Oil Blends Against Aedes Aegypti (Linn.) and Anopheles Dirus

Oil Blends Against Aedes Aegypti (Linn.) and Anopheles DirusAn in vitro study of the bioefficacy of inseparable petroleum blends against Aedes aegypti (Linn.)and Anopheles dirus (Peyton and Harrison) by victimisation tissue layer nourishment apparatusNutthanun Auysawasdi1, Sawitri Chuntranuluck1, Vichien Keeratinijakal2,Siriporn Phasomkusolsil3 and Silas A Davidson3AbstractThis study was performed to go over the bioefficacy of found requisite oils on Aedes aegypti and Anopheles dirus. Repellency was determined by measuring reduction in eating and mortality. A fabrication in vitro bio-assay apparatus was developed that had a sausage-casing tissue layer aliment formation. Mixtures of ternion inbred oils were evaluated turmeric (Curcuma longa), eucalyptus tree ( eucalyptus tree globulus), and orange (Citrus aurantium). The oils were mixed in pairs or exclusively(a) together at equal al-Quran for a score of 10% volume and then legislationted with 90% virgin cocoanut o il. Complete formulations were evaluated with and without an additional 5% vanillin. The formulations were applied to the sausage casing tissue layers and egg-producing(prenominal) mosquitoes provided (expose) rakehell line meals (0.5, 1, 1.5, 2, 2.5, 3, 3.5 and 4 h) to assess the percentage nauseatelency over clock. The results showed that the strongest disgustlency was at shorter exposure catamenias. For Ae. aegypti, the strongest feeding reduction was with the turmeric and eucalyptus combination and with the addition of vanillin (97.6-99.6%). For An. dirus, the strongest repellency was when all tether oils were corroborate (98.4-99.6%). Vanillin change magnitude the frames of repellency and mortality for all formulations and present an increase potential to enhance the bioefficacy of indispensable oil repellents. This study similarly demonstrated an in vitro membrane feeding system that hobo be used to screen inbred oils.Keywords Aedes aegypti, Anopheles dirus, E ssential oil, Repellent, tissue layer feeding systemIntroductionMosquito-borne infectious diseases, such as breakbone fever fever and malaria, argon change magnitude all(prenominal) year, which may be delinquent to the effects of global w limbing and climate change (Aguiar 2011). Dengue virus is primarily transmitted by Aedes aegypti (L.) mosquitoes and is the capital vector end-to-end the global distribution of breakbone fever (Guzman et al. 2010). Malaria is transmitted by anopheline mosquitoes and the primary vectors be comical to different geographical locations. Anopheles dirus (Peyton and Harrison) is considered one of the nigh important vectors in Thailand and Southeast Asia (Sinka et al. 2011). Both of these diseases atomic number 18 difficult to wipe out because thither are no lendable vaccines, and in the case of dengue, there are no therapeutic drugs (Halstead 2014). Efforts to control these diseases often focus on vector control and preventive strategies to minimize mosquito bites.The use of topical sucking louse repellents applied to the splutter is a proven order to reduce mosquito bites. in that respect is a long history of employ plant derived extracts to reduce mosquito bites. However, since the reading of modern synthetical repellents in the 1940s, natural repellents have been largely re hardened by synthetic chemicals (Debboun et al. 2006). Currently there is a renewed interest in development plant-based insect repellents due to concerns slightly safety and the preference for products that are considered more natural (Gerberg et al. 2007). Several essential oils and volatile compounds from a passel of plants have been found to possess repellent properties against arthropods (Curtis et al. 1990). These plant derived chemicals often repel mosquitoes, but there is a wide variability between mosquito species (Kumar et al. 2011). Compounds that repel mosquitoes have been found in the following plant families Graminae (Push panathan et al. 2006), Labiateae (Odalo et al. 2005), Lamiaceae (Ansari et al. 2000), family Myrtaceae (Phukerd Soonwera 2014), Poaceae and Rutaceae (Trongtokit et al. 2005), Umbelliferae (Erler et al. 2006), and Zingiberaceae (Tawatsin et al. 2001).This study evaluated essential oils from the plants turmeric (Curcuma longa L., FamilyZingiberaceae), eucalyptus (Eucalyptus globulus Labill, Family Rutaceae) and orange (Citrus aurantium L. Family Myrtaceae). It is known that turmeric contains the chemical ar-turmerone that is repellent to arthropods (Su et al. 1982). The eucalyptus plant contains important bustling cistron such as 1-8, cineole, - and -pinene that can repel various mosquito species (Yang et al. 2004).Mosquito repellents are often tested by victimisation the fort in cage technique (World wellness Organization 2009). This method allows mosquitoes to feed directly on human volunteers and has several disadvantages, such as the pain and discomfort associated with mosq uito feeding, the requirement for Institutional Review Board (IRB) approval, the circumscribed human operation of candi learn repellents that can be screened at one cadence (Deng et al. 2014). Even though the direct evaluation of repellents on human skin remains essential for evaluating repellents, factitious membrane feeding systems can see as a useful alternative when pre-selecting candidate repellents (Luo 2014). The use of sen clock timental membrane feeding systems is largely dependent on the types of membranes, including animal tissues, Parafilm-M films, and collagen membranes (Friend metalworker 1987 Pothikasikorn et al. 2010).This paper evaluated the efficacy of essential oils from turmeric rhizomes (TU), eucalyptus leaves (EU) and orange peels (OR). These oils were evaluated severally in a previous study using the arm in cage method compared to the synthetic repellent DEET (N,N- viandshyl 1-3 methylbenzamide 25% w/w KOR YOR 15) (Auysawasdi et al. 2016). This study looked at the same leash chemicals but combined them in miscellanys to determine if there was a synergistic effect. Also each admixture was evaluated with or without 5% vanillin extract. Vanillin was added because new(prenominal) studies have found that it extends the core of time that certain natural products are effective against mosquitoes (Tawatsin et al. 2001).Materials and MethodsMosquitoes rearingAe. aegypti and An. dirus were reared in the insectary of the Entomology Department, Armed Forces Research shew of medical examination Sciences (AFRIMS), Bangkok, Thailand. The photoperiod was maintained at 12 h light/12 h dark with a temperature of 252C and a relative humidity of 60-80%.Filter written document containing eggs of Ae. aegypti were laid in waxy trays (30-35-5 cm) with 2,500 ml of distillight-emitting diode water and larvae were provided slant food tablets (HIPPO). after(prenominal)wardwards one day, newly hatched larvae were diluted to about 500 larvae per tray for density and population. For An. dirus, approximately 150 eggs were added to a plastic tray and larvae provided fresh powdered fish food until pupation. The pupae of both species were collected and placed in holding cages until adult emergence. Freshly emerged adults were allowed to feed on strong cotton pads containing a 5% multivitamin solution ad libitum. only testing was performed using five to seven day old post-emergent females that were denied simoleons and only provided water for eight minute of arcs earlier testing.Preparation of plant essential oilsExtracts from many of the plants are available commercially. Eucalyptus leaf oil (New Directions Aromatics Inc.,USA), orangeness peel oil (New Directions Aromatics Inc., USA) and Vanillin (Borregaard Industries Ltd. Company, Norway) were purchased from Chanjao Longevity Co., Ltd., Bangkok, Thailand. Extracts from the turmeric plant were not available commercially. Therefore, turmeric rhizomes were collected from S uwan Farm, Pak Chong, Nakhon Ratchasima Province, Thailand. Essential oils were extracted by water distillation (Charles Simon 1990). The different essential oils were blended at equal ratios for a total volume of 10% and then mixed with virgin coconut oil (Agrilife Co., Ltd., Bangkok, Thailand) using a vortex mixer (Vortex-Genie2, Scientific Industries, Inc., USA) (Table 1). The coconut oil was elect because it created a formulation similar to what would be applied to human skin. totally formulations were kept at room temperature before testing.Repellency assay by feeding membrane apparatusRepellency of essential oil blends was examined for Ae. aegypti and An. dirus under laboratory conditions using a membrane feeding system.Fifty 5-7 day old female mosquitoes were selected and placed in plastic cups (8 cm dia. - 8 cm high) covered with netting. A membrane feeding system was used with a sausage membrane stretched over a standard membrane feeder with a control surface area of 3.14 cm2 (r=1) and secured with a rubber band. Before feeding, either 10 l of each mixture or 10 l of coconut oil (negative control) was pipetted onto the sausage-casing membrane and spread evenly with the tip of the pipette. The treated membranes were allowed to dry and mosquitoes provided blood meals (exposed) at eight different time intervals (0.5, 1, 1.5, 2, 2.5, 3, 3.5 and 4 minute of arcs) after application. A water feeding jacket was used to maintain the temperature of the feeding system at 37C. Approximately 1.5 mL of refrigerated (25C) human blood (Thai Red Cross Society, Bangkok, Thailand) was added to the glass feeder and allowed to warm to 37C. therefore screened plastic cups of 50 female mosquitoes were allowed to feed for five minutes undisturbed. After the 5-min interval the membrane feeder was removed and unengorged mosquitoes were removed. Fully engorged mosquitoes and provided a sugar source and maintained in their containers in the insectary at 252C for 24 hours and then the number of dead mosquitoes counted. information analysisEach mixture of essential oils was replicated five times (n=5) andresults presented as the mean standard deviation (SD). To assess the significance of differences among groups, data were study as a complete randomized design (CRD) with a unidirectional analysis of variance (ANOVA) followed by Duncans multiple range test (DMRT). A p-value of For comparison, percentage repellency was calculated for each test using the following formula% Repellency = - 100(1)Where A is the number of mosquitoes that did not feed on the treated membrane and B is the total number of mosquitoes exposed.alimentation was calculated as% Feeding = - 100 (2)Mortality was calculated as% Mortality = - 100(3)Mortality per hour = (4)Where D is the number of dead mosquitoes 24 hours after blood feeding, F is the total number of mosquitoes which fed on treated membranes, and h is the period of time that the extracts was left on the membrane.The n umber of mosquitoes which not feed on the membrane of each formulation was used to calculate the repellency per hour after application by the following formulaRepellency per hour = (5)Where A is the number of mosquitoes that did not feed on the membrane and h is the period of time that the extracts was on membrane.ResultsThe efficacy of various formulations of selected essential oils (10% Total volume) with and without 5% vanillin, are presented in Figure 1. Shorter exposure times consistently resulted in land feeding rate of both Ae. aegypti and An. dirus to all formulations. Feeding rates increased over time after application of formulations to the membranes. The addition of vanillin change magnitude feeding rates for all formulations. The greatest reduction in Ae. aegypti feeding was with the turmeric and eucalyptus mixture (TUEU, Figure 1c). The greatest reduction in An. dirus feeding was with turmeric, eucalyptus, and orange mixed together (TUEUOR, Figure 1f).Mortality rates are similarly shown in Figure 1. Mortality rates were decreased based on time after application of formulations to the membranes. The greatest and near consistent mortalities for Ae. aegypti were the mixtures of TUOR with and without vanillin (Figure 1a) and the mixture of EUOR with vanillin (Figure 1d). The greatest mortality for An. dirus females was with the mixture of EUOR with vanillin (Figure 1h).The percentage of repellency of all formulations against Ae. aegypti decreased with increasing exposure times (Table 2). The strongest percentage of repellency without vanillin was observed with the mixture of TUEU (90.8-98.4%), followed by EUOR (89.6-98.8%), and TUOR (84.8-98.8%). The lowest percentage of repellency was observed for the mixture of all three plant extracts (TUEUOR, 81.2-94.8%). The percentage of repellency for all essential oil combinations with vanillin was not statistically different between any of the formulations without vanillin. The mixture of TUEU provided t he most repellency at 3.5 and 4 hours with and without vanillin.There were no statistical differences between the repellency of any formulations against An. dirus with or without vanillin, except for EUOR+vanillin after 3 hours (Table 3). The highest percentage of overall repellency was observed for the combination of all three plant extracts (TUEUOR) and with the mixture of TUOR.Overall, there were a positive correlation coefficient for formulations that produced the most repellency and increased mortality (Figure 2). For both Ae. aegypti (Figure 2a) and An. dirus (Figure 2b), the ratio of mortality per hour of formulations with vanillin was greater than without vanillin. Similarly, the repellency per hour of the four formulations with vanillin was stronger than without vanillin.DiscussionEssential oils can have a significant effect on mosquito feeding rates (repellency) and mortality. For all formulations, exposure at 0.5 h after application of plant extracts to the artificial mem brane resulted in greater repellency and higher mortality. Whereas, at 4 h after application there was increased feeding (decreased repellency) and lower mortality. These are consistent with many other studies demonstrating that plant extracts are volatile and draw back their efficacy over time after application (Reifenrath Rutledge 1983 Rutledge Gupta 1999).The different mixtures led to different outcomes. The combination of TUEU resulted in the highest repellency for Ae. aegypti, whilethe combination of all three essential oils (TUEUOR) gave the least repellency. Currently, the combination of all three extracts (TUEUOR) provided the greatest repellency against An. dirus. These results showed that the two mosquito species have different responses to the three plant-derived essential oils tested. It in addition demonstrated that the efficacy of each formulation is based on the compatibility of active ingredients and these compounds produce different effects when combined togethe r. There are other reports showing that essential oils from plants are synergistic. Nerio et al. (2010) reported synergistic actions many plant essential oils used in this study that increased the repellency of formulations that would be evaluate from individual essential oils. Liu et al. (2006) found that the repellent activity of mixing essential oils from Japanese mugwort (Artemisia princeps) and cinnamon (Cinnamomum camphora) was greateragainst Sitophillus oryzae and Bruchus rugimanus (Coleoptera Curculionidae) than that elicited by individual oils.The results also showed that the addition of 5% vanillin significantly decreased feeding rates and increased mortality. Studies have shown that vanillin reduces the evaporation rate of active ingredients and therefore extends the protection time (repellency) (Tawatsin et al. 2001). It is believed that vanillin changes the volatile composition of essential oils and also has an effect on the gustatorial processes of mosquitoes ( downwi nd et al. 2010 Ali et al. 2012). In this study the effect of vanillin was often more evident at increased time after application. It is recommended that vanillin be considered included as an ingredient for future natural product repellents.Finally this study presented a unique method to evaluate repellent formulations using a sausage membrane casing as part of a membrane feeding system. The arm in cage method has been used to evaluate many essential oil formulations (Choochote et al. 2007). While the arm in cage method is the gilt standard for evaluating repellents, a membrane feeding system offers several advantages in that it can be quickly performed, standardly replicated, and does not require human volunteers. (Huang et al. 2015 Cockcroft et al. 1998). This method could be developed to rapidly screen, evaluate, and select the most promising formulations before they are tested on human volunteers.AcknowledgmentsThis study is a explore collaboration between the Department of Bio technology, Faculty of Agro-Industry, Kasetsart University, Bangkok, Thailand and the Armed Forces Research add of medical checkup Sciences (AFRIMS), Bangkok, Thailand. The authors are deeply grateful to the Armed Forces Research Institute of Medical Science (AFRIMS) for facilitating and financial backing the research. Finally, the authors thank the insectary staff Kanchana Pantuwattana, Jaruwan Tawong, Nantaporn Monkanna, Yossasin Kertmanee, Weeraphan Khongtak and Sakon Khaosanorh for teaching and supporting this work. 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