Acetolysis modifications to process small pollen samples swabbed from live bees.

Understanding the resources bees use is essential because we depend greatly on their ecosystem services, and this information could help guide conservation efforts. One way to identify the flowers that bees visit is to collect pollen directly from the bee and then identify the pollen with plant taxa. However, the current method for processing such pollen samples, acetolysis, is designed for samples such as those collected across individuals (e.g., pollen trap), bee nests, or, at the very least, from pollen pellets collected from live bees or from the exhaustive removal of pollen from lethally collected individuals. Smaller samples, including those down to just a few pollen grains sampled from live bees, could facilitate additional opportunities for bee-pollen research, if they can be processed effectively. We present a revised acetolysis methodology designed specifically for processing small pollen samples, so that they can then be used for more accurate identification. Using pollen samples from cotton swabs directly applied to live bees in the field, we demonstrate the effectiveness of our methodology for processing small pollen samples, including samples too small to be visually detected. This methodology can permit nonlethal collections in the field from a greater number of bee species.

Cattle grazing results in greater floral resources and pollinators than sheep grazing in low-diversity grasslands.

Land-use and land-cover change associated with agriculture is one of the main drivers of biodiversity loss. In heavily modified agricultural landscapes, grazing lands may be the only areas that can provide essential resources for native grassland species. Management decisions, such as choice of livestock species, affect the extent to which grazing lands provide suitable habitat for native species such as pollinators.Our study compared how sheep versus cattle herbivory affected floral resources and butterfly abundance across low-diversity, former Conservation Reserve Program (CRP) pastures managed with patch-burn grazing.Across all years (2017-2019), flowering species richness and abundance were significantly higher in cattle pastures than sheep pastures. On average, we recorded 6.9 flowering species/transect in cattle pastures and 3.8 flowering species/transect in sheep pastures. The average floral abundance per transect was 1278 stems/transect in cattle pastures and 116 stems/transect in pastures grazed by sheep.Similarly, we observed higher butterfly species richness, diversity, and abundance in cattle than in sheep pastures. In cattle pastures, we observed an average of 75 butterflies and 6.75 species per transect, compared with an average of 52 butterflies and 3.37 species per transect in sheep pastures. However, the butterfly community composition did not significantly differ between grazing treatments likely because agricultural-tolerant, habitat generalists comprised the majority of the butterfly community. Five generalist butterflies comprised 92.3% of observations; Colias philodice was the most abundant (61% of observations). Speyeria idalia and Danaus plexippus, two butterflies of conservation concern, comprised less than 0.5% of butterfly observations.Our results, which are among the first attempt quantifying butterfly use of post-CRP fields grazed by livestock, show that increased precipitation and cattle grazing promoted higher forb abundance and richness. However, additional interventions may be needed to enhance floral resources to sustain and improve pollinator diversity in these landscapes.

Cattle and sheep differentially alter floral resources and the native bee communities in working landscapes.

Within agricultural landscapes, native bees often rely on limited natural and seminatural lands to provide the majority of the food and nesting resources that sustain them. To understand better how management can affect pollinators in these seminatural areas, we compared how sheep or cattle herbivory influenced floral resources and bee communities in low-diversity, former Conservation Reserve Program (CRP) pastures managed with patch-burn grazing. We sampled bee communities and floral resources three times per season in 2017, 2018, and 2019. We used plant-pollinator line transect sampling and collected bees and counted all flowering stems within 1 m. Across all years, we found that floral abundance, floral richness, floral diversity (Simpson's) and bee richness and abundance were significantly higher in cattle pastures compared to sheep. In cattle pastures, 46 native bee species plus honey bees interacted with 25 of 68 available flowering forbs. In sheep pastures, we recorded 14 native bee species and honey bees interacted with 10 of 34 flowering species. Native bee abundance and native bee richness were best explained by models that included an interaction of floral richness and year. Overall, our results suggest that season-long sheep grazing in low-diversity grasslands greatly reduces available floral resources and correlates with much lower bee abundance and native bee diversity. Given the importance of pollinators to natural and agricultural systems, it is imperative that we take proactive actions to increase forb richness and native flower abundance in seminatural lands to maintain a more diverse and resilient bee community that can continue to support pollination services and global food security.

The expanding role of movement behavior in insect conservation ecology.

Insect conservation will rely on incorporating behavior into management. Dispersal behavior is one such vital behavior for conservation, but it is generally poorly understood at the species level. We reviewed recent literature to identify intricacies that complicate including dispersal behavior in conservation management. Many previous theories used to predict the need to disperse do not explicitly address successful dispersal. Additionally, we found identifying barriers to dispersal as a possible way to improve conservation management, but it is necessary to consider multiple parts of dispersal (emigration, matrix navigation, immigration). Species' dispersal is context-specific. Therefore, to effectively incorporate dispersal behavior into conservation, more research is necessary on individual species' responses to their environment, how they navigate to optimal sites, and their fitness after dispersal events.

Timing alters how a heat shock affects a host-parasitoid interaction.

Abiotic factors' effects on species are now well-studied, yet they are still often difficult to predict, especially for strongly interacting species. If these altered abiotic factors and species interactions occur as discrete events in time, such complications may occur because of the events' relative timing. One such discrete abiotic factor is the short-duration, large magnitude increase in temperature called a heat shock. This study investigates how the timing of heat shocks affects the successful attack and reproduction of a parasitoid wasp (Aphidius ervi) attacking its host, the pea aphid (Acyrthosiphon pisum). We tested three relative timings: 1) heat shock before the wasp attacks hosts, 2) heat shock while the wasp is foraging, and 3) heat shock after the wasp has attacked hosts. In each scenario we compared wasp mummy production (pupal stage) with and without a heat shock. Our results showed that a heat shock had the largest effect when it occurred while wasps actively foraged, with fewer mummies produced when exposed to a heat shock compared to the no heat shock control. Follow-up behavioral tests suggest this was caused by wasps becoming inactive during heat shocks. In contrast, when heat shocks were applied three days before or after foraging, we found no difference in mummy production between the heat shock treatment and no heat shock control. These results show the potential importance of timing when considering the ramifications of an altered abiotic factor, especially with relatively discrete abiotic events and interactions.

Self-perpetuating ecological-evolutionary dynamics in an agricultural host-parasite system.

Ecological and evolutionary processes may become intertwined when they operate on similar time scales. Here we show ecological-evolutionary dynamics between parasitoids and aphids containing heritable symbionts that confer resistance against parasitism. In a large-scale field experiment, we manipulated the aphid's host plant to create ecological conditions that either favoured or disfavoured the parasitoid. The result was rapid evolutionary divergence of aphid resistance between treatment populations. Consistent with ecological-evolutionary dynamics, the resistant aphid populations then had reduced parasitism and increased population growth rates. We fit a model to quantify costs (reduced intrinsic rates of increase) and benefits of resistance. We also performed genetic assays on 5 years of field samples that showed persistent but highly variable frequencies of aphid clones containing protective symbionts; these patterns were consistent with simulations from the model. Our results show (1) rapid evolution that is intertwined with ecological dynamics and (2) variation in selection that prevents traits from becoming fixed, which together generate self-perpetuating ecological-evolutionary dynamics.

Salinity Improves Performance and Alters Distribution of Soybean Aphids.

We know numerous abiotic factors strongly influence crop plants. Yet we often know much less about abiotic effects on closely interacting organisms including herbivorous insects. This lack of a whole-system perspective may lead to underestimating the threats from changing factors. High soil salinity is a specific example that we know threatens crop plants in many places, but we need to know much more about how other organisms are also affected. We investigated how salinity affects the soybean aphid (SBA; Aphis glycines Matsumura; Hemiptera: Aphididae) on soybean plants (Glycine max [L.] Merr.; Fabales: Fabaceae) grown across a range of saline conditions. We performed four complementary greenhouse experiments to understand different aspects of how salinity might affect SBA. We found that as salinity increased both population size and fecundity of SBA increased across electrical conductivity values ranging from 0.84 to 8.07 dS m-1. Tracking individual aphids we also found they lived longer and produced more offspring in high saline conditions compared to the control. Moreover, we found that salinity influenced aphid distribution such that when given the chance aphids accumulated more on high-salinity plants. These results suggest that SBA could become a larger problem in areas with higher salinity and that those aphids may exacerbate the negative effects of salinity for soybean production.

Correction: Aphid symbionts and endogenous resistance traits mediate competition between rival parasitoids.

[This corrects the article DOI: 10.1371/journal.pone.0180729.].

Supply and demand: How does variation in atmospheric oxygen during development affect insect tracheal and mitochondrial networks?

Atmospheric oxygen is one of the most important atmospheric component for all terrestrial organisms. Variation in atmospheric oxygen has wide ranging effects on animal physiology, development, and evolution. This variation in oxygen has the potential to affect both respiratory systems (the supply side) and mitochondrial networks (the demand side) in animals. Insect respiratory systems supplying oxygen to tissues in the gas phase through blind ended tracheal systems are particularly susceptible to this variation. While the large conducting tracheae have previously been shown to respond developmentally to changes in rearing oxygen, the effect of oxygen on the tracheolar network has been relatively unexplored, especially in adult insects. Similarly, mitochondrial networks that meet energy demand in insects and other animals are dynamic and their enzyme activities have been shown to vary in the presence of oxygen. These two systems together should be under selective pressure to meet the aerobic metabolic requirements of insects. To test this hypothesis, we reared Mito-YFP Drosophila under three different oxygen concentrations hypoxia (12%), normoxia (21%), and hyperoxia (31%) and imaged their tracheolar and mitochondrial networks within their flight muscle using confocal microscopy. In terms of oxygen supply, hypoxia increased mean (mid-length) tracheolar diameters, tracheolar tip diameters, the number of tracheoles per main branch and affected tracheal branching patterns, while the opposite was observed in hyperoxia. In terms of oxygen demand, hypoxia increased mitochondrial investment and mitochondrial to tracheolar volume ratios; while the opposite was observed in hyperoxia. Generally, hypoxia had a stronger effect on both systems than hyperoxia. These results show that insects are capable of developmentally changing investment in both their supply and demand networks to increase overall fitness.

Contrasting the potential effects of daytime versus nighttime warming on insects.

Mean increases in temperatures associated with climate change are largely driven by increases in minimum (nighttime) temperatures; however, most climate change studies disproportionately increase maximum (daytime) temperatures. We review current literature to compare the potential effects of increasing daytime and nighttime temperatures on insects and their interactions within ecological communities. Although few studies have explicitly addressed the effects of nighttime warming, we draw from broader literature on how insects are affected by temperature to identify possible mechanisms that the timing (day or night) of warming may affect insects. Specifically, we discuss daily temperature variation, thermal performance curves, behaviour and activity patterns, nighttime recovery from hot days, and bottom-up effects mediated by plants. Although limited, the existing evidence suggests nighttime and daytime warming can have different effects, and thus we encourage scientists to use the most realistic warming treatments possible to truly understand how insects and their communities will be affected by climate change.

Combined effects of night warming and light pollution on predator-prey interactions.

Interactions between multiple anthropogenic environmental changes can drive non-additive effects in ecological systems, and the non-additive effects can in turn be amplified or dampened by spatial covariation among environmental changes. We investigated the combined effects of night-time warming and light pollution on pea aphids and two predatory ladybeetle species. As expected, neither night-time warming nor light pollution changed the suppression of aphids by the ladybeetle species that forages effectively in darkness. However, for the more-visual predator, warming and light had non-additive effects in which together they caused much lower aphid abundances. These results are particularly relevant for agriculture near urban areas that experience both light pollution and warming from urban heat islands. Because warming and light pollution can have non-additive effects, predicting their possible combined consequences over broad spatial scales requires knowing how they co-occur. We found that night-time temperature change since 1949 covaried positively with light pollution, which has the potential to increase their non-additive effects on pea aphid control by 70% in US alfalfa. Our results highlight the importance of non-additive effects of multiple environmental factors on species and food webs, especially when these factors co-occur.

Aphid symbionts and endogenous resistance traits mediate competition between rival parasitoids.

Insects use endogenous mechanisms and infection with protective symbionts to thwart attacks from natural enemies. Defenses that target specific enemies, however, potentially mediate competition between rivals and thereby impact community composition. Following its introduction to North America to control pea aphids (Acyrthosiphon pisum), the parasitoid Aphidius ervi competitively displaced other parasitoids, except for the native Praon pequodorum. The pea aphid exhibits tremendous clonal variation in resistance to A. ervi, primarily through infection with the heritable bacterial symbiont Hamiltonella defensa, although some symbiont-free aphid genotypes encode endogenous resistance. Interestingly, H. defensa strains and aphid genotypes that protect against A. ervi, provide no protection against the closely related, P. pequodorum. Given the specificity of aphid defenses, we hypothesized that aphid resistance traits may contribute to the continued persistence of P. pequodorum. We conducted multiparasitism assays to determine whether aphid resistance traits mediate internal competition between these two solitary parasitoid species, but found this was not the case; P. pequodorum was the successful internal competitor across lines varying in susceptibility to A. ervi. Next, to determine whether resistance traits influence competitive interactions resulting in the stable persistence of P. pequodorum, we established replicated cages varying in the proportion of resistant aphids and recorded successful parasitism for each wasp species over time. As expected, A. ervi outcompeted P. pequodorum in cages containing only susceptible aphids. However, P. pequodorum not only persisted, but was the superior competitor in populations containing any proportion (20-100%) of resistant aphids (20-100%). Smaller scale, better replicated competition cage studies corroborated this finding, and no-competition and behavioral assays provide insight into the processes mediating competition. Genetic variation, including that acquired via infection with protective symbionts, may provide a supply of hosts susceptible only to particular enemies, mediating competition with effects on community richness and stability.

The Effects of Salinity on the Herbivorous Crop Pest Tetranychus urticae (Trombidiformes: Tetranychidae) on Soybean and Corn.

Many environmental factors, including soil characteristics, are critical for plants, herbivorous arthropods, and their interactions. Despite increasing evidence that soil salinity drastically impacts plants, little is known about how salinity affects the herbivorous arthropod pests feeding on those plants. We investigated how soil salinity affects the twospotted spider mite (Tetranychus urticae Koch) feeding on corn (Zea mays L.) and soybean (Glycine max L.). We performed two greenhouse studies, one focusing on the impact of salinity on individual mite fecundity over a period of 3 d and the other focusing on population growth of T. urticae over 7 d. Both experiments were performed across varying salinity levels; electrical conductivity values ranged from 0.84 to 8.07 dS m-1. We also performed the 3-d fecundity experiment in the field, across naturally varying saline conditions. Overall, the twospotted spider mite performed better as salinity increased; both fecundity and population growth tended to have a positive linear correlation with salinity. These studies suggest that salinity can be important for herbivores, just as it is for plants. Moreover, the negative effects of soil salinity on crop plants in agroecosystems may be further compounded by a greater risk of pest problems. Salinity may be another important environmental stressor that can directly influence crop production while also indirectly influencing herbivorous pests.

Specificity of Multi-Modal Aphid Defenses against Two Rival Parasitoids.

Insects are often attacked by multiple natural enemies, imposing dynamic selective pressures for the development and maintenance of enemy-specific resistance. Pea aphids (Acyrthosiphon pisum) have emerged as models for the study of variation in resistance against natural enemies, including parasitoid wasps. Internal defenses against their most common parasitoid wasp, Aphidius ervi, are sourced through two known mechanisms- 1) endogenously encoded resistance or 2) infection with the heritable bacterial symbiont, Hamiltonella defensa. Levels of resistance can range from nearly 0-100% against A. ervi but varies based on aphid genotype and the strain of toxin-encoding bacteriophage (called APSE) carried by Hamiltonella. Previously, other parasitoid wasps were found to commonly attack this host, but North American introductions of A. ervi have apparently displaced all other parasitoids except Praon pequodorum, a related aphidiine braconid wasp, which is still found attacking this host in natural populations. To explain P. pequodorum's persistence, multiple studies have compared direct competition between both wasps, but have not examined specificity of host defenses as an indirectly mediating factor. Using an array of experimental aphid lines, we first examined whether aphid defenses varied in effectiveness toward either wasp species. Expectedly, both types of aphid defenses were effective against A. ervi, but unexpectedly, were completely ineffective against P. pequodorum. Further examination showed that P. pequodorum wasps suffered no consistent fitness costs from developing in even highly 'resistant' aphids. Comparison of both wasps' egg-larval development revealed that P. pequodorum's eggs have thicker chorions and hatch two days later than A. ervi's, likely explaining their differing abilities to overcome aphid defenses. Overall, our results indicate that aphids resistant to A. ervi may serve as reservoirs for P. pequodorum, hence contributing to its persistence in field populations. We find that specificity of host defenses and defensive symbiont infections, may have important roles in influencing enemy compositions by indirectly mediating the interactions and abundance of rival natural enemies.

Multilevel statistical models and the analysis of experimental data.

Data sets from ecological experiments can be difficult to analyze, due to lack of independence of experimental units and complex variance structures. In addition, information of interest may lie in complicated contrasts among treatments, rather than direct output from statistical tests. Here, we present a statistical framework for analyzing data sets containing non-independent experimental units and differences in variance among treatments (heteroscedasticity) and apply this framework to experimental data on interspecific competition among three tadpole species. Our framework involves three steps: (1) use a multilevel regression model to calculate coefficients of treatment effects on response variables; (2) combine coefficients to quantify the strength of competition (the target information of our experiment); and (3) use parametric bootstrapping to calculate significance of competition strengths. We repeated this framework using three multilevel regression models to analyze data at the level of individual tadpoles, at the replicate level, and at the replicate level accounting for heteroscedasticity. Comparing results shows the need to correctly specify the statistical model, with the model that accurately accounts for heteroscedasticity leading to different conclusions from the other two models. This approach gives a single, comprehensive analysis of experimental data that can be used to extract informative biological parameters in a statistically rigorous way.

On their best behavior: how animal behavior can help determine the combined effects of species interactions and climate change.

The increasingly appreciated link between climate change and species interactions has the potential to help us understand and predict how organisms respond to a changing environment. As this connection grows, it becomes even more important to appreciate the mechanisms that create and control the combined effect of these factors. However, we believe one such important set of mechanisms comes from species' behavior and the subsequent trait-mediated interactions, as opposed to the more often studied density-mediated effects. Behavioral mechanisms are already well appreciated for mitigating the separate effects of the environment and species interactions. Thus, they could be at the forefront for understanding the combined effects. In this review, we (1) show some of the known behaviors that influence the individual and combined effects of climate change and species interactions; (2) conceptualize general ways behavior may mediate these combined effects; and (3) illustrate the potential importance of including behavior in our current tools for predicting climate change effects. In doing so, we hope to promote more research on behavior and other mechanistic factors that may increase our ability to accurately predict climate change effects.

Why oviposit there? Fitness consequences of a gall midge choosing the plant's youngest leaf.

For animals that lay eggs, a longstanding question is, why do females choose particular oviposition sites? For insects that lay eggs on plants there are three hypotheses: maximizing suitable habitat for juveniles, maximizing female lifespan, and maximizing egg survival. We investigated the function of the oviposition-site choice behavior of a gall midge, the Hessian fly, Mayetiola destructor (Say). In spite of living less than a day and having hundreds of eggs, the ovipositing female is choosy about the placement of eggs. Choosiness makes sense. The tiny gall-making neonate larva has limited movement and strict requirements for colonization. We examined whether offspring benefit from the Hessian fly female's preference for the plant's youngest leaf. To do this we restricted the female's access to the first, second, or third leaf of a seedling (wheat Triticum aestivum L.) plant. Being placed on older leaves did not impact egg survival or larval survival during migration to attack sites at the base of the plant, but did have negative impacts on egg-to-adult survival (reduced by 48%) and reproductive potential (reduced by 30-45%). These negative impacts appear to come from larvae having to search harder to find the limited number of reactive plant cells that can be reprogrammed to form the gall nutritive tissue. We propose that the ability of larvae to find these reactive cells in spite of being placed on an older leaf is important because it creates leeway for female behavior to evolve in the face of other selection pressures, e.g., attack by egg parasitoids.

Impact of Rag1 aphid resistant soybeans on Binodoxys communis (Hymenoptera: Braconidae), a parasitoid of soybean aphid (Hemiptera: Aphididae).

Multiple strategies are being developed for pest management of the soybean aphid, Aphis glycines Matsumura; however, there has been little published research thus far to determine how such strategies may influence each other, thereby complicating their potential effectiveness. A susceptible soybean (Glycine max L.) variety without the Rag1 gene and a near isogenic resistant soybean variety with the Rag1 gene were evaluated in the laboratory for their effects on the fitness of the soybean aphid parasitoid, Binodoxys communis (Gahan). The presence or absence of the Rag1 gene was verified by quantifying soybean aphid growth. To test for fitness effects, parasitoids were allowed to attack soybean aphids on either a susceptible or resistant plant for 24 h and then aphids were kept on the same plant throughout parasitoid development. Parasitoid fitness was measured by mummy and adult parasitoid production, adult parasitoid emergence, development time, and adult size. Parasitoids that attacked soybean aphids on susceptible plants produced more mummies, more adult parasitoids, and had a higher emergence rate compared with those on resistant plants. Adult parasitoids that emerged from resistant plants took 1 d longer and were smaller compared with those from susceptible plants. This study suggests that biological control by B. communis may be compromised when host plant resistance is widely used for pest management of soybean aphids.

Rag1 aphid resistant soybeans alter the movement and distribution of soybean aphid (Hemiptera: Aphididae).

Herbivorous insects often move and distribute according to the quality of the plant they are on, and this behavior could influence interactions with plants bred for herbivore resistance. However, when an insect is normally considered sedentary, less is known about the potential importance of movement. We performed experiments to determine if a resistant soybean variety alters the movement and distribution, both within and between plants, of the soybean aphid Aphis glycines Matsumura. We did this by counting apterous aphids on leaves of resistant and susceptible soybean plants across several days. In individual plant tests aphid distribution was different between susceptible and resistant soybeans. Most notably aphids on resistant plants were quickly found off the original leaf on which they were placed and were ultimately distributed throughout the resistant soybean. Aphids on susceptible plants, however, tended to stay on their initial leaf of placement. Follow up experiments indicated this was primarily because of the movement of individuals and not differential demography on various plant parts. In experiments where aphids were able to walk to an adjacent plant there appeared to be a net movement of aphids off resistant plants and on to susceptible plants. Aphid populations on susceptible plants were higher when the plant was adjacent to a resistant plant than when adjacent to another susceptible plant. The effect of resistant plants on aphid movement and distribution could lead to unintended side-effects such as greater spread of plant viruses or altered effectiveness of biological control agents.