Symbiotic yeasts on <i>Doubledaya bucculenta</i> eggs and <i>Pleioblastus simonii</i> internodes.

<p>(A) An egg deposited on the inner surface of the internode. Black arrows a to e indicate sites whose cryo-SEM micrographs were taken and from which microbes were isolated. (B) Yeast colonies (black arrowheads) growing at the inner opening of <i>D. bucculenta</i> oviposition hole on a <i>P. simonii</i> internode. Yeast colonies are invisible on the egg surface. White to translucent pieces are fragmented dead pith tissues of <i>P. simonii</i>. (C to H) Cryo-SEM micrographs of (C) the anterior end of an egg (a in Figure A), which came out of the ovipositor first during oviposition and was attached to the inner surface of internode, (D) the lateral side of an egg (b in Figure A), (E) the posterior end of an egg (c in Figure A), which came out of the ovipositor finally during oviposition and was held in the air after oviposition, (F) the edge of the inner opening of an oviposition hole (d in Figure A). h-oviposition hole. (G) The inner surface of internode (e in Figure A) 1 cm distant from an oviposition hole, and (H) the anterior end of an egg when a mite was found at the inner opening of an oviposition hole. White arrowheads indicate the yeast cells. </p

Female adult and behavioral sequence of egg deposition of <i>Doubledaya bucculenta</i>.

<p>(A) A female adult. (B) A female adult inserting the ovipositor into the internode cavity of <i>Pleioblastus simonii</i> for oviposition. (C to H) Behavioral sequence of <i>D. bucculenta</i> egg deposition. (C) An ovipositor being inserted into the internode cavity via the oviposition hole the female made. (D) An egg coming out of the ovipositor. The anterior end of banana-shaped egg is shown. (E, F) Inflated (E) and deflated (F) states of the distal part of ovipositor. Before the ovipositor is detached from the egg, it showed peristaltic movement. (G) Egg detached from the ovipositor completely. (H) Egg left behind on the inner surface of internode cavity. Immediately after depositing an egg, she pulled out the ovipositor. (I) Time course of behavioral events in oviposition. Capitals in parentheses correspond to the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079515#pone-0079515-g001" target="_blank">Figures 1C to 1H</a>. In total it took 83 and 148 sec to complete the oviposition behavior from the appearance to disappearance of the ovipositor inside the cavity in bamboo internodes.</p> <p>Abbreviation: o, the distal part of ovipositor.</p

Phylogenetic placement of the yeast isolates associated with <i>Doubledaya bucculenta</i>.

<p>(A) A neighbor-joining phylogeny inferred from DNA sequences of D1/D2 domain of 26S rRNA gene (530 bps). (B) A neighbor-joining phylogeny inferred from DNA sequences of EF-1α gene (709 bps). (C) A neighbor-joining phylogeny inferred from DNA sequences of ITS/5.8S rRNA gene (360 bps). Bootstrap values (1000 replicates) of 50% or higher are shown at the nodes. Sequence accession numbers are shown in brackets. Yeast isolates obtained from <i>D. bucculenta</i> and <i>Pleioblastus simonii</i> in this study are discriminated by sites of eggs and <i>P. simonii</i> internodes where the isolate was obtained, the number to specify an egg and the internode harboring the egg, and the localities where the isolates were sampled. Abbreviations: AE, the anterior end of an egg; LS, the lateral side of an egg; PE, the posterior end of an egg; OH, the edge of the inner opening of an oviposition hole. ^aMiyazaki Prefecture, Japan; ^bIbaraki Prefecture, Japan; ^ca yeast isolate with colony texture different from <i>Wickerhamomyces anomalus</i>, which was obtained at the inner opening of an oviposition hole.</p

Effects of <i>Doubledaya bucculenta</i> larvae on the dispersal of <i>Wickerhamomyces anomalus</i> yeast cells.

<p>(A) A <i>W. anomalus</i> colony formed by the cells that were inoculated in the center of a potato dextrose agar (PDA) plate in the absence of a <i>D. bucculenta</i> larva. (B) <i>W. anomalus</i> colonies formed by the cells that were inoculated in the center of a PDA plate in the presence of a <i>D. bucculenta</i> larva. (C) No <i>W. anomalus</i> colony formation on an autoclaved <i>Pleioblastus simonii</i> internode strip where a yeast-inoculated dead egg of <i>D. bucculenta</i> was placed. (D) <i>W. anomalus</i> colonies formed on an autoclaved <i>P. simonii</i> internode strip where a yeast-inoculated living egg of <i>D. bucculenta</i> was placed. PDA Plates and internode strips were photographed three and four days after the incubation at 25 °C, respectively. White and black arrows indicate a first instar larva of <i>D. bucculenta</i> and a place where a yeast-inoculated egg of <i>D. bucculenta</i> was placed, respectively. Arrowheads indicate the yeast colonies. Abbreviation: e, egg.</p

The lizard beetle <i>Doubledaya bucculenta</i>, the host bamboo <i>Pleioblastus simonii</i>, and the associated yeast <i>Wickerhamomyces anomalus</i>.

<p>(A) An adult female of <i>D. bucculenta</i>. (B) An oviposition mark of <i>D. bucculenta</i>. An outside view of an oviposited dead bamboo culm is shown. (C) A cross section of an oviposited dead bamboo culm. The oviposition hole is wider outside and narrower inside. (D) A larva of <i>D. bucculenta</i> feeding on fungal layer on the inner surface of the bamboo internode. Arrows indicate the fungal layer. (E) The inner wall of the bamboo internode without oviposition mark. Arrowheads indicate pith tissues of the bamboo. (F) Yeast colonies isolated from <i>D. bucculenta</i> on a potato dextrose agar plate. (G) A light microscopic image of budding yeast cells obtained from <i>D. bucculenta</i>.</p

Mycangium associated with the ovipositor of adult females of <i>Doubledaya bucculenta</i>.

<p>(A) A dorsal view of the tergum of the eighth abdominal segment. (B) A ventral view of the eighth abdominal segment. (C) A lateral view of the eighth abdominal segment that was embedded in paraffin and cut longitudinally. (D) A longitudinal tissue section of the eighth abdominal segment, stained with periodic acid-Schiff reagent and hematoxylin. Arrows in (B), (C) and (D) indicate the opening of mycangium. Solid lines in (A), (C) and (D) correspond to the front edge of mycangial cavity. Abbreviations: M, mycangium; O, ovipositor; Y, yeast cells; AT, anterior; DO, dorsal; PT, posterior; VE, ventral.</p

Effects of the yeast symbiont on larval growth of <i>Doubledaya bucculenta</i>.

<p>(A) Larval growth curves on sterile potato dextrose agar (PDA) plates (open circles), on PDA plates on which the yeast strain was fully grown (filled circles), and on sterilized bamboo strips (filled triangles). Means and standard deviations are shown. (B) A 14-day-old larva reared on a sterile PDA plate. (C) A 14-day-old larva reared on a yeast-inoculated PDA plate.</p

Phylogenetic placement of the yeast strains associated with <i>Doubledaya bucculenta</i>.

<p>(A) A maximum parsimony phylogeny inferred from 26S rRNA gene sequences (530 bps). (B) A maximum parsimony phylogeny inferred from EF-1α gene sequences (802 bps). Bootstrap values of 50% or higher are shown at the nodes. For each yeast strain obtained from <i>D. bucculenta</i>, isolation source and collection locality are indicated. Sequence accession numbers are in brackets. ^a Miyazaki Prefecture, Japan; ^b Ibaraki Prefecture, Japan.</p