Does the Ratio of Compounds in a Plant Volatiles Blend Remain Stable During Transmission by Wind?

For plant volatiles to mediate interactions in tritrophic systems, they must convey accurate and reliable information to insects. However, it is unknown whether the ratio of compounds in plant volatile blends remains stable during wind transmission. In this study, volatiles released from an odor source were collected at different points in a wind tunnel and analyzed. The variation in the amounts of volatiles collected at different points formed a rough cone shape. The amounts of volatiles collected tended to decrease with increasing distance from the odor source. Principal component analyses showed that the volatile proles were dissimilar among different collection points. The proles of volatiles collected nearest the odor source were the most similar to the released odor. Higher wind speed resulted in a clearer distinction of the spatial distribution of volatile compounds. Thus, variations in the ratios of compounds in odor plumes exist even during transport over short distances. available to an organism (Hildebrand 1995; Zimmer and Butman 2000). Further studies are required to clarify the factors affecting the availability of olfactory information, both in terms of the spatiotemporal variations of the odor concentration and the resolution of the insect’s olfactory system. The essential prerequisite for such research is highly sensitive analytical methods for the qualitative and quantitative analysis of instantaneous variations in odor laments.


Introduction
Volatile compounds emitted from plants are a chemical language representing plant signals, and they play an important role in mediating interactions in tritrophic systems ( (Riffell et al. 2008). Ambient motion can transport odorant molecules > 10 3 times quicker than molecular diffusion over equivalent distances and is the principal physical process that controls odor transport at distances > 1 cm (Riffell et al. 2008). Wind in the environment is turbulent, and carries plant odors as a plume. The structure of odor plumes is complex. They contain odor laments (pockets of high odor concentrations) interspaced by pockets of odor-free medium (Mylne et al. 1996;Finelli et al. 1999; Murlis et al. 1992Murlis et al. , 2000, much like the patterns observed in plumes of smoke. Insect antennae receive a odor-laments, and then the insect tracks along wind-borne odor plumes to the source. The physical environment dictates the navigational behavior of an organism through its effects on controlling the information provided by the chemical signal (Willis and Baker 1988; Mafra-Neto and Carde 1994; Vickers and Baker 1994; Kuenen and Carde 1994;Zimmer-Faust et al. 1995;Vickers and Baker 1996). This odormediated insect navigation is vital for interactions in tritrophic systems.
It is important to a recipient insect that the information carried by the plant odor plume should be relatively xed and stable after turbulent transport in air. In other words, the qualitative and quantitative composition of the volatile compounds conveyed in the odor laments should be conserved over long distances (Vickers 2000;Beyaert and Hilker 2014), although the concentration of the odor within the laments may decrease (Voskamp et al. 1998;Murlis et al. 2000). However, little is known about changes in the qualitative and quantitative composition of plant odors as they are transmitted by wind. This is mainly because it is di cult to obtain robust and reliable measurements of the short-term changes in plant odor composition that occur in plant odor laments (Cai et al. 2015). Although we cannot conduct instantaneous measurements, we can analyze the volatiles collected for some time at different xed points to evaluate the stability of the information carried by the plant odor plume. Based on this idea, we measured the spatial variations in the concentrations of nine plant volatile compounds after release in a wind tunnel by a coupled thermal desorption and gas chromatography-mass spectrometry method.
Materials And Methods

Reagents
The tested plant volatile compounds were selected on the basis of the previous studies, which identi ed the compounds that convey chemical information of host location to tea leafhopper (Empoasca onukii) and are attractive to tea leafhopper (Cai et  Odor source The tested compounds were mixed at equal volumes. Forty microliters of the mixture was loaded onto a rubber septum, which was then placed in a refrigerator at 4°C. After the mixture was completely absorbed (about 10 h), the rubber septum was used for tests in the wind tunnel. Before each test, the rubber septum was placed in a fume cupboard at 20 ± 2 ℃ for 3 h.

Wind tunnel
The wind tunnel had a polycarbonate ight section (length × width × height) of 200 cm × 60 cm × 60 cm (Fig. 1). Air was blown into the tunnel by a fan through an activated carbon lter and honeycomb-structured plastic, which were located inside a lter housing. The air exiting the tunnel was passed through a 100-mesh metal screen and a box lled with activated charcoal before being extracted from the room containing the wind tunnel via an exhaust system. Smooth air ow in the wind tunnel was con rmed by burning a mosquito coil at the site of odor source before each test. Hot-lm anemometers (AR866, Dongguan Science & Technology Co. Ltd., Dongguan, China) were used to measure the wind speed at the ve points near the exit of the wind tunnel.

Volatile collection
The rubber septum was threaded onto a string with paper clips. The string was hung in the center of the ight section of the wind tunnel at the upwind end. There was a 20-cm distance between the rubber septum and the honeycomb-structured plastic, and a 30-cm distance between the rubber septum and the top of the wind tunnel. The wind tunnel was lit diffusely from above at about 200 lux. The room was kept at 20 ± 2 ℃, 70-80% relative humidity (R.H.). Before each test, the ight sections were cleaned with ethyl alcohol and maintained in a ventilated environment for 8 h.
Sampling was performed at 12:00 h. Samples were collected at 20 cm, 60 cm, 120 cm and 180 cm away from the odor source in the middle of the wind tunnel (four samples). Another eight samples were collected on the left and right sides of the middle four samples (at a distance of 20 cm from the middle sample). In total, samples were collected from 12 sites in the wind tunnel (see Fig. 1). The air inlet of stainless steel adsorbent tubes (Markes, UK; packed with 200 mg of Tenax™, 60-80 mesh) was at about the same height as the odor source. Air was collected at a ow rate of 100 mL min − 1 for 100 min using a microprocessor-controlled air sampling pump (Mini-pump Σ30; Shibata, Japan).
The trials included three treatments: low wind speed (0.09 m s − 1 ), high wind speed (0.39 m s − 1 ), and a blank control. In the low and high wind speed treatments, the odor sources were the same (rubber septa loaded with 40 µL volatile compound mixture). In the blank control, nothing was loaded on the rubber septum. Each treatment was replicated four times. Different treatments were tested on three consecutive days, and all of the wind tunnel tests were completed within 20 days.
To estimate the emission from the rubber septum, the volatiles emitted from the rubber septum were collected in a push/pull system at the same time of the wind tunnel test. The rubber septum was maintained in a 30-mL glass holding chamber (2.1 cm i.d., 8 cm length). Charcoal-puri ed air entered the holding chamber at a rate of 100 ml min − 1 . After passing over the rubber septum, the air was pulled through the same stainless steel adsorbent tubes as those used in the wind tunnel test. Volatiles were collected at the 4th, 22nd, 40th, 58th, 76th, and 94th minute of the wind tunnel test, and each collection lasted for 2 minutes. Between collections, the owing gas was retained in the chamber containing the rubber septum. The conditions of the collection room were the same as those used in the wind tunnel test.

Volatile analysis
After collection, samples were analyzed immediately as described previously (Cai et al. 2015). All samples were spiked with 5 ng IS, and were analyzed by coupled thermal desorption (TD; TD100, Marks, UK) and GC-MS (GCMS-QP2010, Shimadzu, Japan) with a DB-5 MS capillary column (60 m × 0.25 mm i.d., 0.25 µm lm thickness; J&W Scienti c, USA). The adsorbent tubes were heated at 275°C for 5 min while the desorbed volatile compounds from the tube were focused into the cold trap at 4°C with highpurity helium. Following sample transfer, the cold trap was rapidly heated to 290°C. Then, the desorbed compounds were injected into the GC. The oven temperature of the GC was initially set to 45°C for 2 min, then increased by 5°C min − 1 to 70°C and held for 15 min, increased by 2°C min − 1 to 160°C, and then increased by 30°C min − 1 to 260°C and maintained for 10 min. Ionization was achieved via electron impact at 70 eV and 250°C, and compounds were analyzed in the SIM mode. The calibration curve was established as described previously (Cai et al. 2015) by plotting the abundance ratio of analyte to IS against the mass ratio of analyte to IS. The calibration curve was updated immediately before analyzing each batch of samples.

Statistical analyses
The collection ratio and relative collection ratio were used to describe the dispersal behavior of slowrelease volatiles in the wind tunnel. The formulae used to these ratios were as follows: Collection ratio = (amount collected at a point/amount released from the source) × ‰; Relative collection ratio = (collection ratio at a point/total collection ratio of the three points at the same horizontal distance from the odor source) × %. The amount released from the odor source was estimated as follows: 50 × average amounts detected at the 4th, 22nd, 40th, 58th, 76th, and 94th minute.
All statistical tests were carried out using SAS V8.2 (SAS Institute, Cary, NC, USA). Differences in the collection ratios or relative collection ratios at a collection point between high and low wind speed were

Variation in amounts of collected volatiles among different points
The amount of volatiles collected at the middle four points exponentially decreased with increasing distance from the odor source (Fig. 2). In contrast, the collection ratios at right and left points increased with increasing distance from the odor source (Fig. 2). The collection ratios at M-20 (middle-20 cm) and M-60 (middle-60 cm) were, respectively, 87.1‰ and 27.3‰ under high wind speed, and were signi cantly higher than their corresponding values under low wind speed (P < 0.01, Fig. 2). The collection ratios at L-120 (left-120 cm), L-180 (left-180 cm), R-120 (right-120 cm), and R-180 (right-180 cm) were signi cantly lower under high wind speed than under low wind speed (P < 0.05, Fig. 2). The relative collection ratios at M-20, M-60, M-120 (middle-120 cm) and M-180 (middle-180 cm) were, respectively, 99.8%, 99.3%, 93.4% and 78.2% under high wind speed, and were all signi cantly higher than their corresponding values under low wind speed (P < 0.05, Fig. 3).

Differences in pro les between released and collected volatiles
Although all nine compounds were collected at the 12 collection points, the pro les of the volatiles at the odor source and at the twelve collection points were different. The results of the PCA showed that the collection points were divided into four groups on the basis of the pro les of volatile compounds under

Discussion
It has been reported that the odor plume is roughly cone shaped in the eld, with the main dimension along the wind axis, and the odor concentration decreases with the square of the distance to the source (Conchou et al. 2019). In the present study, measurements of the amounts of volatiles collected in a twodimensional space showed that the slow-released odor formed a rough cone shape in the direction of the blowing wind in the wind tunnel. Moreover, the amounts of volatiles exponentially decreased with increasing distance from the odor source. This spatial outline may indicate that insects are more likely to perceive the odor at sites more distant from the odor source because of the large diffusion area of volatiles. Then, they can follow the higher concentrations of volatiles to easily locate the odor source. In addition, the odor plume became a sharper cone with higher concentrations of volatiles under a higher wind speed. Thus, the odor under a higher wind speed could be detected by insects further from the odor source.
We evaluated the stability of a volatile blend during wind transmission in a wind tunnel. The results of PCA analyses showed that the pro les of volatiles collected at different points were signi cantly dissimilar whether the wind speed was high or low, and that the spatial distribution of the nine volatile compounds was also signi cantly dissimilar. These results indicate that the ratios of compounds in odor laments could change during wind transmission. This is an interesting result. After all, according to previous predictions (Vickers 2000;Beyaert and Hilker 2014), steady and clean air ow in a wind tunnel should lead to relatively xed volatile pro les among different collection points.
Differences in the pro les of the collected volatiles may be related to the physical properties of volatile compounds, such as molecular mass, vapor density, vapor pressure, and etc.. These physical properties might affect the inertial forces, viscous forces, and buoyancy forces of volatile compounds in ambient motion by air (Weissburg 2000;Koehl 2006). In our study, the higher wind speed resulted in more distinct differences in volatile pro les among collection points, and the pro le of volatiles collected at M-20 was most similar to that of the odor source among the collection points. These situation is similar to the different movement speeds of objects with different shapes or weight in owing water, where larger differences in relative distance among objects occur with faster water ow and at places further from the starting point. Our results also showed that the pro le of volatiles collected at M-20 was more dissimilar from that of the source odor under a lower wind speed. This may be because molecular diffusion made a greater contribution to odor distribution during short-distance transport under a low wind speed.
Molecular diffusion by thermal motion is related to molecular mass, concentration differences, and temperature (Curtis and Farrell 1992;Ern and Giovangigli 1999;Rosner et al. 2000;Palle et al. 2005). The nine volatile compounds in this study had different molecular weights, the molecular weight range from 100 to 170, and the maximum differences in odor concentrations were near the odor source. This initial separation of volatile compounds might also play a role in subsequent greater separation during transmission by wind (Bourgoin et al. 2006). More research is required to explore how the physical properties of different compounds affect the ratios of volatile compounds in odor umes.
The variations in the ratios of compounds in odor plumes may be larger in the eld than those in this study, because of turbulent air ow, mixtures of different odors, adsorption by substrates, and atmospheric chemical degradation (Atkinson and Arey 2003; Helmig et al. 2004). For instance, the ratio of volatiles in the plume emanating from owers of Datura wrightii was found to change with distance, as the background volatiles from neighboring vegetation became intermixed with D. wrightii volatiles (Riffell et al. 2014). However, insects such as moths can y over several hundreds of meters navigating upwind through pheromone plumes (Shorey 1976;Cardé and Charlton 1984;Elkinton et al. 1987). It has been postulated that insects can deal with complex odors because of their remarkable capacity of spatial resolution about the olfactory system, which includes active olfactory sampling behaviors and selfgenerated air ows (Baker et al. 1998;Koehl 2006;Szyszka et al. 2012).
The aerodynamic environment can dictate important ecological interactions and be a selective force for the evolution of olfactory systems, because it determines the chemical information available to an organism (Hildebrand 1995;Zimmer and Butman 2000). Further studies are required to clarify the factors affecting the availability of olfactory information, both in terms of the spatiotemporal variations of the odor concentration and the resolution of the insect's olfactory system. The essential prerequisite for such research is highly sensitive analytical methods for the qualitative and quantitative analysis of instantaneous variations in odor laments.  wind speed. Data are mean + SE (n = 4). Asterisks denote signi cant difference in collection ratio at a particular point between low and high wind speed (two-sample t-test for means: P<0.05).  with the odor source. 20, 60, 120, and 180 indicate, respectively, 20 cm, 60 cm, 120 cm, and 180 cm vertical distance of collection point from odor source in the downwind direction. Source indicates the odor source. Score plots from PCA are based on percentages of compounds out of total collected or released volatiles. Data points in the score plot are mean ± SE of principal component scores for four replicates. Figure 5