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\begin{document}

\title{Electrical signalling on Bt and non-Bt cotton plants under stress by
Aphis gossypii}



\author[1]{Jessica Pachu}%
\author[2]{Francynes Macedo}%
\author[3]{Jose Malaquias}%
\author[1]{Wesley Godoy}%
\author[2]{Francisco Ramalho}%
\author[2]{Ricardo Ferraz}%
\affil[1]{USP ESALQ}%
\affil[2]{Affiliation not available}%
\affil[3]{UNESP}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\selectlanguage{english}
\begin{abstract}
Bt cotton is a genetically modified pest-resistant plant that produces
an insecticide from Bacillus thuringiensis (Bt) to control Lepidopteran
species. Surprisingly, there is no study -- yet, that characterizes the
signalling mechanisms in transgenic cotton plants attacked by non-target
insects, such as aphids. In this study, we characterized the production
of electrical signals on Bt and non-Bt cotton plants infested with Aphis
gossypii and, in addition, we characterized the dispersal behaviour of
aphids to correlate this behaviour to plant signalling responses.
Electrical signalling of the plants was recorded with an extracellular
measurement technique. Impressively, our results showed that both Bt and
non-Bt cotton cultivars, when attacked by A. gossypii, emitted potential
variation-type electrical signals and clearly showed the presence of
distinct responses regarding their perception and the behaviour of
aphids, with evidence of delay, in terms of signal amount, and almost
twice the amount of Cry1F protein was observed on Bt cotton plants at
the highest density of insects/plant. We present in our article some
hypotheses that are based on plant physiology and insect behaviour to
explain the responses found on Bt cotton plants under aphid stress%
\end{abstract}%



\sloppy


Electrical signalling on Bt and non-Bt cotton plants under stress by

\emph{Aphis gossypii}

J\selectlanguage{ngerman}éssica K. S Pachú\textsuperscript{1*}, Francynes C.O.
Macedo\textsuperscript{2}, José B Malaquias\textsuperscript{3}, Wesley
A. C. Godoy\textsuperscript{1}, Francisco S.
Ramalho\textsuperscript{4}and Ricardo F. Oliveira\textsuperscript{2}

\textsuperscript{1*}Corresponding author: Jéssica K. S Pachú, Rua Dr
Alvim, 2227, Piracicaba, 13418-060, São Paulo, Brazil, Email:
jessikapachu@gmail.com.\textsuperscript{1}Department of Entomology and
Acarology, Luiz de Queiroz College of Agriculture (ESALQ), University of
São Paulo (USP), Av. Pádua Dias 11, Piracicaba, 13418-900, São Paulo,
Brazil.\textsuperscript{2}Department of Plant Physiology and
Biochemistry, Luiz de Queiroz College of Agriculture (ESALQ), University
of São Paulo (USP), Av. Pádua Dias 11, Piracicaba, 13418-900, São Paulo,
Brazil.\textsuperscript{3}Department of Biostatistics, São Paulo State
University (UNESP), Botucatu, Brazil. \textsuperscript{4}Biological
Control Unit \selectlanguage{english}/ Embrapa Algodao, Av. Osvaldo Cruz, 1143 Campina Grande,
Paraiba 58107-720, Brazil.

\textbf{ABSTRACT} Plants have developed various mechanisms to respond
specifically to each biotrophic attack. It has been shown that the
electrical signals emitted by plants are associated with herbivory
stress responses and can lead to the activation of multiple defences. Bt
cotton is a genetically modified pest-resistant plant that produces an
insecticide from \emph{Bacillus thuringiensis} (Bt) to control
Lepidopteran species. Surprisingly, there is no study -- yet, that
characterizes the signalling mechanisms in transgenic cotton plants
attacked by non-target insects, such as aphids. In this study, we
characterized the production of electrical signals on Bt and non-Bt
cotton plants infested with \emph{Aphis gossypii} and, in addition, we
characterized the dispersal behaviour of aphids to correlate this
behaviour to plant signalling responses. Electrical signalling of the
plants was recorded with an extracellular measurement technique.
Impressively, our results showed that both Bt and non-Bt cotton
cultivars, when attacked by \emph{A. gossypii} , emitted potential
variation-type electrical signals and clearly showed the presence of
distinct responses regarding their perception and the behaviour of
aphids, with evidence of delay, in terms of signal amount, and almost
twice the amount of Cry1F protein was observed on Bt cotton plants at
the highest density of insects/plant. We present in our article some
hypotheses that are based on plant physiology and insect behaviour to
explain the responses found on Bt cotton plants under aphid stress.

\textbf{KEYWORDS} : biotic, response, plant, insect, interaction.

\textbf{1 INTRODUCTION}

An organism's capacity to survive in an ecosystem depends on its ability
to respond quickly and efficiently to external stimuli and to develop
effective and sustainable defences (Zebelo \& Maffei, 2015). For this
reason, plants have developed numerous mechanisms to react specifically
to each biotrophic attack, and cell-to-cell communication between
distant tissues is essential to coordinate activities in response to the
environment. Thus, plants need to produce a signalling mechanism to
integrate perception, transmission, and response to biotic and abiotic
actions that occur in the ecosystem (Baluska et al., 2015; Brenner et
al., 2006; Pelagio-Flores et al., 2011; Maffei et al., 2007). Electrical
signals have been shown to be associated with responses to herbivory
(Pachu, 2020), leading to the activation of multiple organism defences
(Maffei et al., 2004). However, studies that better characterize the
electrical signalling mechanisms in plants attacked by herbivores, such
as aphids, are still scarce.

The aphid \emph{Aphis gossypii} Glover (Hemiptera: Aphididae) is a
cotton-damaging pest (Malaquias et al., 2017a) of cotton and one of the
most important nontarget species of Bt cotton, which is a genetically
modified cultivar expressing proteins derived from \emph{Bacillus
thuringiensis} (Berliner) (Bt), which gives them high efficiency against
some lepidopteran species (Malaquias et al., 2017b; Malaquias et al.,
2020). However, researchers have raised concerns about their potential
impact on nontarget organisms such as aphids (Hagenbucher et al., 2013)
Studies addressing the impact of Bt cotton on the population dynamics of
aphids (Udikeri et al., 2012) or on growth or developmental
characteristics (Zhao et al., 2016) have shown that aphid feeding on Bt
cotton plants can cause morphological, physiological, biochemical and
molecular changes in cotton plants and possibly different biological
response patterns between Bt and non-Bt cotton cultivars.

Different environmental stimuli cause specific responses in living cells
that are capable of transmitting electrical signals (Lautner et al.,
2005). Among the signals involved in electrophysiological responses,
variation potentials are characterized by rapid depolarization and
subsequent slow repolarization. The amplitude and shape of the variation
potentials (VPs) vary with the stimulus intensity. In addition, the
magnitude and speed of responses decrease as the signal moves away from
the stimulus site, and its induction depends on the type of damage
sustained (Stahlberg et al., 2006).

Although the methods used to produce transgenic crops are being
continuously improved, it is currently not possible to control the exact
stability, integration and expression of the gene inserted in plant
genomes. Plant physiological traits may be altered; thus, it is crucial
to understand how plants produce different signs of stress and convert
them into appropriate specific responses. Therefore, it is essential to
characterize the type of electrical signalling of Bt and non-Bt cotton
plants as a function of the stress caused by \emph{A. gossypii} and
provide insights to understand how plants convert these different
signals into appropriate physiological reactions. In our study, we
characterized the production of electrical signals on Bt {[}cultivar
WideStrike(r){]} cotton plants and their non-Bt isoline {[}cultivar FM
993{]} infested with \emph{A. gossypii} in alternating light--dark
cycles. The aphid \emph{A. gossypii} was used as a model insect for the
study because insect feeding occurs at the phloem level, and the
biological interactions between the herbivore and its host plant can be
considered unique. Additionally, we characterized \emph{A.
gossypii}dispersal behaviour to relate this behaviour to plant
signalling responses.

\textbf{2 MATERIAL AND METHODS}

\textbf{2.1 Characterization of the electrical signalling potential of
Bt and non-Bt cotton plants} Bioassays to record electrical signalling
of cotton plants were conducted in the laboratory. The experimental
design was a randomized block design, and each treatment was repeated 10
times. The measurement of electrical signals was made on the plant
surface. A technique was used to detect electrical signalling potential
differences over long periods. At the time of the electrical signal
measurements, the cotton plants were placed in a Faraday cage to ensure
electromagnetic isolation of the environment at 26+-1degC, with a relative
humidity of 60+-10\% and a 12-h photophase.

Measurements were made using electrodes consisting of a 0.25 - 0.5 mm
diameter silver lead wire chlorinated in 3 M KCl solution. Five
electrodes were used, four of which were inserted in different
arrangements along the stem of cotton plants. The fifth electrode is the
reference electrode and was inserted at the base or at the stem apex.
The electrodes are connected to a four-channel data acquisition system
with a built-in amplifier (World Precision Instruments Lab-Trax-4 / 24T
model) that is connected to a computer with LabScribe version 3.0
software that decodes the signal (Zawadzki et al., 1995, with
adaptations).

The recordings of electrical activities in cotton plants were performed
continuously for three days. The following variables were obtained:
amplitude and duration of the signal, number of signals generated, time
of signal concentration and frequency of signals generated by cotton
plants. The electrical signalling profile was contrasted between Bt and
non-Bt cotton plants infested with those not infested with aphids
(\emph{A. gossypii} ). Bt and non-Bt cotton cultivars were planted in
plastic pots containing soil conditioning substrate
(Forth\textsuperscript{(r)}) and kept separately in cages under the same
climate conditions mentioned before.

\textbf{2.2 Dispersal pattern of \emph{A. gossypii} in Bt and non-Bt
cotton plants} Bioassays were performed to study aphid behaviour and
associate it with data obtained from electrical signalling bioassays. A
randomized block design with four treatments was used: \textbf{a1} . Bt
cotton plants infested with 30 aphids/plant; \textbf{a2} . Bt cotton
plants infested with 60 aphids/plant; \textbf{a3} . non-Bt cotton plants
infested with 30 aphids/plant and \textbf{a4} . non-Bt cotton plants
infested with 60 aphids/plant, distributed in 10 blocks. Bt and non-Bt
cotton cultivars were planted in plastic pots containing soil
conditioning substrate (Forth\textsuperscript{(r)}) and kept in the same
climate conditions mentioned before.

Aphid infestations were performed on Bt and non-Bt cotton that reached
the six-leaf stage. After infestations of the Bt and non-Bt cotton
plants with \emph{A. gossypii} , the number of aphids was recorded in
the within-plant regions at 0 (immediately during infestation), 24, 48
and 72 h after infestation. The insect within-plant distribution of each
Bt and non-Bt cotton plant was analyzed at three positions: bottom,
middle and top.

To evaluate aphid dispersal behaviour as a function of cultivars (Bt and
non-Bt cotton) and aphid densities, the negative binomial distribution
parameter \emph{k} was used. There are three basic spatial pattern
distributions: random distribution, regular or uniform distribution, and
aggregate or contagious distribution. This parameter \emph{k} is an
indicator of uniform distribution, where when \emph{k} tends to zero,
the distribution is highly aggregated, \emph{k} ranging from 2 to 8
indicates moderate aggregation, and values greater than 8
(\emph{k}\textgreater{} 8) indicate that the distribution is random
(39). The\emph{k} values were estimated by the method of moments.

\textbf{2.3 Data analyses}

\textbf{2.3.1 Characterization of the electrical signalling potential of
Bt and non-Bt cotton plants} Descriptive analyses were conducted with
boxplots aiming to characterize the quantiles, medians, maximum and
minimum values, and outliers of the variables and time for the emission
of VPs (variation potentials) after the infestations with aphids on Bt
and non-Bt cotton plants and amplitude of VPs.

Correlation analyses were conducted between the variables VP amplitude
and signal emission time after infestation plants within each cotton
cultivar. The degree of correlation between the variables in each
condition was studied using Spearman's rank correlation coefficient (P
\textless{}0.05) using the cor.test function of the R program.

Data on the number of signals per time interval after infestation of Bt
and non-Bt cotton plants with aphids were subjected to deviance
analysis, with the purpose of studying the interaction involving cotton
cultivar, aphid / plant density and time interval. A generalized linear
model with a quasi-Poisson distribution was used. The goodness of fit of
the model was evaluated with a simulated normal envelope using the hnp
package in the R program (Moral et al., 2017).

Deviance analysis was applied to study the interaction involving cotton
cultivar, aphid/plant density and period (photophase / scotophase) in
the number of VPs. Data were divided into four sections, three of which
corresponded to the data recorded during three days of observation, and
the last section corresponded to the accumulated data recorded during
the three days of evaluation. Negative binomial generalized linear
models were used for approximately the 1st and 2nd evaluation days,
while quasi-Poisson models were adopted for data recorded on the 3rd day
and total accumulated over the three days of evaluations. We used a
half-normal plot with a simulated envelope with the hnp package (Moral
et al., 2017) to assess the goodness-of-fit of the models.

\textbf{2.3.2} \textbf{Dispersal pattern of \emph{A. gossypii} in Bt and
non-Bt cotton plants}

The parameter \emph{k} in each cotton cultivar and density was compared
by confidence intervals. Confidence intervals were generated from the
values of \emph{k} for each block. We used the nonparametric bootstrap
technique, with 10,000 pseudoreplications, and for the resampled
parameter in each treatment, we used the R program boot package (Angelo
et al., 2019).

The probability of aphids occurring within each region of Bt and non-Bt
cotton plants in each treatment (cultivar and aphid density) was
estimated and compared with a multinomial linear model. The analyses to
estimate the probabilities and their comparisons were conducted with
nnet (Venables et al., 2012) and emmeans (Lenth, 2020) packages from R.

\textbf{3 RESULTS}

\textbf{3.1 Characterization of the electrical signalling potential of
Bt and non-Bt cotton plants} In descriptive analysis, it was possible to
visualize that Bt cotton plants when infested with \emph{A.
gossypii}emitted the first VPs (minimum value in boxplot) between time
intervals of 0.31 h (60 aphids/plant) and 0.64 h (30 aphids/plant)
(Figure 1). In the absence of aphids, only two Bt cotton plants emitted
these electrophysiological signals. Non-Bt cotton plants emitted the
first VPs after 0.80 h when kept at 30 aphids/plant and after 1.60 h at
the density of 60 aphids/plant, as well as in the control (non-Bt and no
aphids) (\textbf{Fig. 1} ).

The Bt and non-Bt cotton plants infested with aphids emitted signals
after 60 h of aphid infestation (\textbf{Fig. 1} ), while in cotton
plants used as a control, the maximum signal emission values were
observed at 55 and 57 h in non-Bt and Bt cotton plants, respectively
(\textbf{Fig. 1} ). The boxplot with VP amplitude shows that the
response variability (maximum values, not including outliers, and 3rd
quantile) (\textbf{Fig. 2} ) of non-Bt cotton plants at 30 aphids/plant
density was lower than that of the other cotton plants infested with
\emph{A. gossypii} . The maximum VP found in the control cotton plants
was near 28 mV. In general, the mean VP (points within the boxplots) was
near all treatments, ranging from 11.22 mV (non-Bt cotton plants
infested with 30 aphids/plant) to 17 mV (Bt -- control cotton plants).
Outliers (points out of boxplots) occurred for 30 aphid/plant (129
mV)-infested non-Bt cotton plants and 60 aphid/plant (116.60
mV)-infested Bt cotton plants (\textbf{Fig. 2} ).

Spearman rank analysis revealed that there was no correlation between
the amplitude (mV) of VP and time (h) to emission of signals by cotton
plants after aphid infestation at all densities studied within each
cultivar (Bt and not Bt), except at the densities of 30 aphids / non-Bt
cotton plants (\selectlanguage{greek}\emph{ρ} \selectlanguage{english}= -0.2659; \emph{P} = 0.0060) and 60 aphids/Bt
cotton plants (\selectlanguage{greek}\emph{ρ} \selectlanguage{english}= - 0.3528; \emph{P} = 0.00254).

Analyzing the amount of VPs emitted by the cotton plants, we observed
that infestation-free plants emitted few signals, with an average
accumulation of 0.75 (control -- Bt cotton) and 2.50 signals (control --
non-Bt cotton) over 72 h. Only two Bt cotton plants emitted electrical
signals in the absence of aphid stress (\textbf{Table 1} ).

In the accumulated emission of VPs over 72 h, it was verified that Bt
cotton plants exposed to 60 aphids/plant density emitted fewer signals
compared to the other conditions (\emph{P} \textless{}0.05) under aphid
stress. However, by assessing the emission within the intervals, the
deviance analysis revealed that the signal emission pattern in each
cultivar was influenced by the time interval and aphid density, as there
was a significant interaction between these three factors (\emph{P} =
0.0488) (\textbf{Table 1} ).

The highest number of signals emitted by Bt cotton plants when exposed
to aphids occurred in the time interval after infestation of 0-12 h (30
aphids/plant) and 0-12, 36-48 and 60-72 h (60 aphids/plant)
(\textbf{Table 2, Fig. 3} ).

When we compared the signal emission pattern between combined treatments
involving aphid densities and cotton cultivars within each time
interval, it was possible to verify a delay in terms of the production
pattern of signalling on Bt cotton plants under stress with 60
insects/plant because until the time interval of 36 h after infestation,
there was a lower signal emission by Bt cotton plants when exposed to 60
aphids/plant in relation to the other conditions of aphid density/Bt or
non-Bt cotton cultivar (\textbf{Table 2, Fig. 3} ).

In the time interval of 36-48 h, the emission of signals by Bt cotton
plants was lower only in relation to Bt cotton with 30 aphids/plant.
Additionally, in the time interval of 60-72 h, the signal production by
cotton plants was higher when the Bt and non-Bt cotton plants were
exposed to densities of 60 and 30 aphids, respectively, in relation to
other conditions (\textbf{Table 2, Fig. 3} ).

The deviance analysis on the interaction of the factors aphid density
versus cotton cultivar versus light period within each studied day (1st,
2nd or 3rd day) and accumulated over these three days influencing the
number of VPs emitted by plants shows that there was no interaction
(P\textgreater{} 0.05) among the studied factors for the 1st and 2nd day
and the accumulated days of exposure of Bt and non-Bt cotton plants to
aphids. The factor density {[}\emph{F} \textsubscript{density} =
2.29,\emph{P} \textsubscript{density} = 0.1294 (1st day);\emph{F}
\textsubscript{density} = 0.0070,\emph{P} \textsubscript{density} = 0.95
(2nd day);\emph{F} \textsubscript{density} = 0.1734,\emph{P}
\textsubscript{density} = 0.6813 (cumulative total){]}, cultivar
{[}\emph{F} \textsubscript{cultivate} = 0.003,\emph{P}
\textsubscript{cultivate} = 0.95 (1st day);\emph{F}
\textsubscript{cultivate} = 3.0896,\emph{P} \textsubscript{cultivar} =
0.07 (2nd day);\emph{F} \textsubscript{cultivate} = 2.2726,\emph{P}
\textsubscript{cultivate} = 0.1466 (cumulative total){]} and period
{[}\emph{F} \textsubscript{period} = 1.3882,\emph{P}
\textsubscript{period} = 0.2387 (1st day);\emph{F}
\textsubscript{period} = 1.0805, \emph{P} \textsubscript{period}= 0.3679
(2nd day); \emph{F} \textsubscript{period} = 0.2966,\emph{P}
\textsubscript{period} = 0.5918 (cumulative total){]} did not affect the
isolation of VPs emitted by cotton plants.

There was an interaction between the factors aphid density
\emph{versus}cotton cultivar \emph{versus} light/dark phase (\emph{F} =
7.7295,\emph{P} = 0.04150) for the number of VPs observed during the 3rd
day of exposure of Bt cotton plants and non-Bt to aphids. It was found
that on the third day, there was a higher VP production by non-Bt cotton
plants exposed to 30 aphids/plant density than Bt cotton plants exposed
to the same density during the photophase (Table 2). In addition, VP
production by Bt and non-Bt cotton plants exposed to 60 and 30
aphids/plant, respectively, was higher in the light phase than in the
dark phase (\textbf{Table 2} ).

\textbf{3.2 Dispersal pattern of \emph{A. gossypii} in Bt and non-Bt
cotton plants} The behaviour of \emph{A. gossypii} , independent of the
factors of exposure time of plants to aphids, aphid density and cotton
cultivar, followed a highly within-plant aggregated distribution pattern
(\emph{k} \textless{} 2) (\textbf{Table 3} ).

Comparisons of the \emph{k} index, based on confidence interval values,
revealed that the highest \emph{k} index of aphid aggregation with 30
aphids/non-Bt cotton plants was found at 48 h and 72 h after infestation
of cotton plants with \emph{A. gossypii} (\textbf{Table 3} ). However,
non-Bt cotton plants exposed to that density had a lower aphid
\emph{k}aggregation index at 72 h of infestation in relation to Bt
cotton with 60 aphids/plant (\textbf{Table 3} ).

In other words, the dispersal rate of \emph{A. gossypii} was higher on
Bt cotton with 60 aphids/plant than on non-Bt cotton plants with 30
aphids/plant at 72 h (\textbf{Table 3} ). In fact, according to the
multinomial distribution in the within-plant distribution of \emph{A.
gossypii} (\textbf{Fig. 4 a, b, c, d} ), it was confirmed that with 30
aphids/non-Bt cotton plants at 72 h, the highest proportions of aphids
were on the adaxial (0.18) and abaxial (0.49) regions of the leaf (leaf
I); however, there was increased insect dispersal to other positions,
such as leaf II, adjacent leaf I and main meristem (\textbf{Fig. 4 a} ).
On the other hand, on Bt cotton plants infested with 60 aphids/plant, we
observed the most dispersal pattern with 72 h of infestation, where
there was clearly an increased insect dispersal, with 0.16 and 0.41 of
aphids found in the adaxial and abaxial regions of leaf I, respectively,
and 0.20 in the main meristem of the cotton plant.

No significant difference was observed among the treatments within the
infestation times of 0 h, 24 h and 48 h in relation to the \emph{k}index
and multinomial distribution, except for the treatment with 60 aphids/Bt
cotton plants within 48 h, which showed the most dispersal behaviour
because it reached more regions of the cotton plants (\textbf{Fig. 4b}
).

In the comparisons of aggregation level among the time intervals within
non-Bt cotton exposed to 60 aphids/plant, we perceived that the
highest\emph{k} aggregation index was during the infestation time of 48
h (\textbf{Table 3} ). With Bt cotton plants at a density of 30
aphids/cotton plants, it was found that there was no change in the aphid
dispersal pattern at all time intervals (\textbf{Table 3} ).

\textbf{4 DISCUSSION}

The results from our study showed that both cotton cultivars (Bt and
non-Bt), when attacked by \emph{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 \emph{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 (Gall\selectlanguage{ngerman}é 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 \emph{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 \emph{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\selectlanguage{english}-saving (Hilker and Schmulling, 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\selectlanguage{english}-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 \textsuperscript{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 H\textsubscript{2}O\textsubscript{2} (Demidchik \&
Shabala, 2018) system (Pearce et al., 1991), jasmonic acid, abscisic
acid, glutamate, among others. Both
H\textsubscript{2}O\textsubscript{2}and 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 \emph{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.

\textbf{CONFLICTS OF INTEREST:} The authors declare that they have no
competing interests.

\textbf{AUTHOR CONTRIBUTIONS}

JKSP, FCOM, JBM, FSR, RFO and WACG conceived the ideas; JKSP, FCOM and
JBM collected the data; JKSP, FCOM, JBM, FSR, RFO and WACG designed the
methodology, analysed the data and wrote the manuscript. WAC coordinated
the project. All authors contributed critically to the drafts and gave
final approval for publication.

\textbf{ACKNOWLEDGEMENTS} We thank Coordenacao de Aperfeicoamento de
Pessoal de Nivel Superior---Brasil (CAPES)---Finance Code 001, Conselho
Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) and Fundacao
de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) {[}FAPESP process
details are in the Funding section{]}.

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\textbf{Figure Legends, and Tables.}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{FIGURE 1.} Boxplot of time (h) of exposure of Bt and non-Bt
cotton plants to different densities of \emph{A. gossypii} emitting
potentials of variation (VPs) (mV). Bt and non-Bt cotton plants were
infested at densities of 30 and 60 aphids/plant (Bt.30, Bt.60, NBt.30
and NBt.60) and in the absence of aphids (Bt. Control and NBt. Control).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{FIGURE 2.} Boxplot of amplitude (mV) of variation potentials in
Bt and non-Bt (NBt) cotton plants exposed to different densities
of\emph{A. gossypii} Bt and non-Bt cotton plants exposed to densities of
30 and 60 aphids/plant and in the absence of aphids (Bt. Control and
NBt. Control).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{FIGURE 3} . Variation potentials emitted (no.) (MEAN \selectlanguage{ngerman}± SE) by Bt
and non-Bt cotton plants at different time intervals (h) after densities
of 30 and 60 aphids / plant and control (absence of aphid). Mean data
(points) and error bars (SE) predicted by the generalized linear
quasi-Poisson model, except for the plotted values for the control used
in both cotton cultivars (Bt or not Bt).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{FIGURE 4} . Multinomial distribution with occurrence rate
of\emph{A. gossypii} in the regions of non-Bt and Bt cotton plants at
infestation times of 0 h, 24, 48 and 72 h with densities of 30 and 60
aphid/cotton plants. The red circle diameter represents the intensity of
aphid infestation on cotton plants.

\textbf{TABLE 1 -} Number of variation potentials emitted (mean \selectlanguage{ngerman}± SE) by
Bt and non-Bt cotton plants when subjected to different exposure times
and densities of \emph{A. gossypii} /plant.\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
Time interval (h) & Bt cotton & Bt cotton & Bt cotton & non- Bt cotton &
non- Bt cotton & non- Bt cotton\tabularnewline
\midrule
\endhead
& 0 (control) & 30 & 60 & 0 (control) & 30 & 60\tabularnewline
0-12 & 0,00 \selectlanguage{ngerman}± 0,00\emph{\textsuperscript{nia}} & 6,0 \selectlanguage{ngerman}± 2,17 A a & 3,50 \selectlanguage{ngerman}±
,89 A b & 0,50 \selectlanguage{ngerman}± 0,50\emph{\textsuperscript{nia}} & 6,00 \selectlanguage{ngerman}± 2,08 A a &
6,25 \selectlanguage{ngerman}± 2,39 A a\tabularnewline
12\selectlanguage{english}-24 & 0,00 \selectlanguage{ngerman}± 0,00\emph{\textsuperscript{nia}} & 4,50 \selectlanguage{ngerman}± 1,04 B a & 1,75
\selectlanguage{ngerman}± 0,47 C b & 0,00 \selectlanguage{ngerman}± 0,00\emph{\textsuperscript{nia}} & 4,75 \selectlanguage{ngerman}± 1,75 AB a
& 4,50 \selectlanguage{ngerman}± 1,25 BC a\tabularnewline
24\selectlanguage{english}-36 & 0,50 \selectlanguage{ngerman}± 0,25\emph{\textsuperscript{nia}} & 2,75 \selectlanguage{ngerman}± 0,62 C b & 1,00
\selectlanguage{ngerman}± 1,00 D d & 0,75 \selectlanguage{ngerman}± 0,47\emph{\textsuperscript{nia}} & 1,75 \selectlanguage{ngerman}± 0,75 C c &
5,25 \selectlanguage{ngerman}± 1,88 AB a\tabularnewline
36\selectlanguage{english}-48 & 0,00 \selectlanguage{ngerman}± 0,00\emph{\textsuperscript{nia}} & 4,75 \selectlanguage{ngerman}± 1,25 B a & 3,50
\selectlanguage{ngerman}± 1,04 A b & 0,50 \selectlanguage{ngerman}± 0,28\emph{\textsuperscript{nia}} & 3,75 \selectlanguage{ngerman}± 0,94 B ab
& 3,50 \selectlanguage{ngerman}± 0,86 C b\tabularnewline
48\selectlanguage{english}-60 & 0,25 \selectlanguage{ngerman}± 0,25\emph{\textsuperscript{nia}} & 1,25 \selectlanguage{ngerman}± 0,62 D c & 2,50
\selectlanguage{ngerman}± 0,50 B b & 0,75 \selectlanguage{ngerman}± 0,75\emph{\textsuperscript{nia}} & 5,75 \selectlanguage{ngerman}± 0,85 A a &
5,00 \selectlanguage{ngerman}± 1,00 AB a\tabularnewline
60\selectlanguage{english}-72 & 0,00 \selectlanguage{ngerman}± 0,00\emph{\textsuperscript{nia}} & 2,25 \selectlanguage{ngerman}± 1,65 C b & 4,00
\selectlanguage{ngerman}± 0.08 A a & 0,00 \selectlanguage{ngerman}± 0,00\emph{\textsuperscript{nia}} & 4,25 \selectlanguage{ngerman}± 1,54 AB a
& 1,50 \selectlanguage{ngerman}± 0,64 D c\tabularnewline
\selectlanguage{romanian}\emph{Ʃ\textsubscript{acumulated}} (total) & 0,75 \selectlanguage{ngerman}±
0,25\emph{\textsuperscript{nia}} & 22,00 \selectlanguage{ngerman}± 0,17 a & 16,25 \selectlanguage{ngerman}± 0,13 b &
2,50 \selectlanguage{ngerman}± 0,50\emph{\textsuperscript{nia}} & 26,25 \selectlanguage{ngerman}± 0,21 a & 26,00 \selectlanguage{ngerman}± 0,20
a\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

Capital letters compare averages within each column, and lowercase
letters compare averages within each row. Means followed by the same
letters do not differ from each other by overlapping confidence
intervals generated by the quasi-Poisson model (P \textless{}0.05).

\emph{nia} = not incorporated in the analysis.

\textbf{TABLE 2.} Number of variation potentials emitted (mean ± SE) by
Bt and non-Bt cotton plants exposed to different aphid densities /
cotton plant and photophase and scotophase on the 3rd assessment day.\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
Cultivar & Density (aphid/plant) & Photophase &
Scotophase\tabularnewline
\midrule
\endhead
non Bt Cotton & 30 & 6,75 \selectlanguage{ngerman}± 1,43 A a & 2,50 \selectlanguage{ngerman}± 1,50 A b\tabularnewline
Bt cotton & & 1,50 \selectlanguage{ngerman}± 0,86 B a & 1,75 \selectlanguage{ngerman}± 1,03 A a\tabularnewline
non Bt Cotton & 60 & 3,25 \selectlanguage{ngerman}± 1,18 AB a & 4,50 \selectlanguage{ngerman}± 1,18 A a\tabularnewline
Bt cotton & & 5,00 \selectlanguage{ngerman}± 0,70 AB a & 2,00 \selectlanguage{ngerman}± 1,08 A b\tabularnewline
non Bt Cotton & 0 (control) & 0,25 \selectlanguage{ngerman}± 0,25 \emph{\textsuperscript{nic}} &
0,25 \selectlanguage{ngerman}± 0,25 \emph{\textsuperscript{nic}}\tabularnewline
Bt cotton & & 0,00 \selectlanguage{ngerman}± 0,00 \emph{\textsuperscript{nic}} & 0,25 \selectlanguage{ngerman}± 0,25
\emph{\textsuperscript{nic}}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

Uppercase letters compare averages within each column, and lowercase
letters compare averages within each row. Means followed by the same
letters do not differ from each other by overlapping confidence
intervals generated by the quasi-Poisson model (P \textless{}0.05).

\emph{nia} = not incorporated in the analysis.

\textbf{TABLE 3} . Confidence intervals associated with the \emph{A.
gossypii} aggregation index (95\% CI) in Bt and non-Bt cotton plants
submitted to densities of 30 and 60 aphids/cotton plants quantified at
0, 24, 48 and 72 h after the infestation of cotton plants with aphids.\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
Density/Cultivar & Time (h) & Time (h) & Time (h) & Time
(h)\tabularnewline
\midrule
\endhead
& 0 & 24 & 48 & 72\tabularnewline
30 aphids/non-Bt cotton/ & 0.42 - 0.52 A b & 0.47 - 0.74 A ab & 0.55 -
0.85 A a & 0.58 - 0.77 B a\tabularnewline
60 aphids/non-Bt cotton & 0.45 - 0.73 A b & 0.55 - 1.67 A ab & 0.78 -
3.03 A a & 0.67 - 1.48 AB ab\tabularnewline
30 aphids/Bt cotton & 0.45 - 1.16 A a & 0.45 - 0.83 A a & 0.57 - 0.87 A
a & 0.77 - 1.55 AB a\tabularnewline
60 aphids/Bt cotton & 0.42 - 0.90 A b & 0.63 - 0.80 A b & 0.70 - 1.05 A
b & 1.12 - 1.46 A a\tabularnewline
\bottomrule
\end{longtable}

Uppercase letters compare averages within each column, and lowercase
letters compare averages within each row. Means followed by the same
letters do not differ from each other by overlapping confidence
intervals generated by Boostrap (P \textless{}0.05).

\selectlanguage{english}
\FloatBarrier
\end{document}

