Field Observations

Observations were made opportunistically during a field study on the influence of Philornis downsi on the breeding success of Darwin’s finches. This study was conducted at a study site (Los Gemelos, 0°37’34” S, 90°23’10” W) in the humid “Scalesia” forest on the Island of Santa Cruz, between January 11 - April 25 2012, January 9 - April 31 2014 and January 15 - May 30 2015. During this period P. downsi prevalence was very high. In 2012 all investigated nests with chicks of warbler finches (n = 46) and small tree finches (n = 37) were infested by P. downsi larvae. In 2014 and 2015 the prevalence was similar: 2014 warbler finch 97.9% (n = 96), small tree finch 95.8% (n = 72); 2015 warbler finch 94.1% (n = 51), small tree finch 90.2% (n = 51). However, we have no data on the prevalence of mosquitoes in the study area.

Repellent effect of P. galapageium on Mosquitoes

Field-experiment

In the field, the 17 human subjects (10 men, 7 women between 21 and 56 years old, see Table S1) treated one leg and one arm with ten crushed P. galapageium leaves each and left the other arm and leg untreated. The side of treatment was assigned randomly. Participants were standing while the experiment. The number of mosquitoes which were observed biting within 15 min of exposure on treated and non-treated limbs was counted. The experiment was carried out on the 9th of March 2016 at 6:33 pm (participants 1–9) and on the 18th of March 2016 at 6:30 pm (participants 10–17) at the Charles Darwin Station, Puerto Ayora, Galápagos. All participants were given the instruction not to shower two hours previous to testing and not to use repellents. To test whether the choice of the mosquitoes was different from random we used a “test of equal or given proportions” prob.test. library stats, R 3.2.2.,)56. Sample size was defined by the number of treated subjects (17), which were tested only once to avoid pseudoreplication.

Lab-experiment

The experiment was conducted at the Insect Pest Control Laboratory (IPCL) of the Joint FAO/IAEA Division, Seibersdorf, Austria. The Anopheles arabiensis used in this study originated from Dongola in the northern state of Sudan in 2005 and have been maintained and reared since then in the laboratory (for details see ref. 56). Adults were kept in a climate-controlled room maintained at a temperature of 27 ± 1 °C and 60 ± 10% relative humidity on a 12:12 h photoperiod, including dusk (1 h) and dawn (1 h). For the experiments, 20 five-day old female An. arabiensis were placed in a 30 cm cubic insect cage (Megaview Science Education Services Co., Ltd., Taiwan) the day before the experiment started and fed with 5% sugar-solution.

To test the repellent effect of P. galapageium, collagen sausage casings (Edicas 23NC, FIBRAN S.A., Girona, Spain) filled with 50 ml of defibrinated bovine blood heated at 38–39 °C were used. Each sausage was moistened with extract of P. galapageium or control plant or with their respective solvent. To prepare the P. galapageium extract, dried and ground P. galapageium leaves, collected at Los Gemelos, Santa Cruz, Galápagos, were put in 70% ethanol for one week at a weight ratio of 1:10 ground leaves to ethanol. After seven days the extract was filtered and preserved in a sealed glass bottle at room temperature until use.

During the experiment, two sausages were simultaneously presented to the female mosquitoes. One sausage was moistened with repellent while the second sausage was moistened with the control plant or solvent. The sausages were placed in the cage in parallel separated by 10 cm and the experimenter blew 5 times into the cage to motivate the mosquitoes to move. The following combination was tested: P. galapageium extract against ethanol. To test whether the application of a plant extract has a repellent effect per se, we introduced an additional control using Rubus idaeus as a plant species that is not known to have repellent properties and is closely related to the invasive Rubus niveus which was very abundant in our study area. We prepared the R. ideaeus extract in the same way as the P. galapageium extract and tested it against the solvent ethanol. Contrary to our expectation R. ideaus had a repellent effect compared to ethanol. In a subsequent experiment we thus tested P. galapageium extract against R. idaeus extract to assess which of the two plants had the stronger repellent effect. To measure the repellent effect we counted the number of mosquitoes sitting on each sausage after 60 seconds of exposure and repeated this every minute, for nine more minutes. The results presented are based on the number of mosquitoes, which landed on the treated sausage (P. galapageium, R.ideaus) after the first 60 seconds of exposure to the sausages. However, in the subsequent 9 min the percentage of mosquitoes that landed on the sausage treated with P. galapageium was similar to the results from the first 60 seconds (first 60 sec: median 3.3%, range 0–41.5%; subsequent 9 min.: median 0%, range 0–45%).

We conducted ten replicates for each condition. For each replicate new sausages and new cages with different mosquitoes were used. The side of treatment and control was assigned randomly. The number of female mosquitoes, which were still alive when the experiment started, ranged from 15 to 20 (median 19) in each trial. During the first 60 seconds the percentage of mosquitoes which landed on either of the sausages in all trials and conditions ranged from 29–100% (median 66%). All data are shown in Table S2. Again we used a test of equal or given proportion to estimate whether the choice of the mosquitoes in each comparison (P.galapageium – ethanol, R. idaeus – ethanol, P. galapageium – R. idaeus) was different from random (prob.test. library stats, R 3.2.2.57). We have adjusted the significance level to p = 0.017 using a Bonferroni correction

Repellent effect of P. galapageium on P. downsi

Effect on larval growth

In a laboratory experiment, we tested whether P. galapageium leaves reduce larval growth of P. downsi. Larvae were collected from the study site “Los Gemelos” between 07th February and 1st April 2014, which is the main breeding season of Darwin’s finches (Table S3). We only used second instar, small to medium sized larvae from two different size classes: <0.5 cm small larvae, 0.5 cm–1.0 cm medium larvae. Larvae were assigned randomly to test and control group and raised in the laboratory at the Charles Darwin Station, Santa Cruz, Galápagos and fed with chicken blood that was applied to a piece of cotton, which was tamped into a short piece of plastic drinking straw. The tip of the straw was covered with gauze. One group had to feed through gauze on which one drop of crushed P. galapageium leaves was applied. To produce the crushed leaves, four leaves and 1 ml of water were put into an Eppendorf tube (2 ml) and crushed with metal forceps. The other group had to feed through gauze that was treated with water only. Blood and treatment was renewed daily. To test whether the application of a plant extract had a repellent effect per se, we introduced an additional control group with crushed Tradescantia fluminensis leaves, a plant species present on the Galápagos Islands that is not known to have repellent properties. Biosafety regulations severely limit the movement of plants into and out of the Galápagos, therefore we used a different control plant in this lab experiment which was conducted in the Galápagos than that used in the lab-experiment on the repellent effect of P. galapageium on mosquitoes described above which was conducted in Austria. We weighed the larvae before the experiment and after 2 days of treatment and calculated the percentage of weight gain as “weight after 2 days” − “initial weight”)/“initial weight” × 100. Raw data are presented in Table S3. To analyse the effect of P. galapageium leaves on larval growth compared to the two control treatments (water and T. fluminensis) we calculated a linear model with weight after 2 days as predicted variable and treatment and initial weight as predictor variable. We calculated the model with interactions, but since none was significant we excluded them from the final model. We omitted one larva of the P. galapageium treatment group as its weight decreased by the power of ten, likely representing a typing error in data entering. However, the results did not change without this outlier. Weights were log-transformed. The post-hoc tests were done with the R-package multcomp58.

Effect on adult flies

We used a Y-tube laboratory olfactometer to assess the repellent effect of P. galapageium extract on the attractiveness of activated yeast to P. downsi. Olfactometer arms were 30 cm × 6 cm dia. and the angle between the arms was 70°. The y-tube was positioned vertically and a fluorescent lamp with dual bulbs (1.2 m) was centred 50 cm above the olfactometer parallel to a line connecting the ends of the olfactometer arms. Charcoal filtered air was introduced (1.0 L/min) into Erlenmeyer flasks (125 mL) fitted with two-hole rubber stoppers for incurrent and excurrent air flows. Effluent from the flasks was transferred to the olfactometer arms through plastic tubing (5 mm i.d.) and connected to a glass adapter with a barbed fitting on one end and a ground glass fitting (6 cm dia.) on the other end. The positions of the control and treatment were switched after every five trials. Adult P. downsi were released in the centre arm of the olfactometer and allowed to fly upwards towards the light and odour sources. A positive or negative response was recorded if the fly stayed in the distal half of the treatment or control arm for at least one minute. The majority of responding flies chose one arm within ten seconds.

The first experiment was intended to establish the attractiveness of yeast and the veracity of the olfactometer assay. The test stimulus was activated yeast (0.5 g baker’s yeast, 2.5 g sugar, 30 mL water) and the control was deionized water (30 mL). Of the 38 assays of P. downsi males 13 chose the yeast treatment, 9 chose the water control and 16 were unresponsive. The 22 responsive males showed no significant preference for the yeast treatment (binomial test: p = 0.12). Of the 60 assays of P. downsi females 25 chose the yeast treatment, 11 chose the water control and 24 were unresponsive. The 36 responsive females showed a significant preference for the yeast treatment (binomial test: p = 0.01). Thus, only female flies were used for the following experiment.

The purpose of the second experiment was to determine the repellent effect of P. galapageium extracts. Leaves of P. galapageium were collected in the Los Gemelos highlands of Santa Cruz Island on 26 February, 2015, transported to the Charles Darwin Research Station, Puerto Ayora and stored at 4 °C. Leaf extracts were made within 24 hours by placing four crushed P. galapageium leaves in a vial (4 mL) with dichloromethane (4 mL) for 10 minutes and then decanting the solvent extract into a fresh vial. Thus, each assay used 0.1 P. galapageium leaf equivalents (4 leaves in 4 mL DCM = a concentration of 1.0 leaf/mL; the 100 uL aliquots used in the assays would represent 0.1 leaf eq.). It is impossible to say how much volatile material a finch applies to its feathers, but based on our field observations, a tenth of one leaf is reasonably close to the amount used by finches. In Syracuse, extracts were stored at −60 °C until use. An aliquot (100 μL) of the extract was placed on a filter paper strip inside a glass tube that was inserted in the plastic tubing between the Erlenmeyer flask and the olfactometer arm.

Controls consisted of an equal volume of methylene chloride on a filter paper strip. The Erlenmeyer flasks of both the treatment and control contained activated yeast as described above.

Coupled Gas Chromatographic-Electroantennographic Detection (GC-EAD)

In a further experiment coupled Gas Chromatographic-Electroantennographic Detection was carried out to give insight into which chemical compounds of P. galapageium leaves can be detected by adult P. downsi. Coupled GC-EAD analysis was performed using a gas chromatograph (Hewlett-Packard 5890 Series II) equipped with a capillary column (HP5-MS, 30 m × 0.25 mm ID, 0.25 μm film thickness; Agilent Technologies, Wilmington, DE, USA) in splitless mode with 1 min sampling. The oven temperature was programmed for 5 min at 40 °C, then increased at 15 °C/min to 250 °C and then held for 5 min. The injector temperature was 250 °C. Helium was the carrier gas at a flow rate of 1 mL/min. The column effluent was split 1:1 in the oven via a glass Y-connector with nitrogen make-up gas (8 mL/min) introduced through a second glass Y-connector. One arm of the splitter led to the flame ionization detector (FID) (260 °C) and the other to a heated transfer line (260 °C) (Agilent Technologies, Wilmington, DE, USA). The EAD effluent was introduced into a cooled (5 °C) humidified air stream (1 L/min) directed toward the antennae of the mounted fly.

Whole head preparations were made of individual flies, age 3–6 d, for GC-EAD analysis, as described previously from similar studies with other flies59,60,61. The head was excised and the antennae were positioned between two gold-saline (Drosophilaringer solution; 46 mmol NaCl, 182 mmol KCl, 3 mmol CaCl2 and 10 mmol Tris HCl at pH 7.2) electrode micropipettes in an acrylic holder. The output signal from the antenna was amplified (10×) by a custom high input impedance DC amplifier and recorded on an integrator-recorder.

For the GC-EAD analysis of P. galapageium leaf extracts, a total of four antennal pairs from two female and two male flies (1–2 replicated runs/pair) were tested.

To identify the chemical compounds present in the P. galapageium leaf extract to which P. downsi antennae were responding, leaf extracts were analysed by coupled gas chromatography-mass spectrometry (GC-MS; Agilent 7890A GC interfaced to a 5975 mass selective detector in EI mode, 70 eV; Agilent Technologies, Santa Clara, CA, USA). The GC column, temperature program and carrier gas were the same as those used for GC-EAD. Compound identifications were based on matches with spectra in the NIST/EPA/NIH Mass Spectral Library Version 2.0f (2009) and retention index (Table 2).