Properties, production, and applications of camelid single-domain antibody fragments

Camelids produce functional antibodies devoid of light chains of which the single N-terminal domain is fully capable of antigen binding. These single-domain antibody fragments (VHHs or Nanobodies®) have several advantages for biotechnological applications. They are well expressed in microorganisms and have a high stability and solubility. Furthermore, they are well suited for construction of larger molecules and selection systems such as phage, yeast, or ribosome display. This minireview offers an overview of (1) their properties as compared to conventional antibodies, (2) their production in microorganisms, with a focus on yeasts, and (3) their therapeutic applications.
Introduction
The field of recombinant antibody technology has rapidly progressed during the last two decades, mainly because of the interest in their human therapeutic use. The ability to select specific human antibodies by display technologies and to improve their affinity, stability, and expression level by molecular evolution has further boosted the field. Whole antibodies are complex molecules that consist of heavy and light chains (Fig. 1a). They contain an N-linked oligosaccharide attached to the second heavy-chain constant domain (CH2) that is essential for antibody effector functions such as antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytolysis (CDC), and for retaining a long serum half-life.
Fig. 1Schematic diagram of conventional (a) and heavy-chain (b) antibodies and fragments thereof. Variable domains derived from the antibody heavy (VH) and light (VL) chains are shaded dark gray and light gray, respectively, whereas constant domains (CH and CL) are not shaded. Note the absence of the light chain and CH1 domain in heavy-chain antibodies. Antibody domains that pair by noncovalent interactions are indicated by overlaying them. The B-subunits of naturally pentamerizing toxins that are used to generate pentabodies are indicated as hatched spheres
Although isolated antibody heavy (Utsumi and Karush 1964) and light chains (Yoo et al. 1967) can retain antigen-binding specificity, their affinity and solubility is often reduced (Ward et al. 1989). However, the paired N-terminal variable domains of heavy (VH) and light (VL) chains are sufficient for antigen binding (Sundberg and Mariuzza 2002). Such antibody fragments can be produced as monovalent antibody fragment (Fab) or as single-chain Fv (scFv) where the VH and VL domains are joined by a polypeptide linker (Fig. 1a). Their production in microbial cells is often cumbersome, especially when producing multivalent formats, because of the requirement for domain association.
The discovery that camelids (bactrian camels, dromedaries, and llamas) produce functional antibodies devoid of light chains (Hamers-Casterman et al. 1993) formed a further breakthrough because their single N-terminal domain (VHH, also referred to as Nanobody®) binds antigen without requiring domain pairing. These heavy-chain antibodies also lack the CH1 domain, which in a conventional antibody associates with the light chain and to a lesser degree interacts with the VH domain (Fig. 1b). Although single-domain antibodies later were also identified in particular cartilaginous fish (Greenberg et al. 1995), most research on the biotechnological application of single-domain antibodies was done using camelids because of their ease of handling, including immunization. Methods to isolate antigen-specific VHHs from immune (Arbabi-Ghahroudi et al. 1997; Van der Linden et al. 2000a), nonimmune (Tanha et al. 2002; Yau et al. 2003; Verheesen et al. 2006), or semisynthetic (Goldman et al. 2006) libraries using phage, yeast, or ribosome display are now well established. For further reading on these topics, we refer to recently published reviews (Muyldermans 2001; Dufner et al. 2006).
Properties
Sequence analysis (Hamers-Casterman et al. 1993; Muyldermans et al. 1994; Vu et al. 1997; Harmsen et al. 2000) and elucidation of the crystal structure (Desmyter et al. 1996, 2001; Spinelli et al. 1996) has revealed several structural features of VHH domains. Similar to conventional VH domains, VHHs contain four framework regions (FRs) that form the core structure of the immunoglobulin domain and three complementarity-determining regions (CDRs) that are involved in antigen binding. This contrasts with shark single-domain antibodies that have a vestigial CDR2 that does not contribute to antigen binding (Streltsov et al. 2004). As compared to human VH domains, the VHH FRs show a high sequence homology of more than 80%, and their 3D structures can be superimposed (Muyldermans et al. 2001; Holliger and Hudson 2005).
The most characteristic feature of VHHs is the presence of amino acid substitutions at four FR2 positions (positions 37, 44, 45, and 47; Kabat numbering) that are conserved in conventional VH domains and that are involved in forming the hydrophobic interface with VL domains. Occasionally, antigen-binding single-domain antibody fragments that lack these characteristic FR2 substitutions are isolated from camelids. These fall into two groups. The low-affinity binders isolated from a nonimmune library originated from conventional antibodies, presumably because of the polymerase chain reaction crossover cloning artifact, as they were linked to the CH1 domain (Tanha et al. 2002). We refer to these as VHH-like conventional VHs. However, such single-domain antibody fragments with conventional-like FR2 sequences that bind antigen with high affinity are isolated from immune libraries with the high efficiency of about 10% (Conrath et al. 2001a; Saerens et al. 2004; Harmsen et al. 2005a, 2007), which equals their presence in unselected libraries (Harmsen et al. 2000). This is not expected when such clones originate from a cloning artifact. Unlike the clones isolated by Tanha et al. (2002), these clones often contain a hydrophilic residue (mostly arginine) at position 103. This substitution is probably important for their single-domain nature (Desmyter et al. 2001) because conventional antibodies contain a highly conserved hydrophobic residue (tryptophan) at this position that contacts VL. This suggests that these represent functional VHH domains derived from recombination of conventional VH gene segments with heavy-chain constant gene segments during B cell maturation. This was confirmed by the absence of the CH1 domain when such VHH domains were reisolated from the original immune repertoire using a CDR3-specific primer (De Haard, unpublished observation). Therefore, we refer to these as conventional-like VHH domains. Although the increased hydrophilicity of VHHs predominantly relies on the aforementioned changes in the former VL interface, some amino acids at positions that form a slightly hydrophobic patch on conventional VH domains that contacts CH1 (Lesk and Chothia 1988) are also changed into hydrophilic residues in VHHs (Muyldermans et al. 1994; Harmsen et al. 2000).
Furthermore, the CDRs of VHHs contain some characteristic features. Firstly, the N-terminal part of CDR1 is more variable (Vu et al. 1997; Harmsen et al. 2000; Nguyen et al. 2000). Secondly, many dromedary VHHs have an extended CDR3 that is often stabilized by an additional disulfide bond with a cysteine in CDR1 or FR2 (Muyldermans et al. 1994) resulting in the folding of the CDR3 loop across the former VL interface (Desmyter et al. 1996). A particular subfamily of llama VHHs (VHH3) also contains an extended CDR3 that is stabilized by an additional disulfide bond with a cysteine at position 50 in FR2. However, VHHs of this subfamily are rarely isolated, and most llama VHHs have CDR3 loops similar in length to those found in human VHs.
VHHs have many advantages for biotechnological applications, which are summarized in Table 1. An important advantage is their high microbial production level (see next section).
Table 1Advantages of camelid single-domain antibody fragments as compared to conventional antibody fragmentsAdvantageMolecular basisFacile genetic manipulationSingle-domain natureIncreased functional size of immune librariesNo decrease in library size because of reshuffling of VL and VH domainsFacile production of multivalent formatsMore flexible linker design and no mispairing of VL and VH domainsFacile production of oligoclonal preparations from single cellsNo mispairing of VL and VH domainsHigh physicochemical stabilityEfficient refolding due to increased hydrophilicity and single-domain natureHigh solubilityIncreased hydrophilicityRecognition of hidden antigenic sitesSmall size and extended flexible CDR3Rapid tissue penetration, fast clearanceSmall sizeWell expressedEfficient folding due to increased hydrophilicity and single-domain natureSee text for references
Several advantages result from their single domain nature. Thus, VHH libraries generated from immunized camelids retain full functional diversity. This contrasts with the diminished diversity of conventional antibody libraries because of reshuffling of VL and VH domains during library construction. As a result, high-affinity antigen-binding VHHs can be isolated by directly screening a limited number of clones from immune libraries without prior selection using display technologies (Frenken et al. 2000; Harmsen et al. 2005b). Furthermore, the single-domain nature facilitates subsequent molecular manipulation. For example, for many applications, it is advantageous to engineer monovalent antibody fragments into multivalent formats to increase functional affinity (termed avidity) or to produce bispecific antibody fragments that can simultaneously bind to different antigens. Such molecules (diabodies, Fig. 1a) can be produced using conventional recombinant antibodies using linkers between the VH and VL domains of a specific length, although this often results in aggregation and reduced affinity because of mispairing of VH and VL domains (Glockshuber et al. 1990; Whitlow et al. 1993). VHHs are more suitable for production of such formats because they allow more flexible linker design, which is important for simultaneous binding of multivalent antigens, without the problems posed by domain mispairing. Thus, several functional trivalent-bispecific VHHs have been successfully produced (Coppieters et al. 2006; Roovers et al. 2007).
The use of mixtures of a limited number of monoclonal antibodies (oligoclonal antibodies) is advantageous over single monoclonal antibodies for particular applications, such as toxin neutralization (Nowakowski et al. 2002). Because of regulatory requirements, such oligoclonals are preferentially produced from single cells. Again, VHHs are predicted to be more suitable for single-cell production of oligoclonals because of the absence of domain mispairing, although this is yet to be demonstrated experimentally.
Contrary to conventional antibodies, VHHs have been shown to remain functional at 90°C (Van der Linden et al. 1999) or after incubation at high temperatures (Van der Linden et al. 1999; Perez et al. 2001). This high apparent stability is mainly attributed to their efficient refolding after chemical or thermal denaturation and to a lesser extent because of an increased resistance against denaturation (Perez et al. 2001; Dumoulin et al. 2002; Ewert et al. 2002). The increased apparent stability is probably due to an increased hydrophilicity of the former VL interface region because a “camelized” human VH fragment that contains several of the hallmark hydrophilic amino acid residues of VHHs was more stable than the original VH fragment (Davies and Riechmann 1995, 1996), whereas “decamelization” of a VHH to mimic a VH domain reduces its thermodynamic stability (Conrath et al. 2005). In addition to these specific mutations, the packing of extended CDR3 loops against this former VL interface contributes to domain stability (Bond et al. 2003). Furthermore, refolding of VHHs only requires domain refolding, whereas conventional antibodies also require association of VH and VL domains.
VHHs can also recognize antigenic sites that are normally not recognized by conventional antibodies such as enzyme active sites (Lauwereys et al. 1998; De Genst et al. 2006) and conserved cryptic epitopes (Stijlemans et al. 2004). This facilitates their use as enzyme inhibitors or in diagnosis of trypanosome infections. The ability to recognize these recessed antigenic sites has been attributed to their smaller size and the ability of the extended CDR3 loop to penetrate into such sites (Desmyter et al. 1996; De Genst et al. 2006). It is interesting to note that this structure–function relation is also observed in a rare example of a broadly reactive human mAb that recognizes the recessed and conserved CD4-binding cavity of human immunodeficiency virus type 1 gp120 by virtue of an extended CDR3 (Zwick et al. 2003). With respect to antigen binding, the single-domain nature could be a disadvantage for binding to small antigens such as haptens and peptides because these normally bind in a groove or cavity at the VH–VL interface (Sundberg and Mariuzza 2002). Indeed, llamas immunized with clenbuterol developed conventional but not heavy-chain antibodies against this hapten (Lange et al. 2001). However, hapten- and peptide-binding VHHs have been successfully isolated using strong selection systems (Spinelli et al. 2000; Yau et al. 2003; Alvarez-Rueda et al. 2007; Harmsen et al. 2007). The affinities of VHHs are generally comparable to those of conventional antibody fragments (Muyldermans et al. 2001). Occasionally, VHHs with affinity constants (KD) as low as 100 pM are isolated (Saerens et al. 2004; De Genst et al. 2006; Harmsen et al. 2006), which equals the affinity ceiling proposed for natural antibodies (Sundberg and Mariuzza 2002).
Because of their small size of about 15 kDa, VHHs rapidly pass the renal filter, which has a cutoff of about 60 kDa, resulting in their rapid blood clearance. In addition, the small size results in a fast tissue penetration. This is advantageous for targeting of VHHs coupled to toxic substances to tumors (Cortez-Retamozo et al. 2004), in vivo diagnosis using imaging, and treatment of snake bites (Harrison et al. 2006). However, for other therapeutic applications, such as treatment of infectious or inflammatory diseases, the short serum half-life of about 2 h (Cortez-Retamozo et al. 2002; Harmsen et al. 2005a) is a disadvantage.
Production in microorganisms
Although a fully active nonglycosylated IgG was recently produced at high level in Escherichia coli, most functional complete antibodies can only be efficiently produced using mammalian cells, especially when their appropriate glycosylation is required for therapeutic applications. However, antibody fragments that lack the Fc with its N-linked oligosaccharide are preferably produced in microbial systems (Arbabi-Ghahroudi et al. 2005). These have a shorter development time from gene to product and require simple well-established fermentation conditions that can be performed on large-scale resulting in costs of goods that can be as low as $1 per gram heterologous protein (Estell 2006). Most large-scale microbial production systems are based on E. coli, yeasts, or filamentous fungi. Production in E. coli can be done by secretion into the oxidizing periplasmic space or expression in the reducing cytosol. The latter requires the often cumbersome refolding of antibody fragments (Arbabi-Ghahroudi et al. 2005). Using eukaryotic microbial hosts, antibody fragments are generally produced by targeting to the secretory pathway. This enables efficient disulfide bond formation, addition of N-linked oligosaccharide, and secretion of soluble, correctly folded product to the culture medium.
VHHs have often been produced in E. coli (Arbabi-Ghahroudi et al. 1997; Rahbarizadeh et al. 2005). There is only one example of VHH production in filamentous fungi, which resulted in limited proteolytic degradation of the secreted product (Joosten et al. 2005) because of the high levels of proteases secreted by filamentous fungi (Gerngross 2004). VHHs have also often been produced in the yeast Saccharomyces cerevisiae (Frenken et al. 2000; Thomassen et al. 2002; Van der Vaart 2002). VHH production by the favored yeast expression host Pichia pastoris was only recently described (Rahbarizadeh et al. 2006). Occasionally, yeast-produced VHHs are N-glycosylated (Frenken et al. 2000; Harmsen et al. 2005a). This can affect antigen binding (Van der Vaart et al. 2006). Furthermore, it could complicate their therapeutic use because the addition of yeast-specific high-mannose oligosaccharides results in a high immunogenicity and decreased serum half-life because of binding to specific mannose receptors on cells of the reticulo-endothelial system (Sethuraman and Stadheim 2006).
Although VHHs are generally well produced in microorganisms, the production level of different clones can vary by a factor of 100 (Frenken et al. 2000; Harmsen et al. 2005b; Van de Laar et al. 2007). Several VHH sequence patterns can be associated with their production level. First, the presence of a potential N-linked glycosylation site often increases production levels in yeast (Sagt et al. 2000). Second, in our experience (Harmsen, unpublished observations), conventional-like VHHs are generally produced at reduced levels in yeast. This contrasts with the reported efficient production in E. coli of VHH-like VHs (Tanha et al. 2002) but is consistent with the increased production level of “camelized” conventional VH domains in E. coli (Davies and Riechmann 1995). Third, the presence of unpaired C-terminal cysteines reduces expression levels (Simmons et al. 2006). Fourth, replacement of hydrophobic residues of conventional VH domains normally interacting with CH1 increased scFv production in E. coli (Nieba et al. 1997), suggesting that the hydrophilic mutations that naturally occur at these positions in VHHs also contributes to their high expression level. However, there are many examples of VHHs that differ by only a few amino acids and are produced at highly variable levels where the exact amino acid change responsible for the difference in production level is difficult to predict (Frenken et al. 2000; Harmsen et al. 2005b). Furthermore, without such knowledge, VHH production can be improved by random molecular evolution using deoxyribonucleic acid shuffling (Van der Linden et al. 2000b), as has often been done for conventional antibody fragments (Dufner et al. 2006). The high refolding capability of VHHs, which is a consequence of their sequence, has also been correlated with a high production level in E. coli (Jespers et al. 2004; Olichon et al. 2007).
In addition to the nature of the VHH, host factors affecting VHH production have been identified. In baker’s yeast, the specific VHH production rate is correlated with growth rate (Thomassen et al. 2005) and can be up to fivefold increased by growing on ethanol as the carbon source (Van de Laar et al. 2007). Supplementation of the medium with sorbitol, casamino acids, or ethylenediamine tetraacetic acid improves VHH production by P. pastoris (Rahbarizadeh et al. 2006).
In addition to monovalent VHHs, several expression formats for the production of VHH multimers have been described (Fig. 1b). These include genetic fusions of two (Conrath et al. 2001b; Harmsen et al. 2005a) or three VHHs (Coppieters et al. 2006; Roovers et al. 2007) that either recognize different antigens or the same repeating antigen to increase functional affinity. Although such VHH fusions are less efficiently produced than their monovalent versions, their production level exceeds that of their conventional-antibody-based fusion counterparts without aggregation or low solubility. However, antigen binding by the C-terminal VHH in such fusions can be compromised (Conrath et al. 2001b) presumably because of steric hindrance by the N-terminal VHH. The avidity of VHHs has also been strongly increased using genetic fusions to the B-subunits of an E. coli toxin that self-assembles into a homopentamer (Zhang et al. 2004), resulting in pentameric recombinant antibodies (“pentabodies,” Fig. 1b).
VHHs on their own cannot recruit effector functions such as ADCC and CDC. This limits their therapeutic application. Although such effector functions can be indirectly recruited using bispecific (conventional) antibody fragments binding to host immunoglobulin (Holliger et al. 1997), it may be more efficient to recruit these functions by fusing VHHs to host Fc domains. Production of such functional antibodies requires the correct glycosylation of the CH2 domain, which until recently could only be accomplished using higher eukaryotic cells (Nguyen et al. 2003) but not by microbial production. However, this may now be feasible using P. pastoris strains with an engineered glycosylation machinery that are able to produce proteins with a specific human glycoform (Hamilton et al. 2006). Furthermore, transgenic mice containing hybrid llama/human antibody loci that contain llama V regions and human D, J, and C regions have recently been used to generate human heavy-chain antibodies in mice (Janssens et al. 2006).
Therapeutic applications
Although VHHs are highly suited for applications that require a high stability, such as use in shampoo for the prevention of dandruff (Dolk et al. 2005), as capturing reagents in immunoaffinity purification (Verheesen et al. 2003), or use in biosensors (Pleschberger et al. 2004), we would like to focus on their therapeutic applications, which are more challenging. Several VHHs are now being studied for use in various disease areas, including oncology (Revets et al. 2005) and in infectious, inflammatory, and neurodegenerative diseases (Table 2).
Table 2Examples of therapeutic applications of camelid VHHsDiseasePathogenTarget antigenVHH valency for disease targetAdditional fusion partnerReferenceSleeping sicknessTrypanosomesVSG oligomannoseMonovalentApolipoprotein L-IBaral et al. 2006Infant diarrheaRotavirusUnknownMonovalentNoneVan der Vaart et al. 2006Infant diarrheaRotavirusUnknownMonovalentLactobacillus cell-surface anchorPant et al. 2006Piglet diarrheaE. coliF4 fimbriaeMonovalentNoneHarmsen et al. 2006CariesS. mutansI/II adhesionMonovalentNoneKruger et al. 2006FMDFMD virusVP1MonovalentPEGHarmsen et al. 2007SepsisN. meningitidisLPSMonovalentNoneEl Khattabi et al. 2006Cancer–CEAMonovalentβ-LactamaseCortez-Retamozo et al. 2004Cancer–EGF receptorBivalentAnti-albumin VHHRoovers et al. 2007Rheumatoid arthritis–TNFαBivalentAnti-albumin VHHCoppieters et al. 2006Brain disorders–α (2,3)-SialoglycoproteinMonovalentNoneMuruganandam et al. 2002Neurodegenerative diseases–BaxMonovalentNoneGueorguieva et al. 2006
VHHs are especially suited for oral immunotherapy because of their resistance against extremes of pH and the capacity to bind to the target at high concentrations of chaotropic agents (Dumoulin et al. 2002, 2003). Administration to piglets of a VHH that effectively prevents intestinal attachment of E. coli bacteria that cause diarrhea resulted in poor in vivo protection (Harmsen et al. 2005b) because of degradation by gastrointestinal proteases (Harmsen et al. 2006). However, by selection for proteolytic stability, a VHH could be isolated from the original library that was not degraded in vivo (Harmsen et al. 2006). VHHs that successfully prevented diarrhea caused by rotavirus in a mouse model were similarly selected for resistance against the acidic environment of the stomach (Van der Vaart et al. 2006). Alternatively, VHH proteolysis can be prevented by local VHH production using natural gut commensal bacteria. Thus, diarrhea could also be prevented by lactobacilli that produce rotavirus-neutralizing VHHs fused to a cell surface anchor (Pant et al. 2006). Treatment of caries, caused by Streptococcus mutans, with VHHs conferred only limited protection (Kruger et al. 2006). Because these VHHs should function in the oral cavity, the low level of protection cannot be due to proteolytic VHH degradation within the gastrointestinal tract.
The short serum half-life because of a rapid renal clearance limits the efficacy of VHHs in many parenteral applications. Therefore, VHHs have been targeted to normally long-lived serum proteins such as albumin (Coppieters et al. 2006; Roovers et al. 2007) or immunoglobulin (Harmsen et al. 2005a) using bispecific VHHs recognizing these serum proteins in addition to the therapeutic target, resulting in half-lives that equal the half-life of albumin (2 days in mice) and immunoglobulin (9 days). An alternative well-known approach to increase serum half-life of proteins is the chemical addition of polyethylene glycol (PEG). Such PEGylation of foot-and-mouth disease (FMD) virus-neutralizing VHHs not only increased serum half-life but also increased in vitro neutralizing potency to levels above that of the hyperimmune serum (Harmsen et al. 2007). However, in contrast to the full protection afforded by the hyperimmune serum, these VHHs poorly protected guinea pigs from FMD viral challenge infection, suggesting that Fc-mediated effector functions are required for efficient in vivo protection (Harmsen et al. 2007).
Nevertheless, many diseases were successfully treated with VHHs in the absence of Fc-mediated effector functions. These VHHs either are used as targeting devices for toxic enzymes or block a specific molecular interaction. For example, sleeping sickness was successfully treated with VHHs that bind to a trypanosome coat protein and were fused to the apolipoprotein L-1 enzyme, resulting in trypanosome lysis (Baral et al. 2006). In oncology, a VHH directed against carcinoembryonic antigen was used for targeting the genetically fused β-lactamase to tumor cells. This enzyme then converts an injected nontoxic prodrug into a toxic drug in the vicinity of the targeted tumor cells, leading to their killing (Cortez-Retamozo et al. 2004). Several VHH therapies are also being developed for treatment of oncology or inflammatory diseases based on blocking molecular interactions. VHHs binding to epidermal growth factor receptor (EGFR) can block epidermal growth factor (EGF) binding to its receptor, which can be used to treat solid tumors (Roovers et al. 2007). Tenfold more potent EGFR-binding VHHs could be obtained by construction of bivalent formats. It is interesting to note that the recently approved conventional antibody Panitumumab directed against EGFR also blocks EGF binding and is expected to give poor ADCC and CDC (Reichert and Valge-Archer 2007). Furthermore, by blocking receptor interaction, VHHs binding to tumor necrosis factor-α can be used for treatment of rheumatoid arthritis (Coppieters et al. 2006). The potency of bivalent formats was 500-fold increased as compared to monovalent VHHs and even exceeded the potency of clinically used conventional antibodies both in vitro and in a murine arthritis model. Similarly, lipopolysaccharide (LPS)-binding VHHs were isolated that block LPS binding and signaling to host cells for treatment of sepsis (El Khattabi et al. 2006).
The potential immunogenicity of VHHs could compromise their parenteral therapeutic use, especially in treatments that require repeated injections. Until now, multiple injections of VHHs have not shown any immunogenicity in mice, as assessed by the presence of specific antibodies, T cell proliferation, or cytokine levels (Cortez-Retamozo et al. 2002; Coppieters et al. 2006). This could rely on their high sequence homology to conventional VH domains and on their high stability because aggregation of proteins is known to increase immunogenicity (Hermeling et al. 2004). If necessary, technologies developed to decrease immunogenicity of mouse monoclonal antibodies (Presta 2006) could also be applied to VHHs. Alternatively, immunogenicity could be reduced by the use of conventional-like VHHs, which have an even higher structural homology to conventional VH domains.
For their use in targeting drugs across the blood–brain barrier (BBB) into the brain, VHHs were selected that transmigrate the human BBB in an in vitro model and accumulate in the brain after intravenous injection into mice (Muruganandam et al. 2002). These could be used for treatment of neurological disorders. Finally, Bax-specific VHHs have been expressed in the cytoplasm, resulting in so-called intrabodies, to prevent oxidative-stress-induced apoptosis that is implicated in several neurodegenerative diseases (Gueorguieva et al. 2006). Because of their stability, VHHs are especially suited for intrabody production because this requires expression in the reducing environment of the cytoplasm (Gueorguieva et al. 2006; Rothbauer et al. 2006).
Conclusions
Since the discovery of heavy-chain antibodies in 1993, the field of single-domain antibody fragments has been rapidly growing. VHHs have many advantages for biotechnological applications. They can be economically produced in microorganisms and have a high stability. Furthermore, they are highly suited for expression as multivalent, including bispecific, formats or as enzyme fusions. This permits a plug-and-play approach, where, depending on the target, biology potency can be increased by multivalent constructs or bispecific VHH recognizing two different targets can be made. This also enables the tailor-made design of serum half-life using site-directed PEGylation or by targeting to long-lived serum proteins using bispecific VHHs. Although fusions of targeting VHHs to Ig-binding VHHs or Fc can be used to recruit effector functions most current research on VHHs focuses on therapeutic applications where such effector functions are not required. Finally, conventional whole antibodies occasionally give side effects because of their bivalent nature, which can result in target cross-linking, or the presence of the Fc region. Evidently, such side effects are not expected to occur using monovalent VHHs. This, however, is yet to be confirmed as the first VHH has entered phase I clinical trials in 2007 (http://www.ablynx.com).

single-domain
yeast
microbial production
glycosylation


Performances in cerebellar and neuromuscular transmission tests are correlated in migraine with aura

In previous studies, we described subclinical abnormalities of neuromuscular transmission and cerebellar functions in migraineurs. The aim of this study was to search if these two functions are correlated in the same patient. Thirteen migraineurs [five without aura (MO) and eight with aura (MA)] underwent both stimulation-SFEMG and 3D-movement analysis. Single fiber EMG (SFEMG) results were expressed as the “mean value of consecutive differences” (mean MCD). Precision of arm-reaching movements (measured with an infrared optoelectronic tracking system) was expressed as the average deviation in the horizontal plane. Median values of mean MCD and mean horizontal deviation were not different between MO and MA. However, in MA, but not in MO, both variables were positively correlated. Thus, we conclude that neuromuscular transmission and cerebellar functions are correlated in the same patient when affected by migraine with aura. We suggest that this correlation might be due to a common molecular abnormality.
Introduction
Migraine is a paroxysmal neurological disorder with a high prevalence in the general population. There is strong epidemiological and genetic evidence that genetic factors play a major, though variable role, in its pathogenesis. Migraine patients are characterized between attacks on various CNS-evoked responses by a deficit of habituation [1, 2], which may have a familial character [3–5]. The lack of habituation may play a role in migraine pathogenesis [6]. By contrast, other mild abnormalities have been identified that are unlikely to play a pathogenic role: a decreased safety factor at the neuromuscular junction on single fiber EMG (SFEMG) [7–9] and subclinical cerebellar hypermetria in the horizontal plane on opto-electronic analysis of upper arm-reaching movements [10]. It was suggested that these two abnormalities might reflect the same underlying genetic abnormality. If this is correct, one may expect that in the same individual they be of a similar degree. We have therefore compared in the same migraine patient the SFEMG and 3D-movement analysis.
Materials and methods
Patients
Thirteen migraine patients (ICHD-II) [11] were recruited from the Headache Clinic of the Headache Research Unit in Liège, Belgium. Eight patients suffering from typical aura with migraine headache (MTA–ICHD-II code 1.2.1; three women and five men; median age 28.5 years; range 13–67) and five patients with migraine without aura (MO–ICHD-II code 1.1; three women and two men; median age 40.0 years; range 31–51) underwent both stimulation-SFEMG and 3D-movement analysis. All patients were right-handed. None of them had any other medical condition detectable by history and clinical examination; none was taking drugs on a regular basis, nor had taken any drug within 3 days before the recordings. The recordings took place at least 3 days after and before an attack (checked by telephone-interview).
The study was conducted after approval of our Institution’s ethics committee and performed in accordance with the ethical standards of the 1964 Declaration of Helsinki, with the understanding and consent of each involved subject.
SFEMG recordings
A Nicolet Viking IV device (Nicolet® Biomedical, Madison, Wisconsin, USA) was used for stimulation single fiber electromyography [12]. Single muscle fiber activity was recorded with 25-mm-long single fiber needles (Medelec Neurodiagnostic Accessories, ref: 16829, Witney, Oxfordshire, UK), and the motor nerve was stimulated with Nicolet teflon-insulated monopolar needles. We stimulated suprathreshold the motor branch of the radial nerve and assessed the variability in latency, i.e. the jitter (Fig. 1), of single fiber action potentials in m. extensor digitorum communis (EDC) of the right arm. Stimulations–acquisitions were 100 per muscle fiber, and stimulation rate was 10 Hz. We recorded 25 EDC muscle fibers per patient. Off-line analyses of recordings were performed, and on average 18 artifact-free EDC muscle fibers per patient were selected to assess their mean MCD. Results were expressed as the “mean value of consecutive differences” (MCD) of successive interpotential intervals, as usual in SFEMG studies.
Fig. 1Figures representing a normally (on the top) and an abnormally (on the bottom) jittered fibers
3D-movement analysis
Movements were recorded at 100 Hz in three dimensions (3D) using an infrared optoelectronic-tracking-system (ELITE™, Milan, Italy) with a reflective marker attached to the tip of the index finger and another marker to the movement target.
Participants were seated with the target in the medio-sagittal plane on eye level. They were instructed to start with the right arm extended to the right, to touch the target with high precision (but fast) without any trunk movement, to go back to the starting position and to repeat above movements, in a given pace over 15 s (one trial) to result in 8–10 movements (Fig. 2). The used Cartesian coordinate system is head-fixed with a nasooccipital, a horizontal and a vertical axis and the origin in the target. Since in our previous study [10] abnormalities in migraineurs were most pronounced in the horizontal plane, we limited ourselves to an analysis of the mean deviation in the horizontal plane (in millimeters), measured over four trials, each consisting of 8–10 arm movements.
Fig. 2Experimental setup for the movement task
Quantitative variables in each group of migraine patients (MO and MTA) were expressed as medians. Differences between groups were analysed with the Mann–Whitney U test. The Spearman rank order correlation test was used to compare the values of the mean MCD on SFEMG and the mean horizontal deviation on 3D-movement analysis.
Results
On SFEMG, the median value for mean MCD was not significantly different between MO (16.05 μs; range 9.50–22.93) and MTA (18.91 μs; range 11.50–24.55). The median value of mean horizontal deviation was 4.01 mm (range −3.25 to 18.12) in MO and 10.74 mm (range 1.60–17.03) in MTA, a nonsignificant difference.
In MO, there was no significant correlation between mean MCD and mean horizontal deviation. By contrast, in MTA, both variables were positively correlated (R = 0.71, P = 0.046) (Fig. 3).
Fig. 3Scatter plot relating mean MCD on SFEMG (X axis, μs) and mean horizontal deviation on 3D analysis of a reaching arm movement (Y axis, mm), and linear regression lines. Migraine without aura patients (MO): squares, dashed line. Migraine with aura patients (MTA): triangles, continuous line
Discussion
Our within-patient analysis shows that, in migraine with aura, the mean MCD on SFEMG increases significantly with the degree of horizontal deviation in a visually guided reaching movement. This suggests that, in subgroups of migraine patients, neuromuscular transmission (NMT) performance and control of ballistic movements by the lateral cerebellum are similarly influenced by a common biochemical and/or neural mechanism.
Although no gene mutations have been identified till now, the genetic load is thought to be higher in the pathogenesis of migraine with aura than that without aura [13]. In familial hemiplegic migraine 1 (FHM1) [14], mutations have been found in the CACNA1A gene, which codifies for the main subunit of P/Q-type Ca2+ channels, heavily concentrated at the neuromuscular junction and in the cerebellum [15–18]. Thus, we initially hypothesized that the NMJ and cerebellar abnormalities found in some migraine patients might be due to dysfunctioning Ca++ channels. However, SFEMG studies were normal in FHM1 [19], and mutations in the CACNA1A gene are not found in patients suffering from the common forms of migraine, with or without aura [20]. Some evidence that the CACNA1A gene may be involved in migraine with typical aura [21], and the facts that NMT impairment can be found in episodic ataxia type 2, an allelic disorder of FHM1 [22], and normalizes after treatment with acetazolamide [23] are still in favor of a possible involvement of Ca++ channels, particularly in migraine with aura.
Other proteins implicated in migraine pathophysiology may constitute a common link between neuromuscular junctions (NMJ) and the cerebellum. This is unlikely for the alpha-2 subunit of the Na-K ATPase, of which the gene ATP1A2 is mutated in FHM2 [24], because the alpha-2 isoform is found at intracellular membranes [25] and not at the NMJ, where the major role is played by the alpha-3 isoform [26]. Moreover, SFEMG results were found normal in FHM2 patients [19].
By contrast, a different group of calcium channels, the R-type, influence neuromuscular transmission and can compensate for dysfunctioning P/Q channels [27]. Moreover, they seem to play a role in cerebellar functions [28]; thus, their activity may influence in parallel NMJ and cerebellar performances, similarly to what we found in the subgroup of migraineurs with aura. The CACNA1E gene, which codes for the alpha-1E subunit of R-type channels, is precisely located on locus 1q31, for which significant linkage was found in the common forms of migraine [29], and a single nucleotide polymorphism was more frequent in subgroups of patients affected by migraine with aura [30].
In conclusion, the correlation between NMJ and cerebellar performances that we have found in migraine with aura patients might be due to a common genetically determined molecular mechanism, possibly influencing ion channels functions, but this has still to be proven by appropriate genetic studies.

migraine
genetics
neuromuscular junction
cerebellum


Prey and Non-prey Arthropods Sharing a Host Plant: Effects on Induced Volatile Emission and Predator Attraction

It is well established that plants infested with a single herbivore species can attract specific natural enemies through the emission of herbivore-induced volatiles. However, it is less clear what happens when plants are simultaneously attacked by more than one species. We analyzed volatile emissions of lima bean and cucumber plants upon multi-species herbivory by spider mites (Tetranychus urticae) and caterpillars (Spodoptera exigua) in comparison to single-species herbivory. Upon herbivory by single or multiple species, lima bean and cucumber plants emitted volatile blends that comprised mostly the same compounds. To detect additive, synergistic, or antagonistic effects, we compared the multi-species herbivory volatile blend with the sum of the volatile blends induced by each of the herbivore species feeding alone. In lima bean, the majority of compounds were more strongly induced by multi-species herbivory than expected based on the sum of volatile emissions by each of the herbivores separately, potentially caused by synergistic effects. In contrast, in cucumber, two compounds were suppressed by multi-species herbivory, suggesting the potential for antagonistic effects. We also studied the behavioral responses of the predatory mite Phytoseiulus persimilis, a specialized natural enemy of spider mites. Olfactometer experiments showed that P. persimilis preferred volatiles induced by multi-species herbivory to volatiles induced by S. exigua alone or by prey mites alone. We conclude that both lima bean and cucumber plants effectively attract predatory mites upon multi-species herbivory, but the underlying mechanisms appear different between these species.
Introduction
Plants can employ arthropod natural enemies and defend themselves against herbivorous insects and mites. Such arthropod natural enemies can have an impact on local herbivore populations and thereby also on plant fitness (Sabelis and van der Meer 1986; Dicke and Sabelis 1989; Pels and Sabelis 1999; van Loon et al. 2000; Fritzsche Hoballah and Turlings 2001; Kessler and Baldwin 2001). Plants can promote the effectiveness of natural enemies by providing alternative food, shelter, or volatiles (Price et al. 1980; Dicke and Sabelis 1988). The emission of herbivore-induced plant volatiles (HIPV) has been demonstrated in several families (e.g., Dicke et al. 1990a; Turlings et al. 1990; van den Boom et al. 2004; van Poecke and Dicke 2004). Many parasitoid wasps, predatory mites, and predatory insects use HIPV to locate their prey or hosts (reviewed in Dicke 1999; Dicke and Vet 1999; Sabelis et al. 1999). HIPV are thus thought to have an important influence on the interactions between plants, herbivores, and natural enemies in food webs (Turlings et al. 1995; Dicke and Vet 1999).
To date, studies on the production of HIPV by plants and the responses of natural enemies to these have been carried out mostly for systems with one species of plant, herbivore, and natural enemy. Yet, in the field, most plants are likely to be attacked by several herbivore species, or by herbivores and pathogens, at the same time. It is difficult to predict whether or not changes in HIPV blends upon multi-species herbivory affect attraction of natural enemies, and in what direction. Several studies have attempted to fill this gap by analyzing the chemical composition of volatile blends upon multi-species infestation of plants (Shiojiri et al. 2001; Cardoza et al. 2002; Rodriguez-Saona et al. 2003; Rostás et al. 2006; Moayeri et al. 2007; Rasmann and Turlings 2007; Soler et al. 2007). For instance, Shiojiri et al. (2000) showed that the parasitoid wasp Cotesia plutellae prefers the HIPV blend induced by its host Plutella xylostella to the blend induced by multi-species herbivory by its host and the non-host caterpillar Pieris rapae although volatile blends are similar (Shiojiri et al. 2001). Cotesia glomerata, on the other hand, prefers HIPV blends induced by its host P. rapae and non-host caterpillars to blends induced by one caterpillar species feeding alone (Shiojiri et al. 2000; Vos et al. 2001). Cotesia marginiventris and Microplitis rufiventris do not discriminate between the multi-species-induced HIPV blend of plants infested with their host (beet armyworm caterpillars) and a plant pathogen or plants infested with their host alone, although the pathogen significantly suppresses volatile induction (Rostás et al. 2006). Similarly, C. glomerata does not discriminate between mustard plants infested with a root herbivore and its host, or plants infested with its host alone (Soler et al. 2007). The parasitoid C. marginiventris and the entomopathogenic nematode Heterorhabditis megidis exhibited a reduced attraction to volatiles from maize plants that were infested by a non-host in addition to their host, although the non-host was feeding on different tissues from the host, i.e., leaves vs. roots (Rasmann and Turlings 2007).
In this study, we investigated the effects of multi-species herbivory by spider mites (Tetranychus urticae) and beet armyworm caterpillars (Spodoptera exigua) on volatile emissions by two different plant species: lima bean (Phaseolus lunatus) and cucumber (Cucumis sativus), and evaluated the responses of Phytoseiulus persimilis, a specialist natural enemy of spider mites. Both herbivore species are known to induce volatile emission in several plant species, and remarkable differences in HIPV blend composition have been found in lima bean infested by either of these herbivores (e.g., Ozawa et al. 2000; Schmelz et al. 2003; de Boer et al. 2004). We statistically compared volatile emission by multi-species-infested plants with the sum of volatile emissions of plants infested with either herbivore species alone to evaluate the presence of potentially antagonistic or synergistic effects among signal transduction pathways (e.g., Engelberth et al. 2001). Plant responses to multi-species herbivory may depend on feeding modes of the herbivore species involved (Walling 2000), but whether or not plant species differ in these responses is unknown. Therefore, we compared two plant species in their relative responses to multi-species vs. single-species herbivory by using the same two herbivore species to attack both plant species. We also studied the olfactory responses of predatory mites (P. persimilis). In nature, plants are commonly attacked by more than one herbivore. Knowledge on natural enemy responses to volatile blends induced by multi-species vs. single-species herbivory is essential to understand whether differences in volatile composition are relevant in an ecological context.
Methods and Materials
Plants, Herbivores, and Predators Lima bean plants (Phaseolus lunatus L. cv Sieva) and cucumber plants (C. sativus L. cv Lange Groene Giganten) were grown in a greenhouse at 20–30°C, 50–70% R.H. and 16 hr of light. Bean plants were used when their primary leaves had unfolded (and were about 8–12 cm wide), which was 10–15 d after sowing. Cucumber plants were used when their first two leaves were about 8–12 cm wide, which was 15–35 d after sowing.Two-spotted spider mites, Tetranychus urticae Koch (Acari: Tetranychidae), were reared on lima bean plants in a greenhouse under the same conditions as uninfested plants. Eggs of the beet armyworm Spodoptera exigua Hübner (Lepidoptera: Noctuidae) were obtained from a colony reared on artificial diet in the laboratory of Virology, Wageningen University, The Netherlands (Smits et al. 1986). Two to three batches of eggs were placed in a Petri dish together with a lima bean or cucumber leaf in a growth chamber at 23 ± 1°C. Larvae were used in experiments within 24 hr of hatching.A colony of predatory mites, Phytoseiulus persimilis Athias-Henriot (Acari: Phytoseiidae), was kept on spider mite-infested lima bean leaves in a growth chamber at 23 ± 1°C, 50–70% R.H. and continuous light. To obtain females of the same age for olfactometer experiments, gravid females were allowed to oviposit on a spider mite-infested bean leaf for 2 d. The female offspring were used in experiments 9 d after initiation of the colonies, i.e., 1–2 d after their final molt. Before the experiments, females were kept individually in 1.5-ml microcentrifuge tubes with a small droplet of water, for 24 ± 2 hr at 23 ± 1°C.
Plant Treatments Treatments were applied to the two oldest leaves of lima bean or cucumber plants 3 d before an experiment. Plants were incubated in plastic cages in a climate-controlled chamber at 24 ± 1°C, 50–70% R.H. and 16L:8D, each treatment in a separate cage. We used the following four treatments:
T. urticae: Adult female spider mites were transferred to experimental plants. Twenty mites per leaf were used on lima bean, and 100 mites per leaf were used on cucumber because the same number of spider mites leads to lower attraction of P. persimilis to cucumber than to lima bean plants (Dicke et al. 1990b).S. exigua: Newly hatched S. exigua larvae were placed in clip cages to keep caterpillars on the leaves (clip cages were made of two plastic cylinders, 2.5 cm diam., and a hairpin; the upper side was covered with gauze and the underside with Parafilm). Two larvae in one clip cage were used per leaf. Clip cages were moved daily to a new position on the same leaf, and any missing larvae were replaced. The clip cages were supported by sticks to prevent the leaves from bending and incurring damage due to the weight of the cages.Multi-species herbivory: Two newly hatched S. exigua larvae in a clip cage and 20 (lima bean) or 100 (cucumber) T. urticae females were placed on each leaf.Uninfested: Plants without herbivores were treated in the same way as plants of the other treatments. This treatment was only used for volatile collections of cucumber. We did not analyze volatile emission from uninfested lima bean plants because it is documented that they emit low amounts of volatiles (e.g., de Boer et al. 2004).When clip cages were used to keep caterpillars in place, T. urticae-infested or uninfested plants were treated in the same way with empty clip cages to avoid any cage effects.
Volatile Collections and Analyses Just before volatile collections, plants were cut above the soil line. Stems of individual plants were wrapped in moist cotton and aluminum foil to prevent them from wilting during volatile collection, which lasted at most 4 hr. All clip cages, caterpillars, and their products were removed. Five plants of one treatment were transferred to a 5-l glass vessel. A viton O-ring and a metal clamp were used to attach the glass lid to the vessel to make it airtight. Purified air (filtered through silica, a molecular sieve, activated charcoal, and 90 mg Tenax) was split into two air streams of about 100 ml/min each and led into the vessels through teflon tubing. Volatiles from two treatments were collected simultaneously in parallel. The system was purged for 30 min before attaching a collection tube with Tenax (90 mg for bean samples, 200 mg for cucumber samples) to the air outlet in the lid of the vessel. Bean volatiles were collected for 25 min, cucumber volatiles were collected for 3 hr. For lima bean, volatile collection was repeated four times for each treatment, except for uninfested from which volatiles were not sampled. For cucumber, volatile collections were replicated seven times for the uninfested, six times for T. urticae, and five times for S. exigua and multi-species herbivory each. One sample of the T. urticae and one of the S. exigua treatments of cucumber were lost during the analysis.Volatiles from lima bean and cucumber were analyzed on two different gas chromatography-mass spectrometry (GC-MS) systems.
Lima bean Volatiles were released from Tenax traps with a thermodesorption cold trap setup (Chrompack, Middelburg, The Netherlands) by heating at 250°C for 10 min, with a He-flow of 12 ml/min. Desorbed compounds were collected in the cold trap at −90°C. Volatiles were injected in splitless mode into the DB5 column (60 m × 0.25 mm ID, 0.25 μm film thickness) by heating the cold trap to 220°C. After an initial column temperature of 40°C for 4 min, the temperature was raised to 280°C at 4°C/min. The column was directly coupled to the ion source of a Finnigan MAT 95 mass spectrometer, which was operating in the 70-eV electron impact (EI) ionization mode with scanning from mass 24 to 300 at 0.5 scan/sec. Compounds were identified by comparison of mass spectra with those in the NIST 98 library and in the Wageningen Mass Spectral Database of Natural Products, and by checking the retention indices.
Cucumber Volatiles were released from Tenax traps with a thermodesorption cold trap setup (Markes, UK) by heating at 200°C for 10 min, with a He-flow of 30 ml/min. The desorbed volatiles were collected in the cold trap at −10°C. Volatiles were injected in splitless mode into the RTX-5Silms column (Restec, 30 m × 0.32 mm ID, 0.33 μm film thickness) by heating of the coldtrap to 270°C. After an initial column temperature of 40°C for 2 min, the temperature was raised to 95°C at 3°C/min, then to 165°C at 2°C/min, and subsequently to 250°C at 15°C/min. The column was directly coupled to the ion source of a Finnigan quadrupole mass spectrometer, which was operated in the 70-eV EI ionization mode and scanned from mass 33 to 300 at three scans/sec. Compounds were identified by comparison of mass spectra with those in the NIST 98 and Wiley 7th edition spectral libraries, and by checking the retention indices.
Analyses Not all compounds were detected in every replicate of every treatment. We therefore added one peak area unit to all measurements for lima bean and 10 units to all measurements for cucumber. These were the lowest recorded non-zero values for bean and cucumber, respectively. This enabled us to plot volatile emission on a log scale (Figs. 1a and 2a), and to determine ratios among treatments (see below).
Fig. 1Effect of multi-species herbivory on volatile emission by lima bean plants. (a) Volatile emission (mean + SE peak area units) upon single herbivory by Tetranychus urticae (20 mites per leaf) (open bars), single herbivory by Spodoptera exigua (two larvae per leaf) (filled bars), or multi-species herbivory (hatched bars). Asterisks indicate compounds that were significantly affected by herbivore treatment (P < 0.05, Kruskal–Wallis tests). (b) Ratio of emission rates upon multi-species herbivory to the sum per volatile emitted by the two single herbivore treatments (see “Methods and Materials” for detailed explanation). Symbols indicate mean ratio, and error bars indicate upper and lower 95% confidence limits. Asterisks indicate compounds that were significantly more strongly induced by multi-species herbivory than by the sum of T. urticae and S. exigua (lower 95% confidence limit larger than 0). N = 4 for all treatments. Compound numbers: (1) (Z)-3-hexen-1-ol, (2) methyl salicylate, (3) (E)-β-ocimene, (4) (E)-4,8-dimethyl-1,3,7-nonatriene, (5) (3E,7E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene, (6) p-mentha-1,3,8-triene, (7) (Z)-3-hexen-1-ol acetate, (8) 2-methylbutanal-O-methyl oxime, (9) 3-methylbutanal-O-methyl oxime, (10) linalool, (11) 1-octen-3-ol, (12) hexyl acetate, (13) limonene, (14) β-caryophyllene, (15) nonanal, (16) indole, (17) (E)-2-hexen-1-ol acetate, (18) 3-pentanone, (19) 3-octanone, (20) 2-methylbutanal nitrile, (21) (Z)-β-ocimene, (22) rose furan, (23) unknown 95B, 150, (24) 3-methylbutanal nitrile, (25) unknown 91B, 148, (26) unknown 41, 69B, 164, (27) 2-methylpropanal-O-methyl oximeFig. 2Effect of multi-species herbivory on volatile emission by cucumber plants. (a) Volatile emission (mean ± SE peak area units) upon single herbivory by Tetranychus urticae (100 mites per leaf, N = 5, open bars), single herbivory by Spodoptera exigua (2 larvae per leaf, N = 4, filled bars), or multi-species herbivory (N = 4, hatched bars). Asterisks indicate compounds that are significantly affected by herbivore treatment (P < 0.05, Kruskal–Wallis tests). (b) Ratio of emission rates upon multi-species herbivory to the sum per volatile emitted by the two single herbivore treatments (see “Methods and Materials” and for detailed explanation). Symbols indicate mean ratio, and error bars indicate upper and lower 95% confidence limits. Asterisks indicate compounds that were significantly less induced by multi-species herbivory than by the sum of T. urticae and S. exigua (upper 95% confidence limit smaller than 0) (N = 4) Compound numbers: (1) (E)-2-hexenal + (Z)-3-hexanal, (2) (Z)-3-hexen-1-ol acetate, (3) indole, (4) (E,E)-α-farnesene, (5) butyl aldoxime, (6) 3-methylbutanal-O-methyl oxime, (7) (E)-4,8-dimethyl-1,3,7-nonatriene, (8) (3E,7E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene, (9) (E)-β-ocimene. Note that peak area units in Fig. 2a cannot be compared with Fig. 1a because measurements were done on a different GC-MS systemFor cucumber, we first determined which volatile compounds were induced by herbivory by using Kruskal–Wallis tests per compound. Variation in volatile emission levels of uninfested plants was low, indicating that other types of inducing factors (e.g., general stress) were absent or had only minor effects. Only herbivore-induced volatiles were used in subsequent analyses. We assumed that all compounds emitted by infested lima bean plants were induced by herbivores based on previous measurements of volatile emissions by uninfested lima bean plants (e.g., de Boer et al. 2004). Per plant species, we then used Kruskal–Wallis tests to analyze which compounds were differentially induced by the three herbivore treatments (T. urticae, S. exigua, or multi-species herbivory) (α = 0.05). Note that we did not statistically compare absolute levels of volatile emission across plant species because volatiles were collected for different periods of time, and analyses were run on two different GC-MS systems.We also tested whether the effect on HIPV of multi-species herbivory was the same as the combined effect of the two herbivores feeding alone, i.e., T. urticae + S. exigua. Per replicate, the amount of each volatile compound of the multi-species herbivory treatment was divided by the sum of the amounts emitted by T. urticae and S. exigua. The ratios were log-transformed before taking their averages. An average ratio of 0 means the effect of multi-species herbivory and the sum of T. urticae and S. exigua is equal. We concluded that a compound was significantly more strongly induced by multi-species herbivory than by the sum of T. urticae and S. exigua if the lower 95% confidence limit was larger than 0. Similarly, we concluded a significantly weaker effect of multi-species herbivory if the upper 95% confidence limit was smaller than 0 (see Figs. 1 and 2). We also used a χ2 test to test whether the number of compounds with a ratio >0 and a ratio <0 differed from a 50:50 distribution. A 50:50 distribution would be expected if the effect of multi-species herbivory and the sum of T. urticae and S. exigua is equal. One replicate of the T. urticae treatment in cucumber was excluded from this analysis because we had no corresponding replicates of the S. exigua and multi-species herbivory treatments. Note that these tests reflect volatile responses to multi-species herbivory relative to responses to single-species herbivory. This allows us to compare these data between lima bean and cucumber because the data no longer depend on the absolute levels of volatiles detected by the different GC-MS systems.
Y-tube Olfactometer Experiments A closed system Y-tube olfactometer setup was used to test the responses of predatory mites to volatiles induced by multi-species herbivory (Takabayashi and Dicke 1992; de Boer et al. 2004). Odor sources consisted of four leaves of the same treatment that were cut from the plant just before an experiment; the petioles were wrapped in wet cotton wool and aluminum foil. Individual female predators were observed for a maximum of 5 min. A choice was recorded when the finish line, halfway one of the olfactometer arms, was reached within this period. Otherwise, it was recorded as “no-choice”. Sixteen to 23 predators were tested per experimental day, and each experiment was repeated on 4 different days. Per replicate, new groups of predatory mites and new sets of leaves were used. We tested the responses of predatory mites to volatiles from bean or cucumber plants of the multi-species herbivory treatment vs. volatiles from plants of the T. urticae or S. exigua treatments.The choices of predatory mites between two odor sources in the Y-tube olfactometer were analyzed with two-sided binomial tests to investigate whether the distribution of the predators differed from 50:50. Predators that did not make a choice were excluded from statistical analyses.
Results
Volatile Emission by Lima Bean Plants The main compounds emitted by T. urticae-infested bean plants were methyl salicylate (MeSA; compound number 2 in Fig. 1a), (3E)-4,8-dimethyl-1,3,7-nonatriene [(E)-DMNT] (4), and (3E,7E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene [(E,E)-TMTT] (5). Herbivory by S. exigua alone resulted in the emission of similar amounts of (E)-DMNT (4), but amounts of MeSA (2) and (E,E)-TMTT (5) were small. Instead, S. exigua-infested bean leaves emitted large amounts of (E)-β-ocimene (3) and (Z)-3-hexen-1-ol acetate (7). Multi-species herbivory on bean plants induced the same volatile compounds as S. exigua feeding alone. Multi-species-infested plants emitted larger amounts of all volatile compounds than plants infested with a single herbivore species, except for (Z)-3-hexen-1-ol (1) that was emitted in larger amounts by S. exigua-infested plants, and MeSA (2) that was emitted in somewhat larger absolute amounts by T. urticae-infested plants.In lima bean, herbivore treatment had a significant effect on the emission of (Z)-3-hexen-1-ol (1), (E)-β-ocimene (3), (Z)-3-hexen-1-ol acetate (7), 2-methylbutanal-O-methyl oxime (8), linalool (10), 1-octen-3-ol (11), hexyl acetate (12), β-caryophyllene (14), indole (16), (E)-2-hexen-1-ol acetate (17), (Z)-β-ocimene (21), and two unknown compounds (23 and 26) (all P < 0.05, Kruskal–Wallis tests, Fig. 1a). These compounds were all induced in larger amounts by S. exigua and/or multi-species herbivory than by T. urticae. Total volatile emission was also significantly affected by treatment (P < 0.006, Kruskal–Wallis test, Fig. 1a).We compared the ratio of volatiles emitted in the multi-species herbivory treatment to the sum of volatiles emitted by the T. urticae and S. exigua treatments to determine whether infestation by two herbivore species simultaneously had additive, synergistic or antagonistic effects. Figure 1b shows the average ratio of multi-species herbivory to the sum of T. urticae and S. exigua for lima bean. Eight of 27 compounds were significantly more strongly induced by multi-species herbivory than by the sum of the two herbivore species feeding alone, suggesting synergistic effects (Fig. 1b). These compounds included members of various groups of chemicals, for example, (E)-2-hexen-1-ol acetate (17) and (Z)-β-ocimene (21). In addition, more compounds had a ratio >0 than expected (χ2 = 9.80, P = 0.002). None of the compounds was significantly less induced by multi-species herbivory, suggesting the absence of antagonistic effects of T. urticae and S. exigua feeding simultaneously on lima bean.
Volatile Emission by Cucumber Plants Nine compounds were induced by herbivory on cucumber (all P < 0.10, Kruskal–Wallis tests; data not shown). The main compounds emitted by T. urticae-infested cucumber plants were (Z)-3-hexen-1-ol acetate (compound number 2 in Fig. 2a), (E,E)-α-farnesene (4), 3-methylbutanal-O-methyl oxime (6), (E)-DMNT (7), and (E)-β-ocimene (9) (Fig. 2a). Similar compounds were emitted in the largest amounts by S. exigua-infested cucumber plants. Multi-species herbivory resulted in a volatile blend that consisted of the same compounds as the blends induced upon herbivory by each of the herbivore species alone. The multi-species herbivory treatment resulted in the strongest induction of 3-methylbutanal-O-methyloxime (6) and (E)-β-ocimene (9), while T. urticae-infested plants emitted the largest amounts of (Z)-3-hexen-1-ol acetate (2). Herbivory by T. urticae alone and multi-species herbivory induced larger amounts of (E,E)-TMTT (8) than single herbivory by S. exigua alone. No MeSA was detected in any of the cucumber samples.In cucumber, herbivore treatment had an effect on the emission of 3-methylbutanal-O-methyloxime (6) and (E)-DMNT (9) (P < 0.05, Kruskal–Wallis tests, Fig. 2a). Both compounds were induced in larger amounts by T. urticae or multi-species herbivory than by S. exigua. Total volatile emission was not affected by herbivore treatment in cucumber (P > 0.10, Kruskal–Wallis test, Fig. 2a).Two volatile compounds [(E)-2-hexanal/(Z)-3-hexenal (1) and (Z)-3-hexen-1-ol acetate (2)] were induced significantly less by multi-species herbivory than by the sum of T. urticae and S. exigua (Fig. 2b), suggesting the presence of antagonistic effects of the two herbivore species feeding on cucumber. The seven other compounds were equally induced by multispecies herbivory and the sum of the two herbivores feeding alone, indicating an additive effect of T. urticae and S. exigua. The distribution of compounds with a ratio >0 or <0 did not differ from 50:50 (χ2 = 0.06, P = 0.81).
Responses of P. persimilis We tested the preferences of the predatory mite P. persimilis for the volatile blends analyzed above in a Y-tube olfactometer. On bean and cucumber plants, predatory mites preferred HIPV induced by multi-species herbivory to blends induced by either T. urticae or S. exigua alone (Fig. 3, P ≤ 0.02, binomial tests).
Fig. 3Responses of P. persimilis to volatile blends in the Y-tube olfactometer. Odor sources consisted of four leaves from lima bean (a and b) or cucumber plant (c and d) simultaneously infested by Tetranychus urticae and Spodoptera exigua or infested by one of the herbivore species alone (S. exigua: a and c, T. urticae: b and d). Bars present the overall percentages of predators choosing for each odor source. Numbers in bars are the total numbers of predators responding to each odor source. Choices between odor sources were analyzed with a two-sided binomial test (*P < 0.05; ***P < 0.001)
Discussion
We investigated volatile emissions by lima bean and cucumber plants upon single and multi-species herbivory by T. urticae and S. exigua. In lima bean, we found that most compounds were more strongly induced by multi-species herbivory than by the sum of each of the herbivores feeding separately (Fig. 1b). This suggests that the two herbivores feeding together on the same plant have more than an additive effect on volatile emission, potentially indicating that biosynthetic pathways may act synergistically. It is also possible that one or both herbivore species inflicted more damage on multi-species-infested plants than on plants with conspecifics only. However, it is unlikely that spider mite feeding was affected by the presence of S. exigua because in a preliminary experiment oviposition rates of mites, which are correlated with food intake rates, were similar on leaf discs with or without S. exigua damage (de Boer et al., unpublished). Some studies have observed that feeding by S. exigua is enhanced by the presence of aphids or a fungal pathogen (Rodriguez-Saona et al. 2005; Cardoza and Tumlinson 2006), but other studies have found no effect of the presence of whiteflies or a fungus (Rodriguez-Saona et al. 2003; Rostás et al. 2006).
In contrast to bean, we found that in cucumber plants, two volatile compounds were induced less by multi-species herbivory than by the sum of the two herbivores feeding separately, suggesting that their induction was suppressed by simultaneous feeding of T. urticae and S. exigua. Whether this reduction is caused by a negative interaction between the signal transduction pathways induced by spider mites and beet armyworms, or to reduced feeding of one or both herbivore species as a result of feeding on the same plant, remains to be investigated. From our data, it appears that plant species is an important determinant in the way in which herbivore-induced defense pathways interact upon multispecies herbivory.
The plant hormones salicylic acid (SA) and jasmonic acid (JA) play important roles in plant defense pathways and the production of HIPV (e.g., Kessler and Baldwin 2002; van Poecke and Dicke 2004). Interaction between these pathways may occur, for example, when SA inhibits JA-dependent defense pathways (e.g., Doares et al. 1995; Niki et al. 1998; Engelberth et al. 2001). To determine whether synergistic (lima bean) and antagonistic (cucumber) interactions between biosynthetic defense pathways caused the changes in HIPV blends that we observed, endogenous levels of JA and SA should be determined in future studies. Arimura et al. (2002) showed that both JA and SA levels are significantly elevated in lima bean after 3 d of spider mite feeding, whereas several Spodoptera species are known to increase JA levels in corn and broad bean (Blechert et al. 1995; Schmelz et al. 2003). We are not aware of analyses of SA levels in response to Spodoptera feeding. No studies of JA and SA levels in plants upon multi-species herbivory are known to us either.
Another important question that needs to be addressed is the effect of herbivore sequence on volatile emission patterns, since in nature two herbivore species may infest an individual plant sequentially. In fact, some herbivore species are known to be attracted to HIPV themselves (e.g., Bolter et al. 1997; Kalberer et al. 2001), but spider mites are not attracted to S. exigua-infested lima bean plants (Horiuchi et al. 2003).
To determine whether the differences in volatile profiles upon multi-species herbivory and single-species herbivory are relevant in an ecological context, we investigated the behavior of the predatory mite P. persimilis, a specialist natural enemy of spider mites. Our results show that P. persimilis prefers volatiles induced by multi-species herbivory by spider mites and the non-prey caterpillar S. exigua to volatiles induced by one of the herbivore species feeding alone (Fig. 3). The strong preference of P. persimilis for the volatiles induced by multi-species herbivory may be explained by the relatively large amount of some specific compounds. Of the three major compounds emitted by spider mite-infested lima bean, two, namely (E,E)-TMTT and especially (E)-DMNT, were induced more strongly by multi-species herbivory, whereas induction of the third, MeSA, was somewhat reduced (Fig. 1a). Of the five major compounds emitted by spider mite-infested cucumber, four were induced more strongly by multi-species herbivory: (E)-DMNT, (E)-β-ocimene, (E,E)-α-farnesene, and 3-methylbutanal-O-methyl oxime, while (Z)-3-hexen-1-ol acetate emission was reduced (Fig. 2a). (E)-DMNT, (E,E)-TMTT, and (E)-β-ocimene are known attractants for P. persimilis (Dicke et al. 1990a, de Boer et al. 2004), and (E,E)-TMTT can be used by P. persimilis to discriminate between complex blends of HIPV (de Boer et al. 2004). Similarly, when S. exigua-induced volatiles are compared to the blend induced by multi-species herbivory, all of these compounds are produced in larger amounts in the latter. This suggests that a greater emission of (the sum of) attractive compounds may be the main determinant in attracting the predatory mite P. persimilis.
Previous studies on the responses of parasitoid wasps to HIPV also demonstrated a preference for HIPV induced by multi-species herbivory compared to single-species herbivory (Shiojiri et al. 2000; Vos et al. 2001; Cardoza et al. 2003; Moayeri et al. 2007). Rodriguez-Saona et al. (2005) even showed that naïve Cotesia marginiventris females were only attracted to tomato plants infested with their host S. exigua when the same plant was also infested with aphids. The presence of aphids thus made the host-infested plants more detectable. The opposite, i.e., multi-species herbivory-induced volatiles being less attractive than volatiles induced by only the host has been found in two cases: C. plutellae wasps preferred volatiles induced by their host, P. xylostella, to volatiles induced by their host and the non-host caterpillar P. rapae (Shiojiri et al. 2000), and C. marginiventris as well as the entomopathogenic nematode H. megidis were less attracted to volatiles from maize plants that were infested by a non-host in addition to their host (Rasmann and Turlings 2007). In such cases, the presence of non-host or non-prey herbivores may decrease the detectability or reliability of HIPV cues for natural enemies. For example, feeding by a non-prey or non-host herbivore may suppress the induction of volatile compounds used by natural enemies. Multi-species herbivory thus adds a new dimension to the trade-off between reliability and detectability of searching cues used by natural enemies of herbivorous arthropods (Vet and Dicke 1992).

food webs
signal transduction
gc-ms
methyl salicylate
indirect defense
terpenes
tritrophic interactions