Mosquito Repellent Efficacy of Australian Blue Cypress Callitris intratropica Essential Oil and a Topical Formulation under Laboratory and Field Conditions

ABSTRACT.

Mosquito repellents are important for personal protection against nuisance and potentially infectious mosquito bites. Repellent activity of Australian blue cypress essential oil (EO) and a commercially formulated skin lotion containing blue cypress EO (topical formulation) were compared with 20% DEET (N, N-diethyl-3 toluamide) against mosquitoes under laboratory and field conditions in North Queensland, Australia. On a volunteer’s forearm, 1 mL of candidate material was applied to approximately 600 cm² of exposed skin. When blue cypress EO was applied at various concentrations (0.5%–10.5%), it did not fully prevent mosquito landing or biting. However, a dose–dependent increase, approaching 80% protection, was observed at high EO concentrations. On the basis of these results, three concentrations (5%, 10%, and 20%) of blue cypress EO were selected for complete protection time (CPT) experiments. Topical formulation (undiluted) was also included in CPT experiments. Although some protection was afforded, mosquito landing/probing were still recorded immediately after application for both blue cypress EO and its topical formulation. Specifically, protection declined for blue cypress EO from 80% to 70% (laboratory) and from 93% to 50% (field) within 1 hour. For topical formulation, protection declined from 85% to 75% in the laboratory and from 63% to 50% in the field. In comparison, DEET maintained a 100% protection throughout the testing period of up to 1 h, and there was no landing/probing observed in volunteers who had applied DEET. To conclude, both blue cypress products provided some protection against mosquito bites, which decreased soon after application.

INTRODUCTION

Mosquitoes transmit malaria, filariasis, zika, dengue, and other febrile illness and arboviral encephalitis.^1–3 In Australia in particular, the Aedes and Culex mosquitoes are known to transmit several arboviruses including Ross River, Barmah Forest, dengue, Murray Valley encephalitis, Kunjin, and Japanese encephalitis.^4,5 Mosquitoes use a combination of olfactory, visual, and thermal cues to detect suitable hosts.^6–10 Topical repellents are a key personal protection measure when other methods of protection such as bed nets or spatial repellents (e.g., mosquito coils) are not possible or practical.¹¹ Personal protection can reduce infection rates with mosquito-borne diseases—for example, as observed with malaria.¹¹ A repellent is defined as any compound that has an effect on the behavior of mosquitoes including “movement away from the source” (repellency in the strict sense) and “inhibition of attraction” (interference with host detection and/or feeding response).^12,13

The use of insect repellents dates to ancient times, when substances such as plant oils, smokes, and tars were used to kill or repel insects.^14,15 Citronella oil, dimethyl phthalate, Indalone, and Rutgers 612 were the common insect repellents before World War II.¹⁵ However, these products failed to provide the desired protection for military personnel, which led to the screening of 20,000 potential mosquito repellent compounds by the U.S. government and the discovery of N, N-diethyl-m-toluamide (DEET).¹⁵ DEET was first introduced into the market in 1956 and serves as an effective broad-spectrum insect repellent with a long-lasting effect on mosquitoes, ticks, chiggers, and fleas.^15,16 DEET is available in a variety of formulations, including aerosols, creams, lotions, and sprays, and in concentration ranging from 5% to 100%, although most products contain less than 40%.¹⁶ Before they can be registered, most skin-applied repellents are evaluated for their safety and efficacy by the U.S. Environmental Protection Agency.¹⁷ Although not yet registered, citronella, cedar, geranium, peppermint, and soybean oils have been shown to exhibit mosquito repellence.¹⁷

In the past few decades, consumers’ interest in plant-based repellents have increased because they are fully biodegradable, with no ecotoxicology concerns or environmental residues and are considered as “safe” compared with synthetic repellents.¹⁸ This has led to an increase in plant-based ingredients in insect repellents on the shelves in some countries.¹⁹ Plants synthesize and emit a large variety of volatile organic compounds; floral volatiles serve as attractants for pollinators and volatiles extracted from vegetative parts, especially those release after herbivory, protect plants by deterring herbivores.²⁰ Essential oils (EOs) obtained from plants through distillation or mechanical methods such as cold pressing are among the best-studied substances. Some plants whose EOs have reported good repellent activity up to 8 hours (depending on mosquito species) include amyris, broad-leaved eucalyptus, camphor, catnip, carotin, cedarwood, chamomile, cinnamon oil, juniper, cajeput, Curcuma longa, geranium, galbanum, jasmine, litsea, lavender, lemongrass, lemon-scented eucalyptus, narrow-leaved eucalyptus, niaouli, olive, rosemary, sandalwood, soya bean, tagetes, and violet.^14,21–23 EOs contain complex mixtures of volatile organic compounds such as hydrocarbons (terpenes) and oxygenated compounds such as alcohols, aldehydes, esters, ethers, ketones, lactones, and phenols.²³ Monoterpenes (α-pinene, cineole, eugenol, limonene, terpinolene, citronellol, citronellal, camphor) and sesquiterpenes (β-caryophyllene) are common constituents of EOs from plants described in the literature as presenting mosquito repellent properties.²³ Most of these EOs vary in the duration of protection against biting mosquitoes, with effects lasting for few minutes to several hours.^22,24 Their active ingredients are highly volatile and can evaporate quickly from the skin, resulting in brief protection time.¹⁴

In Australia, at least 40 essential oils from native plants, mostly, Eucalyptus spp. and Melaleuca spp., have been evaluated against mosquitoes, march flies, and sand flies.^21,25–28 Indigenous people from northern Australia have used smoke produced from burning of blue cypress (Callistris intratropica R.T. Baker and H.G.) for generations to repel mosquitoes.²⁹ Essential oil from the bark and wood of the blue cypress is composed exclusively of sesquiterpene alcohols (guaiol, bulnesol, and eudesmol) and terpenes (selinene isomers).^30,31 Its antimicrobial activity has been documented, but little is known about its effectiveness to repel insects.^25,30,32 The present study was conducted to determine the repellent effect of Australian blue cypress EO and its topical formulation (a skin lotion, Aussie Blue Off^®) against mosquitoes. Specifically, the estimated effective doses of the blue cypress EO and the complete protection time of both the blue cypress EO and the topical formulation were compared with 20% ethanolic DEET.

MATERIALS AND METHODS

Study design.

This study evaluated the repellency of blue cypress EO and a formulated skin lotion containing blue cypress EO against Aedes aegypti in the laboratory and against wild mosquito populations in North Queensland, Australia following WHO guidelines for topical repellent testing.³³ This involved a blinded randomized trial that compared the repellent effect of the test formulations to 20% ethanolic DEET (Merck & Co., Macquarie Park, NSW, Australia) as positive control and 100% laboratory grade ethanol (Merck & Co., Macquarie Park, NSW, Australia) as negative control. Because Ae. aegypti bites primarily during the day, the tests were conducted between 9 am and 4 pm.

Study participants.

Twenty-three volunteers, 13 males and 10 females, who completed an online survey stating they had low or no known skin reactions against mosquitoes or ingredients of the products were recruited. Twelve participants took part in effective dose estimation, and 11 participants took part in complete protection time (CPT) experiments. The age of the participants ranged from 18 to 65 years. The study was approved by the James Cook University Human Research Ethics Committee (H8476), and the study participants provided written informed consent before repellency testing. To avoid unwanted bias, the participants were asked to avoid the use of fragrance and repellent products for 12 h before and during testing. During the experiments, the participants were instructed to keep their treated arms away from their clothing and other surfaces.

Test formulations.

The Australian blue cypress wood and bark essential oil and a skin lotion containing blue cypress EO marketed as Aussie Blue Off^®, both provided by Australian Blue Cypress Pty. Ltd. (Northern Territory, Australia) were tested. The Australian blue cypress wood and bark essential oil is referred to hereon, as, blue cypress EO and Aussie Blue Off as topical formulation. Different concentrations of blue cypress EO were prepared in laboratory-grade ethanol (100%) before each trial, whereas the topical formulation was used as supplied (undiluted).

LABORATORY EXPERIMENTS USING ARM-IN-CAGE TEST

The laboratory experiments were conducted at the Australian Institute of Tropical Health and Medicine in Smithfield, Queensland, Australia from September to October 2021. The mosquitoes used were 3 to 5 days posteclosion, non–blood-fed nulliparous female Ae. aegypti (Wb_mel strain) from a laboratory-reared colony. The insectary was maintained at 26.0 ± 2.5°C with a relative humidity of around 75% ± 11%.

Effective dose estimation (laboratory).

To find the effective dose to prevent mosquito landing or probing, the following (cumulative) doses of blue cypress EO were tested: 0.5%, 1.5%, 3%, 5%, 7.5%, and 10.5%. The rationale for selecting these doses was that the maximum active ingredient concentration in formulated products was unlikely to exceed 10%. The experiments were repeated three times with each volunteer (N = 12) on different days, so that a total of N = 36 experiments were conducted. The blue cypress EO dilutions were applied in increasing concentrations to one of the forearms of each volunteer with a defined amount (e.g., 1 mL per 600 cm²) and were exposed to N = 50 laboratory-reared Ae. aegypti mosquitoes for 30 seconds. The total number of mosquito landings and probings were counted during that period. Results from the triplicate repetitions conducted with each volunteer were averaged. The negative control (ethanol only; 30-second exposure) tests were conducted on each arm before testing the blue cypress EO. The experiments concluded with a negative control on the other, untreated arm.

Complete protection time estimation (laboratory).

Complete protection time is the number of minutes elapsed between the time of repellent application and the first mosquito landing and/or probing. The blue cypress EO concentration tested, 20%, was based on the results of effective dose laboratory study (as described) and recommendation in the WHO guidelines.³³ On each volunteer (N = 11), 20% blue cypress EO was applied to one forearm, and 20% DEET was applied to the other forearm. After a drying time of 1 minute, the treated forearms were exposed to separate cages (size: 30 cm per side) of N = 200 Ae. aegypti mosquitoes for 3 minutes. The total number of mosquito landings and probings were counted during that period by both the volunteer and the research staff on sectioned forearm. This was done at 0, 30, and 60 minutes postapplication.

The topical formulation (skin lotion, Aussie Blue Off) was tested as provided (without any dilution) in the CPT experiments. Here, we made two modifications to the WHO guidelines due to a substantial biting pressure (more than 100 bites within the 3 minutes of exposure period), causing discomfort to the volunteers. First, the WHO guidelines recommend 200 to 250 mosquitoes per cage,³³ but we reduced the number of mosquitoes per cage to 50. The exposure time was 3 minutes, and the tests were done at 0, 30, and 60 minutes postapplication. Second, the WHO guidelines recommendation is to stop exposure at the time point when the first landing or probing is observed with the candidate product. However, because we never observed complete protection using the blue cypress EO or its topical formulation, even directly after application, we did not stop but conducted the experiments up to 60 minutes postapplication to calculate percent protection.

FIELD EXPERIMENTS USING HUMAN LANDING CATCHES

The field experiments were conducted in February to April 2022 at Cattana Wetlands in Smithfield, North Queensland, Australia, which is an 80-hectare reserve containing regionally significant forests and a diverse range of bird species and wildlife. This site was chosen due to its high abundance of mosquito species as well as a suitable biting rate. A preliminary assessment of collection sites was performed prior to the trial.

Effective dose estimation (field).

The following doses of blue cypress EO in ethanol were tested in the field: 2.5%, 5%, and 10%. The rationale for selecting these doses was that the maximum blue cypress EO concentration in the formulated products usually does not exceed 5%. In addition, we chose 10% because the laboratory results showed that protection increased to ∼80% at the highest blue cypress EO concentration (10.5% cumulative dose). On each trial day, five volunteers participated, and each person was randomly assigned to the five solutions including a negative control (ethanol only), a positive control (20% DEET) and the three doses of the blue cypress EO. The volunteers were fully covered except for the leg (knee to ankle area) where the test solution was applied. The treated leg was exposed for 10 minutes, and any mosquito that landed and/or probed during that period was captured using a handheld aspirator. The sampling container with mosquitoes were removed from the aspirator. New sampling containers were used for each 10-minute collection period and each location. Captured mosquitoes were identified in laboratory. The number of mosquitoes that landed but were not captured were also recorded. A single (1-day) test included rotation of each volunteer at random among all (N = 5) collection sites at 10-minute intervals. The experiments were repeated three times on different days, so that a total of 15 experiments were conducted for blue cypress EO. Results from the triplicate repetitions conducted were averaged.

Complete protection time estimation (field).

For blue cypress EO CPT, four volunteers were recruited per day for testing the two doses of the EO (5% and 10%) and the controls (positive and negative). Human landing catches were performed as described earlier. For the topical formulation, three volunteers were recruited per day for testing the topical formulation and the controls. A single (1-day) test comprised rotation of each test volunteer at random among all collection sites (four for EO and three for topical formulation) at 10-minute intervals. The experiments were repeated three times, for a total of 12 and nine experiments were conducted for the blue cypress EO and the topical formulation, respectively. Results from the triplicate repetitions were averaged.

DATA ANALYSIS

Data was analyzed using Microsoft^® Excel 2016 and GraphPad Prism 9.3.1 (GraphPad Software Inc., www.graphpad.com). The level of protection (% protection) was calculated as the reduction of mosquito landings and/or probing observed on the treated limb relative to the ethanol-only controls using the formula: Protection (p) = (C – T) / C, where T corresponds to the number of landings/probings on repellent treated arm and C is the average of two control arms (ethanol). Complete protection time is calculated as the number of minutes elapsed between the time of repellent application and the first mosquito landing and/or probing.

RESULTS

Effectiveness of Australian blue cypress essential oil at preventing mosquito bites.

Overall, an increasing level of protection with increasing concentrations of blue cypress EO were observed in both the laboratory (Figure 1) and field (Figure 2) experiments. In the laboratory, the median level of protection observed with 12 volunteers was > 50% for all tested blue cypress EO concentrations and increased to around 80% at the highest blue cypress EO concentration (10.5% cumulative dose) (Figure 1). We observed a large degree of variability in the number of landings and probings with different volunteers in both the control and treatment arms. As such, the percent protection calculated was also highly variable—for example, ranging from ∼50% to 100% for a 5% cumulative blue cypress EO concentration. In the field, the highest dose (10% blue cypress EO) provided 81% protection (Figure 2). It should be noted that 100% protection was rarely achieved at 10% concentration.

Figure 1.

Median level of protection (%, black squares) and 95% CI for cumulative doses of 0% to 10.5% of blue cypress essential oil from the laboratory effective dose experiments. The triplicate data for each of the 12 participants was averaged (blue dots) before the calculation of the medians. The red line is an empiric dose response model.

Figure 2.

Percent protection for blue cypress essential oil at 2.5%, 5.0%, and 10.0% doses from the field effective dose experiments. Large blue dots are averages and small, transparent blue dots are results for each replicate. The percent protection for DEET (N, N-diethyl-3 toluamide; not shown) was always at 100%.

Complete protection time.

In the laboratory arm-in-cage tests, CPT for either the 20% blue cypress EO or its topical formulation were < 1 min because mosquito landing and probing were recorded at the exposure immediately after application. Figure 3 shows the percent protection and the absolute number of mosquito landings and probings observed in the 3-minute exposure over time afforded by DEET and blue cypress EO in 11 volunteers (Figure 3A and C), and its topical formulation in eight volunteers (Figure 3B and D). A large degree of variability was observed in the number of landings and probings among volunteers with both the blue cypress EO and its topical formulation. Generally, DEET (as the positive control) prevented probing to 100% but allowed for sporadic landing during the 1-hour observation period. There were instances of probing at 60 minutes after treatment with DEET in the case of two volunteers in the formulated product CPT experiments, but we attribute this to the volunteer not following instructions—that is, rubbing off the repellent during the trial rather than a failure of DEET. The percent protection afforded by 20% blue cypress EO diluted in ethanol against 200 Ae. aegypti mosquitoes was 81% and decreased to 73% after 1 h (Figure 3A and C). This was statistically significantly less protection than the DEET (P < 0.0001) and meant that with 200 mosquitoes per cage, volunteers still received more than an average of 50 landings and/or probings during the 3-minute exposure period. With the topical formulation, we observed a slightly higher initial percent protection value of ∼85%, which then decreased to ∼75% at 1 hour post-application (Figure 3B and D). With 50 mosquitoes per cage, this meant that volunteers still received ∼10 to 20 bites in the 3-minute exposure period.

Figure 3.

Percent protection and absolute number of mosquito bites for 20% blue cypress essential oil (EO) and its topical formulation from the laboratory complete protection time experiments. (A and B) Percent protection (i.e., the number of landings or probings relative to the negative control) over time. (C and D) Absolute number of bites received before (control [CTL]) and after treatment. Large dots are averages and small, transparent dots are results for each replicate (volunteer). Red is N, N-diethyl-3 toluamide (DEET; (positive control), blue is blue cypress EO, green is topical formulation, purple is ethanol (negative control) before application of DEET, and orange is ethanol (negative CTL) after application of candidate products. ***Statistical significance (P < 0.0001) between DEET and candidate product (Mann–Whitney U test).

In the field, 10% blue cypress EO induced 93% protection, which decreased to 56% after 50 minutes, whereas 5% blue cypress EO induced 75% protection, which decreased to 50% protection after 50 min (Figure 4A and B). Similarly, protection decreased from 63% to 50% for the topical formulation (Figure 4C). In comparison, DEET maintained a 100% protection throughout the testing period of 50 minutes, and there were no landings/probings observed in volunteers who had applied DEET. For comparison, we also show the absolute number of mosquito landings and/or probings observed in the 10-minute exposure period for ethanol-only (negative control), DEET, blue cypress EO, and its topical formulation in Figure 4D–F.

Figure 4.

Percent protection and absolute number of mosquito bites for blue cypress essential oil (EO) and its topical formulation in the field complete protection time experiments. (A–C) Percent protection (i.e., the number of landings or probings relative to the negative control) over time. (D–F) Absolute number of landings/probings received by volunteers who applied ethanol (negative control), candidate products and N, N-diethyl-3 toluamide (DEET; positive control). Large dots are averages and small, transparent dots are results for each replicate (volunteer). Red is DEET (positive control), blue is blue cypress EO, green is topical formulation, and purple is ethanol (negative control).

Mosquito species composition.

Altogether 640 mosquitoes belonging to eight species were caught in the field on legs treated with blue cypress EO and its topical formulation. The most common species were Verralina carmenti (63% and 59%) and Ve. lineata (30% and 29%) field experiments of both blue EO (Figure 5A) and the topical formulation (Figure 5B). Other species caught in low numbers (≤ 7%) included Aedes vigilax, Ve. funera, Ae. notoscriptus, Ae. kochi, Culex annulirostris, and Coquillettidia crassipes. The details of collected mosquito species and numbers are in Supplemental Table 1.

Figure 5.

Distribution of mosquito species caught during the field experiments. (A) Percentage of species caught during experiments with blue cypress essential oil. (B) Percentage of species caught during experiments with topical formulation. Mosquito species numbers were combined (negative control and candidate materials) before the percentages were calculated.

DISCUSSION

The presence of sesquiterpenoid and terpene indicate that the Australian blue cypress EO could possess repellent activity (Figures 1 and 2). C. intratropica essential oil is composed mainly of guaiol (26%), bulnesol (16%), eudesmol isomers (9.5%) and selinene isomers (19%–22%) as well as unique lactones (< 1%) such as callitrin, callistrisin, and columellarin.^31,34 However, it should be noted that the composition of bioactive compounds depends on the collection site and the Callistris species. For example, the guaiol in wood essential oil of C. intratropica (blue cypress) is 17% to 21% and, in both C. columellaris (white cypress) and C. endlicheri (black cypress), is 50%.

The blue cypress EO diluted in ethanol (5%, 10%, and 20%) and its topical formulation offered some protection but did not provide complete protection even immediately after application. There is little documentation available on the repellent effect of blue cypress EO against mosquitoes. In addition, because of the variability on methodologies found in the literature to assess repellency, it makes it difficult to compare our results with reported studies. Consequently, we compared our results to studies with methods similar to the WHO guidelines and bioactive compounds in essential oils of plants that have longer duration of repellency.

Greive et al. studied the repellent effect of other Australian Callistris species: C. collumelaris and C. glaucophyla (for both, 5% wood EO were prepared in a cream base) against Ae. aegypti with an exposure time of 1 minute.²⁵ For both Callistris species, repellency post-application was initially high (68.3% for C. collumelaris and 90.6% for C. glaucophyla) but decreased over the 60-minute study period.²⁵ These results are like our study in that the cypress EO offered some protection for a short time but not full protection.

Interestingly, essential oils with major guaiol content have been shown to have larvicidal activity. For instance, mixtures of Australian C. glaucophylla with other botanicals resulted in 100% mortality against Cx. annulirostris larvae and affected emergence of Ae. aegypti adults.³⁵ In addition, sandalwood EO, which contained a higher percentage of guaiol (43.8%), appeared to have the most effective larvicidal activity on Ae. aegypti, Ae. albopictus, and Cx. pipiens compared with cinnamon oil, lemon eucalyptus oil, and turmeric oil.³⁶ Further studies on the effect of blue cypress EO on mosquito larvae are recommended.

Essential oils are highly volatile, and thus diluting them in carrier oils, creams, or lotions may extend the duration of protection time. For example, essential oils of clove, citronella grass, and lemongrass diluted in coconut oil or olive oil have shown to induced protection up to 96 minutes for Ae. aegypti and 165 minutes for Cx. quinquefasciatus.³⁷ In the present study, neither the ethanol (diluent for blue cypress EO) nor the lotion (base for the topical formulation) reduced the ability of the active ingredients to evaporate quickly when applied to the skin.

Further, addition of certain botanicals as synergist to the blue cypress EO can help extend the duration of its repellency. Previous studies have shown that addition of 5% vanillin to EO of turmeric, citronella grass, hairy basil, and eucalyptus oil repelled mosquitoes for up to 8 hours.^21,22,38,39 Also, repellents that have low volatility; for example, PMD, a monoterpene, derived from lemon-scented eucalyptus (Corymbia citriodora), does not tend to evaporate rapidly after application on the skin, resulting in longer protection time.^22,40 The essential oil of Australian C. intratropica is highly volatile resulting in brief protection times, which may be improved with addition of other botanicals as synergists, such as 5% vanillin, but this requires further study.

The positive control, 20% ethanolic DEET, maintained a 100% protection over the entire study duration in both the laboratory (60 min) and the field experiments (30 and 50 minutes) compared with blue cypress EO and its topical formulation. This is because the chemorepellency of DEET is mediated by Ae. aegypti feet when alighting on the skin.³⁷ Interestingly, previous studies have found that essential oils containing oxygenated compounds have a higher repellent activity than DEET against Ae. aegypti and An. gambiae.^41,42

CONCLUSIONS

The results of this study indicate that C. intratropica essential oil offers some protection against biting mosquitoes. Adding a botanical synergist may increase its potential as a short-period repellent or under conditions of low mosquito abundance. However, it is important that public health messages continue to emphasize the greater effectiveness of DEET-based repellents in areas with risks of mosquito-borne disease.

Note: Supplemental material appears at www.ajtmh.org.