4 DISCUSSION
The results from our study showed that both cotton cultivars (Bt and non-Bt), when attacked by A. gossypii , emitted electrical signals of the variation potential type. Abiotic and biotic wounds are perceived differently by plants, as has been shown by other studies on plant-herbivore interactions (Pachu et al., 2020; Mithofer et al., 2019a; Bricchi et al., 2020). Insect damage in plants plays a vital role in recognizing the type of biotic stress to the plant (Wu et al., 2010; Bonaventure et al., 2011). Plants differentiate herbivory from mechanical damage by recognizing compounds present in insect saliva because oral secretion of herbivores can induce ionic flux and promote depolarization of the plant membrane potential (Maischak et al., 2007).
Here, in our research, it was possible to describe how Bt and non-Bt cotton plants react to A. gossypii stress by changing the transmembrane potential by recording extracellular electrical signals. Although plant responses to herbivorous attack are complex and involve a number of signals, it is important to note that different types of stimuli caused by insect action against plants trigger characteristic electrical signals evoked by plants with a specific influence on plant physiological processes (Gallé et al., 2015). The cascade of events involved in plant signalling as a function of stress perception begins at the plasma membrane of cells with changes in transmembrane potential or ion flow; these are the first responses of plants to biotic and abiotic stresses (Shabala, 2006). Attack on herbivorous plants is known to promote membrane potential changes that trigger an electrical signal that can travel to the entire plant or even trigger local plant defence mechanisms (Ebel & Mithofer, 1998).
Our results indicate the presence of VP on Bt and non-Bt cotton plants at all assessed interval times. An aphid continuously inserts its buccal apparatus into the phloem vessels, altering the hydrostatic pressure in these vessels and consequently altering the pressure in the xylem. VP is a signal whose propagation properties vary with the intensity and distance of the stimulus site and is probably a local electrical response, which is induced by a hydraulic signal, chemical signal or the combined action of these signals (Vodeneev et al., 2018). The hydraulic signal is a wave that results from increased hydraulic pressure in the plant, which propagates through the xylem and initiates the generation of a VP by triggering mechanosensitive ion channels present in the cells adjacent to the plant xylem vessels (Dziubinska et al., 2001). Therefore, harmful stimuli such as local damage, burning and mechanical injuries can evoke VPs (Dziubinska et al., 2003; Zimmermann et al., 2009). These kinds of electrical signals emitted by plants when under stress are especially important for hazard perception and response; thus, the plant can become able to mount an appropriate defence response (Davies & Stankovic, 2006).
Distinct response patterns were attributed to the perception and response to A. gossypii by each cotton cultivar and aphid density used in the research. Although we reported the first emission of signals on Bt cotton plants, there was a delay in terms of the propagated signal amount on Bt cotton plants with 60 aphids of infestation with A. gossypii , which produced the smallest numbers of signals between 0 to 36 h. Another important result was the greater dispersal behaviour related to this same treatment, mainly during and after 48 h of infestation. We suggest that the results could be supported by two hypotheses and explained independently or combined.
The first hypothesis is based on the possibility of a trade-off in terms of the defense of the Bt plant; a high dispersal could mean a larger exploitation of food resources by aphids and ease penetration of mouth apparatuses by aphids on Bt cotton plants, which may explain why Bt cotton plants emitted faster electrical signals than non-Bt cotton plants in the first moment, showing that Bt cotton plants may be more susceptible to aphid stress. Inducibility of a plant stress response is the ability to respond to stress only on demand. This is an astrategy that is considered cost‐saving (Hilker and Schmülling, 2019). Therefore, this inducibility of plant defense may indicate a delay in the operation of defensive mechanisms but may also mean a strategy to save energy and prevent self-poisoning (Zhu-Salzman, 2008), how our results show them to save energy with less production of signal until 36 h and producing them later. Since induction of a stress response implies that the plant starts activating resistance mechanisms upon encounter with the stressor, this strategy may lead to delay in mounting an effective response (Hilker et al., 2016; Martinez‐Medina et al., 2016), a first stress experience may prime the organism for an improved response to a subsequent stress.
With an electrical penetration graph (EPG) used for monitoring the penetration of the mouth apparatus by aphids on Bt and non-Bt plants and recording the waveforms that reflect different aphid feeding activities, a lower percentage of waveform np (non-penetration) was observed when the aphid was walking or grabbing the food with the rostrum on Bt cotton plants (Liu et al., 2005). This suggests that aphids spend less time finding suitable places for penetration of their mouthparts on Bt cotton plants, probably due to the suitability of the tissue structure of Bt cotton plants to feed these aphids (Liu et al., 2005).
The second hypothesis is that the higher aphid dispersal on Bt cotton plants may indicate that the first signals emitted by the Bt cotton plants, even in smaller numbers than the non-Bt cotton plants, were enough to activate the Bt cotton plants’ defence, which prevented or hindered aphid feeding, such as occlusion of phloem sieve elements (SEs), which are the main conductive cells in the phloem, by clogging the sieved pores 34(Knoblauch & van Bel, 1998). This is presumed to prevent sap loss (Ever, 1982; Schulz, 1998), and this process is seen as a primary plant defense response (Knoblauch & van Bel, 1998). At the same time, the saliva constituents of sucking insects affect cellular processes (Backus, 2005) and therefore are perceived by cells, leading to the activation of signalling mechanisms, supporting the supposition that local damage induces the propagation of a specific injury substance through the xylem, and this induces the electrical response (Vodeneev et al., 2015). The main candidates for signaling molecules are the H2O2 (Demidchik & Shabala, 2018) system (Pearce et al., 1991), jasmonic acid, abscisic acid, glutamate, among others. Both H2O2and glutamate may activate calcium permeable channels, increasing intracellular calcium concentrations in plants (Toyota et al., 2018) and being an important trigger for the generation and propagation of VP in plants (Mousavi et al., 2013).
The observed delay in the quantitative signalling pattern of Bt cotton plants when exposed to 60 aphids/plant could be attributed to self-preservation under stress and may be supported by the inclusion of resource reallocation for the production of metabolites and defensive structures (first and second hypotheses simultaneously). In the supplementary material, we show that almost twice the amount of Cry1F protein was observed on Bt cotton plants in the presence of aphids than in the absence of the insect. Well-known anti-herbivorous defence proteins include proteinase inhibitors (PIs) and polyphenol oxidases (PPOs), both considered to interfere with digestive processes in herbivore intestines (Zhu-Salzman, 2008). Therefore, simultaneous resource reallocation can serve not only to save resources; thus, the plant can subsequently use them for growth and reproduction but also to deprive the herbivore of food and consequently increase aphid dispersal, as observed in our results on Bt cotton with 60 aphids/plant, in order to search for the available food source. These abovementioned hypotheses generate a basis for further studies that seek to highlight what possible defense mechanisms are involved and how effective they may be as a function of Bt and non-Bt cotton cultivars. In conclusion, the stress caused by the aphid A. gossypii was sufficient to trigger specific responses on Bt and non-Bt plants. Bt cotton plants were faster to propagate VP signals; however, they produced the signals in a smaller quantity with the highest aphid density, also promoting greatest within-plant aphid dispersal. Our results may guide future studies, which aim to elucidate the factors involved in the resistance to stress and plant defense processes and thus assist in the development of successful strategies in integrated pest management.
CONFLICTS OF INTEREST: The authors declare that they have no competing interests.