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- J Arthropod Borne Dis
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- PMC5186743
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J Arthropod Borne Dis. 2016 Dec; 10(4): 528–537.
Published online 2016 Oct 4.
PMCID: PMC5186743
PMID: 28032105
Mona Sharififard,1 Farhad Safdari,2 Amir Siahpoush,3 and Hamid Kassiri1,*
Author information Article notes Copyright and License information PMC Disclaimer
Abstract
Background:
Essential oils, as secondary plant compounds, present a safer alternative to conventional insecticides in insect control programs. So five essential oils including eucalyptus, mint, yarrow, oregano and rosemary oils were evaluated against the brown-banded co*ckroach Supella longipalpa.
Methods:
Evaluation was done against the 3rd and 4th instar nymphs using three bioassay methods; continuous contact toxicity, fumigant toxicity and repellent activity. The study was done in the laboratory of medical entomology, during April 2012 to September 2013.
Results:
Mortality rates by the lowest concentration (2.5%) of rosemary, oregano, yarrow, eucalyptus and mint oils were 100%, 62.2%, 45 %, 36.2% and 5.2% at 24 h after exposure respectively. Rosemary oil was determined as the most toxic oil because of 100 % mortality rate at the concentration range of 2.5% to 30%. The lowest fumigation effect using 50 μl/L air was recorded from mint oil with 97.2 % mortality after 24 h, while the other oils caused 100% mortality. The most repel activity was related to oregano oil which showed 96.5–99.1% repellency at the concentration range of 2.5–30% with a residual effect lasting at least a week after treatment.
Conclusion:
Oregano oil could be used as a potential repellent against S. longipalpa. Also, all five essential oils could be used as the safe compounds for surface treating or fumigation in co*ckroach control programs while rosmary and oregano oils exhibited the most toxicity.
Keywords: Essential oil, Supella longipalpa, Mechnical vector
Introduction
Natural contamination of co*ckroaches with wide range of pathogenic organisms including about 40 species of bacteria, nearly 12 species of pathogenic helminthes, the second largest group of vertebrate pathogens, and also viruses, protozoa and fungi affecting man and other vertebrate animals have been reported by numerous studies (Le Guyader et al. 1989, Rivault et al. 1994, Baumholtz et al. 1997, Cochran, 1999, Eggleston and Arruda 2001, Savoldelli and Luciano 2005). Often their movement between waste and food materials led to acquire, carry, and mechanically transfer of these pathogens. As proven or suspected carries, co*ckroaches play a prominent role in caring and distributing of organisms causing ddiarrhea, dysentery, cholera, leprosy, plague, typhoid fever, and viral diseases such as poliomyelitis are carries by co*ckroaches. They also carry the parasitic worms such as Taenia, Shistosoma, Ascaris and may cause allergic reactions, including dermatitis, itching, swelling of the eyelids, and more serious respiratory conditions (Stankus et al. 1990, Savoldelli and Luciano 2005). The brown-banded co*ckroach, Supella longipalpa known previously as S. supellectilium as a nearly cosmopolitan co*ckroach, has recently become a hygiene problem in the city of Ahvaz, southwestern Iran and it seems to be the dominant species of dwellings particularly in apartments (Vazirianzadeh et al. 2013, Sharififard et al. 2014). This is small co*ckroach measuring 10–14 mm in length with pronotum of male is rather uniformly dark with lateral edges and definitely lacks of the two parallel stripes of Blatella germanica. The adult males appear to be very slender with its wing extending beyond the tip of the abdomen. Adult females have short wings which expose the considerable portion of their stout abdomen. The common name derives from presence of two dark-colored transverse bands on mesonotal and abdominal terga (Cochran 1999).
This co*ckroach species carries a variety of microorganisms (Le Guyader et al. 1989), and is a vector of pathogenic bacteria in urban environments (Rivault et al. 1994). It is also reported as an allergen source (Eggleston and Arruda 2001, Savoldelli and Luciano 2005). Twenty nine bacterial species were isolated from S. longipalpa caught in the hospital (Le Guyader et al. 1989). Its movement from one department to the others, inside the hospital increases potential bacterial contamination risks, for some of the species such as Acinetobacter and Pseudomonas are dangerous for some kind of patients (Le Guyader et al. 1989).
The co*ckroach infestations are common particularly in dwellings without proper ventilation in warm climates, hospitals, and restaurants and in business establishments with a relatively high ambient temperature and humidity (Schal et al. 1984, Le Guyader et al. 1989, Baumholtz et al. 1997, Phillips and Appel 2010). The brown-banded co*ckroach needs nearly much less relative humidity to complete its life cycle and spread the infestation. It may be the main reason for more distribution of this co*ckroach species in Ahwaz City, as a warm area, in recent years compared with same species, the German co*ckroach.
Conventional insecticides are used as main tool to control co*ckroach infestations but there are many concerns about the harmful side-effects of these chemical compounds. Also the insecticide use is restricted in places such food preparation areas, restaurants, storage buildings and apartments. These restrictions of chemical insecticide application increase demand for safer alternatives against co*ckroach infestations (Savoldellis and Suss 2005, Phillips and Appel 2010). Different level of resistance to many compounds of chemical insecticides including organochlorine, organophosphorus and carbamat insecticides have been documented in many field-collected strains of co*ckroaches from Iran. So, application of these insecticides should be stopped and replaced with other safer compounds (Nasirian et al. 2006, Nasirian 2010, Ladonni et al. 2013).
Essential oils, as secondary plant compounds responsible for the aromatic characteristics of plants, present the potential alternative to conventional insecticides (Isman 2000, 2006, Omara et al. 2013). Plant extracts and essential oils are reported to have a wide range of activity against insect and mite pests, plant pathogens, fungi and nematodes (Isman 2006). Recent reports have highlighted antimicrobial, antifungal, anti-cancer and insecticidal properties of plant essential oils (Isman 2000, 2006). They have fumigants, antifeedant and repellent effects as well as inhibiting the reproduction in co*ckroaches and other insects (Omara et al. 2013). They could be used in areas where chemical insecticides are prohibited. The repellent effect of essential oils has been reported against many insect pests such as co*ckroaches, termites, mosquitoes, ticks, ants and houseflies (Chen et al. 2002). Numerous studies have demonstrated the toxicity and repellency of essential oils against co*ckroaches (Ahmad et al. 1995, Apple et al. 2001, Jang et al. 2005, Thavara et al. 2007, Ferrero et al. 2007, Ling et al. 2009, Tunza et al. 2009, Phillips et al. 2010, Phillips and Appel 2010, Zhu et al. 2012, Manzoor et al. 2012, Omara et al. 2013).
Consider to co*ckroaches role in transmission and distribution of many human pathogens, their resistance to many chemical insecticides, the side-effects of insecticide usage in human dwellings, high distribution of the brown-banded co*ckroach in Ahvaz city during recent years, and eventually in order to finding a safe alternative for chemical insecticides, the present study was done to evaluate toxicity and repellency of following five essential oils, Eucalyptus oil (Eucalyptus sp), Mint oil (Mentha piprita), Yarrow oil (Achillea millefolium), Oregano oil (Origanum vulgare) and Rosemary oil (Rosmarinus officinalis) against this co*ckroach species.
Material and Methods
co*ckroach
The brown-banded co*ckroaches were reared in Plexiglas containers and maintained at 27±2 °C, 50±5% RH, and the photoperiod of 12:12 (L: D) h. They were fed with dry crumbled biscuits, bread and water. Pieces of facial tissue were provided as a harborage and surface sticking of ootheca. co*ckroaches were anesthetized by chilling to facilitate handling. The co*ckroach colony had been established at least 2–3 years before the study. The study was done in the laboratory of medical entomology, Department of Medical Entomology and Vector Control, Ahvaz Jundishapure University of Medical Sciences during April 2012 to September 2013.
Essential oils
Five essential oils including, Eucalyptus sp (Eucalyptus oil), Mentha piprita (Mint oil), Achillea millefolium (Yarrow oil), Origanum vulgare (Oregano oil) and Rosmarinus officinalis (Rosemary oil) used in the tests were extracted from fresh or dried plant foliage by the hydrodistillation method using Clevenger apparatus in Herbal and Natural Product Research Center of Ahvaz Jundishpapur University of Medical Science. Plant material was placed in a 2-liter round bottomed flask with distilled water (100 ml for 75g dry material and 400 ml for 200 g fresh material) and the essential oil was extracted by water distillation. The distillation period was 1 h for fresh samples and 1 h 15 min for dried samples (Charles and Simon 1990).
Bioassays
Contact Toxicity: The bioassay method of WHO (World Health Organization) (1975) was used to determine susceptibility or resistance of the co*ckroaches to essential oils. Essential oils were prepared in acetone as the solvent (v/v) at concentrations of 2.5%, 5%, 10%, 15% and 30%. Glass jars (600 ml) were treated uniformly with 2.5 ml of each concentration and left under room conditions to dry. The top inner surfaces of jars were smeared with a thin layer of butter to restrict co*ckroach movement within the jars. Groups of thirty 3rd and 4th nymph instars were anesthetized by chilling and transferred to plastic cups. After recovery, they were transferred to treated jars and left to continuous exposure to treated surface. Control group was exposed to surface treated with acetone. The mortality was calculated 24 h after recovering period.
Fumigant Toxicity: Groups including thirty 3rd and 4th nymphs instars were released in 1-lit glass jars with a 1-cm diameter cotton ball treated with 50 μl pure essential oil. To prevent direct contact of co*ckroaches with essential oil, it was injected to the center of each cotton ball by micro sampler. Control group was exposed to cotton ball treated with acetone. The mortality was calculated 24 h after recovering period.
Repellency Test: The method applied that of Ferrero (2007) and Manzoor (2012) with some modifications. Circular white filter paper No 1 (15 cm diameter, Whatman) was divided in to two approximately equal pieces. Acetone was used as the solvent. One half was treated with 1 ml essential oil solution using a micro sampler and the other half was left untreated. The oils were assayed at the following 5 concentrations: 2.5, 5, 10, 15 and 30% (V/V). After solvent evaporation, filter paper was used to cover the floor of cylindrical Plexiglas jars. Thirty nymphs (3rd and 4th instars) were released into the center of each jar and distribution of the nymphs was calculated 24 h after exposure. For the control group, one half of the filter paper was treated with acetone and the other half was left untreated. All experiments were done with four replicates.
Data Analysis
Means of mortality percentages and standard errors in contact and fumigant toxicity were calculated using SPSS 16 software. Repellency values (RV) were determined using this formula:
Repellency (%) =100−(T×100)/N
Where T stands for the number of co*ckroaches located in the treated area and N stands for the total number of co*ckroaches used (Thavara et al. 2007).
Analyses by ANOVA and comparison of mortality and repellency percentage means was done by Tukey's test (P< 0.05), using SPSS software (Chicago, IL, USA).
Results
Contact Toxicity
Concentrations of 30% and 15% of the five essential oils caused 100% mortality against the co*ckroach nymphs using continuous exposure method. Mortality rates were 100%, 62.2%, 45%, 36.2% and 5.2% respectively at the concentration of 2.5% for the essential oils of rosemary, oregano, yarrow, eucalyptus and mint after 24 h (Table 1) which were significantly different (P< 0.0001). Furthermore, significantly difference was noted between effectiveness of the essential oils with comparison of the total means of mortality (P< 0.0001) against the brown-banded co*ckroach.
Table 1.
Contact toxicity of essential oils against Supella longipalpa at different concentrations at 24 h after recovering period. (Department of Medical Entomology, Ahvaz Jundishapure University of Medical Sciences during April 2012 to September 2013
| Essential oil | Concentrations (%) (acetone was used as solvent) | Mortality Means (%)±SE |
|---|---|---|
| Rosemary oil | 30 | 100 |
| 15 | 100 | |
| 10 | 100 | |
| 5 | 100 | |
| 2.5 | 100 | |
| Oregano oil | 30 | 100 |
| 15 | 100 | |
| 10 | 100 | |
| 5 | 100 | |
| 2.5 | 62.2±1.4 | |
| Eucalyptus oil | 30 | 100 |
| 15 | 100 | |
| 10 | 100 | |
| 5 | 100 | |
| 2.5 | 36.2±1.5 | |
| Yarrow oil | 30 | 100 |
| 15 | 100 | |
| 10 | 93±3 | |
| 5 | 79.9±3.3 | |
| 2.5 | 45±2.9 | |
| Mint oil | 30 | 100 |
| 15 | 100 | |
| 10 | 100 | |
| 5 | 24.7±4.1 | |
| 2.5 | 5.2±1.9 | |
| Control (treated with acetone) | 100 | 0 |
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Rosemary oil was the most toxic oil against S. longipalpa because it caused 100% mortality of the co*ckroach nymphs at all concentrations (Table 1). The next most effective oils were oregano and eucalyptus oils because they killed 100% of the nymphs at the concentration range of 5–30%. At the concentration of 2.5% nymph mortality means were reduced to 62.2% and 36.2% with oregano and eucalyptus oils respectively which showed significantly difference (P< 0.0001). So, oregano oil was more effective than eucalyptus oil for the brown-banded co*ckroach (Table 1). Mortality means of the co*ckroach nymphs varied from 45% to 100% by yarrow oil which were significantly different (P< 0.0001). Mint oil caused 100% mortality at concentrations range of 10–30% but mortality reduced to 24.7% and 5.2% at the treatments of 5% and 2.5% respectively. So, mint oil was clearly determined as the least effective oil against the co*ckroach at lower concentrations compared to the other tested oils but its effect was similar to that of other oils at higher concentrations. All nymphs in the control group remained live 24 h after exposure, and even after a week.
Fumigant Toxicity
No mortality was observed in the control group during 24 h test period. Fumigation of all the five essential oils caused 97.2% to 100% mortality in the brown-banded co*ckroach nymphs at 24 h after exposure or even earlier. Mint oil exhibited lower fumigant effect compared to the other essential oils and it caused a mean mortality rate of 97.2% in nymph population that was not significantly different with the other oils (P> 0.05).
Repellency Effect
All tested essential oils showed high repellency against the brown-banded co*ckroach at the prepared concentrations (Table 2). Evaluations for repellent activity were different according to the essential oil and the concentration. The highest and the lowest repellent effects were recorded in oregano oil and eucalyptus oil respectively compared with the control group. Repellent effect of oregano oil at different concentrations ranged from 96.5 to 99.1% which were not significantly different together (P> 0.05), while the highest repellency (99.1%) was observed at the lower concentration of 2.5% (Table 2). In the repellency test with oregano oil, nearly all the released co*ckroaches were concentrated at the same place in untreated area and they were not close to the treated area even after a week.
Table 2.
Repellency effects of plant essential oils against the brown-banded co*ckroach, S. longipalpa at 24 h after recovery period (Department of Medical Entomology, Ahvaz Jundishapure University of Medical Sciences during April 2012 to September 2013.
| Essential oil | Concentrations (%) (acetone was used as solvent) | Repellency Means (%)±SE |
|---|---|---|
| Origanum vulgare | 30 | 96.5± 3.5 |
| 15 | 96.8 ± 3.8 | |
| 10 | 96.03± 2.4 | |
| 5 | 98.8± 1.2 | |
| 2.5 | 99.1± 0.9 | |
| Eucalyptus sp | 30 | 27.7± 3.9 |
| 15 | 33.3 ± 3.1 | |
| 10 | 43.2± 2.8 | |
| 5 | 51.7. ± 0.9 | |
| 2.5 | 49.8± 4.9 | |
| Mentha piprita | 30 | 63.3± 4.7 |
| 15 | 60.1 ± 3.8 | |
| 10 | 59.01± 2.8 | |
| 5 | 68.8± 3.2 | |
| 2.5 | 63.3± 3.7 | |
| Achillea millefolium | 30 | 86.7 ± 4.9 |
| 15 | 84.2± 3.5 | |
| 10 | 83.3± 4.5 | |
| 5 | 92.8± 1.7 | |
| 2.5 | 79.3± 3.9 | |
| Rosmarinus officinalis | 30 | - |
| 15 | - | |
| 10 | - | |
| 5 | 86.2± 4.3 | |
| 2.5 | 94.5± 0.87 | |
| Control (treated with acetone) | 100 | 6.6± 2.3 |
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Eucalyptus oil was determined as the least repellent compound, it caused 27.7–49.8% repellency at different concentrations against the brown-banded co*ckroach compared to the other oils. Its repel effects were a little significantly different (P< 0.05) at different concentrations.
Mint oil caused 59.1–68.8% repellency against the brown-banded co*ckroach and that repellency of this oil at lower concentration (2.5%) was not significantly different with higher concentrations (P> 0.05).
Repellent effect of yarrow oil was varied from 79.3 to 92.8% that was not significantly different (P> 0.6). The most repellent activity was related to the concentration of 5% that was 92.8% but decreased to 79.3% at the concentration of 2.5% While the different between repellency at the concentration of 2.5% and 5% was not significantly (P= 0.149).
The repellent effect of rosemary oil was tested with only two concentrations. While the highest effect was recorded at the lower concentration of 2.5% (94.3%) but it was not differ significantly with the concentration of 5% (86.2%).
The difference was significant between the repellency of the essential oils with control group (P< 0.0001).
Discussion
There are many studies on evaluation of essential oils against other species of co*ckroaches, but this is the first time to evaluate the efficacy of these components against the brown-banded co*ckroach, S. longipalpa. The tested essential oils showed contact toxicity, fumigant toxicity and repellent activity against the brown-banded co*ckroach compared to control group during this study. Results of continuous contact toxicity showed considerable effect at different concentrations, but yarrow oil was determined as the most toxic oil. It caused 100% mortality even at the lowest concentration of 2.5%. Comparison contact toxicity of rosemary, oregano, yarrow, eucalyptus and mint oils at the lowest concentration (2.5%) showed mortality rates of 100%, 62.2%, 45%, 36.2% and 5.2% respectively at 24 h after treatment. These mortality rates were significantly different together and with control group and confirmed that rosemary oil was the most toxic oil against the brown-banded co*ckroach nymphs.
The highest repellency was related to oregano oil and the most repellent activity was observed at lower concentrations of 2.5% that was 99.1% with a residual effect lasting at least a week after treatment. Oregano and rosemary oils were more efficient at the concentration of 2.5% with 99.1% and 94.5% repellency. Yarrow, mint and eucalyptus oils showed the most repellent effects at the concentration of 5% but the differences were not significant with the repel activity of 2.5% (P values were 0.15 and 0.97 and 0.99 for yarrow, mint and eucalyptus oils respectively). Repellency of these oils was 92.8%, 68.8% and 51.7% at the concentration of 5% and it was 79.3%, 63.3% and 49.8% at the concentration of 2.5%. So it could be concluded that the essential oil concentration of 2.5% is the favorite concentration to recommend for repellency against the brown-banded co*ckroach for further evaluation.
Many previous studies have evaluated the efficacy of different essential oils against co*ckroaches.
Evaluation of repellency and fumigant toxicity of clove (Syzygium aromaticum) and sesame (Sesamum indicum) oils against the American co*ckroach (Periplaneta americana) showed complete repellency (100%) against first nymph at concentration of 2% for clove oil and 6% for sesame oil. Same result was obtained against fourth nymph at concentration of 10% of sesame oil after 48 h. While clove oil completely repelled all fourth nymphs after 24 h at concentration of 8 %. For adult stage, the greatest repellency percentages were recorded by clove oil (90.00±5.77%) and sesame oil (83.33±3.33%) after 48 h at a concentration of 10% (Omara et al. 2013). The repellency value of clove oil at concentration of 8 % against the fourth instar nymphs of the American co*ckroach is nearly close to our repellency value of oregano oil at concentration ranges of 2.5–10% which repel 96.03–99.1% of the brown-banded co*ckroach nymphs after 24 h and event lasted for a week. Both essential oils have considerable repellency against co*ckroaches but the co*ckroach species, essential oil type led to a little difference in the obtained results.
Toxicity and repellent evaluation of Eucalypltus citriodora, Mentha arvensis and Cymbopogen citratus against P. americana. C. citratus exhibited the maximum toxicity and repellency with 20% to 100% toxicity between 2 to 24 h intervals, 100% repellency and 70–100% fumigation after 24 h exposure. Minimum repellency ranged from 11–67% was observed from eucalyptus oil against P. americana (Manzoor et al. 2012). The minimum repellency against S. longipalpa was also recorded for eucalyptus oil in our study that ranged from 27.7–51.7%. Differences in the obtained result could be probably related to difference in co*ckroach species.
In another study, the essential oil of Citrus hystrix exhibited 100 % repellency effect against P. americana and Blatella germanica, and also about 87.5% against Neostylopyga rhombifolia under laboratory conditions. The essential oil caused an 86% reduction in co*ckroaches with a residual effect lasting a week after treatment in the field (Thavara et al. 2007). These results are also similar to repellency values of oregano oil that continued for at least a week or even 10 days after treatment.
Besides, all five essential oils showed nearly complete fumigant toxicity (97.2– 100%) against the brown-banded co*ckroach at the concentration of 50 μl per 1 lit air of pure oil. Apple et al. (2001) investigated that fumigant toxicity of mint oil at the concentration of 50 μl per 1 lit air of pure oil killed 100% of both P. americana and B. germanica after 24 h and caused 92.3–100% repellent effect on both co*ckroach species during each day of the 14-day experiment (Appel et al. 2001). Repel activity of mint oil ranged from 59.1–68.8% against S. longipalpa with the concentration of 2.5–30% and the fumigant toxicity was 97.2% at 24 h after exposure in our study. It seems that the fumigant toxicity of mint oil in the mentioned concentration is not significantly difference against the three co*ckroach species whereas the repellent activity shows considerable difference. This difference could be related to co*ckroach species or essential oil component.
Clove oil provided highly fumigant toxicity against nymphs and adults of P. americana after 24 and 48 h, respectively. Complete mortality (100%) was recorded at a concentration of 7.5 μl/L of air for first nymph, 10 μl for fourth one and 17.5 μl for adults after 48 h of fumigation (Omara et al. 2013).
In the another study, the fumigant toxicity of 12 essential oil components including: carvacrol, 1,8-cineole, trans-cinnamaldehyde, citronellic acid, eugenol, geraniol, S-(−)-limonene, (−)-linalool, (−)-menthone, (+)-alpha-pinene, (−)-beta-pinene, and thymol was determined against adult male, adult female, gravid female, and large, medium, and small nymphs of the German co*ckroach, B. germanica. 1,8-Cineole was the most toxic essential oil component to adult males and females, gravid females, and large nymphs, with LC50 values of 6.8, 8.4, 5.3, and 11.4 mg/liter air at 24 h, respectively (Phillips and Appel 2010).
Evaluation of fumigant toxicity of some plant essential oils by Tunza et al. determined that Allyl isothiocyanae (monoterpen oil component) was the most toxic compound, followed by essential oil of Allium sativum against B. germanica (Tunza et al. 2009).
While all five essential oils exhibited complete fumigant toxicity against S. longipalpa which is comparable with the findings of other studies but it is necessary to test lower concentrations.
The findings of current study are confirmed by all the mentioned studies in that essential oils can be favorite alternative to conventional chemical insecticides to control co*ckroach infestations especially in situations where the use of chemical insecticides has many harmful side effects. Differences in the obtained results could be probably related to difference in co*ckroach species, type of essential oil, bioassay method and exposure time.
Plant essential oils offer the potential repellent agents to co*ckroach infestations in hidden and hard-to-reach areas and to eliminate such infestations (Steltenkamp et al. 1992, Ngoh et al. 1998). This strategy would increase the efficacy of non-repellent, insecticide treated areas because co*ckroaches in the hidden areas will be get out and they will exposed to such treated areas and finally killed (Steltenkamp et al. 1992). Besides, essential oils could be applied as a safe treatment and used to treat surfaces for food preparation in order to deter co*ckroach infestations (Steltenkamp et al. 1992). Essential oil applications may also be effective around co*ckroach infested places that cannot be treated with chemical insecticides, such as food stores, sensitive equipment, clothes lockers and in beds (Koehler et al. 1995). The repellent effects of essential oils could be applied as a flushing agent during inspections of co*ckroach infestation in order to determine the degree of an infestation.
Conclusion
Given that S. longipalpa can be found in all parts of the buildings such as clothes lockers, beds, furniture and cabinets and that ootheca stick in hard-to-reach or obscure places, application of chemical insecticide spraying in such places is hard and unacceptable by the people. However, the use of essential oils presents a safe alternative for indoor application against co*ckroaches because of less toxicity to humans and no toxic residues. Oregano oil showed considerable repellent effect against S. longipalpa, so, it could be recommended as a potential repellent compound for further evaluation on a larger scale and under field conditions. Contact and fumigant toxicity of the five selected essential oils were considerable but additional study is required for the practical application and developing favorable formulation of them against the brown-banded co*ckroach and also other co*ckroach's species.
Acknowledgments
The paper is issued from a research project with project No. U-90200 and financial support was provides by Ahvaz Jundisha-pure University of Medical Sciences. We appreciate experts of the Herbal Medicine and Natural Product Research Center of AJMUS for providing the essential oils. The authors declare that there is no conflict of interests.
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