Jasmonic Acid-Induced Changes in Brassica oleracea Affect Oviposition Preference of Two Specialist Herbivores

Jasmonic acid (JA) is a key hormone involved in plant defense responses. The effect of JA treatment of cabbage plants on their acceptability for oviposition by two species of cabbage white butterflies, Pieris rapae and P. brassicae, was investigated. Both butterfly species laid fewer eggs on leaves of JA-treated plants compared to control plants. We show that this is due to processes in the plant after JA treatment rather than an effect of JA itself. The oviposition preference for control plants is adaptive, as development time from larval hatch until pupation of P. rapae caterpillars was longer on JA-treated plants. Total glucosinolate content in leaf surface extracts was similar for control and treated plants; however, two of the five glucosinolates were present in lower amounts in leaf surface extracts of JA-treated plants. When the butterflies were offered a choice between the purified glucosinolate fraction isolated from leaf surface extracts of JA-treated plants and that from control plants, they did not discriminate. Changes in leaf surface glucosinolate profile, therefore, do not seem to explain the change in oviposition preference of the butterflies after JA treatment, suggesting that as yet unknown infochemicals are involved.
Introduction
Plants can be attacked by many herbivorous insects and have evolved a variety of defense strategies, including morphological barriers, synthesis of toxic or repellent secondary metabolites, and the release of synomones that attract natural enemies of the herbivores. These defenses can be constitutive, i.e., expressed independent of the presence of an attacker, or inducible, in which case defense compounds accumulate in response to attack (Karban and Baldwin, 1997). Herbivores can detect induced defensive compounds and respond by avoiding these plants, which signal lower suitability as a host plant (Landolt, 1993; De Moraes et al., 2001; Kessler and Baldwin, 2001; Meiners et al., 2005). Induced plant defense can affect herbivorous insects directly through the production of toxic compounds or indirectly through the production of cues that indicate intra- or interspecific competition for the herbivores (Schoonhoven et al., 2005). Moreover, induced plant defense signals can reduce the enemy-free space for the herbivores. For parasitoids and predators, induced infochemicals may indicate the presence of their host or prey on the plant (Turlings et al., 1990; Dicke and Vet, 1999). Phenotypic changes in individual plants may therefore affect insects at different trophic levels, and thus, the composition of the insect community and food web associated with the plant (Price et al., 1980; Van Zandt and Agrawal, 2004; Takabayashi et al., 2006).
Already in the 19th century, Kirby and Spence (1863) observed that Pieris brassicae females preferred to lay their eggs on plants devoid of eggs. Later, this was confirmed under more controlled conditions by Rothschild and Schoonhoven (1977) for both P. brassicae and Pieris rapae. This avoidance of infested plants is caused by a physiological response of the plant to oviposition, rather than by compounds excreted by the butterflies themselves (Blaakmeer et al., 1994). The butterflies also avoid egg deposition on leaves with feeding larvae (Rothschild and Schoonhoven, 1977). It was postulated that butterflies avoid laying eggs on herbivore-infested plants because herbivore attack induces defense compounds in plants that can influence the performance of their offspring and to reduce the risk of inter- or intraspecific competition and parasitism (Thompson and Pellmyr, 1991; Shiojiri et al., 2002). Egg-induced chemical changes in Brassica plants are also known to arrest Trichogramma parasitoids that parasitize Pieris eggs (Fatouros et al., 2005).
Oviposition-site selection involves an important behavioral decision in the life cycle of an herbivorous insect because hatching larvae have limited dispersal capacity (Renwick and Chew, 1994). P. rapae is a solitary butterfly that lays one egg at a time, whereas P. brassicae is gregarious and lays batches of about 20–100 eggs. P. rapae appears to spread the risk of larval mortality, laying few eggs within any patch. This has the advantage of being able to exploit isolated plants (Davies and Gilbert, 1985). P. brassicae, however, needs patches of plants because one large egg batch will require more than one plant for all caterpillars to develop into adults. Oviposition-site selection is performed in consecutive phases of searching and contact evaluation. P. rapae butterflies use visual, olfactory and tactile cues during these phases (Rothschild and Schoonhoven, 1977; Renwick and Radke, 1988). Acceptance of a site may be determined by the balance of positive and negative factors (Renwick and Radke, 1988). Renwick and Radke (1988) suggest that olfaction does not play a role in attraction to a host plant, but may be involved in avoidance of non-host plants. P. rapae and P. brassicae are crucifer specialists and are known to use glucosinolates, toxic secondary metabolites characteristic for Brassicaceae, as oviposition stimulants. Glucobrassicin and sinigrin are effective oviposition stimulants for P. brassicae and P. rapae (Renwick et al., 1992; Van Loon et al., 1992a).
A major signal-transduction pathway involved in induced plant defense is the octadecanoid pathway (Arimura et al., 2005). A central compound in the pathway is jasmonic acid (JA), which has an important role in direct and indirect defense in many plant species. In response to JA or methyl jasmonate (MeJA) treatment, increased concentrations of several compounds have been documented in a range of plant species, e.g., proteinase inhibitors (Moura and Ryan, 2001), polyphenol oxidases (Thaler et al., 1996), nicotine (Baldwin et al., 1996), trypsin inhibitors (Cipollini and Sipe, 2001), glucosinolates (Cipollini and Sipe, 2001; Van Dam et al., 2004; Mewis et al., 2005), and increased volatile emission (Boland et al., 1995; Dicke et al., 1999; Koch et al., 1999).
Herbivores are reported to be affected by JA treatment of plants. Several studies have focused on the larval stage of the herbivores and have shown reduced relative growth rates and leaf consumption (Van Dam et al., 2000; Gols et al., 2003; Van Dam et al., 2004). In field experiments, spraying of JA decreased the abundance of caterpillars, flea beetles, aphids, and thrips (Thaler et al., 2001). Other studies addressed the influence of JA application to plants on oviposition-site selection behavior of adult herbivores. These studies showed that JA application can result in induced resistance as well as induced susceptibility (Stanjek et al., 1997; Kessler and Baldwin, 2001; Lu et al., 2004).
Here, we studied how JA application affects oviposition of two specialist herbivores on cabbage, Pieris rapae L. and P. brassicae L. (Lepidoptera: Pieridae) that are closely related, yet differ drastically in the amount of eggs they put on one plant. Our study is the first to compare closely related herbivores with a different oviposition strategy, which might affect the consequences of JA-induced responses. JA is known to mediate the induction of chemical defense responses in plants to feeding damage and deposition of eggs (Dicke and Van Poecke, 2002; Hilker and Meiners, 2006; Mumm and Hilker, 2006). By using JA, we were able to examine the effects of induced defense responses in cabbage. Moreover, JA application has the advantage that visually detectable damage and the presence of herbivores or eggs are avoided. Finally, JA allows control over the strength of induction through controlled dosages. We hypothesized that JA treatment would inhibit the oviposition of the butterflies. We made solvent extracts to address the identity of the active plant compounds that influenced butterfly behavior. Rather than testing whole-leaf extracts, however, we extracted the glucosinolates from the surface of both control and JA-treated plants and tested the oviposition preference of the butterflies for these glucosinolate fractions on a neutral substrate. Furthermore, we included a control experiment to exclude a potential direct effect of JA on oviposition behavior. We addressed the following questions: (1) does JA treatment of cabbage plants affect host plant selection of the two Pieris butterfly species; (2) are there differences between solitary and gregarious butterflies; (3) does JA treatment affect glucosinolate levels in leaf surface extracts; and (4) do changes in glucosinolate levels determine the changes in oviposition preference?
Methods and Materials
Plants and Insects Brussels sprouts plants, Brassica oleracea var. gemmifera L. (Brassicaceae) cultivar Cyrus were grown from seed in a greenhouse in plastic pots (11 × 11 × 11 cm) at 20–28°C, 40–80% RH and a 16-hr light/8-hr dark photoperiod. All experiments were conducted with 6- to 7-wk-old plants. Stock colonies of the large cabbage white P. brassicae and the small cabbage white P. rapae were maintained on Brussels sprouts in a climatized room at 20–22°C, 50–70% RH and a 16-hr light/8-hr dark photoperiod.
Chemical Analysis Brussels sprouts plants were sprayed with 0.1 mM JA or control solution. JA ((±) jasmonic acid, purity >97%; Sigma-Aldrich, St Louis, MO, USA) was applied to the surface of the leaves, i.e., plants were sprayed with a JA solution with 0.1% Tween 20 until run-off or just with 0.1% Tween 20 for the control. The next day, glucosinolates (GLS) were extracted from the surface of the intact Brussels sprouts leaves. Each sample consisted of four leaves (between the third to sixth leaf from the base of a plant) that were cut at the base of the petiole. Directly after cutting, the lamina was dipped for 5 sec in 300 ml of dichloromethane, and after a 5-sec interval, it was dipped for 5 sec in 150 ml of methanol (Städler and Roessingh, 1990; Van Loon et al., 1992a; Griffiths et al., 2001). The methanol was evaporated from the crude methanol dip-volume with a rotary evaporator (IKA-Werke GmbH, Staufen, Germany). For each treatment, 11 plants were sampled. The extract was redissolved in methanol, desulphatased on a DEAE-Sephadex A25 column, and separated on a reverse phase C-18 column by using high performance liquid chromatography (HPLC) as described in Van Dam et al. (2004). Glucosinolate detection was performed with a photodiode array (PDA) detector (200–350 nm) with 229 nm as the integration wavelength. Sinigrin (sinigrin monohydrate, ACROS, NJ, USA) was used as an external standard. We used the correction factors at 229 nm from Buchner (1987) and the EC (EC, 1990) to calculate the concentrations of the glucosinolates. Desulfoglucosinolate peaks were identified by comparison of HPLC retention times and UV spectra with standards kindly provided by M. Reichelt, MPI Chemical Ecology and a certified rape seed standard (Community Bureau of Reference, Brussels, code BCR-367R). The surface area was measured directly after dipping, and the dry mass of the leaves was measured after drying at 50°C for 72 hr. The GLS content was calculated in pmol per cm2 leaf material.
Herbivore Oviposition Preference Test Pieris adults emerged from pupae in a large oviposition cage (67 × 100 × 75 cm) in a greenhouse compartment at 22–24°C and 50–70% RH. Apart from natural daylight, cages were illuminated by sodium vapor lamps (type SON-T, 500 W, Philips, The Netherlands) from 8:00 a.m. until 2:00 p.m. In this cage, they were provided with a 10% sucrose solution and an oviposition substrate; depending on the experiment, a plant or an artificial leaf made of green cardboard paper sprayed with sinigrin. For the experiments, one male and one female butterfly were introduced per oviposition cage (67 × 50 × 75 cm) in the same greenhouse compartment, on the day before the experiment. In these cages, the butterflies were also provided with sucrose solution. At 8:30 a.m., the treated leaves or papers and respective controls were introduced into the cages, and the butterflies were allowed to oviposit until the beginning of the afternoon. At 2:00 p.m., the leaves were removed, and the number of eggs was counted. The experiments were carried out in several cages per day and 3–4 d per treatment with new pairs of butterflies each day, adding up to a total of 24–36 independent replicates.
Surface Application of JA The effect of JA-induced changes in Brussels sprouts plants on butterfly behavior was tested in oviposition experiments with P. brassicae and P. rapae. Three concentrations of JA solution, 0.01, 0.1, and 1 mM, corresponding to approximately 1.25, 12.5, and 125 μg JA/g fresh weight (or 0.25, 2.5, and 25 nmol JA/cm2) respectively, were sprayed on the plants and tested against a control (plants treated with 0.1% Tween 20). The next morning, just before the start of the experiment, the fourth, fifth, and sixth leaves from the base of the plants were cut, and their petioles were placed directly into a vial with tap water and introduced into the cages with butterflies.
Systemic Uptake of JA For P. rapae, two application methods were used to assess the effect of JA-induced changes in Brussels sprouts on oviposition preference. For the second application method, the fourth, fifth, and sixth leaves were cut from untreated plants and placed in a 0.1-mM aqueous JA solution 22 hr before the start of the experiment. Total uptake of the solution was on average 6.3 ± 1.5 ml per control leaf and 6.0 ± 1.6 ml for JA-treated leaves (corresponding to approximately 20 μg JA/g fresh weight or 5 nmol JA/cm2, assuming homogeneous distribution over the leaf tissue after uptake).
Effect of Pure JA on Oviposition Preference In the next experiment, green cardboard paper sprayed with an oviposition stimulant was used to test the effect of pure JA on the oviposition behavior of P. rapae on an inert substrate. Sinigrin has been shown to be a suitable oviposition stimulant for Pieris butterflies (Van Loon et al., 1992a) and was therefore used to stimulate oviposition on the artificial substrate in this experiment. The paper (8 × 11.5 cm) was treated with 1 ml of a 5-mM sinigrin solution (Janssen Pharmaceutica, Tilburg, The Netherlands) by spraying it with a Desaga chromatographic sprayer (Heidelberg, Germany). Subsequently, after drying, papers were sprayed with either 1 ml of a 1-mM JA solution or water (control substrates) just before the test (210 μg JA/carton or 11 nmol JA/cm2).
Bioassays with Purified Glucosinolate (GLS) Fractions GLS were extracted from the leaf surface as described for the chemical analysis. For each treatment, control and 0.1 mM JA, 60 plants were used for the extraction, of which four to five leaves per plant were dipped. Subsequently, the extracts were fractionated following the protocol of Sørensen (Sørensen, 1990). The GLS fractions were stored in the freezer until analysis. The GLS were dissolved in methanol to make two concentrations, one corresponding to the amount of GLS extracted from the material of two plants in 0.8 ml and one concentration corresponding to the amount of GLS from one leaf in 0.8 ml. Hereafter, we will express these concentrations in gram leaf equivalents (gle). One gle corresponds to the amount of GLS extracted from 1 g fresh and intact leaf. With an average weight of 6 g per leaf, the highest concentration corresponds to 48 gle and the lower concentration to 6 gle. With a sprayer, a volume of 0.8 ml of one of the solutions was sprayed on green paper following the same method as described above for the test of pure JA. P. rapae butterflies were offered a two-choice situation, with one paper sprayed with GLS extracted from control plants and one paper with GLS from JA-treated plants.
Performance of P. rapae Caterpillars The development of first instar caterpillars to pupae was observed on control and JA-treated plants. Control plants were sprayed with a 0.1% Tween 20 solution and JA-treated plants with a solution of 0.5 mM JA with 0.1% Tween 20. Thirty newly hatched P. rapae caterpillars were evenly distributed over two plants per treatment, 24 hr after treatment, and were placed in cages (67 × 50 × 75 cm) in a greenhouse compartment at 22–24°C and 50–70% RH. The plants were replaced with new plants twice a week, so that the maximum time between induction and larval feeding never exceeded 5 d. The number of days until pupation and pupal weight were recorded.
Statistical Analyses Each individual butterfly female was subjected to a two-choice situation, in which most individuals oviposited on both control and JA-treated leaves. As the egg load differed between individuals, the number of eggs on each treatment per individual was treated as a paired sample. The oviposition data for P. rapae were normally distributed; therefore, they were analyzed with a paired t-test. The oviposition data for P. brassicae were not normally distributed, and therefore, analyzed with the nonparametric equivalent of the paired t-test, the Wilcoxon matched-pair signed-ranks test. The data on the developmental time of the caterpillars in the performance test were not normally distributed and were analyzed with a Mann–Whitney U test for differences between the treatments. Pupal weight was normally distributed and analyzed with an analysis of variance (ANOVA). Changes in GLS content were analyzed with a Mann–Whitney U test. Statistical analyses were performed with SPSS 11.0.
Results
Chemical Analysis Five GLS were detected in B. oleracea leaf surface samples: glucoiberin, sinigrin, 4-hydroxyglucobrassicin, glucobrassicin, and 4-methoxyglucobrassicin (Table 1). No significant difference was detected between JA-treated and control leaves for the total amount of GLS per cm2. The amounts of glucobrassicin, the most abundant glucosinolate in these samples, 4-methoxyglucobrassicin, and sinigrin, did not significantly differ between control and JA-treated leaves. The amounts of glucoiberin and 4-hydroxyglucobrassicin collected in the leaf surface extracts were significantly lower for JA-treated leaves compared to control leaves (Table 1). The same results were obtained when calculated for the GLS content expressed as nmol per mg dry weight (not shown).
Table 1Glucosinolate content in surface extracts of Brassica oleracea leaves in pmol/cm2 for control and JA-treated plantsCompoundControl Treatment Median (range)a,bJA Treatment Median (range)a,cZPGlucoiberin10.7 (6.3–21.7)0 (0–6.0)−2.7130.007Sinigrin9.3 (0–23.5)4.2 (0–6.8)−1.7740.0764-Hydroxyglucobrassicin0.7 (0–6.1)0 (0–0)−2.2070.027Glucobrassicin34.9 (18.4–98.2)88.6 (38.6–132.5)−1.4790.1394-Methoxyglucobrassicin0.5 (0–2.5)0 (0–1.3)−1.3700.171Total amount of glucosinolates64.1 (27.0–155.7)90.0 (42.9–138.8)−0.5630.573aInterquartile range from first to third quartilebN = 10cN = 11
Herbivore Oviposition Preference: Surface Application of JA As P. brassicae lays its eggs in batches, both the number of egg batches and the number of eggs per leaf were counted. For the 1 mM JA treatment, the number of batches was significantly lower on JA-treated leaves than on control leaves (N = 36, Z = −2.628, P = 0.009, Wilcoxon matched-pair signed-ranks test), and the total number of eggs was significantly lower as well (N = 36, Z = −2.035, P = 0.042, Wilcoxon matched-pair signed-ranks test). For the 0.1-mM JA treatment, the result was similar (N = 27, batches: Z = −2.223, P = 0.026; eggs: Z = −2.138, P = 0.032, Wilcoxon matched-pair signed-ranks test) (Fig. 1). Experiments with 0.01 mM JA application did not show discrimination by the butterflies between the treated and control leaves (results not shown). Also, P. rapae butterflies significantly preferred to oviposit on control leaves compared to JA-treated leaves (Fig. 2). The leaves treated with the two highest concentrations of JA tested, 1 and 0.1 mM, were avoided in favor of the control leaves (paired t-test, respectively, t = 3.805, df = 23, P = 0.001 and t = 3.681, df = 23, P = 0.001). The lowest concentration of JA tested, i.e., 0.01 mM, did not affect the distribution of eggs over the leaves (t = −0.662, df = 23, P = 0.52, paired t-test).
Fig. 1Pieris brassicae oviposition on control and JA-treated plants. Two concentrations of JA were tested against a control. The box represents the interquartile range from first to third quartile, the line across the box indicates the median; asterisks indicate statistical differences between the preference for control and JA-treated plants (*P < 0.05, **P < 0.01, Wilcoxon matched-pair signed-ranks test). a Egg batches per female per leaf. b Eggs per female per leafFig. 2Pieris rapae oviposition preference (measured as the number of eggs per female per leaf) between control and JA-treated B. oleracea. Three concentrations of JA were tested against a control in 24 replicated experiments for each concentration. Mean numbers of eggs per female + SEM are given; asterisks indicate statistical differences between the preference for control and JA-treated plants (n.s. P > 0.05, **P < 0.01, paired t-test)
Systemic Uptake of JA In the experiment with P. rapae that employed systemic uptake of JA through the petiole, the same result was obtained: the number of eggs on the JA-treated leaves (10.0 ± 1.57) was lower than on the control (19.12 ± 2.82) leaves (t = 3.976, df = 31, P < 0.001, paired t-test).
Effect of Pure JA on Oviposition Preference When paper treated with sinigrin and JA was compared to paper with only sinigrin, there was no difference in the number of eggs the butterflies deposited on the two substrates (t = −0.438, df = 26, P = 0.67, paired t-test) (Fig. 3). These results show that the observed effect of the JA treatment on herbivore oviposition behavior was due to induced changes in leaf tissue rather than to a direct repellent or deterrent effect of JA itself.
Fig. 3Oviposition of P. rapae on green paper sprayed with sinigrin plus JA and green paper with sinigrin only. Mean number of eggs per female per paper leaf + SEM (n.s. P > 0.05, paired t-test, N = 27)
Bioassays with Purified Glucosinolate Fractions The butterflies did not discriminate between the two GLS fractions (Table 2), the number of eggs on paper with the GLS from control plants, and the number of eggs on paper with GLS from JA-treated plants was not different for either concentration (concentration 6 gle: Z = −1.514, N = 20, P = 0.130, Wilcoxon matched-pair signed-ranks test; 48 gle: t = −0.523, df = 19, P = 0.607, paired t-test) (Fig. 4).
Table 2Glucosinolate (GLS) content in fractions from leaf surface extracts from control and JA-treated plants in pmol/cm2, Z and P-values of Mann–Whitney U testGlucosinolateGLS from control plantGLS from JA-treated plantGlucoiberin4.93.9Sinigrin3.15.14-HydroxyglucobrassicinNot detectedNot detectedGlucobrassicin16.081.04-MethoxyglucobrassicinNot detected0.8Fig. 4Oviposition of P. rapae on green paper sprayed with purified GLS-fractions from leaf surface extracts of control and JA-treated plants. Mean number of eggs per female per paper leaf + SEM (n.s. P > 0.05, paired t-test, N = 20)
Performance of P. rapae Caterpillars About two-thirds of the caterpillars survived until pupation, and a similar number of caterpillars reached the pupal stage on both treatments, 19 on control and 18 on JA-treated plants (Fig. 5). The caterpillars on JA-treated plants pupated on average after 15 d, while the caterpillars on the control plants pupated significantly sooner, on average after 13 d (Mann–Whitney U, Z = −4.071, P < 0.001). The average pupal weight on the control plants, 165 ± 3.4 mg, was similar to that on the JA-treated plants, 158 ± 3.3 mg (ANOVA, F = 2.665, df = 1, P = 0.112).
Fig. 5Development time of P. rapae caterpillars from hatching until pupation on control [open squares (□)] and JA-treated [closed triangles (▴)] plants. Cumulative number of pupae per treatment
Discussion
Our data show that JA treatment of Brussels sprouts leaves reduces the acceptance of leaves for oviposition by P. rapae and P. brassicae in a similar way. Treatment of leaves with 0.1 or 1 mM JA reduced the proportion of eggs the butterflies laid on these leaves. A concentration of 0.01 mM JA did not change oviposition preference. The former concentrations are comparable to the concentrations of JA or MeJA that were applied to several plant species in other studies and that reduced development of Spodoptera exigua, Trichoplusia ni, Manduca sexta, thrips, and aphids (Thaler et al., 1996; Avdiushko et al., 1997; Van Dam et al., 2000; Omer et al., 2001), and abundance of Manduca quinquemaculata, S. exigua, thrips, and flea beetles in the field (Kessler and Baldwin, 2001; Thaler et al., 2001). For cabbage plants, Lu et al. (2004) found inducible resistance in a susceptible Brassica species (Chinese cabbage, Brassica campestris L.) and induced susceptibility in a resistant Brassica species (common cabbage, B. oleracea) for Plutella xylostella L.
To exclude that JA itself caused the above effect, we tested the phytohormone on an inert substrate and studied two different application methods to the leaf material. We considered this an essential control that is lacking in other studies. The results of these experiments show that it was not JA itself that caused the difference in oviposition preference between control and JA-treated leaves, thus providing proof that processes in the plant induced by the JA treatment changed the acceptability of the leaves. It has also been reported previously that development of cabbage looper or tobacco hornworm larvae is not affected when MeJA is added to an artificial diet, but it is retarded when MeJA was applied to cabbage or tobacco plants (Avdiushko et al., 1997).
In leaf surface extracts of JA-treated and untreated Brussels sprouts, we found five glucosinolates. After JA application, glucobrassicin, the major glucosinolate in the B. oleracea cultivar we used, occurred at a level twice as high as in control plants, and glucoiberin and 4-hydroxyglucobrassicin concentrations decreased. The total glucosinolate content did not change significantly. However, most other studies on glucosinolate content in Brassicaceous plants after induction by JA- or MeJA-treatment or insect attack have reported an increase in glucosinolates, although there is substantial variation among different species, or even genotypes, and type of induction (Bodnaryk, 1994; Cipollini and Sipe, 2001; Mikkelsen et al., 2003; Mewis et al., 2005). Moreover, glucosinolates may not be evenly distributed throughout the leaf. We measured glucosinolate content in a surface extract after 24 hr, whereas most studies have measured glucosinolate content in whole leaf extracts and after a longer induction time. Recently, Reifenrath et al. (2005) postulated that the wax layer does not contain glucosinolates, and the polar glucosinolates that are found by using the solvent extraction method are washed from the inner leaf to the outside through the stomata. Nevertheless, we chose a surface extraction method because the butterflies retrieve chemosensory information from the leaf surface, as they do not damage the leaf before ovipositing. Surface extracts are thus likely to give a better reflection of the chemosensory information used than whole leaf extracts.
Both butterfly species distinguish between induced and non-induced leaves, most likely based on chemical differences, as JA-induced leaves do not display herbivore presence or damage. The different levels of two out of five glucosinolates in the surface extracts may provide a chemosensory basis for the oviposition preference observed, although the isolated GLS from the two treatments yielded no differences in acceptance of the paper for oviposition. While the isolated GLS on paper stimulated oviposition behavior, they appear not to be the main cue to discriminate between the JA-induced and non-induced cabbage plants.
We did not quantify other chemicals, stimulants, deterrents, or precursors that might mediate preference behavior, such as isothiocyanates, terpenoids, other glycosides, or amino acids (Huang et al., 1993; Renwick and Chew, 1994; Soldaat et al., 1996; Agrawal and Kurashige, 2003). Both P. rapae and P. brassicae can perceive a broad range of chemicals (Van Loon et al., 1992b; Hern et al., 1996). Electroantennogram responses to a range of plant volatiles are similar for both species (Van Loon et al., 1992b), although host plant selection by Pieris butterflies appears largely based on contact chemoreception rather than olfaction (Renwick and Chew, 1994). Host plant selection is suggested to depend on a balance of stimulants and deterrents and not just on the detection of presence or absence of particular compounds (Huang et al., 1993; Bruce et al., 2005). Therefore, glucosinolates, in combination with other stimulants or deterrents, may determine the acceptance of a host plant by the butterflies.
The octadecanoid pathway, in which JA is a key molecule, is involved in induction of synomones in response to oviposition and to herbivore damage (Meiners and Hilker, 2000; Dicke and Van Poecke, 2002; Hilker and Meiners, 2006; Mumm and Hilker, 2006). JA treatment of plants results in emission of synomones that attract natural enemies like predatory mites and parasitoids (Dicke et al., 1999; Hilker and Meiners, 2002; Van Poecke and Dicke, 2002; Hilker and Meiners, 2006). This attraction results in a higher natural enemy density around damaged plants, and therefore, it is advantageous for the herbivores to avoid oviposition on induced plants. Moreover, intact plants lack competitors, either intra- or interspecific. Herbivores may use induced plant cues to detect the presence or absence of other herbivores, especially because plant cues are, although less reliable, often easier to detect than cues from the herbivores themselves (Vet and Dicke, 1992).
Furthermore, induced plants may affect herbivores directly by influencing the performance of their offspring. For P. rapae, the development time differed between caterpillars feeding on JA-induced and caterpillars feeding on non-induced plants. Development of the caterpillars to pupae took longer on the induced plants, which exposes them to natural enemies for a longer time and gives them a disadvantage in the competition for resources with other herbivores. These results comply with those of Agrawal and Kurashige (2003), who showed that growth of P. rapae larvae was reduced on herbivore-induced Brassicaceae.
In summary: (1) JA treatment of B. oleracea results in avoidance of host plants by the two Pieris butterflies; (2) the related gregarious and solitary butterfly species tested responded in a similar fashion to JA-treated plants; (3) JA treatment reduced the contents of two out of five glucosinolates in leaf surface extracts of Brussels sprouts; and (4) the purified GLS fractions could not explain the observed avoidance behavior. The results indicate that JA-induced infochemicals play an important role in host plant selection behavior of these butterflies; however, the phytochemicals involved still have to be elucidated.

cabbage
pieris rapae
glucosinolates
pieris brassicae
performance
octadecanoid pathway
host plant selection


Improved renal survival in Japanese children with IgA nephropathy

Since the beginning of the 1990s, Japanese medical practitioners have extensively prescribed angiotensin-converting enzyme (ACE) inhibitors for children with mild IgA nephropathy (IgA-N) and steriods for those with severe IgA-N. We have performed a retrospective cohort study to clarify whether the long-term outcome has improved in Japanese children with IgA-N. Renal survival was defined as the time from onset to end-stage renal disease (ESRD). We divided the study period into two time periods based on the occurrence of the initial renal biopsy:1976–1989 and 1990–2004. Actuarial survivals were calculated by Kaplan–Meier method, and comparisons were made with the logrank test. The Cox proportional hazard model was used for multivariate analysis. Between 1976 and 2004, 500 children were diagnosed as having IgA-N in our hospitals. The actuarial renal survival from the time of apparent disease onset was 96.4% at 10 years, 84.5% at 15 years and 73.9% at 20 years. Renal survival in the 1990–2004 period was significantly better than that in 1976–1989 (p = 0.008), and a marked improvement in renal survival in patients with severe IgA-N was also observed (p = 0.0003). Multivariate analysis indicated that diagnosis year was a significant factor for ESRD-free survival independently of baseline characteristics. The results of this study show that there has been an improvement in terms of renal survival in Japanese children with IgA-N.
Introduction
IgA nephropathy (IgA-N) is the most common primary glomerulonephritis worldwide. It was initially considered to be a benign disease with a favorable prognosis, but data from long-term follow-up studies subsequently revealed that the disease progressed to renal failure in 20–50% of adult patients [1, 2]. Although there has been a prevailing belief that the prognosis of IgA-N is more benign in children, the results of more recent studies do not support this [3]. At the beginning of the 1990s, a strict policy of treatment for IgA-N was adopted by the majority of physicians [4]; this consisted of the extensive use of angiotensin-converting enzyme inhibitors (ACEIs) as an almost universally used therapy for IgA-N, and other therapies, including corticosteroids, for more severe IgA-N [4]. This approach has also been adopted in Japan since the beginning of the 1990s, with ACEIs generally prescribed for children with mild IgA-N showing focal mesangial proliferation and steroids prescribed for children with severe IgA-N showing diffuse mesangial proliferation. Combined therapy with prednisolone, azathioprine, heparin–warfarin and dipyridamole for 2 years in patients with severe IgA-N showing diffuse mesangial proliferation was started at the Kobe University and Wakayama Medical University hospitals in 1990 [5, 6]. At present, the effect of this therapy on long-term renal survival in children with IgA-N remains unknown as there have been few studies on outcome in sufficiently large cohorts of pediatric patients [4].
In the study reported here, we investigated data from 500 children with IgA-N to determine whether the long-term outcome has improved in Japanese children with IgA-N.
Methods
Patients
The medical history of children under the age of 20 years who underwent routine renal biopsies at Kobe University and Wakayama Medical University hospitals between January 1976 and December 2004 were analyzed retrospectively. Clinical data and follow-up information were obtained from the medical records, and if needed, further complementary information was obtained by telephone contact with the patients, family members and or physicians.
A diagnosis of IgA-N was based on the presence of IgA as the sole or predominant immunoglobulin in the glomerular mesangium in the absence of systemic disease [7]. Diffuse or focal mesangial proliferation was defined on the basis of World Health Organization criteria [8]: diffuse mesangial proliferation was defined as more than 80% of glomeruli showing moderate or severe mesangial cell proliferation, i.e. more than three cells per peripheral mesangial area; focal mesangial proliferation was defined as less than 80% of glomeruli showing moderate or severe mesangial cell proliferation. All children with IgA-N were diagnosed by one investigator (N.Y.) using the same criteria during the entire study period. Renal biopsies were performed in children with persistent proteinuria (early morning urine protein/creatinine ratio ≥0.2g/g) with or without hematuria. Renal biopsy criteria were not changed during the entire study period.
Renal survival was defined as the period from the time of apparent disease onset to end-stage renal disease (ESRD) requiring renal replacement therapy.
Given the change of policy for treating IgA-N patients in the early 1990s, we divided the study period when the initial renal biopsy had been performed into two time periods: 1976–1989 (early period) and 1990–2004 (late period).
Treatment
Since we started a strict policy of treatment for IgA-N at the beginning of the 1990s, we investigated the initial treatment profile of children with IgA-N. After a pilot study period, we started the first randomized controlled trial (RCT) by the Japanese Pediatric IgA Nephropathy Treatment Study Group (JPIGANTS) for treatment of children with severe IgA-N showing diffuse mesangial proliferation in 1990 [5]. We started the second RCT by the JPIGANTS for treatment of children with severe IgA-N in 1994 [6]. We started the third RCT by the JPIGANTS for treatment of children with severe IgA-N in 2001. The details of each treatment are shown in Table 1.
Table 1Treatment in three randomized controlled trials for childhood IgA nephropathy with diffuse mesangial proliferationPeriod1990–1993 (first RCT)1994–1998 (second RCT)2001–present (third RCT)Group 1PrednisolonePrednisolone (as in group 1 in the first period)Prednisolone  2 mg/kg/day, 4 weeks  2 mg/kg/day, 4 weeks  2 mg/kg/2 days, 4 weeks  2 mg/kg/2 days, 4 weeks  1.5 mg/kg/2 days, 4 weeks  1.5 mg/kg/2 days, 4 weeks  1 mg/kg/2 days, 21 months  1 mg/kg/2 days, 9 months  0.5 mg/kg/2 days, 12 monthsAzathioprineAzathioprine (as in group 1 in the first period)No azathioprine  2 mg/kg/day, 24 monthsDipyridamoleDipyridamole (as in group 1 in the first period)Dipyridamole  5 mg/kg/day, 24 months  6–7 mg/kg/day, 24 monthsHeparinNo heparin  APTT 60 s, 28 daysWarfarinWarfarinWarfarin  TT 30–50%, 23 months  TT 30–50%, 24 months  TT 20–50%, 24 monthsMizoribine  4 mg/kg/day, 24 monthsGroup 2Heparin-WarfarinDipyridamole (as in group 1)Prednisolone (as in group 1)Prednisolone (as in group 1)Mizoribine (as in group 1)TT, Thrombotest; APTT, activated partial thromboplastin time; RCT, randomized controlled trial
For the treatment of children with mild IgA-N showing focal mesangial proliferation we started a RCT by the JPIGANTS in 1990. During this trial (1990–1993) about half of the children with mild IgA-N in our hospitals received Sairei-to, a Chinese herb, for 24 months, and the other half received no medication. We began to use ACEIs for treatment of mild IgA-N with focal mesangial proliferation as the first choice from the beginning of 2000.
Statistical analyses
The results were analyzed with SAS ver. 9.1.2 (SAS Institute Japan Ltd., Tokyo, Japan). The associations between categorical variables were examined using the Fisher’s exact test. Continuous characteristics of the groups were compared with the Mann-Whitney U-test. Actuarial survival curves were calculated according to the Kaplan-Meier method [9], and comparisons were made with the logrank test [10]. Univariate and multivariate analyses were performed to assess the difference between the two periods in terms of renal survival. For multivariate analysis, we used the Cox proportional hazard model [11]. A two-tailed p value of less than 0.05 was taken as the level of significance.
Results
Patients
Between 1976 and 2004, 1759 children underwent a first renal biopsy examination at the Kobe University and Wakayama Medical University hospitals. Among these, 500 Japanese children (28.4%; 279 boys and 221 girls) were diagnosed as having IgA-N: 219 in 1976–1989 and 281 in 1990–2004. There was no evident change in the number of patients per year between the early (1976–1989) and late (1990–2004) periods. The median patient age at diagnosis was 10.9 years (range 2.5–19.6 years), and the median follow-up period for the patients overall was 5.9 years (range 1.3–20.5 years).
The baseline characteristics of children with IgA-N are shown in Table 2. There were significant differences in some characteristics between the two periods. Age at diagnosis had a tendency to be higher in the late group, while the ratio of asymptomatic proteinuria and hematuria at initial presentation was significantly higher in the late group. The ratio of heavy proteinuria (≥ 1 g/m2 per day) at diagnosis was significantly higher in the early period, but the ratio of patients showing diffuse mesangial proliferation was higher in the late period. Based on these data, we concluded that there was no evident difference in disease severity between the two periods.
Table 2Baseline characteristicsCharacteristicNumber of patients (%)pTotal (n = 500)1976–1989 (n = 219)1990–2004 (n = 281)Sex (M/F)279/221132/87147/1340.08Age at diagnosis, year, median [range]10.9 [2.5–19.6]10.1 [3.4–16.8]11.6 [2.5–19.6]< 0.001Initial presentationAsymptomatic proteinuria and hematuria384 (76.8%)150 (68.5%)234 (83.3%)< 0.001Macroscopic hematuria93 (18.6%)60 (27.4%)33 (11.7%)< 0.001Edema23 (4.6%)9 (4.1%)14 (5.0%)0.67Proteinuria at diagnosis (g/m2/day)  <1361 (72.2%)146 (66.7%)215 (76.5%)0.008  ≥1139 (27.8%)73 (33.3%)66 (23 .5%)Estimate creatinine clearance at diagnosis (ml/min per 1.73 m2)  <6013 (2.6%)3 (1.4%)10 (3.6%)0.16  ≥60487 (97.4%)216 (98.6%)271 (96.4%)Renal biopsy at diagnosis   Diffuse mesangial proliferation171 (34.2%)63 (28.8%)108 (38.4%)0.03   Focal mesangial proliferation329 (65.8%)156 (71.2%)173 (61.6%)
Treatment
The initial treatment for IgA-N showing focal mesangial proliferation or diffuse mesangial proliferation is given in detail in Table 3. There was a clear change of treatment for IgA-N between the early and late periods. This is particularly true in terms of ACEI use for focal mesangial proliferation and combined therapies for diffuse mesangial proliferation, both of which increased dramatically during the late period.
Table 3Change of initial treatment for IgA nephropathy in Japanese childrenTreatmentFocal mesangial proliferationDiffuse mesangial proliferation1976–1989 (n = 156)1990–2004 (n = 173)1976–1989 (n = 63)1990–2004 (n = 108)No treatment96 (61.6%)23 (13.2%)19 (30.2%)1 (0.9%)Antiplatelet and/or anticoagulant35 (22.4%)2 (1.2%)14 (22.2%)7 (6.5%)Prednisolone, (± antiplatelet and/or anticoagulant)4 (2.6%)6 (3.5%)7 (11.1%)25 (23.1%)Prednisolone + immunosuppressant, (± antiplatelet and/or anticoagulant)7 (4.5%)8 (4.6%)19 (30.2%)74 (68.5%)Chinese herb (Sairei-to)14 (9.0%)46 (26.5%)4 (6.3%)0 (0.0%)ACEI and/or ARB0 (0.0%)88 (50.9%)0 (0.0%)1 (0.9%)ACEI, Angiotensin-converting enzyme inhibitor; ARB, angiotensin II receptor blocker
Renal survival
Among a total of 500 patients who had IgA-N therapy, the actuarial renal survival from the time of apparent disease onset was 96.4% at 10 years, 84.5% at 15 years and 73.9% at 20 years (Table 4). As shown in Fig. 1 and Table 4, in children diagnosed as having IgA-N in 1976–1989, the actuarial renal survival from the time of apparent disease onset was 94.0% at 10 years, 80.1% at 15 years and 70.1% at 20 years. In children diagnosed as having IgA-N in 1990–2004, the actuarial renal survival from the time of apparent disease onset was 98.8% at 10 years, 98.8% at 15 years (p = 0.008; Fig. 1, Table 4).
Table 4Actuarial renal survival analysis of 500 children with IgA nephropathy Initial renal biopsy yearNumber of patients10-year renal survival15-year renal survival20-year renal survivalpaTotal1976–200450096.4%84.5%73.9%Total1976–198921994.0%80.1%70.1%0.0081990–200428198.8%98.8%–Diffuse mesangial proliferation1976–19896378.5%68.6%b–0.00031990–200410897.8%97.8%b–Focal mesangial proliferation1976–1989156100.0%97.7%–0.51990–2004173100.0%100.0%–ap values on the logrank testbThe 13-year survivalFig. 1Kaplan–Meier plot of renal survival stratified by the initial biopsy year for children with IgA nephropathy. 95% CI 95% Confidence interval
Figures 2 and 3 show the long-term outcome for children in the two different mesangial proliferation groups. For children with severe IgA-N showing diffuse mesangial proliferation, both the 10- and 13-year renal survivals were 97.8% when the diagnosis was made in the period 1990–2004; when the diagnosis was made in the period 1976–1989, renal survivals were 78.5% and 68.6%, respectively (p = 0.0003; Fig. 2, Table 4). For children with mild IgA-N showing focal mesangial proliferation, both the 10- and 15-year renal survivals were 100.0% when diagnosis was made in 1990–2004, compared with 100.0% and 97.7%, respectively, in 1976–1989 (p = 0.5; Fig. 3, Table 4). Although we observed better renal survival in patients diagnosed in the period 1990–2004 than in the period 1976–1989 for children with IgA-N showing focal mesangial proliferation, the difference did not reach statistical significance. The children with IgA-N showing diffuse mesangial proliferation in 1990–2004 had excellent long-term renal survival.
Fig. 2Kaplan-Meier plot of renal survival stratified by the initial biopsy year for children with severe IgA nephropathy showing diffuse mesangial proliferationFig. 3Kaplan-Meier plot of renal survival stratified by the initial biopsy year for children with mild IgA nephropathy showing focal mesangial proliferation
Table 5 shows the results of the univariate analysis using the logrank test and the multivariate analysis using the Cox proportional hazard model of prognostic factors for ESRD-free survival. Mesangial proliferation degree (focal or diffuse), proteinuria at diagnosis (<1 or ≥1 g/m2 per day), estimate of creatinine clearance at diagnosis (≥60 or <60 ml/min per 1.73 m2) and initial renal biopsy (diagnosis) year (1976–1989 or 1990–2004) were included as factors for analyses. Mesangial proliferation degree and initial renal biopsy year were significant in both the univariate and the multivariate analysis. For children with IgA-N, the most influential prognostic variable was mesangial proliferation degree. Proteinuria at diagnosis was significant in the univariate but not in the multivariate analysis. These results from the multivariate analysis showed that initial renal biopsy year was a significant factor for ESRD-free survival independently of mesangial proliferation degree, proteinuria at diagnosis and estimate of creatinine clearance at diagnosis (hazard ratio = 0.08, 95% CI 0.004–0.43; Table 5).
Table 5Univariate and multivariate analysis of the prognostic value of factors for end-stage renal disease-free survivalFactorUnivariateMultivariateHR95% CIpHR95% CIpMesangial proliferation focal; diffuse10.922.80–72.46<0.00110.272.42–70.750.001Proteinuria at diagnosis <1; ≥1 (g/m2/day)5.271.65–19.770.012.140.57–8.780.26CCl at diagnosis ≥ 60; < 60 (ml/min per m2)14.122.11–57.410.015.580.74–30.220.09Initial renal biopsy year 1976–1989; 1990–20040.140.01–0.740.020.080.004–0.430.002CI, Confidence interval; HR, hazard ratio; CCl, creatinine clearance
Discussion
Although this was a retrospective study, the data seem to provide unique and valuable information about IgA-N in children for several reasons. First, the study subjects were a complete non-selected cohort of patients with IgA-N from among all children who underwent first renal biopsy examinations at Kobe University and Wakayama Medical University hospitals between 1976 and 2004. Five hundred children with IgA-N participated in our study, which to our knowledge is the largest series used to investigate the outcome of IgA-N in children to date. Furthermore, these patients were diagnosed histologically by a single investigator (N.Y.) based on the same criteria during the whole study period. Renal biopsy criteria were also unchanged during the whole study period. Therefore, the cohort in the present study is considered to have been rigidly homogeneous in terms of the analysis of the disease outcome.
One of the conditions that facilitated our study was the school screening program, which was started by the Japanese government in 1974. In Japan, all children between the ages of 6 and 18 years are screened annually, and those found to have urinary abnormalities are referred for further investigation. Thus, in general, treatment for IgA-N is started early in the course of disease because the duration of disease before treatment is short as a result of this school screening program [12]. Therefore, in this study we were able to observe the disease courses of patients during the initial renal biopsy period, 1976–2004, who underwent initial treatments for IgA-N at a relatively homogeneous stage of the disease. Accumulated experience indicates that long-term corticosteroid and/or immunosuppressive treatment during the severely progressive stage of the disease does not confer any benefit in adult patients [2]. In contrast, it is known that centers in countries with active urine screening programs are more likely to diagnose mild disease with a good prognosis, thus favorably influencing the overall outcome of the cohort [13].
Another favorable parameter of this study was the enforcement of consecutive systemic nationwide clinical trials of IgA-N. After a pilot study period, the first RCT for treatment of children with severe IgA-N showing diffuse mesangial proliferation was started by the JPIGANTS in 1990 [5]. To date, two RCTs of treatment for children with severe IgA-N showing diffuse mesangial proliferation by the JPIGANTS have been completed [5, 6], and the third RCT of treatment for children with severe IgA-N showing diffuse mesangial proliferation is currently being conducted by the same group. For the treatment of children with mild IgA-N and focal mesangial proliferation, a RCT was also started by the JPIGANTS in 1990. After completion of the RCT, we have conducted prospective clinical trials of treatment for children with mild IgA-N and focal mesangial proliferation. These prospective trials have enabled for us to clarify the optimum treatment for IgA-N and to collect precise information on patients with IgA-N.
Because of the variable rate of progression to renal failure and the probable multifactorial pathogenesis of IgA-N, it is desirable to evaluate the effectiveness of any treatment by a prospective controlled trial. However, although the ultimate endpoint in any clinical trial of progressive IgA-N is the development of chronic renal insufficiency, most pediatric patients do not develop it during the study period [14]. Therefore, data from a properly analyzed long-term retrospective study may also be important to evaluate disease outcome and treatment effectiveness [4, 9–11]. Retrospective studies with accurate statistical evaluation using life-time analysis and multivariate survivorship analysis according to the Cox regression model are thought to work effectively together with well-designed RCTs.
The primary purpose of this study was to clarify whether long-term outcome was improved in Japanese children with IgA-N retrospectively using adequate statistical methods. Although it seemed that there were no clinically significant differences in the baseline characteristics of the groups, there were some statistically significant differences in the baseline characteristics of the groups between the two periods when initial renal biopsies were performed. These different baseline characteristics may influence the final outcomes reported in this study. To compensate for this limitation, we used a multivariate survivorship analysis according to the Cox regression model. Since we have to adjust for differences of baseline characteristics, which were thought to be important prognostic factors [15], mesangial proliferation degree, proteinuria at diagnosis, estimate of creatinine clearance at diagnosis and the initial renal biopsy (diagnosis) year were included as factors for analyses. This multivariate analysis indicated that the initial renal biopsy year was a significant factor for ESRD-free survival after adjustment of these baseline characteristics. These data support an improvement of renal survival in Japanese children with IgA-N.
There was a notable lack of the usual dominance of males over females in our study and, in addition, there was a difference between the 1976–1989 and the 1990–2004 periods, with more females being recruited in the second period. Although this difference was not statistically significant (p = 0.08), we decided that it be worthwhile discussing possible reasons for the differences in gender representation between the two periods. Therefore, we analyzed males and females separately. However, there was no gender-based factor in the results (data not shown).
At the beginning of the 1990s, a strict policy of treatment was adopted by the majority of physicians in Japan. It is likely that therapeutic intervention early after the onset of clinically apparent disease will provide the best opportunity for improving the outcome of patients with IgA-N, thus reducing the number of patients who develop ESRD [5, 6]. This study showed a clear change in the treatment of IgA-N between the early and late groups (Table 3). In particular, the use of ACEIs for focal mesangial proliferation and combination therapies for diffuse mesangial proliferation increased dramatically in the late group.
Corticosteroids have been widely used to treat moderate to severe IgA-N, particularly in pediatric patients. Some evidence has been obtained recently pertaining to the role of corticosteroids in the treatment of IgA-N [2, 13, 14, 16–18]. With regard to children and based on the results of two multicenter RCTs, we reported previously that the treatment of childhood IgA-N with diffuse mesangial proliferation using prednisolone, azathioprine, warfarin and dipyridamole for 2 years early in the course of disease reduced the severity of immunologic renal injury and prevented any increase in the percentage of sclerosed glomeruli [5, 6]. In contrast, in the first RCT, heparin–warfarin and dipyridamole treatments for 2 years did not reduce urinary protein excretion, serum IgA concentration and mesangial IgA deposition, and they did not prevent any increase of sclerosed glomeruli [5]. The results of the second RCT showed that the treatment of children with severe IgA-N using prednisolone alone for 2 years reduced the severity of immunologic renal injury, but did not prevent any further increase of glomerular sclerosis [6]. Therefore, treatment with the combination therapy using prednisolone and immunosuppressant, such as azathioprine, may be better than the prednisolone monotherapy for patients with severe IgA-N.
We found an improvement of long-term renal survival in Japanese children with IgA-N (Table 4, Figs. 1 and 2). Renal survival in 1990–2004 was significantly better than in 1976–1989 (Fig. 1), and a marked improvement of renal survival in IgA-N showing diffuse mesangial proliferation was observed over time (Fig. 2). This improvement may be related to the 2-year therapy, including corticosteroids, for all patients with IgA-N showing diffuse mesangial proliferation as a treatment policy, although in principle the effectiveness of any treatment can only be evaluated properly by a controlled trial. It is conceivable that the outcome in the period 1976–1989 may have reflected the natural course of disease, whereas that in 1990–2004 may have reflected modification of the disease course by the treatments.
The median follow-up period of the patients overall was 5.9 years (range 1.3–20.5 years). Although this is a considerably long period, it may not be sufficiently long for the follow-up of IgA-N, which has a slow progressive course. Since the elapsed time from apparent disease onset to ESRD was used to evaluate the disease outcome, chronic renal insufficiency before ESRD was not considered in our study. Thus, we do not know whether the observed improvement of outcome means a complete cure of the disease or merely a delay of the disease course. Further follow-up of the cohort for a long period is therefore important.
One aspect which we should consider in our study is the possibility that at least some of the differences in outcome between the early and late groups may be due to general changes in care that have occurred over time. Such changes are frequently not recognized, such as a gradually greater awareness of managing marginally raised blood pressure, among others.
In conclusion, this study has demonstrated a substantial improvement of long-term renal survival in Japanese children with IgA-N. On the basis of the results of this study we were unable to provide the reason for this improved outcome directly; however, it is likely that adequate management using a strict policy of treatment may have been responsible.

corticosteroid
mesangial proliferation
proteinuria
ace inhibitors
immunosuppressive drug


DoD2007: 1082 molecular biology databases

Molecular biology databases are an integral part of biological research. To date, many databases were established with varied options to access associated biological data. Depending on the data being annotated, some are architecturally similar while others are specialized. In order to provide a partial solution to data integration, we report Database of Databases (DoD2007), constructed using html and javascript. The database has a web-based user interface with simple global search, specific database search, keyword help as well as links to abstracts, full-text and database home pages. Majority of data were derived form Nucleic Acids Research database issue and other published resources. The current release includes 15 categories with updated descriptions and links to 1082 databases, of which, 209 are new entries. New databases included in this issue are represented with ‘+’ sign before the name and a ‘*’ symbol provided for those that remained silent.
Background
Molecular Biology databases have become an integral part of scientific research. They are widely used to understand the underlying mechanism of genomes, expression patterns, bimolecular
interactions, metabolism, understanding evolutionary relationships etc. as well as providing knowledge that helps to examine specific state of a disease or condition and assist in drug discovery
and development. This sort of biological knowledge is disseminated to a variety of scientific researchers through specialized databases made possible through internet technologies, software's and
tools. As more and more genomes are being sequenced and annotated, huge amount of data are accumulating. [1] Biological databases designed would
cater to meet the needs of the scientific community. But depending on the data being annotated, some are architecturally similar while the others are specialized. The challenges to develop an integrated
system are due to several factors such as variety and amount of data available and data heterogeneity in different sources. [2] Therefore,
data integration has proved problematic and to provide a partial solution, we report an update on collection of molecular biology databases with a search interface, Database of Databases (DoD2007).
The latest edition, DoD2007 was compiled with similar capabilities [3,4] but with additional features such as a global search for all databases
and links to database entries from category list, added patents database as a new category and one sub-category each in nucleotide, Genomics database and two in Protein Sequence database, respectively.
Methodology
DoD2007 was updated from the reported database issue of Nucleic Acids Research [5] and various journal sources. The 14 categories reported in
our previous update [4] remain unchanged except a new category; ‘patents db’ has been incorporated in DoD2007 update, keeping in view the importance
of patent rights in scientific discipline. Currently, this category includes two databases, patome [6] and DNA patents database. [7]
DoD2007 was constructed using html and javascript. The modes of access of databases listed in DoD2007 vary depending on the content, format and access methods. Majority of the databases are provided with
direct access to search data from DoD2007. Some databases require multiple search items while some others require sequence or annotation to be pasted in the search box and therefore they are provided with a
direct link to the database home page. Over time, DoD2007 has come to enhance the listed molecular biology databases as its use by researchers, educators and students in diverse disciplines has expanded.
The recent release DoD2007 contains three distinct newly developed functionalities, which are outlined below.
First, we have developed a user interface to search databases globally. In other words, a global search can be utilized to search the categories reported in DoD2007, database names and other pages located in
the database. Keywords used to search the database can be general or specific and depends on the terms indexed for search.
Second, an organized category list was created in a separate page. The availability of these databases as a list is intended to promote better understanding of database segregation used to generate
categories and to quickly recognize the new ones with those that are inaccessible. These databases have links to the database entries because a link back to the main page makes it easy for both new and
proficient users to conduct a variety of searches. However, some users may not realize the keywords that need to be used to search a database and even some users may show interest in the published resource.
Hence, a keyword help and links to abstract or full-text articles were created in respective main pages for intuitive and efficient presentation. Finally, a number of sub-categories are created such
as ‘Operons’ and ‘Comparative genomics’ in ‘nucleotide db’ and ‘Genomics db’ respectively along with two sub-categories viz. ‘Protein Interactions’ and ‘Amino Acid Repeats’ in ‘Protein Sequence db’.
Features of DoD2007
DoD2007 is a unique, categorized and easy-to-use interface for all molecular biology databases. New databases in various categories were appended and the number of databases in DOD2007 reached up to 1082 that are 209 databases ahead
from the previous issue. Major additions, 49 new entries, were reported in Genomics database. However, the list is not complete. A brief description of databases under all categories was given as a pop-up window with a provision to view
full-text of the published article and a direct link to the database home page. The search option under ‘link to database’ leads to the respective home pages of the database. Boolean search items are not allowed in the DoD2007 but spaces
between search strings enables the search. During the search, results are displayed in a separate window as this helps in further database scan without losing the home page.
DoD2007 facilitates integrated database search to scan a number of relevant databases from respective categories. A few databases incorporated in DoD2007 require user log-in or restricted access and a few are inaccessible or
discontinued. To distinguish such databases, new entries in DoD2007 are provided with a ‘+’ sign and inaccessible ones are recognized by a ‘*’ sign before the name. The current list of database categories and the number of entries in
each category are given in table 1 (supplementary material). An image of the database is given in figure 1. Since its first appearance in the year 2005, DoD constituted 719 molecular biology databases and the year 2005 resulted in an
addition of 154 entries making up to 873 databases (DoD2006) and the year 2006 has 209 new entries being appended in DoD2007. On the other hand, nearly 4 percent of databases are inaccessible such as HLA Ligand/Motif database
[8] of Immunological database or discontinued while few others remained silent such as European rRNA Database [9]
and Small RNA Database [10] of RNA sequence database etc.
On an average, there is a continuous increase in the number of databases which specifies the importance of such databases to biological community. Therefore, in order to account for the rise in number of databases, a graph as shown in
figure 2 was plotted for each category since year 2004. The displayed graph showed a substantial increase in Nucleotide, Protein and Genomics databases respectively.
Utility
DoD2007 provides the updated descriptions and links to existing and new databases that serves as an interface and user-friendly access to molecular biology scientific community. Number of inaccessible databases reported in the
years 2005 and 2006 remain unchanged in DoD2007 so as to enable the users to know the type of database that once existed.
Conclusion
DoD2007, a freely available web-based database resource enabling ease of access, serves as a general reference source both for community of researchers working in molecular biology and educators who deal with particular databases
of their interest. Brief description about the databases and keyword help makes the users familiar with the contents of the database, respective keywords and database links. The database shall be updated on a yearly basis.
Supplementary material
Data 1

molecular biology
database
data integration
javascript