DOI: 10.1111/eea.12677
1 6 T H I N T E R N AT I O N A L S Y M P O S I U M O N I N S E C T- P L A N T R E L AT I O N S H I P S
Plant response to feeding aphids promotes aphid dispersal Mariangela Coppola1, Elena Manco1, Alessia Vitiello1, Ilaria Di Lelio1, Massimo Giorgini2, Rosa Rao1, Francesco Pennacchio1 & Maria Cristina Digilio1* 1
Dipartimento di Agraria, Universita degli Studi di Napoli Federico II, Portici, Italy, and 2Istituto per la Protezione Sostenibile delle Piante, CNR, IPSP Portici, Portici, Italy Accepted: 19 January 2018
Key words: aphid saliva, plant signalling pathways, plant defence, salicylic acid, gene expression, methyl salicylate, SA-related genes, Aphis gossypii, Cucurbita pepo, Aphididae, Hemiptera, Cucurbitaceae
Abstract
Plant responses against biotic stress agents are affected by a number of environmental conditions, including the presence of other pests and pathogens. Moreover, the impact of infestation on subsequent plant colonization by conspecifics can vary, reflecting the high diversity in the co-evolutionary processes shaping host-plant interactions. Here, we address this issue by studying how aphid–plant interplay can influence the subsequent colonization of zucchini plants (Cucurbita pepo L., Cucurbitaceae) by conspecific Aphis gossypii Glover (Hemiptera: Aphididae, Aphidini). Previous infestation does not impact development time, longevity, and fertility of aphids. However, a previous infestation affects the distribution of the newly produced nymphs on the plant – they actively disperse on the plant, rather than starting their feeding activity where they were originally deposited, as observed in controls. Interestingly, this altered dispersal behaviour is reproduced by saliva application, suggesting the occurrence of an elicitor triggering a plant response affecting the strategy of hostplant colonization by A. gossypii. The hypothesis that salicylic acid (SA) induction can trigger the observed behavioural response in a secondary infestation, was confirmed by exposure to methyl salicylate, a volatile product of the SA pathway. This evidence was further corroborated by analysis of gene expression profiles. Aphid infestation showed a transcriptional up-regulation of genes underlying the biosynthesis of SA and of genes modulating the SA-mediated defence response. Collectively, the experimental data consistently indicate regulation of aphid behaviour, mediated by plant metabolic changes following aphid infestation.
Introduction It has been proposed that herbivore infestations could act as a sort of vaccination against further pest attacks (for an overview, see Karban & Kuc, 1999). A wealth of studies on plant responses to insect herbivory have since been published. In the literature, various types of plant–aphid interactions can be found, characterized in some systems by an induction of resistance (Thackray et al., 1988; Messina et al., 2002; de Vos & Jander, 2009; Sauge et al.,
*Correspondence: Maria Cristina Digilio, Dipartimento di Agraria, Universita degli Studi di Napoli Federico II, Via Universita 100, 80055 Portici, Italy. E-mail:
[email protected]
2012), whereas in other cases the pest may perform better on a stressed plant that has experienced a previous infestation (Jiang, 1996; Prado & Tjallingii, 1997; Gonzales et al., 2002; Takemoto et al., 2013; Cao et al., 2016). Whatever the outcome of this interaction, the underpinning mechanisms are largely mediated by factors released by the feeding aphids through a prolonged and intimate interaction with the plant. During feeding, aphids inject into the plant a large amount of saliva (Miles, 1999) forming, at the beginning of the infestation, a gel that isolates stylets and plant tissue. This structure prevents plant reaction at the feeding site (Felton & Eichenseer, 1999) and facilitates the aphids’ suction of phloem fluids (Rao et al., 2013). In a second step, aphids secrete watery saliva, rich in lytic and digestive enzymes which are able to
© 2018 The Netherlands Entomological Society Entomologia Experimentalis et Applicata 1–9, 2018
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degrade sieve-tube proteins (Will et al., 2007; Furch et al., 2015). After establishing the feeding site, aphids can persist on the plant for hours to weeks. The primary force for stylet penetration and movement is mechanical, but saliva constituents further facilitate this process (Miles, 1999). The limited degree of mechanical damage of tissues caused by phloem-feeding insects influences the plant defence responses. Several studies have now shown that aphids, like other insects, produce and secrete salivary effectors that modulate plant defence responses (Chaudhary et al., 2015; van Bel & Will, 2016; Kettles & Kaloshian, 2016; Mugford et al., 2016; Thorpe et al., 2016; Wang et al., 2016a). In the case of aphids, these effectors are basically perceived through cell membrane receptors, which activate a signal transduction pathway modulated by several cellular messengers, principally ROS, calcium, and stress hormones (Kempema et al., 2007; Kusnierczyk et al., 2008). Jasmonic acid (JA) and salicylic acid (SA) are known to be the two hormones primarily involved in plant defence responses against aphids, although their relative contribution and crossmodulation are still not fully understood. However, several studies suggest a prevailing role of SA at the onset of aphid infestation, which mediates a possible antagonistic cross-talk with JA-signalling pathway and the downstream defence barriers against insects (reviewed in Jaouannet et al., 2014). Cucurbita pepo L. ‘San Pasquale’ (Cucurbitaceae) is a heirloom ecotype of zucchini selected by growers in Campania region (southern Italy), producing a highly appreciated striped and elongated fruit. In this area, zucchini plants suffer heavy attacks in late spring, early summer by the melon aphid, Aphis gossypii Glover (Hemiptera: Aphididae). Here, we study the outcome of the interaction between the zucchini plant and A. gossypii. In particular, we wanted to test whether primary aphid infestation triggers a plant metabolic change that affects a secondary attack by the same aphid species. We investigate both aphid performance and behaviour. We take into consideration not only survival and development, but also a less common parameter such as intra-plant dispersal and aggregation, which is likely controlled by the quality of the feeding substrate, as conditioned by previous feeding. Aphid performance and behaviour were related to the plant transcriptional response following aphid infestation. In addition, we assessed whether the observed aphid–plant interactions were induced by the application of aphid saliva, as this secretion is known to affect a number of symptoms associated with aphid attack (Howe & Jander, 2008). The results provide new interesting information on the molecular interplay between aphids and plants.
Materials and methods Plants and insects
Zucchini plants (cv. San Pasquale) were sown in pots (10 cm diameter) and confined in cages (100 9 20 9 80 cm) made with a wooden frame and anti-insect net (50 mesh). Aphis gossypii was obtained from a field-collected population infesting watermelon in Terracina (Latina, central Italy), and reared on C. pepo in similar cages. Plants and aphids were reared in separate climatic cabinets, at the same environmental conditions (22 1 °C, 75 5% r.h., L16:D8 photoperiod). Aphid infestation assays
Bioassays were designed in order to evaluate the performance and behaviour of A. gossypii after host-plant conditioning by a previous aphid infestation. Zucchini plants had two true leaves at the start of each bioassay (ca. 3 weeks from sowing). Each plant was isolated in an insect breeding cage (30 9 30 9 30 cm) (Vermandel, Sluis, The Netherlands). Aphid development, fertility, and dispersal behaviour (experiment 1). The basal leaf was infested for 48 h by 50 nymphs of A. gossypii, which had attained the third instar; after their removal, a first-instar aphid was placed on the same leaf, and checked daily for survival. Development time and fertility, for 10 days following the onset of reproduction, were measured. The dispersal behaviour was assessed by counting the nymphs that remained fixed on the leaf where they were born or moved towards other feeding sites. The assay was replicated on 10 plants, and 10 controls were set up without previous infestation. Aphid fertility and dispersal behaviour (experiment 2). The basal leaf was infested for 48 h by 50 nymphs of A. gossypii, which had attained the third instar. After nymph removal, a single pre-reproductive adult was placed on the same leaf, and checked daily for survival; the nymphs laid in the following 10 days were counted. The bioassay was replicated on 10 pre-infested plants, and 10 controls (uninfested plants) were set up. Prereproductive adults were synchronized by selecting 0- to 2-h-old newborn nymphs. After 10 days, fertility was measured as the number of nymphs produced by each adult, and dispersal behaviour was assessed by counting the nymphs that remained fixed on the leaf where they were born or moved towards other feeding sites. This assay was replicated twice; 20 plants and 20 prereproductive adults were tested for each experimental condition.
Aphid dispersal behaviour 3
Aphid saliva
In order to assess whether aphid saliva applied to the leaves can reproduce the effect of aphid feeding on the performance and behaviour of A. gossypii, specific bioassays were set up. Saliva was collected in artificial diet (10% sucrose water solution) enclosed in Parafilm sachets, as described by Cooper et al. (2010). Single Parafilm sachets containing 10 ll of diet were mounted in a Petri dish (3.3 cm diameter). Twenty A. gossypii, newly moulted to the adult stage and before the onset of reproduction, were allowed to feed for 24 h at 22 1 °C, 75 5% r.h., and L16:D8 photoperiod. The two layers of Parafilm were carefully separated and the diet with saliva was collected by means of a pipette and frozen (80 °C) in pools of 50 sachets, corresponding to the feeding activity of 1 000 aphids. The controls consisted of diet treated in the same way (i.e., staying 24 h in the sachet, at the same conditions), but in absence of aphids. Preliminary assays showed phytotoxicity when 10% sucrose solution was applied; then, the collected material was diluted by 50%, to a concentration which proved to be safe for plants. The amount of diet applied to leaves in each bioassay was 150 ll, containing the saliva of 150 aphids. Aphid and saliva assays
The basal leaf of zucchini plants was treated with 150 ll of 5% sucrose solution, containing aphid saliva or exposed to the feeding activity of 50 third-instar A. gossypii. After 48 h, the aphids were removed, where present, and a single pre-reproductive adult was placed on the treated leaf, in order to check for fertility and dispersal behaviour of nymphs as described above. The controls consisted of diet (150 ll of 5% sucrose solution) and no treatment, respectively. Each assay was replicated on 10 plants. To assess any possible local or systemic effect of feeding or saliva application, the same experiment was repeated, treating the basal leaf with aphid or saliva, with the difference that the prereproductive adult was placed on the apical leaf. Methyl salicylate assays
Methyl salicylate plant application. To test the hypothesis that methyl salicylate (MeSA) is associated with altered aphid behaviour, and that this effect is plant-mediated, we treated the plant with MeSA (>99% purity, SigmaAldrich, Saint Louis, MO, USA). MeSA was applied as a pure compound, spotted on filter paper (10 ll), in a Perspex cage (4.7 dm3) perfectly sealed and containing a single C. pepo plant, as described in Digilio et al. (2012). After 24 h, the filter paper was removed and the MeSA was allowed to diffuse out of the cage for 20 min. Then, an Eppendorf 1.5-ml tube containing 20 pre-reproductive adult aphids was placed at the base of the plant, so that the
aphids were able to climb the plant and choose a feeding site. Aphid dispersal behaviour was checked at 3, 24, and 48 h and the aphids were assigned to these three categories: ‘remained in the tube’, ‘wandering in the cage’, and ‘feeding on the plant’. Overall, 18 (6 9 3) replicates were performed for each of MeSA and control. Methyl salicylate exposure in the absence of plant. MeSA was used as described above, but in the absence of a plant in the Perspex cage, in order to assess its direct effect on aphids, without the mediation of plant-produced MeSA. The support for the aphids was a Parafilm sachet containing 10 ll of 10% sucrose/water solution. Twenty synchronous adult aphids, before the onset of reproduction, were distributed on each sachet in the vicinity of the diet drop, and 10 ll MeSA was spotted on filter paper inside the cage, which was closed immediately. Six cages per treatment were set up, and three replicates were made. Aphid dispersal behaviour from the diet was checked at 1 and 3 h. Overall, 18 (6 9 3) replicates were performed for each of MeSA and control treatment. RNA isolation and real-time RT-PCR
As the application of MeSA to the plant had proven effective in altering aphid behaviour, we performed a study of the expression profile of genes involved in salicylic acid biosynthesis or responsive to SA. Fully expanded leaves on which 10 young adult aphids had fed for 2 days were cut and frozen immediately in liquid nitrogen, in order to extract high-quality RNA. Total RNA was prepared from leaves by a phenol/chloroform extraction and a lithium chloride precipitation. A second selective RNA precipitation was performed with 0.1 volume of 3M sodium acetate pH 7.2 and 1 volume of 96% ethanol. RNA concentration was calculated by measuring absorbance using Nanodrop (ThermoScientific, Waltham, MA, USA), whereas integrity control was assessed by electrophoresis on a 1.2% agarose gel. DNA contamination was eliminated by treatment with 1 U DNAse I Amplification Grade (Life Technologies, Carlsbad, CA, USA), and first strand cDNA synthesis was performed using SuperScript II Reverse TranscriptaseTM (Life Technologies), according to manufacturer’s protocol. The amplification of the cDNA region coding for EF-1a gene, a ubiquitously expressed gene (Shewmaker et al., 1990), was performed as control of cDNA synthesis. Real-time RT-PCR was performed using Corbett Rotor Gene 6000 (Corbett Research, Hilden, Germany). Amplifications were carried out with two technical and three biological replicates. The thermal cycling program started with a step of 10 min at 95 °C, followed by 45 cycles of a 30 s step at 95 °C, 30 s at Ta temperature, 15 s at 72 °C, followed by a dissociation kinetic analysis to
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assess the specificity of amplification reaction. Primers, designed with Primer Express 2.0 software (Applied Biosystem, Foster City, CA, USA) were chosen to amplify a fragment of ca. 100 bp. Relative quantification of gene expression was carried out using the 2DDCt method (Livak & Schmittgen, 2001). The housekeeping gene EF1a was used as an endogenous reference gene for the normalization of the expression levels of the target genes. Briefly, the average Ct was calculated for target and endogenous reference genes and the ΔCt (Ct target – Ct ref) values were determined. ΔΔCt values for each gene were obtained using samples from uninfested leaves as calibrator: DDCt ¼ DCt test DCt calibrator: Relative quantification values were obtained as 2ΔΔCt (Livak & Schmittgen, 2001). The selected genes were: • Isochorismate synthase 1 (ICS1) (AT1G74710.2) which codes for an enzyme responsible of the chorismate conversion to isochorismate within the plastidial biosynthesis of SA (Loake & Grant, 2007), • 4-coumarate–CoA ligase 1 (4CL1) (AT1G51680.1) which codes for an enzyme responsible of the conversion of 4-cinnamic acid in 4-coumaroyl-CoA within phenylpropanoid biosynthesis (Dempsey et al., 2011; Wang et al., 2016b), • Pathogenesis-related thaumatin-like protein 1b (PR51b) and thaumatin-like protein (PR5) (AT4G38660.1, AT1G18250.2) which are induced by SA (Kusnierczyk et al., 2008), • Glucan endo-1,3-b-glucosidase 12 (PR-2.12) and glucan endo-1,3-b-glucosidase 7 (PR-2.7) (AT2G05790.1, AT4G34480.1) which are SA-induced genes involved in the cell wall degradation (van Loon et al., 2006), and • Nudix hydrolase 8 (NUDX8) (AT5G47240.1), a gene coding for an enzyme with hydrolase activity involved in plant immunity and SA signalling (Fonseca & Dong, 2014).
Results Aphid infestation assays
Development time from first instar to the adult stage was 12 days, on both previously infested and control plants. All the adults started reproduction by 12 h since last moult. In both experiments 1 and 2, differing in the starting instar – first instar vs. adult – no significant difference was obtained in the mean numbers of nymphs deposited on previously infested vs. non-infested leaves (Table S2). Previous infestation elicited dispersal behaviour in newly laid nymphs (Table 1). In both experiments, when transferred on previously infested leaves, about half of the new nymphs were found distributed on various parts of the plant, such as the other leaf, the cotyledons, and the apical sprout (details in Table S3), whereas on control plants the newborn nymphs remained near their mothers. Aphid and saliva assays
In these bioassays, an adult aphid was placed on a salivatreated zucchini leaf. As observed for aphid feeding, the application of saliva to the leaf had no impact on development time and fertility (Table S4), whereas the saliva treatment triggered a distribution of nymphs on the plant similar to that induced by aphid infestation: only 25% of the nymphs remained on the same leaf near their mothers, both in response to aphid feeding and in response to saliva application (Tables 2 and S5). Similarly, when the treated leaf is different from the leaf on which the adult aphid is placed, there is no impact on fertility (Table S6), whereas dispersal behaviour occurs after saliva application and after aphid feeding (Tables 2 and S7). Methyl salicylate assays
Methyl salicylate plant application. Treatment of plants with MeSA resulted in an alteration of aphid dispersal behaviour. A significant difference (Table 3) was evident Table 1 Percentage of Aphis gossypii offspring nymphs (n = 10) that stay on a zucchini leaf over a period of 10 days. The leaf was first infested for 48 h by 50 third instars; after their removal the trial was started by either a first-instar nymph (experiment 1) or a pre-reproductive adult (exp. 2). The control was not infested first
Primers and their main features are listed in Table S1. Distribution of nymphs (%)
Statistical analysis
Fertility data were compared by one-way ANOVA, by means of STATGRAPHICS Plus software (Manugistics, Rockville, MD, USA). The data of distribution of aphids on the different parts of the plant were analysed by G-test of independence (McDonald, 2014). Differences in relative quantities of defence transcripts were analysed by comparing DCt values by a two-tailed t-test.
Starting instar
Aphid feeding
Control
G1 (d.f. = 1)
P
First instar Young adult
49.36 45.81
97.25 97.27
321.71 365.11
6.1E-72 2.2E-81
1
G-test of independence.
Aphid dispersal behaviour 5
Table 2 Percentage of Aphis gossypii offspring nymphs (n = 10) that stay on the leaf where their mother was placed as a pre-reproductive adult. Before the trial, the basal leaf was either uninfested and treated with diet with or without aphid saliva, or it was infested for 48 h by 50 third instars. The basal leaf received the treatment and subsequently (A) the basal leaf received the young adult, or (B) the apical leaf received the young adult Distribution of nymphs (%)
A B
Diet + saliva
Diet
Aphid feeding
No treatment
G1 (d.f. = 3)
P
25.07a 25.38a
96.41b 95.11b
26.61a 24.54a
97.88b 98.09b
839.61 816.18
1.1E-181 1.3E-176
Percentages within a row followed by different letters are significantly different (P
