How accurate these descriptions were, given the resolution of the light microscopy when compared with SEM. However, our SEM work here provided much finer details of the wax pores and cuticular slits found on the circumanal ring of ACP females and/or nymphs. The wax pores we described here, especially in ACP nymphs, are somewhat ultrastructurally similar to openings of the wax glands, described as `canaliculated cuticle’, in the nymphal anal ring of the psyllid A. mori [28]. But they are considerably different from thePsyllid Honeydew: Behavior, Structure CompositionFigure 5. FTIR microscope reflection spectra of the surface of male, female and nymphal honeydew (top three panels) compared to attenuated total reflectance FTIR spectrum of beeswax, a typical ester wax. While all the honeydew spectra are dominated by saccharide peaks (as shown in Fig. 4), the female and nymphal honeydew spectra are markedly different from that of the male. Peaks at 2850 and 2910 cm21 (which are attributable to C bonds in a long chain aliphatic molecule) and the peak at 1732 cm21 (which is attributable to the C = O of an ester carboxyl group) strongly indicate the presence of an ester wax on the surface of honeydew of females and nymphs. doi:10.1371/journal.pone.0064938.g`wax pores’ found on wings of the flatid planthopper Metcalfa pruinosa 16985061 (Fulgoroidae) [33], or from `pores in the wax gland plates’ on the cuticle of some aphid species [39]. The primary role of the wax layer on the honeydew of ACP 4-IBP web nymphs and females is likely to limit/minimize the contamination of nymphs and eggs by the thick, sticky and sugary honeydew. Similarly, Smith [39] suggested that the primary role of the secreted wax on the surface of aphid cuticle is to prevent the aphids becoming contaminated by their own honeydew as well as that of other members of a colony. This may also be the purpose of the difference in behavior, reported here, between ACP males and females in their honeydew excretion behavior. Males deposit their honeydew droplets, devoid of waxy material, immediately behind them, whereas the females cannot do the same (otherwise they will likely smother their deposited eggs and newly hatched nymphs with this sticky material). Hence, females not only produce waxcovered excretions but also propel them some distance to the side away from their bodies, their eggs, and newly hatched nymphs. Heavy feeding and honeydew production by nymphs or adults of another psyllid (Ctenarytaina thysanura) greatly reduced the attractiveness of terminal shoots for oviposition [40]. This deterrence was associated with the production of honeydew resulting in the development of sooty moulds. That ACP nymphs and females have certain mechanisms and adaptations to avoid contaminationwith honeydew whereas ACP males apparently do not is interesting, and suggests that ACP males and females may occupy different sites on citrus plants, especially with regard to egg laying. ACP females normally lay their eggs on very young, feathery and Title Loaded From File folded leaves, or in the axillary corners between young terminal shoots and leaf petioles (Fig. 1H) [2,5,6,7], whereas males were not normally observed feeding on these sites (data not shown). In addition to the wax covering of their honeydew excretions, ACP nymphs seem to have developed two more ways to minimize contamination with honeydew. First, the wax-covered circumabdominal setae (around the abdomen), found in nymphs but not in adults of either sex, appear to keep t.How accurate these descriptions were, given the resolution of the light microscopy when compared with SEM. However, our SEM work here provided much finer details of the wax pores and cuticular slits found on the circumanal ring of ACP females and/or nymphs. The wax pores we described here, especially in ACP nymphs, are somewhat ultrastructurally similar to openings of the wax glands, described as `canaliculated cuticle’, in the nymphal anal ring of the psyllid A. mori [28]. But they are considerably different from thePsyllid Honeydew: Behavior, Structure CompositionFigure 5. FTIR microscope reflection spectra of the surface of male, female and nymphal honeydew (top three panels) compared to attenuated total reflectance FTIR spectrum of beeswax, a typical ester wax. While all the honeydew spectra are dominated by saccharide peaks (as shown in Fig. 4), the female and nymphal honeydew spectra are markedly different from that of the male. Peaks at 2850 and 2910 cm21 (which are attributable to C bonds in a long chain aliphatic molecule) and the peak at 1732 cm21 (which is attributable to the C = O of an ester carboxyl group) strongly indicate the presence of an ester wax on the surface of honeydew of females and nymphs. doi:10.1371/journal.pone.0064938.g`wax pores’ found on wings of the flatid planthopper Metcalfa pruinosa 16985061 (Fulgoroidae) [33], or from `pores in the wax gland plates’ on the cuticle of some aphid species [39]. The primary role of the wax layer on the honeydew of ACP nymphs and females is likely to limit/minimize the contamination of nymphs and eggs by the thick, sticky and sugary honeydew. Similarly, Smith [39] suggested that the primary role of the secreted wax on the surface of aphid cuticle is to prevent the aphids becoming contaminated by their own honeydew as well as that of other members of a colony. This may also be the purpose of the difference in behavior, reported here, between ACP males and females in their honeydew excretion behavior. Males deposit their honeydew droplets, devoid of waxy material, immediately behind them, whereas the females cannot do the same (otherwise they will likely smother their deposited eggs and newly hatched nymphs with this sticky material). Hence, females not only produce waxcovered excretions but also propel them some distance to the side away from their bodies, their eggs, and newly hatched nymphs. Heavy feeding and honeydew production by nymphs or adults of another psyllid (Ctenarytaina thysanura) greatly reduced the attractiveness of terminal shoots for oviposition [40]. This deterrence was associated with the production of honeydew resulting in the development of sooty moulds. That ACP nymphs and females have certain mechanisms and adaptations to avoid contaminationwith honeydew whereas ACP males apparently do not is interesting, and suggests that ACP males and females may occupy different sites on citrus plants, especially with regard to egg laying. ACP females normally lay their eggs on very young, feathery and folded leaves, or in the axillary corners between young terminal shoots and leaf petioles (Fig. 1H) [2,5,6,7], whereas males were not normally observed feeding on these sites (data not shown). In addition to the wax covering of their honeydew excretions, ACP nymphs seem to have developed two more ways to minimize contamination with honeydew. First, the wax-covered circumabdominal setae (around the abdomen), found in nymphs but not in adults of either sex, appear to keep t.
