Skip to main content
PLOS ONE logoLink to PLOS ONE
. 2023 Sep 28;18(9):e0291242. doi: 10.1371/journal.pone.0291242

Drosophila melanogaster as a model for studies related to the toxicity of lavender, ginger and copaiba essential oils

Lucas Matos Martins Bernardes 1, Serena Mares Malta 1, Tamiris Sabrina Rodrigues 1, Luiz Fernando Covizzi 1, Rafael Borges Rosa 2,3, Allisson Benatti Justino 4, Renata Roland Teixeira 4, Foued Salmen Espíndola 4, Débora Oliveira dos Santos 5, Carlos Ueira Vieira 1, Murilo Vieira da Silva 2,3,*
Editor: Efthimios M C Skoulakis6
PMCID: PMC10538661  PMID: 37768921

Abstract

This study addresses the current trend of essential oils in alternative medicine using the non-chordate model Drosophila melanogaster. Following the three R’s principles, it proposes non-chordate models to fill knowledge gaps on essential oil toxicity. Copaiba, lavender, and ginger essential oils are evaluated for effects on D. melanogaster lifespan, climbing ability, and brain structure, while their anti-inflammatory properties are also analyzed. Results show dose-related differences: higher concentrations (0.25% v/v) cause brain deterioration and impaired climbing, while lower concentrations (0.0625% v/v for copaiba and ginger; 0.125% for lavender) have no effect on climbing or brain structure. Lavender oil significantly extends lifespan and maintains anti-inflammatory activity when ingested, underscoring its therapeutic potential. These findings highlight the importance of D. melanogaster as a model for studying essential oil properties, potentially replacing chordate models. In addition, this research advances alternative remedies for currently incurable diseases, with lavender oil emerging as a promising candidate for drug discovery.

Introduction

In 2021, the essential oils (EOs) market size was valued at $20.3 billion, with an estimated growth of 7.4% by 2028, due to their increasing use in industries such as food and beverages, cosmetics, and aromatherapy. In addition, because they are considered more "natural" than most commonly used medicines, their medical interest is increasing, and essential oils are slowly replacing drugs and medicines due to their reduced risk of side effects. Essential oils are basically substances extracted by distillation from different parts of plants, such as leaves, fruits, roots or flowers, and are composed of a mixture of hydrocarbons, ethers, esters, alcohols, aldehydes, phenols and terpenes [1,2].

Essential oils have a variety of different properties, including anti-inflammatory, antioxidant, antibiotic, and antiviral activities. The EOs extracted from lavender (Lavandula angustifolia), copaiba (Copaifera reticulata, officinalis, coriacea, and langsdorffii) and ginger (Zingiber officinale) all exhibited these activities both in vitro and in vivo [39], making them interesting targets for drug discovery that could be used to treat diseases that currently have no cure or treatment, such as neurodegenerative diseases and cancer. However, despite their widespread use and potential benefits, there is still a significant lack of information regarding the safety and efficacy of essential oils. Reports of adverse effects such as neurotoxicity, hepatotoxicity and reproductive abnormalities highlight the need for further research in this area [1014].

To generate this knowledge, it is necessary to use animals as model organisms. However, this has been the subject of intense scientific debate due to ethical and legislative differences between countries. Data collected by the UK government in 2021 showed that more than 3 million procedures were performed on animals, of which 57% were experimental procedures, using animals in studies such as drug safety testing and the development of treatments [15].

Since the use of animals is essential, it is not always possible to avoid animal testing in order to obtain the same results. Considering the replacement principle of the "Three R’s" guidelines established in 1959, the use of non-chordate animals (flies, worms) instead of chordates (mice, dogs, primates) is one of the possibilities to conduct research in a more ethical way, considering that non-chordates are less sentient animals [16]. The Drosophila melanogaster is a model organism that has already been used to test the toxicity of different compounds, such as metals and plant extracts [17,18]. Some of the advantages of using this animal are its fast life cycle, its cheap maintenance in laboratories and a genome with significant human homology [1820]. Another advantage of using this model organism is the UAS-Gal4 system. First introduced in 1993, this system uses the yeast transcriptional activator Gal4, randomly inserted into the D. melanogaster genome, along with genes containing Gal4 binding sites, in this case UAS as an enhancer of the target gene. These genes are activated in cells expressing Gal4, allowing analysis of the phenotype produced by their expression. Expression of the yeast transcriptional activator could also be used alongside a driver gene to target the activation of target genes to specific organs or tissues [21].

In addition, the fruit fly model allows the evaluation of its behavior through assays already well established in the literature, such as the larval crawling assay, the courtship and mating assay, and the climbing assay, also known as the RING (Rapid Iterative Negative Geotaxis) test [22]. Because this test relies on the motor reflex of the flies, it can be used to detect, for example, neurodegeneration that might impair their motor abilities [23].

There have been some experiments using essential oils and fruit flies as a model organism, specifically the species D. melanogaster and Drosophila suzukii [2426]. However, while these studies have analyzed the larvicidal and insecticidal effects of essential oils, our purpose on this study is to evaluate their toxicity and therapeutic potential in this model, aiming to propose this animal model as a replacement for chordate ones.

Therefore, this study aims to establish D. melanogaster as a viable model organism for studying the effects of essential oils in vivo. Specifically, we evaluate the toxicity and potential therapeutic effects of copaiba, lavender, and ginger essential oils on D. melanogaster lifespan, behavior, and brain morphology, as well as their anti-inflammatory activity, contributing to the scientific development and respecting the principles of the use of animals in research, especially the reduction of the number of chordate animals, as well as the replacement in many cases by a non-chordate in animal experiments.

Materials and methods

Drosophila stock and culture

D. melanogaster flies from the Canton S and w1118 (Stock #3605) strains were obtained from the Bloomington Stock Center, Indianapolis, IN. The group also used the GMR-GAL4 > UAS-Eiger stock kindly provided to our laboratory by Dr. Masayuki Miura of the University of Tokyo (Tokyo, Japan).

Animals were reared on a standard cornmeal medium, and the tests were performed on a mashed potato medium containing 75% powdered mashed potato (Yoki®), 15% yeast extract, 9.3% glucose and 0.7% nipagin. The flies were maintained in a BOD incubator at a controlled temperature of 25°C with a 12h/12h light/dark cycle. Flies were anesthetized with cold or ethyl ether for sex determination and mating.

Groups of 30 male adult w1118 flies (0 to 3 days post-emergence) were used for all of the following tests, except for the toxicity evaluation where groups of 15 male and 15 female adult w1118 flies (0 to 3 days post-emergence) were used. Each test lasted for 15 days, since the group aimed at a chronic treatment with the essential oils. All experiments were performed on three independent triplicates.

Essential oils and solutions preparation

Essential oils of copaiba (Copaifera sp.), ginger (Zingiber officinale), and lavender (Lavandula angustifolia) (döTerra, Utah, USA) were used, diluted with water to different concentrations for each assay and treatment (0.0625% v/v, 0.125% v/v, 0.25% v/v, and 0.5% v/v), with the solution being thoroughly mixed by vortexing for 10 seconds to disperse the oil in the water each time it was pipetted. Both EOs and their dilutions were stored at room temperature and protected from light to prevent degradation. Chromatography charts of the EOs provided by döTerra can be found in the Supporting Information (S1S3 Datasets).

Toxicity evaluation of the essential oils

The first step, prior to treating the flies, was to determine the toxicity of each of the oils at different concentrations. Each essential oil was tested at concentrations of 0.0625% v/v, 0.125% v/v, 0.25% v/v, and 0.5% v/v. Groups of 30 adult w1118 flies (0 to 3 days post-emergence) were separated into vials for each treatment in triplicate, with a 1:1 ratio of males to females.

Each vial contained mashed potato medium prepared with 5 mL of water (control group) or 5 mL of the EOs solutions. Flies were fed on the food prepared with these solutions for 15 days and transferred to vials containing fresh food every 2 to 3 days. The number of dead flies was counted each time they were transferred to a new vial.

Lifespan analysis

To understand how consumption of the EOs would alter the lifespan of the flies, a lifespan assay was performed. Male flies of the w1118 stock (0-to-1-day post-emergence) were separated into groups of 30 and fed on mashed potato medium prepared with the solutions at 0.0625% v/v for the copaiba and ginger EOs and 0.125% v/v for the lavender EO.

Similar to the toxicity evaluation, the flies were maintained on vials containing mashed potato medium prepared with 5 mL of water (control group) and 5 mL of the EOs solutions. Flies were transferred to vials containing fresh food every 2 to 3 days, and the number of dead flies was counted at each transfer until all flies had died.

Climbing assay

The Rapid Iterative Negative Geotaxis (RING) test was used to determine whether the essential oils diet would cause any changes in the locomotor ability and behavior of the flies, performed as described by Gargano et. al in 2005 [27]. The only modification is that the digital image used to evaluate the position of the flies on the vials was taken 10 seconds after the apparatus was tapped on the surface. This is due to the strain of flies used, w1118, which has a natural retinal degeneration that makes their climbing slower than other strains [28].

To understand how the animal model used would respond to an essential oil diet, the group performed the RING test using two sets of EO concentrations: lower doses that showed no toxicity in the toxicity test and higher doses that caused a greater number of deaths in the toxicity test. For the first, the concentrations used were 0.0625% v/v for the copaiba and ginger EOs and 0.125% v/v for the lavender EO. For the higher dose test, the concentrations used were 0.25% v/v for each essential oil.

The flies were fed on mashed potato medium prepared with the EO solutions at the above concentrations for 15 days, with the RING test performed every 5 days.

Histological analysis

Knowing that the essential oils have small molecules that can cross the blood-brain barrier, the group decided to analyze whether ingesting the EOs could cause any damage to the flies’ brains. Again, we decided to test both higher and lower doses of the essential oils to understand how the D. melanogaster model would respond to different concentrations of EOs.

Males from the w1118 strain were collected from 0 to 1-day post-emergence and divided into groups of 10 flies in triplicates and fed on mashed potato medium prepared with the solutions of EOs. The animals were kept on this diet for 15 days, euthanized with liquid nitrogen and decapitated for preparation of brain tissue slides. The tissues were fixed in paraffin and stained with hematoxylin and eosin for better visualization, following the protocol used by Malta et. al 2022 [29].

The concentrations used were 0.25% v/v for each essential oil for higher doses and 0.0625% v/v for the copaiba and ginger essential oils and 0.125% v/v for the lavender essential oil for the lower doses.

Analysis of the anti-inflammatory properties of the essential oils

Each of the essential oils used in this project has an anti-inflammatory activity already seen in both in vitro and in vivo tests [39]. However, the most commonly used animal models are mice and rats.

To analyze whether the D. melanogaster model would also respond to treatment with essential oils by ingestion, we used GMR-GAL4 > UAS Eiger flies as a model of inflammation in which eye degeneration is caused by overexpression of Eiger, a Drosophila ortholog of tumor necrosis factor alpha, in the organ. Assays in these animals are performed on larvae rather than adults because inflammation begins as soon as the animal hatches from the egg.

Flies of the GMR-GAL4 > UAS-Eiger strain were mated for 24 hours in oviposition medium. After this time, the parental flies were removed from the flask and the eggs were collected. These eggs were then placed in vials containing mashed potato medium prepared with solutions of the EOs at 0.025% v/v each. The animals were maintained on this medium for 10 days until the adult flies emerged from the pupae. The animals were then anesthetized with ice, and 60 flies from each group were collected for measurement of eye area using ImageJ software. The Canton S strain was used as a wild-type model for comparison of eye morphology.

Statistical analysis

All statistical tests were performed using GraphPad Prism 8 software. The Mantel-Cox test was used to evaluate the statistical significance of the data for toxicity evaluation and lifespan analysis. Two-way ANOVA test was used for the climbing assay. One-way ANOVA test was used to analyze the anti-inflammatory properties of the essential oils. All values of p < 0.05 were considered significant.

Results

Essential oils demonstrate dose-dependent toxicity in Drosophila flies

Toxicity of essential oils was evaluated by exposing flies to food containing a mixture of water and oils at concentrations of 0.0625%, 0.125%, 0.25%, and 0.5% v/v for 15 days. At a concentration of 0.0625% v/v, lavender essential oil was the only oil that resulted in a higher mortality rate compared to the control group (p<0.05) (Fig 1A). At a concentration of 0.125% v/v, flies exposed to copaiba and ginger essential oils had a mortality rate close to 50% (p<0.001 and p<0.05, respectively), while flies exposed to lavender EO had a mortality rate similar to the control (Fig 1B). At. 0.25% v/v, all three essential oils resulted in higher mortality compared to the control group (copaiba: p<0.05, ginger: p<0.001, and lavender: p<0.0001) (Fig 1C). The result is similar at the 5% v/v concentration, where consumption of all essential oils resulted in more deaths compared to the control group (p<0.0001) (Fig 1D).

Fig 1. Toxicity assessment of essential oils.

Fig 1

Lavender essential oil at 0.0625% v/v resulted in a higher mortality rate within 15 days of ingestion compared to the control group (A). At 0.125% v/v, copaiba and ginger essential oils caused more deaths, while no difference was observed for lavender EO (B). Both 0.25% and 0.5% v/v concentrations of EOs resulted in higher mortality rates than the control group (C and D). Statistical significance was determined by Mantel-Cox test (n = 30, * p<0.05, *** p<0.001, **** p<0.0001). Error bars on the graphics represent the standard error. This test was performed on three independent triplicates.

Safe concentrations of essential oils have varying effects on Drosophila melanogaster lifespan

To further investigate the effects of safe concentrations of essential oils on Drosophila melanogaster, we performed a lifespan analysis after prolonged exposure. We used concentrations of 0.0625% v/v for copaiba and ginger essential oils and 0.125% v/v for lavender essential oil. Our results showed that copaiba EO did not alter the lifespan or mortality rate of the flies compared to the control group. However, ginger EO caused a higher mortality rate in the flies from the fifteenth day, suggesting that long-term exposure may be toxic to the animals (p<0.01). In contrast, flies that ingested lavender EO had an increase in lifespan of 12 days compared to the control group (p<0.001) (Fig 2).

Fig 2. Lifespan analysis of D. melanogaster ingesting essential oils.

Fig 2

Copaiba essential oil did not affect the lifespan of the flies, while ginger essential oil decreased it. However, lavender essential oil increased the lifespan by about 12 days. Statistical significance was determined by Mantel-Cox test (n = 30, ** p<0.01, *** p<0.001). Error bars on the graphics represent the standard error. This test was performed on three independent triplicates.

Essential oils at low concentrations do not affect neuromotor function or brain morphology in flies

We evaluated the effects of safer concentrations of essential oils on neuromotor function and brain morphology in Drosophila melanogaster. The results showed that 0.0625% v/v concentration of ginger and copaiba essential oils and 0.125% v/v concentration of lavender essential oil did not affect the climbing ability of the flies. While copaiba EO had no effect on climbing ability, lavender EO increased climbing ability on days 5 (p<0.001) and 10 (p<0.05) of treatment, and ginger EO increased climbing ability only on day 5 (p<0.01) (Fig 3A–3C).

Fig 3. Climbing assay and histological analysis of the brain at lower doses of essential oils intake.

Fig 3

Flies exposed to ginger and lavender essential oils showed improved climbing ability on day 5 (A), while only those treated with lavender essential oil continued to show improvement on day 10 (B). By day 15, there was no significant difference in climbing ability between the groups (C). Statistical significance was determined by two-way ANOVA test (n = 30, * p<0.05, ** p<0.01, *** p<0.001), with graphics demonstrating mean ± standard error of the mean. Figures D to G illustrate the histologic section of the brain of flies from each group (D: control, E: copaiba EO, F: ginger EO and G: lavender EO). Histological analysis of the brain showed no morphological changes in any group. Images were taken at 100X magnification. This test was performed on three independent triplicates.

Furthermore, exposure to these lower doses did not cause any visible brain damage, as there were no vacuoles present in the brain tissue morphology of the treated flies compared to the control group (Fig 3D–3G).

Essential oils cause vacuole formation in Drosophila brain and neuromotor impairment at higher concentrations

During the fifth and tenth days after treatment, the climbing ability of the flies remained relatively unaffected, except for the flies exposed to copaiba essential oil on day five (p<0.05) and ginger essential oil on day ten (p<0.05) (Fig 4A and 4B). However, by day 15, flies exposed to all three essential oils showed a 20% decrease in climbing ability compared to the control group, indicating neuromotor impairment caused by the EOs (copaiba and ginger EOs: p<0.01, and lavender EO: p<0.001) (Fig 4C).

Fig 4. Climbing assay and histological analysis of the brain at higher doses of essential oils.

Fig 4

On day 5 of treatment, only flies exposed to copaiba essential oil had reduced climbing ability, while on day 10, only flies exposed to ginger essential oil had this impairment. However, by day 15 of treatment, flies from all groups had reduced climbing ability compared to the control group. Statistical significance was determined by two-way ANOVA test (n = 30, * p<0.05, ** p<0.01, *** p<0.001), with graphics demonstrating mean ± standard error of the mean. Figures D to G illustrate the histological section of the brain of flies from each group (D: control, E: copaiba EO, F: ginger EO, and G: lavender EO). The presence of vacuoles can be seen in each experimental group, particularly in those treated with lavender essential oil. Pictures were taken at 100X magnification. This test was performed on three independent triplicates.

Histological analysis showed that a concentration of 0.25% v/v of the essential oils for 15 days caused neurological damage in the brains of Drosophila melanogaster flies. The presence of vacuoles in the brain tissue suggests that the essential oils may have caused damage to the nervous system, consistent with the neuromotor impairment observed in the climbing assay. The extent of brain damage varied among the three essential oils, with lavender EO producing more prominent vacuoles than the other two oils (Fig 4D–4G).

Lavender essential oil promotes slight recovery of ocular inflammation caused by Eiger protein overexpression in D. melanogaster

A wild-type D. melanogaster eye phenotype, characterized by its large area and round shape (Canton S strain), is shown in Fig 5A. In contrast, Fig 5B shows the eye of an untreated GMR-GAL4 > UAS-Eiger fly, which shows degeneration due to overexpression of Eiger protein in the organ, resulting in loss of shape and area. Visual analysis of flies fed copaiba essential oil (Fig 5C) or ginger essential oil (Fig 5D) showed no significant evidence of reversal of ocular degeneration. However, when flies were fed lavender essential oil, a slight recovery of shape and increased eye area was observed (Fig 5E).

Fig 5. Effect of essential oils on the eye area of flies.

Fig 5

Figures A through E show the eyes of flies from different groups. The Canton S group (A) had a normal eye, while the negative control group (B) with Eiger protein overexpression had a malformed eye, representing the GMR-GAL4 > UAS-Eiger strain. Experimental groups of GMR-GAL4 > UAS-Eiger flies treated with the essential oils at 0.25% v/v are shown in Figures C to E (C: copaiba EO, D: ginger EO, and E: lavender EO). Images were taken with a stereomicroscope at 3X magnification. Measurements taken with ImageJ show that only lavender essential oil was able to ameliorate the inflammation of the fly eye and slightly restore its area (F). Statistical significance was determined by one-way ANOVA test (n = 60, **** p<0.0001), with graphics demonstrating mean ± standard error of the mean. This test was performed on three independent triplicates.

Quantitative analysis using ImageJ software revealed no statistical difference in eye area between the control group (untreated GMR-GAL4 > UAS-Eiger fly) and flies treated with copaiba essential oil or ginger essential oil. However, flies that ingested lavender essential oil had a significantly larger eye area compared to the control group (p<0.0001) (Fig 5F).

Discussion

The essential oils extracted from Copaifera spp, Zingiber officinale and Lavender angustifolia have shown promising results in previous studies regarding their anti-inflammatory, antioxidant and antimicrobial activities, as mentioned above. These results indicate their potential as alternative treatments for diseases and bacterial, fungal and viral control. However, their toxicity still needs to be evaluated in in vivo models to better understand their limitations as potential drugs. One way to fill this information gap is to test their use in model organisms, in this case Drosophila melanogaster.

In the experiments conducted, the D. melanogaster model showed changes in lifespan depending on the amount of essential oil they ingested. While at higher doses all EOs showed toxicity to the flies and reduced their lifespan, at lower doses they did not change it or, for lavender essential oil, increased it. These results indicate that this model can be a useful tool to assess the potential toxicity of essential oils. Furthermore, the observed increase in lifespan in flies that ingested lavender essential oil at lower doses highlights its potential as a natural product for further exploration in drug discovery.

Our investigation revealed that the ingestion of essential oils had a significant effect on the behavior of fruit flies, with climbing ability being particularly affected in a dose-dependent manner. Higher doses of essential oils resulted in impaired climbing ability, likely due to neurotoxicity, as evidenced by vacuoles observed in brain histology of affected flies. These results are consistent with previous studies showing that essential oils can have neurotoxic effects [3032]. Our results demonstrate that fruit flies can be used to study the effects of essential oils on brain morphology and suggest that neurotoxicity is one of the possible adverse effects of overdosing on copaiba, ginger, and lavender essential oils.

At lower doses of essential oil intake, copaiba and ginger essential oils did not significantly affect the climbing ability of the flies during the 15-day treatment period. However, flies treated with lavender essential oil showed a slight improvement in their climbing ability, consistent with the histological analysis of their brains, where no major changes in morphology were observed. The natural eye degeneration of the w1118 fly strain used in this study leads to slower climbing [28], making the improvement in climbing ability after lavender essential oil treatment particularly noteworthy. These results suggest that lavender essential oil may have a protective effect on retinal degeneration in D. melanogaster. However, further studies are needed to elucidate the underlying mechanisms and to confirm these findings.

Specifically for copaiba, studies analyzing the toxicity of copaiba oleoresin, which shares some of its constituents with the essential oil, found no acute or short-term toxicity in Wistar rats given the oleoresin by gavage at 2000 mg/kg (acute) and 100 mg/kg for 28 days (short-term) [33,34]. It is important to note that although they have the same constituents, essential oils are more concentrated and may have a different effect on animals compared to oleoresin. Unfortunately, studies analyzing the toxicity of essential oils are still scarce, especially in D. melanogaster, so more research is needed in this area.

While previous studies have demonstrated the anti-inflammatory activity of all three essential oils in various applications such as topical and inhalation [7,3538], our investigation revealed that only lavender essential oil had the potential to restore the area and shape of the eye after ingestion. This observation not only demonstrates the ability of flies to respond to essential oil treatment, but also suggests that lavender EO retains its anti-inflammatory properties even after ingestion, unlike ginger and copaiba EO. These findings provide opportunities for further research on lavender EO because several D. melanogaster strains can simulate human diseases for which there are no effective treatments or cures, including neurodegenerative diseases that involve inflammation as a pathological mechanism (e.g., Alzheimer’s, Parkinson’s, and Huntington’s diseases). Notably, these D. melanogaster models are well established in the literature and serve as reliable sources of information on potential treatments for these diseases [3941].

Nevertheless, this study has some limitations. The group analyzed changes in brain morphology which could be useful for future research on essential oils. However, further research is necessary to analyze the impact of essential oils on other organs, particularly the gastrointestinal tract. Moreover, the essential oils used have antimicrobial activity, which implies that they might have disturbed the flies’ gut microbiota, as mentioned above. Dysbiosis of the microbiota is known to not only disrupt intestinal functions but also impact brain function via the gut-brain axis [4247]. The group aims to investigate these questions in future research.

It is also important to mention that since the essential oils were administered to the flies through their food ad libitum, we could not ensure that the flies ingested the same amount of essential oil. A food intake analysis could be performed in the future to avoid this bias and to understand if the presence of the EOs on their food affects the amount of food they consume.

In conclusion, our results highlight the potential of D. melanogaster as a valuable model organism for evaluating the toxicity of essential oils. Our experiments revealed adverse effects of excessive doses of ginger, copaiba, and lavender essential oils, including neurotoxicity. Further investigations are warranted to explore their effects on other organs and their intestinal microbiota. However, our results also demonstrate the therapeutic potential of lavender essential oil, which not only prolonged the lifespan of D. melanogaster, but also induced an anti-inflammatory response without causing any damage to their brains. These observations suggest that lavender essential oil may be a promising candidate for the development of novel drugs.

Conclusion

In conclusion, the experiments with D. melanogaster not only showed that it is a reliable model for studies with essential oils, but also that even though essential oils can have therapeutic potential, they also have adverse effects. While higher doses of essential oils caused toxicity and reduced the lifespan of the animals, lower doses did not cause any significant negative effect and even increased their lifespan, for the lavender essential oil. The main adverse effect observed was neurotoxicity resulting in impaired climbing ability.

Lavender essential oil, on the other hand, proved to be a promising natural product for drug discovery, with the potential to induce an anti-inflammatory response in the flies without causing damage to their brain. These findings highlight the usefulness of D. melanogaster as a model organism for studying the toxicity and therapeutic properties of essential oils, a novelty, given that most tests using this model and essential oils aim at identifying possible larvicidal and insecticidal effects, and indicate that further testing is needed to fully explore their effects on other organs and their potential application in drug development.

Supporting information

S1 Dataset. Chromatographic profile of copaiba essential oil.

Chromatographic analysis of the copaiba essential oil utilized in this study, including the lot number, chromatographic chart, and a table of the constituents of the oil.

(PDF)

S2 Dataset. Chromatographic profile of ginger essential oil.

Chromatographic analysis of the ginger essential oil utilized in this study, including the lot number, chromatographic chart, and a table of the constituents of the oil.

(PDF)

S3 Dataset. Chromatographic profile of lavender essential oil.

Chromatographic analysis of the lavender essential oil utilized in this study, including the lot number, chromatographic chart, and a table of the constituents of the oil.

(PDF)

Acknowledgments

We thank the members of the Genetics Laboratory and the Animal Facility Network of the Federal University of Uberlândia for their support during this project.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

The project was funded by the FAPEMIG agency (Project RED-00103-22). The funding was provided to support Lucas Matos Martins Bernardes as a scholarship holder. The funders did not participate in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

  • 1.Grand View Research. Global Essential Oils Market Size, Share & Trends Analysis Report by Product (Orange, Cornmint, Eucalyptus), Application (Medical, Food & Beverages, Spa & Relaxation), Sales Channel, Region, and Segment Forecasts, 2021–2028. 2021. [Google Scholar]
  • 2.Ali B, Al-Wabel NA, Shams S, Ahamad A, Khan SA, Anwar F. Essential oils used in aromatherapy: A systemic review. Asian Pac J Trop Biomed [Internet]. 2015;5(8):601–11. Available from: 10.1016/j.apjtb.2015.05.007. [DOI] [Google Scholar]
  • 3.Wang X, Shen Y, Thakur K, Han J, Zhang JG, Hu F, et al. Antibacterial Activity and Mechanism of Ginger Essential Oil against Escherichia coli and Staphylococcus aureus. Molecules. 2020;25(17). doi: 10.3390/molecules25173955 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.de Lima DAN, Pelegrini BB, Uechi FAA, Varago RC, Pimenta BB, de Souza Kaneshima AM, et al. Evaluation of antineoplasic activity of Zingiber officinale essential oil in the colorectal region of wistar rats. Asian Pacific J Cancer Prev. 2020;21(7):2141–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.do Carmo Silva L, Miranda MACM, de Freitas JV, Ferreira SFA, de Oliveira Lima EC, de Oliveira CMA, et al. Antifungal activity of Copaíba resin oil in solution and nanoemulsion against Paracoccidioides spp. Brazilian J Microbiol. 2020;51(1):125–34. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Ames-Sibin AP, Barizão CL, Castro-Ghizoni C V., Silva FMS, Sá-Nakanishi AB, Bracht L, et al. β-Caryophyllene, the major constituent of copaiba oil, reduces systemic inflammation and oxidative stress in arthritic rats. Vol. 119, Journal of Cellular Biochemistry. 2018. p. 10262–77. [DOI] [PubMed] [Google Scholar]
  • 7.Bahr T, Allred K, Martinez D, Rodriguez D, Winterton P. Effects of a massage-like essential oil application procedure using Copaiba and Deep Blue oils in individuals with hand arthritis. Complement Ther Clin Pract [Internet]. 2018;33(October):170–6. Available from: doi: 10.1016/j.ctcp.2018.10.004 [DOI] [PubMed] [Google Scholar]
  • 8.Swamy MK, Akhtar MS, Sinniah UR. Antimicrobial properties of plant essential oils against human pathogens and their mode of action: An updated review. Evidence-based Complement Altern Med. 2016;2016. doi: 10.1155/2016/3012462 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Miguel MG. Antioxidant and anti-inflammatory activities of essential oils: A short review. Molecules. 2010;15(12):9252–87. doi: 10.3390/molecules15129252 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Ramsey JT, Shropshire BC, Nagy TR, Chambers KD, Li Y, Korach KS. Essential oils and Health. Yale J Biol Med. 2020;93(2):291–305. doi: 10.1097/DER.0000000000000175 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Ramsey JT, Li Y, Arao Y, Naidu A, Coons LA, DIaz A, et al. Lavender Products Associated with Premature Thelarche and Prepubertal Gynecomastia: Case Reports and Endocrine-Disrupting Chemical Activities. J Clin Endocrinol Metab. 2019;104(11):5393–405. doi: 10.1210/jc.2018-01880 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Dosoky NS, Setzer WN. Maternal reproductive toxicity of some essential oils and their constituents. Int J Mol Sci. 2021;22(5):1–31. doi: 10.3390/ijms22052380 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Lanzerstorfer P, Sandner G, Pitsch J, Mascher B, Aumiller T, Weghuber J. Acute, reproductive, and developmental toxicity of essential oils assessed with alternative in vitro and in vivo systems. Arch Toxicol [Internet]. 2021;95(2):673–91. Available from: doi: 10.1007/s00204-020-02945-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Fouyet S, Olivier E, Leproux P, Dutot M, Rat P. Evaluation of Placental Toxicity of Five Essential Oils and Their Potential Endocrine-Disrupting Effects. Curr Issues Mol Biol. 2022;44(7):2794–810. doi: 10.3390/cimb44070192 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Hobson H. Animal research statistics for Great Britain, 2021 [Internet]. Urdestanding Animal Research. 2022. Available from: https://www.understandinganimalresearch.org.uk/news/animal-research-statistics-for-great-britain-2021. [Google Scholar]
  • 16.Beckmann N, Maier P. Noninvasive Small Rodent Imaging: Significance for the 3R Principles. In: Small Animal Imaging. 2017. p. 47–57. [Google Scholar]
  • 17.Kushalan S, D’Souza LC, Aloysius K, Sharma A, Hegde S. Toxicity Assessment of Curculigo orchioides Leaf Extract Using Drosophila melanogaster: A Preliminary Study. Int J Environ Res Public Health. 2022;19(22). doi: 10.3390/ijerph192215218 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Calap-Quintana P, González-Fernández J, Sebastiá-Ortega N, Llorens JV, Moltó MD. Drosophila melanogaster models of metal-related human diseases and metal toxicity. Int J Mol Sci. 2017;18(7). doi: 10.3390/ijms18071456 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Reiter LT, Potocki L, Chien S, Gribskov M, Bier E. A systematic analysis of human disease-associated gene sequences in Drosophila melanogaster. Genome Res. 2001;11(6):1114–25. doi: 10.1101/gr.169101 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Adams MD, Celniker SE, Holt RA, Evans CA, Gocayne JD, Amanatides PG, et al. The genome sequence of Drosophila melanogaster. Science (80-). 2000;287(5461):2185–95. doi: 10.1126/science.287.5461.2185 [DOI] [PubMed] [Google Scholar]
  • 21.Brand AH, Perrimon N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 1993;118(2):401–15. doi: 10.1242/dev.118.2.401 [DOI] [PubMed] [Google Scholar]
  • 22.Nichols CD, Becnel J, Pandey UB. Methods to Assay Drosophila Behavior. J Vis Exp. 2012;(61):3–7. doi: 10.3791/3795 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Batista LL, Malta SM, Guerra Silva HC, Borges LDF, Rocha LO, da Silva JR, et al. Kefir metabolites in a fly model for Alzheimer’s disease. Sci Rep [Internet]. 2021;11(1):1–12. Available from: 10.1038/s41598-021-90749-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Souza MT de, Souza MT de, Bernardi D, Oliveira D da C, Morais MC, Melo DJ de, et al. Essential Oil of Rosmarinus officinalis Ecotypes and Their Major Compounds: Insecticidal and Histological Assessment Against Drosophila suzukii and Their Impact on a Nontarget Parasitoid. J Econ Entomol. 2022;115(4):955–66. doi: 10.1093/jee/toab230 [DOI] [PubMed] [Google Scholar]
  • 25.De Oliveira MRC, Barros LM, Duarte AE, De Lima Silva MG, Da Silva BAF, Pereira Bezerra AOB, et al. Gc-ms chemical characterization and in vitro evaluation of antioxidant and toxic effects using drosophila melanogaster model of the essential oil of lantana montevidensis (Spreng) briq. Med. 2019;55(5). doi: 10.3390/medicina55050194 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Souza MT de, Souza MT de, Morais MC, Oliveira D da C, Melo DJ de, Figueiredo L, et al. Essential Oils as a Source of Ecofriendly Insecticides for Drosophila suzukii (Diptera: Drosophilidae) and Their Potential Non-Target Effects. Molecules. 2022;27(19). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Gargano JW, Martin I, Bhandari P, Grotewiel MS. Rapid iterative negative geotaxis (RING): A new method for assessing age-related locomotor decline in Drosophila. Exp Gerontol. 2005;40(5):386–95. doi: 10.1016/j.exger.2005.02.005 [DOI] [PubMed] [Google Scholar]
  • 28.Ferreiro MJ, Pérez C, Marchesano M, Ruiz S, Caputi A, Aguilera P, et al. Drosophila melanogaster white mutant w1118 undergo retinal degeneration. Front Neurosci. 2018;11(JAN):1–12. doi: 10.3389/fnins.2017.00732 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Malta SM, Batista LL, Silva HCG, Franco RR, Silva MH, Rodrigues TS, et al. Identification of bioactive peptides from a Brazilian kefir sample, and their anti-Alzheimer potential in Drosophila melanogaster. Sci Rep [Internet]. 2022;12(1):1–12. Available from: doi: 10.1038/s41598-022-15297-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Haditsch U, Roth T, Rodriguez L, Hancock S, Cecere T, Nguyen M, et al. Alzheimer’s Disease-Like Neurodegeneration in Porphyromonas gingivalis Infected Neurons with Persistent Expression of Active Gingipains. J Alzheimer’s Dis. 2020;75(4):1301–17. doi: 10.3233/JAD-200393 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Morris PJ, Burke RD, Sharma AK, Lynch DC, Lemke-Boutcher LE, Mathew S, et al. A comparison of the pharmacokinetics and NMDAR antagonism-associated neurotoxicity of ketamine, (2R,6R)-hydroxynorketamine and MK-801. Neurotoxicol Teratol. 2021;87:1–19. doi: 10.1016/j.ntt.2021.106993 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Tisserand R, Young R. Essential Oil Safety. A Guide for Health Care Professionals. Churchill Livingstone. 2014. 783 p. [Google Scholar]
  • 33.Sachetti CG, Fascineli ML, Sampaio JA, Lameira OA, Caldas ED. Avaliação da toxicidade aguda e potencial neurotóxico do óleo-resina de copaíba (Copaifera reticulata Ducke, Fabaceae). Rev Bras Farmacogn. 2009;19(4):937–41. [Google Scholar]
  • 34.Silva MAC, dos Anjos Melo DF, de Oliveira SAM, Cruz A de C, da Conceição EC, de Paula JR, et al. Acute and a 28-repeated dose toxicity study of commercial oleoresin from Copaifera sp. in rodents. Adv Tradit Med [Internet]. 2022;22(4):739–47. Available from: 10.1007/s13596-021-00601-7. [DOI] [Google Scholar]
  • 35.Akinyemi AJ, Adeniyi PA. Effect of Essential Oils from Ginger (Zingiber officinale) and Turmeric (Curcuma longa) Rhizomes on Some Inflammatory Biomarkers in Cadmium Induced Neurotoxicity in Rats. J Toxicol. 2018;2018. doi: 10.1155/2018/4109491 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Pandur E, Balatinácz A, Micalizzi G, Mondello L, Horváth A, Sipos K, et al. Anti-inflammatory effect of lavender (Lavandula angustifolia Mill.) essential oil prepared during different plant phenophases on THP-1 macrophages. BMC Complement Med Ther [Internet]. 2021;21(1):1–17. Available from: 10.1186/s12906-021-03461-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Horváth A, Pandur E, Sipos K, Micalizzi G, Mondello L, Böszörményi A, et al. Anti-inflammatory effects of lavender and eucalyptus essential oils on the in vitro cell culture model of bladder pain syndrome using T24 cells. BMC Complement Med Ther [Internet]. 2022;22(1):1–14. Available from: 10.1186/s12906-022-03604-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Lucca LG, de Matos SP, Kreutz T, Teixeira HF, Veiga VF, de Araújo B V., et al. Anti - inflammatory Effect from a Hydrogel Containing Nanoemulsified Copaiba oil (Copaifera multijuga Hayne). AAPS PharmSciTech. 2018;19(2):522–30. [DOI] [PubMed] [Google Scholar]
  • 39.Tue NT, Dat TQ, Ly LL, Anh VD, Yoshida H. Insights from Drosophila melanogaster model of Alzheimer’s disease. Front Biosci. 2020;25:134–46. doi: 10.2741/4798 [DOI] [PubMed] [Google Scholar]
  • 40.Bolus H, Crocker K, Boekhoff-Falk G, Chtarbanova S. Modeling neurodegenerative disorders in Drosophila melanogaster. Int J Mol Sci. 2020;21(9). doi: 10.3390/ijms21093055 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Aryal B, Lee Y. Disease model organism for Parkinson disease: Drosophila melanogaster. BMB Rep. 2019;52(4):250–8. doi: 10.5483/BMBRep.2019.52.4.204 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Belvoncikova P, Maronek M, Gardlik R. Gut Dysbiosis and Fecal Microbiota Transplantation in Autoimmune Diseases. Int J Mol Sci. 2022;23(18). doi: 10.3390/ijms231810729 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Chen Y, Zhou J, Wang L. Role and Mechanism of Gut Microbiota in Human Disease. Front Cell Infect Microbiol. 2021;11(March):1–12. doi: 10.3389/fcimb.2021.625913 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Iatcu CO, Steen A, Covasa M. Gut microbiota and complications of type-2 diabetes. Nutrients. 2022;14(1). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Varesi A, Pierella E, Romeo M, Piccini GB, Alfano C, Bjørklund G, et al. The Potential Role of Gut Microbiota in Alzheimer’s Disease: from Diagnosis to Treatment. Nutrients. 2022;14(3):1–27. doi: 10.3390/nu14030668 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Chang L, Wei Y, Hashimoto K. Brain–gut–microbiota axis in depression: A historical overview and future directions. Brain Res Bull [Internet]. 2022;182(January):44–56. Available from: doi: 10.1016/j.brainresbull.2022.02.004 [DOI] [PubMed] [Google Scholar]
  • 47.Klann EM, Dissanayake U, Gurrala A, Farrer M, Shukla AW, Ramirez-Zamora A, et al. The Gut–Brain Axis and Its Relation to Parkinson’s Disease: A Review. Front Aging Neurosci. 2022;13(January):1–17. doi: 10.3389/fnagi.2021.782082 [DOI] [PMC free article] [PubMed] [Google Scholar]

Decision Letter 0

Efthimios M C Skoulakis

12 Jul 2023

PONE-D-23-11609Drosophila melanogaster as a model for studies related to the toxicity of lavender, ginger and copaiba essential oilsPLOS ONE

Dear Dr. Rosa,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Both reviewers found the manuscript interesting and merit in the data and the conclusions. However questions were raised and comments regarding methods, data presentation and manuscript presentation were raised.Please thoroughly address all comments and suggestions as they are aimed at improving the manuscript presentation and clarity

==============================

Please submit your revised manuscript by Aug 26 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Efthimios M. C. Skoulakis, PhD

Academic Editor

PLOS ONE

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating the following financial disclosure:

“Yes. The project was funded by the Fapemig agency (Project RED-00103-22). The funding was given to pay scholarship holder Lucas Matos Martins Bernardes.”

Please state what role the funders took in the study.  If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Essential oils have gained popularity for their potential therapeutic effects, but their safety and efficacy need to be scientifically evaluated. Overall, this study is significant because it provides scientific insights into the safety and efficacy of essential oils, offers an alternative model organism for research, highlights dosage considerations, and identifies lavender essential oil as a promising therapeutic agent. These findings have implications for both the scientific community and individuals interested in utilizing essential oils for health purposes, contributing to the advancement of knowledge and the development of alternative treatment options.

Here are my comments:

Introduction section

1) To strengthen their case, I suggest that the authors cite more papers that have utilized the drosophila model to study the effects of essential oils. Some suggested literature includes PMID: 36751763, PMID: 34865075, and PMID: 36234751.

2) I highly recommend that the authors add information about the gal4-uas system as one of the advantages in the sentence "Some of the advantages of using this animal are its fast life cycle, its cheap maintenance in laboratories, and a genome with significant human homology." This addition will provide a more comprehensive overview of the advantages of using the drosophila model.

Materials and methods section

1) Please add the following information, described in "Toxicity evaluation of the essential oils," to the "Drosophila stock and culture" section: "Groups of 30 adult w1118 flies (0 to 3 days post-emergence) were separated into vials for each treatment in triplicate, with a 1:1 ratio of males to females."

2) Please add the final concentrations after the dilutions of each essential oil, as described in the "Toxicity evaluation of the essential oils" section, to the following sentence: "Essential oils of copaiba (Copaifera sp.), ginger (Zingiber officinale), and lavender (Lavandula angustifolia) (döTerra, Utah, USA) were used, diluted with water to different concentrations for each assay and treatment."

3) I kindly request moving the sentence "Each vial contained mashed potato medium prepared with 5 mL of water (control group) or 5 mL of the EOs solutions. Flies were fed on the food prepared with these solutions for 15 days and transferred to vials containing fresh food every 2 to 3 days. The number of dead flies was counted each time they were transferred to a new vial. The Mantel-Cox survival test was used for statistical analysis." to the “ Lifespan analysis section."

4) It would be helpful if the authors could provide references for the statement: "Each of the essential oils used in this project has demonstrated anti-inflammatory activity in both in vitro and in vivo tests." to the Analysis of the anti-inflammatory properties of the essential oils section.

5) It is recommended to include a dedicated section in the Materials and Methods that describes the statistical analysis methods employed in this study. This would provide transparency and allow readers to understand how the data were analyzed and interpreted. Providing details such as the specific statistical tests used, any assumptions made, and the significance threshold would enhance the rigor and reproducibility of the study.

Results section

1) I recommend that the authors include the statistical significance values directly in the results section, in addition to the figure legends. This will provide readers with a clear and concise understanding of the statistical analysis and outcomes without having to refer back and forth between the figures and the accompanying text.

2) Did the authors investigate the larvicidal activity of the essential oils?

Discussion section

1) Authors must include a paragraph discussing the limitations of the study.

Figures:

1) Please increase the resolution of figures 1 (a, b, c and d), 3, 4 and 5.

2) Figure 3 - I highly recommend the authors to include the corresponding group names at the bottom of each histologic section of the brain (D: control, E: copaiba EO, F: ginger EO, and G: lavender EO). This will provide clarity and enable readers to easily associate each image with the respective treatment group. Additionally, it would be beneficial to indicate the image magnification (100X) to provide a sense of scale and facilitate a better understanding of the histological features.

3) Figure 4 - I highly recommend the authors to include the corresponding group names at the bottom of each histologic section of the brain (D: control, E: copaiba EO, F: ginger EO, and G: lavender EO). This will provide clarity and enable readers to easily associate each image with the respective treatment group. Additionally, it would be beneficial to indicate the image magnification (100X) to provide a sense of scale and facilitate a better understanding of the histological features. Additionally, I suggest that the authors consider including arrows in the histologic sections to clearly indicate the presence of vacuoles. This visual aid will help to emphasize and highlight the specific features being discussed in the image.

Reviewer #2: Comments regarding the manuscript are as follows:

1) Should be checked in italics and abbreviations in the manuscript. Abbreviations: At the first appearance in the abstract and the text, abbreviations should be preceded by words for which they stand.

2) Maybe authors should be added the keywords such as; genotoxicity, environment and health, risk assessment, etc.

3) I think that the concentrations of essential oils (EOs) of copaiba (Copaifera sp.), ginger (Zingiber officinale), and lavender (Lavandula angustifolia) (döTerra, Utah, USA) should be added to the Abstract.

4) Introduction: Authors should discuss the meaningfulness using Drosophila melanogaster compared to other in vivo model organisms. The flow of introduction need to be tuned.

5) The reason for using the in vivo model? What was the rationale aim for choosing D. melanogaster as a model organism? This information must be included in the Introduction section.

6) What is the significance of investigating EOs exposure routes?

7) The researchers need to recommend the dosage range which will be safe for human consumption.

8) I think that it should be mentioned in the Materials and Methods to reference studies for selected concentrations of EOs (v/v)? I think this should be explained and added in the manuscript in order to enable comparisons to other studies. How could they justify the concentrations selection for the study? Are these low or high concentrations? Working with high concentrations reduces the toxicological value of the results since they select the resistant individuals, or simply do not occur in real life. Is the dose limited by toxicity? The authors mention that in the Materials and methods.

9) The Materials and methods section should be referenced from the literature.

10) What's the likelihood for human exposure to used concentrations in EOs? Are they realistic exposure concentrations? Please explain the experimental rationale in discussion section. Please define better the relation between the tested dose and the potential environmental exposure. Moreover, the discussion should be improved with a more stressed comparative analysis of the own research results with literature works, already present.

11) The activation of toxicity mechanisms are not completely clear, and miss of any deep investigation.

12) What were the rationale for choosing the different in vivo assays? This must be included in the Introduction section.

13) How many independent experiments (excluding for replicates) were performed in this study? I think the authors should mention in the Materials and Methods section. Application period projected for groups were chosen according to what? The authors should fully clarify this point.

14) Section Discussion: This section also need to be improved with recent studies. i feel that some irrelevant discussion. Please discuss about the effect of EOs on D. melanogaster as an in vivo model.

15) What is the vehicle control name? Which were used group in the Statistical analysis?

16) What is positive control used for the assays? I think the authors should add values of positive control in the Figures.

17) The correlation between selected compound (or the viability) and the in vivo experiments?

18) In addition in the discussion section comparative evaluation with toxicological effects at cellular levels of EOs could add additional relevance to the work. Please define better the relation between the tested concentration and the potential environmental exposure. Moreover, the discussion should be improved with a more stressed comparative analysis of the own research results with literature works, already present.

19) The authors should include in the discussion some thoughts about how the in vivo findings can translate to human risk.

20) The authors should be added chemical constituents (%) (such as, CAS No, Quality %, Catalog number, etc.)

21) use abbreviations for SI units: d for day.

22) What do authors think about the effects of solubility of EOs on the cytotoxicity?

23) The authors should add some description about the disadvantages or difficulties of using animal in vivo and in vitro models related with EOs.

24) In Discussion: The authors should add new papers and update the literature review. More evidence on the link between toxicity and intestinal damage/life span caused by EOs could support the paper's idea (novelty) better.

25) The rationale for the selection of EOs must be stated at the end of the Discussion section.

26) Running title should be added.

27) In Figure legends, please indicate the following in each Figure legend: (a) the times of each experiments were repeated; (b) the number/replica of each group in each repeat; (c) are the data showed in the figure representative?

28) Please highlight the novelty aspect of the present research in the introduction, abstract and conclusion section.

29) Statistical analysis: The authors need to provide company of stats for the statistical software. There is not enough explanation in statistical analysis. What statistical tests were used in the manuscript? Arithmetic mean ± standard deviation (or arithmetic mean ± standard error) values should be shown in the Figures.

30) What were the rationale for choosing the different assays? This must be included in the Introduction section. Do the assays used by the authors are based on the OECD guidelines?

31) Reported images are really poor and need substantial improvement.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Ana Paula Mendes Silva

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachment

Submitted filename: Comments and questions for manuscript (PONE-D-23-11609).docx

PLoS One. 2023 Sep 28;18(9):e0291242. doi: 10.1371/journal.pone.0291242.r002

Author response to Decision Letter 0


23 Aug 2023

Dear reviewers,

Thank you for reading and reviewing our manuscript, facilitating the improvement of our research to a superior scientific standard. We noted the corrections that you presented, and we have implemented certain changes as a result. Please find attached the revised version of our manuscript, containing all the changes made.

In the following section, we will discuss each point made by the reviewers:

Reviewer #1:

Question 1: To strengthen their case, I suggest that the authors cite more papers that have utilized the drosophila model to study the effects of essential oils. Some suggested literature includes PMID: 36751763, PMID: 34865075, and PMID: 36234751.

Answer: We appreciate the suggestion and acted on it, as we believe it would enhance the quality of our Introduction section. The modifications are available on page 2.

Question 2: I highly recommend that the authors add information about the gal4-uas system as one of the advantages in the sentence "Some of the advantages of using this animal are its fast life cycle, its cheap maintenance in laboratories, and a genome with significant human homology." This addition will provide a more comprehensive overview of the advantages of using the drosophila model.

Answer: It is agreed that the rationale for the choice of Drosophila melanogaster as a model organism would be strengthened by including information on the Gal4-UAS system. Revisions to the Introduction section are located on page 2.

Question 3: Please add the following information, described in "Toxicity evaluation of the essential oils," to the "Drosophila stock and culture" section: "Groups of 30 adult w1118 flies (0 to 3 days post-emergence) were separated into vials for each treatment in triplicate, with a 1:1 ratio of males to females."

Answer: We appreciate the suggestion made by the reviewer and have implemented it. However, the group maintains that this information needs to appear in both the 'Toxicity evaluation of essential oils' and the 'Drosophila stock and culture' sections. Therefore, rather than moving the information to the second section, we have decided to add it to the first section mentioned. Please refer to page 4 for the changes made.

Question 4: Please add the final concentrations after the dilutions of each essential oil, as described in the "Toxicity evaluation of the essential oils" section, to the following sentence: "Essential oils of copaiba (Copaifera sp.), ginger (Zingiber officinale), and lavender (Lavandula angustifolia) (döTerra, Utah, USA) were used, diluted with water to different concentrations for each assay and treatment."

Answer: We appreciate the suggestion and agree that it would enhance the flow of the Materials and Methods sections. The group adopted the suggestion, and you can find the changes made on page 6.

Question 5: I kindly request moving the sentence "Each vial contained mashed potato medium prepared with 5 mL of water (control group) or 5 mL of the EOs solutions. Flies were fed on the food prepared with these solutions for 15 days and transferred to vials containing fresh food every 2 to 3 days. The number of dead flies was counted each time they were transferred to a new vial. The Mantel-Cox survival test was used for statistical analysis." to the “Lifespan analysis section.”

Answer: The group agrees that this information belongs in the “Lifespan Analysis” section, but also finds it relevant to the “Toxicity Evaluation of Essential Oils” section. Rather than relocating the information, the decision is to include it in the “Lifespan Analysis” section since the same methodology is used for both tests, while also ensuring that it can be found in the “Toxicity Evaluation of Essential Oils” section. You can find the changes in the mentioned section.

Question 6: It would be helpful if the authors could provide references for the statement: "Each of the essential oils used in this project has demonstrated anti-inflammatory activity in both in vitro and in vivo tests." to the Analysis of the anti-inflammatory properties of the essential oils section.

Answer: The group expresses gratitude for the suggestion and accepts it. While the references were already present in the Introduction section, we concur that their placement was inappropriate. We have also added the references to the Methodology section. The modifications can be located on page 8. Should the reviewer suggest further changes, we are open to accepting new comments.

Question 7: It is recommended to include a dedicated section in the Materials and Methods that describes the statistical analysis methods employed in this study. This would provide transparency and allow readers to understand how the data were analyzed and interpreted. Providing details such as the specific statistical tests used, any assumptions made, and the significance threshold would enhance the rigor and reproducibility of the study.

Answer: We appreciate the suggestion and agree that including a Statistics section is necessary for a clear analysis of our results and for reproducibility purposes. We added the section, and it is available on page 9.

Question 8: Did the authors investigate the larvicidal activity of the essential oils?

Answer: The main focus of our article was to test the therapeutic effects of essential oils. As such, we did not examine their larvicidal activity. We recognize that investigating the toxicity of essential oils to flies could be a crucial analysis. Given this, we appreciate your inquiry and will incorporate an assessment of the larvicidal activity of essential oil into our next project. This will help us enhance our future research on this essential topic.

Question 9: Authors must include a paragraph discussing the limitations of the study.

Answer: The group agrees that it is necessary to add a paragraph discussing the limitations of the study. We appreciate and accept the suggestion because it will strengthen our discussion. The new paragraphs are located on page 19.

Question 10: Please increase the resolution of figures 1 (a, b, c and d), 3, 4 and 5.

Answer: The group appreciates the suggestion and has made the figures clearer for better analysis of the results obtained. Modifications can be observed in the Results section. If additional adjustments are necessary, we would be pleased to make them.

Question 11: Figure 3 - I highly recommend the authors to include the corresponding group names at the bottom of each histologic section of the brain (D: control, E: copaiba EO, F: ginger EO, and G: lavender EO). This will provide clarity and enable readers to easily associate each image with the respective treatment group. Additionally, it would be beneficial to indicate the image magnification (100X) to provide a sense of scale and facilitate a better understanding of the histological features.

Answer: The figures and/or their subtitles now include both adjustments. Though we recognize the significance of magnification information, we opted to avoid placing it directly on the figure. Instead, we included the details on the subtitle to prevent the image from being too overloaded with information, potentially complicating the analysis of results. If the reviewer believes that the magnification information should be included in the figure, we can make the necessary alteration. Modifications are present in Figure 3.

Question 12: Figure 4 - I highly recommend the authors to include the corresponding group names at the bottom of each histologic section of the brain (D: control, E: copaiba EO, F: ginger EO, and G: lavender EO). This will provide clarity and enable readers to easily associate each image with the respective treatment group. Additionally, it would be beneficial to indicate the image magnification (100X) to provide a sense of scale and facilitate a better understanding of the histological features. Additionally, I suggest that the authors consider including arrows in the histologic sections to clearly indicate the presence of vacuoles. This visual aid will help to emphasize and highlight the specific features being discussed in the image.

Answer: The answer to Question 11 remains the same. Modifications have been made and can be located on Figure 4. The group will make any required updates if necessary.

Reviewer #2:

Question 1: Should be checked in italics and abbreviations in the manuscript. Abbreviations: At the first appearance in the abstract and the text, abbreviations should be preceded by words for which they stand.

Answer: Both italics and abbreviations were double-checked by the group, and we believe that all necessary adjustments have been made. We are happy to make any new correction that might be necessary to improve our article.

Question 2: Maybe authors should add the keywords such as; genotoxicity, environment and health, risk assessment, etc.

Answer: We appreciate the suggestion; however, we decided to keep the chosen keywords. While we recognize the potential use of “environment and health” and “risk assessment,” we believe that these concepts do not align with the article's main theme. Our work did not involve any genetics analysis; therefore, the term “genotoxicity” is irrelevant.

Question 3: I think that the concentrations of essential oils (EOs) of copaiba (Copaifera sp.), ginger (Zingiber officinale), and lavender (Lavandula angustifolia) (döTerra, Utah, USA) should be added to the Abstract.

Answer: The group acknowledges the suggestion, and we believe that it would enhance the quality of our abstract. Therefore, we accepted it and incorporated the suggested change. You can locate it on page two.

Question 4: Introduction: Authors should discuss the meaningfulness using Drosophila melanogaster compared to other in vivo model organisms. The flow of introduction needs to be tuned.

Answer: While the suggestion of discussing the utility of Drosophila melanogaster compared to other in vivo models is appreciated, we assert that our Introduction section clearly presents the reasons for selecting this model organism instead of the more commonly used chordates, as is explained on pages 3 and 4. This information is available on pages 3 and 4. We would be happy to add further details to enrich our article if the reviewer deems it necessary.

Question 5: The reason for using the in vivo model? What was the rationale aim for choosing D. melanogaster as a model organism? This information must be included in the Introduction section.

Answer: The justification for choosing D. melanogaster was already provided in the Introduction section on pages 3 and 4, as we have previously mentioned in response to Question 4. We selected this model to adhere to the Three R’s guidelines, as it allows us to substitute the usual chordate models with a non-chordate model, which helped us refine the experiments by studying the effects of these essential oils on this particular model. By doing so, we aim to reduce the number of chordate animals required for further research. In case the reviewer requests additional information, our group will gladly provide it.

Question 6: What is the significance of investigating EOs exposure routes?

Answer: Essential oils have three exposure routes: topical application, inhalation, and ingestion. The absorption and processing of essential oils depends on the route used. Therefore, it is important to understand the responses that each route produces in the model organism. The model organism utilized in this study is Drosophila melanogaster, which allows for the analysis of ingestion and inhalation through their food. For topical applications, however, chordate model organisms, such as mice or rats, would be more suitable for conducting necessary tests.

Question 7: The researchers need to recommend the dosage range which will be safe for human consumption.

Answer: While we appreciate the suggestion, the goal of our group for this project was to standardize the model and recommend the use of D. melanogaster as a model organism for essential oil screening tests. Although we obtained valuable data for this model, it is still essential to use chordate animal models for more detailed research that enables us to translate the findings to humans. Our primary objective was to recommend the use of fruit flies to decrease the number of chordate animals required. Therefore, recommending a more precise dosage range for humans will be possible after researching chordate models.

Question 8: I think that it should be mentioned in the Materials and Methods to reference studies for selected concentrations of EOs (v/v)? I think this should be explained and added in the manuscript in order to enable comparisons to other studies. How could they justify the concentration selection for the study? Are these low or high concentrations? Working with high concentrations reduces the toxicological value of the results since they select the resistant individuals, or simply do not occur in real life. Is the dose limited by toxicity? The authors mention that in the Materials and methods.

Answer: Few articles published have utilized these specific essential oils on the D. melanogaster model. As a result, we did not have a clear starting point for the concentrations to be used, resulting in the lack of references in our Materials and Methods section. We tested different doses and determined the fruit fly's response to each of them. Based on their toxicity on the model, we selected the concentrations to use.

Question 9: The Materials and methods section should be referenced from the literature.

Answer: The group recognizes the importance of providing references to the Materials and Methods section. Therefore, we accept the suggestion and have added references to that section.

Question 10: What's the likelihood for human exposure to used concentrations in EOs? Are they realistic exposure concentrations? Please explain the experimental rationale in the discussion section. Please define better the relation between the tested dose and the potential environmental exposure. Moreover, the discussion should be improved with a more stressed comparative analysis of the own research results with literature works already present.

Answer: The requested information is significant and requires evaluation in the future. However, given that our research was not focused on extending our findings to humans, we did not deeply analyze and compare the concentrations used to their possible environmental exposures. Moreover, since essential oils are used in diverse ways, it is inaccurate to define an average concentration to which individuals are exposed when using them. Further research is necessary to elucidate this information.

Question 11: The activation of toxicity mechanisms are not completely clear, and miss of any deep investigation.

Answer: Although we believe understanding the toxicity mechanism is important, it is not the focus of our project. Our primary aim in this study was to recommend using Drosophila melanogaster as a model organism for preliminary screening tests on essential oils. We propose this approach to refine the results before testing on chordate animals, thereby reducing the number of animals required. We plan to analyze the toxicity mechanisms in our future research, but we believe they are outside the scope of our current project.

Question 12: What were the rationale for choosing the different in vivo assays? This must be included in the Introduction section.

Answer: It is important to explain why we selected our in vivo assays in both the Introduction and Methodology sections. The group appreciates and acknowledges the suggestion. The changes implemented are located on page 4. If the reviewer feels the necessity to include more information, the group will be happy to provide it.

Question 13: How many independent experiments (excluding for replicates) were performed in this study? I think the authors should mention in the Materials and Methods section. Application period projected for groups were chosen according to what? The authors should fully clarify this point.

Answer: We added information concerning the number of independent experiments to the Materials and Methods section. We welcome the suggestion since it enhances our project. Find the changes on page 4 and in the subtitles for each figure in the Results section. To evaluate the toxicity and therapeutic effects under long-term treatment, we applied the essential oils for 15 days. Our forthcoming objective is to investigate the therapeutic effects of essential oils more profoundly in treating neurodegenerative diseases, specifically Alzheimer's disease, using long-term treatment. We did not include this information in the article. If the reviewer believes it is necessary, we will be happy to add it.

Question 14: Section Discussion: This section also need to be improved with recent studies. i feel that some irrelevant discussion. Please discuss about the effect of EOs on D. melanogaster as an in vivo model.

Answer: Regrettably, there is a significant lack of research using Drosophila melanogaster as a model organism for analyzing the therapeutic effects of essential oils, which is why more research is needed to fill this gap and why there are not many comparisons of our work with previous ones. The present research articles that can be located evaluate the larvicidal and insecticidal effects of essential oils on either D. melanogaster and/or Drosophila suzukii and are referred to in the Introduction section (page 4). In the event that the reviewer deems it necessary, we can also include these articles in the Discussion section.

Question 15: What is the vehicle control name? Which were used group in the Statistical analysis?

Answer: No vehicle was used during our experiments. The essential oil was directly added into the water. To allow for proper mixing, the mixture was vortexed thoroughly every time it was pipetted, to ensure the oil became evenly dispersed in the water.

Question 16: What is positive control used for the assays? I think the authors should add values of positive control in the Figures.

Answer: In light of our previously published work on D. melanogaster, the group believes that a positive control is necessary only for testing the anti-inflammatory activity of essential oils. However, our laboratory could not yet standardize a positive control for the UAS-eiger Drosophila stock we used. We are still searching for a compound that can serve as a positive control. So far, we have tested Leflunomide (6 mg/mL to 0,0117 mg/mL) and Methotrexate (0,75 mg/mL to 0,0058 mg/mL). At the time of writing this letter, additional tests are being conducted, assessing various concentrations of aspirin, ibuprofen, as well as different concentrations of leflunomide. We are excited to obtain the final results so that we can include a positive control in our future research based on these tests.

Question 17: The correlation between selected compound (or the viability) and the in vivo experiments?

Answer: It is uncertain what the reviewer meant with this question; therefore, we are unsure how to respond. We would be pleased to provide a proper answer if they have the option to rewrite it.

Question 18: In addition in the discussion section comparative evaluation with toxicological effects at cellular levels of EOs could add additional relevance to the work. Please define better the relation between the tested concentration and the potential environmental exposure. Moreover, the discussion should be improved with a more stressed comparative analysis of the own research results with literature works, already present.

Answer: As previously stated, our intention with this paper is not to compare the concentrations used for environmental exposure at this time (refer to Question 10). We have included a paragraph in our Discussion section (page 18) that compares our work to previous studies; however, they employ different in vivo models (Wistar rats), as few studies combine Drosophila melanogaster and essential oils. In particular, there is a scarcity of published papers investigating ginger essential oil, so we could not bring in other papers to compare our work with.

Question 19: The authors should include in the discussion some thoughts about how the in vivo findings can translate to human risk.

Answer: The required information has not been added to our Discussion in light of what was reported in Questions 10 and 18. Since our goal was not to translate our results to humans, we believe that this information would be disjointed in the Discussion. If the reviewer really sees the need and believes it would improve our publication, we could add it to the Discussion.

Question 20: The authors should be added chemical constituents (%) (such as, CAS No, Quality %, Catalog number, etc.)

Answer: The chromatography charts previously submitted as supplemental material contain information on the constituents of each essential oil, its catalog number, and its quality assurance. If the author feels the need to add new information, we would be happy to do so.

Question 21: Use abbreviations for SI units: d for day.

Answer: In our prior publications, we had a consistent format for the figures to ensure consistency. We refrained from including abbreviations in our graphs to maintain consistency. We aim to maintain the same pattern in this work, so we prefer not to alter the figures. If the reviewer deems it essential, we are willing to make changes accordingly.

Question 22: What do authors think about the effects of solubility of EOs on the cytotoxicity?

Answer: Based on our current results, our group is unable to provide an accurate answer to this question. Therefore, more research is needed to assess different concentrations of EO application and vehicles for comparison purposes. Although we aim to clarify this in the future, at present, we cannot provide a definitive answer without resorting to speculation.

Question 23: The authors should add some description about the disadvantages or difficulties of using animal in vivo and in vitro models related with EOs.

Answer: This information has not been included in our work because it is deemed inappropriate. We suggest using Drosophila melanogaster as an in vivo model for comparison with other models. This choice was made because an in vivo organism can provide us with comprehensive information about the processing of essential oils in the body, their positive and negative effects, and the possibility of conducting behavioral tests to support the given observations. If deemed necessary by the reviewer, this information can be added to the Introduction.

Question 24: In Discussion: The authors should add new papers and update the literature review. More evidence on the link between toxicity and intestinal damage/life span caused by EOs could support the paper's idea (novelty) better.

Answer: The group included additional papers in the Discussion section to enhance its content. The modifications related to the comparison of our work with previous research can be located on page 19. However, as previously stated, the scarcity of literature in this area limited our ability to compare/discuss our work properly.

Question 25: The rationale for the selection of EOs must be stated at the end of the Discussion section.

Answer: A new paragraph has been added to the Discussion section to explain the reasoning behind our selection of these particular essential oils. We appreciate your suggestion. The made modifications have been recorded on page 17.

Question 26: Running title should be added.

Answer: We are unsure of what is meant by the term 'running title'. Our research shows that it may refer to a shorter version of the title that appears on the top-left corner of each page. However, PLoS One does not specify the inclusion of a running title on their submission guidelines. If the reviewer deems it necessary, we could consider adding one.

Question 27: In Figure legends, please indicate the following in each Figure legend: (a) the times of each experiments were repeated; (b) the number/replica of each group in each repeat; (c) are the data showed in the figure representative?

Answer: The group appreciates the suggestion since it will make our results clearer and easier to understand; therefore, we accept it. Modifications can be located in the Results section.

Question 28: Please highlight the novelty aspect of the present research in the Introduction, abstract and conclusion section.

Answer: Though our Introduction already featured a paragraph quickly touching on the novelty of our research, we elaborated on this topic to strengthen it, as seen on page 4. This topic is also addressed in the Conclusion section. However, as the Abstract has a word-count limit, we were unable to include the topic without sacrificing the essential information it must convey. Therefore, we opted not to make any changes. We are receptive to any new modifications that the reviewer may deem necessary.

Question 29: Statistical analysis: The authors need to provide company of stats for the statistical software. There is not enough explanation in statistical analysis. What statistical tests were used in the manuscript? Arithmetic mean ± standard deviation (or arithmetic mean ± standard error) values should be shown in the Figures.

Answer: To enhance our statistical explanation, we included a paragraph, titled 'Statistical Analysis,' in the Materials and Methods section, providing further details on how the statistical analysis was conducted. We also added requested information to the figures’ subtitles. These changes are present on page 9, and in the figures within the Results section.

Question 30: What were the rationale for choosing the different assays? This must be included in the Introduction section. Do the assays used by the authors are based on the OECD guidelines?

Answer: The group employed assays commonly used in research papers incorporating Drosophila melanogaster as a model organism. We have appended the references to our methodology to enable the reviewer to cross-check them if needed. The team attempted to acquire information on OECD guidelines for research on Drosophila melanogaster, but the only guidelines obtained were for experiments on humans and mice/rat models. However, we have elaborated on the reasons for using the chosen assays, inscribing the explanation within our Introduction section to enrich clarity (page 4).

Question 31: Reported images are really poor and need substantial improvement.

Answer: We appreciate the suggestion. The group realized upon further inspection that the image quality was not up to our standards. The resolution of the images was heightened to improve clarity. If additional changes are necessary, kindly notify us so we can improve our work. The Results section reflects the changes made.

Best regards,

Lucas Matos Martins Bernardes

Rafael Borges Rosa

Murilo Vieira da Silva

Attachment

Submitted filename: Point by Point Response Letter.docx

Decision Letter 1

Efthimios M C Skoulakis

25 Aug 2023

Drosophila melanogaster as a model for studies related to the toxicity of lavender, ginger and copaiba essential oils

PONE-D-23-11609R1

Dear Dr. Rosa,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Efthimios M. C. Skoulakis, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have diligently taken into account all of the comments, and I am confident that the manuscript is now in a commendable state for acceptance and publication.

Reviewer #2: I have checked the revised manuscript (Manuscript ID: PONE-D-23-11609R1, the paper entitled “Drosophila melanogaster as a model for studies related to the toxicity of lavender, ginger and copaiba essential oils”) and responses and I do not see anything critically wrong with this manuscript. The author has responded to the comments and made the revisions accordingly. Thus this manuscript can be accepted for publication.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Ana Paula Mendes-Silva

Reviewer #2: No

**********

Acceptance letter

Efthimios M C Skoulakis

19 Sep 2023

PONE-D-23-11609R1

Drosophila melanogaster as a model for studies related to the toxicity of lavender, ginger and copaiba essential oils

Dear Dr. Vieira da Silva:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Efthimios M. C. Skoulakis

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Dataset. Chromatographic profile of copaiba essential oil.

    Chromatographic analysis of the copaiba essential oil utilized in this study, including the lot number, chromatographic chart, and a table of the constituents of the oil.

    (PDF)

    S2 Dataset. Chromatographic profile of ginger essential oil.

    Chromatographic analysis of the ginger essential oil utilized in this study, including the lot number, chromatographic chart, and a table of the constituents of the oil.

    (PDF)

    S3 Dataset. Chromatographic profile of lavender essential oil.

    Chromatographic analysis of the lavender essential oil utilized in this study, including the lot number, chromatographic chart, and a table of the constituents of the oil.

    (PDF)

    Attachment

    Submitted filename: Comments and questions for manuscript (PONE-D-23-11609).docx

    Attachment

    Submitted filename: Point by Point Response Letter.docx

    Data Availability Statement

    All relevant data are within the paper and its Supporting Information files.


    Articles from PLOS ONE are provided here courtesy of PLOS

    RESOURCES