J. Appl. Entomol. Haemolymph defence of an invasive herbivore: its breadth of effectiveness against predators J. G. Lundgren1, S. Toepfer2,3, T. Haye2 & U. Kuhlmann2 1 USDA-ARS, North Central Agricultural Research Laboratory, Brookings, SD, USA 2 CABI Europe-Switzerland, Delemont, Switzerland 3 CABI Europe, c/o Plant Health Service, CABI Europe, Hodmezovasarhely, Hungary Keywords Abstract Tetramorium caespitum, Zea mays, biological control, Carabidae, diel cycle, Lycosidae Defensive characteristics of organisms affect the trophic linkages within food webs and influence the ability of invasive species to expand their Correspondence range. Diabrotica v. virgifera is one such invasive herbivore whose preda- Jonathan Lundgren (corresponding author), tor community is restricted by a larval haemolymph defence. The effec- NCARL, USDA-ARS, 2923 Medary Avenue, tiveness of this haemolymph defence against a range of predator Brookings, SD, USA. E-mail: functional and taxonomic guilds from the recipient biota was evaluated [email protected] in a series of experiments. Eight predator species (Carabidae, Lycosidae, Received: August 5, 2009; accepted: October Formicidae) were fed D. v. virgifera 3rd instars or equivalent-sized mag- 28, 2009. gots in the laboratory, and the mean times spent eating, cleaning their mouthparts, resting and walking following attacks on each prey were doi: 10.1111/j.1439-0418.2009.01478.x compared. Prey species were restrained in five Hungarian maize fields for 1 h periods beginning at 09:00 and 22:00 hours. The proportion of each species attacked and the number and identity of predators consum- ing each prey item were recorded. All predators spent less time eating D. v. virgifera larvae than maggots in the laboratory, and four of the eight predator species spent more time cleaning their mouthparts. The differ- ential responses in the predator species indicate differences in suscepti- bility to the D. v. virgifera haemolymph defence. The predator communities (numerically dominated by Tetramorium caespitum) in the field showed clear diel patterns in their foraging behaviour, and D. v. vir- gifera was consumed by fewer predators than maggots. The defence of D. v. virgifera may partly explain how invasive insects that are exposed to an extensive predator community overcome biotic resistance to the invasion process. invader depends in part on its defensive capabilities Introduction against predation. But, predator diversity within Invasive insects have profound and lasting effects on even the most disturbed subterranean arthropod the food webs of recipient habitats, and how food communities is significant (Duelli and Obrist 1998; webs respond to the introduction of an exotic species Downie et al. 1999; Duelli et al. 1999; Lundgren has important implications for the ultimate establish- et al. 2006, 2009b; Juen and Traugott 2007), and ment and integration of these invasives. One way predators rely differentially on a given herbivore that resident food webs confront invasion by exotic within these systems. Thus, a key aspect of the inva- species is through biotic resistance, in part through sion process is how predator constituents of a food predation of the invader (Parker et al. 2006; Nunez web are relatively affected by the defensive capabili- et al. 2008; Harwood et al. 2009). The success of an ties of an invasive pest. J. Appl. Entomol. 134 (2010) 439–448 ª 2009 Blackwell Verlag, GmbH 439 Anti-predator defence of rootworms J. G. Lundgren et al. Haemolymph-based defence (Happ and Eisner former are defended from predation by their hae- 1961; de Jong et al. 1991; Peck 2000; Boeve´ and molymph. Thus, the current research is a first step Schaffner 2003; Mu¨ ller and Arand 2007; Vleiger in addressing how the D. v. virgifera haemolymph et al. 2007) in an invasive insect is one physiological defences affect the strength of trophic interactions factor that influences the biotic resistance to inva- that this pest has with its predator community sion, and ultimately the trophic connectedness within maize agroecosystems. within even endeamic food webs. These haemol- ymph defences can act against predators by making Methods the prey species sticky, toxic, or distasteful (Pasteels et al. 1983; Bowers 1992; Laurent et al. 2005). Breadth of predators affected by D. v. virgifera Because of the intense predation pressure experi- haemolymph defence enced in soil-based food webs (Lundgren et al. 2006) and reduced mobility of immature stages of herbivo- Eight species of field-collected predators commonly rous insects (relative to the adult stage), there is found in European cornfields were selected; these often a disproportionate number of subterranean, predators represent a range of different feeding ecol- herbivorous insects that possess chemical defences ogies and natural histories present in the habitats of against predation (Pasteels et al. 1983). One group of D. v. virgifera larvae. Chewing predators with differ- insects that is particularly well known for both their ent dietary specializations included Carabus monilis herbivorous lifestyle and their chemical haemol- Fabricius (Carabidae), Harpalus pensylvanicus (DeG- ymph-based defences are beetles in the Chrysomeli- eer) (Carabidae), Harpalus rufipes (DeGeer) (Carabi- dae (Pasteels et al. 1982, 2004; Dettner 1987; Hilker dae), Poecilus cupreus (L.) (Carabidae), Pterostichus et al. 1992; Laurent et al. 2005). The intricacies of anthracinus (Panzer) (Carabidae) and Pterostichus mel- how these anti-predator defences influence biotic anarius (Illiger) (Carabidae). Sucking predators were resistance to invasion by exotic insects remains to be represented by Trochosa ruricola (DeGeer) (Aranae: well established. Lycosidae). Ant workers were classified as a separate As an invasive pest of international importance, feeding category (described below). Predators were Diabrotica virgifera virgifera LeConte (western corn captured from maize fields in northwestern Switzer- rootworm; Coleoptera: Chrysomelidae) shapes the land using dry pitfall traps (except H. pensylvanicus, maize ecosystem through its consumption of maize which was collected from farmland in Brookings, root systems. Recent research has revealed that pred- SD, USA). Following collection, they were main- ator taxonomic and feeding guilds consume D. v. vir- tained in the laboratory in dampened field soil and gifera immatures to varying degrees under field fed moistened cat food (Iam’s Original with Chicken, conditions (Lundgren et al. 2009c; Toepfer et al. The Iam’s Company, Cincinatti, OH, USA). 2009). In part, predation intensity is driven by the Each of these predators was fed a D. v. virgifera 3rd spatio-temporal occurrences of D. virgifera and mem- instar or an equivalent-sized (based on mass) surro- bers of its predator community (Lundgren et al. gate prey item (Calliphora vicina L. maggot; Diptera: 2009b,c). But a recent discovery has shown that Calliphoridae), and their feeding behaviours were D. v. virgifera larvae possess predator-repellent prop- monitored. Pterostichus anthracinus was fed 2nd instar erties in their haemolymph (Lundgren et al. 2009a) D. v. virgifera and equivalent-sized maggots, as 3rd which are unrelated to the previously identified cu- instars were too large for this predator to consis- curbitacin-based defences discussed by Tallamy et al. tently attack. Harpalus pensylvanicus received maggots (2005). The larval defence of D. v. virgifera is based of Sarcophaga bullata Parker (Diptera: Sarcophagidae), on offensive chemistry and the haemolymph’s ten- a native North American fly of similar larval biology dency for rapid coagulation on predator mouthparts to the European C. vicina. These maggots were deter- (Lundgren et al. 2009a). Here, in addition to describ- mined to be a suitable prey for a wide range of pre- ing the diverse predator community present during dators, and functioned as a control to observe the day and night in Hungarian maize fields, specific predator behaviour under ideal circumstances. experiments were designed to (i) investigate whether Twenty-four hours prior to the assay, predators were predators are differentially affected by the haemol- isolated into empty 100 mm diameter Petri dishes, ymph defence of D. v. virgifera in the laboratory, and where they were starved with only water to ensure (ii) substantiate that D. v. virgifera are attacked less hunger at the time of the assay (C. monilis was intensively than maggots of C. vicina of equal size in starved for 48 h, due to their large size and ability to Central European maize fields, in part because the withstand starvation). At the time of the assay, a 440 J. Appl. Entomol. 134 (2010) 439–448 ª 2009 Blackwell Verlag, GmbH J. G. Lundgren et al. Anti-predator defence of rootworms predator was placed into a clean and empty Petri plastic tub on sugar water and moistened cat food dish that contained either a 3rd instar D. v. virgifera, for several weeks. For the assay, 15 worker ants or a maggot of equivalent size. Each predator was were placed into a 100 mm diameter plastic Petri observed for 10 min or until it attacked the prey dish containing either a 3rd instar D. v. virgifera or item. The amounts of time devoted to eating, clean- an equivalent-sized C. vicina maggot. The arenas ing of mouthparts, resting, and walking were were watched constantly for 10 min, and the recorded for 2 min following the initial predation maximum number of ants attacking the prey item event. Cleaning of mouthparts was defined as wiping was recorded during each minute of observation. the mouthparts with the palps or legs to clear the A total of 15 assays were conducted for each oral cavity of congealed haemolymph, or wiping of treatment. the mouthparts on the dish. Observations were ceased after this period, because this was the mini- Larval defence of D. v. virgifera under field conditions mum duration it took to entirely consume the mag- got prey (D. v. virgifera were seldom entirely Predation rates on sentinel D. v. virgifera larvae and consumed). An exception to this is that the amount C. vicina maggots were measured in five convention- of time spent eating the D. v.