The Effect of Rosmarinus officinalis Essential Oil Fumigation on Biochemical, Behavioral, and Physiological Parameters of Callosobruchus maculatus

This study explores the influence of rosemary, Rosmarinus officinalis (L.) essential oil (EO) on the biochemical (acetylcholinesterase, catalase, and glutathione S-transferase), physiological (oxygen consumption), and behavioral (open field test, repellency) parameters of an important stored product insect: cowpea weevil, Callosobruchus maculatus (F.). R. officinalis EO exhibited effective insecticidal action against C. maculatus even at relatively low concentrations. LC50 = 15.69 μL/L air, and was highly repellent at concentrations equal to or above LC25. Statistically significant inhibition in locomotor activity occurred only after the acute exposure to the EO at LC12.5 and LC25. The oxygen consumption test showed metabolism increase only at LC50. An increase in activity was observed in the case of all three enzymes examined. The presented data provides a potentially valuable resource in designing more environmentally friendly and safer insecticide agents.


Introduction
Rosemary, Rosmarinus officinalis (L.), is an herbaceous member of the mint family Lamiaceae, and is native to the Mediterranean region. However, it is well-known, and globally and widely cultured because of its cultural, culinary, aesthetic, and folk medicinal usage [1]. A distinctive characteristic of R. officinalis is its fragrance, originating from the high content of volatile compounds in the plant material. These compounds can be extracted by steam distillation and concentrated in the form of R. officinalis essential oil (EO).
One of the most widely recognized hypotheses of the mode of action R. officinalis EO toxicity is its ability to inhibit the acetylcholinesterase (AChE). AChE inhibition is also the primary effect of

Fumigation Mortality
Fumigation mortality was assessed in four replications per volume, each comprising 30 concentrations (0.1-3.0 µL by 0.1 µL increments). Each replication consisted of ten mixed-sex insects (n = 40; Σn = 1240) that were put into 50 mL non-hermetic, conical, plastic containers with tight-fitting lids (30 mm height, 55 and 48 mm diameter). Tested EO (or ultrapure water in the control group) was applied on a cotton pad attached to the cover of the container. Dead beetles were counted after 24 h. Insects were considered dead when no movement for 1 h was observed. The bioassays were conducted under constant conditions, i.e., 30 ± 1 • C, 50% relative humidity. LC 50 was determined by fitting a model to the data obtained from the mortality assay. The model was fitted with the least-squares fit method. The LC50 was calculated from the slope of the fitted curve using GraphPad Prism v6.00 software (GraphPad Software, San Diego, CA, USA).

Repellency
To assess the repellent activity of the R. officinalis EO, a repellency test described by Francikowski et al. [13] was adapted. For every volume (volume corresponding to LC 12.5 ; LC 25 ; LC 50 ), 40 beetles were used. Insects were placed individually in rectangular chambers (3 mm height, 15 mm width, 160 mm long, made of transparent Lucite). In each of the chambers, a flow of air from both sides was maintained. The inlet air for the odor side was pumped through the cotton wool (placed inside the Insects 2020, 11, 344 3 of 11 gas bubbler in place of liquid) with applied EO (the volumes of applied EO have been recalculated to match the volume of the gas bubbler-200 cm 3 ) while for the opposite site through cotton wool with an equal volume of ultra-pure water. The airflow was kept constant at 10 L/h. The insects were able to explore the chambers freely, and their movement was recorded with Microsoft LifeCam Studio and AMCap software. Recordings lasted for 10 min at 15 fps frame rate with 640 × 860 px resolution. The obtained data was analyzed as described in Baran et al. [14], which allowed the calculation of the Preference Index (PI).

Open Field Test
The locomotor response of C. maculatus to R. officinalis EO fumigation was assessed in the open field test-performed in the glass Petri dish (ø = 100 mm, h = 20 mm) arenas. The assessments were conducted in four replications per volume corresponding to LC 12.5 ; LC 25 ; LC 50 (the volumes have been recalculated to match the volume of the Petri dish-157.08 cm 3 ). For each replication ten mixed-sex insects were used (n = 40; Σn = 320). Open field assessments were conducted in two variants: prolonged exposition (24 h of fumigation) and acute (insect movement was recorded directly during the fumigation). The fumigation was performed in the Petri dishes lined with filter paper disks (Whatman N • 1). On each paper filter, a specific volume of the EO was applied.
The locomotor activity recording lasted for 50 min. The analysis of the video was conducted in five intervals, each consisting of ten minutes. Trajectories of insects' movement were extracted with SwissTrack 4 software [15] and subsequently analyzed in the R environment with trajr [16] package. The time spent on resting and distance traveled were analyzed.

Oxygen Consumption
An oxygen consumption test was conducted in four replications, using a SiLab data acquisition unit and oxygen sensor (sampling rate: 1/sec) tightly fitted into the 50 mL Falcon ® tube (BD Biosciences, San Jose, CA, USA). For each experiment, ten mixed-sex individuals (n = 40; Σn = 160) were put into airtight tubes containing 15 g of mung beans. To avoid the contact of the insects with the tested EO, cotton wool wetted with EO was placed in meshed three-dimensional (3D) printed containers (cylindrical, d = 20 mm, h = 26 mm fabricated using photocurable resin) mounted on the bottom of Falcon ® tubes. To each of the containers, the volume corresponding to LC 12.5 ; LC 25 ; LC 50 of the tested EO (ultrapure water in the control group) was applied. Measurements were started immediately after putting insects into the Falcon ® tube and lasted for 1 h. The bioassays were conducted in constant conditions, i.e., 30 ± 1 • C, 50% relative humidity.

Enzyme Assays
For each replication, 10 mixed-sex insects were weighed and homogenized in Sorensen's buffer (0.05 M; pH 7.4) in a 1:10 ratio. Thereafter, the homogenate was centrifuged (10,000 RPM, 10 min, 4 • C). Blind tests were prepared using buffers instead of homogenates. All measurements were performed with the Tecan M200 spectrophotometer (Tecan Group Ltd., Männedorf, Switzerland) in the Corning ® 96-well UV-Transparent microplates (Corning, Tewksbury, MA, USA). For each well, five measurements in different regions were taken, and the obtained results were averaged. In the samples the protein content was determined using the Bradford method, and then the enzyme activity was converted into ∆/min/mg of protein [17].  The catalase activity was determined using a modified Orr's [19] spectrophotometric assay based on the changes of the absorbance of 230 nm UV light by H 2 O 2 over time in the presence of catalase. The samples were diluted 40 fold in Sorensen's medium (0.05 M; pH 7.4). The reaction mixture consists of 40 µL 0.03 M H 2 O 2 and 80 µL of the diluted sample. The six consecutive measurements were performed every 10 s.

Glutathione S-Transferase (GST) Activity Assay
The GST activity was determined using Yu's spectrophotometric assay [20] based on the changes of the absorbance of 340 nm light by CDNB (GST substrate) over time in the presence of GST. The reaction mixture consists of 10 µL of the sample, 5 µL CDNB (15 mM), and 200 µL GSH (1 mM). The six consecutive measurements were performed every 30 s.

Statistical Analysis
All statistical analysis were conducted using GraphPad Prism v6.00 software on Windows OS. For all the obtained data, the normality test was performed (Shapiro-Wilk normality test). The repellent effect was analyzed with the usage of a nonparametric test-Kruskal-Wallis with multiple comparisons. For the analysis of oxygen consumption, locomotor activity, and enzymatic activity, an ANOVA multiple comparisons test (Tukey test, Holm-Sidak test, p < 0.05) was used. Additionally, for the locomotor activity the Two-way RM (reapeted measures) ANOVA was performed with p < 0.05.

Repellency
The results of the preference assay using three determined concentrations (LC12.5, LC25, LC50) showed differentiated reactions depending on the used concentration ( Figure 2). For insects treated with LC12.5 of R. officinalis EO, the PI was close to zero. However, groups treated with LC25 and LC50 differed significantly from the LC12.5 group. The repellent effects of EO in the groups treated with LC50 are demonstrated by the insects avoiding the region of the system with odor inlet.

Open Field Test
The effect of R. officinalis EO on the locomotor activity of the C. maculatus varied significantly depending on the time between treatment and measurement. Immediately after the treatment, the activity measured in the first interval is comparable in all the groups. The differences emerged with the second interval where insects treated with LC12.5 and LC25 displayed a significant decrease in

Repellency
The results of the preference assay using three determined concentrations (LC 12.5 , LC 25 , LC 50 ) showed differentiated reactions depending on the used concentration ( Figure 2). For insects treated with LC 12.5 of R. officinalis EO, the PI was close to zero. However, groups treated with LC 25 and LC 50 differed significantly from the LC 12.5 group. The repellent effects of EO in the groups treated with LC 50 are demonstrated by the insects avoiding the region of the system with odor inlet.

Repellency
The results of the preference assay using three determined concentrations (LC12.5, LC25, LC50) showed differentiated reactions depending on the used concentration ( Figure 2). For insects treated with LC12.5 of R. officinalis EO, the PI was close to zero. However, groups treated with LC25 and LC50 differed significantly from the LC12.5 group. The repellent effects of EO in the groups treated with LC50 are demonstrated by the insects avoiding the region of the system with odor inlet.

Open Field Test
The effect of R. officinalis EO on the locomotor activity of the C. maculatus varied significantly depending on the time between treatment and measurement. Immediately after the treatment, the activity measured in the first interval is comparable in all the groups. The differences emerged with the second interval where insects treated with LC12.5 and LC25 displayed a significant decrease in

Open Field Test
The effect of R. officinalis EO on the locomotor activity of the C. maculatus varied significantly depending on the time between treatment and measurement. Immediately after the treatment, the activity measured in the first interval is comparable in all the groups. The differences emerged with the second interval where insects treated with LC 12.5 and LC 25 displayed a significant decrease in activity in comparison to control. The group treated with LC 50 displayed activity at the level of between the control and groups treated with lower concentrations but did not differ from those groups until the fourth interval. LC 50 differed significantly from the control in the fifth interval, where the activity decreased to the level of the groups treated with lower concentrations. Activity after 24 h was significantly lower, and the groups did not differ from each other (Figure 3) throughout the measurement period.
Insects 2020, 11, 344 6 of 11 activity in comparison to control. The group treated with LC50 displayed activity at the level of between the control and groups treated with lower concentrations but did not differ from those groups until the fourth interval. LC50 differed significantly from the control in the fifth interval, where the activity decreased to the level of the groups treated with lower concentrations. Activity after 24 h was significantly lower, and the groups did not differ from each other (Figure 3) throughout the measurement period.

Oxygen Consumption
The treatment with R. officinalis EO significantly affected the level of oxygen consumption of the exposed insects. The treatment with LC50 resulted in a significant increase in oxygen consumption, while groups treated with LC12.5 and LC25 did not differ significantly from the controls (Figure 4).

Acetylcholinesterase (AChE) Activity Assay
The analysis of acetylcholinesterase activity revealed changes caused by the treatment with R. officinalis EO. The two lowest LC12.5 and LC25 concentrations did not differ significantly from the

Oxygen Consumption
The treatment with R. officinalis EO significantly affected the level of oxygen consumption of the exposed insects. The treatment with LC 50 resulted in a significant increase in oxygen consumption, while groups treated with LC 12.5 and LC 25 did not differ significantly from the controls (Figure 4).
Insects 2020, 11, x 6 of 12 activity in comparison to control. The group treated with LC50 displayed activity at the level of between the control and groups treated with lower concentrations but did not differ from those groups until the fourth interval. LC50 differed significantly from the control in the fifth interval, where the activity decreased to the level of the groups treated with lower concentrations. Activity after 24 h was significantly lower, and the groups did not differ from each other (Figure 3) throughout the measurement period.

Oxygen Consumption
The treatment with R. officinalis EO significantly affected the level of oxygen consumption of the exposed insects. The treatment with LC50 resulted in a significant increase in oxygen consumption, while groups treated with LC12.5 and LC25 did not differ significantly from the controls (Figure 4).

Acetylcholinesterase (AChE) Activity Assay
The analysis of acetylcholinesterase activity revealed changes caused by the treatment with R. officinalis EO. The two lowest LC12.5 and LC25 concentrations did not differ significantly from the

Acetylcholinesterase (AChE) Activity Assay
The analysis of acetylcholinesterase activity revealed changes caused by the treatment with R. officinalis EO. The two lowest LC 12.5 and LC 25 concentrations did not differ significantly from the control (however, differed significantly from each other), while the strongest stimulation of activity was observed again in the group treated with LC 50 (Figure 5).
Insects 2020, 11, x 7 of 12 control (however, differed significantly from each other), while the strongest stimulation of activity was observed again in the group treated with LC50 ( Figure 5).

Catalase (CAT) Activity Assay
Catalase activity in the control group was the lowest of all groups. Increased concentration of EO increased catalase activity. The groups treated with LC12.5 and LC25 did not differ from each other, but they differed from the control and LC50 groups. The highest activity was observed in the group treated with LC50 concentration (Figure 6).

Catalase (CAT) Activity Assay
Catalase activity in the control group was the lowest of all groups. Increased concentration of EO increased catalase activity. The groups treated with LC 12.5 and LC 25 did not differ from each other, but they differed from the control and LC50 groups. The highest activity was observed in the group treated with LC 50 concentration ( Figure 6).

Catalase (CAT) Activity Assay
Catalase activity in the control group was the lowest of all groups. Increased concentration of EO increased catalase activity. The groups treated with LC12.5 and LC25 did not differ from each other, but they differed from the control and LC50 groups. The highest activity was observed in the group treated with LC50 concentration (Figure 6).

Glutathione S-Transferase (GST) Activity Assay
The Glutathione S-transferase activity did not change in the two lowest applied concentrations (LC12.5 and LC25), compared to, the control group. Significant differences were observed only in the group treated with LC50 concentration compared to control (Figure 7).

Glutathione S-Transferase (GST) Activity Assay
The Glutathione S-transferase activity did not change in the two lowest applied concentrations (LC 12.5 and LC 25 ), compared to, the control group. Significant differences were observed only in the group treated with LC 50 concentration compared to control (Figure 7).

Discussion
Contemporary agriculture relies heavily on pesticides, including insecticides. However, due to their extensive (and frequently inappropriate) usage, these substances are permeating into soil and ground waters, posing a risk to non-target organisms, including humans [22]. Therefore, there is a growing emphasis on developing safe alternatives that could potentially replace commonly used insecticides, while also being cheaper and more convenient to use.
One group of substances possibly possessing required insecticide characteristics to which increasing attention is being given are volatile plant extracts-essential oils. Their insecticidal activity, biodegradability by the soil microorganisms, and low toxicity to mammals, make them a good candidate as suitable pest management agents [23]. Moreover, many of these substances have been declared as generally recognized as safe (GRAS) by the United States Food and Drug Administration and are widely used in the food industry as flavoring agents [24].
Based on a wide range of studies and pilot tests conducted by authors, herein the R. officinalis EO presented high insecticidal potential against C. maculatus is an extract from R. officinalis. In a fumigant mortality assay, we showed that LC50 of this EO against C. maculatus is 19.4 μL/L air. The presented data coalesce with previously reported results published by Gudek and Cetin [7], where the LC50 value was 15.69 μL/L air, and support the potential of the application of the said EO as the pest management agent. In addition, they demonstrated that R. officinalis EO does not affect the germination capacity of protected commodities [4].
Most of available articles on insecticidal potential of EOs focus mainly on the mortality assessments or EOs' influence on biochemical parameters, while the presented article aims to examine the influence of the rosemary EO also on physiological and behavioral parameters. Analysis of a broad spectrum of parameters allows a better understanding of the influence of EO on the tested insect.
Repellency is a behavioral measure describing the extent of the aversive action of the tested substance on the tested organism. Stronger the repellent action-the further the extent to which the animal would avoid the space treated with the tested substance. Most of the commonly used

Discussion
Contemporary agriculture relies heavily on pesticides, including insecticides. However, due to their extensive (and frequently inappropriate) usage, these substances are permeating into soil and ground waters, posing a risk to non-target organisms, including humans [22]. Therefore, there is a growing emphasis on developing safe alternatives that could potentially replace commonly used insecticides, while also being cheaper and more convenient to use.
One group of substances possibly possessing required insecticide characteristics to which increasing attention is being given are volatile plant extracts-essential oils. Their insecticidal activity, biodegradability by the soil microorganisms, and low toxicity to mammals, make them a good candidate as suitable pest management agents [23]. Moreover, many of these substances have been declared as generally recognized as safe (GRAS) by the United States Food and Drug Administration and are widely used in the food industry as flavoring agents [24].
Based on a wide range of studies and pilot tests conducted by authors, herein the R. officinalis EO presented high insecticidal potential against C. maculatus is an extract from R. officinalis. In a fumigant Insects 2020, 11, 344 8 of 11 mortality assay, we showed that LC 50 of this EO against C. maculatus is 19.4 µL/L air. The presented data coalesce with previously reported results published by Gudek and Cetin [7], where the LC50 value was 15.69 µL/L air, and support the potential of the application of the said EO as the pest management agent. In addition, they demonstrated that R. officinalis EO does not affect the germination capacity of protected commodities [4].
Most of available articles on insecticidal potential of EOs focus mainly on the mortality assessments or EOs' influence on biochemical parameters, while the presented article aims to examine the influence of the rosemary EO also on physiological and behavioral parameters. Analysis of a broad spectrum of parameters allows a better understanding of the influence of EO on the tested insect.
Repellency is a behavioral measure describing the extent of the aversive action of the tested substance on the tested organism. Stronger the repellent action-the further the extent to which the animal would avoid the space treated with the tested substance. Most of the commonly used insecticides, e.g., pyrethroids, do not exhibit such action, acting solely as biocides. This characteristic, while in some situations beneficial, renders them (if used alone) inadequate for integrated pest management; therefore analyzing possible repellent activity is crucial for designing pull-push systems. R. officinalis has been reported to possess a repellent effect against many insect species [13]. Similar results were observed in the presented study as the R. officinalis were found to significantly repel the insects in the concentration equal to or higher than LC 12.5 ; the repellency observed after the exposition to LC 50 , was stronger albeit, insignificantly. This result could potentially relate to another behavioral parameter analyzed, the locomotor activity, which provides a more generalized overview of baseline insect activity. The locomotor activity measured in the open field paradigm is affected not only by external stimuli as it is analyzed in the case of repellency assay but also in an internal state of the insects' physiology. We observed significant inhibition of insects' locomotion, directly after the exposition, in all treated groups except the one treated with LC 50 . Therefore, the observed repellent effect could not be straightforwardly explained by inhibition of movement but the active avoidance of the region with the vapors of R. officinalis EO. The significantly lowered locomotor activity in treated groups may indicate various underlying effects, from the general sedation, through the disruption of gait (which could be further corroborated by observations (Video S1) to the breakage of connectivity synapses (either neuro neuronal, neuromuscular or both). Nevertheless, at baseline conditions, the lowered locomotor activity should result in decreased oxygen consumption; however, the contrary was observed, as oxygen consumption was either comparable to or elevated in comparison to the control. Such results suggest that while the insects were not involved in walking, they may have been exhibiting spontaneous muscle contractions or other metabolically intensive processes related to the EO action (Video S1). In contrary to the activity directly after the exposition, no statistically significant differences between the examined groups were observed after 24 h. This may be since only alive insects were used in the assessment. This indicates that those individuals belonged to a subpopulation that effectively manages exposure to R. officinalis EO.
One of the potential mechanisms underlying the observed reactions in insects caused by the rosemary EO could be AChE inhibition [25]. The changes in its activity cause the disruption of the cholinergic transmission in head ganglia and ventral nerve cord causing uncoordinated legs movement [10]. Numerous articles describing the insecticidal effect of EO also point to the AChE inhibitory effect [26][27][28][29][30]. The same effect was reported by Abdelgaleil at al. (2009) where the influence of various monoterpenes was tested on the Sitophilus oryzae and Tribolium castaneum [26]. One of the substances that has inhibited enzymatic activity the most was 1,8-cineole. Additionally it was reported that the two major constituents of R. officinalis EO-aforementioned 1,8-cineole and camphor ( Table 2) act synergistically with each other. The potent insecticidal effect (against the cabbage looper, Trichoplusia ni, (Hübner) (Lepidoptera: Noctuidae)) of both substances combined were attributed to the enhancement of the cuticular permeability. Such a mechanism is most relevant in regards to the apical delivery; however, similar effects (increased absorption on the tracheae level) could also occur in the case of fumigation with said substances [31]. However, such results were mainly reported on Sitophilus species, and the assays were conducted exclusively in vitro, while the presented experiments herein were performed on living insects (in vivo). Additionally, contrary to the results in the aforementioned articles, we observed increased AChE activity in insects exposed to the EO. Such a result may be associated with the experimental design, where the enzyme activity was assessed after 24 h using only the live insects (dead individuals were excluded from the experiment because of possible post mortem enzyme degradation). This may have resulted in the assessment being carried out only on the insects that had high initial enzyme activity levels and/or were able to adapt physiologically.
The primary line of defense of insects against xenobiotics includes the CAT and GST enzymes [32]. The elevated activity levels of these enzymes are characteristic of various insect strains resistant to a wide range of insecticides. The main functions of the aforementioned enzymes include metabolizing the xenobiotics (to reduce their toxicity or to increase their solubility in water) or reduce oxidative stress caused by the accumulation of free radicals. Moreover, in case of the toxic effects of EOs, the abovementioned enzymes are involved in the defense mechanisms. Depending on the tested EOs, a decrease, increase, or no changes in activity was reported in available studies [33][34][35][36]. The reduction of activity may indicate the inhibitory activity of the EOs' components. This may imply that the composition of the EO and, thus, the action of these components on the biochemical level, will differentiate the reactions on the level of detoxification enzymes. In the presented experiments, an increase in the activity of both enzymes was observed. Additionally, the changes in CAT and GST activity indicate the occurrence of oxidative stress as a result of EO exposition [32]. Differences in the change in activity, depending on the applied EO concentration, indicate higher sensitivity of CAT than GST (statistically significant increase in activity of the former at lower concentration: LC 12.5 ). This may also be due to the fact that free radicals appear earlier than the xenobiotic itself at a concentration high enough to activate GST. The results of changes in activity combined with oxygen consumption indicate a complex multi-aspect metabolic and biochemical response of the insect to contact with rosemary EO.

Conclusions
The present study demonstrates the toxicity of R. officinalis EO against C. maculatus and its influence on the selected physiological, biochemical, and behavioral parameters. The tested EO exhibited significant repellency in concentrations equal to or exceeding LC 25 . Additionally, we observed an increase of activity of all tested enzymes and oxygen consumption in LC 50 concentration. However, the locomotor activity was initially inhibited, but after the 24 h, the effect reverted to the baseline levels. The observed effects may be caused by the action of the main constituents of the EO: 1,8-cineol, camphor, and α-pinene. The acquisition of in-depth knowledge on how the EOs affect insect organisms could allow for the more informed approach in designing precisely targeted insecticidal agents (i.e., designing composite insecticide formulations with inhibitors of detoxification enzymes) and, thus, provide a valuable weapon in the fight against the development of resistance.