NITRIC OXIDE LEVELS AND CELL PROFILE ANALYSIS PRESENT IN THE HEMOLYMPH OF Aedes aegypti LARVAE EXPOSED TO THE ESSENTIAL OIL OF Perlargonium graveolens L’Her.
Thalita Barbosa Andrade1
Luís Fellipe Alves Silva2
Renan Tavares Leite3
Maria de Fátima Agra4
Fabíola da Cruz Nunes5
1PhD student of the Multicenter Postgraduate Program in Physiological Sciences at the Federal University of Paraíba, Biotechnology Center, Federal University of Paraíba, João Pessoa, Brazil.
2Master's student of the Multicenter Postgraduate Program in Physiological Sciences at the Federal University of Paraíba, Biotechnology Center, Federal University of Paraíba, João Pessoa, Brazil.
3Undergraduate of the Bachelor's Degree in Biotechnology at the Federal University of Paraíba, Biotechnology Center, Federal University of Paraíba, João Pessoa, Brazil.
4Professor associated with the Department of Biotechnology, Biotechnology Center, Federal University of Paraíba, João Pessoa, Brazil.
5Professor associated with the Department of Cellular and Molecular Biology, Biotechnology Center, Federal University of Paraíba, João Pessoa, Brazil.
INTRODUCTION: Arboviruses are human infections that have arthropods as their vector. The prevalence of these diseases is greater in tropical and subtropical regions, as climatic factors such as temperature, rainfall and humidity can influence the reproduction and spread of these insects. Conditions lacking basic sanitation, social factors and an unplanned growth in the expansion of urbanization are aspects that are also related to the spread of arboviruses in these regions (Shanmugam, et al., 2022). The Pan American Health Organization (PAHO), in an international consensus on the time period for reporting epidemiological events, created the division of the 365 days of the year into 52 or 53 epidemiological weeks. Counting from SE 52 of 2022, the highest number of dengue cases in the Region of the Americas brings Brazil in first place with 2,363,490 cases (84.1%), Nicaragua with 97,541 cases (3.5%), Peru with 72,851 cases (2.6%), Colombia with 69,497 cases (2.5%) and Mexico with 59,918 cases (2.1%). Brazil followed ahead with the highest number of Chikungunya cases in the Region of the Americas, presenting 265,289 cases (96.9%) and also in Zika cases, presenting 34,176 cases (84.9%). Numerous studies have been produced in search of alternative methods for controlling Aedes aegypti. The investigation of new elements obtained from medicinal plants stimulates the production of natural, renewable and degradable insecticides (Pinto, 2015; Zara et al., 2016). The genus Pelargonium, belonging to the Geraniaceae family, has an average of 750 species that are widely distributed throughout the world. Perlagonium graveolens is a durable plant growing up to 1 meter in height. P. Graveolens essential oil is constantly used in the cosmetic industry mainly in flavorings, but some studies also demonstrate repellent and insecticidal activity (Asgarpanah et al., 2015; Cavar et al., 2012; Rabelo et al., 2020). Conformational limits and competent cellular responses are important properties that make up the insect immune system and act against foreign agents. Within these conformational limits are the inflexible exoskeleton, digestive system and respiratory system that establish the insects' first defense barrier. When there is a rupture in these mechanisms, foreign elements reach the hemocoel, activating cellular and humoral complexes and processes. In hemolymph, free flowing cells called hemocytes act by generating active cellular responses such as phagocytosis, nodulation, encapsulation and cytotoxicity where the success of these processes depends on the quantity and specificity of hemocytes linked in these systems (Ribeiro, 2010). The enzyme nitric oxide synthase (NOS) is responsible for the catalyzing reaction, where in the presence of molecular oxygen, the nitrogenous guanidino terminal of L-arginine will biosynthesize a small and simple molecule, nitric oxide (Cerqueira & Yoshida, 2002; Dusse et al. al., 2003). Nitric oxide in insects is involved in mediation processes in the nervous system and in the immunological response where it has activity against infectious agents, stimulating the destruction of amino acid residues, causing changes in the secondary structure of proteins, leading to the incapacity of the pathogen (Perin, 2018). AIMS: The objective of this work is to determine the levels of nitric oxide and cellular profile in the hemolymph of Aedes aegypti larvae exposed to LC50 of the test substance after 24 hours and analyze the composition of Pelargonium graveolens L’Her essential oil by gas chromatography. METHODS: Aedes aegypti mosquitoes were obtained from a cyclic colony maintained at LAPAVET, called LAPAVET-SD. The Aedes aegypti cycle is maintained inside a climate-controlled chamber of the Biological Oxygen Demand (BOD) type, under controlled temperature conditions of 27 ± 2°C, relative humidity of 75 ± 5% and a 12-hour photoperiod of light and dark. (WHO, 2013; IMAM et al. 2014; NUNES et al. 2015). The use of standardized conditions for maintaining the mosquito life cycle for laboratory tests is essential to ensure the reliability and reproducibility of data (WHO, 2013). Aedes aegypti larvae in the fourth stage of development (L4) were used in the experiments. Pelargonium gravelolens essential oil (China) was obtained through purchase on the Laszlo© Group website. The essential oil extraction method carried out by the company was by steam distillation. The stock solution (mother) was prepared at 10,000 ppm of essential oil in water with 4% Tween 80 (diluent). With the aid of a macerator, dilution was carried out. The production of NO by larvae exposed to the test substance was determined based on the Griess reagent (GREEN et al., 1981). Comparisons were made of nitrite ion (NO2-) concentrations in the hemolymph pool collected at intervals of 3, 6 and 24 hours after treatment with the LC50 of the substance under study. In the negative control group, larvae were exposed to dechlorinated water and Tween 80 (4%). Assays were performed in duplicate. Each sample was composed of hemolymph from 20 larvae (L4), diluted in 1.5 mL of PBS buffer. After refrigerated centrifugation (4 °C) at 1500 rpm for 10 minutes, the supernatant was transferred to quartz cuvettes containing a solution of PBS and Griess reagent (naphthylenamide 0.1% w/v, in orthophosphoric acid 5% v/v , and 10 ?L sulfanilamide 1%) in a 1:1 ratio. To determine NO2- concentrations, an aliquot of each sample/interval/treatment was analyzed using spectrophotometry. Absorbance was measured by analyzing the individual wavelengths of each test substance, scanning from 190 to 562 nm. Nitric oxide was quantified using a NaNO2 standard curve as a reference. In cytotoxicity tests, larvae (L4) of Aedes aegypti were exposed to LC50 of the test substance for 24 hours. After this period in an aseptic laminar flow hood, equipped with a UV lamp, 30 live larvae were washed in PBS buffer and placed under refrigeration (1-2 minutes) for immobilization. The larvae were placed in a petri dish, under a magnifying glass, where their heads were decapitated with the aid of a scalpel blade and the hemolymph collected with the aid of a glass microcapillary and placed in a 1.5 mL eppendorf container containing 20 µL of PBS buffer. The hemolymph pool was then centrifuged under refrigeration (4 °C) at 1500 rpm for 10 minutes. Subsequently, the cell bud was transferred to another tube containing 20 ?L of PBS and 20 ?L of propidium iodide was added and in another tube, annexin-FITC was added, completing the volume to 40 ?L. It was then incubated for 15 minutes in the dark. With the help of a micropipette, a 10 ?L aliquot was transferred to the Neubauer chamber to evaluate cell integrity and viability with the aid of a fluorescence microscope (SILVA, 2007). Gas chromatography was performed by the Institute of Research in Drugs and Medicines of the Federal University of Paraíba in the Multiuser Characterization and Analysis Laboratory (model: GCMS-QP2010 Ultra | Brand: 69 Shimadzu. Column: brand: RTX-5MS capillary (5% Diphenyl / 95% dimethyl polysiloxane) Size: 30 m (length) / 0.25 mm / Inner Diameter 0.25). Statistical analysis and LC50 calculation will be performed using the GraphPad Prism version 5.0 program for Windows (GraphPad Software, San Diego, CA). Significant differences between groups will be analyzed by ANOVA and Tukey's post-test (P < 0.05). RESULTS AND DISCUSSION: Pelargonium graveolens essential oil inhibited the production of nitric oxide after 24 hours in larvae (L4) of Aedes aegypti. After exposing 20 larvae (L4) to LC50 (181.5 ppm), analysis of the 3, 6 and 24 hour groups was carried out by spectrophotometry (562) nm. In the negative control group and at intervals of 3 and 6 hours there was no statistically significant difference, that is, in this case, there was no decrease in nitric oxide production when compared to the control group. Only in the 24-hour interval was there a marked decrease in nitric oxide production, showing a statistically significant difference in relation to the negative control (p <0.05). The results found by Elmann et al., 2009, confirm this finding, since in their study, geranium essential oil inhibited the production of NO, as well as the expression of pro-inflammatory enzymes such as cyclooxygenase-2 (COX-2 ) and nitric oxide synthase (iNOS) induced in microglial cell cultures evaluated after 20 hours. In order to understand which constituents of the oil were directly linked to the anti-inflammatory activity found, 6 of the main compounds were tested (citronellol, citronellil formate, linalool, geraniol, isomenthone and menthone). It was seen that when tested in their natural concentrations, none of the constituents significantly inhibited the production of NO, this demonstrated that there may be a synergistic action between the chemical compounds of the essential oil or that the responsible constituent was not tested. Elmann et al., 2009, then evaluated the effect of citronellol at higher concentrations and it was possible to identify an attenuation of NO production in a dose-dependent manner. Gas Chromatography coupled to a Mass Spectrometer of Pelargonium graveolens essential oil identified the following major chemical constituents present in the oil: Citronellol (38.78%); Citronellyl format (12.57%); Cyclohexanone, 5-methyl-2-(1-methylethyl)-, cis (8.20%); geraniol (5.93%); 1,6-Octadien-3-ol, 3,7-dimethyl- (4.66%); Guaia-6,9-diene (4.00%); CIS-Rose Oxide (3.47%); p-Menthone (2.49%); Geranyl format (2.19%); CIS-Rose oxide (1.33%); Ledene (CAS) (1.09%); Cadinene delta (1.00%). Citronellol and citronellil formate were the main constituents also found in the study by Elmann et al., 2009, a result that confirms the findings of this study. The total count of cells present in the hemolymph of larvae (L4) exposed to LC50 (181.5) of Pelargonium graveolens essential oil was performed. The test group where larvae (L4) were exposed to LC50 of Perlagonium graveolens essential oil showed 3x105 cells/mL and a decrease in total cell count of 85% when compared to the negative control group. After 24 hours of exposure of the larvae (L4) to LC50, hemolymph was collected, centrifugation and analysis of the cell bud in the differential count in a Neubauer Chamber. In the test group, prohemocytes correspond to 74.81%, granulocytes to 11.35% and oenocytoids to 13.83%. The adipohemocyte, plasmatocyte and thrombocytoid cell types were not visualized for quantification. In the control group, prohemocytes correspond to 55.84%, granulocytes to 4.88%, and oenocytoids to 35.35%. Adipohemocytes, plasmatocytes and thrombocytoids accounted for 1.30%. Pelargonium graveolens essential oil induces apoptosis and necrosis in hemocytes of larvae (L4) of Aedes aegypti. Through the cytotoxicity assay, it was possible to visualize under fluorescent light the hemocytes present in the hemolymph of larvae (L4) that were exposed to LC50 of Pelargonium graveolens essential oil. It was possible to visualize two cell populations with different colors. Green-colored cells are apoptotic cells that display this color due to the dye used (Annexin-FITC). Red-colored cells are necrotic cells that present this color due to the dye used (Propidium iodide) (Menezes, 2013). An important fact found was the double labeling of hemocytes. This event occurs due to the lack of phagocytes during the cell death process, consequently causing a process called secondary necrosis or late apoptosis (Menezes, 2013). The classification of insect hemocytes is still very controversial. Due to the lack of staining in optical microscopy, the methodologies used to collect these cells, physiological effects such as differentiation in circulating hemolymph and rapid transformation in bleeding. Another important aspect is the subjectivity in the evaluation of each professional, where it becomes difficult to make comparisons using electron microscopes (Brehélin, 1986). Seeking to define in a more enlightening way the types of hemocytes found in the hemolymph of invertebrates, Brehélin, 1986, described the types according to their reliable morphological characteristics of the ultrastructure of each type. cell phone. In this way, nine types of insect hemocytes were described: prohemocytes, plasmatocytes, oenocytoids, spherical cells, thrombocytoids and four types of granular hemocytes (GH1, GH2, GH3 and GH4). In the study by Araújo, 2011, the predominance of the types of hemocytes present in the hemolymph of Aedes aegypti and Aedes albopictus followed the following order: prohemocytes, oenocytoids, plasmatocytes, granulocytes, adipohemocytes and thrombocytoids. These results contributed to the finding in this study where prohemocytes and oenocytoids were predominant in the hemolymph of larvae (L4) of Aedes aegypti. The decrease in the total cell count of hemocytes was possibly due to the induction of apoptosis, necrosis and secondary necrosis caused by the chemical constituents of Pelargonium graveolens essential oil. In an apoptotic process, Annexin V is used as an early marker of apoptosis, as it strongly binds to a phospholipid on the inner leaflet of the cell plasma membrane, called phosphatidylserine, which is externalized during the apoptotic process. When Annexin V is labeled with fluorochromes such as fluorescein isothiocyanate (FITC), it can be used as a marker for phosphatidylserine. In marking necrotic cells, propidium iodide (PI) is used. PI is a fluorescent marker of DNA impermeable to the intact cell membrane, thus acting as an important marker of increased membrane permeability, a characteristic episode in necrotic cells. Double-labeled cells characterize a process called secondary necrosis or late apoptosis. Sets of apoptotic elements, which, due to lack of phagocytes to complete the cell death process, enter secondary necrosis (Menezes, 2013). Westman et al., 2020, describes secondary necrosis as an event that can occur even after apoptosis. When apoptotic cells are not destroyed within a short period, the plasma membrane decays and loses its integrity, causing its contents to leak, an important characteristic similar to what occurs in a primary necrotic cell. CONCLUSION: Pelargonium graveolens essential oil induced a marked decrease in the production of nitric oxide in the hemolymph of larvae (L4) exposed to LC50 after 24 hours and showed cytotoxicity in hemocytes, where apoptosis, necrosis and secondary necrosis were identified in Aedes aegypti hemocytes exposed to LC50 for 24 hours using fluorescent microscopy. According to the results found, Pelargonium graveolens essential oil can be used as an alternative for the creation of new products of plant origin with activity against the Aedes aegypti mosquito.
Keywords: geranium, essential oil, mosquito, vector, arboviruses.
ACKNOWLEDGMENT: LAPAVET, IPeFarM.
REFERENCES:
ARAÚJO, Helena Rocha Corrêa de. Morphological characterization of Aedes aegypti and Aedes albopictus hemocytes and the immune response of Aedes aegypti hemocytes after Dengue virus infection. 2011. Doctoral Thesis.
ASGARPANAH, Jinous; RAMEZANLOO, Fereshteh. An overview on phytopharmacology of Pelargonium graveolens L. 2015.
BREHÉLIN, Michel. Immunity in Invertebrates. Cells, Molecules and Defense Reactions. Edition: 1986. Montpellier - France. 1986.
CAVAR, Sanja; MAKSIMOVIC, Milka. Antioxidant activity of essential oil and aqueous extract of Pelargonium graveolens L’Her. Food control, vol. 23, no. 1, p. 263-267, 2012.
CERQUEIRA, Nereide Freire; YOSHIDA, Winston Bonetti. Nitric oxide: review. Acta Cirúrgica Brasileira, v. 17, p. 417-423, 2002.
Available at: <https://www3.paho.org/data/index.php/en/mnu-topics/indicadores-dengue-en/annual-arbovirus-bulletin-2022.html> Accessed on: 06/07/2023 .
Available at: <https://www.paho.org/hq/dmdocuments/2016/2016-cha-epidemiological-calendar.pdf> Accessed on: 06/07/2023.
DUSSE, Luci Maria Sant'Ana; VIEIRA, Lauro Mello; CARVALHO, Maria das Graças. Nitric oxide review. Brazilian Journal of Pathology and Laboratory Medicine, v. 39, p. 343-350, 2003.
ELMANN, Anat et al. Anti-neuroinflammatory effects of geranium oil in microglial cells. Journal of Functional Foods, vol. 2, no. 1, p. 17-22, 2010.
GREEN, L. C.; RUIZ DE LUZURIAGA, K.; WAGNER, D.A.; RAND, W.; ISTFAN, N.; YOUNG, V.R.; TANNENBAUM, S.R. Nitrate biosynthesis in man. Proceedings of the National Academy of Sciences, vol. 78, p. 7764–7768, 1981.
IMAM, H.; ZARNIGAR, D.; SOFI, G.; AZIZ, S. The basic rules and methods of mosquito rearing (Aedes aegypti). Tropical Parasitology, vol. 4, p. 53-55, 2014.
MENEZES, Ramon Róseo Paula Pessoa Bezerra de. Study of the enzymatic activity and effects of Bothropoides insularis snake venom on RAW 264.7 macrophages in vitro. 2013. Master's Thesis.
NUNES, F.C.; LEITE, J.A.; OLIVEIRA, L.H.G.; SOUSA, P.A.P.S.; MENEZES, M.C.; MORAES, J.P.S.; MASCARENHAS, S.R.; BRAGA, V.A. The larvicidal activity of Agave sisalana against L4 larvae of Aedes aegypti is mediated by internal necrosis and inhibition of nitric oxide production. Parasitol Res. v. 114, p. 543-549, 2015.
PERIN, Ana Paula. Jaburetox, peptide derived from ureases: effects on the enzymatic pathways of the cockroach Nauphoeta cinerea. 2018.
PINTO, Zeneida Teixeira. Chemical characterization and insecticidal activity of essential oils from aromatic plants from Brazil and Cuba on the post-embryonic development of muscoid dipterans. 2015. Doctoral Thesis.
RABELO, Paulo Gonçalves et al. Geranium seedlings: bud positions in the stake and substrates. Brazilian Journal of Development, v. 6, no. 5, p. 30988-30997, 2020.
RIBEIRO, Lilian Maria da Solidade. Immunological and mechanical responses in a susceptible and resistant population Plutella xylostella (L.) (Lepidoptera: Plutellidae) against commercial formulations based on Bacillus thuringiensis Berliner. 2010. 76 f. Dissertation (Postgraduate Program in Agricultural Entomology) - Federal Rural University of Pernambuco, Recife.
SHANMUGAM, Lakshmi et al. Arboviruses in human diseases: An Indian perspective. International Journal of Advanced Medical and Health Research, vol. 9, no. 2, p. 69-77, 2022.
SILVA, B.G. Quantification of apoptosis and necrosis using fluorescent dyes and image analysis in insect cell culture: the case of Drosophila melanogaster. Dissertation (Master’s degree in Chemical Engineering). São Carlos, SP, 2007.
WESTMAN, Johannes; GRINSTEIN, Sergio; MARQUES, Pedro Elias. Phagocytosis of necrotic debris at sites of injury and inflammation. Frontiers in immunology, vol. 10, p. 3030, 2020.
WHO - World Health Organization. Guidelines for Efficacy testing of Spatial repellents. Control of neglected tropical diseases. Who pesticide evaluation scheme. WHO/CDS/CPC/ MAL/13.12., Geneva, 58 pp, 2013.
ZARA, Ana Laura de Sene Amâncio et al. Aedes aegypti control strategies: a review. Epidemiology and Health Services, v. 25, p. 391-404, 2016.
Comissão Organizadora
Francisco Mendonça Junior
Pascal Marchand
Teresinha Gonçalves da Silva
Isabelle Orliac-Garnier
Gerd Bruno da Rocha
Comissão Científica
Ricardo Olimpio de Moura
Complete tool for submission, evaluation and publication of proceedings for scientific events.
