Donald C. Weber

Detection of predation using qPCR: Effect of prey quantity, elapsed time, chaser diet, and sample preservation on detectable quantity of prey DNA

Abstract Using quantitative PCR that amplified a prey-specific mtDNA 214 bp amplicon from the COI mitochondrial gene of the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), prey eggs of known age and number were fed to larvae of the generalist predator lady beetle Coleomegilla maculata (De Geer) (Coleoptera: Coccinellidae), to elucidate the effects of time and diet since consumption, number of prey eggs, and methods for sample fixation and preservation, on the quantity of target DNA detected. Signal was strongly attenuated directly after cessation of feeding, even when predators were immediately frozen at -20°C. However, the quantity of target detected was significantly related to the number of eggs consumed and the time elapsed since eating. Decrease in detected prey DNA was consistent with a negative exponential model. The target DNA sequence disappeared from starved predators (quantitative half-life estimate of 59 min) more slowly than those fed potato aphids after consuming the target prey eggs (half-life estimate 16 min), whereas those fed C. maculata eggs as a chaser were intermediate in the rate at which they degraded the target prey DNA sequence. Fixative protocols are of critical importance in proper use of the qPCR technique. Among seven methods tested, storing the predator immediately in 70% ethanol prechilled to -20°C yielded the highest amount of target sequence, 22.8% of that recovered directly from a single intact prey egg. Samples frozen without solvent at -80°C and -20°C yielded only 6.0% and 2.3% of the target DNA respectively, and room temperature ethanol and ethylene glycol-based antifreeze averaged below 1% recovery of target DNA. Nevertheless, target prey was detected in more than 80% of antifreeze-stored predators. Predators killed and held at room temperature for 4 h or 5 days yielded no target prey DNA in 18 of 20 cases. These results emphasize both the value and the complexities of application of the qPCR technique to field predation studies.

Conservation tillage issues: Cover crop-based organic rotational no-till grain production in the mid-Atlantic region, USA

Organic producers in the mid-Atlantic region of the USA are interested in reducing tillage, labor and time requirements for grain production. Cover crop-based, organic rotational no-till grain production is one approach to accomplish these goals. This approach is becoming more viable with advancements in a system for planting crops into cover crop residue flattened by a roller–crimper. However, inability to consistently control weeds, particularly perennial weeds, is a major constraint. Cover crop biomass can be increased by manipulating seeding rate, timing of planting and fertility to achieve levels(>8000kgha �1 ) necessary for suppressing summerannual weeds. However, while cover crops are multi-functional tools, when enhancing performance for a given function there are trade-off with other functions. While cover crop management is required for optimal system performance, integration into a crop rotation becomes a critical challenge to the overall success of the production system. Further, high levels of cover crop biomass can constrain crop establishment by reducing optimal seed placement, creating suitable habitat for seed- and seedling-feeding herbivores, and impeding placement of supplemental fertilizers. Multi-institutional and -disciplinary teams have been working in the mid-Atlantic region to address system constraints and management trade-off challenges. Here, we report on past and current research on cover crop-based organic rotational no-till grain production conducted in the mid-Atlantic region.

Discovery of the Aggregation Pheromone of the Brown Marmorated Stink Bug (Halyomorpha halys) through the Creation of Stereoisomeric Libraries of 1‑Bisabolen-3-ols

We describe a novel and straightforward route to all stereoisomers of 1,10-bisaboladien-3-ol and 10,11-epoxy-1-bisabolen-3-ol via the rhodium-catalyzed asymmetric addition of trimethylaluminum to diastereomeric mixtures of cyclohex-2-enones 1 and 2. The detailed stereoisomeric structures of many natural sesquiterpenes with the bisabolane skeleton were previously unknown because of the absence of stereoselective syntheses of individual stereoisomers. Several of the bisabolenols are pheromones of economically important pentatomid bug species. Single-crystal X-ray crystallography of underivatized triol 13 provided unequivocal proof of the relative and absolute configurations. Two of the epoxides, (3S,6S,7R,10S)-10,11-epoxy-1-bisabolen-3-ol (3) and (3R,6S,7R,10S)-10,11-epoxy-1-bisabolen-3-ol (4), were identified as the main components of a male-produced aggregation pheromone of the brown marmorated stink bug, Halyomorpha halys, using GC analyses on enantioselective columns. Both compounds attracted female, male, and nymphal H. halys in field trials. Moreover, mixtures of stereoisomers containing epoxides 3 and 4 were also attractive to H. halys, signifying that the presence of additional stereoisomers did not hinder attraction of H. halys and relatively inexpensive mixtures can be used in monitoring, as well as control strategies. H. halys is a polyphagous invasive species in the U.S. and Europe that causes severe injury to fruit, vegetables, and field crops and is also a serious nuisance pest.

Synergy of Aggregation Pheromone with Methyl (E,E,Z)-2,4,6-Decatrienoate in Attraction of Halyomorpha halys (Hemiptera: Pentatomidae)

ABSTRACT The reported male-produced aggregation pheromone of the brown marmorated stink bug, Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), identified as a mixture of (3S,6S,7R,10S)-10,11-epoxy-1-bisabolen-3-ol and (3R,6S,7R,10S)-10,11-epoxy-1-bisabolen-3-ol, offers new opportunities for its management. We found that black pyramid traps deployed along crop borders in Maryland and West Virginia, containing lures with both stereoisomers of this reported aggregation pheromone combined with methyl (E,E,Z)-2,4,6-decatrienoate (MDT) lures, attracted more adult and nymphal H. halys than either the aggregation pheromone or MDT alone. In season-long totals, combined lures acted synergistically by catching 1.9–3.2 times more number of adults, and 1.4–2.5 times more number of nymphs, than expected from an additive effect of the lures deployed individually. There were no significant differences in patterns of male and female captures. MDT alone was not significantly attractive to adults during most of the growing season, but became increasingly attractive to adults and especially nymphs in autumn. Mixed-isomer lures containing eight stereoisomers of 10,11-epoxy-1-bisabolen-3-ol, including the two active stereoisomers, were as effective at catching adults and nymphs with or without MDT as were lures loaded only with the two active stereoisomers in the natural ratio ((3S,6S,7R,10S)-10,11-epoxy-1-bisabolen-3-ol: (3R,6S,7R,10S)-10,11-epoxy-1-bisabolen-3-ol) of 3.5:1. These results identify a combination of semiochemicals that is attractive season-long for detection, monitoring, and potential control of this polyphagous invasive pest of North America and Europe.

The detectability half‐life in arthropod predator–prey research: what it is, why we need it, how to measure it, and how to use it

Molecular gut‐content analysis enables detection of arthropod predation with minimal disruption of ecosystem processes. Most assays produce only qualitative results, with each predator testing either positive or negative for target prey remains. Nevertheless, they have yielded important insights into community processes. For example, they have confirmed the long‐hypothesized role of generalist predators in retarding early‐season build‐up of pest populations prior to the arrival of more specialized predators and parasitoids and documented the ubiquity of secondary and intraguild predation. However, raw qualitative gut‐content data cannot be used to assess the relative impact of different predator taxa on prey population dynamics: they must first be weighted by the relative detectability periods for molecular prey remains for each predator–prey combination. If this is not carried out, interpretations of predator impact will be biased towards those with the longest detectabilities. We review the challenges in determining detectability half‐lives, including unstated assumptions that have often been ignored in the performance of feeding trials. We also show how detectability half‐lives can be used to properly weight assay data to rank predators by their importance in prey population suppression, and how sets of half‐lives can be used to test hypotheses concerning predator ecology and physiology. We use data from 32 publications, comprising 97 half‐lives, to generate and test hypotheses on taxonomic differences in detectability half‐lives and discuss the possible role of the detectability half‐life in interpreting qPCR and next‐generation sequencing data.

Trapping Phyllophaga spp. (Coleoptera: Scarabaeidae: Melolonthinae) in the United States and Canada using sex attractants.

Abstract The sex pheromone of the scarab beetle, Phyllophaga anxia, is a blend of the methyl esters of two amino acids, L-valine and L-isoleucine. A field trapping study was conducted, deploying different blends of the two compounds at 59 locations in the United States and Canada. More than 57,000 males of 61 Phyllophaga species (Coleoptera: Scarabaeidae: Melolonthinae) were captured and identified. Three major findings included: (1) widespread use of the two compounds [of the 147Phyllophaga (sensu stricto) species found in the United States and Canada, males of nearly 40% were captured]; (2) in most species intraspecific male response to the pheromone blends was stable between years and over geography; and (3) an unusual pheromone polymorphism was described from P. anxia. Populations at some locations were captured with L-valine methyl ester alone, whereas populations at other locations were captured with L-isoleucine methyl ester alone. At additional locations, the L-valine methyl ester-responding populations and the L-isoleucine methyl ester-responding populations were both present, producing a bimodal capture curve. In southeastern Massachusetts and in Rhode Island, in the United States, P. anxia males were captured with blends of L-valine methyl ester and L-isoleucine methyl ester. Resumen La feromona sexual del escarabajo, Phyllophaga anxia, es una mezcla de los ésteres metílicos de dos aminoácidos, L-valina y L-isoleucina. Se condujo un estudio de campo usando diferentes mezclas de los dos componentes en 59 sitios de Estados Unidos y Canada. Más de 57,000 machos de 61 especies dePhyllophaga fueron capturados e identificados. Tres de los resultados más importantes incluyen: (1) el extenso uso de los dos componentes [de las 147 especies de Phyllophaga (sensu stricto), en Estados Unidos y Canada, fueron capturados machos de cerca del 40% de ellas.]; (2) para la mayoría de las especies, la respuesta intraespecífica de los machos a las combinaciones de los dos aminoácidos fue consistente entre años diferentes, y en todos los sitios geográficos; y (3) un inusual polymorfismo de la feromona fue descrito para P. anxia. Poblaciones de algunos sitios fueron atrapados sólo con valina, mientras que poblaciones de otros sitios fueron atrapados sólo con isoleucina. También se encontraron sitios donde las poblaciones responden a ambos componentes, valina e isoleucina, produciendo una curva de captura bimodal. En el sureste del estado de Massachusetts y en Rhode Island, en Estados Unidos, machos de P. anxia fueron atrapados en trampas con mezclas de valina e isoleucina.

A review of the natural enemies of beetles in the subtribe Diabroticina (Coleoptera: Chrysomelidae): implications for sustainable pest management

Abstract Diabroticina is a speciose subtribe of New World Chrysomelidae (Subfamily Galerucinae: Tribe Luperini) that includes pests such as corn rootworms, cucumber beetles and bean leaf beetles (e.g. Diabrotica, Acalymma, Cerotoma species). The evolution and spread of pesticide resistance, the European invasion of Diabrotica v. virgifera LeConte, and possible development of resistance due to the large-scale deployment of Diabrotica-active Bt maize in North America have generated a sense of urgency in developing biological control options against Diabroticina pests. In the present study, we review available knowledge on biological control options, including 290 publications on natural enemy–Diabroticina associations in the New World. Several natural enemy species or groups appear to be promising candidates for control strategies with different ecological rationales. We propose that future research should pursue: (1) development of inundative biological control products, particularly mass-produced entomopathogenic nematodes and fungi, (2) understanding of specific natural enemies of Diabroticina larvae throughout the Americas and of adults particularly in higher altitudes of Central America or northern South America including potential classical biological control agents against D. v. virgifera; (3) enhancement of natural enemies through cultural practices, i.e., reduced tillage, reduced weed control, cover crops, diversified crop rotations or soil amendments. Research and action must be coordinated to accelerate the exploration of biological control options.

Attraction of the invasive halyomorpha halys (Hemiptera: Pentatomidae) to traps baited with semiochemical stimuli across the United States

ABSTRACT A recent identification of the two-component aggregation pheromone of the invasive stink bug species, Halyomorpha halys (Stål), in association with a synergist, has greatly improved the ability to accurately monitor the seasonal abundance and distribution of this destructive pest. We evaluated the attraction of H. halys to black pyramid traps baited with lures containing the pheromone alone, the synergist methyl (2E,4E,6Z)-decatrienoate (MDT) alone, and the two lures in combination. Traps were deployed around areas of agricultural production including fruit orchards, vegetables, ornamentals, or row crops in Delaware, Maryland, North Carolina, New Jersey, New York, Ohio, Oregon, Pennsylvania, Virginia, and West Virginia from mid-April to mid-October, 2012 and 2013. We confirmed that H. halys adults and nymphs are attracted to the aggregation pheromone season long, but that attraction is significantly increased with the addition of the synergist MDT. H. halys adults were detected in April with peak captures of overwintering adults in mid- to late May. The largest adult captures were late in the summer, typically in early September. Nymphal captures began in late May and continued season long. Total captures declined rapidly in autumn and ceased by mid-October. Captures were greatest at locations in the Eastern Inland region, followed by those in the Eastern Coastal Plain and Pacific Northwest. Importantly, regardless of location in the United States, all mobile life stages of H. halys consistently responded to the combination of H. halys aggregation pheromone and the synergist throughout the entire season, suggesting that these stimuli will be useful tools to monitor for H. halys in managed systems.

Changes in digestive rate of a predatory beetle over its larval stage: implications for dietary breadth.

Prey and non-prey foods differ substantially in their suitability for zoophytophagous omnivores, but the relative quality of these foods depends on the stage-specific digestive capabilities of the organism in question. Quantitative (or real-time) PCR was used to amplify food-specific DNA and measure consumption rates and digestion efficiencies of four foods - two prey (Aphis glycines and Leptinotarsa decemlineata eggs) and two non-prey (Zea mays pollen and the yeast Saccharomyces cerevisiae) species - over different larval stages of Coleomegilla maculata. The amount of Z. mays pollen consumed increased as larvae aged, but not proportionately with larval size, such that consumption rates decreased uniformly with insect age. While aging larvae fed A. glycines had a similar pattern in their diminishing consumption rates, they consumed similar amounts of A. glycines regardless of age, suggesting a negative feedback mechanism for consumption of this species of aphids. Older larvae digested three of the four foods significantly more efficiently than younger larvae, the exception being larvae fed A. glycines which was digested at a similar rate throughout the larval stage. There was a significant effect of time on food quantity detected for all four species of food. We conclude that C. maculata expands its physiological capacity for digesting prey and non-prey foods as they age in order to better accommodate the increased nutritional needs of the older larvae. This strategy has important implications for the life history strategies of zoophytophagous insects and how they function within foods webs.

Attack and Success of Native and Exotic Parasitoids on Eggs of Halyomorpha halys in Three Maryland Habitats.

Egg parasitoids of the exotic invasive brown marmorated stink bug, Halyomorpha halys (Stål), were investigated using lab-reared fresh (live) and frozen (killed) lab-reared sentinel egg masses deployed for 72h on foliage in three habitats—woods, orchard, and soybean field—in Maryland, USA, in summer 2014. Four native hymenopteran species, Telenomus podisi Ashmead (Scelionidae), Trissolcus euschisti (Ashmead) and Tr. brochymenae Ashmead (Scelionidae), and Anastatus reduvii (Howard) (Eupelmidae), developed and emerged from H. halys eggs. One exotic parasitoid, Trissolcus japonicus (Ashmead), emerged, providing the first known occurrence of this species in North America. Native parasitoids emerged from frozen eggs significantly more often than from fresh eggs (89.3% of egg masses and 98.1% of individual eggs), whereas the exotic Tr. japonicus did not show a similar difference, strongly suggesting adaptation to H. halys as a host by Tr. japonicus but not by the native species. Parasitoids were habitat-specific: all three Trissolcus species were significantly more likely to occur in the woods habitat, whereas Te. podisi was found exclusively in the soybean field. Further investigations are required to elucidate evolving host-parasitoid relationships, habitat specificity, and non-target effects of Tr. japonicus over the expanded range of H. halys in North America.