Invasive Japanese beetle (Popillia japonica Newman) larvae alter structure and carbon distribution in infested surface soil.

Invasive macrofauna influence the biophysical state and function of soil, helping to drive ecological changes over time. Many soil-dwelling invertebrates affect soil stability by facilitating or hindering the soil aggregation process, changing the availability of plant and soil organic matter (SOM) for aggregate incorporation, and shifting the predominant mechanisms by which carbon is incorporated into soil aggregates. Using mass fractionation and stable carbon isotope techniques, this 17-month experimental study examined silt-clay-loam mesocosms either infested or not infested with soil-dwelling larvae of the invasive Japanese beetle, Popillia japonica Newman (JB). We hypothesized that larval root-herbivory would promote a pathway of large aggregate formation that features the mixing of digested root tissue with mineral soil and subsequent fecal deposition. These newly deposited, large soil aggregates will then grow by agglomeration of particles, thereby occluding a larger pool of fresh organic carbon, or be broken apart, exposing fresh organic inputs to microbial activity and mineralization processes, depending on soil conditions. Findings show a proportional increase of larger soil size fractions (2- 8 mm) in the rhizosphere of infested soil after 1½ life cycles of the beetle, but a decrease in the smaller soil size fractions (0.053-2 mm). In infested bulk surface soil (0-2.5 cm) carbon increased, primarily due to greater carbon content in the largest size fractions. Carbon also increased in all size fractions, although the proportion of total carbon in fractions was greater only in the largest fractions due to their greater relative abundance. There may also be an increase of microbially derived carbon in the largest size fractions, possibly indicating significant priming effects associated with JB larval herbivory. The implications of these findings for relative stabilization of the bulk surface soil carbon pool in JB-infested soil likely depends on the residence time of, and stable microaggregate formation within these large size fractions.

Larvae of an invasive scarab increase greenhouse gas emissions from soils and recruit gut mycobiota involved in C and N transformations.

Background: Soil-derived prokaryotic gut communities of the Japanese beetle Popillia japonica Newman (JB) larval gut include heterotrophic, ammonia-oxidizing, and methanogenic microbes potentially capable of promoting greenhouse gas (GHG) emissions. However, no research has directly explored GHG emissions or the eukaryotic microbiota associated with the larval gut of this invasive species. In particular, fungi are frequently associated with the insect gut where they produce digestive enzymes and aid in nutrient acquisition. Using a series of laboratory and field experiments, this study aimed to (1) assess the impact of JB larvae on soil GHG emissions; (2) characterize gut mycobiota associated with these larvae; and (3) examine how soil biological and physicochemical characteristics influence variation in both GHG emissions and the composition of larval gut mycobiota. Methods: Manipulative laboratory experiments consisted of microcosms containing increasing densities of JB larvae alone or in clean (uninfested) soil. Field experiments included 10 locations across Indiana and Wisconsin where gas samples from soils, as well as JB and their associated soil were collected to analyze soil GHG emissions, and mycobiota (ITS survey), respectively. Results: In laboratory trials, emission rates of CO2, CH4, and N2O from infested soil were ≥ 6.3× higher per larva than emissions from JB larvae alone whereas CO2 emission rates from soils previously infested by JB larvae were 1.3× higher than emissions from JB larvae alone. In the field, JB larval density was a significant predictor of CO2 emissions from infested soils, and both CO2 and CH4 emissions were higher in previously infested soils. We found that geographic location had the greatest influence on variation in larval gut mycobiota, although the effects of compartment (i.e., soil, midgut and hindgut) were also significant. There was substantial overlap in the composition and prevalence of the core fungal mycobiota across compartments with prominent fungal taxa being associated with cellulose degradation and prokaryotic methane production/consumption. Soil physicochemical characteristics such as organic matter, cation exchange capacity, sand, and water holding capacity, were also correlated with both soil GHG emission, and fungal a-diversity within the JB larval gut. Conclusions: Results indicate JB larvae promote GHG emissions from the soil directly through metabolic activities, and indirectly by creating soil conditions that favor GHG-associated microbial activity. Fungal communities associated with the JB larval gut are primarily influenced by adaptation to local soils, with many prominent members of that consortium potentially contributing to C and N transformations capable of influencing GHG emissions from infested soil.

Soil Environments Influence Gut Prokaryotic Communities in the Larvae of the Invasive Japanese Beetle Popillia japonica Newman.

Invasive scarab beetles, like the Japanese beetle Popillia japonica Newman (JB), spend most of their lives as larvae feeding in the soil matrix. Despite the potential importance of the larval gut microbial community in driving the behavior, physiology, and nutritional ecology of this invasive insect, the role of soil biological and physicochemical characteristics in shaping this community are relatively unknown. Our objectives were to (1) characterize the degree to which larval gut microbial communities are environmentally acquired, (2) examine the combined effects of the gut region (i.e., midgut, hindgut) and local soil environments on gut microbial communities, and (3) search for soil physicochemical correlates that could be useful in future studies aimed at characterizing gut microbial community variation in soil-dwelling scarabs. Gut communities from neonates that were never in contact with the soil were different from gut communities of third instar larvae collected from the field, with neonate gut communities being significantly less rich and diverse. The influence of compartment (soil, midgut, or hindgut) on prokaryotic α- and ß-diversity varied with location, suggesting that JB larval gut communities are at least partially shaped by the local environment even though the influence of compartment was more pronounced. Midgut microbiota contained transient communities that varied with the surrounding soil environment whereas hindgut microbiota was more conserved. Prokaryotic communities in the hindgut clustered separately from those of soil and midgut, which displayed greater interspersion in ordination space. Soil cation exchange capacity, organic matter, water holding capacity, and texture were moderately correlated (≥29%) with gut prokaryotic microbial composition, especially within the midgut. Findings suggest that microbial communities associated with the JB gut are partially a function of adaptation to local soil environments. However, conditions within each gut compartment appear to shape those communities in transit through the alimentary canal.

Characterizing Billbug (Sphenophorus spp.) Seasonal Biology Using DNA Barcodes and a Simple Morphometric Analysis.

Billbugs (Sphenophorus spp.) are a complex of grass-feeding weevil species that reduce the aesthetic and functional qualities of turfgrass. Effective billbug monitoring and management programs rely on a clear understanding of their seasonal biology. However, our limited understanding of regional variation in the species compositions and seasonal biology of billbugs, stemming primarily from our inability to identify the damaging larval stage to species level, has hindered efforts to articulate efficient IPM strategies to growers. We used a combination of DNA barcoding methods and morphometric measures to begin filling critical gaps in our understanding of the seasonal biology of the billbug species complex across a broad geographic range. First, we developed a DNA barcoding reference library using cytochrome oxidase subunit 1 (COI) sequences from morphologically identified adult billbugs collected across Indiana, Missouri, Utah and Arizona. Next, we used our reference library for comparison and identification of unknown larval specimens collected across the growing season in Utah and Indiana. Finally, we combined our DNA barcoding approach with larval head capsule diameter, a proxy for developmental instar, to develop larval phenology charts. Adult COI sequences varied among billbug species, but variation was not influenced by geography, indicating that this locus alone was useful for resolving larval species identity. Overlaid with head capsule diameter data from specimens collected across the growing season, a better visualization of billbug species composition and seasonal biology emerged. This approach will provide researchers with the tools necessary to fill critical gaps in our understanding of billbug biology and facilitate the development of turfgrass pest management programs.

Volatile and Contact Chemical Cues Associated with Host and Mate Recognition Behavior of Sphenophorus venatus and Sphenophorus parvulus (Coleoptera: Dryophthoridae).

Beetles in the genus Sphenophorus Schönherr, or billbugs, potentially utilize both volatile and non-volatile behavior-modifying chemical signals. These insects are widely distributed across North America, often occurring in multi-species assemblages in grasses. However, details about their host- and mate-finding behavior are poorly understood. This study tested the hypothesis that volatile organic compounds from host-plants and conspecifics direct the dispersal behavior of hunting billbug S. venatus Say. Further, we characterized the cuticular hydrocarbon profiles of two widespread pest species, S. venatus and bluegrass billbug S. parvulus Gyllenhaal, to assess the potential role of contact pheromones in mate-recognition. In Y-tube olfactometer bioassays, S. venatus males were attracted to a combination of conspecifics and Cynodon dactylon host-plant material, as well as C. dactylon plant material alone. S. venatus females were attracted to a combination of male conspecifics and host-plants but were also attracted to male conspecifics alone. Field evaluation of a putative male-produced aggregation pheromone, 2-methyl-4-octanol, identified from two congeners, S. levis Vaurie and S. incurrens Gyllenhaal, did not support the hypothesis that S. venatus and S. parvulus were also attracted to this compound. Gas chromatography-mass spectrometry analysis of S. venatus and S. parvulus whole-body cuticular extracts indicated a series of hydrocarbons with qualitative and quantitative interspecific variation in addition to intraspecific quantitative variation between males and females. This study provides the first evidence that S. venatus orients toward host- and insect-derived volatile organic compounds and substantiates the presence of species-specific cuticular hydrocarbons that could serve as contact pheromones for sympatric Sphenophorus species.

Billbug (Coleoptera: Dryophthoridae: Sphenophorus spp.) Seasonal Biology and DNA-Based Life Stage Association in Indiana Turfgrass.

Eleven species of billbugs (Coleoptera: Dryophthoridae: Sphenophorus spp. Schönherr) infest managed turfgrass in North America. However, the regional variation in species composition remains unresolved and the seasonal phenology of several species has not been well documented. The latter gap is largely due to the inability to identify the larval stage to species-a confounding problem with several sympatric insect species. We used field trapping (adults) and soil sampling (larvae and pupae) surveys along with a DNA-based life-stage association to characterize the biology of billbugs associated with turfgrass in the Midwestern United States. Pitfall trapping at four locations in Indiana revealed four billbug species: S. venatus Say, S. parvulus Gyllenhaal, S. minimus Hart, and S. inaequalis Say. Sphenophorus venatus was the most abundant species on warm-season turfgrass while S. parvulus was most abundant on cool-season turfgrass. Investigation of S. venatus seasonal biology revealed two overwintered life stages-larva and adult-which resulted in two overlapping cohorts and two larval generations. Degree-day models describing S. venatus activity were more accurate for first-generation adults and larvae than for overwintering life stages. Maximum-likelihood analyses provided the first molecular species identification of billbug larvae and direct evidence that S. venatus larvae are capable of overwintering above 40°N latitude. Findings clarify the utility of molecular markers (CO1, 18S, and ITS2) for describing billbug larval population dynamics and seasonal phenology in regions where several sympatric billbug species occur. These results support the development of sustainable management strategies based on billbug seasonal phenology in different regions of North America.

Larval western bean cutworm feeding damage encourages the development of Gibberella ear rot on field corn.

BACKGROUND: A 2 year study was conducted to determine whether western bean cutworm (Striacosta albicosta Smith) (WBC) larval feeding damage increases severity of the fungal disease Gibberella ear rot [Fusarium graminearum (Schwein.) Petch] in field corn (Zea mays L.). The effect of a quinone-outside inhibiting fungicide, pyraclostrobin, on Gibberella ear rot severity and mycotoxin production, both with and without WBC pressure, was also evaluated. The impact of each variable was assessed individually and in combination to determine the effect of each upon ear disease severity. RESULTS: There was a positive correlation between the presence of WBC larvae in field corn and Gibberella ear rot severity under inoculated conditions in the 2 years of the experiment. An application of pyraclostrobin did not impact Gibberella ear rot development when applied at corn growth stage R1 (silks first emerging). CONCLUSION: Feeding damage from WBC larvae significantly increases the development of F. graminearum in field corn. We conclude that an effective integrated management strategy for Gibberella ear rot should target the insect pest first, in an effort to limit disease severity and subsequent mycotoxin production by F. graminearum in kernels. © 2016 Society of Chemical Industry.

Evaluation of Metarhizium brunneum F52 (Hypocreales: Clavicipitaceae) for Control of Japanese Beetle Larvae in Turfgrass.

Experimental and commercial preparations of Metarhizium brunneum (Petch) strain F52 were evaluated for control of Japanese beetle, Popillia japonica Newman (Coleoptera: Scarbaeidae), larvae (white grubs) in the laboratory and under field conditions. Experimental preparations consisted of granule and liquid formulations made using in vitro produced microsclerotia, which are intended to produce infective conidial spores after application. These formulations were compared against commercial insecticides (imidacloprid and trichlorfon), and commercial formulations of M. brunneum F52 (Met 52) containing only conidia. Field-collected grubs were susceptible to infection in a dosage-dependent relationship when exposed to potting soil treated with experimental microsclerotia granules in the laboratory. The LC(50) for field-collected larvae was 14.2 mg of granules per cup (∼15 g granules/m(2)). Field plots treated with experimental and commercial formulations of M. brunneum F52 after 10 September (targeting second and third instar grubs) had significantly lower grub densities compared with untreated plots, providing 38.6-69.2% control, which sometimes equaled levels of control with chemical insecticides. Fungal treatments made prior to 21 August provided 14.3-69.3% control, although grub densities resulting from these treatments were often not significantly lower than those in untreated control plots. By comparison, chemical insecticide treatments provide 68-100% grub control, often providing better control when applied earlier in the season. In conclusion, P. japonica larvae are susceptible to infection by M. brunneum, and grub densities were reduced most consistently by fall applications targeting later instars.

Association of candidate genes with drought tolerance traits in diverse perennial ryegrass accessions.

Drought is a major environmental stress limiting growth of perennial grasses in temperate regions. Plant drought tolerance is a complex trait that is controlled by multiple genes. Candidate gene association mapping provides a powerful tool for dissection of complex traits. Candidate gene association mapping of drought tolerance traits was conducted in 192 diverse perennial ryegrass (Lolium perenne L.) accessions from 43 countries. The panel showed significant variations in leaf wilting, leaf water content, canopy and air temperature difference, and chlorophyll fluorescence under well-watered and drought conditions across six environments. Analysis of 109 simple sequence repeat markers revealed five population structures in the mapping panel. A total of 2520 expression-based sequence readings were obtained for a set of candidate genes involved in antioxidant metabolism, dehydration, water movement across membranes, and signal transduction, from which 346 single nucleotide polymorphisms were identified. Significant associations were identified between a putative LpLEA3 encoding late embryogenesis abundant group 3 protein and a putative LpFeSOD encoding iron superoxide dismutase and leaf water content, as well as between a putative LpCyt Cu-ZnSOD encoding cytosolic copper-zinc superoxide dismutase and chlorophyll fluorescence under drought conditions. Four of these identified significantly associated single nucleotide polymorphisms from these three genes were also translated to amino acid substitutions in different genotypes. These results indicate that allelic variation in these genes may affect whole-plant response to drought stress in perennial ryegrass.

The effect of urbanization on ant abundance and diversity: a temporal examination of factors affecting biodiversity.

Numerous studies have examined the effect of urbanization on species richness and most studies implicate urbanization as the major cause of biodiversity loss. However, no study has identified an explicit connection between urbanization and biodiversity loss as the impact of urbanization is typically inferred indirectly by comparing species diversity along urban-rural gradients at a single time point. A different approach is to focus on the temporal rather than the spatial aspect and perform "before and after" studies where species diversity is cataloged over time in the same sites. The current study examined changes in ant abundance and diversity associated with the conversion of natural habitats into urban habitats. Ant abundance and diversity were tracked in forested sites that became urbanized through construction and were examined at 3 time points - before, during, and after construction. On average, 4.3 ± 1.2 unique species were detected in undisturbed plots prior to construction. Ant diversity decreased to 0.7 ± 0.8 species in plots undergoing construction and 1.5 ± 1.1 species in plots 1 year after construction was completed. With regard to species richness, urbanization resulted in the permanent loss of 17 of the 20 species initially present in the study plots. Recovery was slow and only 3 species were present right after construction was completed and 4 species were present 1 year after construction was completed. The second objective examined ant fauna recovery in developed residential lots based on time since construction, neighboring habitat quality, pesticide inputs, and the presence of invasive ants. Ant diversity was positively correlated with factors that promoted ecological recovery and negatively correlated with factors that promoted ecological degradation. Taken together, these results address a critical gap in our knowledge by characterizing the short- and long-term the effects of urbanization on the loss of ant biodiversity.

Survival and Infectivity of the Insect-Parasitic Nematode Heterorhabditis bacteriophora Poinar in Solutions Containing Four Different Turfgrass Soil Surfactants.

This laboratory study examined viability and infectivity of the entomopathogenic nematode (EPN) Heterorhabditis bacteriophora Poinar in solutions containing four different turfgrass soil surfactants: Revolution (Aquatrols Corp., Paulsboro, NJ), Aqueduct (Aquatrols Corp., Paulsboro, NJ), Cascade Plus (Precision Laboratories Inc., Waukegan, IL) and OARS (Aqua-Aid Inc., Rocky Mount, NC). Infective juvenile H. bacteriophora were added to solutions containing each of the four surfactants, and nematode viability and infectivity were monitored over time. In one of two trials, nematode survival in solutions containing the surfactants Aqueduct and Cascade Plus was consistently higher compared to the water control and solutions containing Revolution or OARS. Surfactants had no significant influence on nematode infectivity in either trial. Findings indicate that most of the common turfgrass soil surfactants examined should be compatible with EPNs and that some may potentially enhance nematode survival. Results also imply that tank-mixing of EPNs with most turfgrass soil surfactants should not pose a significant risk to the nematodes. The influence of soil surfactants on EPN performance remains to be examined in the field.

Endophyte-mediated resistance to black cutworm as a function of plant cultivar and endophyte strain in tall fescue.

To improve Neotyphodium endophyte-mediated resistance to black cutworm Agrotis ipsilon (Hufnagel) (BCW), a series of experiments was conducted by using several different cultivars of tall fescue, Schedonorus arundinaceus (Schreb.) Dumort. in combination with several different haplotypes of the endophyte Neotyphodium coenophialum (Morgan-Jones & Gams) (plant cultivar × endophyte haplotype = plant line), each producing unique alkaloid profiles. BCW settling response, survival at 5 and 10 d, and larval biomass varied significantly among plant lines. In general, greater variation BCW performance was observed within a single plant cultivar infected with different endophyte haplotypes than among different plant cultivars infected with the same endophyte haplotype, but comparisons among the former were far more numerous. Although five endophyte-mediated alkaloids representing three alkaloid classes were quantified in the plants, the pyrrolizidine alkaloid N-acetyl norloline was consistently the single best predictor of BCW performance. BCW settling response, 5-d survival, and 10-d survival decreased as levels of the alkaloid N-acetyl norloline increased. The same three response variables also decreased with increasing levels of peramine, but increased with increasing levels of ergovaline. Minor variation in endophyte infection levels occurring among infected plant lines had no significant influence on BCW performance. Results indicate a potentially important role for N-acetyl norloline and peramine in providing resistance to black cutworm whereas ergovaline appears to be much less important. Therefore, endophyte haplotypes expressing high levels of N-acetyl norloline and peramine may be of particular importance for developing 'friendly' endophyte-enhanced turf and pasture grasses that resist challenging lepidopteran pests, although remaining safe for wildlife and grazing mammals.

Ground beetle (Coleoptera: Carabidae) phenology, diversity, and response to weed cover in a turfgrass ecosystem.

Despite being fragmented and highly disturbed habitats, urban turfgrass ecosystems harbor a surprising diversity of arthropods. The suitability of turf as arthropod habitat, however, likely depends on the extent and types of pesticides and fertilizers used. For example, moderate levels of weed cover in low-input lawns may provide alternative food resources. We conducted a 2-yr field study to: 1) characterize the ground beetle (Carabidae) species assemblage in turfgrass, and 2) assess the direct and indirect effects of lawn management on carabid communities. Weed cover and beetle activity were compared among four lawn management programs: 1) consumer/garden center, 2) integrated pest management (IPM), 3) natural organic, and 4) no-input control. Nearly 5,000 carabid beetles across 17 species were collected with the predator Cyclotrachelus sodalis LeConte numerically dominating the trap catch (87% and 45% of individuals in 2005 and 2006, respectively). Populations of C. sodalis underwent a distinct peak in activity during the third week of June, whereas omnivorous and granivorous species tended to occur at far lower levels and were less variable over the season. We found no evidence for direct effects of lawn management on carabid species diversity; however, we detected an indirect effect mediated by variation in weed cover. Seed-feeding species were positively correlated with turf weeds early in 2006, whereas strictly predaceous species were not. Thus, turf management programs that lead to changes in plant species composition (i.e., herbicide regimes) may indirectly shape epigeal arthropod communities more strongly than the direct effects of insecticide use.

A novel method for estimating soybean herbivory in western corn rootworm (Coleoptera: Chrysomelidae).

The western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is the key pest of corn, Zea mays L., in North America. The western corn rootworm variant is a strain found in some parts of the United States that oviposits in soybean, Glycine max (L.) Merr., thereby circumventing crop rotation. Soybean herbivory is closely associated with oviposition; therefore, evidence of herbivory could serve as a proxy for rotation resistance. A digital image analysis method based on the characteristic green abdominal coloration of rootworm adults with soybean foliage in their guts was developed to estimate soybean herbivory rates of adult females. Image analysis software was used to develop and apply threshold limits that allowed only colors within the range that is characteristic of soybean herbivory to be displayed. When this method was applied to adult females swept from soybean fields in an area with high levels of rotation resistance, 54.3 +/- 2.1% were estimated to have fed on soybean. This is similar to a previously reported estimate of 54.8%. Results when laboratory-generated negative controls were analyzed showed an acceptably low frequency of false positives. This method could be developed into a management tool if user-friendly software were developed for its implementation. In addition, researchers may find the method useful as a rapid, standardized screen for measuring frequencies of soybean herbivory.