Chemically mediated intraguild predator
avoidance by aphid parasitoids: Interspecific
variability in sensitivity to semiochemical
trails of ladybird predators
著者（英）
Nakashima Yoshitaka, Birkett Michael A., Pye
Barry J., Powell Wilf
journal or
publication title
Journal of Chemical Ecology
volume
32
number
9
page range
1989-1998
year
2006-09
Chemically-mediated intraguild predator avoidance by aphid parasitoids:
inter-specific variability in sensitivity to semiochemical trails of ladybird
predators
YOSHITAKA NAKASHIMA1, MICHAEL A. BIRKETT2, BARRY J. PYE2and WILF POWELL2
1Laboratory of Entomology, Obihiro University of Agriculture and Veterinary Medicine,
Obihiro, Hokkaido, 080-8555, Japan
2
Rothamsted Research, Harpenden, Herts., AL5 2JQ, UK.
Abstract – The avoidance responses of aphid parasitoids with varying host ranges (Aphidius eadyi, Aphidius ervi and Praon volucre) to chemical trails deposited by intraguild predatory ladybirds, Coccinella septempunctata and Adalia bipunctata, were investigated. Females of all three parasitoid species significantly avoided leaves previously visited by C. septempunctata or A. bipunctata adults. The avoidance responses shown by the two Aphidius species were stronger to trails of
C. septempunctata than to thoseof A. bipunctata. However, P. volucre
avoided trails of both ladybird species to a similar degree. Dose-responses of these three parasitoid species to the hydrocarbons
n-tricosane (C23H48), n-pentacosane (C25H52) and n-heptacosane
(C27H56), which are components of the trails of both C. septempunctata
and A. bipunctata, were evaluated.Dual-choice bioassays indicated that 1) A. eadyi showed more sensitive avoidance responses to n-tricosane than did the other two parasitoid species, 2) all three species showed
similar responses to n-pentacosane across a range of doses, and 3) only
P. volucre showed avoidance responses to n-heptacosane. Quantitative analyses of each hydrocarbon in the trails of the two ladybird species showed that n-pentacosane and n-heptacosane occur in significantly greater amounts in C. septempunctata trails than in those of A. bipunctata. The trails of the two species also differ qualitatively in the other hydrocarbons present.
Key Words– Intraguild predation, predator avoidance, trail, oviposition decision, host range, habitat similarity, dose-response.
INTRODUCTION
Intraguild interactions between carnivores that share the same trophic resources include intraguild predation (IGP), which has been well documented (Rosenheim et al., 1993; Colfer and Rosenheim, 1995; Rosenheim et al., 1998; Ferguson and Stiling, 1996: Raymond et al., 2000; Snyder and Ives 2001), but also may involve interactions that serve to avoid intraguild predation (Doumbia et al., 1998; Taylor et al., 1998; Nakashima and Senoo, 2003). Immature stages of parasitoids often become intraguild prey in interactions between predators and parasitoids operating in the same guild, and so adult parasitoids would benefit by developing avoidance responses to cues from intraguild predator species living in the same habitats in order to reduce predation risks to their offspring. Aphids are associated with a large assemblage of insect natural enemies (Mackauer and Finlayson, 1967; Takada, 1968; Wheeler, 1977; Wratten and Powell, 1991; Ekbom, 1994), and so adult aphid parasitoids would encounter different types and species of intraguild predator during host searching. The more generalist aphid parasitoids, which can forage in a range of habitats, might be expected to develop a generalized response to predator cues. Semiochemicals mediating intraguild interactions are poorly defined. Hydrocarbons have been reported previously as components of chemical trails left on substrates by seven-spot ladybirds, Coccinella septempunctata (Kosaki and Yamaoka, 1996). The same study suggested strongly that large amounts of these chemicals were secreted from the tarsi. Recently, two aliphatic hydrocarbons,
responses by the aphid parasitoid, Aphidius ervi, towards C. septempunctata
(Nakashima et al., 2004).These hydrocarbons were present in the chemicaltrails left by the ladybirds on plant surfaces. Adult A. ervi females spent a significantly shorter time foraging on plants treated with these chemicals, resulting in significantly lower parasitism rates of the pea aphid, Acyrthosiphon pisum, than on untreated plants (Nakashima et al., 2004).
In this study we compare the responses to ladybird trails of three parasitoid species
that attack A. pisum but which differ greatly in their host and habitat ranges. A. eadyi
is a specialist on A. pisum (Pennacchio, 1989), and so forages predominantly in habitats containing legume plants, whereas eight aphid species belonging to seven genera, including A. pisum, have been recorded as host species of A. ervi (Pennacchio, 1989), allowing it to forage in a wider range of habitats. Praon volucre is an even more polyphagous parasitoid, attacking at least30 species belonging to 13 genera (Stary, 1976), which occur in a greater variety of habitats including forests. Therefore, each parasitoid species may be at risk from different species and numbers of intraguild predators due to these differences in the range of habitats in which they forage. For example, C. septempunctata occurs predominantly on herbaceous plants, whilst the two-spot ladybird, Adalia bipunctata, is found on both herbaceous plants and trees (Honék, 1985), suggesting that P. volucre would regularly encounter both C. septempunctata and A. bipunctata, but the two Aphidius species would probably encounter A. bipunctata much less frequently than C. septempunctata.It is therefore
ladybird trails than would the two Aphidius species, and that the Aphidius species should be more sensitive to the particular chemicals in trails of ladybirds that are dominant species in their preferred habitat.
The aims of our study were to compare the avoidance responses of these three parasitoid species to the chemical trails of the two ladybird species, and to measure their sensitivity to the different hydrocarbons present in the trails. The variability in avoidance responses among aphid parasitoids is discussed from the aspects of differences in the relative amounts of chemical compounds in the different ladybird trails and adaptive responses in accordance withthe parasitoids’ habitat/host ranges.
MATERIALS AND METHODS
Insects. The three aphid parasitoids, A. eadyi,A. ervi and P. volucre, were obtained from laboratory colonies that had been initiated with mummies of pea aphids, A. pisum. The first two species were initially collected from pea fields in Harpenden, Hertfordshire and Sharnbrook, Bedfordshire, UK during spring 2001, whilst P. volucre was collected in Harpenden during spring 2002. Overwintered, adult, seven-spot ladybirds, C. septempunctata, were collected from evergreen shrubs at Rothamsted Research during March and April, 2002. Overwintering, adult, two-spot ladybirds, A. bipunctata, were collected from window frames at Horticultural Research International (HRI) in East Malling, Kent, UK in November 2001. Parasitoids and ladybirds were both kept at 20 IC and a LD 16 : 8 h photoperiod, and provided with A. pisum, reared on broad bean plants, Vicia faba L. (Fabaceae) (var.
Sutton), as food. Parasitoids were removed from colony cages at the mummy stage, and were kept in Petri dishes (9 cm diameter, 1.5 cm height) containing honey solution on cotton wool as adult food until emergence. Two days after the first adult emergence was observed, females were individually confined in small Petri dishes (5.0 cm x 1.5 cm) with approximately 50 A. pisum and honey solution on cotton wool. All females used in bioassays were 4-5 days old.
Ladybird Chemicals. Extracts of ladybird chemical trails were prepared by confining a single C. septempunctata or A. bipunctata adult in a glass Petri dish (9 cm x 1.5 cm) for 18 hr at 20oC, then removing the ladybirds and washing the dishes with distilled hexane (10 ml per dish). Glass surfaces provide an ideal substrate for the
isolation and collection of insect chemical trails. The extracts were evaporated to
100 µl under a gentle stream of high purity nitrogen and stored in microvials at -20oC until further use. These procedures were repeated 6 times for adults of each ladybird species.
Gas Chromatography (GC). The ladybird footprint hexane extracts were analyzed on a Hewlett-Packard 6890A gas chromatograph (GC) equipped with a cool on-column injector, a flame-ionization detector (FID), and a 50 m x 0.32 mm (0.52
µm film thickness) i.d. HP-1 bonded-phase fused-silica capillary column (J & W Scientific). The oven temperature was maintained at 30oC for 0.5 minute, then programmed at 5oC/minute to 150oC, held at this temperature for 0.1 minute, then
programmed at 10oC/minute to 280oC. The carrier gas was hydrogen. Quantities of
n-tricosane, n-pentacosane and n-heptacosane in the extracts were determined by comparison of GC peak areas with those obtained from authentic samples (100 ng).
Coupled GC-Mass spectrometry (GC-MS). A capillary GC column (50 m x 0.32 mm i.d. HP-1) fitted with a cool on-column injector was directly coupled to a mass spectrometer (Thermo-Finnigan MAT95XP, Bremen, Germany). Ionization was by electron impact at 70eV, 200oC. The oven temperature was maintained at 30oC for 5 minutes, then programmed at 5oC/minute to 280oC. The carrier gas was helium. Tentative identifications were made by comparison of MS data with published spectra (NIST, 2002). Confirmation of tentative identifications was accomplished by peak enhancement on GC with authentic samples obtained from commercial sources (Pickett, 1990).
Chemicals. n-Tricosane (C23H48), n-pentacosane (C25H52) and n-heptacosane
(C27H56) (all 99% purity) were purchased from the Aldrich Chemical Company
(Gillingham, UK). For behavioral studies, individual solutions of these chemicals were prepared in distilled ethanol at the following concentrations: 10, 1, 0.1 and 0.01
µg/ml. All solvents were distilled prior to use.
Application of Chemicals on Plants. Pure authentic compounds in ethanol were applied on broad bean plants, V. faba, for use in behavioral bioassays, as previously
described (Nakashima et al., 2004). These solutions were applied to the plants (6-8 leaf stage, 1 plant per pot) using a rotary atomizer mounted on a multi-speed track. The atomizer operated at 4500 rpm and produced a drop size of approximately 110µm VMD. Applications were made at a velocity of 0.4 ms-1 at a height of 25 cm above the plants, providing an application rate of 1.04 mls m2. Control leaves were prepared in a similar manner using distilled ethanol. Using the ethanolic solutions of compounds prepared at the concentrations described above, and based on the application rate and the dimensions of the leaf squares used (see below), the leaf squares generated (1.5 cm x 1.5 cm) were coated with 4, 0.4, 0.04 and 0.004 ng test compound respectively.
Behavioral bioassays. The effects of C. septempunctata and A. bipunctata trails and trail extracts on aphid parasitoid responses were investigated using a dual-choice bioassay. Treatment leaves with ladybird trails were prepared by inserting a leaf from a bean plant (6-8 leaf stage) into a plastic container via a slit, releasing a ladybird adult into the container, allowing it to walk upon the leaf for 24 hr, and then removing it from the plant. Control leaves were left untouched for a similar length of time. Leaves were used for experiments immediately after exposure ended. Treated and control leaf squares (1.5 cm x 1.5 cm) were taken from the plants and placed 0.5 cm apart in a Petri dish (5 cm diameter), into which a single parasitoid female was then released. After allowing the parasitoid to settle (1 minute), the time spent foraging on each leaf square was measured for a period of 10 minutes. Each
experiment was repeated twenty times. To account for potential temporal effects, equal numbers of each of the three parasitoid species were tested each day, randomizing the order of testing between days.
Statistical analysis. The durations of visits by parasitoids were analyzed using Wilcoxon's signed rank tests and Mann-Whitney U test. The degree of avoidance was expressed as time allocated on leaf squares with treatments divided by total residence time on control and treatment leaf squares. The index of avoidance responses were analyzed by an ANOVA with chemical compounds, their concentrations and parasitoid species as main effects; the proportional data were arcsine transformed to stabilize the variance before this analysis. The amounts of trail chemical compounds were compared by Mann-Whitney U test. For the latter, the data were transformed to logarithms to stabilize the variance before analysis.
RESULTS
In dual-choice leaf square bioassays conducted immediately after leaf exposure to
C. septempunctata and A. bipunctata adults, parasitoids significantly avoided the leaf squares treated with ladybird trails in all combinations of parasitoid and ladybird species (Wilcoxon’s signed rank test, P< 0.05) (Figure 1). The degree of avoidance of C. septempunctata trails was stronger than that of A. bipunctata trails for A. eadyi
and A. ervi (Mann-Whitney U test, P< 0.05), but it was statistically similar for the trails of both ladybirds in the case of P. volucre (Figure 1).
To identify the chemicals responsible for parasitoid avoidance, hexane extracts of footprint trails from adult C. septempunctata and A. bipunctata were analyzed by high resolution GC and coupled GC-MS. Samples from both species comprised almost entirely of aliphatic hydrocarbons (Figure 2; Table 1). Components identified in both species were n-tricosane, n-pentacosane and n-heptacosane. By comparison with authentic samples using GC, the levels of n-tricosane, n-pentacosane and
n-heptacosane in the footprint trails of C. septempunctata and A. bipunctata adults were determined. Significantly larger amounts of n-pentacosane and n-heptacosane were found in the trails of C. septempunctata than in those of A. bipunctata
(Mann-Whitney U test, P< 0.05), but no significant difference was found in the levels of n-tricosane (Table 2). Other major aliphatic hydrocarbons were identified tentatively as branched hydrocarbons by comparison with MS data from similar studies elsewhere (Table 1; Nakashima et al., 2004; Hemptinne et al., 2001). Compounds identified specifically for C. septempunctata included 13-, 9- and 7-methylheptacosane, 9,13-, 7,11-dimethylheptacosane and 7,11,15- trimethyl- heptacosane. Compounds identified specifically for A. bipunctata were
n-heneicosane, 9- and 7-methyltricosane.
An ANOVA of proportions of time spent by parasitoids on leaf squares treated with individual hydrocarbons from ladybird trails indicated highly significant effects of both hydrocarbon type and concentration (Table 3). The interaction between hydrocarbon type and parasitoid species was also significant (Table 3), indicating that parasitoids differed in their responses to different hydrocarbons.
The total residence times of A. eadyi on leaf squares treated with n-tricosane were significantly shorter than those on control leaves for all treatment concentrations (Wilcoxon’s signed rank test, P< 0.05), but significant effects were only found at higher doses (0.4 and 4 ng) for A. ervi and only at the highest dose (4 ng) for P. volucre (Wilcoxon’s signed rank test, P < 0.05) (Figure 3). In the case of
n-pentacosane, the residence times for all three parasitoid species were significantly reduced by the hydrocarbon only at the two highest doses (0.4 and 4 ng)(Wilcoxon’s signed rank test, P< 0.05) (Figure 3). There were no significant differences between residence times on control leaf squares and those treated with n-heptacosane, at any concentrations used, in the cases of the two Aphidius species, but P. volucre spent significantly less time on treated leaf squares at the two highest doses (0.4 and 4 ng) (Wilcoxon’s signed rank test, P< 0.05) (Figure 3).
DISCUSSION
To our knowledge, this is the first published report that adult parasitoids vary in their avoidance of different species of intraguild predators and that this variability potentially relates to differences in the amounts of specific chemical compounds in the predator trails. The two Aphidius species tested responded more strongly to trails of C.septempunctata than to trails of A. bipunctata, but there were no statistically significant differences in the responses of P. volucre to the trails of each of the two ladybird species. Components identified in both species were n-tricosane,
been demonstrated previously (Nakashima et al., 2004). All three parasitoid species showed avoidance responses to n-tricosane and n-pentacosane, as shown in a previous study (Nakashima et al., 2004), but only P. volucre responded to
n-heptacosane. Other chemicals found specifically in C. septempunctata and A. bipunctata trails were not investigated for avoidance activity in this study, as previous work (Nakashima et al., 2004) showed that n-tricosane, n-pentacosane and
n-heptacosane accounted for parasitoid avoidance responses.
The differences in the levels of avoidance behavior shown towards the two different ladybird trails by both Aphidius species could be due to differences in the amounts of specific hydrocarbons present in the two trails (Table 2). n-Tricosane and
n-pentacosane are known to be the main ladybird avoidance stimuli for A. ervi, and these two chemicals additively increase avoidance responses (Nakashima et al., 2004). This finding, together with the present results suggest that larger amounts of
n-pentacosane in the trails of C. septempuncata may induce the stronger avoidance responses recorded for A. ervi and A. eadyi to trails of this ladybird than to those of A. bipunctata (Figure 1); the same chemical compounds stimulating the avoidance behaviour in these closely related species. P. volucre showed no difference in the level of response to trails of the two ladybird species, but this parasitoid responded to all three hydrocarbons and so may have a more general sensitivity to hydrocarbons in ladybird chemical trails.
The presence of aliphatic hydrocarbons specific to C. septempunctata and A.
ecology of the ladybirds. Studies elsewhere have implied that such compounds may play a role in mediating intraspecific ladybird interactions (Hemptinne et al., 2001), specifically the oviposition deterrent response of adult A. bipunctata following detection of intraspecific larval footprints. Previous work on avoidance of C. septempuncata footprints by A. ervi (Nakashima et al., 2004) revealed virtually identical hydrocarbon profiles for adults and larvae. Thus, it can be expected that these compounds do indeed play a role in C. septempunctata oviposition deterrent behavior, and that a similar outcome can be expected for A. bipunctata. Further studies are required to confirm the role of these compounds.
As a specialist parasitoid of pea aphid, A. eadyi is likely to restrict its foraging to habitats containing legume plants (Pennacchio, 1989). Thus, A. eadyi would encounter a limited number of ladybird species, and principally C. septempunctata, which is a dominant species in legume crops (Ekbom, 1994; Nakashima and Akashi, 2005). A. ervi also forages commonly on legume plants, but also forages on other arable crops, including cereals, where C. septempunctata is also the dominant aphidophagous ladybird (Wratten and Powell, 1991). Therefore, it may be adaptive for females of these two Aphidius species to optimize their sensitivity to the chemical trails of the ladybirds that they are most likely to encounter as intraguild predators, thereby maximizing the avoidance behavior that reduces the predation risk to their offspring. However, little is known about encounter rates in natural habitats and such studies are needed to help elucidate the evolutionary development of intraguild interactions.
A. eadyi was the most sensitive of the three parasitoids to n-tricosane, responding to very low concentrations. Detection of early instar larval predators, which deposit much smaller amounts of trail chemicals than adults and mature larvae, could be important because ladybird larvae are likely to stay and complete development within an aphid patch (Dixon, 2000). Additionally, ladybird trails appear to have relatively short active periods, possibly due to absorption of the hydrocarbons into the plant cuticular lipid layer, or even evaporation/sublimation from the leaf surface over time (Nakashima et al., 2004). Increased sensitivity to one of the chemical components of ladybird trails may help A. eadyi to perceive trails which A. ervi and
P. volucre cannot detect, giving this extreme specialist species a competitive advantage in the restricted range of habitats in which it forages. In contrast, P. volucre is a much more generalist parasitoid at both the host species and host plant levels (Pennacchio, 1989). In this case, a more generalized sensitivity to trail chemicals may be advantageous as parasitoids with broader host/habitat ranges would face a predation risk from a larger number of predator species.
The overall results showed that hydrocarbon types and their concentrations affected parasitoid avoidance responses, and the level of avoidance responses shown to the three chemical compounds differed among the parasitoid species (Figure 3). P. volucre avoided all three hydrocarbons in contrast to the Aphidius species, which avoided only n-tricosane and n-pentacosane. Thus, the range of trail chemicals detected by the generalist P. volucre was wider than that detected by the two
which is only known to attack the pea aphid, A. pisum, was the most sensitive to n-tricosane, responding to this chemical at the lowest concentration tested.
The results of the present study strongly suggest that oviposition preferences by aphid parasitoids in response to intraguild predators are common in Aphidius and
Praon species. Nakashima and Senoo (2003) and Nakashima et al. (2004) suggested that both intraguild predation and its avoidance play important roles in determining top-down forces by predator guilds, and consequently affect herbivore populations in local areas. Although several reports have revealed that aphidophagous predators such as coccinellids and chrysopids are deterred from ovipositing when exposed to areas where heterospecific competitors are present (Ruzicka, 1998; Ruzicka, 2001; Agarwala et al, 2003), there was little evidence for this behavior in parasitoids. We predict that intraguild predator avoidance by adult parasitoids should be widespread in other systems because immature parasitoids in/on hosts are always at risk from predators. Additionally, host/prey density is known to affect the degree of predator avoidance by parasitoids (Nakashima and Senoo, 2003) as well as affecting intraguild predation (Lucas et al., 1998). Further research should determine how extrinsic (e.g. prey density) and intrinsic (e.g. experience and physiological state) factors affect the magnitude of intraguild predation and its avoidance, in order to understand the role of these interactions in the dynamics of predator-parasitoid-herbivore systems.
supported by a Research Fellowship (to Y.N.) through the Japan Society for the Promotion of Science, and by the United Kingdom Department for Food, Environment and Rural Affairs (DEFRA). Rothamsted Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council (BBSRC) of the United Kingdom.
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TABLE 1. COMPOUNDS IDENTIFIED FROM COUPLED GC-MS ANALYSIS OF SEVEN-SPOT LADYBIRD, Coccinella septempunctata, AND
TWO-SPOT LADYBIRD, Adalia bipunctata, FOOTPRINT EXTRACTS ________________________________________________________________
Peak number (see Fig. 1) Compound
_________________________________________________________________ 1 n-Heneicosanea,c 2 n-Tricosaneb,c,d 3 9-Methyltricosanea,c 4 7-Methyltricosanea,c 5 n-Pentacosaneb,c,d 6 n-Heptacosaneb,c,d 7 13-Methylheptacosanea,d 8 9-Methylheptacosanea,d 9 7-Methylheptacosanea,d 10 9-13-Dimethylheptacosanea,d 11 7,11-Dimethylheptacosanea,d 12 7,11-15-Trimethylheptacosanea,d _____________________________________________________________________ a
Tentative identification, based upon fragmentation patterns and MS data published elsewhere (Hemptinne et al., 2001) b
Identification confirmed by peak enhancement on GC using authentic samples cCompound identified in adult two-spot ladybird, Adalia bipunctata
dCompound identified in adult seven-spot ladybird, Coccinella septempunctata
TABLE 2. MEAN LEVELSa(NANOGRAMS ± SE) OF THREE HYDROCARBONS FOUND IN CHEMICAL TRAIL EXTRACTS OF THE LADYBIRDS Coccinella
--- Log amount ± SE
---
Compound C. septempunctata A. bipunctata
---
n-Tricosane 2.77 ± 0.06 a 2.73 ± 0.14 a
n-Pentacosane 2.52 ± 0.11 a 2.13 ± 0.04 b
n-Heptacosane 2.69 ± 0.10 a 2.40 ± 0.06 b
--- Values followed by different letters in the same row are significantly different
(Mann-Whitney U test, P< 0.05).
a
TABLE 3. ANOVA FOR EFFECTS OF DIFFERENT HYDROCARBONS FROM LADYBIRD TRAILS, HYDROCARBON CONCENTRATION AND PARASITOID SPECIES ON PROPORTION OF TIME SPENT FORAGING BY FEMALE PARASITOIDS ON TREATED LEAF SQUARES
--- Factor df F P --- Hydrocarbon 2 8.1 <0.01 Concentration 3 5.9 <0.01 Parasitoid species 2 0.9 0.40 Hydrocarbon x Concentration 6 0.7 0.67
Hydrocarbon x Parasitoid species 4 2.6 0.036
Concentration x Parasitoid species 6 0.4 0.87
Hydrocarbon x Concentration 12 0.8 0.66
x Parasitoid species
---Figure legends
FIG. 1. Effect of seven-spot ladybird, Coccinella septempunctata, and two-spot ladybird,
Adalia bipunctata, chemical trails on time spent foraging by female aphid parasitoids,
Aphidius ervi,Aphidius eadyi and Praon volucre on broad bean, Vicia faba, leaf squares. Data are expressed as time spent on leaf squares treated with ladybird trails divided by total residence time on control and treated leaf squares in choice bioassays. Vertical lines indicate ± SE.
FIG. 2. Typical gas chromatograms (GC) of adult (a) seven-spot ladybird, Coccinella septempunctata (b) two-spot ladybird, Adalia bipunctata, footprint extracts. Peak numbers correlate to compounds listed in Table 1 identified by coupled GC-mass spectrometry (GC-MS). Extracts were collected from individual C. septempunctata and A. bipunctata
adults over 18hr in a glass Petri dish. Flame ionization detector (FID) responses are provided on the same scale for comparison.
FIG. 3. Response of female aphid parsitoids, Aphidius ervi, Aphidius eadyi and Praon volucre to leaf squares treated with either n-tricosane (C23H48), n-pentacosane (C25H52) or
n-heptacosane (C27H56). For each compound, concentrations of 10, 1, 0.1 and 0.01 µg/ml
were applied, equivalent to doses of 4, 0.4, 0.04 and 0.004 nanograms (ng) per leaf square. Data are expressed as time spent on leaf squares treated with ladybird trail hydrocarbons divided by total residence time on control and treated leaf squares. Vertical lines indicate ± SE. Asterisks indicate significant difference from control (Wilcoxon’s signed rank test, P<
0.25
0.3
0.35
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P. volucre
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Figure 1
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FIG. 2
Retention Time (min)
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ID Res
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(b)
0.2 0.3 0.4 0.5 0.6
