United States 

Department of 

Agriculture 

Forest 

Service 

Northern 

Region 

200 East Broadway 

P.O. Box 7669 

Missoula, MT 59807 

Numbered Report 06-08 July 2006

Evaluation of Establishment of Cyphocleonus achates and its Potential 

Impact on Spotted Knapweed 

Nancy Sturdevant, Entomologist 

USDA Forest Service, Forest Health Protection 

Northern Region, Missoula, MT 

Sandy Kegley, Entomologist 

USDA Forest Service, Forest Health Protection 

Northern Region, Coeur d’Alene, ID 

Yvette Ortega, Wildlife Biologist 

Rocky Mountain Research Station, Missoula, MT 

Dean Pearson, Research Ecologist 

Rocky Mountain Research Station, Missoula, MT 

INTRODUCTION 

Environmental characteristics of a site may 

greatly influence the establishment and impact of 

biological control agents on the target weed. A 

biological control agent that is highly successful 

in one region may be virtually ineffective in 

another (Gurr and Wratten 2000). To maximize 

the use of limited availability of agents and 

financial resources, we should try to improve the 

success of our biological control releases by 

understanding the site requirements of individual 

biological control agents. 

Thirteen insects have been released on spotted 

knapweed in the United States since the early 

1970s. Seven insect species appear to be well 

established and of these, four show promise for 

biological control of spotted knapweed (Corn et 

al. 2006). Cyphocleonus achates (Fahraeus) was 

first introduced into Montana in 1988 by (Story 

et al. 1994) and is now well established in many 

counties in the state. 

European screening studies showed that C. 

achates attacks spotted and diffuse knapweed 

(Stinson et al. 1994). The larvae mine the roots 

of knapweed plants in Montana between about 

mid-July through late spring. Larval mining 

causes extensive root damage, reductions in 

shoot biomass (Steinger & Muller-Scharer 1992) 

and plant mortality (Corn et al. 2006). 

Clark et al. (2001a) found that the probability of 

establishment of C. achates was greatest in 

continuous knapweed infestations, larger 

infestations, and at mid-elevational sites as 

compared to lower and higher elevations. 
2

Sturdevant and Dewey (2003) reported that size 

of plants, weather conditions during and 

following the insect release, number of years 

since the release and number of insects released 

can influence the probability of establishment of 

C. achates. Recent declines of spotted knapweed 

in western Montana have been coincidental with 

the release of C. achates and the recent drought. 

Beginning in 1997, Forest Health Protection in 

cooperation with other USDA Forest Service and 

county weed cooperators began to monitor 

impact of weevils on knapweed at previous 

release sites. 

The objectives of this project were to: 1) 

evaluate the relationship between environmental 

variables and the establishment of C. achates at 

release sites to try and identify factors that may 

determine establishment success, and 2) examine 

the relationship between C. achates presence and 

abundance and changes in spotted knapweed 

over time to assess the short-term efficacy of this 

agent in suppression of its target weed. 

METHODS 

1997/2002 Data 

The primary dataset analyzed was based on 48 

study sites sampled in both 1997-98 and 2002-

03. Records indicated that C. achates had been 

released at 46 of these sites between 1991 and 

1997, with most releases occurring in the 1993-

1996 period. An average of 150 weevils was 

released per site (range 26-445). Two additional 

sites with no record of C. achates release were 

included in analyses since weevils were detected 

on these sites during initial sampling. According 

to records, the biological control agent Agapeta 

zoegana was also released and/or detected at 41 

of the study sites within the same timeframe as 

C. achates. 

In 1997 and 2002, densities of C. achates and A. 

zoegana per study site were assessed by counting 

larvae in roots of knapweed plants collected at 

points equally spaced along four transects 

radiating from the central release point 

(n=52/site in 1997-98 and n=20/site in 2002-3). 

Root sample size was reduced because a separate 

study showed no significant differences in the 

average number of larvae when sampling 20 

versus 52 roots (Sturdevant and Dewey 2002). 

Counts of adult C. achates from sweep net 

sampling were used to assess presence of 

weevils, but otherwise were not considered in 

analyses since sampling of larvae in roots 

provides a more consistent measure of 

abundance (Clark et al. 2001a).

Environmental variables describing site-level 

characteristics were assessed once during the 

study period. Variables included: elevation, 

aspect (reclassified as warm [ S, SW, W] versus 

cold [N, NE, E]), slope, topography (reclassified 

as valley bottom versus not), disturbance level 

(reclassified as low [scores 0-1] versus high 

[scores 2-31), and primary soil type (reclassified 

as sandy versus not). Categorical variables were 

reclassified to reduce the number of classes and 

increase the number of observations per class, 

thereby facilitating analyses. However, before 

doing so, preliminary relationships with C. 

achates prevalence were considered to assure 

that patterns were not lost as a result of 

collapsing classes. 

Additional environmental variables described 

vegetation. In 1997 and 2003, cover of bare 

ground, shrubs, spotted knapweed, and 

remaining vegetation were visually estimated 

from the release point within a 1/300-acre 

circular plot. In addition, density of knapweed 

stems was measured in both sampling periods 

within 16 1-m plots radiating out from the 

release point in the four cardinal directions. In 

2002 & 2003 only, average height of mature 

knapweed plants, density of mature knapweed 

plants, and cover of knapweed seedlings were 

also recorded within 16, 1-m plots. 
3

2002-2004 Data 

Additional data relating C. achates prevalence to 

knapweed characteristics, 2002-2004, were 

obtained from a report to FHP by Sturdevant and 

Dewey (2003). Study sites (n=15) had been 

chosen from a set of C. achates release sites 

(first monitored in 2002) because they had 

moderate to high numbers of weevils. At each of 

these sites, relative abundance of adult C. 

achates was estimated by sweeping a total of 300 

meters of knapweed per site. Sweeping began 

near the release point and continued through the 

densest portions of knapweed surrounding the 

release point. Knapweed characteristics 

including height of mature plants, density of 

stems, and density of mature plants were 

measured in 16 1-m plots that radiated out from 

the release point in the four cardinal directions. 

All sampling was done approximately during the 

first 2 weeks in August. 

Statistical Analyses 

We used the 1997/2002 dataset to relate the 

degree of C. achates establishment to 

environmental site variables. The degree of C. 

achates establishment per release site was 

defined and independently analyzed in two ways: 

1) presence of C. achates as determined by 

detection of C. achates larvae or adults in 2002, 

and 2) density of C. achates larvae in 2002. We 

used logistic regression to evaluate the presence 

of weevils as a function of background 

environmental variables (i.e., excluding 

knapweed characteristics). Similarly, we 

evaluated the relationship between density of 

weevils and background environmental variables 

using mixed linear models. 

In a second analysis, we examined knapweed 

characteristics measured in 1997 and 2002, 

testing for: 1) association with C. achates 

establishment in terms of presence of weevils 

and density of larvae, respectively, 2) population 

trends (i.e., differences between years), and 3) 

influence of C. achates establishment on 

population trends (i.e., interaction of C. achates

variables with year effects). Mixed linear 

models were used to conduct tests, with year 

treated as a repeated measure. 

Knapweed characteristics measured from 2002-

2004 were analyzed with the same mixed model 

used in the 1997/2002 analysis, i.e., testing 

knapweed variables for association with C. 

achates, for population trends, and for influence 

of C. achates on population trends. Total 

number of adult weevils caught in sweep nets 

was used to represent the prevalence of C. 

achates per site and year since larval counts were 

not available. 

All statistical analyses were conducted with SAS 

(SAS Institute 1999). We report least squares 

means from mixed models instead of raw means 

to adjust for imbalances in sample size and 

account for influence of covariates. 

RESULTS 

1997/2002 Data 

Densities of C. achates increased substantially at 

release sites between 1997-98 and 2002-03, from 

an average of 0.08 ± SE of 0.067 larvae/root to 

0.3 ± 0.068 larvae/root (P=0.012). Neither 

release date, number of C. achates released, nor 

number of releases were significantly related to 

densities of C. achates larvae in 2002 (P>0.4). 

However, densities of C. achates were positively 

associated with densities of A. zoegana (P=0.01). 

C. achates was detected at 36 of 48 release sites 

in 2002. Multivariate analysis of background 

environmental variables indicated that primary 

soil type and topography were associated with 

the presence of C. achates in 2002: weevils 

tended to occur at sites with sandy soils 

(P=0.026) located in valley bottoms (P=0.046). 

Remaining background environmental variables 

showed no significant relationships with the 

presence of weevils (P>0.37). Sites with sandy 

soils tended to have higher densities of C. 

achates larvae in 2002, but the difference was 

only marginally significant in a multivariate test 
4

(P=0.061) and less so in a bivariate test 

(P=0.14). Remaining background environmental 

variables showed no association with the density 

of C. achates larvae (P>0.16). 

There was a trend of less knapweed cover at sites 

where C. achates was present but it was not 

significantly different (P=0.57) and did not vary 

significantly with the density of weevil larvae 

(P=0.49). Knapweed cover declined 

significantly between years, from an average of 

49.7 ± 3.94% in 1997 to 41.7 ± 4.09% in 2002 

(P=0.02). The decline in knapweed cover over 

this time period tended to be greater at sites 

where C. achates was present compared to sites 

where it was absent, but this difference was not 

statistically significant (P=0.16; Fig. 1). When 

soil type and topography (i.e., variables that were 

significantly associated with the presence of C. 

achates) were included in statistical models, 

results were not altered. 

0

10

20

30

40

50

60

70

+ C. achates - C. achates

Knapweed % cover

1997

2002

Figure 1. Declines in mean percent cover of spotted knapweed from 1997 to 2002 at release sites where 

C. achates was present (+ C. achates) versus absent (- C. achates) as determined by detection of weevils 

during 2002 sampling. 

Density of mature spotted knapweed stems 

across years did not differ significantly between 

sites where C. achates was present versus absent 

(P=0.47) and did not vary significantly with the 

density of weevil larvae (P=0.72). Stem density 

declined between 1997 and 2002, from an 

average of 31.4 ± 3.42 stems/m2

 to 11.1 ± 2.51 

stems/m2 (P<0.001). The decline in stem density 

from 1997 to 2002 was not affected by the 

presence of weevils (P=0.44; Fig. 2) or by larval 

densities (P=0.63). Inclusion of soil type and 

topography in statistical models did not affect 

results. 
5

0

5

10

15

20

25

30

35

40

+ C. achates - C. achates

Stem density (no./m2

)

1997

2002

Figure 2. Declines in mean stem density of spotted knapweed from 1997 to 2002 at release sites where 

C. achates was present (+ C. achates) versus absent (- C. achates) as determined by detection of weevils 

during 2002 sampling. 

2002-2004 Data 

Average number of adult C. achates detected per 

site was 11.6 ± 5.38 weevils in 2002, 21.4 ± 5.38 

weevils in 2003, and 6.9 ± 5.53 weevils in 2004, 

but differences among years were only 

marginally significant (P=0.08). For each site, 

counts of weevils conducted in 2002 and 2003 

were highly correlated (r=0.7, P=0.004). 

However, weevil counts from 2004 showed no 

relationship with prior years (r<0.3, P>0.3), 

likely reflecting the fact that 

sampling conditions in 2004 were particularly 

unfavorable, as noted by Sturdevant and Dewey 

(2003). Given results of correlation analysis and 

the tendency for higher counts in 2003, we used 

the 2003 sample to characterize the relative 

abundance of C. achates at study sites in 

remaining analyses. 

Height of spotted knapweed plants across years 

did not vary significantly with the relative 

abundance of C. achates at sites (P=0.62). 

Although plant height had a tendency to decline 

between 2002 and 2004, the trend was nonsignificant (P=0.88). Furthermore, the yearly 

variation in plant height was not affected by the 

relative abundance of C. achates (P=0.59). 

Neither stem density nor plant density across 

years showed a significant relationship with the 

relative abundance of weevils at sites (P>0.4). 

Both stem and plant density declined 

significantly between 2002 and 2004 (P<0.03), 

but the degree of decline was not significantly

influenced by the relative abundance of C. 

achates (P>0.5). 

DISCUSSION 

This study showed that Cyphocleonus achates

can establish across a wide variety of sites if 

given time. C. achates also appears to have 

generally increased in numbers at most release 

sites since its introduction. In the 1997/2002 

dataset, C. achates increased by nearly four-fold 

over the study period, as measured by larval 

densities within knapweed roots. There was also 
6

some evidence of an increase in C. achates in the 

2002-2004 dataset, as measured by sweepnetting adult weevils. Although weevil counts 

were low in 2004, this was likely due to the 

prevalence of cool, rainy conditions during the 

sampling period which likely caused C. achates 

to be inactive (Sturdevant and Dewey 2003). In 

contrast, sampling during more normal 

conditions in 2002 and 2003 indicated that 

weevils had roughly doubled between years. 

Our observations and analysis of the 1997/2002 

dataset indicated that establishment of C. achates

tended to be associated with sandy soils and 

valley bottom sites, but not with elevation, 

aspect, slope, disturbance level, or cover of bare 

ground, shrubs, or other non-knapweed 

vegetation. In their study of the same general set 

of monitoring sites that we examined, Clark et al. 

(2001b) reported that C. achates establishment 

was most commonly associated with midelevations and found no relationship with soil 

conditions. However, their analyses were based 

on data collected in 1997-1998 only, i.e., early in 

the study period. In the current study, C. achates

was detected at only 19 of the 48 (40%) release 

sites in 1997 compared to 36 of the 48 (75%) 

release sites in 2002. Also, 4 of the 19 sites with 

evidence of establishment in 1997 showed no 

evidence of weevils in 2002. Given this 

substantial difference in the subset of sites where 

C. achates appeared to have established in 1997 

versus 2002, it is not surprising that Clark et al.’s 

(2001b) results differed from ours. 

Several measures of spotted knapweed viability 

suggested that knapweed declined from 1997 to 

2002 and from 2002 to 2004. Percent cover of 

knapweed and knapweed stem density showed 

significant reductions from 1997 to 2002, and 

stem densities and plant densities declined 

significantly from 2002 to 2004. Knapweed 

cover from 1997 to 2002 appeared to decline 

more markedly at sites where C. achates was 

present (Fig. 1), but this difference was not 

statistically significant. Knapweed cover was 

assessed at a relatively broad spatial scale (1/300 

acre), which may have resulted in less precise 

estimates, making resolution of patterns more 

difficult. However, in contrast to apparent trends 

in knapweed cover, declines in stem density over 

the study period were strong whether or not C. 

achates was present at study sites. Corn et al. 

(2006) found no difference between number of 

stems and seedheads between treatments with 

high versus low numbers of weevils. Other 

studies have shown that number of stems is not a 

good indicator of knapweed vigor. Finally, we 

found no significant relationships between 

measures of C. achates abundance and knapweed 

declines in either dataset. 

We also examined the potential effects of 

Agapeta zoegana, another agent for which data 

were collected, on knapweed decline. The 

analysis showed no significant association with 

knapweed decline. The other most prominent 

biological control agents at the sites would be the 

ubiquitous gall flies (Story et al. 1987). 

However, several intensive studies have 

indicated that these agents do not reduce spotted 

knapweed populations (e.g., Maddox 1982, 

Stanley 2005). The seedhead weevils, Larinus

species, would be the only other biological 

control agents likely to occur in any numbers at 

multiple sites, but even now they are not well 

distributed. For example, Larinus was only 

present at 23% of the C. achates release sites in 

the 1997/2002 dataset. 

From these data, it appears that spotted 

knapweed has declined over the monitoring 

period and that a combination of factors may 

have influenced this decline. Numerous authors 

across the region have recently reported declines 

in spotted knapweed abundance corresponding 

with spring drought conditions occurring in the 

early 2000s (Ortega et al. 2004, Stanley 2005, 

Pearson and Callaway 2006, and historical 

studies have also reported declines in spotted 

knapweed in association with past droughts 

occurring prior to the introduction and 

establishment of most knapweed biological 

control agents (e.g., Schirman 1981). 
7

In light of these conclusions, it is important to 

note the limitations of these monitoring data for 

evaluating cause and effect relationships. For 

example, to be able to separate the effects of C. 

achates establishment from other factors 

affecting knapweed, control sites lacking weevils 

are needed to quantify background trends in 

knapweed populations. In our analysis of the 

1997/2002 dataset, control sites were limited to 

those sites where C. achates had failed to 

establish. This resulted in a smaller sample than 

would be ideal given what appeared to be a 

confounding effect of other factors such as 

drought. Furthermore, measures of relative 

abundance derived from sweep netting (as in the 

case of the 2002-2004 dataset), were sensitive to 

weather conditions, and therefore may not have 

adequately captured trends in C. achates density. 

Future studies should rely on larval monitoring 

in roots for providing a consistent population 

measurement. 

We found that C. achates has increased at 

monitoring sites since it was introduced and that 

spotted knapweed has declined during this same 

period. Trends found in this study showed that 

there was some evidence that C. achates may 

have impacted spotted knapweed cover, as 

discussed above. Story et al. (2006) showed that 

spotted knapweed density declined significantly 

over an eleven-year period at two sites after C. 

achates increased dramatically at both sites. 

Although the trend in our study was not 

statistically significant, it is possible that C. 

achates was beginning to weakly impact spotted 

knapweed, since it was released for a relatively 

short period of time prior to the start of this 

study. Also, our study included sites where 

weevil numbers ranged from low to high. Corn 

et al. (2006) found that plants may survive initial 

attacks by large numbers of weevils, but may be 

stressed and continue to die over time. They also 

found that knapweed mortality occurred only 

when weevils were found in high numbers 

and/or over multiple years of attack. Our study 

showed a decline in plant density over time in 

the 2002/2004 data set. All of the sites in this 

data set had moderate to high numbers of 

weevils. 

In summary, C. achates established at most sites 

(75%) over five years and establishment was 

highest at sites with sandy soils and located on 

valley bottoms. Knapweed density decreased 

across sites, but decreases were not significantly 

correlated with C. achates. However, the effects 

of the biological control agents may have been 

overshadowed by the effects of the recent 

drought which probably also played a role in the 

decrease in knapweed plant density. 
8

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