CHEMICAL ECOLOGY Sex Attraction in cockerelli (: )

1 CHRISTELLE GUE´ DOT, DAVID R. HORTON, AND PETER J. LANDOLT

U.S. Department of Agriculture, Agricultural Research Service, 5230 Konnowac Pass Rd., Wapato, WA 98951

Environ. Entomol. 39(4): 1302Ð1308 (2010); DOI: 10.1603/EN10048 ABSTRACT The potato psyllid, Bactericera (ϭParatrioza) cockerelli (Sˇulc) (Hemiptera: Triozidae), is a major pest of potato. We examined the role of chemical signals in sex attraction, assessing male and female response to male- and female-produced volatile chemicals. In laboratory olfactometer assays, potato psyllid males were attracted to odorants emitted from live females and from solvent extract of females. These results indicate that the female-produced chemicals responsible for attracting males may be isolated by means of extractions. Males were also attracted to volatile chemicals from males and extracts of males, providing the Þrst example of maleÐmale attraction in the . Males exposed simultaneously to odorants from conspeciÞc females and males were preferentially attracted to female odorants, suggesting the presence of a female-speciÞc sex attractant for males. Potato psyllid females avoided volatile chemicals emitted by females and extracts of females and by volatile chemicals emitted by males and extracts of males. Possible explanations for avoidance of conspeciÞcs by females are discussed. This study is the Þrst report of male attraction to volatile chemicals emitted by females and female extracts in the Triozidae and more speciÞcally in the potato psyllid.

KEY WORDS mate location, olfactometer, potato, insect extract, pheromone

The potato psyllid, also known as the tomato psyl- with the Psylloidae have recently shown male attrac- lid, Bactericera (ϭParatrioza) cockerelli (Sˇ ulc) tion to female-produced volatile chemicals in three (Hemiptera: Triozidae), is native to North America species of Psyllidae, the pear psylla Cacopsylla bidens and is found in western north America, Mexico, Cen- (Sˇulc) (Soroker et al. 2004), the pear psylla C. pyricola tral America, and New Zealand (Liu and Trumble (Fo¨rster) (Horton and Landolt 2007; Horton et al. 2007, Teulon et al. 2009) on a variety of host species, 2007, 2008; Gue´dot et al. 2009), and the Asian citrus with a preference for plants in the (Wallis psyllid, Diaphorina citri (Kuwayama) (Wenninger et 1955). It has been a major pest of potato (Solanum al. 2008). Despite the economic importance of the tuberosum L.) and tomato (Solanum lycopersicum L.) potato psyllid as a vector of psyllid yellows and zebra for many years (Pletsch 1947, Wallis 1955). The potato chip, no studies have investigated the role of chemical psyllid has long been associated with the psyllid yel- signals in mate-Þnding and mate selection with this lows disease of potato (Richards 1928, Richards and pest. Blood 1933, Wallis 1955). The potato psyllid is closely Previous work with the pear psylla C. pyricola and associated with zebra chip, an emerging disease of C bidens indicated that males were attracted to fe- commercial potatoes (Secor and Rivera-Varas 2004), male-infested pear shoots and to females even in the and was recently identiÞed as a vector for the patho- absence of the host plant, suggesting that the females gen of this disease (Munyaneza et al. 2007a, b, 2008; are the source of the attraction rather than the infested Secor et al. 2009). Potato psyllids transmit the bacte- foliage (Soroker et al. 2004, Horton and Landolt 2007, rium Candidatus solanacearum also known as Candidatus Liberibacter psyllaurous, which Horton et al. 2007, Gue´dot et al. 2009). It has long been is presumed to be a causative agent for zebra chip established that the insect cuticle is composed of a disease (Liefting et al. 2008, 2009; Hansen et al. 2008; mixture of lipids and constitutes a reservoir of chem- Abad et al. 2009, Secor et al. 2009, Sengoda et al. 2010). icals that can play an important role in chemical com- Current management practices for the potato psyl- munication (for review, see Singer 1998; Howard and lid rely primarily on the use of insecticides (Goolsby Blomquist 1982, 2005; Howard 1993). Gue´dot et al. et al. 2007). The identiÞcation of semiochemical at- (2009) showed with C. pyricola that cuticular extracts tractants for potato psyllids could provide additional of females were attractive to males and that these tools for monitoring and management of this pest. extracts were as attractive as live females in choice Studies on the role of chemical signals in mate location assays. These results suggested that the chemicals col- lected through insect extractions include the compo- 1 Corresponding author, e-mail: [email protected]. nents of the female-produced pheromone attractant. August 2010 GUE´DOT ET AL.: SEX ATTRACTION IN POTATO PSYLLID 1303

In this study, our Þrst objective was to test whether in Horton and Landolt (2007), was constructed of a potato psyllid males are attracted to female-produced 2.5-cm-diameter glass tube 27 cm in length, having two volatile chemicals. We tested both live females and arms each 7 cm in length and oriented at an angle of extracts of females as odor sources, as was done with 135Њ to one another. The Y-tube was used horizontally C. pyricola (Gue´dot et al. 2009). We also compared with a Ϸ15Њ incline. Compressed air (Oxarc, Spokane, extracts of females against live females to test whether WA) provided airßow through a charcoal Þlter, an air the female extracts were as effective at attracting humidiÞer, and paired 1-liter glass jars containing odor males as the live females. sources. Each jar was connected to an arm of the Behavioral assays with C. pyricola have suggested Y-tube olfactometer by a 25-cm-length of 2-mm-di- that males avoid volatiles emitted by conspeciÞc males ameter polytetraßuoroethylene hose (Cole Parmer (Horton et al. 2008, Gue´dot et al. 2009). Male-pro- Instrument, Vernon Hills, IL). During the assays, air- duced sex pheromones can act in conspeciÞc inter- ßow was maintained at 50 ml/min through each arm actions as attractants (Landolt and Heath 1990, Leal et of the olfactometer. Preceding each assay, air was al. 1998, Kirk and Hamilton 2004) or as repellents passed through the whole system at 50 ml/min into (Zhang and Aldrich 2003). Our second objective was each arm, including the jars containing the odor to assess male response to odorants from live males and sources, for 15 min. from extracts of males to determine whether the maleÐ Psylla Extracts. Extractions were done between male repellency reported for C. pyricola (Horton et al. 1200 and 1630 hours (P.S.T.). Each extraction con- 2008, Gue´dot et al. 2009) also occurs in B. cockerelli. sisted of 25 psyllids transferred into a 10-ml glass vial Several studies have suggested that females in the containing 300 ␮l pentane for a period of 5 min, during Psyllidae are not attracted to volatiles from either sex. which the glass vial was agitated by hand. The solvent For example, females of the pear psylla C. bidens and was then transferred by Pasteur pipette to another the citrus psyllid D. citri did not respond to either male clean glass vial (extract). Simultaneously with each or female conspeciÞcs in laboratory bioassays (Soro- extraction, 300 ␮l of pentane with no psylla was agi- ker et al. 2004, Wenninger et al. 2008). In Þeld exper- tated by hand for 5 min to act as the control treatment. iments, C. pyricola females did not show a preference All pentane samples (both extracts and controls) were for either male- or female-baited traps compared with stored at 0ЊC until the assays were conducted. Ap- unbaited traps (Brown et al. 2009). Our third objective proximately 1 h before the bioassays were conducted, was to assess the response of female B. cockerelli to the pentane samples were warmed at room tempera- male- and female-produced volatile chemicals. We ture. Extracts and solvent controls were applied in tested female response to both live females and males their entirety to Þlter paper disks (55 mm ID; What- and to extracts of females and males. We also com- mann #1 Cat. 1001 055; Whatmann, Maidstone, United pared these extracts with live to determine Kingdom) with glass syringes (Hamilton Company, whether female responses to extracts were similar to Reno, NV) and allowed to evaporate in a fume hood female responses to live insects. for 1 min. Each Þlter paper disk was folded and placed into the jars, and within 3 min the jars were attached to the olfactometer. Each olfactometer assay con- Materials and Methods sisted of paired 1-liter glass jars containing either Source of Insects. Assays were completed using lab- the extract or solvent control. oratory-reared potato psyllids. Cultures were started Assay Methods. Six experiments were conducted. by collecting adult potato psyllids from potato Þelds in Within each experiment, multiple two-treatment Weslaco, TX, and Dalhart, TX. To obtain insects for comparisons were run in random order. Each com- use in the assays, adult psyllids were removed from parison of paired odor sources consisted of 15 repli- culture and placed on potato plants (5Ð10 cm in cates, with each replicate comprising 10 psyllids as- height) at densities of 25Ð30 psyllids per plant. Plants sayed one at a time. Psyllids to be assayed were placed and insects were reared under long-day conditions (16 in a 50-ml holding vial Ϸ30 min preceding an assay. L:8 D) at 25ЊC. The psyllids were allowed to oviposit Psyllids were individually allowed to enter the olfac- for 7Ð10 d. Adults were removed from each plant and tometer and were given 10 min to enter an arm of the discarded. Egg-laden plants were examined every 2 d Y-tube. Choice was recorded when a psyllid came into to collect newly eclosed adults, thus not preventing contact with the upwind end of an arm at the point of mating to occur. Psyllids were separated by sex within insertion for the Teßon hose (Horton and Landolt 1 h of collection to eliminate opportunities for any 2007). For each replicate, after the Þrst Þve psyllids additional mating and moved onto clean potato ter- were assayed, the arms of the olfactometer were ro- minal shoot clippings bearing four to Þve leaves (5Ð10 tated 180Њ horizontally, and the last Þve psyllids were cm in height) in groups of 25Ð30 individuals of the assayed. After each replicate, glassware and Teßon same sex and similar ages. The plants and adult psyllids hoses were soaked in hot soapy water, rinsed with were enclosed in 135-ml ventilated plastic cages and water, acetone, and hexane, and baked in an oven at stored under long-day conditions for 3Ð6 d until the 150ЊC for at least 2 h. insects were assayed. Experiment 1: Male Response to Live Females and Y-Tube Olfactometer. A Y-tube olfactometer was Males. Three comparisons were conducted: (1) 25 used to assess the response of male and female potato females versus empty jar; (2) 25 males versus empty psyllids to olfactory cues. The olfactometer, described jar; and (3) 25 females versus 25 males. Psyllids were 1304 ENVIRONMENTAL ENTOMOLOGY Vol. 39, no. 4

Fig. 1. Numbers of potato psyllid males (mean Ϯ SEM) that chose paired odor sources. For each paired comparison, n ϭ 15 groups of 10 males assayed individually. (A) Responses of males to live females versus empty jar, live males versus empty jar, and live females versus live males. (B) Responses of males to extract of females versus solvent control and to extract of males versus solvent control. (C) Responses of males to live females versus extract of females and to live males versus extract of males. *Choice is signiÞcant at P Ͻ 0.05. **Choice is signiÞcant at P Ͻ 0.001. moved into the 1-liter glass jars for eventual attach- The Shapiro-Wilk statistic in PROC UNIVARIATE ment to the olfactometer 2 h before each assay. was used to test the normality assumption. When the Experiment 2: Male Response to Extracts of Fe- normality assumption was not met, the paired differ- males and Males. Two comparisons were made: (1) ences were analyzed using a signed-ranks test in female extract versus control and (2) male extract PROC UNIVARIATE (Zar 1999; Horton et al. 2007, versus control. Each extract consisted of 25 psyllids 2008). In all comparisons, the signed-ranks test yielded extracted in pentane for 5 min. signiÞcant differences, possibly leading to the com- Experiment 3: Male Response to Live Females Ver- mission of a type I error. We thus opted to use results sus Extracts of Females and to Live Males Versus obtained with paired sample t-tests. Extracts of Males. Two comparisons were assayed: (1) live females versus female extract and (2) live Results males versus male extract. In each treatment, 25 psyl- lids were used. For the live psyllid treatments, each jar Experiment 1: Male Response to Live Females and contained a control Þlter paper onto which a control Males. All 450 males that were assayed made a choice pentane sample was applied. (entered one arm or the other of the olfactometer) Experiment 4: Female Response to Live Females within the 10-min cut-off time. SigniÞcantly more and Males. Three comparisons were assayed: (1) 25 males (65%) chose the jar containing live females females versus empty jar; (2) 25 males versus empty compared with the empty jar (t ϭ 6.49; df ϭ 14; P ϭ jar; and (3) 25 females versus 25 males. Psyllids were 0.0001; Fig. 1A, upper bar). Sixty-seven percent of moved into the 1-liter glass jars for eventual attach- males chose the live males when paired with an empty ment to the olfactometer 2 h before each assay. jar (t ϭ 5.23; df ϭ 14; P ϭ 0.0001; Fig. 1A, middle bar). Experiment 5: Female Response to Extracts of Fe- SigniÞcantly more males (64%) chose live females males and Males. Two comparisons were made: (1) when paired with live males (t ϭ 4.37; df ϭ 14; P ϭ female extract versus control and (2) male extract 0.0006; Fig. 1A, lower bar). versus control. Each extract consisted of 25 psyllids Experiment 2: Male Response to Extracts of Fe- extracted in pentane for 5 min. males and Males. All 300 males that were assayed Experiment 6: Female Response to Live Females made a choice within 10 min. Sixty-four percent of Versus Extracts of Females and to Live Males Versus males selected the jar containing the female extract Extracts of Males. Two comparisons were conducted: when paired with an empty jar (t ϭ 4.37; df ϭ 14; P ϭ (1) live females versus female extract and (2) live 0.0006; Fig. 1B, upper bar). Similarly, 63% of males males versus male extract. Jars containing live psyllids chose the male extract when paired with an empty jar each contained a Þlter paper disk onto which a control (t ϭ 5.29; df ϭ 14; P ϭ 0.0001; Fig. 1B, lower bar). pentane sample had been applied. Experiment 3: Comparison of Male Response to Data Analysis. SAS version 9.1 for Windows was Live Females Versus Extracts of Females and to Live used for all statistical analyses (SAS Institute 2002). Males Versus Extracts of Males. All 300 males that Bioassay data were analyzed using paired sample t- were assayed made a choice within the 10-min cut-off tests in PROC TTEST (Horton et al. 2007, 2008). The time. Statistically, more males (58% of males) chose t-test assumes a normal distribution of the arithmetic the live females when paired against the female ex- differences between paired observations (Zar 1999). tracts (t ϭ 2.57; df ϭ 14; P ϭ 0.02; Fig. 1C, upper bar). August 2010 GUE´DOT ET AL.: SEX ATTRACTION IN POTATO PSYLLID 1305

Fig. 2. Numbers of potato psyllid females (mean Ϯ SEM) that chose paired odor sources. For each paired comparison, n ϭ 15 groups of 10 females assayed individually. (A) Responses of females to live females versus empty jar, live males versus empty jar, and live females versus live males. (B) Responses of females to extract of females versus solvent control and to extract of males versus solvent control. (C) Responses of females to live females versus extract of females and to live males versus extract of males. *Choice is signiÞcant at P Ͻ 0.05. **Choice is signiÞcant at P Ͻ 0.001.

Males did not show a signiÞcant preference between emitted by potential mates, could also be involved in live males (55%) and male extracts (t ϭ 1.95; df ϭ 14; mate location in psyllids. Krysan (1990) suggested that P ϭ 0.07; Fig. 1C, lower bar). male C. pyricola use visual recognition to orient to- Experiment 4: Female Response to Live Females ward a female. Additionally, observations indicated and Males. All 450 females that were assayed made a that mating was reduced in total darkness with C. choice within the 10-min cut-off time. SigniÞcantly pyricola (Krysan 1990) and D. citri (Wenninger and more females selected the empty jar when paired with Hall 2007). A number of psyllid species produce live females (t ϭ 6.32; df ϭ 14; P Ͻ 0.0001; Fig. 2A, acoustic signaling by means of substrate-borne vibra- upper bar) or live males (t ϭ 2.95; df ϭ 14; P ϭ 0.01; tions (Ossiannilsson 1950; Tishechkin 1989, 2006; Fig. 2A, middle bar), with 37% of females choosing the Percy et al. 2006; Wenninger et al. 2009). In these live females and 43% choosing the live males. Females species, acoustic signals are caused by wing vibrations did not show a preference between live males (47%) of the insect and are probably used to mediate sexual compared with live females (53%) (t ϭ 1.29; df ϭ 14; encounters (Tishechkin 1989, 2006; Percy et al. 2006; P ϭ 0.22; Fig. 2A, lower bar). Wenninger et al. 2009). However, acoustic signaling Experiment 5: Female Response to Extracts of Fe- does not always precede successful pairing of the sexes males and Males. All 300 females that were assayed (Tishechkin 2006, 2007; Brown 2008), suggesting that made a choice within 10 min. Fifty-nine percent of additional signals, such as olfactory cues, are involved females selected the jar containing the solvent control in mate location. when paired with the female extract (t ϭ 2.58; df ϭ 14; The results presented here showed that B. cockerelli P ϭ 0.022; Fig. 2B, upper bar). Similar results were males are attracted to female-produced volatile chem- obtained with the male extract (t ϭ 3.38; df ϭ 14; P ϭ icals. Such an attraction has also been shown with 0.0045; Fig. 2B, lower bar), with 39% of females choos- three other species of psyllids: C. bidens, C. pyricola, ing the male extract. and D. citri (Soroker et al. 2004; Horton and Landolt Experiment 6: Comparison of Female Response to 2007; Horton et al. 2007, 2008; Wenninger et al. 2008, Live Females Versus Extracts of Females and to Live Gue´dot et al. 2009). This is the Þrst report showing Males Versus Extracts of Males. All 300 females that male attraction to volatile chemicals emitted by fe- were assayed made a choice within 10 min. Females males in the Triozidae and more speciÞcally in the did not show a signiÞcant preference for either the live potato psyllid. This study investigated male response females (t ϭ 1.10; df ϭ 14; P ϭ 0.29; Fig. 2C, upper bar) to odorants from females in the absence of a host plant or the live males (t ϭ 0.74; df ϭ 14; P ϭ 0.5; Fig. 2C, and did not address the potential for interaction be- lower bar) when paired with the female extract or the tween host plant and female odorants on male re- male extract, respectively. In both comparisons, 53% sponse. Previous studies have shown with the pear of females chose the live females and the live males. psylla C. pyricola that the presence of the pear host did not enhance male attraction to female-produced vol- atile chemicals in similar laboratory assays (Horton et Discussion al. 2008, Gue´dot et al. 2009). However, the attractive- This study focused on the use of olfactory signals ness of C. bidens females to males was enhanced when involved in mate Þnding in the potato psyllid. Other females were in the presence of a host plant (Soroker types of signaling, such as visual and acoustic signals et al. 2004). Further studies are needed to address the 1306 ENVIRONMENTAL ENTOMOLOGY Vol. 39, no. 4 possible role of host plant volatiles on female attrac- gered stronger antennal responses in C. bidens females tiveness and male attraction to potato psyllid female compared with volatile collections from uninfested odorants. plants, suggesting that females can perceive the pres- Potato psyllid males were attracted to extracts of ence of females (Soroker et al. 2004). Whether the females that had been applied to Þlter paper disks. increased antennal response is indicative of any be- Although live females were slightly more attractive to havioral response between females remains to be an- males than an extract of females (58 versus 42%, re- swered in this species. In potato psyllids, the femaleÐ spectively), these results suggest that the female-pro- female repellency could be an epideictic type duced chemicals responsible for male attraction might pheromone, which might limit competition between be isolated by means of extraction of females with a females for feeding or oviposition sites. Potato psyllid nonpolar solvent. Cuticular lipids have been identiÞed females have been reported to produce Ϸ200Ð300 for numerous insect species (Singer 1998). Although eggs on average (Knowlton and Janes 1931, Wallis the primary role of insect cuticular hydrocarbons is to 1955, Abdullah 2008), and the femaleÐfemale repel- minimize water loss (Nelson 1978, Lockey 1988, lency could also limit competition for resources be- Howard 1993), cuticular hydrocarbons can also act as tween their offspring (Hodges and Dobson 1998). intraspeciÞc recognition signals, communicating in- Potato psyllid females also avoided volatile chemi- formation such as sex, species, and physiological state cals from live males and extracts of males. This result (for review, see Singer 1998; Howard and Blomquist seems to be consistent with behavioral observations 1982, 2005). Generally, these hydrocarbons are much made by Soroker et al. (2004) for a different psyllid, less volatile than volatilized attractants that are per- C. bidens. Wenninger et al. (2009) reported that Asian ceived at some distance from the insect. However, citrus psyllid females did not respond to male-pro- pheromones may be released from the cuticle, or re- duced volatile chemicals when paired with a blank. It leased through the cuticle, and be extracted by insect is not clear why potato psyllid females would be re- solvent washes. More speciÞcally, in the Psylloidea, pelled by males. Observations of mating behavior with cuticular extractions were recently used to isolate and a number of psyllid species have indicated that females identify a female-produced sex attractant pheromone remain stationary, whereas males are actively search- for males of the pear psylla C. pyricola (Gue´dot et al. ing for females (Burts and Fischer 1967, Krysan 1990, 2009, 2010). Tishechkin 2006, Percy et al. 2006, Brown, 2008). In Potato psyllid males were attracted to male-pro- laboratory assays with highly male-biased sex ratios, duced chemicals from live males and extract of males. oviposition in C. pyricola was reduced (Burts and Previous studies with the pear psylla C. pyricola indi- Fischer 1967), and females moved away from host cated that males avoided male-produced volatiles plants (Horton and Lewis 1995). These studies attrib- (Horton et al. 2008, Gue´dot et al. 2009). However, C. uted reduced oviposition rates and increased female bidens males did not respond to conspeciÞc male- movement to harassment by males (Burts and Fischer produced volatile chemicals (Soroker et al. 2004), and 1967, Horton and Lewis 1995). Additionally, the fe- similar results were obtained with the Asian citrus male to male repellency may occur when females are psyllid (Wenninger et al. 2009). Several roles have dispersing in search for a new feeding or oviposition been suggested for male-produced pheromones, in- site and are thus not seeking a mate. cluding female and/or male attractants (Leal et al. In conclusion, this study showed that potato psyllid 1998, Kirk and Hamilton 2004), aggregation phero- males are attracted to odorants emitted by females and mones (reviewed in Wertheim et al. 2005), male re- extractable volatile chemicals from females. This is the pellents (Zhang and Aldrich 2003), female aphrodisi- Þrst report of male attraction to conspeciÞc males in acs (Hillier and Vickers 2004), and inhibitors of female the Psylloidea. This is also the Þrst indication of fe- attractiveness (Andersson et al. 2003, Schulz et al. maleÐfemale repellency in psyllids. Future research 2008). This study reports the Þrst indication of maleÐ will address the identiÞcation of the speciÞc chemicals male attraction in the Psylloidea and suggests that that are attractive or repellent to B. cockerelli. male-produced volatile chemicals might be used as male aggregation pheromones in this species. Overall, Acknowledgments our results showed that potato psyllid males are at- tracted to both female- and male-produced volatile Assistance from M. Bayer and D. Broers in collecting and chemicals. However, males exposed simultaneously to maintaining insects and conducting the olfactometer trials odor sources from conspeciÞc females and males were was greatly appreciated. We thank B. Ohler for help with preferentially attracted to female-produced volatile determining the sex of the psyllids. We also thank L. Stelinski chemicals, which may be evidence for the existence of and R. S. Mann for improving an earlier version of this manuscript and two anonymous reviewers for comments. a female-speciÞc sex attractant for males. females avoided female-pro- duced volatile chemicals from live females and from References Cited extracts of females. Previous studies with C. bidens and Abad, J. A., M. Bandla, R. D. French-Monar, L. W. Liefting, D. citri indicated that females did not respond to and G.R.G. Clover. 2009. First report of the detection of volatile chemicals emitted by conspeciÞc females (So- ÔCandidatus LiberibacterÕ species in Zebra chip disease- roker et al. 2004, Wenninger et al. 2009). Nevertheless, infected potato plants in the United States. Plant Dis. 93: volatile collections from female-infested plants trig- 108. August 2010 GUE´DOT ET AL.: SEX ATTRACTION IN POTATO PSYLLID 1307

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