Introduction

Plants produce a variety of compounds that provide certain protection against insect attack (Argandona et al. 1983; Leszczynski et al. 1989, 2003; Felton and Gatehouse 1996; Ridsdill-Smith et al. 2004). Many of these compounds affect aphid behavior, physiology, and metabolism, and, as a result, can reduce aphid populations on resistant plants. Saponins also have been suggested as possible chemical defensive agents of plants against generalist herbivores (Oleszek et al. 1990; Nozzolillo et al. 1997; Osbourn 2003).

Saponins are widely distributed secondary plant metabolites and occur among ca 100 plant families (Oleszek 2002). They can be classified into two groups based on the nature of their aglycone structure. The first group consists of steroidal saponins; the second is composed of triterpenoid saponins. Some authors also distinguish a third group, the so-called steroidal amines that can be classified as steroidal alkaloids (Bruneton 1995). Steroidal saponins consist of a steroidal aglycone, a C27 spirostane, generally composed of a six-ring structure (Fig. 1a). Within fresh plant tissues, the hydroxyl group in the 26-position participates in a glycosidic linkage, and the aglycone structure remains as pentacyclic furostane (Fig. 1b). Triterpenoid saponins consist of a triterpenoid aglycone composed of a C30 pentacyclic structure (Fig. 1c; Sparg et al. 2004).

Fig. 1
figure 1

Aglycone frames of saponins: a steroidal spirostane; b steroidal furostane; and c triterpenoid saponins. R Sugar moiety

Full size image

Saponin concentrations in different alfalfa varieties have been reported and range from 0.8–2% (Pedersen and Wang 1971; Majak et al. 1980). Pecetti et al. (2006) also showed saponin differences among alfalfa cultivars with compounds such as medicagenic acid and zanhic acid.

Saponins are toxic to herbivorous insects (Ishaaya et al. 1969; Shany et al. 1970; Oleszek et al. 1992), are reported to be important resistance factors to the pea aphid, and have been suggested a potentially useful for alfalfa resistance breeding programs (Horber et al. 1974). High saponin content in some alfalfa cultivars is related to resistance against the pea aphid, Acyrthosiphon pisum (Pedersen et al. 1976). Saponins have also been shown to interfere with aphid feeding behavior (Golawska et al. 2006).

In this paper, the effects of isolated alfalfa saponins on pea aphid feeding behavior are examined in detail. Three major alfalfa saponins—(1) 3GlcA, 28AraRhaXyl medicagenic acid glycoside, (2) 3GlcA, 28AraRha medicagenic acid glycoside, and (3) zanhic acid tridesmoside—were used in in vitro experiments. Saponin effects on aphid probing of the plant peripheral tissues and phloem phase were studied.

Methods and Materials

Aphid Culture

The pea aphid, A. pisum Harris, wingless females were used in all feeding behavior experiments and came from a stock culture kept at the University of Podlasie, Siedlce, Poland. Aphids were collected from a laboratory culture reared on broad been (Vicia faba L. var. Start) and maintained in an environmental chamber at about 21°C, L16:D8 photoperiod, and 70% RH.

Chemicals

Individual saponins were obtained from the Institute of Soil Science and Plant Cultivation, Pulawy, Poland. Compounds 1, 2 and 3 (Fig. 2) were isolated from alfalfa as described by Oleszek et al. (1990).

Fig. 2
figure 2

Structures of compounds 1–3

Full size image

Application of Tested Compounds

The effect of saponins on pea aphid feeding behavior was investigated in vitro, using sucrose–agarose gels. Control gels (without saponins) were prepared by incorporating 1.25% agarose (Sigma A-0169) into 30% sucrose solution. Experimental gels (containing saponins) were prepared by incorporating individual saponins at concentrations of 50 or 100 ppm. After stirring, the mixtures were heated in a water bath (75°C for 30 min) and then poured into plastic rings covered by a stretched Parafilm M® membrane. Transparent gels formed after 1–2 min and offered to aphids for probing.

Electrical Penetration Graph Recordings

The probing behavior of adult apterous aphids was recorded using a DC electrical penetration graph (EPG) amplifier (type Giga-4). Aphids were connected to the EPG system by a 2-cm gold wire (20 μm in diameter) and attached with conductive silver point (Demetron, L2027, Darmstadt, Germany). A second electrode was introduced into the gel. EPG recordings were performed inside a Faraday cage under laboratory conditions (ca 24°C for 4 hr). Collected insects were starved for 2 hr and then placed onto gels. Pea aphid probing behavior was recorded as follows: One adult apterous aphid was placed onto each gel and EPG recordings were made for 10 aphids on 10 different gels without saponins (control) and 10 gels for each concentration of the tested compounds. Aphid feeding behavior was monitored for 4 hr.

EPG Analysis

Acquisition and analysis of EPG signals were done with STYLET 2.2 software (ref). Waveform patterns were identified according to Tjallingii (1990). Insect feeding behavior was based on aphid activities and noted as follows: non-probing (np pattern, aphids cannot start probing), path (C pattern—pathway; penetration of peripheral tissues—epidermis and mesophyll), salivation into sieve elements (E1 pattern), ingestion of phloem sap (E2 pattern—aphid feeding), and ingestion of xylem sap (G pattern). EPG parameters were measured in each group and recalculated per one insect. Data obtained with experimental and control insects were subjected to one-way analysis of variance followed by Duncan’s test.

Results

Saponins clearly affect feeding behavior of A. pisum. EPG recordings indicated that the pea aphid behaved in a variable manner while probing artificial diets containing test compounds. On each occasion, aphid-probing activity was composed of major EPG patterns: pathways (C), sieve element salivation (E1), phloem sap ingestion (E2), and xylem sap ingestion (G) (Table 1). Among the saponins studied, 2 and 3 were more effective modulators of probing behavior than 1. The total number of gel penetrations by the pea aphids and duration of the pathways were reduced by higher concentrations of compounds (Table 2). Compounds 1 (at 100 ppm), 2 (at both 50 and 100 ppm), and 3 (at 50 ppm) reduced the number of gel penetrations by approximately half. At 100 ppm, compound 3 decreased the number of penetrations by four times (Table 2). Saponins also prolonged the first probe by experimental aphids compared to controls—about twice the duration for saponins 1 and 2 at concentrations of 50 and 100 ppm, and over seven times in the case of saponin 3 at 100 ppm (Table 2). For all tested saponins, reduction in average duration of probing activity in relation to control gels was observed (Table 2).

Table 1 Probing behavior of A. pisum on artificial diets
Full size table
Table 2 Effect of different concentrations of saponins on probing behavior of A. pisum
Full size table

A similar tendency was found for aphid salivation into sieve elements and phloem sap ingestion (Table 3). At a concentration of 50 ppm, all saponins reduced duration until the first E1 pattern and for 3GlcA, 28AraRhaXyl medicagenic acid up to seven times. Prolongation of such aphid activity was observed for 1 and 2 at a concentration of 100 ppm and 3 at 50 ppm. Generally, the tested concentrations of 2 and 3 reduced total time of pea aphid salivation into the gels (Table 3). Saponins also modified the duration of the first phloem sap ingestion (Table 3). In addition, the total time of this aphid activity was prolonged in the presence of all 3 saponins at 50 ppm. Higher concentrations (100 ppm) reduced the duration of phloem sap ingestion except for 1 (Table 3).

Table 3 Effect of different concentrations of saponins on feeding behavior of A. pisum at phloem phase during probing of artificial diets
Full size table

Compound 1 did not affect xylem sap ingestion at the tested concentrations (Table 4). When 2 was added to gels, pea aphid activity corresponding to xylem sap ingestion did not occur (Table 4). At the lower concentration, compound 3 reduced this phase of aphid feeding behavior and at the higher concentration completely stopped this activity.

Table 4 Effect of different concentrations of saponins on feeding behavior of A. pisum at xylem phase during probing of artificial diets
Full size table

Discussion

The EPG recordings of pea aphid probing of sucrose–agarose gels containing different concentrations of saponins showed clear differences in feeding behavior. Generally, higher concentrations caused reduction of aphid activities that corresponded to ingestion of phloem and xylem sap. Similar findings have been reported previously for saponins isolated from seeds of Barringtonia asiatica in relation to Epilachna sp. larvae (Herlt et al. 2002). EPG results indicated moreover that the pea aphid avoided ingesting saponins. The concentrations of saponins used in this study corresponded to levels within a low-saponin line of alfalfa and were lower than those found in high-saponin lines of the Radius cultivar (Staszewski et al. 1994). Thus, the antibiotic effect of alfalfa saponins on pea aphid might be even stronger in a high-saponin line.

Synergism among the tested saponins was not studied in these experiments; however, synergistic interactions among saponins may occur within alfalfa tissues. Horber et al. (1974) and Adel et al. (2000) reported that saponins may act synergistically. Moreover, they may also act synergistically with other groups of alfalfa allelochemicals as reported for other plants and insects (Sutherland et al. 1982; Berenbaum 1985).

The results here obtained did not confirm earlier reports by Oleszek et al. (1992). The tested compounds may be antifeedant compounds for pea aphid—Fabaceae relationships. Szynkarczyk et al. (2000) showed that A. pisum fed on high-saponin alfalfa lines reduced pea aphid performance and phloem sap ingestion. Golawska et al. (2006) further showed differences in pea aphid feeding behavior on alfalfa with low and high saponin content. Aphids fed on high-saponin lines had prolonged penetration of the epidermis and mesophyll (pattern C) and showed a significant reduction in phloem sap ingestion. On the other hand, negative effects of saponins on herbivore performance, e.g., reduction of growth and pupal mass, could be a consequence of shortening or suppressing the feeding process. Thus, the results presented here confirm that alfalfa saponins are natural feeding barriers for phytophagous insects (Sutherland et al. 1982; Meisner and Mitchell 1983; Potter and Kimmerer 1989; Jain and Tripathi 1991; Nozzolillo et al. 1997). Agrell et al. (2003) demonstrated that alfalfa showed an herbivore-induced resistance based on saponins and that such induction affected feeding behavior and exerted negative effects on herbivore physiology. We documented that saponin 3 is important in chemical interactions between host-plants and herbivores. Moreover, a similar reduction in insect food consumption by saponins has been reported by Adel et al. (2000). They were the major cause of diminished body growth and possibly extension of the feeding period. Alfalfa saponins administered to larval Spodoptera littoralis through diet caused prolongation of both larval and pupal stages, retarded growth, increased mortality, and reduced fecundity and fertility. There are data also that indicate that saponins slow the passage of food through the gut, perhaps by reducing digestibility, and may secondarily influence food uptake. Inhibition of digestive enzymes (Ishaaya and Birk 1965) and interference with sterol metabolism (Ishaaya et al. 1969; Shany et al. 1970) also may be involved with the effects of alfalfa saponins on food processing in S. littoralis. In addition, extension of pupal instars and reduction of adult fertility have been reported by Hubrecht et al. (1989). Szynkarczyk et al. (2001) and Golawska et al. (2005) found a negative relationship between saponin concentration and development of pea aphids on alfalfa lines. Horber (1972) used commercial saponins isolated from alfalfa and found that even low concentrations may exert an adverse effect on the pea aphid. Finally, there are reports of the high toxicity of saponins to other insects (Bondi and Birk 1968; Applebaum et al. 1969; Thorp and Briggs 1972; Sutherland et al. 1975a, b).

The current studies suggest that saponins 1, 2, and 3 are toxic toward the pea aphid and may have potential as alfalfa resistance factors toward the pea aphid. Thus, it might be worthwhile to modify saponin levels in the breeding of modern alfalfa lines with biotechnologically introduced genes that regulate saponin concentration and composition.