Current Distribution of the Olive Psyllid, Euphyllura olivina, in California and Initial Evaluation of the Mediterranean Parasitoid Psyllaephagus euphyllurae as a Biological Control Candidate.

The olive psyllid, Euphyllura olivina, is a newly invasive species to California with the potential to become an economical pest if it reaches the olive production regions of California's Central Valley. Here, we report on surveys undertaken in California to assess the psyllid's current distribution and the occurrence of parasitism. Additionally, we present results of foreign collections of its parasitoids and initial non-target studies of a possible biological control agent, the Mediterranean parasitoid Psyllaephagus euphyllurae. The current distribution of the psyllid appears to be limited to the California coast between Monterey and San Diego; there have been no reports of infestations on olives in the major production areas of central and northern California. Psyllaephagus euphyllurae was the major primary parasitoid found in our foreign collections. The potential non-target impact of P. euphyllurae was tested on three native North American psyllid species: Neophyllura arctostaphyli, Euglyptoneura nr. robusta, and Calophya nigrella. No P. euphyllurae developed on the non-target species during no-choice tests. Behavioral observations in choice tests confirmed no attack on the non-target hosts, although the parasitoid did remain longer on N. arctostaphyli-infested manzanita plants, and revealed no host feeding behavior.

Evaluation of Olive as a Host of Xylella fastidiosa and Associated Sharpshooter Vectors.

Olive (Olea europaea) trees exhibiting leaf scorch or branch dieback symptoms in California were surveyed for the xylem-limited, fastidious bacterium Xylella fastidiosa. Only approximately 17% of diseased trees tested positive for X. fastidiosa by polymerase chain reaction, and disease symptoms could not be attributed to X. fastidiosa infection of olive in greenhouse pathogenicity assays. Six strains of X. fastidiosa were isolated from olive in Southern California. Molecular assays identified strains recovered from olive as belonging to X. fastidiosa subsp. multiplex. Pathogenicity testing of olive strains on grapevine and almond confirmed that X. fastidiosa strains isolated from olive yield disease phenotypes on almond and grapevine typical of those expected for subsp. multiplex. Mechanical inoculation of X. fastidiosa olive strains to olive resulted in infection at low efficiency but infections remained asymptomatic and tended to be self-limiting. Vector transmission assays demonstrated that glassy-winged sharpshooter (Homalodisca vitripennis) could transmit strains of both subspp. multiplex and fastidiosa to olive at low efficiency. Insect trapping data indicated that two vectors of X. fastidiosa, glassy-winged sharpshooter and green sharpshooter (Draeculacephala minerva), were active in olive orchards. Collectively, the data indicate that X. fastidiosa did not cause olive leaf scorch or branch dieback but olive may contribute to the epidemiology of X. fastidiosa-elicited diseases in California. Olive may serve as an alternative, albeit suboptimal, host of X. fastidiosa. Olive also may be a refuge where sharpshooter vectors evade intensive areawide insecticide treatment of citrus, the primary control method used in California to limit glassy-winged sharpshooter populations and, indirectly, epidemics of Pierce's disease of grapevine.

Overwintering survival of olive fruit fly (Diptera: Tephritidae) and two introduced parasitoids in California.

The overwintering survival and development of olive fruit fly, Bactrocera oleae (Rossi), and the endoparasitoids, Psyttalia humilis Silvestri and P. lounsburyi (Silvestri), were investigated at sites in California's interior valley and coastal region. In the interior valley, adult flies survived up to 4-6 mo during the winter when food was provided. Adult female flies could oviposit in late fall and early winter on nonharvested fruit and, although egg survival was low (0.23-8.50%), a portion of the overwintered cohort developed into adults the following spring; percentage of survival was negatively correlated to daily minimum temperature. P. humilis and P. lounsburyi successfully oviposited into host larvae in late fall, and their progeny developed into adults the following spring, although with a low percentage (0-11.9%) survivorship. Overwintering survival of puparia of the olive fruit fly and immature larvae of P. humilis and P. lounsburyi (inside host puparia), buried in the soil, were tested at an interior valley and coastal site. Survival of olive fruit fly ranged from 0 to 60% and was affected by the trial date and soil moisture. Overwintering survival of both the fruit fly and tested parasitoids was lower at the colder interior valley than the coastal site; P. humilis immature stages had the highest mortality levels while B. oleae pupae had the lowest mortality levels. The spring emergence pattern of the tested insects was well predicted by a degree-day model. We discuss factors potentially impeding establishment of introduced olive fruit fly parasitoids in California and elsewhere.

Pattern of stylet penetration activity by Homalodisca vitripennis (Hemiptera: Cicadellidae) adults in relation to environmental temperature and light conditions.

Effects of ambient spring air temperature and light intensity on stylet penetration activities of the glassy-winged sharpshooter, Homalodisca vitripennis (Germar), were studied under field conditions by using an electrical penetration graph. Electrical penetration graph waveforms representing salivary sheath formation and searching (pathway phase), xylem contact (X waves), and ingestion of xylem fluid (waveform C) were analyzed. Previous research supported the concept that acquisition of Xylella fastidiosa, the Pierce's disease bacterium, occurs during ingestion, whereas inoculation occurs during xylem contact periods (X waves). Diel patterns of H. vitripennis stylet activity showed that, regardless of light condition, xylem ingestion occurred for the longest duration when temperature remained above the feeding threshold (10°C), and only occurred at temperatures below the threshold when ingestion was continued from a preceding, warmer time. Regression analysis indicated that mean waveform durations per insect (WDI) for combined stylet activities (pathway and ingestion) as well as X wave frequencies were significantly influenced by temperature, but there was no significant impact of light intensity or interaction between temperature and light intensity. The relationship between temperature and stylet activities in terms of WDI and X wave frequency was described using linear and nonlinear models. Validation of the nonlinear models indicated that they well predicted the WDIs for both ingestion and combined stylet activities, using temperature only as a single input. Overall, findings clearly demonstrate that temperature is an important factor that influences the H. vitripennis feeding behaviors responsible for transmission (acquisition and inoculation) of the Pierce's disease bacterium, with implications for vector ecology and management, as well as disease epidemiology.

Performance of Psyttalia humilis (Hymenoptera: Braconidae) reared from irradiated host on olive fruit fly (Diptera: Tephritidae) in California.

The parasitoid Psyttalia humilis (Silvestri) was reared on Mediterranean fruit fly, Ceratitis capitata (Wiedemann), third instars irradiated at 0-70 Gy at the USDA, APHIS, PPQ, Moscamed biological control laboratory in San Miguel Petapa, Guatemala, and shipped to the USDA, ARS, Parlier, CA. Irradiation dose did not affect the parasitoid's offspring sex ratio (53-62% females), percentage of unemerged adults (12-34%), number of progeny produced per female (1.4-1.8), and parasitism (19-24%). Host irradiation dose had no significant effect on the forewing length of female P. humilis and its parasitism on olive fruit fly, Bactrocera oleae (Rossi) and offspring sex ratio, but dissection of 1-wk-old female parasitoids reared from hosts irradiated with 70 Gy had a significantly lower number of mature eggs than females from nonirradiated hosts. Longevity of P. humilis adults decreased with increased temperature from 15 to 35°C, regardless of food provisions, gender, and host irradiation dose. Females survived 37-49 d at 15°C with water and food, and only 1-2 d at 35°C without food, whereas males lived shorter than females at all temperatures and food combinations tested. Adult P. humilis reared from fertile C. capitata and aspirated for dispensing in cups lived significantly longer after shipment than those specimens chilled and dispensed by weight. At 21 and 32°C, 50% of parasitoids departed release cages after 180 and 30 min, respectively, but none departed at 12°C. Thirteen shipments of P. humilis (2,980-21,922 parasitoids per shipment) were received between September and December 2009, and seven shipments (7,502-22,560 parasitoids per shipment) were received between October and December 2010 from San Miguel Petapa, Guatemala. Daily number of olive fruit fly adult and percentage female trap captures ranged <1-19 and 8-58% in 2009, and <1-11 and 0-42% in 2010, respectively. The number of parasitoids released ranged 848-12,257 in 2009 and 3,675-11,154 in 2010. Percentage parasitism of olive fruit fly third instars at all locations ranged 0-9% in 2009 and 0-36% in 2010.

Plant water stress effects on the net dispersal rate of the insect vector Homalodisca vitripennis (Hemiptera: Cicadellidae) and movement of its egg parasitoid, Gonatocerus ashmeadi (Hymenoptera: Mymaridae).

Homalodisca vitripennis (Germar), a vector of Xylella fastidiosa, is associated with citrus plantings in California. Infested citrus orchards act as a source of vectors to adjacent vineyards where X. fastidiosa causes Pierce's disease. An analysis of the pattern and rate of movement of H. vitripennis and its egg parasitoid, Gonatocerus ashmeadi Girault, was conducted in a citrus orchard by using a protein mark-capture technique to quantify movement and net dispersal rates in the experimental areas. Treatments included irrigation at 100% of the crop evapotranspiration rate (ET(c)), 80, and 60% ET(c). Sex-specific net dispersal rates showed that H. vitripennis males and females moved consistently and contributed equally to the level of population change within treated areas. Trees irrigated at 60% ET(c) were the least preferred by H. vitripennis. Among all protein-marked individuals captured in the 60% ET(c) treatment, ≈ 75 and 88% in 2005 and 2006, respectively, were inflow individuals. Movement toward less preferable plants indicates that in agricultural landscapes dominated by perennial monocultures, there is a random component to H. vitripennis movement, which may result from the inability of H. vitripennis to use plant visual cues, olfactory cues, or both to make well-informed long-range decisions. The 80% ET(c) areas were a significant source of adult H. vitripennis and G. ashmeadi compared with the other treatments. Colonization rates by parasitoids were synchronized with the spatiotemporal distribution of H. vitripennis eggs. Results suggest that H. vitripennis movement from citrus into adjacent vineyards could be a result of random dispersal rather than oriented movement in response to host-plant characteristics.

Estimation of feeding threshold for Homalodisca vitripennis (Hemiptera: Cicadellidae) and its application to prediction of overwintering mortality.

The glassy-winged sharpshooter, Homalodisca vitripennis (Germar), vectors the bacterium Xylella fastidiosa that induces Pierce's disease of grape. This study determined the effect of temperature on the feeding activity of H. vitripennis adults and the resulting production of excreta. The Logan type I model described a nonlinear pattern that showed excreta production increased up to an optimal temperature (33.1°C), followed by an abrupt decline near an estimated upper threshold (36.4°C). A temperature threshold for feeding, at or below which adults cease feeding, was estimated to be 10°C using a linear regression model based on the percentage of adults producing excreta over a range of constant temperatures. A simulated winter-temperature experiment using fluctuating thermal cycles confirmed that a time period above the temperature threshold for feeding was a critical factor in determining adult survival. Using data from the simulated temperature study, a predictive model was constructed by quantifying the relationship between cumulative mortality and cooling degree-hours. In field validation experiments, the model accurately predicted the temporal pattern of overwintering mortality of H. vitripennis adults held under winter temperatures simulating conditions in Bakersfield and Riverside, California, in 2006-2007. Model prediction using winter temperature data from a Riverside weather station indicated that H. vitripennis adults would experience an average of 92% overwintering mortality before reproduction in the spring, but levels of mortality varied depending on winter temperatures. The potential for temperature-based indices to predict temporal and spatial dynamics of H. vitripennis overwintering is discussed.

Olive fruit fly: managing an ancient pest in modern times.

Olive fruit fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae), is the major pest of commercial olives worldwide. Various aspects of its biology, ecology, management, and impact on olive production are highlighted. With the discovery of insecticidal resistance in some populations frequently treated with organophosphates, old and new control options are being investigated. The potential of biological control is examined. Surveys suggest that a small group of braconids in the Opiinae subfamily best represent the primary parasitoids attacking olive fruit fly in its native range. These species include Psyttalia lounsburyi, P. dacicida, P. concolor, P. ponerophaga, and Utetes africanus. Bracon celer, another braconid but in the Braconinae subfamily, is also reared from the fruit fly in its native range. The potential of these and other natural enemies is discussed with respect to olive fruit fly biology, commercial olive production, and biological constraints that may limit their success. We suggest that numerous species exist that should be further investigated as control agents for olive fruit fly in the many climatic regimes where the pest is found.

Influences of temperature on Homalodisca vitripennis (Hemiptera: Cicadellidae) survival under various feeding conditions.

Survival of the glassy-winged sharpshooter, Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae), was studied under various constant temperatures and feeding conditions. When provided a host plant (Citrus limon L. Burm. f.) to feed on during a 21-d trial, 100% mortality occurred at 0.1, 3.2, and 40.1 degrees C, whereas an average of 74-76% of adults survived in the 13.2-24.5 degrees C range. When individually confined with moist cotton, adult longevity was greatest (16.3 d) at 13.3 degrees C, but it was <3 d at -2.4 and 36.2 degrees C. In a companion study comparing the presence versus absence of a host plant, the presence of a host plant was not a significant factor influencing survival at temperatures < or =7.8 degrees C but was at temperatures > or =18.9 degrees C. The relationship between temperature and survival was described by a nonlinear function that estimated the optimum temperature in each feeding regimen: no host plant or moist cotton (5.5 degrees C), moist cotton (9.9 degrees C), and accessible host plant (25.1 degrees C). The model quantitatively predicted that H. vitripennis would survive longer periods at a wider temperature regimen when provided with a host plant than when provided with water alone (moist cotton) or when provided with neither plant host nor water. Our results suggest that continuous exposure to either low (<5 degrees C) or high (>30 degrees C) temperatures are detrimental for adult survival. Specifically, low temperatures caused early mortality because of inhibition of feeding activity and presumably this threshold lies between 7.8 and 13.2 degrees C. Furthermore, this study clearly shows that temperature may influence the survival of H. vitripennis adults regardless of feeding regimens, and its implications for population dynamics are discussed.

High summer temperatures affect the survival and reproduction of olive fruit fly (Diptera: Tephritidae).

The olive fruit fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae), is an invasive pest in California. Identifying environmental constraints that affect the geographic distribution and abundance of any invasive insect pest is fundamental to its effective management. California's Central Valley, where most commercial olives are grown, is extremely hot during the summer, with maximum daily temperatures consistently >35.0 degrees C. This study examined the effects of two diurnal temperature regimens (low 18.3 degrees C, high 35.0 or 37.8 degrees C) reflecting summer conditions in the valley, and one control temperature regimen (low 18.3 degrees C, high 23.9 degrees C) on the fly's survival and reproductive success in the laboratory. The temperature regimen of 18.3-35.0 degrees C resulted in delayed egg maturation and reduced production of mature eggs compared with the control temperature regimen. Egg maturation was possible at the higher temperature regimen when females were provided with water and food, and egg-laying occurred during the cold phase of the temperature cycle. Access to olive fruit and oviposition itself further promoted egg maturation. Under exposure to the 18.3-35.0 degrees C temperature regimen, approximately 50% of eggs died, and the remainder that hatched died as first instars. No egg hatch occurred at the temperature treatment of 18.3-37.8 degrees C. We confirmed these laboratory results through field cage studies with adult B. oleae, conducted in the summer of 2007 and 2008. Under ambient summer temperatures, adult B. oleae survived for 1-2 wk, and females readily laid eggs when provided water and food. No offspring developed in midsummer of 2007, and <2% of the offspring developed to adults in summer 2008 trials. These results suggest that high summer temperatures limit the fly's abundance in California's Central Valley.

Seasonal population dynamics of Homalodisca vitripennis (Hemiptera: Cicadellidae) in sweet orange trees maintained under continuous deficit irrigation.

A 2-yr study was conducted in a citrus orchard (Citrus sinensis L. Osbeck cultivar Valencia) to determine the influence of plant water stress on the population dynamics of glassy-winged sharpshooter, Homalodisca vitripennis (Germar). Experimental treatments included irrigation at 100% of the crop evapotranspiration rate (ET(c)) and continuous deficit-irrigation regimens at 80 and 60% ET(c). Microclimate and plant conditions monitored included temperature and humidity in the tree canopy, leaf surface temperature, water potential, and fruit quality and yield. Glassy-winged sharpshooter population densities and activity were monitored weekly by a combination of visual inspections, beat net sampling, and trapping. Glassy-winged sharpshooter populations were negatively affected by severe plant water stress; however, population densities were not linearly related to decreasing water availability in plants. Citrus trees irrigated at 60% ET(c) had significantly warmer leaves, lower xylem water potential, and consequently hosted fewer glassy-winged sharpshooter eggs, nymphs, and adults than trees irrigated at 80% ET(c). Citrus trees irrigated at 100% ET(c) hosted similar numbers of glassy-winged sharpshooter stages as trees irrigated at 60% ET(c) and a lower number of glassy-winged sharpshooter nymphs than the 80% ET(c) treatment, specifically during the nymphal density peak in mid-April to early July. Irrigation treatments did not affect populations of monitored natural enemies. Although the adult glassy-winged sharpshooter population was reduced, on average, by 50% in trees under severe water stress, the total number of fruit and number of fruit across several fruit grade categories were significantly lower in the 60% ET(c) than in the 80 and 100% ET(c) irrigation treatments.

Effects of Peganum harmala (Zygophyllaceae) seed extract on the olive fruit fly (Diptera: Tephritidae) and its larval parasitoid Psyttalia concolor (Hymenoptera: Braconidae).

Peganum harmala L. (Zygophyllaceae) is an herb native to arid and semiarid regions of Central Asian deserts. This study investigated the effects of ethanol extracts of P. harmala seeds on the olive fruit fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae), i.e., adult repellency, reproductive activity, and larval growth, as well as parasitism levels by Psyttalia concolor (Szépligeti). Olive fruit treated with 2% extract reduced B. oleae oviposition. In choice tests, female B. oleae spent >99% of their time foraging on untreated fruit rather than P. harmala-treated fruit. These changes in ovipositional behavior resulted in a nearly 30-fold decrease in oviposition marks on treated fruit compared with untreated fruit during a 48 h exposure period. When female B. oleae were fed liquid diet containing 0.2% P. harmala extract, there was no effect on the number of ovipositional marks on exposed fruit, but up to 21.4% of the deposited eggs were deformed. SDS-polyacrylamide gel electrophoresis analyses of deformed eggs revealed that some protein bands were missing. Consequently, the number of offspring produced by treated females was lower than by untreated females. Neither the sex ratio nor body size of the fly's offspring were affected by adults fed diet containing 0.2% P. harmala extract. However, there was a slightly prolonged developmental time from egg to adult. Parasitism of larval B. oleae by P. concolor was not affected by infested fruit treatment with 2% P. harmala extract. P. harmala extracts as a potential control for insect pest species are discussed.

Seasonal population dynamics of Draeculacephala minerva (Hemiptera: Cicadellidae) and transmission of Xylella fastidiosa.

The grass sharpshooter, Draeculacephala minerva Ball (Hemiptera: Cicadellidae), is a very common and often abundant grass-feeding leafhopper in California. Its population dynamics and ability to transmit Xylella fastidiosa were monitored over a 2-yr period in California's San Joaquin Valley. Collections of individuals from natural populations in irrigated pastures and alfalfa, Medicago savita L. fields adjacent to X. fastidiosa-infected almond (Prunus spp.) orchards indicated the occurrence of three discrete generations per year that peaked during the summer. Population densities varied significantly among experimental field survey sites. Insects captured on intercepting mesh traps, yellow sticky cards, and UV-light traps indicated local movement of these insects into and surrounding X. fastidiosa-infected, almond orchards. Local movement and seasonal transmission of X. fastidiosa from infected almonds to Catharanthus roseus (L.) G. Don indicated that this insect may be partly responsible for the slow spread of almond leaf scorch now recently observed in California's San Joaquin Valley.

Behavioral responses of Homalodisca vitripennis (Hemiptera: Auchenorrhyncha: Cicadellidae) on four Vitis genotypes.

Pierce's disease is a major threat to the California grape industry. The disease-causing bacterium Xylella fastidiosa is vectored by a number of leafhoppers including Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae). Experiments were conducted to study H. vitripennis preference, feeding, and survivorship in response to four Vitis genotypes. Plants of V. vinifera ('Chardonnay'), V. girdiana, V. candicans, and a V. rupestris x V. arizonica/candicans hybrid (D8909-17) were grown in pots in the greenhouse and transferred to laboratory conditions for experiments with field-collected H. vitripennis. A choice test without prior insect acclimation on grapes revealed that H. vitripennis selected Chardonnay over V. candicans throughout the duration of the experiment, whereas a shift in preference between D8909-17 and V. girdiana was observed over time. In a second set of choice tests, which were preceded by an acclimation on one of the four grape genotypes, significant genotype, time, and acclimation x genotype effects were observed. Chardonnay was preferred over V. candicans independent of acclimation genotype. Although H. vitripennis confined on D8909-17 excreted 1.8-fold (dry-weight corrected) the amount of insects feeding on V. candicans, differences in the rate of excreta production per insect or insect dry weight were not significant among grape genotypes. Adult mortality was greatest on V. candicans when H. vitripennis were confined in parafilm sachets for excreta collection as well as in a no-choice test. Grape genotype affected the behavior of adult H. vitripennis under controlled conditions, which may influence Pierce's disease epidemiology under field conditions.

Multiple plant exploiters on a shared host: testing for nonadditive effects on plant performance.

The combined impact of multiple plant parasites on plant performance can either be additive (the total damage equals the sum of the individual effects) or nonadditive (synergistic or antagonistic damage). Two statistical models are available for testing the independent (=additive) effects of two factors. Here we suggest that the natural history of the plant-parasite system should motivate the choice of a statistical model to test for additivity. Using in-field, manipulative experiments, we examined the interactions between the herbivorous mite Calacarus flagelliseta Fletchmann, De Moraes and Barbosa (Acari: Eriophyidae), the fungal pathogen Oidium caricae F. Noack (a powdery mildew), and their host plant Carica papaya L. in Hawaii. First, we found that herbivorous mites had a moderate negative effect on powdery mildew: when mites were absent, powdery mildew colonies were larger and more numerous. Second, we showed that each plant parasite, when evaluated alone, significantly reduced several measures of plant performance. Third, we found that the combined impact of mites and mildew on plant performance is mostly additive and, for a few variables, less than additive. Finally, we explored compensatory responses and found no evidence for nonlinearities in the relationship between plant performance and cumulative parasite impact. Plants are almost universally subject to attack by multiple herbivores and pathogens; thus a deeper understanding of how multiple plant parasites shape each other's population dynamics and plant performance is essential to understanding plant-parasite interactions.

Population dynamics and within-plant distribution of the mite Calacarus flagelliseta (Acari: Eriophyidae) on papaya in Hawaii.

An important element in developing a management strategy for a new pest is the study of its seasonal dynamics and within-plant distribution. Here, we studied the mite Calacarus flagelliseta Fletchmann, De Moraes & Barbosa on papaya, Papaya carica L. (Caricaceae), in Hawaii to quantify 1) patterns of seasonal abundance, 2) its distribution across different vertical strata of the papaya canopy, and 3) shifts in its use of the upper versus the lower surfaces of papaya leaves. Nondestructive sampling conducted in two papaya plantings revealed that 1) populations of C. flagelliseta peak during the summer; 2) mites are most abundant in the middle and lower strata of the plant canopy, and least abundant on the youngest leaves found in the upper canopy; and 3) mites are found more predominantly on the upper leaf surfaces when overall population density peaks, suggesting that individuals move from the lower to the upper leaf surfaces when food resources on the lower leaf surface have been exploited by conspecifics. These results have significant implications for the development of sampling plans for C. flagelliseta in papaya.

Herbivorous mites as ecological engineers: indirect effects on arthropods inhabiting papaya foliage.

We examined the potential of a leaf roller to indirectly influence a community of arthropods. Two mite species are the key herbivores on papaya leaves in Hawaii: a spider mite, Tetranychus cinnabarinus Boisduval, and an eriophyid mite, Calacarus flagelliseta, which induces upward curling of the leaf margin at the end of the summer when populations reach high densities. A survey and three manipulative field experiments demonstrated that (1) leaf rolls induce a consistent shift in the spatial distribution of spider mites and their predators, the coccinellid Stethorus siphonulus Kapur, the predatory mites Phytoseiulus spp., and the tangle-web building spider Nesticodes rufipes Lucas; (2) the overall abundance of spiders increases on leaves with rolls; (3) the specialist predators Stethorus and Phytoseiulus inhabit the rolls in response to their spider mite prey; and (4) the spider inhabits the rolls in response to the architecture of the roll itself. This study shows the importance of indirect effects in structuring a terrestrial community of herbivores.

The genetic properties of the primary endosymbionts of mealybugs differ from those of other endosymbionts of plant sap-sucking insects.

Mealybugs (Hemiptera, Coccoidea, Pseudococcidae), like aphids and psyllids, are plant sap-sucking insects that have an obligate association with prokaryotic endosymbionts that are acquired through vertical, maternal transmission. We sequenced two fragments of the genome of Tremblaya princeps, the endosymbiont of mealybugs, which is a member of the beta subdivision of the Proteobacteria. Each of the fragments (35 and 30 kb) contains a copy of 16S-23S-5S rRNA genes. A total of 37 open reading frames were detected, which corresponded to putative rRNA proteins, chaperones, and enzymes of branched-chain amino acid biosynthesis, DNA replication, protein translation, and RNA synthesis. The genome of T. princeps has a number of properties that distinguish it from the genomes of Buchnera aphidicola and Carsonella ruddii, the endosymbionts of aphids and psyllids, respectively. Among these properties are a high G+C content (57.1 mol%), the same G+C content in intergenic spaces and structural genes, and similar G+C contents of the genes encoding highly and poorly conserved proteins. The high G+C content has a substantial effect on protein composition; about one-third of the residues consist of four amino acids with high-G+C-content codons. Sequence analysis of DNA fragments containing the rRNA operon and adjacent regions from endosymbionts of several mealybug species suggested that there was a single duplication of the rRNA operon and the adjacent genes in an ancestor of the present T. princeps. Subsequently, in one mealybug lineage rpS15, one of the duplicated genes, was retained, while in another lineage it decayed. These results extend the diversity of the types of endosymbiotic associations found in plant sap-sucking insects.