Mammalian serum stimulates <i>S. aureus</i> colony-spreading activity.

<p>(A) Stimulation of colony-spreading activity by calf serum. Overnight culture of <i>S. aureus</i> MRSA NI-15 was spotted on soft agar supplemented with serially diluted calf serum and incubated for 8 h at 37°C. Each plate contained 20 ml soft agar medium. (B) Stimulation of colony-spreading by porcine serum. Porcine serum or calf serum was serially diluted 2-fold and applied to 20 ml soft agar medium and its colony-spreading stimulatory activity was measured. Open circles indicate the halo diameters of giant colonies supplemented with porcine serum and filled circles indicate those supplemented with calf serum. Horizontal axis represents the volume of serum added to 20 ml soft agar medium in a plate. (C) Stimulation of colony-spreading activity by silkworm hemolymph. Hemolymph was collected from fifth instar larvae of silkworms and applied to the soft agar plates in 2-fold serial dilutions and its colony-spreading stimulatory activity was measured. Open circles indicate the halo diameters of giant colonies supplemented with silkworm hemolymph and filled circles indicate those supplemented with calf serum. (D) Growth curves of MRSA NI-15, MW2, or FRP3757 in tryptic soy broth supplemented with or without 1.25% (v/v) calf serum.</p

Gel filtration column chromatography of calf serum.

<p>(A) Elution profile of gel filtration column chromatography using a Superdex 200. Open circles indicate diameters of colonies. Filled circles indicate absorbance at 280 nm. Molecular weight markers were eluted in the fraction described below. Catalase (250-kDa) was eluted in fraction 21, bovine serum albumin (66-kDa) in fractions 25–26, and cyanocobalamin (1.3-kDa) in fraction 38. 250-µl aliquots of each sample were applied to soft agar medium and their colony-spreading stimulatory activity was measured. (B) SDS-PAGE analysis of gel filtration fractions. The gel was stained with Coomassie Brilliant Blue. A 66-kDa protein coincided with the colony-spreading stimulatory activity in fractions 24–29.</p

Stimulation of colony-spreading by bovine serum albumin.

<p>(A) Colony-spreading stimulation by purified bovine serum albumin. Purified bovine serum albumin of 96% grade (open triangles, Nacalai, cat. no. 01861-26) or 99% grade (open circles, Sigma, cat. no. A0281) was applied to soft agar by 2-fold serial dilution and their colony-spreading stimulatory activities against MRSA NI-15 strain were measured. (B) Elution profile of DEAE-cellulose column chromatography using bovine serum albumin (96% grade, Nacalai). Open circles indicate the colony-spreading stimulatory activity. Filled circles indicate absorbance at 280 nm. (C) SDS-PAGE analysis of DEAE-cellulose column chromatography fractions. The gel was stained with Coomassie Brilliant Blue. The 66-kDa protein coincided with colony-spreading stimulatory activity in fractions 10–20. (D) Bovine serum albumin (Nacalai), fatty acid-free albumin from bovine serum (Wako Chemicals, cat. no. 017-15146), casein from bovine milk (Sigma, cat. no. C4032), or fetuin from calf serum (Sigma, cat. no. F2379) was applied to soft agar by 2-fold serial dilution and their colony-spreading stimulatory activities on the MRSA NI-15 strain were measured.</p

Purification of colony-spreading stimulatory factor in calf serum.

<p>Calf serum (19 ml) was used as the starting medium. To measure the colony-spreading stimulatory activity, samples were serially diluted 2-fold and applied to 20 ml of soft agar and incubated for 8 h at 37°C. The diameters of giant colonies were measured from each dish, and stimulatory activities were calculated.</p

Diverse colony-spreading response to calf serum among HA-MRSA and CA-MRSA strains.

<p>(A) Representative images of the colony-spreading activity of MRSA NI strains stimulated by calf serum. Overnight cultures of MRSA NI strains were spotted onto soft agar plates supplemented with or without calf serum (250 µl/plate) and incubated for 8 h. (B) Colony-spreading response of CA-MRSA strains to calf serum. Overnight cultures of MW2 (USA400, open circles) or FRP3757 (USA300, filled circles) were spotted onto soft agar plates supplemented with 2-fold serially diluted calf serum and incubated for 8 h. The halo diameter was measured. (C) Amount of PSMα3 in MRSA strains cultured in the presence or absence of calf serum. MRSA strains with high colony-spreading response against calf serum (NI-15, MW2, FRP3757, NI-5, NI-7, NI-27, NI-29, NI-36, and NI-38) were cultured in the presence or absence of 1.25% (v/v) calf serum and the culture supernatants were analyzed by HPLC as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097670#pone.0097670-Kaito4" target="_blank">[6]</a>. Asterisks means not detected.</p

Lipoprotein and albumin-depleted serum decreases colony-spreading stimulatory activity.

<p>(A) Stimulatory activities of calf serum, lipoprotein-depleted serum, lipoprotein- and albumin-depleted serum were examined. Fractionation was performed from a 50-ml volume of calf serum containing 3850 units of stimulatory activity. Each fraction was applied to soft agar by 2-fold serial dilution and their colony-spreading stimulatory activities on the MRSA NI-15 strain were measured. Relative total activities of each fraction compared with that of calf serum are presented in graph. (B) SDS-PAGE analysis of the lipoprotein-depleted serum and Affi-Gel blue gel column effluent. 10 µg protein of the lipoprotein-depleted serum and the Affi-Gel blue gel column effluent was electrophoresed in 12.5% SDS-polyacrylamide gel.</p

Identification of lipoprotein particles as a colony-spreading stimulator.

<p>(A) Protein from each purification step was electrophoresed by SDS-PAGE. The gel was stained with Coomassie Brilliant Blue. (B) The final fraction (Fraction IV) was serially diluted 2-fold, and the colony-spreading stimulatory activity was measured. (C) Elution profile of DEAE-cellulose column chromatography using Fraction III. <i>Open circles</i> indicate diameters of colony-spreading. <i>Filled circles</i> indicate absorbance at 280 nm. 500-µl aliquots of each sample were applied to the soft agar medium and their colony-spreading stimulatory activity was measured. (D) SDS-PAGE analysis of DEAE-cellulose column chromatography fractions. The 25-kDa protein coincided with colony-spreading stimulatory activity in fractions 46-58. The 25-kDa protein was identified as apolipoprotein A1 by peptide-mass fingerprinting. (E) Colony-spreading stimulation by HDL particles. Purified HDL, delipidated HDL, or recombinant human apolipoprotein A1 (Wako Chemicals, cat. no. 019-20731) was applied to soft agar by 2-fold serial dilution and their colony-spreading stimulatory activities on the MRSA NI-15 strain were measured. Human apolipoprotein A1 shares 78% amino acids identity with calf apolipoprotein A1. (F) Agarose gel electrophoresis analysis of calf serum, HDL, LDL, and lipoprotein-depleted serum. Agarose gel electrophoresis was performed according to a previous report <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097670#pone.0097670-Noble1" target="_blank">[23]</a>. Each fraction (4 µg phospholipid) was electrophoresed in agarose 0.5%, and then lipoprotein bands were detected by lipid-specific staining using Oil Red O. The electrophoresed protein amount of calf serum, HDL, LDL, and lipoprotein-depleted serum was 500 µg, 5 µg, 5 µg, and 1800 µg, respectively. (G) Colony-spreading stimulation by phosphatidylcholine. Purified HDL, lipid extract from HDL, phosphatidylcholine from egg yolk (99% purity, Sigma, cat. no. P3556), or cholesterol (99% purity, Sigma, cat. no. C8667) was applied to soft agar by 2-fold serial dilution and their colony-spreading stimulatory activities on the MRSA NI-15 strain were measured. (H) Colony-spreading stimulation by HDL, LDL, and VLDL. Fractionated HDL, LDL, or VLDL was applied to soft agar by 2-fold serial dilution and their colony-spreading stimulatory activities on the MRSA NI-15 strain were measured.</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

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