Understanding the meaning of medications for patients: The medication experience

Objective: To understand and describe the meaning of medications for patients. Methods: A metasynthesis of three different, yet complementary qualitative research studies, was conducted by two researchers. The first study was a phenomenological study of patients’ medication experiences that used unstructured interviews. The second study was an ethnographic study of pharmaceutical care practice, which included participant observation, in-depth interviews and focus groups with patients of pharmaceutical care. The third was a phenomenological study of the chronic illness experience of medically uninsured individuals in the United States and included an explicit aim to understand the medication experience within that context. The two researchers who conducted these three qualitative studies that examined the medication experience performed the meta-synthesis. The process began with the researchers reviewing the themes of the medication experience for each study. The researchers then aggregated the themes to identify the overlapping and similar themes of the medication experience and which themes are sub-themes within another theme versus a unique theme of the medication experience. The researchers then used the analytic technique, “free imaginative variation” to determine the essential, structural themes of the medication experience. Results: The meaning of medications for patients was captured as four themes of the medication experience: a meaningful encounter; bodily effects; unremitting nature; and exerting control. The medication experience is an individual’s subjective experience of taking a medication in his daily life. It begins as an encounter with a medication. It is an encounter that is given meaning before it occurs. The experience may include positive or negative bodily effects. The unremitting nature of a chronic medication often causes an individual to question the need for the medication. Subsequently, the individual may exert control by altering the way he takes the medication and often in part because of the gained expertise with the medication in his own body. Conclusion: The medication experience is a practice concept that serves to understand patients’ experiences and to understand an individual patient’s medication experience and medication-taking behaviors in order to meet his or her medication-related needs.
Introduction
Medications are one of the main options in the cure, treatment, and prevention of numerous medical conditions. However, there is still a gap in society regarding who must be held accountable for the outcomes of pharmacotherapy. Pharmacists have recently recognized the societal need to take responsibility for drug therapy outcomes [1]. However, if a health care practitioner strives to meet a patient’s drug-related needs then that practitioner must understand the meaning of medications for that patient [1–3]. However, the medication experience as a practice concept has not been comprehensively researched in pharmacy.
Medical and social scientists have examined the subjective experience of medications for patients, typically within the illness experience. Studies have been conducted on the meaning of medications for patients with specific medical conditions including: asthma [4]; hypertension [5]; and schizophrenia [6]. Moreover, studies have explored the meaning of therapeutic classes including: selective serotonin reuptake inhibitors [7]; hormone-replacement therapy [8, 9]; and antipsychotic medications [10]. Studies have also focused on patients’ medication practices or medication-taking behaviors [10, 11]; decision-making [8]; and cultural ideas of drug use [12]. Conrad [13] introduced the concept of medication practices or how patients manage their medications. He found that patients with epilepsy interpret the prescribed regimen and create medication practices that may vary from the prescribed one. Though many studies have examined the meaning of medications, most have focused on specific diseases or classes of drugs. The authors surmise that there could be a common experience of taking medications chronically that transcends the specificity of diseases and medications. Additionally, most studies on the meaning of medications have focused on patients’ decision-making, medication-taking behaviors, or compliance. Synthesizing the findings from three studies that investigate the meaning of taking medications for patients with many chronic conditions could provide a deeper level of understanding about a concept of importance to pharmacy practitioners. These three studies focused on understanding how patients experience taking medications on a daily basis; meaning how they feel, react and think about medications. This understanding may help pharmacists to better comprehend how and why patients make the decisions and take the actions they do. It is the goal of this article to present an understanding of the meaning of medications for patients, introduced here as the medication experience.
Methods
This paper is the result of a meta-synthesis of three different yet complementary qualitative studies that included aims to understand the medication experience. These three studies were conducted by the two authors. Table 1 describes each study’s method and participants’ demographics.
Table 1Studies’ methods and participant attributesStudyMedication experiencePharmaceutical care practiceIllness experience of medically uninsuredMethodologyPhenomenologyEthnographyInterpretive phenomenologyMethods InterviewsInterviews, focus groups, participant observationInterviewsSetting of interviewsUniversity conference rooms and officesParticipants’ homes and clinic exam roomsParticipants’ homes, public library rooms, & university roomsParticipantsStaff and faculty at public universityPatients of pharmaceutical care (PC)Medically uninsured individualsSelection criteria6+ months taking a chronic medication (actual 3–15 years)2+ years experience with PC1+ year uninsured, 2+ medical conditions, prescribed medsNumber of participants52511Age range24–6036–7427–64Gender of participants4 women, 1 man17 women, 8 men8 women, 3 menAverage number of medical conditions per participant2.844.5
The first study was a phenomenological study of individuals’ experiences taking at least two prescription medications. The methods were guided by Van Manen’s [14] insights using in-depth interviews, which were audio-taped and transcribed. The analysis involved reading and coding each interview transcript for the meaning units of each participant’s experience. Meaning units are phrases or text sections that illustrate a segment of the meaning distinct from the adjacent text. Then the codes were reviewed to identify the common themes of the medication experience for all participants.
The second study was an ethnography that used a triangulation of methods to reach a comprehensive understanding of pharmaceutical care practice [3]. The study revealed patients’ medication experiences as a significant part of patients’ experiences with pharmaceutical care. The study was conducted over a period of eight months in six clinics and one community pharmacy. The sampling was theoretical sampling, which involved selecting participants who would yield information that was relevant to the understanding of experiences of those in the pharmaceutical care practices. The data analysis was conducted following the techniques of Wolcott [15].
The third study was an interpretive phenomenological study that used in-depth interviews to understand medically uninsured individuals’ (in the United States) chronic illness experiences, including the medication experience [16]. The interviews were audiotaped and transcribed. The interview transcripts and field notes were segmented and labeled for one of the three experiential units - the illness, medication, and uninsured experience. A thematic analysis of the lived experience of each segment of text was conducted coding each segment with a descriptive code that captured the meaning conveyed. The codes were iteratively refined, and removed as appropriate. This was in part conducted using an analytic method called free imaginative variation. Free imaginative variation involves removing a theme, then asking if the essence of the phenomenon withstands [14]. The resultant essential codes became the themes of the illness and medication experience of medically uninsured individuals.
The researchers used meta-synthesis of the three studies to reach a greater breadth of understanding about individuals’ medication experiences. Meta-synthesis is an analytic method that facilitates a fuller understanding of a phenomenon by providing an interpretive integration of qualitative findings. The meta-synthesis of qualitative findings is increasingly seen as essential to enhance the generalizability of qualitative research [17]. The two researchers who conducted these three qualitative studies performed the meta-synthesis. The process began with the researchers reviewing the themes of the medication experience for each study. The researchers then aggregated the themes to identify the overlapping and similar themes of the medication experience. The researchers used free imaginative variation to determine the essential themes of the medication experience and from these the prevailing themes were identified. The findings from the meta-synthesis of the three studies informed the meaning of medications (i.e., the medication experience) that is presented in this paper.
Results
The meaning of medications for participants was revealed as the following themes: a meaningful encounter, bodily effects, unremitting nature, and exerting control.
A meaningful encounter
The medication experience is first revealed as an encounter with a medication. It is an encounter that is embedded with meaning long before it occurs. The meaningful encounter can be revealed as a sense of losing control, a sign of getting older, cause questioning, and a meeting with stigma. Each of these sub-themes of the meaningful encounter is described below.
The encounter with a chronic medication for the first time can be experienced as a sense of losing control, as one participant states: I used to think I was immune to disease. This body is so tremendous that it will never let me down, but here I was—I had to start taking medication.
One participant explicitly characterized her medical need for chronic medications as a loss of control:I lost control of my health. I wish I could handle it myself.
Another participant describes a similar desire to treat her condition without medication:I felt like if I took lorazepam that I’m failing. If I could fight it myself, get over it myself without the drug then I’m getting better.
Participants commonly ascribed a negative meaning to medications when they first encountered them or when they began taking an increasing number of medications chronically. Another study found loss of control as part of the meaning of medications for patients [18].
Many participants described medications as signifying aging. One participant characterized starting a medication as depressing because it indicated he was “getting older.” However, for a participant in her mid-twenties using several medications, the significance was even greater:I feel like a 90-year-old woman. Like my grandpa, he’s on all these pills. If you’re an old person that’s okay, but if you’re young it’s really odd.
Medications distinguish the healthy from the sick and the young from the old. Taking chronic medications often made participants feel that they were now part of that group of people who take medications because they are getting older.
As part of the initial encounter with a chronic medication, many participants question the actual need for the medication they are prescribed.I’m not sure what I’ll get from these medications.
Uncertainty arises when participants realize they are supposed to take a medication chronically.I thought about what would happen to me if I just decided not to take it.
Questioning the need for a medication when it is first prescribed can be interpreted as resistance by health care professionals, whereas for participants it is a way to reclaim a degree of control. Participants sensed that their individual autonomy was undermined when taking chronic medications until the point they questioned the taken-for-granted notion that medications are the right option.
The first reactions to initiating a medication can also be shaped by the social views of the medical condition. For example, the reaction of individuals to initiating psychotropic medications can be a response to the stigma of mental illness. As expressed by participants in these studies, taking an antidepressant carries the shame associated with mental illness.Maybe I’m just embarrassed, even if nobody would know. I think people would see me as weak.
When initiating a chronic medication, participants encountered different meanings that shaped their initial experience of the medication. Participants experienced the meaningful encounter with a chronic medication as a sense of losing control, a sign of getting older, causing them to question, and a meeting with stigma.
Bodily effects
Medications have expected pharmacological benefits as well as anticipated side effects and unanticipated adverse events, all of which are bodily experiences. When a patient’s experience of their body in illness is debilitating, a medication can provide relief and allow the patient to regain their “healthy” body. However, medications also can cause negative bodily sequelae that are part of a patient’s medication experience. The bodily effects of medications theme was revealed as the experience of a magic elixir and trade-offs.
The medication can “normalize” the patient or bring her body as a whole together: At first I was very grateful that insulin existed—it saved my life. I am grateful that I’m alive. It is scary that you’re reliant on this magic elixir to be alive.
Participants who characterized their medications as “magic elixirs” often had medical conditions that incapacitated them.I’ve struggled with depression all my life. My psychiatrist started me on antidepressants. I’m very grateful for these drugs. I wouldn’t be able to work if it wasn’t for them.
Sometimes the body, disrupted by a disease, can be brought back into balance with medications. Conrad [13] found that epileptic patients perceived taking their anticonvulsants as a ‘ticket’ to normality.
An important component of the bodily effects stemmed from what the medications did to improve their lives. In contrast, other participants experienced the negative sequelae of a medication. However, if the benefit experienced was sufficiently good they would be willing to accept the side effects as a trade-off. One participant indicates:You learn to decide if you’re gonna take the side effect over how well it helps with your depression. Citalopram worked so well for me that I didn’t care that I was peeing my pants.
Another participant describes:I’m not worried about taking them, mostly because if they’re going to kill me earlier I don’t care because I just want the problem to go away.
Another study identified that patients are aware of the trade-offs that must be made while trying to maximize their well-being [10]. Participants also expressed concern about taking a medication until they experienced the benefits.Even though I was hesitant at the beginning, after seeing the benefits and realizing how life can be with “just a medication” and feel good…
This theme illustrates how patients may rationally approach the decision to take or continue a medication and that they are willing to take a medication once they believe that the medication’s benefits outweigh its risks.
Unremitting nature
The unremitting nature of a chronic medication, like a chronic condition, is a burden.The first time somebody told me I would have to take that for the rest of my life I got mad.
Another participant states: I feel that part of my life now is going to be me taking more and more medicine and relying on medications to continue living.
The expectation of taking a medication regularly positions the patient as a passive agent, and the medication then becomes a symbol of dependence.I think, what if I went a couple months without taking anything? But I don’t feel I have that choice. Those pills keep you prisoner. If you go on a trip, that’s one of the major factors - it’s more important than packing enough underclothes.
This participant has lost autonomy and in her words she is held captive by her medications. Additionally, due to the chronicity of medications participants have to take responsibility.It’s this constant thing you wish you could have a break from, but you can’t.
In order to achieve normal function some participants have to take medications indefinitely, yet they still wish that wasn’t the case.You feel sorry for yourself because you’re still on these pills. I don’t want to think - it’s been this long and I’m still on them.
Exerting control
The last theme of the medication experience was revealed as participants exerting control over their medications. After encountering the meaning of a medication, questioning it, realizing the bodily effects and the continuous nature of medications, participants experimented with becoming the managers of their treatment regimens. After taking chronic medications for a period of time participants became familiar with the effects of medications on their bodies. They discovered creative ways to manage their medications and exert control over them; in part because they were now knowledgeable.I don’t think people understand how much you have to think about it. I call it thinking like a pancreas. You have to do all the work. You have to determine how much you take [insulin] and that changes from day to day and from hour to hour...
Participants have their own method of learning pharmacology–through experience. They know their bodies and perceive the changes produced by medications. As a result, exerting control is a common practice in individuals taking medications chronically.I know how to go off of it, decrease it very slowly or you get sick.
Health care professionals rarely acknowledge this practice as a form of exerting control; instead it is labeled as noncompliance. Another participant offers a description of being attuned to her body and taking control: As the doctor upped the dose I felt like I was high … I started getting a really weird feeling in my head, I even had pains in my chest. I thought to myself, I’m just gonna quit taking this. So I quit, and then as the week went on I was slowly getting better.
Participants also self manage their medications by reducing the dose particularly when they are uncertain about the effects or concerned about becoming “dependent.” I like being able to adjust my sertraline as I find necessary. I like to have that control.
As the participants in these studies convey and Carrick et al. [10] also observed, patients are the ultimate managers of their treatments and create their own medication practices. From the patient’s perspective, such behaviors are rational and help them preserve control. Ramalho de Oliveira and Shoemaker [2] argued that pharmacists typically take a paternalistic perspective, viewing patients’ non-compliant behavior as an irrational act. The understanding of patients exerting control over their medications provides a context in which to challenge the notion that patients’ medication practices are illogical.
Discussion
The authors define the medication experience as an individual’s subjective experience of taking a medication in his daily life. It begins as an encounter with a chronic medication. It is an encounter that is given meaning before it happens and is often a reaction to the symbol that medication holds. The experience may include positive or negative bodily effects. The unremitting nature of a chronic medication often causes an individual to question the need for the medication. Subsequently, the individual may exert control by altering the way he takes the medication and often in part because of the gained expertise with the medication in his own body.
In this paper, the themes of the medication experience are described as being essentially independent of one another; however there was a temporal location in which the meanings seemed to arise in the participants’ stories. It appeared that the four themes are essentially stages of the medication experience or the experience of taking a chronic medication has a natural progression from a meaningful encounter to participants exerting control. Other scholars have recognized a pattern or stages that patients go through with specific medications. A study of young women using selective-serotonin reuptake inhibitors (SSRIs) for depression revealed that SSRI users passed through stages corresponding to how they felt about themselves [7]. Further research is needed to explore the validity of patients’ medication experiences as a progression through stages or a journey.
Since medications are among the most common options in the treatment and mitigation of diseases, it is essential for health care practitioners to acknowledge an individual’s medication experience in order to positively influence patients’ medication-taking behaviors. Patients’ decisions, which at first appear irrational, might be seen as intelligent when a practitioner understands a patient’s unique medication experience. Cipolle et al. [1] state “a practitioner cannot make sound clinical decisions without a good understanding of the patient’s medication experience” and urge practitioners to take responsibility for improving each patient’s medication experience. The findings of this study provide pharmacists insights on the meanings that medications can have for patients, which may explain and certainly impact their medication-taking behavior. For example, understanding the potential meaning of initiating a chronic medication (e.g., losing control) may help explain a patient’s reticence to taking the medication.
A limitation of this study is that the synthesis of findings was from studies that explored the medication experience across multiple chronic conditions, which makes it difficult to discern the aspects of participants’ experiences related to the unique illness experience of a specific disease. Additionally, the participants were all in the U.S. (though not all native born) and the medication experiences may vary in different cultures. Even though meta-synthesis addresses the issue of small sample sizes in qualitative research, this study still has a relatively small sample size (n = 41), which limits the applicability of these findings to others’ experiences. Lastly, the authors re-analyzed their own work, which could have led to bias and reinforcement of the original findings.
Conclusions
The medication experience is a practice concept that serves to understand patients’ experiences and to understand an individual patient’s medication experience in order to meet his or her medication-related needs. The understanding of the medication experience presented in this paper can serve as a guide for health care practitioners who are interested in meeting a patient’s medication-related needs and provide a foundation from which to interpret each individual’s unique medication experience. Additional research is needed to further understand the medication experience and examine how it can be used to optimize patients’ medication-taking behaviors as well as if there are stages of the medication experience that patients pass through.

patients
medication experience
qualitative research
pharmaceutical care
united states
pharmacy services
medication management
patient-centered care


Genome-wide microRNA profiling in human fetal nervous tissues by oligonucleotide microarray

Objects Our objective was to develop an oligonucleotide DNA microarray (OMA) for genome-wide microRNA profiling and use this method to find miRNAs, which control organic development especially for nervous system.
Introduction
To date, 326 microRNAs (miRNAs), a ubiquitous family of about 22-nt noncoding regulatory RNAs, have been identified in the human genome and published in a database (http://www.sanger.ac.uk/Software/Rfam/mirna/index.shtml) [1]. Although more than 200 distinct miRNAs are predicted in the human genome, little is known about their biological functions, especially during embryonic development. MiRNAs regulate target genes at the post-transcriptional level and play important roles in development and cell lineage decisions. However, in vertebrates, neither the targets of miRNAs nor their expression profiles during development are well understood. Some miRNAs are specifically expressed in individual tissues and at particular developmental stages. The developmental or tissue-specific patterns of miRNA expression observed may suggest analogous roles in regulating human development or cellular differentiation [2–4].
A major obstacle in the study of miRNA function is the lack of methods for quantitative expression profiling. The miRNA microarray method, a powerful tool for global analysis of miRNA expression first reported by Krichevsky et al. [5], is based on a membrane array spotted with specific antisense mature miRNA oligonucleotides. Since then, other types of oligonucleotide array methods have been published, including arrays on glass slides and on beads [6–9].
In this study, a robust microarray-based technique was established and used to identify the expression of 158 miRNAs in human fetal organs. The oligonucleotide microarray (OMA) was designed according to the method of Liu [7], with modifications. We used miRNA microarrays to study the profiles of miRNA expression in nervous tissue samples and other organic samples from two fetuses. Some clusters of miRNA families were co-expressed, providing clues about the maturation processes of miRNAs. At the same time, we found a high concordance between our array results and those from Northern blots. The microarray described here offers more comprehensive coverage and higher throughput than other methods, represents a powerful tool to better understand miRNA expression profiles in human tissues, and provides clues to the mechanisms for regulating protein translation.
Materials and methods
Samples and RNA extraction
Two fetal abortuses [12 weeks (G12w) and 24 weeks (G24w) gestational age] tissues were obtained from the National Infrastructure Program of the Chinese Genetic Resources after obtaining informed consent. The G12w tissues were liver, kidney, cerebrum, lung, and heart, and the G24w tissues were the same, plus ovary, spleen, hypothalamus, pancreas, and cervical, thoracic, lumbar, and sacral spinal cord. Total RNA was isolated using TRIZOL reagent (Invitrogen) according to the manufacturer’s standard protocol, and mRNA was purified by oligotex (Qiagen).
Microarray design
The miRNA oligonucleotide microarray design was based on that of spotted OMAs by Liu et al. [7], with significant modifications. The mature miRNA sequences, which ranged from 19 to 24 nt, were too short, so two probes were designed for several miRNAs, one containing active sites that detected both the mature and the precursor forms (probe a), while the other did not contain the active sites located in the precursor (probe b). An oligo probe was designed that was not homologous to any human sequence and was used as normalization control (control oligo); the sequence was 5′ ATGTCATCTTGCGCGGCAGCTCGTCGACCGTGGCGAGAGT GTCTCGTCGATCATC 3′. All oligos were 40 nt long. In theory, the mature miRNA expression level can be determined by subtracting the (a) signal from the (b) signal. Nine probes for nine tRNA genes were used as positive controls and six probes for six types of miRNA genes from Arabidopsis thaliana were used as negative controls.
Microarray fabrication
All probes were dissolved in 150 mM phosphate acid buffer (pH 7.5–8.0) to a final concentration of 25 pmol/μl. Then the control oligo was added to each miRNA probe at 2 pmol/μl. The OMAs were spotted by a GeneMachine OmniGrid 100 Microarrayer in a 1×4 pin and 9×8 spot configuration for each subarray with triplicates. The spotting conditions were 75% humidity and 20°C. After spotting, slides were hydrated overnight in saturated salt solution and then crosslinked with UV light at 600 mJ/cm2 (UVP CL1000).
Sample labeling
The cDNA was labeled during first strand synthesis by using a fluorescence-tagged (Cy5) random octameric primer. Briefly, 10 μg total RNA and 1 μg random primer were mixed and incubated at 70°C for 10 min, then 5 × first-strand buffer, 0.1 M DTT, 5 mM unlabeled dNTP mix, Cy5-dCTP, RNA inhibitor, and Superscript II (200 U/μl) were added and incubated at 42°C for 2 h, then denatured at 70°C for 10 min. NaOH was added to hydrolyze RNA to stop the reaction of reverse transcription, and then HEPES was added to neutralize it. The labeled cDNA was purified by using the QIAquick Nucleotide Removal Kit (Qiagene).
Microarray hybridization
The labeled cDNAs and Cy3-tagged oligonucleotides complementary to the control probes were dissolved in 6 × SSPE/5 × Denhardt hybridization buffer and were hybridized with the miRNA oligonucleotide microarray for 16 h at 42°C. Then the slides were washed with buffer I (2 × SSC/0.5% SDS) for 15 min at 42°C, buffer II (1 × SSC/0.1% SDS) for 10 min at 42°C, buffer III (0.1 × SSC) for 5 min at room temperature, dipped in double-distilled water for 1 min at room temperature, and then dried. The slides were scanned by an Agilent scanner (G2565AA) at 535 and 635 nm.
Statistical analysis
The images were split into two, Cy3 and Cy5 channels, and each channel was imported into the Imagene Software 7.0 to read the signal value. The Cy3 signal was used as reference for the spot size of each miRNA oligo on the slides. The expression level of each miRNA in the sample labeled by Cy5 was normalized by a median method according to the Cy3 signal between two microarrays. So, the Cy5 signal, after normalization, gave the expression level of each miRNA. Clustering was carried out by Genespring Software 8.0 according to Cy5 intensity.
Northern blot analysis
Forty micrograms of total RNA from each sample was separated on 15% acrylamide denaturing gels (8 M urea) and then transferred to Hybond N+ membranes (Amersham) by electrophoresis for Northern blots. The filters were crosslinked with 150 mJ of UV (Bio-Rad) and baked at 80°C for 1 h. The specific oligo probes complementary to the corresponding miRNAs were labeled at the 5′ end by using T4 polynucleotide kinase with 32P-γ-ATP (Amersham). The sequence list of probes for Northern blots were: miR-92-2: 5′- CAGGCCGGGACAAGTGCAATA-3′; miR-9: 5′- TCATACAGCTAGATAACCAAAGA-3′; miR-9*: 5′-ACTTTCGGTTATCTAGCTTTA-3′; miR-15a: 5′- CACAAACCATTATGTGCTGCTA-3′; miR-17: 5′- ACAAGTGCCTTCACTGCAGT-3′; miR-20: 5′-CTACCTGCACTATAAGCACTTTA-3′; miR-106a: 5′- GCTACCTGCACTGTAAGCACTTTT-3′, and U6: 5′-CGTTCCAATTTTAGTATATGTGCTGCCGAAGCGA-3′ [10]. U6 on the membrane served as loading control. Prehybridization and hybridization were carried out using ExpressHyb Hybridization Solution (Clontech) according to the manufacturer’s instructions. Membranes were washed at room temperature, twice with 2 × SSC, 0.1% SDS and twice with 0.5 × SSC, 0.1% SDS. The blots were exposed on Molecular Dynamics Phosphorimager screens and signals were quantified using ImageQuant (Molecular Dynamics). For reuse, blots were stripped by boiling in 0.5% SDS for 20 min and scanned on Phosphorimager screens. Blots without radioactive signals were re-hybridized and re-used up to six times without influencing the quality of radioactive signals.
Results
MiRNA oligo nucleotide microarray construction
We designed 243 oligonucleotide probes and covered 158 human miRNAs from http://microrna.sanger.ac.uk/cgi-bin/sequences/browse.pl and from publications. Nine tRNAs served as positive controls. The oligonucleotide probes were 40 nt in length. For 67 miRNAs, two different probes were designed, one containing the active site and the other specific for the precursor. Using this strategy, we were able to analyze the miRNA and pre-miRNA in the same experiment. A control oligo probe, which was not homologous to any human gene sequence, was designed for spotting and quality control. All miRNA probes were dissolved in phosphate buffer at 25 μmol/ml and the control probe was mixed with each miRNA probe at 2 μmol/ml. The microarray slides were printed on an Ominigride 100 machine (Gene Machine). Each oligo probe was spotted in triplicate.
To develop the optimal conditions for the OMA, we compared mRNA and total RNA as the starting sample. Two micrograms of mRNA and 10 μg of total RNA were separately labeled during the reverse transcription reaction and hybridized to the OMA. The results indicated that mRNA had a relatively low signal compared to the total RNA, suggesting that mRNA had little influence on the results (Fig. 1d), so total RNA could be used for the microarray experiment. The microarray hybridization time was tested at 4, 6, and 16 h. The hybridization signals at 6 h were considerably higher than those at 4 h, while the signals at 16 h were only slightly higher than those at 6 h (Fig. 1a). To test the OMA sensitivity, quantities of total RNA ranging from 5 to 20 μg were labeled and then hybridized to the OMA (Fig. 1c). The strongest signal was obtained at 10 μg total RNA. To find the optimal temperature, we tested hybridization at 42, 50, and 55°C. The signal was very low at 50 and 55°C (Fig. 1b). From the above results, we chose 10 μg total RNA and 42°C hybridization for 16 h in all OMA experiments.
Fig. 1miRNA oligonucleotiede microarrary construction. a The signal at hybridization times of 6 and 16 h was much higher than that at 4 h. b A hybridization temperature of 42°C was optimal for the microarray. c On testing quantities of total RNA from 5 to 20 μg, the signal was highest at 10 μg. d Comparison of total RNA and mRNA as the starting sample. The 100 highest signal pairs were chosen, and signal values greater than 3,000 were shown as 3,000. The results indicated that mRNA had little influence on the signal, so total RNA could be used as the starting material
Distinct miRNA profiling in different organs during human development
Total RNAs of samples from human fetal organs at G12w and G24w were labeled during reverse transcription. The labeled cDNAs hybridized to the OMA. After data processing, we found that 72–83% of the miRNAs were expressed in human fetal organs, in which the most miRNAs were expressed in G24w cerebrum. Distinct miRNA expression patterns were found in different organs at each developmental stage (Fig. 2a). Some miRNAs were seen in all samples from both embryonic stages (e.g., let-7d, miR-193, miR-7-3, miR-185, and miR-328, among others); they represent a type of restricted spatial and temporal expression pattern. These miRNAs were uniformly expressed over time, which suggests more general roles in gene regulation.
Fig. 2Profiles of miRNome expression in human G12w and G24w fetal organs. a Distinct patterns of miRNA expression in different human fetal organs (abbreviation listed below). All of the data represent the average of at least three replicates from the same organ. b The same G24w heart sample triplicate chip results showed good reliability. Each result was from three replicates of the array. c Three stage-specific miRNAs, miR-92-2, miR-25, and miR-321, were expressed in G24w and G12w liver and lung. The bar represents the microarray signal intensity from 1,000 to 10,000. The blue line on the left indicates that the four miRNAs were clustered by cluster software analysis. C cerebrum, Hr heart, K kidney, Li liver, Lu lung, O ovary, P pancreas, S spleen, Vc cervical spinal cord, Vl lumbar spinal cord, Vs sacral spinal cord, Vt thoracic spinal cord
To test the replicability of the microarray, the G24w heart sample was labeled and hybridized in triplicate. The results from different detection batches were highly reproducible (Fig. 2b). Similar miRNA expression patterns were seen in samples from developmentally proximal organs, indicating that a miRNA expression profile can be a marker of developmental stage in organs originating from the same endodermal layer. Three different miRNAs, miR-92-2, miR-25, and mirR-321, were predominantly expressed in liver and lung at G24w compared with the same organs at G12w (Fig. 2c). As an indicator of miRNA expression level, the color of samples from G24w organs was brighter than those from G12w organs. So, the miRNA expression profile can be used as a biomarker of developmental stage.
CNS specifically and higher expressed microRNAs
In our study, we found four CNS specifically expressed microRNAs, miR-9, miR-9*, miR-124a, and miR-125b, in which miR-124a and miR-125b are expressed higher in G24w cerebrum than in G12w. At the same time, miR-15a, miR-106a, and miR-17–92 cluster members were found expressed relatively higher in CNS tissues than other tissues. To validate the reliability of the microarray data, we selected seven CNS specifically and higher expressed miRNAs and designed specific probes for Northern blot analysis. The miRNAs were: miR-9, miR-9*, miR-15a, miR-106a, and miR-17–92 cluster members including miR-17, miR-20 and miR-92-1, which are located in the genomic site of both terminals and in the middle of the cluster. We found good consistency between the Northern blot and microarray data, as shown by the results from the selected miR-15a and miR-106a (Fig. 3).
Fig. 3Expression of miR-15a and miR-106a, detected by Northern blot. were in accord with the signals detected by microarray. The abbreviations are as in the legend of Fig. 2. The miR-15a and miR-106a are shown on the left, indicating the corresponding expression detected by Northern blot. The microarray results are presented numerically, indicated by Oligo. U6 snRNA detected by Northern blot was used as detection control
MiR-9, miR-9*, miR-124a, and miR-125b known to be specifically expressed in rat nervous system, are also only expressed in the human fetal nervous system found by microarray and Northern blot at the same time (Fig. 4). Analysis of the array results indicated that miR-9 and miR-9* probably originated from the expressed pre-mi-RNA of chromosome 5 and may play key roles in human nervous system development. So it can be concluded that miRNA microarrays can identify different pre-miRNA origins by using different oligo probes.
Fig. 4miR-9 and miR-9* were expressed specifically in the human nervous system. a miR-9 and miR-9* can be expressed from human chromosomes 1, 5, and 15 and form three types of pre-miRNA. The secondary structures of pre-miRNAs from the Sanger miRNA register website were predicted with mfold software. The pre-miRNAs can be excised by Dicer into the same mature miRNAs—miR-9 and miR-9*. b Northern blot results probed with miR-9 and miR-9*. The abbreviations are as in the legend of Fig. 2. The microarray results are presented numerically, indicated by Oligo. U6 snRNA detected by Northern blot was used as detection control
Members of the miR-17–92 cluster, which contains six pre-miRNAs within about 1 kb on human chromosome 13, share the same expression profile [11]. The microarray and Northern blot results showed that these six miRNAs had the same expression pattern (Fig. 5), suggesting that clustered miRNAs have the same transcript and share the same promoter element.
Fig. 5Co-expressed miR-17-92 cluster miRNAs. a The three known human miRNA clusters. The miR-17–92 cluster contains six pre-miRNAs within about 1 kb on chromosome 13. b The miR-17, miR-20, and miR-92-1 expression profiles detected by Northern blot (the name of the miRNA and pre-miRNA are given on the left and the abbreviations are as in the legend of Fig. 2.) and microarray (results given numerically and marked as miR-17-oligo, miR-20-oligo, and miR-92-1-oligo). U6 snRNA detected by Northern blot was used as detection control
Discussion
The small size of miRNAs requires more sensitive tools for quantitative analysis. Although the optimized method of RT-Q-PCR can indirectly detect mature miRNAs [12], the efficiency of detection is relatively low. Currently, the most reliable method for the study of miRNA expression is Northern blot analysis with polyacrylamide gels. This method can distinguish pre-miRNA and miRNA at the same time, although the technique is relatively insensitive owing to the large total RNA volume needed, and it is labor-intensive.
The miRNA oligonucleotide microarray provides a novel method to carry out genome-wide microRNA profiling in human samples. We used total RNA as the sample for the microarray test, not just labeling filtered low molecular weight RNA, which could change the ratio of pre-miRNA to miRNA. So the profile we generated was that of pre-miRNAs and miRNAs. Owing to its high throughput and small sample requirement, the miRNA OMA can be used as screening method in miRNA research.
Some microRNAs are within the introns of host genes. Intronic miRNAs are usually expressed in coordination with their host gene mRNA, suggesting that they are generally derived from a common transcript [13]. Some human microRNAs are even processed from capped, polyadenylated transcripts and can function as mRNAs [14]. Therefore, recognition of these miRNA gene families should help in the identification of putative mRNA targets and in understanding the pathways of miRNA biogenesis. Through GeneCluster software analysis, we found that miR-17, miR-18, miR-19a, miR-19b, miR-20, and miR-92-1 form a cluster.
Based on the bioinformatics study and previous work, we attempted to verify the hypothesis that the miR-17–92 cluster may share the same expression unit. We searched for the genome location and possible co-expressed mRNA and found the six pre-miRNAs within about 1 kb on human chromosome 13; the possible co-expressed mRNA is human chromosome 13 open reading frame 25 (C13 orf25), transcript variant 2 mRNA, indicating that the miR-17–92 cluster members are intronic miRNAs. Analysis of clustered miRNA expression profiles suggested that the six clustered miRNAs may have the same promoter element.
In our study, we found that four miRNAs were only expressed in the human fetal nervous system, indicating that they may play important roles in human nervous system development. Before our study, Krichevsky et al. found miR-9 and miR-9* specifically expressed in mouse brain, and they were de-regulated in presenilin-1 null mice, which exhibited severe developmental defects of the brain [5]. MiR-9 and miR-9* may modulate critical development processes in human brain development and changes in stage and level of expression may induce major developmental errors. Giraldez et al. showed that miR-430 regulates brain morphogenesis in zebrafish and MZdicer mutants undergo axis formation and differentiate multiple cell types but display abnormal morphogenesis during gastrulation, brain formation, somitogenesis, and heart development. Injection of miR-430 rescues the brain defects in MZdicer mutants, revealing essential roles for miRNAs during morphogenesis [15].
No human miR-430 is registered online at present, but miR-17, miR-20, and miR-106a have the same sequence at nucleotides 2–8. Giraldez thought this is most important for recognition. In our study, members of the miR-17–92 cluster were highly expressed in the nervous system, but no human miRNA expression homologous to miR-430 was detected by Northern blot with a pre-miR-430 probe (data not shown). In humans, it is not miR-430 that plays a critical role in nervous system morphogenesis. So these results indicated that there are differences in the mechanisms of brain morphogenesis between human and zebra fish.
This raises the question of whether the specifically expressed miR-9, miR-9*, and higher expressed miR-17–92 cluster are functionally linked, or perhaps this just reflects higher nonspecific Dicer activity. He et al. compared B-cell lymphoma samples and cell lines to normal tissues and found that the levels of the primary or mature miRNAs derived from the miR-17–92 locus are often substantially increased in these cancers, implicating the miR-17–92 cluster as a potential human oncogene [16]. O’Donnell et al. found that c-Myc-regulated miR-17 and miR-20 modulate E2F1 expression [17]. These findings indicate that the miR-17–92 cluster may be a common channel to regulate cell differentiation.

microrna
oligonucleotide microarray
expression profile
nervous system development


Performance of Generalist and Specialist Herbivores and their Endoparasitoids Differs on Cultivated and Wild Brassica Populations

Through artificial selection, domesticated plants often contain modified levels of primary and secondary metabolites compared to their wild progenitors. It is hypothesized that the changed chemistry of cultivated plants will affect the performance of insects associated with these plants. In this paper, the development of several specialist and generalist herbivores and their endoparasitoids were compared when reared on a wild and cultivated population of cabbage, Brassica oleracea, and a recently established feral Brassica species. Irrespective of insect species or the degree of dietary specialization, herbivores and parasitoids developed most poorly on the wild population. For the specialists, plant population influenced only development time and adult body mass, whereas for the generalists, plant populations also affected egg-to-adult survival. Two parasitoid species, a generalist (Diadegma fenestrale) and a specialist (D. semiclausum), were reared from the same host (Plutella xylostella). Performance of D. semiclausum was closely linked to that of its host, whereas the correlation between survival of D. fenestrale and host performance was less clear. Plants in the Brassicaceae characteristically produce defense-related glucosinolates (GS). Levels of GS in leaves of undamaged plants were significantly higher in plants from the wild population than from the domesticated populations. Moreover, total GS concentrations increased significantly in wild plants after herbivory, but not in domesticated or feral plants. The results of this study reveal that a cabbage cultivar and plants from a wild cabbage population exhibit significant differences in quality in terms of their effects on the growth and development of insect herbivores and their natural enemies. Although cultivated plants have proved to be model systems in agroecology, we argue that some caution should be applied to evolutionary explanations derived from studies on domesticated plants, unless some knowledge exists on the history of the system under investigation.
Introduction
The occurrence of pests in agroecosystems has long promoted the study of insect–plant interactions in crop plants, such as cabbage, lima bean, maize, cotton, and tomato (Takabayashi et al. 1994; Turlings et al. 1995; De Moraes et al. 1998; Geervliet et al. 2000; Thaler et al. 2001). Studies with crop plants have generated a wealth of data, highlighting a number of important mechanisms that influence the structure and function of multitrophic interactions and communities (Root 1973; Sheehan 1986; Khan et al. 1997; Gols et al. 2005). However, critics have argued that the biology and ecology of crop plants is often dramatically different from wild populations, thus bringing into some question the evolutionary relevance of the conclusions generated from data that rely on crop plants (Benrey et al. 1998; van der Meijden and Klinkhamer 2000). For instance, plant breeding programs have been reported to disrupt the original plant defense strategies that were present in the wild progenitors of cultivated species (Evans 1993). Artificial selection of some crop plants, aimed at accentuating a specific plant trait or group of traits (e.g., the production of edible structures), has been shown to reduce the level of undesired constituents, such as defense compounds, while enhancing others (such as primary metabolites including proteins and sugars). Many of the undesired secondary plant compounds (SPC) are known to have evolved and function as putative defenses against herbivores, whereas the desired primary plant compounds act as nutrients and thus may actually enhance the performance of herbivores (Schoonhoven et al. 2005).
Levels of SPC are dynamic and vary with such factors as season, soil conditions, and leaf age (reviewed by Schoonhoven et al. 2005). Moreover, plants may increase their levels of defenses in response to feeding damage (Karban and Baldwin 1997; Agrawal 1999a), which may reduce the costs of defenses by avoiding the allocation of resources to defense when the attacker is absent. Secondary plant compounds have also been shown to affect negatively the development of higher trophic levels that attack these herbivores, such as predators, parasitoids, and even hyperparasitoids (Barbosa et al. 1986, 1991; Francis et al. 2001; Harvey et al. 2003, 2005, 2007; Ode et al. 2004). Consequently, changes in plant chemistry, mediated by artificial selection, may influence the behavior and development of consumers over several trophic levels, and this may ultimately lead to broader effects on the communities associated with these plants (Harvey et al. 2003; Ode 2006). Most importantly, in wild plants, defense mechanisms have not been constrained by the “directional selection” that characterizes many crop plants. Therefore, to understand the evolution of plant defenses against insect herbivores, multitrophic interactions should also be studied in wild conspecifics of the cultivated plant species where plant defenses are the likely result of a range of biotic and abiotic selection pressures.
One appropriate plant family for studying the effects of artificial versus natural selection on multitrophic interactions is the Brassicaceae, which contains such important crops as cabbages and various types of mustard (Gómez-Campo and Prakash 1999). Of all plants that have been domesticated, few have been manipulated to produce so many different cultivars as Brassica oleracea L. (e.g., cabbage, broccoli, cauliflower, and Brussels sprout; Gómez-Campo and Prakash 1999). Wild types of B. oleracea grow naturally along rocky coastlines of Britain and France (Mitchell and Richards 1979). It has been speculated that the wild populations in the UK are derived from plants that were cultivated by the Greeks and Romans in the Mediterranean region between 1,000 and 2,000 BC (Mitchell 1976). These early cultivated forms were introduced to Britain, but have been naturalized for centuries (Mitchell 1976). However, more recent evidence points also at an Atlantic origin of domestication (Song et al. 1990).
Plants in the Brassicaceae characteristically produce secondary metabolites called glucosinolates (hereafter GS) (Fahey et al. 2002). After tissue damage, myrosinases catalyze the hydrolysis of GS into (iso)thiocyanates and nitriles (Mithen 2001; Fahey et al. 2002), which play a role in defense against insect herbivores (Rask et al. 2000). Generalist herbivores produce enzymes that can detoxify a wide range of substrates (Krieger et al. 1971), whereas specialists have evolved enzyme systems that can detoxify specific plant compounds that are associated with herbivore diet (e.g., Johnson 1999; Ratzka et al. 2002). Thus, generalist herbivores are usually more sensitive to high levels of specific allelochemicals compared to specialists. (see e.g., Blau et al. 1978; Giamoustaris and Mithen 1995).
Specialist feeding on brassicaceous plants are adapted to plants containing GS, and they detoxify, excrete, or even sequester these harmful metabolites (Müller et al. 2001; Ratzka et al. 2002; Wittstock et al. 2004). Moreover, some insects use these compounds as indicators of food plant suitability (Nayar and Thorsteinson 1963; Renwick and Lopez 1999). Not all GS are equally effective as stimulants, and high levels of GS can reduce the performance of herbivores that are specialized on brassicaceous species (Stowe 1998; Li et al. 2000; Traw and Dawson 2002; Agrawal and Kurashige 2003). In addition, GS concentrations can increase in response to herbivore feeding damage (Bodnaryk 1992; Agrawal 1999b) and negatively affect subsequent herbivory by both generalists and specialists (Agrawal 1999b; Bartlet et al. 1999; Traw and Dawson 2002).
This study compares the development of several species of herbivores and endoparasitoids when reared on three Brassica populations that differ in their degree of domestication. Insects were reared on a cultivated and a wild population of B. oleracea, and a recently escaped (feral) Brassica species. Levels of GS were measured as indicators of direct plant defense. Initially, the development of two specialists on Brassicaceae, Plutella xylostella L. (Lepidoptera: Plutellidae) and Pieris rapae L. (Lepidoptera: Pieridae), and a generalist herbivore, Mamestra brassicae L. (Lepidoptera: Noctuidae), were examined when reared on the three populations. Finally, the development of a specialist and generalist parasitoid reared on the same host, P. xylostella, were compared. Separate cohorts of P. xylostella were parasitized by two species of endoparasitoids, Diadegma semiclausum Hellén (Hymenoptera: Ichneumonidae) and a related species, D. fenestrale Holmgren (Hymenoptera: Ichneumonidae). These two parasitoids differ in host specialization, with D. semiclausum restricted to P. xylostella and D. fenestrale attacking several other hosts that feed on non-brassicaceous species (Legaspi 1986; Azidah et al. 2000).
The following hypothesis was tested: specialist herbivores and parasitoids are less affected than generalists by differences in host plant chemistry between various Brassica populations that differ in their degree of domestication. It is proposed that changes in plant biology via domestication have significant effects on community level interactions and processes.
Methods and Materials
Plants The B. oleracea variety gemmifera (Brussels sprout) cv. Cyrus was used. Compared to other vegetable crops of B. oleracea, Brussels sprout cultivars contain relatively high levels of GS (Kushad et al. 1999; Rosa 1999), but considerably lower levels than the wild B. oleracea populations in Dorset, Great Britain (Moyes et al. 2000; Gols et al. 2008). Seeds from several plants (>10) were collected from a wild population of B. oleracea growing on chalk cliffs along the south coast of Great Britain, near Swanage, Dorset (“Old Harry,” 50°38′N, 1°55′E). This population contains intermediate levels of GS compared to other Dorset wild populations (Moyes et al. 2000). A feral Brassica population, which was found in a roadside hollow about 15 km east of Wageningen (51°95′N, 5°78′E, The Netherlands), was also included.
In addition to comparing the development of different herbivores, we also compared the development of a specialist and generalist parasitoid reared on the same host (see section on Plutella xylostella and Diadegma species). To discriminate between food-plant quality mediated through the host and host quality itself, a second closely related wild brassicaceous plant species, black mustard, Brassica nigra L., was included in one of the experiments. Seeds of B. nigra were collected from a natural population growing in a small patch along the River Rhine, near Wageningen, The Netherlands (51°94′N, 5°62′E).
Seeds from the different populations were germinated in the first week of March 2005. Seedlings were transferred to 1.1 l pots filled with potting soil (“Lentse potgrond” no. 4, Lent, The Netherlands). Plants were grown in a greenhouse at 20–30°C, 40–80% r.h, with a photoperiod of at least 16 hr. If the light dropped below 500 μmol photons/m2/sec during the 16-hr photoperiod, supplementary illumination was supplied by high-pressure mercury lamps. Plants were watered daily. After the plants were 4 wk old, they were fertilized once a week with Kristalon Blauw (N–P–K) 19–6–20–3 micro (2.5 mg/l), which was applied to the soil. B. oleracea plants were 7 wk old when they were used in experiments and attained similar amounts of biomass (25–30 g per plant). Plants from all three populations were in the vegetative state and developed new leaves during the experiments. Fertilization and watering continued during the experiments. Brassica nigra plants were 5 wk old and were not fertilized because the soil still contained enough nutrients for optimal growth. B. nigra matures much faster than B. oleracea.
Insects All insects used originated from cabbage fields in the vicinity of Wageningen. Cultures of all the herbivores have been maintained in the laboratory on Brussels sprouts cv. Cyrus for many years in climate rooms at 22 ± 2°C, 40–80% r.h, with a light regime of 16:8 L/D. The two parasitoid species were collected in the summer of 2004 and were thereafter reared on plants heavily infested with P. xylostella larvae for several generations. After pupation on the walls of the rearing cage, parasitoid cocoons were carefully removed and transferred to a clean cage. Emerged adult wasps were provided with water and honey ad libitum. For parasitism, we used females that were 5 to 10 d old after adult emergence.
Glucosinolate Analyses As an indicator of direct defense, GS concentrations in leaf tissues of B. oleracea were measured. Leaf samples were taken during the performance experiments (see below) from three treatment groups: plants that were undamaged, plants damaged by unparasitized P. xylostella, and plants damaged by larvae that had been parasitized by D. semiclausum. When leaf samples were taken, the damaged plant groups had been exposed to herbivore feeding for 7 d. Undamaged control plants were maintained in the same greenhouse, but were physically separated from the plants with caterpillars. Larvae, feces, and pupae were removed from leaves. All fully developed leaves were harvested with the exception of the oldest leaves, which had turned yellow and did not contain feeding damage. Leaves were removed with a razor blade, pooled per plant, and stored at −80°C. Samples were later freeze-dried and pulverized with a mortar and pestle. Fifty milligram aliquots of freeze-dried material were weighed in 2-ml centrifuge tubes. GS were extracted and purified as described in van Dam et al. (2004) and were separated on a reverse phase C-18 column (Alltima C-18, 3 μm,150 × 4.6 mm, Alltech, Deerfield, IL, USA) on HPLC (Dionex, Sunnyvale, CA, USA) with an acetonitrile water gradient. Detection was performed with a DIONEX PDA-100 Photodiode array detector set to scan from 200 to 350 nm. For quantification, sinigrin (Sigma, St. Louis, MO, USA) was used as an external standard. Peaks were integrated at 229 nm, for which standard response factors have been defined (EC 1990). The different GS were identified based on their retention times, and UV spectra were compared to those of pure compounds (sinigrin, Sigma, St. Louis, MO, USA; glucotropaeolin and glucobrassicin were kindly provided by M. Reichelt, Max Planck Institute for Chemical Ecology, Jena, Germany), or compared to a certified oil seed reference (EC Community Bureau of Reference BCR-367R, Fluka, Buchs, Switzerland).
Insect Performance To investigate the effect of domestication on plant direct defenses, the different herbivores and parasitoids (see below) were reared on the three plant populations. For all insects, egg-to-adult development time, adult dry mass, and survival (to adult) were determined when reared on the different populations. Adult dry mass was obtained by weighing adults on a Cahn C-33 microbalance (Cahn Instruments, Cerritos, CA, USA) that had been dried to constant weight at 80°C (3 d). Plants with insects were maintained in a greenhouse under the same conditions as described in the Plant section. Plants of the same population that received the same insect treatment were placed together, and caterpillars were allowed to develop and move around freely on plants until they reached the final instar. Different herbivore treatments were randomly positioned in a greenhouse, but were all placed in a similar position relative to the light sources to minimize microclimatic differences among plant populations and treatments.
Plutella xylostella and Diadegma Species To obtain eggs of P. xylostella, more than 150 adult moths were released with a 50:50 sex ratio in a plastic cage (37 × 40 × 30 cm). Folded strips of Parafilm served as substrate for females to lay eggs on. Females were allowed to oviposit on the Parafilm overnight. Subsequently, the strips with eggs were incubated for 4 d at 22°C until the eggs hatched. Pieces of Parafilm with neonate larvae were placed on top of individual plants of each of the three plant populations. Larvae were allowed to feed on these plants until they reached the third instar (L3).
For each plant population, one cohort of 60 larvae was transferred to new plants and served as an unparasitized control. A second cohort of 130 larvae was parasitized by D. semiclausum, and a third cohort of 180 larvae was parasitized by D. fenestrale. For parasitism, individual female wasps of both species were presented with a L3 P. xylostella host. A host was considered as parasitized when the female wasp was observed to insert into and remove her ovipositor from the larva. Individual female wasps were allowed to oviposit in up to 10 separate hosts. After this, they were removed. Parasitized larvae were transferred to new plants of the same population on which the larvae had fed previously. Five plants were used for the unparasitized controls and nine for each of the parasitoid treatments. The number of plants provided ample food for all larvae to complete their development.
When caterpillars molted into L4, strips of corrugated cardboard were placed on top of the plants, as P. xylostella prefers to pupate in secluded areas. After pupation, cocoons were collected and stored in labeled vials until adult emergence. When the moths or wasps emerged, the time of eclosion and sex were recorded. Individuals were killed by freezing at −20°C and stored for dry mass determination. Vials with cocoons ready to emerge were checked every 2 hr. Development time for P. xylostella was measured in full days, as the exact time of oviposition had not been recorded. In the case of the two Diadegma species, the median time point of the period needed to parasitize the hosts (3–4 hr) was used as the time of oviposition.
To further investigate whether food plant quality or host quality was a more important factor in the development of D. fenestrale, the experiments described above with P. xylostella were repeated on a second (and closely related) wild brassicaceous species, B. nigra. A separate study (Gols et al. unpublished) has shown that B. nigra is a qualitatively superior plant for the development of P. xylostella, compared with B. oleracea. We reared 33 unparasitized larvae on three B. nigra plants, and 180 larvae parasitized by D. fenestrale on 10 plants. We recorded egg-to-adult development time, adult biomass, and survival as before. The plants provided ample food for all the larvae to complete their development.
Pieris rapae Neonate larvae were obtained from the general culture and transferred to seven plants of each population with a distribution of six larvae per plant. When larvae had developed into L5, they were transferred to plastic containers that contained some leaf material from the plants they had fed on previously. After pupation, pupae were collected and placed in new plastic containers lined with filter paper. At adult emergence, the time of eclosion and sex were recorded, and the individuals were killed by freezing, followed by dry mass determination (as above). Containers with pupae ready to emerge were checked every 2 hr. Development time was measured in full days, as the exact time of oviposition had not been recorded.
Mamestra brassicae Like P. xylostella, adult M. brassicae are primarily nocturnal. Females do not need plants as an oviposition substrate and readily lay batches of eggs onto the surface of paper. From the general culture, we obtained paper sheets with M. brassicae eggs that were laid the previous night. These sheets were incubated at 22 ± 2°C (5 d) until the eggs hatched. Neonate larvae were transferred to 10 plants of each population, with a density of five larvae per plant. Once they had reached late L5, M. brassicae larvae were collected from the three populations, counted, and transferred to plastic containers (15 × 12 × 6 cm) that contained 2 cm of potting soil mixed with vermiculite (1:1) and some leaf material from the plant on which they had been feeding previously. After the larvae had pupated, they were collected and placed in new plastic containers filled with a layer of vermiculite. At moth emergence, the date of eclosion was recorded, and the individuals were killed by freezing, followed by dry mass determination. Containers with pupae ready to emerge were checked every 2 h. Development was measured in full days as the exact time of oviposition had not been recorded.
Statistical Analysis Data on adult dry mass and development time were analyzed by using ANOVA with plant population and sex and their interaction as factors. All larvae within one plant population were considered as independent samples. The Tukey–Kramer method was used for multiple comparisons of the means. For each insect species, a G test for heterogeneity was performed on survival rates of the three plant populations with H0: survival on each of the three plant populations is equal.
Concentrations of individual GS compounds were log(x+1) transformed to meet assumptions of normality. To examine differences in GS content, a Mixed Model was used with plant population and plant treatment (intact, damaged by unparasitized Plutella, damaged by parasitized Plutella) as the fixed factors in the model. There was no random factor in the analysis, and the estimation of effects in the model was based on restricted likelihood maximization. When the main factors or their interactions were significant, specific linear contrasts were applied to separate further factor levels. When necessary, correction for unbalanced sample sizes was carried out by using the Satterthwaite correction. Analysis was carried out with SAS 8.02 (1999–2001 ©SAS Institute, Inc).
Results
Glucosinolate Analyses GS analyses of leaf tissues revealed considerable quantitative and some qualitative variation among the different Brassica populations (Fig. 1). Three pentyl-derived (C5) GS, glucoalyssin, gluconapoleiferin, and glucobrassicanapin were only detected in the feral population, whereas the other 10 compounds were present in all three populations (Fig. 1). Total GS concentrations in undamaged plants were 3.2 and 1.4 times higher in plants of the wild population than in the cultivated and feral populations, respectively (Fig. 1). Furthermore, concentrations of all individual compounds were significantly different among the three populations (statistics not shown, but all significance levels were lower than 0.05).
Fig. 1Glucosinolate (GS) concentrations (mean ± SE) in leaf tissues of a cultivated (a), feral (b), and wild (c) Brassica population. Concentrations were measured in leaf tissue from plants that were undamaged (black bars), damaged by unparasitized P. xylostella larvae (white bars) and damaged by parasitized (D. semiclausum) larvae (gray bars). GS abbreviations and scientific names: TOT total GS concentration, GBC glucobrassicin (= indol-3-ylmethyl GS), IBE glucoiberin (= 3-methylsulfinyl propyl GS), GNA gluconapin (= 3-butenyl GS), NEO neoglucobrassicin (= 1-methoxyindol-3-ylmethyl GS), RAP glucoraphanin (= 4-methylsulfinyl butyl GS), SIN sinigrin (= 2-propenyl GS), PRO progoitrin (= 2(R)-2-hydroxy-3-butenyl GS), GBN glucobrassicanapin (= 4-pentenyl GS), GNL gluconapoleiferin (= 2-hydroxy-4-pentenyl GS), ALY glucoallyssin (= 5-methylsulfinyl pentyl GS), 4OH 4-hydroxyglucobrassicin (= 4-hydoxyindol-3-ylmethyl GS), 4MeOH, 4-methoxyglucobrassicin (= 4-methoxyindol-3-ylmethyl GS), and NAS gluconasturcin (= 2-phenylethyl GS). ND not detectable
Differences between the cultivated and feral populations on the one side, and the wild population on the other side, became even more pronounced after the plants were induced by larval P. xylostella feeding (Fig. 1). In the cultivated and feral plants, total levels of GS remained at similar levels before and after induction by P. xylostella feeding (cultivar: t50 = 0.82, P = 0.42; feral population: t50 = 0.39, P = 0.70), whereas in the wild population, concentrations were 1.5–2 times higher after herbivore feeding (t50 = 3.11, P < 0.001). Levels of individual GS changed differentially in response to herbivore feeding. The indole GS, glucobrassicin, was induced by P. xylostella feeding in all the plant populations (t50 = 6.90, P < 0.001). Moreover, glucobrassicin accounted for almost 70% of the GS composition in the wild population after induction, and for only 53% and 35% in the cultivated and feral population, respectively. In contrast, the relative amount of glucobrassicin in undamaged plants was only 21%, 5%, and 25% in the cultivated, feral, and wild populations, respectively. A second indole GS, neoglucobrassicin, was also induced in response to herbivory in the feral (t50 = 2.55, P = 0.01) and the wild population (t50 = 4.76, P < 0.001), but not in the cultivated population (t50 = 0.37, P = 0.71) in which levels of this compound were very low.
Not all GS concentrations increased after herbivory. Sinigrin was reduced after P. xylostella larval feeding in the cultivated (t50 = 2.4, P = 0.02) and the wild population (t50 = 3.67, P < 0.001), but not in the feral population (t50 = 0.07, P = 0.95). Similarly, levels of glucoiberin decreased in response to P. xylostella feeding in the cultivar (t50 = 2.79, P = 0.007) and the wild population (t50 = 4.22, P < 0.001), but not in the feral population (t50 = 0.02, P = 0.99). In the feral population, both sinigrin and glucoiberin were present in much lower concentrations than in the other two populations (Fig. 1). In plants damaged by parasitized and unparasitized larvae of P. xylostella, concentrations of individual GS were not significantly different (statistics not shown, but all significance levels were lower than 0.05).
Insect Performance: Herbivores In P. xylostella, plant population and sex had an effect on egg-to-adult development time (plant population: F2, 131 = 5.95, P = 0.003; sex: F1, 131 = 5.52, P = 0.02; Fig. 2a). Female P. xylostella developed faster than males; the fastest development time was observed for females reared on the cultivated and the feral population. For adult biomass, the interaction between plant population and sex was significant (F2, 132 = 11.1, P < 0.001). Plant population had a strong effect on female but not on male biomass (Fig. 2b). The heaviest females were recovered from the cultivar, and the lightest from the wild population. In contrast, males were significantly lighter than females (F1, 132 = 317, P < 0.001) and obtained similar biomasses on all three plant populations (Fig. 2b). For P. xylostella, plant population did not affect larval survival to the adult stage (χ2 = 0.96, df = 2, P = 0.62, Fig. 3).
Fig. 2Egg-to-adult development time (a) and adult dry mass (b) of P. xylostella males (white bars) and females (gray bars) reared on either a cultivated, a feral, or wild Brassica population. Bars (mean ± SE) with different letters are significantly different from each other (Tukey multiple comparisons, α = 0.05). Numbers of individuals (N) were on the cultivar, escape, and wild population, respectively: males, 21, 22, and 31; females 23, 22, 18Fig. 3Larval to adult survival of two specialist herbivores, P. xylostella and P. rapae, and one generalist, Mamestra brassica, when reared on either a cultivated, feral or wild Brassica population. P. xylostella was also reared on a wild population of B. nigra
Development time also varied with the population on which the P. rapae larvae had been reared (F2, 105 = 7.8, P < 0.001, Fig. 4a). Egg-to-adult development time of males was shortest on the cultivar, longer on the feral, and longest on the wild population (Fig. 4a). Males took longer to complete their development than females (F1, 105 = 7.5, P = 0.007). Plant population also had a significant effect on adult biomass (F2, 101 = 17.5, P < 0.001, Fig. 4b). Whereas adult biomass in P. rapae did not differ between the cultivated and the feral line, biomass of butterflies reared on the wild population was lower (Fig. 4b). On average, females were marginally heavier than males (F1, 101 = 3.2, P = 0.08). Irrespective of plant population, more than 92% of all P. rapae larvae successfully developed into adults, and survival rates were not significantly different (χ2 = 0.22, df = 2, P = 0.90, Fig. 3).
Fig. 4Egg-to-adult development time (a) and adult dry mass (b) of P. rapae males (white bars) and females (gray bars) reared on either a cultivated, feral, or wild Brassica population. Bars (mean ± SE) with different letters are significantly different from each other (Tukey multiple comparisons, α = 0.05). Numbers of individuals (N) were on the cultivated, feral, and wild population, respectively: males, 22, 10, and 19; females 16, 20, 20
In the case of the generalist herbivore, M. brassicae, the effect of host-plant population on adult biomass was more pronounced compared to the two specialists. It is difficult to determine the sex of adult moths, therefore, the data were pooled. Adult mass on the cultivated line was twice as high, compared to the wild population, and was also significantly higher on the feral population (F2, 49 = 8.28, P < 0.001, Fig. 5b). Plant population did not affect development time (F2, 49 = 0.33, P = 0.72, Fig. 5a). Unlike the two specialists, survival of M. brassicae was affected by plant population (χ2 = 20.4, df = 2, P < 0.001, Fig. 3). The percentage of M. brassicae larvae that developed successfully into adults was highest on the cultivated population (58%), slightly lower on the feral population (42%), and the smallest (4%) on the wild population.
Fig. 5Egg-to-adult development time (a) and adult dry mass (b) of M. brassicae reared on either a cultivated, feral, or wild Brassica population. Bars (mean ± SE) with different letters are significantly different from each other (Tukey multiple comparisons, α = 0.05). Numbers of individuals (N) were on the cultivated, feral, and wild population, respectively: 21, 29, and 2
Insect Performance: Parasitoids In the specialist parasitoid, D. semiclausum, plant population had an effect on egg-to-adult development time (F2, 275 = 6.49, P = 0.002, Fig. 6a). The parasitoid developed fastest on the feral population and developed more slowly on the cultivated and wild populations of B. oleracea (Fig. 6a). Egg-to-adult development time was longer in females than in males (F1, 275 = 13.0, P < 0.001). Furthermore, plant population had an effect on adult biomass in D. semiclausum, (F2, 277 = 11.3, P < 0.001, Fig. 6b). The heaviest D. semiclausum wasps emerged from hosts that were reared on the cultivated population, whereas the lightest emerged from host reared on the wild and feral populations. D. semiclausum females were heavier than males (F1, 277 = 57.4, P < 0.001). Between 67 and 80% D. semiclausum successfully completed development to eclosion on the three Brassica populations (Fig. 7). Survival rates were not significantly different on the three plant populations (χ2 = 3.77, df = 2, P = 0.15).
Fig. 6Egg-to-adult development time (a) and adult dry mass (b) of D. semiclausum males (white bars) and females (gray bars) reared on P. xylostella feeding on either a cultivated, feral, or wild Brassica population. Bars (mean ± SE) with different letters are significantly different from each other (Tukey multiple comparisons, α = 0.05). Numbers of individuals (N) were on the cultivated, feral, and wild population, respectively: 87, 69, and 81 for males and 11, 12, 10 for femalesFig. 7Egg-to-adult survival of a specialist parasitoid, D. semiclausum, and a generalist parasitoid, D. fenestrale, when reared from the host, P. xylostella, on either a cultivated, feral, or wild Brassica population. D. fenestrale was also reared from P. xylostella on a wild population of B. nigra
As with D. semiclausum, egg-to-adult development time in D. fenestrale varied with plant population (F2, 69 = 5.27, P = 0.007, Fig. 8a). However, dry mass in male D. fenestrale wasps did not vary significantly with the population on which the hosts had been reared (F2, 69 = 0.25, P = 0.78, Fig. 8b). As only thirteen D. fenestrale females in total successfully developed, data on females were excluded from the analysis and are not presented in the figures. The most dramatic effect of plant population was on the survival of D. fenestrale (Fig. 7), which was significantly different for the three plant populations (χ2 = 12.2, df = 2, P = 0.002). Only 9% of the parasitized hosts reared on the wild population successfully produced D. fenestrale wasps. By contrast, 33% survived on the cultivar and 16% on the feral population. Moreover, the lowest number of surviving D. fenestrale parasitoids, eight out of 172 (or 4.6%), was obtained on B. nigra. Male wasps (N = 5) developing in hosts reared on B. nigra plants were lighter (0.432 ± 0.033 mg, mean ± SE) and developed slower (17.8 ± 0.37 d) than males reared from hosts on B. oleracea (see Fig. 6). In contrast, healthy P. xylostella moths performed better on B. nigra than on B. oleracea, in terms of survival (97%, Fig. 3) and adult mass, 1.985 ± 0.068 mg (N = 16) and 1.104 ± 0.068 mg (N = 16), for females and males, respectively (see also Fig. 2b). However, development time, 17.8 ± 0.26 d for both female and male moths, was slightly longer (see also Fig. 2a).
Fig. 8Egg-to-adult development time (a) and adult dry mass (b) of D. fenestrale males reared on P. xylostella feeding on either a cultivated, feral, or wild Brassica population. Bars (mean ± SE) with different letters are significantly different from each other (Tukey multiple comparisons, α = 0.05). Numbers of individuals (N) were on the cultivar, escape, and wild population, respectively: 44, 18, and 14
Discussion
Through artificial selection via domestication, levels of primary and secondary compounds in domesticated plants are often altered compared with their progenitors. The results of this study revealed that GS concentrations in leaf tissue varied significantly among the three different plant populations. Much higher concentrations of GS were recorded in wild B. oleracea than in cultivated and feral population. GS levels in the wild population studied in this paper were similar to concentrations reported earlier in other wild populations of B. oleracea (Mithen et al. 1995; Moyes et al. 2000). The most striking differences in GS concentrations were observed after induction by herbivore feeding, especially in the wild population. In wild plants, total GS concentrations were 1.7 times higher in induced plants than in uninduced conspecific plants, and were 2.7 and 4.9 times higher after herbivory than in induced plants of the cultivated and feral population, respectively. By contrast, the total GS concentrations in induced plants remained at the same level as in undamaged plants in the cultivated and feral populations, although differences were found for levels of individual compounds.
The feral population differed from the other two populations with respect to pentyl-derived GS, which were absent in the cultivated and wild population. These C5 GS usually are not found in B. oleracea crops (Rosa 1999), indicating that this population may have crossed with a closely related species such as B. napus or B. rapa, both of which contain pentyl GS (Giamoustaris and Mithen 1995). Moreover, both B. napus and B. rapa are cultivated and grow naturally in the Netherlands. In all plant populations, concentrations of the indole GS, glucobrassicin, increased the most after herbivore feeding, but this compound was dominant only in the wild population, accounting for 70% of the GS content. Several studies have reported on herbivore-induced changes in GS concentrations in both cultivated and wild brassicaceous species. In line with our results, previous studies have shown that levels of indole GS increase in response to insect wounding (Bodnaryk 1992; Agrawal et al. 1999; Bartlet et al. 1999; Gols et al. 2008). We found that concentrations of some of the aliphatic GS were lower in plants that had been damaged by P. xylostella than in undamaged plants. Previous studies that used different herbivores reported that levels of aliphatic GS were either unaffected (Bodnaryk 1992; Bartlet et al. 1999; Traw and Dawson 2002; Gols et al. 2008) or even increased (Traw and Dawson 2002) in response to insect wounding. These results suggest differential induction of aliphatic GS by different herbivores (see also Traw and Dawson 2002).
Glucosinolates also play a major role in determining a plant’s nutritional quality, not only for humans and livestock but also for pathogens and insect herbivores (Chew 1988; Mithen 1992). This study shows that plants from a wild population of B. oleracea are less suitable for the development of several herbivores than plants from a cultivated and a feral population. However, the severity of these effects differed between the specialists P. rapae and P. xylostella on one hand, and the generalist M. brassicae on the other. In P. rapae, emerging adult butterflies were smaller and took longer to complete development when reared on wild plants than on the other two populations. These effects were less pronounced in P. xylostella; however, where adult body mass was more negatively affected than development time. Importantly, in both of the specialist herbivores, survival was high, irrespective of the plant population on which the larvae had been reared. By contrast, adult body mass and survival in the generalist herbivore, M. brassicae, were significantly lower when reared on the wild B. oleracea strain. This reveals that costs in terms of reduced fitness are higher for generalist herbivores than for specialists when they feed on the more toxic wild plants. Similarly, Giamoustaris and Mithen (1995) found a negative relationship between GS content in oilseed rape (B. napus) and the amount of leaf damage by generalist herbivores, but a positive relationship for specialists. Levels of GS, especially indole GS, were higher in the wild population of B. oleracea than levels of GS found in B. napus by Giamoustaris and Mithen (1995). These results suggest that indole GS, especially neoglucobrassicin, which is present in very low concentrations in the cultivar, may play a role in reducing the performance of insect herbivores.
Specialist insect herbivores, in contrast with generalists, may use GS as indicators of host plant suitability. For example, GS serve as feeding stimulants for insect herbivores specialized on plants belonging to the Brassicaceae (Nayar and Thorsteinson 1963; David and Gardiner 1966; Renwick and Lopez 1999). However, not all GS are equally active as feeding stimulants (e.g., Nayar and Thorsteinson 1963), and high levels of GS can even be toxic for specialists (Agrawal and Kurashige 2003). As such, high levels of specific GS may be responsible for the reduced performance of the specialists P. xylostella and P. rapae when reared on the wild population. Furthermore, levels of the enzyme myrosinase, which catalyzes hydrolysis of GS into the more toxic (iso)thiocyanates and nitriles (Rask et al. 2000; Mithen 2001), may also have differed among the populations. In addition, host plant quality is not determined only by the presence of allelochemicals. Nutrients, such as proteins and carbohydrates, as well as digestibility reducers, also play a role (Slansky 1993). It is possible that levels of limiting nutrients such as nitrogen and other defense-related compounds also vary across the three populations and thus amplify differences in performance caused by GS (Slansky and Feeny 1977).
The performance of the two parasitoid species reared on P. xylostella also varied with the plant population on which the host had been reared. However, there were also significant differences in performance between the specialist parasitoid, D. semiclausum, and the congeneric generalist parasitoid, D. fenestrale. The development of D. semiclausum was directly affected by the development of the host. Adult body mass was reduced when developing on wild B. oleracea plants, whereas development time and survival were unaffected, revealing that D. semiclausum ontogeny is affected by quantitative changes in host quality as mediated through the diet of their host. Alternatively, the development of D. fenestrale was characterized less by direct differences in host quality than by indirect population-related variations in plant quality. Although P. xylostella survival was high (>80%) on all B. oleracea populations, as well as on B. nigra plants, mortality in D. fenestrale was much higher on the wild Brassica populations.
As in most endoparasitoids, larvae of D. fenestrale primarily consume host hemolymph and fat body during early development. They only begin to indiscriminately attack other tissues later during development (the so-called “destructive feeding phase”). In this way, they do not kill the host until the last possible moment. Larvae of P. xylostella are known to utilize enzymes that convert GS into desulfo-GS in their gut, which are then excreted with their feces (Ratzka et al. 2002). Because of their polarity, GS presumably do not permeate the host-gut membrane but effectively remain in the gut before they are excreted. Consequently, the larvae of D. fenestrale probably ingest little, if any, GS when feeding on hemolymph. However, during the destructive feeding phase, the parasitoid larvae undoubtedly consume the host gut and its contents, and this is when the toxic effects of plant allelochemicals on non-adapted parasitoids may be realized. In hosts parasitized by D. fenestrale, mortality mainly occurred just before the parasitoids would have been expected to pupate (personal observation) supporting this argument. Furthermore, the gut of endoparasitoid larvae is not externally connected until after emergence from the host. The excretion of wastes into internal host tissues would facilitate bacterial infection and precocious death of both the host and the developing parasitoid (Harvey et al. 2003). Thus, low concentrations of allelochemicals that are ingested by parasitoid larvae are stored and presumably accumulate in their tissues. This may account for the high mortality recorded here with D. fenestrale that developed in P. xylostella caterpillars reared on mustard and wild cabbage plants, which contain high levels of GS.
All insects used in this study have been reared on the Cyrus cultivar for many generations and may have adapted to this plant population. However, P. xylostella developed more successfully on B. nigra plants than on the cultivar, with moths enjoying higher survival and larger body mass. Furthermore, the performance of Mamestra brassicae and Pieris rapae was almost similar on the feral and the cultivated population. We cannot exclude that the observed differences are the result of rearing history rather than true plant effects, but the fact that the insects have no history with the recently cultivated feral population of B. oleracea and developed with equal success as on the cultivar suggests that plant quality is affected by domestication. Moreover, the development of the insects was more strongly negatively affected on the wild “Old Harry” population than on the feral population.
In summary, this study has shown that plant quality in terms of development of herbivores and their natural enemies differs significantly between wild and cultivated populations of B. oleracea. The identity of the food plant and the degree of specialization exhibited by the herbivores and their parasitoids influenced the degree to which plant population affected performance. Most importantly, these results demonstrate that artificial selection in which certain plant traits are accentuated at the expense of others can alter a significant part of a plant’s evolved physiology. This may in turn have large impacts on insect communities that are associated with these plants. In wild plants, defense mechanisms have evolved under natural selection pressures from herbivores and pathogens and by the effects of natural enemies on herbivore populations. The reduction of the levels of direct defenses in cultivated plants could partly explain why these plants have often become more susceptible to attack from a wide range of herbivores and pathogens. To better understand the relative contribution of insect herbivory as a selective agent on the evolution of plant defenses, these traits should be studied in wild populations in which defense mechanisms have not been constrained by the “directional selection” that characterizes many species of crop plants. Future studies should examine insect communities associated with plant populations, including cultivars that differ in resistance against insect herbivores in plots in which the structural heterogeneity is also manipulated. This will facilitate a better understanding of the role that artificial selection has played in shaping the structure of communities associated with cropping systems.

diadegma fenestrale
plutella xylostella
glucosinolates
pieris rapae
mamestra brassicae
diadegma semiclausum
direct defense
induction
crucifer