Variations in terrestrial arthropod DNA metabarcoding methods recovers robust beta diversity but variable richness and site indicators.

Terrestrial arthropod fauna have been suggested as a key indicator of ecological integrity in forest systems. Because phenotypic identification is expert-limited, a shift towards DNA metabarcoding could improve scalability and democratize the use of forest floor arthropods for biomonitoring applications. The objective of this study was to establish the level of field sampling and DNA extraction replication needed for arthropod biodiversity assessments from soil. Processing 15 individually collected soil samples recovered significantly higher median richness (488-614 sequence variants) than pooling the same number of samples (165-191 sequence variants) prior to DNA extraction, and we found no significant richness differences when using 1 or 3 pooled DNA extractions. Beta diversity was robust to changes in methodological regimes. Though our ability to identify taxa to species rank was limited, we were able to use arthropod COI metabarcodes from forest soil to assess richness, distinguish among sites, and recover site indicators based on unnamed exact sequence variants. Our results highlight the need to continue DNA barcoding local taxa during COI metabarcoding studies to help build reference databases. All together, these sampling considerations support the use of soil arthropod COI metabarcoding as a scalable method for biomonitoring.

Author Correction: DNA metabarcoding and morphological macroinvertebrate metrics reveal the same changes in boreal watersheds across an environmental gradient.

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DNA metabarcoding and morphological macroinvertebrate metrics reveal the same changes in boreal watersheds across an environmental gradient.

Cost-effective, ecologically relevant, sensitive, and standardized indicators are requisites of biomonitoring. DNA metabarcoding of macroinvertebrate communities is a potentially transformative biomonitoring technique that can reduce cost and time constraints while providing information-rich, high resolution taxonomic data for the assessment of watershed condition. Here, we assess the utility of DNA metabarcoding to provide aquatic indicator data for evaluation of forested watershed condition across Canadian eastern boreal watersheds, subject to natural variation and low-intensity harvest management. We do this by comparing the similarity of DNA metabarcoding and morphologically derived macroinvertebrate metrics (i.e. richness, % Ephemeroptera, Plecoptera and Trichoptera, % chironomid), and the ability of DNA metabarcoding and morphological metrics to detect key gradients in stream condition linked to forested watershed features. Our results show consistency between methods, where common DNA metabarcoding and morphological macroinvertebrate metrics are positively correlated and indicate the same key gradients in stream condition (i.e. dissolved oxygen, and dissolved organic carbon, total nitrogen and conductivity) linked to watershed size and shifts in forest composition across watersheds. Our study demonstrates the potential usefulness of macroinvertebrate DNA metabarcoding to future application in broad-scale biomonitoring of watershed condition across environmental gradients.

Analytical study of azadirachtin and 3-tigloylazadirachtol residues in foliage and phloem of hardwood tree species by liquid chromatography-electrospray mass spectrometry.

A rapid and sensitive LC-ESI-MS method has been developed and validated for the quantitation of azadirachtin and 3-tigloylazadirachtol in deciduous tree matrices. The method involves automated extraction and simultaneous cleanup using an accelerated solvent technique with the matrix dispersed in solid phase over a layer of primary-secondary amine silica. The limits of quantification were 0.02 mg/kg for all matrices with the exception of Norway maple foliage (0.05 mg/kg). Validation at three levels (0.02, 0.1, and 1 mg/kg), demonstrated satisfactory recoveries (71-103%) with relative standard deviation <20%. Two in-source fragment ions were used for confirmation at levels above 0.1 mg/kg. Over a period of several months, quality control analyses showed the technique to be robust and effective in tracking the fate of these natural botanical insecticides following systemic injection into various tree species for control of invasive insect pest species such as the emerald ash borer and Asian longhorned beetle.

Foliar residue dynamics of azadirachtins following direct stem injection into white and green ash trees for control of emerald ash borer.

BACKGROUND: Azadirachtins are natural insecticides derived from the neem tree. The emerald ash borer (EAB) is an exotic invasive insect pest that infests various ash tree species and has the potential for significant economic, aesthetic and ecological impacts throughout North America. The initial translocation and foliar residue dynamics of azadirachtins were examined following direct injection into white and green ash trees growing in urban scenarios as a potential control for EAB. RESULTS: Substantial concentrations of azadirachtins A and B [mean maxima > 0.98 mg kg(-1) fresh weight (f.w.)] were observed within 2 days of injecting a specifically designed formulation of azadirachtins. Foliar residues declined exponentially through time, with half-life estimates ranging from 5.1 to 12.3 days. At the time of leaf senescence, foliar residue levels approximated 0.01 mg kg(-1) f.w., strongly mitigating the potential effects of non-target biota in soil or aquatic compartments. CONCLUSION: The magnitude and duration of exposures observed in this field study were considered to be above the thresholds required for biological effectiveness against both larval and adult life stages of EAB. Results support the use of azadirachtins as an environmentally acceptable systemic insecticide for control of EAB and protection of high-value ash trees in urban environments.

Environmental safety to decomposer invertebrates of azadirachtin (neem) as a systemic insecticide in trees to control emerald ash borer.

The non-target effects of an azadirachtin-based systemic insecticide used for control of wood-boring insect pests in trees were assessed on litter-dwelling earthworms, leaf-shredding aquatic insects, and microbial communities in terrestrial and aquatic microcosms. The insecticide was injected into the trunks of ash trees at a rate of 0.2 gazadirachtin cm(-1) tree diameter in early summer. At the time of senescence, foliar concentrations in most (65%) leaves where at or below detection (<0.01 mg kg(-1) total azadirachtin) and the average concentration among leaves overall at senescence was 0.19 mg kg(-1). Leaves from the azadirachtin-treated trees at senescence were added to microcosms and responses by test organisms were compared to those in microcosms containing leaves from non-treated ash trees (controls). No significant reductions were detected among earthworm survival, leaf consumption rates, growth rates, or cocoon production, aquatic insect survival and leaf consumption rates, and among terrestrial and aquatic microbial decomposition of leaf material in comparison to controls. In a further set of microcosm tests containing leaves from intentional high-dose trees, the only significant, adverse effect detected was a reduction in microbial decomposition of leaf material, and only at the highest test concentration (∼6 mg kg(-1)). Results indicated no significant adverse effects on litter-dwelling earthworms or leaf-shredding aquatic insects at concentrations up to at least 30 × the expected field concentrations at operational rates, and at 6 × expected field concentrations for adverse effects on microbial decomposition. We conclude that when azadirachtin is used as a systemic insecticide in trees for control of insect pests such as the invasive wood-boring beetle, emerald ash borer, resultant foliar concentrations in senescent leaf material are likely to pose little risk of harm to decomposer invertebrates.

Are leaves that fall from imidacloprid-treated maple trees to control Asian longhorned beetles toxic to non-target decomposer organisms?

The systemic insecticide imidacloprid may be applied to deciduous trees for control of the Asian longhorned beetle, an invasive wood-boring insect. Senescent leaves falling from systemically treated trees contain imidacloprid concentrations that could pose a risk to natural decomposer organisms. We examined the effects of foliar imidacloprid concentrations on decomposer organisms by adding leaves from imidacloprid-treated sugar maple trees to aquatic and terrestrial microcosms under controlled laboratory conditions. Imidacloprid in maple leaves at realistic field concentrations (3-11 mg kg(-1)) did not affect survival of aquatic leaf-shredding insects or litter-dwelling earthworms. However, adverse sublethal effects at these concentrations were detected. Feeding rates by aquatic insects and earthworms were reduced, leaf decomposition (mass loss) was decreased, measurable weight losses occurred among earthworms, and aquatic and terrestrial microbial decomposition activity was significantly inhibited. Results of this study suggest that sugar maple trees systemically treated with imidacloprid to control Asian longhorned beetles may yield senescent leaves with residue levels sufficient to reduce natural decomposition processes in aquatic and terrestrial environments through adverse effects on non-target decomposer organisms.

Effects on litter-dwelling earthworms and microbial decomposition of soil-applied imidacloprid for control of wood-boring insects.

BACKGROUND: Imidacloprid is an effective, systemic insecticide for the control of wood-boring insect pests in trees. Systemic applications to trees are often made by soil injections or drenches, and the resulting imidacloprid concentrations in soil or litter may pose a risk of harm to natural decomposer organisms. The authors tested effects of imidacloprid on survival and weight gain or loss of the earthworms Eisenia fetida (Savigny) and Dendrobaena octaedra (Savigny), on leaf consumption rates and cocoon production by D. octaedra and on microbial decomposition activity in laboratory microcosms containing natural forest litter. RESULTS: Dendrobaena octaedra was the most sensitive of the two earthworm species, with an LC(50) of 5.7 mg kg(-1), an LC(10) of about 2 mg kg(-1) and significant weight losses among survivors at 3 mg kg(-1). Weight losses resulted from a physiological effect rather than from feeding inhibition. There were no effects on cocoon production among survivors at 3 mg kg(-1). The LC(50) for E. fetida was 25 mg kg(-1), with significant weight losses at 14 mg kg(-1). There were no significant effects on microbial decomposition of leaf material at the maximum test concentration of 1400 mg kg(-1). CONCLUSION: The results indicate that, when imidacloprid is applied as a systemic insecticide to the soil around trees, it is likely to cause adverse effects on litter-dwelling earthworms if concentrations in the litter reach or exceed about 3 mg kg(-1).

Non-target effects on aquatic decomposer organisms of imidacloprid as a systemic insecticide to control emerald ash borer in riparian trees.

Imidacloprid is effective against emerald ash borer when applied as a systemic insecticide. Following stem or soil injections to trees in riparian areas, imidacloprid residues could be indirectly introduced to aquatic systems via leaf fall or leaching. Either route of exposure may affect non-target, aquatic decomposer organisms. Leaves from ash trees treated with imidacloprid at two field rates and an intentionally-high concentration were added to aquatic microcosms. Leaves from trees treated at the two field rates contained imidacloprid concentrations of 0.8-1.3 ppm, and did not significantly affect leaf-shredding insect survival, microbial respiration or microbial decomposition rates. Insect feeding rates were significantly inhibited at foliar concentrations of 1.3 ppm but not at 0.8 ppm. Leaves from intentionally high-dose trees contained concentrations of about 80 ppm, and resulted in 89-91% mortality of leaf-shredding insects, but no adverse effects on microbial respiration and decomposition rates. Imidacloprid applied directly to aquatic microcosms to simulate leaching from soils was at least 10 times more toxic to aquatic insects than the foliar concentrations, with high mortality at 0.13 ppm and significant feeding inhibition at 0.012 ppm.

Effect of release herbicide on mortality, avoidance response, and growth of amphibian larvae in two forest wetlands.

Effects of Release herbicide (triclopyr butoxyethyl ester, [TBEE]) on mortality, avoidance response, and growth of larval amphibians (Rana clamitans, Rana pipiens) were investigated using in situ enclosures deployed in two forest wetlands in northern Ontario, Canada. Release was applied at nominal concentrations ranging from 0.26 to 7.68 mg TBEE acid equivalents (AE)/L. No significant deleterious effects of this herbicide on larval growth were detected. However, concentration-dependent mortality and abnormal avoidance response were observed. Most mortality occurred within 96 h following treatment. Median lethal concentration (LC50) values for each species and experimental site ranged from 2.79 to 3.29 mg AE/L, while median effective concentration (EC50) values (abnormal avoidance response) ranged from 1.67 to 3.84 mg AE/L. The LC10 and EC10 endpoints approximated aqueous concentrations (0.59 mg AE/L) expected under direct aerial overspray scenarios, indicating a potential risk of impacts for a small proportion of native amphibian larvae. However, given the low frequency and limited use of this herbicide formulation in Canadian forestry, these risks are considered negligible. Changes in usage patterns would require concurrent chemical and biological monitoring of operational spray programs to accurately quantify the probability and magnitude of real-world exposures and to relate these exposure levels to concentration-response relationships including those described in this study.

Effects of Vision herbicide on mortality, avoidance response, and growth of amphibian larvae in two forest wetlands.

The effects of Vision (glyphosate, 356 mg acid equivalents (a.e.)/L) on mortality, avoidance response, and growth of larval amphibians (Rana clamitans and Rana pipiens) were investigated using in situ enclosures deployed in two forest wetlands of northern Ontario, Canada. In addition to untreated controls, Vision was applied to yield initial concentrations ranging from 0.29 to 14.3 mg a.e./L (0.94-46.1 mg/L of Vision). Resultant 96-h median lethal concentration (LC50) values ranged from 2.70 to 11.5 mg a.e./L (8.71-37.1 mg/L of Vision) depending on the species or site involved. Substantial mortality and incidences of abnormal avoidance response occurred only at concentrations exceeding the expected environmental concentrations (EEC) (1.43 mg a.e./L, or 4.61 mg/L of Vision) as calculated by Canadian regulatory authorities. The concentration dependence of larval growth rate and maximum size varied depending on site and species. Mean growth rates and maximum sizes exposed to 1.43 mg a.e./L (EEC) treatments were the same or greater than controls. Experimental site and biotic/abiotic factors therein, such as pH and suspended sediments, substantially affected the expression of Vision herbicide toxicity in the amphibian larvae tested. Overall, results suggest that the silvicultural use of Vision herbicide in accordance with the product label and standard Canadian environmental regulations should have negligible adverse effects on sensitive larval life stages of native amphibians.

Chemical and biomonitoring to assess potential acute effects of Vision herbicide on native amphibian larvae in forest wetlands.

In conjunction with operational forest herbicide spray programs in Ontario, Canada, chemical and biological monitoring studies were conducted in 51 different wetlands to quantify the probability and magnitude of contamination by a glyphosate herbicide formulation (Vision). Wetlands were classified as oversprayed, adjacent, or buffered in relation to the operational target spray blocks. Results show that vegetated buffers significantly mitigated against exposure and thus potential for acute effects. Aqueous concentrations of glyphosate in buffered wetlands were below analytical limits of quantitation (0.02 mg acid equivalent [a.e.]/L) in 14 of 16 cases, with mean concentration (0.03 +/- 0.02 mg a.e./L) significantly (p < 0.05) less than that of either adjacent (0.18 +/- 0.06 mg a.e./L) or oversprayed wetlands (0.33 +/- 0.11 mg a.e./L). Biomonitoring with caged amphibian larvae showed no significant differences among mean mortality (48 h) of either Rana pipiens (p = 0.194) or Rana clamitans larvae (p = 0.129) exposed in situ to Vision under these various wetland conditions. Percent mortality was not significantly (p = 0.05) correlated with exposure concentrations for either amphibian species tested. Results suggest that exposures typically occurring in forest wetlands are insufficient to induce significant acute mortality in native amphibian larvae.

Fate of spinosad in litter and soils of a white spruce plantation in central Ontario.

Spinosad is a natural insecticide with potential as a novel biorational control agent for spruce budworm (Choristoneura fumiferana [Clem]), the most destructive insect defoliator of spruce and balsam fir in Canada. Concurrent terrestrial fate experiments were conducted under full coniferous canopy and in a natural opening of a mature white spruce (Piecea glauca [Moench]) plantation of central Ontario to examine the fate and persistence of spinosad in the forest floor and underlying soils. Mean initial residues of spinosyn A and D were approximately 0.2 and 0.02 microgram g-1, respectively, in thatch and exposed soils, but were substantially higher, 2.72 and 0.36 micrograms g-1, in litter under coniferous canopy. Results demonstrated that spinosad residues in spruce litter, graminaceous thatch and exposed sandy loam soils dissipated rapidly, following hyperbolic or exponential decline models. Dissipation time (DT50) values ranged from 2.0 to 7.8 days, depending on matrix and experimental conditions. Transient increases in demethylated metabolite residues confirmed that the parent product was degraded in situ. No evidence of vertical mobility of any of the analytes was observed.

Fate of spinosad in litter and soils of a mixed conifer stand in the Acadian forest region of New Brunswick.

Spinosad is a natural insecticide, produced via fermentation culture of the actinomycete Saccharopolyspora spinosa, with potential use against a number of forest pests including spruce budworm (Choristoneura fumiferana [Clem]). Persistence of spinosad was determined in terrestrial fate experiments conducted within a semimature stand of black spruce (Picea mariana [Mill.]) and balsam fir (Abies balsamea [L]) in the Acadian forest region of New Brunswick, Canada. Results of experiments established under full coniferous canopy and in a canopy opening indicated that spinosad dissipated rapidly following hyperbolic kinetics in both litter and soils and was not susceptible to leaching. Time to 50% dissipation estimates for spinosyn A ranged from 2.0 to 12.4 days depending upon matrix and experimental conditions. Spinosyn D dissipated to levels below quantitation limits (0.02 microg/g of dry mass) within 7 days in all cases. Sporadic low-level detection of the demethylated metabolites suggested that parent compounds were degraded in situ.