The contribution of insects to global forest deadwood decomposition.

The amount of carbon stored in deadwood is equivalent to about 8 per cent of the global forest carbon stocks1. The decomposition of deadwood is largely governed by climate2-5 with decomposer groups-such as microorganisms and insects-contributing to variations in the decomposition rates2,6,7. At the global scale, the contribution of insects to the decomposition of deadwood and carbon release remains poorly understood7. Here we present a field experiment of wood decomposition across 55 forest sites and 6 continents. We find that the deadwood decomposition rates increase with temperature, and the strongest temperature effect is found at high precipitation levels. Precipitation affects the decomposition rates negatively at low temperatures and positively at high temperatures. As a net effect-including the direct consumption by insects and indirect effects through interactions with microorganisms-insects accelerate the decomposition in tropical forests (3.9% median mass loss per year). In temperate and boreal forests, we find weak positive and negative effects with a median mass loss of 0.9 per cent and -0.1 per cent per year, respectively. Furthermore, we apply the experimentally derived decomposition function to a global map of deadwood carbon synthesized from empirical and remote-sensing data, obtaining an estimate of 10.9 ± 3.2 petagram of carbon per year released from deadwood globally, with 93 per cent originating from tropical forests. Globally, the net effect of insects may account for 29 per cent of the carbon flux from deadwood, which suggests a functional importance of insects in the decomposition of deadwood and the carbon cycle.

Effects of Eucalyptus wood and leaf litter on saproxylic insects in the southeastern United States.

Although Eucalyptus is widely planted outside its native range for timber and pulp production, the effects of these exotic plantations on biodiversity relative to native semi-natural forests or plantations of native tree species remain incompletely understood. Here, we compare the diversity of saproxylic beetles (Coleoptera) and true bugs (Hemiptera) between non-native Eucalyptus benthamii Maiden and Cambage (Camden white gum) and native Pinus taeda L. (loblolly pine) stands on the upper Coastal Plain of South Carolina, U.S.A. We sampled insects emerging from logs of both species placed in both stand types after 1, 2, 6, and 12 months in the field. Beetle and true bug richness and diversity were both significantly lower from eucalypt than from pine wood. Moreover, the two communities were compositionally distinct. Whereas pine supported many species of host-specific phloeoxylophagous beetles, most species collected from eucalypts were mycophagous or predatory taxa capable of utilizing a wide range of hosts. Species richness did not differ between logs placed in eucalypt vs. pine stands but Shannon's diversity was significantly higher in the eucalypt stands, possibly due to greater sun exposure in the latter. Contrary to a previous study, we found no support for the idea that eucalypt litter reduces the diversity of saproxylic insects. Our findings add to the growing body of evidence that non-native plantations are less favorable to biodiversity than those consisting of native tree species.

The effects of invertebrates on wood decomposition across the world.

Invertebrates and microorganisms are important but climate-dependent agents of wood decomposition globally. In this meta-analysis, we investigated what drives the invertebrate effect on wood decomposition worldwide. Globally, we found wood decomposition rates were on average approximately 40% higher when invertebrates were present compared to when they were excluded. This effect was most pronounced in the tropics, owing mainly to the activities of termites. The invertebrate effect was stronger for woody debris without bark as well as for that of larger diameter, possibly reflecting bark- and diameter-mediated differences in fungal colonisation or activity rates relative to those of invertebrates. Our meta-analysis shows similar overall invertebrate effect sizes on decomposition of woody debris derived from angiosperms and gymnosperms globally. Our results suggest the existence of critical interactions between microorganism colonisation and the invertebrate contribution to wood decomposition. To improve biogeochemical models, a better quantification of invertebrate contributions to wood decomposition is needed.

Evidence of cue synergism in termite corpse response behavior.

Subterranean termites of the genus Reticulitermes are known to build walls and tubes and move considerable amounts of soil into wood but the causes of this behavior remain largely unexplored. In laboratory assays, we tested the hypothesis that Reticulitermes virginicus (Banks) would carry more sand into wooden blocks containing corpses compared to corpse-free controls. We further predicted that the corpses of predatory ants would elicit a stronger response than those of a benign beetle species or nestmates. As hypothesized, significantly more sand was carried into blocks containing corpses and this material was typically used to build partitions separating the dead from the rest of the colony. Contrary to expectations, however, this behavior did not vary among corpse types. We then tested the hypothesis that oleic acid, an unsaturated fatty acid released during arthropod decay and used by ants and other arthropod taxa in corpse recognition, would induce a similar building response in R. virginicus. To additionally determine the role of foreign objects in giving rise to this behavior, the experiment was carried out with and without imitation corpses (i.e., small glass beads). As predicted, oleic acid induced building (a tenfold increase) but only when applied to beads, suggesting strong synergism between tactile and chemical cues. Oleic acid also significantly reduced the amount of wood consumed by R. virginicus and may possess useful repellent properties.

Pine savanna restoration on agricultural landscapes: The path back to native savanna ecosystem services.

Restoration of savanna ecosystems within their historic range is expected to increase provision of ecosystem services to resident human populations. However, the benefits of restoration depend on the degree to which ecosystems and their services can be restored, the rate of restoration of particular services, and tradeoffs in services between restored ecosystems and other common land uses. We use a chronosequence approach to infer multi-decadal changes in ecosystem services under management aimed at restoring fire-dependent pine savannas, including the use of frequent prescribed fire, following abandonment of row-crop agriculture in the southeastern U.S. We compare ecosystem services between restored pine savannas of different ages and reference pine savannas as well as other common land uses (row-crop agriculture, improved pasture, pine plantation, unmanaged forest). Our results suggest that restoring pine savannas results in many improvements to ecosystem services, including increases in plant species richness, perennial grass cover, tree biomass, total ecosystem carbon, soil carbon and C:N, reductions in soil bulk density and predicted erosion and sedimentation, shifts from soil fungal pathogens to fungal symbionts, and changes in soil chemistry toward reference pine savanna conditions. However, the rate of improvement varies widely among services from a few years to decades. Compared to row-crop agriculture and improved pasture, restored savannas have lower erosion, soil bulk density, and soil pathogens and a higher percentage of mycorrhizal fungi and ecosystem carbon storage. Compared to pine plantations and unmanaged forests, restored pine savannas have lower fire-prone fuel loads and higher water yield and bee pollinator abundance. Our results indicate that restoration of pine savanna using frequent fire provides a broad suite of ecosystem services that increase the landscape's overall resilience to climate change. These results are likely relevant to other savannas dominated by perennial vegetation and maintained with frequent fire.

Quantifying wood decomposition by insects and fungi using computed tomography scanning and machine learning.

Wood decomposition is a central process contributing to global carbon and nutrient cycling. Quantifying the role of the major biotic agents of wood decomposition, i.e. insects and fungi, is thus important for a better understanding of this process. Methods to quantify wood decomposition, such as dry mass loss, suffer from several shortcomings, such as destructive sampling or subsampling. We developed and tested a new approach based on computed tomography (CT) scanning and semi-automatic image analysis of logs from a field experiment with manipulated beetle communities. We quantified the volume of beetle tunnels in wood and bark and the relative wood volume showing signs of fungal decay and compared both measures to classic approaches. The volume of beetle tunnels was correlated with dry mass loss and clearly reflected the differences between beetle functional groups. Fungal decay was identified with high accuracy and strongly correlated with ergosterol content. Our data show that this is a powerful approach to quantify wood decomposition by insects and fungi. In contrast to other methods, it is non-destructive, covers entire deadwood objects and provides spatially explicit information opening a wide range of research options. For the development of general models, we urge researchers to publish training data.

Effect of trap type, trap position, time of year, and beetle density on captures of the redbay ambrosia beetle (Coleoptera: Curculionidae: Scolytinae).

The exotic redbay ambrosia beetle, Xyleborus glabratus Eichhoff (Coleoptera: Curculionidae: Scolytinae), and its fungal symbiont Raffaellea lauricola Harrington, Fraedrich, and Aghayeva are responsible for widespread redbay, Persea borbonia (L.) Spreng., mortality in the southern United States. Effective traps and lures are needed to monitor spread of the beetle and for early detection at ports-of-entry, so we conducted a series of experiments to find the best trap design, color, lure, and trap position for detection of X. glabratus. The best trap and lure combination was then tested at seven sites varying in beetle abundance and at one site throughout the year to see how season and beetle population affected performance. Manuka oil proved to be the most effective lure tested, particularly when considering cost and availability. Traps baited with manuka oil lures releasing 5 mg/d caught as many beetles as those baited with lures releasing 200 mg/d. Distributing manuka oil lures from the top to the bottom of eight-unit funnel traps resulted in similar numbers of X. glabratus as a single lure in the middle. Trap color had little effect on captures in sticky traps or cross-vane traps. Funnel traps caught twice as many beetles as cross-vane traps and three times as many as sticky traps but mean catch per trap was not significantly different. When comparing height, traps 1.5 m above the ground captured 85% of the beetles collected but a few were caught at each height up to 15 m. Funnel trap captures exhibited a strong linear relationship (r2 = 0.79) with X. glabratus attack density and they performed well throughout the year. Catching beetles at low densities is important to port of entry monitoring programs where early detection of infestations is essential. Our trials show that multiple funnel traps baited with a single manuka oil lure were effective for capturing X. glabratus even when no infested trees were visible in the area.

Role of emerald ash borer (Coleoptera: Buprestidae) larval vibrations in host-quality assessment by Tetrastichus planipennisi (Hymenoptera: Eulophidae).

The biological control agent Tetrastichus planipennisi Yang (Hymenoptera: Eulophidae) is a gregarious larval endoparasitoid of the emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), an invasive cambium-feeding species responsible for recent, widespread mortality of ash (Fraxinus spp.) in North America. T. planipennisi is known to prefer late-instar emerald ash borer, but the cues used to assess host size by this species and most other parasitoids of concealed hosts remain unknown. We sought to test whether vibrations produced by feeding emerald ash borer vary with larval size and whether there are any correlations between these cues and T. planipennisi progeny number (i.e., brood size) and sex ratio. The amplitudes and rates of 3-30-ms vibrational impulses produced by emerald ash borer larvae of various sizes were measured in the laboratory before presenting the larvae to T. planipennisi. Impulse-rate did not vary with emerald ash borer size, but vibration amplitude was significantly higher for large larvae than for small larvae. T. planipennisi produced a significantly higher proportion of female offspring from large hosts than small hosts and was shown in previous work to produce more offspring overall from large hosts. There were no significant correlations, however, between the T. planipennisi progeny data and the emerald ash borer sound data. Because vibration amplitude varied significantly with host size, however, we are unable to entirely reject the hypothesis that T. planipennisi and possibly other parasitoids of concealed hosts use vibrational cues to assess host quality, particularly given the low explanatory potential of other external cues. Internal chemical cues also may be important.

Field-cage methodology for evaluating climatic suitability for introduced wood-borer parasitoids: preliminary results from the emerald ash borer system.

Field-cage methods were developed to evaluate the abilities of Tetrastichus planipennisi Yang (Hymenoptera: Eulophidae) and Spathius agrili Yang (Hymenoptera: Braconidae), biocontrol agents of Agrilus planipennis Fairmaire (Coleoptera: Buprestidae), to parasitize, develop and overwinter following three late-season releases at both a northern (Michigan) and a southern (Maryland) location within the current North American range of A. planipennis. In August, September and October of 2009, five young green ash trees were selected at each location. Tetrastichus planipennisi and S. agrili were each randomly assigned to one of two cages attached to each tree, surrounding separate sections of trunk in which late-instar A. planipennis had been inserted. The following April, the caged trunk sections were dissected to determine the fate of each A. planipennis larva and the developmental stages of all recovered parasitoid progeny. At both locations, T. planipennisi and S. agrili were able to parasitize hosts and successfully overwinter (i.e., reach adulthood the following spring). For T. planipennisi, successful parasitism (i.e., parasitoid progeny reached adulthood) occurred for all caged releases in Maryland, but only for the August and September releases in Michigan. At both locations, percent parasitism by T. planipennisi was higher in August and September than in October. For S. agrili, successful parasitism occurred for all caged releases in Maryland, but only for the August release in Michigan. In Maryland, percent parasitism by S. agrili in August and September was higher than in October. The caging method described here should be useful in determining the climatic suitability of other regions before proceeding with large-scale releases of either species and may have utility in other wood-borer parasitoid systems as well.

The importance of termites and fire to dead wood consumption in the longleaf pine ecosystem.

Microbes, insects, and fire are the primary drivers of wood loss from most ecosystems, but interactions among these factors remain poorly understood. In this study, we tested the hypothesis that termites and fire have a synergistic effect on wood loss from the fire-adapted longleaf pine (Pinus palustris Mill.) ecosystem in the southeastern United States. We predicted that the extensive galleries created by termites would promote the ignition and consumption of logs by fire. We exposed logs from which termites had or had not been excluded to prescribed fire after 2.5 years in the field. We found little support for our hypothesis as there was no significant interactive effect of termites and fire on wood mass loss. Moreover, there was no significant difference in mass loss between burned and unburned logs. Termites were responsible for about 13.3% of observed mass loss in unprotected logs, a significant effect, while microbial activity accounted for most of the remaining mass loss. We conclude that fire has little effect on wood loss from the longleaf pine ecosystem and that termite activity does not strongly promote wood combustion. However, longer term research involving multiple burn cycles, later stages of decay, and differing fire intensities will be needed to fully address this question.

Suitability and accessibility of immature Agrilus planipennis (Coleoptera: Buprestidae) stages to Tetrastichus planipennisi (Hymenoptera: Eulophidae).

Tetrastichus planipennisi Yang (Hymenoptera: Eulophidae), a gregarious larval endo-parasitoid, is one of three biocontrol agents from Asia currently being released in the United States to combat the invasive emerald ash borer, Agrilus planipennis Fairmaire (Coleoptera: Buprestidae). The current protocol for rearing T. planipennisi involves presenting the wasps with artificially infested ash sticks made by placing field-collected larvae into shallow grooves beneath flaps of bark. Although third and fourth instars are readily accepted by T. planipennisi in these exposures, the suitability of younger or older developmental stages, which are often more readily available in the field, has not been tested. In this study, we used both artificially infested ash sticks and naturally infested ash logs to test which emerald ash borer developmental stages (second to fourth instars, J larvae [preprepupae], prepupae, and pupae) are most suitable for rearing T. planipennisi. T. planipennisi parasitized all stages except for pupae, but parasitized fewer J larvae and prepupae in naturally infested logs than in artificially infested ash sticks. This is probably because, in naturally infested ash logs, these stages were confined to pupal chambers excavated in the sapwood and may have been largely beyond the reach of ovipositing T. planipennisi. The number of T. planipennisi progeny produced was positively correlated (logarithmic) with host weight, but this relationship was stronger when J larvae and prepupae were excluded from the data set. Fourth instars yielded the most parasitoid progeny, followed by, in approximately equal numbers, J larvae, prepupae, and third instars. Second instars yielded too few parasitoid progeny to benefit rearing efforts.

Effects of prescribed fire and social insects on saproxylic beetles in a subtropical forest.

We tested the immediate and delayed effects of a low-intensity prescribed fire on beetles, ants and termites inhabiting log sections cut from moderately decomposed pine trees in the southeastern United States. We also explored co-occurrence patterns among these insects. Half the logs were placed at a site scheduled for a prescribed fire while the rest were assigned to a neighboring site not scheduled to be burned. We then collected insects emerging from sets of logs collected immediately after the fire as well as after 2, 6, 26 and 52 weeks. The fire had little effect on the number of beetles and ants collected although beetle richness was significantly higher in burned logs two weeks after the fire. Both beetle and ant communities differed between treatments, however, with some species preferring either burned or unburned logs. We found no evidence that subterranean termites (Reticulitermes) were influenced by the fire. Based on co-occurrence analysis, positive associations among insect species were over two times more common than negative associations. This difference was significant overall as well for ant × beetle and beetle × beetle associations. Relatively few significant positive or negative associations were detected between termites and the other insect taxa, however.

A comparison of bee communities between primary and mature secondary forests in the longleaf pine ecosystem.

Much of the once-dominant longleaf pine (Pinus palustris Mill.) ecosystem has been lost from the Coastal Plain of the southeastern United States and only a few scattered remnants of primary forest remain. Despite much interest in understanding and restoring this ecosystem, relatively few studies have attempted to characterize or assess the conservation status of the longleaf bee fauna. The objective of this study was to compare the diversity and composition of bee communities between primary and mature secondary (>100 years old) fire-maintained forests in Georgia and Florida. We used colored pan traps to sample bees at three primary and four secondary locations divided between two regions characterized by sandy (Eglin Air Force Base) or clayey (Red Hills) soils. There were no overall differences between primary and secondary forests in bee richness, diversity, evenness or abundance. Community composition differed among locations but we found no evidence that primary remnants provide critical habitat to sensitive bee species.

Responses of arthropods to large-scale manipulations of dead wood in loblolly pine stands of the southeastern United States.

Large-scale experimental manipulations of dead wood are needed to better understand its importance to animal communities in managed forests. In this experiment, we compared the abundance, species richness, diversity, and composition of arthropods in 9.3-ha plots in which either (1) all coarse woody debris was removed, (2) a large number of logs were added, (3) a large number of snags were added, or (4) no coarse woody debris was added or removed. The target taxa were ground-dwelling arthropods, sampled by pitfall traps, and saproxylic beetles (i.e., dependent on dead wood), sampled by flight intercept traps and emergence traps. There were no differences in total ground-dwelling arthropod abundance, richness, diversity, or composition among treatments. Only the results for ground beetles (Carabidae), which were more species rich and diverse in log input plots, supported our prediction that ground-dwelling arthropods would benefit from additions of dead wood. There were also no differences in saproxylic beetle abundance, richness, diversity, or composition among treatments. The findings from this study are encouraging in that arthropods seem less sensitive than expected to manipulations of dead wood in managed pine forests of the southeastern United States. Based on our results, we cannot recommend inputting large amounts of dead wood for conservation purposes, given the expense of such measures. However, the persistence of saproxylic beetles requires that an adequate amount of dead wood is available in the landscape, and we recommend that dead wood be retained whenever possible in managed pine forests.

Wood decomposition as influenced by invertebrates.

The diversity and habitat requirements of invertebrates associated with dead wood have been the subjects of hundreds of studies in recent years but we still know very little about the ecological or economic importance of these organisms. The purpose of this review is to examine whether, how and to what extent invertebrates affect wood decomposition in terrestrial ecosystems. Three broad conclusions can be reached from the available literature. First, wood decomposition is largely driven by microbial activity but invertebrates also play a significant role in both temperate and tropical environments. Primary mechanisms include enzymatic digestion (involving both endogenous enzymes and those produced by endo- and ectosymbionts), substrate alteration (tunnelling and fragmentation), biotic interactions and nitrogen fertilization (i.e. promoting nitrogen fixation by endosymbiotic and free-living bacteria). Second, the effects of individual invertebrate taxa or functional groups can be accelerative or inhibitory but the cumulative effect of the entire community is generally to accelerate wood decomposition, at least during the early stages of the process (most studies are limited to the first 2-3 years). Although methodological differences and design limitations preclude meta-analysis, studies aimed at quantifying the contributions of invertebrates to wood decomposition commonly attribute 10-20% of wood loss to these organisms. Finally, some taxa appear to be particularly influential with respect to promoting wood decomposition. These include large wood-boring beetles (Coleoptera) and termites (Termitoidae), especially fungus-farming macrotermitines. The presence or absence of these species may be more consequential than species richness and the influence of invertebrates is likely to vary biogeographically.

Abundance of volatile organic compounds in white ash phloem and emerald ash borer larval frass does not attract Tetrastichus planipennisi in a Y-tube olfactometer.

Many natural enemies employ plant- and/or herbivore-derived signals for host/prey location. The larval parasitoid Tetrastichus planipennisi Yang (Hymenoptera: Eulophidae) is 1 of 3 biocontrol agents currently being released in an effort to control the emerald ash borer (EAB), Agrilus planipennis Fairmaire (Coloeptera: Burprestidae) in North America. To enhance its efficiency, allelochemicals that attract it need to be assessed. In this study, ash phloem volatile organic compounds (VOCs) of black, green, and white ash, and EAB larval frass were compared. Foraging behavior of T. planipennisi females in response to VOCs of white ash or frass from EAB larvae feeding on white ash phloem was tested using a Y-tube olfactometer. Results indicated that the 3 ash species had similar VOC profiles. EAB larval frass generally contained greater levels of VOCs than phloem. Factor analysis indicated that the 11 VOCs could be broadly divided into 2 groups, with α-bisabolol, ß-caryophyllene, (E)-2-hexenal, (Z)-3-hexenal, limonene, methyl benzoate, methyl indole-3-acetic acid, methyl jasmonate, methyl salicylate as the first group and the rest (i.e., methyl linoleate and methyl linolenate) as a second. Abundance of VOCs in white ash phloem tissue and frass, nevertheless, did not attract T. planipennisi females. The concealed feeding of EAB larvae might explain the selection for detectable and reliable virbrational signals, instead of undetectable and relatively unreliable VOC cues from phloem and frass, in short-range foraging by T. planipennisi. Alternatively, it is possible that T. planipennisi is not amenable to the Y-tube olfactometer assay employed.

Interacting effects of insects and flooding on wood decomposition.

Saproxylic arthropods are thought to play an important role in wood decomposition but very few efforts have been made to quantify their contributions to the process and the factors controlling their activities are not well understood. In the current study, mesh exclusion bags were used to quantify how arthropods affect loblolly pine (Pinus taeda L.) decomposition rates in both seasonally flooded and unflooded forests over a 31-month period in the southeastern United States. Wood specific gravity (based on initial wood volume) was significantly lower in bolts placed in unflooded forests and for those unprotected from insects. Approximately 20.5% and 13.7% of specific gravity loss after 31 months was attributable to insect activity in flooded and unflooded forests, respectively. Importantly, minimal between-treatment differences in water content and the results from a novel test carried out separately suggest the mesh bags had no significant impact on wood mass loss beyond the exclusion of insects. Subterranean termites (Isoptera: Rhinotermitidae: Reticulitermes spp.) were 5-6 times more active below-ground in unflooded forests compared to flooded forests based on wooden monitoring stakes. They were also slightly more active above-ground in unflooded forests but these differences were not statistically significant. Similarly, seasonal flooding had no detectable effect on above-ground beetle (Coleoptera) richness or abundance. Although seasonal flooding strongly reduced Reticulitermes activity below-ground, it can be concluded from an insignificant interaction between forest type and exclusion treatment that reduced above-ground decomposition rates in seasonally flooded forests were due largely to suppressed microbial activity at those locations. The findings from this study indicate that southeastern U.S. arthropod communities accelerate above-ground wood decomposition significantly and to a similar extent in both flooded and unflooded forests. Seasonal flooding has the potential to substantially reduce the contributions of these organisms to wood decomposition below-ground, however.

Measuring the impact of biotic factors on populations of immature emerald ash borers (Coleoptera: Buprestidae).

Cohorts of emerald ash borer larvae, Agrilus planipennis Fairmaire, were experimentally established in July of 2008 on healthy green ash (Fraxinus pennsylvanica) trees in two wooded plots at each of three sites near Lansing, MI, by caging gravid emerald ash borer females or placing laboratory-reared eggs on trunks (0.5-2 m above the ground) of selected trees. One plot at each site was randomly chosen for release of two introduced larval parasitoids, Tetrastichus planipennisi Yang (Hymenoptera: Eulophidae) and Spathius agrili Yang (Hymenoptera: Braconidae), whereas the other served as the control. Stage-specific mortality factors and rates were measured for all experimentally established cohorts and for associated wild (i.e., naturally occurring) emerald ash borer immature stages via destructive sampling of 2.5 m (above the ground) trunk sections of cohort-bearing trees in the spring and fall of 2009. Host tree defense was the most important mortality factor, causing 32.0 to 41.1% mortality in the experimental cohorts and 17.5 to 21.5% in wild emerald ash borer stages by spring 2009, and 16.1 to 29% for the remaining experimental cohorts, and 9.9 to 11.8% for wild immature emerald ash borer stages by fall 2009. Woodpecker predation was the second most important factor, inflicting no mortality in the experimental cohorts but causing 5.0 to 5.6% mortality to associated wild emerald ash borer stages by spring 2009 and 9.2 to 12.8% and 3.2 to 17.7%, respectively, for experimental cohorts and wild emerald ash borer stages by fall 2009. Mortality from disease in both the experimental and wild cohorts was low (<3%) in both the spring and fall sample periods. In the fall 2009 samples, ≈ 1.5% of experimental cohorts and 0.8% of the wild emerald ash borer stages were parasitized by T. planipennisi. While there were no significant differences in mortality rates because of parasitism between parasitoid-release and control plots, T. planipennisi was detected in each of the three release sites by the end of the study but was not detected in the experimental cohorts or associated wild larvae in any of the three control plots.