Relationship between forewing mirror size and whole body size.

<p><b>(A)</b> Male forewings were dissected as shown in the panel. Measurements of the area (mm²) of the harp and mirror regions were obtained as indicated in magenta and blue, respectively. (B) Correlations between the harp size (magenta circle) or mirror size (blue circle) and whole body mass. The vertical axis shows the area (mm²) and the horizontal axis shows the cricket mass (mg). Both areas correlated significantly with the body mass. The correlation coefficient (r) and p values are presented in the graph.</p

No Effect of Body Size on the Frequency of Calling and Courtship Song in the Two-Spotted Cricket, <i>Gryllus bimaculatus</i>

<div><p>The relationship between body size and vocalization parameters has been studied in many animal species. In insect species, however, the effect of body size on song frequency has remained unclear. Here we analyzed the effect of body size on the frequency spectra of mating songs produced by the two-spotted cricket, <i>Gryllus bimaculatus</i>. We recorded the calling songs and courtship songs of male crickets of different body sizes. The calling songs contained a frequency component that peaked at 5.7 kHz. On the other hand, courtship songs contained two frequency components that peaked at 5.8 and 14.7 kHz. The dominant frequency of each component in both the calling and courtship songs was constant regardless of body size. The size of the harp and mirror regions in the cricket forewings, which are the acoustic sources of the songs, correlated positively with body size. These findings suggest that the frequency contents of both the calling and courtship songs of the cricket are unaffected by whole body, harp, or mirror size.</p></div

Analysis of the frequency spectra in calling and courtship songs, and body-size effects in <i>G</i>. <i>bimaculatus</i>.

<p><b><i>(A)</i></b><i>Upper panel</i>. The spectrogram of the calling song from a representative male. The vertical axis shows the frequency in kHz, and the horizontal axis shows the time in seconds. The color indicates the energy of each frequency component in dB (dBFS, decibels relative to full scale). The color scale is shown in the right panel. <i>Lower panel</i>. An oscillogram of the calling song from the representative male corresponding to the above spectrogram. The vertical axis shows the sound amplitude, and the horizontal axis shows the time [s]. (B) Distribution of the frequency component of calling songs (n = 50). The vertical axis shows the density, and the horizontal axis shows the frequency (kHz). The histogram (gray) and distribution curve from the kernel density estimate (magenta). The distribution curve shows a single peak at 5.7 kHz. The frequency band analyzed in the following experiment (Fig 1C) is indicated in orange. (C) Evaluation of the body-size effect on the dominant frequency of the calling song. The dominant frequency of the 2-s calling song was calculated for each male (n = 50) and plotted against the individual body mass. The vertical axis shows the dominant frequency (kHz), and the horizontal axis shows the body mass (mg). The background color of the band corresponds to the orange band shown in Fig 1B. No significant effect was detected (p = 0.572). Statistical information is summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146999#pone.0146999.t001" target="_blank">Table 1</a>. The significance level was adjusted by Bonferroni correction. (D) <i>Upper panel</i>. The spectrogram of the courtship song from a representative male. The vertical axis shows the frequency in kHz, and the horizontal axis shows the time in seconds. The color indicates the energy of each frequency component in dB (dBFS, decibels relative to full scale). The color scale is shown on the right panel. <i>Lower panel</i>. An oscillogram of the courtship song from the representative male corresponding to the above spectrogram. The vertical axis shows the sound amplitude, and the horizontal axis shows the time [s]. (E) Distribution of the frequency component of courtship songs (n = 53). The vertical axis shows the density, and the horizontal axis shows the frequency (kHz). The histogram (gray) and distribution curve from the kernel density estimate (magenta). The distribution curve shows two peaks at 5.8 and 14.7 kHz. The frequency bands analyzed in the following experiment (Fig 1C) are indicated in green or in blue. (F) Evaluation of the body-size effect on the dominant frequency of the courtship song. The dominant frequency of the 2-s courtship song was calculated for each male (n = 53) and plotted against the individual body mass. The vertical axis shows the dominant frequency (kHz), and the horizontal axis shows the body mass (mg). Each background color of the band corresponds to the green or blue band shown in Fig 1E. No significant effect was detected (p = 0.241 for lower band, p = 0.042 for higher band). Statistical information is summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146999#pone.0146999.t001" target="_blank">Table 1</a>. The significance level was adjusted by Bonferroni correction.</p

Colony-spreading activities of <i>S</i>. <i>aureus</i> strains expressing different PSMs in different localization.

<p>A. The <i>S</i>. <i>aureus</i> PSMα1-4/δ-toxin knockout strain was transformed with genes encoding PSMα1, PSMα2, PSMα3, PSMα4, PSMα1–4, and δ-toxin, which were placed under a strong or weak promoter. Overnight cultures of <i>S</i>. <i>aureus</i> Newman strain (Parent), the PSMα1-4/δ-toxin knockout strain transformed with an empty vector (pND50K), and the strains transformed with different PSM genes were spotted on soft agar plates and incubated at 37°C. The photograph was obtained at 9 h after incubation. B. The diameter of the giant colonies in <i>A</i> was measured. Data are means±standard errors from three independent experiments. Asterisks indicate Student’s t-test p value between PSMs expressing plasmid and pND50K less than 0.05.</p

Competitive binding assay of PSMs against <i>S</i>. <i>aureus</i> cell surface.

<p><b>A.</b> Inhibitory activity of δ-toxin against PSMα2 binding to the <i>S</i>. <i>aureus</i> cell surface of the PSMα1-4/δ-toxin knockout strain was measured. Binding assay of PSMα2 (10 nmol) to the cell surface of the PSMα1-4/δ-toxin knockout strain was performed in the absence or presence of δ-toxin (0, 10, 20, and 30 nmol) and the amount of PSMα2 bound to the cell surface was measured (left graph). In the competition assay, the binding of δ-toxin to the <i>S</i>. <i>aureus</i> cell surface was also measured (center graph) and the binding of total PSM (PSMα2 and δ-toxin) is presented (right graph). In all graphs, horizontal axis represents the amount of PSM added to <i>S</i>. <i>aureus</i> cells and vertical axis represents the amount of PSM bound to <i>S</i>. <i>aureus</i> cells (3 x 10⁸ CFU). B. Inhibitory activity of PSMα2 against δ-toxin binding to the <i>S</i>. <i>aureus</i> cell surface was measured. Binding assay of δ-toxin (10 nmol) to the cell surface of the PSMα1-4/δ-toxin knockout strain was performed in the absence or presence of PSMα2 (0, 10, 20, and 30 nmol) and the amount of δ-toxin bound to the cell surface was measured (left graph). In the competition assay, binding of PSMα2 to the <i>S</i>. <i>aureus</i> cell surface was also measured (center graph) and the binding of total PSM (δ-toxin and PSMα2) is presented (right graph). C. Inhibitory activity of δ-toxin against PSMα3 binding to the <i>S</i>. <i>aureus</i> cell surface was measured. Binding assay of PSMα3 (10 nmol) to the cell surface of the PSMα1-4/δ-toxin knockout strain was performed in the absence or presence of δ-toxin (0, 10, 20, and 30 nmol) and the amount of PSMα3 bound to the cell surface was measured (left graph). In the competition assay, binding of δ-toxin to the <i>S</i>. <i>aureus</i> cell surface was also measured (center graph) and the binding of total PSM (PSMα3 and δ-toxin) is presented (right graph). D. Inhibitory activity of PSMα3 against δ-toxin binding to <i>S</i>. <i>aureus</i> cell surface was measured. Binding assay of δ-toxin (10 nmol) to the cell surface of the PSMα1-4/δ-toxin knockout strain was performed in the absence or presence of PSMα3 (0, 10, 20, and 30 nmol) and the amount of δ-toxin bound to the cell surface was measured (left graph). In the competition assay, binding of PSMα3 to the <i>S</i>. <i>aureus</i> cell surface was also measured (center graph) and the binding of total PSM (δ-toxin and PSMα3) is presented (right graph).</p

Summary of the cell surface PSMα1–4 and the colony spreading in <i>S</i>. <i>aureus</i> gene knockout strains.

<p>PSMα1–4 and δ-toxin are presented as orange and blue dots, respectively. Knockout of δ-toxin increases the amount of cell surface PSMα1–4. In contrast, knockout of PSMα1–4 does not affect the amount of cell surface δ-toxin. The amount of cell surface PSMα1–4 and the colony-spreading activity in the wild-type strain, the δ-toxin knockout strain, the PSMα1–4 knockout strain, and the PSMα1-4/δ-toxin knockout strain is summarized in the lower part of this figure. The amount of cell surface PSMα1–4 is a determinant of colony-spreading activity.</p

Presence of phenol soluble modulins on the <i>S</i>. <i>aureus</i> cell surface.

<p>A. S. <i>aureus</i> Newman overnight cultured cells were washed in water, 5 M NaCl, 2% CHAPS, 8 M urea, 6 M guanidine HCl, or 3 M LiCl. In another sample, <i>S</i>. <i>aureus</i> cells were digested with lysostaphin and treated with 2% CHAPS. Samples were centrifuged and the amount of PSMα3 or PSMα1+δ-toxin in the supernatant was measured by HPLC. Vertical axis represents the amounts of PSM recovered from <i>S</i>. <i>aureus</i> cells (1.33 ml bacterial culture). Data are means±standard errors from three independent experiments. B. The centrifuged supernatants obtained in <i>A</i> were analyzed by SDS-PAGE. Proteins in the supernatants were precipitated with 10% trichloroacetic acid and electrophoresed on a 12.5% SDS polyacrylamide gel. The gel was stained by Coomassie brilliant blue. Each lane contains proteins from the same number of <i>S</i>. <i>aureus</i> cells (0.09 ml bacterial culture). C. <i>S</i>. <i>aureus</i> Newman overnight cultured cells were washed in 6 M guanidine HCl or 2% SDS. Samples were centrifuged and the amount of PSMα3 in the supernatant was measured by HPLC. Vertical axis represents the amount of PSMα3 recovered from <i>S</i>. <i>aureus</i> cells (1.33 ml bacterial culture). Data are means±standard errors from triplicate experiments.</p

Amount of PSMα1–4 and δ-toxin on the <i>S</i>. <i>aureus</i> cell surface and in the culture supernatant.

<p>A. <i>S</i>. <i>aureus</i> Newman strain was cultured for 19 h. Cells were washed with 6 M guanidine HCl and PSMs on the cell surface were obtained. PSMs on the cell surface (from 1.33 ml bacterial culture) and in the culture supernatant (from 0.267 ml bacterial culture) were analyzed by HPLC. Dotted line indicates the respective PSMs. B. The amount of PSMs on the cell surface or in the culture supernatant was measured. Vertical axis represents the amount of each PSM per 1 ml bacterial culture. Data are means±standard errors from three independent experiments.</p

Correlation analysis between the amount of cell surface PSMs and the colony-spreading activity in <i>S</i>. <i>aureus</i> clinical isolates.

<p>HA-MRSA isolates (n = 40), CA-MRSA isolates (n = 14), and Newman strain were cultured for 19 h. The total amount of PSMα1, PSMα2, and PSMα3 (PSMα1–3) (<i>A</i>) or the amount of δ-toxin (<i>B</i>) in each strain was measured by HPLCs and the mean value from three independent experiments was plotted on the horizontal axis as the relative value against that of Newman strain. The colony-spreading activity of each strain, which was reported in our previous study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164523#pone.0164523.ref018" target="_blank">18</a>], was plotted on the vertical axis. A linear approximation and correlation coefficient are presented in the graph.</p

Summary of the cell surface PSMα1–4 and the colony spreading in <i>S</i>. <i>aureus</i> gene knockout strains.

<p>PSMα1–4 and δ-toxin are presented as orange and blue dots, respectively. Knockout of δ-toxin increases the amount of cell surface PSMα1–4. In contrast, knockout of PSMα1–4 does not affect the amount of cell surface δ-toxin. The amount of cell surface PSMα1–4 and the colony-spreading activity in the wild-type strain, the δ-toxin knockout strain, the PSMα1–4 knockout strain, and the PSMα1-4/δ-toxin knockout strain is summarized in the lower part of this figure. The amount of cell surface PSMα1–4 is a determinant of colony-spreading activity.</p