Why Do the Galls Induced by Calophya Duvauae Scott on Schinus Polygamus (Cav.) Cabrera (Anacardiaceae) Change Colors?

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Biochemical Systematics and Ecology 48 (2013) 111–122

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Biochemical Systematics and Ecology

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Why do the galls induced by Calophya duvauae Scott on Schinus polygamus (Cav.) Cabrera (Anacardiaceae) change colors?

Graciela Gonçalves Dias a, Gilson Rudinei Pires Moreira b, Bruno Garcia Ferreira a, Rosy Mary dos Santos Isaias a,*

a Universidade Federal de Minas Gerais, Instituto de Ciências Biológicas, Departamento de Botânica, Av. Antônio Carlos 6627, Campus Pampulha, Postal Ofﬁce Box: 286, 31270-901 Belo Horizonte, MG, Brazil b Universidade Federal do Rio Grande do Sul, Instituto de Biociências, Departamento de Zoologia, Av. Bento Gonçalves 9500, Campus do Vale, 91501-970 Porto Alegre, RS, Brazil

article info abstract

Article history: One of the galling herbivores associated to the superhost Schinus polygamus (Cav.) Cabrera Received 29 August 2012 (Anacardiaceae) is Calophya duvauae Scott (Hemiptera: Calophyidae). The galls are located Accepted 1 December 2012 on the adaxial surface of leaves and vary from red to green. The levels of their pigments Available online 16 January 2013 were herein investigated in relation to age. Samples were collected between June 2008 and March 2009, in a population of S. polygamus at Canguçu municipality, Rio Grande do Sul, Keywords: Brazil. Galls were separated by color, measured, dissected, and the instar of the inducer Extended phenotype was determined. The levels of photosynthetic (chlorophyll a and b, total chlorophylls, and Gall color changes Anthocyanin carotenoids) and protective pigments (anthocyanins) were also evaluated. Red galls were Chlorophyll more numerous than the green ones, and the induction should occur preferentially on Psylloid bugs young leaves, but may also occur on mature leaves. Immature stages of C. duvauae were Anacardiaceae observed in all samples throughout the year, characterizing its life cycle as multivoltine. There was a signiﬁcant correlation between the instar of the inducer and the size of the gall (r ¼ 0.675, p < 0.001), with larger galls corresponding to more advanced instars. The ratio between red and green galls varied over the samples, with the highest (96%) and lowest (38%) frequency of red galls observed in September, and December 2008, respectively. The average amounts of chlorophyll b and total chlorophylls were 70% lower in the gall tissues when compared to non-galled portions of galled leaves. There is a notable reduction in the contents of all pigments in the galls when compared to non-galled leaves, especially for total anthocyanins in green galls. The red galls presumably had the constant stimuli of healthy gall inducers, whereas in green galls they died prematurely, due to the interference of parasitoids and inquilines. The alterations in gall structure and color were related to the gradual decreasing in galling stimuli. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction

The changing of color from green to red has already been observed in some galls (Inbar et al., 2010). However, the causes and adaptive signiﬁcance of this feature have not been determined. Schinus polygamus (Cav.) Cabrera (Anacardiaceae) is a superhost plant, native to temperate South America, where several insects induce different gall morphotypes, including

* Corresponding author. Tel.: þ55 3134092687; fax: þ55 3134092671. E-mail address: [email protected] (R.M.S. Isaias).

Diptera (Cecidomyiidae), Hemiptera (Psylloidea) and Lepidoptera (Cecidosidae) (Burckhardt and Basset, 2000; Moreira et al., 2012; Sáiz and Núñez, 1997). Within the psylloids, Tainarys Brèthes (Psyllidae) and Calophya Löw (Calophyidae) have species associated with Schinus L. (Burckhardt and Basset, 2000). The leaf galls induced by Calophya duvauae Scott on S. polygamus,in particular, vary from red to green, supposedly due to changes in pigment production in response to either abiotic or biotic factors (Dias et al., in press). This leads to interesting questions from both theoretical (e.g. physiology level) and practical (e.g. taxonomy level) perspectives, since galls are recognized as extended phenotypes of their inducers (Abrahamson and Weis, 1997; Dawkins, 1982; Isaias and Oliveira, 2011). In other words, holding this concept true in theory, unless gall are modified by other external agent, a given inducer in any gall system should be always associated with only one gall phenotype. The constant stimuli from the gall inducers are responsible for maintaining the structure of galls (Mani, 1992) and therefore, any corresponding change caused by pathogens, parasitoids and/or inquilines may alter the biochemical and structural patterns. There is no indication that pathogens expressively occur in the S. polygamus – C. duvauae gall system. Parasitoids have been registered in the galls of C. duvauae on Schinus fasciculatus, of Calophya mammifex on Schinus latifolius and S. polygamus, and of Calophya terebinthifolii on Schinus terebinthifolius (Burckhardt and Basset, 2000), and are known to reduce the fitness of the galling herbivores. Even though inquilines (sensu Noort et al., 2007) are not capable of inducing galls, they take advantages on gall structure either by feeding upon or modifying the gall tissues induced by another organism. Also, the success of insect galls is commonly related to the ability of the inducer to change the concentrations of primary and secondary metabolites (Motta et al., 2005), as well as of photosynthetic pigments (Castro, 2007; Yang et al., 2003, 2007), transforming its gall into strong sinks (Fay et al., 1996). This is a potential mechanism by which galls induced by C. duvauae on S. polygamus may change their phenotype during ontogenesis. The alterations in pigment content are particularly interesting to be investigated in this host plant-gall inducer system because of the concomitant occurrence of green and red galls on the host leaves. The color of the galls should be product of the balance of plant pigments, such as chlorophyll, carotenoids and anthocyanins. It is expected that the anthocyanin content of red galls should be higher than those of the green galls and of the non-galled leaves. This high content of anthocyanin could be related to high production, resulting in protection of plant tissues against oxidative stress generated by the gall inducer (Vanderauwera et al., 2009). A lower concentration of this pigment in regions near the red galls should also be expected, because of the establishment of a sink of anthocyanins precursors. This study focuses on the causes of the color variability of this gall by analyzing (1) the developmental stages of the gall and those of C. duvauae; and (2) the concentrations of anthocyanins, chlorophylls (a, b and total) and carotenoids of non- galled leaves, non-galled portions of host leaves, red, and green galls. The occurrence of a preferential site for oviposition, the level of infestation and its impact on leaf area were also taken into account, based on Price and Roininen (1993) hypothesis of an evolutionary trend of preferential oviposition in the central regions of the host plant. This should contribute to the adaptive advantages of the gall to the galling herbivore, by avoiding the reactions of abscission and intra-specific competition, as well as providing a better control of the flow of nutrients (Nyman et al., 2000).

2. Materials and methods

2.1. Sampling and leaf morphometry

The study was carried out in a population of S. polygamus located in Canguçu municipality, Rio Grande do Sul state, Brazil (32 1500000S, 65 5800000W). The area consists of typical grassland, part of the Pampa Biome (described in detail in Overbeck et al., 2007), where individuals of the host plants occur either isolated or in small groups generally located in hilltops. A total of 426 totally expanded galled or non-galled leaves, randomly obtained from branches of 38 different individuals (10–12 leaves per branch) were sampled in June, September and December 2008, and March 2009. To verify the inﬂuence of gall induction on leaf area, the area of non-galled (103) and galled (323) leaves were estimated, using the graphics software Quantikov Image Analyzer (Pinto, 1996). Vouchers of fertile plant material (ICN 42884) are deposited at the herbarium of the Instituto de Biociências, Universidade Federal do Rio Grande do Sul, Brazil. Identiﬁcation of the inducer was performed by using the morphological criteria described in Burckhardt and Basset (2000). Corresponding vouchers (LMCI 202) are deposited in the tissue collection of Laboratório de Morfologia e Comportamento de Insetos, Departamento de Zoologia of the Universidade Federal do Rio Grande do Sul.

2.2. Gall size and instar of the inducer

Ò The largest diameter and height of galls were measured with a digital caliper (Digimess model 100.174B). Based on the heights, artificial categories of size (with an interval of 1 mm) were established. The material was fixed either in 4% Karnovsky in 0.1 mM phosphate buffer for 24 h (O’Brien and McCully, 1981), modified to pH ¼ 7.2 or in FAA (37% formaldehyde, glacial acetic acid and 50% ethanol) for 48 h (Johansen, 1940), and subsequently stored in 70% ethanol. Galls larger than 1 mm Ò (n ¼ 633) were dissected with the aid of a razor blade under a stereomicroscope (Olympus SZH ) and regrouped, considering the size and color of the gall, the instar of the inducer and the presence of insects of other trophic levels. The ultrastructural morphology of the inducer hind wing pads (instars 3–5) was characterized as the following protocol: Ò acetone series, critical point with carbon dioxide (BAL-TEC CPD-030), metallization with 30 nm of gold (Paim et al., 2004)in G.G. Dias et al. / Biochemical Systematics and Ecology 48 (2013) 111–122 113

Ò Ò a sputter coater BAL-TEC SCD-050, observation and imaging in either a JEOL scanning electron microscope LEO EVO or Ò JSM6060 40.

2.3. Dosage of pigments

Photosynthetic (chlorophylls a and b, total chlorophyll and carotenoids), and photoprotective pigments (anthocyanins) of non-galled leaves (NL), non-galled portions of host leaves (NGL), red galls (RG), and green galls (GG) were extracted (n ¼ 80) after maceration and centrifugation. Discs of 0.44 cm2 were removed, weighed, and used for dosage of pigments. Chlorophyll and carotenoids were extracted in 80% acetone. The quantiﬁcation was obtained according to the equations proposed by Lichtenthaler and Wallburn (1983), and the values were expressed in unit of fresh mass (FM). From the data obtained, the ratio of chlorophyll a/b was calculated. Anthocyanins were extracted in 95% ethanol: 1.5 N HCl and quantiﬁed as established by Lees and Francis (1972). The data were adjusted for mgg 1FM.

2.4. Site of oviposition, proportion of galls and level of infestation

For all samples, the position of galls on leaf was assessed by direct observation, and registered as follows: top, middle and basal regions, secondary veins, and internervural region. The level of infestation was determined by counting the number of galls, and those of galled and non-galled leaves (n ¼ 150 leaves) from 10 individuals in a non-destructive sampling in December 2008 (n ¼ 2895 galls). From these data, the percentage of galled and non-galled leaves, and of red and green galls was calculated.

2.5. Statistical analysis

Ò Measurements of gall dimensions were performed with Axion Vision Zeiss software, and subjected to statistical analyses in JMP (SAS Institute, U.S.A., 1989–2002). Parametric data were analyzed either by Student’s t tests or ANOVA followed by Tukey’s multiple comparison tests. The data that did not pass normality tests (Shapiro–Wilk tests) were compared by a nonparametric Kruskal–Wallis test, followed by Dunn’s multiple comparison tests. Pearson correlation test was adopted to Ò compare the sizes of the insects and of the galls. Analyses were performed and graphs generated by using GraphPad Prism (a ¼ 0.05).

3. Results

3.1. Host plant and galls

S. polygamus is a shrub (Fig. 1A), whose leaves have spiral phyllotaxy (Fig. 1B). The leaves vary in size and shape within and among individuals and are usually elliptic, with entire blade, acute apex, attenuate base, and mostly glabrous. Gall induction occurs on the abaxial surface, in the internervural region of newly expanded leaves, but might reach the secondary veins (Table 1). They are sessile, globose, mainly located in the median leaf portion, followed by basal and apical regions, isolated or grouped, with only one inducer per nymphal chamber. The adaxial surface color varies from red to green with intermediate tones (Fig. 1C–E) in young and mature galls. All senescent galls are green, and open fully on abaxial surface (Fig. 1F). Even red galls are green on the abaxial surface. C. duvauae has five larval instars (Fig. 2), all with dorso-ventrally flattened bodies. The first instar is round shaped, with no conspicuous appendages in dorsal view (Fig. 2A). The abdomen is less long than wide, slightly distinct from the thorax. As already showed for other Hemiptera (e.g. Southwood, 1956; Rodrigues and Moreira, 2005), in the second instar, the wing pads are not apparent, and the abdomen is longer than wide (Fig. 2B). In the third instar, the wing pads are incipient, represented as small bumps that not overlap in the pterothorax, (Fig. 2C–D). In the fourth instar, the forewing pads are well developed, laterally directed, overriding less than half the length of the metathorax (Fig. 2E–F). In the fifth instar, these structures are directed posterior, and the forewing pads exceed the total length of the metathorax (Fig. 2G–H). The adult stage is free leaving, being characterized by the presence of functional wings (Fig. 2I–J), eventually found within the gall dissected just after molting. The male have a characteristic aedeagus (Fig. 2I) and the female, an appendicular short ovipositor (Fig. 2J). More than half of the leaves were galled, with 62.4% being GG (green gall) in December 2008. The area of host leaves was similar to that of non-galled leaves (Student’s t test, p ¼ 0.05) (Table 1). The highest occurrence of senescent galls (17.9%) was registered in June 2008, and they were absent in September 2008. In all samples, 3% of closed galls had neither gall inducers nor other associated trophic level. Unidentified species of an inquiline and of an endoparasitoid (Hymenoptera) were found in 11.3% of the galls. The former was found free leaving in the gall, feeding upon the plant tissues. The later was located within the inducer’s hoemocele, being located through the transparent tegument (Fig. 3).

3.2. Gall size and color, and C. duvauae ontogeny

Red and green galls were sampled in all size classes, except those smaller than 1 mm that were absent in September 2008 (Table 2). Gall size was signiﬁcantly related to C. duvauae instar (y ¼ 0.8524 x þ 0.6218, r ¼ 0.675, n ¼ 578, p < 0.001). More 114 G.G. Dias et al. / Biochemical Systematics and Ecology 48 (2013) 111–122

Fig. 1. Galls of Calophya duvauae Scott (Hemiptera: Calophyidae) on Schinus polygamus (Cav.) Cabrera (Anacardiaceae). A) Habitus of the host plant. B) Apical and young leaves, with galls in growth and development stage. C) Stem branch with galls with different colors. D) Red gall. E) Green galls. F) Senescent gall. Bars: B, D, E, F ¼ 0.5 cm; C ¼ 1 cm (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.).

advanced instars correspond to galls with larger diameter and height (Fig. 4). In 1–2 size class galls, second and third instar nymphs predominated, in 3 size class galls, all instars are equally observed, while in 4–5 size class galls, mainly fifth instar and adults were found. The presence of different instars of C. duvauae in all samples indicates a multivoltine life cycle (Fig. 5). First instar insects were rarely found. Third instar nymphs prevailed on June and December 2008, and on March 2009, while in September, there was a higher number of fifth instar nymphs and adults. The ratio between red and green galls varied greatly among the samples. In December, 21% of green galls had malformed individuals of C. duvauae (with the shape of body parts altered, presumably by the endoparasitoidism not detected by transparency yet). The remaining 69% green galls had either an inquiline or an endoparasitoid, conspicuously identified by tegument transparency during gall dissections.

3.3. Photosynthetic and photoprotective pigments

The average contents of chlorophyll a and b and total chlorophylls were signiﬁcantly lower in the galls when compared to the NGL (non-galled portion of host leaf) (Table 3). There was no signiﬁcant difference between these pigments in GG (green galls) and RG (red galls), or between NL (non-galled leaf) and NGL for chlorophyll a and total chlorophyll. Chlo- rophyll b differs between NL and NGL. The contents of carotenoids were similar in NL, NGL and GG, with a reduction of about 32% in RG, when compared to the average value for NL. The ratio of chlorophyll a/b was higher in NL and similar in NGL, RG and GG. G.G. Dias et al. / Biochemical Systematics and Ecology 48 (2013) 111–122 115

Table 1 Features of the galls of Calophya duvauae Scott (Hemiptera: Calophyidae) on leaves of Schinus polygamus (Cav.) Cabrera (Anacardiaceae), Canguçu, RS, Brazil.

2008 2009

June September December March Mean Position of galls on the leaf (%) Apex 16 20.6 20.9 12.3 17.4 Mid portion 50.3 61.3 46.3 56.5 53.6 Base 33.7 18.1 32.8 31.2 29 Internervural region 33.4 20.2 35.6 57.9 36.8 Secondary veins 66.6 79.8 64.4 42.1 63.2 Leaf area in cm2 (Mean SE) Galled leaves 1.96 1.23 Non-galled leaves 1.71 1.16 Infestation (%) Galled leaves 53.2 Green galls 62.4 Red galls 37.6 Gall color (%) Green galls 24.6 3.6 15.9 25 Red galls 75.4 96.4 84.1 75 Samples composition (%) Galls with inducer 65.2 83 88.9 77.5 78.7 Galls with other trophic level 12.1 12.2 7.5 13.2 11.3 Senescent galls 17.9 0 3.2 7 7 Empty galls 4.8 4.4 0.4 2.3 3

The average contents of total anthocyanins differed between samples, and are considerably higher in NL and NGL when compared to the galls. The NL had the highest content of anthocyanins, followed by NGL, RG and GG. The GG had at about four times less anthocyanin than the NL.

4. Discussion

4.1. Gall color and the development of the gall inducer

In S. polygamus–C. duvauae system, there is no evidence of more than one species of gall inducer, contrasting to what was suggested by Sáiz and Núñez (2000). In the present study, we clearly showed for such a sap-feeding gall inducer that, even for a variable character such as color, there is a constancy throughout gall ontogeny, providing further support in a fine scale that galls are extended phenotypes of their inducers (Abrahamson and Weis, 1997; Dawkins, 1982; Isaias and Oliveira, 2011). Furthermore, changes in color during gall ontogeny may be associated with the additional species, which later on occupy the gall system, such endoparasitoids and inquilines. These additional trophic levels alter the morphophysiological natural course of the gall. The implications from a practical taxonomic perspective are that, by taking into account such characteristic variation in color on the gall external surface, one should be able to identify with relative certainty, which biological agent is inside. Sáiz and Núñez (1997) related gall color to its age in galls induced by other psylloids, with young galls being green and mature galls being red. Our data revealed red galls with young instars and green galls with fifth instars. Even the youngest galls were red on the adaxial surface. Also, there was no correlation between the color of the gall and its size, with red and green galls belonging to all size classes. So in S. polygamus–C. duvauae system, there is no relation between gall color, and its age and size, except for senescent galls which are totally green. The feeding activity of C. duvauae continually stimulates and maintains the gall structure, resulting in a successive increase in size, which is proportional to the development of the instars. The first stage of development, attributed to the galls with less than 1.0 mm, occurred all over the year except for September 2008, which might represent the moment of adults eclosion and mating. At the stage of growth and development, (1.1–4.0 mm), the galls greatly increased in biomass, and the insects go through second to fourth instars. At the maturation phase (4.1 mm), the insect is in the last nymphal stage, and demands the largest amount of energy. At the senescent phase, the adult emerges, and significant physiological and chemical changes occur in the gall tissues. Similar relationships were found by Ferreira et al. (1990) in galls of Euphalerus ostreoides, another Psylloidea, in Lonchocarpus guilleminianus (Fabaceae). Even though there is more than one instar per gall size class, we observed a significant correspondence between the size of the gall and the instar of the inducer, with the larger galls corresponding to the more advanced ones. In fact, there were seasonal variations in the proportion of red and green galls, always with a predominance of red galls, except for the samples of December 2008. In this month, the higher proportion of green galls coincided with both the greatest attack by parasitoids and inquilines (about 70%), and also with the morphological malformations of C. duvauae, as already discussed, probably the result of underscoring the effect of parasitoidism in its early stage. Thus, whereas the stimulation of 116 G.G. Dias et al. / Biochemical Systematics and Ecology 48 (2013) 111–122 G.G. Dias et al. / Biochemical Systematics and Ecology 48 (2013) 111–122 117

Fig. 3. Endoparasitoids in nymphs of Calophya duvauae (Hemiptera: Calophyidae) and inquilines found in galls induced on Schinus polygamus (Cav.) Cabrera (Anacardiaceae). A–F) Endoparasitoids inside the nymphs; G, H) Ecloded endoparasitoids; I, J) Inquilines. Bars: A–D ¼ 100 mm; E–F ¼ 200 mm; G–J ¼ 250 mm.

the gall inducer is responsible for maintaining the structure (Abrahamson and Weis, 1997; Mani, 1964, 1992) and the red color of the gall, parasitoids or inquilines seems to trigger the gall physiological senescence, once all senescent galls of C. duvauae were green.

4.2. Relation between gall color and pigment contents

Although there was no correlation between gall size and color, alterations in the levels of pigments were verified. The contents of chlorophyll a and total chlorophylls were similar between NL and NGL, and between RG and GG, but they were higher in leaf laminas when compared to the galls. Moreover, regarding the contents of chlorophyll b and carotenoids, there were differences between NL and NGL, reflecting not only the local influence of the gall but also its effect in the adjacent tissues. In fact, biochemical and physiological changes may occur at the local level (Mani, 1964; Motta et al., 2005)orin regions close to the gall, depending on the analyzed compounds (Nyman and Julkunen-Tiitto, 2000). A significant reduction in the levels of total chlorophylls in the galls demonstrated its poor ability to synthesize photo- assimilates. The reduction in photosynthetic pigments has been reported in other systems (Castro, 2007; Fleury, 2009;

Fig. 2. Calophya duvauae (Hemiptera: Calophyidae) in dorsal view under light and scanning electron microscopy (SEM). A) First instar. B) Second instar. C) Third instar. D) Left wing pads of the third instar. E) Fourth instar. F) Right wing pads of the fourth instar. G) Fifth instar. H) Right wing pads of the ﬁfth instar. I) Adult stage, male. J) Female. Details of the ﬁgures I and J with the corresponding genitalia. Bars: A, D, F and H ¼ 100 mm; B, C and E ¼ 200 mm; G ¼ 400 mm; I and J ¼ 1 mm. 118 G.G. Dias et al. / Biochemical Systematics and Ecology 48 (2013) 111–122

Table 2 Variation in size (means standard deviations) of Calophya duvauae Scott (Hemiptera: Calophyidae) galls on Schinus polygamus (Cav.) Cabrera (Ana- cardiaceae) in Canguçu, RS, Brazil.

Height class (mm)

Samples N 0–1.0 1.1–2.0 2.1–3.0 3.1–4.0 4.1–5.0 5.1 Jun/2008 207 0.79 0.08 1.50 0.22 2.64 0.26 3.42 0.27 4.36 0.30 5.60 0.44 Sep/2008 159 – 1.86 0.11 2.58 0.39 3.66 0.24 4.37 0.26 5.44 0.31 Dec/2008 252 0.87 0.10 1.58 0.24 2.51 0.31 3.49 0.26 4.38 0.31 5.49 0.44 Mar/2009 129 0.96 0.04 1.63 0.25 2.47 0.29 3.36 0.30 4.46 0.29 5.36 0.29

Khattab and Khattab, 2005; Oliveira et al., 2011) and may be due to the reallocation of nitrogen and carbon skeletons for the synthesis of secondary metabolites (Fleury, 2009; Yang et al., 2003). Then these galls functions as a sink of resources, as proposed by several authors (Álvarez et al., 2009; Burstein et al., 1994; Raman et al., 2006; Schrönrogge et al., 2000; Weis et al., 1988). NL and NGL had similar contents of photosynthetic pigments and different levels of anthocyanins. These pigments have been related to signaling for pollinators and dispersers, and to protection against stress factors such as ultraviolet radiation, free radicals, extreme temperatures, drought, fungal and viral infections, injuries and herbivory (Hatier and Gould, 2009; Zuanazzi, 2000). The highest concentration of anthocyanins occurs in NL when compared to the other samples. Despite their lower content, the regions adjacent to the galls (NGL) retained their ability to synthesize relatively large quantities of these pigments. The galls had reduced anthocyanin contents, but the stimulation of the gall inducer is responsible for maintaining approximately the double of its level in RG than in GG. In the GG, this stimulus has ceased or diminished considerably, resulting in lower levels of anthocyanins and, therefore, the green color observed. The RG, therefore, present this color due to the reduction of total chlorophyll content, which hides the anthocyanins in NL. This pattern of changing in color is reported for deciduous leaves, with the reddish senescent leaves corresponding to chlorophyll degradation (Matile, 2000). These changes in relative levels of chlorophyll and anthocyanin, reﬂected in RG and GG, seem to be related to the low rates of parasitoidism observed. Such signaling molecules might be aposematic, as suggested by Inbar et al. (2010), reducing predation and parasitism in the RG, avoiding some potential enemies, which remains to be tested.

4.3. Oviposition site preference, level of infestation and impact on leaf area

Most galls induced by C. duvauae on S. polygamus are located in the median portion of the leaves, what according to Price and Roininen (1993) would be a derived character within the galling habit. The choice of a region for oviposition could influence the allocation of nutrients (Price and Roininen, 1993), and improve the sink-strength generated by the gall (Larson and Whitham, 1997). In this system, the preferential site of oviposition seems to be related to a better nutritional intake, which reflects on the observed levels of infestation. This result is similar to those of Ferreira et al. (1990) in galls induced by Psyllidae and Formiga et al. (2009) in galls of Cecidomyiidae, which also demonstrated the success of the galling insects to colonize their host plants. The C. duvauae–S. polygamus system has a relatively low level of infestation (53.2%) when compared to other systems, such as Aspidosperma spruceanum-Cecidomyiidae (64–87%) (Formiga et al., 2009) and Rollinia laurifolia–Pseudotectococcus rolliniae (93.8%) (Gonçalves et al., 2005). Two aspects seem to be responsible for the few galls of S. polygamus along 1-year time: the availability of induction sites throughout the year, and the presence of natural enemies (Dias, 2010). When galls are induced on deciduous host plants, the time when suitable oviposition sites are available is very limited. Hence, new inductions might occur in synchronized way, in large quantities but in a short period of time, resulting in rates similar to those reported by Gonçalves et al. (2005). S. polygamus allows C. duvauae to oviposit and develop throughout the whole year, which is common for other perennial host plants (Weis et al.,1988; Lara and Fernandes,1994). Parasitoids and inquilines in this system cause the mortality of the galling insects, and somehow should regulate the population at certain levels, as found for other gall inducers (Ito and Hijii, 2004; László and Tóthmérész, 2006; Noort et al., 2007). Natural enemies may be responsible for up to 96% of mortality in some insect gall systems (e.g. Abrahamson and Weis, 1997). C. duvauae preferentially induces galls on young newly expanded leaves of S. polygamus. However, inductions were also observed on mature tissues, which return to the meristematic condition necessary for gall formation (Dias et al., in press). It contradicts the assumption that the induction must necessarily occur in still undifferentiated tissues (Abrahamson and Weis, 1997; Weis et al., 1988), which receive major input of nutrients and amino acids (Andrade et al., 1995). Inductions on mature leaves were also observed on galls of Lantana camara (Verbenaceae) (Moura et al., 2009) and of Copaifera langsdorffii Desf. (Fabaceae) (Oliveira and Isaias, 2009), where some differences in gall phenotype related to the age of the host tissues were reported (Oliveira and Isaias, 2009). Even though alterations in leaf area are a common effect of parasitism (Castro et al., in press; Gonçalves et al., 2005), on S. polygamus the high values of standard deviation observed either for galled or non-galled leaves may be due to the large phenotypic variability on leaves of this species as described by Fleig (1985). Thus, the differences on galled leaf area would not be due to the galls, but to differences in the patterns of leaf expansion. In addition, the oviposition can occur in fully expanded leaves where the leaf area expansion has ended. G.G. Dias et al. / Biochemical Systematics and Ecology 48 (2013) 111–122 119

Fig. 4. Temporal variation in proportion among the developmental stages of Calophya duvauae (Hemiptera: Calophyidae) in galls induced on Schinus polygamus (Cav.) Cabrera (Anacardiaceae). (A) 1 size class galls (1.1–2.0 mm). (B) 2 size class galls (2.1–3.0 mm). (C) 3 size class galls (3.1–4.0 mm). (D) 4 size class galls (4.1–5.0 mm). (E) 5 size class galls (5.1 mm). 1) First instar. 2) Second instar. 3) Third instar. 4) Fourth instar. 5) Fifth instar. 6) Adult stage.

The occurrence of a large number of senescent galls in June 2008 suggests a large number of adult emergences before. The absence of senescent galls in September of the same year indicates the beginning of a new phase of growth and/or infestation between June and September. In summary, in the S. polygamus–C. duvauae system red galls were more numerous than the green ones. The green color results from a reduction in galling stimuli or even the death of the gall inducer due to parasitism and/or inquilinism. 120 G.G. Dias et al. / Biochemical Systematics and Ecology 48 (2013) 111–122

Fig. 5. Variation among proportions of developmental stages of Calophya duvauae (Hemiptera: Calophyidae) from June 2008 to March 2009, in a population of Canguçu municipality, Brazil. 1) First instar. 2) Second instar. 3) Third instar. 4) Fourth instar. 5) Fifth instar. 6) Adult stage.

Table 3 Pigment contents (means standard errors; mg.g 1FM) in non-galled leaves (NL), non galled portions of host leaves (NGL), red galls (RG) and green galls (GG) of Calophya duvauae Scott (Hemiptera: Calophyidae) (n ¼ 20) on Schinus polygamus (Cav.) Cabrera (Anacardiaceae) Canguçu, RS, Brazil.

Sample type Chlorophyll a Chlorophyll b Carotenoids Chlorophyll a/b Total chlorophyll Total anthocyanins NL 759.52 12.06a 148.13 8.29a 10.3 1.82a 7.26 2.5a 917.95 11.93a 21.09 1.79 a NGL 688.56 10.92a 234.83 7.46b 10.82 1.62a 3.06 0.93b 934.22 12.39a 17.04 1.98b RG 194.70 11.79b 46.36 4.33c 7.02 1.45b 4.39 1.62b 218.82 6.44b 9.68 1.64c GG 208.94 11.88b 61.94 4.86c 9.22 1.43a 3.34 1.35b 251.54 8.4b 5.13 0.97d

Means followed by the same letter in column are not signiﬁcantly different (Kruskal–Wallis test, followed by Dunn’s multiple comparison tests; a ¼ 0.05). NL ¼ Non-galled leaf; NGL ¼ Non-galled portion of host leaf; GG ¼ Green galls; RG ¼ Red galls; NL ¼ Non-galled leaf NL ¼ Non-galled leaf NGL ¼ Non-galled portion of host leaf GG ¼ Greeg galls RG ¼ Red galls.

Anthocyanin and chlorophyll contents were reduced in galls, and color changes are stimulated by variations in the balance chlorophyll-anthocyanin. The induction of galls on the median portion of the leaves seems to increase the sink strength, as areflection of the reduction of photosynthetic and photoprotective pigments. The galling activity of C. duvauae directly influences gall size, but does not cause significant changes in leaf area. G.G. Dias et al. / Biochemical Systematics and Ecology 48 (2013) 111–122 121

Acknowledgments

We thank Cibele Bedetti de Souza, Maria do Socorro Silva, Thiago Alves Magalhães and Wagner Afonso Rocha (UFMG) for technical assistance. G.G. Dias and B.G. Ferreira were ﬁnancially supported by Capes and CNPq fellowships, respectively. R.M.S. Isaias and G.R.P. Moreira beneﬁted from CNPq grants (process numbers 307488/2009-8 and 304458/2008-2, respectively).

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