Genetic polymorphisms in the nucleotide excision repair pathway and lung cancer risk: A meta-analysis

Various DNA alterations can be caused by exposure to environmental and endogenous carcinogens. Most of these alterations, if not repaired, can result in genetic instability, mutagenesis and cell death. DNA repair mechanisms are important for maintaining DNA integrity and preventing carcinogenesis. Recent lung cancer studies have focused on identifying the effects of single nucleotide polymorphisms (SNPs) in candidate genes, among which DNA repair genes are increasingly being studied. Genetic variations in DNA repair genes are thought to modulate DNA repair capacity and are suggested to be related to lung cancer risk. We identified a sufficient number of epidemiologic studies on lung cancer to conduct a meta-analysis for genetic polymorphisms in nucleotide excision repair pathway genes, focusing on xeroderma pigmentosum group A (XPA), excision repair cross complementing group 1 (ERCC1), ERCC2/XPD, ERCC4/XPF and ERCC5/XPG. We found an increased risk of lung cancer among subjects carrying the ERCC2 751Gln/Gln genotype (odds ratio (OR) = 1.30, 95% confidence interval (CI) = 1.14 - 1.49). We found a protective effect of the XPA 23G/G genotype (OR = 0.75, 95% CI = 0.59 - 0.95). Considering the data available, it can be conjectured that if there is any risk association between a single SNP and lung cancer, the risk fluctuation will probably be minimal. Advances in the identification of new polymorphisms and in high-throughput genotyping techniques will facilitate the analysis of multiple genes in multiple DNA repair pathways. Therefore, it is likely that the defining feature of future epidemiologic studies will be the simultaneous analysis of large samples.
1. Introduction
Sporadic cancer is a multifactorial disease that results from complex interactions between many genetic and environmental factors 1. This means that there will not be a single gene or single environmental factor that has large effects on cancer susceptibility. Environmental factors (e.g. tobacco smoke, dietary factors, infectious agents and radiation) add to the carcinogenic load to which humans are exposed, but exact numbers for added risk are generally less well established.
Cancer is the result of a series of DNA alternations in a single cell or clone of that cell, which leads to a loss of normal function, aberrant or uncontrolled cell growth and often metastasis. Several of the genes that are frequently lost or mutated have been identified, including genes that function to induce cell proliferation under specific circumstances (e.g. the ras and myc proto-oncogenes) and those which are programmed to halt proliferation in damaged cells (e.g. the TP53 and RB1 tumor suppressor genes). Other mutations in genes involved in DNA repair are also necessary. About 150 human DNA repair genes have been identified to date 2, but the real number is probably higher, since less than 50% of known and putative genes have an identified function. The association between defects in DNA repair and cancer was established by Cleaver in 1968 3, who showed that xeroderma pigmentosum (XP) is caused by deficient nucleotide excision repair (NER). For more than a quarter of a century after that it was thought that only rare syndromes, such as XP, Cockayne syndrome (CS) and ataxia telangiectasia, were associated with DNA repair defects 4. Novel, common polymorphisms in DNA repair genes are continuously being identified, and these polymorphisms may play a pivotal role in sporadic carcinogenesis. A growing body of literature, including observations of inter-individual differences in measures of DNA damage, suggests that these polymorphisms may alter the functional properties of DNA repair enzymes.
At least four pathways of DNA repair operate on specific types of damaged DNA. Base excision repair (BER) operates on small lesions, while the NER pathway repairs bulk lesions. Mismatch repair corrects replication errors. Double-strand DNA break repair (DSBR) actually consists of two pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ). The NHEJ repair pathway involves direct ligation of the two double strand break ends, while HR is a process by which double-strand DNA breaks are repaired through the alignment of homologous sequences of DNA. The following sections review the literature on DNA repair genes in more detail, specifically those involved in the NER pathway.
NER is a versatile DNA repair system that removes a wide range of DNA lesions including UV-induced lesions. There are two subpathways in NER. One is transcription-coupled DNA repair (TCR), which preferentially removes DNA damage that blocks ongoing transcription in the transcribed DNA strand of active genes. The other is global genome repair (GGR), which removes lesions throughout the genome, including those from the nontranscribed strand in the active gene 5. Three rare, autosomal recessive inherited human disorders are associated with impaired NER activity: XP, CS and trichothiodystrophy (TTD) 6. XP has been studied most extensively. XP patients develop skin tumors at an extremely high frequency (1000 fold increased incidence as compared to normal individuals) because of their inability to repair UV-induced DNA lesions. These clinical findings are associated with cellular defects, including hypersensitivity to killing and the mutagenic effects of UV and the inability of XP cells to repair UV-induced DNA damage 7. Approximately 80% of XP patients who have been classified have a defect in the NER pathway. These patients are said to have "classical" XP, in contrast to the remaining 20% of patients who are designated as XP variants (XPV) and most likely have a defect in post-replication repair. In XPV patients, DNA replication stops or is interrupted at sites of UV-damage. Furthermore, de novo DNA synthesis opposite cyclobutane pyrimidine dimer lesions is prone to errors, leading to the fixation of multiple DNA mutations and ultimately to cancer. Seven different DNA NER genes, which correct seven distinct genetic XP complementation groups (XPA, XPB (ERCC3), XPC, XPD (ERCC2), XPE, XPF (ERCC4) and XPG (ERCC5, this gene causes CS)) and XPV have been identified 6. XPA, ERCC3/XPB, ERCC2/XPD, ERCC4/XPF and ERCC5/XPG have a defect in TCR and GGR, while XPC and XPE have a defect in GGR only. ERCC6 and ERCC8 are also known as CS type B (CSB) and CSA, respectively. Approximately 20% of patients have been assigned to the CSA complementation group Essentially CS shows some overlap with certain forms of XP. In contrast to XP and TTD, however, the NER defect in CS is limited to the TCR pathway. As with XP, TTD involves mutations in XP genes, usually XPD, which encodes a component of the transcription factor TFIIH 8. However, it has been suggested that the functions of XPD associated with TTD are distinct from those of XPD associated with XP. Approximately half of the patients with TTD display photosensitivity, correlated with the NER defect.
The aim of this article is to review and evaluate associations between genes in the NER pathway and lung cancer risk, focusing on genes encoding five key enzymes in this pathway: XPA, ERCC1, ERCC2/XPD, ERCC4/XPF and ERCC5/XPG.
2. Materials and methods
2-1. Identification and eligibility of relevant studies
We conducted MEDLINE, Current Contents and Web of Science searches using "XPA", "ERCC1", "ERCC2/XPD", "ERCC4/XPF", "ERCC5/XPG", "lung cancer" and "polymorphism" as keywords to search for papers published (from January 1, 1966 through May 31, 2006). Additional articles were identified through the references cited in the first series of articles selected. Articles included in the meta-analysis were in any language, with human subjects, published in the primary literature and had no obvious overlap of subjects with other studies. We excluded studies with the same data or overlapping data by the same authors. Case-control studies were eligible if they had determined the distribution of the relevant genotypes in lung cancer cases and in concurrent controls using a molecular method for genotyping. Using the MEDLINE database, we identified 5 genetic epidemiological studies 9-13 that provided information on lung cancer occurrence associated with the XPA G23A polymorphism (one of the identified 6 candidate studies was excluded due to overlapping data 11). We identified 5 studies of the ERCC1 T19007C polymorphism (all of 5 candidate studies were independent 13-17). We gathered 18 articles on the ERCC2 312/751 polymorphisms found through literature searches and checked their references for additional relevant studies. Of the relevant 18 studies, 2 studies appeared to be on populations already reported 14, 18, 19, leaving 15 independent studies (11 studies for the Asp312Asn polymorphism 11, 13, 14, 17-24 and 14 studies for the Lys751Gln polymorphism 11, 13, 14, 17-19, 21-28. Less than 5 studies each have been reported on the ERCC1 C8092A, ERCC4/XPF Arg415Gln, ERCC4/XPF Ser835Ser, ERCC5/XPG His46His, ERCC5/XPG Asp1104His SNPs.
2-2. Data extraction and assessment of study quality
For each study, characteristics such as authors, year of publication, ethnic group of the study population, source of control population, number of genotyped cases and controls, crude odds ratio (OR) and the method for quality control of genotyping were noted. For studies including subjects of different ethnic groups, data were extracted separately for each ethnic group whenever possible.
Methods for defining study quality in genetic studies are more clearly delineated than those for observational studies. We assessed the homogeneity of the study population (Caucasian or Asian).
2-3. Meta-analysis
Data were combined using both a fixed effects (the inverse variance-weighted method) and a random effects (DerSimonian and Laird method) models 29. The Cochrane Q statistics test is used for the assessment of heterogeneity. The fixed effects model is used when the effects are assumed to be homogenous, while the random effects model is used when they are heterogenous. In the absence of between-study heterogeneity, the two methods provide identical results. The presence of heterogeneity can result from differences in the selection of controls, age distribution, prevalence of lifestyle factors, histologic type of lung cancer, stage of lung cancer and so on. The random effects model incorporates an estimate of the between-study variance and tends to provide wider CIs when the results of the constituent studies differ among themselves. As the random effects model is more appropriate when heterogeneity is present 29, the summary OR and prevalence were essentially based on the random effects model. The meta-analyses were performed on crude ORs, since the adjusted ORs were not comparable because of the inclusion of different covariates in the multivariate regression models. Using individuals with the homozygous common genotype as the reference group, we calculated ORs for individuals with the heterozygous genotype and homozygous rare genotype separately whenever possible (information available in at least two studies). In some cases, we combined the heterozygous genotype with the homozygous rare genotype due to a low prevalence of the rare allele in several polymorphisms. The Q statistic was considered significant for P<0.10 30, 31. Publication bias is always a concern in meta-analysis. The presence of publication bias indicates that nonsignificant or negative findings remain unpublished. To test for publication bias, both Begg's 32 and Egger's 33 tests are commonly used to assess whether smaller studies reported greater associations than larger studies. Publication bias is considered significant for P<0.10. Publication bias may be always a possible limitation of combining data from various sources as in a meta-analysis. The idea of adjusting the results of meta-analyses for publication bias and imputing "fictional" studies into a meta-analysis is controversial at the moment 34. Sutton et al. concluded that publication or related biases did not affect the conclusions in most meta-analyses because missing studies changed the conclusions in less than 10% of meta-analyses 34. All of the calculations were performed using STATA Version 8.2 (Stata Corporation, College Station, TX) software.
3. Results
3-1. DNA repair capacity and lung cancer risk
Cigarette smoke contains several thousand chemicals that are known to chemically modify DNA 35 and lead to the formation of mutations 36. Most of these compounds are procarcinogens that must be activated by Phase I enzymes, such as cytochrome P450s. All activated carcinogens can bind to DNA and form DNA adducts that are capable of inducing mutations and initiating carcinogenesis. The capacity to repair DNA damage induced by activated carcinogens appears to be one of the host factors that may influence lung cancer risk. A critical cellular response that counteracts the carcinogenic effects of DNA damage is DNA repair. As stated earlier, there are several known pathways of DNA repair, all of which act to remove DNA lesions and prevent mutations, thereby restoring genetic integrity.
Several studies have investigated whether reduced DNA repair capacity (DRC) is associated with an increased risk of cancer 37. The reduced DRC of benzo(a)pyrene-7,8-diol-9,10-epoxide (an active form of benzo(a)pyrene)-DNA adducts is associated with an increased risk of lung cancer (2.1-fold, 95% confidence interval (CI) = 1.5 - 3.0) 38. The reduced DRC has been shown to be associated with a 5.7-fold (95% CI = 2.1 - 15.7) increased risk of developing lung cancer 39. Likewise, the reduced DRC of bleomycin-induced damage was found to be associated with an increased risk of lung cancer 40. These studies suggested that a low DRC of various DNA repair mechanisms predisposes individuals to lung cancer, and this realization prompted us to search for defined DNA repair activities that may be risk factors for lung cancer. Polymorphisms in DNA repair genes may be associated with differences in the DRC of DNA damage and may influence an individual's risk of lung cancer, because the variant genotype in those polymorphisms might destroy or alter repair function.
3-2. XPA G23A polymorphism and lung cancer risk
The heterotrimeric replication protein A (RPA) is required for NER and may play an important role in the damage recognition process. The XPA protein is required for NER and is involved in the DNA damage recognition process. Both RPA and XPA preferentially bind damaged DNA, and because RPA and XPA directly interact in the absence of DNA, the RPA-XPA complex has been implicated as a key component in the earliest stage of damage recognition 41. There is also evidence that the XPC-hHR23B protein complex may initiate recognition of DNA damage for the global genomic repair pathway of NER 42. Recent evidence also implicates the damaged DNA binding protein heterodimer in damage recognition, because the complex binds damaged DNA with high affinity 43 and can dramatically increase the repair rate of certain DNA adducts, including cyclobutane pyrimidine dimers, in conjunction with XPA and RPA 44.
The XPA maps on chromosome 9, at 9q22.3. In the XPA gene, a polymorphic site was identified that was in the 5' untranslated region (UTR) of the gene and which consisted of a G-to-A (or A-to G) substitution in the fourth nucleotide before the ATG start codon (dbSNP rs 1800975) 45. SNP alleles with higher frequencies are more likely to be ancestral than less frequently occurring alleles although there may be some exceptions. As the 23G allele was more prevalent than the 23A allele (Table 1), we regarded the 23G allele as ancestral (wild-type or major) allele for descriptive purposes (the XPA 23 polymorphism caused by the G-to-A substitution is the XPA G23A polymorphism). The polymorphism, termed the XPA G23A polymorphism (at position 23 in the transcript, four nucleotides upstream of the start codon), is in the Kozak sequence near the start codon and thus may affect the XPA protein levels in cells 46. A functional association between the XPA G23A polymorphism and DRC has been reported 10. It has been shown that healthy subjects with at least one 23G allele have significantly higher DRC. When the combined A/A and A/G genotype was used as the reference, the G/G genotype was associated with a significantly decreased risk of lung cancer (adjusted OR = 0.56, 95% CI = 0.35 - 0.90) in Koreans 9. A significant protective effect of the combined G/A and G/G genotypes on lung cancer risk was reported in Americans (adjusted OR = 0.69, 95% CI = 0.53 - 0.90) and Mexican-Americans (adjusted OR = 0.32, 95% CI = 0.12 - 0.83) 10. Likewise, a protective and nonsignificant effect was seen among Germans 11 and Danes 12. As compared with the combined G/A and A/A genotypes, the G/G genotype was, however, associated with a significantly increased risk of lung cancer (adjusted OR = 1.59, 95% CI =1.12 - 2.27) in a Norwegian population 13. Summary frequencies of the 23A allele among all and Caucasian populations, based on the random effects model, were 0.368 (95% CI = 0.308 - 0.429) and 0.352 (95% CI = 0.277 - 0.428), respectively (Table 1). Summary ORs for the G/A genotype and G/G genotype among 5 studies in 7 populations were 0.73 (95% CI = 0.61 - 0.89) and 0.75 (95% CI = 0.59 - 0.95), respectively (Table 1). Evidence for heterogeneity was absent in both analyses. Among Caucasian studies, the summary ORs for the G/A genotype and the A/A genotype were 0.72 (95% CI = 0.58 - 0.89) and 0.82 (95% CI = 0.61 - 1.11), respectively. The Cochrane Q test for heterogeneity did not show a statistical significance. The Egger's test was statistically significant for publication bias in a subgroup analysis of Caucasians (P = 0.073, G/A genotype vs. G/G genotype).
Two studies investigated associations between cigarette smoking and the G23A polymorphism in relation to lung cancer. When stratifying by smoking status, there was a significant protective effect for current smokers who possessed the G/G genotype (adjusted OR = 0.23, 95% CI = 0.07- 0.71) but not for former or never smokers 9. Ever smokers (current and former) with at least one copy of the 23G allele showed a significantly reduced risk of lung cancer (adjusted OR = 0.68, 95% CI = 0.51 - 0.91) among Caucasians 10. The presence of the 23A polymorphism, however, was associated with a statistically significant reduced risk in subjects who smoked >29 pack-years (OR = 0.53, 95% CI = 0.17 - 0.97) 13. Interactions between cigarette smoking and the polymorphism were not determined in the studies 9, 10, 13. No associations were seen between the G23A polymorphism and any histologic types of lung cancer 11, while the G/G genotype was associated with a significantly decreased risk for small cell lung cancer (OR = 0.23, 95% CI = 0.07 - 0.71) 9.
The XPA G23A polymorphism may, thus, be a promising SNP for lung cancer. It is thought that cigarette smoking modifies the association between DNA repair polymorphisms, as well as metabolic polymorphisms, and lung cancer risk. Since interactions between the G23A polymorphism and smoking have not been fully elucidated, further studies are needed to better understand the associations between the XPA G23A polymorphism and lung cancer risk.
3-3. ERCC1 polymorphisms and lung cancer risk
The ERCC1 coding region is 1.1 kb long and comprises 10 exons. This gene is located on 19q13.2 - q13.3. Shen et al. 47 have identified polymorphisms of three of the exons of the ERCC1 gene, all of which resulted in silent mutations. No amino acid substitutions were observed among the ERCC1 polymorphisms 48. The functional effects of the silent polymorphisms in ERCC1 have not been fully elucidated; however, some of the variant alleles of the polymorphisms in DNA repair genes may be associated with the reduced DRC. The studies have focused on polymorphisms of the 3′ UTR (C8092A, dbSNP no. rs3212986) and codon 118 (Asn118Asn, T19007C, dbSNP no. rs11615) in ERCC1.
For the T19007 C (Asn118Asn) polymorphism, although the T/T genotype generates the less commonly associated triplet codon sequence encoding the amino acid and has been termed the "variant" by convention, the T/T genotype indeed has been reported to occur at higher frequencies. Hence, the T/T genotype is used as reference in this paper. The C/C genotype of the C8092A polymorphism is used as reference on the same score.
The C/C genotype of the T19007C polymorphism was associated with a significantly decreased risk of lung cancer (adjusted OR = 0.32, 95% CI = 0.19 - 0.55) in a Norwegian population 13. A lack of association between the T19007C polymorphism and lung cancer risk was observed in a Danish population 14, a large American population 15, a Chinese population 16 and a nonsmoking European population 17. As shown in Table 2, summary frequencies of the 19007T allele among all and Caucasian populations, based on the random effects model, were 0.499 (95% CI = 0.387 - 0.611) and 0.575 (95% CI = 0.529 - 0.622), respectively. The summary ORs for the T/C genotype and the C/C genotype were 0.82 (95% CI = 0.62 - 1.08) and 0.72 (95% CI = 0.46 - 1.11), respectively. Even if the analysis was restricted to Caucasian studies, the ORs did not materially change. The Cochrane Q test for heterogeneity showed a statistical significance in any analysis. In comparison of the T/C genotype with the T/T genotype, the Begg's test was statistically significant in an overall analysis (P = 0.086) and a subgroup analysis of Caucasians (P = 0.089).
Two studies examined an interaction between the T19007C polymorphism and cigarette smoking. When stratified by smoking status, the interaction between smoking and the polymorphism was not statistically significant 15, 16. Only one study provides information on the T19007C polymorphism and lung cancer risk in histologic types. There was no difference in risk estimates according to the histological type of lung cancer 16.
As for the C8092A polymorphism, no association was found between the polymorphism and lung cancer risk in Norwegians 13 and Americans 15. The C8092A and T19007C polymorphisms have been reported to be in linkage disequilibrium 15.
Although harboring at least one 19007C allele may be associated with a deceased risk of lung cancer, the protective effect of the 19007C allele needs to be confirmed in other independent studies. Furthermore, additional studies are needed to detect the function of the ERCC1 polymorphisms.
3-4. ERCC2/XPD polymorphisms and lung cancer risk
The ERCC2/XPD protein plays a role in the NER pathway, which recognizes and repairs a wide range of structurally unrelated lesions such as bulky adducts and thymidine dimers. ERCC2/XPD works as an ATP-dependent (5'→3') helicase joined to the basal TFIIH complex used to separate the double helix. The ERCC2/XPD protein is necessary for normal transcription initiation and NER. ERCC2/XPD maps on chromosome 19, at 19q13.3 and covers 21.14 kb. Mutations in the ERCC2 gene can diminish the activity of TFIIH complexes, giving rise to repair defects, transcription defects and abnormal responses to apoptosis 49.
A number of polymorphisms in the ERCC2/XPD gene have been reported. Whereas polymorphisms in the codons 199, 201 and 575 are rare, those in codons 156, 312, 711 and 751 are common. Two ERCC2/XPD polymorphisms, Asp312Asn (db SNP no. rs1799793) and Lys751Gln (db SNP no. rs13181), have mainly been investigated in relation to phenotypic endpoints relevant to lung carcinogenesis. With regard to the Asp312Asn polymorphism, most of the reported data indicate a higher level of DNA adducts in subjects with the Asn allele. The interpretation of this finding is a lower DRC for the Asn allele than the Asp allele. This is also true for the ERCC2/XPD Lys751Gln polymorphism. The Gln allele is associated with a higher DNA adduct level or lower DRC.
The Asp/Asp genotype of the ERCC2/XPD Asp312Asn polymorphism was found to have an increased risk of lung cancer when the combined Asp/Asn and Asn/Asn genotypes served as reference (OR = 1.86, 95% CI =1.02 - 3.40) in Polish men 20. A large American lung-cancer study also reported an elevated risk (adjusted OR = 1.5, 95% CI = 1.1 - 2.0; Asn/Asn genotype vs. Asp/Asp genotype) 18. Likewise, Chinese subjects homozygous for the Asn/Asn genotype had an increased risk of lung cancer (adjusted OR = 10.33, 95% CI = 1.29 - 82.50) compared with subjects homozygous for the Asp/Asp genotype 19. No association with this polymorphism was seen in an admixed population 21, a small Swedish population 22 and among Finnish smoking men 23. Two meta-analyses have been published in 2004 50 and 2005 51, respectively. Both of them are based on the same published data from 6 individual case-control studies 18-23. The first meta-analysis showed that individuals with the Asn/Asn genotype had a 27% (95% CI = 1.04 - 1.56) increased risk of lung cancer compared with individuals with the Asp/Asp genotype. The results supported the hypothesis that individuals with the Asn/Asn genotype are at higher risk of developing lung cancer 50. The second meta-analysis was somewhat different from the first one, because unadjusted ORs were summarized in the first one. The summary OR associated with the Asn/Asn genotype was 1.18 (95% CI = 0.84 - 1.67). No significant association between the ERCC2/XPD Asp312Asn polymorphism and lung cancer was found in the second meta-analysis 51. Regardless, these meta-analyses indicate that the excess lung cancer risk from the Asn/Asn genotype may be less than 30%.
Five studies have been reported since the publication of these two meta-analyses. They revealed that the Asp312Asn polymorphism was not associated with lung cancer risk in Germans 11, Norwegians 13, Danes 14, Europeans 17 and Chinese 24.
As shown in Table 3, the summary frequency of the 312Asp allele among Caucasians (0.645, 95% CI = 0.572 - 0.719) was significantly lower than that among Asians (0.936, 95% CI = 0.925 - 0.946). Summary ORs associated with the ERCC2/XPD Asp312Asn polymorphism are also shown in Table 3. No significant association between lung cancer and the heterozygous Asp/Asn genotype was found for all of the studies combined or by ethnicity. The Cochrane Q test for heterogeneity did not show a statistical significance in all analyses. Although no evidence of publication bias was found in overall analyses, both Begg's (P= 0.035) and Egger's (P = 0.003) tests showed a statistical significance in a subgroup analysis of Caucasians (Asn/Asn genotype vs. Asp/Asp genotype).
When stratifing by smoking dose, the risk of lung cancer was significantly higher in light-smokers with the Asp/Asp genotype than in those with the Asn/Asn genotype 20. Similar findings were not seen for never-smoker or heavy-smokers 20. A significant interaction between smoking (smoking status, pack-years and duration) and the polymorphism was observed in one study 18 but not in two other studies 16,19. Stratification analysis revealed that the increased risk was mainly confined to squamous cell carcinoma of the lung, with the ORs being 20.50 (95% CI = 2.25 - 179.05) for the 312Asn/Asn genotype 19.
Table 4 shows the association between the ERCC2 Lys751Gln polymorphism and lung cancer risk. The Gln/Gln genotype was associated with an increased risk for lung cancer compared with the 751Lys/Lys genotype (adjusted OR = 2.71, 95% CI = 1.01 - 7.24) in Chinese 19. Stratification analysis revealed that the increased risk was mainly confined to lung squamous cell carcinoma, with the OR being 4.24 (95% CI = 1.34 - 13.38) for the Gln/Gln genotype 19, however. Although David-Beades et al. reported that the Gln/Gln genotype was associated with a significantly increased risk of lung cancer in Caucasians (USA), a multivariate-adjusted OR was no longer significant 25. No association with the Lys751Gln polymorphism was seen in two Caucasian populations 18, 22, an admixed population 21, a Finnish population 23, African-Americans 25, a Chinese population 26 and a Korean population 27. The meta-analysis by Hu et al. (2004) showed that the Gln/Gln genotype had a 21% (95% CI = 1.02 - 1.43) increased risk of lung cancer compared with individuals with the Lys/Lys genotype 51. The meta-analysis by Benhamou and Sarasin (2005) reported that the summary OR for the Gln/Gln genotype was 1.18 (95% CI = 0.95 - 1.47) 51. Both of the meta-analyses were based on the same published data from 8 individual case-control studies 18, 19, 21-23, 25-27. No significant association between the Lys751Gln polymorphism and lung cancer was found in the two meta-analyses 51. These meta-analyses indicate that the excess lung cancer risk from the Gln/Gln genotype may be about 20%. Six studies 11, 13, 14, 17, 24, 28 have been reported after the two meta-analysis. Danish subjects with the Gln/Gln genotype were at a 2.01-fold (95% CI = 1.20 - 3.35) higher risk of lung cancer risk than those with the Lys/Lys genotype 14. Similarly, the Gln/Gln genotype was associated with significantly increased risk of lung cancer (adjusted OR = 1.60, 95% CI = 1.10 - 2.30) in Norwegians 13. German individuals with the Gln/Gln genotype were at a borderline increased risk (adjusted OR = 1.59, 95% CI = 0.95 - 2.67) 11. However, individuals with the Gln allele had a 61% (95% CI = 14 - 83) reduction of lung cancer risk in a Chinese population 24. No association with the Lys751Gln polymorphism was seen in a European cohort 17 and in non-Hispanic Caucasians (USA) 28.
The summary frequency of the 751Lys allele among Caucasians (0.634, 95% CI = 0.614 - 0.655) was significantly lower than that among Asians (0.843, 95% CI = 0.763 - 0.924). A statistically significant ethnic difference was observed between Caucasians and Asians. Summary ORs for the Gln/Gln genotype and Lys/Gln genotype were 1.06 (95% CI = 0.97 - 1.16) and 1.30 (95% CI = 1.14 - 1.49), respectively. Evidence of publication bias was absent in all of the analyses. The effect of the Gln/Gln genotype on lung cancer risk was stronger in Caucasians (OR = 2.25, 95% CI = 0.97 - 5.23) than in Asians (OR = 1.02, 95% CI = 0.20 - 5.27). This may only be due to a difference in sample sizes. Reasons for this difference in risk among different ethnic populations are as yet unknown but, if real, may be related to other genetic or environmental factors. The Cochrane Q test for heterogeneity showed a statistical significance among Asian studies (P = 0.040, Gln/Gln genotype vs. Lys/Lys genotype).
There was no interaction between smoking (smoking status, pack-years and duration) and the polymorphism 14, 19, 26, 27. Although the Lys/Lys genotype was associated with a statistically significant increased risk (OR = 2.0, 95% CI = 1.15 - 3.41) among subjects who smoked>29 pack-years, an interaction between cigarette smoking and the polymorphism was not determined 13. When stratified by histological type, no statistically significant association between the polymorphism and lung cancer risk was found 26, 27.
Several studies have investigated the possible association of ERCC2/XPD Asp312Asn and Lys751Gln polymorphisms with lung cancer with inconsistent results. The Lys751Gln polymorphism has been more studied than the Asp312Asn polymorphism, because the frequency of the 751Gln allele is more prevalent than the 312Asn allele. The Asp312Asn polymorphism is in linkage disequilibrium with the Lys751Gln polymorphism 19, 20, 21, however. The inconsistent associations in previous studies of the ERCC2/XPD polymorphisms could be due to differences in study populations, the small sample sizes of earlier studies and possible environmental interactions.
3-5. ERCC4/XPF polymorphisms and lung cancer risk
ERCC4/XPF is an essential protein in the NER pathway, which is responsible for removing UV-C photoproducts and bulky adducts from DNA. Among the NER enzymes, ERCC4/XPF and ERCC1 are also uniquely involved in removing DNA interstrand cross-linking damage. The ERCC4/XPF-ERCC1 complex, which makes incisions at the 5′ end of DNA loops, may contribute to the repair of large trinucleotide repeat containing loops that are generated due to replication slippage and that are too long to be repaired by the postreplicative DNA mismatch repair system 52. Polymorphisms in enzymes involved in large loop repair could be responsible for the observed variation in the stability of similar-sized trinucleotide repeat disease alleles among different individuals. The ERCC4/XPF gene is evolutionarily conserved. Extensive homology exists between human ERCC4/XPF, Drosophila Mei-9, Saccharomyces cerevisiae RAD1, and S. pombe Rad16 53, all of which have similar functions in NER.
The ERCC4/XPF gene contains 11 exons, spans 28.2 kb and is located on chromosome 16p13.2 - p13.13. Several polymorphisms exist in the coding region of ERCC4/XPF, a few of which have been associated with cancer risks. Genetic instability of simple repeated sequences might also be influenced by the ERCC4/XPF polymorphisms. The ERCC4/XPF G1244A polymorphism is a G-to-A change in exon 8 (Arg415Gln, dbSNP no. rs1800067) that results in a change from arginine to glutamine. The ERCC4/XPF polymorphism in exon 8 has been reported to be associated with an increased risk for developing breast cancer 54. The T2505C polymorphism is a T-to-C change in exon 11 (Ser835Ser, dbSNP no. rs1799801) that results in no amino acid change (serine is conserved) 55. Functionally significant SNPs in the ERCC4/XPF gene may also contribute to individual differences in the fine details of DNA repair. A lack of association was found between the G1244A (Arg415Gln) polymorphism and lung cancer risk (adjusted OR = 1.11, 95% CI = 0.59 - 2.07; Arg/Gln genotype vs. Arg/Arg genotype) in Koreans 9. The C/C genotype of the T2505C polymorphism was nonsignificantly associated with an increased risk of lung cancer (adjusted OR = 1.71, 95% CI = 0.52 - 5.58) in Chinese 24.
3-6. ERCC5/XPG polymorphisms and lung cancer risk
ERCC5/XPG is responsible for a 1186 amino acid structure-specific endonuclease activity that is essential for the two incision steps in NER. The ERCC5/XPG nuclease has been suggested to act on the single-stranded region created as a result of the combined action of the XPB helicase and the ERCC2/XPD helicase at the DNA damage site. In human cells, ERCC5/XPG catalyses an incision approximately 5 nucleotides 3' to the site of damage but is also involved non-enzymatically in the subsequent 5' incision. It is further involved in the stabilization of a pre-incision complex on the damaged DNA.
The ERCC5/XPG gene contains 17 exons, spans 32 kb and is located on chromosome 13q32.3 -q33.1. Several polymorphisms in the coding sequence of the EECC5/XPG gene have been identified. The association between lung cancer and two common polymorphisms, T335C (His46His, dbSNP no. rs1047768) and G3507C (Asp1104His, dbSNP no. rs17655), have been investigated. The functional effects of these two SNPs are still unknown. However, it is likely that the SNPs in the coding DNA sequences may result in a subtle structural alteration of the ERCC5/XPG activity and modulation of lung cancer susceptibility.
The Asp/Asp genotype of the Asp1104His polymorphism was associated with a significantly decreased risk of lung cancer (adjusted OR = 0.60, 95% CI = 0.38 - 0.95) in a Korean population 56. Similarly, the Asp/Asp genotype was inversely associated with lung cancer (adjusted OR = 0.65, 95% CI = 0.39 - 1.1) in an admixed population (composed mostly composed of whites) 57. However, the Asp/Asp genotype was not associated with lung cancer risk in a Chinese population 24. As for T335C polymorphism, the C/C genotype was associated with a significantly increased risk of lung cancer (adjusted OR = 1.79, 95% CI = 1.19 - 2.63) in Norwegians 13 but not in Chinese 24.
4. Discussion
Epidemiological studies of common polymorphisms in DNA repair genes, if large and unbiased, can provide insight into the in vivo relationships between DNA repair genes and lung cancer risk. Such studies may identify empirical associations which indicate that a polymorphism in a gene of interest has an impact on lung cancer, independent of metabolic regulatory mechanisms and other genetic and environmental variability. Findings from epidemiological studies can complement in vitro analyses of the various polymorphisms, genes, and pathways. In addition, epidemiological studies of common polymorphisms can lead to an increased understanding of the public health dimension of DNA-repair variation.
We conducted a systematic literature review to evaluate the associations between sequence variants in DNA repair genes and lung cancer risk. We found an increased risk of lung cancer among subjects carrying the ERCC2/XPD 751Gln/Gln genotype (OR = 1.30, 95% CI = 1.14 - 1.49). The Gln allele of the ERCC2/XPD Lys751Gln polymorphism is associated with a higher DNA adduct level or lower DNA repair efficiency, except in research published by Duell et al. (2000) who found no correlation between the ERCC2/XPD Lys751Gln polymorphism and the level of polyphenol-DNA adducts in human blood samples 58. Matullo et al. (2003) demonstrated a higher level of DNA adducts, measured by 32P-postlabeling, in lymphocytes from nonsmokers with the ERCC2/XPD 751Gln/Gln genotype 59. Similarly, Palli et al. (2001) reported a higher level of DNA adducts in workers with at least one Gln allele who were exposed to traffic pollution in comparison with workers with the two common alleles 60. An increased number of aromatic DNA adducts was found by Hou et al. (2002) in peripheral blood lymphocytes from subjects with the ERCC2/XPD 312Asn and ERCC2/XPD 751Gln alleles 22. The combined Asn/Asn and Gln/Gln genotypes showed a higher level of DNA lesions than did other genotypes.
In contrast, we found a protective effect of the XPG G23A G/G genotype (OR = 0.75, 95% CI = 0.59 - 0.95) on lung cancer risk. The G23A polymorphism itself may alter the transcription and/or translation of the gene. Because this polymorphism is located in the vicinity of the translation initiation codon, it may alter translation efficiency. The nearby proximal nucleotides to the AUG initiation codon are important for the initiation of translation because the 40S ribosomal subunit binds initially at the 5'-end of the mRNA 61. The consensus sequence around the start codon is GCCRCCAUGG, which is known as the Kozak consensus sequence 62. The R at position -3 and the G just downstream of the start codon are especially important, and the lack of these bases leads to read-through of the start codon 63. However, there has been no precise explanation of the mechanism by which the recognition of the start codon is aided by a purine at position -3 62, which is the core nucleotide of the Kozak consensus. The polymorphism XPA G23A is a G/A transversion occurring 4 nucleotides upstream of the start codon of XPA and possibly improving the Kozak sequence 9. The sequences (CCAGAGAUGG) around the predicted initiator methionine codon of the XPA gene agree with the Kozak's consensus sequence at positions -3 and +4 64. Although both the A and polymorphic variant G nucleotides at the -4 position of the XPA gene do not correspond to the original consensus Kozak sequence containing the nucleotide C at position -4, it is possible that a nucleotide substitution of A to G at position -4 preceding the AUG codon may affect ribosomal binding and thus alter the efficiency of XPA protein synthesis. To investigate whether the transition from G to A changes the translation efficiency, an in vitro transcription/translation analysis and a primer extension assay of the initiation complex will be necessary in the future. Furthermore, a functional association between the G23A polymorphism and DRC was reported 10, which showed significantly higher repair efficiency in healthy subjects with at least one G allele. An alternative explanation could be that the protective XPA 23G allele is in linkage disequilibrium with an allele from an adjacent gene which is the true susceptibility gene.
Several DNA repair pathways are involved in the maintenance of genetic stability. The most versatile and important one is the NER pathway, which detects and removes bulky DNA adducts, including those induced by cigarette smoking 65. However, there are several conflicting reports on the association between this polymorphism and lung cancer risk among various populations. Although the reasons for the inconsistencies in the studies are not clear, possible explanations are: 1) low frequency of the "at-risk" genotype, which would reduce the statistical power of the studies and 2) small size of the studies. Ethnic differences in the roles of the polymorphism may be caused by gene-gene interactions, different linkages to the polymorphisms determining lung cancer risk and different lifestyles.
The most important problems facing lung cancer research are identifying "at-risk" individuals and implementing clinical surveillance, prevention practices, and follow-up care. Repair pathways play an important role in lung cancer risk, and genetic variations may contribute to decreased DRC and lung cancer susceptibility. Although the increased/decreased risk associated with individual DNA repair SNPs may be small compared to that conferred by high-penetrance cancer genes, their public health implication may be large because of their high frequency in the general population. It is thus essential that epidemiological investigations of DNA repair polymorphisms are adequately designed. Unfortunately a fairly good number of studies are limited by their sample size and subsequently suffer from too low power to detect effects that may truly exist. Also, given the borderline significance of some associations and multiple comparisons that have been carried out, there is a possibility that one or more findings are false-positives 66. Large and combined analyses may be preferred to minimize the likelihood of both false-positive and false-negative results. In addition, controls should be chosen in such a way that, if they were cases, they would be included in the case group; when controls are matched to cases, it is essential to account for matching in the analysis. When appropriate, confounding factors should be controlled for, with particular consideration of race and ethnicity. An additional major concern is the grouping of genotypes for calculation of ORs. Without functional data to dictate genotype groupings, it seems prudent to present two ORs per polymorphism (one for heterozygotes vs. common-allele homozygotes and one for rare-allele homozygotes vs. common-allele homozygotes) so that dominant, codominant, or recessive patterns may be elucidated.
Continued advances in SNP maps and in high-throughput genotyping methods will facilitate the analysis of multiple polymorphisms within genes and the analysis of multiple genes within pathways. The effects of polymorphisms are best represented by their haplotypes. Data from multiple polymorphisms within a gene can be combined to create haplotypes, the set of multiple alleles on a single chromosome. None of the studies reviewed here reported haplotype associations, although several studies analyzed multiple polymorphisms within a gene, sometimes with inconsistent results. The analysis of haplotypes can increase the power to detect disease associations because of higher heterozygosity and tighter linkage disequilibrium with disease-causing mutations. In addition, haplotype analysis offers the advantage of not assuming that any of the genotyped polymorphisms is functional; rather, it allows for the possibility of an ungenotyped functional variant to be in linkage disequilibrium with the genotyped polymorphisms 67. An analysis of data from multiple genes within the same DNA-repair pathway (particularly those known to form complexes) can provide more comprehensive insight into the studied associations. Such an analysis may shed light on the complexities of the many pathways involved in DNA repair and lung cancer development, providing hypotheses for future functional studies. Because of concerns over inflated type I error rates in pathway-wide or genome-wide association studies, methods of statistical analysis seeking to obviate this problem are under development 68. The ability to include haplotype information and data from multiple genes, and to model their interactions, will provide more powerful and more comprehensive assessments of the DNA repair pathways.
This review, which is limited by the bias against publication of null findings, highlights the complexities inherent in epidemiological research and, particularly, in molecular epidemiological research. There is evidence that some polymorphisms in DNA repair genes play a role in carcinogenesis, most notably the ERCC2/XPD Lys751Gln and XPA G23A polymorphisms. The variant allele of each of the three polymorphisms was associated with about a 30% decrease or increase in lung cancer risk. Although the summary risk for developing lung cancer in individuals of each genotype may not be large, lung cancer is such a common malignancy that even a small increase in risk can translate to a large number of excess lung cancer cases. Therefore, polymorphisms, even those not strongly associated with lung cancer, should be considered as potentially important public health issues. In addition, it is important to keep in mind that a susceptibility factor in one population may not be a factor in another. There are differences in the prevalence of DNA repair polymorphisms across populations. In a population where the prevalence of an "at-risk" genotype in a given polymorphism is very low, the "at-risk" allele or "at-risk" genotype may be too infrequent to assess its associated risk. At a population level, the attributable risk must be small simply because it is an infrequent allele. Finally, the major burden of lung cancer in the population probably results from the complex interaction between many genetic and environmental factors over time. Most environmental carcinogens first require metabolic activation by Phase I enzymes to their ultimate forms which then bind to DNA, forming aromatic-DNA adducts that are thought to be an early step in tumorigenesis. On the other hand, these activated forms are detoxified by Phase II enzymes. Thus, genetically determined susceptibility to lung cancer may depend on the metabolic balance among Phase I enzymes, Phase II enzymes and DNA repair enzymes 69. Further investigations of the combined effects of polymorphisms between DNA repair genes and drug-metabolizing genes may also help to clarify the influence of genetic variation in the carcinogenic process. Consortia and international collaborative studies, which may be a way to maximize study efficacy and overcome the limitations of individual studies, are needed to help further illuminate the complex landscape of lung cancer risk and genetic variations.

genetic polymorphism
nucleotide excision repair
lung cancer
meta-analysis


Barbarea vulgaris Glucosinolate Phenotypes Differentially Affect Performance and Preference of Two Different Species of Lepidopteran Herbivores

The composition of secondary metabolites and the nutritional value of a plant both determine herbivore preference and performance. The genetically determined glucosinolate pattern of Barbarea vulgaris can be dominated by either glucobarbarin (BAR-type) or by gluconasturtiin (NAS-type). Because of the structural differences, these glucosinolates may have different effects on herbivores. We compared the two Barbarea chemotypes with regards to the preference and performance of two lepidopteran herbivores, using Mamestra brassicae as a generalist and Pieris rapae as a specialist. The generalist and specialist herbivores did not prefer either chemotype for oviposition. However, larvae of the generalist M. brassicae preferred to feed and performed best on NAS-type plants. On NAS-type plants, 100% of the M. brassicae larvae survived while growing exponentially, whereas on BAR-type plants, M. brassicae larvae showed little growth and a mortality of 37.5%. In contrast to M. brassicae, the larval preference and performance of the specialist P. rapae was unaffected by plant chemotype. Total levels of glucosinolates, water soluble sugars, and amino acids of B. vulgaris could not explain the poor preference and performance of M. brassicae on BAR-type plants. Our results suggest that difference in glucosinolate chemical structure is responsible for the differential effects of the B. vulgaris chemotypes on the generalist herbivore.
Introduction
Glucosinolates and their breakdown products are involved in plant defense against a wide variety of potential plant enemies. They can also serve as feeding and oviposition stimulants for specialist herbivores (Chew 1988; Louda and Mole 1991; Wittstock et al. 2003). The composition of glucosinolates is genetically variable within plant species, and influences the feeding choices of insect herbivores (Kroymann et al. 2001; Lambrix et al. 2001). A heritable glucosinolate polymorphism in Barbarea vulgaris results in two discrete chemotypes (Van Leur et al. 2006). In this study, we investigate the effect of these two chemotypes on the preference and performance of two lepidopterans.
The most common and genetically dominant chemotype of B. vulgaris forms mainly (S)-2-hydroxy-2-phenylethylglucosinolate (glucobarbarin, BAR-type). Although depending on the presence of cofactors like ESP, pH, or metal ions (Burow et al. 2006), the most likely initial hydrolysis product of glucobarbarin is an isothiocyanate. Due to the 2-hydroxylation of the glucosinolate side chain, it spontaneously cyclizes to 5-phenyloxazolidine-2-thione (Kjaer and Gmelin 1957). This glucosinolate breakdown product is known to reduce infection by the soil fungus Plasmodiophora brassicae (Ludwig-Müller et al. 1999). Hardly anything is known about other ecological activities of oxazolidine-2-thiones (Wittstock et al. 2003).
The less abundant and genetically recessive chemotype of B. vulgaris contains mainly 2-phenylethyl glucosinolate (gluconasturtiin, NAS-type). 2-Phenylethyl glucosinolate is also present in Arabidopsis thaliana and other Brassicaceae (Reichelt et al. 2002), predominantly in root tissues (Sang et al. 1984). Its most likely breakdown product is 2-phenylethyl isothiocyanate. Isothiocyanates are the predominant breakdown products of glucosinolates and are generally toxic to various herbivores (Wittstock et al. 2003). 2-Phenylethyl isothiocyanate negatively affects a broad range of phytophages, e.g., nematodes (Potter et al. 1999, 2000; Serra et al. 2002; Lazzeri et al. 2004), snails (Kerfoot et al. 1998), flies, aphids, mites (Lichtenstein et al. 1962), fungi (Sarwar and Kirkegaard 1998), and several generalist and specialist Lepidoptera (Wadleigh and Yu 1988; Borek et al. 1998). Despite counter-adaptations of specialists to reduce or circumvent negative effects of glucosinolates (Ratzka et al. 2002; Wittstock et al. 2003), isothiocyanates can still reduce survival and growth, and increase development time of specialists (Agrawal and Kurashige 2003). In contrast to the oxazolidine-2-thiones formed in BAR-type plants, which can increase the incidence of goiter in mammals, the 2-phenylethyl isothiocyanate formed in NAS-type plants has chemopreventive effects against tumorigenesis in mammalian organisms (Canistro et al. 2004).
Based on the specific biological effects of the expected breakdown products of glucobarbarin and gluconasturtiin, we hypothesized that NAS-type and BAR-type B. vulgaris plants have differential effects on insect herbivores. Generally, it is expected that chemical plant defenses are more effective against generalist herbivores than against specialists (Cornell and Hawkins 2003). Therefore, we compared the preference and performance on the two chemotypes of a generalist (Mamestra brassicae) and a specialist (Pieris rapae) herbivore, which are both well-studied, important crucifer pests (Theunissen et al. 1985; Finch and Kienegger 1997).
M. brassicae is a generalist feeding on plants in 70 species and 22 families, of which Brassicaceae are among the most preferred (Rojas et al. 2000). Even though M. brassicae can detect glucosinolates by receptor cells on the sensilla (Wieczorek 1976), and its oviposition is stimulated by damaged cabbage plants (Rojas 1999), no physiological adaptations of this species to glucosinolates have been yet described. The larvae of P. rapae, on the other hand, can detoxify glucosinolates by shifting hydrolysis products from isothiocyanates to less toxic nitriles by using the myrosinase directing nitrile-specifier protein (NSP). This enables P. rapae to consume foliage that is otherwise well defended (Wittstock et al. 2003, 2004). Female adults of P. rapae can detect intact glucosinolates in leaves of Brassicaceae with specialized receptor cells, and are stimulated to lay eggs on glucosinolate containing plants.
We assessed herbivore preference on the level of adult oviposition and larval feeding. Female oviposition preference initially determines the host of the larvae (Akhtar and Isman 2003). As the isothiocyanates from NAS-type plants are expected to be more toxic than the oxazolidine-2-thiones produced by BAR-type plants, we expected M. brassicae to be repelled by NAS-type plants and to prefer BAR-type plants for oviposition. Oviposition of P. rapae is affected by glucosinolates at the leaf surface (Renwick et al. 1992; Van Loon et al. 1992) and known to be stimulated by glucobarbarin as well as by gluconasturtiin in a dose-dependent way (Chew 1988; Huang and Renwick 1994; Huang et al. 1994a, b).
As larvae can eventually leave their initial host (van Dam et al. 2000), we also assessed larval preference. If the B. vulgaris chemotypes differ in toxicity or palatability, we expect to see the largest effect on larval preference and larval performance for the unadapted generalist M. brassicae and no or minor effects on the glucosinolate specialist P. rapae.
Although we observed no differences in morphology, growth, or germination between the chemotypes (van Leur and van Dam, unpublished results), pleiotropic effects or close linkages could possibly cause other genes and metabolites to be consistently different between the chemotypes. As the nutritional value of plants is important for herbivore performance and preference (Simpson and Simpson 1990; Berenbaum 1995), additionally, we analyzed the sugar content and amino acid level of the tissue on which the herbivores were feeding.
Materials and Methods
Plant Material B. vulgaris seeds were collected from 10 individual BAR and their nearest neighbor NAS-type maternal plants, which were freely cross pollinated in a natural population of B. vulgaris. The population was located in Elderveld, The Netherlands (51.95°N; 5.87°E) and consisted of 22% NAS-type plants (Van Leur et al. 2006). We selected offspring of maternal NAS-type plants “EL44” (68% BAR-type offspring) and “EL13” (62% BAR-type offspring). Plants were grown in a glasshouse, at 21°C (day) and 16°C (night), with 60% relative humidity and natural daylight supplemented with sodium lamps to maintain the minimum PAR at 225 μmol m−2 s−1 with a photoperiod of 16:8 (L:D). One week after germination on glass beads, seedlings were transplanted to a mixture of peat soil (Potgrond 4, Lentse Potgrond BV., Lent, The Netherlands) and 20% sand. After 2 weeks, the seedlings were transplanted to 1.1-l pots, watered, and fertilized regularly with half strength Hoagland’s nutrient solution with a doubled KH2PO4 content.
Chemical Analysis For quantification of glucosinolates, soluble sugars, and amino acids, one global extraction was used. In a 2-ml Eppendorf tube, 50.0 mg of lyophilized finely ground plant material were dissolved in 1.0 ml 70% MeOH in water (v/v), vortexed, and immediately boiled for 5 min to kill the remaining myrosinase activity. Tubes were placed in an ultrasonic bath for 15 min and centrifuged (10 min 10,000 rpm). The extraction was repeated for the pellet omitting the boiling step. For each sample, both supernatants were combined in a new 2-ml Eppendorf tube and supplemented individually with 70% MeOH to attain the average mass (N = 3) of a 2-ml Eppendorf tube that contained 2.0 ml 70% MeOH. This “stock” extract was stored at −20°C until further analysis.Half (1.0 ml) of the stock extract was used for glucosinolate analysis and applied to a DEAE-Sephadex A 25 column (EC 1990), desulfated with arylsulfatase (Sigma, St. Louis, MO, USA) and separated on a reversed phase C-18 column on HPLC with a CH3CN–H2O gradient as described in van Dam et al. (2004). Glucosinolate analysis was performed with a PDA detector (200–350 nm) with 229 nm as the integration wavelength. Desulfoglucosinolate peaks were identified by comparison of HPLC retention times and UV spectra with authentic standards isolated from B. vulgaris as previously described (Agerbirk et al. 2001), as well as standards kindly provided by M. Reichelt, MPI Chemical Ecology, and a certified rape seed standard (Community Bureau of Reference, Brussels, code BCR-367R). The same response factor was used for glucobarbarin as for gluconasturtiin (Buchner 1987). To calculate glucosinolate concentrations in the plant tissue, the obtained values were multiplied by two and divided by dry mass.To analyze soluble sugar content, a 10-μl aliquot of the stock extract was diluted with 990 μl MilliQ water. Soluble sugars were analyzed by injecting 5 μl of the diluted extract on Dionex HPLC system, equipped with a Carbopac PA1 column (2 × 250 mm) and a Carbopac PA1 guard column (2 × 50 mm, Dionex, Sunnyvale, CA, USA). An isocratic gradient mixture of 10% 1 M NaOH and 90% MilliQ water was used to separate sugars at a flow rate of 0.25 ml/min. Column temperature was kept at 20°C. A “10-ppm” reference solution that contained 54.9 μM sorbitol and manitol, 29.21 μM trehalose, sucrose, and melbiose, and 55.51 μM glucose and fructose, was diluted to obtain 7.5, 5, and 2.5 ppm calibration standards to obtain a reference curve. After every 10 samples, an additional standard was injected to check for deviations of retention times and the calibration curve. To calculate the molar concentration of sugars in the plant tissue, the concentration values were multiplied by 200 and divided by dry mass.Amino acids were analyzed on a Dionex HPLC system by integrated pulsed amperometric detection. An aliquot of 20 μl of the stock extract was diluted with 980 μl MilliQ. Of this diluted extract, 25 μl were injected, and amino acids were separated with a ternary gradient (see DIONEX application update 152, Method 1, standard AAA gradient; condition 60/2 in Hanko and Rohrer 2004) on a 2 × 250 mm AminoPac© PA10 column with a 2 × 50 mm AminoPac© PA10 Guard column (Dionex, Sunnyvale, CA, USA). Eluents, flow rates, waveform, and working electrode conditions were all as specified under Method 1 in Dionex application update 152 and in Hanko and Rohrer (2004). The Sigma AA-S-18 amino acid standard (Sigma, St Louis, MO, USA) that contained 17 amino acids was supplemented with asparagine, glutamine, and tryptophane (2.5 μmol/ml each) to obtain a reference sample that contained the 20 most common amino acids. This reference solution was diluted to obtain calibration standard ranging from 1–8 μM for each amino acid, except for cysteine, which had a range of 0.5–4 μM. After every 10 samples, an additional standard was injected to check for deviations of retention times and the calibration curve. To calculate the molar concentration of the amino acids in the plant tissue, the concentration values were multiplied by 200 and divided by dry mass.To determine the chemotype of each plant, glucosinolates were extracted from the first full grown leaf and analyzed on HPLC as described above. When the peak area of glucobarbarin divided by the peak area of gluconasturtiin was >10, the plant was considered a BAR-type. When this ratio was <0.1, it was considered a NAS-type.
Oviposition Insects were obtained from the Laboratory of Entomology of Wageningen University the Netherlands. Stock colonies of P. rapae and M. brassicae were maintained on Brassica oleracea var. gemnifera L., cultivar Cyrus, in a climatized room at 20–22°C, 50–70% relative humidity, and a photoperiod of 16:8 (L:D). We used ca. 4-month-old half-sib plants of family EL44 that were clipped 1 month before use to ensure abundant fresh leaf material. At the start of the experiment, plants were moved from the glasshouse to a climatized room at 21°C (day) 16°C (night), 60% relative humidity, illuminated to 200 PAR at plant height (Philips Master TLD 50W/840 HF and 60 W lights) and a photoperiod of 16:8 (L:D). In the same room, 1- to 3-day-old adult insects were held in a mesh cage (40 × 45 × 65 cm3) provided with sugar solution. Males and females were held together to mate for at least 24 h. Oviposition preference was assessed by introducing individual pairs into one of the 11 oviposition mesh cages (40 × 45 × 65 cm3) each containing one BAR-type and one NAS-type plant and a source of sugar. Oviposition was checked every day at 10.30 and 15.30 h. M. brassicae oviposition was recorded 24 h after the first egg up to 7 days after introduction (N = 68). P. rapae oviposition was recorded for at least 5 h after the first egg and up to a maximum of 24 h in total (N = 35). Eggs deposited on the cage, pot, or on the label were not included in the analyses. To obtain sufficient replicates, four randomly chosen plants were used twice for M. brassicae, but oviposition preference was never tested on the same combination of plants.
Larval Preference For P. rapae as well as for M. brassicae, we used plants from EL44 and EL13 half sibs (one plant per chemotype per half-sib family and per herbivore). Two 0.9 cm diameter discs of each chemotype were cut from fully expanded leaves and placed in a circle (in an alternated design) in a 12 cm diameter Petri dish. We tested neonate larvae and 5-day-old larvae, which, until use, were feeding on Brassica oleracea var. gemnifera L., cultivar Cyrus leaves. The larvae were released individually at the center of the Petri dish with equal distance to all leaf discs (N = 20 per species per age). After 4 h, the amount of leaf material consumed was recorded visually and categorized as follows: 0 = no damage, 1 = only consumption of the lower leaf layers, 2 = less then 10% was consumed, 3 = between 10% and 50% of the leaf disc was consumed, and 4 = more than 50% was consumed. We were not able to assign consumption categories in the experiment with neonates because they caused so little damage, but 5-day-old larvae showed distinct feeding patterns.
Larval Performance Larval performance was tested by forcing neonate larvae to stay on selected plants and measuring larval biomass and survival every other day. We selected forty 6-week-old plants (20 EL44 half sibs and 20 EL13 half sibs; 10 of each chemotype per half-sib family). To obtain a total of 80 plants, every plant was multiplied by cutting it into two halves and growing each plant in fresh pots. After 3 weeks, the plants were used to test performance of M. brassicae. Larvae were kept on the plant using 25 mm diameter meshed clip cages. After 4 days, these clip cages were replaced by 55 mm diameter meshed clip cages. After 8 days, M. brassicae larvae had consumed such large amounts of leaf tissue that the experiment had to be stopped. The remaining larvae were removed and weighed. All plants were clipped and after 3 weeks of regrowth used to asses performance of Pieris in the same way as we did for M. brassicae. However, P. rapae larvae were kept on the plants till day 18 when most larvae had pupated. Until day 4, each larva was feeding on a single leaf that we harvested to measure the consumed leaf area and sugar, glucosinolate, and amino acid content. These samples were considered to provide an estimate for the chemotype differences over the first 8 days of feeding.The long-term performance of M. brassicae larvae was studied in a second experiment, in which three neonate larvae were placed on one plant (15 BAR and 15 NAS-type plants of EL 13). The larvae could freely move on the plant, but could not move to other plants due to a plastic cylinder and a water barrier around each plant. After 8 and 13 days, we determined larval mass and the number of dead or lost larvae. After 13 days on BAR-type plants, all but five larvae had died or were lost, so that we stopped the experiment. As dead larvae had often dried out, we excluded their biomass from all calculations.
Statistical Analysis In the oviposition preference experiments, total egg load may differ among individuals. Therefore, the number of eggs on each chemotype per individual female was treated as a paired sample. Per paired sample, we considered the chemotype with the highest egg load as the preferred type. Overall preference was tested with a Sign test. The number of eggs per plant and number of clusters per plant were not normally distributed, and were analyzed with the Wilcoxon matched-pair signed-ranks test. As there were no paired observations for cluster size, these data were analyzed with the Mann–Whitney U test.To analyze larval preference, we compared the average food consumption category between the two chemotypes by using the Wilcoxon matched-pair signed-ranks test.In the larval performance experiments, the larval masses were not normally distributed and therefore analyzed for differences between the chemotypes with separate Mann–Whitney U tests for each day followed by Bonferroni correction for multiple comparisons (for M. brassicae α = 0.05/4 = 0.0125; for P. rapae α = 0.05/8 = 0.0062). Data on the leaf consumption of each herbivore on the two chemotypes were also tested with the Mann–Whitney U test. The relationships between larval biomass and leaf consumption and between larval biomass and total glucosinolate concentration were tested by using Pearson product–moment correlations. The data were log-transformed to get a normal distribution. Before analysis of variance (ANOVA) total glucosinolate, sugar, and amino acid content data were log-transformed to meet the assumptions of ANOVA. Statistical analyses were performed with STATISTICA (data analysis software system), version 7.1. (StatSoft (2005); http://www.statsoft.com).
Results
Oviposition Preference Generalist and specialist adult herbivores did not prefer one chemotype over the other. Of the 68 M. brassicae females tested, 33.8% oviposited on the plants, 47.1% preferred to oviposit on the cage, whereas 19.1% did not oviposit at all within 1 week. The chemotypes did not receive significantly different numbers of eggs (Table 1). M. brassicae, which is a gregarious species, deposited a total of 19 clusters on BAR-type and 19 on NAS-type plants. The number of clusters per plant and the average cluster size, ranging from 6 to 465 eggs per cluster, were not significantly different between chemotypes (Table 1).
Table 1Oviposition of Mamestra brassicae and Pieris rapae on BAR-type and NAS-type Barbarea vulgaris  Mamestra brassicaePieris rapaeBARNASPZBARNASPZPreferencea14110.8380.20412190.4720.720Eggsb220 ± 40.9193 ± 51.70.3990.84326.9 ± 18.226.6 ± 14.50.9220.100Clusters/plantc1.36 ± 0.21.73 ± 0.30.9510.061Cluster sized162 ± 23.4112 ± 32.90.8000.254aTimes when that chemotype received most eggs in a pairwise comparisonbAverage number of eggs per plant ± SEcAverage number of clusters per plant ± SEdAverage number of eggs per cluster ± SEOf the 35 P. rapae pairs tested, 32 females oviposited on plants, two females did not oviposit at all, and one female died. With an average of 53.5 ± 5.1 eggs per female, 61% deposited most eggs on NAS-type plants, and one deposited an equal amount of eggs on both chemotypes. The average numbers of eggs deposited on each chemotype did not significantly differ between chemotypes (Table 1). In total, BAR-type plants received 861 eggs and NAS-type plants 850 (Table 1).
Larval Preference Of the 20 5-day-old M. Brassicae larvae tested, only one preferred to feed on BAR-type leaf discs, whereas 19 preferred the NAS-type (Sign test: Z = 3.67, P < 0.001). This preference for NAS-type leaf discs was confirmed when corrected for the consumed quantity (feeding categories). M. brassicae larvae consumed more from NAS-type leaf discs than from BAR-type (Fig. 1; Wilcoxon matched-pairs test on feeding categories: M. brassicae N = 20, Z = 3.743, P < 0.001). In contrast, the 20 5-day-old P. rapae larvae did not show a significant preference. Five larvae preferred NAS-type, 11 BAR-type, and four consumed from BAR-type as well as NAS-type leaf discs (Sign test: Z = 1.25, P = 0.211). Although P. rapae larvae consumed more from BAR-type leaves, the difference in feeding categories was not significant (Fig. 1; Wilcoxon matched-pairs test on feeding categories: P. rapae N = 20, Z = 1.629, P = 0.103). Consequently, we found no significant preference of P. rapae larvae for either chemotype.
Fig. 1Consumption of BAR-type (grey bars) and NAS-type (white bars) leaf discs by 5-day-old larvae of Mamestra brasscicae and Pieris rapae (feeding category averaged per Petri dish ± SE)
Larval Performance and Chemotypes Over the first 8 days, the larval biomass accumulation of the generalist M. brassicae was affected by chemotype (Mann–Whitney U test: P < 0.001 for each day of recording). When forced to stay on NAS-type plants, M. brassicae larvae grew exponentially (Fig. 2a). On BAR-type plants, however, M. brassicae larvae hardly increased in biomass and were moribund. This difference in performance was positively correlated to the difference in leaf consumption after 4 days of feeding (correlation analysis: r = 0.942, r2 = 0.887, P < 0.01). Consequently, M. brassicae larvae caused more damage to NAS-type leaves than to BAR-type (Fig. 3a; Mann–Whitney U test: U = 12, Z = –7.583, P < 0.001). Larval survival after 8 days on the plants was in line with these results. On NAS-type plants, all larvae were still alive, whereas on BAR-type plants 37.5% of the M. brassicae larvae had died. The results of the second experiment, in which the larvae could move freely on the plants, confirmed the results of the clip cage experiment. After 13 days, 89% ±10 of the larvae had stayed and survived on NAS-type plants and weighed 42.13 ± 2.69 mg, whereas only 20% ±10 of the larvae had stayed and survived on BAR-type plants and weighed only 4.11 ± 0.02 mg.
Fig. 2Biomass accumulation of aMamestra brassicae and bPieris rapae larvae during 8 days of feeding BAR-type (grey bars) and NAS-type (white bars) Barbarea vulgaris plants ± SEFig. 3Leaf characteristics after 4 days of larval feeding of Mamestra brassicae (left graphs) or Pieris rapae (right graphs) on Barbarea vulgaris ± SEM: a and b consumed leaf area (cm2), c and d total glucosinolate content, e and f total sugar content, g and h total amino acid content. Results are depicted per chemotype (grey bars BAR-type plants; white bars NAS-type plants) and per half-sib family (no hatching EL13, hatching EL44)In contrast to M. brassicae larvae, P. rapae larvae grew and survived equally well on both chemotypes. P. rapae larval biomass accumulation over time showed no significant difference between chemotypes over the first 18 days (Fig. 2b; Mann–Whitney U test: P > 0.05 for each day of recording) and was positively correlated to leaf consumption after 4 days (correlation: r = 0.768, r2 = 0.589, P < 0.01). Leaf damage due to P. rapae feeding was not different between the chemotypes (Fig. 3b; Mann–Whitney U test: U = 673, Z = −1.217, P = 0.223). After 8 days, larval mortality was 7.5% on NAS-type and 12.5% on BAR-type plants. On day 14, the first P. rapae larvae started pupating. After 16 days on BAR-type plants, 45% had pupated, and on NAS-type plants this was 47.5%. At the end of the experiment, after 18 days, larval weights were still not significantly different on the chemotypes (on BAR-type: 161.9 ± 7.37 mg, on NAS-type: 175.24 ± 5.63 mg; Mann–Whitney U test: U = 238, Z = −1.131, P = 0.265).
Larval Performance and Total Glucosinolate Content The leaves on which larvae were feeding differed in glucosinolate composition (glucobarbarin vs. gluconasturtiin) and in total glucosinolate content (Fig. 3c,d). In both chemotypes, we detected glucobarbarin, gluconasturtiin, glucosibarin, glucobrassicin, 4-methoxyglucobrassicin, and neoglucobrassicin. In both families, BAR-type leaves had on average 1.5 times higher total glucosinolate levels than NAS-type leaves (Fig. 3c; ANOVA type effect: F1,143 = 22.831, P < 0.001). Because M. brassicae fed more on NAS plants, overall a negative—but weak—correlation of total glucosinolate content with the consumed leaf area was observed (correlation: r(X,Y) = −0.26, r2 = 0.065, P < 0.05). To analyze in more detail whether total glucosinolate level determines larval consumption and performance, we also examined the correlation within each chemotype. Within chemotypes, the range of glucosinolate concentrations was substantial (factor 6–7), but there was no negative correlation between glucosinolate level and larval biomass (Fig. 4; Correlation: within BAR, r(X,Y) = 0.15, r2 = 0.024, P = 0.932; within NAS, r(X,Y) = 0.28, r2 = 0.078, P = 0.090). Moreover, larvae on BAR-type leaves with similar low levels of glucosinolates as NAS-types consumed and performed considerably worse (Fig. 4; glucosinolate levels <35 μmol g−1 dry mass). There was also no difference in total glucosinolate level from BAR-type leaves on which the larvae survived or died (with living larvae: 31.32 ± 2.95 μmol g−1, with dead larvae: 32.81 ± 6.49 μmol g−1; ANOVA: F1,29 = 0.059, P > 0.05; Fig. 4). As the performance of P. rapae larvae was similar among chemotypes and families, there was no correlation of larval biomass with total glucosinolate level.
Fig. 4Correlation of total glucosinolate content and the larval biomass after 4 days feeding of Mamestra brassicae experiment on NAS-type (white dots), BAR-type (black dots), and dead larvae of on BAR-type (black crosses) Barbarea vulgaris plants with their correlations per chemotype (black line) and their 95% confidence intervals (dotted lines)
Larval Performance and Nutritional Value Total sugar content of both chemotypes was composed of five consistently detected sugars, which were glucose, sucrose, sorbitol, fructose, and trehalose (from high to low average concentration). Total amino acid content of both chemotypes was composed of 11 consistently detected amino acids, which were threonine, isoleucine, arginine, serine, glutamate, aspartate, glutamine, asparagine, phenylalanine, tyrosine, and histidine (from high to low average concentration). Because the individual sugars and amino acids showed effects similar to the total levels (results not shown), we only present data on total levels.In contrast to the total glucosinolate level, sugar and amino acid content did not differ between the chemotypes (Fig. 3e–h). Although in the M. brassicae experiment the total sugar content was overall higher in BAR-type plants, there was a significant interaction between chemotype and family (Fig. 3e; ANOVA type effect: F1, 75 = 12.014, P < 0.001; type × family F1, 75 = 10.252, P < 0.05). Similar interactions between chemotype and family were shown for amino acid content of the plants in the M. brassicae experiment and for sugars and amino acid content of plants in the P. rapae experiment. In all chemotype × family interactions, the BAR-type of the EL13 half-sib family had higher levels of primary metabolites than the NAS-type of that family (Fig. 3e–h). In the EL44 half-sib family, the sugar and amino acid level differences of the chemotypes were negligible in plants during the M. brassicae experiment or even opposite to those of the EL13 during the P. rapae experiment. As sugar and amino acid content did not vary consistently with chemotype, these cannot explain the differences in larval performance and preference between the chemotypes.
Discussion
NAS and BAR chemotypes of B. vulgaris differentially affected preference and performance of the generalist herbivore M. brassicae but not the specialist P. rapae. Larvae of M. brassicae grew exponentially and had 100% survival on NAS-type leaves, but hardly grew and had a high mortality when feeding on BAR-type plants. As the dose-dependent effect of total glucosinolate content on herbivores is well-known (Li et al. 2000; Agrawal and Kurashige 2003; Mewis et al. 2006), the poor performance of M. brassicae could have been caused by the on average 1.5 times higher total glucosinolate content of BAR-type plants compared to NAS-type plants (Van Leur et al. 2006). However, we did not observe any dose-dependent effect within the chemotypes. Moreover, M. brassicae larvae on BAR-type plants with total glucosinolate levels similar to NAS-type plants still performed significantly worse. Hence, we exclude total glucosinolate level as explanatory factor for the differences found between chemotypes on larval biomass and leaf consumption.
In addition to total glucosinolate content, sugar and amino acid contents differed between chemotypes, but the pattern was inconsistent among half-sib families. The statistical interaction between chemotype and family found for sugar and amino acid levels was not found for herbivore performance and preference (Fig. 3). Therefore, total sugar content and total amino acid content can not explain the observed effects on M. brassicae.
In a different B. vulgaris polymorphism, described by Agerbirk et al. (2003b), there were P- and G-type plants that differed in resistance to Plutella xylostella. Other than our BAR/NAS polymorphism, the P-type and G-types also differed in trichome density, did not co-occur in natural populations, were hard to cross, and neither of the Danish types had a high content of gluconasturtiin in the leaves (Agerbirk, personal communication).The resistance of the G-type to P. xylostella was due to a difference in saponin content (Agerbirk et al. 2003a). Based on these findings, we compared saponin levels between BAR and NAS-type plants by using LC-TOF-MS (see supplemental data). This analysis revealed that the levels of the saponin described by Agerbirk et al. (2003a) and a saponin described by Shinoda et al. (2002) did not differ between the NAS and BAR chemotypes (supplemental data). Therefore, we exclude these saponins as an explanatory factor for the poor performance of M. brassicae on BAR-type plants.
The observed effects are likely caused by difference in glucosinolate structures and their break-down products. BAR-type plants were more toxic and deterrent to M. brassicae than the NAS-type plants. This suggests that M. brassicae can deal effectively with gluconasturtiin and isothiocyanates, but not with glucobarbarin and resulting oxazolidine-2-thiones. The latter compounds have received hardly any attention in chemical–ecological literature. Generalist herbivores possess broad-spectrum detoxification enzymes such as P450 enzymes and mixed-function oxidases (MFO) that enable them to deal with a wide range of allelochemicals, including glucosinolates (Schoonhoven et al. 1998; Li et al. 2000). Which detoxification mechanism is present in M. brassicae and why it can handle the presumably more toxic isothiocyanates, but not the oxazolidine thiones is unknown. The non-different performance of P. rapae on the chemotypes may indicate that NSP enzymes are equally effective in redirecting the hydrolysis pathways of both chemotypes towards the generally less toxic nitriles (Wittstock et al. 2004). Identification of the bioactive compounds could be acquired by using bioassay-guided fractionation.
Neither herbivore species significantly preferred one of the chemotypes as a host plant for oviposition. Even though isothiocyanates are known to elicit anemotaxis in herbivores at extremely low concentrations (Finch and Skinner 1982), the concentrations of volatile cues emitted by our undamaged plants may have been below the detection limit (Finch et al. 1978). Upon damage or induction by herbivores, volatile levels may rise and affect oviposition preference (Rothschild and Schoonhoven 1977; Bruinsma et al. 2007). Next to chemical suitability, the surface on which to oviposit can be an important factor (Renwick and Chew 1994). In our experiment, however, leaf surface structure was unlikely to affect oviposition preference between chemotypes because the chemotypes did not have apparent differences in leaf surface (e.g., trichomes) in contrast to the completely different B. vulgaris polymorphism reported by Agerbirk et al. (2003b).
As M. brassicae larvae performed poorly on BAR-type plants, the lack of oviposition preference of adults seems to be non-adaptive. The discrepancy between larval performance and oviposition preference on B. vulgaris has also been shown for the Diamondback moth (Plutella xylostella) (Serizawa et al. 2001; Badenes-Perez et al. 2006). It may occur when insects or plants are new to an area and there has not been enough time for evolutionary adaptation (Agosta 2006). Whether this is the case for M. brassicae and the B. vulgaris chemotypes is unknown.
Co-evolutionary theory suggests that the variation of plant defense compounds is maintained by sequential adaptations of specialist herbivores and plants (Agrawal et al. 1999; Cornell and Hawkins 2003). The good performance of P. rapae on both chemotypes suggests that neither chemotype is effectively defended against this specialist. On the contrary, BAR-type plants are defended against the generalist M. brassicae. Although we have no experimental evidence indicating which chemotype is the newest form, the effective defense of BAR-type plants against M. brassicae suggests an evolutionary adaptive step in which BAR-type evolved from NAS-type. This matches with the biosynthetic origin of these glucosinolates. Gluconasturtiin is presumably the precursor that is hydroxylated to produce glucobarbarin. Moreover, gluconasturtiin occurs in five times more genera than glucobarbarin (Fahey et al. 2001). This does not preclude, however, that the NAS-types found locally in the Netherlands are due to a loss-of-function mutation from the BAR-type. In natural populations of B. vulgaris, there are only 0–22% NAS-type plants (Van Leur et al. 2006). This results in a potentially limited genetic basis for NAS-type plants compared to BAR-type plants, which could contribute to linkage disequilibrium between the BAR/NAS locus and other loci (for instance closely linked loci). However, the natural population from which we selected seed batches for these experiments had a phenotype frequency close to a Hardy–Weinberg equilibrium (22% potential NAS-type fathers observed instead of 25% expected). We estimate the chance of linkage disequilibrium effects in our experiments to be relatively small. Additionally, there are no a priori indications of closely linked loci that affect herbivore performance.
For the plant, the benefit of increasing its defense against M. brassicae will be larger than increasing its defense against P. rapae. Being gregarious and larger, M. brassicae larvae are more harmful to the chosen individual (many larvae feeding from one plant) and will more strongly reduce plant fitness than the solitary P. rapae (only one or few larvae feeding on a plant). Therefore, irrespective of the degree of specialization of the herbivores, it will be more important for B. vulgaris to be defended against M. brassicae than against P. rapae.
Based on our results, we would expect BAR-type plants in the field to suffer less from herbivory than NAS-type plants. Besides being the chemotype that is most severely damaged by M. brassicae larvae, the NAS chemotype is also the recessive genotype. Therefore, if herbivorous insects are the only selective force, we expect that natural selection will drive natural populations towards 100% BAR-types. European populations of B. vulgaris indeed are mainly dominated by BAR-type plants, but a minority number of populations has still up to 22% NAS-type plants. This indicates that in these populations there may be other factors that play a role in maintaining this chemical polymorphism. Other factors that may differ between the two types and that determine plant fitness, e.g., belowground herbivory, higher trophic level interactions, and inter- and intraspecific competition, need to be included in future studies.
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barbarea vulgaris
glucosinolates
performance
mamestra brassicae
pieris rapae
oviposition
polymorphism
co-evolution