Metabarcoding and applied ecology with hyperdiverse organisms: Recommendations for biological control research.

Metabarcoding is revolutionizing fundamental research in ecology by enabling large-scale detection of species and producing data that are rich with community context. However, the benefits of metabarcoding have yet to be fully realized in fields of applied ecology, especially those such as classical biological control (CBC) research that involve hyperdiverse taxa. Here, we discuss some of the opportunities that metabarcoding provides CBC and solutions to the main methodological challenges that have limited the integration of metabarcoding in existing CBC workflows. We focus on insect parasitoids, which are popular and effective biological control agents (BCAs) of invasive species and agricultural pests. Accurately identifying native, invasive and BCA species is paramount, since misidentification can undermine control efforts and lead to large negative socio-economic impacts. Unfortunately, most existing publicly accessible genetic databases cannot be used to reliably identify parasitoid species, thereby limiting the accuracy of metabarcoding in CBC research. To address this issue, we argue for the establishment of authoritative genetic databases that link metabarcoding data to taxonomically identified specimens. We further suggest using multiple genetic markers to reduce primer bias and increase taxonomic resolution. We also provide suggestions for biological control-specific metabarcoding workflows intended to track the long-term effectiveness of introduced BCAs. Finally, we use the example of an invasive pest, Drosophila suzukii, in a reflective "what if" thought experiment to explore the potential power of community metabarcoding in CBC.

Effects of phenological mismatch under warming are modified by community context.

Climate change is altering the relative timing of species interactions by shifting when species first appear in communities and modifying the duration organisms spend in each developmental stage. However, community contexts, such as intraspecific competition and alternative resource species, can prolong shortened windows of availability and may mitigate the effects of phenological shifts on species interactions. Using a combination of laboratory experiments and dynamic simulations, we quantified how the effects of phenological shifts in Drosophila-parasitoid interactions differed with concurrent changes in temperature, intraspecific competition, and the presence of alternative host species. Our study confirmed that warming shortens the window of host susceptibility. However, the presence of alternative host species sustained interaction persistence across a broader range of phenological shifts than pairwise interactions by increasing the degree of temporal overlap with suitable development stages between hosts and parasitoids. Irrespective of phenological shifts, parasitism rates declined under warming due to reduced parasitoid performance, which limited the ability of community context to manage temporally mismatched interactions. These results demonstrate that the ongoing decline in insect diversity may exacerbate the effects of phenological shifts in ecological communities under future global warming temperatures.

A Coordinated Sampling and Identification Methodology for Larval Parasitoids of Spotted-Wing Drosophila.

We provide recommendations for sampling and identification of introduced larval parasitoids of spotted-wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae). These parasitoids are either under consideration for importation (aka classical) biological control introductions, or their adventive (presumed to have been accidentally introduced) populations have recently been discovered in North America and Europe. Within the context of the ecology of D. suzukii and its parasitoids, we discuss advantages and disadvantages of estimating larval parasitism levels using different methods, including naturally collected fruit samples and sentinel baits. For most situations, we recommend repeated sampling of naturally occurring fruit rather than using sentinel baits to monitor seasonal dynamics of host plant-Drosophila-parasitoid associations. We describe how to separate Drosophilidae puparia from host fruit material in order to accurately estimate parasitism levels and establish host-parasitoid associations. We provide instructions for identification of emerging parasitoids and include a key to the common families of parasitoids of D. suzukii. We anticipate that the guidelines for methodology and interpretation of results that we provide here will form the basis for a large, multi-research team sampling effort in the coming years to characterize the biological control and nontarget impacts of accidentally and intentionally introduced larval parasitoids of D. suzukii in several regions of the world.

DROP: Molecular voucher database for identification of Drosophila parasitoids.

Molecular identification is increasingly used to speed up biodiversity surveys and laboratory experiments. However, many groups of organisms cannot be reliably identified using standard databases such as GenBank or BOLD due to lack of sequenced voucher specimens identified by experts. Sometimes a large number of sequences are available, but with too many errors to allow identification. Here, we address this problem for parasitoids of Drosophila by introducing a curated open-access molecular reference database, DROP (Drosophila parasitoids). Identifying Drosophila parasitoids is challenging and poses a major impediment to realize the full potential of this model system in studies ranging from molecular mechanisms to food webs, and in biological control of Drosophila suzukii. In DROP, genetic data are linked to voucher specimens and, where possible, the voucher specimens are identified by taxonomists and vetted through direct comparison with primary type material. To initiate DROP, we curated 154 laboratory strains, 856 vouchers, 554 DNA sequences, 16 genomes, 14 transcriptomes, and six proteomes drawn from a total of 183 operational taxonomic units (OTUs): 114 described Drosophila parasitoid species and 69 provisional species. We found species richness of Drosophila parasitoids to be heavily underestimated and provide an updated taxonomic catalogue for the community. DROP offers accurate molecular identification and improves cross-referencing between individual studies that we hope will catalyse research on this diverse and fascinating model system. Our effort should also serve as an example for researchers facing similar molecular identification problems in other groups of organisms.

Experimental warming influences species abundances in a Drosophila host community through direct effects on species performance rather than altered competition and parasitism.

Global warming is expected to have direct effects on species through their sensitivity to temperature, and also via their biotic interactions, with cascading indirect effects on species, communities, and entire ecosystems. To predict the community-level consequences of global climate change we need to understand the relative roles of both the direct and indirect effects of warming. We used a laboratory experiment to investigate how warming affects a tropical community of three species of Drosophila hosts interacting with two species of parasitoids over a single generation. Our experimental design allowed us to distinguish between the direct effects of temperature on host species performance, and indirect effects through altered biotic interactions (competition among hosts and parasitism by parasitoid wasps). Although experimental warming significantly decreased parasitism for all host-parasitoid pairs, the effects of parasitism and competition on host abundances and host frequencies did not vary across temperatures. Instead, effects on host relative abundances were species-specific, with one host species dominating the community at warmer temperatures, irrespective of parasitism and competition treatments. Our results show that temperature shaped a Drosophila host community directly through differences in species' thermal performance, and not via its influences on biotic interactions.

Geographic and Seasonal Variation in Species Diversity and Community Composition of Frugivorous Drosophila (Diptera: Drosophilidae) and their Leptopilina (Hymenoptera: Figitidae) Parasitoids.

Many studies have investigated species diversity patterns across space and time, but few have explored patterns of coexistence of tightly interacting species. We documented species diversity patterns in a host-parasitoid system across broad geographic location and seasons. We calculated species diversity (H and eH   ') and compared the relationship between community similarity and geographic distances of frugivorous Drosophila host (Diptera: Drosophilidae) and Leptopilina parasitoid (Hymenoptera: Figitidae) communities across Eastern North America, from New Hampshire to Florida, at two time points during the breeding season. We also analyzed the influence of environmental factors on species assemblages via constrained correspondence analysis and lastly calculated cluster dendrograms to identify potential host-parasitoid interactions. We found that the composition of Drosophila-Leptopilina communities varied significantly with latitude. Interestingly, diversity increased with increasing latitude, a trend counter to latitudinal patterns of diversity observed in many other taxa. We also found seasonal effects of monthly temperature range and precipitation on host biodiversity patterns across geographic locations. Cluster dendrograms nominated potential parasitoid-hosts and competitive interactions to be validated in the future studies. The present study fills an important gap of knowledge in North American Drosophila-Leptopilina species diversity patterns and lays the groundwork for future ecological and evolutionary studies in this system.

Knockdown expression of Syndecan in the fat body impacts nutrient metabolism and the organismal response to environmental stresses in Drosophila melanogaster.

The heparan sulfate proteoglycan syndecans are transmembrane proteins involved in multiple physiological processes, including cell-matrix adhesion and inflammation. Recent evidence from model systems and humans suggest that syndecans have a role in energy balance and nutrient metabolism regulation. However, much remains to be learned about the mechanisms through which syndecans influence these phenotypes. Previously, we reported that Drosophila melanogaster Syndecan (Sdc) mutants had reduced metabolic activity compared to controls. Here, we knocked down endogenous Sdc expression in the fat body (the functional equivalent of mammalian adipose tissue and liver) to investigate whether the effects on metabolism originate from this tissue. We found that knocking down Sdc in the fat body leads to flies with higher levels of glycogen and fat and that survive longer during starvation, likely due to their extra energy reserves and an increase in gluconeogenesis. However, compared to control flies, they are also more sensitive to environmental stresses (e.g. bacterial infection and cold) and have reduced metabolic activity under normal feeding conditions. Under the same conditions, fat-body Sdc reduction enhances expression of genes involved in glyceroneogenesis and gluconeogenesis and induces a drastic decrease in phosphorylation levels of AKT and extracellular signal regulated kinase 1/2 (ERK1/2). Altogether, these findings strongly suggest that Drosophila fat body Sdc is involved in a mechanism that shifts resources to different physiological functions according to nutritional status.