Fipronil (Fpl), an insecticide belonging to the class of phenylpyrazoles, is associated with the widespread mortality of pollinator insects worldwide. Based on studies carried out on residual concentrations of Fpl commonly found in the environment, in this study, we evaluated the sublethal effects of Fpl on behavior and other neurophysiological parameters using the cockroach Nauphoeta cinerea as a biological model. Sublethal doses of Fpl (0.1–0.001 μg g−1) increased the time spent grooming and caused dose-dependent inhibition of exploratory activity, partial neuromuscular blockade in vivo and irreversible negative cardiac chronotropism. Fpl also disrupted learning and olfactory memory formation at all doses tested. These results provide the first evidence that short-term exposure to sublethal concentrations of Fpl can significantly disrupt insect behavior and physiology, including olfactory memory. These findings have implications for current pesticide risk assessment and could be potentially useful in establishing a correlation with pesticide effects in other insects, such as honey bees.

The exponential growth of the human population in the post-war period required a major expansion in crop production and resulted in a massive increase in the use of pesticides applied to crops before or after harvest to protect the commodities from deterioration (Tudi et al., 2021). The use of pesticides has increased ∼50-fold since 1950 and ∼2.5 million tons (corresponding to US$15 billion) are currently used worldwide every year (Gro Intelligence, 2018; Zaller, 2020).

Many sprayed insecticides and herbicides reach non-target species, in addition to contaminating air, water and soil (Mali et al., 2020). Pesticides are also responsible for reducing the general biodiversity and pollinator insect populations (Wells, 2007), degrading habitats by reducing plant pollination and reducing insect resources for birds (Palmer et al., 2007), and threatening endangered species (Gill and Garg, 2014). In addition, pesticide use can damage neighboring agricultural activity by forcing pests towards nearby crops that are free of pesticides, thereby causing harm and potential losses in crop yield (Dyck et al., 2021). Insecticides cause both lethal and sublethal effects in invertebrates and some vertebrates (Rani et al., 2021). Hence, there is a need for a better understanding of the potential risks posed by sublethal doses of insecticides on non-target insect species.

Fipronil (Fpl) is a broad-spectrum insecticide, belonging to the phenylpyrazole family, that acts primarily by blocking gamma-aminobutyric acid (GABA) receptors in insects (Gupta and Anadón, 2018). Fpl is a systemic pesticide that distributes amply throughout plant tissues and is toxic to any insect (and, potentially, other organisms) that feed upon the plant (Simon-Delso et al., 2015). The high to moderate solubility of Fpl and its persistence and leaching potential in soil means that this compound can easily contaminate aqueous environments and affect non-target invertebrates such as honey bees and other important pollinators that drink contaminated water (Bonmatin et al., 2015). Fpl has been found in honey bee hives in southern Queensland (Australia), where it has caused a massive loss of colonies involving the death of >600,000 honey bees (Robinson and Sanders, 2021). This finding agrees with evidence that exposure to sublethal doses of Fpl disrupts learning, memory and orientation in gregarious insects such as honey bees (Pisa et al., 2015). Navigation and foraging behavior are also impaired as Fpl reduces the proportion of active bees in the hive and causes behavioral changes, such as a deficiency in visual learning, that reduce the efficacy of foraging flights (Pisa et al., 2015).

Whereas numerous studies have investigated the neurophysiological effects of Fpl in insects (Durham et al., 2001; Narahashi et al., 2010; Kostromytska et al., 2011; Gols et al., 2020), the influence of sublethal doses of this pesticide, such as commonly encountered in the environment, on insects is still poorly understood. Several studies have examined the distribution and degradation of Fpl in soil and other environments (Gunasekara et al., 2007). Degradation typically takes 111–350 days and in soil, concentrations ranging from 0.000636 to 0.0248 mg g−1 have usually been found (Demcheck and Skrobialowski, 2003). In view of studies on bioaccumulation and residual doses of Fpl in the environment (Tingle et al., 2003; Bhatti et al., 2019; Holder et al., 2018), in this work we investigated the ability of sublethal concentrations of Fpl to disrupt olfactory memory and other behavioral parameters in Nauphoeta cinerea cockroaches. To our knowledge, this is the first study to examine the effect of Fpl on these parameters in cockroaches.

Reagents and solutions

All reagents and solutions were of high purity and were purchased from Sigma-Aldrich Chemical Co. (St Louis, MO, USA). Fipronil 800 WG® (Fpl) was purchased from BASF Agri-Production SAS (Borborema, São Paulo, Brazil). Fpl was dissolved in 0.9% NaCl (saline solution; stock solution) and diluted (working solutions) in saline solution at pre-set concentrations. All solutions used in the tests were prepared daily, before use, to ensure the stability of the compound to be tested.

Cockroaches

All experiments were carried out on adult Nauphoeta cinerea (Olivier 1789) cockroaches of both sexes (3–4 months after the adult molt), with an average weight of 500±30 mg measured during the early morning, that were reared in polyethylene plastic boxes (20 cm×50 cm) in sawdust substrate in a controlled environment at ±25°C, and a 12 h light:dark cycle (on at 07:00 h and off at 19:00 h), with water and food ad libitum (basic food composition: bone and meat meal, viscera meal, animal lipids, ground corn, wheat bran, sodium chloride, flavoring, antioxidant, folic acid, iron, copper sulfate, zinc sulfate, iodine, selenium, choline chloride, potassium, magnesium, niacin, pantothenic acid, vitamin E, vitamin B6, vitamin A, vitamin D3, vitamin K3, vitamin B1, vitamin B2 and vitamin B12). The animals were kept in the above conditions, in groups, until the beginning of each experiment. In the case of the animals subjected to memory assays, the selected insects were kept in the same conditions, but partially deprived of water and food for 15 days. The purpose of this deprivation period was to increase the animals' appeal to the solutions used in the olfactory memory tests. Thus, food and water were offered only once a day, in minimal amounts that were completely consumed, to ensure the necessary resting nutrition for the well-being of the animals.

Biological assays

The effect of Fpl on the biological activity of N. cinerea cockroaches was assessed by injecting the insects with sublethal doses of the insecticide (0.1, 0.01 or 0.001 µg g−1 body mass) directly into the hemocoel, via the third abdominal segment, using a Hamilton syringe in a final volume of 10 µl, unless otherwise specified. Insects were injected with Fpl or with saline solution alone in control experiments.

Assay for grooming behavior

The grooming behavior of the cockroaches was monitored based on the activity of the legs and antennae, which was recorded for 30 min in a controlled environment at 24–25°C, essentially as described by Stürmer et al. (2014). The tests were carried out individually and for each treatment, including the control saline group, n=30 animals were used in multiple trials and never reused again in other protocols.

Assay for locomotory activity

The locomotory activity was assessed by recording the trajectory of the cockroaches as described by Leal et al. (2018). To accomplish this, four insects each time were randomly selected and placed individually in a white polystyrene box (25 cm in length×15 cm in width×7 cm in height). Their exploratory behavior was recorded over 10 min with a Logitech® HD Webcam connected to a desktop computer (Dell, São Paulo, Brazil) that recorded and retrieved the videos for posterior analysis. Animal tracking was observed using the software idTracker (Stoelting, Denver, CO, USA) and the data were analyzed using the Insect Locomotion Tracking Program ILTP®, a freely available software developed by our research group that can be downloaded at http://sites.unipampa.edu.br/gomndi. With a script specially developed for this purpose, the following were analyzed: distance traveled (cm); number of immobile episodes (n) and time spent immobile (%). The control and treated groups were injected with saline solution or Fpl 10 min before the beginning of data acquisition. The tests were carried out individually in groups of 4 animals, each one in an individual box and for each treatment including the control saline group, n=30 animals were used and never reused again in other protocols.

Metathoracic coxal-adductor nerve–muscle preparation

The in vivo metathoracic coxal-adductor nerve–muscle preparation was mounted as described by Carrazoni et al. (2017). For this, the cockroaches were immobilized by chilling (5–7 min) and then fixed ventral-side up on a stage covered with soft rubber. After fixation, one of the third metathoracic legs was tied to an isometric force transducer (AVS Instruments, São Carlos, SP, Brazil) and indirectly electrically stimulated via the nerve fiber (0.5 Hz/5 ms). Muscle twitches were recorded for 120 min using a data acquisition and analysis system (AQCAD and ANCAD, respectively; AVS Instruments). The tests were carried out individually and for each treatment including the control saline group, n=6 animals were used and never reused again in other protocols.

Cockroach semi-isolated heart preparation

The effect of Fpl on N. cinerea heart rate was evaluated as described by Leal et al. (2020). Briefly, adult cockroaches were immobilized by chilling (5–7 min) and placed ventral-side up on a dissection plate. The lateral margins of the abdomen were cut along each side, and the ventral abdominal body wall was pulled out to show the viscera. After moving the viscera carefully aside, the heart was exposed still contracting, while attached to the dorsal body wall. The heart preparations were washed by bathing in 200μl of saline solution at room temperature (21–24°C). After 5 min of heartbeat stabilization, the treatments were delivered by exchanging the bathing solution. The average heart rate (beats min−1) in the first 5 min was taken as a reference. Heart rate (frequency) was monitored for 35 min, under a 1000× Digital Microscope (Shenzhen Huishixin Technology Co., Shenzhen, China), connected to a desktop computer (Dell) that recorded and retrieved the videos for posterior analysis. The responses to saline alone or Fpl were monitored from the 5th to the 30th minute, and the preparation was washed several times from the 30th to the 35th minute to remove the saline or Fpl and to verify the possible reversal of the toxic effects. The tests were carried out individually and for each treatment including the control saline group, n=3 animals were used and never reused again in other protocols.

Memory assay

The olfactory memory assays were done as described by Matsumoto and Mizunami (2000) with a few modifications. Before the assays, the cockroaches were deprived of food and water for 15 days. The tests were subsequently done in an apparatus (dimensions: length: 29 cm; width: 14.4 cm; height: 6.8 cm) developed specifically for olfactory tests (Fig. 1). Initially, the cockroaches were placed in a compartment at one end (labeled A) and allowed to adapt to the surroundings for 5 min. Subsequently, a gate (B) was lifted, giving access to the arena (C) where a rotating cylinder (D) simultaneously offered two odor sources placed inside a well (E).

Fig. 1.

Memory apparatus. Schematic drawing of memory apparatus, showing the compartment where the cockroaches were kept for 5 min before starting the experiment (A), the gate (B), the arena in which the experiment was performed (C), the rotating cylinder containing the odor sources (D) and the odor sources (E).

Fig. 1.

Memory apparatus. Schematic drawing of memory apparatus, showing the compartment where the cockroaches were kept for 5 min before starting the experiment (A), the gate (B), the arena in which the experiment was performed (C), the rotating cylinder containing the odor sources (D) and the odor sources (E).

The odor sources used were vanilla essence and citronella diluted 1:3 in milli-Q water. Inside each well was placed a piece of cotton (1 cm×1 cm) soaked with 500 µl of the odor sources alternately in the cylinders. Each cylinder was capped with a circle of absorbent paper. For the memory tests, 100 µl of 40% sucrose solution was placed on the absorbent paper of cylinders with citronella odor source and 100 µl of 40% NaCl solution on the paper of cylinders with vanilla odor source. This way, the animals associated the repulsive odor with the reward (40% sucrose solution). The assay began as soon as the cockroach entered the arena and involved recording the time spent probing each odor source during a 10 min period, with the position of the wells being inverted every minute. The memory test was basically divided into two phases that consisted of preference tests and olfactory memory tests. The preference tests served as positive controls to confirm the hypothesis that cockroaches are naturally attracted to vanilla odor and repelled by citronella odor. The tests were carried out individually and for each treatment including the control saline group, n=30 animals were used and never reused again in other protocols.

Statistical analysis

The results are expressed as the mean±s.e.m. As indicated in the figures, statistical comparisons between two experimental groups were done using Student's t-test; one-way ANOVA followed by Dunnett's test was used to compare treatments only with the control group; for comparisons when the y-axis had more than one variable, two-way ANOVA followed by the Bonferroni post hoc test was used, with all groups being compared with the saline control. P≤0.05 indicated significance. All statistical analyses were done using Prism v.7.0 software (GraphPad, San Diego, CA, USA).

Grooming activity

The exposure of N. cinerea to sublethal doses of Fpl (0.1, 0.01 and 0.001 μg g−1 body mass) significantly increased the leg grooming time, but did not markedly affect the antennal grooming time (Fig. 2).

Fig. 2.

Modulation of leg and antennal grooming activity in Nauphoeta cinerea cockroaches exposed to sublethal doses of fipronil. Fipronil (Fpl; 0.1, 0.01 and 0.001 μg g−1 body mass) was injected directly into the hemocoel, via the third abdominal segment, using a Hamilton syringe in a final volume of 10 µl. Fpl significantly increased leg grooming activity with minimal effect on antennal grooming activity. The columns represent the mean±s.e.m. (n=30) of the total time spent grooming. ***P≤0.001 compared with the corresponding saline control (one-way ANOVA followed by Dunnett's post hoc test). For details of statistics, see Table S1.

Fig. 2.

Modulation of leg and antennal grooming activity in Nauphoeta cinerea cockroaches exposed to sublethal doses of fipronil. Fipronil (Fpl; 0.1, 0.01 and 0.001 μg g−1 body mass) was injected directly into the hemocoel, via the third abdominal segment, using a Hamilton syringe in a final volume of 10 µl. Fpl significantly increased leg grooming activity with minimal effect on antennal grooming activity. The columns represent the mean±s.e.m. (n=30) of the total time spent grooming. ***P≤0.001 compared with the corresponding saline control (one-way ANOVA followed by Dunnett's post hoc test). For details of statistics, see Table S1.

Locomotor activity

Fpl caused a dose-dependent locomotory deficit (Fig. 3D), with an overall reduction of 46% in the total distance traveled by the cockroaches after injection of the highest dose (0.1 µg g−1) (Fig. 3A). All three doses markedly increased the number of immobile episodes (defined as more than 3 s without movement; ∼200% increase in this parameter at the highest dose) (Fig. 3B), in addition to causing lethargy in the cockroaches (∼80% increase in time spent immobile, i.e. time without movement during the total experiment, at the highest dose compared with the saline control) (Fig. 3C).

Fig. 3.

Locomotory deficit induced by sublethal doses of Fpl in N. cinerea cockroaches. The effect of Fpl (0.1, 0.01 and 0.001 μg g−1 body mass) on locomotor activity was assessed based on: (A) the total distance traveled, (B) the number of immobile episodes, (C) the percentage of time spent immobile and (D) analysis of the trajectory of the insect (blue line indicates the trajectory followed by the insect during the experiment and green dots indicate that the insect remained immobile for at least 3 s). Fpl was injected directly into the hemocoel, via the third abdominal segment, using a Hamilton syringe in a final volume of 10 µl. The columns represent the mean±s.e.m. (n=30). ***P≤0.001 (one-way ANOVA followed by Dunnett's post hoc test). For details of statistics, see Table S2.

Fig. 3.

Locomotory deficit induced by sublethal doses of Fpl in N. cinerea cockroaches. The effect of Fpl (0.1, 0.01 and 0.001 μg g−1 body mass) on locomotor activity was assessed based on: (A) the total distance traveled, (B) the number of immobile episodes, (C) the percentage of time spent immobile and (D) analysis of the trajectory of the insect (blue line indicates the trajectory followed by the insect during the experiment and green dots indicate that the insect remained immobile for at least 3 s). Fpl was injected directly into the hemocoel, via the third abdominal segment, using a Hamilton syringe in a final volume of 10 µl. The columns represent the mean±s.e.m. (n=30). ***P≤0.001 (one-way ANOVA followed by Dunnett's post hoc test). For details of statistics, see Table S2.

Effect of Fpl on cockroach metathoracic coxal-adductor nerve–muscle preparations

In cockroach metathoracic coxal-adductor neuromuscular preparations, only the highest dose of the pesticide (0.1 µg g−1) significantly affected the twitch tension and caused a maximal decrease of ∼45% in the contractile response in 50 min of experiment (Fig. 4A). Incubation with Fpl did not affect the baseline tension of the preparations, i.e. there was no muscle contracture independent of electrical stimulation (Fig. 4B).

Fig. 4.

Alterations in muscle twitch tension induced by sublethal doses of Fpl in cockroach neuromuscular preparations. Fpl (0.001, 0.01 and 0.1 mg g−1 body mass) was added to the preparations and the changes in twitch tension were monitored for 2 h. (A) Changes in twitch tension (expressed as a percentage of the basal tension) throughout the experiment. Data points represent the mean±s.e.m. (n=6). (B) Representative traces of neuromuscular twitches. In A and B, the arrows indicate the moment of Fpl application. *P≤0.05, **P≤0.01 and ***P≤0.001 compared with the corresponding intervals in the saline (control) group (two-way ANOVA followed by the Bonferroni post hoc multiple comparisons test). For details of statistics, see Table S3.

Fig. 4.

Alterations in muscle twitch tension induced by sublethal doses of Fpl in cockroach neuromuscular preparations. Fpl (0.001, 0.01 and 0.1 mg g−1 body mass) was added to the preparations and the changes in twitch tension were monitored for 2 h. (A) Changes in twitch tension (expressed as a percentage of the basal tension) throughout the experiment. Data points represent the mean±s.e.m. (n=6). (B) Representative traces of neuromuscular twitches. In A and B, the arrows indicate the moment of Fpl application. *P≤0.05, **P≤0.01 and ***P≤0.001 compared with the corresponding intervals in the saline (control) group (two-way ANOVA followed by the Bonferroni post hoc multiple comparisons test). For details of statistics, see Table S3.

Effect of Fpl on semi-isolated cockroach heart preparations

In cockroach semi-isolated heart preparations, Fpl (0.001–0.1 µg 200 µl−1) caused irreversible, dose-dependent bradycardia at all doses. In control conditions, in the absence of Fpl, the heart rate (frequency) was considered to be 100%. Thus, the maximal mean heart rates were 70.3±10.6%, 75.8±23.4% and 70.3±28.8% after 30 min for doses of 0.001, 0.01 and 0.1 µg 200 µl−1, respectively (Fig. 5). Extensive washing of the preparations did not lead to a recovery of heart rate, indicating the irreversible nature of the cardiac effect.

Fig. 5.

Irreversible negative chronotropic effect of Fpl on N. cinerea semi-isolated heart preparations. Fpl (0.001, 0.01 and 0.1 μg 200 μl−1 in 0.9% saline) was added to the preparations and the changes in heart rate (frequency) were monitored for 30 min followed by extensive washing (W), which failed to reverse the bradycardia. The data points represent the mean±s.e.m. (n=3). *P≤0.05, **P≤0.01 and ***P≤0.001 compared with the corresponding intervals in the saline (control) group (two-way ANOVA followed by the Bonferroni post hoc multiple comparisons test). For details of statistics, see Table S4.

Fig. 5.

Irreversible negative chronotropic effect of Fpl on N. cinerea semi-isolated heart preparations. Fpl (0.001, 0.01 and 0.1 μg 200 μl−1 in 0.9% saline) was added to the preparations and the changes in heart rate (frequency) were monitored for 30 min followed by extensive washing (W), which failed to reverse the bradycardia. The data points represent the mean±s.e.m. (n=3). *P≤0.05, **P≤0.01 and ***P≤0.001 compared with the corresponding intervals in the saline (control) group (two-way ANOVA followed by the Bonferroni post hoc multiple comparisons test). For details of statistics, see Table S4.

Memory test

The preference test confirmed that N. cinerea cockroaches naturally prefer the odor of vanilla rather than citronella (Fig. 6A). However, when citronella was offered in a 40% sucrose solution, the olfactory memory test showed that the odor preference became similar between citronella and vanilla (Fig. 6B). Treatment with sublethal doses of Fpl (0.001, 0.01 and 0.1 µg g−1) markedly decreased the olfactory memory for all compounds offered at all doses tested (Fig. 6C).

Fig. 6.

Effect of sublethal doses of Fpl on the olfactory memory of N. cinerea. (A) A preference test using 0.9% saline (control) showing the natural preference of the cockroaches for vanilla odor. The columns represent the mean±s.e.m. (n=30). ***P≤0.001 (Student's t-test). (B) The olfactory memory test with 0.9% saline (control) showing reversal of the insect's preference for vanilla over citronella when citronella was associated with a reward (sucrose solution). The columns represent the mean±s.e.m. (n=30). ***P≤0.001 (Student's t-test). (C) Treatment with Fpl (0.1, 0.01 and 0.001 µg g−1) reduced the length of time that cockroaches spent searching for odors associated with a reward (sucrose solution). The columns represent the mean±s.e.m. (n≥30). *P≤0.05, **P≤0.01 and ***P≤0.001 compared with the corresponding saline control (one-way ANOVA followed by Dunnett's post hoc test). For details of statistics, see Table S5.

Fig. 6.

Effect of sublethal doses of Fpl on the olfactory memory of N. cinerea. (A) A preference test using 0.9% saline (control) showing the natural preference of the cockroaches for vanilla odor. The columns represent the mean±s.e.m. (n=30). ***P≤0.001 (Student's t-test). (B) The olfactory memory test with 0.9% saline (control) showing reversal of the insect's preference for vanilla over citronella when citronella was associated with a reward (sucrose solution). The columns represent the mean±s.e.m. (n=30). ***P≤0.001 (Student's t-test). (C) Treatment with Fpl (0.1, 0.01 and 0.001 µg g−1) reduced the length of time that cockroaches spent searching for odors associated with a reward (sucrose solution). The columns represent the mean±s.e.m. (n≥30). *P≤0.05, **P≤0.01 and ***P≤0.001 compared with the corresponding saline control (one-way ANOVA followed by Dunnett's post hoc test). For details of statistics, see Table S5.

The findings described here indicate that sublethal doses of Fpl adversely affect several physiological systems of N. cinerea cockroaches and cause important behavioral changes. Exposure to Fpl increased the frequency of leg grooming but not that of antennal grooming. Grooming in insects is a fundamental activity associated with body cleanliness, courtship, social signaling, movement activity and bodily excitement (Spruijt et al., 1992; Zhukovskaya et al., 2013), and is modulated primarily by octopaminergic and dopaminergic neuronal pathways (Weisel-Eichler et al., 1999; Libersat and Pflueger, 2004; Leal et al., 2018, 2020). Changes in grooming behavior are related to fundamental factors associated with the survival and persistence of insects in the environment and can influence a species’ fertility and longevity (França et al., 2017). The alterations in grooming behavior observed here suggest a direct effect of Fpl on the central nervous system that involves the modulation of octopaminergic and dopaminergic neurotransmission, possibly in a similar manner to other toxins that we have studied (Stürmer et al., 2014; Barreto et al., 2020).

The influence of Fpl on exploratory activity was assessed by examining changes in the locomotor behavior of cockroaches. Specifically, Fpl reduced the distance walked by cockroaches and increased the number of immobile episodes and time spent immobile. Fpl also affected leg grooming behavior. These findings strongly suggest that sublethal doses of Fpl adversely affect the activity of the central nervous system, leading to locomotory and exploratory deficits. Alterations in the locomotion and exploratory behavior of insects can increase their susceptibility to predators (Marliére et al., 2015) as locomotion is the greatest defensive strategy used to avoid and escape predators in the environment (Adedara et al., 2016). In bees, for example, locomotion, including flight, is responsible for maintaining colony homeostasis through behavioral mechanisms and pheromones (Bortolotti and Costa, 2014). Flight activity is also responsible for recognition of the environment, including biotic factors (pheromones, diseases, stress, etc.) and abiotic factors (rain, food, temperature, etc.) that are important for colony survival, and its failure is associated with swarming and hive abandonment. Thus, behavior and locomotion are essential for insect maintenance and survival (Mizutani et al., 2021).

Although leg grooming behavior is mostly associated with octopaminergic neurotransmission, motivation to walk is dependent on dopamine, an important excitatory neurotransmitter (Stürmer et al., 2014; Borges et al., 2020). Fpl is a well-known agonist of the main inhibitory neurotransmitter, GABA (Byrne, 2019), and sublethal doses of this pesticide can persist in the insect neuromuscular junction (Chapman, 2012). For this reason, we cannot exclude the possibility that the mechanism behind the Fpl-mediated decrease in exploratory activity may include an inhibitory effect on peripheral neuronal activity.

Indeed, experiments with neuromuscular preparations showed that even very small sublethal doses of Fpl caused a reduction in the contractile activity of N. cinerea leg muscle. The effects observed here in cockroaches resemble those seen with sublethal doses of Fpl in the behavior of gregarious insects such as bees. In the case of bees, the remaining activity of sublethal Fpl doses might affect motor activity, which is important for foraging and orientation during the ‘dance’ used to indicate the location of resources to the colony (El Hassani et al., 2005). Thus, a Fpl-induced decrease in the contractile response would disturb the exploratory activity of those pollinators, causing disorientation and interference with the maintenance of honey bee colonies (Bovi et al., 2018).

The bradycardia caused by sublethal doses of Fpl suggests the involvement of octopamine, the main neurotransmitter associated with the modulation of heart rate in insects (Hillyer, 2018). High concentrations of octopamine cause tachycardia, whereas low concentrations are associated with bradycardia (Papaefthimiou and Theophilidis, 2011). In contrast, the role of other neurotransmitters such as acetylcholine (ACh) in insect heart rate is still controversial. Some studies have shown that ACh increases heart rate, while others suggest the opposite effect (Claros-Guzmán et al., 2020). Resolution of the relative contributions of these neurotransmitters to cardiac function will require the use of pharmacological interventions to modulate the octopaminergic and cholinergic pathways, as well as an assessment of acetylcholinesterase activity.

Another important finding of this work was that sublethal doses of Fpl interfered with olfactory memory learning and formation in N. cinerea cockroaches. Olfaction is an important sense used by insects, such as cockroaches and bees, and is closely related to behavior and physiological homeostasis. In cockroaches, which are essentially nocturnal animals, communication occurs mainly through strategies based on smell (involving pheromones and odorant bacteria) and touch (Gullan and Cranston, 2014). The insect olfactory system is one of the most developed among animals and acts by converting a chemical into an electrical signal. Physiologically, the electrical impulses generated through excitation of the system by odorous molecules are conducted from the antennal nerve to the antennal lobe (glomeruli). The information captured through this pathway is directed to a region of the animal's brain known as the ‘mushroom body’ that is associated with learning and memory processes (Modi et al., 2020). Adult cockroaches can associate aversive odors, such as mint, with a reward, such as sucrose solution, and preferential odors, such as vanilla, with a punishment, such as hypertonic saline solution (Watanabe et al., 2003).

Studies have shown that the reward-associated processing is mainly related to octopaminergic neuromodulation, whereas aversion behavior is related to dopaminergic neuromodulation (Gauthier and Grünewald, 2012). Glutamatergic receptors are also involved in learning processes, memory formation and retrieval (Gauthier and Grünewald, 2012; Leboulle, 2012). In the mushroom body, third-order neurons (Kenyon cells, KCs), which play critical roles in olfactory learning (Menzel et al., 2006; Liu et al., 2012), respond only to specific odors with a few spikes and result in sparse odor coding (Gupta and Stopfer, 2014; Lin et al., 2014). One mechanism that contributes to the formation of spatially and temporally sparse odor representations in populations of KCs is a widespread and broadly tuned GABAergic inhibition that feeds signals from KCs back to KCs (Szyszka et al., 2005; Papadopoulou et al., 2011).

The precise mechanism by which sublethal Fpl disturbs olfactory memory learning and formation is unclear but may be associated with GABAergic interneurons and the blockage of chloride channels in GABAergic and glutamatergic neurons. Thus, if sublethal doses of Fpl cause small disturbances in GABAergic interneurons, this could create hyperexcitation, thereby preventing feedback signals to KCs, leading to a destabilization of physiological processes involved in olfaction and associated memory formation. Future experiments involving site-specific intracellular recordings in mushroom bodies and other central structures of the N. cinerea central nervous system may help to elucidate the mechanism involved in the Fpl-induced disturbance of olfactory memory.

Conclusions

The results of this work demonstrate that sublethal doses of Fpl cause striking alterations in the physiology and behavior of N. cinerea cockroaches that affect the cardiovascular system together with olfactory, exploratory and locomotory behavior. These alterations involve mostly central nervous system pathways, probably by affecting octopaminergic and GABAergic neurotransmission. Overall, these findings show that the effects of sublethal doses of Fpl on insect behavior should not be neglected and they reinforce the need to examine the toxicological effects of sublethal doses of insecticides prior to their approval for general and agricultural use.

We thank Dr João Batista Teixeira da Rocha of the Federal University of Santa Maria (UFSM), Santa Maria, RS, Brazil, for providing some of the reagents used in this work. We also thank the Centro Integrado de Pesquisas em Biotecnologia-CIPBIOTEC for offering all the necessary infrastructure in terms of equipment and technical staff for the proper conduct of this work.

Author contributions

Conceptualization: C.A.D.B.; Methodology: M.E.R., L.C., B.T.B., Y.C.B., V.Q.d.S., C.A.D.B.; Software: B.T.B., C.A.D.B.; Validation: D.R.d.A., S.H., V.Q.d.S., C.A.D.B.; Formal analysis: S.S., Y.C.B., S.H., V.Q.d.S., C.A.D.B.; Investigation: M.E.R., L.C., B.T.B., S.S., Y.C.B., C.A.D.B.; Resources: L.V., C.A.D.B.; Data curation: M.E.R., V.Q.d.S., C.A.D.B.; Writing - original draft: M.E.R., D.R.d.A., S.H., L.V., C.A.D.B.; Writing - review & editing: D.R.d.A., S.H., C.A.D.B.; Visualization: C.A.D.B.; Supervision: L.V., C.A.D.B.; Project administration: C.A.D.B.; Funding acquisition: L.V., C.A.D.B.

Funding

This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Finance code 001), the Universidade Federal do Pampa (UNIPAMPA), and grants from Edital de Apoio a Grupos de Pesquisa-2019, PRONEM/FAPERGS/CNPq (003/2011) and Toxinologia/CAPES (063/2010). S.H. was supported by a research fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnologia (CNPq, grant no. 313273/2018-9). D.R.d.A. is supported by the European Union's Horizon 2020 Research and Innovation Programme under the Marie Skłodowska-Curie Grant Agreement no. 801133.

Data availability

All relevant data can be found within the article and its supplementary information.

Adedara
,
I. A.
,
Rosemberg
,
D. B.
,
de Souza
,
D.
,
Farombi
,
E. O.
,
Aschner
,
M.
,
Souza
,
D. O.
and
Rocha
,
B. T. D.
(
2016
).
Neurobehavioral and biochemical changes in Nauphoeta cinerea following dietary exposure to chlorpyrifos
.
Pestic. Biochem. Physiol.
130
,
22
-
30
.
Barreto
,
Y. C.
,
Rosa
,
M. E.
,
Zanatta
,
A. P.
,
Borges
,
B. T.
,
Hyslop
,
S.
,
Vinadé
,
L. H.
and
Dal Belo
,
C. A.
(
2020
).
Entomotoxicity of jaburetox: revisiting the neurotoxic mechanisms in insects
.
J. Venom Res.
10
,
38
-
44
.
Bhatti
,
S.
,
Satyanarayana
,
G. N. V.
,
Patel
,
D. K.
and
Satish
,
A.
(
2019
).
Bioaccumulation, biotransformation and toxic effect of fipronil in Escherichia coli
.
Chemosphere
231
,
207
-
215
.
Bonmatin
,
J.
,
Giorio
,
C.
,
Girolami
,
V.
,
Goulson
,
D.
and
Kreutzweiser
,
D. P.
(
2015
).
Environmental fate and exposure: neonicotinoids and fipronil
.
Environ. Sci. Pollut. Res.
22
,
35
-
67
.
Borges
,
B. T.
,
de Brum Vieira
,
P.
,
Leal
,
A. P.
,
Karnopp
,
E.
,
Ogata
,
B. A. B.
,
Rosa
,
M. E.
,
Barreto
,
Y. C.
,
Oliveira
,
R. S.
,
Dal Belo
,
C. A.
and
Vinadé
,
L.
(
2020
).
Modulation of octopaminergic and cholinergic pathways induced by Caatinga tree Manilkara rufula chemical compounds in Nauphoeta cinerea cockroaches
.
Pestic. Biochem. Physiol.
169
,
104651
.
Bortolotti
,
L.
and
Costa
,
C.
(
2014
).
Chemical communication in the honey bee society
. In
Neurobiology of Chemical Communication
(ed.
C.
Mucignat-Caretta
).
Boca Raton, FL
:
CRC Press/Taylor & Francis
. https://www.ncbi.nlm.nih.gov/books/NBK200983/
Bovi
,
T. S.
,
Zaluski
,
R.
and
Orsi
,
R. O.
(
2018
).
Toxicity and motor changes in Africanized honey bees (Apis mellifera L.) exposed to fipronil and imidacloprid
.
An. Acad. Bras. Sci.
90
,
239
-
245
.
Byrne
,
J. H.
(
2019
).
The Oxford Handbook of Invertebrate Neurobiology
.
New York
:
Oxford University Press
.
Carrazoni
,
T.
,
de Brum Vieira
,
P.
,
da Silva
,
P. G.
,
Heberle
,
M. A.
,
Sturmer
,
G. D.
,
Correa
,
M. S.
,
Batista
,
P. A.
,
Moura
,
S.
,
Mendez
,
A. S.
and
Dal Belo
,
C. A.
(
2017
).
Mechanism of the entomotoxic activity induced by Araucaria angustifolia methanolic extract in Nauphoeta cinerea lobster cockroaches
.
J. Bot. Res.
1
,
38
-
49
.
Chapman
,
R. F.
(
2012
).
The Insects: Structure and Function
, 5th edn.
Cambridge
:
Cambridge University Press
.
Claros-Guzmán
,
A.
,
Rodríguez
,
M. G.
,
Heredia-Rivera
,
B.
and
González-Segovia
,
R.
(
2020
).
Three-dimensional analysis of the heart function and effect of cholinergic agonists in the cockroach Gromphadorhina portentosa
.
J. Comp. Physiol. A
206
,
857
-
870
.
Demcheck
,
D. K.
and
Skrobialowski
,
S. C.
(
2003
).
Fipronil and degradation products in the rice-producing areas of the Mermentau River Basin, Louisiana, February-September 2002
.
Fact Sheet 010-03
.
Dyck
,
V. A.
,
Hendrichs
,
J.
and
Robinson
,
A. S.
(
2021
).
Sterile Insect Technique. Principles and Practice in Area-Wide Integrated Pest Management
, 2nd edn.
CRC Press, Taylor and Francis Group
.
Durham
,
E. W.
,
Scharf
,
M. E.
and
Siegfried
,
B. D.
(
2001
).
Toxicity and neurophysiological effects of fipronil and its oxidative sulfone metabolite on European corn borer larvae (Lepidoptera: Crambidae)
.
Pestic. Biochem. Phys
.
El Hassani
,
A. K.
,
Dacher
,
M.
,
Gauthier
,
M.
and
Armengaud
,
C.
(
2005
).
Effects of sublethal doses of fipronil on the behavior of the honeybee (Apis mellifera)
.
Pharmacol. Biochem. Behav.
82
,
30
-
39
.
França
,
S. M.
,
Breda
,
M. O.
,
Barbosa
,
D. R. S.
,
Araujo
,
A. M. N.
and
Guedes
,
C. A.
(
2017
).
The sublethal effects of insecticides in insects
. In
Biological Control of Pest and Vector Insects
(ed.
V. D. C.
Shields
), pp. 23-39.
IntechOpen
.
Gauthier
,
M.
and
Grünewald
,
B.
(
2012
).
Neurotransmitter systems in the honey bee brain: functions in learning and memory
. In
Honeybee Neurobiology and Behavior
(ed.
C.
Galizia
,
D.
Eisenhardt
and
M.
Giurfa
), pp. 155-169.
Berlin, Heidelberg, New York
:
Springer Verlag
.
Gill
,
H. K.
and
Garg
,
H.
(
2014
).
Pesticides: environmental impacts and management strategies
. In
Pesticides: Toxic Aspects
(ed.
M. L.
Larramendy
and
S.
Soloneski
), pp. 187-230.
Rijeka, Croatia: Intech
.
Gols
,
R.
,
WallisDeVries
,
M. F.
and
van Loon
,
J. J. A.
(
2020
).
Reprotoxic effects of the systemic insecticide fipronil on the butterfly Pieris brassicae
.
Proc. R. Soc.
287
,
20192665
.
Gro Intelligence
(
2018
).
A look at fertilizer and pesticide use in the US
. (
Accessed 01 May 2022
).
Gullan
,
P. J.
and
Cranston
,
P. S.
(
2014
).
The Insects: an Outline of Entomology
, 5th edn.
London
:
Wiley-Blackwell
.
Gunasekara
,
A. S.
,
Truong
,
T.
,
Goh
,
K. S.
,
Spurlock
,
F.
and
Tjeerderma
,
R. S.
(
2007
).
Environmental fate and toxicology of fipronil
.
J. Pestic. Sci.
32
,
189
-
199
.
Gupta
,
R. C.
and
Anadón
,
A.
(
2018
).
Fipronil
. In
Veterinary Toxicology: Basic and Clinical Principles
, 3rd edn (ed.
R. C.
Gupta
), pp.
533
-
538
.
New York
:
Academic Press
.
Gupta
,
N.
and
Stopfer
,
M.
(
2014
).
A temporal channel for information in sparse sensory coding
.
Curr. Biol.
24
,
2247
-
2256
.
Hillyer
,
J. F.
(
2018
).
Insect heart rhythmicity is modulated by evolutionarily conserved neuropeptides and neurotransmitters
.
Curr. Opin. Insect Sci.
29
,
41
-
48
.
Holder
,
P. J.
,
Jones
,
A.
,
Tyler
,
C. R.
and
Cresswell
,
J. E.
(
2018
).
Fipronil pesticide as a suspect in historical mass mortalities of honeybees
.
Proc. Natl. Acad. Sci. USA
115
,
13033
-
13038
.
Kostromytska
,
O. S.
,
Buss
,
E. A.
and
Scharf
,
M. E.
(
2011
).
Toxicity and neurophysiological effects of selected insecticides on the mole cricket, Scapteriscus vicinus (Orthoptera: Gryllotalpidae)
.
Pestic. Biochem. Phys.
100
,
27
-
34
.
Leal
,
A. P.
,
Oliveira
,
R. S.
,
Perin
,
A. P. A.
,
Borges
,
B. T.
,
de Brum Vieira
,
P.
,
dos Santos
,
T. G.
,
Vinadé
,
L.
,
Valsecchi
,
C.
and
Dal Belo
,
C. A.
(
2018
).
Entomotoxic activity of Rhinella icterica (Spix, 1824) toad skin secretion in Nauphoeta cinerea cockroaches: an octopamine-like modulation
.
Pest. Biochem. Physiol.
148
,
175
-
181
.
Leal
,
A. P.
,
Karnopp
,
E.
,
Barreto
,
Y. C.
,
Oliveira
,
R. S.
,
Rosa
,
M. E.
,
Borges
,
B. T.
,
Goulart
,
F. L.
,
de Souza
,
V. Q.
,
Laikowski
,
M. M.
,
Moura
,
S.
, et al. 
(
2020
).
The insecticidal activity of Rhinella schneideri (Werner, 1894) paratoid secretion in Nauphoeta cinerea cockroaches
.
Toxins
12
,
630
.
Leboulle
,
G.
(
2012
).
Glutamate neurotransmission in the honey bee central nervous system
. In
Honeybee Neurobiology and Behavior
(ed.
C.
Giovanni Galizia
,
D.
Eisenhardt
and
M.
Giurfa
), pp.
171
-
184
.
Dordrecht
:
Springer
.
Libersat
,
F.
and
Pflueger
,
H.-J.
(
2004
).
Monoamines and the orchestration of behavior
.
Bioscience.
54
,
17
-
25
.
Lin
,
A. C.
,
Bygrave
,
A. M.
,
Calignon
,
A.
,
Tzumin
,
L.
and
Miesenböck
,
G.
(
2104
).
Sparse, decorrelated odor coding in the mushroom body enhances learned odor discrimination
.
Nat. Neurosci.
17
,
559
-
568
.
Liu
,
C.
,
Plaçais
,
P. Y.
,
Yamagata
,
N.
,
Pfeiffer
,
B. D.
,
Aso
,
Y.
,
Friedrich
,
A. B.
,
Siwanowicz
,
I.
,
Rubin
,
G. M.
,
Preat
,
T.
and
Tanimoto
,
H.
(
2012
).
A subset of dopamine neurons signals reward for odour memory in Drosophila
.
Nature
488
,
512
-
516
.
Tudi
,
M.
,
Daniel
,
R. H.
,
Wang
,
L.
,
Lyu
,
J.
,
Sadler
,
R.
,
Connell
,
D.
,
Chu
,
C.
and
Phung
,
D. T.
(
2021
).
Agriculture development, pesticide application and its impact on the environment int
.
J. Environ. Health Res.
18
,
1112
.
Mali
,
S. C.
,
Raj
,
S.
and
Trivedi
,
R.
(
2020
).
Nanotechnology: a novel approach to enhance crop productivity
.
Biochem. Biophys. Rep.
24
,
100821
.
Marliére
,
N. P.
,
Latorre-Estivalis
,
J. M.
,
Lorenzo
,
M. G.
,
Carrasco
,
D.
,
Alves-Silva
,
J.
,
Rodrigues
,
J. d. O.
,
Ferreira
,
L. L.
,
Lara
,
L. M.
,
Lowenberger
,
C.
and
Guarneri
,
A. A.
(
2015
).
Trypanosomes modify the behavior of their insect hosts: effects on locomotion and on the expression of a related gene
.
PLoS Negl. Trop. Dis.
9
,
e0003973
.
Matsumoto
,
Y.
and
Mizunami
,
M.
(
2000
).
Olfactory learning in the cricket Gryllus bimaculatus
.
J. Exp. Biol.
203
,
2581
-
2588
.
Menzel
,
R.
,
Leboulle
,
G.
and
Eisenhardt
,
D.
(
2006
).
Small brains, bright minds
.
Cell
124
,
237
-
239
.
Mizutani
,
H.
,
Tagai
,
K.
,
Habe
,
S.
,
Takaku
,
S.
,
Uebi
,
T.
,
Kimura
,
T.
,
Hariyama
,
T.
and
Ozaki
,
M.
(
2021
).
Antenna cleaning is essential for precise behavioral response to alarm pheromone and nestmate–nonnestmate discrimination in Japanese carpenter ants (Camponotus japonicus)
.
Insects
12
,
773
.
Modi
,
M. N.
,
Shuai
,
Y.
and
Turner
,
G. C.
(
2020
).
The Drosophila mushroom body: from architecture to algorithm in a learning circuit
.
Ann. Rev. Neurosci.
43
,
465
-
484
.
Narahashi
,
T.
,
Zhao
,
X.
,
Ikeda
,
T.
,
Salgado
,
V. L.
and
Yeh
,
J. Z.
(
2010
).
Glutamate-activated chloride channels: Unique fipronil targets present in insects but not in mammals
.
Pestic. Biochem. Phys.
97
,
149
-
152
.
Palmer
,
W. E.
,
Bromley
,
P. T.
and
Brandenburg
,
R. L.
(
2007
).
Wildlife and pesticides – peanuts. North Carolina Cooperative Extension Service. Pesticide Risk Assessment
.
Papaefthimiou
,
C.
and
Theophilidis
,
G.
(
2011
).
Octopamine – a single modulator with double action on the heart of two insect species (Apis mellifera macedonica and Bactrocera oleae): acceleration vs. inhibition
.
J. Insect Physiol.
57
,
316
-
325
.
Papadopoulou
,
M.
,
Cassenaer
,
S.
,
Nowotny
,
T.
and
Laurent
,
G.
(
2011
).
Normalization for sparse encoding of odors by a wide-field interneuron
.
Science.
332
,
721
-
725
.
Pisa
,
L. W.
,
Belzunces
,
L. P.
,
Bonmatin
,
J. M.
,
Downs
,
C. A.
,
Goulson
,
D.
,
Kreutzweiser
,
D. P.
,
Krupke
,
C.
,
Liess
,
M.
,
Mcfield
,
M.
,
Morrissey
,
C. A.
et al. 
(
2015
).
Effects of neonicotinoids and fipronil on non-target invertebrates
.
Environ. Sci. Pollut. Res. Int.
22
,
68
-
102
.
Rani
,
L.
,
Thapa
,
K.
,
Kanojia
,
N.
,
Sharma
,
N.
,
Singh
,
S.
,
Grewal
,
A. S.
,
Srivastav
,
A. L.
and
Kaushal
,
J.
(
2021
).
An extensive review on the consequences of chemical pesticides on human health and environment
.
J. Clean. Prod.
283
,
124657
.
Robinson
,
L.
and
Sanders
,
B.
(
2021
).
Bee deaths spark investigation after traces of chemical Fipronil found in hives
.
ABC News
. . (
accessed on August 15, 2022
).
Simon-Delso
,
N.
,
Amaral-Rogers
,
V.
,
Belzunces
,
L. P.
,
Bonmatin
,
J.-M.
,
Chagnon
,
M.
,
Downs
,
C.
,
Furlan
,
L.
,
Gibbons
,
D. W.
,
Giorio
,
C.
and
Girolami
,
V.
(
2015
).
Systemic insecticides (neonicotinoids and fipronil): trends, uses, mode of action and metabolites
.
Environ. Sci. Pollut. Res. Int.
22
,
3
-
5
.
Spruijt
,
B. M.
,
van Hooff
,
J. A.
and
Gispen
,
G. H.
(
1992
).
Ethology and neurobiology of grooming behavior
.
Physiol. Rev.
3
,
825
-
852
.
Stürmer
,
G. D.
,
de Freitas
,
T. C.
,
Heberle
,
M. A.
,
Assis
,
D. R.
,
Vinadé
,
L.
,
Pereira
,
A. B.
,
Franco
,
J. L.
and
Dal Belo
,
C. A.
(
2014
).
Modulation of dopaminergic neurotransmission induced by sublethal doses of the organophosphate trichlorfon in cockroaches
.
Ecotoxicol. Environ. Safety
109
,
56
-
62
.
Szyszka
,
P.
,
Ditzen
,
M.
,
Galkin
,
A.
,
Galizia
,
C. G.
and
Menzel
,
R.
(
2005
).
Sparsening and temporal sharpening of olfactory representations in the honeybee mushroom bodies
.
J. Neurophysiol.
94
,
3303
-
3313
.
Tingle
,
C. C. D.
,
Rother
,
J. A.
,
Dewhurst
,
C. F.
,
Lauer
,
S.
and
King
,
W. J.
(
2003
).
Fipronil: environmental fate, ecotoxicology, and human health concerns
.
Rev. Environ. Contam. Toxicol.
176
,
1
-
66
.
Zaller
,
J. G.
(
2020
).
Daily Poison – Pesticides – an Underestimated Danger
.
Springer
.
Zhukovskaya
,
M.
,
Yanagawa
,
A.
and
Forschler
,
B.
(
2013
).
Grooming behavior as a mechanism of insect disease defense
.
Insects
4
,
609
-
630
.
Watanabe
,
H.
,
Kobayashi
,
Y.
,
Sakura
,
M.
,
Matsumoto
,
Y.
and
Mizunami
,
M.
(
2003
).
Classical olfactory conditioning in the cockroach Periplaneta americana
.
Zool. Sci.
20
,
1447
-
1454
.
Weisel-Eichler
,
A.
,
Haspel
,
G.
and
Libersat
,
F.
(
1999
).
Venom of a parasitoid wasp induces prolonged grooming in the cockroach
.
J. Exp. Biol.
202
,
957
-
964
.
Wells
,
M.
(
2007
).
Vanishing bees threaten U.S. crops
.
BBC news
(
accessed March 14, 2022
).

Competing interests

The authors declare no competing or financial interests.

Supplementary information