Organization of the macroinvertebrate community in a tropical annual agroecosystem into modules.

The structure of macroinvertebrate communities in agroecosystems has been assumed to be modular and organized around key herbivore pests. We characterized the macroinvertebrate community in the annual organic brassica agroecosystem in tropical central Brazil to determine if the community was a random assemblage of independent populations or was organized into repeatable multi-species components. We sampled 36 macroinvertebrate taxa associated with six organic brassica farms at biweekly intervals during the dry season during two years in the Distrito Federal, Brazil. We used an unconstrained ordination based on latent variable modeling (boral) with negative binomial population counts to analyze community composition independent of variation in sample abundance. We evaluated observed community structure by comparing it with randomized alternatives. We found that the community was not a random assemblage and consistently organized itself into two modules based around the major herbivores; one with lepidoptera and whiteflies and their associated natural enemies which was gradually replaced during the season by one with brassica aphids, aphid parasitoids and coccinellids. This analysis suggests that the historical and present-day focus on pest herbivores and their associated species in agroecosystems may be justified based on community structure.

A simple definition of food resources for the highly polyphagous Lygus lineolaris (Hemiptera: Miridae) enables prediction of adult abundance.

Lygus lineolaris (Palisot de Beauvois) is a highly polyphagous herbivore with more than 300 known host plants. The high polyphagy has created logistical challenges for understanding its population dynamics. I hypothesized that the primary food resource of this species can be characterized simply, cutting across the multiple host plant species, and enabling a simpler understanding of its population dynamics. The food resource was defined as the apical buds and meristematic tissue and terminal flowers and young seeds. Adult abundance in a habitat was related to the relative abundance of food in the habitat, abundance on a host plant stem was related to the amount of food resource on the stem, and the rate of emigration was lower from host plant patches with higher amounts of food resources. These results suggest that the population dynamics of L. lineolaris depends less on the identity of the host plants and more on the amount of food resource the various host plant species provide.

Estimating predation rates from molecular gut content analysis.

Several methods have been published to estimate per capita predation rates from molecular gut content analysis relying on intuitive understanding of predation, but none have been formally derived. We provide a theoretical framework for estimating predation rates to identify an accurate method and lay bare its assumptions. Per capita predation can be estimated by multiplying the prey decay rate and the prey quantity in the predators. This assumes that variation in per capita predation rate is approximately normally distributed, prey decay occurs exponentially, and predation is in steady state. We described several ways to estimate steady state predation, including using only qualitative presence-absence data to estimate the decay rate and in addition, we provided a method for estimating per capita predation rate when predation is not in steady state. We used previously published data on aphid consumption by a ladybird beetle in a feeding trial to calculate the predation rate and compare published methods with this theoretically derived method. The estimated predation rate (3.29 ± 0.27 aphids/h) using our derived method was not significantly different from the actual predation rate, 3.11 aphids/h. In contrast, previously published methods were less accurate, underestimating the predation rate (0.33 ± 0.02 to 1.66 ± 0.8 aphids/h) or overestimating it (3.64 ± 0.30 aphids/h). In summary, we provide methods to estimate predation rates even when variation in predation rates is not exactly normally distributed and not in steady state and demonstrate that the prey decay rate, and not the prey detection period, is required.

Quantitative prey species detection in predator guts across multiple trophic levels by mapping unassembled shotgun reads.

Quantifying species trophic interaction strengths is crucial for understanding community dynamics and has significant implications for pest management and species conservation. DNA-based methods to identify species interactions have revolutionized these efforts, but a significant limitation is the poor ability to quantify the strength of trophic interactions, that is the biomass or number of prey consumed. We present an improved pipeline, called Lazaro, to map unassembled shotgun reads to a comprehensive arthropod mitogenome database and show that the number of prey reads detected is quantitatively predicted from the prey biomass consumed, even for indirect predation. Two feeding bioassays were performed: starved coccinellid larvae consuming different numbers of aphids (Prey Quantity bioassay), and starved coccinellid larvae consuming a chrysopid larvae that had consumed aphids (Direct and Indirect Predation bioassay). Prey taxonomic assignment against a mitochondrial genome database had high accuracy (99.8% positive predictive value) and the number of prey reads was directly related to the number of prey consumed and inversely related to the elapsed time since consumption with high significance (r2  = .932, p = 4.92E-6). Aphids were detected up to 6 h after direct predation plus 3 h after indirect predation (9 h in total) and detection was related to the predator-specific decay rates. Lazaro enabled quantitative predictions of prey consumption across multiple trophic levels with high taxonomic resolution while eliminating all false positives, except for a few confirmed contaminants, and may be valuable for characterizing prey consumed by field-sampled predators. Moreover, Lazaro is readily applicable for species diversity determination from any degraded environmental DNA.

Predation on sentinel prey increases with increasing latitude in Brassica-dominated agroecosystems.

In natural ecosystems, arthropod predation on herbivore prey is higher at lower latitudes, mirroring the latitudinal diversity gradient observed across many taxa. This pattern has not been systematically examined in human-dominated ecosystems, where frequent disturbances can shift the identity and abundance of local predators, altering predation rates from those observed in natural ecosystems. We investigated how latitude, biogeographical, and local ecological factors influenced arthropod predation in Brassica oleracea-dominated agroecosystems in 55 plots spread among 5 sites in the United States and 4 sites in Brazil, spanning at least 15° latitude in each country. In both the United States and Brazil, arthropod predator attacks on sentinel model caterpillar prey were highest at the highest latitude studied and declined at lower latitudes. The rate of increased arthropod attacks per degree latitude was higher in the United States and the overall gradient was shifted poleward as compared to Brazil. PiecewiseSEM analysis revealed that aridity mediates the effect of latitude on arthropod predation and largely explains the differences in the intensity of the latitudinal gradient between study countries. Neither predator richness, predator density, nor predator resource availability predicted variation in predator attack rates. Only greater non-crop plant density drove greater predation rates, though this effect was weaker than the effect of aridity. We conclude that climatic factors rather than ecological community structure shape latitudinal arthropod predation patterns and that high levels of aridity in agroecosystems may dampen the ability of arthropod predators to provide herbivore control services as compared to natural ecosystems.

Metabarcoding versus mapping unassembled shotgun reads for identification of prey consumed by arthropod epigeal predators.

BACKGROUND: A central challenge of DNA gut content analysis is to identify prey in a highly degraded DNA community. In this study, we evaluated prey detection using metabarcoding and a method of mapping unassembled shotgun reads (Lazaro). RESULTS: In a mock prey community, metabarcoding did not detect any prey, probably owing to primer choice and/or preferential predator DNA amplification, while Lazaro detected prey with accuracy 43-71%. Gut content analysis of field-collected arthropod epigeal predators (3 ants, 1 dermapteran, and 1 carabid) from agricultural habitats in Brazil (27 samples, 46-273 individuals per sample) revealed that 64% of the prey species detections by either method were not confirmed by melting curve analysis and 87% of the true prey were detected in common. We hypothesized that Lazaro would detect fewer true- and false-positive and more false-negative prey with greater taxonomic resolution than metabarcoding but found that the methods were similar in sensitivity, specificity, false discovery rate, false omission rate, and accuracy. There was a positive correlation between the relative prey DNA concentration in the samples and the number of prey reads detected by Lazaro, while this was inconsistent for metabarcoding. CONCLUSIONS: Metabarcoding and Lazaro had similar, but partially complementary, detection of prey in arthropod predator guts. However, while Lazaro was almost 2× more expensive, the number of reads was related to the amount of prey DNA, suggesting that Lazaro may provide quantitative prey information while metabarcoding did not.

Diverse patterns of constitutive and inducible overexpression of detoxifying enzyme genes among resistant Aphis glycines populations.

Understanding the mechanisms of pyrethroid resistance is essential to the effective management of pesticide resistance in Aphis glycines Matsumura (Hemiptera: Aphididae). We mined putative detoxifying enzyme genes in the draft genome sequence of A. glycines for cytochrome oxidase P450 (CYP), glutathione-S-transferase (GST) and esterases (E4 and carboxylesterases-CES). Aphids from clonal populations resistant to pyrethroids from three sites in Minnesota, USA, were screened against a diagnostic LC99 concentration of either λ-cyhalothrin or bifenthrin and detoxifying enzyme genes expression in survivors was analyzed by qPCR. Their expression profiles were compared relative to a susceptible clonal population. We found 61 CYP (40 full-length), seven GST (all full-length), seven E4 (five full-length) and three CES (two full-length) genes, including 24 possible pseudogenes. The detoxifying enzymes had different expression profiles across resistant aphid populations, possibly reflecting differences in the genetic background and pyrethroid selection pressures as the number of constitutively overexpressed detoxifying enzyme genes was correlated with the level of resistance. Our findings will strengthen the understanding of the pyrethroid resistance mechanisms in A. glycines.

Investigating the Movement Components of Host Preference in a Highly Mobile Insect Herbivore, Nephotettix cincticeps (Hemiptera: Cicadellidae).

Effective insect management strategies require a firm understanding of the factors determining host preference, particularly in highly mobile insect herbivores. Host preference studies commonly employ average or first position as a proxy for preference. Yet few studies have explored host preference in relation to transitory attraction and leaving rates, yet these are both components of host plant selection. We investigated the transitory dynamics of preference by the green rice leafhopper, Nephotettix cincticeps (Uhler) (Hemiptera: Cicadellidae) by conducting experiments on groups of females, males, or mixed-sex leafhoppers, and recording their position over time between low-N and normal-N rice plants. Utilizing a log-linear model and variants of a biostatistical model we used these positional data to extract attraction, leaving and tenure rates to better understand the process of host-plant selection. We found a general preference for normal-N over low-N plants at equilibrium. However, between sexes there was variation in the relative significance of attraction or leaving rates on that preference. Female leafhoppers were more attracted to host plants with higher nitrogen content. Male leafhoppers were less discriminate in their initial attraction to hosts but left low-N hosts at a faster rate. Whereas estimated tenure times on both normal- and low-N plants exceeded transmission times for the leafhopper-transmitted rice dwarf virus, longer tenure on normal-N plants likely increases the likelihood of virus acquisition from these plants. Our findings support previous recommendations that growers can mitigate the risks of leafhopper damage and pathogen transmission by optimizing their application of nitrogenous fertilizers.

Transfer of Cry1F from Bt maize to eggs of resistant Spodoptera frugiperda.

The intergenerational transfer of plant defense compounds by aposematic insects is well documented, and since 2006, has been shown for Cry toxins. Cry toxins are proteins naturally produced by the soil bacterium Bacillus thuringiensis (Bt) and its genes have been expressed in plants to confer insect pest resistance. In this work we tested if non-aposematic larvae of a major maize pest, Spodoptera frugiperda, with resistance to Cry1F, could transfer Cry1F from a genetically engineered maize variety to their offspring. Resistant 10-day-old larvae that fed on Cry1F Bt maize until pupation were sexed and pair-mated to produce eggs. Using ELISA we found that Cry1F was transferred to offspring (1.47-4.42 ng Cry1F/10 eggs), a toxin concentration about 28-83 times less than that detected in Cry1F Bt maize leaves. This occurred when only one or both sexes were exposed, and more was transferred when both parents were exposed, with transitory detection in the first five egg masses. This work is an unprecedented demonstration that a non-aposematic, but resistant, species can transfer Cry1F to their offspring when exposed to Bt host plant leaves as immatures.

Landscape Effects on Solenopsis invicta (Hymenoptera: Formicidae) and Geocoris spp. (Hemiptera: Geocoridae), Two Important Omnivorous Arthropod Taxa in Field Crops.

The economically important brown stink bug, Euschistus servus (Say) (Hemiptera: Pentatomidae), is a native pest of many crops in southeastern United States and insecticide applications are the prevailing method of population suppression. To elucidate biological control of E. servus populations, we investigated two egg predators' (red imported fire ants, Solenopsis invicta Buren (Hymenoptera: Formicidae), and Geocoris spp. (Hemiptera: Geocoridae)) responses to both local and landscape factors that may have influenced their combined ability to cause mortality in immature E. servus. We estimated the density of fire ants and Geocoris spp. on four major crop hosts-maize, peanut, cotton, and soybean-in 16 landscapes over 3 yr in the coastal plain of Georgia, USA. Both Geocoris spp. and fire ant populations were concentrated on specific crops in this study, maize and soybean for Geocoris spp. and peanut and cotton for fire ants, but the percentage area of specific crops and woodland and pasture in the landscape and year also influenced their density in focal fields. The crop specific density of both taxa, the influence of the percentage area of specific crops and woodland in the landscape, and the variability in density over years may have been related to variable alternative resources for these omnivores in the habitats. Despite the variability over years, differential habitat use of fire ants and Georcoris spp. may have contributed to their combined ability to cause E. servus immature mortality.

Landscape Effects on Reproduction of Euschistus servus (Hemiptera: Pentatomidae), a Mobile, Polyphagous, Multivoltine Arthropod Herbivore.

Landscape factors can significantly influence arthropod populations. The economically important brown stink bug, Euschistus servus (Say) (Hemiptera: Pentatomidae), is a native mobile, polyphagous and multivoltine pest of many crops in southeastern United States and understanding the relative influence of local and landscape factors on their reproduction may facilitate population management. Finite rate of population increase (λ) was estimated in four major crop hosts-maize, peanut, cotton, and soybean-over 3 yr in 16 landscapes of southern Georgia. A geographic information system (GIS) was used to characterize the surrounding landscape structure. LASSO regression was used to identify the subset of local and landscape characteristics and predator densities that account for variation in λ. The percentage area of maize, peanut and woodland and pasture in the landscape and the connectivity of cropland had no influence on E. servus λ. The best model for explaining variation in λ included only four predictor variables: whether or not the sampled field was a soybean field, mean natural enemy density in the field, percentage area of cotton in the landscape and the percentage area of soybean in the landscape. Soybean was the single most important variable for determining E. servus λ, with much greater reproduction in soybean fields than in other crop species. Penalized regression and post-selection inference provide conservative estimates of the landscape-scale determinants of E. servus reproduction and indicate that a relatively simple set of in-field and landscape variables influences reproduction in this species.

First detection of a Sesamia nonagrioides resistance allele to Bt maize in Europe.

The Ebro Valley (Spain) is the only hotspot area in Europe where resistance evolution of target pests to Cry1Ab protein is most likely, owing to the high and regular adoption of Bt maize (>60%). The high-dose/refuge (HDR) strategy was implemented to delay resistance evolution, and to be effective it requires the frequency of resistance alleles to be very low (<0.001). An F2 screen was performed in 2016 to estimate the frequency of resistance alleles in Sesamia nonagrioides from this area and to evaluate if the HDR strategy is still working effectively. Out of the 137 isofemale lines screened on Cry1Ab maize leaf tissue, molted larvae and extensive feeding were observed for two consecutive generations in one line, indicating this line carried a resistance allele. The frequency of resistance alleles in 2016 was 0.0036 (CI 95% 0.0004-0.0100), higher but not statistically different from the value obtained in 2004-2005. Resistance does not seem to be evolving faster than predicted by a S. nonagrioides resistance evolution model, but the frequency of resistance is now triple the value recommended for an effective implementation of the HDR strategy. Owing to this, complementary measures should be considered to further delay resistance evolution in the Ebro Valley.

Co-occurrence Among Intraguild Predators: Avoidance or Aggregation?

Intraguild predation (IGP) may interact with prey availability to structure predator communities. However, if predators are able to avoid each other, its effect on predator community structure may be minimized or absent. To determine whether co-occurrence among IG predators is limited, we estimated co-occurrence among predators in experimental plots of maize and soybean. These crops provide high densities of shared resources (aphids) as well as known IG predators (primarily coccinellids). Despite documented intraspecific and interspecific avoidance behaviors, aggregation to patchy resources could bring IG predators into contact. We, therefore, hypothesized that despite documented intraspecific avoidance behaviors, aphidophagous IG coccinellid predators would not significantly avoid co-occurrence, making IGP likely. Co-occurrence was estimated from visual counts of aphid predators and their prey on randomly selected plants over the course of the growing season. For each habitat, we used maximum likelihood analysis to determine whether observed co-occurrence deviated significantly from that expected for each possible pairwise combination of IG predators. We repeated this analysis using published data on co-occurrence among aphid predators on tansy. We found that most co-occurrence among IG predators was random, suggesting that avoidance does not limit co-occurrence. Failure to limit co-occurrence could be the result of ineffective avoidance mechanisms or because predators balance aggregating on shared resources with avoiding IG predators.

Optimal management strategy of insecticide resistance under various insect life histories: Heterogeneous timing of selection and interpatch dispersal.

Although theoretical studies have shown that the mixture strategy, which uses multiple toxins simultaneously, can effectively delay the evolution of insecticide resistance, whether it is the optimal management strategy under different insect life histories and insecticide types remains unknown. To test the robustness of this management strategy over different life histories, we developed a series of simulation models that cover almost all the diploid insect types and have the same basic structure describing pest population dynamics and resistance evolution with discrete time steps. For each of two insecticidal toxins, independent one-locus two-allele autosomal inheritance of resistance was assumed. The simulations demonstrated the optimality of the mixture strategy either when insecticide efficacy was incomplete or when some part of the population disperses between patches before mating. The rotation strategy, which uses one insecticide on one pest generation and a different one on the next, did not differ from sequential usage in the time to resistance, except when dominance was low. It was the optimal strategy when insecticide efficacy was high and premating selection and dispersal occur.

Effect of male mating history and age on remating by female European corn borer.

If mating with an inferior male has high fitness costs, females may try to avoid mating with these males. Alternatively, females may accept an inferior male to ensure they have obtained at least one mate, and/or to avoid the costs of resisting these males. We hypothesized that females compensate for mating with an inferior male by remating. We tested this hypothesis by measuring remating propensity in females that had mated with an old, multiply-mated male, a 9-day-old virgin male, or a young, virgin male. Females were more likely to remate when they had mated with multiply-mated males than when they had mated with a 9-day-old or young virgin male. We discuss the observed mating behavior by females in terms of sexual selection for multiple mating.

Is a larger refuge always better? Dispersal and dose in pesticide resistance evolution.

The evolution of resistance against pesticides is an important problem of modern agriculture. The high-dose/refuge strategy, which divides the landscape into treated and nontreated (refuge) patches, has proven effective at delaying resistance evolution. However, theoretical understanding is still incomplete, especially for combinations of limited dispersal and partially recessive resistance. We reformulate a two-patch model based on the Comins model and derive a simple quadratic approximation to analyze the effects of limited dispersal, refuge size, and dominance for high efficacy treatments on the rate of evolution. When a small but substantial number of heterozygotes can survive in the treated patch, a larger refuge always reduces the rate of resistance evolution. However, when dominance is small enough, the evolutionary dynamics in the refuge population, which is indirectly driven by migrants from the treated patch, mainly describes the resistance evolution in the landscape. In this case, for small refuges, increasing the refuge size will increase the rate of resistance evolution. Our analysis distils major driving forces from the model, and can provide a framework for understanding directional selection in source-sink environments.

Spatio-Temporal Variation in Landscape Composition May Speed Resistance Evolution of Pests to Bt Crops.

Transgenic crops that express insecticide genes from Bacillus thuringiensis (Bt) are used worldwide against moth and beetle pests. Because these engineered plants can kill over 95% of susceptible larvae, they can rapidly select for resistance. Here, we use a model for a pyramid two-toxin Bt crop to explore the consequences of spatio-temporal variation in the area of Bt crop and non-Bt refuge habitat. We show that variability over time in the proportion of suitable non-Bt breeding habitat, Q, or in the total area of Bt and suitable non-Bt habitat, K, can increase the overall rate of resistance evolution by causing short-term surges of intense selection. These surges can be exacerbated when temporal variation in Q and/or K cause high larval densities in refuges that increase density-dependent mortality; this will give resistant larvae in Bt fields a relative advantage over susceptible larvae that largely depend on refuges. We address the effects of spatio-temporal variation in a management setting for two bollworm pests of cotton, Helicoverpa armigera and H. punctigera, and field data on landscape crop distributions from Australia. Even a small proportion of Bt fields available to egg-laying females when refuges are sparse may result in high exposure to Bt for just a single generation per year and cause a surge in selection. Therefore, rapid resistance evolution can occur when Bt crops are rare rather than common in the landscape. These results highlight the need to understand spatio-temporal fluctuations in the landscape composition of Bt crops and non-Bt habitats in order to design effective resistance management strategies.

Uncovering Trophic Interactions in Arthropod Predators through DNA Shotgun-Sequencing of Gut Contents.

Characterizing trophic networks is fundamental to many questions in ecology, but this typically requires painstaking efforts, especially to identify the diet of small generalist predators. Several attempts have been devoted to develop suitable molecular tools to determine predatory trophic interactions through gut content analysis, and the challenge has been to achieve simultaneously high taxonomic breadth and resolution. General and practical methods are still needed, preferably independent of PCR amplification of barcodes, to recover a broader range of interactions. Here we applied shotgun-sequencing of the DNA from arthropod predator gut contents, extracted from four common coccinellid and dermapteran predators co-occurring in an agroecosystem in Brazil. By matching unassembled reads against six DNA reference databases obtained from public databases and newly assembled mitogenomes, and filtering for high overlap length and identity, we identified prey and other foreign DNA in the predator guts. Good taxonomic breadth and resolution was achieved (93% of prey identified to species or genus), but with low recovery of matching reads. Two to nine trophic interactions were found for these predators, some of which were only inferred by the presence of parasitoids and components of the microbiome known to be associated with aphid prey. Intraguild predation was also found, including among closely related ladybird species. Uncertainty arises from the lack of comprehensive reference databases and reliance on low numbers of matching reads accentuating the risk of false positives. We discuss caveats and some future prospects that could improve the use of direct DNA shotgun-sequencing to characterize arthropod trophic networks.

Artificial tritrophic exposure system for environmental risk analysis on aphidophagous predators.

We evaluated an artificial tritrophic exposure system for use in ecotoxicological evaluations of environmental stressors on aphidophagous predators. It consists of an acrylic tube with a Parafilm M sachet containing liquid aphid diet, into which can be added environmental stressors. Immature Cycloneda sanguinea, Harmonia axyridis and Chrysoperla externa, and adult H. axyridis were reared on Myzus persicae. Larval and pupal development and survival of all species and reproductive parameters of H. axyridis were similar to published results. The system provides a suitable tritrophic exposure route, enables ex-ante evaluation of stressors, and improves the accuracy of the assessment.

Competitive release and outbreaks of non-target pests associated with transgenic Bt cotton.

The adoption of transgenic Bt cotton has, in some cases, led to environmental and economic benefits through reduced insecticide use. However, the distribution of these benefits and associated risks among cotton growers and cotton-growing regions has been uneven due in part to outbreaks of non-target or secondary pests, thereby requiring the continued use of synthetic insecticides. In the southeastern USA, Bt cotton adoption has resulted in increased abundance of and damage from stink bug pests, Euschistus servus and Nezara viridula (Heteroptera: Pentatomidae). While the impact of increased stink bug abundance has been well-documented, the causes have remained unclear. We hypothesize that release from competition with Bt-susceptible target pests may drive stink bug outbreaks in Bt cotton. We first examined the evidence for competitive release of stink bugs through meta-analysis of previous studies. We then experimentally tested if herbivory by Bt-susceptible Helicoverpa zea increases stink bug leaving rates and deters oviposition on non-Bt cotton. Consistent with previous studies, we found differences in leaving rates only for E servus, but we found that both species strongly avoided ovipositing on H. zea-damaged plants. Considering all available evidence, competitive release of stink bug populations in Bt cotton likely contributes to outbreaks, though the relative importance of competitive release remains an open question. Ecological risk assessments of Bt crops and other transgenic insecticidal crops would benefit from greater understanding of the ecological mechanisms underlying non-target pest outbreaks and greater attention to indirect ecological effects more broadly.