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.