Melanie F Kho

Audrey Bellier

Venkatasamy Balasubramani

Yan Hu

Wayne Hsu

Christina Nielsen-Leroux

Shauna M Mcgillivray

Victor Nizet

Raffi V Aroian
email: raroian@ucsd.edu

The Pore-Forming Protein Cry5B Elicits the Pathogenicity of Bacillus sp. against Caenorhabditis elegans

The soil bacterium Bacillus thuringiensis is a pathogen of insects and nematodes and is very closely related to, if not the same species as, Bacillus cereus and Bacillus anthracis. The defining characteristic of B. thuringiensis that sets it apart from B. cereus and B. anthracis is the production of crystal (Cry) proteins, which are pore-forming toxins or pore-forming proteins (PFPs). Although it is known that PFPs are important virulence factors since their elimination results in reduced virulence of many pathogenic bacteria, the functions by which PFPs promote virulence are incompletely understood. Here we study the effect of Cry proteins in B. thuringiensis pathogenesis of the nematode Caenorhabditis elegans. We find that whereas B. thuringiensis on its own is not able to infect C. elegans, the addition of the PFP Cry protein, Cry5B, results in a robust lethal infection that consumes the nematode host in 1-2 days, leading to a ''Bob'' or bag-of-bacteria phenotype. Unlike other infections of C. elegans characterized to date, the infection by B. thuringiensis shows dose-dependency based on bacterial inoculum size and based on PFP concentration. Although the infection process takes 1-2 days, the PFP-instigated infection process is irreversibly established within 15 minutes of initial exposure. Remarkably, treatment of C. elegans with Cry5B PFP is able to instigate many other Bacillus species, including B. anthracis and even ''non-pathogenic'' Bacillus subtilis, to become lethal and infectious agents to C. elegans. Co-culturing of Cry5B-expressing B. thuringiensis with B. anthracis can result in lethal infection of C. elegans by B. anthracis. Our data demonstrate that one potential property of PFPs is to sensitize the host to bacterial infection and further that C. elegans and probably other roundworms can be common hosts for B. cereusgroup bacteria, findings with important ecological and research implications.

Introduction

The Bacillus cereus group of bacteria comprises six species, including three highly related species, B. cereus sensu stricto, B. anthracis and B. thuringiensis, which are sometimes considered a single species [START_REF] Helgason | Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis-one species on the basis of genetic evidence[END_REF][START_REF] Kolsto | What sets Bacillus anthracis apart from other Bacillus species?[END_REF][START_REF] Rasko | Genomics of the Bacillus cereus group of organisms[END_REF]. Although closely related, these bacteria are associated with very different diseases. B. anthracis is the causative agent of anthrax [4], B. cereus can cause food poisoning and various opportunistic and nosocomial infections [START_REF] Drobniewski | Bacillus cereus and related species[END_REF], and B. thuringiensis is an invertebrate-specific pathogen [START_REF] Schnepf | Bacillus thuringiensis and its pesticidal crystal proteins[END_REF]. The relationship between these three closely related species of Bacillus in the wild, e.g. how they replicate in the soil environment and what environmental niches they occupy relative to one another, has been the subject of much speculation [START_REF] Jensen | The hidden lifestyles of Bacillus cereus and relatives[END_REF][START_REF] Vilas-Boas | Biology and taxonomy of Bacillus cereus, Bacillus anthracis, and Bacillus thuringiensis[END_REF].

The defining characteristic of B. thuringiensis is the production of Crystal (Cry) proteins, a large family of related proteins that kill insects and nematodes [START_REF] Vilas-Boas | Biology and taxonomy of Bacillus cereus, Bacillus anthracis, and Bacillus thuringiensis[END_REF][START_REF] Griffitts | Many roads to resistance: how invertebrates adapt to Bt toxins[END_REF][START_REF] De Maagd | How Bacillus thuringiensis has evolved specific toxins to colonize the insect world[END_REF]. Each B. thuringiensis strain found in the wild can produce one or multiple (typically 2-4) Cry proteins. These Cry proteins are pore-forming proteins (PFPs), and thus are members of the single largest class of bacterial virulence factors [START_REF] Alouf | Molecular features of the cytolytic pore-forming bacterial protein toxins[END_REF]. Indeed, roughly 25-30% of all protein toxins made by human bacterial pathogens are PFPs, many with proven roles in disease pathogenesis. Purified Cry proteins alone, even in the absence of B. thuringiensis, produce dose-dependent lethality in invertebrates, and as such can be expressed in transgenic crops to control insect pests.

Here for the first time we characterize in detail the effect of Cry protein PFP administration on the outcome of B. thuringiensis infection in the nematode Caenorhabditis elegans, and uncover an unexpected interaction of Cry proteins with other Bacillus species bacteria during C. elegans infection.

Results

Infectivity of C. elegans by Bacillus species in presence of Cry5B pore-forming protein

In our numerous studies of nematicidal Cry proteins in the absence of B. thuringiensis but in the presence of its standard laboratory food source, Escherichia coli, we have found that Cry proteins produce a dose-dependent mortality in nematodes but do not promote an E. coli infection. Likewise, non-Cry proteinproducing B. thuringiensis strains, such as HD1 cry-, do not cause an infection when fed to C. elegans in the absence of Cry proteins (Figure 1A; Table 1; [START_REF] Marroquin | Bacillus thuringiensis (Bt) toxin susceptibility and isolation of resistance mutants in the nematode Caenorhabditis elegans[END_REF]). We therefore investigated what would happen if we fed C. elegans Cry5B, a Cry protein that forms pores in membranes [START_REF] Kao | Global functional analyses of cellular responses to pore-forming toxins[END_REF], in the presence of B. thuringiensis. Purified Cry5B produces mortality in C. elegans over a 6 day time course at 25uC with a calculated LD 50 of ,8 mg/ml [START_REF] Hu | Discovery of a highly synergistic anthelmintic combination that shows mutual hypersusceptibility[END_REF]. Under these conditions, E. coli is used as the food source and no infections (internal multiplication of E. coli) are seen; the nematodes die from Cry protein intoxication. Conversely, when we fed Cry5B to C. elegans at 10 mg/ml in the presence of B. thuringiensis, we observed accelerated killing of nematodes in 24-48 h, with the internal structures of the nematodes completely digested by the proliferating bacteria (Figure 1B, Table 1). With B. thuringiensis, the majority of the killing takes place in the first 24 h, although we typically let the assays run for 48 h to ensure maximum killing was reached. Nematodes killed by B. thuringiensis with Cry5B are filled with vegetative B. thuringiensis cells or B. thuringiensis spores (Figure 1B), a phenotype we have named ''Bob'' for Bag of bacteria, where the undigested cuticle of the nematode represents the bag that contains the bacteria.

Quantitation of infected animals in the presence and absence of Cry5B demonstrates that the PFP is absolutely required for the infection process-in the absence of Cry5B no B. thuringiensis infections are seen whereas in the presence of Cry5B a very high percentage of the nematodes succumb to accelerated lethal infections (Table 1). To confirm that the infections seen are independent of internal hatching of progeny in hermaphroditic C. elegans, we performed similar studies using the C. elegans mutant glp-4(bn2), which are sterile at the restrictive temperature and do not produce progeny although they do have a similar response to Cry5B as wildtype C. elegans [START_REF] Huffman | Mitogen-activated protein kinase pathways defend against bacterial poreforming toxins[END_REF]. Similar robust Cry5B-mediated infections by B. thuringiensis are found in this genetic background as well (Table 1). In these experiments and other experiments with other Bacillus sp., Cry5B purified from B. thuringiensis was added exogenously so that all the experiments could be directly compared. We have confirmed that B. thuringiensis HD-1 endogenously producing Cry5B also produces a robust number of Bobs (32/33) whereas an isogenic HD-1 strain of B. thuringiensis lacking Cry5B production does not (0/51). Thus Cry5B PFP is an essential virulence factor required for lethal infection of C. elegans by B. thuringiensis.

Cry5B PFP intoxicates and kills C. elegans via binding to glycolipid receptors found in the intestine [START_REF] Griffitts | Glycolipids as receptors for Bacillus thuringiensis crystal toxin[END_REF]. To confirm that Cry5B-stimulated B. thuringiensis infections require the established PFP intoxication pathways, we repeated the challenge experiments in glycolipid-receptor deficient bre-4(ye13) C. elegans animals [START_REF] Griffitts | Glycolipids as receptors for Bacillus thuringiensis crystal toxin[END_REF][START_REF] Griffitts | Resistance to a bacterial toxin is mediated by removal of a conserved glycosylation pathway required for toxin-host interactions[END_REF]. As predicted, nematodes lacking the receptor for Cry5B are not infected by B. thuringiensis even in the presence of Cry5B (Table 1).

Dependency of the infection process upon PFP dose and bacterial inoculum size

Our experiments demonstrate that infectivity is fully dependent both upon Cry5B sensitization and the presence of a pathogenic bacterium (B. thuringiensis). We next set out to determine the relationship between each of these elements and their dosage. Infection assays were performed with either highly infective B. thuringiensis or weakly infective B. megaterium (as a negative control; see below) at increasing doses of Cry5B (Figure 2A). As demonstrated above, in the absence of Cry5B, no infections were seen with either bacterium (Figure 2A). As the dose of Cry5B increases, the percent of worms infected by B. thuringiensis HD-1 increases until maximum infectivity is reached at ,10 mg/ml Cry5B (Figure 2A). Thus, B. thuringiensis lethal infection depends upon Cry5B levels in a dose-dependent manner. Because it was the smallest dose that still achieved penetrant infectivity, we utilized 10 mg/mL as our Cry5B dose in all our fixed dose experiments.

Many key epidemiological processes are inoculum-dependent, e.g., the chance of infection in a given host increases with increasing pathogen inoculum (reviewed in [START_REF] Regoes | Dose-dependent infection rates of parasites produce the Allee effect in epidemiology[END_REF]). To determine if this relationship between pathogen and host are true for B. thuringiensis infections in C. elegans, we varied the dose of inoculum by diluting B. thuringiensis with varying amounts of weakly infective B. megaterium. Two different B. thuringiensis strains were used for this study. We found a dose-dependent relationship in which the C. elegans infection rate increases when the proportion of B. thuringiensis in the inoculum is raised (Figure 2B). This wellbehaved inoculum-dependent infectivity of B. thuringiensis in Cry5B-sensitized C. elegans is rather unique, as C. elegans-pathogenic bacterial studies generally focus on percent animals killed at different time points at fixed doses of bacteria versus percent animals killed at different bacterial concentrations at a fixed time.

Temporal requirements of the infection process

Our data demonstrate that incubation of B. thuringiensis and Cry5B continually with C. elegans results in a lethal infection within 48 h. We next set out to study the temporal requirements for the infection process-e.g., does B. thuringiensis and Cry5B need to be continually fed to C. elegans during this entire time period or are shorter incubation periods also effective in establishing an infection? To determine when the infection process was established, a B. thuringiensis pulse-chase experiment was designed (Figure 3A, schematic). Young adult C. elegans hermaphrodites were exposed to pulses of the B. thuringiensis and Cry5B PFP for varying lengths of time that ranged from 5 minutes to 8 hours. Following these pulses, the nematodes were passed through a series of five washes containing B. megaterium (no Cry5B) to dilute out both the infectious bacteria and Cry5B. Animals were incubated in the final wash for a total of 48 hours and then scored for Bobs (Figure 3A, graph). Surprisingly, high levels of lethal infections are established in as little as 15 minutes of exposure to the pathogen plus Cry PFP. Indeed, the high levels of infections seen upon exposure to pathogenic conditions for any of the time points between 15 minutes and 8 hours are not statistically different (ANOVA, P.0.05, Tukey's post test).

To confirm that the infections seen in the pulse-chase experiment were not the result of residual B. thuringiensis and Cry5B in the final well containing B. megaterium, the experiment was set up as above but no worms were placed in the initial B. thuringiensis well. The same volume as pipetted in the above experiments (without nematodes) was then passaged from the initial well through four B. megaterium dilution wells to the final B. megaterium well as above. Nematodes and Cry5B were then added to this final well. No infections were ever detected in four repetitions of this experiment. Thus, the amount of B. thuringiensis transferred from the first to the final well is not sufficient on its own to cause infection and the infections seen in Figure 3 are the result of interactions between the worms and bacteria exposed to one another in the first well.

It is known that the intestinal pores formed by a short pulse of Cry5B on C. elegans can last for up to 1 d [START_REF] Los | RAB-5-and RAB-11-dependent vesicle-trafficking pathways are required for plasma membrane repair after attack by bacterial pore-forming toxin[END_REF]. Thus, it is possible that the effects of Cry5B PFP on the infection process occurs late even though the pulse used in the above experiments was early and brief. To address the issue of whether Cry5B PFP is acting late or early, we first exposed the animals to the infectious bacteria, washed these away, and then added Cry5B (Figure 3B, schematic). If Cry5B were only acting late in the process, we rationalized that there should be no difference between adding Cry5B with the bacteria in the very beginning or adding Cry5B after a slight delay of ,10 min. In fact, we found that adding Cry5B subsequent to the bacteria leads to a significant drop in infection levels. These data indicate that the PFP instigates infection of B. thuringiensis in C. elegans at the earliest stages.

To see if we could detect visual cues of the beginning of the infection process, green-fluorescent protein (GFP)-expressing B. thuringiensis was fed to C. elegans along with Cry5B PFP. The earliest and most common phenotype we could see (,3-6 h after initiation of the experiment) was the formation of a cluster of vegetative bacteria in the interior lumen of the intestine in many of the nematodes (Figure 3C). These data suggest that, at least by this time-point, the focus point of the infection process is in the anterior intestine, from which it likely spreads.

Cry5B PFP potentiates the infectivity of many Bacillus sp.

Our data demonstrate that exposure to the crystal PFP Cry5B allows B. thuringiensis to initiate a lethal infection in C. elegans. We tested that the PFP might also potentiate the infectivity of other Bacillus sp.. First, we tested Bacillus subtilis. B. subtilis is nonpathogenic towards C. elegans, and C. elegans have increased longevity when fed on B. subtilis than when fed on E. coli OP50, the standard laboratory C. elegans food source [START_REF] Garsin | A simple model host for identifying Gram-positive virulence factors[END_REF][START_REF] Garsin | Long-lived C. elegans daf-2 mutants are resistant to bacterial pathogens[END_REF]. We quantitated the ability of B. subtilis (two different strains) to infect C. elegans in the absence and presence of Cry5B PFP. Whereas B. subtilis alone is unable to infect C. elegans, this ''nonpathogenic'' bacterium is able to infect C. elegans in the presence of Cry5B PFP (Table 1). As with B. thuringiensis, B. subtilis infections occur even in the sterile glp-4(bn2) C. elegans background (Table 1). Thus, internal hatching of progeny is not required for infection by B. subtilis, although some contribution may be reflected by a drop-off in infection rate in glp-4(bn2) animals (Table 1). As with B. thuringiensis, Cry5B intoxication via the glycolipid receptor is required for subsequent infection by B. subtilis, as bre-4(ye13) receptor-less mutants are not infected by B. subtilis in the presence of PFP (Table 1).

To look closer at the specificity on the Bacillus species, we repeated these Cry5B PFP-mediated infection assays using a broader range of Bacillus sp. for these experiments and the C. elegans strain glp-4(bn2) to avoid any complication in interpretation from internal hatching of larvae (Figure 4). We found that the bacteria from the B. cereus group-B. cereus, B. thuringiensis 407-gfp, B. thuringiensis 4Q7 and B. anthracis Sterne are the most virulent, each infecting .25% of the nematodes (Figure 4). As reported in Table 1, B. subtilis is able to infect at an appreciable level and B. megaterium at a barely detectable level (Figure 4). Interestingly, B. circulans, which is phylogenetically quite divergent from the other Bacillus, is also capable of infection, but B. sphaericus, which is used as an insecticidal bacterium like B. thuringiensis, is not (Figure 4). B. anthracis-infected nematodes look similar to B. thuringiensis-infected nematodes under low and high-magnification microscopy (Figure 1C).

It has been previously shown that B. thuringiensis is capable of causing lethal infections in insects and that this infectivity is greatly dependent on the master regulator of virulence, PlcR [START_REF] Agaisse | PlcR is a pleiotropic regulator of extracellular virulence factor gene expression in Bacillus thuringiensis[END_REF][START_REF] Salamitou | The plcR regulon is involved in the opportunistic properties of Bacillus thuringiensis and Bacillus cereus in mice and insects[END_REF]. In Galleria mellonella larvae, co-ingestion of the B. thuringiensis parental strain with a sublethal concentration of Cry1C toxin caused 70% mortality whereas only 7% mortality was recorded if a DplcR mutant strain were used [START_REF] Salamitou | The plcR regulon is involved in the opportunistic properties of Bacillus thuringiensis and Bacillus cereus in mice and insects[END_REF]. Interestingly, in Cry5B-sensitized C. elegans, loss of PlcR from B. thuringiensis still produces a robust, albeit reduced, percentage of infected dead animals (Figure 4; 44% of Bobs with B. thuringiensis 407DplcR versus 73% with the B. thuringiensis-407-gfp parental strain). Moreover, B. anthracis Sterne, which lacks a functional PlcR [START_REF] Agaisse | PlcR is a pleiotropic regulator of extracellular virulence factor gene expression in Bacillus thuringiensis[END_REF], is able to produce a level of infection in Cry5B-sensitized C. elegans comparable to one of the B. thuringiensis strain (4Q7) (Figure 4). PlcR is therefore dispensable for infection in C. elegans and much less important for infection in C. elegans than in Galleria. Taken together, Cry5B PFP is able to potentiate the infectivity of many, but not all, Bacillus towards C. elegans, with the tightly knit B. cereus group as the most potentiated.

B. anthracis, which naturally lacks crystal proteins, can infect in the presence of Cry5B-B. thuringiensis

By definition B. thuringiensis is the bacterium that encodes for and produces Cry proteins whereas none of the other Bacillus sp., including the other B. cereus family members, do. We therefore wondered whether there might be any instance in which a Cry protein-minus Bacillus bacterium, such as B. anthracis, might be able to infect C. elegans with the help of B. thuringiensis. This question is important because it is still a mystery how B. anthracis maintains its life cycle in the soil so that it can be periodically transmitted to herbivore hosts [START_REF] Jensen | The hidden lifestyles of Bacillus cereus and relatives[END_REF][START_REF] Vilas-Boas | Biology and taxonomy of Bacillus cereus, Bacillus anthracis, and Bacillus thuringiensis[END_REF][START_REF] Blackburn | Confirmation of Bacillus anthracis from flesh-eating flies collected during a West Texas anthrax season[END_REF]. We hypothesized that B. anthracis might be able to infect C. elegans if it was present coincidently with B. thuringiensis-i.e. B. anthracis could cause a super infection when B. thuringiensis was present.

We took a B. thuringiensis strain with a Cry5B plasmid, as might be found in the soil, and sporulated it to express Cry5B. We then mixed these B. thuringiensis spore-crystal lysates with sporulated B. anthracis expressing green-fluorescent protein (GFP). In these experiments, the bacteria were mixed at a ratio of about 16 B. thuringiensis spores for one B. anthracis-GFP spore. After 48 h of incubation at 25uC, we counted the number of Bobs and looked at them under the fluorescence microscope. We found that some of these infected worms were full of the gfp-anthrax (Figure 5; Table 2). Thus, even with an initially much larger load of B. thuringiensis being given to the animals and at a temperature (25uC) where B. thuringiensis might be expected to perform better, on occasion B. anthracis is able to overtake and drive the infection process.

Discussion

Here for the first time we characterize in detail the infectivity of Bacillus family bacteria in a nematode, C. elegans. Beginning with the invertebrate-specific pathogen, B. thuringiensis, we find that the infection of C. elegans by B. thuringiensis is dependent upon the presence of a PFP crystal protein virulence factor, Cry5B. The Cry5B pore-forming protein either can be added exogenously or can be naturally expressed by B. thuringiensis in order to elicit infection. The B. thuringiensis strains used in this study cannot infect the in the absence of added Cry5B (either exogenous or elegans has been seen before [START_REF] Borgonie | Germination of Bacillus thuringiensis spores in bacteriophagous nematodes (Nematoda: Rhabditida)[END_REF][START_REF] Schulenburg | Natural variation in the response of Caenorhabditis elegans towards Bacillus thuringiensis[END_REF], the dependence of this infection on crystal protein has not been studied. We furthermore demonstrate that the PFP acts early in the process to instigate infection. The process is likely to be distinct from B. thuringiensis infections characterized in insects since the dependence upon the PlcR virulence regulator, which has been shown to be central for infectivity in an insect [START_REF] Salamitou | The plcR regulon is involved in the opportunistic properties of Bacillus thuringiensis and Bacillus cereus in mice and insects[END_REF], is minimal in the nematode system.

Surprisingly we find that many Bacillus species also possess the ability to infect C. elegans in the presence of Cry5B. This list of Bacillus sp. that can infect C. elegans includes B. subtilis, a bacterium considered non-pathogenic to C. elegans and even more benign than the standard laboratory E. coli fed to it [START_REF] Garsin | A simple model host for identifying Gram-positive virulence factors[END_REF][START_REF] Garsin | Long-lived C. elegans daf-2 mutants are resistant to bacterial pathogens[END_REF]. Of all Bacillus sp. tested, B. cereus group bacteria show the highest level of infectivity in the presence of the Cry5B PFP. The relative role of Cry toxin to various Bacillus species has also been investigated in the larvae of the greater wax moth (G. mellonella). In this insect Cry1C toxin induced higher mortality to B. thuringiensis and B. cereus strains and to a much lower level to B. subtilis and B. megaterium and not at all with B. anthracis [START_REF] Salamitou | The plcR regulon is involved in the opportunistic properties of Bacillus thuringiensis and Bacillus cereus in mice and insects[END_REF][START_REF] Fedhila | Comparative analysis of the virulence of invertebrate and mammalian pathogenic bacteria in the oral insect infection model Galleria mellonella[END_REF], suggesting that B. anthracis develops better in nematode than in insects. We speculate that infectivity of nematodes is an ancient hallmark of the Bacillus genus and that either many Bacillus sp. have subsequently lost the ability to infect nematodes or in the wild factors other than crystal proteins (e.g., environmental stress, starvation of the nematodes, virulence factors not normally expressed in the laboratory) instigate infection.

This work establishes the Bacillus-Cry5B-C. elegans interaction system as a model for studying specific requirements of the nematode infection process of Bacillus, most notably B. cereus-family bacteria such as B. anthracis, for studying the role of PFPs in promoting bacterial pathogenesis, and for studying host responses to B. cereus family bacteria. The fact that both the bacteria and the host are amenable to genetics makes it possible to investigate the interactions of B. cereus family bacteria and an animal host at a level not previously attained.

This research also begins to address an important question in the B. anthracis field-what are the potential reservoirs of this bacterium in nature? B. anthracis causes periodic, large-scale episodes of infection in ruminants. The question has remained: where does it reside and how does it propagate between episodes? One suggestion is that earthworms are reservoirs for B. anthracis and B. cereus multiplication and persistence [START_REF] Schuch | Prevalence of Bacillus anthracis-like organisms and bacteriophages in the intestinal tract of the earthworm Eisenia fetida[END_REF]. Our data suggest that nematodes are potential targets too, when present with B. thuringiensis, B. anthracis is able to replicate within a nematode. Therefore, nematodes might provide a key, and heretofore elusive, natural reservoir for B. cereus and B. anthracis along with earthworms and could play a specific epidemiological role in anthrax outbreaks in the wild. Furthermore, nematodes may provide a key ecological niche linking the three bacteria in the B. cereus-group and facilitating genetic exchange, such as the many that have already been observed [START_REF] Hoffmaster | Identification of anthrax toxin genes in a Bacillus cereus associated with an illness resembling inhalation anthrax[END_REF][START_REF] Rasko | The genome sequence of Bacillus cereus ATCC 10987 reveals metabolic adaptations and a large plasmid related to Bacillus anthracis pXO1[END_REF] and thus helping shape the common evolution of these bacteria.

Materials and Methods

Bacterial strains

Bacteria were cultured in Brain Heart Infusion (BHI) media (BD, Sparks, MD, USA) at 30uC (37uC for B. anthracis Sterne). For sporulation, B. anthracis was cultured in Difco Sporulation Medium [START_REF] Steichen | Non-uniform assembly of the Bacillus anthracis exosporium and a bottle cap model for spore germination and outgrowth[END_REF]

Nematode strains

C. elegans were maintained using standard techniques [START_REF] Sulston | Methods[END_REF]. Unless otherwise mentioned, glp-4(bn2) worms were used in all assays to prevent matricidal death due to premature hatching of embryos. At 25uC, this strain lacks germline and does not produce progeny. This strain is often used in C. elegans assays involving bacterial pathogens [START_REF] Huffman | Mitogen-activated protein kinase pathways defend against bacterial poreforming toxins[END_REF][START_REF] Breger | Antifungal chemical compounds identified using a C. elegans pathogenicity assay[END_REF][START_REF] Kim | A conserved p38 MAP kinase pathway in Caenorhabditis elegans innate immunity[END_REF][START_REF] Moy | Identification of novel antimicrobials using a live-animal infection model[END_REF][START_REF] Shapira | A conserved role for a GATA transcription factor in regulating epithelial innate immune responses[END_REF][START_REF] Tenor | Caenorhabditis elegans-based screen identifies Salmonella virulence factors required for conserved host-pathogen interactions[END_REF]. We found that many fertile C. elegans hermaphrodites can die from the internal hatching of larvae when exposed to Bacillus spp. under conditions of the infection model. Invariably, these dead worms would subsequently become infected. Under these circumstances, it was not easy to differentiate between worms that had become infected before dying versus worms that had died from internal hatching of larvae before becoming infected. Thus, we use glp-4(bn2) in many of our assays to avoid this ambiguity. We do not believe the use of glp-4(bn2) significantly alters our findings. Similarly robust infections are achieved using wild-type N2 C. elegans as with glp-4(bn2), infected animals look the same under high power magnification regardless of which strain is used, and a completely different sterile strain, fer-1(ba576), shows similar levels of infection to glp-4(bn2) (data not shown). glp-4(bn2) worms were maintained at 15uC and shifted at the appropriate times to 25uC when sterility was desired.

Expression and Purification of Cry5B protein

Purified Cry5B protein was prepared using a modified sucrose gradient [START_REF] Cappello | A purified Bacillus thuringiensis crystal protein with therapeutic activity against the hookworm parasite Ancylostoma ceylanicum[END_REF]. Purified Cry5B protein was precipitated, resuspended in double-distilled H 2 O, frozen into aliquots using liquid nitrogen, and stored at 280uC until needed. On the day of use, aliquots were thawed at room temperature and centrifuged at 16,000 g to pellet the protein and allow aspiration of the supernatant. Pellets were then solubilized in 20 mM citrate buffer (pH 3.0) to a final concentration of 0.4 mg/ml.

Bacillus infection assays

Unless otherwise specified, infection assays were carried out in 48-well assay plates. The final volume in each assay well was 200 ml. Standard S-medium was used to maintain C. elegans in liquid cultures [START_REF] Sulston | Methods[END_REF].

Preparation of bacteria. Bacteria were grown overnight in 5 ml liquid BHI in a standard large test tube (256150 mm) at 250 rpm, 30uC (37uC for B. anthracis). The overnight culture was then centrifuged in 1.5-ml tubes in a microcentrifuge at 16,000 g for 4 min to pellet cells and the supernatant was aspirated. Bacterial cells were resuspended in S-medium for a final OD 600 of 0.3+/20.02. 190 ml of bacteria in S-media was added to the assay well. This methodology allows for relatively easy normalization amongst the different bacterial strains.

For assays with mixing of B. anthracis and Cry5B-expressing B. thuringiensis, B. anthracis-gfp, spores were used since B. thuringiensis produces Cry5B during sporulation. Bacteria were grown in 5 ml liquid Difco Sporulation Medium for 48 hours at 37uC [START_REF] Steichen | Non-uniform assembly of the Bacillus anthracis exosporium and a bottle cap model for spore germination and outgrowth[END_REF]. Cultures were checked using a compound microscope for sproulation and were found to be .99% spores. 0.1 ml of Cry5B spore crystal lysates, containing about 4.0610 6 spores were added with 10 ml of B. anthracis-gfp spores (containing about 2.5 10 5 spores) to 190 ml of S-media in a 48-well plate. glp-4(bn2) animals were prepared as described below.

Addition of Cry5B protein. 5 ml of 0.4 mg/ml Cry5B in 20 mM citrate buffer (pH 3.0) was added to appropriate wells for a final well concentration of 10 mg/ml Cry5B. Citrate buffer (pH 3.0) alone was used as a negative control.

Preparation of nematodes. Synchronized populations of glp-4(bn2) nematodes were prepared using standard techniques [START_REF] Cappello | A purified Bacillus thuringiensis crystal protein with therapeutic activity against the hookworm parasite Ancylostoma ceylanicum[END_REF]. Two days prior to setting up the infection assay, a synchronous population of L1s was seeded onto standard 3.5-cm NG plates containing OP50. The worms were incubated at 25uC for 52 h to sterilize the hermaphrodites and allow them to reach the young adult stage. The synchronized nematodes were then washed off the plates with M9 and rinsed three times; twice with M9 and the final time with S-medium. Worms were centrifuged at 500 g for 40 sec to pellet worms for removal of wash supernatants. Worms were then resuspended in S-medium for the desired concentration of 8-12 worms per 5 ml, and 5 ml of this suspension was then added to wells. The assay plates were incubated at 25uC for 48 h, after which they were scored for dead infected worms with a dissecting microscope. Although the majority of nematodes that would end up infected were infected and dead in one day, the assays were allowed to go for two days to be sure the infections had gone to completion. Worms were only counted as infected worms if they were filled with bacteria, which can be detected by dark, rigid, and immobile worms. We have confirmed numerous times by high-resolution microscopy that such worms represent terminally infected worms.

Cry5B dose-response assay

Preparation of bacteria and worms was identical to the described procedure of the C. elegans infection assay. Different doses of the Cry5B PFP were prepared by serial dilution so that the same volume of protein would be added to each well. Each condition was tested in multiple wells (from 4-6 wells).

Bacillus dose-response assay

Bacteria were prepared in S-medium according to the C. elegans infection assay protocol. Different doses of pathogenic Bacillus were prepared by mixing varying amounts of pathogenic Bacillus (i.e. B. thuringiensis HD-1 or B. thuringiensis 407-gfp) with the nonpathogenic B. megaterium, all at the same OD 600 . The inoculum amount of pathogenic Bacillus was expressed as the percentage of the mixed bacteria by volume. These doses were pipetted into appropriate wells and the rest of the assay was set up per the C. elegans infection assay protocol. Each condition was tested in quadruplicate wells.

Bacillus pulse-chase assay

This pulse-chase assay was designed to test the minimum length of time needed for the bacteria to establish an infection. To allow for ease of manipulation, 24-well plates were used. Each row was dedicated to one condition. The total volume in each assay well was 500 ml. The Bacillus strains, purified Cry5B, and nematodes were prepared in an identical manner to the Bacillus infection assay. The first well of each row was designated the 'Pulse' well and contained 475 ml pathogenic Bacillus and 12.5 ml purified Cry5B protein (final concentration 10 mg/ml). The remaining 5 wells of each row were designated the 'Chase' wells, each containing 450 ml non-pathogenic B. megaterium. At t 0 12.5 ml of glp-4 young adults in S-media (approximately 40 worms) were added to the 'Pulse' well and the plate was incubated at 25uC for 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, or 8 h. At the end of the exposure time, the worms were serially transferred through the 'Chase' wells in a volume of 50 ml (spending less than 1 min in each intermediate well), such that the final chase well had a total volume of 500 ml. During the chase transfers, the plates were kept tilted at a 60u angle on the bench top to allow worms to more rapidly settle to the bottom and collect them for pipetting to the next well. Plates were then returned to 25uC. At t 48 the final chase wells were scored for dead infected worms. Worms that were not transferred in the 50 ml serial washes and thus did not make it into the final 'Chase' well were excluded from analysis.

Microscopy

Images of worms in wells were taken with a Nikon Coolpix digital camera on an Olympus dissecting microscope. Images of the infection process were captured using differential interference contrast (DIC) and fluorescence optics on a Zeiss AxioImager A1 microscope using an AxioCam HRm camera and a 6361.4 NA PlanApochromat lens. Images of colony formation were taken on an Olympus IX70 DeltaVision microscope (Applied Precision, Issaquah, WA) with a Nikon 606, 1.4 NA PlanApo oil objective lens. Nematodes were mounted on glass slides with 2% agarose pads with 15 mM sodium azide as an anesthetic prior to imaging. Images were recorded and deconvolved using the SoftWorx program (Applied Precision).

Statistics

Statistical analyses were performed with GraphPad Prism (GraphPad Software, San Diego). For multiple-group comparisons, ANOVA was performed followed by Tukey's posttest. Values of p,0.05 were considered statistically significant.

Figure 1 .

 1 Figure 1. Infection of C. elegans by B. thuringiensis and B. anthracis. Top row: Dissecting microscope view of nematodes cultured under various conditions. Scale bar of all images in top row is 500 mm. Bottom row: Compound microscope view of nematodes cultured under various conditions. For all images in the bottom row, anterior of the worm is top right and scale bar is 50 mm. (A) C. elegans cultured in a well with B. thuringiensis without Cry5B. Top row: None of the six nematodes are infected. All are healthy. The blur associated with some of the worms in the top row is due to their movement in the well. Bottom row: The internal structures of C. elegans fed B. thuringiensis without Cry5B, including the pharynx and intestine, are all intact. (B) C. elegans cultured in a well with B. thuringiensis and Cry5B. Top row: Five of the six worms are completely infected (rigid, lack of internal structures and normal coloration); one is not. Bottom row: Infected animals show complete or near complete digestion of internal structures by the bacteria. Vegetative and sporulated bacteria can be seen in these lethally infected animals. (C) Similar images as in (B) except the bacterium cultured with the nematodes is Bacillus anthracis. doi:10.1371/journal.pone.0029122.g001

Figure 2 .

 2 Figure 2. Dose-dependency of lethal infections by Bacillus and Cry5B. (A) Infections increase in a dose-dependent manner with increasing Cry5B. Doses from 0 to 20 mg/ml Cry5B were prepared and standard infection assays with B. thuringiensis HD-1 carried out. No worms were infected at any of the Cry5B concentrations when exposed to the relatively non-pathogenic B. megaterium in the negative control experiment. Error bars indicate standard error. n = 3 trials, with an average of 40 worms per condition per trial. The glp-4(bn2) strain was used to eliminate any concerns of internal hatching of larvae as the source of death. (B) The lethal infection rate induced by Cry5B increases with increasing doses of B. thuringiensis or B. anthracis. Varying volumes of B. thuringiensis or B. anthracis were mixed with volumes of the nonpathogenic B. megaterium to create different 'doses' of the pathogen while presenting a constant total bacterial load to the worms. Error bars indicate the SEM. n = 3 trials, with an average of 60 worms per condition per trial. The right-most data point is 100% B. thuringiensis or B. anthracis. The data for 0% B. thuringiensis or B. anthracis were collected but cannot be displayed on this log plot; there were no lethal infections at this dose. doi:10.1371/journal.pone.0029122.g002

Figure 3 .

 3 Figure 3. Temporal aspects of the infection process. (A) Infections are established upon a short exposure to pathogen. Upper: schematic of experiment. Worms are added to the well on the left containing B. thuringiensis and Cry5B. Following either 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, or 8 h, the worms were then moved through a series of five wells lacking Cry5B protein and containing non-infectious B. megaterium instead of B. thuringiensis. Infection outcomes in the final well were scored 48 h later. Lower: results of the experiment depicted in upper schematic. Data shown represent a total of three independent experiments with ,30 animals per time point per experiment. Error bars indicate SEM. Only the infection rate for a 5-minute pulse is significantly different from the other data points (ANOVA, p,0.05, Tukey's post test). (B) Cry5B acts early in the infection process as temporal addition of Cry5B protein after, and separate from, pathogen exposure results in a significant drop in infections. Upper: schematic of experiment in which worms are exposed to pathogen first and then to Cry5B. Lower: results in which two sets of experiments were set up simultaneously-a normal 15 minute pulse chase with both Cry5B PFP and pathogen added together (light gray bars; see (A) for set up) and pulse chase in which Cry5B was not added until the end (dark gray bars). Error bars represent SEM. ,30 worms/condition/trial; 3-4 independent trials per condition. (C) The infection process appears to begin with colonization of the anterior intestine. The anterior intestine of a nematode 3 hr after exposure to B. thuringiensis (407-gfp) under pathogenic conditions. The left panel is a DIC image; the right panel deconvolved fluorescent (FITC) of the same animal. Images taken at 6006. Anterior is down. doi:10.1371/journal.pone.0029122.g003

Figure 4 .

 4 Figure 4. Infection of C. elegans by different Bacillus species. The percent-infected worms by different Bacillus are shown along with standard error. Means below the same letter were not significantly different at P,0.05; means below different letters are significantly different at P,0.05. Each bar represents the mean for 3-4 independent trials. The total number of animals screened from left to right are: 117, 209, 117, 122, 107, 142, 96, 96, 96, 72, 96, and 216. doi:10.1371/journal.pone.0029122.g004

Figure 5 .

 5 Figure 5. Infection of C. elegans by B. anthracis-gfp in presence of B. thuringiensis expressing Cry5B spores (non-gfp) and B. anthracis-gfp spores. (A) Transmitted-light view of the worm cuticle filled with vegetative and sporulated Bacillus. (B) View of the same worm using the fluorescence light: rod-shaped bacteria expressing GFP are visible, indicating that B. anthracis is capable of infecting C. elegans in presence of Cry5B expressing-B. thuringiensis. In this animal and all similar animals examined, the vast majority of bacteria inside the nematode cuticle were GFP positive. Scale bar is 50 mm. doi:10.1371/journal.pone.0029122.g005

Table 1 .

 1 Infectivity of Bacillus sp. on C. elegans in the presence/absence of pore-forming Cry5B.

		N2 wild-type animals
		Without Cry5B PFP		With Cry5B PFP
	Bacillus species	Total number of animals	Number of Bobs	Total number of animals	Number of Bobs
	Bt HD1	107	0	133	102
	B. subtilis PY79	119	0	128	45
	B. subtilis 6051	115	0	122	53
	B. megaterium	132	0	149	8
		glp-4(bn2) mutant animals
		Without Cry5B PFP		With Cry5B PFP
	Bacillus species	Total number of animals	Number of Bobs	Total number of animals	Number of Bobs
	Bt HD1	80	0	72	68
	B. subtilis PY79	71	0	72	5
	B. subtilis 6051	90	0	90	7
	B. megaterium	70	0	76	1
		bre-4(ye13) animals
		Without Cry5B PFP		With Cry5B PFP
	Bacillus species	Total number of animals	Number of Bobs	Total number of animals	Number of Bobs
	Bt HD1	115	0	100	0
	B. subtilis PY79	117	0	94	0
	B. subtilis 6051	103	0	95	0
	B. megaterium	106	0	101	0

doi:10.1371/journal.pone.0029122.t001

  . The Bacillus strains used in this work were: B. cereus (ATCC 14579), B. thuringiensis (HD-1), B. thuringiensis (407-gfp), B. thuringiensis (407-DPlcR-gfp), B. thuringiensis (4Q7), B. anthracis Sterne, B. anthracis 7702 harboring pUTE610 (a gift from Theresa Koehler, University of Texas), B. subtilis PY79 (a gift from Richard Losick, Harvard University), B. subtilis 3610, B. subtilis 6051, B. sphaericus (ATCC 4525), B. circulans (ATCC 4513), and B. megaterium (ATCC 14581). B. thuringiensis 407-gfp and B. thuringiensis (407-DPlcR-gfp) contain a constitutively expressed Mut1-green fluorescent protein (GFP) carried on a multicopy plasmid (pHT315 papha3::gfp) with an erythromycin selection factor [32].

Table 2 .

 2 Infectivity of B. anthracis in the presence of Cry-plus B. thuringiensis.

	Condition	Number animals screened	Number Bobs seen	No. GFP-filled Bobs
	B. anthracis-gfp	110	0	0
	B. anthracis-gfp plus Cry5B-Bt	281	72	19
	doi:10.1371/journal.pone.0029122.t002

PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e29122

PLoS ONE | www.plosone.org

December 2011 | Volume 6 | Issue 12 | e29122

Funding for this project was provided by National Institutes of Health grants to RVA (NIAID R01 AI056189) and to RVA/VN (NIAID R21 AI065993). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author Contributions


methods can address fundamental questions in ecology and practical aspects of the management of biological invasions in agricultural settings. We provide a brief description of the methods used to reconstruct invasion routes and describe their main characteristics.

In particular, we focus on a scenario -the bridgehead invasion scenario -, which had been overlooked until recently. We show that this scenario, in which an invasive population is the source of other invasive populations, is evolutionarily parsimonious and may have played a crucial role in shaping the distribution of many recent agricultural pests. ß 2011 Acade ´mie des sciences. Published by Elsevier Masson SAS. All rights reserved. R E ´S U M E Ĺa biologie de l'invasion et l'agriculture sont intimement lie ´es pour plusieurs raisons et en particulier parce que de nombreuses espe `ces de ravageurs agricoles sont des envahisseurs re ´cents. Nous sugge ´rons que la reconstruction des routes d'invasion par des me ´thodes de ge ´ne ´tique des populations permet d'aborder des questions e ´cologiques fondamentales et des aspects pratiques de la gestion des invasions biologiques en agriculture. Nous fournissons une bre `ve description des me ´thodes utilise ´es pour reconstruire les routes d'invasion et de ´crivons leurs principales caracte ´ristiques. En particulier, nous nous concentrons sur un sce ´nario -le sce ´nario d'invasion « te ˆte de pont » -qui n'avait pas e ´te ćonside ´re ´jusqu'a `pre ´sent. Nous montrons que ce sce ´nario, dans lequel une population envahissante est la source d'autres populations envahissantes, est parcimonieux du point de vue e ´volutif et a probablement joue ´un ro ˆle crucial dans l'e ´laboration de la distribution ge ´ographique de nombreux ravageurs des cultures re ´cents.

ß 2011 Acade ´mie des sciences. Publie ´par Elsevier Masson SAS. Tous droits re ´serve ´s.

Contents lists available at ScienceDirect

Comptes Rendus Biologies significant element in global change and have been accused of having adverse effects on public health, the economy and biodiversity [START_REF] Sax | Species Invasions: insights into Ecology, Evolution, and Biogeography[END_REF]. The development of human trade and transport since the 15th Century, which has accelerated over the last 200 years, has increased the importance of invasions as a cause of human-induced global change [START_REF] Jeschke | Invasion success of vertebrates in Europe and North America[END_REF]. Invasive species are important vectors of emerging diseases [START_REF] Eritja | Worldwide invasion of vector mosquitoes: present European distribution and challenges for Spain[END_REF], agricultural pests [START_REF] Work | Arrival rate of nonindigenous insect species into the United States through foreign trade[END_REF] and responsible for many species extinctions and changes in biodiversity worldwide (e.g. [START_REF] Alyokhin | Changes in a lady beetle community following the establishment of three alien species[END_REF]). Many studies on biological invasions have been published since the 1990s, but the definition of an invasive species remains vague. The terminology relating to biological invasions includes a plethora of terms and a wide variety of uses. For example, terms such as ''introduction'', ''establishment'' or ''invasive species'' have been used in different ways in previous publications [START_REF] Colautti | A neutral terminology to define 'invasive' species[END_REF][START_REF] Richardson | Naturalization and invasion of alien plants: concepts and definitions[END_REF]. The vocabulary associated with biological invasions suffers from two flaws: polysemy (multiple meanings for one word, e.g. ''invasive'') and synonymy (several words for one meaning, e.g. ''alien'', ''exotic'', ''non indigenous'', ''introduced''). These problems partly account for the difficulties involved in finding a definition acceptable to most biologists. In addition, one of the problems encountered when trying to define the term ''invasive species'' arises from the tendency of the word ''invasion'' to evoke anthropocentric concepts (e.g. ''Barbarian Invasions'', assault, attack, intrusion, incursion, raid, etc.) [START_REF] Richardson | Naturalization and invasion of alien plants: concepts and definitions[END_REF] associated with negative connotations that may not necessarily apply to ecological phenomena. Current definitions differ in the relative importance attributed to three major components: 'range expansion' [START_REF] Facon | A general eco-evolutionary framework for understanding biological invasions[END_REF], 'high local abundance' [START_REF] Suarez | Behavioral and genetic differentiation between native and introduced populations of the Argentine ant[END_REF] and 'disruption of ecosystem function' [START_REF] Mooney | Invasive species in a changing world[END_REF]. We will use the following definition here: an invasion may be considered to have occurred when a group of individuals has been introduced into a new area, in which they have established themselves, increased in number and spread geographically. This definition does not necessarily imply a spread into new ecological conditions and does not necessarily result in negative effects on the invaded ecosystem.

Many studies on this topic have been published, but only very few biological invasions have been properly described, studied and understood, due to conceptual, methodological and experimental limitations (e.g. [START_REF] Facon | A general eco-evolutionary framework for understanding biological invasions[END_REF]). Consequently, most of the hypotheses formulated concerning the key factors determining the probability of success or failure of invasions, such as propagule size, genetic variability or hybridization, have never been tested. One of the key scientific questions concerning biological invasions that has yet to be answered concerns the reasons why some species become successful invaders whereas others do not [START_REF] Sax | Species Invasions: insights into Ecology, Evolution, and Biogeography[END_REF][START_REF] Cadotte | Conceptual Ecology and Invasion Biology: reciprocal Approaches to Nature[END_REF]. The general characteristics of species (such as dispersal, competitiveness) may determine the probability of the species becoming invasive [START_REF] Sakai | The population biology of invasive species[END_REF]. There is, however, some intraspecific variation for this probability as illustrated by the observation that only a small fraction of populations becomes invasive in many ''invasive species'' (e.g. [START_REF] Ciosi | Invasion of Europe by the western corn rootworm, Diabrotica virgifera virgifera: multiple transatlantic introductions with various reductions of genetic diversity[END_REF][START_REF] Kelly | Contrasting patterns in genetic diversity following multiple invasions of fresh and brackish waters[END_REF][START_REF] Kang | Testing the role of genetic factors across multiple independent invasions of the shrub Scotch broom (Cytisus scoparius)[END_REF]). There is therefore still a need to identify explanatory evolutionary and environmental factors at the population level. We argue that the precise descriptions of biological invasions, including their history, geography, demography and genetics -referred to here as invasion routes -represent a first step toward identifying these factors.

We focus here on biological invasions in agricultural settings. We briefly review the specificity of invasions in agricultural settings and explain why the reconstruction of invasion routes can be used to address fundamental questions about the determinants of invasions and practical aspects of biological invasion management. We then consider the methodological challenges associated with studies of invasion routes and describe the main evolutionary and environmental insights drawn to date from the large set of published studies dealing with the reconstruction of invasion routes.

Invasion in agricultural settings

The history of agriculture is intimately linked to biological invasions. The invention and development of agriculture allowed the worldwide spread of human populations [START_REF] Ammerman | The Neolithic transition and the genetics of populations in Europe[END_REF]. It also led to the invasion, as defined above, of areas by cultivated plants and livestock animals [START_REF] Mack | Biotic invasions: causes, epidemiology, global consequences, and control[END_REF]. As a result, a few animal and plant species are now found throughout the world. Pimentel et al. [START_REF] Pimentel | Economic and environmental threats of alien plant, animal, and microbe invasions[END_REF] observed that agricultural activities have led to 90% of the food of the world's human population being provided by a mere 15 plant species, and eight animal species accounting for most of the animal proteins consumed by humans. Species such as maize (Zea mays) and chicken (Gallus gallus domesticus) are found all over the world (with the exception of hot and cold deserts) and their populations are much larger than that of humans (references in Pimentel et al. [START_REF] Pimentel | Economic and environmental threats of alien plant, animal, and microbe invasions[END_REF]).

A large number of animals, plants and microbes living in agricultural ecosystems decrease the quality and/or quantity of the cultivated resource and are therefore considered to be pest species. These pests are often recently introduced organisms capable of taking advantage of the extraordinarily large amount of resources provided by cultivated crops or animals for settlement and spread. In the absence of predators and parasites, invaders often undergo explosive population increases, with severe consequences for the crop plants and domesticated animals concerned. This results in the invader being classified as a major pest species. For example, the oomycete Phytophthora infestans, the causal agent of potato blight, was introduced into Europe from America around 1843 [START_REF] Andrivon | The origin of Phytophthora infestans populations present in Europe in the 1840s: a critical review of historical and scientific evidence[END_REF]. It invaded large cultivated areas of Europe and was the cause of the Great Famine in Ireland in the mid-19th Century. Another famous example is grape phylloxera, Daktulosphaira vitifoliae, which is a worldwide pest of grapevine [START_REF] Granett | Biology and management of grape phylloxera[END_REF]. This insect devastated European vineyards after its introduction in the region of Bordeaux, France, and its spread across Europe in the second half of the 19th Century. Similarly, Ceratitis capitata, the Mediterranean fruit fly, is a famous pest of fruit crops originating from Africa. It invaded the Americas and Australia during the late 19th and 20th centuries and is now one of the world's most threatening agricultural pests, attacking over 200 different cultivated plants [START_REF] Davies | Bioinvasions of the medfly Ceratitis capitata: source estimation using DNA sequences at multiple intron loci[END_REF]. Ten of the 16 invasive terrestrial invertebrates present in the DAISIE (a European consortium of researchers studying invasive species in Europe) ''100 of the worst'' list are crop pests [START_REF] Daisie | Handbook of Alien Species in Europe[END_REF].

The invasion of agricultural settings by pests may have tremendous economic and social consequences. Such invasions may generate costs due to production loss, decreases in the value of the product and the need for control practices (survey, containment, eradication). The estimated world cost of biological invasions in agriculture reaches the astronomical range of 50 to 250 billion US dollars per year [START_REF] Pimentel | Economic and environmental threats of alien plant, animal, and microbe invasions[END_REF]. Many social and economic activities have also developed to deal with this problem, from the chemical industry, genetic engineering development and agricultural advisory services to public and private agronomy research.

Invasion and biological control

Biological control is a promising approach to the control of pest species in agriculture, because it has few if any adverse effects on the environment and human health. Classical biological control (CBC) is a component of both integrated pest management and organic farming. It involves the introduction of an organism -often a predator or a parasite of the pest species targeted -into an area in which it was not previously found, in the hope of establishing stable populations capable of reducing the density of a specific pest [START_REF] Eilenberg | Suggestions for unifying the terminology in biological control[END_REF]. CBC and invasion biology are intimately linked for at least two reasons: the target of CBC is often an invading species that has recently acquired pest status. A successful example of CBC against an invasive pest species is provided by the glassy-winged sharpshooter, Homalodisca vitripennis. This large leafhopper is a xylem-feeding pest that transmits Xelella fastidiosa, a parasitic bacterium responsible for a lethal infection in plants. This species originates from the South East USA and Northern Mexico, and has invaded a number of sites in the Pacific, including several archipelagos in French Polynesia, since 1999 [START_REF] Grandgirard | Classical biological control of the glassy-winged sharpshooter, Homalodisca vitripennis, by the egg parasitoid Gonatocerus ashmeadi in the Society. Marquesas and Austral archipelagos of French Polynesia[END_REF][START_REF] Petit | Invasion dynamics of the glassy-winged sharpshooter Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae) in French Polynesia[END_REF][START_REF] Petit | Shortdistance dispersal behavior and establishment of the parasitoid Gonatocerus ashmeadi (Hymenoptera: Mymaridae) in Tahiti: Implications for its use as a biological control agent against Homalodisca vitripennis (Hemiptera: Cicadellidae)[END_REF]. A CBC operation was implemented in 2004, with Gonatocerus ashmeadi, a parasitoid wasp that parasitizes the eggs of the pest species. After the release of more than 10,000 individuals in Tahiti in 2005, invasive glassy-winged sharpshooter populations decreased in size by about 90% around the release sites [START_REF] Petit | Shortdistance dispersal behavior and establishment of the parasitoid Gonatocerus ashmeadi (Hymenoptera: Mymaridae) in Tahiti: Implications for its use as a biological control agent against Homalodisca vitripennis (Hemiptera: Cicadellidae)[END_REF]; CBC and biological invasions have similar properties. CBC aims to establish and spread populations of beneficial species, through ecological processes resembling those occurring during unintentional invasions [START_REF] Ehler | Invasion biology and biological control[END_REF][START_REF] Fagan | Invasion theory and biological control[END_REF][START_REF] Roderick | Genes in new environments: genetics and evolution in biological control[END_REF]. Biological invasions, which may be unintentional and detrimental (in the case of pest species) or intentional and beneficial (in the case of CBC), have enough characteristics in common to be considered as a single eco-evolutionary process. Thus, an understanding of the ecological and genetic factors underlying efficient biological control may help us to understand and to manage detrimental biological invasions. Conversely, the information obtained from descriptions of accidental biological invasions may help us to design more effective biological control. In a CBC operation, the initial demographic (number of individuals, number of release points, timing of releases) and genetic (genetic variance and adaptive traits of the introduced population) parameters of the invasion -the introduction parameters -can be controlled experimentally [START_REF] Grevstad | Experimental invasions using biological control introductions: the influence of release size on the chance of population establishment[END_REF][START_REF] Memmott | The effect of propagule size on the invasion of an alien insect[END_REF]. Meta-analyses of ancient CBC successes and failures have been used to address certain questions [START_REF] Hopper | Mate finding, dispersal, number released, and the success of biological introduction[END_REF], but CBC can also be used in natura, in the design of specific experiments testing biological invasion hypotheses. This approach has seldom been used, but has recently begun to drive the use of CBC in model experiments. A recent example is provided by the work of Fauvergue et al. [START_REF] Fauvergue | Invading parasitoids suffer no allee effect: a manipulative field experiment[END_REF], who manipulated the demographic characteristics of a parasitoid introduced in a CBC context to test for a positive effect of the size of the population introduced on the success of establishment and, hence, of invasion. These authors introduced the North American parasitoid Neodryinus typhlocybae into Southern France, to control the North American invasive flatid planthopper, Metcalfa pruinosa, and demonstrated a total absence of the expected positive demographic effect on the success of settlement [START_REF] Fauvergue | Invading parasitoids suffer no allee effect: a manipulative field experiment[END_REF].

Factors promoting invasions

Role of humans in shaping invasion routes

Human activities are responsible for a large proportion of recent biological invasions [START_REF] Lockwood | The role of propagule pressure in explaining species invasions[END_REF] in two main ways. First, human activities serve as a vector for the introduction of propagules (canals, marine ballast, air, road and train traffic) into new, geographically disconnected areas and for geographic expansion of populations that have already been introduced (see p. 21 of [START_REF] Cox | Alien Species and Evolution: the evolutionary ecology of exotic plants, animals, microbes, and interacting native species[END_REF]). The second role of human activities in promoting biological invasions involves environmental modification. The disturbance of natural habitats by human activities is thought to facilitate bioinvasions [START_REF] Lake | Invasion success of exotic in natural ecosystems: the role of disturbance, plant attributes and freedom from herbivores[END_REF] and disturbances due to agriculture development may play a particular role in this respect [START_REF] Lockwood | Invasion ecology[END_REF][START_REF] Tilman | Forecasting agriculturally driven global environmental change[END_REF]. Agriculture has a particular consequence in terms of habitat disturbance: it has homogenized the environment worldwide. The cultivation of domesticated plants, such as maize, has homogenized habitats, decreasing ecological differences between regions in different parts of the world, from Africa to Asia, and from North and South America to Europe [START_REF]Anonymous, Maize in human nutrition, Food and Agriculture Organization of the United nations[END_REF]. When a species is introduced into a new and remote area, the expected mismatch between its phenotypic characteristics and local ecological conditions is greatly attenuated by this homogenization.

Adaptation

Natural selection and adaptation probably play key roles in determining the success of invasion during the establishment phase [START_REF] Reznick | The population ecology of contemporary adaptations: what empirical studies reveal about the conditions that promote adaptive evolution[END_REF][START_REF] Schierenbeck | The role of evolutionary genetics in studies of plant invasions[END_REF]. In the absence of strong environmental homogenization, the new geographic area into which individuals are introduced may have ecological conditions very different from those of the native area. A large additive genetic variance in the introduced population should increase adaptability, thereby increasing the probability of settlement and subsequent demographic growth and geographic spread (e.g. [START_REF] Facon | A general eco-evolutionary framework for understanding biological invasions[END_REF]). However, adaptation may also occur in populations with low levels of genetic variation, provided that ''good'' genetic combinations are present [START_REF] Facon | A general eco-evolutionary framework for understanding biological invasions[END_REF]. This is particularly true in Comment citer ce document : Guillemaud, T. (Auteur de correspondance), Ciosi, M., Lombaert, E., Estoup [START_REF] Cox | Alien Species and Evolution: the evolutionary ecology of exotic plants, animals, microbes, and interacting native species[END_REF]). For example, pesticide resistance may be the main prerequisite for the settlement and spread of introduced pests in areas commonly treated with one or more pesticides to control resident pests. In the aphid Aphis gossypii, a pest of many cultivated plants (cotton, melon, potato, pepper, eggplant, citrus, etc.), a few genetic clones have spread worldwide. These clones are adapted both to their host plants -they display an intimate degree of host specialization [START_REF] Carletto | Ecological specialization of the aphid Aphis gossypii glover on cultivated host plants[END_REF] -and to the most common pesticide treatments, with most clones resistant to organophosphate and pyrethroid insecticides [START_REF] Carletto | Insecticide resistance traits differ among and within host races in Aphis gossypii[END_REF]. The spread of the western corn rootworm, Diabrotica virgifera virgifera, in the US during the 1960s probably resulted from the appearance and spread of allelic forms conferring insecticide resistance [START_REF] Metcalf | Implications and prognosis of resistance to insecticides[END_REF]. This insect pest of maize has been present in the Great Plains since at least 1867, and by 1955, its geographic distribution remained limited to parts of Kansas, Colorado and Nebraska [START_REF] Gray | Adaptation and invasiveness of Western Corn rootworm: intensifying research on a worsening pest[END_REF]. Cyclodiene insecticides were introduced in 1952 and were massively used, and the first reported case of resistance occurred in 1959 [START_REF] Metcalf | Implications and prognosis of resistance to insecticides[END_REF]. Resistant rootworm rapidly spread throughout the Corn Belt and reached Northwest Indiana in 1968. In 1979, it was present throughout most of the US Corn Belt, from Nebraska to Ohio and from Minnesota to Missouri [START_REF] Metcalf | Implications and prognosis of resistance to insecticides[END_REF][START_REF] Gray | Adaptation and invasiveness of Western Corn rootworm: intensifying research on a worsening pest[END_REF]. It has not been demonstrated that insecticide resistance was responsible for accelerating the geographic spread of the western corn rootworm in the 1960s [START_REF] Gray | Adaptation and invasiveness of Western Corn rootworm: intensifying research on a worsening pest[END_REF], but it is nonetheless clear that this spread would not have been possible if the insects had not evolved cyclodiene resistance.

Routes of invasion

An approach to tackling academic issues

The genetic variability of invading populations depends on the history and demography of the populations or groups of individuals, from their emigration from the source population to their introduction and spread [START_REF] Dlugosch | Founding events in species invasions: genetic variation, adaptive evolution, and the role of multiple introductions[END_REF]. The description of this history depicts invasion routes. It includes information about source populations (number and genetic composition), the number of introductions from the sources, the number of individuals involved in each introduction, the occurrence of admixture between independently introduced populations, the number of intermediate invasive populations between the initial introduction point and the invasive population studied and demographic dynamics at each step in the history of the invasion. In a recent review on this topic, Estoup and Guillemaud [START_REF] Estoup | Reconstructing the routes of invasion: what for, how and so what?[END_REF] argued that knowledge of invasion routes was required to decipher the factors responsible for the success of invasions. More specifically, information about sources and invasion pathways is essential if we are to avoid making erroneous conclusions when testing the hypothesis that a particular environmental or evolutionary factor affects invasion success. Keller and Taylor [START_REF] Keller | History, chance and adaptation during biological invasion: separating stochastic phenotypic evolution from response to selection[END_REF] argued that adaptive evolution could not be inferred from the simple observation of changes in the distribution of phenotypic traits between the invaded and the native area. They pointed out that the hypothesis of neutral evolution during invasion processes could only be rejected if ancestor-descendent comparisons or Qst-Fst analyses are carried out. Estoup and Guillemaud [START_REF] Estoup | Reconstructing the routes of invasion: what for, how and so what?[END_REF] also argued that such analyses need to compare ''comparable entities'' (here, the invasive populations and their precise source(s)), which requires a basic knowledge of invasion routes.

An approach to tackling practical issues in agricultural settings

The reconstruction of routes of invasion can contribute to the development or optimization of measures for preventing invasions, particularly in an agricultural context, in two main ways. Firstly, invasion routes basically describe the geographic origins of invasive pests until their introduction. This geographic information can be used as the basis of management actions directed against the main steps of the invasion process: exit from the native area (emigration), vector transport or migration and entry into the invasion site (see Introduction). In the case of recurrent introductions, as demonstrated for the chrysomelid D. virgifera [START_REF] Miller | Multiple transatlantic introductions of the western corn rootworm[END_REF], identification of the precise location of the escape path in the native area (e.g. a specific airport, harbor, ecosystem, region etc.) can lead to the design of specific monitoring and quarantine measures targeting the sources [START_REF] Mack | Biotic invasions: causes, epidemiology, global consequences, and control[END_REF]. The same rationale can be applied to the vectors responsible for recurrent introductions and to the entry portals for the pest: control strategies focusing on specific vectors (e.g. freight containers of a particular crop seed or a specific human mode of transportation) or entry locations (e.g. a specific airport, harbor, ecosystem, region etc. as in the case of ''exit doors''). By contrast, for pests arriving in a new area through a single or a small number of introduction events, eradication or containment strategies may be efficient if applied shortly after the arrival of the pest [START_REF] Mack | Biotic invasions: causes, epidemiology, global consequences, and control[END_REF].

Secondly, identification of the invasion route provides information about the original environment and the genetic properties of the source population of the invading pest. A knowledge of the biotic and abiotic environment to which the pest is adapted may make it easier to design an effective control strategy. This is particularly true when choosing pesticides, as this choice must take into account the potential resistance of the source population. This simple rationale applies to all strategies for which susceptibility varies within the source populations of the pest (e.g. parasite or predator use, crop rotation). In the context of biological control, the choice of natural enemy to be introduced may depend on what we know about the source populations of the invader. Generally, the aim is to choose species or populations of a species with the same geographic origin as the pest population [START_REF] Cox | Alien Species and Evolution: the evolutionary ecology of exotic plants, animals, microbes, and interacting native species[END_REF]. The probability of an invasive pest being controlled by a natural enemy depends on the level of adaptation of the two protagonists to their environment, their adaptation to each other and their ability to evolve [START_REF] Roderick | Genes in new environments: genetics and evolution in biological control[END_REF][START_REF] Parker | The evolutionary ecology of novel plantpathogen interactions[END_REF]. In particular, biological control agents may be more effective against the native populations with which they coevolved and to which they have adapted than against other populations (see for a complication of the simple case [START_REF] Kang | Testing the role of genetic factors across multiple independent invasions of the shrub Scotch broom (Cytisus scoparius)[END_REF][START_REF] Roderick | Genes in new environments: genetics and evolution in biological control[END_REF]). accounts for the success of invasion. Without a bridgehead population, the framework of Facon et al. [START_REF] Cadotte | Conceptual Ecology and Invasion Biology: reciprocal Approaches to Nature[END_REF] requires multiple genetic shifts (one in each introduced area, Fig. 2A). In a bridgehead invasion scenario, only one evolutionary shift toward invasiveness has to occur in the bridgehead population (Fig. 2B). This scenario is therefore evolutionarily more parsimonious than that without a bridgehead. Two illustrative example of a bridgehead invasion scenario are given in Boxes 2 and 3. How general are bridgehead invasion scenarios? Apart from the studies on D. virgifera and H. axyridis (Boxes 2 and 3), other studies have shown that intermediate invasive populations may be the sources of other, often distant, invasive populations [START_REF] Kolbe | Genetic variation increases during biological invasion by a Cuban lizard[END_REF][START_REF] Downie | Locating the sources of an invasive pest, grape phylloxera, using a mitochondrial DNA gene genealogy[END_REF][START_REF] Grapputo | The voyage of an invasive species across continents: genetic diversity of North American and European colorado potato beetle populations[END_REF]. However, it is possible to demonstrate the occurrence of such scenarios only in very well documented cases of biological invasions: demonstrating the existence of a ''bridgehead'' population requires a good knowledge of the geographic distribution of the species (native and invaded areas) and of the routes of invasion of the species. Crop pests probably commonly establish bridgehead populations. We found several examples of pests or pathogens for which precise documentation of the invasion made it possible to identify a bridgehead population, or at least to show that the likely scenario involved an intermediate invasive population giving rise to several secondary invasions. A non-exhaustive list of examples is provided below: potato blight, Phytophthora infestans, was introduced into Europe in the 1840 s from America (references in [START_REF] Andrivon | The origin of Phytophthora infestans populations present in Europe in the 1840s: a critical review of historical and scientific evidence[END_REF]). Goodwin et al. [START_REF] Goodwin | Panglobal Distribution of a Single Clonal lineage of the irish potato famine fungus[END_REF] suggested that the European population then served as the source population for a number of other introductions, resulting in the worldwide distribution of this oomycete; the causal agent of apple scab, Venturia inaequalis, invaded the world by following its host [START_REF] Gladieux | On the origin and spread of the scab disease of apple: out of central Asia[END_REF]. It originated in Central Asia, and was first introduced into Europe during the Ancient history. Europe then acted as a secondary source for world colonization by the pathogen over the last 500 years; the European corn borer, Ostrinia nubilalis first invaded Europe, establishing a bridgehead from which it was subsequently introduced into North America [START_REF] Kaster | European corn borers and western corn rootworms: old and new invasive maize pests challenge farmers on European and North American continents[END_REF][START_REF] Kim | Spatial and temporal genetic analyses show high gene flow among European corn borer (Lepidoptera: Crambidae) populations across the central US corn belt[END_REF] The Harlequin ladybeetle or multicolored Asian lady beetle, Harmonia axyridis, a native of Asia, has long been used as a biological control agent to control aphid populations. Despite repeated attempts at introduction since 1916, the establishment of this species was not observed until recently (references in Lombaert et al. [START_REF] Lombaert | Bridgehead effect in the worldwide invasion of the biocontrol harlequin ladybird[END_REF]). In 1988 and 1999, the first invasive populations were recorded in Louisiana and Oregon, respectively. Invasive populations of the Asian ladybeetle were then observed in Europe (Belgium in 2001) and South America (Argentina in 2001), followed by South Africa in 2004. Using molecular markers, historical information and ABC methods, Lombaert et al. [START_REF] Lombaert | Bridgehead effect in the worldwide invasion of the biocontrol harlequin ladybird[END_REF] showed that the invasive populations in western and eastern North America arose from two introductions from Asia, either through biological control or accidental introductions, and that the European, South American and African outbreaks all originated independently from eastern North America. In addition, evidence of an admixture between the eastern North American population and the native Asian population were found in Europe. The invasion routes summarized in Fig. 3 indicate that the eastern North American invasive population acted as a bridgehead population in the worldwide invasion of H. axyridis. The role of eastern North America in the sudden invasion of Europe, South America and Africa, the long history of unsuccessful introductions of the ladybeetle from its native range for biological control, and the apparent absence of invasive populations originating from western North America suggest that an evolutionary change or a change in emigration regime probably occurred in eastern North America. Additional quantitative genetics studies of key life history traits are underway.

Box 3. Bridgehead invasion of the western corn rootworm, Diabrotica virgifera virgifera.

This chrysomelid, a pest that attacks the root system of maize, is one of the most important pests of maize in the USA and is sometimes referred to as the ''billion dollar bug'' [START_REF] Sappington | Coordinated Diabrotica genetics research: accelerating progress on an urgent insect pest problem[END_REF]. It originates from what we now call Mexico, was first observed in the US in 1867, in the Great Plains, and invaded North America during the second half of the 20th century [START_REF] Gray | Adaptation and invasiveness of Western Corn rootworm: intensifying research on a worsening pest[END_REF]. It was first observed in Europe in 1992, in the former Yugoslavia, and rapidly invaded a large part of Central and South Eastern Europe. A number of isolated outbreaks have been detected almost every year since 1998, in various countries, including Italy, France, Switzerland, Belgium, the United Kingdom, the Netherlands and Germany [START_REF] Edwards | New WCR 2006 General spread map for Europe[END_REF]. Using molecular markers and historical information, Miller et al. [START_REF] Miller | Multiple transatlantic introductions of the western corn rootworm[END_REF] and Ciosi et al. [START_REF] Ciosi | Invasion of Europe by the western corn rootworm, Diabrotica virgifera virgifera: multiple transatlantic introductions with various reductions of genetic diversity[END_REF] showed that the invasion of Europe by D. virgifera was very probably due to multiple introductions from North America and, more precisely, from the Northern US [START_REF] Ciosi | Invasion of Europe by the western corn rootworm, Diabrotica virgifera virgifera: multiple transatlantic introductions with various reductions of genetic diversity[END_REF]. The invasions of North America and Europe by this agricultural pest thus form a succession of introductions and geographic expansions and correspond to a bridgehead invasion scenario: the native Mexican population gave rise to an invasive bridgehead population in the US, which acted as the source of all the other invasive populations in Europe. We propose the following hypotheses for invasion by D. virgifera: after its introduction from the Mexican source population into the US, invasion by the North American bridgehead population was triggered by one or more adaptive changes, such as specialization on a widespread resource (in this case, maize). A change in emigration regime in the bridgehead population when it reached the North East US was then sufficient to initiate remote invasions in Europe.

Comment citer ce document : Guillemaud, T. (Auteur de correspondance), Ciosi, M., Lombaert, E., Estoup the grape phylloxera, Daktulosphaira vitifoliae, in addition to invading the whole of Europe from France, probably initially invaded California, subsequently being introduced, from the American population, into Australia, New Zealand and Peru [START_REF] Downie | Locating the sources of an invasive pest, grape phylloxera, using a mitochondrial DNA gene genealogy[END_REF]; the Guatemalan potato moth, Tecia solanivora, first invaded the southern part of Central America and, from there, was introduced into South America and the Canary Islands [START_REF] Puillandre | Genetic bottleneck in invasive species: the potato tuber moth adds to the list[END_REF].

Most of the species mentioned above, like D. virgifera (see Box 2), probably achieved pest status following an evolutionary shift allowing the bridgehead population to become invasive: adaptation to a cultivated plant. D. virgifera and the European corn borer probably left several wild herbaceous hosts to adapt to maize. The Colorado beetle, the Guatemalan potato moth and the potato blight probably all moved onto the cultivated potato from wild tuber-bearing plants. Phylloxera may not have adapted: this aphid became a major pest on grapes because the Vitis species used happened to be susceptible. In this case, a change in migration regime was probably the cause of the secondary introductions worldwide from California. Once it was discovered that the American Vitis species could be used as a rootstock for the efficient control of phylloxera, the intensive collection and exchange of American native Vitis plants occurred, undoubtedly increasing the number of phylloxera introductions worldwide [START_REF] Downie | Locating the sources of an invasive pest, grape phylloxera, using a mitochondrial DNA gene genealogy[END_REF]. In the case of apple scab, the global codispersal of apple and its pathogen probably provides the best explanation of the current worldwide distribution of Venturia inaequalis. This scenario reflects a major change in the migration regime of the pathogen due to the colonization of the rest of the world by European settlers.

Conclusion

The histories of invasions and agriculture are intimately linked, with many crop and livestock pests being invasive species and vice versa. In addition, some pest management practices essentially constitute intentional and beneficial invasions. Such practices require a precise knowledge of invasion biology and, conversely, their application may provide valuable information about invasion biology. Recent methods based on analyses of genetic markers have provided tools for the retracing of invasion routes -the history of the invading populations from their geographic origin to their final spread in the invaded area. The examples for which a precise description of invasion routes at the global level is available provide new insight into invasion biology and have highlighted previously unsuspected global trends. In particular the bridgehead invasion scenario seems to apply to many cases of pest invasions and merits more thorough consideration when trying to explain the distribution of other invasive species in agricultural settings. The number of publications on the invasion routes of crop and livestock pests and pathogens is growing, and future studies in this field will undoubtedly provide valuable information challenging the generalization of this evolutionary scenario.

  w w w . s c i e n c e d i r e c t . c o m 1631-0691/$ -see front matter ß 2011 Acade ´mie des sciences. Published by Elsevier Masson SAS. All rights reserved. doi:10.1016/j.crvi.2010.12.008

  Comment citer ce document : Guillemaud, T. (Auteur de correspondance), Ciosi, M., Lombaert, E.,Estoup, A. (2011). Biological invasions in agricultural settings: insights from evolutionary biology and population genetics. Comptes Rendus. Biologies, 334 (3), 237-246. DOI : 10.1016/j.crvi.2010.12.008 Comment citer ce document : Guillemaud, T. (Auteur de correspondance), Ciosi, M., Lombaert, E., Estoup, A. (2011). Biological invasions in agricultural settings: insights from evolutionary biology and population genetics. Comptes Rendus. Biologies, 334 (3), 237-246. DOI : 10.1016/j.crvi.2010.12.008 Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Version définitive du manuscrit publiée dans / Final version of the manuscript published in : Comptes Rendus Biologies (2011), Vol. 334, N°.3, p. 237-246, DOI: 10.1016/j.crvi.2010.12.008 Journal homepage: www.sciencedirect.com

Comptes Rendus Biologies (2011), Vol. 334, N°.3, p. 237-246, DOI: 10.1016/j.crvi.2010.12.008 Journal homepage: www.sciencedirect.com

, A. (2011). Biological invasions in agricultural settings: insights from evolutionary biology and population genetics. Comptes Rendus. Biologies, 334 (3), 237-246. DOI : 10.1016/j.crvi.2010.12.008 Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Version définitive du manuscrit publiée dans / Final version of the manuscript published in : agricultural contexts, in which the selection pressure exerted by pest control strategies may be very strong (see p. 128 of

  ; the Colorado beetle, Leptinotarsa decemlineata first invaded the United States. It was then introduced into Europe [69]; Box 2. Bridgehead invasion in the biocontrol agent Harmonia axyridis.

Comptes Rendus Biologies (2011), Vol. 334, N°.3, p. 237-246, DOI: 10.1016/j.crvi.2010.12.008 Journal homepage: www.sciencedirect.com

	Manuscrit d'auteur / Author manuscript
	Manuscrit d'auteur / Author manuscript
	Manuscrit d'auteur / Author manuscript

, A. (2011). Biological invasions in agricultural settings: insights from evolutionary biology and population genetics. Comptes Rendus. Biologies, 334 (3), 237-246. DOI : 10.1016/j.crvi.2010.12.008 Version définitive du manuscrit publiée dans / Final version of the manuscript published in :

T.Guillemaud et al. / C. R. Biologies 334 (2011) 237-246

Acknowledgments

This work was supported by grants from the Agence nationale de la recherche (ANR-06-BDIV-008-01 and ANR-09-BLAN-0145-01) and from the Agropolis Fondation (RTRA-Montpellier, BIOFIS project).

. Multiple introductions

Recent papers on invasion routes suggest that invasions are often associated with multiple introductions (i.e. several introductions from one or several sources into one or several remote areas). This scenario has been demonstrated in the cases of the maize pest D. virgifera [START_REF] Ciosi | Invasion of Europe by the western corn rootworm, Diabrotica virgifera virgifera: multiple transatlantic introductions with various reductions of genetic diversity[END_REF][START_REF] Miller | Multiple transatlantic introductions of the western corn rootworm[END_REF], the false brome Brachypodium sylvaticum [START_REF] Rosenthal | Evidence for multiple sources of invasion and intraspecific hybridization in Brachypodium sylvaticum (Hudson) Beauv. in North America[END_REF], the spotted knapweed Centaurea stoebe micranthos [START_REF] Marrs | Evidence for multiple introductions of Centaurea stoebe micranthos (spotted knapweed, Asteraceae) to North America[END_REF], the shrub Scotch broom [START_REF] Kang | Testing the role of genetic factors across multiple independent invasions of the shrub Scotch broom (Cytisus scoparius)[END_REF], the mosquito Culex quiquefasciatus [START_REF] Fonseca | Bottlenecks and multiple introductions: population genetics of the vector of avian malaria in Hawaii[END_REF], the amphipod Gammarus tigrinus [START_REF] Kelly | Contrasting patterns in genetic diversity following multiple invasions of fresh and brackish waters[END_REF], the Cuban lizard [START_REF] Kolbe | Genetic variation increases during biological invasion by a Cuban lizard[END_REF], the freshwater snail M. tuberculata [START_REF] Facon | A molecular phylogeography approach to biological invasions of the New World by parthenogenetic thiarid snails[END_REF], and many other invasive species [START_REF] Bossdorf | Phenotypic and genetic differentiation between native and introduced plant populations[END_REF].

Admixture

Studies using indirect genetic methods to reconstruct invasion routes have suggested that admixtures between different source populations often occur in invasions, as demonstrated principally in plants (e.g. [START_REF] Rosenthal | Evidence for multiple sources of invasion and intraspecific hybridization in Brachypodium sylvaticum (Hudson) Beauv. in North America[END_REF]) although this scenario has also been reported for a number of invasive animals, including the Cuban lizard [START_REF] Kolbe | Differential admixture shapes morphological variation among invasive populations of the lizard Anolis sagrei[END_REF], the freshwater snail Melanoides tuberculata [START_REF] Facon | Hybridization and invasiveness in the freshwater snail Melanoides tuberculata: hybrid vigour is more important than increase in genetic variance[END_REF], and the harlequin ladybeetle Harmonia axyridis [START_REF] Lombaert | Bridgehead effect in the worldwide invasion of the biocontrol harlequin ladybird[END_REF].

Bridgehead effect

In a number of articles describing invasion routes, successful invasive populations appear to originate from an intermediate population, which is, itself, a successful invasive population [START_REF] Miller | Multiple transatlantic introductions of the western corn rootworm[END_REF][START_REF] Kolbe | Genetic variation increases during biological invasion by a Cuban lizard[END_REF][START_REF] Downie | Locating the sources of an invasive pest, grape phylloxera, using a mitochondrial DNA gene genealogy[END_REF][START_REF] Floerl | The importance of transport hubs in stepping-stone invasions[END_REF][START_REF] Hanfling | mt-DNA sequences and possible invasion pathways of the Chinese mitten crab[END_REF]. In this scenario, the intermediate invasive population at the origin of the secondary invasive populations plays the role of a bridgehead. As described below, this ''invasion bridgehead'', is particularly important for an understanding of how and why invasions occur in agricultural settings. As suggested by Facon et al. [START_REF] Cadotte | Conceptual Ecology and Invasion Biology: reciprocal Approaches to Nature[END_REF], several factors are required to account for the occurrence of an invasion:

Conflicts of interest statement

The authors declared no conflict of interest.