Claims about the role of predator diversity in maintaining ecosystem function and providing ecosystem services such as pest control are controversial, but evaluative tests are beginning to accumulate. Empirical and experimental comparisons of species-rich versus species-poor assemblages of entomophagous arthropods and vertebrates range from strong suppression to facilitative release of herbivorous arthropod prey. Top-down control can be strengthened when natural enemies complement each other, dampened by negative interactions, balanced by both factors, and driven by single influential species. A meta-analytic synthesis shows a significant overall effect of enemy richness increasing top-down control of herbivores, which is consistent in agricultural studies conducted in tropical versus temperate zones, in studies using caged versus open-field designs, but not so in nonagricultural habitats. Synthetic analyses address theory and help set precautionary policy for conserving ecological services broadly, while characterizing uncertainty associated with herbivore response to changes in enemy diversity.

Next-generation sequencing (NGS) technology has extraordinarily enhanced the scope of research in the life sciences. To broaden the application of NGS to systems that were previously difficult to study, we present protocols for processing faecal and swab samples into amplicon libraries amenable to Illumina sequencing. We developed and tested a novel metagenomic DNA extraction approach using solid phase reversible immobilization (SPRI) beads on Western Bluebird (Sialia mexicana) samples stored in RNAlater. Compared with the MO BIO PowerSoil Kit, the current standard for the Human and Earth Microbiome Projects, the SPRI-based method produced comparable 16S rRNA gene PCR amplification from faecal extractions but significantly greater DNA quality, quantity and PCR success for both cloacal and oral swab samples. We furthermore modified published protocols for preparing highly multiplexed Illumina libraries with minimal sample loss and without post-adapter ligation amplification. Our library preparation protocol was successfully validated on three sets of heterogeneous amplicons (16S rRNA gene amplicons from SPRI and PowerSoil extractions as well as control arthropod COI gene amplicons) that were sequenced across three independent, 250-bp, paired-end runs on Illumina's MiSeq platform. Sequence analyses revealed largely equivalent results from the SPRI and PowerSoil extractions. Our comprehensive strategies focus on maximizing efficiency and minimizing costs. In addition to increasing the feasibility of using minimally invasive sampling and NGS capabilities in avian research, our methods are notably not avian-specific and thus applicable to many research programmes that involve DNA extraction and amplicon sequencing.

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The emerging field of molecular scatology enables critical testing of food web theory. The non-invasive application of molecular tools allows for sequencing of prey DNA from predator fecal matter, evaluating diet breadth and foraging guild. While insectivorous bats are obscure foragers compared to most insectivorous birds, more is known about which arthropod species bats consume because molecular techniques have been optimized for mammalian systems, not avian physiology. Our research objective was to use molecular tools to detect arthropod prey in the fecal matter of an insectivorous avian predator. We used Western Bluebird (Sialia mexicana) as a model predator due to its generalist foraging strategy. We compared two fecal DNA extraction kits: (1) Qiagen's DNA stool mini kits, used widely in dietary studies on bats and (2) Zymo's Soil/Fecal DNA MiniPrep kits, not currently cited in the molecular scatology literature. We successfully extracted DNA only with the Zymo kit, amplified mitochondrial cytochrome oxidase c subunit I genes, sequenced, and identified the arthropod prey. A spiked PCR experiment showed evidence of possible inhibitors remaining in the Qiagen kit extractions. Overall, arthropod prey from seven different orders and five different classes were identified. We discuss the ecological implications of these data and suggest areas of future research applying molecular techniques to avian fecal matter.Consistent methodological advancement will enable molecular scatology to identify ecosystem services provided by insectivorous birds, develop ecological theory, and inform predator conservation efforts.

Insectivorous Western Bluebirds (Sialia mexicana) occupy vineyard nest boxes established by California winegrape growers who want to encourage avian conservation. Experimentally, the provision of available nest sites serves as an alternative to exclosure methods for isolating the potential ecosystem services provided by foraging birds. We compared the abundance and species richness of avian foragers and removal rates of sentinel prey in treatments with songbird nest boxes and controls without nest boxes. The average species richness of avian insectivores increased by over 50 percent compared to controls. Insectivorous bird density nearly quadrupled, primarily due to a tenfold increase in Western Bluebird abundance. In contrast, there was no significant difference in the abundance of omnivorous or granivorous bird species some of which opportunistically forage on grapes. In a sentinel prey experiment, 2.4 times more live beet armyworms (Spodoptera exigua) were removed in the nest box treatment than in the control. As an estimate of the maximum foraging services provided by insectivorous birds, we found that larval removal rates measured immediately below occupied boxes averaged 3.5 times greater than in the control. Consequently the presence of Western Bluebirds in vineyard nest boxes strengthened ecosystem services to winegrape growers, illustrating a benefit of agroecological conservation practices. Predator addition and sentinel prey experiments lack some disadvantages of predator exclusion experiments and were robust methodologies for detecting ecosystem services.