Erik J. Wenninger

Roles of Olfactory Cues, Visual Cues, and Mating Status in Orientation of Diaphorina citri Kuwayama (Hemiptera: Psyllidae) to Four Different Host Plants

ABSTRACT Diaphorina citri Kuwayama (Hemiptera: Psyllidae) is an important worldwide pest of citrus that vectors bacteria (Candidatus Liberibacter spp.) responsible for huanglongbing (citrus greening disease). We examined the behavioral responses of mated and unmated D. citri of both sexes to odors from host plants in a Y-tube olfactometer, with and without visual cues. The host plants tested were ‘Duncan’ grapefruit (Citrus paradisi Macfayden), sour orange (Citrus aurantium L.), navel orange (C. sinensis L.), and Murraya paniculata L. Jack. Responses varied by plant species, psyllid sex and mating status, and the presence of a visual cue. Evidence of attraction generally was stronger in females and in mated individuals of both sexes relative to virgins. The presence of a visual cue typically enhanced attractiveness of olfactory cues; in no case did unmated individuals show evidence of attraction to host plant odors in the absence of a visual cue. In the absence of visual cues, mated females and males showed evidence of attraction only to odors from sour orange and navel orange, respectively. Psyllids exhibited anemotactic responses when assayed with plant odors alone but showed strong evidence of attraction only when olfactory and visual cues were combined, suggesting that olfactory cues facilitate orientation to host plants but may be insufficient alone. Antennal responses to citrus volatiles were confirmed by electroantennogram. The results reported here provide evidence that D. citri uses olfactory and visual cues in orientation to host plants and suggest the possibility of using plant volatiles in monitoring and management of this pest.

Daily Timing of Mating and Age at Reproductive Maturity in Diaphorina citri (Hemiptera: Psyllidae)

Abstract The psyllid Diaphorina citri Kuwayama vectors a bacterium, Candidatus Liberibacter asiaticus, in Florida which is responsible for citrus greening disease (huanglongbing), one of the most serious diseases of citrus. Despite the great economic importance of D. citri to citrus production, little is known about the reproductive biology and behavior of this insect. We conducted studies to determine the copula duration, the age at which both males and females reach reproductive maturity, the pre-oviposition period, the daily timing of mating activity in a laboratory colony, and the temporal dynamics in the sex ratio of a cohort of newly eclosed adults. The emergence patterns of males and females were strikingly similar, with no evidence for protandry or protogyny. Both sexes reached reproductive maturity by 2-3 d post-eclosion. Oviposition generally began within 1 d after mating, but was longer when females were mated at 2 d of age. Mean ± SEM copula duration was 48.3 ± 8.4 minutes (range: 15.2-98.0). Mating on orange jasmine was observed almost exclusively on flush shoots during daylight hours, with no obvious peak of daily mating activity. Mating activity may be constrained during scotophase in part by cooler temperatures and lack of light. Methods for carrying out mating experiments with this species are described.

Effects of a Particle Film on Biology and Behavior of Diaphorina citri (Hemiptera: Psyllidae) and Its Infestations in Citrus

Abstract Studies were conducted to investigate the effects of a kaolin-based hydrophilic particle film, Surround WP, on the biology and behavior of the psyllidDiaphorina citri Kuwayama (Hemiptera: Psyllidae) and to assess population densities ofD.citri in citrus subjected to monthly applications of Surround WP. Laboratory investigations indicated a 3% (wt:vol) suspension of Surround WP in water applied directly was not acutely toxic to eggs, older nymphs or adults. Presence of the dried particle film on leaves interfered with the ability of adults to grasp and walk on citrus leaves. During a 30-s period, adults spent an average of 5 s moving on leaves with particle film compared with 16 s on leaves without particle film. When leaves were inverted, a significantly higher percentage of adults fell or flew from treated leaves (53%) than untreated leaves (16%). In a 12-mo study investigating infestations ofD.citri on citrus treated monthly with Surround WP, cumulative reductions of 78% in adult numbers on mature leaves and of 60% in adult numbers on flush shoots (immature leaves) were observed in treated trees compared with untreated trees. Numbers of eggs and nymphs per flush shoot were reduced by 85 and 78%, respectively, in trees treated with particle film. Reductions in infestation levels ofD.citri in treated trees were attributed to the negative effects of the particle film on the ability of adults to grasp, move, and oviposit. The suppressive effects of a Surround treatment against adult psyllids were degraded by rain.

Vibrational Communication Between the Sexes in Diaphorina citri (Hemiptera: Psyllidae)

ABSTRACT We examined the substrate-borne vibrational signals used in communication between the sexes in Diaphorina citri Kuwayama (Hemiptera: Psyllidae), a vector of huanglongbing (an economically devastating disease of citrus), in an anechoic chamber and an olfactometer. Males and females both primarily produced simple, low-amplitude vibrational signals at multiples of 170-250 Hz, ranging in duration from 140 to 700 ms. The vibrational frequencies of males and females were negatively correlated with mass, but the correlation was statistically significant only for males. Females replied to male calls within an interval of 0.3-1.2 s. Such signals are within the ranges of signals observed for other psyllids. Female-female interactions were occasionally observed. Intervals between male calls were not significantly different whether or not females replied. In an olfactometer, calling rate did not differ between virgin males exposed to odors from virgin females on citrus versus those exposed only to clean air. However, the latent period for initialization of calling was significantly shorter for males exposed to clean air, suggesting that in the absence of olfactory cues psyllids might be more inclined to use acoustic signals to communicate with conspecifics. Moreover, calling rate and latency to initialization of calling were positively and negatively correlated, respectively, with male age, suggesting that males are more likely to call the longer they remain unmated.

Temperature Studies with the Asian Citrus Psyllid, Diaphorina citri: Cold Hardiness and Temperature Thresholds for Oviposition

Abstract This study was conducted to obtain information on the cold hardiness of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), in Florida and to assess upper and lower temperature thresholds for oviposition. The psyllid is an important pest in citrus because it transmits the bacterial pathogens responsible for citrus greening disease, Huanglongbing, considered the most serious citrus disease worldwide. D. citri was first found in Florida during 1998, and the disease was discovered during 2005. Little was known regarding cold hardiness of D. citri, but Florida citrus is occasionally subjected to notable freeze events. Temperature and duration were each significant sources of variation in percent mortality of D. citri subjected to freeze events. Relatively large percentages of adults and nymphs survived after being exposed for several hours to temperatures as low as -5 to -6° C. Relatively large percentages of eggs hatched after being exposed for several hours to temperatures as low as -8° C. Research results indicated that adult D. citri become cold acclimated during the winter through exposure to cooler winter temperatures. There was no evidence that eggs became cold acclimated during winter. Cold acclimation in nymphs was not investigated. Research with adult D. citri from laboratory and greenhouse colonies revealed that mild to moderate freeze events were usually nonlethal to the D. citri irrespective of whether they were cold acclimated or not. Upper and lower temperature thresholds for oviposition were investigated because such information may be valuable in explaining the geographic distribution and potential spread of the pest from Florida as well as how cooler winter temperatures might limit population growth. The estimated lower and upper thresholds for oviposition were 16.0 and 41.6° C, respectively; the estimated temperature of peak oviposition over a 48 h period was 29.6° C.

Daily and Seasonal Patterns in Abdominal Color in Diaphorina citri (Hemiptera: Psyllidae)

Abstract Diaphorina citri Kuwayama, a psyllid vector of huanglongbing (citrus greening disease), exhibits three more or less distinct abdominal colors in the adult psyllid: gray/brown, blue/green, and orange/yellow. We explored the daily (in individuals in the laboratory) and seasonal (in a field population) patterns in abdominal color of adult D. citri to clarify the biology of this species in relation to abdominal color and investigated the relationship between abdominal color and the reproductive state of adults (i.e., whether an individual is reproductively mature, has mated, or—in females—is gravid). Females were predominantly blue/green throughout their lives, with a small portion of individuals being gray/brown, especially just after emergence. Approximately 86% of mated females developed an orange/yellow abdominal color after mating, but they ultimately turned back to blue/green within several days to 1 mo after mating. Only 31% of virgin females turned orange/yellow. Males were predominantly blue/green early in life, but a greater portion of males relative to females were gray/brown. The orange/yellow color in females reflected the presence of eggs in the abdomen; in males it seemed to derive from the color of the internal reproductive organs, and it was generally only evident in older males. The preponderance of blue/green females, rarity of gray/brown females relative to gray/brown males, and rarity of orange/yellow males and females was largely reflected in sticky card trap captures from the field. Abdominal color is of essentially no value in discerning the state of sexual maturity and of only limited value in discerning whether females have mated.

Relationships Between Adult Abdominal Color and Reproductive Potential in Diaphorina citri (Hemiptera: Psyllidae)

ABSTRACT Diaphorina citri Kuwayama (Hemiptera: Psyllidae), a vector of huanglongbing (citrus greening disease), exhibits three more or less distinct abdominal colors in the adult stage: gray/brown, blue/green, and orange/yellow. A previous report showed that, relative to blue/green individuals, gray/brown individuals of both sexes have lower body mass, and gray/brown females may have lower reproductive output. In the present investigation, we directly examined the relationships between female and male abdominal color and reproductive output. We also assessed how psyllid body mass was influenced by transfer to fresh plant material and how female and male abdominal color affected behavioral responses of males to crushed females in a petri dish assay. Relative to blue/green females, gray/brown females exhibited lower fecundity over the first 2–5 d after mating, but fecundity did not differ over the remainder of a 21-d observation period. Regardless of abdominal color, females mated with gray/brown males showed an earlier, sharper peak in fecundity, whereas females mated with blue/green males showed a later, more gradual peak in fecundity. Gray/brown males elicited lower egg fertility in their mates, but egg fertility did not vary according to female abdominal color. Males—irrespective of abdominal color—exhibited stronger evidence of attraction to crushed blue/ green females than to crushed gray/brown females. Gray/brown individuals of both sexes showed an increase in body mass 5–6 d after transfer to a new citrus seedling, suggesting that abdominal color (which is closely related to body mass) may be influenced at least in part by plant quality.

Abdominal color of the Asian citrus psyllid, Diaphorina citri, is associated with susceptibility to various insecticides

BACKGROUND Color morphs of an insect species are known to vary in activities of detoxifying enzymes and associated susceptibility to insecticides. In Diaphorina citri, three color morphs are known to occur. In the present study, susceptibility to four insecticides was compared among gray/brown, blue/green and orange/yellow color morphs of field-collected D. citri. RESULTS The orange/yellow morph was significantly more susceptible to fenpropathrin than the blue/green morph, and imidacloprid and chlorpyrifos caused higher mortality in the orange/yellow morph than in the blue/green and gray/brown morphs. To confirm the genetic basis of variable levels of susceptibility, the relative expression of five CYP4 genes was compared among the color morphs. CYP4C67 was expressed at significantly higher levels in the blue/green than in the orange/yellow and gray/brown morphs. CYP4DA1, CYP4C68, CYP4G70 and CYP4DB1 were expressed at significantly higher levels in the blue/green and gray/brown morphs than in the orange/yellow morph. Lower expression of CYP4 genes in the orange/yellow morph as compared with the others was correlated with reduced signal of 45 kD cytochrome P450 proteins, as determined by the western blot. CONCLUSIONS The results indicate differential susceptibility of D. citri color morphs to insecticides, which will need to be accounted for in future insecticide monitoring programs and may affect management programs.

Comparison of transmission efficiency of various isolates of Potato virus Y among three aphid vectors

Potato virus Y (PVY) strains are transmitted by different aphid species in a non‐persistent, non‐circulative manner. Green peach aphid (GPA), Myzus persicae Sulzer, is the most efficient vector in laboratory studies, but potato aphid (PA), Macrosiphum euphorbiae Thomas (both Hemiptera: Aphididae, Macrosiphini), and bird cherry‐oat aphid (BCOA), Rhopalosiphum padi L. (Hemiptera: Aphididae, Aphidini), also contribute to PVY transmission. Studies were conducted with GPA, PA, and BCOA to assess PVY transmission efficiency for various isolates of the same strain. Treatments included three PVY strains (PVYO, PVYN:O, PVYNTN) and two isolates of each strain (Oz and NY090031 for PVYO; Alt and NY090004 for PVYN:O; N4 and NY090029 for PVYNTN), using each of three aphid species as well as a sham inoculation. Virus‐free tissue‐cultured plantlets of potato cv. Russet Burbank were used as virus source and recipient plants. Five weeks post inoculation, recipient plants were tested with quantitative DAS‐ELISA to assess infection percentage and virus titer. ELISA‐positive recipient plants were assayed with RT‐PCR to confirm presence of the expected strains. Transmission efficiency (percentage infection of plants) was highest for GPA, intermediate for BCOA, and lowest for PA. For all aphid species, transmission efficiency did not differ significantly between isolates within each strain. No correlations were found among source plant titer, infection percentage, and recipient plant titer. For both GPA and BCOA, isolates of PVYNTN were transmitted with greatest efficiency followed by isolates of PVYO and PVYN:O, which might help explain the increasing prevalence of necrotic strains in potato‐growing regions. Bird cherry‐oat aphid transmitted PVY with higher efficiency than previously reported, suggesting that this species is more important to PVY epidemiology than has been considered.

Apple and sugar feeding in adult codling moths, Cydia pomonella: effects on longevity, fecundity, and egg fertility.

Abstract Attraction of adult codling moths, Cydia pomonella (L.) (Lepidoptera: Tortricidae), to sweet baits has been well documented. However, beneficial effects of sugar feeding on moth fitness have not been demonstrated. Longevity, fecundity, and egg fertility were examined for female/male pairs of moths maintained with the following food regimens: water, sucrose water, honey water, apple juice, apple flesh, or starved, i.e., no food or water provided. Longevity and total fecundity were enhanced in all treatments relative to the starved treatment moths. Sucrose water, honey water, and apple juice treatments yielded the highest longevity, but total fecundity was highest for moths maintained on honey water or apple juice. Total egg fertility did not differ among treatments. However, egg fertility declined more gradually over the female lifespan for the three aqueous solution diets of sucrose water, honey water, and apple juice. Similarly, fecundity per day declined more gradually over time for honey water and apple juice treatments. Performance of moths maintained with apple flesh was generally intermediate between that of moths with water and the three aqueous solution treatments. This suggests that moths benefit from feeding on ripe apple flesh, although apple may be more difficult to ingest or its nutrients less concentrated compared to aqueous solutions. The results presented here may explain attraction of adult moths to sweet baits as well as to odors from ripe fruit, which may be a natural source of food in the fall.