W. Rodney Cooper

Comparisons of Salivary Proteins from Five Aphid (Hemiptera: Aphididae) Species

ABSTRACT Aphid (Hemiptera: Aphididae) saliva, when injected into host plants during feeding, causes physiological changes in hosts that facilitate aphid feeding and cause injury to plants. Comparing salivary constituents among aphid species could help identify which salivary products are universally important for general aphid feeding processes, which products are involved with specific host associations, or which products elicit visible injury to hosts. We compared the salivary proteins from five aphid species, namely, Diuraphis noxia (Kurdjumov), D. tritici (Gillette), D. mexicana (Baker), Schizaphis graminum (Rondani), and Acyrthosiphon pisum (Harris). A 132-kDa protein band was detected from the saliva of all five species using sodium dodecyl sulfate Polyacrylamide gel electrophoresis. Alkaline phosphatase activity was detected from the saliva of all five species and may have a universal role in the feeding process of aphids. The Diuraphis species cause similar visible injury to grass hosts, and nine electrophoretic bands were unique to the saliva of these three species. S. graminum shares mutual hosts with the Diuraphis species, but visible injury to hosts caused by S. graminum feeding differs from that of Diuraphis feeding. Only two mutual electrophoretic bands were visualized in the saliva of Diuraphis and S. graminum. Ten unique products were detected from the saliva of A. pisum, which feeds on dicotyledonous hosts. Our comparisons of aphid salivary proteins revealed similarities among species which cause similar injury on mutual hosts, fewer similarities among species that cause different injury on mutual hosts, and little similarity among species which feed on unrelated hosts.

Localization of ‘Candidatus Liberibacter solanacearum' (Rhizobiales: Rhizobiaceae) in Bactericera cockerelli (Hemiptera: Triozidae)

ABSTRACT ‘Candidatus Liberibacter solanacearum’ is a pathogen of solanaceous crops (Solanales: Solanaceae) that causes zebra chip disease of potato (Solanum tuberosum L.) and plant dieback in tomato (S. lycopersicum L.) and pepper (Capsicum spp.). This pathogen is vectored by the potato/ tomato psyllid Bactericera cockerelli (Šulc) (Hemiptera: Triozidae), but little is known about the interactions between B. cockerelli and ‘Ca. Liberibacter solanacearum.’ Fluorescence in situ hybridization was used to assess the incidence of ‘Ca. Liberibacter solanacearum’ in the hemolymph, bacteriomes, alimentary canals, and salivary glands of B. cockerelli. Liberibacter was observed in 66% of alimentary canals, 39% of salivary glands, and 40% of bacteriomes dissected from adult psyllids. Compared with adults, the organs of fifth instars appeared less likely to harbor Liberibacter, which was observed in 52% of alimentary canals, 10% of salivary glands, and 6% of bacteriomes dissected from the nymphs. Results of real-time polymerase chain reaction confirmed that fewer fifth instars were infected with Liberibacter compared with adults and indicated that fifth instars were less likely to transmit the pathogen to noninfected host plants. These observations of the localization of ‘Ca. Liberibacter solanacearum’ in the organs and tissues of B. cockerelli adults and nymphs will aid the study of Liberibacter-psyllid interactions and the epidemiology of ‘Ca. Liberibacter solanacearum.’

Salivary Proteins of Lygus hesperus (Hemiptera: Miridae)

ABSTBACT Lygus hesperus Knight (Hemiptera: Miridae) causes economic damage to a wide range of crops in the western United States. While stylet-probing, L. hesperus discharge saliva consisting of lytic enzymes that facilitate extraoral digestion of host tissues. L. hesperus saliva primarily consists of polygalacturonases, &agr;-amylases, and proteases, but salivary constituents other than these enzymes have not been documented. Improved knowledge of L. hesperus salivary proteins could lead to a better understanding of Lygus-host interactions, and aid in the development of crops that are resistant or tolerant to injury caused by Lygus spp. The objective of our study was to use mass spectrometry to identify salivary proteins of L. hesperus adults. Multiple polygalacturonases and proteases were identified from diet that was stylet-probed and fed upon by L. hesperus. Laccase, alkyl hydroperoxide reductase-like protein, glucose dehydrogenase, and xanthine dehydrogenase were identified in styletprobed diets by using mass spectrometry, and laccase activity was confirmed using a colorimetric assay. This is the first study to identify laccase, glucose dehydrogenase, and xanthine dehydrogenase from L. hesperus saliva; we propose these enzymes target plant-defense compounds. Nearly 100 proteins from the bacterial plant pathogen, Pantoea ananatis and nine proteins from Serratia spp. were identified in stylet-probed diets. These results suggest L. hesperus may vector these plant pathogens. Our study adds to the list of documented L. hesperus salivary proteins, and provides information that will be useful for the further study of L. hesperus saliva and Lygus-microbe interactions.

Use of Electrical Penetration Graph Technology to Examine Transmission of ‘Candidatus Liberibacter solanacearum’ to Potato by Three Haplotypes of Potato Psyllid (Bactericera cockerelli; Hemiptera: Triozidae)

The potato psyllid, Bactericera cockerelli (Šulc) (Hemiptera: Triozidae), is a vector of the phloem-limited bacterium ‘Candidatus Liberibacter solanacearum’ (Lso), the putative causal agent of zebra chip disease of potato. Little is known about how potato psyllid transmits Lso to potato. We used electrical penetration graph (EPG) technology to compare stylet probing behaviors and efficiency of Lso transmission of three haplotypes of potato psyllid (Central, Western, Northwestern). All haplotypes exhibited the full suite of stylet behaviors identified in previous studies with this psyllid, including intercellular penetration and secretion of the stylet pathway, xylem ingestion, and phloem activities, the latter comprising salivation and ingestion. The three haplotypes exhibited similar frequency and duration of probing behaviors, with the exception of salivation into phloem, which was of higher duration by psyllids of the Western haplotype. We manipulated how long psyllids were allowed access to potato (“inoculation access period”, or IAP) to examine the relationship between phloem activities and Lso transmission. Between 25 and 30% of psyllids reached and salivated into phloem at an IAP of 1 hr, increasing to almost 80% of psyllids as IAP was increased to 24 h. Probability of Lso-transmission was lower across all IAP levels than probability of phloem salivation, indicating that a percentage of infected psyllids which salivated into the phloem failed to transmit Lso. Logistic regression showed that probability of transmission increased as a function of time spent salivating into the phloem; transmission occurred as quickly as 5 min following onset of salivation. A small percentage of infected psyllids showed extremely long salivation events but nonetheless failed to transmit Lso, for unknown reasons. Information from these studies increases our understanding of Lso transmission by potato psyllid, and demonstrates the value of EPG technology in exploring questions of vector efficiency.

Laboratory Studies of Variations in Feeding Behaviors Among Lygus hesperus (Hemiptera: Miridae) of Different Gender and Reproductive States

ABSTRACT Lygus hesperus Knight (Hemiptera: Miridae) is a key pest of cotton (Gossypium spp.) in the western United States that injures floral buds (squares) and developing fruit (bolls). Levels of Lygus-induced damage to cotton can vary by Lygus ages or gender, and these variations complicate interpretation of studies designed to elucidate Lygus and cotton interactions. Variations in observed injury may reflect different behaviors among Lygus gender or ages. We compared times allocated to feeding and trivial movement between male and female adult L. hesperus of different reproductive states: prereproductive, reproductive and mated, and reproductive and unmated. Prereproductive adults exhibited less trivial movement and spent more time stylet-probing compared with reproductive unmated and mated adults. Mated females stylet-probed more times than other classes of adults, whereas mated and unmated reproductive females exhibited more test probes (≤10 s duration) than prereproductive females. Reproductive females probed the anther region of squares less than prereproductive females. Instead, reproductive females tended to stylet-probe squares below the bracts, which is also where they oviposited. Each oviposition event was preceded by a short duration stylet-probe at the oviposition location. Unmated reproductive males exhibited more test probes but fewer ingestion probes (>1 min) compared with prereproductive and mated males. These results indicate a pattern in which prereproductive adults are less active and feed more compared with reproductive adults, but behaviors vary among reproductive adults of different gender and mating states. We propose that differences in behaviors exhibited among adult L. hesperus are related to the different requirements imposed by mate seeking, mate attraction, and oviposition.

Temperature dependent development of Lygus hesperus (Hemiptera: Miridae) nymphs.

ABSTRACT Lygus hesperus Knight (Hemiptera: Miridae) is a key pest of fruit and vegetable crops, forages, and cotton (Gossypium spp.) in the western United States. Accurate models describing relationships between temperature and L. hesperus development are critical to the study of seasonal L. hesperus population dynamics. Development of L. hesperus nymphs was assessed at nine constant temperatures from 10 to 37.8°C. The relationships between temperature and development for each L. hesperus instar, and for the entire nymphal stage, were best described by six-parameter biophysical models indicating both low- and high-temperature inhibition of development. Development rates asymptotically approached zero with decreasing temperature in the lower thermal range, and decreased with increasing temperatures above 32.2°C. Nymphs did not survive from egg hatch to adulthood at either 10 or 37.8°C, and nymph mortality was >90% at both 12.8 and 35.0°C. The fifth instar exhibited the longest stadium, whereas the shortest stadia were associated with the second and third instars. Development rates of males and females did not differ, and the ratio of males to females was not different from 1:1 at any temperature. Our temperature-dependent development rate models for L. hesperus nymphs will facilitate control of insect physiological age in controlled laboratory experiments, and should be useful in planning and interpreting field studies on L. hesperus population dynamics.

External Visibility of Spermatophores as an Indicator of Mating Status of Lygus hesperus (Hemiptera: Miridae) Females

Abstract Mated females of the western tarnished plant bug, Lygus hesperus Knight, are distinguished from unmated females by the presence of one or more spermatophores. The presence of a spermatophore is normally determined by dissection. A simple and nondestructive method to distinguish mated L. hesperus females from unmated females would facilitate laboratory studies that require mated insects. Spermatophores are visible through the abdominal sternites of recently mated L. hesperus females, but the consistency and persistence with which spermatophores are externally visible has not been previously documented. The overall objective of this study was to evaluate whether examination for the presence of externally visible spermatophores is a reliable method to determine whether L. hesperus females have mated. The presence of externally visible spermatophores correctly discerned 99% of recently mated (≤24 h) females from unmated females. None of the unmated females were misclassified as mated. The apparency of visible spermatophores decreased with increasing time after mating until the spermatophores were no longer visible. The period during which spermatophores were externally visible decreased linearly with increasing temperature from 15.6 - 29.4°C. Females continued to oviposit fertile eggs after spermatophores were no longer externally visible. Results indicate that examination for the presence of externally visible spermatophores is a reliable method to discern mated female L. hesperus from unmated females in controlled laboratory studies. Because spermatophores become less apparent with increasing time after mating, this method is not suitable for the classification of mating states of field-collected insects or others of unknown history.

Injury to Cotton by Adult Lygus hesperus (Hemiptera: Miridae) of Different Gender and Reproductive States

ABSTRACT Lygus hesperus Knight (Hemiptera: Miridae) is a key pest of cotton (Gossypium spp.) in the western United States that injures floral buds (squares) and developing fruit (bolls). However, no clear relationship between Lygus population level and plant injury has been established. Agedependent feeding activity by L. hesperus is a possible source of variation that has not been examined for its influence in studies of the impact of Lygus on cotton. Recent video-based laboratory studies indicated that feeding behaviors and trivial movement varied among L. hesperus adults of different gender and reproductive states (prereproductive; reproductive and unmated; and reproductive and mated). We compared within-plant distributions and accumulations of feeding injury to intact cotton plants corresponding to adult L. hesperus of different gender and reproductive states. Adult females, regardless of reproductive state, were observed on squares and axillary buds more often than were males. Additionally, prereproductive adults were observed on squares and axillary buds more often than were mated or unmated reproductive adults, regardless of gender. Plants that were exposed to prereproductive adults exhibited more abscised squares and more squares with injured anthers compared with plants exposed to reproductive adults. However, feeding injury did not differ by insect mating status or gender. These results are consistent with results of our previous video-based assays, and indicate adult reproductive state represents a source of variation that should be controlled in studies to evaluate Lygus-induced injury to cotton and other crop plants.

Temperature-dependent egg development of Lygus hesperus (Hemiptera: Miridae).

ABSTRACT Lygus hesperus Knight (Hemiptera: Miridae) is a key agricultural pest in the western United States, but certain aspects of its temperature-dependent development are poorly defined. Accurate models describing the relationships between temperature and development of L. hesperus would facilitate the study of Lygus seasonal population dynamics and overwintering ecology. We used nonlinear biophysical models to describe the development of L. hesperus eggs oviposited in green bean pods and semitransparent agarose and held under constant temperatures from 10 to 37.8°C. The agarose substrate was used because it is less susceptible than green beans to deterioration at low and high temperature extremes, and because it allowed the observation of phases of embryonic development that are hidden from view from eggs developing in plant material. The models indicated that both low and high temperature inhibited development of each observed embryonic phase regardless of oviposition substrate. Developmental rates asymptotically approached zero with decreasing temperature in the lower thermal range, and decreased with increasing temperatures above 32.2°C. Eggs oviposited in agarose developed slower than eggs oviposited in green bean pods suggesting that egg developmental rates were influenced by the type of host substrate. Our temperature-dependent developmental rate models for L. hesperus eggs supplement nonlinear models recently reported for L. hesperus nymphal and adult reproductive development and should be useful in planning and interpreting field studies of L. hesperus population dynamics and overwintering ecology.

Variation in Bactericera cockerelli (Hemiptera: Triozidae) Oviposition, Survival, and Development on Solanum bulbocastanum Germplasm

The potato psyllid, Bactericera cockerelli, is a key pest of potato and important vector of the pathogen that causes zebra chip disease. Control of zebra chip relies entirely on the use of insecticides to reduce populations of this vector. The development of potato varieties resistant to B. cockerelli would contribute to cost-effective control of this insect. Wild potato germplasm are key sources for desirable traits including pest resistance to develop new potato cultivars. Our objective was to screen Solanum bulbocastanum germplasm for resistance to B. cockerelli. The combined use of choice and no-choice assays demonstrated considerable variability among S. bulbocastanum populations in their susceptibility to psyllids. At least six S. bulbocastanum populations exhibited resistance to B. cockerelli: PI 243512, PI 243513, PI 255518, PI 275194, PI 275197, and PI 283096. The documentation of the variability among S. bulbocastanum germplasm populations in their susceptibility to B. cockerelli can aid the development of potato cultivars that are naturally resistant to the potato psyllid.ResumenEl psílido de la papa, Bactericera cockerelli, es una plaga clave de la papa y un vector importante del patógeno que causa la enfermedad del tubérculo rayado o “zebra chip”. El control de esta enfermedad se confía completamente en el uso de insecticidas para reducir las poblaciones de este vector. El desarrollo de variedades de papa resistentes a Bactericera cockerelli contribuirá al control costeable de este insecto. El germoplasma silvestre de papa es una fuente clave para características deseables incluyendo resistencia a plagas para el desarrollo de nuevas variedades. Nuestro objetivo fue evaluar germoplasma de Solanum bulbocastanum para resistencia a B. cockerelli. El uso combinado de ensayos de selección y no selección demostró variabilidad considerable entre poblaciones de S. bulbocastanum en su susceptibilidad a los psílidos. Por lo menos seis poblaciones de S. bulbocastanum exhibieron resistencia a B. cockerelli: PI 243512, PI 243513, PI 255518, PI 275194, PI 275197, y PI 283096. La documentación de la variabilidad entre las poblaciones del germoplasma de S. bulbocastanum respecto a su susceptibilidad a B. cockerelli puede ayudar en el desarrollo de variedades de papa que son resistentes de forma natural al psílido de la papa.