field study results on gut bacteria and fat content of wild populations of lady beetles of gut contents of lady beetles revealed potential symbionts (Paula et al., 2016). However, facultative gut symbionts were also detected in omnivorous ground beetles (Lundgren, Lehman, & Chee-Sanford, 2007) and distinct gut communities in predatory ants (Anderson et al., 2012) and wasps (Mrázek, Strosová, Fliegerová, Kott, & Kopecný, 2008).  Another potential explanation is that species-specific chemophysical characteristics of the gut select for colonization by certain bacteria (Dillon & Dillon, 2004;Nelson, Rogers, Carlini, & Brown, 2012).
However, if this was a strong influencing factor, then we would expect that shared evolutionary history of beetles would result in the gut bacterial communities of closely related species to be more similar than distantly related species (Sanders et al., 2014). However, this was not the case for the three species of the genus Hippodamia in our study which had distinct bacterial assemblages more associated with whether they were exotic or native to the Midwestern USA. Although this study was not specifically designed to test for systematic differences in bacterial communities as a function of evolutionary relatedness or their exotic vs. native status, the patterns found in the most widespread beetle species in this area were strong and warrant additional study.
A third explanation for our findings of species-specific differences in gut bacteria relates to differences in their diets, which could result in different sets of prey-related bacteria. The laboratory experiment demonstrated that beetle gut communities could change relatively rapidly even within one species. Similar to our findings, H. axyridis gut microbes were enriched by aphid symbionts shortly after aphid ingestion (Paula et al., 2015). This hypothesis is further supported by a study on fruit fly species with distinct feeding habits, whose gut communities were different in wild populations but became similar on the same diet under laboratory conditions (Chandler et al., 2011). Thus, it is likely that at least some of the bacterial variation between lady beetle species was due to dietary differences maybe as a result of resource partitioning through differences in the dietary breadth, prey preferences, the ability to locate prey, preferred areas on a plant to forage, and the likelihood of switching habitats (Forbes & Gratton, 2011;Hodek & Honěk, 1996;Iperti, 1999;Schellhorn & Andow, 2005;Sloggett & Majerus, 2000). Studies that simultaneously identify food remains and microbes in gut contents (Paula et al., 2015;Tiede et al., 2016) could further illuminate the relation between diet and the gut microbiome.

| Landscape effects on bacterial richness and composition
Other studies have shown that exotic species often dominate lady beetle communities in arable land. In this region, native species are mainly found in perennial grasslands and other seminatural habitats (Gardiner et al. 2009b;Diepenbrock & Finke 2013;Grez et al. 2013).
A similar pattern was found for native and exotic spider communi- ties. An increasing amount of arable land is often associated with seminatural habitat fragmentation and more distant remnant patches are expected to harbor more dissimilar communities than close ones . Thus, native beetles might have sampled a greater beta diversity of microbes from isolated natural habitat patches when located in landscapes with a high proportion of cropland. The preference of exotic beetles for homogenous agricultural habitats (i.e., crops fields) could have led to a reduced exposure to bacteria in the environment and therefore a lower gut bacterial richness. Additionally, a higher pathogen load in agricultural landscapes combined with higher antimicrobial defense in exotic species could contribute to the pattern of increasing microbial richness with increasing amount of cropland in native but not exotic lady beetles.
The specific habitat type in which beetles were collected, soy compared to prairie, was another strong predictor for bacterial richness.
In contrast, to our expectation that beetles from prairie would have a richer gut community, we found more bacterial diversity in the guts of beetles from soy. This finding could be partly attributed to a drought that affected the soybean plants and aphid populations in southern Wisconsin (Mallya, Zhao, Song, Niyogi, & Govindaraju, 2013). The low availability of soybean aphids, the principal prey of lady beetles in this crop, likely increased the consumption of alternative prey (Iperti, 1999;Sloggett & Majerus, 2000). A broader diet in soybean would expose the beetles to a greater variability of environmental bacteria compared to a diet of mainly aphids. In H. axyridis, aphid-symbionts were detected up to 96 hr after aphid consumption (Paula et al., 2015). Prairie plant communities were more resilient to the drought than row crops (Joo et al., 2016) and likely allowed the aphidophagous lady beetles in our study to be more selective in their prey choice.
Additionally, differences in local food availability between the two habitat types could have led to differences in residency time. The beetles we collected in soybean might have switched from another (crop-) habitat not long before (Forbes & Gratton, 2011) and carried over bacteria and higher food availability in prairie could have increased smallscale foraging. The lack of information on how much time a beetle has spent in the field where it was sampled may to some degree confound the local and the landscape scale used in our study.

| Microbe and landscape effects on ladybeetle fat content
We posit that the higher gut bacterial richness in beetles from soy fields compared to prairies is an indicator of consumption of mixed alternative resources in absence of soybean aphids. This interpretation is consistent with the findings that beetles collected in prairie had a higher fat content compared to soy-collected beetles, indicating superior feeding conditions and a better outcome for body condition in prairie compared to aphid-depauperate soy. Landscape context on a broad scale had no effect itself but mediated the effect of bacterial richness on body fat of beetles: As bacterial richness increased, beetles became fatter in agriculturally dominated landscapes, while for beetles collected in landscapes with few crops, higher bacterial richness was associated with lower fat content. Generalist predators can benefit from some proportion of cropland, which periodically provides abundant food resources (Rand & Tscharntke, 2007) but may benefit more from the inclusion of alternative resource with complementary nutrients in simplified landscapes in which they mainly find crop pests. Other studies on predatory beetle body condition found positive effects of landscape heterogeneity (Östman et al., 2001) and succession-related food supply and diversity of wildflower habitats (Barone & Frank, 2003).
Although landscape context clearly had an impact on gut microbiota, and landscape context and gut microbial richness together affected the fat content of lady beetles, the ultimate causal mechanisms remain to be explored. We propose that food resource abundance and diversity in the local habitat could be one of the main drivers for both gut bacterial richness and host fat content. Further, diet-related bacteria can potentially affect host fitness directly when they serve as a supplemental food source, temporarily contribute to digestion processes (Bouchon et al., 2016) or facilitate adaption to novel food sources (Chu, Spencer, Curzi, Zavala, & Seufferheld, 2013).
However, if and to what extend a predator benefits from a mixed diet (Evans, Stevenson, & Richards, 1999;Harwood et al., 2009;Lefcheck, Whalen, Davenport, Stone, & Duffy, 2012;Lundgren, 2009) and diverse gut bacteria depends on host species: In our study, the two native beetles H. convergens and H. parenthesis had more body fat when their guts harbored many different bacterial. In contrast, the exotic C. septempunctata and H. axyridis were fatter when their gut bacterial communities were species poor. This finding might reflect that exotic species are better adapted to homogenous conditions in cropland than native species and therefore often dominate coccinellid communities in cultivated habitats (Bahlai, Colunga-Garcia, Gage, & Landis, 2013).

| CONCLUSION
A key finding of this study is that mobile predatory insects have a species-specific set of gut bacteria that is stable over a range of environmental conditions. However, landscape and habitat-associated differences in where they are collected can alter this base assemblage.
Although the mechanisms for these patterns are not resolved, the strong differences between exotic and native species and the contrasting effects of landscape context on gut bacteria suggest inherent differences in habitat and prey use among these groups. Moreover, that landscape context can also affect host performance as indicated by fat content, both directly and indirectly via gut microbiota, potentially indicates a novel mechanism through which human-altered landscapes can affect invertebrate predators. The method we used to analyze gut bacterial communities allowed us to rapidly compare samples from multiple species and locations but does not provide information on taxon identity. Sequencing-based technologies in combination with reference databases for taxon identification are an ideal next step. This could help identify the core microbes of different species, their relationship to the host and response to environmental factors. We focused on bacterial microbes which are thought to comprise the greatest fraction of organisms in the guts of many insect (Engel & Moran, 2013), but further studies could expand the range to other potential interaction partners, like fungi, protists, and archaea. Overall, our study illustrates the importance of both resource and landscapebased influences on gut microbiota and their interactions with speciesspecific traits including foraging behavior and physiology.

ACKNOWLEDGMENTS
We thank the landowners who allowed us to sample lady beetles on their land and the field assistants who helped to collect samples (C. Bergstrom, C. Fritz, A. Rudie, C. Schwantes, and A. Wenninger).
We also thank C. Mason

DATA ACCESSIBILITY
The data and R scripts used for data analysis are provided in the in Appendices S2, S3, and S4.