Plant structural changes due to herbivory: Do changes in Aceria-infested coconut fruits allow predatory mites to move under the perianth?

Being minute in size, eriophyoid mites can reach places that are small enough to be inaccessible to their predators. The coconut mite, Aceria guerreronis, is a typical example; it finds partial refuge under the perianth of the coconut fruit. However, some predators can move under the perianth of the coconut fruits and attack the coconut mite. In Sri Lanka, the phytoseiid mite Neoseiulus baraki, is the most common predatory mite found in association with the coconut mite. The cross-diameter of this predatory mite is c. 3 times larger than that of the coconut mite. Nevertheless, taking this predator’s flat body and elongated idiosoma into account, it is—relative to many other phytoseiid mites—better able to reach the narrow space under the perianth of infested coconut fruits. On uninfested coconut fruits, however, they are hardly ever observed under the perianth. Prompted by earlier work on the accessibility of tulip bulbs to another eriophyoid mite and its predators, we hypothesized that the structure of the coconut fruit perianth is changed in response to damage by eriophyoid mites and as a result predatory mites are better able to enter under the perianth of infested coconut fruits. This was tested in an experiment where we measured the gap between the rim of the perianth and the coconut fruit surface in three cultivars (‘Sri Lanka Tall’, ‘Sri Lanka Dwarf Green’ and ‘Sri Lanka Dwarf Green × Sri Lanka Tall’ hybrid) that are cultivated extensively in Sri Lanka. It was found that the perianth-fruit gap in uninfested coconut fruits was significantly different between cultivars: the cultivar ‘Sri Lanka Dwarf Green’ with its smaller and more elongated coconut fruits had a larger perianth-fruit gap. In the uninfested coconut fruits this gap was large enough for the coconut mite to creep under the perianth, yet too small for its predator N. baraki. However, when the coconut fruits were infested by coconut mites, the perianth-rim-fruit gap was not different among cultivars and had increased to such an extent that the space under the perianth became accessible to the predatory mites.
Introduction
To reduce the predation risk, some organisms show behavioural or morphological changes that are induced by their predators (Wiackowski and Starońska 1999; Buskirk and McCollum 2000; Oku et al. 2003). Some have adapted to find refuge in such a way that predators cannot reach them. Eriophyoid mites have a worm-like body with a very small cross-section diameter (40–100μm) that allows them to reach concealed plant parts or to live in self-induced, small plant galls where they find protection from biotic and abiotic stresses (Sabelis and Bruin 1996). The coconut mite, Aceria guerreronis Keifer (Acari: Eriophyidae), feeds on the meristematic tissue beneath the perianth covering the base of the coconut fruit. Here it not only profits from the relatively nutritious value of this tissue, but also from the reduced risk of predation due to the perianth. Among the predatory mites that have been reported to be associated with the coconut mite in Sri Lanka, Neoseiulus baraki Athias-Henriot (Acari: Phytoseiidae) is the most frequently found species (Fernando et al. 2003; Moraes et al. 2004). This species was previously referred to as N. aff. paspalivorus (Fernando et al. 2003), but was later confirmed as N. baraki (Moraes et al. 2004). It has a flat and elongated idiosoma (Moraes et al. 2004) and we suggest this might make this predator—relative to other phytoseiid mites—better suited to creep into narrow spaces.
When the coconut mites are outside the perianth they are exposed and vulnerable to predators, but under the perianth of the coconut fruit they face less risk of being eaten. In the absence of natural enemies, coconut mite populations may grow exponentially and, consequently, the development of the coconut fruit will be impaired. Therefore, we expect the coconut palms to defend themselves directly against the coconut mites and/or indirectly by promoting the efficiency of predators against these herbivores (e.g. Sabelis et al. 2007). In this article, we investigate whether coconut fruits exhibit a mode of indirect defence that is similar to that observed in tulip bulbs by Lesna et al. (2004). These authors have found that, when tulip bulbs are attacked from within by the eriophyoid mite Aceria tulipae Keifer (Acari: Eriophyidae), bulbs increase the gap between scales to such an extent that predatory mites can enter the interior of the bulbs. This prompted us to hypothesize that mite-infested coconut fruits undergo a change in perianth structure with the effect that predatory mites have better access to the space underneath the perianth and thereby to the coconut mites. To test this hypothesis we measured the gap between the perianth and the surface of coconut fruit (“perianth-fruit gap”) when uninfested and when infested by coconut mites, and compared the size of the gap with the size of the predatory mite N. baraki.
The perianth functions as a protective cover to the female flower and the tender meristematic zone of the growing coconut fruit. In young coconut fruits (i.e. 1–2 months after fertilization) the perianth is tightly appressed to the surface of the coconut fruit (Howard and Abreu-Rodriguez 1991), but, as the coconut fruit grows, the perianth-fruit gap increases slightly, but apparently just sufficient for the coconut mites to move under the perianth and feed on the meristematic zone of the coconut fruit. Tightness of the perianth (Howard and Abreu-Rodriguez 1991), bract arrangement (Moore 1986) and shape (Mariau 1986) of the coconut fruit have been shown to affect the susceptibility of coconut fruit to the coconut mites. Thus, perianth structure affects the probability of coconut mite infestation, but the extent of the effect depends on the growth phase of the coconut and on the palm cultivar. Coconut mites usually do not infest the meristematic zone of unfertilized coconut flowers (Mariau and Julia 1970; Hall and Espinosa-Becerril 1981; Moore and Alexander 1987). After fertilization, coconut fruits of all stages are susceptible to mite attack but in general, peak populations occur in 3- to 7-months-old coconut fruits (Moore and Alexander 1987; Ramaraju et al. 2002; Fernando et al. 2003). For our experiments we used 4-month-old coconut fruits of three cultivars, commonly grown in Sri Lanka. We measured the perianth-fruit gap for each of these cultivars when uninfested and when infested by coconut mites. Finally, we compared the size of the gap with that of the predatory mite, N. baraki, to make inferences on accessibility of the space under the perianth to the predators of coconut mites.
Materials and methods
Perianth-fruit gap measurement
Four-month-old coconut fruits (i.e. 4 months after fertilization) were collected from palms of the three cultivars: (1) cultivar ‘Sri Lanka Dwarf Green’ (DG), which has usually small, elongated coconut fruits, (2) cultivar ‘Sri Lanka Tall’ (SLT) with larger and more round-shaped coconut fruits, and (3) a hybrid ‘Sri Lanka Dwarf Green × Sri Lanka Tall’ (DGT). After bringing the coconut fruits to the laboratory they were first split transversely into two halves to remove nut water. This made it easier to dissect the coconut fruit into four longitudinal sections across the perianth (Fig. 1). Dissected coconut fruits with disturbed perianth structure and loosened fibres at the coconut fruit surface were discarded from the measurements.
Fig. 1Bract arrangement of the perianth on a coconut fruit. Longitudinal sections were taken along line A and B. OB-Outer bracts of the perianth, IB-Inner bracts of the perianth
After splitting coconut fruits into four sections, the perianth-fruit gap was measured at two different places on each section (Fig. 2) using a stereomicroscope with a graticule. The first measurement (L1) was made at the rim of the perianth where it touches the coconut fruit surface (Fig. 2). The edge of each bract of the perianth has two different positions: (1) the edge that directly touches the surface of the coconut fruit, (2) the edge that overlaps (or is overlapped by) another bract. Measurements were not taken at the latter position, as it was difficult to dissect the coconut fruits along this position of the bract without disturbing the structure of the perianth. The second measurement (L2) was taken 1 cm away from position L1, higher up along the surface of the coconut fruit (Fig. 2). These two perianth-fruit gaps were measured in 157 infested (61 from SLT, 39 from DG and 57 from DGT) and 114 uninfested (43 from SLT, 28 from DG and 43 from DGT) coconut fruits.
Fig. 2Longitudinal section of a coconut fruit showing position L1 at the edge of the bract touching the coconut fruit and position L2, 1 cm away from L1 along the surface of the coconut fruit
Mite census
After measuring the gap between the surface of the coconut fruit and the perianth, bracts of each coconut fruit were removed to count the number of mites on the lower surface of each of them as well as on the underlying surface of the fruit. Counts of total number of mobile stages were done under a stereomicroscope. Total number of N. baraki was counted, whereas the population level of A. guerreronis was estimated by counting the total number of mites from six randomly selected circular (1 cm Ø) patches, three on the lower side of the perianth and three on the surface of the coconut fruit under the perianth.
Size of the predatory mites
For c. 30 min predatory mites were kept in a Petri dish on wet cotton wool, placed on ice to lower the temperature (to 3°C), thereby reducing mite activity. Thickness of the soma was taken as a measure of size. This was assessed for 12 female deutonymphs, just before their last moult, and for 20 adult females, ten of which were 1-day-old since their last moult and the other ten were more-than-5-days-old. Measurements in each stage of predatory mites were replicated four times. Because the migratory population of N. baraki mainly consisted of adult females (Kumara, unpublished data) and they are more likely to be the first invaders under the perianth of infested coconut fruits, sizes of the larval and protonymphal stages of the predatory mites were not used in our analysis. The other predatory mite that is commonly found under the perianth is N. paspalivorus which occurs mainly in the wet zone of Sri Lanka (Fernando, unpublished data). We did not measure the size of N. paspalivorus in our study since it does not occur in the dry-intermediate zone where we collected the coconuts.
Statistical analysis
The perianth-fruit gap (L1) appeared to vary with the position along the perianth rim. The mean thickness of adult female N. baraki exceeded the mean value of the measurements taken at four positions along the perianth rim in most coconut fruits. Yet, the soma was less thick than the widest perianth-fruit gap on each coconut fruit. We hypothesized that the predatory mites can find the entrance to the interior of the perianth if there are places with a sufficiently large gap between fruit and perianth. Thus, the mean value of the perianth-rim-fruit gap (L1) is less relevant if it concerns perianth accessibility to the predatory mite. Therefore, it was assumed that the widestgap observed from data obtained from four sites on each coconut fruit was the most relevant variable to be taken into account in the data analysis. Generalized Linear Models (GLM) were used to test differences in the perianth-fruit gap between main factors (category of coconut fruits i.e. infested and uninfested coconut fruits and cultivar) and to assess the interactions between the main factors.
Using only the data on infested coconut fruits it was further investigated whether the perianth-fruit gap is a predictor of predator/herbivore mite density under the perianth. Regression analyses were performed to assess the relation between the widest perianth-fruit gap and the per nut density of coconut mites under the perianth and the per nut density of predatory mites under the perianth. The difference in the density of coconut mites and predatory mites among cultivars were analyzed using one-way ANOVA on log-transformed data. All analyses were carried out using Minitab®, Version 11.
Results
The mean of the widest perianth-fruit gaps at L1 and L2 in infested and uninfested coconut fruits in three cultivars are shown in Fig. 3. In uninfested coconut fruits these were 41, 68 and 40 μm at L1 and 39, 78 and 45 μm at L2 in SLT, DG and DGT, respectively, whereas in infested coconut fruits these were 80, 75 and 99 μm at L1 and 84, 107 and 94 at L2 in SLT, DG and DGT, respectively. Thus, the perianth-fruit gap at L1 and L2 was significantly higher in infested coconut fruits than in uninfested coconut fruits (Table 1; see also Figs. 3 and 4). No significant difference was observed in the perianth-fruit gap among cultivars at L1. However, the perianth-fruit gap was significantly affected by the cultivar at L2 (Table 1). The interaction between category of the coconut fruit (infested and uninfested) and cultivar was significant at L1, but not at L2 (Table 1). This significant category-cultivar interaction at L1 arises because the increase in perianth-fruit gap due to coconut mite infestation is significant in two cultivars (SLT, DGT), but not in the third cultivar (DG). It should be noted that the perianth-fruit gap of uninfested fruits from the latter cultivar (DG) is very similar to the gap size of infested fruits in the two former cultivars (SLT, DGT) (Fig. 3).
Fig. 3Mean (±SE) of the widest gap at L1 and L2 in infested and uninfested coconut fruits of three cultivars: SLT = ‘Sri Lanka Tall’ (white rectangles), DG = ‘Sri Lanka Dwarf Green’ (black rectangles), DGT = ‘Sri Lanka Dwarf Green × Sri Lanka Tall’ hybrid (grey rectangles)Table 1Analysis of variance of the gap (L1 and L2) between the perianth and the surface of the coconut fruitSources of variancedfMSFPAt L1Category1254,67033.88<0.001Cultivar25,9080.790.457Category × Cultivar240,2875.36<0.01Residual2657,516At L2Category1582,23549.27<0.001Cultivar256,5866.870.001Category × Cultivar211,8311.440.239Residual2658,232Category refers to classification into infested and uninfested coconuts; df = Degrees of freedom; MS = Mean of squares; P = Critical levelFig. 4Frequency distribution of coconut fruits over classes of widest L1 gaps in infested (black rectangles) and uninfested (white rectangles) coconut fruits (all three cultivars together)
To test whether the widest perianth-fruit gap (L1) is a predictor of mite density under the perianth regression analysis was carried out. Because most infested coconut fruits had predatory mites under their perianth, it was not possible to obtain a sufficiently large sample of coconut fruits that had coconut mites only. A scatter plot of the few data points did not show any conspicious trend. Regression of the number of predatory mites under the perianth on L1 measured on the same coconut fruit did not reveal a significant relation (R2 = 0.04, slope = 0.011, P = 0.436). This means that among the infested coconut fruits the perianth-fruit gap is not a reliable predictor of the density of predatory mites under the perianth. The absence of a correlation is not unexpected because the density records are snapshots of a dynamic process involving interactions between predator and prey. Sampling randomly over the time course of such an interaction is bound to show no correlation.
The frequency distribution of coconut fruits over different gap classes showed different patterns in infested and uninfested coconut fruits (Fig. 4). At L1, 79% of the uninfested coconut fruits had a widest perianth-fruit gap less than 100 μm whereas 68% of the infested coconut fruits had a highest perianth-fruit gap exceeding 100 μm. These percentages, i.e. 21% for uninfested fruits and 68% for infested fruits, are significantly different according to Chi-square analysis of the 2 × 2 frequency table that can be constructed from the table below Fig. 4 (X2 = 58.63 >> 10.83 at α = 0.001). The frequency distribution of perianth-fruit gap at L2 followed the same pattern as that at L1. In conclusion, the gap between perianth and surface of the coconut fruits increased dramatically when infested by coconut mites.
The mean thickness (±SE) of the female deutonymphs of N. baraki was estimated to be 95 ± 4 μm (n = 12). In adult female predatory mites, the mean thickness was 100 ± 4 μm in mites 1-day-old after their last moult (n = 10) and 110 ± 7 μm in mites older than 5 days after their last moult (n = 10). However, the sizes of deutonymphs and two age classes of adult females were not statistically different from each other.
The number of A. guerreronis varied from 0 to 6491 with means of 1,178, 1,302 and 1,093 in six circular patches of 1 cm diameter in SLT, DG and DGT, respectively (Fig. 5). Their density (per six circular patches of 1 cm diameter) was not significantly different among the infested cultivars (P = 0.322, df = 2 and F = 1.14 for log-transformed data). The number of N. baraki ranged from 0 to 55 with means of 9, 13 and 8 mites per coconut fruit in SLT, DG and DGT, respectively (Fig. 5). There was a significant difference in the density of N. baraki among infested cultivars (P = 0.024, df = 2 and F = 3.82 for log transformed data): cultivar DG had a significantly higher density of predatory mites than the other two cultivars. We can therefore not exclude the possibility that the densities of coconut mites are blurred by effects of predatory mites. Unfortunately, sample sizes of coconut fruits ‘with coconut mites but no predatory mites’ were far too small to warrant further analysis (e.g. in DG there were only five coconut fruits of this type).
Fig. 5Mean (±SE) of the number of coconut mites (white rectangles) and predatory mites (black rectangles) per coconut fruit from three coconut cultivars (SLT, DG, DGT; see legend of Fig. 3)
Discussion
We found that the perianth-fruit gap was different among cultivars SLT, DG and DGT of the coconut palm. Except at L1 in infested coconut fruits, among the three cultivars used in our study, cultivar DG had the widest mean gap. In general DG bears smaller and more elongated coconut fruits than the other two cultivars. As observed by Mariau (1977), varieties with small coconut fruits are more susceptible to coconut mites than varieties with large coconut fruits. This may well be because the perianth of the smaller coconut fruits is less firmly attached to the coconut fruit, giving mites better access to the space under the perianth. In our study the highest mean number of coconut mites was also found on DG. However, the density of coconut mites did not significantly differ among cultivars. Therefore, the results of our study did not firmly support the idea that the coconut fruits with larger gap between perianth and the surface of the coconut fruit (in other words fruits with loosely attached perianth) are more susceptible to the coconut mite. It should be noted that the mean widest gap between the perianth and the surface of the uninfested coconut fruits exceeded 40 μm in all three cultivars, which is larger than the cross-diameter of adult female coconut mites (36–52 μm; Keifer 1965). Hence, the perianth-fruit gap in uninfested coconut fruits of any of the three cultivars is large enough for mature coconut mites to enter the space under the perianth. Hence, we expect the sub-perianth space of the three cultivars in our study to be equally accessible to mature coconut mites.
The widest perianth-fruit gap per uninfested coconut fruit was much less than 100 μm (see Fig. 3) in 79% of the coconut fruits we observed in our study (Fig. 4). However, the mean cross-diameter of the soma of adult female N. baraki was c. 110 μm. Therefore, it is clear that the perianth-fruit gap of most uninfested coconut fruits is not large enough for adult females of N. baraki to enter the space under the perianth. This largely applies to the deutonymphs of N. baraki as well, since their mean soma cross-diameter is close to 100 μm. Possibly, larvae and protonymphs of N. baraki have a soma small enough to pass the perianth-fruit gap of uninfested coconut fruits. However, juvenile predatory mites are unlikely to be the first invaders under the perianth, because they are much less mobile than adults and because female predatory mites can only lay eggs when there is a sufficient supply of prey. Adult females are therefore more likely to be the first to colonize a coconut, but they cannot enter sub-perianth space unless coconut mites have established. Hence, initially coconut mites experience reduced risk of being eaten by predatory mites when feeding on the tissue beneath the perianth.
Interestingly, we found that the gap in coconut fruits infested by coconut mites was significantly wider than that in uninfested coconut fruits (Fig. 4). Of the infested coconut fruits in our study, 68% had a perianth-fruit gap above 100 μm at L1 which is large enough for N. baraki to creep under the perianth. This suggests the following mechanistic hypothesis to be tested by future experimental analysis: when infested by coconut mites, the coconut fruits increase the gap between the perianth and the surface of the coconut fruit to such an extent that the predators can reach their otherwise-concealed prey. Our results on coconuts are consistent with observations by Lesna et al. (2004) on tulip bulbs. When infested by the rust mite, A. tulipae, tulip bulbs become attractive to the predatory mite, Neoseiulus cucumeris Oudemans (Acari: Phytoseiidae) (Aratchige et al. 2004). Having a small cross-sectional area, rust mites can easily move into the spaces in between bulbs scales where they are in refuge because the bulb scales in the apex (so called ‘nose’) of the bulbs are too tightly packed for predators to move in. However, in response to damage by rust mites, bulbs start to produce ethylene which triggers the bulbs to widen the gap between scales in the ‘nose’ of the bulb, apparently to an extent that is just enough to allow the predatory mites to enter the inside of the bulb (Lesna et al. 2004). More importantly, treating the tulip bulbs with an ethylene blocker causes the space between bulb scales to be more narrow: tight enough to prevent access by predatory mites, but definitely not tight enough for the rust mite to enter the inside. Thus, it is not just the feeding damage to the bulb scale tissue that causes widening of the space between bulb scales (Lesna et al. unpublished data). Whether the structural changes in the perianth of coconut fruits in response to coconut mite attack is herbivore-induced plant response or a by-product of necrosis and suberization of the coconut fruit surface due to herbivory, remains to be elucidated, however. The ‘by-product’ hypothesis is not supported by the observation that the perianth-fruit gap of uninfested fruits from cultivar DG is very similar to the gap size of infested fruits from the same cultivar, as well as the two other cultivars (SLT, DGT) under study. Thus, coconut mite infestation has no significant effect on perianth-fruit gap in cultivar DG and the space beneath the perianth is always accessible to the predatory mite N. baraki, irrespective of coconut mite infestation. Whereas this shows that increased gap size is not a simple consequence of necrosis and suberization of the meristimatic tissue beneath the perianth, it does not disprove this hypothesis (e.g. feeding effects on perianth structure may depend on the structure itself) and—as yet—there is no evidence for the ‘induction’ hypothesis.
Determining whether or not the changes in perianth structure are induced or not, has wider implications. An induced response indicates a net benefit to the coconut palm and hence a reason why it may have been favoured by natural selection. Such an evolutionary response of the plant requires that the association between the eriophyids and the coconut palm has existed for a long enough time and that eriophyids have represented a selective factor of importance. This may not be true for the Americas because Cocos nucifera was introduced some 500 years ago, whereas A. guerreronis, while native to the Americas, has been first described and discovered as a pest in 1965 (Navia et al. 2005). Moreover, there is insufficient data to make any inference on mite-palm associations for the Asian region. Thus, any speculation on the evolution of induced plant responses in perianth structure rests on the assumption of a sufficiently long association with some eriophyoid species or other herbivore (e.g. tarsonemid mites; see Lawson-Balagbo et al. 2007) small enough to reach the space beneath the perianth. Alternatively, plants ancestral to coconut palms have been selected to induce a generalized ‘open-the-gap’ response to attack by any herbivore small enough to enter and feed in otherwise tight folds of plant tissue. Another crucial assumption is that there are predators small enough to enter sub-perianth space and capable of controlling coconut mites. This may hold for the predatory mite N. baraki since it is frequently found associated with coconut mites (Fernando et al. 2003), but critical tests to prove its biocontrol capacity are still to be done.
For herbivore-induced increase of the perianth-fruit gap to be a plant defense strategy favoured by selection, the benefits should exceed the costs. Clearly, when the perianth-fruit gap is increased, coconut fruits may not only get assistance from predatory mites to combat coconut mites, but they would also become more vulnerable to herbivores that are somewhat larger than eriophyoid mites. This cost may be offset by providing sub-perianth access to a generalist predatory mite, like N. baraki, that can feed on (juvenile stages of) slightly larger herbivorous arthropods as well. However, the increased gap width may also provide intraguild predators, such as Proctolaelaps bickleyi (Acari: Ascidae), with access beneath the perianth, as recently shown in an extensive survey in Brazil (Lawson-Balagbo et al. 2007). Most intraguild predators, including P. bickleyi, are larger than their intraguild prey, and this may either lead to partitioning of refuge space under the perianth depending on predator and refuge size or even to exclusion from the sub-perianth space (Lawson-Balagbo et al. 2007). We therefore hypothesize that the maximum perianth-fruit gap created by the coconut palm represents a compromise between benefits in terms of protection by predatory mites and costs in terms of desiccation, increased diversity of herbivores and impact of intraguild predators of the effective predators of coconut mites.

coconut
predatory mites
perianth
coconut mites
eriophyidae
phytoseiidae
induced response
indirect plant defence


Subtypes of oligodendroglioma defined by 1p,19q deletions, differ in the proportion of apoptotic cells but not in replication-licensed non-proliferating cells

Oligodendrogliomas may be divided into those with deletion of chromosomes 1p and 19q (Del+), and those without (Del−). Del+ tumours show better survival and chemoresponsiveness but the reason for this difference is unknown. We have investigated whether these subgroups differ in (a) apoptotic index, (b) the proportion of cells licensed for DNA replication but not in-cycle, and (c) the relative length of G1-phase. Fluorescence in situ hybridisation with probes to 1p and 19q was used to determine the deletion status of 54 oligodendrogliomas, including WHO grades II and III. The apoptotic index was determined using counts of apoptotic bodies. Replication-licensed non-proliferating cells were determined from the Mcm2 minus Ki67 labelling index, whilst the geminin to Ki67 ratio was used as a measure of the relative length of G1. Del+ oligodendrogliomas showed a higher apoptotic index than Del− tumours (P = 0.037); this was not accounted for by differences in tumour grade or in proliferation. There were no differences in the Mcm2 − Ki67 index or in the geminin/Ki67 ratio between the subgroups, but grade III tumours showed a higher proportion of licensed non-proliferating cells than grade II tumours (P = 0.001). An increased susceptibility to apoptosis in oligodendrogliomas with 1p ± 19q deletion may be important in their improved clinical outcome compared to Del− tumours.
Introduction
Oligodendrogliomas are tumours composed of cells that morphologically resemble oligodendrocytes and that range in spectrum from well differentiated to anaplastic tumours. The current WHO classification of CNS tumours recognises two histological grades, WHO grades II and III, and grading is a significant predictor of survival [23]. Oligodendrogliomas have generally been considered to have a better prognosis than the corresponding grades of diffuse astrocytoma, and recent data continues to support this [21]. However, oligodendrogliomas, like astrocytomas, are diffusely infiltrating tumours, precluding complete surgical resection, and they may show malignant progression over time. There is therefore a role for adjuvant radiotherapy and/or chemotherapy, particularly for anaplastic tumours.
Approximately two-thirds of oligodendrogliomas show allelic losses from chromosomes 1p and 19q [24, 25], a genetic profile that appears to be associated with clinical outcome. Some anaplastic oligodendrogliomas respond to combination chemotherapy [2, 3], and better progression free survival and chemo-responsiveness appear to be linked to the presence of these deletions [4, 34]. Results from two recent large-scale trials provide support for the view that the presence of 1p 19q deletion identifies a subgroup with better clinical outcome [1, 41]. 1p 19q deletion may also identify better prognosis in low-grade oligodendrogliomas [13].
There may be some histopathological differences between the two genetic subgroups. Classical oligodendroglioma features, such as perinuclear haloes (in >50% of cells), uniform rounded nuclei, chickenwire vascular pattern and calcification, are associated with 1p 19q loss [14, 27, 42], suggesting that classical features, such as perinuclear haloes associated with tumour cellularity, can predict genotype [14]. However, there are cases of histologically typical oligodendrogliomas without 1p 19q deletion and more astrocytic appearing tumours with deletion. For these cases clinical behaviour appears to be better predicted by genotype than phenotype, so that 1p 19q deletion provides additional information to conventional histopathological analysis [27]. Some studies suggest that oligodendrogliomas with 1p 19q deletion have lower activity of O6-methylguanine DNA methyltransferase (MGMT) due to promoter hypermethylation and decreased expression. MGMT is a DNA repair enzyme associated with resistance to alkylating agents, so this may contribute to chemosensitivity [18], although other studies have not found a clear association [42]. At present, therefore, the biological basis for the intrinsically better prognosis of the 1p 19q deletion subgroup and the possible basis of a different interaction with adjuvant therapies remain poorly understood.
In this study we hypothesise that the difference in behaviour of tumours with and without these deletions is due to differences in kinetic properties of the tumours related to proliferation and apoptosis. Apoptotic bodies are often a conspicuous feature in oligodendrogliomas and levels of apoptosis increase with grade [43]. Tumour growth depends on the net effects of cell proliferation and loss [35] so that a subgroup of tumours with a greater propensity to apoptosis versus proliferation may have slower growth. It is thus possible that increased susceptibility of tumour cells to undergo apoptosis might underlie both a better intrinsic biological behaviour and a greater sensitivity to adjuvant therapy. We hypothesise therefore that tumours with 1p,19q deletions show a greater tendency to apoptosis that is reflected in increased basal levels of apoptosis in the tumours.
Differences in kinetic parameters related to DNA replication licensing are also candidates for relevance to growth capacity and chemosensitivity. The initiation of chromosomal replication is a crucial decision point in cell proliferation that lies at the point of convergence of all oncogenic signalling and transduction pathways that trigger proliferation. DNA replication initiation is precisely controlled through a core set of licensing factors (Orc1–6, Cdc6, Cdt1, Mcm2–7) that sequentially assemble into pre-replicative complexes (pre-RCs) at ∼30,000 replication origins scattered along the chromosomes, resulting in chromatin being ‘licensed’ for replication in the subsequent S phase. Licensed replication origins are activated by the concerted action of S phase promoting kinases (Cdk2) and the ASK-dependent Cdc7 kinase in a process commonly known as ‘origin firing’. Origin firing results in local melting of the DNA helix at replication origins and the recruitment of DNA polymerases (reviewed in [40]). Notably the Mcm2–7 replication licensing factors, which have been shown to act as a replicative DNA helicase, are present within the nucleus in cells in-cycle, but are down-regulated in out-of-cycle states [10, 38].
We have previously shown that higher grade oligodendrogliomas show a greater proportion of cells that are licensed for DNA replication, as determined by the Mcm2 labelling index, which is of prognostic significance [44]. Certain tissues contain a population of licensed but non-proliferating cells [10, 38] and higher labelling indices for Mcm2 than for the conventional proliferation marker Ki67 (which marks all four phases of the cell-cycle) suggest that this may be true for oligodendrogliomas [44]. A population of licensed but non-proliferating cells might conceivably resist cell-cycle dependent cytotoxicity and form a viable cell cache for tumour re-population. We have used the difference between the Mcm2 and Ki67 labelling indices (Mcm2 − Ki67 LI) as an assay of this population.
It is essential for faithful propagation of the genome that origins are fired once and only once in each cell cycle to avoid re-replication events, which would lead to gene amplification. Geminin, a 23.5 kDa nuclear protein, is an endogenous regulator of chromosomal replication, absent during G1 phase but allowed to accumulate during S, G2 and M phase [17]. Geminin acts to prevent a second, inappropriate firing of replication origins by competitively binding to Cdt1 and thereby blocking recruitment of the Mcm2–7 helicase to replication origins [39, 48]. Thus pre-RC re-assembly and firing are prevented. At the metaphase to anaphase transition in mitosis, the anaphase promoting complex (APC/C) becomes activated and geminin is targeted for polyubiquitination and degradation via the 26S proteosome pathway, lowering levels to the point where the protein no longer interferes with assembly of pre-RCs during the following G1 phase [17]. Since geminin is present in S–G2–M phase, the ratio of geminin to Ki67 may provide an index of the relative length of G1-phase. We have previously shown that this ratio decreases in higher-grade oligodendrogliomas, which may be a reflection of a shortened G1 in anaplastic tumours [45]. The length of G1-phase may be relevant to chemotherapeutic agents targeting the cell cycle machinery. We have therefore determined whether cytogenetic subgroups of oligodendrogliomas differ in their geminin/Ki67 ratio.
Materials and methods
A total of 55 cases of oligodendroglioma were identified and paraffin blocks retrieved from the archives of the Histopathology Department of the Royal Hallamshire Hospital, Sheffield, from a 12 year period from 1985 to 1997. As previously described [45], 25 cases were graded as WHO grade II (mean age 37.4 years) and 30 as grade III (mean age 44.6 years). The series comprised 22 female and 33 male patients. All of the cases were resection specimens; stereotactic and other small biopsies were excluded. Of these cases, 47 were first resections. Eight cases were recurrent resections that fell within the study period; the initial resections on these cases were prior to the study period and not included. All of the cases were used for the labelling index analyses but only the 47 first resections were used for survival analysis. Clinical follow-up data and data on radiotherapy and chemotherapy were obtained from the Clinical Oncology Department at Western Park Hospital, Sheffield. Approval for the study was granted by the Local Research Ethics Committee.
Immunohistochemistry
Immunohistochemistry was performed on serial sections from a representative paraffin block from each case using a standard ABC method and the signal visualised using diaminobenzidine. Immunostaining was performed to Ki67 (Novocastra), Mcm2 (BD Transduction Laboratories) and geminin (G95, rabbit polyclonal) on this series of tumours as previously described [45]. Apoptotic bodies were counted on haematoxylin and eosin stained sections. Areas of highest cellularity were used for quantitation. The percentage of labelled cells, derived from a count of at least 1000 cells, was determined for each marker using an eyepiece graticule [44, 45].
Fluorescence in situ hybridisation
Fluorescence in situ hybridisation (FISH) was performed on 4 μ thick sections using the technique previously described [7]. The Vysis dual colour probe sets LSI 1p36/LSI 1q25 and LSI 19p13/LSI 19q13 were used according manufacturers instructions; probes were hybridised overnight using a PTC Peltier Thermal Cycler. Analysis was performed independently by two scientists, both scoring 30 cells for each probe set.
Statistical analyses
Statistical analyses were performed using the statistical package for the social sciences (SPSS v10.1). Comparison of age was made using an unpaired t test. Data on labelling indices was either not normally distributed and/or did not satisfy criteria for equality of variance between groups (Levene’s test). Therefore comparisons of labelling indices between groups were performed using the non-parametric Mann–Whitney U test. Fisher’s exact test was used for assessment of distribution between groups. For comparison of survival between groups, Kaplan Meier plots were constructed and the log rank test used. Multivariable linear regression analysis was used to examine the relationship of apoptotic index (AI) to proliferation and deletion status. Ki67 labelling index (LI) was used as measure of the former, rather than Mcm2 LI, as the latter also includes replication licensed but non-proliferating cells. To model a difference in the slope of the relationship between AI and Ki67 LI in the two subgroups, an interactive term between Ki67 LI and deletion status was included [Model: AI = β0 + (β1 × Ki67 LI) + (β2 × deletion status) + (β3 × Ki67 LI × deletion status)].
Results
Characteristics of the deletion sub-groups
FISH analysis for 1p and 19q deletion was successfully performed in 54 out of 55 cases (Fig. 1). Cases were assigned to the Del+ group if they had deletion of both 1p and 19q, or 1p alone. Cases were assigned to the Del− group if they had no deletion or deletion of 19q only. There were 34 Del+ cases and 20 Del− (Table 1). The Del+ group had an older mean age than the Del− group (Table 2) (P = 0.002). There was no difference in the distribution of histological grade between the two groups (P = 1).
Fig. 1Representative FISH images using the VYSIS dual colour probe sets; a relative loss of 1p (red) to 1q (green) with probable increase of chromosome number, b simple deletion of 1p (red), c relative loss of 19q (red) to 19p (green) with probable increase of chromosome number, d simple deletion of 19q (red)Table 1Results of FISH analysisGroupDeletionNumberNo. in Del group% of casesDel+1p 19q3434631p 0Del−None17203719q3Table 2Charateristics of cytogenetic sub-groupsGroupDel+Del−Mean age, SD46.0, 11.5 33.1, 17.9Grade II169Grade III1811% of grade II47.145.0
Survival analysis
Survival for the 47 cases included in the survival analysis was taken from the date of the surgical resection. The eight (recurrent) cases excluded from this analysis were distributed evenly between Del+ and Del− groups. The Del+ group showed a trend to better survival (Fig. 2) but this did not reach significance (P = 0.092), probably due to the number of censored cases. Further survival analysis to include multivariate parameters was therefore not attempted.
Fig. 2Kaplan Meier survival curves for Del+ (solid line) and Del− (dashed line) oligodendroglioma subgroups. Censored cases shown as cross marks
Comparison of indices between cytogenetic subgroups
The labelling index for apoptotic bodies (AI), assessed from H&E stained sections was used as a measure of apoptosis. Higher levels of apoptosis were seen in the Del+ group, carrying either deletions of 1p and 19q, or 1p alone (P = 0.037) (Table 3, Fig. 3). To further analyse the difference in apoptosis between the two cytogenetic subgroups, we considered whether higher levels of apoptosis in the Del+ group were a reflection of higher cell turnover. However, there was no difference in replication-licensed or proliferating cells between the two subgroups as assessed by Mcm2 (P = 0.87) or Ki67 (P = 0.77), respectively. Inspection of scatterplots of AI against Ki67 labelling index for the two subgroups suggested that with increasing tumour proliferation, apoptosis shows a greater increase in the Del+ subgroup (Fig. 4). Because this suggested a steeper relationship of AI to proliferation in the Del+ subgroup, multivariable regression analysis was carried out with a model, incorporating Ki67 LI, deletion status and a term for interaction between these two variables as predictors of apoptosis index. This gave an overall significance of P = 0.002 for the model. The interaction between Ki67 LI and deletion status was significant (P = 0.031) and AI rose more steeply against Ki67 LI in the Del+ group (AI α 0.58xKi67LI) than in the Del− group (AI α 0.13 Ki67 LI).
Table 3Descriptive data for kinetic measuresSubgroupMeasureAIMcm2 − Ki67Gem/Ki67Del−Mean (SD)0.72 (0.38)8.31 (22.21)0.39 (0.25)Median (IQR)0.60 (0.48)6.65 (23.18)0.38 (0.44)Del+Mean (SD)1.10 (0.69)12.51 (15.11)0.29 (0.15)Median (IQR)0.95 (1.00)7.15 (21.88)0.26 (0.22)AI apoptotic index, Mcm2 − Ki67 Mcm2 minus Ki67 labelling index, Gem/Ki67 ratio of geminin to Ki67 indices, SD standard deviation, IQR interquartile rangeFig. 3Boxplot showing apoptotic indices in Del− and Del+ subgroupsFig. 4Scatterplots of apoptosis index (AI) versus Ki67 labelling index for Del− (left) and Del+ (right) cases
The difference between Mcm2 and Ki67 labelling indices (Mcm2 − Ki67 LI) was used as a measure of the fraction of tumour cells that were licensed but not proliferating. In general tumours showed a higher labelling index for Mcm2 than Ki67, indicating the presence of a population of licensed cells that are not actively replicating. The Mcm2 − Ki67 LI was higher in grade III than in grade II tumours (P = 0.001, Fig. 5). However, we demonstrated no difference in the proportion of licensed, non-cycling cells by this method between the two cytogenetic subgroups of oligodendrogliomas (P = 0.46). We also demonstrated no difference in the geminin/Ki67 ratio (P = 0.22).
Fig. 5Boxplot of Mcm2 minus Ki67 labelling index according to histological grade
Discussion
The basis for the difference in clinical behaviour between oligodendrogliomas with deletions of 1p and 19q (Del+) and those without (Del−) remains unclear, but its elucidation may yield important new avenues for improved therapeutic approaches. In this study we have investigated whether there are differences in parameters related to tumour cell proliferation and death in these two cytogenetic subgroups of tumours.
The tumours were typed using FISH with probes to 1p and 19q. In our series, 63% of cases showed deletions in 1p and 19q, or 1p alone, a similar proportion to that reported in series of oligodendrogliomas in the literature where figures for combined loss from approximately 50 to 70% of cases have been described (sometimes higher in classical appearing WHO grade II tumours) [13, 19, 24, 25, 33]. Del+ and Del− had a similar distribution of histological grades whilst the older average age in the Del+ group is also unlikely to be of relevance to our findings as age is in general associated with a worse clinical behaviour, and opposite to the effects here. A trend towards better overall survival in Del+ cases in our series did not reach significance, probably due to the number of total and censored cases.
Analysis of the FISH probes in this cohort of cases revealed a pattern of multiple signals in some cases, indicating that increased copy number of chromosomes 1 and 19 were present, but that there was still effective loss of heterozygosity of short arm chromosome 1 and long arm chromosome 19. (Fig. 1) These cases were interpreted Del+. One other subgroup was identified in the cohort: three cases in which there was 19q deletion present without concomitant 1p deletion. There was no significant association of these subgroups in our series with an obvious pathological feature, but more cases should be studied to determine any clinico-pathological correlation. The multiple signal cases, which would indicate complex aneuploid karyotypes, usually found in tumours with more malignant status, may be of particular interest. One other group has identified the single 19q deletion status cases and have suggested that 19q deletion alone may also predict a favourable prognosis [22].
We have previously reported values for apoptosis, Mcm2, Ki67 and geminin in this series of tumours [45], but we now define indices and relate these to cytogenetic subtype. We have investigated three selected parameters that we hypothesise may affect survival and therapeutic response. Apoptosis is an important process in tumour biology as defective apoptosis induction may contribute to cancer development and the susceptibility of tumour cells to apoptosis may affect rate of growth. Apoptosis may be induced by adjuvant therapies and is likely to become an increasingly important molecular therapeutic target as specific pathways are defined (reviewed in [26]). Apoptosis is variable between tumour types and a number of factors may contribute to apoptosis induction in a tumour. The apoptotic index often appears to be a function of the proliferation rate, with higher levels in more rapidly cycling tumours. In oligodendrogliomas, we and others have previously shown that apoptosis tends to increase with proliferation and with tumour grade [29, 43], though it has not been shown to be an independent prognostic factor. A similar relationship of apoptosis to histological grade and proliferation has also been demonstrated in the diffuse astrocytomas [8]. Although quantification of apoptosis, and so comparison between tumour types and studies, is beset with methodological difficulties, there is evidence that oligodendrogliomas show higher levels of apoptosis than other brain tumours of comparative WHO grade [15, 28]. Alterations in regulatory factors related to molecular genetic changes are likely to be important in setting the susceptibility to apoptosis in a given tumour type, whilst other factors such as ischaemia and the influence of tumoural inflammatory cells may also contribute to apoptosis induction. Thus variation in apoptosis is a candidate to explain differences in clinical behaviour of tumour subtypes.
In this series of oligodendrogliomas we have assessed apoptosis using counts of apoptotic bodies. This method has been used for other tumour types, including astrocytomas [8]. In a previous oligodendroglioma series we showed that the apoptotic index correlates with an assessment of apoptosis using the TUNEL method [43], but it is a much simpler method. In addition, the TUNEL technique can be capricious and lacks specificity as it may detect DNA damage in cells from processes other than apoptosis, including necrosis [12]. Immunohistochemical detection of caspase 3 has also been used to detect apoptotic cells in histological sections [5]. We have previously carried out caspase 3 immunohistochemistry on this series [45], but this showed weaker relationships to grade and proliferation than apoptotic index using counts of apoptotic bodies. Therefore, for this study, apoptotic index based on counts of apoptotic bodies was selected a priori for analysis.
We have demonstrated that Del+ oligodendrogliomas show a higher apoptotic index than Del- tumours, supporting the hypothesis that tumour cells from Del+ cases may have a greater susceptibility to undergo apoptosis. We have not investigated the molecular pathways that might lead to this difference. However, it is not simply a reflection of increased cell turnover as Del+ cases show no increase in either Mcm2 or Ki67 labelling indices compared to Del- cases (although a difference in cell-cycle length is not formally excluded). The greater rate of increase in apoptotic index against Ki67 labelling index for Del+ than Del- tumours suggests that oligodendrogliomas with 1p, 19q deletion have a greater tendency to the induction of apoptosis at higher rates of proliferation.
Immunohistochemical demonstration and quantification of components of the DNA replication licensing machinery have recently been used for prognostic [16, 30, 32, 44] and surveillance [37, 46, 47] studies in human tumours. Theoretically demonstration of MCM proteins may be considered superior to Ki67 as it identifies not only cycling cells, but also those non-cycling cells with proliferative potential [10, 38], and this superiority is born out in many of these studies. Combined assessment of these markers with other cell cycle related markers may be used to obtain information on the relative length of cell cycle phases in archival biopsy material [9, 20, 31]. In this study, which adds to these applications, we have used a variable generated from the difference between the Mcm2 and Ki67 labelling indices (Mcm2-Ki67 LI) for each case to assess the proportion of tumour cells that are licensed to replicate but not actually in cycle. This index has been previously used in renal cell cancer, where it increases with tumour grade [6]. We now also demonstrate that this index increases with tumour grade in oligodendrogliomas, suggesting that the pool of cells that are licensed but not proliferating is larger in more anaplastic tumours, evidence for greater dysregulation of the origin licensing pathway with anaplasia. Such a licensed, non-proliferating fraction might conceivably act as a treatment-resistant population that could re-grow after adjuvant therapy. There is increasing evidence for tumour stem cells in brain tumours (reviewed in [11]) and it is intriguing to speculate whether a stem cell population, responsible for perpetuating tumour growth, might reside in this fraction. Its assay is therefore of potential interest and we hypothesised that the size of this fraction might be greater in Del- tumours. However, we did not demonstrate any difference in Mcm2 − Ki67 LI between Del+ and Del− tumours, suggesting that this is not important in determining their clinical differences.
We have previously shown that the ratio of geminin to Ki67 labelling indices is increased in grade III versus grade II oligodendrogliomas [45], implying a shortened G1-phase in higher grade tumours (assuming that other cell cycle phases are not substantially altered in duration). In this study, however, we have not demonstrated evidence for a differing G1-phase duration between Del+ and Del− tumours.
Molecular differences between these two cytogenetic subtypes of oligodendroglioma are beginning to be defined, which will allow therapeutically relevant molecular classification [24]. Some of these genetic differences likely relate to the regulation of apoptosis, and may differ in the two subgroups. This study has shown that those oligodendrogliomas with 1p ± 19q deletions have higher apoptotic indices than those without. Differences in cell death susceptibility may affect prognosis through a treatment independent effect on intrinsic growth rate. But apoptosis may also be induced by adjuvant therapies, and amongst the gliomas this may be particularly important for oligodendroglial tumours [36]. This study does not allow distinction between intrinsic growth rate and treatment response contributions of apoptosis. It has also been limited to histological methods and has not provided functional data on susceptibility or defined its molecular basis. However, the findings support the hypothesis that differences in clinical outcomes between these cytogenetic subgroups is related to differing susceptibility to apoptosis induction and suggest that detailed comparative studies of apoptotic pathways in molecular subgroups of oligodendrogliomas are warranted.

oligodendroglioma
replication
apoptosis
dna replication licensing
cytogenetic