Roups, and other large bands from 1100 to 1000 cm21 attributed to the carbon-oxygen single bonds of sugars, including sucrose, fructose and Beta-D-(+)-glucose (Fig. 4). Peaks in the region from 2850 to 3050 cm21 due to C bonds were weak and ill-defined, which is characteristic of wet sugar samples. Given these findings, we reasoned that if wax was present in the honeydew of ACP females and nymphs, as our SEM studies abovePsyllid Honeydew: Behavior, Structure CompositionFigure 2. Structure of the anal area in nymphs and adults of the Asian citrus psyllid. A . Scanning electron micrographs (SEM) of nymphs (A , ventral views) and adult females (D . dorsal views) showing position of the circumanal ring (cr) around the anus (an) near the posterior end of the abdomen (boxed areas in A and D). In B C (nymphs), note the ornate cuticular ridges around that ring (arrows), wax pores (wp), and the narrow cuticular slits (sl) with wax filament (wf) oozing out. In E F (females), note the outer and inner rows (or ir, respectively) of wax pores and the waxy Title Loaded From File material (wm) coming out of these pores (in E). G . Light and scanning electron micrographs (lateral, dorso-lateral and dorsal views, respectively) of D. citri males showing the anal tube (at), anus (an), aedeagus (ae), and lateral plates (lp); note lack of the circumanal ring 16985061 or any cuticular ridges or wax pores around the anus. J. SEM of a male’s honeydew droplet (on a citrus leaf) showing no filamentous structures on the surface like those found on the honeydew of nymphs or females. Other abbreviations: at, antenna; cs, circumabdominal setae; st, stylets. doi:10.1371/journal.pone.0064938.gPsyllid Honeydew: Behavior, Structure CompositionFigure 3. Scanning electron micrographs of waxy structures on the surface of honeydew and circumabdominal setae of the Asian citrus psyllid. A. Honeydew tube (arrow) attached to the exuvia (ex) of a fifth instar nymph (dorsal view); note long circumabdominal setae (cs) around the abdomen. B C. Higher magnifications of the wax filaments (wf) found on the surface of the nymphal honeydew oozing out of wax pores in the circumanal ring (cr). D E. Details of the bases of the circumabdominal setae (cs) of 5th instar nymphs, showing the waxy material covering these setae (arrows). F. Adult female pellet (slightly open at the center) showing the wax structures (ws) on the surface, and gluey material devoid of filamentous structures inside (asterisks). G H. Higher magnifications of the wax structures (ws) as filaments or ribbons on the surface of female pellets. doi:10.1371/journal.pone.0064938.gsuggested, it must be only on the surface of the honeydew pellets or tubes produced by females and nymphs respectively. Thus, the T-bronchodilator spirometry 10 minutes later. These patients were nebulized for 30 seconds with samples were subjected to FTIR reflectance microscopy, in whichintact honeydew samples were not crushed but simply scanned after the microscope is carefully focused on the upper surface of the honeydew structures. While the large peaks mentioned abovePsyllid Honeydew: Behavior, Structure CompositionFigure 4. FTIR microscope reflection spectra of the surface of male, female and nymphal honeydew excretions (top three panels) compared to attenuated total reflectance FTIR spectra of typical monosaccharides. The honeydew spectra contain many peaks attributable to simple saccharides, especially the broad peak centered at 3300 cm21 attributable to the O bonds in sugars and water, and broad poorly resolved peaks at frequencies lower than 1500.Roups, and other large bands from 1100 to 1000 cm21 attributed to the carbon-oxygen single bonds of sugars, including sucrose, fructose and Beta-D-(+)-glucose (Fig. 4). Peaks in the region from 2850 to 3050 cm21 due to C bonds were weak and ill-defined, which is characteristic of wet sugar samples. Given these findings, we reasoned that if wax was present in the honeydew of ACP females and nymphs, as our SEM studies abovePsyllid Honeydew: Behavior, Structure CompositionFigure 2. Structure of the anal area in nymphs and adults of the Asian citrus psyllid. A . Scanning electron micrographs (SEM) of nymphs (A , ventral views) and adult females (D . dorsal views) showing position of the circumanal ring (cr) around the anus (an) near the posterior end of the abdomen (boxed areas in A and D). In B C (nymphs), note the ornate cuticular ridges around that ring (arrows), wax pores (wp), and the narrow cuticular slits (sl) with wax filament (wf) oozing out. In E F (females), note the outer and inner rows (or ir, respectively) of wax pores and the waxy material (wm) coming out of these pores (in E). G . Light and scanning electron micrographs (lateral, dorso-lateral and dorsal views, respectively) of D. citri males showing the anal tube (at), anus (an), aedeagus (ae), and lateral plates (lp); note lack of the circumanal ring 16985061 or any cuticular ridges or wax pores around the anus. J. SEM of a male’s honeydew droplet (on a citrus leaf) showing no filamentous structures on the surface like those found on the honeydew of nymphs or females. Other abbreviations: at, antenna; cs, circumabdominal setae; st, stylets. doi:10.1371/journal.pone.0064938.gPsyllid Honeydew: Behavior, Structure CompositionFigure 3. Scanning electron micrographs of waxy structures on the surface of honeydew and circumabdominal setae of the Asian citrus psyllid. A. Honeydew tube (arrow) attached to the exuvia (ex) of a fifth instar nymph (dorsal view); note long circumabdominal setae (cs) around the abdomen. B C. Higher magnifications of the wax filaments (wf) found on the surface of the nymphal honeydew oozing out of wax pores in the circumanal ring (cr). D E. Details of the bases of the circumabdominal setae (cs) of 5th instar nymphs, showing the waxy material covering these setae (arrows). F. Adult female pellet (slightly open at the center) showing the wax structures (ws) on the surface, and gluey material devoid of filamentous structures inside (asterisks). G H. Higher magnifications of the wax structures (ws) as filaments or ribbons on the surface of female pellets. doi:10.1371/journal.pone.0064938.gsuggested, it must be only on the surface of the honeydew pellets or tubes produced by females and nymphs respectively. Thus, the samples were subjected to FTIR reflectance microscopy, in whichintact honeydew samples were not crushed but simply scanned after the microscope is carefully focused on the upper surface of the honeydew structures. While the large peaks mentioned abovePsyllid Honeydew: Behavior, Structure CompositionFigure 4. FTIR microscope reflection spectra of the surface of male, female and nymphal honeydew excretions (top three panels) compared to attenuated total reflectance FTIR spectra of typical monosaccharides. The honeydew spectra contain many peaks attributable to simple saccharides, especially the broad peak centered at 3300 cm21 attributable to the O bonds in sugars and water, and broad poorly resolved peaks at frequencies lower than 1500.
