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

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

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

Primed Immune Responses Triggered by Ingested Bacteria Lead to Systemic Infection Tolerance in Silkworms

<div><p>In the present study, we examined whether microorganisms collaterally ingested by insects with their food activate the innate immune system to confer systemic resistance against subsequent bacterial invasion. Silkworms orally administered heat-killed <i>Pseudomonas aeruginosa</i> cells showed resistance against intra-hemolymph infection by <i>P</i>. <i>aeruginosa</i>. Oral administration of peptidoglycans, cell wall components of <i>P</i>. <i>aeruginosa</i>, conferred protective effects against <i>P</i>. <i>aeruginosa</i> infection, whereas oral administration of lipopolysaccharides, bacterial surface components, did not. In silkworms orally administered heat-killed <i>P</i>. <i>aeruginosa</i> cells, <i>P</i>. <i>aeruginosa</i> growth was inhibited in the hemolymph, and mRNA amounts of the antimicrobial peptides cecropin A and moricin were increased in the hemocytes and fat body. Furthermore, the amount of paralytic peptide, an insect cytokine that activates innate immune reactions, was increased in the hemolymph of silkworms orally administered heat-killed <i>P</i>. <i>aeruginosa</i> cells. These findings suggest that insects sense bacteria present in their food by peptidoglycan recognition, which activates systemic immune reactions to defend the insects against a second round of infection.</p></div

Resistance of silkworms orally administered heat-killed <i>P</i>. <i>aeruginosa</i> cells against <i>P</i>. <i>aeruginosa</i> infection.

<p>A. Experimental scheme of the study. Heat-killed bacterial cells were mixed with artificial diet and administered to silkworms for 2 d. Live bacteria were then injected into the silkworm hemolymph. B. Silkworms were fed a diet containing heat-killed <i>P</i>. <i>aeruginosa</i> cells (heat-killed PAO1 diet, n = 51) or a normal diet (n = 50) for 2 d and then injected with <i>P</i>. <i>aeruginosa</i>. Results from five independent trials (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130486#pone.0130486.s003" target="_blank">S1 Table</a>, exp.1-3 and 5–6) were combined into a single analysis. The combined survival curve is shown in the figure. Survival of silkworms fed the diet containing heat-killed <i>P</i>. <i>aeruginosa</i> was significantly higher than that of silkworms fed the normal diet (p = 4.59E-11). None of the mock-infected silkworms died (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130486#pone.0130486.s001" target="_blank">S1 Fig</a>).</p

Model of primed immune responses triggered by ingestion of bacteria.

<p>Silkworms ingest bacteria with their food. The ingested bacteria activate systemic immune responses in the gut, which leads to tolerance against bacterial invasion into the hemolymph.</p

Activation of systemic immunity in silkworms by oral administration of heat-killed <i>P</i>. <i>aeruginosa</i>.

<p>A. Hemolymph samples were collected from fifth instar silkworms (n = 3) fed a diet containing heat-killed <i>P</i>. <i>aeruginosa</i> cells for 23 h, and living <i>P</i>. <i>aeruginosa</i> was inoculated into the hemolymph and incubated at 37°C. After incubation, samples were diluted and spread on agar plates for measurement of viable cell numbers. Data are shown as mean ± SD. Asterisk indicates p < 0.05 (Student’s t-test). A representative result from two independent experiments is shown. B, C, D. Total RNAs were extracted from the midgut, fat body, or hemocytes of fifth instar silkworms fed diet containing heat-killed <i>P</i>. <i>aeruginosa</i> cells for 2 d. The amounts of cecropin A, moricin, and IKKγ mRNA in the extracted RNA fractions were then measured. Each RNA amount was normalized by the mRNA amount of elongation factor-2. Values in the silkworms fed with heat-killed <i>P</i>. <i>aeruginosa</i> cells relative to that in the silkworms fed with normal diet were log-transformed, and the mean values ± SD from three independent experiments are shown. Asterisks indicate significant difference compared with silkworms on the normal diet (p < 0.05, Student’s t-test). The log-transformed values were used in the statistical analysis because they exhibited higher normality than the non-transformed values. E. Hemolymph samples were collected from silkworms (n = 4) fed a normal diet or diet containing heat-killed <i>P</i>. <i>aeruginosa</i> cells. The samples, containing 28 μg protein, were electrophoresed using 16.5% tricine sodium dodecyl sulfate-polyacrylamide gels. Proteins in the gels were then transferred to a membrane and analyzed by Western blotting using an anti-PP antibody. Asterisk indicates the active-form of PP. A representative result from two independent experiments is shown.</p

Primers used in this study.

<p>Primers used in this study.</p

Resistance of silkworms orally administered with heat-killed cells of various microorganisms against <i>P</i>. <i>aeruginosa</i> infection.

<p>A. Silkworms were fed a normal diet (n = 20) or a diet containing heat-killed cells of <i>S</i>. <i>marcescen</i>s (n = 20) for 2 d, and then infected with <i>P</i>. <i>aeruginosa</i> in hemolymph. Results from two independent trials (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130486#pone.0130486.s003" target="_blank">S1 Table</a>, exp. 3–4) were combined into a single analysis. The combined survival curve is shown in the figure. Survival of silkworms fed a diet containing heat-killed <i>S</i>. <i>marcescens</i> cells was significantly higher than that of silkworms fed a normal diet (p = 0.0219). None of the mock-infected silkworms died (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130486#pone.0130486.s001" target="_blank">S1 Fig</a>). B. Silkworms were fed a normal diet (n = 20) or a diet containing heat-killed <i>S</i>. <i>aureus</i> cells (n = 20) for 2 d, and then injected with <i>P</i>. <i>aeruginosa</i>. Results from two independent trials (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130486#pone.0130486.s003" target="_blank">S1 Table</a>, exp. 5–6) were combined into a single analysis. The combined survival curve is shown in the figure. Survival of silkworms fed a diet containing heat-killed <i>S</i>. <i>aureus</i> cells was not significantly different from that of silkworms fed a normal diet (p = 0.235). None of the mock-infected silkworms died (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130486#pone.0130486.s001" target="_blank">S1 Fig</a>). C. Silkworms were fed a normal diet (n = 20) or a diet containing heat-killed <i>C</i>. <i>albicans</i> cells (n = 20) for 2 d, and then injected with <i>P</i>. <i>aeruginosa</i>. Results from two independent trials (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130486#pone.0130486.s003" target="_blank">S1 Table</a>, exp. 5–6) were combined into a single analysis. The combined survival curve is shown in the figure. Survival of silkworms fed a diet containing heat-killed <i>C</i>. <i>albicans</i> cells was significantly higher than that of silkworms fed a normal diet (p = 0.0494). None of the mock-infected silkworms died (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130486#pone.0130486.s001" target="_blank">S1 Fig</a>).</p