Assessing the Influence of Low Doses of Sucrose on Memory Deficits in Fish Exposed to Common Insecticide Based on Fipronil and Pyriproxyfen

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Following are some questions and suggestions that need to be discussed in the manuscript by the author;

1.      What underlying mechanisms might be responsible for the large drop in alternating tetragram sequences (LRLR, RLRL) observed in the group that was given sucrose and insecticide, and how can this be related to certain cognitive dysfunctions?

2.      Given that there was no significant difference between the groups regarding the number of arm entries (exploratory activity), what does this imply about the specificity of the effects of insecticide or sucrose exposure on cognitive processes as opposed to motor activities?

3.      In what ways might the findings of this T-maze test contribute to our comprehension of the influence that pesticides have on short-term spatial memory, particularly when they are employed in conjunction with sucrose?

4.      What might account for the delayed latency to enter the illuminated compartment, particularly in the group that was given sucrose plus insecticide, and how does this behavior relate to increased levels of anxiety?

5.      Because the sucrose group exhibits more frequent entry into the illuminated compartment, why does the sucrose + insecticide group show a significant drop in the frequency of entries into the lighted compartment compared to the sucrose group?

6.      The group exposed to insecticides displayed a higher level of risk-taking behavior, including frequent trips back to the dark compartment. What are the implications of this finding regarding the anxiogenic effects of insecticides, and how might sucrose influence the reactions associated with anxiety?

7.      In what ways are the results of the light/dark test regarding anxiety different from those that were observed in the mirror biting test? Furthermore, what conclusions can be drawn regarding the interaction between sucrose and insecticide with regard to anxiety and aggression?

8.      Why did the group that received sucrose and insecticide exhibit a much shorter amount of time spent in contact with the mirror than the groups who received either insecticide or sucrose? How might this reflect the interaction between anxiety and aggression?

9.      Regarding the observed difference in freezing behavior (immobility) in the pesticide group, what neurobiological mechanisms could explain this difference? Furthermore, how could the absence of freezing behavior in the sucrose + insecticide group provide insights into the moderating effects of sucrose?

10.  How does the large increase in superoxide dismutase (SOD) and catalase activity in the pesticide and insecticide + sucrose groups suggest that the body is attempting to counteract oxidative damage, and what does this imply about the overall oxidative stress response to these chemicals?

11.  What is the reason for the lack of substantial changes in superoxide dismutase (SOD) levels in the sucrose group when compared to the control group, and what does this say about the antioxidant capability of sucrose individually as opposed to when it is combined with insecticides?

12.  An excessive amount of oxidative stress is indicated by the considerable decrease in GPx activity that was observed in the pesticide and insecticide plus sucrose groups. Which of the following are some of the more widespread physiological implications that could result from these groups' weakened antioxidant defense systems?

13.  For cellular damage, what are the potential consequences of elevated MDA levels in the pesticide and insecticide + sucrose groups, and how can this correlation be related to the observed behavioral alterations (such as anxiety and aggression)?

14.  The group exposed to insecticides displayed decreased AChE activity, which is a sign of neural toxicity. What is the possible correlation between this and the behavioral effects reported in the T-maze and mirror biting tests, and what does this tell us about the neurotoxic impact that pesticides have on cholinergic signaling?

15.  Please enhance the resolution of Figs. 1, 2, 3, and 4. The axes in the figures are not visible.

Author Response

Thank you very much for taking the time to review this manuscript (see attachment).

We deeply appreciate your thoughtful comments and suggestions, which have greatly contributed to enhancing the quality of our work. Below, you will find our detailed responses to each of your points. The corresponding revisions have been made directly in the manuscript and are clearly visible using track changes.

1. What underlying mechanisms might be responsible for the large drop in alternating tetragram sequences (LRLR, RLRL) observed in the group that was given sucrose and insecticide, and how can this be related to certain cognitive dysfunctions?

Thank you for your feedback. We have carefully considered your suggestion and incorporated relevant details into the discussion to provide a more comprehensive explanation of the observed behavioral changes. Specifically, we have included in the discussion section aspects that highlight potential mechanisms underlying the significant decrease in alternating tetragram sequences (LRLR, RLRL) observed in Silver crucian carp exposed to sucrose and insecticide.

In our revised text, lines 617-624, we explain that the decrease in alternating tetragram sequences can be attributed to the combined effects of neurotoxicity and oxidative stress. Thus, insecticides, such as organophosphates, inhibit acetylcholinesterase, can disrupt neurotransmission impairing memory and decision-making processes [93]. Furthermore, we have shown that sucrose consumption may exacerbate these effects by inducing metabolic stress and neuroinflammation, potentially impacting the hippocampus, a critical region for spatial memory and exploratory behavior [94]. These insights provide a stronger link between the combined exposure and the observed cognitive dysfunctions.

2. Given that there was no significant difference between the groups regarding the number of arm entries (exploratory activity), what does this imply about the specificity of the effects of insecticide or sucrose exposure on cognitive processes as opposed to motor activities?

The lack of significant differences in the number of arm entries (exploratory activity) suggests that insecticide or sucrose exposure primarily affects cognitive processes rather than motor functions. While the fish maintained their overall exploratory behavior, their performance in tasks requiring memory and cognitive flexibility, such as alternating tetragram sequences, was impaired. This indicates that the exposure did not impact basic motor activity but did disrupt higher-order cognitive functions.

3. In what ways might the findings of this T-maze test contribute to our comprehension of the influence that pesticides have on short-term spatial memory, particularly when they are employed in conjunction with sucrose?

Thank you for this insightful question. We have added the following (lines 578-595): “The results of the T-Maze test provide key insights into how pesticide exposure, particularly in combination with sucrose, could affect cognitive functions such as short-term spatial memory. Thus, the antagonistic action of Fipronil on GABAergic neurotransmission may disrupt inhibitory signals necessary for proper cognitive processing, as GABA plays a crucial role in modulating hippocampal activity, which is essential for the formation and retention of spatial memory [84,85]. Although sucrose may provide some buffering effects against anxiety through its impact on the HPA axis and serotonergic pathways [86,87], it may also exacerbate neurocognitive dysfunction due to its known metabolic effects. Excessive sucrose intake has been shown to induce hippocampal insulin resistance and oxidative stress, both of which have a negative impact on spatial memory and synaptic plasticity [88,89]. Furthermore, the interaction between insecticide-induced oxidative damage and sucrose-induced metabolic stress could lead to combined effects that affect neurogenesis and memory-related pathways in the brain. As such, the combination of insecticide and sucrose highlights a broader concern about the ecological and environmental implications of pesticide use. Studies in zebrafish have shown that exposure to sublethal concentrations of pesticides can lead to persistent memory and learning deficits [90], suggesting that similar disruptions may occur in other aquatic species.”

4. What might account for the delayed latency to enter the illuminated compartment, particularly in the group that was given sucrose plus insecticide, and how does this behavior relate to increased levels of anxiety?

Thank you for your question. To answer this, we add the following in the Discussion section (lines 541- 550): “The delayed latency to enter the lighted compartment in the sucrose plus insecticide group likely reflects increased levels of anxiety. This behavior is consistent with the anxiogenic effects observed in the study, where prolonged exposure to insecticides disrupts GABAergic signaling, leading to CNS hyperactivity and increased stress responses [77] Sucrose exacerbates this by inducing metabolic stress and oxidative damage, further affecting neurotransmitter regulation and amplifying anxiety-like behaviors. The reluctance to explore the lighted area indicates an increased aversion to perceived risk, a hallmark of anxiety, corroborated by other observed markers such as reduced exploratory activity and elevated freezing times [78].”

This addition highlights potential mechanisms driving the observed behavioral response and links it to the broader context of anxiety-related behaviors. It also aligns with the overall findings of the study, further strengthening the discussion.

 

5. Because the sucrose group exhibits more frequent entry into the illuminated compartment, why does the sucrose + insecticide group show a significant drop in the frequency of entries into the lighted compartment compared to the sucrose group?

The sucrose group’s more frequent entry into the illuminated compartment suggests that sucrose alone may positively influence exploratory behavior, potentially by providing an energy boost or modulating stress responses. However, the significant drop in the frequency of entries into the illuminated compartment observed in the sucrose + insecticide group compared to the sucrose group likely reflects the combined effects of insecticides and sucrose. Specifically, the neurotoxic impact of insecticides disrupts central nervous system (CNS) function, particularly through GABA receptor antagonism, which leads to heightened anxiety and impaired decision-making. When combined with the metabolic stress induced by sucrose, these effects appear to outweigh sucrose’s potential benefits, reducing exploratory behavior and increasing anxiety-like responses.

Additionally, we have added the following in Discussions (lines 650-658): „The significant decrease in the frequency of entries into the illuminated compartment in the sucrose + insecticide group compared to the sucrose only group likely arises from the combined neurotoxic effects of insecticides and the metabolic stress induced by sucrose. While sucrose alone may enhance exploratory behavior by providing an energy boost, the addition of insecticides disrupts CNS function, particularly through GABA receptor antagonism, leading to increased anxiety and impaired decision making [77,79]. This combined effect likely outweighs the influence of sucrose, resulting in reduced exploratory behavior and a preference to avoid the illuminated area, indicating increased anxiety and impaired cognitive function [79].”

 

6. The group exposed to insecticides displayed a higher level of risk-taking behavior, including frequent trips back to the dark compartment. What are the implications of this finding regarding the anxiogenic effects of insecticides, and how might sucrose influence the reactions associated with anxiety?

Frequent trips back to the dark compartment in the insecticide exposed group suggest a heightened state of risk assessment and hypervigilance. This behavior aligns with the anxiogenic effects of insecticides, which disrupt GABAergic neurotransmission and induce central nervous system hyperactivity, resulting in heightened stress responses. The dark compartment likely functions as a perceived safe zone, reflecting a conditioned avoidance preference commonly associated with anxiety disorders. Sucrose, although it may modulate stress through serotonergic signaling, appears to interact with the neurotoxic effects of insecticides in a manner that exacerbates metabolic stress and oxidative damage. This combination may enhance avoidance behaviors because the combined stressors increase the individual's sensitivity to perceived threats.

We have added the following in the discussion section (lines 558-566) to expand on these implications: “In addition, the increased frequency of returns to the dark compartment may represent enhanced risk-assessment behavior triggered by the anxiogenic effects of insecticides. Thus, this trend aligns with observations of hypervigilance and repetitive risk-avoidance actions observed in anxiety disorders [80]. In this context, the dark compartment likely serves as a perceived safe zone, as heightened anxiety often leads to a preference for environments with less perceived threat [81]. While sucrose may provide a buffering effect against stress by modulating serotonergic signaling [82], metabolic stress and the oxidative damage it induces, especially when combined with insecticides, may amplify avoidance responses.”

 

7. In what ways are the results of the light/dark test regarding anxiety different from those that were observed in the mirror biting test? Furthermore, what conclusions can be drawn regarding the interaction between sucrose and insecticide with regard to anxiety and aggression?

The light/dark test primarily assesses anxiety-related behaviors, such as latency to enter and time spent in the illuminated compartment. In contrast, the mirror-biting test focuses on aggressive behaviors, which are linked to dominance or territoriality. In the light/dark test, the sucrose + insecticide group displayed heightened anxiety levels, as evidenced by reduced exploration of the illuminated area and frequent retreats to the dark compartment. These behaviors suggest a hypervigilant state driven by disrupted GABAergic signaling from insecticide exposure and oxidative stress induced by sucrose.

In the mirror-biting test, however, the same group showed significantly reduced aggression compared to the sucrose only or insecticide only groups. This suggests that the heightened anxiety from the combined sucrose and insecticide exposure may suppress aggression, as persistent anxiety often inhibits circuits in the hypothalamus and amygdala that regulate aggressive behaviors. This pattern aligns with findings that hyperglycemia and insecticide induced neurotoxicity disrupt mesocorticolimbic systems critical for aggression modulation.

The interaction between sucrose and insecticide thus appears to amplify anxiety while concurrently inhibiting aggression, likely through combined oxidative stress, altered neurotransmitter systems, and hyperactivation of the HPA axis.

 

We added in the discussions (lines 632-658): „The relationship between anxiety and aggressiveness seems to be influenced by the levels of anxiety and may be mediated by various neuronal and hormonal mechanisms [96–98]. In our view, the sucrose group enters a state of enhanced arousal linked with increased anxiety caused by sucrose exposure. Long term sucrose consumption in animals is reported to produce neuronal alterations of amygdala [99] or accumbens nucleus [100] which are mesolimbic regions modulating fear responses. Other reports on the effects of chronic consumption of sucrose in rodents show altered steroid levels and dopamine signalling in the brain, particularly in the mesocorticolimbic system and hypothalamus [101]. Arguablly, hyperglycemia may lead to disruptions in these brain circuitries, which were previously suggested to be associated with impulsivity and violence in humans [102]. Remarkably, a very recent study showed that hyperglycaemic rats engage in hyper-sociable and hyper-aggressive encounters more often than controls. The key mechanisms underlying this abnormal behaviour would include hyperglycemia induced neuronal oxidative stress damage in brain areas, such as midbrain, striatum, frontal cortex, and hippocampus [103]. In the case of the insecticide-sucrose group, it is likely that high levels of anxiety - the result of the combined exposure to insecticide and sucrose - caused a disruption along the stress-regulation pathways or other systems that support aggression. In our study, the neurotoxic insecticide, which alters GABAergic and glutamatergic neurotransmitter systems, caused reduced exploration and frequent sudden returns to the safe areas in the light-dark test. Physiologically, anxiety is related to activating the stress responses and circuits, the HPA [104], that could induce withdrawal and escape responses. Several papers indicate hyperglycaemia as eliciting anxiety-like behaviors and robust stress in zebrafish [105]. Significant increases in levels of cortisol are showed to be correlated with higher visceral fat deposits, insulin resistance and also increased sugar consumption [106]. Correlations between persistent anxiety and reduced aggression are reported in the context of elevated stress hormone levels that inhibit aggression-related circuits in the hypothalamus and amygdala [107].”

 

8. Why did the group that received sucrose and insecticide exhibit a much shorter amount of time spent in contact with the mirror than the groups who received either insecticide or sucrose? How might this reflect the interaction between anxiety and aggression?

The significantly reduced time spent in contact with the mirror by the sucrose + insecticide group reflects a suppression of aggression due to heightened anxiety. Insecticide exposure, particularly fipronil, disrupts GABAergic and glutamatergic neurotransmitter pathways, causing increased stress and hypervigilance. When combined with sucrose, which induces hyperglycemia, oxidative stress, and disruptions in mesocorticolimbic circuits, this results in an overactivation of anxiety-related pathways that inhibit aggression-related circuits.

Physiologically, the combined stressors likely dysregulate the hypothalamus, amygdala, and HPA axis, which are critical for balancing aggression and anxiety responses. Elevated cortisol levels, associated with chronic stress from the combined exposure, are known to suppress aggression-related circuits, as corroborated by studies in zebrafish and rodents.

The interplay between anxiety and aggression becomes evident in this group: anxiety dominates the behavioral response, suppressing the impulsive and proactive behaviors required for aggressive encounters, as seen in the mirror-biting test. This outcome underscores how the interaction between sucrose and insecticide intensifies anxiety while impairing aggression-related neural mechanisms.

Because question 8 refers to the same thing, to answer this question and clarify the subject I used the same text from the discussions, lines 632-658: „The relationship between anxiety and aggressiveness seems to be influenced by the levels of anxiety and may be mediated by various neuronal and hormonal mechanisms [96–98]. In our view, the sucrose group enters a state of enhanced arousal linked with increased anxiety caused by sucrose exposure. Long term sucrose consumption in animals is reported to produce neuronal alterations of amygdala [99] or accumbens nucleus [100] which are mesolimbic regions modulating fear responses. Other reports on the effects of chronic consumption of sucrose in rodents show altered steroid levels and dopamine signalling in the brain, particularly in the mesocorticolimbic system and hypothalamus [101]. Arguablly, hyperglycemia may lead to disruptions in these brain circuitries, which were previously suggested to be associated with impulsivity and violence in humans [102]. Remarkably, a very recent study showed that hyperglycaemic rats engage in hyper-sociable and hyper-aggressive encounters more often than controls. The key mechanisms underlying this abnormal behaviour would include hyperglycemia-induced neuronal oxidative stress damage in brain areas, such as midbrain, striatum, frontal cortex, and hippocampus [103]. In the case of the insecticide-sucrose group, it is likely that high levels of anxiety - the result of the combined exposure to insecticide and sucrose - caused a disruption along the stress-regulation pathways or other systems that support aggression. In our study, the neurotoxic insecticide, which alters GABAergic and glutamatergic neurotransmitter systems, caused reduced exploration and frequent sudden returns to the safe areas in the light-dark test. Physiologically, anxiety is related to activating the stress responses and circuits, the HPA [104], that could induce withdrawal and escape responses. Several papers indicate hyperglycaemia as eliciting anxiety-like behaviors and robust stress in zebrafish [105]. Significant increases in levels of cortisol are showed to be correlated with higher visceral fat deposits, insulin resistance and also increased sugar consumption [106]. Correlations between persistent anxiety and reduced aggression are reported in the context of elevated stress hormone levels that inhibit aggression-related circuits in the hypothalamus and amygdala [107].”

9. Regarding the observed difference in freezing behavior (immobility) in the pesticide group, what neurobiological mechanisms could explain this difference? Furthermore, how could the absence of freezing behavior in the sucrose + insecticide group provide insights into the moderating effects of sucrose?

The reviewer’s question regards these lines: We observed also the installation of significant freezing durations in the Insecticide group (p = 0.033 vs control) in which approximately 1/3 of the total number of the tested individuals exhibited prolonged immobility during the six-minutes test. Given that no individual in the sucrose + Insecticide group entered states of total immobility, a question arises whether sucrose in low concentrations has a beneficial effect in counteracting Insecticide-induced anxiogeny.

We insert these in the discussion text at lines 677-680, and 686-692: “Fipronil impairing GABA's role as an inhibitory neurotransmitter can lead to hyperactivation of the HPA axis and elevated cortisol levels [111], which in turn can impact on amygdala and hippocampal function [112,113] with subsequent exacerbates anxiety responses, including freezing….[116]. The lack of freezing in the insecticide-sucrose group can be linked with a protective effect of sucrose along the stress-induced anxiogenesis pathway. Previously, sucrose has been showed to attenuate HPA axis responses to stress in humans and animals [86,117], possibly by activating serotonergic signaling in several reward-regulatory brain sites which can counteract the effects of stress [87]. Clearly, the interplay between sucrose and fipronil, or other insecticides, suggests more complex aspects to be elucidated by further in-depth studies.”

10. How does the large increase in superoxide dismutase (SOD) and catalase activity in the pesticide and insecticide + sucrose groups suggest that the body is attempting to counteract oxidative damage, and what does this imply about the overall oxidative stress response to these chemicals?

The significant increase in SOD and catalase activity in the insecticide + sucrose group indicates that the organism is responding to heightened oxidative stress by upregulating its antioxidant defenses. This suggests a robust protective mechanism aimed at neutralizing ROS and reducing cellular damage. However, the substantial increase in enzyme activity also points to considerable oxidative stress, implying that while these responses help maintain redox balance, they may not fully prevent damage, potentially leading to long-term health implications.

 We add these in the discussions (lines 720-733): “The pronounced increase in SOD and catalase activity observed in the insecticide + sucrose group indicates that the organism actively responds to the increased OS by upregulating its antioxidant defense mechanisms. This suggests that these groups are subjected to a significant oxidative load, leading to a strong adaptive response to neutralize ROS and mitigate potential cellular damage. The increased catalase activity, especially in the insecticide + sucrose group, indicates an intensified response, probably determined by the combined effects of insecticide and sucrose. Thus, we can say that sucrose may contribute to an exacerbated oxidative environment, requiring a more robust activation of antioxidant enzymes [122]. The upregulation of both SOD and catalase reflects an adaptive mechanism aimed at counteracting oxidative damage and preserving cellular redox balance. However, the marked increase in enzymatic activity suggests that the organism is subjected to considerable OS. Although these adaptive responses are protective, they may not be sufficient to completely prevent cellular damage, which can lead to long-term repercussions for cellular and systemic health.”

 

11. What is the reason for the lack of substantial changes in superoxide dismutase (SOD) levels in the sucrose group when compared to the control group, and what does this say about the antioxidant capability of sucrose individually as opposed to when it is combined with insecticides?

Our findings indicate that SOD levels in the sucrose-only group did not show significant differences compared to the control group, suggesting that sucrose alone does not strongly activate antioxidant pathways or upregulate SOD expression to mitigate oxidative stress. This means that sucrose administered alone may not be sufficient to induce a significant protective effect against oxidative stress. However, when sucrose is combined with insecticides, it appears to contribute to an increased oxidative load, which triggers a more pronounced activation of antioxidant defenses, such as increased SOD activity. These observations indicate the need for further research to fully understand the potential roles and mechanisms of sucrose in regulating oxidative stress. This context-dependent behavior suggests that sucrose may not function individually as an antioxidant when administered alone.

We added into the discussion text lines 704-714: “In particular, the lack of significant changes in SOD levels in the sucrose-alone group may indicate that sucrose does not directly trigger an antioxidant response or activate pathways that regulate SOD expression. This suggests that sucrose alone may not be sufficient to induce a significant protective effect against oxidative stress. However, when combined with insecticides, sucrose may play a role in increasing oxidative damage, leading to an increased SOD response as the body attempts to counteract the higher oxidative load [120]. This finding highlights the differential impact of sucrose on antioxidant defense mechanisms depending on its context. While sucrose alone does not appear to significantly activate SOD, on the other hand, its presence in combination with insecticides appears to amplify the response to oxidative stress, leading to a stronger upregulation of antioxidant enzymes as a compensatory mechanism.”

12. An excessive amount of oxidative stress is indicated by the considerable decrease in GPx activity that was observed in the pesticide and insecticide plus sucrose groups. Which of the following are some of the more widespread physiological implications that could result from these groups' weakened antioxidant defense systems?

Decreased GPx activity in the pesticide and insecticide + sucrose groups indicate weakened antioxidant defenses, leading to increased oxidative stress and cellular damage. This can impair mitochondrial function, reduce energy production, and promote apoptosis, contributing to systemic inflammation and an increased risk of chronic diseases. Additionally, oxidative stress can damage brain regions involved in emotional regulation, such as the amygdala and prefrontal cortex, disrupting neurotransmitter balance and leading to behaviors like anxiety and aggression.

We again revised the text at 739-754: “Elevated MDA levels, as a marker of lipid peroxidation, reflect significant damage to cell membranes caused by oxidative stress. This membrane damage can disrupt cellular signaling and neuronal integrity, which are critical for normal brain function. Weakened antioxidant defense systems, as indicated by reduced GPx activity, can have broader physiological consequences beyond neuronal damage. For example, oxidative stress can impair mitochondrial function, leading to reduced energy production and increased apoptosis, which are critical for tissue homeostasis [123–125]. Additionally, oxidative damage to DNA, proteins, and lipids can contribute to systemic inflammation and accelerate aging processes, potentially predisposing organisms to chronic diseases such as neurodegeneration, cardiovascular disorders, and metabolic dysregulation [126, 127]. In the pesticide and insecticide + sucrose groups, the oxidative damage may interfere with synaptic transmission and neuronal plasticity, leading to alterations in neural circuits associated with anxiety and aggression [128]. Behavioral manifestations such as increased anxiety or aggression could result from damage to brain regions involved in emotional regulation, such as the amygdala and prefrontal cortex, where oxidative stress may impair neurotransmitter balance or receptor functionality [129, 130].”

 

13. For cellular damage, what are the potential consequences of elevated MDA levels in the pesticide and insecticide + sucrose groups, and how can this correlation be related to the observed behavioral alterations (such as anxiety and aggression)?

Elevated MDA levels in the pesticide and insecticide + sucrose groups indicate significant oxidative stress and lipid peroxidation, which can compromise neuronal membrane integrity and disrupt synaptic signaling. This oxidative damage is strongly linked to behavioral alterations such as anxiety and aggression, as it affects neurotransmitter systems (e.g., cholinergic, dopaminergic) and brain regions like the amygdala and hippocampus involved in mood and behavior regulation. In the insecticide + sucrose group, the combined oxidative stress likely exacerbates neuroinflammation, amplifying these behavioral deficits. Thus, MDA elevation directly correlates with the observed neurotoxic and behavioral effects.

We insert these observations in the discussion text at lines 762-771: “The observed behavioral deficits, such as those recorded in the T-maze and mirror biting tests, may be directly linked to the decrease in AChE activity. In the T-maze, reduced AChE activity and subsequent acetylcholine accumulation could impair the cholinergic pathways critical for learning and memory, resulting in disorganized behavior or di-minished cognitive performance. In the mirror biting test, overstimulation of cholinergic receptors due to inhibited AChE may lead to increased impulsivity, heightened anxiety, or hyperactivity. These behavioral changes reinforce the hypothesis that the neurotoxic effects of the insecticide, amplified by sucrose, disrupt normal cholinergic signaling and contribute to the observed cognitive and affective impairments.”

 

14. The group exposed to insecticides displayed decreased AChE activity, which is a sign of neural toxicity. What is the possible correlation between this, and the behavioral effects reported in the T-maze and mirror biting tests, and what does this tell us about the neurotoxic impact that pesticides have on cholinergic signaling?

The observed decrease in AChE activity in the insecticide-exposed group indicates a disruption in cholinergic signaling due to the accumulation of acetylcholine, which leads to overstimulation of cholinergic receptors. This neurotoxic mechanism is closely linked to the behavioral effects observed in the T-maze and mirror biting tests.

In the T-maze test, cognitive functions such as learning and memory rely heavily on precise cholinergic signaling. The overstimulation caused by inhibited AChE activity could impair these processes, leading to disorganized behavior or diminished decision-making abilities.

Similarly, in the mirror biting test, which evaluates impulsivity and exploratory behavior, prolonged activation of cholinergic pathways may result in increased hyperactivity, anxiety, or aggressive behaviors. These symptoms are consistent with overstimulation of the nervous system, a hallmark of the neurotoxic effects induced by insecticides. Thus, the findings suggest a strong correlation between decreased AChE activity, disrupted cholinergic signaling, and the behavioral deficits observed in these tests. This highlights the significant impact of neurotoxic pesticides on cognitive and affective functions, emphasizing the importance of further research into their effects on both animal and human health.

We insert these observations in the discussion text at lines 762-771: “The observed behavioral deficits, such as those recorded in the T-maze and mirror biting tests, may be directly linked to the decrease in AChE activity. In the T-maze, reduced AChE activity and subsequent acetylcholine accumulation could impair the cholinergic pathways critical for learning and memory, resulting in disorganized behavior or diminished cognitive performance [130]. In the mirror biting test, overstimulation of cholinergic receptors due to inhibited AChE may lead to increased impulsivity, heightened anxiety, or hyperactivity. These behavioral changes reinforce the hypothesis that the neurotoxic effects of the insecticide, amplified by sucrose, disrupt normal cholinergic signaling and contribute to the observed cognitive and affective impairments.”

15. Please enhance the resolution of Figs. 1, 2, 3, and 4. The axes in the figures are not visible.

We have changed the images.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The aim of the manuscript entitled “Assessing the Influence of Low-Doses of Sucrose on Memory Deficits in Fish Exposed to Common Insecticide Based on Fipronil and Pyriproxyfen” is to investigate the behavioral effects of exposure to a commercial insecticide formulation containing fipronil, pyriproxyfen, and other additives, as well as sucrose, individually and in combination, on Silver crucian carp (Carassius auratus gibelio). Moreover, the study assesses how these environmental and metabolic stressors influence memory, anxiety, and aggression behaviors, with a focus on the potential synergistic effects of their interaction in contributing to memory deficits and other behavioral alterations.

The topic addressed in the manuscript is highly important, as the effects of low doses of pesticides on health receive relatively little attention. The manuscript is interesting and fits well with the scope of the Journal. It is generally well-prepared, but some issues should be resolved. My specific comments are given below.

In the abstract, the statement "Pesticides represent an emerging issue of great interest in the public domain in the last decade" is broad and could be rephrased for precision, as pesticides have been a concern for much longer.

The introduction is overly detailed and should be condensed, particularly the sections from lines 74 to 133, to improve clarity and focus.

At the end of the introduction, the novelty of this study needs to be clearly defined.

The subtitles in Section 2 should be numbered.

Figure 1 is poorly visible. It should be larger. Also, the figure caption should be given below the figure.

The other figures are also too small, particularly the font, making it difficult to follow the results clearly. They should be enlarged and organized in a way that enhances readability and clarity.

The statement regarding the increased risk of developing AD due to acetylcholinesterase inhibition by organophosphates (lines 510-513) may appear counterintuitive, given that acetylcholinesterase inhibitors are commonly used as treatments for AD. It would be helpful if the authors could clarify this distinction, emphasizing how chronic pesticide exposure leads to neurodegeneration through mechanisms such as prolonged cholinergic dysfunction, oxidative stress, and neuroinflammation, contrasting this with the controlled therapeutic use of acetylcholinesterase inhibitors.

In the end, I would like to emphasize that this manuscript represents a valuable contribution to the existing literature, addressing an important and often underexplored topic. Investigating the effects of low-dose pesticide exposure, particularly in combination with metabolic stressors like sucrose, provides critical insights into the complex interplay between environmental and metabolic factors affecting health. The study's findings have the potential to advance our understanding of the broader implications of pesticide exposure. 

Author Response

Thank you very much for taking the time to review this manuscript (see attachment).

We deeply appreciate your thoughtful comments and suggestions, which have greatly contributed to enhancing the quality of our work. Below, you will find our detailed responses to each of your points. The corresponding revisions have been made directly in the manuscript and are clearly visible using track changes.

The aim of the manuscript entitled “Assessing the Influence of Low-Doses of Sucrose on Memory Deficits in Fish Exposed to Common Insecticide Based on Fipronil and Pyriproxyfen” is to investigate the behavioral effects of exposure to a commercial insecticide formulation containing fipronil, pyriproxyfen, and other additives, as well as sucrose, individually and in combination, on Silver crucian carp (Carassius auratus gibelio). Moreover, the study assesses how these environmental and metabolic stressors influence memory, anxiety, and aggression behaviors, with a focus on the potential synergistic effects of their interaction in contributing to memory deficits and other behavioral alterations.

The topic addressed in the manuscript is highly important, as the effects of low doses of pesticides on health receive relatively little attention. The manuscript is interesting and fits well with the scope of the Journal. It is generally well prepared, but some issues should be resolved. My specific comments are given below.

In the abstract, the statement "Pesticides represent an emerging issue of great interest in the public domain in the last decade" is broad and could be rephrased for precision, as pesticides have been a concern for much longer.”

Thank you for your suggestion! We have revised the sentence to read: “Although pesticides have been a constant concern for decades, in the last ten years, public discussions and scientific research have emphasized their impact on human health and the environment, drawing increased attention to the problems associated with their use.”

 

The introduction is overly detailed and should be condensed, particularly the sections from lines 74 to 133, to improve clarity and focus.

We modified.

At the end of the introduction, the novelty of this study needs to be clearly defined.

We have modified and added so as to focus on the novelty of this study.

The subtitles in Section 2 should be numbered.

We have numbered section 2.

Figure 1 is poorly visible. It should be larger. Also, the figure caption should be given below the figure.

We modified.

 

The other figures are also too small, particularly the font, making it difficult to follow the results clearly. They should be enlarged and organized in a way that enhances readability and clarity.

We modified.

 

The statement regarding the increased risk of developing AD due to acetylcholinesterase inhibition by organophosphates (lines 510-513) may appear counterintuitive, given that acetylcholinesterase inhibitors are commonly used as treatments for AD. It would be helpful if the authors could clarify this distinction, emphasizing how chronic pesticide exposure leads to neurodegeneration through mechanisms such as prolonged cholinergic dysfunction, oxidative stress, and neuroinflammation, contrasting this with the controlled therapeutic use of acetylcholinesterase inhibitors.

 

Thank you for your thoughtful comment regarding the statement on the increased risk of developing Alzheimer's disease (AD) due to acetylcholinesterase inhibition by organophosphates (lines 510-513). We appreciate the opportunity to clarify this point, as we understand how it may appear counterintuitive given the therapeutic use of acetylcholinesterase inhibitors in AD treatment.

To address this, we have revised the statement to emphasize the difference between chronic pesticide exposure and the controlled therapeutic use of acetylcholinesterase inhibitors. Chronic exposure to organophosphates can lead to prolonged cholinergic dysfunction, oxidative stress, and neuroinflammation, which contribute to neurodegeneration and an increased risk of AD. This is distinct from the short-term, regulated use of acetylcholinesterase inhibitors, which are designed to temporarily enhance cholinergic function and manage symptoms of AD without inducing the same prolonged negative effects on the nervous system.

 

In the end, I would like to emphasize that this manuscript represents a valuable contribution to the existing literature, addressing an important and often underexplored topic. Investigating the effects of low-dose pesticide exposure, particularly in combination with metabolic stressors like sucrose, provides critical insights into the complex interplay between environmental and metabolic factors affecting health. The study's findings have the potential to advance our understanding of the broader implications of pesticide exposure. 

Thank you for your valuable feedback and thoughtful comments.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Well done