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Growing evidence reveals strong overlap between microbiomes of flowers and bees, suggesting that flowers are hubs of microbial transmission. Whether floral transmission is the main driver of bee microbiome assembly, and whether functional importance of florally sourced microbes shapes bee foraging decisions are intriguing questions that remain unanswered. We suggest that interaction network properties, such as nestedness, connectedness, and modularity, as well as specialization patterns can predict potential transmission routes of microbes between hosts. Yet microbial filtering by plant and bee hosts determines realized microbial niches. Functionally, shared floral microbes can provide benefits for bees by enhancing nutritional quality, detoxification, and disintegration of pollen. Flower microbes can also alter the attractiveness of floral resources. Together, these mechanisms may affect the structure of the flower-bee interaction network.

The spotted-wing drosophila, Drosophila suzukii Matsumura, is an exotic species in North America and represents a major threat to fruit production. Efforts to manage D. suzukii have focused primarily on insecticides, but such controls may, at times, be unreliable, given that D. suzukii larvae are often ensconced within fruit. The fruit interior, however, may represent suitable foraging substrates for carnivorous/entomopathogenic nematodes. In preliminary trials, a rare nematode species, Oscheius onirici Torrini et al., was shown to be highly virulent against D. suzukii when the nematodes were applied directly to fly larvae. To address the more important question of whether this nematode would be as virulent when applied to fruit, we set up assays in which blueberries were infested with D. suzukii larvae and then sprayed with O. onirici infective juveniles (IJs). Across two laboratory trials, O. onirici IJs suppressed D. suzukii puparia by 78.2%. Oscheius onirici IJs were able to search effectively within fruit substrates, find the fly larvae therein, and kill the flies before they could pupariate. Oscheius onirici, therefore, may represent a viable new bio-control agent for D. suzukii management and should be field-tested across a broader diversity of cropping systems.

As pollen and nectar foragers, bees have long been considered strictly herbivorous. Their pollen provisions, however, are host to abundant microbial communities, which feed on the pollen before and/or while it is consumed by bee larvae. In the process, microbes convert pollen into a complex of plant and microbial components. Since microbes are analogous to metazoan consumers within trophic hierarchies, the pollen-eating microbes are, functionally, herbivores. When bee larvae consume a microbe-rich pollen complex, they ingest proteins from plant and microbial sources and thus should register as omnivores on the trophic "ladder." We tested this hypothesis by examining the isotopic compositions of amino acids extracted from native bees collected in North America over multiple years. We measured bee trophic position across the six major bee families. Our findings indicate that bee trophic identity was consistently and significantly higher than that of strict herbivores, providing the first evidence that omnivory is ubiquitous among bee fauna. Such omnivory suggests that pollen-borne microbes represent an important protein source for larval bees, which introduces new questions as to the link between floral fungicide residues and bee development.

Microbial symbiotic interactions, mediated by small molecule signaling, drive physiological processes of higher order systems. Metabolic analytic technologies advancements provide new avenues to examine how chemical ecology, or conversion of existing biomass to new forms, changes over a symbiotic lifecycle. We examine such processes using the tripartite relationship between nematode host Steinernema carpocapsae, its obligate mutualist bacterium, Xenorhabdus nematophila, and the insects they infect together. We integrate trophic, metabolomics, and gene regulation analyses to understand insect biomass conversion to nematode or bacterium biomass. Trophic analysis established bacteria as the primary insect consumers, with nematodes at trophic position 4.37, indicating consumption of bacteria and likely other nematodes. Significant, discrete metabolic phases were distinguishable from each other, indicating the insect chemical environment changes reproducibly during bioconversion. Tricarboxylic acid cycle components and amino acids were significantly affected throughout infection. These findings contribute to an ongoing understanding of how symbiont associations shape chemical environments.TeaserEntomopathogenic nematodes act as an apex predator in some ecosystems through altering chemical environments of their prey.

Cranberry fruitworm (Acrobasis vaccinii Riley (Lepidoptera: Pyralidae)) and blackheaded fireworm (Rhopobota naevana Hubner (Lepidoptera: Tortricidae)) threaten cranberry production annually by causing significant fruit damage. Up to four pesticide applications are made each year to control these insects, which are costly to producers and elevate pesticide residues in fruit. Pheromone-based mating disruption technology can provide control of these pests in cranberry production, with the potential to minimize, or eliminate, pesticide applications. In 2016, an uncrewed aerial vehicle (UAV) was investigated to apply a thick paraffin emulsion containing insect sex pheromones. Traditional agricultural equipment is not capable of applying the paraffin emulsion to cranberry beds due to the product’s viscous, paste-like consistency. The first objective of this study was to retrofit an UAV (octocopter) with a novel extrusion device that had been engineered to deliver the pheromone-loaded paraffin at regular intervals during flight. The second objective was to confirm adequate distribution of the pheromones by measuring the mating disruption efficacy by monitoring male moth trap catches. The UAV was able to fly autonomously along a prescribed itinerary, deploying the paraffin product uniformly; however, the increased mass of the retrofitted UAV limited flight times to ~12 min. The number of male cranberry fruitworm and blackheaded fireworm moths caught in lure-baited traps were reduced in the paraffin-treated beds compared with untreated beds, indicating adequate distribution of the pheromones. The UAV-applied pheromones concept could be developed into a production scale application method in the future, although issues of battery life and lifting capacity will need to be resolved.

The pollen stores of bumble bees host diverse microbiota that influence overall colony fitness. Yet, the taxonomic identity of these symbiotic microbes is relatively unknown. In this descriptive study, we characterized the microbial community of pollen provisions within captive-bred bumble bee hives obtained from two commercial suppliers located in North America. Findings from 16S rRNA and ITS gene-based analyses revealed that pollen provisions from the captive-bred hives shared several microbial taxa that have been previously detected among wild populations. While diverse microbes across phyla Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Ascomycota were detected in all commercial hives, significant differences were detected at finer-scale taxonomic resolution based on the supplier source. The causative agent of chalkbrood disease in honey bees, Ascosphaera apis, was detected in all hives obtained from one supplier source, although none of the hives showed symptoms of infection. The shared core microbiota across both commercial supplier sources consisted of two ubiquitous bee-associated groups, Lactobacillus and Wickerhamiella/Starmerella clade yeasts that potentially contribute to the beneficial function of the microbiome of bumble bee pollen provisions.

Biological control plays an important role in many integrated pest management programmes, but can be disrupted by other control strategies, including chemical and cultural controls. In commercial cranberry (Vaccinium macrocarpon Aiton; Ericaceae) production, a spring flood can replace an insecticide application, providing an opportunity to study the compatibility of the flood (a cultural control) with biological control. We suspect that chemical controls will tend to reduce the number of natural enemies, while the flood, through removal of detritus and detritivores, may cause generalist predators to prey-switch to consume proportionally more pest individuals. We measured the abundance of herbivores (Lepidoptera), detritivores, Arachnida, and parasitoids (Hymenoptera) every week for six weeks in Wisconsin (United States of America) cranberry beds following either an insecticide spray or a cultural control flood. We found that detritivore populations rapidly declined in both flood and spray treatments; conversely, carnivore populations (spiders and parasitoids) were more abundant in the flooded beds than in sprayed beds. Populations of key cranberry pests were similar between flooded and sprayed beds. Our results showed that early-season flooding preserved more natural enemies than an insecticide application. This increase in natural enemy abundance after the flood may allow for greater continuity in herbivore suppression, potentially providing a basis for long-term cranberry pest management.

Compound-specific isotopic analysis of amino acids (CSIA-AA) has emerged in the last decade as a powerful approach for tracing the origins and fate of nitrogen in ecological and biogeochemical studies. This approach is based on the empirical observation that source amino acids (SAAs) (i.e., phenylalanine), fractionate 15N very little (

Compound-specific nitrogen stable isotope analysis provides an approach for estimating animal trophic position that may overcome key issues associated with stable isotope analysis of bulk tissue. Yet compound-specific trophic discrimination factors have not been estimated for a broad range of habitats, taxa, and diets. We conducted a controlled-feeding experiment to characterize the variation in compound-specific (TDFAA) and bulk (TDFBulk) trophic discrimination factors of four freshwater fish species fed on three distinct diets. We also compared TDFAA of fish muscle and scale to evaluate the viability of scales for making food web inferences. Mean ± 1 SD TDFBulk was 2.2‰ ± 0.9‰, and there were significant effects of species and diet trophic position on TDFBulk. Mean ± 1 SD TDFAA was 6.9‰ ± 0.8‰. Although there was no effect of species on TDFAA, there were significant differences in TDFAA across the three diets. TDFAA from fish scales were not significantly different from those of muscle. Our study illustrates the advantages of estimating trophic position using compound-specific stable isotopes and the need for continued investigation of factors resulting in variation in TDF values.

Detritivory is the dominant trophic paradigm in most terrestrial, aquatic, and marine ecosystems, yet accurate measurement of consumer trophic position within detrital (=“brown”) food webs has remained unresolved. Measurement of detritivore trophic position is complicated by the fact that detritus is suffused with microbes, creating a detrital complex of living and nonliving biomass. Given that microbes and metazoans are trophic analogues of each other, animals feeding on detrital complexes are ingesting other detritivores (microbes), which should elevate metazoan trophic position and should be rampant within brown food webs. We tested these hypotheses using isotopic (15N) analyses of amino acids extracted from wild and laboratory‐cultured consumers. Vertebrate (fish) and invertebrate detritivores (beetles and moths) were reared on detritus, with and without microbial colonization. In the field, detritivorous animal specimens were collected and analyzed to compare trophic identities among laboratory‐reared and free‐roaming detritivores. When colonized by bacteria or fungi, the trophic positions of detrital complexes increased significantly over time. The magnitude of trophic inflation was mediated by the extent of microbial consumption of detrital substrates. When detrital complexes were fed to vertebrate and invertebrate animals, the consumers registered similar degrees of trophic inflation, albeit one trophic level higher than their diets. The wild‐collected detritivore fauna in our study exhibited significantly elevated trophic positions. Our findings suggest that the trophic positions of detrital complexes rise predictably as microbes convert nonliving organic matter into living microbial biomass. Animals consuming such detrital complexes exhibit similar trophic inflation, directly attributable to the assimilation of microbe‐derived amino acids. Our data demonstrate that detritivorous microbes elevate metazoan trophic position, suggesting that detritivory among animals is, functionally, omnivory. By quantifying the impacts of microbivory on the trophic positions of detritivorous animals and then tracking how these effects propagate “up” food chains, we reveal the degree to which microbes influence consumer groups within trophic hierarchies. The trophic inflation observed among our field‐collected fauna further suggests that microbial proteins represent an immense contribution to metazoan biomass. Collectively, these findings provide an empirical basis to interpret detritivore trophic identity, and further illuminate the magnitude of microbial contributions to food webs.

The differential discrimination of nitrogen isotopes (N-15/N-14) within amino acids in consumers and their diets has been routinely used to estimate organismal tropic position (TP). Analogous isotopic discrimination can occur within plants, particularly in organs lacking chloroplasts. Such discrimination likely arises from the catabolic deamination of amino acids, resulting in a numerical elevation of estimated TP, within newly synthesized biomass. To investigate this phenomenon, we examined the 15N/14N of amino acids (delta N-15(AA)) in spring leaves and flowers from eight deciduous and two annual plants. These plants were classified on the basis of their time of bloom, plants that bloomed when their leaves were absent (Type I) versus plants that bloomed while leaves were already present (Type II). Based on the delta N-15(AA) values from leaves, both plant types occupied comparable and ecologically realistic mean TPs (= 1.0 +/- 0.1, mean +/- 1 sigma). However, the estimated TPs of flowers varied significantly Type I: 2.2 +/- 0.2; Type II: 1.0 +/- 0.1). We hypothesize that these results can be interpreted by the following sequence of events: (1) Type I floral biomass is synthesized in absence of active photosynthesis; (2) the catabolic deamination of amino acids in particular, leaves behind N-15 in the residual pool of amino acids; and (3) the incorporation of these N-15-enriched amino acids within the biomass of Type I flowers results in the numerical elevation of the TPs. In contrast, the actively photosynthesizing Type II leaves energetically sustain the synthesis of Type II flower biomass, precluding any reliance on catabolic deamination of amino acids. Amino acids within Type II flowers are therefore isotopically comparable to the Type II leaves. These findings demonstrate the idiosyncratic nature of the delta N-15(AA) values within autotrophic organs and have implications for interpreting trophic hierarchies using primary producers and their consumers.

Quantifying resource partitioning is central to community and food web ecology and of increasing interest in an era of rapid global change disrupting biotic interactions. Multichannel feeding – consuming resources from both green and brown food webs – can be a stabilizing force in communities. While multichannel feeding has been well-documented in invertebrate and aquatic systems, it has been relatively under-studied in terrestrial vertebrate populations. Applied ecologists are seeking approaches to assess niche partitioning and cryptic trophic pathways, like multichannel feeding, which have been difficult to quantify, especially among vertebrates. Using both bulk (δ13C and δ15N) and compound specific stable isotope ratios (δ15N glutamic acid and phenylalanine), we tested how three common and competing tree squirrel partition resources. Our complementary analyses revealed that squirrels partitioned niche space and, because of differences in multichannel foraging, possessed different trophic identities. While all squirrels consumed food items from green and brown food webs, their dependence on each differed, revealing an important, yet cryptic, mechanism behind apparent stable co-occurrence of these competitors. Our work supports multichannel feeding as a potential mechanism promoting coexistence in this guild of terrestrial vertebrates, and provides a framework to quantify resource partitioning in other ecological communities.

Host plant resistance, an important strategy of integrated pest management, was examined in the American cranberry, Vaccinium macrocarpon Aiton (Ericaceae). Despite the pressure on cranberry growers to reduce pesticide usage, host plant resistance is not used to help manage insect populations. This study measured field population densities of the three most economically important pest insects in Wisconsin, namely, cranberry fruitworm (Acrobasis vaccinii Riley), sparganothis fruitworm (Sparganothis sulfureana Clemens), and blackheaded fireworm (Rhopobota naevana Hübner), in five different cranberry cultivars, i.e., 'Stevens', 'Ben Lear', 'GH1', 'Mullica Queen', and 'HyRed'. Population densities of male moths of all three species were assessed using pheromone traps in beds of the different cranberry cultivars in commercial marshes in central Wisconsin. For each cultivar, damaged cranberries were collected, and the number of damaged berries and the number of larvae feeding within berries were compared among cultivars. More than 99% of larvae collected were cranberry fruitworm. Mullica Queen and Ben Lear had more damaged berries than Stevens or GH1, and had more larvae than GH1. Conversely, fewer adult male sparganothis fruitworm were found in Ben Lear and Mullica Queen beds than in beds of Stevens or GH1. Adult populations of cranberry fruitworm and blackheaded fireworm were not different among cultivars. Our findings provide evidence of different levels of resistance in common cranberry cultivars, which should inform future plantings and breeding programs.

We embarked on a large project designed to help enhance biological control in apple, pear and walnut orchards in the western U.S., where management programs were in the midst of a transition from older organo-phosphate insecticides to mating disruption and newer reduced-risk insecticides. A “pesticide replacement therapy” approach resulted in unstable management programs with unpredictable outbreaks of spider mites and aphids. Our project was designed to provide growers and pest managers with information on the effects of newer pesticide chemistries on a suite of representative natural enemies in both the laboratory and field, potential of new monitoring tools using herbivore-induced plant volatiles and floral volatiles, phenology of the key natural enemy species, economic consequences of using an enhanced biological control program, and value of an outreach program to get project outcomes into the hands of decision-makers. We present an overview of both the successes and failures of the project and of new projects that have spun off from this project to further enhance biological control in our systems in the near future.

In this study we used sticky traps baited with plant volatile lures to monitor the biodiversity of natural enemies in orchard ecosystems in the western U.S. We compared the diversity of predator genera from season total trap catches in 37 different orchards (apple, cherry, pear and walnut) over a two-year period (2010−2011) using standardized Hill number biodiversity indices and community similarity profiles. For a subset of 23 of these orchards we were also able to monitor the change in biodiversity of predator genera over the full growing season in the different orchard crops. A total of 37,854 individuals from 31 different genera of foliage-active generalist predators were collected from all orchards combined. Mean sample coverage was high (0.98) and richness, diversity and evenness differed between crops in 2010, but not in 2011. There was more than 90% similarity in the richness of predator genera among crops and among orchards within crops, but a greater level of differentiation was observed among orchards when variation in their relative abundance and dominance in the communities was taken into account. There was a consistent rise in predator generic richness and diversity through the season in both years for apple, cherry and pear orchards, but in walnut orchards, a steep rise from March to May was followed by a decline through the rest of the season. In an additional component of the study, the species level similarity of predator and parasitoid communities was analyzed for total season trap catch data from six walnut orchards. The rarefied species richness of parasitoids was much greater than that for predators, although the diversity, evenness and dominance of the parasitoid species varied considerably among orchards. The results from this study highlight the fact that natural enemy communities in orchard ecosystems can be effectively monitored using plant volatile traps, and that these communities are surprisingly diverse despite frequent disturbance from pest management intervention.

The ability to estimate natural enemy abundance is crucial to the integration of biological control into IPM programs. Traditional sampling approaches for natural enemies are few and most are inefficient, but recent studies suggest attraction of natural enemies to plant volatiles may be a useful proxy for direct sampling. We evaluated various combinations of herbivore-induced plant volatiles and floral volatiles as monitoring tools for natural enemies found in apple, pear, and walnut orchards in California, Oregon, and Washington. In 2010 we used a full factorial experimental designs to evaluate lures for all combinations of acetic acid (AA), acetophenone (AP), phenylacetaldehyde (PAA) and 2-phenylethanol (PE). Of nine natural enemy taxa analyzed, we found syrphid flies responded strongly to PE, but combining AA with PE attenuated trap catch and combining PAA to PE eliminated the activity of PE. Chrysoperla spp. (Chrysopidae) responded strongly to most of the individual compounds and the various interactions between the components allowed multiple ways to achieve roughly the same trap catch. All of the hymenopteran taxa collected responded strongly to PAA, and PAA containing lures were nearly always a component of the top eight lures. A smaller factorial experiment testing all possible combinations of AA, PAA and methyl salicylate (MS) showed that single component AA or MS lures were generally not significantly different than the controls for all taxa tested, but for the hymenopteran taxa, traps baited with MS+PAA performed the best or were not significantly different than the best performing lure. A 2011 trial was conducted to test the influence of the addition of AA and/or MS on previously tested lures. Combining AA or MS with other lures, improved the capture of Chrysoperla spp.; Scaeva pyrastri (L.) (Syrphidae) capture was enhanced when MS was used with PE; and PE was attractive to the three syrphid flies, Chrysoperla spp., and the parasitoid Aphelinus mali (Haldeman) (Aphelinidae). The differential responses to various blends exhibited among taxa show that combinations of plant volatiles can be chosen to increase specificity of attraction to a few taxa or increase the number of species attracted. This flexibility should add to the general value and breadth of use of plant volatile monitoring lures.

Simple sequence repeats (SSRs) in chloroplast and mitochondrial DNA, which have not been previously developed in the Ericaceae or, more specifically, in the genus Vaccinium, can be powerful tools for determining evolutionary relationships among taxa. In this study, 30 chloroplast, 23 mitochondrial, and 1 mitochondrion-like SSRs were identified in cranberry (V. macrocarpon), and primer-pairs were developed and tested for each locus. Although no polymorphisms were detected for any of the 54 SSR loci in nine diverse cranberry genotypes, all primers were cross-transferable to some extent to a panel of 12 additional Vaccinium taxa and four non-Vaccinium Ericaceae species. A Neighbor-Joining tree of the estimated average squared distances resolved the species by genus and by section within Vaccinium. Similar topologies with increased branch support were observed in Bayesian inference trees constructed from the DNA sequences of six plastid and two mitochondrial SSR loci. Two multiplexing/poolplexing panels of M13 fluorescently labeled primers, which amplify 24 of the 54 markers, were developed and can serve as an efficient, cost-effective means for characterizing the basic molecular phylogeny of Vaccinium. Increased understanding of evolutionary relationships among Vaccinium species should facilitate interspecific hybridization and introgression efforts to improve economically important traits of commercial berry crops.

Although solitary bees provide crucial pollination services for wild and managed crops, this species-rich group has been largely overlooked in pesticide regulation studies. The risk of exposure to fungicide residues is likely to be especially high if the spray occurs on, or near host plants while the bees are collecting pollen to provision their nests. For species of Osmia that consume pollen from a select group of plants (oligolecty), the inability to use pollen from non-host plants can increase their risk factor for fungicide-related toxicity. This manuscript describes protocols used to successfully rear oligolectic mason bees, Osmia ribifloris sensu lato, from egg to prepupal stage within cell culture plates under standardized laboratory conditions. The in vitro-reared bees are subsequently used to investigate the effects of fungicide exposure and pollen source on bee fitness. Based on a 2 × 2 fully crossed factorial design, the experiment examines the main and interactive effects of fungicide exposure and pollen source on larval fitness, quantified by prepupal biomass, larval developmental time, and survivorship. A major advantage of this technique is that using in vitro-reared bees reduces natural background variability and allows the simultaneous manipulation of multiple experimental parameters. The described protocol presents a versatile tool for hypotheses testing involving the suite of factors affecting bee health. For conservation efforts to be met with significant, lasting success, such insights into the complex interplay of physiological and environmental factors driving bee declines will prove to be critical.

Although solitary bees provide crucial pollination services for wild and managed crops, this species-rich group has been largely overlooked in pesticide regulation studies. The risk of exposure to fungicide residues is likely to be especially high if the spray occurs on, or near host plants while the bees are collecting pollen to provision their nests. For species of Osmia that consume pollen from a select group of plants (oligolecty), the inability to use pollen from non-host plants can increase their risk factor for fungicide-related toxicity. This manuscript describes protocols used to successfully rear oligolectic mason bees, Osmia ribifloris sensu lato, from egg to prepupal stage within cell culture plates under standardized laboratory conditions. The in vitro-reared bees are subsequently used to investigate the effects of fungicide exposure and pollen source on bee fitness. Based on a 2 × 2 fully crossed factorial design, the experiment examines the main and interactive effects of fungicide exposure and pollen source on larval fitness, quantified by prepupal biomass, larval developmental time, and survivorship. A major advantage of this technique is that using in vitro-reared bees reduces natural background variability and allows the simultaneous manipulation of multiple experimental parameters. The described protocol presents a versatile tool for hypotheses testing involving the suite of factors affecting bee health. For conservation efforts to be met with significant, lasting success, such insights into the complex interplay of physiological and environmental factors driving bee declines will prove to be critical.

Carnivores indirectly protect plants by reducing herbivory. This important ecosystem service can be undermined, however, as carnivores feed upon fellow carnivores. Such intraguild predation is exceedingly common, yet measurement of the degree to which this occurs has remained elusive due to difficulties in measuring the trophic tendencies of free-roaming animals. Conventional molecular methods, such as bulk-isotopic analyses, do not produce reliably accurate trophic position estimates, and often the inaccuracy is substantial. With the advent of compound-specific isotopic analysis (CSIA), it is now possible to accurately quantify the lifetime trophic tendencies of wild carnivore populations. Unfortunately, CSIA is extraordinarily expensive and time-consuming, limiting the number of samples that can be analyzed. The need for high-quality trophic information has to be balanced with the inaccessibility of CSIA. Here, we propose coupling CSIA-derived trophic position estimates with conventional bulk-15N analysis, effectively calibrating site-specific bulk-15N data and thereby allowing for trophic position estimation using bulk data alone. We also create a framework that uses trophic position as a basis to characterize carnivores as beneficial for crop protection. Within an agricultural field, we demonstrated the utility of this new approach by measuring the trophic positions of six common arthropod species. We then compare these trophic position estimates to those deriving from conventional bulk-15N analysis. Our hybrid approach produced more accurate trophic position estimates than the stand-alone bulk-15N method. Ultimately, we were able to examine enough specimens to determine which carnivore populations were likely beneficial for plant protection, and which were not.

In most ecosystems, microbes are the dominant consumers, commandeering much of the heterotrophic biomass circulating through food webs. Characterizing functional diversity within the microbiome, therefore, is critical to understanding ecosystem functioning, particularly in an era of global biodiversity loss. Using isotopic fingerprinting, we investigated the trophic positions of a broad diversity of heterotrophic organisms. Specifically, we examined the naturally occurring stable isotopes of nitrogen ((15)N:(14)N) within amino acids extracted from proteobacteria, actinomycetes, ascomycetes, and basidiomycetes, as well as from vertebrate and invertebrate macrofauna (crustaceans, fish, insects, and mammals). Here, we report that patterns of intertrophic (15)N-discrimination were remarkably similar among bacteria, fungi, and animals, which permitted unambiguous measurement of consumer trophic position, independent of phylogeny or ecosystem type. The observed similarities among bacterial, fungal, and animal consumers suggest that within a trophic hierarchy, microbiota are equivalent to, and can be interdigitated with, macrobiota. To further test the universality of this finding, we examined Neotropical fungus gardens, communities in which bacteria, fungi, and animals are entwined in an ancient, quadripartite symbiosis. We reveal that this symbiosis is a discrete four-level food chain, wherein bacteria function as the apex carnivores, animals and fungi are meso-consumers, and the sole herbivores are fungi. Together, our findings demonstrate that bacteria, fungi, and animals can be integrated within a food chain, effectively uniting the macro- and microbiome in food web ecology and facilitating greater inclusion of the microbiome in studies of functional diversity.

Bees provide vital pollination services to the majority of flowering plants in both natural and agricultural systems. Unfortunately, both native and managed bee populations are experiencing declines, threatening the persistence of these plants and crops. Agricultural chemicals are one possible culprit contributing to bee declines. Even fungicides, generally considered safe for bees, have been shown to disrupt honey bee development and impair bumble bee behavior. Little is known, however, how fungicides may affect bumble bee colony growth. We conducted a controlled cage study to determine the effects of fungicide exposure on colonies of a native bumble bee species (Bombus impatiens). Colonies of B. impatiens were exposed to flowers treated with field-relevant levels of the fungicide chlorothalonil over the course of one month. Colony success was assessed by the number and biomass of larvae, pupae, and adult bumble bees. Bumble bee colonies exposed to fungicide produced fewer workers, lower total bee biomass, and had lighter mother queens than control colonies. Our results suggest that fungicides negatively affect the colony success of a native bumble bee species and that the use of fungicides during bloom has the potential to severely impact the success of native bumble bee populations foraging in agroecosystems.

Stable nitrogen isotopic composition of amino acids (δ (15)NAA) has recently been employed as a powerful tool in ecological food web studies, particularly for estimating the trophic position (TP) of animal species in food webs. However, the validity of these estimates depends on the consistency of the trophic discrimination factor (TDF; - Δδ (15)NAA at each shift of trophic level) among a suite of amino acids within the tissues of consumer species. In this study, we determined the TDF values of amino acids in tadpoles (the Japanese toad, Bufo japonicus) reared exclusively on one of three diets that differed in nutritional quality. The diets were commercial fish-food pellets (plant and animal biomass), bloodworms (animal biomass), and boiled white rice (plant carbohydrate), representing a balanced, protein-rich, and protein-poor diet, respectively. The TDF values of two "source amino acids" (Src-AAs), methionine and phenylalanine, were close to zero (0.3-0.5‰) among the three diets, typifying the values reported in the literature (∼0.5‰ and ∼0.4‰, respectively). However, TDF values of "trophic amino acids" (Tr-AAs) including alanine, valine, leucine, isoleucine, and glutamic acid varied by diet: for example, the glutamic acid TDF was similar to the standard value (∼8.0‰) when tadpoles were fed either the commercial pellets (8.0‰) or bloodworms (7.9‰), but when they were fed boiled rice, the TDF was significantly reduced (0.6‰). These results suggest that a profound lack of dietary protein may alter the TDF values of glutamic acid (and other Tr-AAs and glycine) within consumer species, but not the two Src-AAs (i.e., methionine and phenylalanine). Knowledge of how a nutritionally poor diet can influence the TDF of Tr- and Src-AAs will allow amino acid isotopic analyses to better estimate TP among free-roaming animals.

The American cranberry, Vaccinium macrocarpon Ait., is an economically important North American fruit crop that is consumed because of its unique flavor and potential health benefits. However, a lack of abundant, genome-wide molecular markers has limited the adoption of modern molecular assisted selection approaches in cranberry breeding programs. To increase the number of available markers in the species, this study identified, tested, and validated microsatellite markers from existing nuclear and transcriptome sequencing data. In total, new primers were designed, synthesized, and tested for 979 SSR loci, 697 of the markers amplified allele patterns consistent with single locus segregation in a diploid organism and were considered polymorphic. Of the 697 polymorphic loci, 507 were selected for additional genetic diversity and segregation analyses in 29 cranberry genotypes. More than 95% of the 507 loci did not display segregation distortion at the p &lt, 0.05 level, and contained moderate to high levels of polymorphism with a polymorphic information content &gt, 0.25. This comprehensive collection of developed and validated microsatellite loci represents a substantial addition to the molecular tools available for geneticists, genomicists, and breeders in cranberry and Vaccinium.

In a search for an entomopathogenic nematode to control cranberry insect pests, three Oscheius populations (Rhabditidae) were recovered through the Galleria-bait method from one sample taken in a wild cranberry marsh in Jackson County, Wisconsin, USA. Morphological studies with light microscopy and scanning electron microscopy, as well as molecular analyses of the near-full-length small subunit rDNA gene, D2/D3 expansion segments of the large subunit rDNA gene, internal transcribed spacer, and mitochondrial cytochrome oxidase subunit 1 (CoxI) genes revealed this as Oscheius onirici, a species recently described from a karst cave soil of central Italy. The species belongs to the dolichura-group and is characterized by its DNA sequences; hermaphroditic reproduction; and males not found. A Bacillus-like bacterium appears to be associated with this nematode based on our microscopic and SEM observations; however its identity and persistent association with the nematode has not been confirmed. Nonetheless, this nematode is capable of infecting and killing the sparganothis fruitworm Sparganothis sulfureana Clemens (Lepidoptera: Tortricidae), the brown-banded cockroach Supella longipalpa Fabricius (Blattodea: Ectobiidae), and the cranberry fruitworm Acrobasis vaccinii Riley (Lepidoptera: Pyralidae), under laboratory conditions, and each in less than 72 hr. The mealworm Tenebrio molitor Linnaeus (Coleoptera: Tenebrionidae) and the greater wax moth Galleria mellonella Linnaeus (Lepidoptera: Pyralidae), are also susceptible, but take 3.5 and 5.2 days to die, respectively. This species is a new potential bio-control agent on insects.

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered "bee-safe," there is mounting evidence that fungicide residues in pollen are associated with bee declines (for both honey and bumble bee species). While the mechanisms remain relatively unknown, researchers have speculated that bee-microbe symbioses are involved. Microbes play a pivotal role in the preservation and/or processing of pollen, which serves as nutrition for larval bees. By altering the microbial community, it is likely that fungicides disrupt these microbe-mediated services, and thereby compromise bee health. This manuscript describes the protocols used to investigate the indirect mechanism(s) by which fungicides may be causing colony decline. Cage experiments exposing bees to fungicide-treated flowers have already provided the first evidence that fungicides cause profound colony losses in a native bumble bee (Bombus impatiens). Using field-relevant doses of fungicides, a series of experiments have been developed to provide a finer description of microbial community dynamics of fungicide-exposed pollen. Shifts in the structural composition of fungal and bacterial assemblages within the pollen microbiome are investigated by next-generation sequencing and metagenomic analysis. Experiments developed herein have been designed to provide a mechanistic understanding of how fungicides affect the microbiome of pollen-provisions. Ultimately, these findings should shed light on the indirect pathway through which fungicides may be causing colony declines.

Organizational structure for the proposed IsoBank. A central executive group would oversee four subcommittees (SC): Information technology, integrative disciplinary, education and training, and analytical expertise. GNIP, Global Network of Isotopes in Precipitation; IAEA, International Atomic Energy Association; QA/QC, quality assurance/quality control.

Pheromone-based mating disruption has proven to be a powerful pest management tactic in many cropping systems. However, in the cranberry system, a viable mating disruption program does not yet exist. There are commercially available pheromones for several of the major pests of cranberries, including the cranberry fruitworm, Acrobasis vaccinii Riley (Lepidoptera: Pyralidae) and blackheaded fireworm, Rhopobota naevana (Hübner) (Lepidoptera: Tortricidae). Previous studies have shown that mating disruption represents a promising approach for R. naevana management although carrier and delivery technologies have remained unresolved. The present study examined the suitability of Specialized Pheromone & Lure Application Technology (SPLAT; ISCA Technologies, Inc., Riverside, CA), a proprietary wax and oil blend, to serve as a pheromone carrier in the cranberry system. In 2013 and 2014, we tested a blend of pheromones targeting A. vaccinii and R. naevana in field-scale, replicated trials. Pheromones were loaded into SPLAT and the resulting “SPLAT BFW CFW” formulation was deployed in commercial cranberry marshes. We compared moth trap-catch counts within SPLAT-treated blocks to those of conventionally managed blocks. In 2013, applications of SPLAT BFW CFW resulted in highly successful disruption of R. naevana and promising, though inconsistent, disruption of A. vaccinii. To improve disruption of A. vaccinii, the pheromone load was increased in 2014, providing 92% and 74% reductions in trap-catch for R. naevana and A. vaccinii, respectively. Importantly, larval infestation rates in SPLAT-treated blocks were lower than those of conventionally managed blocks. These results suggest that a multispecies mating disruption system (SPLAT BFW CFW) may represent an effective pesticide-alternative for serious pests of cranberries.

Sparganothis sulfureana Clemens is a severe pest of cranberries in the Midwest and northeast United States. Timing for insecticide applications has relied primarily on calendar dates and pheromone trap-catch; however, abiotic conditions can vary greatly, rendering such methods unreliable as indicators of optimal treatment timing. Phenology models based on degree-day (DD) accrual represent a proven, superior approach to assessing the development of insect populations, particularly for larvae. Previous studies of S. sulfureana development showed that the lower and upper temperature thresholds for larval development were 10.0 and 29.9°C (49.9 and 85.8°F), respectively. We used these thresholds to generate DD accumulations specific to S. sulfureana, and then linked these DD accumulations to discrete biological events observed during S. sulfureana development in Wisconsin and New Jersey cranberries. Here, we provide the DDs associated with flight initiation, peak flight, flight termination, adult life span, preovipositional period, ovipositional period, and egg hatch. These DD accumulations represent key developmental benchmarks, allowing for the creation of a phenology model that facilitates wiser management of S. sulfureana in the cranberry system.

American cranberry (Vaccinium macrocarpon Ait.) is a perennial, woody plant species, native to North American bogs and wetlands. Cranberries represent one of the few agriculturally important native plants in which wild gene pools are still readily available within the undeveloped wetlands the northern U.S. and Canada. Past studies have reported low genetic variation in V. macrocarpon at the species and population level. However, in this study, we characterized 229 samples of wild V. macrocarpon and V. oxycoccos (small cranberry) and 22 control accessions using microsatellite markers and observed substantial genetic variation and differentiation within and among populations and species. While V. macrocarpon was analyzed using 108 alleles from eleven microsatellite loci revealing 42 unique genotypes, V. oxycoccos was analyzed using 156 alleles from eight loci revealing 28 unique genotypes. There were a total of 182 alleles found in both species combined with 156 of those alleles present in V. oxycoccos and 84 alleles found in V. macrocarpon. All eight loci possessed species-specific alleles with V. oxycoccos possessing 98 private alleles versus 26 private alleles found V. macrocarpon, and 58 alleles were found in common between both species. Our data will be valuable not only for future wild cranberry diversity and population genetics research, but for other cranberry breeding and genetics studies.

Food webs are known to have myriad trophic links between resource and consumer species. While herbivores have well-understood trophic tendencies, the difficulties associated with characterizing the trophic positions of higher-order consumers have remained a major problem in food web ecology. To better understand trophic linkages in food webs, analysis of the stable nitrogen isotopic composition of amino acids has been introduced as a potential means of providing accurate trophic position estimates. In the present study, we employ this method to estimate the trophic positions of 200 free-roaming organisms, representing 39 species in coastal marine (a stony shore) and 38 species in terrestrial (a fruit farm) environments. Based on the trophic positions from the isotopic composition of amino acids, we are able to resolve the trophic structure of these complex food webs. Our approach reveals a high degree of trophic omnivory (i.e., noninteger trophic positions) among carnivorous species such as marine fish and terrestrial hornets.This information not only clarifies the trophic tendencies of species within their respective communities, but also suggests that trophic omnivory may be common in these webs.

This is the first de novo assembly and annotation of a complete mitochondrial genome in the Ericales order from the American cranberry (Vaccinium macrocarpon Ait.). Moreover, only four complete Asterid mitochondrial genomes have been made publicly available. The cranberry mitochondrial genome was assembled and reconstructed from whole genome 454 Roche GS-FLX and Illumina shotgun sequences. Compared with other Asterids, the reconstruction of the genome revealed an average size mitochondrion (459,678 nt) with relatively little repetitive sequences and DNA of plastid origin. The complete mitochondrial genome of cranberry was annotated obtaining a total of 34 genes classified based on their putative function, plus three ribosomal RNAs, and 17 transfer RNAs. Maternal organellar cranberry inheritance was inferred by analyzing gene variation in the cranberry mitochondria and plastid genomes. The annotation of cranberry mitochondrial genome revealed the presence of two copies of tRNA-Sec and a selenocysteine insertion sequence (SECIS) element which were lost in plants during evolution. This is the first report of a land plant possessing selenocysteine insertion machinery at the sequence level.

Honey bees are the most important managed pollinator in the world. Recent trends suggest, however, that the demand for their pollination services is growing faster than the available supply. Therefore, it is critical to determine the most efficient management practices to maximize their use for crop production. One factor known to influence the efficiency of crop pollination is the availability of alternative, non-crop floral resources. These resources can vary as a function of the landscape surrounding a farm as well as local management practices within a farm. However, little is known about how the foraging behavior of honey bees on the target crop responds to the spatial arrangement of hives or the composition of the surrounding landscape. In this study, we collected pollen from pollen traps on honey bee hives placed on commercial cranberry marshes in central Wisconsin (USA). Individual marshes were selected to fall across a gradient of surrounding landscape from high- to low-woodland. Within each marsh, hives were placed either adjacent to wooded habitat, adjacent to a water reservoir, or in the center of the marsh. Honey bees from hives near water reservoirs collected a lower proportion of cranberry pollen than honey bees near the wooded area or at the center of the marsh. However, honey bees collected the same number of cranberry pollen grains and total pollen biomass irrespective of hive location or the surrounding landscape. Honey bees from hives located near water reservoirs, relative to the other two hive locations, tended to collect more pollen from fewer plants (low evenness). Cranberry yield did not vary as a function of the proportion of cranberry pollen collected or total number of cranberry pollen grains collected, but yield was higher at marshes located in low-woodland landscapes relative to those in high-woodland landscapes. We conclude that the location of hives on a cranberry marsh in relation to non-crop habitat does not affect yield allowing growers to place hives where it is convenient, although placing hives near water reservoirs should provide bees with a more diverse pollen diet.

In 2015 and 2016, surveys of entomopathogenic nematodes were conducted in the wild cranberry habitats of Wisconsin. The purpose of the survey was to find a biological control agent for control of insect pests of cranberries. Two nematodes, Oscheius onirici Torini (Rhabditida: Rhabditidae) and Heterorhabditis georgiana Nguyen (Rhabditida: Rhabditidae), were collected in this survey. Laboratory trials show that applications of either O. onirici or H. georgiana resulted in 75% and 65% mortality rates, respectively, when applied to sparganothis fruitworm Sparganothis sulfureana Clemens (Lepidoptera: Tortricidae) larvae. In replicated field trials, both nematode species were also shown to significantly suppress the redheaded flea beetle, Systena frontalis Fabricius (Coleoptera: Chrysomelidae) populations. Collectively, these findings suggest that these nematodes may be promising bio-insecticides for cranberries.

Teeming within pollen provisions are diverse communities of symbiotic microbes, which provide a variety of benefits to bees. Microbes themselves may represent a major dietary resource for developing bee larvae. Despite their apparent importance in sustaining bee health, evidence linking pollen-borne microbes to larval health is currently lacking. We examined the effects of microbe-deficient diets on the fitness of larval mason bees. In a series of diet manipulations, microbe-rich maternally collected pollen provisions were replaced with increasing fractions of sterilized, microbe-deficient pollen provisions before being fed to developing larvae. Convergent findings from amino acid and fatty acid trophic biomarker analyses revealed that larvae derived a substantial amount of nutrition from microbial prey and occupied a significantly higher trophic position than that of strict herbivores. Larvae feeding on increasingly sterile diets experienced significant adverse effects on growth rates, biomass and survivorship. When completely deprived of pollen-borne microbes, larvae consistently exhibited marked decline in fitness. We conclude that microbes associated with aged pollen provisions are central to bee health, not only as nutritional mutualists, but also as a major dietary component. In an era of global bee decline, the conservation of such bee–microbe interactions may represent an important facet of pollinator protection strategies.

Detritivory represents the dominant trophic mode on Earth, and within this vast consumer group, microbes tend to predominate. Virtually all detritus is replete with microbes, many of which consume the detrital substrate, rendering it a complex of living and non-living components. A detrital mass, therefore, can be considered an ‘undead’ food-web in which the resident microbes busily displace non-living biomass with their own, while carnivorous microbes prey upon other organisms. In this food-chain, bacteria and fungi have been shown to register just as animals do, demonstrating that microbes are analogous to animals within trophic hierarchies. Given that microbial consumers are often suffused throughout detrital substrates, the detritus becomes a complex of multiple trophic groups. It has long been suggested that microbial consumers within soil systems represent distinct trophic groups, and in recent studies, these hypotheses have been tested empirically. The detrital complex, then, can be considered a dynamic microcosm of the broader food-web, and the trophic identities within the complex (as well as the metazoans that consume the complex), can be measured and interpreted. This multi-trophic aspect of ‘undead’ detritus tends to elevate the trophic positions of animals (i.e., trophic inflation), which derives from the assimilation of heterotrophic (microbial) and autotrophic proteins. Importantly, inclusion of microbes in trophic hierarchies provides a more comprehensive framework within which to interpret organismal-, population-, and community-scale trophic identities. However, in recalibrating our trophic ‘lens’ to include microbial consumers, there remain some long-standing concepts, questions, and definitions that will need to be revisited.

Drosophila suzukii Matsumura (Diptera: Drosophilidae), commonly referred to as the spotted wing drosophila, is an exotic species that has proven a troublesome pest of fruit production in the United States. The fly targets small fruit and thus represents a concern for the U.S. cranberry industry. Two studies were conducted to assess whether cranberries may serve as hosts for D. suzukii. In the first study, the suitability of ripe, unripe, and over-ripe cranberries were assayed by examining adult oviposition and larval development in no-choice trials. In the second study, wounded and unwounded fruit were examined as potential hosts in choice and no-choice trials. Our first study showed that ripe, unripe, and over-ripe cranberries were unsuitable hosts (few eggs were laid, with no surviving puparia). In the wounded and unwounded berry study, no larvae survived to adulthood among unwounded berries. Within wounded fruit, D. suzukii readily fed and developed into adults. Together, these results suggest...

The cranberry fruitworm (Acrobasis vaccinii Riley), sparganothis fruitworm (Sparganothis sulfureana Clemens), and blackheaded fireworm (Rhopobota naevana Hubner) are historically significant pests of cranberries (Vaccinium macrocarpon Aiton) in the Upper Midwest (Wisconsin), USA. Their respective natural histories are well documented but correlations between developmental benchmarks (e.g., larval eclosion) and degree-day accruals are not yet known. Treatment timings are critical to the optimization of any given control tactic, and degree-day accrual facilitates optimization by quantifying the developmental status of pest populations. When key developmental benchmarks in the pest life cycle are linked to degree-days, real-time weather data can be used to predict precise treatment timings. Here, we provide the degree-day accumulations associated with discrete biological events (i.e., initiation of flight and peak flight) for the three most consistent moth pests of cranberries in Wisconsin. Moths were trapped each spring and summer from 2003 to 2011. To characterize flight dynamics and average timing of flight initiation, pheromone-baited trap-catch data were tallied for all three pest species within each of seven growing seasons. These flight dynamics were then associated with the corresponding degree-day accumulations generated using the cranberry plant’s developmental thresholds. Finally, models were fit to the data in order to determine the peak flight of each species. The initiation of the spring flight among all three moth species was highly synchronous, aiding in the timing of control tactics; however, there were substantial differences in the timing of peak flight among the moth species. Characterization of the relationship between temperature and pest development allows pest management professionals to target specific life stages, improving the efficacy of any given pest control tactic.

Background The application of genotyping by sequencing (GBS) approaches, combined with data imputation methodologies, is narrowing the genetic knowledge gap between major and understudied, minor crops. GBS is an excellent tool to characterize the genomic structure of recently domesticated (~200 years) and understudied species, such as cranberry (Vaccinium macrocarpon Ait.), by generating large numbers of markers for genomic studies such as genetic mapping. Results We identified 10842 potentially mappable single nucleotide polymorphisms (SNPs) in a cranberry pseudo-testcross population wherein 5477 SNPs and 211 short sequence repeats (SSRs) were used to construct a high density linkage map in cranberry of which a total of 4849 markers were mapped. Recombination frequency, linkage disequilibrium (LD), and segregation distortion at the genomic level in the parental and integrated linkage maps were characterized for first time in cranberry. SSR markers, used as the backbone in the map, revealed high collinearity with previously published linkage maps. The 4849 point map consisted of twelve linkage groups spanning 1112 cM, which anchored 2381 nuclear scaffolds accounting for ~13 Mb of the estimated 470 Mb cranberry genome. Bin mapping identified 592 and 672 unique bins in the parentals and a total of 1676 unique marker positions in the integrated map. Synteny analyses comparing the order of anchored cranberry scaffolds to their homologous positions in kiwifruit, grape, and coffee genomes provided initial evidence of homology between cranberry and closely related species. Conclusions GBS data was used to rapidly saturate the cranberry genome with markers in a pseudo-testcross population. Collinearity between the present saturated genetic map and previous cranberry SSR maps suggests that the SNP locations represent accurate marker order and chromosome structure of the cranberry genome. SNPs greatly improved current marker genome coverage, which allowed for genome-wide structure investigations such as segregation distortion, recombination, linkage disequilibrium, and synteny analyses. In the future, GBS can be used to accelerate cranberry molecular breeding through QTL mapping and genome-wide association studies (GWAS). Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2802-3) contains supplementary material, which is available to authorized users.

Degree-day models link ambient temperature to the development of insects, making such models valuable tools in integrated pest management. These models increase management efficacy by quantifying and predicting pest phenology. In Wisconsin, the top insect pest of cranberry production is the cranberry fruitworm, Acrobasis vaccinii Riley (Lepidoptera: Pyralidae). Control of this species is often complicated by the fact that the larvae feed entirely within the fruit. Timing of control tactics, therefore, is critical and generally targets the adult and egg stages. However, the commencement of oviposition and egg hatch are extremely difficult to track empirically, forcing pest management strategies to rely on proxy events that are more apparent but less informative as indicators of cranberry fruitworm egg presence. This research provides the upper and lower temperature-mediated growth thresholds of this pest, which represents the first steps toward the creation of a degree-day model. Using field-collected A. vaccinii, we reared the larvae within cranberry fruit and monitored larval growth at nine different constant temperatures. We determined the average growth rate at each temperature and modeled growth rates as a function of temperature. We then calculated the precise upper and lower developmental temperature thresholds of this species. Future work will be able to use these thresholds to generate degree-day accumulations that correspond to phenological events in the field, providing a powerful predictive tool for pest management in cranberry production.