Predator diversity and bromeliad communities: experimental results

<div><p>We tested effects of both single and multiple predator species on community responses with a manipulative experiment where identical prey communities were exposed to treatments of either a single predator, or pairs of predators representing increasing phylogenetic diversity. In this experiment we focused on the four most abundant large predators found in<br>the community: <em>Leptagrion andromache</em> and <em>Leptagrion elongatum</em> (Odonata:<br>Coenagrionidae), a predatory Tabanid fly (Diptera:Tabanidae:<em>Stibasoma</em> sp.) and<br>a predatory leech. We combined these species in eight treatments: predator-free<br>control (no predators), each of the four predator species alone (3a) and<br>pairs of predator species chosen to maximize variation in phylogenetic distance<br>(3b). Specifically, these pairs were: two congeneric damselflies<br>(<em>Leptagrion andromache</em> and <em>Leptagrion elongatum</em>), two insects (<em>L.<br>elongatum</em> and <em>Stibasoma</em>), and two invertebrates (<em>L. elongatum</em><br>and a predatory leech). We used five replicate bromeliads for each of these 8<br>treatments (8 treatments, n=5). This experiment, therefore, allows the estimation<br>of the effect of each predator species (single-species treatments), as well as the<br>detection of non-additive effects in predator combinations.</p> <p>We created bromeliad communities that were as similar as possible to each other, and also to the average composition of a bromeliad. In February 2011 we collected bromeliads with a volume between 90 and 200ml,<br>thoroughly washed the plants to remove organisms and detritus, and soaked them for 12 hours in a<br>tub of water. We then hung all bromeliads for 48 hours to dry. This procedure was intended to remove all existing macroinvertebrates; one bromeliad dissected<br>afterwards contained no insects . We simulated natural detritus inputs from the canopy by adding a standard mass of dried leaves of the species <em>Plinia cauliflora</em> (Jabuticaba, Myrtaceae; a common Brazilian tree). In order to<br>track the effects of detrital decomposition on bromeliad N cycling, we enriched<br>these leaves with ¹⁵N by fertilizing five plants with 40ml pot^-1 day^-1 of 5g L^-1 ammonium sulphate containing 10% atom excess of ¹⁵N. After 21 days we then collected <em>P. cauliflora</em> leaves,<br>air-dried until constant weight, and then soaked them for three days. This procedure removes excess nutrients from the artificial fertilization. Because some of our prey species consume fine detritus, not coarse, we also added a standard amount of dried fine detritus to our bromeliads. We separated coarse and fine detritus by passing water from bromeliads through two sieves (as above for observational work, 150 and 850 µm). We defined “coarse detritus” as anything retained on the 850 µm sieve, and “fine detritus” as anything found on the 150 µm sieve.</p> <p>Each bromeliad was stocked with a representative insect community (See supplementary material). The densities<br>of each prey taxon were calculated from the observational dataset (Hypothesis 1), using<br>data from bromeliads of similar size to those in our experiment. We ran this experiment in two temporal blocks for logistical reasons: three complete replicates of all treatments were set up on 20 February 2011, and two on 08 March 2011. We first placed the prey species into the bromeliad, allowed two days for the prey to adjust, then added predators. After 26 days from the beginning of each block, we added the same prey community a second time to simulate the continuous<br>oviposition that characterizes the system. We concluded the experiment 43 days from the first addition of prey (20 April 2011). Throughout the experiment, all bromeliads were enclosed<br>with a mesh cage topped with a malaise trap and checked daily for emergence of<br>adults. At the end of the experiment we completely dissected our bromeliads, collecting all invertebrates and detritus remaining inside.</p> <p>We used a substitutive design, maintaining the same predator<br>metabolic capacity in all replicates (see below). In a substitutive experiment, all experimental units receive the same “amount” of predators — usually standardized by abundance — and only species composition varies. However, when species differ substantially in body<br>size - as in this experiment - abundance does not standardize the their effects on the community. We chose to standardize using metabolic capacity instead. Metabolic capacity is equal to individual body<br>mass raised to the power of 0.69; this reflects the nonlinear relationship between feeding rate and body size across many invertebrate taxa. </p> <p>To quantify the effect of predators on ecosystem function, at the end of the experiment we measured five<br>community and ecosystem response variables: decomposition of coarse detritus,<br>production of fine particulate organic matter (FPOM), bromeliad growth,<br>uptake of detrital nitrogen into bromeliad tissue, and survival of<br>invertebrate prey (emerged adults + surviving larvae). We measured decomposition by once again passing the bromeliad water through a 850 µm sieve, collecting the retained detritus and determining the mass of this detritus after oven-drying it at approximately 70C. We measured the<br>production of FPOM by taking the remaining liquid and filtering it on pre-weighed coffee filters, which were then dried and reweighed. We measured bromeliad growth as the average increase in length of five leaves per plant. We<br>tracked the uptake of labeled detrital nitrogen by analyzing three innermost<br>(closest to meristem) bromeliad leaves at the end of the experiment. Finally, we quantified the species<br>composition and survivorship of invertebrate prey by combining counts of<br>emerging adult insects and surviving larvae. </p> <p>We measured decomposition by collecting all <em>Plinia</em> leaves from bromeliads; these<br>were oven-dried at 70C before their mass was determined. At the end of<br>experiment, we sampled three new bromeliad leaves for isotopic (¹⁵N) and<br>nitrogen concentration analyses. These analyses were performed at the Stable<br>Isotope Facility laboratory (UC Davis, CA, USA) using continuous flow isotope<br>ratio mass spectrometer (20-20 mass spectrometer; PDZ Europa, Sandbach,<br>England) after sample combustion to N~2~ at 1000C by an on-line elemental<br>analyzer (PDZ Europa ANCA-GSL).</p> <div>​</div></div

Feeding trials of bromeliad invertebrates

To test whether related predators eat similar prey, we fed prey to predators in laboratory feeding trials. We conducted 314 feeding trials of 10 predator taxa and 14 prey taxa between March and April 2011. We performed as many feeding trials as possible, however many combinations between rare taxa are still missing. We tested 56 pairwise combinations. Most trials were replicated at least five times, but the number of replicates ranged from 1 to 11. To conduct the trials, we placed predators together with prey in a 50ml vial, with a stick for substrate. The only exception was the tabanid larvae, which we placed between two vertical surfaces to imitate the narrow space found in bromeliad leaf axils (their preferred microhabitat, necessary for successful feeding). Generally our trials contained a single predator and a single prey individual, except in the case of very small prey (_Elpidium_ sp.) or predators (_Monopelopia_ sp.) in which case we increased the density of the small taxon to five. We recorded whether prey was consumed after 24 hours. <br><br>All tests were performed at the Parque Estadual da Ilha do Cardoso, in São Paulo State, Brazil (25° 03' S, 47° 53' W)<br

Final_data_set

Final_data_se

Additional file 1: Table S1. of Successful isolation of Leishmania infantum from Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) collected from naturally infected dogs

Complete data of diagnostic tests applied to samples from 48 dogs and ticks collected from them. Brasilia, Federal District. Brazil. (DOCX 33 kb

Site characteristics in Old World settings.

<p>Site characteristics in Old World settings.</p

Agreement on key parameters by OWCL and NWCL clinical researchers.

<p>‘Standardized’ criteria are those as proposed in the reference paper Olliaro et al, 2013⁹; ‘updated’ criteria are those resulting from the consultation.</p

Site characteristics in New World settings.

<p>Site characteristics in New World settings.</p