Introduction
Canada and the United States of America are currently working to combat invasions of two exotic pests, one arthropod and one weed, the control measures for which are linked. Cabbage seedpod weevil, Ceutorhynchus obstrictus (Marsham) (Coleoptera: Curculionidae: Ceutorhynchinae), is a pest of canola (oilseed rape), Brassica napus L. (Brassicaceae), in Europe and was accidentally introduced to North America where it is now widespread (Kuhlmann et al. Reference Kuhlmann, Dosdall, Mason, Mason and Huber2002). European hoary cress, Cardaria draba (L.) Desv. (Brassicaceae), is a widespread introduced weed in North America that is often found in habitats surrounding canola fields (Schwarzlaender et al. Reference Schwarzlaender, McKenney and Cripps2002; Fox and Dosdall Reference Fox and Dosdall2003; Cripps et al. Reference Cripps, Hinz, McKenney, Harmon, Merickel and Schwarzlaender2006). Ceutorhynchus obstrictus and hoary cress are currently controlled with chemical pesticides (Dosdall et al. Reference Dosdall, Moisey, Cárcamo and Dunn2001; Cárcamo et al. Reference Cárcamo, Dosdall, Johnson and Olfert2005; Anonymous 2011). Alternative control strategies are needed to reduce pesticide use and overcome difficulties in current management strategies.
Classical biological control for invasive alien species is cost-effective, permanent, self-sustaining, and ecologically safe when host-specific agents are established (Wittenberg and Cock Reference Wittenberg and Cock2001). In Europe, C. obstrictus populations are suppressed by natural enemies, including the ectoparasitoids Trichomalus perfectus (Walker) and Mesopolobus morys (Walker) (Hymenoptera: Chalcidoidea: Pteromalidae), which may reduce populations of C. obstrictus by as much as 90% (Williams Reference Williams and Alford2003). Both species were released in British Columbia in the 1950s but failed to establish (Gillespie et al. Reference Gillespie, Mason, Dosdall, Bouchard and Gibson2006), and they are being re-evaluated for introduction to Canada (Kuhlmann et al. Reference Kuhlmann, Dosdall, Mason, Mason and Huber2002, Reference Kuhlmann, Mason, Hinz, Blossey, De Clerck-Floate and Dosdall2006).
Ceutorhynchus turbatus Schultze is widespread in natural habitats in Europe. Because it undergoes larval development within hoary cress siliques, it is being studied as a hoary cress biological control agent for potential introduction to North America (Cripps et al. Reference Cripps, Hinz, McKenney, Harmon, Merickel and Schwarzlaender2006).
Hoary cress is a reservoir for various economically important pests in North America, including C. obstrictus (food source for adults) (Fox and Dosdall Reference Fox and Dosdall2003; Dosdall and Moisey Reference Dosdall and Moisey2004; Cripps et al. Reference Cripps, Hinz, McKenney, Harmon, Merickel and Schwarzlaender2006). Reducing populations of hoary cress should benefit C. obstrictus control by decreasing the availability of one of its food plants, especially in spring, when adults emerge from overwintering sites, until canola crops become suitable for adult feeding and larval development (Dosdall and Moisey Reference Dosdall and Moisey2004). However, introduction of T. perfectus and (or) M. morys to control C. obstrictus could affect control of hoary cress if lack of host specificity enables the parasitoids to attack C. turbatus. Another related nontarget seed-feeding species, Ceutorhynchus typhae (Herbst), could also be adversely affected. It is widespread in eastern Canada in Ontario, Quebec, New Brunswick, Nova Scotia, and Newfoundland (Bousquet Reference Bousquet1991; Bouchard et al. Reference Bouchard, Lesage, Goulet, Bostanian, Vincent, Zmudzinska and Lasnier2005) and is associated with shepherd's purse, Capsella bursa-pastoris (L.) Medik. (Brassicaceae), an economically important weed in canola and other crops in North America (Moss Reference Moss1959; Budd and Best Reference Budd and Best1969). Consequently, these potential interactions must be considered when predicting nontarget effects of biological control agents for C. obstrictus.
Assessment of the potential impacts of C. obstrictus parasitoids on C. turbatus and C. typhae requires surveys to determine parasitoid assemblages associated with the hosts in their natural ranges. Such studies can also provide crucial host ecology information for use in selecting nontarget species for host-specificity testing and improving prediction of potential risks associated with introduction of biocontrol agents. Our objectives were to study C. typhae and C. turbatus to determine the (i) phenologies of the species (and potential overlap with phenology of C. obstrictus), (ii) levels of parasitism of their larvae, and (iii) parasitoids associated with these two weevils in their native ranges in Europe.
Materials and methods
Field collections of insects and plants (Table 1)
Shepherd's purse and hoary cress plants were field collected in fallow fields, at field margins, or along road sides at various European localities from May to July 2003 and 2004. Surveys for C. typhae were made at seven sites in Germany, Switzerland, and France, where C. obstrictus is common. Ceutorhynchus turbatus specimens were collected at 14 sites in Switzerland and in two regions of Hungary where populations being evaluated for introduction to North America had been collected previously (Gassmann et al. Reference Gassmann, Hinz, de Meij, Heinlein, Spiewak and Yaworski2001). Insect collections were made in wastelands or abandoned fields because the weevils are generally more abundant in those habitats than they are in recently disturbed sites.
* Collections made in 2003.
† Collections made in 2004.
Samples of 10–20 hoary cress or shepherd's purse plants were collected from each site on each sampling date with 2- to 10-day intervals between collections. Larvae of C. typhae and C. turbatus are morphologically indistinguishable but are specialists found in pods of shepherd's purse (Baur et al. Reference Baur, Muller, Gibson, Mason and Kuhlmann2007) and hoary cress (Cripps et al. Reference Cripps, Hinz, McKenney, Harmon, Merickel and Schwarzlaender2006), respectively; thus, host-plant association was used to identify larvae of each species. Twenty pods per plant were dissected to detect weevil eggs and larvae; larvae were then determined to instar. Mature third instars of both species leave seed pods to pupate in soil. Thus, pod exit hole counts were also used for estimating numbers of third-instar larvae.
Estimates of parasitism level
For each plant dissected, parasitoid eggs and larvae found on or near host larvae and pupae found near host bodies were recorded to estimate parasitism levels on each collection date for every field site. Mortality due to host feeding by parasitoid adults (as evidenced, e.g., by brownish punctures on larval integument) was recorded separately as an indirect effect of parasitism. Parasitism was calculated for each site as follows:
where Σn1 paras. and Σn1 avail.hosts are the total numbers within a plant of parasitoids and of hosts (second- and third-instar larvae) available for parasitism, respectively; Σlarvpar and Σlarv† are the numbers of parasitized larvae and of dead larvae with traces of host feeding, respectively; Σparalone is the number of parasitoids (pupae and newly eclosed adults) found alone near dead hosts; Σlarvhealthy is the number of healthy host larvae; and Σholes is the number of exit holes found on shoots or pods during dissections.
A representative parasitism level was estimated for each site on the date when the maximum number of larvae still available for parasitism was present in the plant (i.e., just before exit holes appeared in the pods). Host density was determined by considering the maximum number of hosts parasitized or available for parasitism during the season. Only second- and third-instar larvae were used for calculations. At high larval infestation levels, a maximum of two larvae can develop within the same pod, but they are separated from each other by a partition (replum). Occasionally, two third-instar larvae may exit a plant through the same hole; this may have led to a slight under-estimation of larval numbers.
Parasitoids
Parasitoids were reared on filter paper discs in 5.5 cm diameter Petri dishes in a controlled-environment chamber at 20 ± 2°C, 70% ± 10% RH, and 16L:8D and were checked daily until emergence of adults. Parasitoid eggs and larvae were reared on the host larvae upon which they were collected; pupae were reared individually. Adults were killed with ethyl acetate, card-mounted, and labelled. Voucher specimens were deposited in the Natural History Museum of Bern, Switzerland; the Canadian National Collection of Insects, Ottawa, Canada; the National Museum of Natural History, Washington D.C., United States of America; and at CABI Europe-Switzerland, Delémont, Switzerland.
Results
Ceutorhynchus typhae on shepherd's purse
Phenological data for C. typhae are presented for three study sites for 2003 (Fig. 1). Some phenological characteristics of C. typhae varied between sites and years, apparently in relation to the developmental biology of shepherd's purse. Shepherd's purse flowered throughout the season, so fresh pods were available until late summer (Muller Reference Muller2006). Nevertheless, some weevil phenological trends were consistent across sites and years. For instance, eggs of C. typhae were laid from May to the end of June, but egg numbers declined from early to late in the sampling period as a proportion of all life stages observed. Eggs typically comprised more than 70% of all life stages in May to early June, but declined to less than 10% by the end of June. First-instar larvae were present in pods of shepherd's purse on every sampling date from mid-May onwards; their proportion of all life stages present usually did not decline over time. Numbers of second and third instars increased beginning in the third week of May, and densities ranged from 0.15 ± 0.08 larvae per plant in the southern Rhine Valley of Germany (site 4) to 4.60 ± 0.29 in the Swiss Jura near Alle (site 5) (Table 2). Third-instar larvae were first found in early to mid-June, and mature larvae began to exit plants beginning in the second week of June (Fig. 1).
* Collections made in 2003.
† Collections made in 2004.
Parasitoids associated with Ceutorhynchus typhae
Parasitoids were observed within siliques of shepherd's purse for about 3 weeks beginning in the first week of June (Fig. 1). Parasitism increased over time at most (e.g., sites 1, 5), but not all (e.g., site 7) sites (Fig. 1). In Germany, parasitism was highest at site 2 near Neuenburg in the southern Rhine Valley (Table 2). In Switzerland, parasitism reached a maximum of 59.5% at site 5 near Alle, in the Swiss Jura on 21 June 2003, and parasitism at the site in France was 16.7%.
Thirty-eight specimens of Mesopolobus gemellus Baur and Muller (37) and Stenomalina gracilis (Walker) (1) (Hymenoptera: Pteromalidae) were reared in association with C. typhae. Mesopolobus gemellus oviposited on late-instar larvae of C. typhae in May and June. Adults of the new generation emerged and left the senesced pods in late June of the same year.
Ceutorhynchus turbatus on hoary cress
Phenological data for C. turbatus are presented for two study sites for 2004 (Fig. 2). As for C. typhae, phenological details varied somewhat across sites and years, but some observations were consistent. Eggs of C. turbatus typically comprised greater than 90% of all life stages from early to late May. First-instar larvae were first observed from the last week of May to the first week of June; second-instar larvae were present beginning in the second week of June. Densities of second and third instars per plant in the Swiss Valais ranged from 0.75 ± 0.23 at Aproz (site 11) to 11.25 ± 0.64 at Sion (site 14) (Table 3). In Hungary, densities tended to be lower on average at all sites surveyed, with a maximum of 7.50 ± 0.70 second- and third-instar larvae per plant observed at site 15 near Hödmezövasarhely and a minimum of no larvae at another site near the same town (site 16) (Table 3). Mean densities of second and third instars per plant in 2003 and 2004 were 6.8 ± 1.7 in Switzerland and 2.1 ± 1.6 in Hungary.
* Collections made in 2003.
† Collections made in 2004.
Parasitoids associated with Ceutorhynchus turbatus
Parasitoids were present earlier in the season in Hungary than in Switzerland at all sites surveyed. In 2004, parasitism was first evident in the second week of June in Hungary, as soon as second-instar larvae were observed; in the Swiss Valais, parasitism was first evident in the last week of June (Fig. 2). All larvae had exited plants by 24 June 2004 in Hungary at site 20 near Gyal; at the same time in Switzerland, all larval instars were found in plants. Parasitism levels in Switzerland ranged from 15.4% to 80.0% in 2003, and 18.2% to 44.0% in 2004. Similar parasitism levels (11.1%–41.8%) were observed in Hungary, except for site 18 near Hödmezövasarhely, where no parasitoids were found.
Ten chalcidoid species (Table 4) were associated with C. turbatus collected from hoary cress: Eurytoma curculionum Mayr (Eurytomidae), Mesopolobus morys (Walker), Pteromalus sequester Walker, and Trichomalus cf. perfectus (Walker) (Pteromalidae), as well as an unidentified species of each of Baryscapus Förster and Closterocerus Westwood (Eulophidae), Eupelmus Dalman (Eupelmidae), Pteromalus Swederus, Syntomopus Walker, and Trichomalus Thomson (Pteromalidae). Parasitoid species compositions varied considerably among sites, with M. morys found at all sites except site 16 in Hungary (Table 4). None of the parasitoids of C. turbatus were confirmed as T. perfectus.
* Chalcidoidea: Pteromalidae.
† Chalcidoidea: Eulophidae.
‡ Chalcidoidea: Eurytomidae.
§ Chalcidoidea: Eupelmidae.
Discussion
Distributions of the three weevil species, C. obstrictus, C. typhae, and C. turbatus, are sympatric in Europe (Bonnemaison Reference Bonnemaison1957; Cripps et al. Reference Cripps, Hinz, McKenney, Harmon, Merickel and Schwarzlaender2006), and our observations indicate that some overlap also occurs in their phenological development. For instance, females of C. turbatus and C. typhae oviposit in pods of their host plants during May and June. Larvae develop through three instars and, when mature, each chews a hole in its pod wall, drops to the ground, and pupates in the soil. Weevils of the new generation emerge during June and July of the same year. Both species overwinter as adults and resume activity the following spring. Larvae of C. obstrictus also develop from May through July (Bonnemaison Reference Bonnemaison1957; Dosdall and Moisey Reference Dosdall and Moisey2004), confirming that their pre-imaginal development coincides temporally with that of larvae of C. turbatus and C. typhae in the field.
Host-plant phenologies also overlap in Europe and North America. On both continents, shepherd's purse can overwinter as a seedling or a seed, and flowering occurs from March to June or July (Defelice Reference Defelice2001; Iannetta et al. Reference Iannetta, Begg, Hawes, Young, Russell and Squire2007). Hoary cress flowers from March to July in Europe and North America (Mulligan and Findlay Reference Mulligan and Findlay1974; Larina Reference Larina2003), a period overlapping with the range of flowering time of canola on the two continents (Lefol et al. Reference Lefol, Danielou and Darmency1996; Thomas Reference Thomas2002). As a consequence of this phenological overlap, C. turbatus and C. typhae could be attacked by a nonhost-specific parasitoid introduced for biological control of C. obstrictus.
The phenologies of larval parasitoids collected from C. turbatus and C. typhae were closely linked with host phenologies. All parasitoids encountered during our surveys oviposited on late-instar host larvae. Parasitoid larvae fed on their hosts for 2–4 weeks and pupated within the siliques. Pupation required 1–2 weeks, and adults of the new parasitoid generation emerged through openings in the senesced pods. This development occurred primarily from early to late June (on C. typhae) and mid-June to early July (on C. turbatus) (Figs. 1, 2), a period that coincides with the development of T. perfectus and M. morys on C. obstrictus (Williams Reference Williams and Alford2003; Baur et al. Reference Baur, Muller, Gibson, Mason and Kuhlmann2007; Muller et al. Reference Muller, Baur, Gibson, Mason and Kuhlmann2007).
The parasitoid assemblage associated with C. typhae was considerably less diverse than that of C. turbatus. Mesopolobus gemellus was responsible for 98% of C. typhae parasitism at all sites studied, and only a single specimen of another parasitoid, S. gracilis, was reared from this host. On average, approximately 25% of all C. typhae hosts were parasitized. Because all C. typhae life stages were present when sampling was terminated (Fig. 1), it is possible that additional parasitoid species that attack later in the season were missed in our study. Mesopolobus gemellus appears to be host specific in C. typhae: Baur et al. (Reference Baur, Muller, Gibson, Mason and Kuhlmann2007) reared many specimens from C. typhae but only a single female (of questionable identity) from C. turbatus. By contrast, S. gracilis has a broad host range, reported from approximately 25 host species including C. obstrictus and several other ceutorhynchine weevils (Dosdall et al. Reference Dosdall, Gibson, Olfert and Mason2009).
In contrast to C. typhae on shepherd's purse, C. turbatus on hoary cress had a more diverse parasitoid assemblage with 10 species recovered. Mesopolobus morys, a candidate agent for C. obstrictus biological control, was the most common parasitoid of C. turbatus and accounted for approximately 40% of total C. turbatus parasitism. This species was also widespread, occurring at almost all sites in Switzerland and Hungary where C. turbatus was collected on hoary cress. Pteromalus sequester, the second most important larval parasitoid attacking C. turbatus, represented 37% of the parasitoid assemblage and was also present at nearly all sites studied. Our rearing of Eurytoma curculionum, previously associated with C. obstrictus (Dmoch Reference Dmoch1975), is the first record of its association with C. turbatus on hoary cress. Interestingly, although this parasitoid is known to occur in France (Thompson Reference Thompson1955; Herting Reference Herting1973) and Germany (Freese Reference Freese1995; Vidal Reference Vidal1997), we only observed it on C. turbatus in Hungary. Several other parasitoid species (e.g., Closterocerus sp., Baryscapus sp., Trichomalus sp., Pteromalus sp., and Syntomopus sp.) were also encountered only in Hungary during our surveys and species of these genera have not previously been associated with C. turbatus. Two specimens of Trichomalus cf. perfectus were collected, together representing less than 1% of all parasitoids obtained during our surveys.
Concerns regarding nontarget effects in arthropod biological control are well documented (Howarth Reference Howarth1991; Simberloff and Stiling Reference Simberloff and Stiling1996; Thomas and Willis Reference Thomas and Willis1998; Stiling and Simberloff Reference Stiling, Simberloff, Follet and Duan.2000; Louda et al. Reference Louda, Pemberton, Johnson and Follett2003; Stiling Reference Stiling2004). Consequently, new methods to assess the risks associated with the introduction of non-native species for biological control of arthropods have been proposed (Babendreier et al. Reference Babendreier, Bigler and Kuhlmann2005; Simberloff Reference Simberloff2005; Wright et al. Reference Wright, Hoffmann, Kuhar, Gardner and Pitcher2005; Kuhlmann et al. Reference Kuhlmann, Mason, Hinz, Blossey, De Clerck-Floate and Dosdall2006). It is clear that the pre-imaginal development of ceutorhynchine weevils that comprise pest (C. obstrictus), adventive (C. typhae), and potential weed biocontrol agents (C. turbatus) overlap, and increased risk to the nontarget species is associated with release of biological agents for control of C. obstrictus unless the agents are host specific. Mesopolobus morys, an important parasitoid of C. obstrictus (Reference Williams and AlfordWilliams 2003), was the most common species parasitizing C. turbatus in Switzerland and Hungary. In the event of separate introductions of M. morys and C. turbatus as biocontrol agents against C. obstrictus and hoary cress, respectively, potential conflicts might occur between the two programmes. In contrast, because T. perfectus appears to be host specific and does not attack C. turbatus or C. typhae, this species should pose minimal risk if it is introduced in Canada for biological control of C. obstrictus. Therefore, to avoid conflicts with weed biological control our results suggest that further evaluation of T. perfectus for classical biological control of C. obstrictus would be appropriate.
Acknowledgments
We thank E. Cuenot, V. Larraz, L. Lovis, G. Nagy, F. Janos, A. Leroux, and L. Andreassen for technical assistance in the field and laboratory. We thank H. Hinz and S. Toepfer for helpful advice and information on collection sites of C. draba in various European countries. Hannes Baur (Natural History Museum, Bern, Switzerland) and Gary Gibson (Canadian National Collection, Ottawa, Ontario) provided pteromalid identifications, Michael Gates (United States Department of Agriculture, Agriculture Research Service, National Museum of Natural History, Washington D.C.) identified the eurytomids. This work was funded by Agriculture and Agri-Food Canada, its Pest Management Research Centre Project PRR03-370, and the Alberta Agricultural Research Institute.
