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Individual and combined effects of Trichosirocalus horridus and Rhinocyllus conicus (Coleoptera: Curculionidae) on musk thistle
Biological Control 30 (2004) 418–429 www.elsevier.com/locate/ybcon
Individual and combined effects of Trichosirocalus horridus and Rhinocyllus conicus (Coleoptera: Curculionidae) on musk thistle Lindsey R. Milbrath* and James R. Nechols Department of Entomology, Kansas State University, 123 W. Waters Hall, Manhattan, KS 66506-4004, USA Received 19 August 2003; accepted 16 December 2003
Abstract Field experiments were conducted in Northeast Kansas under conditions of limited plant competition to evaluate the individual and combined impact of the imported weevils Rhinocyllus conicus Froelich and Trichosirocalus horridus (Panzer) on the introduced weed, musk thistle (Carduus nutans L.). No effects on seed production occurred when low larval densities of T. horridus (<20 per plant) fed in rosettes, with or without the later-arriving, head-feeding weevil, R. conicus, present. In contrast, under high T. horridus larval densities (66 per plant), production of flower heads was reduced compared to uninfested plants. This indirect effect resulted in a 30% loss in viable seed per plant (with R. conicus present). Plant survival was not affected by T. horridus within the range of larval densities tested. R. conicus, when present alone, reduced viable seed by approximately 45%, whereas high densities of both weevils reduced viable seed by 59%. Thus, under conditions of low plant competition, neither weevil substantially limited seed production by musk thistle. This suggests the need for a multi-faceted management program for musk thistle involving additional forms of stress, such as interspecific plant competition. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Seed production; Classical biological control; Weeds; Trichosirocalus horridus; Rhinocyllus conicus; Musk thistle; Carduus nutans
1. Introduction Musk thistle (Carduus nutans L. species group) is a Eurasian weed that was first recorded in North America in the 1850s (Stuckey and Forsyth, 1971). It is most problematic in pastures and rangeland, especially in overgrazed situations (Dunn, 1976; Fick and Peterson, 1995). The presence of musk thistle can disrupt grazing by cattle, and competition with grasses reduces forage production (Batra, 1978; Fick and Peterson, 1995; Kates et al., 1975). Musk thistle grows as a biennial or winter annual and can be a prolific seed producer, although only one-third of the seed may germinate (McCarty, 1982; McCarty and Scifres, 1969). In addition, dormant *
Corresponding author. Present address: United States Department of Agriculture, Agricultural Research Service, Grassland, Soil, and Water Research Laboratory, 808 E. Blackland Road, Temple, TX 76502, USA. Fax: 1-254-770-6561. E-mail address: [email protected] (L.R. Milbrath). 1049-9644/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2003.12.005
seed can remain viable for 15 years (Burnside et al., 1981). During the 1960s and 1970s, two species of weevils— Rhinocyllus conicus Froelich and Trichosirocalus horridus (Panzer)—were imported into the United States and Canada as classical biological control agents of musk thistle (see Kok, 2001). In Northeast Kansas, releases of weevils originally collected in France and Italy resulted in the establishment of both species (Hilbert and Brooks, 2000). Larvae of T. horridus feed in the plant crown, which often results in the production of multiple flower stems. Plants are rarely killed by T. horridus and its indirect impact on seed production appears to be variable (Cartwright and Kok, 1985; Sieburth et al., 1983; Woodburn, 1997). R. conicus attacks flower heads. Larval feeding in the receptacle causes the abortion of developing ovules, as well as the destruction of young achenes (Kok, 2001; Shorthouse and Lalonde, 1984). As a result, the production of high quality seed may be reduced by as much as 78% (McCarty and Lamp, 1982).
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In Virginia and other parts of the eastern United States, substantial declines in musk thistle densities have been attributed, at least in part, to one or both weevils (Kok, 1986; Kok and Pienkowski, 1985). A similar claim has more recently been made for musk thistle in Oklahoma, based primarily on anecdotal information (Roduner et al., 2003). At other locations, reductions have not been observed (Andres and Rees, 1995), especially where plant competition with musk thistle was low (Harris, 1981). Thus, it remains unclear how efficacious the weevils are relative to other stress factors in suppressing and maintaining low musk thistle densities (but see Kok et al., 1986). A challenge to the efficacy of these biological control agents also has been made (Louda et al., 1998) because of the widespread feeding by R. conicus on nontarget, native thistles (Louda et al., 1997; Turner and Herr, 1996; Turner et al., 1987). The potential for T. horridus to do the same (McAvoy et al., 1987), should it be widely redistributed and established, is unclear given the recent report that T. horridus is a species complex in which each species may specialize on a single genus of thistles (Alonso-Zarazaga and Sanchez-Ruiz, 2002). Because each weevil feeds on different plant parts and at different times of the year, it is assumed that R. conicus and T. horridus are complementary biological control agents. Therefore, there has been general interest in establishing both weevils at the same localities. However, there have been no experimental evaluations to quantify the individual versus combined effects of the two weevils on viable seed production (Nechols, 2000). Such baseline data are important, not only because of plans for redistributing the weevils, but because several other species have been released in North America on musk and related thistles, some of which are now established. These include the stem-feeding fly, Cheilosia corydon (Harris) (Diptera: Syrphidae), a flea beetle, Psylliodes chalcomera (Illiger) (Coleoptera: Chrysomelidae), a gall-forming fly, Urophora solstitialis (L.) (Diptera: Tephritidae), and a rust fungus, Puccinia carduorum Jacky (Uredinales: Pucciniaceae) (Baudoin and Bruckart, 1996; De Quattro, 1997; Nechols, 2000). The objective of this study was to determine the short-term impact of T. horridus and R. conicus, alone and in combination, on musk thistle flower head and seed production under conditions of minimal plant competition in the field.
2. Materials and methods 2.1. Insect colony Adult T. horridus were field-collected annually in the fall from musk thistle and overwintered at 5 or 10 °C with a photoperiod of 10:14 h (L:D) in an environmental
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growth chamber. Adults were held in ventilated, plexiglass boxes containing bouquets of musk thistle leaves in cotton-plugged, water-filled, shell vials. Eggs were obtained by placing 15–30 weevils (1:2 male:female) in ventilated, 2-liter paper containers with thistle bouquets. Adults were held at a thermoperiod of 20:10 °C (10:14 h) with a photoperiod of 10:14 h (L:D). Leaves were dissected every 2–7 days for eggs, which were then placed in petri dishes lined with moist filter paper layered over wet cotton. Eggs were stored at 2–3 °C in darkness for 1–4 months (Kok and McAvoy, 1983). Subsequently, the eggs were transferred to 25 °C and allowed to develop. When the embryonic head capsule had darkened (24–48 h pre-hatch), batches of 10–13 eggs were aspirated with water into individual Pasteur pipettes. The pipette tip was wrapped with Parafilm ÔMÕ (American National Can, Chicago, IL) and the pipettes were refrigerated for approximately 1 week until eggs were needed to artificially infest musk thistle plants in field experiments. Preliminary tests showed that neither refrigeration nor storage in water affected the hatching rate. Voucher specimens of T. horridus and R. conicus were deposited in the Kansas State University Museum of Entomological and Prairie Arthropod Research under Lot No. 140. 2.2. Field experiments Between 1998 and 2001, three field experiments were conducted as follows. 2.2.1. 1998–1999, Keats, KS A field site was selected near Keats, Riley, KS (39°14.920 N, 96°43.500 W, 380 m) in fall, 1998. It had a history of musk thistle infestation and populations of R. conicus and T. horridus. In October, a 0.25-ha plot was fenced off to exclude cattle and 0.8 0.8 0.2 m (length width height) frame cages were placed over naturally occurring musk thistle rosettes (one plant per cage) to protect them from field populations of T. horridus. Cages had a window screen top and were large enough to allow for the expansion of rosettes. The density of experimental thistles was 0.04 musk thistle per m2 . The initial diameter of thistles ranged from 15 to 30 cm. The phenological development of caged thistles through bolting was similar to nonexperimental thistles growing near the field plot. To minimize interspecific plant competition on musk thistles, grasses and other vegetation within the cages and in the surrounding areas were clipped or mowed to maintain an average height of 10 cm. The experimental design was a 3 3 factorial treatment structure in a Latin square design. Because of the varying terrain at the field site, treatments were grouped by location within the site (first blocking factor). Rosette diameters measured in December (when fall growth had
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ceased and prior to freezing of the ground) served as a second blocking factor because the growth response of thistles to T. horridus damage had previously been shown to interact with thistle size (Sieburth et al., 1983). There were nine replications (blocks), each having three levels of T. horridus infestation (0, 25, or 50 eggs per plant) and three levels of R. conicus infestation (excluded with a closed bag, natural infestation with an open or sham bag, and natural infestation with no bag) for a total of 81 experimental units. In late March, 1999, musk thistle plants designated for T. horridus treatments were infested by inserting the tips of two or four Pasteur pipettes (depending on infestation level) that contained 12–13 mature (within 1 day of hatching) eggs into the main vein of thistle leaves. Eggs were collected as described above. This procedure was used because naturally deposited eggs and larvae cannot be counted in the field without destroying the plant. To account for possible effects on thistle plants from mechanical injury, three empty pipette tips were inserted into each control thistle. Thistle plants were not available to establish an unwounded control. Six additional caged thistles (nonexperimental) also were infested with 25 or 50 eggs. These thistles were dissected for larvae after 1 month to estimate the actual infestation levels of the experimental thistles. Similar densities of about 16 larvae per plant were recovered (mean SD [range]: 15.8 5.8 [5–21]). In addition, to obtain estimates of natural infestations of T. horridus, 10 musk thistle plants were dug from the ground in late April/early May at each of three locations in Northeast Kansas. The plants were dissected and T. horridus larvae were counted. When bolting (stem elongation) began in late April 1999, frame cages were opened and a 0.25% solution of permethrin [(3-phenoxyphenyl)methyl 3-(2,2-dichloroethenyl)-2,2-dimethylcyclopropanecarboxylate, Eliminator, Gro Tec, Madison, GA] was applied to the grass immediately outside the cages to exclude late-ovipositing T. horridus. Frame cages were removed by mid-May when R. conicus began ovipositing. All of the flower buds (capitula) that developed on each plant were individually covered by (1) closed or (2) open (sham) nylon mesh bags (23 23 mesh), or (3) left unbagged. To exclude R. conicus, bags were tied off at the top and bottom (on the rachis just below the bud) with twist ties. To test for unintended effects resulting from closed bags, sham bags were tied to the rachis only, leaving the top and most of the bottom open. To provide additional access for R. conicus adults, a 3 13-cm square was cut in the side of the bag. Flowers in all treatments were hand-pollinated with a small paintbrush to ensure uniform pollination. The number of R. conicus eggs per flower head was counted at least once per week. Cages remained in place until 1 week before seed heads were harvested. All seed heads were then covered with a closed bag to prevent seed loss due to birds and storms.
To quantify the impact of the weevils on thistle development and seed production, the number of flower heads per plant was counted about once per week beginning in the spring and continuing until plant death. Receptacle diameters for only heads that formed during the period of R. conicus reproduction were measured to the nearest 0.1 mm at full bloom or abortion of the head. Seed heads were clipped and placed in 0.5-liter containers 10–14 days after full bloom (just prior to seed dispersal) or when they had aborted. Heads were held in the laboratory at 25 °C (range 23–30 °C) to allow flower heads to dry. Musk thistles were infested by the sunflower moth, Homoeosoma electellum (Hulst), beginning in June and the infestation intensified as the season progressed. Flower heads produced after R. conicus adults were no longer active (July onward) were not harvested for seed because of extensive damage by sunflower moths. Seed heads from individual plants present during the period of R. conicus activity were combined into two groups based on the dates of flower head production and intensity of sunflower moth infestation. The first group consisted of flower heads produced between 28 April and 28 May that experienced little to no damage by sunflower moth. The second group contained flower heads produced from 29 May to 30 June during which time higher levels of sunflower moth infestation occurred. Seeds were detached from flower heads and poured into a 4-liter pitcher along with 0.25 liter of 1-cm gravel. The pitcher was covered and shaken for 5–10 s to separate seed from the pappus. The contents were then sieved. The seed was separated into four different weight classes using a South Dakota Seed Blower model B (E.L. Erickson Products, Brookings, SD) by varying the air flow according to the procedure of McCarty and Lamp (1982). The different classes of seed were defined by McCarty and Lamp (1982) as follows: class I—unfilled and not viable, class II—partially filled with a germination rate of 2%, class III—better filled with a 32% germination rate, and class IV—wellfilled with a 96% germination rate. Samples of seed masses obtained for each weight class were found to be similar to those of McCarty and Lamp (1982). For each weight class, the number of seeds was counted on a per-plant basis. Long-term storage of experimental seed may have compromised germination rates. Therefore, data relating percentage germination by weight class from McCarty and Lamp (1982) were used to estimate viable seed production per plant among treatments. 2.2.2. 1999–2000, Westmoreland, KS The experiment was established at a 0.18-ha field site near Westmoreland, Pottawatomie, KS (39°26.090 N, 96°20.430 W, 409 m). This site had a history of musk thistle and both weevils were present. The experimental design was a 4 2 factorial treatment structure in a randomized complete block design. There were 10 blocks (replications), each of which had four levels of
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T. horridus infestation (artificial infestation in the fall, spring, both fall and spring, or excluded) and two R. conicus infestation levels (natural infestation or excluded) for a total of 80 experimental units. Musk thistle rosettes were transplanted for increased uniformity of plot conditions and plant size. In late September, 2 weeks prior to transplanting, a 0.64-m2 area was cleared of vegetation for each experimental plant by applying glyphosate (N-(phosphonomethyl)glycine, Roundup, Monsanto, St. Louis, MO) at 4.7 liter/ha. This zone was established to reduce effects of competing grasses on thistles without disrupting the soil profile. In addition, surrounding grass and other vegetation were mowed at regular intervals to maintain an average height of 10 cm. Frame cages were placed in the ground 1 week before transplanting and a 0.25% permethrin solution was applied inside the cages to eliminate any T. horridus adults. Greenhouse-germinated musk thistle seedlings, 10–15 cm in diameter, were transplanted on 8 October. Plants were spaced 2 m apart, resulting in a density of 0.25 musk thistle per m2 . Musk thistle plants designated for T. horridus infestation were inoculated with five mature eggs from a laboratory colony on 7 December or 15 mature eggs on 13 March, or both. The number of T. horridus eggs used for each treatment was based on the quantity of eggs that could be harvested from the laboratory colony. However, the majority of eggs are oviposited in the spring. Eggs were placed directly in the crown of the thistles and covered by moist cotton to prevent desiccation before hatching. Six additional caged thistles were infested with T. horridus at experimental rates in the fall or spring and then dissected in April to provide estimates of actual densities of T. horridus in the experimental plants. The numbers of larvae per plant recovered in the spring were about 2 [n ¼ 1] (fall), 9.8 3.3 [n ¼ 5] (spring) and an estimated 12 (fall and spring). Four uninfested thistles were left in frame cages until April and then dissected. Results confirmed that the cages were effective in preventing the invasion of natural populations of T. horridus. Rhinocyllus conicus was excluded from plants by applying sprays of 0.25% permethrin to the developing flower heads at 2-week intervals. Sprays were discontinued on heads that were about to flower to prevent interfering with pollinators, and no spraying was done after R. conicus adults were no longer present in the field. Applications of Bacillus thuringiensis Berliner subsp. kurstaki (DiPel ES, Valent BioSciences, Walnut Creek, CA) at 1.8 liter/ha were applied weekly to flower heads in all treatments to minimize infestations of the sunflower moth. Applications began on individual heads just prior to bloom and continued until head senescence. Data collection and analyses were similar to the 1998–1999 experiment. Head receptacle diameters were measured 4 days after full bloom (when the maximum
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head size was achieved). Seed heads from each plant were placed into two groups: (1) flower heads produced during the period of oviposition by R. conicus (through 12 June) and (2) flower heads produced after R. conicus activity had ceased. 2.2.3. 2000–2001, Keats, KS This experiment used natural infestations of T. horridus and R. conicus and was conducted on a fenced, 0.02-ha site located at 39°14.690 N, 96°43.660 W (366 m) and approximately 0.5 km from the 1998–1999 experimental site. The experimental design was a oneway treatment structure in a randomized complete block design. There were 20 blocks (replications), each of which had two levels of T. horridus infestation (natural infestation or excluded) for a total of 40 experimental units. Two treatment combinations used in the previous experiments (neither weevil present, T. horridus present but R. conicus absent) were not included because this experiment was designed primarily to evaluate the interactions between the two musk thistle weevils. A parallel study that incorporated the absence of R. conicus could not be run because of very low R. conicus densities at that field site. The field plots were established as described previously except that no frame cages were used. Naturally germinated thistles surrounding the field plots were destroyed and additional T. horridus adults were released into the plot area in March to ensure high infestation levels of T. horridus. Greenhouse-germinated musk thistle seedlings, 10– 15 cm in diameter, were transplanted to experimental plots on 28 September. T. horridus was excluded by applying sprays of 0.25% permethrin, and sunflower moth was excluded with applications of DiPel ES at 1.8 liter/ha at 2- or 1-week intervals, respectively. Five additional transplanted thistles (nonexperimental) were dug from the ground in late April and dissected to quantify levels of natural infestation by T. horridus. An average of 66.2 22.9 larvae was found per plant. Data were collected using procedures described previously with the following change. Seed heads from each plant were placed into five groups based on the dates of flower-head production and intensity of R. conicus infestation, the last two groups experiencing no damage from R. conicus: (1) flower heads produced from 27 April to 23 May, (2) 24 May to 7 June, (3) 8 to 26 June, (4) 27 June to 25 July, and (5) 26 July to 10 September. Due to the very large volume of seed produced in 2001, seed numbers were estimated by mass instead of by counting. Twenty samples of 100 seed each for weight classes II, III, and IV were weighed to the nearest 0.1 mg (10 samples from plants infested by both T. horridus and R. conicus and 10 samples from plants infested only by R. conicus). Analysis of variance indicated there were no significant differences (P > 0:05) in seed mass between the two
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treatments, so the data were pooled to produce a mean mass per seed for each weight class (II—1.3 mg, III— 1.7 mg, and IV—2.9 mg). Samples of total seed for each weight class were weighed to the nearest 0.1 mg and divided by the respective mass per seed to derive a seed number. Estimates of viable seed were made as previously described. Weight class I (unfilled seed) was not included because of technical difficulty in removing seed from nonseed material, and because McCarty and Lamp (1982) had reported 0% germination for this seed class. 2.3. Statistical analyses Plants were omitted from analysis if they had died prematurely (e.g., in the rosette stage) from unknown causes, were contaminated by naturally occurring (nonexperimental) infestations of T. horridus, or displayed no signs of damage and developed a central, primary flower stem after having been artificially infested with T. horridus eggs. Data on flower-head production, receptacle diameters, the number of R. conicus eggs per flower head, the percentage of flower heads that received R. conicus eggs, and seed production were analyzed with Analysis of Variance (PROC MIXED, SAS Institute, 1999). Percentage data were subjected to arcsine square root transformation before analysis. Adjustments could not be made for the effect of contamination by sunflower moth on the 1999 seed data using Analysis of Covariance (PROC MIXED, SAS Institute, 1999). For the 1999 and 2000 data, treatments were pooled to create four treatments, utilizing a 2 2 factorial analysis, involving the infestation of musk thistle by no insects, T. horridus only, R. conicus only, or both species combined. Data involving flower-head production from individual sampling dates were combined to represent up to five periods of R. conicus seasonal activity: (1) 50% of R. conicusÕ seasonal egg production oviposited and high adult R. conicus densities (first 3 to 4 weeks), (2) an additional 40–45% eggs oviposited and decreasing R. conicus numbers (next 1 to 2 weeks), (3) remaining 5– 10% eggs oviposited and disappearance of R. conicus adults (1–3 weeks), (4) reproductive R. conicus absent from the field, flower heads produced through late July ( ¼ plant death in 1999 and 2000, 4–6 weeks), and (5) reproductive R. conicus absent from field, flower heads produced from late July until plant death (2001 only, 6 weeks).
3. Results 3.1. Seasonal dynamics of musk thistle and weevils In Northeast Kansas, average spring densities of T. horridus on naturally infested musk thistle ranged
from 17 to 34 larvae per plant, with individual plants having from zero to 77 larvae per plant. In all years examined, musk thistle plants began producing flower buds during the first week of May. Flowering continued until plant death, which ranged from mid July to mid September. Adult R. conicus were present on thistles by late April and rapidly reached high densities (averages of 2–10 adults per plant). Adults began ovipositing as soon as flower buds were accessible, and populations remained high for about 4 week before declining over a 3- to 4-week period. Low numbers of R. conicus adults were observed until mid or late June. 3.2. 1999 Neither weevil species, individually or together, had an effect on the seasonal production of flower heads or head size (P > 0:05; Table 1). The proportion of flower heads infested by R. conicus, and the egg densities on infested flower heads, were similar on T. horridus-infested and uninfested thistles (Table 1). Infestation of musk thistle rosettes by T. horridus had no effect on total or viable seed production by musk thistle at any time of the season. In contrast, fewer total and viable seeds were produced during May when R. conicus was present than when it was excluded (P < 0:001; Table 2). Reductions in per-plant total and viable seed attributable to R. conicus were 50 and 58%, respectively. When R. conicus was present in flower heads, the proportion of class IV seed (highest germination rate, comprising 92–99% of viable seeds) was reduced. There was a corresponding increase in the proportions of class II and III seed, which have considerably lower germination potential. There was no added impact on seed production or viability when plants were infested by both T. horridus and R. conicus (Table 2). In June, there were no reductions in total or viable seeds produced when R. conicus was present compared to when it was absent (Table 2). However, during this period, musk thistle heads were moderately to severely infested by larvae of the sunflower moth. 3.3. 2000 Musk thistle infested by T. horridus produced more flower heads at the beginning of the season compared to uninfested thistles (P < 0:02). Otherwise, neither T. horridus nor R. conicus affected the number of flower heads produced (Table 3). The presence of R. conicus resulted in smaller flower heads early in the season than for plants not infested by R. conicus (P < 0:002), and T. horridus-infested thistles produced smaller heads in May than uninfested thistles (P < 0:05; Table 3). Similar densities of R. conicus eggs were deposited on T. horridus-infested and uninfested thistles (Table 3).
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Table 1 Seasonal flower-head production and R. conicus egg densities (mean SE or 95% CI) on musk thistle plants infested or not infested by T. horridus and/or R. conicus, Keats, KS, 1999a Dates
T. horridus
R. conicus
New flower heads per plant
Maximum receptacle diameter (mm)
R. conicus eggs per infested head
% Heads with eggs
28 April–28 May
)b + ) +
)b ) + +
6.0 1.8 3.5 1.6 6.6 1.4 6.8 1.3
27.0 1.4 25.9 1.2 26.6 1.0 24.5 0.9
—
—
) + ) +
) ) + +
5.4 2.1 5.8 1.8 8.5 1.6 7.6 1.4
18.4 1.1 19.1 0.9 17.6 0.8 18.1 0.8
) + ) +
) ) + +
2.7 2.0 1.5 1.7 4.9 1.4 4.7 1.2
13.6 1.4 16.2 1.3 15.8 0.8 14.8 0.7
) + ) +
) ) + +
6.9 2.6 5.1 2.3 7.1 2.0 9.9 1.8
29 May–9 June
10–30 June
1–28 July
—
—
31.4 2.7 26.4 2.3
100 (99.9–100) 100 (100–100)
—
—
—
—
7.9 1.2 7.9 1.0
99.1 (93.9–99.6) 95.0 (87.6–99.1)
—
—
—
—
1.8 0.3 2.0 0.2
40.4 (10.5–75.0) 45.1 (15.5–76.7)
c
—
—
c
—
—
c
c
c
c
c
c
a Neither the T. horridus nor R. conicus as main effects, or the interaction term, were significant (P > 0:05) for means within columns and for each set of dates (n ¼ 4–26). From 29 May to 28 July, thistles also infested by sunflower moths. b ) Absent, + present. c Head diameters not measured and no reproductive R. conicus present in the field.
There was no effect of T. horridus on total or viable seed production by musk thistle at any time of the season (P > 0:05; Table 4). In contrast, infestation by R. conicus during the period of its reproductive activity (29 April to 12 June), reduced the number of total and viable seeds per plant by 32 and 45%, respectively, compared to plants protected from R. conicus (P < 0:02; Table 4). The number of class IV seed was reduced and the number of class II seed was increased for R. conicusinfested heads (Table 4). There was no additional effect on seed production when both T. horridus and R. conicus infested musk thistle plants (Table 4). Following the absence of R. conicus in the field (13 June to 24 July), there were no differences in seed production between plants that had been previously infested by R. conicus or those that had not (Table 4). A low percentage of heads flowered (5–12%) due to plant senescence and few numbers of viable seed were produced, averaging up to four seeds per plant (Table 4). This was less than 0.1% of all viable seed produced for each plant.
Infestation of musk thistle by T. horridus resulted in fewer numbers of total and viable seed produced during May and June than for thistles not infested by T. horridus (P < 0:05). Depending on the particular time, total seed per plant was reduced from 28 to 44%, and viable seed was reduced from 31 to 45% (Table 6). However, the proportion of class IV seed (80–90% of total seed) did not vary with T. horridus infestation. There were no differences in seed production per plant between T. horridus-infested and uninfested thistles from late June onward (Table 6). A high percentage of heads continued to flower for several weeks afterwards (around 75%), with each plant producing an average of 4180 viable seed or 14–19% of viable seed production (Table 6). The total and viable seed per plant produced for the season were therefore reduced by 28 and 30%, respectively, for T. horridus-infested musk thistles compared to thistles protected from T. horridus (Table 6).
3.4. 2001 4. Discussion Plants infested by T. horridus produced fewer flower heads than plants protected from T. horridus during May and June, which was the period of oviposition by R. conicus (Table 5). The earliest flower heads were slightly smaller on T. horridus-infested thistles than uninfested thistles (Table 5). Also, T. horridus-infested plants received fewer numbers of R. conicus eggs per head early in the season (Table 5).
Under conditions of little or no plant competition and high weevil densities, each species of weevil contributed to the reduction of viable seed in musk thistle. In addition, the combined impact of T. horridus and R. conicus was greater than that of either weevil alone. However, these effects were not observed at low population densities of T. horridus.
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Table 2 Seasonal seed production (mean SE) for musk thistle plants infested or not infested by T. horridus and/or R. conicus, Keats, KS, 1999 Dates of flower-head production
T. horridus
28 April–28 May
)b + ) + T. horridusc R. conicusc
29 May–30 Juned
) + ) + T. horridus R. conicus
R. conicus
No. of seed per plant by weight class and totala I
II
III
IV
Total
)b ) + +
1014 187 819 161 419 141 382 125 0.36 <0.01
49 51 32 44 128 38 107 33 0.57 0.03
27 40 34 35 100 30 85 27 0.89 0.02
1141 217 1052 180 429 152 418 126 0.75 <0.01
2236 430 1920 371 1076 325 987 287 0.49 <0.01
) ) + +
1281 313 764 260 595 222 724 186 0.40 0.12
18 17 34 14 52 11 33 9 0.90 0.20
31 19 39 16 42 14 55 12 0.45 0.33
416 231 360 195 373 170 486 147 0.86 0.80
1755 500 1184 423 1063 367 1302 316 0.64 0.41
Estimated number of viable seed per plante 28 April–28 May
29 May–30 Juned
) + ) + T. horridus R. conicus
) ) + +
e
) + ) + T. horridus R. conicus
) ) + +
e
e e e
e e e
11 11 31 21 0.57 0.03
10 15 13 13 38 12 32 10 0.89 0.02
1096 209 1010 172 412 146 401 121 0.75 <0.01
1106 222 1022 184 452 156 435 130 0.76 <0.01
00 10 10 10 0.90 0.20
12 7 15 6 16 5 21 5 0.45 0.33
399 222 346 187 358 163 466 141 0.86 0.80
411 226 361 191 375 166 488 143 0.84 0.77
a
Total seed numbers may not equal the sum of all weight classes due to differences in least squares means estimates. ) Absent, + present. c P values for main effects. No interaction terms were significant (P > 0:05) (n ¼ 7–26). d Affected by sunflower moth infestation. e Based on 0% germination for seed of weight class I, 2% (II), 38% (III), and 96% (IV) (McCarty and Lamp, 1982). b
4.1. Trichosirocalus horridus Because T. horridus feeds only on vegetative (nonfloral) tissues, its potential for having an impact on musk thistle is restricted to two categories: direct mortality of rosettes and indirect effects on reproductive fitness (e.g., reductions in the size and/or number of flower heads produced). In our experiments, there were no plant deaths attributable to T. horridus and the capacity of this herbivore for doing so appears to be generally low unless larval densities are very high (Sheppard et al., 1995; Sieburth et al., 1983; Woodburn, 1997). However, it is possible that combined stress from T. horridus and plant competition could cause direct mortality to musk thistle, although this has not been shown. With respect to sublethal impacts, our findings agree with previous studies that have shown no consistent, significant effect of T. horridus on musk thistle head size (Cartwright and Kok, 1985; Woodburn, 1997). On the other hand, high densities of T. horridus did cause a 15% reduction in the number of flower heads per plant—an effect that was evident only during the first half of the
season. These data suggest that in the absence of plant competition, T. horridus may cause marginal reductions in musk thistle seed production, but only during growing seasons when hotter, drier weather shortens the thistle flowering period, and only when sporadically high T. horridus densities occur. The impact of T. horridus would diminish even further in years when milder temperatures and sufficient rainfall allow thistles to produce flower heads for a longer period of time, as occurs in some years in Kansas. The lower densities (<20 larvae per plant) of T. horridus that we observed in 1999 and 2000, in combination with little or no intra- or interspecific plant competition, had no effect on either the seasonal production of flower heads or seed by musk thistle. The only detectable effect was in stem number; low larval densities of T. horridus are correlated with altered thistle architecture but not reproduction (Sheppard et al., 1995). Furthermore, the effect of T. horridus was not altered by the additional stress imposed by R. conicus. Plant size can influence the effect that T. horridus has on seed production. For example, Cartwright and Kok
L.R. Milbrath, J.R. Nechols / Biological Control 30 (2004) 418–429
425
Table 3 Seasonal flower-head production and R. conicus egg densities (mean SE or 95% CI) on musk thistle plants infested or not infested by T. horridus and/or R. conicus, Westmoreland, KS, 2000 Dates
T. horridus
R. conicus
New flower heads per plant
Maximum receptacle diameter (mm)
R. conicus eggs per infested head
% Heads with eggs
29 April–22 May
)a + ) + T. horridusb R. conicusb
)a ) + +
4.0 1.0 6.4 0.7 3.6 1.1 5.8 0.8 0.01 0.59
34.3 1.6 29.4 1.1 27.6 1.6 26.7 1.2 0.04 <0.01
—
—
—
—
22.0 4.0 15.0 3.0 0.09
84.3 (55.7–99.2) 97.0 (82.5–99.3) 0.15
—
—
) + ) + T. horridus R. conicus
) ) + +
3.9 1.3 4.0 0.9 4.1 1.3 5.7 1.0 0.40 0.39
24.3 1.6 17.7 1.1 22.2 1.7 19.6 1.1 <0.01 0.92
—
—
) + ) + T. horridus R. conicus
) ) + +
3.6 1.0 2.0 0.6 2.8 1.0 4.2 0.7 0.90 0.41
) + ) + T. horridus R. conicus
) ) + +
5.2 1.2 4.0 0.9 3.9 1.2 5.8 0.9 0.72 0.81
23 May–1 June
2–12 June
13 June–24 July
—
—
6.4 1.3 4.6 0.9 0.30
53.4 (24.2–81.4) 31.0 (5.5–65.5) 0.12
—
—
12.8 1.5 12.0 1.2 12.3 1.5 12.6 1.2 0.84 0.96
—
—
c
—
c
—
—
c
c
c
c
c
c
—
—
—
0.02 ()0.13–0.45) 0.27 (0.05–1.54) 0.41
—
— —
) Absent, + present. P values for main effects. No interaction terms were significant (P > 0:05) (n ¼ 7–23). c Head diameters not measured and no reproductive R. conicus present in the field. a
b
(1985) reported both decreased and increased seed production for smaller thistles infested by T. horridus compared to uninfested thistles, but not for larger thistles (rosette diameters >25 cm). The mean rosette diameters of our experimental thistles ranged from 32 to 64 cm, which fall into a large size class. Therefore, our results for musk thistles that were infested at low T. horridus densities are consistent with those of Cartwright and Kok (1985). 4.2. Rhinocyllus conicus The seasonal pattern of host exploitation that we observed for R. conicus on musk thistle in Northeast Kansas agrees with the findings of researchers in other regions (Andres and Rees, 1995; Kok and Pienkowski, 1985; McCarty and Lamp, 1982; Rees, 1977). That is, R. conicus oviposits heavily on the earliest flower heads (main terminal and early lateral heads), causing significant damage (including aborted heads) and a substantial reduction in viable seed produced. Subsequently, adult weevil numbers and oviposition rates decline such that egg densities and the percentage of flower heads attacked are lower on the lower parts of the plant that are produced later in the season. Thus, although the earliest flower heads may be completely destroyed (Kok and
Surles, 1975), most heads are able to produce moderate to high numbers of viable seed. Furthermore, in Kansas and other locations, because R. conicus ceases oviposition by early summer, well before the potential end of the flowering period of musk thistle, variable numbers of heads escape herbivory from R. conicus altogether. In some locations and years, few flower heads will set seed after R. conicus activity has ceased and the amount of viable seed produced will be minimal at this time. This may have much to do with individual site characteristics as well as conditions of moisture stress in summer, whether due to drought or normal weather patterns (Hodgson and Rees, 1976; McCarty and Lamp, 1982; Roduner, 2001). In contrast, conditions that are presumably more optimal for musk thistle can prolong flowering well beyond the period of R. conicus activity (Surles et al., 1974). As a result of these different factors, the greatest overall reduction in total seed and viable seed attributed to R. conicus were 32 and 45%, respectively. Therefore, despite localized and occasionally heavy infestations by other insects like the sunflower moth (McCarty and Lamp, 1982; Rees, 1977), a large amount of viable seed can be produced by musk thistle plants under conditions favorable for growth and development (e.g., weather, low levels of plant competition).
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Table 4 Seasonal seed production (mean SE) for musk thistle plants infested or not infested by T. horridus and/or R. conicus, Westmoreland, KS, 2000 Dates of flower-head production
T. horridus
R. conicus
29 April–12 June
) + ) + T. horridusc R. conicusc
13 June–24 July
) + ) + T. horridus R. conicus
b
No. of seed per plant by weight class and totala I
II
III
IV
Total
) ) + +
722 205 852 139 706 216 963 148 0.28 0.79
54 25 76 18 109 26 116 20 0.48 0.03
148 75 144 51 174 79 277 54 0.45 0.22
3602 534 3222 412 1815 558 1809 429 0.64 <0.01
4526 739 4271 562 2823 774 3159 586 0.94 0.02
) ) + +
72 27 34 18 50 28 47 19 0.40 0.85
42 21 02 21 0.72 0.22
62 22 03 12 0.53 0.15
22 11 02 32 0.56 0.89
84 30 39 20 50 32 54 21 0.43 0.70
b
Estimated no. of viable seed per plantd 29 April–12 June
13 June–24 July
) + ) + T. horridus R. conicus
) ) + +
d
) + ) + T. horridus R. conicus
) ) + +
d
d d d
d d d
11 20 21 20 0.48 0.03
56 28 55 19 66 30 105 21 0.45 0.22
3458 513 3093 396 1742 536 1736 411 0.64 <0.01
3515 526 3143 407 1816 549 1844 423 0.67 <0.01
00 00 00 00 0.72 0.22
21 11 01 01 0.53 0.15
22 11 02 31 0.56 0.89
43 22 03 32 0.84 0.54
a
Total seed numbers may not equal the sum of all weight classes due to differences in least squares means estimates. ) Absent, + present. c P values for main effects. No interaction terms were significant (P > 0:05) (n ¼ 9–23). d Based on 0% germination for seed of weight class I, 2% (II), 38% (III), and 96% (IV) (McCarty and Lamp, 1982). b
Table 5 Seasonal flower-head production and R. conicus egg densities (mean SE or 95% CI) on musk thistle plants infested or not infested by T. horridus and R. conicus, Keats, KS, 2001a Dates
T. horridus
R. conicus
27 April–23 May
)b +
+b +
24 May–7 June
) +
8–26 June 27 June–25 July 26 July–10 September
New flower heads per plant
Maximum receptacle diameter (mm)
R. conicus eggs per infested head
% Heads with eggs
19.9 2.1 a 9.7 2.1 b
31.2 0.8 a 29.2 0.8 b
41.8 1.7 a 22.2 1.7 b
100.0 (99.8–100) a 99.9 (99.6–100) a
+ +
45.6 4.9 a 34.8 5.0 b
25.1 0.6 a 25.6 0.6 a
7.7 0.4 a 6.4 0.4 b
89.5 (83.8–94.0) a 84.4 (77.8–90.0) b
) +
+ +
61.1 6.5 a 42.8 6.6 b
20.0 0.7 a 20.3 0.7 a
2.6 0.4 a 2.0 0.4 a
11.6 (7.7–16.2) a 12.8 (8.6–17.6) a
) +
+ +
114.2 21.3 a 99.2 21.3 a
c
c
c
c
c
c
) +
+ +
20.0 9.1 a 12.9 9.1 a
c
c
c
c
c
c
a
Means within columns and for each set of dates followed by different letters were significantly different (P < 0:05) (n ¼ 19–20). ) Absent, + present. c Head diameters not measured and no reproductive R. conicus present in the field. b
Our data suggest that at locations and during years when both R. conicus and T. horridus occur at high densities, the impact on musk thistle seed production would be greater than if only one species of weevil were present.
An estimate of the additional percentage reduction in viable seed due to the combined impact of both weevils can be obtained by multiplying the percentage reduction attributable to R. conicus [45% based on 2000 results,
L.R. Milbrath, J.R. Nechols / Biological Control 30 (2004) 418–429
427
Table 6 Seasonal seed production (mean SE) for musk thistle plants infested or not infested by Trichosirocalus horridus and Rhinocyllus conicus, Keats, KS, 2001a No. of seed per plant by weight class and totalb
Dates of flower-head production
T. horridus
R. conicus
II
III
27 April–23 May
)c +
+c +
500 70 a 319 70 b
414 51 a 254 51 b
4207 534 a 2294 534 b
5121 625 a 2867 625 b
24 May–7 June
) +
+ +
685 139 a 858 139 a
723 82 a 723 82 a
14,891 1566 a 10,167 1566 b
16,289 1681 a 11,748 1681 b
8–26 June
) +
+ +
480 87 a 399 87 a
525 69 a 407 69 a
7866 991 a 5126 991 b
8871 1100 a 5932 1100 b
27 June–25 July
) +
+ +
461 108 a 387 108 a
420 96 a 353 96 a
4413 1194 a 3579 1194 a
5294 1376 a 4319 1376 a
26 July–10 September
) +
+ +
46 24 a 34 24 a
37 20 a 25 20 a
271 166 a 94 166 a
354 206 a 153 206 a
IV
Total
Estimated no. of viable seed per plantd 27 April–23 May
) +
+ +
10 1 a 61 b
157 19 a 97 19 b
4039 512 a 2202 512 b
4206 530 a 2305 530 b
24 May–7 June
) +
+ +
13 3 a 17 3 a
275 31 a 275 31 a
14,295 1503 a 9760 1503 b
14,583 1527 a 10,052 1527 b
8–26 June
) +
+ +
10 2 a 82 a
200 26 a 155 26 a
7551 951 a 4921 951 b
7761 972 a 5084 972 b
27 June–25 July
) +
+ +
92 a 82 a
160 36 a 134 36 a
4237 1146 a 3436 1146 a
4406 1182 a 3578 1182 a
26 July–10 September
) +
+ +
10 a 10 a
14 7 a 97 a
260 159 a 91 159 a
275 167 a 101 167 a
a
Means within columns and for each set of dates followed by different letters were significantly different (P < 0:05) (n ¼ 19). Weight class I was not counted or included in analyses. c ) Absent, + present. d Based on 2% germination for seed of weight class II, 38% (III), and 96% (IV) (McCarty and Lamp, 1982). b
which is in general agreement with the findings of others (Shea and Kelly, 1998 and comments therein)] with the T. horridus-associated percentage reduction in seed that occurs beyond that of R. conicus (30%). Therefore, an additional 14, or 59% total, reduction in viable seed may occur when both weevils are present at high densities. However, whether the actual combined effect of the two weevils is more or less efficient than the sum of the individual impacts could not be determined. The effect of R. conicus on musk thistle reproduction may vary among years because of differences in weather conditions, indirect interactions with T. horridus (Milbrath and Nechols, 2004) , or other factors. For example, oviposition by R. conicus was lower on thistles that were heavily infested by T. horridus than on uninfested plants. If reduced oviposition by R. conicus results in greater seed production, T. horridus would be indirectly responsible for reducing the impact of R. conicus. 4.3. Recommendations The experimental data presented here suggest that the presence of both weevils may lead to a net reduction in
viable musk thistle seed. However, the magnitude of that effect is difficult to estimate because musk thistle occurs at times and in locations where other stresses may modify the impact of herbivores. For example, a potentially different outcome might occur when musk thistle is subject to interspecific plant competition (e.g., Kok et al., 1986). The role of intraspecific thistle competition, particularly in enhancing plant stress or creating better synchrony with R. conicus, has received no attention. Prior to the release of either weevil, musk thistle densities could be very high, ranging from 2 to 43 plants per m2 (Harris, 1981; Kok, 1986, 2001; Lee and Hamrick, 1983). In addition, the long-term impact of R. conicus and T. horridus on musk thistle population dynamics is unknown because musk thistle plants continue to produce large amounts of seed, despite significant reductions in viable seed production. Much of that seed may accumulate in seed banks. For example, the large thistles we observed in 2001 produced over 20,000 viable seeds, despite high densities of both R. conicus and T. horridus. Similarly, Woodburn (1997) reported that musk thistles infested by very high densities of T. horridus produced about 8000 seeds, even after a 72%
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reduction in seed set. Thus, even under high levels of weevil pressure, there may be more seed available than is needed to replace the plant population (see Shea and Kelly, 1998). This scenario may help explain, in part, the lack of thistle suppression reported in some areas that have had moderate to high populations of weevils and experienced only moderate to no reductions in thistle populations over time (Andres and Rees, 1995; Harris, 1981). The efficacy of T. horridus appears to depend on the larval densities it can attain. The presence of both R. conicus and T. horridus at high densities appears to provide greater reductions in viable seed than when R. conicus is present alone. However, high densities of T. horridus can have a negative effect on the short-term, and possibly long-term, population dynamics of R. conicus (Milbrath and Nechols, 2004). Therefore, the benefit of greater seed reduction from the combination of both weevils is potentially offset by reduced populations of R. conicus. However, based on direct observations and published reports, it is questionable whether consistently high T. horridus densities will occur in most areas of the United States (Andres and Rees, 1995; Kok and Mays, 1989). The low densities of T. horridus likely to occur appear to be of limited usefulness in the biological control of musk thistle, whether alone or in combination with R. conicus, especially when other types of stress are not acting on musk thistle. Low-stress environmental conditions may occur frequently and at many locations where vegetation has been disturbed (e.g., from overgrazing). These conditions are typical of many musk thistle sites in Kansas. Therefore, the continued redistribution of this weevil within the United States, either as a complementary biological control to R. conicus or as a single agent, does not seem warranted at this time.
Acknowledgments We thank Darrel Westervelt and Karen Krouse for the use of their land; Lucas Robison, Jason Sweet, Nicole Sweet, Mary Milbrath, Shauna Dendy, Xiaoli Wu, Neil Miller, Megan Murphy, Justin Schmitz, Soledad Villamil, Erika Jensen, and Vincent Gregorio for technical assistance; Tom Loughin for statistical advice; and David Margolies, Walter Fick, Randall Higgins, Sonny Ramaswamy, Walter Dodds (all from Kansas State University), and two anonymous reviewers for providing helpful suggestions on earlier drafts of the manuscript. We also acknowledge the following colleagues who contributed information and ideas during discussions: Ernst Horber (Kansas State University), Loke Kok (Virginia Polytechnic Institute and State University), Svata Louda (University of Nebraska), and Ben Puttler (University of Missouri). This research was
funded in part by a grant to L.R.M. from the Sigma Xi Grants-in-Aid of Research program, and by Regional Research Project W-185. This is Contribution No. 04005-J from the Kansas Agricultural Experiment Station.
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rocalus horridus, for control of musk thistle, Carduus nutans, in Oklahoma. M.S. thesis, Oklahoma State University, Stillwater. Roduner, M., Cuperus, G., Mulder, P., Stritzke, J., Payton, M., 2003. Successful biological control of the musk thistle in Oklahoma using the musk thistle head weevil and the rosette weevil. Am. Entomol. 49, 112–120. SAS Institute, 1999. SAS/STAT UserÕs Guide, version 8th ed, SAS Institute, Cary, NC. Shea, K., Kelly, D., 1998. Estimating biocontrol agent impact with matrix models: Carduus nutans in New Zealand. Ecol. Appl. 8, 824–832. Sheppard, A.W., Aeschlimann, J.-P., Sagliocco, J.-L., Vitou, J., 1995. Below-ground herbivory in Carduus nutans (Asteraceae) and the potential for biological control. Biocontrol Sci. Technol. 5, 261–270. Shorthouse, J.D., Lalonde, R.G., 1984. Structural damage by Rhinocyllus conicus (Coleoptera: Curculionidae) within the flowerheads of nodding thistle. Can. Entomol. 116, 1335–1343. Sieburth, P.J., Kok, L.T., Lentner, M., 1983. Factors influencing the effectiveness of Trichosirocalus horridus (Panzer) in the control of Carduus thistles. Crop Prot. 2, 143–151. Stuckey, R.L., Forsyth, J.L., 1971. Distribution of naturalized Carduus nutans (Compositae) mapped in relation to geology in northwestern Ohio. Ohio J. Sci. 71, 1–15. Surles, W.W., Kok, L.T., Pienkowski, R.L., 1974. Rhinocyllus conicus establishment for biocontrol of thistles in Virginia. Weed Sci. 22, 1– 3. Turner, C.E., Herr, J.C., 1996. Impact of Rhinocyllus conicus on a nontarget, rare, native thistle (Cirsium fontinale) in California. In: Moran, V.C., Hoffmann, J.H. (Eds.), Proc. IX Int. Symp. Biol. Contr. Weeds, 19–26, January 1996, Stellenbosch, South Africa, University of Cape Town, Cape Town, South Africa, p. 103. Turner, C.E., Pemberton, R.W., Rosenthal, S.S., 1987. Host utilization of native Cirsium thistles (Asteraceae) by the introduced weevil Rhinocyllus conicus (Coleoptera: Curculionidae) in California. Environ. Entomol. 16, 111–115. Woodburn, T.L., 1997. Establishment in Australia of Trichosirocalus horridus, a biological control agent for Carduus nutans, and preliminary assessment of its impact on plant growth and reproductive potential. Biocontrol Sci. Technol. 7, 645–656.
Individual and combined effects of Trichosirocalus horridus and Rhinocyllus conicus (Coleoptera: Curculionidae) on musk thistle Lindsey R. Milbrath* and James R. Nechols Department of Entomology, Kansas State University, 123 W. Waters Hall, Manhattan, KS 66506-4004, USA Received 19 August 2003; accepted 16 December 2003
Abstract Field experiments were conducted in Northeast Kansas under conditions of limited plant competition to evaluate the individual and combined impact of the imported weevils Rhinocyllus conicus Froelich and Trichosirocalus horridus (Panzer) on the introduced weed, musk thistle (Carduus nutans L.). No effects on seed production occurred when low larval densities of T. horridus (<20 per plant) fed in rosettes, with or without the later-arriving, head-feeding weevil, R. conicus, present. In contrast, under high T. horridus larval densities (66 per plant), production of flower heads was reduced compared to uninfested plants. This indirect effect resulted in a 30% loss in viable seed per plant (with R. conicus present). Plant survival was not affected by T. horridus within the range of larval densities tested. R. conicus, when present alone, reduced viable seed by approximately 45%, whereas high densities of both weevils reduced viable seed by 59%. Thus, under conditions of low plant competition, neither weevil substantially limited seed production by musk thistle. This suggests the need for a multi-faceted management program for musk thistle involving additional forms of stress, such as interspecific plant competition. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Seed production; Classical biological control; Weeds; Trichosirocalus horridus; Rhinocyllus conicus; Musk thistle; Carduus nutans
1. Introduction Musk thistle (Carduus nutans L. species group) is a Eurasian weed that was first recorded in North America in the 1850s (Stuckey and Forsyth, 1971). It is most problematic in pastures and rangeland, especially in overgrazed situations (Dunn, 1976; Fick and Peterson, 1995). The presence of musk thistle can disrupt grazing by cattle, and competition with grasses reduces forage production (Batra, 1978; Fick and Peterson, 1995; Kates et al., 1975). Musk thistle grows as a biennial or winter annual and can be a prolific seed producer, although only one-third of the seed may germinate (McCarty, 1982; McCarty and Scifres, 1969). In addition, dormant *
Corresponding author. Present address: United States Department of Agriculture, Agricultural Research Service, Grassland, Soil, and Water Research Laboratory, 808 E. Blackland Road, Temple, TX 76502, USA. Fax: 1-254-770-6561. E-mail address: [email protected] (L.R. Milbrath). 1049-9644/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2003.12.005
seed can remain viable for 15 years (Burnside et al., 1981). During the 1960s and 1970s, two species of weevils— Rhinocyllus conicus Froelich and Trichosirocalus horridus (Panzer)—were imported into the United States and Canada as classical biological control agents of musk thistle (see Kok, 2001). In Northeast Kansas, releases of weevils originally collected in France and Italy resulted in the establishment of both species (Hilbert and Brooks, 2000). Larvae of T. horridus feed in the plant crown, which often results in the production of multiple flower stems. Plants are rarely killed by T. horridus and its indirect impact on seed production appears to be variable (Cartwright and Kok, 1985; Sieburth et al., 1983; Woodburn, 1997). R. conicus attacks flower heads. Larval feeding in the receptacle causes the abortion of developing ovules, as well as the destruction of young achenes (Kok, 2001; Shorthouse and Lalonde, 1984). As a result, the production of high quality seed may be reduced by as much as 78% (McCarty and Lamp, 1982).
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In Virginia and other parts of the eastern United States, substantial declines in musk thistle densities have been attributed, at least in part, to one or both weevils (Kok, 1986; Kok and Pienkowski, 1985). A similar claim has more recently been made for musk thistle in Oklahoma, based primarily on anecdotal information (Roduner et al., 2003). At other locations, reductions have not been observed (Andres and Rees, 1995), especially where plant competition with musk thistle was low (Harris, 1981). Thus, it remains unclear how efficacious the weevils are relative to other stress factors in suppressing and maintaining low musk thistle densities (but see Kok et al., 1986). A challenge to the efficacy of these biological control agents also has been made (Louda et al., 1998) because of the widespread feeding by R. conicus on nontarget, native thistles (Louda et al., 1997; Turner and Herr, 1996; Turner et al., 1987). The potential for T. horridus to do the same (McAvoy et al., 1987), should it be widely redistributed and established, is unclear given the recent report that T. horridus is a species complex in which each species may specialize on a single genus of thistles (Alonso-Zarazaga and Sanchez-Ruiz, 2002). Because each weevil feeds on different plant parts and at different times of the year, it is assumed that R. conicus and T. horridus are complementary biological control agents. Therefore, there has been general interest in establishing both weevils at the same localities. However, there have been no experimental evaluations to quantify the individual versus combined effects of the two weevils on viable seed production (Nechols, 2000). Such baseline data are important, not only because of plans for redistributing the weevils, but because several other species have been released in North America on musk and related thistles, some of which are now established. These include the stem-feeding fly, Cheilosia corydon (Harris) (Diptera: Syrphidae), a flea beetle, Psylliodes chalcomera (Illiger) (Coleoptera: Chrysomelidae), a gall-forming fly, Urophora solstitialis (L.) (Diptera: Tephritidae), and a rust fungus, Puccinia carduorum Jacky (Uredinales: Pucciniaceae) (Baudoin and Bruckart, 1996; De Quattro, 1997; Nechols, 2000). The objective of this study was to determine the short-term impact of T. horridus and R. conicus, alone and in combination, on musk thistle flower head and seed production under conditions of minimal plant competition in the field.
2. Materials and methods 2.1. Insect colony Adult T. horridus were field-collected annually in the fall from musk thistle and overwintered at 5 or 10 °C with a photoperiod of 10:14 h (L:D) in an environmental
419
growth chamber. Adults were held in ventilated, plexiglass boxes containing bouquets of musk thistle leaves in cotton-plugged, water-filled, shell vials. Eggs were obtained by placing 15–30 weevils (1:2 male:female) in ventilated, 2-liter paper containers with thistle bouquets. Adults were held at a thermoperiod of 20:10 °C (10:14 h) with a photoperiod of 10:14 h (L:D). Leaves were dissected every 2–7 days for eggs, which were then placed in petri dishes lined with moist filter paper layered over wet cotton. Eggs were stored at 2–3 °C in darkness for 1–4 months (Kok and McAvoy, 1983). Subsequently, the eggs were transferred to 25 °C and allowed to develop. When the embryonic head capsule had darkened (24–48 h pre-hatch), batches of 10–13 eggs were aspirated with water into individual Pasteur pipettes. The pipette tip was wrapped with Parafilm ÔMÕ (American National Can, Chicago, IL) and the pipettes were refrigerated for approximately 1 week until eggs were needed to artificially infest musk thistle plants in field experiments. Preliminary tests showed that neither refrigeration nor storage in water affected the hatching rate. Voucher specimens of T. horridus and R. conicus were deposited in the Kansas State University Museum of Entomological and Prairie Arthropod Research under Lot No. 140. 2.2. Field experiments Between 1998 and 2001, three field experiments were conducted as follows. 2.2.1. 1998–1999, Keats, KS A field site was selected near Keats, Riley, KS (39°14.920 N, 96°43.500 W, 380 m) in fall, 1998. It had a history of musk thistle infestation and populations of R. conicus and T. horridus. In October, a 0.25-ha plot was fenced off to exclude cattle and 0.8 0.8 0.2 m (length width height) frame cages were placed over naturally occurring musk thistle rosettes (one plant per cage) to protect them from field populations of T. horridus. Cages had a window screen top and were large enough to allow for the expansion of rosettes. The density of experimental thistles was 0.04 musk thistle per m2 . The initial diameter of thistles ranged from 15 to 30 cm. The phenological development of caged thistles through bolting was similar to nonexperimental thistles growing near the field plot. To minimize interspecific plant competition on musk thistles, grasses and other vegetation within the cages and in the surrounding areas were clipped or mowed to maintain an average height of 10 cm. The experimental design was a 3 3 factorial treatment structure in a Latin square design. Because of the varying terrain at the field site, treatments were grouped by location within the site (first blocking factor). Rosette diameters measured in December (when fall growth had
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ceased and prior to freezing of the ground) served as a second blocking factor because the growth response of thistles to T. horridus damage had previously been shown to interact with thistle size (Sieburth et al., 1983). There were nine replications (blocks), each having three levels of T. horridus infestation (0, 25, or 50 eggs per plant) and three levels of R. conicus infestation (excluded with a closed bag, natural infestation with an open or sham bag, and natural infestation with no bag) for a total of 81 experimental units. In late March, 1999, musk thistle plants designated for T. horridus treatments were infested by inserting the tips of two or four Pasteur pipettes (depending on infestation level) that contained 12–13 mature (within 1 day of hatching) eggs into the main vein of thistle leaves. Eggs were collected as described above. This procedure was used because naturally deposited eggs and larvae cannot be counted in the field without destroying the plant. To account for possible effects on thistle plants from mechanical injury, three empty pipette tips were inserted into each control thistle. Thistle plants were not available to establish an unwounded control. Six additional caged thistles (nonexperimental) also were infested with 25 or 50 eggs. These thistles were dissected for larvae after 1 month to estimate the actual infestation levels of the experimental thistles. Similar densities of about 16 larvae per plant were recovered (mean SD [range]: 15.8 5.8 [5–21]). In addition, to obtain estimates of natural infestations of T. horridus, 10 musk thistle plants were dug from the ground in late April/early May at each of three locations in Northeast Kansas. The plants were dissected and T. horridus larvae were counted. When bolting (stem elongation) began in late April 1999, frame cages were opened and a 0.25% solution of permethrin [(3-phenoxyphenyl)methyl 3-(2,2-dichloroethenyl)-2,2-dimethylcyclopropanecarboxylate, Eliminator, Gro Tec, Madison, GA] was applied to the grass immediately outside the cages to exclude late-ovipositing T. horridus. Frame cages were removed by mid-May when R. conicus began ovipositing. All of the flower buds (capitula) that developed on each plant were individually covered by (1) closed or (2) open (sham) nylon mesh bags (23 23 mesh), or (3) left unbagged. To exclude R. conicus, bags were tied off at the top and bottom (on the rachis just below the bud) with twist ties. To test for unintended effects resulting from closed bags, sham bags were tied to the rachis only, leaving the top and most of the bottom open. To provide additional access for R. conicus adults, a 3 13-cm square was cut in the side of the bag. Flowers in all treatments were hand-pollinated with a small paintbrush to ensure uniform pollination. The number of R. conicus eggs per flower head was counted at least once per week. Cages remained in place until 1 week before seed heads were harvested. All seed heads were then covered with a closed bag to prevent seed loss due to birds and storms.
To quantify the impact of the weevils on thistle development and seed production, the number of flower heads per plant was counted about once per week beginning in the spring and continuing until plant death. Receptacle diameters for only heads that formed during the period of R. conicus reproduction were measured to the nearest 0.1 mm at full bloom or abortion of the head. Seed heads were clipped and placed in 0.5-liter containers 10–14 days after full bloom (just prior to seed dispersal) or when they had aborted. Heads were held in the laboratory at 25 °C (range 23–30 °C) to allow flower heads to dry. Musk thistles were infested by the sunflower moth, Homoeosoma electellum (Hulst), beginning in June and the infestation intensified as the season progressed. Flower heads produced after R. conicus adults were no longer active (July onward) were not harvested for seed because of extensive damage by sunflower moths. Seed heads from individual plants present during the period of R. conicus activity were combined into two groups based on the dates of flower head production and intensity of sunflower moth infestation. The first group consisted of flower heads produced between 28 April and 28 May that experienced little to no damage by sunflower moth. The second group contained flower heads produced from 29 May to 30 June during which time higher levels of sunflower moth infestation occurred. Seeds were detached from flower heads and poured into a 4-liter pitcher along with 0.25 liter of 1-cm gravel. The pitcher was covered and shaken for 5–10 s to separate seed from the pappus. The contents were then sieved. The seed was separated into four different weight classes using a South Dakota Seed Blower model B (E.L. Erickson Products, Brookings, SD) by varying the air flow according to the procedure of McCarty and Lamp (1982). The different classes of seed were defined by McCarty and Lamp (1982) as follows: class I—unfilled and not viable, class II—partially filled with a germination rate of 2%, class III—better filled with a 32% germination rate, and class IV—wellfilled with a 96% germination rate. Samples of seed masses obtained for each weight class were found to be similar to those of McCarty and Lamp (1982). For each weight class, the number of seeds was counted on a per-plant basis. Long-term storage of experimental seed may have compromised germination rates. Therefore, data relating percentage germination by weight class from McCarty and Lamp (1982) were used to estimate viable seed production per plant among treatments. 2.2.2. 1999–2000, Westmoreland, KS The experiment was established at a 0.18-ha field site near Westmoreland, Pottawatomie, KS (39°26.090 N, 96°20.430 W, 409 m). This site had a history of musk thistle and both weevils were present. The experimental design was a 4 2 factorial treatment structure in a randomized complete block design. There were 10 blocks (replications), each of which had four levels of
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T. horridus infestation (artificial infestation in the fall, spring, both fall and spring, or excluded) and two R. conicus infestation levels (natural infestation or excluded) for a total of 80 experimental units. Musk thistle rosettes were transplanted for increased uniformity of plot conditions and plant size. In late September, 2 weeks prior to transplanting, a 0.64-m2 area was cleared of vegetation for each experimental plant by applying glyphosate (N-(phosphonomethyl)glycine, Roundup, Monsanto, St. Louis, MO) at 4.7 liter/ha. This zone was established to reduce effects of competing grasses on thistles without disrupting the soil profile. In addition, surrounding grass and other vegetation were mowed at regular intervals to maintain an average height of 10 cm. Frame cages were placed in the ground 1 week before transplanting and a 0.25% permethrin solution was applied inside the cages to eliminate any T. horridus adults. Greenhouse-germinated musk thistle seedlings, 10–15 cm in diameter, were transplanted on 8 October. Plants were spaced 2 m apart, resulting in a density of 0.25 musk thistle per m2 . Musk thistle plants designated for T. horridus infestation were inoculated with five mature eggs from a laboratory colony on 7 December or 15 mature eggs on 13 March, or both. The number of T. horridus eggs used for each treatment was based on the quantity of eggs that could be harvested from the laboratory colony. However, the majority of eggs are oviposited in the spring. Eggs were placed directly in the crown of the thistles and covered by moist cotton to prevent desiccation before hatching. Six additional caged thistles were infested with T. horridus at experimental rates in the fall or spring and then dissected in April to provide estimates of actual densities of T. horridus in the experimental plants. The numbers of larvae per plant recovered in the spring were about 2 [n ¼ 1] (fall), 9.8 3.3 [n ¼ 5] (spring) and an estimated 12 (fall and spring). Four uninfested thistles were left in frame cages until April and then dissected. Results confirmed that the cages were effective in preventing the invasion of natural populations of T. horridus. Rhinocyllus conicus was excluded from plants by applying sprays of 0.25% permethrin to the developing flower heads at 2-week intervals. Sprays were discontinued on heads that were about to flower to prevent interfering with pollinators, and no spraying was done after R. conicus adults were no longer present in the field. Applications of Bacillus thuringiensis Berliner subsp. kurstaki (DiPel ES, Valent BioSciences, Walnut Creek, CA) at 1.8 liter/ha were applied weekly to flower heads in all treatments to minimize infestations of the sunflower moth. Applications began on individual heads just prior to bloom and continued until head senescence. Data collection and analyses were similar to the 1998–1999 experiment. Head receptacle diameters were measured 4 days after full bloom (when the maximum
421
head size was achieved). Seed heads from each plant were placed into two groups: (1) flower heads produced during the period of oviposition by R. conicus (through 12 June) and (2) flower heads produced after R. conicus activity had ceased. 2.2.3. 2000–2001, Keats, KS This experiment used natural infestations of T. horridus and R. conicus and was conducted on a fenced, 0.02-ha site located at 39°14.690 N, 96°43.660 W (366 m) and approximately 0.5 km from the 1998–1999 experimental site. The experimental design was a oneway treatment structure in a randomized complete block design. There were 20 blocks (replications), each of which had two levels of T. horridus infestation (natural infestation or excluded) for a total of 40 experimental units. Two treatment combinations used in the previous experiments (neither weevil present, T. horridus present but R. conicus absent) were not included because this experiment was designed primarily to evaluate the interactions between the two musk thistle weevils. A parallel study that incorporated the absence of R. conicus could not be run because of very low R. conicus densities at that field site. The field plots were established as described previously except that no frame cages were used. Naturally germinated thistles surrounding the field plots were destroyed and additional T. horridus adults were released into the plot area in March to ensure high infestation levels of T. horridus. Greenhouse-germinated musk thistle seedlings, 10– 15 cm in diameter, were transplanted to experimental plots on 28 September. T. horridus was excluded by applying sprays of 0.25% permethrin, and sunflower moth was excluded with applications of DiPel ES at 1.8 liter/ha at 2- or 1-week intervals, respectively. Five additional transplanted thistles (nonexperimental) were dug from the ground in late April and dissected to quantify levels of natural infestation by T. horridus. An average of 66.2 22.9 larvae was found per plant. Data were collected using procedures described previously with the following change. Seed heads from each plant were placed into five groups based on the dates of flower-head production and intensity of R. conicus infestation, the last two groups experiencing no damage from R. conicus: (1) flower heads produced from 27 April to 23 May, (2) 24 May to 7 June, (3) 8 to 26 June, (4) 27 June to 25 July, and (5) 26 July to 10 September. Due to the very large volume of seed produced in 2001, seed numbers were estimated by mass instead of by counting. Twenty samples of 100 seed each for weight classes II, III, and IV were weighed to the nearest 0.1 mg (10 samples from plants infested by both T. horridus and R. conicus and 10 samples from plants infested only by R. conicus). Analysis of variance indicated there were no significant differences (P > 0:05) in seed mass between the two
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treatments, so the data were pooled to produce a mean mass per seed for each weight class (II—1.3 mg, III— 1.7 mg, and IV—2.9 mg). Samples of total seed for each weight class were weighed to the nearest 0.1 mg and divided by the respective mass per seed to derive a seed number. Estimates of viable seed were made as previously described. Weight class I (unfilled seed) was not included because of technical difficulty in removing seed from nonseed material, and because McCarty and Lamp (1982) had reported 0% germination for this seed class. 2.3. Statistical analyses Plants were omitted from analysis if they had died prematurely (e.g., in the rosette stage) from unknown causes, were contaminated by naturally occurring (nonexperimental) infestations of T. horridus, or displayed no signs of damage and developed a central, primary flower stem after having been artificially infested with T. horridus eggs. Data on flower-head production, receptacle diameters, the number of R. conicus eggs per flower head, the percentage of flower heads that received R. conicus eggs, and seed production were analyzed with Analysis of Variance (PROC MIXED, SAS Institute, 1999). Percentage data were subjected to arcsine square root transformation before analysis. Adjustments could not be made for the effect of contamination by sunflower moth on the 1999 seed data using Analysis of Covariance (PROC MIXED, SAS Institute, 1999). For the 1999 and 2000 data, treatments were pooled to create four treatments, utilizing a 2 2 factorial analysis, involving the infestation of musk thistle by no insects, T. horridus only, R. conicus only, or both species combined. Data involving flower-head production from individual sampling dates were combined to represent up to five periods of R. conicus seasonal activity: (1) 50% of R. conicusÕ seasonal egg production oviposited and high adult R. conicus densities (first 3 to 4 weeks), (2) an additional 40–45% eggs oviposited and decreasing R. conicus numbers (next 1 to 2 weeks), (3) remaining 5– 10% eggs oviposited and disappearance of R. conicus adults (1–3 weeks), (4) reproductive R. conicus absent from the field, flower heads produced through late July ( ¼ plant death in 1999 and 2000, 4–6 weeks), and (5) reproductive R. conicus absent from field, flower heads produced from late July until plant death (2001 only, 6 weeks).
3. Results 3.1. Seasonal dynamics of musk thistle and weevils In Northeast Kansas, average spring densities of T. horridus on naturally infested musk thistle ranged
from 17 to 34 larvae per plant, with individual plants having from zero to 77 larvae per plant. In all years examined, musk thistle plants began producing flower buds during the first week of May. Flowering continued until plant death, which ranged from mid July to mid September. Adult R. conicus were present on thistles by late April and rapidly reached high densities (averages of 2–10 adults per plant). Adults began ovipositing as soon as flower buds were accessible, and populations remained high for about 4 week before declining over a 3- to 4-week period. Low numbers of R. conicus adults were observed until mid or late June. 3.2. 1999 Neither weevil species, individually or together, had an effect on the seasonal production of flower heads or head size (P > 0:05; Table 1). The proportion of flower heads infested by R. conicus, and the egg densities on infested flower heads, were similar on T. horridus-infested and uninfested thistles (Table 1). Infestation of musk thistle rosettes by T. horridus had no effect on total or viable seed production by musk thistle at any time of the season. In contrast, fewer total and viable seeds were produced during May when R. conicus was present than when it was excluded (P < 0:001; Table 2). Reductions in per-plant total and viable seed attributable to R. conicus were 50 and 58%, respectively. When R. conicus was present in flower heads, the proportion of class IV seed (highest germination rate, comprising 92–99% of viable seeds) was reduced. There was a corresponding increase in the proportions of class II and III seed, which have considerably lower germination potential. There was no added impact on seed production or viability when plants were infested by both T. horridus and R. conicus (Table 2). In June, there were no reductions in total or viable seeds produced when R. conicus was present compared to when it was absent (Table 2). However, during this period, musk thistle heads were moderately to severely infested by larvae of the sunflower moth. 3.3. 2000 Musk thistle infested by T. horridus produced more flower heads at the beginning of the season compared to uninfested thistles (P < 0:02). Otherwise, neither T. horridus nor R. conicus affected the number of flower heads produced (Table 3). The presence of R. conicus resulted in smaller flower heads early in the season than for plants not infested by R. conicus (P < 0:002), and T. horridus-infested thistles produced smaller heads in May than uninfested thistles (P < 0:05; Table 3). Similar densities of R. conicus eggs were deposited on T. horridus-infested and uninfested thistles (Table 3).
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Table 1 Seasonal flower-head production and R. conicus egg densities (mean SE or 95% CI) on musk thistle plants infested or not infested by T. horridus and/or R. conicus, Keats, KS, 1999a Dates
T. horridus
R. conicus
New flower heads per plant
Maximum receptacle diameter (mm)
R. conicus eggs per infested head
% Heads with eggs
28 April–28 May
)b + ) +
)b ) + +
6.0 1.8 3.5 1.6 6.6 1.4 6.8 1.3
27.0 1.4 25.9 1.2 26.6 1.0 24.5 0.9
—
—
) + ) +
) ) + +
5.4 2.1 5.8 1.8 8.5 1.6 7.6 1.4
18.4 1.1 19.1 0.9 17.6 0.8 18.1 0.8
) + ) +
) ) + +
2.7 2.0 1.5 1.7 4.9 1.4 4.7 1.2
13.6 1.4 16.2 1.3 15.8 0.8 14.8 0.7
) + ) +
) ) + +
6.9 2.6 5.1 2.3 7.1 2.0 9.9 1.8
29 May–9 June
10–30 June
1–28 July
—
—
31.4 2.7 26.4 2.3
100 (99.9–100) 100 (100–100)
—
—
—
—
7.9 1.2 7.9 1.0
99.1 (93.9–99.6) 95.0 (87.6–99.1)
—
—
—
—
1.8 0.3 2.0 0.2
40.4 (10.5–75.0) 45.1 (15.5–76.7)
c
—
—
c
—
—
c
c
c
c
c
c
a Neither the T. horridus nor R. conicus as main effects, or the interaction term, were significant (P > 0:05) for means within columns and for each set of dates (n ¼ 4–26). From 29 May to 28 July, thistles also infested by sunflower moths. b ) Absent, + present. c Head diameters not measured and no reproductive R. conicus present in the field.
There was no effect of T. horridus on total or viable seed production by musk thistle at any time of the season (P > 0:05; Table 4). In contrast, infestation by R. conicus during the period of its reproductive activity (29 April to 12 June), reduced the number of total and viable seeds per plant by 32 and 45%, respectively, compared to plants protected from R. conicus (P < 0:02; Table 4). The number of class IV seed was reduced and the number of class II seed was increased for R. conicusinfested heads (Table 4). There was no additional effect on seed production when both T. horridus and R. conicus infested musk thistle plants (Table 4). Following the absence of R. conicus in the field (13 June to 24 July), there were no differences in seed production between plants that had been previously infested by R. conicus or those that had not (Table 4). A low percentage of heads flowered (5–12%) due to plant senescence and few numbers of viable seed were produced, averaging up to four seeds per plant (Table 4). This was less than 0.1% of all viable seed produced for each plant.
Infestation of musk thistle by T. horridus resulted in fewer numbers of total and viable seed produced during May and June than for thistles not infested by T. horridus (P < 0:05). Depending on the particular time, total seed per plant was reduced from 28 to 44%, and viable seed was reduced from 31 to 45% (Table 6). However, the proportion of class IV seed (80–90% of total seed) did not vary with T. horridus infestation. There were no differences in seed production per plant between T. horridus-infested and uninfested thistles from late June onward (Table 6). A high percentage of heads continued to flower for several weeks afterwards (around 75%), with each plant producing an average of 4180 viable seed or 14–19% of viable seed production (Table 6). The total and viable seed per plant produced for the season were therefore reduced by 28 and 30%, respectively, for T. horridus-infested musk thistles compared to thistles protected from T. horridus (Table 6).
3.4. 2001 4. Discussion Plants infested by T. horridus produced fewer flower heads than plants protected from T. horridus during May and June, which was the period of oviposition by R. conicus (Table 5). The earliest flower heads were slightly smaller on T. horridus-infested thistles than uninfested thistles (Table 5). Also, T. horridus-infested plants received fewer numbers of R. conicus eggs per head early in the season (Table 5).
Under conditions of little or no plant competition and high weevil densities, each species of weevil contributed to the reduction of viable seed in musk thistle. In addition, the combined impact of T. horridus and R. conicus was greater than that of either weevil alone. However, these effects were not observed at low population densities of T. horridus.
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Table 2 Seasonal seed production (mean SE) for musk thistle plants infested or not infested by T. horridus and/or R. conicus, Keats, KS, 1999 Dates of flower-head production
T. horridus
28 April–28 May
)b + ) + T. horridusc R. conicusc
29 May–30 Juned
) + ) + T. horridus R. conicus
R. conicus
No. of seed per plant by weight class and totala I
II
III
IV
Total
)b ) + +
1014 187 819 161 419 141 382 125 0.36 <0.01
49 51 32 44 128 38 107 33 0.57 0.03
27 40 34 35 100 30 85 27 0.89 0.02
1141 217 1052 180 429 152 418 126 0.75 <0.01
2236 430 1920 371 1076 325 987 287 0.49 <0.01
) ) + +
1281 313 764 260 595 222 724 186 0.40 0.12
18 17 34 14 52 11 33 9 0.90 0.20
31 19 39 16 42 14 55 12 0.45 0.33
416 231 360 195 373 170 486 147 0.86 0.80
1755 500 1184 423 1063 367 1302 316 0.64 0.41
Estimated number of viable seed per plante 28 April–28 May
29 May–30 Juned
) + ) + T. horridus R. conicus
) ) + +
e
) + ) + T. horridus R. conicus
) ) + +
e
e e e
e e e
11 11 31 21 0.57 0.03
10 15 13 13 38 12 32 10 0.89 0.02
1096 209 1010 172 412 146 401 121 0.75 <0.01
1106 222 1022 184 452 156 435 130 0.76 <0.01
00 10 10 10 0.90 0.20
12 7 15 6 16 5 21 5 0.45 0.33
399 222 346 187 358 163 466 141 0.86 0.80
411 226 361 191 375 166 488 143 0.84 0.77
a
Total seed numbers may not equal the sum of all weight classes due to differences in least squares means estimates. ) Absent, + present. c P values for main effects. No interaction terms were significant (P > 0:05) (n ¼ 7–26). d Affected by sunflower moth infestation. e Based on 0% germination for seed of weight class I, 2% (II), 38% (III), and 96% (IV) (McCarty and Lamp, 1982). b
4.1. Trichosirocalus horridus Because T. horridus feeds only on vegetative (nonfloral) tissues, its potential for having an impact on musk thistle is restricted to two categories: direct mortality of rosettes and indirect effects on reproductive fitness (e.g., reductions in the size and/or number of flower heads produced). In our experiments, there were no plant deaths attributable to T. horridus and the capacity of this herbivore for doing so appears to be generally low unless larval densities are very high (Sheppard et al., 1995; Sieburth et al., 1983; Woodburn, 1997). However, it is possible that combined stress from T. horridus and plant competition could cause direct mortality to musk thistle, although this has not been shown. With respect to sublethal impacts, our findings agree with previous studies that have shown no consistent, significant effect of T. horridus on musk thistle head size (Cartwright and Kok, 1985; Woodburn, 1997). On the other hand, high densities of T. horridus did cause a 15% reduction in the number of flower heads per plant—an effect that was evident only during the first half of the
season. These data suggest that in the absence of plant competition, T. horridus may cause marginal reductions in musk thistle seed production, but only during growing seasons when hotter, drier weather shortens the thistle flowering period, and only when sporadically high T. horridus densities occur. The impact of T. horridus would diminish even further in years when milder temperatures and sufficient rainfall allow thistles to produce flower heads for a longer period of time, as occurs in some years in Kansas. The lower densities (<20 larvae per plant) of T. horridus that we observed in 1999 and 2000, in combination with little or no intra- or interspecific plant competition, had no effect on either the seasonal production of flower heads or seed by musk thistle. The only detectable effect was in stem number; low larval densities of T. horridus are correlated with altered thistle architecture but not reproduction (Sheppard et al., 1995). Furthermore, the effect of T. horridus was not altered by the additional stress imposed by R. conicus. Plant size can influence the effect that T. horridus has on seed production. For example, Cartwright and Kok
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Table 3 Seasonal flower-head production and R. conicus egg densities (mean SE or 95% CI) on musk thistle plants infested or not infested by T. horridus and/or R. conicus, Westmoreland, KS, 2000 Dates
T. horridus
R. conicus
New flower heads per plant
Maximum receptacle diameter (mm)
R. conicus eggs per infested head
% Heads with eggs
29 April–22 May
)a + ) + T. horridusb R. conicusb
)a ) + +
4.0 1.0 6.4 0.7 3.6 1.1 5.8 0.8 0.01 0.59
34.3 1.6 29.4 1.1 27.6 1.6 26.7 1.2 0.04 <0.01
—
—
—
—
22.0 4.0 15.0 3.0 0.09
84.3 (55.7–99.2) 97.0 (82.5–99.3) 0.15
—
—
) + ) + T. horridus R. conicus
) ) + +
3.9 1.3 4.0 0.9 4.1 1.3 5.7 1.0 0.40 0.39
24.3 1.6 17.7 1.1 22.2 1.7 19.6 1.1 <0.01 0.92
—
—
) + ) + T. horridus R. conicus
) ) + +
3.6 1.0 2.0 0.6 2.8 1.0 4.2 0.7 0.90 0.41
) + ) + T. horridus R. conicus
) ) + +
5.2 1.2 4.0 0.9 3.9 1.2 5.8 0.9 0.72 0.81
23 May–1 June
2–12 June
13 June–24 July
—
—
6.4 1.3 4.6 0.9 0.30
53.4 (24.2–81.4) 31.0 (5.5–65.5) 0.12
—
—
12.8 1.5 12.0 1.2 12.3 1.5 12.6 1.2 0.84 0.96
—
—
c
—
c
—
—
c
c
c
c
c
c
—
—
—
0.02 ()0.13–0.45) 0.27 (0.05–1.54) 0.41
—
— —
) Absent, + present. P values for main effects. No interaction terms were significant (P > 0:05) (n ¼ 7–23). c Head diameters not measured and no reproductive R. conicus present in the field. a
b
(1985) reported both decreased and increased seed production for smaller thistles infested by T. horridus compared to uninfested thistles, but not for larger thistles (rosette diameters >25 cm). The mean rosette diameters of our experimental thistles ranged from 32 to 64 cm, which fall into a large size class. Therefore, our results for musk thistles that were infested at low T. horridus densities are consistent with those of Cartwright and Kok (1985). 4.2. Rhinocyllus conicus The seasonal pattern of host exploitation that we observed for R. conicus on musk thistle in Northeast Kansas agrees with the findings of researchers in other regions (Andres and Rees, 1995; Kok and Pienkowski, 1985; McCarty and Lamp, 1982; Rees, 1977). That is, R. conicus oviposits heavily on the earliest flower heads (main terminal and early lateral heads), causing significant damage (including aborted heads) and a substantial reduction in viable seed produced. Subsequently, adult weevil numbers and oviposition rates decline such that egg densities and the percentage of flower heads attacked are lower on the lower parts of the plant that are produced later in the season. Thus, although the earliest flower heads may be completely destroyed (Kok and
Surles, 1975), most heads are able to produce moderate to high numbers of viable seed. Furthermore, in Kansas and other locations, because R. conicus ceases oviposition by early summer, well before the potential end of the flowering period of musk thistle, variable numbers of heads escape herbivory from R. conicus altogether. In some locations and years, few flower heads will set seed after R. conicus activity has ceased and the amount of viable seed produced will be minimal at this time. This may have much to do with individual site characteristics as well as conditions of moisture stress in summer, whether due to drought or normal weather patterns (Hodgson and Rees, 1976; McCarty and Lamp, 1982; Roduner, 2001). In contrast, conditions that are presumably more optimal for musk thistle can prolong flowering well beyond the period of R. conicus activity (Surles et al., 1974). As a result of these different factors, the greatest overall reduction in total seed and viable seed attributed to R. conicus were 32 and 45%, respectively. Therefore, despite localized and occasionally heavy infestations by other insects like the sunflower moth (McCarty and Lamp, 1982; Rees, 1977), a large amount of viable seed can be produced by musk thistle plants under conditions favorable for growth and development (e.g., weather, low levels of plant competition).
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Table 4 Seasonal seed production (mean SE) for musk thistle plants infested or not infested by T. horridus and/or R. conicus, Westmoreland, KS, 2000 Dates of flower-head production
T. horridus
R. conicus
29 April–12 June
) + ) + T. horridusc R. conicusc
13 June–24 July
) + ) + T. horridus R. conicus
b
No. of seed per plant by weight class and totala I
II
III
IV
Total
) ) + +
722 205 852 139 706 216 963 148 0.28 0.79
54 25 76 18 109 26 116 20 0.48 0.03
148 75 144 51 174 79 277 54 0.45 0.22
3602 534 3222 412 1815 558 1809 429 0.64 <0.01
4526 739 4271 562 2823 774 3159 586 0.94 0.02
) ) + +
72 27 34 18 50 28 47 19 0.40 0.85
42 21 02 21 0.72 0.22
62 22 03 12 0.53 0.15
22 11 02 32 0.56 0.89
84 30 39 20 50 32 54 21 0.43 0.70
b
Estimated no. of viable seed per plantd 29 April–12 June
13 June–24 July
) + ) + T. horridus R. conicus
) ) + +
d
) + ) + T. horridus R. conicus
) ) + +
d
d d d
d d d
11 20 21 20 0.48 0.03
56 28 55 19 66 30 105 21 0.45 0.22
3458 513 3093 396 1742 536 1736 411 0.64 <0.01
3515 526 3143 407 1816 549 1844 423 0.67 <0.01
00 00 00 00 0.72 0.22
21 11 01 01 0.53 0.15
22 11 02 31 0.56 0.89
43 22 03 32 0.84 0.54
a
Total seed numbers may not equal the sum of all weight classes due to differences in least squares means estimates. ) Absent, + present. c P values for main effects. No interaction terms were significant (P > 0:05) (n ¼ 9–23). d Based on 0% germination for seed of weight class I, 2% (II), 38% (III), and 96% (IV) (McCarty and Lamp, 1982). b
Table 5 Seasonal flower-head production and R. conicus egg densities (mean SE or 95% CI) on musk thistle plants infested or not infested by T. horridus and R. conicus, Keats, KS, 2001a Dates
T. horridus
R. conicus
27 April–23 May
)b +
+b +
24 May–7 June
) +
8–26 June 27 June–25 July 26 July–10 September
New flower heads per plant
Maximum receptacle diameter (mm)
R. conicus eggs per infested head
% Heads with eggs
19.9 2.1 a 9.7 2.1 b
31.2 0.8 a 29.2 0.8 b
41.8 1.7 a 22.2 1.7 b
100.0 (99.8–100) a 99.9 (99.6–100) a
+ +
45.6 4.9 a 34.8 5.0 b
25.1 0.6 a 25.6 0.6 a
7.7 0.4 a 6.4 0.4 b
89.5 (83.8–94.0) a 84.4 (77.8–90.0) b
) +
+ +
61.1 6.5 a 42.8 6.6 b
20.0 0.7 a 20.3 0.7 a
2.6 0.4 a 2.0 0.4 a
11.6 (7.7–16.2) a 12.8 (8.6–17.6) a
) +
+ +
114.2 21.3 a 99.2 21.3 a
c
c
c
c
c
c
) +
+ +
20.0 9.1 a 12.9 9.1 a
c
c
c
c
c
c
a
Means within columns and for each set of dates followed by different letters were significantly different (P < 0:05) (n ¼ 19–20). ) Absent, + present. c Head diameters not measured and no reproductive R. conicus present in the field. b
Our data suggest that at locations and during years when both R. conicus and T. horridus occur at high densities, the impact on musk thistle seed production would be greater than if only one species of weevil were present.
An estimate of the additional percentage reduction in viable seed due to the combined impact of both weevils can be obtained by multiplying the percentage reduction attributable to R. conicus [45% based on 2000 results,
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427
Table 6 Seasonal seed production (mean SE) for musk thistle plants infested or not infested by Trichosirocalus horridus and Rhinocyllus conicus, Keats, KS, 2001a No. of seed per plant by weight class and totalb
Dates of flower-head production
T. horridus
R. conicus
II
III
27 April–23 May
)c +
+c +
500 70 a 319 70 b
414 51 a 254 51 b
4207 534 a 2294 534 b
5121 625 a 2867 625 b
24 May–7 June
) +
+ +
685 139 a 858 139 a
723 82 a 723 82 a
14,891 1566 a 10,167 1566 b
16,289 1681 a 11,748 1681 b
8–26 June
) +
+ +
480 87 a 399 87 a
525 69 a 407 69 a
7866 991 a 5126 991 b
8871 1100 a 5932 1100 b
27 June–25 July
) +
+ +
461 108 a 387 108 a
420 96 a 353 96 a
4413 1194 a 3579 1194 a
5294 1376 a 4319 1376 a
26 July–10 September
) +
+ +
46 24 a 34 24 a
37 20 a 25 20 a
271 166 a 94 166 a
354 206 a 153 206 a
IV
Total
Estimated no. of viable seed per plantd 27 April–23 May
) +
+ +
10 1 a 61 b
157 19 a 97 19 b
4039 512 a 2202 512 b
4206 530 a 2305 530 b
24 May–7 June
) +
+ +
13 3 a 17 3 a
275 31 a 275 31 a
14,295 1503 a 9760 1503 b
14,583 1527 a 10,052 1527 b
8–26 June
) +
+ +
10 2 a 82 a
200 26 a 155 26 a
7551 951 a 4921 951 b
7761 972 a 5084 972 b
27 June–25 July
) +
+ +
92 a 82 a
160 36 a 134 36 a
4237 1146 a 3436 1146 a
4406 1182 a 3578 1182 a
26 July–10 September
) +
+ +
10 a 10 a
14 7 a 97 a
260 159 a 91 159 a
275 167 a 101 167 a
a
Means within columns and for each set of dates followed by different letters were significantly different (P < 0:05) (n ¼ 19). Weight class I was not counted or included in analyses. c ) Absent, + present. d Based on 2% germination for seed of weight class II, 38% (III), and 96% (IV) (McCarty and Lamp, 1982). b
which is in general agreement with the findings of others (Shea and Kelly, 1998 and comments therein)] with the T. horridus-associated percentage reduction in seed that occurs beyond that of R. conicus (30%). Therefore, an additional 14, or 59% total, reduction in viable seed may occur when both weevils are present at high densities. However, whether the actual combined effect of the two weevils is more or less efficient than the sum of the individual impacts could not be determined. The effect of R. conicus on musk thistle reproduction may vary among years because of differences in weather conditions, indirect interactions with T. horridus (Milbrath and Nechols, 2004) , or other factors. For example, oviposition by R. conicus was lower on thistles that were heavily infested by T. horridus than on uninfested plants. If reduced oviposition by R. conicus results in greater seed production, T. horridus would be indirectly responsible for reducing the impact of R. conicus. 4.3. Recommendations The experimental data presented here suggest that the presence of both weevils may lead to a net reduction in
viable musk thistle seed. However, the magnitude of that effect is difficult to estimate because musk thistle occurs at times and in locations where other stresses may modify the impact of herbivores. For example, a potentially different outcome might occur when musk thistle is subject to interspecific plant competition (e.g., Kok et al., 1986). The role of intraspecific thistle competition, particularly in enhancing plant stress or creating better synchrony with R. conicus, has received no attention. Prior to the release of either weevil, musk thistle densities could be very high, ranging from 2 to 43 plants per m2 (Harris, 1981; Kok, 1986, 2001; Lee and Hamrick, 1983). In addition, the long-term impact of R. conicus and T. horridus on musk thistle population dynamics is unknown because musk thistle plants continue to produce large amounts of seed, despite significant reductions in viable seed production. Much of that seed may accumulate in seed banks. For example, the large thistles we observed in 2001 produced over 20,000 viable seeds, despite high densities of both R. conicus and T. horridus. Similarly, Woodburn (1997) reported that musk thistles infested by very high densities of T. horridus produced about 8000 seeds, even after a 72%
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reduction in seed set. Thus, even under high levels of weevil pressure, there may be more seed available than is needed to replace the plant population (see Shea and Kelly, 1998). This scenario may help explain, in part, the lack of thistle suppression reported in some areas that have had moderate to high populations of weevils and experienced only moderate to no reductions in thistle populations over time (Andres and Rees, 1995; Harris, 1981). The efficacy of T. horridus appears to depend on the larval densities it can attain. The presence of both R. conicus and T. horridus at high densities appears to provide greater reductions in viable seed than when R. conicus is present alone. However, high densities of T. horridus can have a negative effect on the short-term, and possibly long-term, population dynamics of R. conicus (Milbrath and Nechols, 2004). Therefore, the benefit of greater seed reduction from the combination of both weevils is potentially offset by reduced populations of R. conicus. However, based on direct observations and published reports, it is questionable whether consistently high T. horridus densities will occur in most areas of the United States (Andres and Rees, 1995; Kok and Mays, 1989). The low densities of T. horridus likely to occur appear to be of limited usefulness in the biological control of musk thistle, whether alone or in combination with R. conicus, especially when other types of stress are not acting on musk thistle. Low-stress environmental conditions may occur frequently and at many locations where vegetation has been disturbed (e.g., from overgrazing). These conditions are typical of many musk thistle sites in Kansas. Therefore, the continued redistribution of this weevil within the United States, either as a complementary biological control to R. conicus or as a single agent, does not seem warranted at this time.
Acknowledgments We thank Darrel Westervelt and Karen Krouse for the use of their land; Lucas Robison, Jason Sweet, Nicole Sweet, Mary Milbrath, Shauna Dendy, Xiaoli Wu, Neil Miller, Megan Murphy, Justin Schmitz, Soledad Villamil, Erika Jensen, and Vincent Gregorio for technical assistance; Tom Loughin for statistical advice; and David Margolies, Walter Fick, Randall Higgins, Sonny Ramaswamy, Walter Dodds (all from Kansas State University), and two anonymous reviewers for providing helpful suggestions on earlier drafts of the manuscript. We also acknowledge the following colleagues who contributed information and ideas during discussions: Ernst Horber (Kansas State University), Loke Kok (Virginia Polytechnic Institute and State University), Svata Louda (University of Nebraska), and Ben Puttler (University of Missouri). This research was
funded in part by a grant to L.R.M. from the Sigma Xi Grants-in-Aid of Research program, and by Regional Research Project W-185. This is Contribution No. 04005-J from the Kansas Agricultural Experiment Station.
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