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Effect of light intensity on colour morph formation and performance of the grain aphid Sitobion avenae F. (Homoptera: Aphididae)
Journal of Insect Physiology 56 (2010) 1999–2005
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Effect of light intensity on colour morph formation and performance of the grain aphid Sitobion avenae F. (Homoptera: Aphididae) Hussein Alkhedir a, Petr Karlovsky b, Stefan Vidal a,* a b
Agricultural Entomology, Department of Crop Sciences, Georg-August-University Goettingen, Grisebachstrasse 6, 37077 Goettingen, Germany Molecular Phytopathology and Mycotoxin Research, Department of Crop Sciences, Georg-August-University Goettingen, Grisebachstrasse 6, 37077 Goettingen, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 October 2009 Received in revised form 30 August 2010 Accepted 31 August 2010
The grain aphid Sitobion avenae F., one of the major pest aphids of cereals in Central Europe, exhibits colour polymorphism, even within the same clones. Although there is evidence that green and brown morphs of S. avenae contain different carotenoids, the mechanisms determining the induction of colour morphs are unknown. The common understanding is that the formation of colour morphs is controlled by light and affected by genetic and environmental factors and by host plant species. So far, there is no unequivocal evidence that light intensity, photoperiod, or a mixture of several variables are involved in the induction of S. avenae colour formation, resulting in the induction of S. avenae colour formation and carotenoid synthesis. Here we determined the effect of light intensity on the colour formation and performance of ten clones of S. avenae with experiments that controlled for the effects of host plant and genetic factors. We found that some clones remained green under all test conditions. In other clones, colour morph formation was controlled by light. The synthesis of carotenoids correlated with changes in colour formation. Host plant did not affect colour formation in the ten clones studied. Although colour of the aphid clones did not affect their performance, high light intensity increased the fecundity and fresh weight of S. avenae clones, while low light intensity stimulated the production of alatae. ß 2010 Elsevier Ltd. All rights reserved.
Keywords: Carotenoids Light intensity Colour polymorphism
1. Introduction Aphids exhibit polymorphism, i.e., a population of identical genotypes (Loxdale and Lushai, 2003) can have two or more morphologically distinct individuals, termed morphs. These morphs are adapted to specific ecological conditions and are characterised by differences in their life cycles and colour (Blackman and Eastop, 1984; Dixon, 1998). The colour of these morphs results from the interaction of two factors: the colour of the cuticle and the pigmentation of the haemolymph (Mu¨ller, 1961). The colour formation of the cuticle is affected by temperature alone (Dixon, 1973), whereas the pigmentation of the haemolymph is affected by temperature, photoperiod, and light intensity (Markkula and Pulliainen, 1965; Jenkins et al., 1999). Glucosidic haemolymph pigments in aphids are synthesised as aphins or protoaphins, which are unique to Aphididae (Bowie et al., 1966). Other haemolymph pigments also include carotenoids, which cannot be synthesized by aphids or other animals but which are found in higher plants and microorganisms (Goodwin, 1986).
* Corresponding author. Tel.: +49 551 39 39744; fax: +49 551 39 12105. E-mail address: [email protected] (S. Vidal). 0022-1910/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2010.08.025
Aphins are responsible for the yellow, orange, or red colour in aphid morphs (Bowie et al., 1966). As mentioned above, differences in colour morphs of the same aphid species can result from differences in carotenoids (Andrewes et al., 1971; Weisgraber et al., 1971; Jenkins et al., 1999). Because animals, including aphids, are unable to synthesise carotenoids, precursors are needed for the synthesis of these compounds. The obligate bacterial endosymbiont Buchnera aphidicola has been proposed to provide these metabolites to their aphid hosts (Moran et al., 1994; Douglas, 1998). Colour polymorphism has been documented in many aphid species (Dixon, 1998) and is regarded as a selective advantage with regard to natural enemies and environmental factors. For example, red colour morphs of the pea aphid Acyrthosiphon pisum (Harris) produced more alate and escaped from predators more rapidly than green morphs (Braendle and Weisser, 2001). Seven spot ladybird beetles Coccinella septempunctata L. consumed more aphid morphs when the morph colour contrasted with rather than matched the background colour (Harmon et al., 1998). Ankersmit et al. (1981) reported that colour morphs of S. avenae differed in their susceptibility to the parasitoid Aphidius rhopalosiphi De Stefani Perez, the brown morphs being more resistant than the green morphs. In this host–parasitoid system, partial resistance was assumed to be related to the increased handling time of brown
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morphs by female parasitoids combined with an increase in the development time and mortality of the larvae produced by females that consumed brown morphs. These effects were significant even for morphs derived from the same clone (i.e. asexual progeny from a known founder stem mother) (Ankersmit et al., 1986). In addition to affecting the interaction between aphids and their natural enemies, colour polymorphisms resulting from light intensity, photoperiod, or temperature can also affect aphid fecundity (Markkula and Pulliainen, 1965; Markkula and Rautapa¨a¨, 1967). The grain aphid Sitobion avenae F. produces colour morphs ranging from green (the most common colour) to red, brown, and pink (Phillips, 1916; Mu¨ller, 1961; Markkula and Myllyma¨ki, 1963; Markkula and Pulliainen, 1965; Markkula and Rautapa¨a¨, 1967; Jenkins, 1991; Jenkins et al., 1999). Although the basis for this colour polymorphism mainly depends on differences in the carotenoide content in the haemolymph (Jenkins, 1991; Jenkins et al., 1999). Jenkins (1991) also demonstrated that anholocyclic clones are of green colour and holocyclic clones are of brown colour, but the mechanisms determining the induction of colour morphs in S. avenae are incompletely understood. In this study, we tested whether colour morph formation and aphid performance of S. avenae clones are influenced by light intensity and host plant species. Moreover, we tested the synthesis of specific carotenoids in S. avenae colour morphs is influenced by light intensity using Thin layer chromatography (TLC). 2. Materials and methods 2.1. Aphid cultures and light intensity The ten clones of S. avenae used in this study were established from single asexual female aphid, collected in different regions of Germany and from different host plants. At the time of collection, the clones displayed different colour morphs. The clones relate to different genotypes and harbour different bacterial endosymbionts (Alkhedir, unpublished data) (Table 1). In the laboratory, these clones were reared on wheat seedlings (winter wheat cv ‘‘Bussard’’, Lochow Petkus Comp., Germany) grown in 11-cm diam. pots (10 seedlings per pot) containing a 2:1 mixture of soil (Fruhstorfer Typ P) and sand. Each plant was covered with a transparent ventilated cylinder, which was 10 cm wide and 30 cm high. Aphids were transferred to newly grown plants at two-week intervals. Cultures were kept at 20 8C, and at a 16:8 L:D hour photoperiod, 15 mEinstein (mE) flux intensity, and 60–80% humidity in climate chambers (WB 750 KFL; Mytron Bio-Und Solartechnik GmbH, Germany). Light intensity was measured with a LI-COR Quantum Radiometer/Photometer (Inc. Bioscience). Plants were watered from below twice each week. Given these conditions, all clones reproduced parthenogenetically, and the colour of all clones and individuals in these cultures were light green. Experiments with high light intensity (200 mE; see Section 2.3) were performed in a climate chamber (Viessmann Company, Germany), while
experiments with low light intensity or darkness were performed in the climate chambers mentioned above. The lamps installed in the climate chambers were OSRAM L 36W/25 and OSRAM FQ 54 W/840 HO, respectively. Specifications of wavelengths and light spectrum are given in Supplementary files 1 and 2. 2.2. Colour of clones as affected by host plant and light intensity (experiments 1 and 2) Seven host plant species were used in two main experiments (designated as 1 and 2) viz Wheat Triticum aestivum, cv ‘‘Bussard’’ from Lochow Petkus Comp.; and the grasses Poa annua, Lolium perenne cv. ‘‘Herault’’. Avena sp., Phalaris arundinacea (culture form), Agropyron repens from Appels Wilde Samen Comp., Germany; and Dactylis glomerata cv. ‘‘Neva’’ from the National Agricultural Research Centre for the Hokkaido Region, Japan. For experiment 1, wheat was grown as described previously, and seedlings were transplanted into pots when 7 days old. All other host plants were germinated in the greenhouse in trays and transplanted when 1 month old and about 10–13 cm tall. The seedlings were transplanted into pots as described in Section 2.1, with 10 wheat seedlings or three seedlings of the other host plants per pot. Each of the 10 S. avenae clones was reared on all seven host plants, with 12 replicate pots per combination of clone and host plant species. Plants with aphids were divided into two sub-groups and randomly placed in growth chambers at 20 8C, 60–80% humidity, 18:6 L:D photoperiod, and one of two light intensities (15 mE vs. 200 mE). The colour formation of the aphid clones was visually assessed during 1 month; thereafter, the aphids were transferred to fresh host plants as described for experiment 2 below. In experiment 2, those aphids reared at 15 mE in experiment 1 were divided into two groups. The first group was thereafter continuously reared at 200 mE with 18:6 L:D photoperiod, while the second group was reared in darkness. A similar procedure was followed for the aphids previously reared at 200 mE in the first experiment but these were either continuously reared at 15 mE with 18:6 L:D illumination periods or reared in darkness. This experimental set-up was established separately for each host plant species. The colour of the aphid specimens was visually determined and recorded as pale green, green, pink, brown, or dark brown (supplementary 3). By the end of both experiments 1 and 2 all aphid were used for carotenoids analysis. 2.3. Colour morph formation in clone 6 (experiment 3) Five synchronized first instar green nymphs of clone 6 were propagated on one wheat seedling (7 cm tall) whose roots and the lower stem were immersed in a transparent 250-ml flask containing 200 ml of water. The seedling with aphids was covered with a transparent ventilated cylinder and kept at 20 8C and 60–80% humidity, with a constant light intensity of 200 mE. This set up ensured that all aphids were well exposed to light from all
Table 1 Origin and collection details of the S. avenae clones used in the study. Clone
Collection site
Host plant
Developmental stage collected
Colour at collection time
1 2 3 4 5 6 7 8 9 10
Goettingen Kassel Kassel Goettingen Giessen Kassel Giessen Giessen Kassel Kassel
Wheat Cocksfoot Bromus sp. Cocksfoot Wheat Cocksfoot Wheat Wheat Cocksfoot Cocksfoot
Virginoparae Virginoparae 2nd nymph Virginoparae Virginoparae Virginoparae Virginoparae Virginoparae 4th, winged 4th nymph
Green Green–brown Green–brown Green–brown Green–brown Green–brown Green Green Brown Brown
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sides. Aphid colour formation on five replicate seedlings was recorded daily for 28 days. 2.4. Colour morph formation in clone 9 (experiment 4) Ten green adults of clone 9 were chosen randomly from the aphid culture on wheat. One adult was placed in each of 10 Petri dishes containing a small piece of wheat seedling leaf sitting on a moist filter paper. The Petri dishes were placed under 200 mE at 20 8C. After 5 days, half of the Petri dishes were covered with black boards to produce dark conditions. The offspring of the initial adult aphids were left to become adults and produce offspring. The colour of all aphids was observed daily, and the experiment was terminated after 21 days. 2.5. Performance of clones at different light intensities (experiment 5) Synchronized first-stage nymphs of the 10 clones were added to 7-day-old wheat seedlings (10 nymphs per pot) growing in pots and covered with transparent ventilated cylinders as described earlier. After 2 weeks in growth chambers at 20 8C, 60–80% humidity, 18:6 L:D illumination periods, and two different light intensities (15 mE vs. 200 mE), the experiment was terminated and clonal performance was assessed. There were seven replicate pots for each combination of aphid clone and light intensity. Clonal performance was assessed as the number of aphids per pot, total fresh weight of aphids per pot, and the number of alatae per pot. 2.6. Carotenoide analysis of clones in experiments 1 and 2 (experiment 6) Aphids were collected at the end of experiments (1 and 2), and were separated into four groups. The first group contained aphids from clones 7 and 8 collected from plants grown at 15 mE and were pale green. The second group contained aphids from clones 7 and 8 collected from plants grown at 200 mE and were green. The third and fourth groups included all remaining clones collected from plants grown at 15 and 200 mE; the third group (from plants grown at 15 mE) was pale green and the fourth (from plants grown at 200 mE) was red-brown. Carotenoids were extracted and separated with TLC as described by Jenkins et al. (1999). They were extracted from each sample, containing 0.5–1 g specimens, using 2 ml of pre-chilled acetone three times, 2 ml of methanol/acetone (1:1, v:v) (once), and finally 2 ml of petroleum ether (twice). All extracts were pooled in a plastic vial, centrifuged at 2000 rpm for 20 min, and then placed in separation funnels containing 5 ml of petroleum ether. A sufficient quantity of distilled water was added to aid in the separation of the phases. The bulked extracts were partitioned in two phases: the extract of the brown clone separated into a blue-green aqueous phase and an orange epiphase; the extract of the green clone separated into the same blue-green hypophase, but the epiphase was yellow. The epiphase was collected into new plastic vials, and petroleum ether was added to retain pigments. The collected epiphase (10 ml) was washed three times with distilled water and then dried under vacuum. The extract containing pigments in various stages of esterification with fatty acids was saponified under nitrogen gas for 16 h at 5 8C in 5 ml of petroleum ether with 6% KOH. The saponified extractions were placed in separatory funnels with 5 ml distilled water; the organic phase was then collected in new vials, and petroleum ether added again to the bulk extracts containing the pigments. These extractions were dried using a rotary vacuum. Thereafter, they were dissolved in 200 ml of petroleum ether. The pigments were separated with TLC using either silica gel or aluminum oxide foils (20 20 cm) (Fluka, Germany) pre-washed with 3% KOH in
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methanol. The TLC solvent was diethyl ether in petroleum ether (5% v:v) or diethyl ether in hexane (5%, v:v). TLC was performed in the dark at 5 8C. The extracts were co-chromatographed with bcarotene (Sigma), and the Rf values for each band recorded. Lycopene was analysed according to the TLC method decribed in Jenkins et al. (1999), following the experimental set-up described by these authors. 2.7. Statistical analysis A Kruskal–Wallis one-way analysis of variance was used to compare the performance of each clone under the different light intensity regimes. Bonferroni’s adjustments were included to compare the clonal performance for each variable under each of light intensity. SYSTAT for Windows version 11.00.01 (SYSTAT, 2004) was used to perform these analyses. Multivariate analysis of variance (MANOVA) tests were done to analyse the effect of light intensity on the performance of S. avenae clones; the number of aphids per pot, total aphid fresh weight per pot, and the number of alatae per pot were the dependent variables, and clone, light intensity, and interactions between them the independent, categorical variables. MANOVA was also used to analyze the effect of colour morph on performance of S. avenae clones; the number of aphids per seedling, total aphid fresh weight per seedling, and numbers of alatae per seedling were the dependent variables, and clones and colour morphs the independent, categorical variables. 3. Results 3.1. Colour morph formation as affected by host plant and light intensity (experiments 1 and 2) Host plant did not affect the colour of Sitobion clones in experiments 1 and 2, i.e., the colour of each S. avenae clone at low light intensity was the same on all host plant species. Similarly, the colour of each S. avenae clone at high light intensity was the same on all host plant species. Although the colour of S. avenae clones was not affected by host plant, the colour was affected by light intensity in experiment 1 (Table 2). Clones that were pale green at the beginning of the experiment remained pale green at 15 mE throughout the experiment. In contrast clones that were pale green at the beginning became pink, or brown when reared at 200 mE between day 1 and 10. Thereafter these clones turned dark brown, except for clones 7 and 8, which turned dark green; these colours remained constant for all clones after day 10. The effect of light intensity in experiment 2 is described in the text without tabular support. When aphids previously kept at 200 mE were transferred to 15 mE in experiment 2, all specimens of all clones became pale green. When clones previously kept at 15 mE were transferred to the 200 mE treatment, all specimens of all clones turned dark brown, except clones 7 and 8, which became dark green. When aphids previously kept at 200 or 15 mE were transferred to darkness, all specimens of all clones turned pale green. After 2 weeks in darkness, plants became yellow and died, and mortality of aphids on these plants rapidly increased. At this time, the colour of all aphid clones in this treatment was pale green to yellow. There were differences among the clones in experiment 2. At the high light intensity, some clones changed their colour earlier than others. For example, clone 6 started to change colour after 3 days at 200 mE compared with clone 9 which change its colour after 5–6 days. The colour of clones responding to high light intensities gradually changed from pale green to green, and pink. After 10 days, all aphids initially exposed to 15 mE in experiment 1
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Table 2 Influence of light intensity on colour morph formation in 10 clones of Sitobion avenae (experiment 1). G: green; B: brown; M: green-brown; PG: pale green; Y: yellow; DG: dark green; DB: dark brown; P: pink. Clone
Original colour
1 2 3 4 5 6 7 8 9 10
G M M M M M G G B B
Colour immediately after 6 days of exposure to 0, 15, or 200 mE
Final colour after 10 days of exposure to 15 or 200 mE
0 mE
15 mE
200 mE
15 mE
200 mE
PG-Y PG-Y PG-Y PG-Y PG-Y PG-Y PG-Y PG-Y PG-Y PG-Y
PG PG PG PG PG PG PG PG PG PG
P-B P-B P-B P-B P-B P-B DG DG P-B P-B
PG PG PG PG PG PG PG PG PG PG
DB DB DB DB DB DB DG DG DB DB
and then exposed to 200 mE in experiment 2 turned dark brown except clones 7 and 8 which became dark green. In experiments 1 and 2, colour formation of the aphid clones at the high light intensity was faster when the plants were small (during the first 2 weeks). As the plants grew larger, some leaves created shadows in some areas within the cylinders. At this time, different aphid specimens within one clone showed different colours; aphids that were not directly exposed to the light became green or exhibited mixed colours, while aphids exposed to the full light displayed the colour equivalent to the one found when reared at 200 mE. The green of clones 7 and 8 was less intense at 15 mE than at 200 mE. 3.2. Colour formation in clone 6 at 200 mE (experiment 3) The colour of clone 6 nymphs kept at 200 mE ranged from green to pink during the experiment, the final colour being dark brown. This change in colour took place during 4 days; during this period, the nymphs developed into stage-four nymphs. The adults (both apterae and alate) and their offspring were brown. After 3 weeks, the wheat seedlings had become completely yellow except for the leaf tips, which had turned brown. At this time, the aphid population on each seedling included the original nymphs that were added at the start and a second generation of nymphs. In general, the aphids and their offspring in this transparent system at 200 mE were darker than aphids kept at 200 mE on the regular culture of wheat seedlings. 3.3. Colour morph formation in clone 9 at 0 and 200 mE (experiment 4) All clone 9 adults kept at 200 mE laid new nymphs on the first day. At birth, these nymphs were green. The colour of the adults began to change after 2 days. This colour change began at the head and spread into the middle part of the abdomen first and then to the sides of the abdomen. The colour of the offspring began to change within one day after birth. After five days, nymphs had become pink to dark brown. When these nymphs became adults, their offspring were not born green but were pink to dark brown. Even the visible embryos inside the adults were pink to dark brown. Aphids directly exposed to light on the upper surface of the leaf pieces changed colour sooner than those feeding beneath the leaf pieces. Those aphids that were moved to dark conditions after 5 days became green after 3–5 days and produced green offspring after becoming adults; the embryos inside the adults were also green. 3.4. Effect of host plant on colour of clones Host plants did not affect the colour formation of Sitobion clones. The colour of each S. avenae clone was the same at each of light intensity tested on all host plant species.
3.5. Effect of light intensity on performance of clones (experiment 5) Population size, fresh weight, and alatae production of S. avenae differed significantly among the 10 clones in experiment 3 (F9,120 = 58.443, P < 0.001). These variables were also affected by light intensity (F1,120 = 115.839, P < 0.001), and the interaction between clone and light intensity was also significant (F9,120 = 37.570, P < 0.001). Population sizes of clones 1, 2, 7, 8, and 10 were larger under high light intensity than under low light intensity (Fig. 1A); the opposite was true for clones 4, 5, and 6. Population sizes of clones 3 and 9 were unaffected by light intensity. The fresh weights of clones 1, 2, 3, 7, 8, and 10 were greater under high light intensity than under low light intensity (Fig. 1B); the opposite was true for clones 4, 5, and 6. The fresh weight of clone 9 was unaffected by light intensity. Fresh weight of all clones was positively correlated with population size (P < 0.001; R2 = 0.920). Alatae production of clones 1, 2, 3, 5, 6, 7, 8, and 10 was greater under low light intensity than under high light intensity (Fig. 1C). Alatae production of the other clones (clones 4 and 9) was unaffected by light intensity but tended to be greater under low light intensity. Clone 9 produced significantly fewer alatae than the other clones at the low light intensity, whereas clone 4 produced significantly more alatae than the other clones at the high light intensity. Morph colour (green vs. other colours) was unrelated to performance in experiment 3. Thus, the performance of clones 7 and 8, which were green, did not differ from the performance of the other clones, which were not green, regardless of light intensity (Fig. 1A–C). 3.6. Carotenoids in S. avenae morphs (experiment 6) We compared several TLC systems for the detection of carotenoids in aphid extracts. The best resolution was achieved on silica gel plates developed in diethyl ether/hexane (5:95) (Table 3). When aphids were harvested from 200 mE light conditions and diethyl ether/petroleum ether (5:95) was used as the solvent, the pigments of clones 7 and 8 generated one yellow band while the pigments of the all other clones separated into five bands. When diethyl ether/hexane (5:95) was the solvent, the pigments of clones 7 and 8 separated into four yellow bands while the pigments of the other clones separated into seven bands. Extracts of all clones reared at 15 mE generated one yellow band with diethyl ether/petroleum ether and two yellow bands with diethyl ether/ hexane. To understand the dynamics of carotenoid synthesis during colour change, we analyzed extracts of clones (using the same procedures and solvents as described in Jenkins et al., 1999) at
[(Fig._1)TD$IG]
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4. Discussion 4.1. Impact of light intensity on colour formation in S. avenae clones
Fig. 1. Effect of light intensity on performance of Sitobion avenae clones (experiment 5). (A) Offspring production (number of aphids per pot); (B) fresh weight (total weight of aphids per pot); and (C) alatae production (number of alatae per pot). Values are means SE for 15 mE (white bars) and 200 mE (black bars). Black bars with the same uppercase letter and white bars with the same lowercase letter are not significantly different at P 0.05. Statistical comparison of light intensity within each clone is indicated by NS (not significant, P > 0.05) and * (significant, P 0.05).
different stages of colour change during 5 days following transfer from 200 mE to 15 mE, when most aphids turned from dark brown to pink to green. While all compounds analyzed remained constant during this time period, we found that the carotenoid lycopene gradually declined.
The initial colour of the S. avenae clones (their colour when collected in the field) changed when they were reared in the laboratory, where the light intensity was lower than in the field, even before our formal experiments were started (unpubl. obs.). This observation thus already indicated that aphids exposed to more intensive light will display more intensive colours. This observation was corroborated by the colour changes of aphid clones in the transparent rearing system. Here the changes were more rapid than in the normal rearing system, where colour changed after 5–6 days. Our results allow us to separate S. avenae clones into two main groups that differ in their ability to produce coloured morphs. Clones of the first group were green at all light intensities, while clones of the second group changed their colour from green at low light intensity (15 mE) to red and brown at high light intensity (200 mE). The aphids that fed on the upper leaves became coloured in a shorter time and their final colour was more intense than that of aphids partially protected from the light on the lower parts of the plants. These patterns of colour change, observed under controlled laboratory conditions, explain the prevalence of green morphs in spring and pink-brown morphs in summer and autumn (Chroston, 1983). In winter, light intensity in temperate regions is very low, and thus all aphids in the field are green, regardless of whether they are of parthenogenetic origin or gynoparae. In spring, aphid specimens of early generations are still green. Following the increase of the light intensity in late spring and summer, some aphid clones become pink-brown while others remain green. In autumn, the decreasing light intensity remains sufficiently high to maintain the pink-brown colour of those aphid clones, becoming brown in the summer. In our study some aphid clones (clones 7 and 8) changed colour but did not become brown, and this change was also affected by light intensity. These clones were pale green under low light intensity but dark green under high light intensity. Thus, our observations corroborate previous work of Phillips (1916) and Chroston (1983), who reported that green morphs occurred at all times but that brown morphs only occurred during summer and autumn. We found that a specific S. avenae clone (e.g. clone 9) may comprise two colour morphs that contain different carotenoids. Carotenoids protect non-photosynthetic bacteria from lethal photodynamic processes at natural light conditions (Mathews and Sistrom, 1959). Because B. aphidicola is considered a free living bacteria before becoming symbiotic in aphids (Moran et al., 1994), the synthesis of carotenes in aphids may be regarded an ancestral attribute of their primary bacteria (Moran et al., 1994; Douglas, 1998). However, although the genome of B. aphidicola has been fully sequenced, no genes have been identified so far coding for carotenoids or their precursors. This has also been corroborated by Jenkins (1991) who reared aposymbiotic S. avenae clones on artificial diets free of carotenoids and did not change their colour following this treatment. He speculated that their colour had already been determined genetically in the ovarioles. Support for the hypothesis that genes coding for carotene or at least their precursors are totally or partially integrated in the aphid genome can be found when looking at other bacteria. Erwinia sp. and some bacteria in the family Enterobacteriaceae are able to synthesize carotenoids (Armstrong et al., 1993). Furthermore, Escherichia coli transformed with the carotenoid synthesis genes (crt) of E. herbicola become more resistant to near-UV irradiation and phototoxic agents, indicating the functional role of carotenoide synthesis under natural conditions (Tuveson et al., 1988).
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Table 3 Thin layer chromatography of the carotenoids of different morphs of S. avenae. Morph colour morph
Banda
Colour of band
Rf silica gel plates
Rf aluminium plates
Brown (all clones except 7 and 8) at 200 mE
1 2 3 4 5 6 7
Yellow Yellow Orange/pink Pink Pink Yellow Yellow
0.92 0.86 0.76 0.64 0.41 0.21 0.16
0.83 0.79 0.75 0.70 0.68 0.43 0.23
Green (clones 7 and 8) at 200 mE
1 2 3 4
Yellow Yellow Yellow Yellow
0.94 0.85 0.24 0.19
0.89 0.84 0.43 0.24
All clones at 15 mE
1 2
Yellow Yellow
0.91 0.93
0.87 0.83
a
The bands are ranked from top to bottom on silica gel plates. Diethyl ether in hexane (5%, v/v) was used as solvent.
Further evidence for a genomic interaction or gene absorption has been reported by Hundle et al. (1994) who reported that E. herbicola devoid of the gene lycopene cyclase became pink in colour. We speculate that the green morphs of S. avenae (or their symbiotic bacteria) have no gene(s) encoding for lycopene cyclase, whereas the brown morph still posses these genes. However, the fact that green morphs remain green does not indicate that green morphs do not synthesize carotenoids. The results of the TLC analyses confirm that the amount of carotenoids in the green morphs was higher when reared at 200 mE as compared to 15 mE. Jenkins et al. (1999) found, by using HPLC analyses, that green morphs contain many unknown carotenoids. Our findings are consistent with the results of Markkula and Rautapa¨a¨ (1967), who reported that brown morphs changed to green morphs at a photoperiod of 0.5 h. However, these authors did not control for the effects of light on the host plants, suffering from light deficiency. In another study, Markkula and Pulliainen (1965) investigated the impact of temperature, length of photoperiod, and light intensity on colour formation in S. avenae clones. In this study, however, they did not characterise the clones of S. avenae used and did not separate the effects of three independent variables contained in their experiments. The effects of light intensity on the influence of colour change in S. avenae reported by Markkula and Pulliainen (1965) were not confirmed by Ankersmit et al. (1986) or by Jenkins (1991). By separately controlling light intensity and host plants in our study, we have demonstrated that host plants did not affect the colour formation in S. avenae clones. For instance, rearing the clones on different host plants did not affect the colour formation and the colour of the dark brown clone 6 remained constant, even when reared for one week on yellow wheat leaves. 4.2. Impact of light intensity on performance of clones The performance of S. avenae clones was affected by light intensity. All clones, except clones 4, 5, and 6, produced more offspring and had a higher fresh weight under high than low light intensity. A previous study (Alkhedir, unpubl. data) indicated that clones 4, 5 and 6 may rapidly adapt to wheat at 200 mE, in that the clones have quite small populations in their first generation under these conditions but relatively large populations in their second and third generations. These clones also were able to adapt to feeding on wheat at 15 mE, though the populations were larger at 200 than at 15 mE (unpubl. obs.). Previous studies have shown that aphids produce more alatae morphs in short than in long photoperiods (Dixon, 1998). Moreover, clones of S. avenae are known to produce more alatae
when they are crowded or poorly nourished, and green S. avenae morphs produce more alate morphs than brown morphs (Ankersmit and Dijkman, 1983). Crowding and poor nutrition, however, have the opposite effect on alatae production by the pea aphid A. pisum (Weisser and Braendle, 2001). In our experiments, crowding and nutrition were kept constant (they were controlled), enabling us to separate the effects of light intensity on alatae production. The results demonstrated that all colour morphs of S. avenae produced more alatae at low than at high light intensity. In addition, our results demonstrate that S. avenae clones benefit from responding to variation in light intensity. When light intensity becomes low in early spring or late autumn aphids produce more alatae, allowing to migrate to either their summer or winter host plants. Production of alatae also varied within the S. avenae clones tested. For example, clone 4 produced more alatae as compared to clone 9, the former being more variable and more rapidly adapting to wheat cultivars (Alkhedir, unpubl.). Weber (1985) reported that during summer, brown clones of S. avenae clones increased faster than green clones on their host plants, barley and oat. Our results did not support this finding. For example, the performance of clones 7 and 8, which produced green morphs at all light intensities, did not differ from the performance of clone 1 and other brown clones. Light intensity could affect the performance of S. avenae clones by two pathways. In the first pathway, light intensity could indirectly affect the aphids by changing the physiology and chemical composition of the host plants. In the second pathway, light intensity could directly affect the aphids by affecting carotenoids synthesis and alatae production (this study). With respect to the indirect effect of light on aphids, light could influence aphids by altering plant hormones and enzymes. Aphids feed on phloem sap containing large quantities of different sugars. Phloem sugar levels are often 1.1–2.0 times greater in the day than at night and are also influenced by temperature, light radiation, plant species, and plant developmental age (Winter et al., 1992; Geiger and Servaites, 1994; Kehr et al., 1998). Levels of phytohormones in plants are also affected by light conditions (Kraepiel and Miginiac, 1997). Casaretto and Corcuera (1998) observed that proteinase inhibitors accumulated in barley leaves in the course of aphid infestations, thus reducing the numbers of some aphid species. Moreover, the induction of proteinase inhibitors is regulated by endogenous chemical factors including phytohormones, such as abscisic acid and jasmonic acid, and these phytohormones increase the induction of proteinase inhibitors in barley leaves when applied exogenously (Casaretto et al., 2004). Because light affects levels of phytohormones and therefore of proteinase inhibitors, light (including photoperiod and intensity) could affect aphid reproduction and survival.
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In summary, our study provides new information about the effect of light intensity on both colour formation and clonal performance of S. avenae. Further studies are needed regarding the characterisation of Sitobion pigments and the nature (mechanism) of their induction/expression. The rearing of aposymbiotic clones on artificial diets for at least three generations would help clarify the origin of the pigments and prove whether this is due to genetically based factors alone or whether the symbionts are involved.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jinsphys.2010.08.025. References Andrewes, A.G., Kjosen, H.S.L.-J., Weisgraber, K.H., Lousberg, R.J., Weiss, U., 1971. Animal carotenoids. 7. Carotenes of two colour variants of the aphid Macrosiphum liriodendri—identification of natural gamma, gamma-carotene. Acta Chemica Scandinavica 25, 3878–3880. Ankersmit, G.W., Acreman, T.M., Dijkman, H., 1981. Parasitism of colour forms in Sitobion avenae. Entomologia Experimentalis et Applicata 29, 362–363. Ankersmit, G.W., Bell, C., Dijkman, H., Mace, N., Rietstra, S., Schro¨der, J., De Visser, C., 1986. Incidence of parasitism by Aphidius rhopalosiphi in colour forms of the aphid Sitobion avenae. Entomologia Experimentalis et Applicata 40, 223–229. Ankersmit, G.W., Dijkman, H., 1983. Alatae production in the cereal aphid Sitobion avenae. Netherlands Journal of Plant Pathology 89, 105–112. Armstrong, G.A., Hundle, B.S., Hearst, J.E., 1993. Evolutionary conservation and structural similarities of carotenoid biosynthesis gene products from photosynthetic and nonphotosynthetic organisms. Methods in Enzymology 214, 297–311. Blackman, R.T., Eastop, V.F., 1984. Aphids on the World’s Crops: An Identification Guide. Wiley, New York. Bowie, J.H., Cameron, D.W., Findlay, J.A., Quartey, J.A.K., 1966. Haemolymph pigments of aphids. Nature 210, 395–397. Braendle, C., Weisser, W.W., 2001. Variation in escape behavior of red and green clones of the pea aphid. Journal of Insect Behavior 14, 497–509. Casaretto, J.A., Corcuera, L.J., 1998. Proteinase inhibitor accumulation in aphidinfested barley leaves. Phytochemistry 49, 2279–2286. Casaretto, J.A., Zuniga, G.E., Corcuera, L.J., 2004. Abscisic acid and jasmonic acid affect proteinase inhibitor activities in barley leaves. Journal of Plant Physiology 161, 389–396. Chroston, J.R., 1983. Colour polymorphism of the English grain aphid (Sitobion avenae F.). Ph.D. Thesis, University of East Anglia, Norwich, U.K. Dixon, A.F.G., 1973. Metabolic acclimatization to seasonal changes in temperature in the sycamore aphid, Drepanosiphum platanoides (Schr.), and lime aphid, Eucallipterus tiliae L. Oecologia 13, 205–210. Dixon, A.F.G., 1998. Aphid Ecology. Chapman and Hall, London, United Kingdom. Douglas, A.E., 1998. Nutritional interactions in insect–microbial symbioses: Aphids and their symbiotic bacteria Buchnera. Annual Review of Entomology 43, 17–37. Geiger, D.R., Servaites, J.C., 1994. Diurnal regulation of photosynthetic carbon metabolism in C3 plants. Annual Review of Plant Physiology and Plant Molecular Biology 45, 235–256.
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Goodwin, T.W., 1986. Metabolism, nutrition, and function of Carotenoids. Annual Review of Nutrition 6, 273–297. Harmon, J.P., Losey, J.E., Ives, A.R., 1998. The role of vision and color in the close proximity foraging behavior of four coccinellid species. Oecologia 115, 287– 292. Hundle, B., Alberti, M., Nievelstein, V., Beyer, P., Kleinig, H., Armstrong, G.A., Burke, D.H., Hearst, J.E., 1994. Functional assignment of Erwinia herbicola Eho10 carotenoid genes expressed in Escherichia coli. Molecular and General Genetics 245, 406–416. Jenkins, R.L., 1991. Colour and symbionts of aphids. PhD Thesis, University of East Anglia, U.K. Jenkins, R.L., Loxdale, H.D., Brookes, C.P., Dixon, A.F.G., 1999. The major carotenoid pigments of the grain aphid, Sitobion avenae (F.) (Hemiptera: Aphididae). Physiological Entomology 24, 171–178. Kehr, J., Hustiak, F., Walz, C., Willmitzer, L., Fisahn, J., 1998. Transgenic plants changed in carbon allocation pattern display a shift in diurnal growth pattern. The Plant Journal 16, 497–503. Kraepiel, Y., Miginiac, E., 1997. Photomorphogenesis and phytohormones. Plant, Cell & Environment 20, 807–812. Loxdale, H.D., Lushai, G., 2003. Rapid changes in clonal lines: the death of a ‘sacred cow’. Biological Journal of the Linnean Society 79, 3–16. Markkula, M., Myllyma¨ki, S., 1963. Biological studies on cereal aphids, Rhopolosiphum padi (L.), Macrosiphum avenae (F.) and Acyrthosiphum dirhodum (Wlk.) (Hom., Aphididae). Annales Agriculturae Fenniae 2, 33–43. Markkula, M., Pulliainen, E., 1965. The effect of temperature on the lengths of the life periods of the English grain aphid Macrosiphum avenae (F.) (Hom., Aphididae) and on the number and colour of its progeny. Annales Entomologia Fennica 31, 39–45. Markkula, M., Rautapa¨a¨, J., 1967. The effect of light and temperature on the colour of the English grain aphid Macrosiphum avenae (F.) (Hom., Aphididae). Annales Entomologia Fennica 33, 1–13. Mathews, M.M., Sistrom, W.R., 1959. Function of carotenoid pigments in nonphotosynthetic bacteria. Nature 184, 1892–1893. Moran, N.A., Baumann, P., Van Dohlen, C., 1994. Use of DNA sequences to reconstruct the history of the association between members of the sternorrhyncha (Homoptera) and their bacterial endosymbionts. European Journal of Entomology 91, 79–83. Mu¨ller, F.P., 1961. Stabilita¨t und Vera¨nderlichkeit der Fa¨rbung bei Blattla¨usen. Archiv der Freunde der Naturgeschichte in Mecklenburg (Rostock) 7, 228– 239. Phillips, W.J., 1916. Macrosiphum granarium, the English grain aphid. Journal of Agricultural Research 7, 463–480. SYSTAT, 2004. Release 11.00.01, Systat Software, Inc., 1735, Technology Drive, Ste 430 San Jose, CA 95110. Tuveson, R.W., Larson, R.A., Kagan, J., 1988. Role of cloned carotenoid genes expressed in Escherichia coli in protecting against inactivation by near-UV light and specific phototoxic molecules. Journal of Bacteriology 170, 4675– 4680. Weber, G., 1985. On the ecological genetics of Sitobion avenae (F.) (Hemiptera, Aphididae). Journal of Applied Entomology 100, 100–108. Weisgraber, K.H., Lousberg, R.J.J.C., Weiss, U., 1971. The chemical basis of the color dimorphism of an aphid, Macrosiphum liriodendri (monell), and a locust, Amblycorypha sp. novel carotenoids. Experientia 27, 1017–1018. Weisser, W.W., Braendle, C., 2001. Body colour and genetic variation in winged morph production in the pea aphid. Entomologia Experimentalis et Applicata 99, 217–223. Winter, H., Lohaus, G., Heldt, H.W., 1992. Phloem transport of amino acids in relation to their cytosolic levels in barley leaves. Plant Physiology 99, 996– 1004.
Contents lists available at ScienceDirect
Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys
Effect of light intensity on colour morph formation and performance of the grain aphid Sitobion avenae F. (Homoptera: Aphididae) Hussein Alkhedir a, Petr Karlovsky b, Stefan Vidal a,* a b
Agricultural Entomology, Department of Crop Sciences, Georg-August-University Goettingen, Grisebachstrasse 6, 37077 Goettingen, Germany Molecular Phytopathology and Mycotoxin Research, Department of Crop Sciences, Georg-August-University Goettingen, Grisebachstrasse 6, 37077 Goettingen, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 October 2009 Received in revised form 30 August 2010 Accepted 31 August 2010
The grain aphid Sitobion avenae F., one of the major pest aphids of cereals in Central Europe, exhibits colour polymorphism, even within the same clones. Although there is evidence that green and brown morphs of S. avenae contain different carotenoids, the mechanisms determining the induction of colour morphs are unknown. The common understanding is that the formation of colour morphs is controlled by light and affected by genetic and environmental factors and by host plant species. So far, there is no unequivocal evidence that light intensity, photoperiod, or a mixture of several variables are involved in the induction of S. avenae colour formation, resulting in the induction of S. avenae colour formation and carotenoid synthesis. Here we determined the effect of light intensity on the colour formation and performance of ten clones of S. avenae with experiments that controlled for the effects of host plant and genetic factors. We found that some clones remained green under all test conditions. In other clones, colour morph formation was controlled by light. The synthesis of carotenoids correlated with changes in colour formation. Host plant did not affect colour formation in the ten clones studied. Although colour of the aphid clones did not affect their performance, high light intensity increased the fecundity and fresh weight of S. avenae clones, while low light intensity stimulated the production of alatae. ß 2010 Elsevier Ltd. All rights reserved.
Keywords: Carotenoids Light intensity Colour polymorphism
1. Introduction Aphids exhibit polymorphism, i.e., a population of identical genotypes (Loxdale and Lushai, 2003) can have two or more morphologically distinct individuals, termed morphs. These morphs are adapted to specific ecological conditions and are characterised by differences in their life cycles and colour (Blackman and Eastop, 1984; Dixon, 1998). The colour of these morphs results from the interaction of two factors: the colour of the cuticle and the pigmentation of the haemolymph (Mu¨ller, 1961). The colour formation of the cuticle is affected by temperature alone (Dixon, 1973), whereas the pigmentation of the haemolymph is affected by temperature, photoperiod, and light intensity (Markkula and Pulliainen, 1965; Jenkins et al., 1999). Glucosidic haemolymph pigments in aphids are synthesised as aphins or protoaphins, which are unique to Aphididae (Bowie et al., 1966). Other haemolymph pigments also include carotenoids, which cannot be synthesized by aphids or other animals but which are found in higher plants and microorganisms (Goodwin, 1986).
* Corresponding author. Tel.: +49 551 39 39744; fax: +49 551 39 12105. E-mail address: [email protected] (S. Vidal). 0022-1910/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2010.08.025
Aphins are responsible for the yellow, orange, or red colour in aphid morphs (Bowie et al., 1966). As mentioned above, differences in colour morphs of the same aphid species can result from differences in carotenoids (Andrewes et al., 1971; Weisgraber et al., 1971; Jenkins et al., 1999). Because animals, including aphids, are unable to synthesise carotenoids, precursors are needed for the synthesis of these compounds. The obligate bacterial endosymbiont Buchnera aphidicola has been proposed to provide these metabolites to their aphid hosts (Moran et al., 1994; Douglas, 1998). Colour polymorphism has been documented in many aphid species (Dixon, 1998) and is regarded as a selective advantage with regard to natural enemies and environmental factors. For example, red colour morphs of the pea aphid Acyrthosiphon pisum (Harris) produced more alate and escaped from predators more rapidly than green morphs (Braendle and Weisser, 2001). Seven spot ladybird beetles Coccinella septempunctata L. consumed more aphid morphs when the morph colour contrasted with rather than matched the background colour (Harmon et al., 1998). Ankersmit et al. (1981) reported that colour morphs of S. avenae differed in their susceptibility to the parasitoid Aphidius rhopalosiphi De Stefani Perez, the brown morphs being more resistant than the green morphs. In this host–parasitoid system, partial resistance was assumed to be related to the increased handling time of brown
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morphs by female parasitoids combined with an increase in the development time and mortality of the larvae produced by females that consumed brown morphs. These effects were significant even for morphs derived from the same clone (i.e. asexual progeny from a known founder stem mother) (Ankersmit et al., 1986). In addition to affecting the interaction between aphids and their natural enemies, colour polymorphisms resulting from light intensity, photoperiod, or temperature can also affect aphid fecundity (Markkula and Pulliainen, 1965; Markkula and Rautapa¨a¨, 1967). The grain aphid Sitobion avenae F. produces colour morphs ranging from green (the most common colour) to red, brown, and pink (Phillips, 1916; Mu¨ller, 1961; Markkula and Myllyma¨ki, 1963; Markkula and Pulliainen, 1965; Markkula and Rautapa¨a¨, 1967; Jenkins, 1991; Jenkins et al., 1999). Although the basis for this colour polymorphism mainly depends on differences in the carotenoide content in the haemolymph (Jenkins, 1991; Jenkins et al., 1999). Jenkins (1991) also demonstrated that anholocyclic clones are of green colour and holocyclic clones are of brown colour, but the mechanisms determining the induction of colour morphs in S. avenae are incompletely understood. In this study, we tested whether colour morph formation and aphid performance of S. avenae clones are influenced by light intensity and host plant species. Moreover, we tested the synthesis of specific carotenoids in S. avenae colour morphs is influenced by light intensity using Thin layer chromatography (TLC). 2. Materials and methods 2.1. Aphid cultures and light intensity The ten clones of S. avenae used in this study were established from single asexual female aphid, collected in different regions of Germany and from different host plants. At the time of collection, the clones displayed different colour morphs. The clones relate to different genotypes and harbour different bacterial endosymbionts (Alkhedir, unpublished data) (Table 1). In the laboratory, these clones were reared on wheat seedlings (winter wheat cv ‘‘Bussard’’, Lochow Petkus Comp., Germany) grown in 11-cm diam. pots (10 seedlings per pot) containing a 2:1 mixture of soil (Fruhstorfer Typ P) and sand. Each plant was covered with a transparent ventilated cylinder, which was 10 cm wide and 30 cm high. Aphids were transferred to newly grown plants at two-week intervals. Cultures were kept at 20 8C, and at a 16:8 L:D hour photoperiod, 15 mEinstein (mE) flux intensity, and 60–80% humidity in climate chambers (WB 750 KFL; Mytron Bio-Und Solartechnik GmbH, Germany). Light intensity was measured with a LI-COR Quantum Radiometer/Photometer (Inc. Bioscience). Plants were watered from below twice each week. Given these conditions, all clones reproduced parthenogenetically, and the colour of all clones and individuals in these cultures were light green. Experiments with high light intensity (200 mE; see Section 2.3) were performed in a climate chamber (Viessmann Company, Germany), while
experiments with low light intensity or darkness were performed in the climate chambers mentioned above. The lamps installed in the climate chambers were OSRAM L 36W/25 and OSRAM FQ 54 W/840 HO, respectively. Specifications of wavelengths and light spectrum are given in Supplementary files 1 and 2. 2.2. Colour of clones as affected by host plant and light intensity (experiments 1 and 2) Seven host plant species were used in two main experiments (designated as 1 and 2) viz Wheat Triticum aestivum, cv ‘‘Bussard’’ from Lochow Petkus Comp.; and the grasses Poa annua, Lolium perenne cv. ‘‘Herault’’. Avena sp., Phalaris arundinacea (culture form), Agropyron repens from Appels Wilde Samen Comp., Germany; and Dactylis glomerata cv. ‘‘Neva’’ from the National Agricultural Research Centre for the Hokkaido Region, Japan. For experiment 1, wheat was grown as described previously, and seedlings were transplanted into pots when 7 days old. All other host plants were germinated in the greenhouse in trays and transplanted when 1 month old and about 10–13 cm tall. The seedlings were transplanted into pots as described in Section 2.1, with 10 wheat seedlings or three seedlings of the other host plants per pot. Each of the 10 S. avenae clones was reared on all seven host plants, with 12 replicate pots per combination of clone and host plant species. Plants with aphids were divided into two sub-groups and randomly placed in growth chambers at 20 8C, 60–80% humidity, 18:6 L:D photoperiod, and one of two light intensities (15 mE vs. 200 mE). The colour formation of the aphid clones was visually assessed during 1 month; thereafter, the aphids were transferred to fresh host plants as described for experiment 2 below. In experiment 2, those aphids reared at 15 mE in experiment 1 were divided into two groups. The first group was thereafter continuously reared at 200 mE with 18:6 L:D photoperiod, while the second group was reared in darkness. A similar procedure was followed for the aphids previously reared at 200 mE in the first experiment but these were either continuously reared at 15 mE with 18:6 L:D illumination periods or reared in darkness. This experimental set-up was established separately for each host plant species. The colour of the aphid specimens was visually determined and recorded as pale green, green, pink, brown, or dark brown (supplementary 3). By the end of both experiments 1 and 2 all aphid were used for carotenoids analysis. 2.3. Colour morph formation in clone 6 (experiment 3) Five synchronized first instar green nymphs of clone 6 were propagated on one wheat seedling (7 cm tall) whose roots and the lower stem were immersed in a transparent 250-ml flask containing 200 ml of water. The seedling with aphids was covered with a transparent ventilated cylinder and kept at 20 8C and 60–80% humidity, with a constant light intensity of 200 mE. This set up ensured that all aphids were well exposed to light from all
Table 1 Origin and collection details of the S. avenae clones used in the study. Clone
Collection site
Host plant
Developmental stage collected
Colour at collection time
1 2 3 4 5 6 7 8 9 10
Goettingen Kassel Kassel Goettingen Giessen Kassel Giessen Giessen Kassel Kassel
Wheat Cocksfoot Bromus sp. Cocksfoot Wheat Cocksfoot Wheat Wheat Cocksfoot Cocksfoot
Virginoparae Virginoparae 2nd nymph Virginoparae Virginoparae Virginoparae Virginoparae Virginoparae 4th, winged 4th nymph
Green Green–brown Green–brown Green–brown Green–brown Green–brown Green Green Brown Brown
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sides. Aphid colour formation on five replicate seedlings was recorded daily for 28 days. 2.4. Colour morph formation in clone 9 (experiment 4) Ten green adults of clone 9 were chosen randomly from the aphid culture on wheat. One adult was placed in each of 10 Petri dishes containing a small piece of wheat seedling leaf sitting on a moist filter paper. The Petri dishes were placed under 200 mE at 20 8C. After 5 days, half of the Petri dishes were covered with black boards to produce dark conditions. The offspring of the initial adult aphids were left to become adults and produce offspring. The colour of all aphids was observed daily, and the experiment was terminated after 21 days. 2.5. Performance of clones at different light intensities (experiment 5) Synchronized first-stage nymphs of the 10 clones were added to 7-day-old wheat seedlings (10 nymphs per pot) growing in pots and covered with transparent ventilated cylinders as described earlier. After 2 weeks in growth chambers at 20 8C, 60–80% humidity, 18:6 L:D illumination periods, and two different light intensities (15 mE vs. 200 mE), the experiment was terminated and clonal performance was assessed. There were seven replicate pots for each combination of aphid clone and light intensity. Clonal performance was assessed as the number of aphids per pot, total fresh weight of aphids per pot, and the number of alatae per pot. 2.6. Carotenoide analysis of clones in experiments 1 and 2 (experiment 6) Aphids were collected at the end of experiments (1 and 2), and were separated into four groups. The first group contained aphids from clones 7 and 8 collected from plants grown at 15 mE and were pale green. The second group contained aphids from clones 7 and 8 collected from plants grown at 200 mE and were green. The third and fourth groups included all remaining clones collected from plants grown at 15 and 200 mE; the third group (from plants grown at 15 mE) was pale green and the fourth (from plants grown at 200 mE) was red-brown. Carotenoids were extracted and separated with TLC as described by Jenkins et al. (1999). They were extracted from each sample, containing 0.5–1 g specimens, using 2 ml of pre-chilled acetone three times, 2 ml of methanol/acetone (1:1, v:v) (once), and finally 2 ml of petroleum ether (twice). All extracts were pooled in a plastic vial, centrifuged at 2000 rpm for 20 min, and then placed in separation funnels containing 5 ml of petroleum ether. A sufficient quantity of distilled water was added to aid in the separation of the phases. The bulked extracts were partitioned in two phases: the extract of the brown clone separated into a blue-green aqueous phase and an orange epiphase; the extract of the green clone separated into the same blue-green hypophase, but the epiphase was yellow. The epiphase was collected into new plastic vials, and petroleum ether was added to retain pigments. The collected epiphase (10 ml) was washed three times with distilled water and then dried under vacuum. The extract containing pigments in various stages of esterification with fatty acids was saponified under nitrogen gas for 16 h at 5 8C in 5 ml of petroleum ether with 6% KOH. The saponified extractions were placed in separatory funnels with 5 ml distilled water; the organic phase was then collected in new vials, and petroleum ether added again to the bulk extracts containing the pigments. These extractions were dried using a rotary vacuum. Thereafter, they were dissolved in 200 ml of petroleum ether. The pigments were separated with TLC using either silica gel or aluminum oxide foils (20 20 cm) (Fluka, Germany) pre-washed with 3% KOH in
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methanol. The TLC solvent was diethyl ether in petroleum ether (5% v:v) or diethyl ether in hexane (5%, v:v). TLC was performed in the dark at 5 8C. The extracts were co-chromatographed with bcarotene (Sigma), and the Rf values for each band recorded. Lycopene was analysed according to the TLC method decribed in Jenkins et al. (1999), following the experimental set-up described by these authors. 2.7. Statistical analysis A Kruskal–Wallis one-way analysis of variance was used to compare the performance of each clone under the different light intensity regimes. Bonferroni’s adjustments were included to compare the clonal performance for each variable under each of light intensity. SYSTAT for Windows version 11.00.01 (SYSTAT, 2004) was used to perform these analyses. Multivariate analysis of variance (MANOVA) tests were done to analyse the effect of light intensity on the performance of S. avenae clones; the number of aphids per pot, total aphid fresh weight per pot, and the number of alatae per pot were the dependent variables, and clone, light intensity, and interactions between them the independent, categorical variables. MANOVA was also used to analyze the effect of colour morph on performance of S. avenae clones; the number of aphids per seedling, total aphid fresh weight per seedling, and numbers of alatae per seedling were the dependent variables, and clones and colour morphs the independent, categorical variables. 3. Results 3.1. Colour morph formation as affected by host plant and light intensity (experiments 1 and 2) Host plant did not affect the colour of Sitobion clones in experiments 1 and 2, i.e., the colour of each S. avenae clone at low light intensity was the same on all host plant species. Similarly, the colour of each S. avenae clone at high light intensity was the same on all host plant species. Although the colour of S. avenae clones was not affected by host plant, the colour was affected by light intensity in experiment 1 (Table 2). Clones that were pale green at the beginning of the experiment remained pale green at 15 mE throughout the experiment. In contrast clones that were pale green at the beginning became pink, or brown when reared at 200 mE between day 1 and 10. Thereafter these clones turned dark brown, except for clones 7 and 8, which turned dark green; these colours remained constant for all clones after day 10. The effect of light intensity in experiment 2 is described in the text without tabular support. When aphids previously kept at 200 mE were transferred to 15 mE in experiment 2, all specimens of all clones became pale green. When clones previously kept at 15 mE were transferred to the 200 mE treatment, all specimens of all clones turned dark brown, except clones 7 and 8, which became dark green. When aphids previously kept at 200 or 15 mE were transferred to darkness, all specimens of all clones turned pale green. After 2 weeks in darkness, plants became yellow and died, and mortality of aphids on these plants rapidly increased. At this time, the colour of all aphid clones in this treatment was pale green to yellow. There were differences among the clones in experiment 2. At the high light intensity, some clones changed their colour earlier than others. For example, clone 6 started to change colour after 3 days at 200 mE compared with clone 9 which change its colour after 5–6 days. The colour of clones responding to high light intensities gradually changed from pale green to green, and pink. After 10 days, all aphids initially exposed to 15 mE in experiment 1
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Table 2 Influence of light intensity on colour morph formation in 10 clones of Sitobion avenae (experiment 1). G: green; B: brown; M: green-brown; PG: pale green; Y: yellow; DG: dark green; DB: dark brown; P: pink. Clone
Original colour
1 2 3 4 5 6 7 8 9 10
G M M M M M G G B B
Colour immediately after 6 days of exposure to 0, 15, or 200 mE
Final colour after 10 days of exposure to 15 or 200 mE
0 mE
15 mE
200 mE
15 mE
200 mE
PG-Y PG-Y PG-Y PG-Y PG-Y PG-Y PG-Y PG-Y PG-Y PG-Y
PG PG PG PG PG PG PG PG PG PG
P-B P-B P-B P-B P-B P-B DG DG P-B P-B
PG PG PG PG PG PG PG PG PG PG
DB DB DB DB DB DB DG DG DB DB
and then exposed to 200 mE in experiment 2 turned dark brown except clones 7 and 8 which became dark green. In experiments 1 and 2, colour formation of the aphid clones at the high light intensity was faster when the plants were small (during the first 2 weeks). As the plants grew larger, some leaves created shadows in some areas within the cylinders. At this time, different aphid specimens within one clone showed different colours; aphids that were not directly exposed to the light became green or exhibited mixed colours, while aphids exposed to the full light displayed the colour equivalent to the one found when reared at 200 mE. The green of clones 7 and 8 was less intense at 15 mE than at 200 mE. 3.2. Colour formation in clone 6 at 200 mE (experiment 3) The colour of clone 6 nymphs kept at 200 mE ranged from green to pink during the experiment, the final colour being dark brown. This change in colour took place during 4 days; during this period, the nymphs developed into stage-four nymphs. The adults (both apterae and alate) and their offspring were brown. After 3 weeks, the wheat seedlings had become completely yellow except for the leaf tips, which had turned brown. At this time, the aphid population on each seedling included the original nymphs that were added at the start and a second generation of nymphs. In general, the aphids and their offspring in this transparent system at 200 mE were darker than aphids kept at 200 mE on the regular culture of wheat seedlings. 3.3. Colour morph formation in clone 9 at 0 and 200 mE (experiment 4) All clone 9 adults kept at 200 mE laid new nymphs on the first day. At birth, these nymphs were green. The colour of the adults began to change after 2 days. This colour change began at the head and spread into the middle part of the abdomen first and then to the sides of the abdomen. The colour of the offspring began to change within one day after birth. After five days, nymphs had become pink to dark brown. When these nymphs became adults, their offspring were not born green but were pink to dark brown. Even the visible embryos inside the adults were pink to dark brown. Aphids directly exposed to light on the upper surface of the leaf pieces changed colour sooner than those feeding beneath the leaf pieces. Those aphids that were moved to dark conditions after 5 days became green after 3–5 days and produced green offspring after becoming adults; the embryos inside the adults were also green. 3.4. Effect of host plant on colour of clones Host plants did not affect the colour formation of Sitobion clones. The colour of each S. avenae clone was the same at each of light intensity tested on all host plant species.
3.5. Effect of light intensity on performance of clones (experiment 5) Population size, fresh weight, and alatae production of S. avenae differed significantly among the 10 clones in experiment 3 (F9,120 = 58.443, P < 0.001). These variables were also affected by light intensity (F1,120 = 115.839, P < 0.001), and the interaction between clone and light intensity was also significant (F9,120 = 37.570, P < 0.001). Population sizes of clones 1, 2, 7, 8, and 10 were larger under high light intensity than under low light intensity (Fig. 1A); the opposite was true for clones 4, 5, and 6. Population sizes of clones 3 and 9 were unaffected by light intensity. The fresh weights of clones 1, 2, 3, 7, 8, and 10 were greater under high light intensity than under low light intensity (Fig. 1B); the opposite was true for clones 4, 5, and 6. The fresh weight of clone 9 was unaffected by light intensity. Fresh weight of all clones was positively correlated with population size (P < 0.001; R2 = 0.920). Alatae production of clones 1, 2, 3, 5, 6, 7, 8, and 10 was greater under low light intensity than under high light intensity (Fig. 1C). Alatae production of the other clones (clones 4 and 9) was unaffected by light intensity but tended to be greater under low light intensity. Clone 9 produced significantly fewer alatae than the other clones at the low light intensity, whereas clone 4 produced significantly more alatae than the other clones at the high light intensity. Morph colour (green vs. other colours) was unrelated to performance in experiment 3. Thus, the performance of clones 7 and 8, which were green, did not differ from the performance of the other clones, which were not green, regardless of light intensity (Fig. 1A–C). 3.6. Carotenoids in S. avenae morphs (experiment 6) We compared several TLC systems for the detection of carotenoids in aphid extracts. The best resolution was achieved on silica gel plates developed in diethyl ether/hexane (5:95) (Table 3). When aphids were harvested from 200 mE light conditions and diethyl ether/petroleum ether (5:95) was used as the solvent, the pigments of clones 7 and 8 generated one yellow band while the pigments of the all other clones separated into five bands. When diethyl ether/hexane (5:95) was the solvent, the pigments of clones 7 and 8 separated into four yellow bands while the pigments of the other clones separated into seven bands. Extracts of all clones reared at 15 mE generated one yellow band with diethyl ether/petroleum ether and two yellow bands with diethyl ether/ hexane. To understand the dynamics of carotenoid synthesis during colour change, we analyzed extracts of clones (using the same procedures and solvents as described in Jenkins et al., 1999) at
[(Fig._1)TD$IG]
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4. Discussion 4.1. Impact of light intensity on colour formation in S. avenae clones
Fig. 1. Effect of light intensity on performance of Sitobion avenae clones (experiment 5). (A) Offspring production (number of aphids per pot); (B) fresh weight (total weight of aphids per pot); and (C) alatae production (number of alatae per pot). Values are means SE for 15 mE (white bars) and 200 mE (black bars). Black bars with the same uppercase letter and white bars with the same lowercase letter are not significantly different at P 0.05. Statistical comparison of light intensity within each clone is indicated by NS (not significant, P > 0.05) and * (significant, P 0.05).
different stages of colour change during 5 days following transfer from 200 mE to 15 mE, when most aphids turned from dark brown to pink to green. While all compounds analyzed remained constant during this time period, we found that the carotenoid lycopene gradually declined.
The initial colour of the S. avenae clones (their colour when collected in the field) changed when they were reared in the laboratory, where the light intensity was lower than in the field, even before our formal experiments were started (unpubl. obs.). This observation thus already indicated that aphids exposed to more intensive light will display more intensive colours. This observation was corroborated by the colour changes of aphid clones in the transparent rearing system. Here the changes were more rapid than in the normal rearing system, where colour changed after 5–6 days. Our results allow us to separate S. avenae clones into two main groups that differ in their ability to produce coloured morphs. Clones of the first group were green at all light intensities, while clones of the second group changed their colour from green at low light intensity (15 mE) to red and brown at high light intensity (200 mE). The aphids that fed on the upper leaves became coloured in a shorter time and their final colour was more intense than that of aphids partially protected from the light on the lower parts of the plants. These patterns of colour change, observed under controlled laboratory conditions, explain the prevalence of green morphs in spring and pink-brown morphs in summer and autumn (Chroston, 1983). In winter, light intensity in temperate regions is very low, and thus all aphids in the field are green, regardless of whether they are of parthenogenetic origin or gynoparae. In spring, aphid specimens of early generations are still green. Following the increase of the light intensity in late spring and summer, some aphid clones become pink-brown while others remain green. In autumn, the decreasing light intensity remains sufficiently high to maintain the pink-brown colour of those aphid clones, becoming brown in the summer. In our study some aphid clones (clones 7 and 8) changed colour but did not become brown, and this change was also affected by light intensity. These clones were pale green under low light intensity but dark green under high light intensity. Thus, our observations corroborate previous work of Phillips (1916) and Chroston (1983), who reported that green morphs occurred at all times but that brown morphs only occurred during summer and autumn. We found that a specific S. avenae clone (e.g. clone 9) may comprise two colour morphs that contain different carotenoids. Carotenoids protect non-photosynthetic bacteria from lethal photodynamic processes at natural light conditions (Mathews and Sistrom, 1959). Because B. aphidicola is considered a free living bacteria before becoming symbiotic in aphids (Moran et al., 1994), the synthesis of carotenes in aphids may be regarded an ancestral attribute of their primary bacteria (Moran et al., 1994; Douglas, 1998). However, although the genome of B. aphidicola has been fully sequenced, no genes have been identified so far coding for carotenoids or their precursors. This has also been corroborated by Jenkins (1991) who reared aposymbiotic S. avenae clones on artificial diets free of carotenoids and did not change their colour following this treatment. He speculated that their colour had already been determined genetically in the ovarioles. Support for the hypothesis that genes coding for carotene or at least their precursors are totally or partially integrated in the aphid genome can be found when looking at other bacteria. Erwinia sp. and some bacteria in the family Enterobacteriaceae are able to synthesize carotenoids (Armstrong et al., 1993). Furthermore, Escherichia coli transformed with the carotenoid synthesis genes (crt) of E. herbicola become more resistant to near-UV irradiation and phototoxic agents, indicating the functional role of carotenoide synthesis under natural conditions (Tuveson et al., 1988).
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Table 3 Thin layer chromatography of the carotenoids of different morphs of S. avenae. Morph colour morph
Banda
Colour of band
Rf silica gel plates
Rf aluminium plates
Brown (all clones except 7 and 8) at 200 mE
1 2 3 4 5 6 7
Yellow Yellow Orange/pink Pink Pink Yellow Yellow
0.92 0.86 0.76 0.64 0.41 0.21 0.16
0.83 0.79 0.75 0.70 0.68 0.43 0.23
Green (clones 7 and 8) at 200 mE
1 2 3 4
Yellow Yellow Yellow Yellow
0.94 0.85 0.24 0.19
0.89 0.84 0.43 0.24
All clones at 15 mE
1 2
Yellow Yellow
0.91 0.93
0.87 0.83
a
The bands are ranked from top to bottom on silica gel plates. Diethyl ether in hexane (5%, v/v) was used as solvent.
Further evidence for a genomic interaction or gene absorption has been reported by Hundle et al. (1994) who reported that E. herbicola devoid of the gene lycopene cyclase became pink in colour. We speculate that the green morphs of S. avenae (or their symbiotic bacteria) have no gene(s) encoding for lycopene cyclase, whereas the brown morph still posses these genes. However, the fact that green morphs remain green does not indicate that green morphs do not synthesize carotenoids. The results of the TLC analyses confirm that the amount of carotenoids in the green morphs was higher when reared at 200 mE as compared to 15 mE. Jenkins et al. (1999) found, by using HPLC analyses, that green morphs contain many unknown carotenoids. Our findings are consistent with the results of Markkula and Rautapa¨a¨ (1967), who reported that brown morphs changed to green morphs at a photoperiod of 0.5 h. However, these authors did not control for the effects of light on the host plants, suffering from light deficiency. In another study, Markkula and Pulliainen (1965) investigated the impact of temperature, length of photoperiod, and light intensity on colour formation in S. avenae clones. In this study, however, they did not characterise the clones of S. avenae used and did not separate the effects of three independent variables contained in their experiments. The effects of light intensity on the influence of colour change in S. avenae reported by Markkula and Pulliainen (1965) were not confirmed by Ankersmit et al. (1986) or by Jenkins (1991). By separately controlling light intensity and host plants in our study, we have demonstrated that host plants did not affect the colour formation in S. avenae clones. For instance, rearing the clones on different host plants did not affect the colour formation and the colour of the dark brown clone 6 remained constant, even when reared for one week on yellow wheat leaves. 4.2. Impact of light intensity on performance of clones The performance of S. avenae clones was affected by light intensity. All clones, except clones 4, 5, and 6, produced more offspring and had a higher fresh weight under high than low light intensity. A previous study (Alkhedir, unpubl. data) indicated that clones 4, 5 and 6 may rapidly adapt to wheat at 200 mE, in that the clones have quite small populations in their first generation under these conditions but relatively large populations in their second and third generations. These clones also were able to adapt to feeding on wheat at 15 mE, though the populations were larger at 200 than at 15 mE (unpubl. obs.). Previous studies have shown that aphids produce more alatae morphs in short than in long photoperiods (Dixon, 1998). Moreover, clones of S. avenae are known to produce more alatae
when they are crowded or poorly nourished, and green S. avenae morphs produce more alate morphs than brown morphs (Ankersmit and Dijkman, 1983). Crowding and poor nutrition, however, have the opposite effect on alatae production by the pea aphid A. pisum (Weisser and Braendle, 2001). In our experiments, crowding and nutrition were kept constant (they were controlled), enabling us to separate the effects of light intensity on alatae production. The results demonstrated that all colour morphs of S. avenae produced more alatae at low than at high light intensity. In addition, our results demonstrate that S. avenae clones benefit from responding to variation in light intensity. When light intensity becomes low in early spring or late autumn aphids produce more alatae, allowing to migrate to either their summer or winter host plants. Production of alatae also varied within the S. avenae clones tested. For example, clone 4 produced more alatae as compared to clone 9, the former being more variable and more rapidly adapting to wheat cultivars (Alkhedir, unpubl.). Weber (1985) reported that during summer, brown clones of S. avenae clones increased faster than green clones on their host plants, barley and oat. Our results did not support this finding. For example, the performance of clones 7 and 8, which produced green morphs at all light intensities, did not differ from the performance of clone 1 and other brown clones. Light intensity could affect the performance of S. avenae clones by two pathways. In the first pathway, light intensity could indirectly affect the aphids by changing the physiology and chemical composition of the host plants. In the second pathway, light intensity could directly affect the aphids by affecting carotenoids synthesis and alatae production (this study). With respect to the indirect effect of light on aphids, light could influence aphids by altering plant hormones and enzymes. Aphids feed on phloem sap containing large quantities of different sugars. Phloem sugar levels are often 1.1–2.0 times greater in the day than at night and are also influenced by temperature, light radiation, plant species, and plant developmental age (Winter et al., 1992; Geiger and Servaites, 1994; Kehr et al., 1998). Levels of phytohormones in plants are also affected by light conditions (Kraepiel and Miginiac, 1997). Casaretto and Corcuera (1998) observed that proteinase inhibitors accumulated in barley leaves in the course of aphid infestations, thus reducing the numbers of some aphid species. Moreover, the induction of proteinase inhibitors is regulated by endogenous chemical factors including phytohormones, such as abscisic acid and jasmonic acid, and these phytohormones increase the induction of proteinase inhibitors in barley leaves when applied exogenously (Casaretto et al., 2004). Because light affects levels of phytohormones and therefore of proteinase inhibitors, light (including photoperiod and intensity) could affect aphid reproduction and survival.
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In summary, our study provides new information about the effect of light intensity on both colour formation and clonal performance of S. avenae. Further studies are needed regarding the characterisation of Sitobion pigments and the nature (mechanism) of their induction/expression. The rearing of aposymbiotic clones on artificial diets for at least three generations would help clarify the origin of the pigments and prove whether this is due to genetically based factors alone or whether the symbionts are involved.
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