Quantitative prey species detection in predator guts across multiple trophic levels by mapping unassembled shotgun reads.

Quantifying species trophic interaction strengths is crucial for understanding community dynamics and has significant implications for pest management and species conservation. DNA-based methods to identify species interactions have revolutionized these efforts, but a significant limitation is the poor ability to quantify the strength of trophic interactions, that is the biomass or number of prey consumed. We present an improved pipeline, called Lazaro, to map unassembled shotgun reads to a comprehensive arthropod mitogenome database and show that the number of prey reads detected is quantitatively predicted from the prey biomass consumed, even for indirect predation. Two feeding bioassays were performed: starved coccinellid larvae consuming different numbers of aphids (Prey Quantity bioassay), and starved coccinellid larvae consuming a chrysopid larvae that had consumed aphids (Direct and Indirect Predation bioassay). Prey taxonomic assignment against a mitochondrial genome database had high accuracy (99.8% positive predictive value) and the number of prey reads was directly related to the number of prey consumed and inversely related to the elapsed time since consumption with high significance (r2  = .932, p = 4.92E-6). Aphids were detected up to 6 h after direct predation plus 3 h after indirect predation (9 h in total) and detection was related to the predator-specific decay rates. Lazaro enabled quantitative predictions of prey consumption across multiple trophic levels with high taxonomic resolution while eliminating all false positives, except for a few confirmed contaminants, and may be valuable for characterizing prey consumed by field-sampled predators. Moreover, Lazaro is readily applicable for species diversity determination from any degraded environmental DNA.

Uncovering Trophic Interactions in Arthropod Predators through DNA Shotgun-Sequencing of Gut Contents.

Characterizing trophic networks is fundamental to many questions in ecology, but this typically requires painstaking efforts, especially to identify the diet of small generalist predators. Several attempts have been devoted to develop suitable molecular tools to determine predatory trophic interactions through gut content analysis, and the challenge has been to achieve simultaneously high taxonomic breadth and resolution. General and practical methods are still needed, preferably independent of PCR amplification of barcodes, to recover a broader range of interactions. Here we applied shotgun-sequencing of the DNA from arthropod predator gut contents, extracted from four common coccinellid and dermapteran predators co-occurring in an agroecosystem in Brazil. By matching unassembled reads against six DNA reference databases obtained from public databases and newly assembled mitogenomes, and filtering for high overlap length and identity, we identified prey and other foreign DNA in the predator guts. Good taxonomic breadth and resolution was achieved (93% of prey identified to species or genus), but with low recovery of matching reads. Two to nine trophic interactions were found for these predators, some of which were only inferred by the presence of parasitoids and components of the microbiome known to be associated with aphid prey. Intraguild predation was also found, including among closely related ladybird species. Uncertainty arises from the lack of comprehensive reference databases and reliance on low numbers of matching reads accentuating the risk of false positives. We discuss caveats and some future prospects that could improve the use of direct DNA shotgun-sequencing to characterize arthropod trophic networks.

Detection and decay rates of prey and prey symbionts in the gut of a predator through metagenomics.

DNA methods are useful to identify ingested prey items from the gut of predators, but reliable detection is hampered by low amounts of degraded DNA. PCR-based methods can retrieve minute amounts of starting material but suffer from amplification biases and cross-reactions with the predator and related species genomes. Here, we use PCR-free direct shotgun sequencing of total DNA isolated from the gut of the harlequin ladybird Harmonia axyridis at five time points after feeding on a single pea aphid Acyrthosiphon pisum. Sequence reads were matched to three reference databases: Insecta mitogenomes of 587 species, including H. axyridis sequenced here; A. pisum nuclear genome scaffolds; and scaffolds and complete genomes of 13 potential bacterial symbionts. Immediately after feeding, multicopy mtDNA of A. pisum was detected in tens of reads, while hundreds of matches to nuclear scaffolds were detected. Aphid nuclear DNA and mtDNA decayed at similar rates (0.281 and 0.11 h(-1) respectively), and the detectability periods were 32.7 and 23.1 h. Metagenomic sequencing also revealed thousands of reads of the obligate Buchnera aphidicola and facultative Regiella insecticola aphid symbionts, which showed exponential decay rates significantly faster than aphid DNA (0.694 and 0.80 h(-1) , respectively). However, the facultative aphid symbionts Hamiltonella defensa, Arsenophonus spp. and Serratia symbiotica showed an unexpected temporary increase in population size by 1-2 orders of magnitude in the predator guts before declining. Metagenomics is a powerful tool that can reveal complex relationships and the dynamics of interactions among predators, prey and their symbionts.

Ancient associations of aquatic beetles and tank bromeliads in the Neotropical forest canopy

Water reservoirs formed by the leaf axils of bromeliads are a highly derived system for nutrient and water capture that also house a diverse fauna of invertebrate specialists. Here we investigate the origin and specificity of bromeliad-associated insects using Copelatinae diving beetles (Dytiscidae). This group is widely distributed in small water bodies throughout tropical forests, but a subset of species encountered in bromeliad tanks is strictly specialized to this habitat. An extensive molecular phylogenetic analysis of Neotropical Copelatinae places these bromeliadicolous species in at least three clades nested within other Copelatus. One lineage is morphologically distinct, and its origin was estimated to reach back to 12-23 million years ago, comparable to the age of the tank habitat itself. Species of this clade in the Atlantic rainforest of southern Brazil and mountain ranges of northern Venezuela and Trinidad show marked phylogeographical structure with up to 8% mtDNA divergence, possibly indicating allopatric speciation. The other two invasions of bromeliad water tanks are more recent, and haplotype distributions within species are best explained by recent expansion into newly formed habitat. Hence, bromeliad tanks create a second stratum of aquatic freshwater habitat independent of that on the ground but affected by parallel processes of species and population diversification at various temporal scales, possibly reflecting the paleoclimatic history of neotropical forests.