INTRODUCTION

Since ancient times, flies have been in contact with humans. The species of the genus Chrysomya (Robineau-Desvoidy, 1830) (Diptera: Calliphoridae) are commonly known as blowflies and, like other blowflies, Chrysomya putoria (Wiedemann, 1818) is now distributed through the New World (Guimarães et al., 1978). Blowflies are of medical and veterinary importance worldwide due to their feeding habits, such as on animal and human excrement, urban garbage, decomposing meat and fresh food (Tomberlin et al., 2017). These flies are synanthropic and could act as important physical carriers of various pathogens to man and domestic animals such as bacteria (Escherichia coli, Proteus mirabilis, Citrobacter sp., Klebesiella sp., Morganella sp., Pseudomona sp., Enterobacter sp., Salmonella sp.), viruses (West Nile Virus), protozoa (Entamoeba coli, Entamoeba histolytica, Giardia lamblia and Cryptosporidium sp.) and helminths (Ascaris lumbricoides, Trichuris trichiura, Strongyloides stercoralis, Taenia spp.) (Getachew et al., 2007; Johnson et al., 2010; Nayduch and Burrus, 2017). Chrysomya putoria are commonly found in latrines and larvae can cause secondary myiasis in animals and humans (Barbosa and Vasconcelos, 2015).

The current use of chemical insecticides could cause untold toxicological damage to humans and animals, in the short and long terms. Moreover, insecticide resistance has become an increasing problem in agriculture, in the economy and in public health. Botanical insecticides provide an alternative to synthetic pesticides because the excessive use of synthetic insecticides resulted in a progressive resistance of the pests to these chemicals, diminishing their effectiveness and generating consequences with negative environmental impact (Valente et al., 2007).

In the last decades, thousands of plants have been studied to detect activity as repellents and insecticides. Secondary metabolites often have a potential to control insects, because they are naturally feeding deterrents, toxic agents or developmental disruptors. It has been shown that numerous plant species growing in different geographical areas around the world cause lethal and sub-lethal effects on various insects (Cabral et al., 2007; Mendonça et al., 2011).

Ocimum sanctum L. (Lamiaceae) also known as Ocimum tenuiflorum L., holy basil, tulasi or tulsi is an aromatic plant native to the Indian subcontinent and cultivated throughout the tropical Southeast Asia, cultivated and naturalized also in other tropical areas (Al Rashid et al., 2013). The plant is less known in western countries where a lack of information on cultural practices exists (Sims et al., 2014). In Cuba, it is cultivated in yards and gardens like aromatic herb. Its seeds easily propagate by air and then could be found spontaneously in the neighborhoods of the towns and the peasant dwellings (Roig, 1998).

The extracts of this species have potential in treatments for coughs, dysentery, diarrhea, worms and kidney malfunction, bronchitis, liver diseases, genitourinary disorders, catarrhal fever, otalgia, lumbago, hiccough, ophthalmia, painful eye disease, gastric disorders, diabetes, skin diseases, various forms of poisoning and psychosomatic stress disorders, arthritis, chronic fever and insect sting (Al Rashid et al., 2013; Gradinariu et al., 2015; Jamal et al., 2015).

Previous reports sign the essential oil of O. sanctum as insecticide, but those studies were developed only against mosquitoes’ species (Anopheles stephensi Liston, Aedes aegypti Linnaeus and Culex quinquefasciatus) under laboratory conditions (Bhatnagar et al., 1993), but in the literature reviewed there is not reports regarding their actions over flies.

The aim of this study was to evaluate the insecticidal effect of the essential oil of Ocimum sanctum L. var. cubensis Gomes on the post-embryonic development of the blowfly Chrysomya putoria.

MATERIAL AND METHODS

Plant material

The leaves of Ocimum sanctum were collected in September 2016, in the municipality of San Luis (20°11’13.88’’ N; 75°50’51.31’’ O), province of Santiago de Cuba, Cuba. The plants were in a vegetative state. To collect the plant a region was selected where a population of more than 20 plants of the species existed. The collected plants were randomly selected and taxonomically identified by Mr. Jainer Costa Acosta, specialist at the Eastern Center for Ecosystems and Biodiversity (BIOECO) in the province of Santiago de Cuba. A voucher specimen was checked and confirmed with a previous one archived at the BSC Herbarium of the same center under No. 3247.

Essential oil extraction

The extraction of the essential oil from Ocimum sanctum L. fresh leaves was carried out until exhaustion by hydrodistillation, using a conventional Clevenger apparatus. The essential oil chemical composition was determined in a Gas Chromatography Mega 2 series coupled to a mass spectrometer (GC/MS) Hewlett Packard model 5890 (USA), using the same conditions declared in a previous work conducted by the research group. A DB-5 MS capillary column (Agilent Technologies, USA) and helium as carrier gas were used. The program temperature condition was 60°C (2 min), with an increment of 3°C/min until 110°C, of 15°C/min until 150°C and finally with an increment of 17°C/min until 290°C (Chil Núñez et al., 2017). Electron impact ionization at -70 eV was used to characterize the compounds that were identified comparing their mass spectral data with the National Institute of Standards and Technology mass spectrometry library and according with their Kovats retention indexes.

Colonies of Diptera

The flies were obtained from the Collection of flies of the laboratory “Laboratório de Entomologia Médica e Forense, Instituto Oswaldo Cruz/Funda-ção Oswaldo Cruz”, Rio de Janeiro, RJ, Brazil. The adult flies were kept in wooden cages with nylon screens on the sides and a front opening containing a black fabric sleeve for insect handling. The volume of the cage was approximately 2.7 m3 (30 x 30 x 30 cm).

Bioassays

For the topical application, four groups of 50 newly-hatched larvae were placed in Petri dishes and get in contact with 1 µL/larva applied on the larvae bodies using an automatic pipette. Six level of concentration of the essential oil were tested and defined as experimental groups A, B, C, D, E and F: (A = 4.13 mg/mL, B = 8.25 mg/mL, C = 20.63 mg/mL, D = 41.25 mg/mL, E = 61.87 mg/mL and F = 80.25 mg/mL) were prepared using dimethylsulfoxide (DMSO, ACS reagent ≥ 99.9%; 472301 Sigma-Aldrich) as solvent. Three control groups were considered: solvent control (just with pure DMSO), classic or pure control where no substances were added and a natural insecticide essential oil as positive control. With this purpose, Cymbopogon citratus essential oil at 83,3 mg/mL was used according to previous experiences at laboratory with this substance on this biological model (Pinto et al., 2015). Bioassays were performed in quadruplicate.

After application of the different concentrations of the essential oil, the larvae were transferred to a vessel containing 50 g of bovine putrefied meat. These 50 mL vials were then placed in 500 mL vials (Copaza, Brazil) containing vermiculite (Vermiculite Expanded Medium, expansion volume 0.1 m³, Brazil) as substrate for pupation and covered with a nylon web clamped with a rubber band. The experiments were maintained in acclimatized chambers setting the temperature at 27 ± 1°C, 70 ± 10% of relative humidity and with a 12:12 light cycle (light/darkness). Mortality and changes in life cycle were recorded daily. After reaching maturity, the larvae spontaneously abandoned the diet and were collected. These larvae were individually weighed and transferred to glass tubes containing vermiculite and sealed with cotton plugs. After emergence, adults were separated by gender selected by the distance between the eyes (Dübendorfer et al., 2002). The bioassays were performed in quadruplicate with the second laboratory generation. Corrected mortality and duration of each developmental period (larval, pupal and newly-hatched larvae to adult) were analyzed. With this intention, a corrected mortality parameter was calculated using the Abbot equation (equation 1). The weight of mature larvae and the sex ratio (equation 2) were also considered.

[equation 1]

Morphological alterations

All insects were observed under an optical stereoscope (Zeiss, model Stemi SV 6, Germany) to investigate possible morphological alterations in head, wings and abdomen. Pictures were taken using a digital camera (Canon, model PowerShot A630, Japan) attached to the stereoscope.

Statistical analysis

The duration of development periods of this fly species and larval weight were analyzed by ANOVA (p ≤ 0.05), mean values were compared with the Newman-Keuls test at a significance level of 0.05, while sex ratio and mortality were analyzed with the chi-square test. Mortality was corrected using Abbot’s formula (Abbott, 1995). Probit analysis of concentration-mortality data was conducted to estimate the LC₅₀ value using the SPSS for Windows 18.0/2009 statistical package. For this intention, data normalization was done using the square root of the concentrations tested.

RESULTS

The chemical composition showed β-caryophyllene, β-selinene and eugenol as the main compounds (Table 1). These results are similar to those obtained in previous studies matching two of the three main compounds (β-caryophyllene and eugenol) according to previous experiences of the research group (Chil Núñez et al., 2017). The compound bicyclogermacrene (that appears as majority in the previous study with 20.38%) in this case is present in 7.81% as fourth place after the three-main declared. In total, seven compounds are common between both samples representing more than the fifty percent (Table 1).

The six concentrations evaluated affected the life cycle of flies. It is noted that all concentrations of the essential oil O. sanctum showed a significant decrease in the duration of the larval period of C. putoria when compared to control group and control with DMSO, but not significant regarding to the positive control C. citratus essential oil (Table 2). The duration of the pupal period was also significantly lower for insects treated with all concentrations of essential oil when compared to the control group and control with DMSO, except for the group treated with 20.63 mg/mL. On the other hand, these times reduction were different statistically than those obtained for the positive control same tendency of time reduction with statistical differences is described when analyzing the newly hatched larvae to adult period (Table 2).

Larval weight was also directly affected by basil essential oil. According to Fig. 1, larvae from pure control group (36.5 ± 10.17 mg) and DMSO control (34.7 ± 5.24 mg) were lighter than the larvae treated with O. sanctum essential oil in all concentrations (all above 50 mg), with highly significant differences (p < 0.001) as well happen with the positive control with which not statistical behavior is found. Therefore, the use of holly basil essential oil besides reducing the larval period (Table 2) also caused the larvae to gain more weight in a shorter time.

Figure 1
Effects on Chrysomya putoria larval weight when topically treated with Ocimum sanctum var. cubensis Gomes essential oil.
Data represent mean ± SD of n = 200 larvae per each group. Values with different upper letters were significantly different (Newman-Keuls test; p < 0.05). Groups A-F: Ocimum sanctum var. cubensis Gomes essential oil at different concentrations: A = 4.13 mg/mL, B = 8.25 mg/mL, C = 20.63 mg/mL, D = 41.25 mg/mL, E = 61.87 mg/mL and F = 80.25 mg/mL dissolved in dimethylsulfoxide (DMSO). Cymbopogon citratus essential oil (C. citratus) was used as a reference compound.

Mortality effects of holly basil oil were observed in all concentrations tested (Fig. 2). In the larvae stage, the most effective concentration of the essential oil was 4.13 mg/mL and the least effective was a concentration of 61.87 mg/mL. The mortality in the pupal period was higher than in the larval and newly hatched larvae to adult periods for five of six concentrations tested as well as happen with positive control. The only exception was for the group treated with 80.25 mg/mL essential oil (Fig. 2).

Figure 2
Mortality (%), corrected using Abbot’s formula (1925), of Chrysomya putoria (Diptera: Calliphoridae) topically treated with different concentrations of essential oil extracted from Ocimum sanctum var. cubensis Gomes.
Groups A-F: Ocimum sanctum var. cubensis Gomes essential oil at different concentrations: A = 4.13 mg/mL, B = 8.25 mg/mL, C = 20.63 mg/mL, D = 41.25 mg/mL, E = 61.87 mg/mL and F = 80.25 mg/mL dissolved in dimethylsulfoxide (DMSO). Cymbopogon citratus essential oil (C. citratus) was used as a reference compound. L1 to adult → Newly hatched larvae to adults.

Regarding to the sexual ratio parameter, no statistical difference was found, according to the chi-square test, similar as happen with all control groups including the positive one. By this way, the use of this natural extract has not a measured influence on this biological variable.

Besides all the modifications of the postembryonic cycle, O. sanctum also caused morphological alterations in adults whose larvae where treated with 4.13 mg/mL and 8.25 mg/mL concentrations (Fig. 3A-B).

Figure 3
Morphological alterations of Chrysomya putoria (Diptera: Calliphoridae) adults topically treated with 5 and 10% concentrations of essential oil extracted from Ocimum sanctum var. cubensis Gomes.
A: Abdomen contracted, and wings twisted (x16). B: Ptilinum not retracted (x40). C: Insect from control group (x16).

DISCUSSION

As can be observed in Table 1, similarities and differences can be appreciated in O. sanctum essential oil. The fact that the main compounds remains almost unaltered in both studies could mean that they can be considered as important chemical patterns of the Ocimum sanctum var. cubensis essential oil that grow up in Cuba, giving them an important chemotaxonomic role. This is in agreement with other studies within the American continent presenting eugenol and and β-caryophyllene as the major components (Machado et al., 1999; Sims et al., 2014). On the other hand, the different composition regarding the minor compounds are in agreement with the suggestion that aromatic character of each type of basil depends on the genetics of the vegetable, its state of vegetative development, agroclimatological factors and chemical compounds in the synthesis of essential oils as is well described in the literature (Sims et al., 2014; Jamal et al., 2015).

Eugenol and β-caryophyllene are frequently found in high concentrations in essential oils with insecticidal properties (Jantan and Zaki, 1998). Eugenol is repeatedly described as a good insecticide compound in many kinds of insects and also in flies, such as Musca domestica L. and Drosophila melanogaster (Meigen, 1830), as well as sesquiterpenes (Koul et al., 2008).

It is internationally accepted that the larval period is the most important in the life cycle of the blowfly. This is the period where the larvae get to eat as much food as possible in the shortest time. Furthermore, larvae remain on the diet for a shorter time if the quantity and quality of nutrients is enough for pupation and if they reach the minimum weight needed for pupation. Nevertheless, the standard conditions in which all experimental and control groups are conducted allowed us to infer that the effects observed were due to the effect of the holly basil essential oil, that is the same when comparing with the positive control. A previous study tested the larvicidal effects of aqueous crude extract from Pouteria sapota (Jacq.) (Sapotaceae) on the same blowfly species and found the same behavior: a time decreasing larval period (Carriço et al., 2014). This finding is important, once the larval period is one of the most vulnerable in flies’ development cycle. During this period, the insects are more susceptible to predation.

The pupal period is when the most important hormonal changes are taking place in holometabolous insects, such as the blowflies. Cabral et al. (2007) suggested that compounds extracted from plants and tested to control insects could modify specific physiological processes such as the endocrine control of insect growth, the neuroendocrine system or the production of some hormones. Holy basil essential oil could be, somehow, affected those physiological processes. Kostyukovsky et al. (2002) stated that treatments with essential oils or their purified constituents resulted in visible symptoms suggesting a neurotoxic mechanism of action similar to that produced by carbamates and organophosphates. In another study, it was observed that the terpenes of essential oil have been shown to be competitive inhibitors of the acetylcholinesterase isolated from the electric eels, as well as the cholinesterase of the domestic fly and cockroach heads (Ryan and Byrne, 1998).

The newly hatched larvae adult period or the complete developmental period is the most efficient parameter to evaluate the substance efficacy as an insecticide, because it prevents distortions between the larval and pupal periods (d’Almeida et al., 2001). According to Table 2, higher concentrations affected this variable less than the lower ones, which is not usual. A possible explanation for this fact is to consider the nature of the substance applied. In this particular case, the application of a pure essential oil makes it more susceptible to evaporation under lab conditions than when dissolutions in DMSO are used. Thus, the larvae treated with lower concentrations could be in contact with the tested substance for longer periods of time, while the higher concentrations could lose the active substances through evaporation. Another possibility can be related with the DMSO use. Some authors have suggested the ability of DMSO to increase the membrane permeability. Notman et al. (2006) referred that DMSO was able to induce pores in the membrane, while Gurtovenko and Anwar (2007) referred that this substance induced thinning and expansion of a phospholipid bilayer increasing their fluidity. This hydrophobic core can explain why DMSO promotes the permeation of solutes, particularly in hydrophobic entities. Nevertheless, and in spite of any theoretical explanation, the essential oil tested at all concentrations proved to be effective against the insect development.

The reduction of the larval period was also observed by Carriço et al. (2014) using crude aqueous extract of P. sapota on the same blowfly species. Further, Carvalho et al. (2012) when testing the effects of cocaine on the developmental rate of Chrysomya albiceps (Wiedemann, 1819) and C. putoria, found the same effects. They considered that this performance was due to some effect on the endocrine system of the flies; this could also be the cause of this particular behavior in the present study.

The corrected mortality was superior that 50% in the lower concentrations reaching a top of 70% for the experimental group C. This C group as well as group B showed values that exceed the obtained for the positive control, demonstrating a good activity. Once again, the groups with higher concentrations of essential oil were less effective than groups with lower concentrations. The possibility of evaporation and/or the DMSO effect again helps to explain this particular behavior. Considering the non-linear tendency of the most concentrated experimental groups (E, F) those mortality values were skipped while computing the LC₅₀, rendering a 7.47 mg/mL value.

In the group A, from a total of 92 flies that emerges as adults, the 13.04% (12 flies) results in some kind of morphological alteration; while for the group B, seven flies from 67 (10.44%) appear also with some handicap. This behavior appears just in the groups treated at minor concentrations, indicating how toxic could be the essential oil to the postembryonic cycle of this fly since when; if is not able to kill, produces some alteration affecting their normal development. The most common morphological alterations found were a contraction of the abdomen, ptilinum not retracted, and a twisted hemolymph affecting the wingspan (Fig. 3A-B).

All insects from control group were normal (Fig. 3C). The morphological alterations affected directly the ability to fly, and consequently to find food and to reproduce. So, these results demonstrated that basil essential oils may represent an effective alternative method to control blowfly.

CONCLUSIONS

The results obtained in this work indicate that the essential oil of Ocimum sanctum var. cubensis Gomes is a good option for the control of the blowfly Chrysomya putoria, in some cases with better performance than the positive control Cymbopogon citrates essential oil. The principal strength of the present work consists not only in the first report of the effect on the post-embryonic development of this blowfly, but also that it is possible to have a good activity with low concentrations of this essential oil on this species of fly with medical and sanitary importance, reaching 70% mortality with a 20.63 mg/mL concentration and a LC₅₀ estimated as 7.47 mg/mL.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Acknowledgments

This work was supported by the Instituto Oswaldo Cruz (IOC/FIOCRUZ); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ); and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior with Ministerio de Educación Superior de Cuba (CAPES–MES Cuba, number 130/11).

References

Abbott WS (1995) A method of computing the effectiveness of an insecticide. J Econ Entomol 18: 265–267.

Al Rashid MH, Banerjee A, Maiti NJ (2013) The queen of herb with potent therapeutic constituent in various disease states: A reappraisal. Intern J Phytomed 5: 125–130.

Barbosa TM, Vasconcelos SD (2015) An updated checklist of myiasis-inducing Diptera species in livestock in Northeastern Brazil. Arch Zootec 64(246): 187–190.

Bhatnagar M, Kapur KK, Jalees S, Sharma SK (1993) Laboratory evaluation of insecticidal properties of Ocimum basilicum Linnaeus and O. sanctum Linnaeus plant's essential oils and their major constituents against vector mosquito species. J Entomolog Res 17: 21–29.

Cabral MMO, Mendonça PM, Gomes CMS, Barbosa-Filho JM, Queiroz MMC, Mello RP (2007) Biological activity of neolignans on the post-embrionic development of Chrysomya megacephala. Fitoterapia 78: 20–24.

Carriço C, Pinto ZT, Dutok CMS, Caetano RL, Pessanha RR, Chil I, Mendonça PM, Escalona JC, Reyes-Tur B, Queiroz MMC (2014) Biological activity of Pouteria sapota leaf extract on postembryonic development of blowfly Chrysomya putoria (Wiedemann, 1818) (Calliphoridae). Rev Bras Farmacogn 24: 304–308.

Carvalho LMC, Linhares AX, Palhares FAB (2012) The effect of cocaine on the development rate of immatures and adults of Chrysomya albiceps and Chrysomya putoria (Diptera: Calliphoridae) and its importance to postmortem interval estimate. Forensic Sci Inter 220: 27–32.

Chil Núñez I, Escalona Arranz JC, Berenger Rivas CA, Mendonça PM, Mateo Pérez K, Dutok Sánchez CM, Cortinhas LB, Silva CF, Carvalho MG, Queiroz MMC (2017) Chemical composition and toxicity of Ocimum sanctum var. cubensis essential oil up-growing in the Eastern of Cuba. Intern J Pharmacogn Phytochem Res 9(7): 1021–1028.

d’Almeida JM, Ferro CL, Fraga MB (2001) Desenvolvimento pós-embrionário de Chrysomya putoria (Diptera: Calliphoride) em dietas artificiais. Acta Biol Leopoldensia 23: 4–7.

Dübendorfer A, Hediger M, Burghardt G, Bopp D (2002) Musca domestica, a window on the evolution of sex-determining mechanisms in insects. Int J Dev Biol 46: 75–79.

Getachew S, Gebre-Michael T, Erko B, Balkew M, Medhin G (2007) Non-biting cyclorrhaphan flies (Diptera) as carriers of intestinal human parasites in slum areas of Addis Ababa, Ethiopia. Acta Trop 103: 186–194.

Gradinariu V, Cioanca O, Hritcu L, Trifan A, Gille E, Hancianu M (2015) Comparative efficacy of Ocimum sanctum L. and Ocimum basilicum L. essential oils against amyloid beta (1–42)-induced anxiety and depression in laboratory rats. Phytochem Rev 14(4): 567–575.

Guimarães JH, Prado AP, Linhares AX (1978) Three newly introduced blowfly species in Southern Brazil (Diptera: Calliphoridae). Rev Bras Entomol 22: 53–60.

Gurtovenko AA, Anwar J (2007) Modulating the structure and properties of cell membranes: The molecular mechanism of action of dimethyl sulfoxide. J Physiol Chem 111: 10453–10460.

Jamal M, Kamyab AA, Khatoon N, Zomorodian K, Pakshir K, Javad M (2015) Chemical compositions and antimicrobial activities of Ocimum sanctum L. essential oils at different harvest stages. Jundishapur J Microbiol 8(1): 13720.

Jantan I, Zaki MZ (1998) Development of environment friendly insect repellents from the leaf oils of selected Malaysian plants. Rev Biodivers Environ Conserv 1: 1–7.

Johnson G, Panella N, Hale K, Komar N (2010) Detection of West Nile virus in stable flies (Diptera: Muscidae) parasitizing juvenile American white pelicans. J Med Entomol 47: 1205–1211.

Kostyukovscky M, Rafaeli A, Gileadi C, Demchenko N, Shaaya E (2002) Activation of octopaminergic receptors by essential oil constituents isolated from aromatic plants: possible mode of action against insect pests. Pest Manag Sci 58: 1101–1106.

Koul O, Walia S, Dhaliwa GS (2008) Essential oils as green pesticides: Potential and constraints. Biopest Internat 4: 63–84.

Machado MIL, Silva MGV, Matos FJA, Craverio AA, Alencar WJ (1999) Volatile constituents from leaves and inflorescence oil of Ocimum tenuiflorum L. f. (syn. Ocimum sanctum L.) grown in Northeastern Brazil. J Essent Oil Res 11: 324–326.

Mendonça PM, Lima MG, Albuquerque LRM, Queiroz MMC (2011) Effects of látex from “Amapazeiro” Parahancornia amapa (Apocynaceae) on blowfly Chrysomya megacephala (Diptera: Calliphoridae) post-embryonic development. Vet Parasitol 178: 379–382.

Nayduch D, Burrus RG (2017) Flourishing in filth: House fly–microbe interactions across life history. Ann Entomol Soc Am 110(1): 6–18.

Notman R, Noro MG, O’Malley B, Anwar J (2006) Molecular basis for dimethylsulfoxide (DMSO) action on lipid membranes. J Am Chem Soc 128: 13982–1398.

Pinto ZT, Fernández-Sánchez F, Santos AR, Amaral AC, Ferreira JL, Escalona-Arranz JC, Queiroz MMC (2015) Effect of Cymbopogon citrates (Poaceae) oil and citral on post-embryonic time of blowflies. J Entomol Nematol 7(6): 54–64.

Roig JT (1998) Plantas medicinales aromáticas o venenosas de Cuba. La Habana: Editorial Ciencia y Técnica.

Ryan MF, Byrne O (1998) Plant-insect coevolution and inhibition of acetylcholinesterase. J Chem Ecol 14: 1965–1975.

Sims AC, Juliani HR, Mentreddy SR, Simon JE (2014) Essential oils in holy basil (Ocimum tenuiflorum L.) as influenced by planting dates and harvest times in North Alabama. J Med Active Plant 2(3): 33–41.

Tomberlin JK, Crippen TL, Tarone AM, Chaudhury MFB, Sigh B, Cammack JA, Meisel RP (2017) A review of bacterial interactions with blow flies (Diptera: Calliphoridae) of medical, veterinary, and forensic importance. Ann Entomol Soc Am 110(1): 19–36.

Valente M, Barranco A, Sellaive-Villaroel AB (2007) Eficácia do extrato acuoso de Azadiracta indica no controle de Boophilus microplus em bovino. Arq Bras Med Vet Zootec 59: 1341–1343.

Notas de autor

Información adicional

Citation Format: Chil-Nuñez I, Martins Mendonça P, Escalona-Arranz JC, Barbosa Cortinhas L, Dutok-Sánchez CM, De Carvalho Queiroz MM (2018) Insecticidal effects of Ocimum sanctum var. cubensis essential oil on the diseases vector Chrysomya putoria. J Pharm Pharmacogn Res 6(3): 148–157.

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