DNA metabarcoding diet analysis in a generalist omnivore: feeding trials reveal the efficacy of extraction kits and a multi-locus approach for identifying diverse diets.

Metabarcoding-based diet analysis is a valuable tool for understanding the feeding behavior of a wide range of species. However, many studies using these methods for wild animals assume accuracy and precision without experimental evaluation with known positive control food items. Here, we conducted a feeding trial experiment with a positive control community in pasture-raised chickens and assessed the efficacy of several commonly used DNA extraction kits and primer sets. We hand-fed 22 known food items, including insects and plants, to six backyard laying hens and collected their excreta for eight h. We evaluated the efficacy of three DNA extraction kits, three primer sets for plant identification (targeting rbcL, trnL, and internal transcribed spacer 2 [ITS2]), and three primer sets for arthropod identification (targeting cytochrome oxidase subunit I [COI]). The detection success rate of our positive control food items was highly variable, ranging from 2.04% to 93.88% for all kit/primer combinations and averaging 37.35% and 43.57% for the most effective kit/primer combination for plants and insects, respectively. Extraction kits using bead-based homogenization positively affected the recovery proportion of plant and insect DNA in excreta samples. The minimum time to detect known food items was 44 min post-feeding. Two COI primer sets significantly outperformed the third, and both recovery proportion and taxonomic resolution from ITS2 were significantly higher than those from rbcL and trnL. Taken together, these results display the potential variability that can be inherently present in DNA-based diet analyses and highlight the utility of experimental feeding trials in validating such approaches, particularly for omnivores with diverse diets.

Interactive effects of multiscale diversification practices on farmland bird stress.

Farmland diversification practices (i.e., methods used to produce food sustainably by enhancing biodiversity in cropping systems) are sometimes considered beneficial to both agriculture and biodiversity, but most studies of these practices rely on species richness, diversity, or abundance as a proxy for habitat quality. Biodiversity assessments may miss early clues that populations are imperiled when species presence does not imply persistence. Physiological stress indicators may help identify low-quality habitats before population declines occur. We explored how avian stress indicators respond to on-farm management practices and surrounding seminatural area (1-km radius) across 21 California strawberry farms. We examined whether commonly used biodiversity metrics correlate with stress responses in wild birds. We used ∼1000 blood and feather samples and body mass and wing chord measurements, mostly from passerines, to test the effects of diversification practices on four physiological stress indicators: heterophil to lymphocyte ratios (H:L), body condition, hematocrit values, and feather growth rates of individual birds. We then tested the relationship between physiological stress indicators and species richness, abundance, occurrence, and diversity derived from 285 bird point count surveys. After accounting for other biological drivers, landscape context mediated the effect of local farm management on H:L and body condition. Local diversification practices were associated with reduced individual stress in intensive agricultural landscapes but increased it in landscapes surrounded by relatively more seminatural area. Feathers grew more slowly in landscapes dominated by strawberry production, suggesting that nutritional condition was lower here than in landscapes with more crop types and seminatural areas. We found scant evidence that species richness, abundance, occurrence, or diversity metrics were correlated with the individual's physiological stress, suggesting that reliance on these metrics may obscure the impacts of management on species persistence. Our findings underscore the importance of considering landscape context when designing local management strategies to promote wildlife conservation.

Algunas veces se considera a las prácticas de diversificación agrícola (es decir, los métodos usados para producir alimentos de manera sustentable mediante el enriquecimiento de la biodiversidad en los sistemas de cultivo) como benéficas para la agricultura y la biodiversidad, pero la mayoría de los estudios sobre estas prácticas dependen de la riqueza, abundancia o diversidad de especies como indicadores de la calidad del hábitat. Las valoraciones de la biodiversidad pueden ignorar las señales tempranas de una población en peligro cuando la presencia de la especie no implica persistencia. Los indicadores de estrés fisiológico pueden auxiliar en la identificación de hábitats de baja calidad antes de que ocurra la declinación poblacional. Analizamos las respuestas de los indicadores de estrés en aves al manejo en las granjas y áreas seminaturales circundantes (1 km de radio) de 21 cultivos de fresas en California. Evaluamos si las medidas comunes de biodiversidad se correlacionan con las respuestas al estrés de las aves silvestres. Usamos aproximadamente mil muestras de sangre y plumas y medidas de masa corporal y cuerda alar, la mayoría de paseriformes, para analizar los efectos de las prácticas de diversificación sobre cuatro indicadores de estrés fisiológico: la relación heterófilos/linfocitos (H:L), condición corporal; valores hematocritos; y la tasa de crecimiento de las plumas en aves individuales. Después probamos la relación entre los indicadores de estrés fisiológicos y la riqueza, abundancia, presencia y diversidad de especies tomadas de 285 conteos por puntos de aves. Después de considerar otros factores biológicos, el contexto del paisaje medió el efecto de la gestión de las granjas locales sobre la H:L y la condición corporal. Las prácticas locales de diversificación estuvieron asociadas con una reducción en el estrés individual en los paisajes con agricultura intensiva; sin embargo, el estrés aumentó en los paisajes rodeados por áreas relativamente más seminaturales. Encontramos poca evidencia que respalde que las medidas de riqueza, abundancia, presencia y diversidad de especies estuvieran correlacionadas con el estrés fisiológico de los individuos, lo que sugiere que depender de estas medidas puede nublar el impacto de la gestión sobre la persistencia de las especies. Nuestros descubrimientos apuntalan lo importante que es considerar el contexto del paisaje cuando se diseñan las estrategias de gestión local para promover la conservación de la fauna.

A trait-based framework for predicting foodborne pathogen risk from wild birds.

Recent foodborne illness outbreaks have heightened pressures on growers to deter wildlife from farms, jeopardizing conservation efforts. However, it remains unclear which species, particularly birds, pose the greatest risk to food safety. Using >11,000 pathogen tests and 1565 bird surveys covering 139 bird species from across the western United States, we examined the importance of 11 traits in mediating wild bird risk to food safety. We tested whether traits associated with pathogen exposure (e.g., habitat associations, movement, and foraging strategy) and pace-of-life (clutch size and generation length) mediated foodborne pathogen prevalence and proclivities to enter farm fields and defecate on crops. Campylobacter spp. were the most prevalent enteric pathogen (8.0%), while Salmonella and Shiga-toxin producing Escherichia coli (STEC) were rare (0.46% and 0.22% prevalence, respectively). We found that several traits related to pathogen exposure predicted pathogen prevalence. Specifically, Campylobacter and STEC-associated virulence genes were more often detected in species associated with cattle feedlots and bird feeders, respectively. Campylobacter was also more prevalent in species that consumed plants and had longer generation lengths. We found that species associated with feedlots were more likely to enter fields and defecate on crops. Our results indicated that canopy-foraging insectivores were less likely to deposit foodborne pathogens on crops, suggesting growers may be able to promote pest-eating birds and birds of conservation concern (e.g., via nest boxes) without necessarily compromising food safety. As such, promoting insectivorous birds may represent a win-win-win for bird conservation, crop production, and food safety. Collectively, our results suggest that separating crop production from livestock farming may be the best way to lower food safety risks from birds. More broadly, our trait-based framework suggests a path forward for co-managing wildlife conservation and food safety risks in farmlands by providing a strategy for holistically evaluating the food safety risks of wild animals, including under-studied species.

Environmental factors influencing fine-scale distribution of Antarctica's only endemic insect.

Species distributions are dependent on interactions with abiotic and biotic factors in the environment. Abiotic factors like temperature, moisture, and soil nutrients, along with biotic interactions within and between species, can all have strong influences on spatial distributions of plants and animals. Terrestrial Antarctic habitats are relatively simple and thus good systems to study ecological factors that drive species distributions and abundance. However, these environments are also sensitive to perturbation, and thus understanding the ecological drivers of species distribution is critical for predicting responses to environmental change. The Antarctic midge, Belgica antarctica, is the only endemic insect on the continent and has a patchy distribution along the Antarctic Peninsula. While its life history and physiology are well studied, factors that underlie variation in population density within its range are unknown. Previous work on Antarctic microfauna indicates that distribution over broad scales is primarily regulated by soil moisture, nitrogen content, and the presence of suitable plant life, but whether these patterns are true over smaller spatial scales has not been investigated. Here we sampled midges across five islands on the Antarctic Peninsula and tested a series of hypotheses to determine the relative influences of abiotic and biotic factors on midge abundance. While historical literature suggests that Antarctic organisms are limited by the abiotic environment, our best-supported hypothesis indicated that abundance is predicted by a combination of abiotic and biotic conditions. Our results are consistent with a growing body of literature that biotic interactions are more important in Antarctic ecosystems than historically appreciated.

Floral resource availability from groundcover promotes bee abundance in coffee agroecosystems.

Patterns of bee abundance and diversity across different spatial scales have received thorough research consideration. However, the impact of short- and long-term temporal resource availability on biodiversity has been less explored. This is highly relevant in tropical agricultural systems for pollinators, as many foraging periods of pollinators extend beyond flowering of any single crop species. In this study, we sought to understand how bee communities in tropical agroecosystems changed between seasons, and if short- and long-term floral resource availability influenced their diversity and abundance. We used a threshold analysis approach in order to explore this relationship at two time scales. This study took place in a region dominated by coffee agroecosystems in Southern Mexico. This was an ideal system because the landscape offers a range of coffee management regimes that maintain heterogeneity in floral resource availability spatially and temporally. We found that the bee community varies significantly between seasons. There were higher abundances of native social, solitary and managed honey bees during the dry season when coffee flowers. Additionally, we found that floral resources from groundcover, but not trees, were associated with bee abundance. Further, the temporal scale of the availability of these resources is important, whereby short-term floral resource availability appears particularly important in maintaining high bee abundance at sites with lower seasonal complementarity. We argue that in addition to spatial resource heterogeneity, temporal resource heterogeneity is critical in explaining bee community patterns, and should thus be considered to promote pollinator conservation.

Local and Landscape Constraints on Coffee Leafhopper (Hemiptera: Cicadellidae) Diversity.

The intensification of agriculture drives many ecological and environmental consequences including impacts on crop pest populations and communities. These changes are manifested at multiple scales including small-scale management practices and changes to the composition of land-use types in the surrounding landscape. In this study, we sought to examine the influence of local and landscape-scale agricultural factors on a leafhopper herbivore community in Mexican coffee plantations. We sampled leafhopper (Hemiptera: Cicadellidae) diversity in 38 sites from 9 coffee plantations of the Soconusco region of Chiapas, Mexico. While local management factors such as coffee density, branches per coffee bush, tree species, and density were not important in explaining leafhopper abundance and richness, shade management at the landscape level and elevation significantly affected leafhoppers. Specifically, the percentage of low-shade coffee in the landscape (1,000-m radius surrounding sites) increased total leafhopper abundance. In addition, Shannon's diversity of leafhoppers was increased with coffee density. Our results show that abundance and diversity of leafhoppers are greater in simplified landscapes, thereby suggesting that these landscapes will have higher pest pressure and may be more at-risk for diseases vectored by these species in an economically important crop.

Changes in species diversity of arboreal spiders in Mexican coffee agroecosystems: untangling the web of local and landscape influences driving diversity.

Agricultural intensification is implicated as a major driver of global biodiversity loss. Local management and landscape scale factors both influence biodiversity in agricultural systems, but there are relatively few studies to date looking at how local and landscape scales influence biodiversity in tropical agroecosystems. Understanding what drives the diversity of groups of organisms such as spiders is important from a pragmatic point of view because of the important biocontrol services they offer to agriculture. Spiders in coffee are somewhat enigmatic because of their positive or lack of response to agricultural intensification. In this study, we provide the first analysis, to our knowledge, of the arboreal spiders in the shade trees of coffee plantations. In the Soconusco region of Chiapas, Mexico we sampled across 38 sites on 9 coffee plantations. Tree and canopy connectedness were found to positively influence overall arboreal spider richness and abundance. We found that different functional groups of spiders are responding to different local and landscape factors, but overall elevation was most important variable influencing arboreal spider diversity. Our study has practical management applications that suggest having shade grown coffee offers more suitable habitat for arboreal spiders due to a variety of the characteristics of the shade trees. Our results which show consistently more diverse arboreal spider communities in lower elevations are important in light of looming global climate change. As the range of suitable elevations for coffee cultivation shrinks promoting arboreal spider diversity will be important in sustaining the viability of coffee.

Biodiversity conservation in agriculture requires a multi-scale approach.

Biodiversity loss--one of the most prominent forms of modern environmental change--has been heavily driven by terrestrial habitat loss and, in particular, the spread and intensification of agriculture. Expanding agricultural land-use has led to the search for strong conservation strategies, with some suggesting that biodiversity conservation in agriculture is best maximized by reducing local management intensity, such as fertilizer and pesticide application. Others highlight the importance of landscape-level approaches that incorporate natural or semi-natural areas in landscapes surrounding farms. Here, we show that both of these practices are valuable to the conservation of biodiversity, and that either local or landscape factors can be most crucial to conservation planning depending on which types of organisms one wishes to save. We performed a quantitative review of 266 observations taken from 31 studies that compared the impacts of localized (within farm) management strategies and landscape complexity (around farms) on the richness and abundance of plant, invertebrate and vertebrate species in agro-ecosystems. While both factors significantly impacted species richness, the richness of sessile plants increased with less-intensive local management, but did not significantly respond to landscape complexity. By contrast, the richness of mobile vertebrates increased with landscape complexity, but did not significantly increase with less-intensive local management. Invertebrate richness and abundance responded to both factors. Our analyses point to clear differences in how various groups of organisms respond to differing scales of management, and suggest that preservation of multiple taxonomic groups will require multiple scales of conservation.

Food webs in the litter: effects of food and nest addition on ant communities in coffee agroecosystems and forest.

Community assembly is driven by multiple factors, including resource availability and habitat requirements. Litter nesting ants respond to food and nest site availability, and adding food and nests may increase ant species richness and abundance. However, litter decomposers share food resources with ants, and increasing food availability may speed decomposition processes, eliminating twigs and seeds in which litter ants nest. We manipulated ant food and nest resources in three habitat types (forest, high-shade coffee, and low-shade coffee) to determine ant community responses after 1 and 2 mo. We examined changes in numbers of ant species, colonies, workers, brood, colony growth rate, and ant species composition. Habitat type strongly affected ant communities, influencing ant species richness, numbers of colonies and workers, and ant species composition. However, food addition and nest addition did not affect these community characteristics. Colony growth rate did not differ with food addition but was greater in forest and low-shade coffee compared with high-shade coffee. Habitat differences in colony growth may be because of presence of an aggressive species (Wasmannia auropunctata Roger) in high-shade coffee plots or naturally low arthropod densities during a time when ant colonization was low. Thus, in coffee landscapes, habitat type impacts litter nesting ant community structure, composition, and colony growth rate; however, food and nest addition had small impacts.

Cryptic biodiversity effects: importance of functional redundancy revealed through addition of food web complexity.

Interactions between predators and the degree of functional redundancy among multiple predator species may determine whether herbivores experience increased or decreased predation risk. Specialist parasites can modify predator behavior, yet rarely have cascading effects on multiple predator species and prey been evaluated. We examined influences of specialist phorid parasites (Pseudacteon spp.) on three predatory ant species and herbivores in a coffee agroecosystem. Specifically, we examined whether changes in ant richness affected fruit damage by the coffee berry borer (Hypothenemus hampei) and whether phorids altered multi-predator effects. Each ant species reduced borer damage, and without phorids, increasing predator richness did not further decrease borer damage. However, with phorids, activity of one ant species was reduced, indicating that the presence of multiple ant species was necessary to limit borer damage. In addition, phorid presence revealed synergistic effects of multiple ant species, not observed without the presence of this parasite. Thus, a trait-mediated cascade resulting from a parasite-induced predator behavioral change revealed the importance of functional redundancy, predator diversity, and food web complexity for control of this important pest.

Do herbivores eavesdrop on ant chemical communication to avoid predation?

Strong effects of predator chemical cues on prey are common in aquatic and marine ecosystems, but are thought to be rare in terrestrial systems and specifically for arthropods. For ants, herbivores are hypothesized to eavesdrop on ant chemical communication and thereby avoid predation or confrontation. Here I tested the effect of ant chemical cues on herbivore choice and herbivory. Using Margaridisa sp. flea beetles and leaves from the host tree (Conostegia xalapensis), I performed paired-leaf choice feeding experiments. Coating leaves with crushed ant liquids (Azteca instabilis), exposing leaves to ant patrolling prior to choice tests (A. instabilis and Camponotus textor) and comparing leaves from trees with and without A. instabilis nests resulted in more herbivores and herbivory on control (no ant-treatment) relative to ant-treatment leaves. In contrast to A. instabilis and C. textor, leaves previously patrolled by Solenopsis geminata had no difference in beetle number and damage compared to control leaves. Altering the time A. instabilis patrolled treatment leaves prior to choice tests (0-, 5-, 30-, 90-, 180-min.) revealed treatment effects were only statistically significant after 90- and 180-min. of prior leaf exposure. This study suggests, for two ecologically important and taxonomically diverse genera (Azteca and Camponotus), ant chemical cues have important effects on herbivores and that these effects may be widespread across the ant family. It suggests that the effect of chemical cues on herbivores may only appear after substantial previous ant activity has occurred on plant tissues. Furthermore, it supports the hypothesis that herbivores use ant chemical communication to avoid predation or confrontation with ants.