1. Introduction
Hesperidin (3,5,7-trihydroxyflavanone 7-rhamnoglucoside) (
Figure 1) belongs to the diverse group of flavonoids. In general, flavonoids occur in most plants and are involved in all kinds of ecological interactions, mainly in defenses against various abiotic and biotic stresses, but also as infochemicals in extraorganismal plant signaling [
1,
2,
3,
4,
5]. Flavonoids are present in various tissues, cells, and sub-cellular compartments [
3] and their biological functions in plants include the defense against UV-B radiation and pathogen infection, nodulation, and pollen fertility [
6]. The roles of many flavonoids in plant–herbivore relationships have been well documented [
1,
7,
8,
9,
10]. These plant metabolites can have negative effects on non-adapted herbivores, may reduce the nutritive value of the food, or may act as feeding deterrents or toxins [
11,
12,
13]. The flavonoid mode of action on microorganisms and insects probably arises from an interference with important cellular processes and structures, yet this is not fully understood [
14]. In view of these activities, flavonoids have been considered as prospective biopesticides [
14,
15,
16].
Hesperidin occurs mainly in the flavedo, albedo, segment membranes and juice sacs of the fruits of plants of the genus
Citrus (Rutaceae) [
17,
18], but it has also been recorded from Fabaceae, Betulaceae, and Lamiaceae [
19,
20,
21]. Hesperidin has attracted a lot of attention as one of the most interesting and promising bioflavonoids for application in traditional medicines and as a combination product [
20,
21,
22]. It is safe in application to humans and without side effects even during pregnancy [
21]. Various pharmacological activities of hesperidin have been reported, including antioxidant, antibacterial, antimicrobial, antiviral, anti-inflammatory, and anticarcinogenic properties [
20,
22,
23].
In contrast, the roles of hesperidin in ecological interactions and its effects on herbivores in particular have rarely been explored and reported. At the molecular level, the available reports provide information that hesperidin alleviates oxidative stress in
Drosophila melanogaster Meigen (Diptera: Drosophilidae) caused by the groundwater pollutant trichloroethylene [
24]. At the organismal level, the proven activities of hesperidin in plant–herbivore interactions include its roles as host plant recognition cue and oviposition stimulant for
Papilio protenor Cramer and
P. xuthus L. (Lepidoptera: Papilionidae) [
1,
7,
25], defense chemical in
Citrus x
sinensis (L.) Osbeck against
Xylella fastidiosa Wells et al. (Bacteria: Xanthomonadaceae) [
26],
Phytophthora citrophthora (R.E. Sm. and E.H. Sm.) Leonian (Oomycetes: Peronosporaceae) and
Candidatus liberibacter Fagen et al. (Bacteria: Rhizobiaceae) [
27,
28], and in
C. aurantium L. against
Aphis punicae Passerini (Hemiptera: Aphididae) and
Planococcus citri Risso (Hemiptera: Pseudococcidae) [
29]. Hesperidin, applied as hesperidin-Mg complex, showed insecticidal activity against
Spodoptera frugiperda (Lepidoptera: Noctuidae) and
Bemisia tabaci (Hemiptera: Aleyrodidae) [
30,
31]. In the same study [
30], the repellent activity of the hesperidin-Mg complex towards
Myzus persicae (Hemiptera: Aphididae) was reported with the reservation that it required further evaluation. Hesperidin was also reported as a weak antifeedant when offered to aphids (Hemiptera: Aphididae)
M. persicae and
Schizaphis graminum in artificial diets [
32]. The practical lack of knowledge on the effect of hesperidin on insect herbivores is surprising: the data are fragmentary and refer mainly to herbivores associated with citrus plants. Hesperidin is a rather unique flavonoid, the natural occurrence of which is limited to a narrow range of plant species. As such, it should be considered as a prospective herbivore-limiting factor, especially in relation to monophagous and oligophagous insects that do not use this flavonoid as the host plant recognition cue.
The aim of the present study was to investigate in detail the effect of hesperidin on host plant selection behavior of three species of aphids that vary in host plant specialization that determines their sensitivities to plant allelochemicals. The species studied were the oligophagous pea aphid Acyrthosiphon pisum Harris, which is the non-host alternating specialist on Fabaceae, the bird cherry-oat aphid Rhopalosiphum padi (L.), which is the host-alternating oligophagous specialist on Poaceae, and the highly polyphagous green peach aphid Myzus persicae (Sulz.). We hypothesized that hesperidin, absent in their preferred host plants, may alter the ability of aphids to recognize and accept these plants as hosts. We concentrated on behavioral aspects of aphid host plant selection process, specifically on aphid probing and settling behaviors. Our studies were carried out under semi-natural conditions in no-choice and choice situations. In the no-choice experiment, tethered aphids were offered their preferred host plants untreated and treated with hesperidin. Then, the aphid stylets’ movements in plant tissues were monitored using the technique of electropenetrography (Electrical Penetration Graph, EPG). This experiment was to reveal whether hesperidin had any deterrent influence on individual phases of probing in specific plant tissues. In the choice experiment, the freely moving aphids could choose between hesperidin-treated and untreated plant leaves. This experiment was designed to establish the potency and durability of hesperidin antifeedant activity.
3. Discussion
Secondary plant compounds can adversely affect three major phases in insect activities associated with feeding: the pre-ingestive, ingestive, and post-ingestive phases [
33]. The impact of plant allelochemicals during the pre-ingestive phase is associated with host finding and host selection processes and involves gustatory receptors, while during the ingestive phase, the effects are related to the transport of food as well as the release and digestion by salivary enzymes. The post-ingestive effects are usually delayed in time and refer to various aspects of digestion and absorption of food. The presented classification of deterrent effects of allelochemicals was originally established for insect herbivores that consume plant material using their chewing mouthparts equipped with chemosensillae [
33]. Aphids are plant sap-consuming herbivores with specialized sucking-piercing mouthparts that lack external contact chemoreceptors; the gustatory organ is located in the hypopharynx [
34]. Henceforth, the pre-ingestive and ingestive phases of host plant selection by aphids require the insertion of the mouthparts’ stylets into plant tissues. Consequently, this activity and the possible immediate responses of aphids to plant allelochemicals are hidden from the human eye. Electropenetrography is the technique which allows an insight into pre-ingestive and ingestive aphid behaviors [
35,
36]. The parameters describing aphid behavior during probing such as the total time of probing, the duration and frequency of phloem sap ingestion events, the number of probes, etc., are good indicators of plant suitability or the interference in probing by chemical or physical factors present in individual plant tissues or administered exogenously on plant surface [
37]. The post-ingestive effects of plant allelochemicals on aphid foraging behavior can be determined by monitoring aphid probing and preferences of the free-moving aphids in settling on plants [
38].
The present study showed that the pre-probing behavior was similar in all aphids: all aphids initiated probing shortly after gaining access to experimental plants. This finding is consistent with previous reports on aphid predisposition to probe in any substrate, provided that no deterrent constituents are present on the surface [
39]. Significant alterations in the foraging behavior occurred after the aphids inserted stylets into plant leaves treated with 0.1% and/or 0.5% ethanolic solutions of hesperidin.
The modification of aphid behavior during the pre-ingestive phase concerned different aspects of probing in non-vascular tissues epidermis and mesophyll. In the case of
A. pisum, the time to cross the barrier of epidermis and mesophyll to reach phloem vessels and start sustained feeding was significantly longer on hesperidin-treated
P. sativum. This was caused by the discontinuation of the relatively long probes, i.e., probes including pathway longer than 3 min, which means that aphids reached beyond epidermis before the stylets were withdrawn [
40]. At the same time, the pea aphids spent more time on no probing activities on hesperidin-treated plants than on control. Nevertheless, the probes were repeated and finally, most of the aphids reached phloem on all plants. The successful probes—those that ended in the sieve elements—included the pathway of similar duration on treated and untreated plants. No individual of
A. pisum showed activity ‘F’ on any plant, which reflects the lack of difficulties in mechanical work of the stylets in the apoplast [
35]. No effects of hesperidin concentration on the pea aphid probing in non-vascular tissues were observed. In
R. padi, statistical analysis did not detect significant differences in the time to reach sieve elements from the onset of probing, but a trend towards an increase in the duration of the pre-phloem period occurred in aphids on hesperidin-treated
A. sativa. Nevertheless, the proportion of aphids that reached sieve elements was reduced on hesperidin-treated plants as compared to control. Evident differences were recorded when analyzing the duration of the pathway that directly preceded the first contact with sieve elements within a probe: it was twice as long on 0.1% hesperidin-treated plants, but similar on 0.5% hesperidin-treated plants in respect to control. The incidence of activity ‘F’ in apoplast was the highest on control and the lowest on 0.5% hesperidin-treated plants. In
M. persicae, no differences in the probing behavior occurred during the pre-phloem phase, except the frequency of activity ‘F’, which was the highest on control and the lowest on 0.5% hesperidin-treated plants. The pathway activity comprises extracellular movements of stylets and brief punctures of cells adjacent to stylet route for gustatory purposes [
35,
41]. It may be stated that the more frequent events of termination of pathway probes on hesperidin-treated plants in relation to control were caused by the deterrent properties of this flavonoid. Hesperidin might have been detected in the sap samples acquired by aphids during the pathway cell punctures, as it has been found in the cases of various exogenously applied allelochemicals [
36,
37,
38].
The ingestive phase in aphid probing embraces the uptake of sap from phloem and/or xylem vessels [
35]. The uptake of the phloem sap is always preceded by a shorter or longer bout of watery saliva secretion into the sieve element [
42]. The role of the watery saliva is to prepare and adjust the sieve element for aphid feeding by blocking or eliminating plant defense mechanisms [
43]. The duration of salivation and the contribution of this activity to the phloem phase reflects the potency of plant defense factors located in the phloem [
42,
43,
44]. In
A. pisum, the contribution of salivation to the phloem phase was the lowest on control
P. sativum and the highest on 0.5% hesperidin-treated plants, while in
R. padi and
M. persicae, no differences among treatments occurred. In
A. pisum and
R. padi, the total and mean durations and the contribution of sap ingestion activity to all probing activities were twice as low on hesperidin-treated plants than on control, and no effect of hesperidin concentration was recorded. In
M. persicae, no significant differences related to sap ingestion activities occurred among treatments.
While the ingestion of the nutrient-rich phloem sap is actually the feeding activity and reflects plant acceptance, the ingestion of the xylem sap that contains mostly water occurs usually under stress and probably reflects the inability to use phloem resources due to the presence of negative factors in the plant at the stage before the aphids reach the phloem [
44]. In
A. pisum,
R. padi, and
M. persicae, the xylem sap ingestion activity was the most frequent in aphids on 0.1% hesperidin-treated plants. However, the duration of individual bouts and the contribution to the total probing activities differed among aphid species, and in some cases, these variables were hesperidin concentration-dependent. In
A. pisum, the duration of individual bouts of xylem phase was extended in aphids on 0.1% and on 0.5% hesperidin-treated plants, while in
R. padi and
M. persicae, the bouts of xylem sap ingestion were longer on hesperidin-treated plants, but no effect of hesperidin concentration was observed.
The behavior of aphids during the post-ingestive phase reflects the level of suitability of a plant for feeding, settling, and reproduction [
33]. On suitable hosts, aphids may continue phloem sap ingestion for many hours without interruption, while on less accepted plants or on non-hosts, the bouts of phloem sap ingestion are relatively short and interrupted by periods of pathway activities within the same probe, or even by periods of no probing which follow the withdrawal of the stylets [
45]. The EPG experiment in the present study showed that the proportion of time spent on phloem sap ingestion after the beginning of the first bout of sustained sap ingestion (the ‘potential E2 index’) changed depending on the treatment with hesperidin and was aphid species-dependent. In
A. pisum and
R. padi, the values of pE2 index were reduced on hesperidin-treated plants, while in
M. persicae, it was not the case. In
A. pisum and
R. padi, no hesperidin concentration-effect was observed. The free-choice experiment showed noticeable differences in response to hesperidin treatments among aphid species. Significant differences in preference to settle on untreated plants were recorded 24 h after treatment with 0.1% hesperidin in
A. pisum and 1 and 2 h in
R. padi. In
M. persicae, the deterrent effects of both 0.1% and 0.5% hesperidin were recorded 1 h, 2 h, and 24 h after aphids gained access to plants. The avoidance of the treated leaves during settling might have been the delayed effect of consuming the toxic sap from hesperidin-treated leaves, as the ingestion of the phloem sap was not obstructed. This explanation, though, needs further study.
In summary, the results of the present study indicate that hesperidin can be ascribed to all three functional groups of feeding deterrents, respectively, the pre-ingestive, ingestive, and the post-ingestive groups, depending on aphid species and the applied concentration. Hesperidin can be applied as a pre-ingestive, ingestive, and post-ingestive deterrent against A. pisum, as an ingestive deterrent against R. padi, and as a post-ingestive deterrent against M. persicae. In all cases, hesperidin can be applied at a relatively low 0.1% concentration, as an increase in the amount of hesperidin did not evoke significantly stronger effects on aphid probing behavior as compared to 0.1% concentration. The results of the present study also demonstrate that the oligophagous A. pisum was the most sensitive to the application of hesperidin, and the polyphagous M. persicae was the least sensitive. While in A. pisum the deterrent effects of hesperidin were manifested as early as during aphid probing in peripheral plant tissues, in M. persicae, the avoidance of plants was probably the consequence of consuming the hesperidin-containing phloem sap.
4. Materials and Methods
4.1. Cultures of Plants and Aphids
Laboratory clones of Acyrthosiphon pisum, Myzus persicae, and Rhopalosiphum padi were maintained on Pisum sativum cv. Milwa (Hodowla Roślin Smolice Sp. z o.o. Grupa IHAR, Smolice 146, 63-740 Kobylin, Poland), Brassica rapa ssp. pekinensis cv. Hilton (World of Flowers Sp. z o.o., ul. Sulejkowska 56/58, 215, 04-157 Warszawa, Poland), and Avena sativa cv. Komfort (Hodowla Roślin Strzelce Sp. z o.o. Grupa IHAR, ul. Główna 20, 99-307 Strzelce, Poland), respectively, in the laboratory at 20 °C, 65% r.h., and L16:D8 photoperiod. Aphid clones have been maintained in the laboratory of Department of Botany and Ecology, University of Zielona Góra, Poland for at least 10 years. One- to seven-day old apterous aphid females and three-week-old plants were used for the experiments. Plants used for experiments were the same plant species and cultivars that were used for the rearing of aphids. All experiments were carried out under the same conditions of temperature, relative humidity, and photoperiod. The bioassays were started at 10–11 a.m.
4.2. Application of Hesperidin
Hesperidin (≥80% HPLC) was purchased from Sigma–Aldrich (Poznań, Poland). The flavonoid was dissolved in 70% ethanol to obtain 0.1% and 0.5% solutions. For the aphid probing behavior experiment (no-choice test), hesperidin was applied on the adaxial and abaxial leaf surfaces by immersing one leaf of an intact plant in the ethanolic solution of a given concentration for 30 s. Control leaves of similar size on the control intact plants were immersed in 70% ethanol that was used as a solvent for the studied compound. For the aphid settling success experiment (choice-test), hesperidin was applied on the adaxial and abaxial leaf surfaces by immersing the cut leaves in the ethanolic solution of a given concentration for 30 s. Control leaves of similar size were immersed in 70% ethanol.
All experiments were performed 1 h after the compound application to allow for the evaporation of the solvent.
4.3. Aphid Probing Behavior (No-Choice Experiment)
Aphid probing (aphid stylet penetration in plant tissues) was monitored using the electronic penetration graph technique (electropenetrography) known as EPG, which is frequently employed in insect–plant relationship studies considering insects with sucking-piercing mouthparts. In this experimental setup, aphids and plants are parts of an electric circuit, which is completed when the aphid inserts its stylets into the plant. Weak voltage is supplied in the circuit, and all changing electric properties are recorded as EPG waveforms that can be correlated with aphid activities and stylet position in plant tissues. In the present study, aphids were attached to a golden wire electrode with conductive silver paint and starved for 1 h prior to the experiment. Probing behavior of 20 apterous females per studied flavonoid concentration/aphid combination was monitored for 8 h continuously with four-channel DC EPG recording equipment. Each aphid was given access to a freshly prepared plant leaf of an intact plant. Each plant–aphid set was considered as a replication and was tested only once. The number of replications (EPG recordings) for each plant treatment was 24. Recordings that terminated due to aphid falling from the plant or where EPG signal was unclear were discarded from analysis. Only the replications that included complete 8 h recordings were kept for analysis. All experiments were carried out under the same conditions of temperature, relative humidity (r.h.), and photoperiod as those used for the rearing of plants and aphids. All bioassays started at 10:00–11:00 h MEST (Middle European Summer Time).
Signals were saved on the computer and analyzed using the PROBE 3.1 software provided by W.F. Tjallingii (
www.epgsystems.eu, accessed on 20 August 2022; Wageningen 6703 CJ, The Netherlands). The following aphid behaviors were distinguished: no penetration (waveform ‘np’—aphid stylets outside the plant), pathway phase-penetration of non-phloem tissues (waveforms ‘ABC’), derailed stylet movements (waveform ‘F’), salivation into sieve elements (waveform ‘E1’), ingestion of phloem sap (waveform ‘E2’), and ingestion of xylem sap (waveform ‘G’). The E1/E2 transition pattern was split in two between E1 and E2. The waveform patterns that were not terminated before the end of the experimental period (8 h) were included in the calculations. All variables were processed using the EPG Excel Data Workbook produced by Sarria et al. [
46]. The parameters derived from EPGs were analyzed according to their frequency and duration in a configuration related to activities in peripheral and vascular tissues. In non-sequential parameters, when a given waveform had not been recorded for an individual, the duration of that waveform was given the value of 0. In sequential parameters, when parameters related to phloem phase (E1 or E2) were involved, only aphids that reached phloem phase were included in the statistical analysis.
4.4. Aphid Settling Success (Choice-Experiment)
Aphids settle on a plant only when they accept it as a food source [
47]. Therefore, the number of aphids that settle and feed on a given substrate is a good indicator of its suitability. This bioassay allows studying aphid host preferences under semi-natural conditions. Aphids are given free choice between control and treated leaves. In the present study, aphids were placed in the Petri dish along the line that divided the arena into two halves so that aphids could choose between treated (on one half of a Petri dish) and control leaves (on the other half of the dish). Aphids that settled, i.e., they did not move and the position of their antennae indicated feeding [
48] on each leaf were counted at 1 h, 2 h, and 24 h intervals after access to the leaves (8 replicates, 20 viviparous apterous females/replicate). Aphids that did not settle on any of the leaves were discarded from calculations.
4.5. Statistical Analysis
EPG parameters describing aphid probing behavior (no-choice test) were calculated manually and individually for every aphid, and the mean and standard errors were subsequently calculated using the EPG analysis Excel worksheet created for this study. The results were statistically analyzed using ANOVA (Statistica 13.3 package) [
13]. Fisher’s least significant differences (LSDs) were estimated at the 0.05 significance level to identify significant differences between individual traits. Homogeneous groups were designated based on these LSD values. The data deriving from the choice-test for freely moving aphids (aphid settling deterrent activity) were analyzed using Student’s
t-test. If aphids showed clear preference for the leaf treated with the tested compound (
p < 0.05), the compound was described as having attractant properties. If aphids settled mainly on the control leaf (
p < 0.05), the compound tested in the respective choice-test was stated a deterrent. The relative index of deterrence (DI) was calculated according to the formula DI = (C − T)/(C + T), where C is the number of aphids that remained on control leaf, and T is the number of aphids that remained on the treated leaf. The value of DI ranged between “+1” (ideal deterrent) and “−1” (ideal attractant) [
38].