Does S. carpocapsae prefer or naturally navigate towards milkweed roots or milkweed-feeding insects by using CGs or other chemicals as cues? Drosophila was previously shown to be susceptible to all three of these EPN species . Larvae were fed a non-toxic diet or a diet containing the purified CG ouabain . We chose the hydrophilic CG ouabain since we could deliver millimolar levels of this CG into this non-CG-sequestering insect via its diet to mimic the high CG levels that can be found in the hemolymph of monarch caterpillars without needing levels of a solvent such as DMSO that would have adverse effects on insects and nematodes . Fly food containing ouabain was created by preparing Nutri-Fly food packets in a flask. Once cooled, 15 mM of ouabain was added to the flask. Fly food was poured into vials and allowed time to cool, then stored at 4 °C. Non-toxic fly food, prepared as described above without the addition of ouabain, served as the control. Six to eight adult males and six to eight adult females were placed into each vial and allowed to mate for 3–4 days. Adults were removed and larvae were left to hatch. Twelve-well plates were prepared with filter paper or NGM agar and one second-instar fly larva in each well. Twenty EPNs in 10 µL of water were then added to the wells with fly larvae that were either feeding on nontoxic or ouabain-containing food. Plates were covered with parafilm. Larval mortality was recorded at 2, 12, 24, and 48 h post infection to assess whether CGs influence the ability of EPNs to kill their insect hosts. The experiment was performed in triplicate.Asclepias curassavica seeds were germinated in seedling trays containing organic planting mix . Seedlings were maintained in a growth chamber at 26 °C with a 16 h light / 8 h dark phase at a light intensity of 200 µM m–2 s–1 until the first true leaf was observed.

Seedling roots were then thoroughly rinsed with water to eliminate soil particles. Subsequently, plastic planters bulk the roots were flash-frozen in liquid nitrogen and pulverized into a fine powder using a mortar and pestle. The resulting powdered root tissues were then dissolved in three volumes of 5% methanol solution and centrifuged at 10,000 g for 10 min. The resulting supernatant was carefully removed, and the pellet was suspended in H2O before being stored at 4 °C until further use .Chemotaxis plates were set up according to a previously published protocol . Chemotaxis agar media was poured into small Petri dishes . Then, using a pipette, a small crater-like shape was created in the middle, forming a higher level of agar referred to as the volcano deck. IJs were exposed to wax worm host cuticle for a duration of 20 min to allow host stimulation, which allows for higher participation rates of EPNs . A quantity of 20 µL of root extract was placed onto the volcano deck, followed by the addition of 4 µL of the paralytic agent sodium azide at 0.5 M. The NaN3 solution was made by adding 500 µL of 1-M stock to 500 µL of milliQ water. The paralytic agent was used to visualize the EPNs’ initial directional movement. Subsequently, a suspension containing 100–200 IJs in 20 µL of H2O was carefully dispensed around the perimeter of the deck slope. Plates were then stacked into groups of three in opposite orientations, placed in a box with a lid on a vibration-resistant platform and stored in the dark for 24 h. After 24 h, the numbers ofIJs on and below the deck as well as the number of IJs that displayed a coiling phenotype were recorded. The experiment was performed in triplicate.Sand was autoclaved and then washed repeatedly with tap water followed by DI water. The assays were performed in olfactometers , the setup of which has been described previously .

The measurements of the pipes and glass were as follows: each tube measured 9 cm in diameter and 5 cm in length, the pipe was 7 cm in length and 6 cm in height, and the entire set-up was 15 cm in length. Sand was dried at 60 °C overnight and moistened to 12% with tap water. Filter paper was spotted with 20 µL of tap water as a control or with 20 µL of test solution, and then placed at the end of the tubes. A consistent weight of 28 g of sand per tube was used for each replicate and trial. Root extracts were protected from light. Prenol is a known repellent for EPNs and served as a negative control test solution . A 2-M solution of prenol was made by adding 203 µL of prenol to 797 µL of DI water. Asclepias curassavica milkweed root extracts were prepared using the method described above. The region near the middle of the olfactometer consisted of sand moistened to 12% with tap water, which was the same as the conditions on the control side of the olfactometer to ensure that no biases were introduced. One thousand IJs in 100 µL of H2O were carefully dispensed into the center of the olfactometer. IJs were collected from fresh white traps followed by host stimulation prior to each experiment. For host stimulation, three wax worms were placed on Petri dishes with filter paper and the IJs were allowed to interact with host cuticle for 15–20 min before being collected and used . Each olfactometer was placed horizontally on a foam pad to suppress any vibrations. These were then placed in the dark in random orientations to avoid any potential directionality biases. Each assay ran for 24 h before the caps were removed from each side of the olfactometer separately. Nematodes were collected using the Baermann funnel technique followed by counting their numbers. Each replicate had three biological replicates for each condition and each EPN species. Choice percentages were calculated by counting the number of nematodes in the control area or the test area, dividing these by the total number of IJs inoculated, and multiplying the result by 100.

The experiment was performed in triplicate within each of three biological replicates. The figures were graphed using means across biological replicates, and a chi-squared analysis was conducted on the average of each replicate, with the number in the control treatment serving as the expected value and the number in the test treatment serving as the observed value.Chemotaxis plates were prepared as described previously : 17 g agar was dissolved in 1,000 mL dH2O and autoclaved for 30 min; this was followed by the addition of 5 mL filThered potassium phosphate buffer, 1 mL filThered MgSO₄, and 1 mL filThered CaCl₂. Plates were left at room temperature for 12 h before the experiment. EPNs were collected from fresh white traps to ensure healthy IJs were used in assays. IJs were collected and washed with DI water before 500 µL of IJ suspension at a density of one IJ per µL was placed onto a wax worm. IJs were given 20 min to have contact with the host cuticle for host stimulation . They were then collected and left at a density of eight IJs per µL, collection pot ready to be used for chemotaxis assays. A 2-M solution of the EPN repellent prenol was made by adding 203 µL of prenol to 797 µL of DI water. A tetrahydrofuran solution was made by adding 14.4 µL of THF to 985.6 µL of DI water. THF is a known attractant for S. carpocapsae and S. feltiae . A 0.5-M solution of the paralytic NaN3 was made by adding 500 µL of 1-M stock to 500 µL of milliQ water. Ouabain solutions of 15 mM or 100 µM were made by dissolving ouabain in DI water with 0.3% DMSO. Templates for chemotaxis assays were printed and placed under each chemotaxis plate. On the test side, 5 µL of chemical solution was placed in the test circle. On the control side, 5 µL of DI water with 0.3% DMSO was placed in the control circle. Chemicals were given 15–20 min to diffuse. Then, 2 µL of 0.5 M NaN3 was placed in each scoring circle. A 15-µL suspension of IJs at a density of 5 IJs per µL was placed in the center of the plate, containing a total of 70–170 nematodes. Plates were then stacked into groups of three in opposite orientations, placed in a box with a lid on a vibration-resistant platform and stored in the dark. The assay was run for two hours, after which data was taken on where nematodes were found: the test side, the control side or in the middle. Choice percentages were calculated by counting the number of nematodes in the control area or the test area, dividing these by the total number of IJs inoculated, and multiplying the result by 100. The figures were graphed using means across biological replicates, and a chi-squared analysis was conducted on the average of each replicate, with the number in the control area serving as the expected value and the number in the test area serving as the observed value.Plant functional traits have proved useful in identifying life history strategies for predicting plant community assembly and for assessing the impact of vegetation composition and diversity on ecosystem functioning . Consequently, vegetation models including coupled climate–vegetation models benefit from a better representation of plant trait variation to adequately analyse terrestrial biosphere dynamics under global change .

Today, in combination with advanced gap-filling techniques , databases of plant traits have sufficient coverage to allow quantitative analyses of plant form and function at the global scale . Analysing six fundamental traits, Díaz and colleagues revealed that essential patterns of form and function across the plant kingdom can be captured by two main axes. The first reflects the size spectrum of whole plants and plant organs. The second axis corresponds to the ‘leaf economics spectrum’ emerging from the necessity for plants to balance leaf persistence against plant growth potential. The concept of a global spectrum of plant form and function has since been investigated from various perspectives . It has been shown, for instance, that orthogonal axes of variation in size and economics traits emerge even in the extreme tundra biome or at the scale of plant communities . However, it remains unclear whether the two axes remain dominant for extended sets of traits or when differentiating among growth forms. A particular knowledge gap is what environmental controls determine these two axes of plant form and function. There is ample evidence that large-scale variation of individual plant traits is related to environmental gradients. Early plant biogeographers suggested that climate and soils together shape plant form and function but could not propose a more precise theoretical framework describing these fundamental relationships. Over the last decades, examples have thus accumulated without an overall framework in which to place them . For instance, tree height depends on water availability while leaf economics traits depend on soil properties, especially soil nutrient supply, as well as on climatic conditions reflected in precipitation . Leaf size, leaf dark respiration rate, specific leaf area , leaf N and P concentration, seed size and wood density, all show broad-scale correlations with climate or soil . It has also been reported that many of these traits show latitudinal patterns . Generalizing such insights is, however, not trivial, as soil properties partly mirror climate gradients, as a consequence of long-term soil formation through weathering, leaching and accumulation of organic matter—processes related to temperature and precipitation ; however,climate-independent features reflecting geology and surface morphology also contribute to soil fertility . Soil may furthermore buffer climate stresses; for example, by alleviating water deficit in periods of low precipitation . Combining the insights suggests that the global spectrum of plant traits reveals two internally correlated orthogonal groups and that many plant traits are individually linked to environmental gradients, we expect that both trait groups should closely follow gradients of climate and soil properties. Here, we investigate to what extent the major dimensions underpinning the global spectrum of plant form and function can be attributed to global gradients of climate and soil conditions; and to what extent these factors can jointly or independently explain the global spectrum of form and function.