Trachymyrmex septentrionalis ants promote fungus garden hygiene using Trichoderma-derived metabolite cues.

Fungus-growing ants depend on a fungal mutualist that can fall prey to fungal pathogens. This mutualist is cultivated by these ants in structures called fungus gardens. Ants exhibit weeding behaviors that keep their fungus gardens healthy by physically removing compromised pieces. However, how ants detect diseases of their fungus gardens is unknown. Here, we applied the logic of Koch's postulates using environmental fungal community gene sequencing, fungal isolation, and laboratory infection experiments to establish that Trichoderma spp. can act as previously unrecognized pathogens of Trachymyrmex septentrionalis fungus gardens. Our environmental data showed that Trichoderma are the most abundant noncultivar fungi in wild T. septentrionalis fungus gardens. We further determined that metabolites produced by Trichoderma induce an ant weeding response that mirrors their response to live Trichoderma. Combining ant behavioral experiments with bioactivity-guided fractionation and statistical prioritization of metabolites in Trichoderma extracts demonstrated that T. septentrionalis ants weed in response to peptaibols, a specific class of secondary metabolites known to be produced by Trichoderma fungi. Similar assays conducted using purified peptaibols, including the two previously undescribed peptaibols trichokindins VIII and IX, suggested that weeding is likely induced by peptaibols as a class rather than by a single peptaibol metabolite. In addition to their presence in laboratory experiments, we detected peptaibols in wild fungus gardens. Our combination of environmental data and laboratory infection experiments strongly support that peptaibols act as chemical cues of Trichoderma pathogenesis in T. septentrionalis fungus gardens.

Combining surface and soil environmental DNA with artificial cover objects to improve terrestrial reptile survey detection.

Reptiles are increasingly of conservation concern due to their susceptibility to habitat loss, emerging disease, and harvest in the wildlife trade. However, reptile populations are often difficult to monitor given the frequency of crypsis in their life history. This difficulty has left uncertain the conservation status of many species and the efficacy of conservation actions unknown. Environmental DNA (eDNA) surveys consistently elevate the detection rate of species they are designed to monitor, and while their use is promising for terrestrial reptile conservation, successes in developing such surveys have been sparse. We tested the degree to which inclusion of surface and soil eDNA sampling into conventional artificial-cover methods elevates the detection probability of a small, cryptic terrestrial lizard, Scincella lateralis. The eDNA sampling of cover object surfaces with paint rollers elevated per sample detection probabilities for this species 4-16 times compared with visual surveys alone. We readily detected S. lateralis eDNA under cover objects up to 2 weeks after the last visual detection, and at some cover objects where no S. lateralis were visually observed in prior months. With sufficient sampling intensity, eDNA testing of soil under cover objects produced comparable per sample detection probabilities as roller surface methods. Our results suggest that combining eDNA and cover object methods can considerably increase the detection power of reptile monitoring programs, allowing more accurate estimates of population size, detection of temporal and spatial changes in habitat use, and tracking success of restoration efforts. Further research into the deposition and decay rates of reptile eDNA under cover objects, as well as tailored protocols for different species and habitats, is needed to bring the technique into widespread use.

El interés por la conservación de los reptiles es cada vez mayor debido a su susceptibilidad ante la pérdida del hábitat, enfermedades emergentes y la captura para el mercado de fauna. Sin embargo, las poblaciones de reptiles son difíciles de monitorear por lo frecuente que es la cripsis en sus historias de vida. Esta dificultad deja incierto el estado de conservación de muchas especies y desconocida la eficacia de las acciones de conservación. Los censos de ADN ambiental (DNAa) elevan sistemáticamente la tasa de detección de las especies que monitorean, y aunque su uso es prometedor para la conservación de los reptiles terrestres, han sido escasos los éxitos en el desarrollo de dichos censos. Analizamos el grado al que la inclusión del muestreo de DNAa superficial y del suelo a los métodos convencionales de cobertura artificial eleva la probabilidad de detección de una pequeña lagartija terrestre críptica: Scincella lateralis. El muestreo de DNAa de las superficies con cobertura de objetos con rodillos de pintura elevó las probabilidades de detección por muestra para esta especie 4-16 veces más que los censos visuales. Detectamos fácilmente el DNAa de S. lateralis bajo los objetos de cubierta hasta dos semanas después de la última detección visual y en algunos objetos de cubierta en donde no se había observado en los meses previos a S. lateralis. Con suficiente intensidad de muestreo, el análisis de DNAa del suelo bajo objetos de cubierta produjo probabilidades de detección por muestra comparables como métodos de rodillo superficial. Nuestros resultados sugieren que la combinación del DNAa y los métodos de objetos de cobertura puede incrementar considerablemente el poder de detección de los programas de monitoreo de reptiles, lo que permite estimaciones más precisas del tamaño poblacional, detección de los cambios espaciales y temporales en el uso de hábitat y el éxito de rastreo de los esfuerzos de restauración. Además, se necesita la investigación sobre las tasas de depósito y descomposición del DNAa de reptiles bajo objetos de cubierta, así como los protocolos hechos para diferentes especies y hábitats, para que la técnica entre al uso difundido.

Chemical Gradients of Plant Substrates in an Atta texana Fungus Garden.

Many ant species grow fungus gardens that predigest food as an essential step of the ants' nutrient uptake. These symbiotic fungus gardens have long been studied and feature a gradient of increasing substrate degradation from top to bottom. To further facilitate the study of fungus gardens and enable the understanding of the predigestion process in more detail than currently known, we applied recent mass spectrometry-based approaches and generated a three-dimensional (3D) molecular map of an Atta texana fungus garden to reveal chemical modifications as plant substrates pass through it. The metabolomics approach presented in this study can be applied to study similar processes in natural environments to compare with lab-maintained ecosystems. IMPORTANCE The study of complex ecosystems requires an understanding of the chemical processes involving molecules from several sources. Some of the molecules present in fungus-growing ants' symbiotic system originate from plants. To facilitate the study of fungus gardens from a chemical perspective, we provide a molecular map of an Atta texana fungus garden to reveal chemical modifications as plant substrates pass through it. The metabolomics approach presented in this study can be applied to study similar processes in natural environments.

Moving eDNA surveys onto land: Strategies for active eDNA aggregation to detect invasive forest insects.

The use of environmental DNA (eDNA) surveys to monitor terrestrial species has been relatively limited, with successful implementations still confined to sampling DNA from natural or artificial water bodies and soil. Sampling water for eDNA depends on proximity to or availability of water, whereas eDNA from soil is limited in its spatial scale due to the large quantities necessary for processing and difficulty in doing so. These challenges limit the widespread use of eDNA in several systems, such as surveying forests for invasive insects. We developed two new eDNA aggregation approaches that overcome the challenges of above-ground terrestrial sampling and eliminate the dependency on creating or utilizing pre-existing water bodies to conduct eDNA sampling. The first, "spray aggregation," uses spray action to remove eDNA from surface substrates and was developed for shrubs and other understorey vegetation, while the second, "tree rolling," uses physical transfer via a roller to remove eDNA from the surface of tree trunks and large branches. We tested these approaches by surveying for spotted lanternfly, Lycorma delicatula, a recent invasive pest of northeastern USA that is considered a significant ecological and economic threat to forests and agriculture. We found that our terrestrial eDNA surveys matched visual surveys, but also detected L. delicatula presence ahead of visual surveys, indicating increased sensitivity of terrestrial eDNA surveys over currently used methodology. The terrestrial eDNA approaches we describe can be adapted for use in surveying a variety of forest insects and represent a novel strategy for surveying terrestrial biodiversity.