Donald J. Baird

Environmental barcoding: A next-generation sequencing approach for biomonitoring applications using river benthos

Timely and accurate biodiversity analysis poses an ongoing challenge for the success of biomonitoring programs. Morphology-based identification of bioindicator taxa is time consuming, and rarely supports species-level resolution especially for immature life stages. Much work has been done in the past decade to develop alternative approaches for biodiversity analysis using DNA sequence-based approaches such as molecular phylogenetics and DNA barcoding. On-going assembly of DNA barcode reference libraries will provide the basis for a DNA-based identification system. The use of recently introduced next-generation sequencing (NGS) approaches in biodiversity science has the potential to further extend the application of DNA information for routine biomonitoring applications to an unprecedented scale. Here we demonstrate the feasibility of using 454 massively parallel pyrosequencing for species-level analysis of freshwater benthic macroinvertebrate taxa commonly used for biomonitoring. We designed our experiments in order to directly compare morphology-based, Sanger sequencing DNA barcoding, and next-generation environmental barcoding approaches. Our results show the ability of 454 pyrosequencing of mini-barcodes to accurately identify all species with more than 1% abundance in the pooled mixture. Although the approach failed to identify 6 rare species in the mixture, the presence of sequences from 9 species that were not represented by individuals in the mixture provides evidence that DNA based analysis may yet provide a valuable approach in finding rare species in bulk environmental samples. We further demonstrate the application of the environmental barcoding approach by comparing benthic macroinvertebrates from an urban region to those obtained from a conservation area. Although considerable effort will be required to robustly optimize NGS tools to identify species from bulk environmental samples, our results indicate the potential of an environmental barcoding approach for biomonitoring programs.

Biomonitoring 2.0: a new paradigm in ecosystem assessment made possible by next-generation DNA sequencing

Biological monitoring has failed to develop from simple binary assessment outcomes of the impacted/unimpacted type, towards more diagnostic frameworks, despite significant scientific effort over the past fifty years. It is our assertion that this is largely because of the limited information content of biological samples processed by traditional morphology‐based taxonomy, which is a slow, imprecise process, focused on restricted groups of organisms. We envision a new paradigm in ecosystem assessment, which we refer to as ‘Biomonitoring 2.0’. This new schema employs DNA‐based identification of taxa, coupled with high‐throughput DNA sequencing on next‐generation sequencing platforms. We discuss the transformational nature of DNA‐based approaches in biodiversity discovery and ecosystem assessment and outline a path forward for their future widespread application.

Influence of genetic and environmental factors on the tolerance of Daphnia magna Straus to essential and non-essential metals.

The ultimate aim of ecotoxicological studies is to predict how natural populations respond to contaminant exposure. Thus, it is crucial to understand how genetic and environmental factors in the field modify responses measured in the laboratory. In the present study the authors determine the genetic and environmental components of variability in acute responses among four Daphnia magna clones exposed to both essential (Zn and Cu) and non-essential (Cd and U) metals in waters with varying water hardness. The authors postulate that genotype acute responses to physiologically non-essential metals may be more variable than responses to essential metals and that this may be explained by hypothesizing that acute responses to non-essential substances are subject to intermittent selection (since the substances may not always be present in biologically significant amounts), whereas responses to essential substances are subject to continuous directional selection (since essential substances by definition co-occur with biota in biologically significant amounts). D. magna clones were exposed to single-metal solutions of varying concentration at two or three levels of water hardness (soft, moderate–hard and hard) for periods ranging from 12–96 h (12 h increments). LC50 values for each metal×genotype×water hardness×exposure period combination were determined for (i) total metal concentration and (ii) the free hydrated metal ion concentration (predicted using geochemical speciation modeling) and compared using analysis of covariance with environment (water hardness) and genotype (clone) as fixed factors and exposure time as a covariate. The results showed that Zn–Cd were consistent, but Cu–U inconsistent, with the essentiality hypothesis. In addition, the small, or even non-existent, genotype-environment interaction effect on the inheritance of metal tolerance supports the argument that genetic variability in resistance to trace metals is inherited through single major genes. Implications of these results for the prediction of population tolerance ranges are discussed in relation to the problem of predicting metal speciation and the biological effects of metals in fresh waters with varying hardness.

Determining the ecotoxicological mode of action of chemicals from measurements made on individuals: results from instar-based tests with Daphnia magna Straus

A short-term Daphnia toxicity test design was used to assess the ecotoxicological mode of action of pollutants. It was demonstrated that by exposing Daphnia females over a single instar to three toxic substances (3,4-dichloroaniline, cadmium and fluoranthene) it was possible to successfully measure both lethal effects on egg and adult stages and nonlethal effects on food acquisition and production rates. Dichloroaniline exposure reduced egg survival during development at concentrations below those which affected adult survival or production rates. For cadmium, however, concentration effects on production were almost an order of magnitude lower than those which affected adult survival, and no reductions in egg survival were observed. Responses to fluoranthene showed a different pattern again, with egg survival during development and production equally affected at concentrations which affected adult survival. Thus the three pollutants chosen showed contrasting dominant ecotoxicological modes of action, in terms of the relative importance of production and mortality effects, and this could be easily assessed using a novel toxicity test design. These results have important implications for risk assessment since with a relatively short, and thus cost-effective test, the functional relationship between effects on lethality and production rates and concentration can be determined.

Fear and loathing in the benthos: Responses of aquatic insect larvae to the pesticide imidacloprid in the presence of chemical signals of predation risk.

The influence of interactions between pesticide exposure and perceived predation risk on the lethal and sub-lethal responses of two aquatic insects was investigated using the pesticide imidacloprid, and a combination of predator-release kairomones from trout and alarm substances from conspecifics. Laboratory experiments examined feeding and respiration rates of the caddisfly Sericostoma vittatum as well as the growth, emergence and respiration rates of the midge Chironomus riparius, exposed to sub-lethal concentrations of imidacloprid. The effects of the two stressors on burrowing behaviour of both species were also assessed. The results show significant effects of environmentally relevant concentrations of imidacloprid on all endpoints studied. Perceived predation risk also elicited sub-lethal effects in C. riparius and S. vittatum, the latter species being less responsive to predation cues. The effects of simultaneous exposure to both types of stressors were assessed using two different approaches: analysis of variance and conceptual models [concentration addition (CA) and independent action (IA)] normally used for the evaluation of contaminant mixture exposure. Both statistical approaches showed no significant interactions on responses in simultaneous exposures in the majority of parameters assessed with only a signification deviation from the reference CA and IA models being found for C. riparius respiration data contrary to the ANOVA results. Exposure to imidacloprid also compromised antipredator behavioural responses of both insect species, with potential negative consequences in terms of mortality from predation in the field. The results obtained demonstrate that natural and anthropogenic stressors can be treated within the same framework providing compatible data for modelling. For an improved interpretation of ecological effects it will be important to expand the mechanistic study of effects of combined exposure to pesticides and perceived predation risk by measuring different endpoints over a wider range of pesticide concentrations.

A new method for ranking mode-specific sensitivity of freshwater arthropods to insecticides and its relationship to biological traits.

The problem of how to deal with species sensitivity differences to toxic substances has been addressed successfully with the species sensitivity distribution (SSD), yet this has not increased understanding about the underlying mechanisms of sensitivity. Other researchers have identified the mode of action of chemicals and also biological traits of species as determinants for sensitivity, yet no systematic approach combines these factors. To achieve this, first existing data on organophosphate, carbamate, and pyrethroid toxicity and mode of action and also species trait information were mined. Second, we linked taxon sensitivity to their traits at the family level to generate empirical and mechanistic hypotheses about sensitivity-trait relationships. In this way, a mode-specific sensitivity (MSS) ranking method was developed, and tested at the taxonomic level of family and genus. The application of several quality criteria indicated overall confidence in rankings, but confidence in exact taxon rank was less certain, due to data insufficiency for certain groups. The MSS rankings were found to be applicable for trait-based approaches and were successfully linked to existing trait data to identify traits with predictive potential. Although this empirical analysis cannot test causality relationships between traits and sensitivity, testable hypotheses were generated, for further experimental investigation. Single traits as well as combinations of traits can be used to predict laboratory sensitivity to the substances tested, although associations were not as strong as in previous studies. We conclude that existing trait data are not suitable for every trait-based research question and that important traits remain to be identified and quantified in relation to the processes of toxicity, i.e., the toxicokinetics and toxicodynamics.

Avoidance of copper contamination by field populations of Daphnia longispina

Almost all terrestrial and aquatic assays that are accepted as standardized, or that have been proposed, involve the exposure of confined organisms to toxicants. If avoidance (sensu evasion, displacement) of contaminants occurs in real situations, then bioassays involving forced exposure severely underestimate pernicious effects of contamination. Two main objectives were achieved in this study: To verify the occurrence of avoidance of copper by cladocerans; to determine the association between avoidance and other toxicity endpoints (lethality and feeding depression), and therefore, to ascertain if fully acclimated individuals from a reference field population exhibited a genetically determined avoidance markedly different from those belonging to a historically metal-stressed population. Twelve cloned lineages of the cladoceran Daphnia longispina, collected from two field populations, were selected according to their lethal sensitivity to copper and acclimated to controlled conditions for more than 30 generations. A 1.1-m test chamber with five compartments was built, allowing the establishment of a dissolved toxicant gradient and the free movement of individuals. In the absence of any toxicant, juveniles from each cloned lineage distributed themselves randomly along the test chamber and furthermore, no significant differences were observed between the two replicates, attesting the repeatability of this novel assay. All lineages showed significant avoidance to copper when exposed to a gradient from 3 to 87 microg/L. The most sensitive lineages to lethal levels of copper began avoiding this metal earlier than resistant ones. An intense association was observed between other endpoints and avoidance; furthermore, avoidance was determined to be much more sensitive than lethality. Therefore, avoidance assays should be recommended as a complementary tool in ecological risk assessments and effluent biomonitoring because such assays can provide cost-effective and ecologically relevant information.

In situ-based effects measures: Determining the ecological relevance of measured responses

Abstract The aim of this review is to examine how the choice of test species and study design employed in the use of in situ approaches in ecological risk assessment can maximize the ecological relevance of data. We provide a framework to define and assess ecological relevance that permits study designs to remain focused on the ecological question being addressed. This framework makes explicit the linkages between effects at lower levels of biological organization and higher-order ecological effects at the population, community, and ecosystem levels. The usefulness of this framework is illustrated by reference to specific examples from aquatic ecotoxicology. The use of models as both interpretive and predictive tools is discussed, with suggestions of appropriate methods for different protection goals.

Behavioural versus physiological mediation of life history under predation risk

Predator-generated variation in prey energy intake remains the dominant explanation of adaptive response to predation risk in prey life history, morphology and physiology across a wide range of taxa. This “behavioural hypothesis” suggest that chemical or visual signals of predation risk reduce prey energy intake leading to a life history characterized by a small size and late age at maturity. However, size-selective predation can induce either smaller size–early age or large size–late age life history. The alternative “physiological hypothesis” suggests that size-selective cues decouple the relationship between energy and life history, acting instead directly on development. Here we use a series of experiments in a fish–daphnid predator–prey system to ask whether size-selective predator cues induce a physiological mediation of development, overshadowing behaviourally based changes in food intake. We found fish chemical cues reduce the net energy intake in Daphnia magna, suggesting a behaviourally mediated reduction in energy. Experimental manipulation of food levels show further that reductions in food lead to later but smaller size at maturity. However, in line with the physiological hypothesis, we show that D. magna matures earlier and at a smaller size when exposed to fish predation cues. Furthermore, our data shows that they do this by increasing their development rate (earlier maturity) for a given growth rate, resulting in a smaller size at maturity. Our data, from a classic size-selective predation system, indicate that predator-induced changes in this system are driven by physiological mediation of development rather than behavioural mediation of energy intake.

A DNA Barcode Library for North American Ephemeroptera: Progress and Prospects

DNA barcoding of aquatic macroinvertebrates holds much promise as a tool for taxonomic research and for providing the reliable identifications needed for water quality assessment programs. A prerequisite for identification using barcodes is a reliable reference library. We gathered 4165 sequences from the barcode region of the mitochondrial cytochrome c oxidase subunit I gene representing 264 nominal and 90 provisional species of mayflies (Insecta: Ephemeroptera) from Canada, Mexico, and the United States. No species shared barcode sequences and all can be identified with barcodes with the possible exception of some Caenis. Minimum interspecific distances ranged from 0.3–24.7% (mean: 12.5%), while the average intraspecific divergence was 1.97%. The latter value was inflated by the presence of very high divergences in some taxa. In fact, nearly 20% of the species included two or three haplotype clusters showing greater than 5.0% sequence divergence and some values are as high as 26.7%. Many of the species with high divergences are polyphyletic and likely represent species complexes. Indeed, many of these polyphyletic species have numerous synonyms and individuals in some barcode clusters show morphological attributes characteristic of the synonymized species. In light of our findings, it is imperative that type or topotype specimens be sequenced to correctly associate barcode clusters with morphological species concepts and to determine the status of currently synonymized species.