Seasonal Abundance and Spatio-Temporal Distribution of Dominant ...

COMMUNITY AND ECOSYSTEM ECOLOGY

Seasonal Abundance and Spatio-Temporal Distribution of Dominant Xylem Fluid-Feeding Hemiptera in Vineyards of Central Texas and Surrounding Habitats ISABELLE LAUZIÈRE,1 SIMON SHEATHER,2

AND

FORREST MITCHELL3

Environ. Entomol. 37(4): 925Ð937 (2008)

ABSTRACT A survey of xylem ßuid-feeding insects (Hemiptera) exhibiting potential for transmission of Xylella fastidiosa, the bacterium causing PierceÕs disease of grapevine, was conducted from 2004 to 2006 in the Hill Country grape growing region of central Texas. Nineteen insect species were collected from yellow sticky traps. Among these, two leafhoppers and one spittlebug comprised 94.57% of the xylem specialists caught in this region. Homalodisca vitripennis (Germar), Graphocephala versuta (Say), and Clastoptera xanthocephala Germar trap catches varied signiÞcantly over time, with greatest counts usually recorded between May or June and August and among localities. A comparison of insect counts from traps placed inside and outside vineyards indicated that G. versuta is always more likely captured on the vegetation adjacent to the vineyard. C. xanthocephala was caught inside the vineyard during the summer. Between October and December, the natural habitat offers more suitable host plants, and insects were absent from the vineyards after the Þrst freezes. H. vitripennis was caught in higher numbers inside the vineyards throughout the grape vegetative season. However, insects were also caught in the habitat near the affected crop throughout the year, and residual populations overwintering near vineyards were also recorded. This study shed new light on the fauna of xylem ßuid-feeding insects of Texas. These results also provide critical information to vineyard managers for timely applications of insecticides before insect feeding and vectoring to susceptible grapevines. RE´ SUME´ Cette e´tude, reáliseé entre avril 2004 et mai 2006, a pris place dans la re´gion viticole de Hill Country au centre du Texas et a examine´ les insectes phytophages du xyle`me qui sont vecteurs potentiels de la bacte´rie Xylella fastidiosa responsable de la maladie de Pierce de la vigne. Dix neuf espe`ces dÕhe´mipte`res ont e´te´ collecteés sur de pie`ges jaunes collants, parmi lesquels, deux cicadelles et un cercope constituaient 94.57% des spećialistes du xyle`me de cette re´gion. LÕabondance de Homalodisca vitripennis (Germar), Graphocephala versuta (Say) et Clastoptera xanthocephala Germar variait signiÞcativement a` travers le temps, leurs maxima ayant e´te´ enregistre´s entre mai ou juin et aouˆ t, ainsi quÕentre les diffe´rentes localite´s. La comparaison des comptes dÕinsectes des pie`ges situe´s dans le vignoble et ceux place´s en dehors indiquait que G. versuta est toujours plus souvent pie´ge´ pre`s de la ve´ge´tation qui borde le vignoble. Clastoptera xanthocephalaae´te´ pie´ge´ danslavignedurantlape´riodeestivalealorsquÕentreoctobreetdećembre,lÕhabitatnaturel offre plus de plantes hoˆtes convenables. Cette espe`ce est absente apre`s les premiers gels. Homalodisca vitripennis est capture´ en beaucoup plus grands nombres a` lÕinte´rieur des vignobles durant toute la saison ve´ge´tative de la vigne. Cependant, cet insecte est aussi pie´ge´ toute lÕanneé dans lÕhabitat proximal de la culture affecteé et des populations re´siduelles hivernant pre`s des vignobles ont aussi e´te´ de´tecteés. Cette e´tude enrichit notre niveau de connaissance de la faune connue des insectes suceurs du xyle`me du Texas Central. Les re´sultats sont aussi critiques aux viticulteurs en ce qui concerne lÕapplication bien cibleé dÕinsecticide avant lÕexploitation et la transmission de la maladie par les insectes aux vignes susceptibles. KEY WORDS leafhopper, glassy-winged sharpshooter, PierceÕs disease, Auchenorrhyncha, Hexapoda

The insects from the suborder Auchenorrhyncha of the order Hemiptera that feed on xylem ßuid have the This article presents the results of research only. Mention of a commercial or proprietary product does not constitute an endorsement or recommendation for its use by the Texas Agricultural Experiment Station or Texas A&M University. 1 Corresponding author: Texas PierceÕs Disease Research and Education Program, 259 Business Court, Texas AgriLife Research Fredericksburg, TX 78624 (e-mail: [email protected]).

ability to acquire from infected plants, carry, and transmit to healthy host plants a bacterium, Xylella fastidiosa Wells et al., responsible for several plant diseases. Different strains of this bacterium are patho2 Department of Statistics, Texas A&M University, 3143 TAMU, College Station, TX 77843. 3 Texas AgriLife Research 1229 North U.S. Highway 281, Stephenville, TX 76401.

0046-225X/08/0925Ð0937$04.00/0 䉷 2008 Entomological Society of America

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genic in plants such as grapevine (PierceÕs disease), stone fruits (phony peach disease), almond, coffee, oleander, elm, oak, and sycamore (leaf scorchs), alfalfa (alfalfa dwarf disease), and citrus (citrus variegated chlorosis) (Blua et al. 1999, Hopkins and Purcell 2002). Susceptible plants infected with X. fastidiosa die relatively slowly from restriction of sap movement in the plantÕs conductive tissue (Freitag 1951, Purcell 1981). In the past 100 yr, grape production in California experienced several epidemics of PierceÕs disease known to be transmitted by three relatively small leafhopper vectors, Draeculacephala minerva Ball, Graphocephala atropunctata (Signoret), and Xyphon fulgida Nottingham (Cicadellini), common in riparian/weedy habitats (Wrinkler 1949, Young 1977, Purcell 1980, 1981, Hopkins and Purcell 2002). The accidental introduction of the glassy-winged sharpshooter, Homalodisca vitripennis (Germar) (Proconiini), formerly H. coagulata (Takiya et al. 2006), a much larger leafhopper, into southern California during the late 1990s resulted in a very serious threat to the grape agricultural system in that state (Phillips 1999). This newly introduced species was a challenge because it exhibits different movement and feeding behaviors within the vineyards (Redak et al. 2004). A multiinstitutional program to manage the new outbreaks was quickly implemented starting in 2000 Ð2001 to prevent further devastation to the grape acreage and potential spread to the coastal and northern grape producing counties in the state. In this aggressive program, any population of this insect pest is immediately treated with chemicals to prevent further spread to vineyards and other host plants grown in close proximity to vineyards (Wendel et al. 2002, Redak et al. 2004). Similarly, grape growers in Texas have been dealing with this disease since the introduction of Vitis vinifera varieties over a century ago. During the 1990s, the grape growing region of central Texas witnessed an increase in the incidence and severity of PierceÕs disease (McEachern et al. 1997, Texas PierceÕs disease Task Force 2004). The potential vector species were not thoroughly identiÞed but glassy-winged sharpshooters were among suspected vectors. According to Young (1968), H. vitripennis is native to the southeastern United States, including Texas. In addition, the gulf coast states are known as the area of origin of X. fastidiosa (Hewitt 1958). Researchers in Texas therefore were provided an opportunity to study glassywinged sharpshooters in their natural habitat. This was impossible in California where the insect pest is not endemic and where an active suppression program was initiated. Since 2003, detailed and intensive observations have been conducted on xylem ßuid-feeding Hemiptera associated with grapes in Texas. This study, conducted in 2004 Ð2006, was meant to shed light on the seasonality and spatio-temporal distribution of putative vectors of PierceÕs disease throughout the growing season. Limited information was available in Texas when we initiated this work. A general key to identify members

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of the Cicadellidae captured near vineyards in north and central Texas was developed by Medley in 1997 (Medley 2004). It was also reported that over the past years, glassy-winged sharpshooters appear in vineyards the last week of May. In the robust agricultural setting found in California, glassy-winged sharpshooters move year-round from one preferred host to another. This rotation includes grapevine, citrus, and a variety of agricultural crops and ornamentals used throughout the year. Perennial and row crops are rarely grown adjacent to vineyards in Texas or with the same intensity as found in California. Most Texas vineyards are much smaller than those found in California and the natural vegetation growing adjacent to the vineyards, sometimes along bodies of water, may provide suitable food sources, sites for oviposition, and shelter to a variety of insect species at different times of the year. Among our program objectives are the identiÞcation of factors that contribute to the spread of PierceÕs disease infestations and the determination of insect management needs. This study provides evidence of seasonal changes in insect behavior and populations and also serves as a basis for in-depth studies of the overwintering behavior of select vectors of X. fastidiosa and the characterization of the mechanisms involved in host plant selection. Knowledge of the ecology of xylem ßuid-feeding insect populations in their natural environment, the species involved with PierceÕs disease, their behavior throughout the year, and when and where they acquire the bacterium is critical to the development of timely management strategies. Materials and Methods Texas is the Þfth largest wine producer in the United States, and the economic impact of this industry in the state is more than $200 million (Dodd et al. 2006). Although grape and wine production is expanding throughout the state, most vineyards and wineries are found in the Hill Country and the north-central (Dallas-Fort Worth Metroplex) regions where tourism contributes to their economic viability (Dodd et al. 2006). In these regions, PierceÕs disease has increased in intensity and severity and has become a serious threat to the production of preferred V. vinifera varieties. Commercial vineyards (n ⫽ 20) producing wine grape varieties selected for this study were located in 13 counties in the Hill Country region of central Texas on the eastern half of the Edwards Plateau (Fig. 1). The area where observations were carried out is delimited east and west by Interstate 35 and Interstate 10 and ranges as far north as San Saba and as far south as Bulverde, near San Antonio. At these selected locations, elevations varied between 235 and 550 m above sea level (Meridian Magellan GPS receiver; Thales Navigation Consumer Products, San Dimas, CA). Vineyard geographic location and main characteristics as they relate to this insect survey are listed in Table 1. Vineyards were monitored for xylem ßuid-feeding Hemiptera for a period of 2 yr, between April 2004 and

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LAUZIE` RE ET AL.: DISTRIBUTION OF THREE HEMIPTERA IN VINEYARDS

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Fig. 1. Number of traps monitored in each county where commercial vineyards were surveyed for xylem ßuid-feeding Hemiptera in central Texas.

May 2006, depending on the starting date of observations at individual locations. Monitoring in one of the originally selected vineyards was suspended during 2004 and at another during 2005 (vineyards closed). Traps were renewed every 2 wk. Monitoring of insect populations took place using standard double-sided traps (Seabright Laboratories, Emeryville, CA), each 23 by 14 cm in size, bright yellow in color (Pantone Matching System [PMS] 102), and coated with Stikem Special glue. Traps were tightly stapled to a 1.8-m bamboo stake driven into the ground a little lower than grapevine canopy. Between 6 and 13 traps were placed in each vineyard. Traps were located on each cardinal compass face of the vineyard, at the edge of the Þrst plant row, and at half the length of the row. One trap was also placed at the very center of the vineyard. These traps are referred to as “inside traps.” Within property boundaries, where natural habitat surrounded the vines, additional traps were set at 50 and 100 m. These traps are referred to as “outside traps.” Overall, the trapping pattern formed a cross passing through the center of the vineyard with traps at 50-m intervals outside the vineyard. Exposed traps were retrieved and placed individually in plastic bags. Traps were refrigerated at 10⬚C

until reviewed. Each trap was reviewed in the laboratory under a stereomicroscope (⫻6; Stemi SV 11; Zeiss MicroImaging, Thornwood, NY). All adult xylem ßuid-feeding Hemiptera were identiÞed to species and counted. Specimens that could not be identiÞed immediately were extracted from the glue using undiluted Histo-Clear II (National Diagnostics, Atlanta, GA) to dissolve the glue. Specimens were photographed, coded, placed in alcohol, and labeled. Unknown Hemiptera were sent to the Center for Biodiversity, Illinois Natural History Survey (Champaign, IL), for further identiÞcation to species by taxonomist R. Rakitov. Voucher specimens of each species were placed at this museum. Trap count data were analyzed using a generalized additive Poisson regression model allowing for overdispersion because many of the data points were zeroes, especially in winter months. All computations were performed using the statistical package R (R Development Core Team 2006). Interest centered on testing whether signiÞcant differences existed between trap counts: (1) over time; (2) inside and outside vineyards; and (3) across vineyards. Total sample size was 10,865 traps. Three separate sets of counts, i.e., H. vitripennis, Graphocephala versuta, and Clastoptera

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Table 1. Geographical information, varieties cultivated, general vineyard and survey characteristics, and mean total no. of xylem fluid-feeding Hemiptera captured in vineyards of the Edwards Plateau County Bexar Blanco Blanco Burnet Comal Gillespie Gillespie Hays Kendall Llano Llano Mason Mason Mason Mason McCullock Menard San Saba Travis

Elevation (m)

Grape variety

Acreage

PierceÕs disease

Insecticide treatment

Traps

Insects/trap/da (mean ⫾ SD)

329 332 473 331 333 465 545 336 373 271 319 487 527 401 523 488 548 335 235

BS; CS; Sh C; CB; CS; CF; M; OM; PG; PV; Sa M; Sa C; CB; CF; CS; M; Ma; PV; Sh BS; N; V C; CS; M; SB CS; M; Ma C; CB; CS; MB; PN; Sa; Sh; Vi BS CS; Sh CS; M; Sh G; Mo; Pr; Sa; Sh B; Ma; PG; Pr; PV; Sh Sh CS; G; Ma; Mo; PG; PV; Sh C; CS; M; MC B; C; CB; CF; CS; M; PN; R; SB; Sh; Z CS; G; MaB; Mo; Sa; Sh; T; Vi MB; MC; PB; PG; Pr; Sa; Sh; T; TM

5.0 18.9 1.8 8.8 5.3 20.0 27.0 14.1 4.0 3.0 6.0 7.5 11.5 12.0 31.3 29.1 212.0 13.0 15.2

Yes Yes Yes Yes Yes Yes No No Yes No Yes No No No No No No Yes No

Yes Yes Yes Yes No Yes No Yes Yes Yes No Yes Yes Yes No Yes Yes No Yes

8 9 11 10 8 7 10 13 8 6 10 6 6 12 11 7 7 8 10

0.06 ⫾ 0.13 0.09 ⫾ 0.34 0.08 ⫾ 0.20 0.05 ⫾ 0.20 0.26 ⫾ 0.63 0.20 ⫾ 0.90 0.19 ⫾ 0.74 0.06 ⫾ 0.19 0.51 ⫾ 1.01 0.20 ⫾ 0.43 0.40 ⫾ 1.45 0.04 ⫾ 0.09 0.16 ⫾ 0.46 0.20 ⫾ 0.45 0.18 ⫾ 035 0.18 ⫾ 0.43 0.03 ⫾ 0.06 0.19 ⫾ 0.49 0.07 ⫾ 0.29

a

Xylem ßuid-feeding Hemiptera with potential to transmit PierceÕs disease. B, Barbera; BS, Black Spanish; C, Chardonnay; CB, Chenin Blanc; CS, Cabernet Sauvignon; CF, Cabernet Franc; G, Grenache; M, Merlot; Ma, Malbec; MaB, Malvasia Bianca; MB, Muscat Blanc; MC, Muscat Canneli; Mo, Mourvedre; N, Norton; OM, Orange Muscat; PB, Pinot Blanc; PN, Pinot Noir; PG, Pinot Grigio; Pr, Primitivo Di Gioia; PV, Petit Verdot; R, Riesling; Sa, Sangiovese; SB, Sauvignon Blanc; Sh, Shiraz; T, Tempranillo; V, Vignoles; Vi, Viognier; Z, Zinfandel.

xanthocephala, were analyzed after we determined these were the preponderant insect species in the region (see Results). If we denote these counts by Y, the generalized additive Poisson regression model is of the form: Y ⬃ Poisson{exp[␤0 ⫹ s(Time) ⫹ ␤1x 1 ⫹ . . . ⫹ ␤px p]} where x1, . . . , xp are predictor variables and s denotes a smooth function that is estimated from the data. Smooth functions are a feature of a class of analyses termed general additive models (GAMs) (Hastie and Tibshirani 1990). In linear systems, the Þt of the predictor variables is constrained to known functions, which is where much of the power of these models lies. Under generalized linear model (GLM) theory, squared, cubic, and fourth-order power transformations of predictor variables allow for model Þts to response surfaces that are not linear. When no a priori information can be asserted for a predictor variable or transformation of such, it can instead be modeled as a smoothed function, similar in concept to a moving average. The smoothing function is often a spline, in this case a penalized regression spline, and such is nonparametric. Although there is considerable exploratory value in accepting this approach, some power is sacriÞced because, in a GAM, the smoothed predictor variables become explanatory variables, due to the absence of a deÞned parametric Þt. However, because the data determine the shape of the response curve by the smoothed Þt rather than a preset parametric function of a predictor variable, a GAM may be more suitable for Þnding asymmetry and multipeak responses such as bimodality than would a comparable GLM (Yee and Mitchell 1991). Once a response is

deÞned, it may be possible to replace the smoothed variable with a parametric variable, ultimately resulting in a more descriptive model. Model 1 was Þt using the GAM function from the R-library mgcv (multiple generalized cross-validation) with the default settings. The smooth function s is a penalized regression spline with the smoothing parameter selected by generalized cross-validation (gcv). The smoothing parameter will determine the degree to which surrounding data are incorporated into the smoothing of a single point and is objectively determined by the gcv algorithm from the data. In doing this, the Þt can be adjusted by the smoothing parameter and can vary from completely smoothed, a straight line, to completely unpenalized, the actual model Þt without smoothing the variable (Wood 2006). This means that an unpenalized GAM is a GLM and it follows from this that the choice of linear predictor is the major difference between the GAM and GLM (Barry and Welsh 2002). To standardize the data, insect captures were expressed as the number per trap per day. The fact that the number of days varied across months was taken into account using what is commonly referred to as an offset in the form of ln(d). This is a general approach when data are expressed as rates (Simonoff 2003). One of the advantages of using a Poisson regression model is that the estimated coefÞcient for the jth predictor can be interpreted as follows: 100 ⫻ [exp(␤ˆ j) ⫺ 1] is the estimated expected percentage change in Y associated with a one unit change in the jth predictor variable, holding all else in the model Þxed. The additive model Þt to each of the three separate sets of counts is based on the following predictor variables: (1) x1 is a dummy variable that is 1 if the trap was

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Table 2. Taxonomical identification, count, and percent xylem fluid-feeding Hemiptera captured in and around central Texas vineyards between April 2004 and May 2006 using glue-coated traps Superfamily

Family

Tribe

Species

N

% Total

Membracoidea Membracoidea Cercopoidea Membracoidea Membracoidea Cercopoidea Membracoidea Membracoidea Membracoidea Membracoidea Membracoidea Membracoidea Membracoidea Cercopoidea Membracoidea Cicadoidea Membracoidea Cercopoidea Membracoidea

Cicadellidae Cicadellidae Clastopteridae Cicadellidae Cicadellidae Clastopteridae Cicadellidae Cicadellidae Cicadellidae Cicadellidae Cicadellidae Cicadellidae Cicadellidae Clastopteridae Cicadellidae Tibicinidae Cicadellidae Cercopidae Cicadellidae

Proconiini Cicadellini Clastopterini Cicadellini Cicadellini Clastopterini Proconiini Cicadellini Proconiini Cicadellini Cicadellini Proconiini Proconiini Clastopterini Cicadellini Fidicinini Cicadellini Lepyroniini Cicadellini

Homalodisca vitripennis (Germar) Graphocephala versuta (Say) Clastoptera xanthocephala Germar Xyphon sagittifera (Uhler) Draeculacephala navicula Hamilton Clastoptera lawsoni Doering Homalodisca insolita (Walker) Graphocephala hieroglyphica (Say) Cuerna costalis (Fabricius) Xyphon flaviceps (Riley) Sibovia occatoria (Say) Oncometopia sp. (undescribed) Paraulacizes irrorata (Fabricius) Clastoptera lineatocollis Stål Graphocephala coccinea (Forster) Pacarina puella Davis Ciminius harti (Ball) Lepyronia quadrangularis (Say) Draeculacephala robinsoni Hamilton

12,841 4,571 2,492 269 258 145 116 76 75 62 33 32 19 18 12 11 11 4 4

61.01 21.72 11.84 1.28 1.23 0.69 0.55 0.36 0.36 0.29 0.16 0.15 0.09 0.09 0.06 0.05 0.05 0.02 0.02

outside the vineyard and 0 otherwise and (2) x2 to x19 are variables representing the effect of each vineyard. It is possible to assess the statistical signiÞcance of the terms in model 1 using F-tests, which are extensions of those used in analysis of variance (ANOVA) (Wood 2006). GLM hypothesis tests based on likelihood ratios and ␹2 distributions can be generalized to GAM, using effective degrees of freedom for the penalized spline rather than the real degrees of freedom (to avoid too many inßections in the smoothed result). This generalization results in F-tests rather than ␹2 tests, because in GAM, the scale parameter is not Þxed but has to be estimated (Hastie and Tibshirani 1990, Wood 2006). Temperature and relative humidity were monitored at each location every 15 min using HOBO data loggers (Onset Computer, Bourne, MA) placed in a threesided white wooden box hung in the vineyard above the canopy. Climatological data of interest were summarized and presented as an additional tool to understand variations in insect densities and seasonal changes in insect behavior. Results Insect populations in the Hemiptera were monitored twice a month in 18 vineyards for 2 yr. Our interest focused on insect species that ingest primarily xylem ßuid and may be involved in the transmission of X. fastidiosa. Data indicate the presence of three groups of xylem specialists: leafhoppers (Cicadellidae), spittlebugs (Clastopteridae), and cicadas (Tibicinidae) (Table 2). Of 155 Hemiptera species captured in the vineyards and adjacent natural habitat, 19 species in 12 genera were identiÞed as xylem specialists. Captured insects in the family Cicadellidae (leafhoppers) were the most abundant, with a total of 14 species recovered; this family contributed to 87.33% of all individuals caught in 2004 Ð2006. Predominant species in the samples were H. vitripennis (Germar), G. versuta (Say), and C. xanthocephala Germar, two leafhoppers and a spittlebug. These species together com-

prised 94.57% of all xylem ßuid-feeding insects identiÞed, with 61.01, 21.72, and 11.84% abundance attributed to each species, respectively (Table 2). H. vitripennis and C. xanthocephala were present at each of the surveyed locations. However, no G. versuta were collected at location 1, and only one adult was collected at location 5. Insect species number varied from 7 to 15 per location, with an average of 10.33 ⫾ 2.59 species per location. However, at certain locations, a speciÞc Hemiptera species clearly dominated. For example, at Þve locations, trap counts for H. vitripennis exceeded 80%, at two locations, trap counts for G. versuta exceeded 50%, and at four locations, trap counts for C. xanthocephala exceeded 25%, well above the respective regional averages. The average total number of adult xylem ßuid-feeding Hemiptera per trap per day caught over the 2-yr period of time was 0.17 ⫾ 0.62 (n ⫽ 10,865; range: 0.00 Ð14.33). The mean number (all years combined, all traps combined, all species combined) of insects caught at each location is reported in Table 1. Insect abundance varied through time, and the insect counts were cyclical, increasing in the spring and early summer (AprilÐJuly), and progressively decreasing in the late summer and fall (AugustÐNovember) to remain low thereafter. At most locations, xylem ßuid-feeding Hemiptera trap catches remained relatively low throughout the vineÕs vegetative season (MarchÐOctober; 0.22 ⫾ 0.01 insects/trap/d), except for June when adult counts were the highest (0.51 ⫾ 0.03 insects/trap/d). In this region, temperatures peaked during the months of May through August, and mean temperatures were 26.88 ⫾ 5.91⬚C (n ⫽ 720,139; range: 3.31Ð 47.96⬚C). Between November and February, mean temperatures of 12.17 ⫾ 8.09⬚C (n ⫽ 457,155; range: ⫺13.49 to 36.13⬚C) were registered. Climatological data were summarized and indicate that, during both winters (2004 Ð2005 and 2005Ð2006), temperatures remained below 0⬚C for a total of 100 Ð140 and 200 Ð240 h, respectively. On average, temperatures remained below 0⬚C for 8.63 ⫾ 7.65 consecutive h at

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Fig. 2. Nonparametric estimate of the time effect (smooth curve) with ⬃95% conÞdence bands (dashed curves) for H. vitripennis.

a time (n ⫽ 490; range: 1.00 Ð71.00 h), usually somewhere between 1900 and 0700 hours, and minimal temperatures of ⫺2.83 ⫾ 2.23⬚C (n ⫽ 18,487; range: ⫺13.49 to ⫺0.16⬚C) were observed at that time. F-tests showed that H. vitripennis counts differed signiÞcantly over time (P ⬍ 0.001) and across vineyards (P ⬍ 0.001). The overall difference between H. vitripennis counts inside and outside the vineyard was not statistically signiÞcant (P ⫽ 0.874). Figure 2 displays the nonparametric estimate of the time effect for H. vitripennis counts (the smooth curve) along with ⬃95% conÞdence bands (the dashed curves) for the mean at each time point and partial residuals (i.e., H. vitripennis counts adjusted for differences caused by effects other than time) (Wood 2006). Interpretation of Fig. 2 shows one of the features of a GAM. The y-axis label s(Time) refers to the Þtted smooth function of the variable Time achieved by means of the penalized spline. The function is normalized ⬇0 and ßuctuates between positive and negative values depending on its contribution to the model at that point in time. The term s(Time) is put into the model the same way a linear term is. Instead of a calculated coefÞcient for a predictor variable (i.e., ␤pxp), a function f(xp), in this case s(Time), is inserted. This could also be referred to as f(Time). Although confusing, the term s(variable name) is a convention that has been adopted and is also in use with the software packages that can calculate it. To illustrate this application of the function, in June 2004 (Fig. 2), an additional 1.9 units will be added to the model output to reßect the increased number of H. vitripennis present at that time of year.

Conversely, 1 unit is subtracted from the model output in January 2005 to reßect the absence of H. vitripennis in the vineyard vicinity at that time of year. Elaboration on the application of smooth functions can be found in Zuur et al. (2007). Because of the exploratory approach taken using a smooth function, the quality of the function relative to use in the model is most easily interpreted graphically. If the function is not understandable or useful, it can either be removed from the model or retried by changing the degrees of freedom, the spline or smoothing function used to generate it, or the span used to Þt the spline. The function in Fig. 2 is intuitive because the populations of H. vitripennis rise in the warm months and decline in the colder months, and the contribution of Time to H. vitripennis abundance can be estimated. Data were further analyzed to see if the difference between H. vitripennis counts inside and outside the vineyard was consistent over time. An F-test found that it was not (P ⬍ 0.001). Therefore, separate nonparametric estimates of the time effect for adult counts of this species inside and outside the vineyards (the solid curves) were graphed along with ⬃95% conÞdence bands (the dashed curves) for the mean at each time point (Fig. 3). Interpreting the results as with Fig. 2, the 2004 summer peak inside the vineyard was found to be higher than the 2004 summer peak outside the vineyard and that the 2004 Ð2005 winter trough inside the vineyard was lower than the same winter trough outside the vineyard (Fig. 3). However, the summer peak and winter trough in 2005Ð2006 were much more similar for counts inside and outside the vineyard. We

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Fig. 3. Estimates of the time effects for H. vitripennis counts inside and outside vineyards.

next studied over which months the difference between inside and outside the vineyard differed significantly from the average difference in H. vitripennis counts. Figure 4 depicts the estimated change in the average difference between outside and inside traps over time, compared with the average. Only statisti-

cally signiÞcant changes are shown (P ⬍ 0.05). The difference between outside and inside counts was minimized in the summer, whereas it was increased in the colder months (the positive percent change in the average difference between outside and inside glassywinged sharpshooter trap counts increased signiÞ-

Percent Change in the average difference between outside and inside trap counts

3000

2500

2000

1500

1000

500

0

May 06

September 05

January 05

May 04

−500

Time

Fig. 4. Estimated percent change in the average between inside and outside traps for H. vitripennis (P ⬍ 0.05).


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0 −4

−2

s(Time)

2

4

932

May 2004

Jan 2005

Sep 2005

May 2006

Time

Fig. 5. Nonparametric estimate of the time effect (smooth curve) with ⬃95% conÞdence bands (dashed curves) for G. versuta.

cantly in October, November, February, and March) because there were signiÞcantly more adults caught in the habitat adjacent to the vineyard during those months. In December and January 2004 Ð2005 and 2005Ð2006, there also were positive differences observed in the difference between inside and outside trap counts. However, this pattern was only close to signiÞcant in December (2004: P ⫽ 0.060; 2005: P ⫽ 0.092) and not signiÞcant in January (2005: P ⫽ 0.150; 2006: P ⫽ 0.120). The explanation lies in the fact that because of the very small number of insects caught during those months, the SEs, especially for January, are very large. The highest adult counts were observed in June for all 3 consecutive yr (0.41 ⫾ 0.03; n ⫽ 10,865; range: 0.00 Ð14.33). Adult trap catches were ⬍0.01/ trap/d (equivalent to one insect per vineyard of 13 traps/wk) in April, October, November, and December 2004, February, March, April, and November 2005, and February and April 2006. Zero counts in the vineyard were seen in January and December 2005 and January and March 2006. Adult counts in the natural habitat bordering the vineyard were lower during colder months than during summer months but never reached zero. These fell below 0.01/trap/d only in April 2004, 2005, and 2006. In the case of G. versuta, the highest trap counts were observed between May and August. Counts from traps placed inside the vineyard were signiÞcantly lower (272%) than the corresponding glassywinged sharpshooter counts (P ⬍ 0.001). Overall, F-tests showed that counts of G. versuta differed statistically over time (P ⬍ 0.001), across vineyards (P ⬍ 0.001), and between cultivated and natural

habitats (P ⬍ 0.001). Furthermore, the difference between G. versuta counts inside (cultivated) and outside (natural) the vineyard did not differ significantly over time (P ⬎ 0.5). In other words, the difference was consistent over time, the estimated percent change in G. versuta counts outside the vineyard relative to inside counts was 152%, i.e., these counts were always signiÞcantly higher on outside traps. Figure 5 shows the nonparametric estimate of the time effect for G. versuta counts along with ⬃95% conÞdence bands for the mean at each time point and partial residuals. A decreasing trend in trap catches over time is noticeable (Fig. 5). Finally, F-tests showed that C. xanthocephala counts differed statistically over time (P ⬍ 0.001) and across vineyards (P ⬍ 0.01), but the overall difference between counts inside (cultivated) and outside (natural habitat) the vineyard was not statistically signiÞcant (P ⫽ 0.227). Figure 6 shows the nonparametric estimate of the time effect for C. xanthocephala counts along with ⬃95% conÞdence bands for the mean at each time point and partial residuals. As in previous cases, data were further analyzed to see whether the difference between adult counts inside and outside the vineyard was consistent over time. An F-test found that this difference was not consistent over time (P ⬍ 0.001). Figure 7 shows separate nonparametric estimates of the time effect for C. xanthocephala counts inside and outside the vineyards, along with ⬃95% conÞdence bands for the mean at each time point. It is clear that C. xanthocephala counts were cyclical both inside and outside the vineyard, increasing in


933

0 −10

−5

s(Time)

5

10

August 2008

May 2004

Jan 2005

Sep 2005

May 2006

Time

Fig. 6. Nonparametric estimate of the time effect (smooth curve) with ⬃95% conÞdence bands (dashed curves) for C. xanthocephala.

outside the vineyard differed signiÞcantly from the average difference in C. xanthocephala counts. In May 2004, C. xanthocephala counts outside the vineyards were signiÞcantly higher, whereas in October and November 2004, there were signiÞcantly more insects on traps set inside the vineyards. The apparent difference observed in the counts of the late

Outside

−5

Inside

−5 −15

−10

s(Time)

0

5

−15

−10

s(Time)

0

5

the summer and decreasing in the winter. It seems from Fig. 7 that the 2006 low counts inside the vineyard were lower than the 2006 low counts outside the vineyard. However, the summer peaks in 2004 Ð2005 were much more similar for counts inside and outside the vineyard. We next studied over which months the difference between inside and

May 2004

Jan 2005

Sep 2005

May 2006

Time

Fig. 7. Estimates of the time effects for C. xanthocephala counts inside and outside vineyards.

934


spring 2006 outside and inside the vineyards was found to be not statistically signiÞcant, because of large SEs at these time points when C. xanthophala counts were very low. Discussion Among insects, xylem-ßuid feeding specialists are found only in the order Hemiptera where this feeding mode is exhibited by all spittlebugs (Cercopoidea) and cicadas (Cicadoidea), and also by leafhoppers (Cicadellidae) from the subfamily Cicadellinae, commonly referred to as sharpshooters (Riley and Howard 1893). In our study, representatives of all of these groups were collected (Table 2). Insect counts and identiÞcation from 10,865 double-sided traps detected 19 xylem ßuid-feeding species belonging to 12 genera. These data underestimate the actual number of xylem ßuid-feeding taxa in the area. Traps were used as indicators of the presence of Hemipteran species and insect densities knowing that trap color and trap height play a signiÞcant role in the effectiveness with which insects get caught (Ozanne 2005). Therefore, various degrees of attractiveness can be expected for the many insect species we encountered. Color traps essentially collect active ßiers, and the standardized height selected can be out of ßying ranges for some of the xylem ßuid-feeding Hemiptera species identiÞed. In 2006, on-site observations using a sweep net for insect collection showed the existence of three species, Tylozygus bifida Say (Cicadellidae), Lepyronia gibbosa Ball (Cercopidae), and Prosapia bincinta (Say) (Cercopidae), in the natural habitat surrounding some of these vineyards that were never caught on sticky traps (I. L., unpublished data). Triapitsyn and Phillips (2000) established that the natural geographic distribution of the glassy-winged sharpshooter in the United States ranges from the southeastern Gulf coast states to Texas. The known geographic distribution of G. versuta and C. xanthocephala is outdated and not very reliable (R. Rakitov, personal communication). Young (1977) reports a range for G. versuta from the United States to Costa Rica. Oman (1949) recorded G. versuta from the central and southeastern subregions of the United States. Metcalf (1962) reported a range for C. xanthocephala from the United States to Mexico. Doering (1928) indicated that this spittlebug is one of three species that have the greatest range of distribution in the United States, but it is most abundant in the southern states of Georgia, Alabama, Mississippi, and Texas. Among the three species under discussion, one, the glassy-winged sharpshooter, is a known vector of X. fastidiosa. The other two have not yet been conÞrmed as vectors of PierceÕs disease in grape, but both have been determined to be associated with X. fastidiosa in polymerase chain reaction (PCR) assays designed to detect the bacterium from Þeld-collected specimens (F.M. and I.L., unpublished data). In addition, G. versuta is a proven vector of X. fastidiosa associated with phony peach disease (Turner and Pollard 1959a,b, Wells et al. 1983). The disease vectoring ability of H.

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vitripennis has been attributed to several of its biological characteristics, including its polyphagous feeding behavior, size, longevity, and ßying capacity/high mobility (Redak et al. 2004, Almeida et al. 2005). Its foraging behavior certainly provides vectoring opportunities throughout a vineyard, including possibilities of both primary and secondary transmission of X. fastidiosa bacteria and is not limited to an edge effect that is characteristic of other xylem ßuid-feeding insects known in northern California such as Graphocephala atropunctata, Draeculacephala minerva, or Xyphon fulgida (Purcell 1974, 1981, Purcell and Frazier 1985). It is interesting to note that G. versuta and C. xanthocephala were caught at most locations monitored and that their numbers are sufÞciently high to justify additional studies, including molecular genetic techniques and potential contribution to maintaining infection cycles outside susceptible crops. Glassy-winged sharpshooter population abundance in the vineyard cycled throughout the vineÕs vegetative season, from the highest adult counts of June to near complete or complete absence in the fall, winter, and early spring (Fig. 2). The positive percent change in the average difference between outside and inside glassy-winged sharpshooter trap counts increased signiÞcantly in October, November, and December (Fig. 4) because there were signiÞcantly more adults caught in the uncultivated habitat adjacent to the affected crop during those months. Observed counts in that habitat were lower during colder months but never reached zero. They were, however, found below 0.01/ trap/d only in April 2004, 2005, and 2006. From these observations, we deduce that environmental conditions recorded in this region may allow for a residual adult population to overwinter on suitable vegetation. Some seasonal migration out of the vineyard takes place that may be attributed to adult glassy-winged sharpshooter progressively leaving physiologically changing grapevines that are preparing for their fallwinter dormancy for more suitable host plants to overwinter on, plants that are found in other habitats, whether directly adjacent to the vineyards or not. Similarly, in FebruaryÐMarch, activity by H. vitripennis adults was noted by positive percent change in the average difference between outside and inside trap counts (Fig. 4). At that time of the year, grapevines are breaking dormancy, and growth starts very slowly under mean temperatures of 10 Ð15⬚C (Winkler et al. 1974). These vines are not yet attractive to glassywinged sharpshooter adults and we suggest that the signiÞcantly higher counts reßect an increased ßight activity of the overwintering adults searching the vegetation for suitable host plants that become progressively available in the spring for feeding and oviposition. Search for mating partners may take place as well during those warmer sunny days of March. Reproductively mature glassy-winged sharpshooter females exhibiting masses of brochosomes on the forewings (Hix 2001, Rakitov 2004) caught in mid-March indicated the onset of the oviposition period (I.L., unpublished data). In the study region, starting at the beginning of March, several hosts plants become rapidly available

August 2008


in the natural habitat surrounding the vineyards as mean daily temperatures quickly warm up. The uncultivated hosts plants selected early in the year for oviposition remain unidentiÞed, however, except for ornamental plants growing in urban areas (I.L., unpublished data). Ambient temperatures encountered in April and May are favorable to glassy-winged sharpshooter embryonic and nymphal development (I.L., unpublished data). We believe a new adult generation is produced at the time when grapevine growth and shoot elongation is vigorous and accelerated, making those plants very attractive to the young emerging adults. SigniÞcantly higher trap catches inside the vineyard (negative percent change in the average difference between outside and inside trap counts) may coincide with this inßux of adults into the vineyards during the Þrst days of June through July. Observed percent change in the average difference between H. vitripennis outside and inside trap counts are not signiÞcant during the summer months, which suggests that host preference for native host plants and grape plants is similar or that the adults constantly ßy in and out of the cultivated areas during that time of the year in search of different feeding or oviposition hosts. These key behavioral aspects are currently being studied in this insectÕs native habitat. Data analyses for G. versuta indicated that the highest trap counts can be observed once summer temperatures have been reached. In the vineyard, insect abundance indicate that grapevines, and possibly other vegetation growing between rows of vines, become attractive hosts to G. versuta adults starting in May. Grapevine host selection occurs well after shoots have established and also a month earlier than observed for glassy-winged sharpshooters. However, G. versuta counts in the vineyard were signiÞcantly lower (272%) than corresponding glassy-winged sharpshooter counts. The vines and other vegetation found in the cultivated area are abandoned in October. The vines become less succulent and attractive at that time of the year, and other key host plants may go to seed, devitalize, or vanish. It was on the outside traps that G. versuta adults were caught in the greatest numbers, and in July and August 2004, in numbers greater than those of H. vitripennis. The highly signiÞcant positive percent change in the average difference between outside and inside trap counts (152%) indicates that this species is always caught in greater numbers in the native habitat adjacent to the vineyard. This difference suggests a rather strong preference for plants found near the affected crop, which would be consistent with general observations of the biology and behavior of species in this genus by Turner and Pollard (1959b) but inconsistent with the observation of Purcell (1981) in California for the related species G. atropunctata. In California, G. atropunctata ßies into the vineyards from natural vegetation growing along streams and canyons when those plants begin to senesce and their populations reach high levels on grapevines between June and August (Purcell 1981). Our observations in central Texas suggest that suitable vegetation for host feeding and oviposition by G. ver-

935

suta is available year-round near vineyards, even during the coldest or warmest months of the year. A broad diversity of native wild vegetation, i.e., wild ßowers, weedy grasses, succulents, shrubs, and trees, ßourish because of the presence of streams, creeks, ponds, seasonal ponds, and lakes that irrigate the land in the study area. Throughout the vineÕs vegetative season, trap counts for C. xanthocephala, either inside or outside the vineyard, were comparable and also comparable to those of G. versuta. These observations indicate the suitability of both grapevines and native plants as hosts. No major population peak was observed for this species. Adults of these species may be relatively abundant between October and December on plants growing nearby permanent creeks and rivers, after which counts approach zero (I.L., unpublished data). Unlike the other two species, C. xanthocephala was not captured between December and April. Severin (1950) reported that the species C. brunnea overwinters in the egg stage on certain shrubs. The biology of spittlebugs in general, and C. xanthocephala in particular, is poorly known but our observations are consistent with this possibility. Our statistical approach was guided by the numerical importance of the three most common insect species and wanting to explore their spatio-temporal distribution near the vines under study, while accounting for numerous zero values for trap counts during certain time periods in either one of the habitats (cultivated versus natural). General additive models are commonly used in ecological studies but have not been explored as a tool in applied entomology. This type of modeling has been used by life science researchers from Europe, Australia, New Zealand, and South America, as evidenced by the 17 case studies in Zuur et al. (2007). A number of studies and books are available that document the use and theory behind GAMs, some of which are cited in this publication. The ßexibility of using such statistics in descriptive and exploratory studies provides insight into the biology of populations using census data. Surveys to obtain such data are underpinnings of integrated pest management (IPM) programs, yet in and of themselves are not inherently publishable. By using GAMs in an exploratory fashion, inferences may be made about population biology that have a degree of statistical probity, allowing for further modeling with the data set or hypothesis construction for designing the next experimental study. Using this analytical protocol, important information has been extracted while carrying out this large study in central Texas. Our survey was not exhaustive; however, putative vectors of PierceÕs disease of grapevine in central Texas were identiÞed, and the most abundant candidates were determined and will be the focus of extended research. Extensive state-wide sampling activities have taken place since 2005, which will broaden this picture (I.L., F.M., S.S. and J. Brady unpublished data). Intensive molecular work is being carried out to conÞrm which of these insects do in fact carry the grape strain of X. fastidiosa. Additional research is needed to better

936


understand the biology and ecology of the vectors and how populations ßuctuate both in the vineyards and once the insects vacate the cultivated areas. The feeding and oviposition habits of these species are complex, and acceptable host plants are diverse. The scarcity of insects on natural vegetation after grapevines have entered dormancy makes observations even more laborious. New techniques are being developed to take a closer look at the overwintering habits of H. vitripennis and the host plants involved. The highly ßuctuating abundance of xylem ßuid-feeding species at different locations is not yet thoroughly understood, but the differences observed among vineyards could be attributed to differences in the vegetative assemblage/composition (I.L., F.M., S.S. and J. Brady unpublished data). Acknowledgments The authors thank R. Rakitov (Center for Biodioversity, Champaign, IL) and L. Wendel (formely USDAÐAPHIS, Edinburg, TX) for constructive comments on a previous version of the manuscript; R. Rakitov for assistance with insect identiÞcation; A. Klein (Texas A&M University, College Station, TX) for creating the map; and A. Redd (Texas A&M University, College Station, TX) for editing the graphs. The Þrst author acknowledges the assistance received during part of the 2004Ð2005 trapping effort by personnel from Texas AgriLife Extension (Fredericksburg, TX) and USDAÐAPHIS (San Saba, TX) during 2005Ð2006. Technical assistance in the laboratory was provided by cooperative students J. Arriola and M. Gonzalez (Edinburg, TX), A. Hassell (Texas AgriLife Fredericksburg, TX), and D. McDonald and L. Heimer (Texas AgriLife Stephenville, TX), whose work is greatly appreciated. This research was supported in part by U.S. Department of AgricultureÕs Animal and Plant Health Inspection Service cooperative agreement 05-8500-0955-CA.

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