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Biocontrol efficacy of bay essential oil against housefly, Musca domestica (Diptera: Muscidae)

Biocontrol efficacy of bay essential oil against housefly, Musca domestica (Diptera: Muscidae) Background: The synanthropic housefly, Musca domestica, augments the transmission of several detrimental diseases like cholera and avian flu. Consequently, during the last century, many physico-chemical methods including synthetic compounds have been applied for its control. But these methods have proven to be prohibitive due to their side effects and serious issues like resistance development, environmental contamination, and detrimental effects on non-target fauna. Therefore, in view of these objectives, we investigated the effects of bay essential oil (EO) against M. domestica. Methods: The attractant/repellent assays were conducted by double choice technique. Different enzyme assays evaluating the effect of LC concentration of the tested essential oil on larval gut were taken into consideration. To determine the composition, the tested oil was subjected to GC-MS/MS analysis. Further, the morphological alterations caused by EO treatment to third instar larvae were observed in a Nova Nano SEM machine. Data was statistically analyzed by one-way ANOVA using Tukey’stest (p < 0.001). The LC and LC values were calculated by probit 50 90 analysis. Results: The adulticidal bioassay revealed significant effects with LC concentration as 43.03 mg/dm against the newly emerged adult flies while in larvicidal assay mortality was dose dependent showing maximum effect at LC 0.0629 μg/cm . The pupicidal activity was more effective at a dose of LD 64.09 μl/0.25 L of air which either killed the pupae or caused deformity in the emerged adults. Likewise total sugar, protein, glycogen, and lipid contents of larvae were reduced after treatment with EO when compared with the normal larvae along with some gut enzymes. The EO reduced the acetylcholinesterase activity from 0.013 U/mg protein in normal larvae to 0.0093 U/mg protein after EO treatment. The GC-MS/MS analysis of the bay EO showed the abundance of myrcene, linalool, eugenol, chavicol, and anethole along with diterpenoid, geranylgeraniol. However, the insecticidal activity of tested EO might be majorly imparted by eugenol content. The FESEM analysis showed shrinkage of integument and distortion to intersegmental regions caused by the tested compound. Conclusion: The present study concludes the significant efficacy of bay EO against M. domestica which could be employed to breakdown its population below threshold levels to prevent the menace of vector-borne diseases. Keywords: Biocontrol, Essential oil, Laurus nobilis, Musca domestica,FESEM, GC-MS * Correspondence: mudasir.dar@unipune.ac.in; panditrao499@gmail.com Department of Zoology, Savitribai Phule Pune University, Pune, Maharashtra 411007, India © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Chintalchere et al. The Journal of Basic and Applied Zoology (2020) 81:6 Page 2 of 12 Background Materials and methodology The housefly, Musca domestica (Diptera: Muscidae), is a Chemicals and reagents well-known pest of livestock and human health import- The EO of the Laurus nobilis was supplied by Sigma Al- ance. It constitutes a worldwide problem wherever poor drich, USA. The oil doses were prepared freshly by dilu- sanitation and bad hygienic conditions exist (Khan et al., tion in acetone solvent and immediately used for tests. 2013). Moreover, the biology and ecology of Musca All other chemicals/reagents used in this study were of domestica makes it an ideal organism to carry and dis- highest purity and molecular or analytical grade unless seminate human and animal pathogens, such as hel- defined. minth parasites, protozoan cysts, viruses, and bacteria (Fotedar et al., 1992; Greenberg, 1973; Kobayashi et al., Rearing of housefly colony 1999). Recently, it has been inculpated to transmit avian The laboratory-reared colonies of the model organism, flu (Wanaratana et al., 2013) also. Therefore, hitherto, a i.e., M. domestica, were generously provided by the En- large variety of synthetic compounds such as DDT and tomology Section, National Chemical Laboratory (NCL), cyromazine have been used against this vector to prevent Pune (M.S.), India, which were free from insecticides the epidemics. However, indiscriminate use of these and pathogens. The culture of housefly was maintained chemical insecticides has resulted in many serious prob- in vitro at a temperature of 28 ± 2 °C and 60–70% rela- lems like insect resistance, persistence of chemicals in tive humidity (RH) in plastic jars (35 × 15 cm), covered the environment, and biomagnifications through trophic with cheese cloth for several generations. A cotton swab levels leading to detrimental effects on human beings soaked in milk (10% w/v) was offered as a food to adult (Tabashnik & Johnson, 1999). Therefore, researchers are flies which also served as a substratum for oviposition. continuously prospecting for active natural products of The eggs were transferred to another set of jars contain- plant origin as potential alternatives to conventional ing animal feed and water or cotton swab soaked in milk insecticides. for hatching and larval development. Similarly, pupae The eco-toxicological property of plants such as lower were collected and kept in another container for adult toxicity to humans, cheap, easy cultivation and degrad- emergence. All stages of the fly such as eggs, larvae, ation along with reduced environmental impact makes pupae, and adults were continuously available for the them promising candidates for the management of in- experiments. sect pests (Kumar et al., 2011). Additionally, aromatic plants have proved effective insecticides and their essen- Larvicidal and adulticidal bioassays tial oils (EOs) often constitute the bioactive fraction The larvicidal and adulticidal bioassays were carried out (Regnault-Roger, 1997). Plant metabolites act by either by following the methods of Busvine (Busvine, 1971) and exerting their effect on octopaminergic nervous system Palacios et al. (2009), respectively, with few modifica- of insect pests (El-Zayyat et al., 2017) or interfering with tions. To determine the effect of EO, 10 individuals of GABA-gated chloride channels (Khater, 2012). Further the third instar larvae were exposed to different concen- effects can be seen in behavioral modifications (attrac- trations of EO in each test. Primarily, a range of desir- tion/repellency) and contact toxicity for different life able doses were tested to determine the LC and LC 50 90 stages of the targeted organisms. Similarly, some natural values. For residual film method, 1 ml each of different oils are complexes of many biologically active constitu- concentrations of EOs was applied on filter paper discs ents including terpenes, acyclic monoterpene alcohols, kept inside the glass petri dish of 90 mm diameter. The monocyclic alcohols, aliphatic aldehydes, aromatic phe- EO doses were applied uniformly in order to make a uni- nols, monocyclic ketones, bicyclic monoterpenic ke- form film over the filter paper. Initially, the treated petri tones, acids, and esters (Koul, 2008). dishes were air dried for few minutes to allow the solvent In the past few years, there is a strong impetus to develop evaporation, followed by release of larvae (n = 10) and EOs of botanical origin as potential agents for pest control then incubating the plates under laboratory conditions for strategies. Since aromatic angiosperm, Laurus nobilis L., is 24 h. widely exploited for treatment of gastrointestinal disorders In case of adulticidal assay, 10 adult flies were placed (Lorenzi & Matos, 2008), the aqueous extracts of this plant in plastic jars (1.2 dm ) containing a 7-cm-long cotton are also used for the treatment of many open wounds from yarn suspended from the cap of the jar. Different dos- ancient times (Nayak et al., 2006). In view of these proper- ages of EO ranging from 10 to 100 mg/dm were used ties, the present study aimed to determine the chemical for the tests. Each dose was applied after being dissolved composition of Laural nobilis essential oil and evaluate its in 10 μl of acetone then applied to a cotton yarn. In the efficiency against Musca domestica.Further,weinvesti- control jar, the cotton yarn was treated with 10 μlof gated the larvicidal effect and oviposition deterrent activities acetone only. The jars were then sealed tightly and kept posedbythisoil to thedifferent lifestagesof the fly. at 28 ± 2 °C for 30 min. All tests were replicated five Chintalchere et al. The Journal of Basic and Applied Zoology (2020) 81:6 Page 3 of 12 times with acetone solution being used as a control The percent inhibition rate (% IR) on pupal emergence treatment. The mortality rate of the treated larvae and was calculated by using the below given formula no. 4. flies was calculated after 24 h of treatment by using below mentioned formula 1. Where X denotes the per- %inhibition rate ¼ Cn−Tn=Cn  100 ð4Þ centage of larvae that survived in the control test and Y represents the percentage survival of larvae in the tested doses. where Cn represents the number of newly emerged in- sects in the control set and Tn depicts the number of in- Mortalityð%Þ¼½X−Y =X100 ð1Þ sects emerged after treatment. The data were pooled and analyzed by standard probit analysis to obtain LC and LC values. However, the 50 90 Effect of essential oil on biochemical aspects of housefly actual dose of EO present in 1 ml mixture was calculated larvae by using the formula 2. In larvicidal bioassays, 10 individuals of third instar larvae Dose=cm ¼ value present in 1 ml=area of petridish of the fly were exposed to LC concentrations of essential oil for 24 h. After the treatment, the exposed larvae were ð2Þ collected and subjected to biochemical analyses such as determination of total sugars, glycogen, lipids, and pro- Attractant/repellant bioassays teins by following the Anthrone method specified by The attractant and repellant bioassays were carried out Plummer (Plummer, 1988). However, proteins and lipids by using the double choice method (Campbell, 1983) were estimated by the methods of Bradford (Bradford, where 20 newly emerged adults of mixed sexes were re- 1976) and Van Handel and Day (Van Handel & Day, leased in a cage (size 20 × 12 × 8 cm) with two conical 1988), respectively. Results obtained were analyzed with flasks. One flask contained 0.1% test oil in 5 ml of milk, one-way ANOVA, followed by Tukey’s test (mean com- while the other contained acetone as a solvent and 5 ml parison) using GraphPad software version 3.6. of milk to serve as control. The conical flasks were fitted with funnel (10 cm in diameter) to avoid the escape of the flies. The assays were replicated five times, and the Enzyme assays numbers of flies attracted towards the tested oil flask Different enzyme assays were carried out by treating the and in the control flask were counted after 24 h to deter- larvae with LC concentration approximately as de- mine the percentage of repellency. The results were scribed in the larvicidal bioassay. The whole guts of the expressed in terms of percentage (%) attraction whereas treated larvae were suspended in lysis buffer and homog- percentage repellency (% R) was calculated by the fol- enized on ice using a glass homogenizer. The homogen- lowing formula. ate was centrifuged at 10,000 rpm for 10 min at 4 °C. The supernatant obtained was treated as enzyme extract %R ¼½ 100ðÞ C  T =C ð3Þ and used for different enzyme assays. The α-amylase ac- where C is the number of flies trapped in the control tivity was determined by dinitrosalicylic acid (DNSA) flask and T indicates the number of flies trapped in the procedure described by (Bigham et al. 2010), using 1% treated flask. (w/v) starch as substrate. Briefly, 20 μl of the enzyme ex- tract was mixed with 50 μl of PBS (pH 7.4) and 25 μlof Pupicidal bioassay 1% starch solution (w/v). The mixture was incubated at Pupicidal bioassays were carried out by following the 35 °C for 30 min to allow the reaction to occur which method described by Kumar et al. (Kumar et al., 2011) was subsequently terminated by adding 85 μl DNSA re- with slight modifications. The bioassays were performed agent. The terminated reactions were heated in a boiling with 20 pupae (3 days old) in a 250-ml conical flask. The water bath for 10 min. Similarly, α- and β-glucosidase pupae were exposed to different doses in the range of activities were determined by measuring the amount of 20 μl/0.25 l to 100 μl/0.25 l of air to assess the effect of p-nitrophenol released from 5 mM p-nitrophenyl–α–D- fumigation through impregnation on a cotton swab. In glucopyranoside and p-nitrophenyl–β–D-glucopyrano- control treatment, the pupae were exposed to acetone side, respectively. Here the assay mixtures were incu- only. These pupicidal assays were performed at 28 ± bated at 25 °C for 30 min followed by termination with 2 °C and RH 65 ± 5%. The observation was recorded till 100 μl of NaOH (0.1 M) solution. The absorbance was th the 6 day after exposure to EOs during which emer- read spectrophotometrically at 540 nm, and the reducing gence into adults was documented. These assays were sugars released were estimated by using glucose as carried out with five or more independent replicates. standard. Chintalchere et al. The Journal of Basic and Applied Zoology (2020) 81:6 Page 4 of 12 Inhibition of acetylcholinesterase (AChE) activity drying (CPD) in hexamethylenedisilazane (HMDS). The To determine the acetylcholinesterase (AChE) activ- specimens were mildly coated with gold (100 Å) followed ity caused by the EO treatment, the sample preparation by observation at a voltage of 5.0 kV in a FEI NOVA was carried out as described above except in place of the Nano SEM (NPEP303, FEI USA). whole gut only head regions were taken for inhibition ana- lysis. The activity was assayed by the modified method of Statistical analysis Ellman and colleagues (Ellman et al., 1961). Briefly, 10 μl Data obtained was subjected to statistical analysis. Re- of enzyme extract was mixed with 10 μl deionized water, sults were reported as mean ± standard error means incubated at room temperature for 15 min followed by (SEM) of five or more independent replicates which addition of 20 mM acetylthiocholine iodide and 10 μlof were subjected to one-way ANOVA using Tukey’s test 0.5 M DTNB [5,5′-dithiobis (2-nitrobenzoic acid)]. The (p < 0.001). The LC and LC values with their 95% 50 90 control was treated by taking acetone in place of the en- confidence limits were determined by probit analysis zyme extract. The absorbances were recorded at 412 nm, which helped to analyze the dose–mortality response and enzyme activity was measured in U/min/mg of (Finney, 1952). Analysis of data was carried out in SPSS protein. software version 19 as well as Microsoft Excel (Microsoft office suite, 2013). Gas chromatography-mass spectrometric (GC-MS) analysis of essential oil Results The chemical composition of tested EO was analyzed by Larvicidal and adulticidal efficacy gas chromatography-mass spectrometric (GC-MS) The larvicidal assay carried out by the residual film method. The liquid phase used in the chromatographic method showed that LC concentration of the bay EO procedures helps to characterize the oil constituents re- shows higher larval mortality at 4 mg/ml with an actual sponsible for the insecticidal property of the oils. For dose 0.0629 μg/cm which is a very low concentration. GC-MS, analysis of bay EO was carried out by the The results observed with the larvicidal assay are method of Peris and Blazquez (Peris & Blázquez, 2015). depicted in Table 1. However, the LC concentration of The column temperature in the program was 60 °C dur- the EO was observed to be 9.231 mg/ml with an actual ing 5 min, with 6 °C/min increase up to 180 °C, followed dose of 0.144 μg/cm (Fig. 1a). As the dose concentra- by a 20 °C/min increase up to 280 °C, which was main- tion of EO increases, the percent (%) mortality of the tained for 10 min. The carrier gas used was helium at a treated larvae also increased. The efficacy of the tested flow rate of 1 ml/min and a split ratio of 1:30. The ana- oil was very significant when compared with the control lysis was performed using a GCMS-TQ8030 apparatus, tests indicating short-term exposure of larvae to lethal equipped with RTX 5MS column. Finally, the compo- doses can markedly increase their mortality over time, nent identification was elucidated based on the compari- thereby reducing the number of viable adults, leading to son of their relative retention time and mass spectra possible significant diminution in overall population of analysis with those of NIST11 library data (GC-MS sys- M. domestica. The effect of tested doses of the EO on tem), and available literature. adult insects showed significant mortality (Fig. 1b) vary- ing with the dose range. However, the LC and LC 50 90 Field emission scanning electron microscopy (FESEM) concentrations for adult mortality by bay EO were found 3 3 In order to have a clear and discernible observation of to be 43.03 mg/dm and 84.42 mg/dm , respectively the effects of oil treatment on larval body, 10 larvae of (Table 1). the third instar stage were treated with LC concentra- tion of tested EO. The sacrificed (treated) larvae and Attractant/repellant bioassay control larvae were prepared for FESEM visualization by The EOs derived from plants are volatile, natural, and using the protocol of Kumar et al. (Kumar et al., 2011) complex organic compounds characterized by a strong with few modifications to compare the structural odor formed as secondary metabolites. The repellency changes caused by the oil treatment. The specimens potential of the EO from L. nobilis was evaluated against were primarily fixed in 2.5% glutaraldehyde (v/v) in dis- housefly in glass chambers. In this assay, EO showed tilled water for 15–17 h. Thereafter, sample specimens 27.58% repellency at a concentration of 0.1% (Table 2). were washed with distilled water for 20 min, followed by Among the tested essential oil concentrations, 0.5% was fixation in 4% osmium tetroxide for about 2 h. The sam- found to be the most effective with 100% repellency ples were washed with deionized water for 20 min and whereas no repellency was observed on a dose of 0.1% then dehydrated serially with different grades (30, 50, 70, after 1 h of continuous observations. The repellency po- 90, and 100% v/v) of alcohol each for 10 min. After de- tential of EO doses followed the pattern 0.1% > 0.5% > hydration, samples were subjected to critical point 1.0%. The tested laurel oil elicited a strong and abrupt Chintalchere et al. The Journal of Basic and Applied Zoology (2020) 81:6 Page 5 of 12 Table 1 Larvicidal and adulticidal activities of bay essential oil on M. domestica # # Essential LC (mg/ml) 95% confidential limit (LC ) Regression LC (mg/ml) Chi 50 50 90 3 ## 3 ## oil (mg/dm ) equation (mg/dm ) square LCL UCL Larvicidal 4 3.48 4.45 y = 0.245 − 0.98x 9.231 4.37*(5) Adulticidal 43.03 34.833 50.866 Y = 0.31 − 1.33X 84.42 8.051*(5) Each value represents the mean of five replicates. LC and LC are lethal concentrations at which 50% and 90% population dies, respectively. *Significant at p < 0.001. 50 90 # ## Unit used for larval mortality; Unit used for adulticidal assay. The mortality rate of larvae or adult flies in control sets was negligible when compared with the treatment assays effect against the adults of M. domestica at concentra- content of the treated larvae as compared with the con- tions higher than 0.5% indicating 0.5% sufficient for trol set. Further, the biochemical assays revealed that 100% mortality of the flies. bay EO showed notable changes in total sugar, glycogen, lipid, and protein contents of third instars of M. domes- Pupicidal bioassays tica (Table 3). The tested oil remarkably reduced the The pupicidal assay carried out by fumigation and con- total sugar content from 3.169 ± 0.191 μg/larva in the tact toxicity protocols against the tested organism exhib- control to 0.448 ± 0.036 μg/larva in the treated larva. ited significant variation in growth inhibition with Not only sugars, the treatment also reduced total glyco- different doses of the bay EO (Table 2). The results of gen content, i.e., 0.117 ± 0.007 μg/larva after treatment the fumigation assay revealed that a LD dose of bay comparable to normal larva (7.302 ± 0.131 μg/larva). A EO for the pupal death was 64.09 μl/0.25 l of air (Fig. 2) similar trend was observed with lipid content of the in- whereas LD concentration was found to be over sect where its amount was decreased from 1400.2 ± 104.35 μl/0.25 l of air. In case of higher doses, a complete 38.87 μg/larva in normal individual to 474.62 ± 57.35 μg/ inhibition of pupal emergence was noted (Additional file larva after treatment. In case of total protein content of 1: Figure S1). Further, the increase in the dose concen- the treated and control larvae, the proteins were signifi- tration caused increased percent inhibition rate (PIR) cantly reduced from 1000.6 ± 11.47 μg/larva to 636.72 ± which eventually lead to the deformities in adults in case 26.98 μg/larva due to EO treatment. of survived pupae. Similarly, in contact toxicity, we found that not a single pupa can transform into adult Effect of EO on gut enzymes successfully at all tested doses indicating 100% mortality Since digestive tract is the main interface between insect of the pupae. and the environment, given that nutrition is a decisive factor in the evolutionary process of these organisms, Effect of EO on biochemistry of housefly the main aspect of the pest control is the selective inhib- The secondary metabolites of the plant origin cause a di- ition of digestive enzymes secreted by the alimentary verse effect on the metabolism of the insects. Therefore, canal of pests. Keeping that in mind, we checked the ef- we tested the effect of different concentrations of the fect of tested EO on gut enzymes such as α-amylase, bay EO on the larvae of housefly. The inferences of the α and β-glucosidases. There was a significant difference present study elucidate that LC dose of the bay EO in- (Fig. 3a–c) in enzyme activities between treated and con- duced marked reduction in the level of total protein trol larvae in case of both the enzymes under Fig. 1 Bioefficacy of bay essential oil against M. domestica. a Percent mortality of larvae after a treatment of 24 h. b Adulticidal activity of the tested EO against newly emerged M. domestica after 24-h treatment. LC and LC are lethal concentrations at which 50% and 90% population 50 90 dies, respectively. Each value represents the mean of five replicates. Asterisk indicates significance at p < 0.05 Chintalchere et al. The Journal of Basic and Applied Zoology (2020) 81:6 Page 6 of 12 Table 2 Percent (%) inhibition repellency of the adult houseflies by different doses of the EO using the fumigation method Dose (μl/ % LD (μl/ 95% confidential limit (LC ) Regression LD (μl/ Chi 50 50 90 0.25 l of air) IR 0.25 l of air) equation 0.25 l of air) square LCL UCL 20 14 40 22 60 44 64.09 55.79 73.58 Y = 0.032x − 2.040 104.35 9.44*(5) 80 68 100 88 Each value represents the mean of five replicates. LC and LC are lethal concentrations at which 50% and 90% population dies, respectively. df degree of 50 90 freedom. *Significant at p < 0.001. Negligible repellency or inhibition rate was observed for the control set consideration showing a p < 0.001 only. The bay essen- GC-MS analysis of essential oil tial oil significantly decreased α-amylase activity (0.092 ± The GC-MS analysis of the bay EO revealed an abun- 0.002 μg/larva) when compared with control test (0.214 dance of seven major components (Fig. 4). The compo- ± 0.004 μg/larva). We observed a substantial decrease of nents observed (Table 4) were terpenes such as 57.1% in the activity of α-amylase indicating the effect of myrcene, linalool (3, 7-dimethyl-1, 6-octadien-3-ol), and bay EO on M. domestica. Similarly, the effect of EO on diterpenoids (E,E,E)-3,7,11,15-tetramethylhexadeca-1,3,6. α-glucosidase showed considerable reduction (1.878 ± Some of the phenolic compounds observed were eu- 0.043 μg/larva) in its secretion by fly larvae as compared genol, chavicol (phenol,4- (2-propenyl)) and anethole. with control set (l2.375 ± 0.074 μg/larva) marking a dif- The highest amount found was of the eugenol (58.95%) ference of 21%. It also showed 1.3-fold reduction in β- which proved to be the main component followed by glucosidase activity from 3.066 ± 0.071 μg/larva to 2.19 phenol (13.96%), tetra methyl hexadeca (10.57%), and β- ± 0.024 μg/larva after treatment with the EO (Additional myrcene (6.03%). The insecticidal property of this plant file 1: Table S1). The tested EO significantly affected the EO could be attributed majorly to eugenol content and α-amylase of the gut extract upon treatment showing a to some extent linalool compound. However, the com- decrease of over 51% activity (Fig. 3a). pounds like β-myrcene, eugenol, and linalool comprise only about 3% of the total chemical compounds present in this plant. Acetylcholinesterase (AchE) activity As shown in Additional file 1: Figure S2, the bay EO was found to inhibit the AchE activity of M. domestica larvae Field emission scanning electron microscopy (FESEM) after 24 h of treatment. The AchE activity in treated lar- In order to clearly visualize the effect of EO treatment on vae was 0.0093 U/mg of protein compared with control the larval body surface, we employed FESEM technique to larvae where it was much higher (0.013 U/mg of protein) compare the ultra-structural changes in larval integument indicating significant reductions caused by the exposure due to EO treatment with normal larvae. Upon analysis, a to EO. clear observation showed the toxic effect of bay oil in the form of integument shrinkage particularly at spinose rings with distorted intersegmental regions. However, control/ normal larvae of M. domestica showed normal appearance with a smooth textured integument and well-shaped inter- segment spines (Fig. 5a–c). Therefore, it can be concluded Table 3 Effect of bay EO on nutritional reserves of M. domestica larvae Sr. no. Analysis Control larvae (μg/larva) Treated larvae (μg/larva) 1 Sugar 3.169 ± 0.191 0.448 ± 0.036* 2 Glycogen 7.302 ± 0.131 0.117 ± 0.007* 3 Lipid 1400.2 ± 38.87 474.62 ± 57.35* Fig. 2 Pupicidal assay showing percent (%) mortality of M. domestica 4 Protein 1000.6 ± 11.47 636.72 ± 26.98* larvae by fumigation method with bay EO. Significant *p < 0.001 Values are mean ± SE (standard error). Data were analyzed by one-way when compared with the control. Each value represents the mean ANOVA, followed by Tukey’s test and significance at *p < 0.001 compared with of five replicates the control Chintalchere et al. The Journal of Basic and Applied Zoology (2020) 81:6 Page 7 of 12 Fig. 3 Effect of bay essential oil on the gut enzymes of M. domestica larvae. a α-Amylase, b α-glucosidase, and c β-glucosidase activities of M. domestica. Values are means ± SE (standard error). Data analyzed by one-way ANOVA, followed by Tukey’s test and significance at *p < 0.001 compared with the control from the observed results that fly larvae upon contact with quite higher than the concentration used in this study. EO show superficial alterations to the integument. Similarly, Asid and co-workers stated that Citrullus colo- cynthis extracts exhibit potential efficacy against all Discussion stages of M. domestica at 50% concentration (Asid et al., Pesticides derived from plants are organic in nature, 2015). Previously, many authors have reported the anti- considered to be biodegradable, non-mutagenic or non- bacterial (Evrendilek, 2015), antifungal (Gumus et al., toxic to animals, and more particularly target specific 2010), antioxidant (Inan et al., 2012), analgesic and anti- than their chemically synthesized counterparts. Some of inflammatory properties (Sayyah et al., 2003), apart from them also inhibit or reduce the insecticide detoxifying some acaricidal (Senfi et al., 2014), larvicidal (Pavela, enzymes of the pests thereby could be important from 2008), and insecticidal (Sertkaya et al., 2010) activities by the agriculture viewpoint (Wang et al.,2016). Therefore, laurel EO. keeping in mind the importance of plant-based EOs, we In order to ascertain the potential of bioactive com- tested the efficacy of EO from L. nobilis against the dif- pounds present in the bay EO, we used the GC-MS ana- ferent stages of the nuisance-causing housefly, M. lysis that revealed dominance of eugenol and phenolic domestica. We observed higher larval mortalities at very content which was in congruence with previous reports low doses of the compound which was only 0.0629 μg/ that have stated the chemical composition of the EO cm . The efficacy of Laurus EO against M. domestica as from the leaves of L. nobilis (Senfi et al., 2014; Sertkaya observed in this study is much higher than the insecti- et al., 2010). Further insecticidal property of many com- cidal property of EOs derived from various other plants pounds present in EO has been conducted topically by (Pavela, 2008). Recently, the efficiency of the EOs from Rozman and co-workers (2007) wherein eugenol was L. nobilis has been tested on several other insects such found highly toxic to all tested beetles. Eugenol is also as stored grain pests, where authors have suggested known to show higher repellency against beetles (Andro- species-specific dose requirements for the control of nikashvili & Reichmuth, 2002; Huang et al., 2002; Roz- pests (Drapeau et al., 2009; Pavela, 2008; Sertkaya et al., man et al., 2007) and other stored grain pests 2010). Ours is the pioneering study to report the effects (Cosimi et al., 2009; Lee et al., 2003). Further, these au- of L. nobilis EO on the adults of M. domestica. Recently, thors concluded the complete destruction of the eggs Pavela and his colleagues (Pavela, 2008) tested more and immature stages of T. castaneum by eugenol. Our than 30 plant extracts against M. domestica and con- results were in line with Nehir et al. (2014) who found cluded that EO from Pogostemon cablin is the most effi- eugenol; 1,8-cineole; linalool; methyl eugenol; α-terpinyl cient against fly at a concentration of 3 μg/fly which is acetate; α-pinene; and β-pinene as major components of Fig. 4 Chromatogram obtained from GC-MS/MS analysis shows the chemical composition and abundance of some compounds of the bay EO Chintalchere et al. The Journal of Basic and Applied Zoology (2020) 81:6 Page 8 of 12 Table 4 GC-MS analysis of bay essential oil revealing the abundance of certain compounds Peak R. time Area Area % Height Height % Name 1 9.048 8,758,593 6.03 2,457,962 10.47 Beta myrcene 2 12.304 5,117,500 3.52 1,400,514 5.97 1,6-Octadien-3-ol,3,7-dimethyl 3 16.540 20,281,918 13.96 2,876,306 12.26 Phenol,4-(2-propenyl) 4 19.233 85,635,465 58.95 6,319,156 26.93 Eugenol 5 29.826 15,347,881 10.57 5,460,173 23.27 (E,E,E)-3,7,11,15-Tetramethylhexadeca-1,3,6 6 30.114 5,874,016 4.04 3,065,184 13.06 (E,E,E)-3,7,11,15-Tetramethylhexadeca-1,3,6 7 33.403 4,246,485 2.92 1,887,660 8.04 Anethole laurel EO. Most of the terpenoids and phenols of plant monoterpenes alter the physiological functions by competi- origin have minimal vertebrate toxicity and are regarded tive inhibition of acetylcholine esterase in houseflies and as safe by United States Food and Drug Administration cockroaches (Grundy & Still, 1985; Ryan & Byrne, 1988). (USFDA). These compounds apparently cause hyperactiv- The toxic effects exerted by EO of L. nobilis could be attrib- ity, tremors, and convulsions followed by knockdown of uted to the toxic constituent like eugenol, linalool, myrcene pests similar to the symptoms by insecticides such as or- and other phenolic compounds present therein. Besides the ganophosphates and carbamates. However, previously simi- fumigant and repellent activities of bay EO against M. lar signs have been described by insects upon exposure to domestica, it has also proven insecticidal against red flour pure monoterpenes (Coats et al., 1991). Some beetle, T. castaneum (Cosimi et al., 2009; Lee et al., 2003). Fig. 5 The ultra-structural and superficial changes induced by bay EO to the larvae of M. domestica after a treatment for 24 h. a, c, and e show the anterior, posterior, and abdominal segments respectively of the control larva exhibiting normal appearance with smooth integument and well-defined or swollen intersegment spines, whereas b, d, and f show the FESEM micrograph anterior, posterior, and abdominal segments depicting the ultra-structural changes like shrinkage, rupture of body fluids, and corrugation of the integument induced by bay EO during treatment Chintalchere et al. The Journal of Basic and Applied Zoology (2020) 81:6 Page 9 of 12 Further, monoterpenoids abundantly distributed in EOs of whereas β-glucosidases catalyze the hydrolysis of β- aromatic plants are lipophilic in nature thereby inter- glycosides into respective monosaccharides (Sezginturk vene with metabolic, biochemical, physiological, or be- & Dinckaya, 2008). Many insects are known to show β- havioral functions of insects (Brattsten et al., 1983). glucosidase activities required for metabolism of Similar to our study, Palacios et al. (2009b) examined ingested food such as Diatraea saccharalis (Azevedo et al. the efficacy of EOs from several medicinal and edible 2003) and Parnassiusapollos sp. (Nakonieczny, Michalc- plants against housefly and stated that orange peel and zyk, & Kedziorski, 2006). The tested EO depicted neuro- eucalyptus leaves are the most toxic to flies which had toxic effects by inhibiting AchE activity of the M. limonene (92.5%) and 1,8-cineole (56.9%), as the princi- domestica larvae after 24 h of treatment with LC doses pal compounds. However, Kumar and co-workers only. Many secondary metabolites such as EOs and (Kumar et al., 2011) have observed that EOs of Mentha monoterpenes of aromatic plants are known to inhibit piperita and Eucalyptus globules are the most effective the AchE activity of insects (Senthilm et al., 2008). plant extracts against M. domestica showing both Our observations were in accordance with Rajashekar repellent and insecticidal properties. From time imme- and co-workers (2014) who concluded the inhibition morial, plant-derived substances have been used to repel of AchE in M. domestica by Coumaran, extracted or kill mosquitoes before the advent of synthetic chemi- from L. camara. Therefore, for designing a biocontrol cals (Curtis et al., 1990). The tested EO also showed strategy, it becomes imperative to understand the higher repellency against adult flies preventing them interaction of pesticide with digestive enzymes and from oviposition in the nearby vicinity which is also AchE of thetargetorganism. Hencewedetermined stated by other researchers against rust-red flour beetle the effect of EO on gut enzymes of M. domestica Tribolium castaneum (Andronikashvili & Reichmuth, where drastic changes in expression levels were ob- 2002), Sitophilus zeamais, Cryptolestes ferrugineus, served. The treatment with EO reduced the activities Tenebriomolitor (Cosimi et al., 2009), and adult females of the α-amylase and α-and β-glucosidases by 57.1, of Culex pipiens (Erler et al.2006). The repellency of 21, and over 30%, respectively, in treated larvae as these compounds appears due to the presence of volatile compared with larvae without treatment (control). monoterpenoids (Buescher et al., 1982; Curtis et al., The reduction in enzyme activity could be a conse- 1990;Rutledge et al.,1983). Besides, Papachristos and Stam- quence of the cytotoxic effect of plant compounds on poulos (2002) showed that EO from bay plant presented a epithelial cells of the gut that are responsible for the syn- repellent activity against Acanthoscelides obtectus. thesis of α-amylase (Hichri et al.t, 2019). Cytotoxicity ap- The pupicidal effect exerted by the tested EO was also pears to include membrane damage by causing significant for the bio control of housefly. Besides vari- coagulation of the cytoplasm (Gustafson et al., 1998) ous fumigant and repellent activities of bay leaf EO thereby damaging its lipid and protein contents against stored grain pest (Cosimi et al., 2009; Lee et al., (Ultee et al., 2000) or disrupting cell membrane leading 2003), there are few reports available about the insecti- to leakage of macro molecules and ultimately cell lysis cidal potential of bay leaf oil and its fractions against T. (Oussalah et al., 2007). Some plant EOs are also known castaneum. In the present study, significant mortality or to form stable complexes with digestive enzymes, mak- inhibition of pupal emergence was observed by both ing dissociation difficult. In case of biochemical con- contact and fumigation pupicidal bioassays. In contrast, tents, decreased levels of proteins, lipids, and glycogen Kumar et al. (2011) observed 100% mortality of the same were observed due to treatment of EO which could be insect by contact toxicity method only. primarily due to blocking of gut hydrolases, which leads Since most insects feed on carbohydrate-rich food to poor nutrient utilization, retarded growth, and conse- products such as seed, fruits, crops, and stored grains, quent death by starvation (Jongsma & Bolter, 1997). their guts are efficient natural biochemical reactors se- However, the reduction in body protein content may be creting a suite of hydrolytic enzymes such as amylase attributed to inhibition of DNA and RNA synthesis at and α- and β-glucosidases. These enzymes hydrolyze α- molecular levels which then reflects the decrease in en- D-1, 4-glucan linkages of starch, glycogen, and some zymatic activities. Our results were in congruence with other carbohydrates (Ferreira & Terra, 1989; Strobl earlier reports (Nathan et al., 2005) where authors have et al., 1998). The amylase enzyme transforms starch into stated similar reduction in protein and metabolite con- maltose, which is then converted to glucose by α- tents in moth, Cnaphalocrocis medinalis, after treatment glucosidase and thus utilized as an energy source by the with plant compounds. The changes in total lipid con- insect. In insects, only α-amylases have been found to tents were also caused by the treatment of EOs. Some hydrolyze long α-1, 4-glucan chains of starch or glyco- authors have suggested that decreased lipid content may gen (Terra et al., 1988). Likewise, α-glucosidases break be a result of transformation of lipids into proteins to non-reducing 1, 4-linked α-D-glucose ends of glycogen substitute the reduction in protein content or produce Chintalchere et al. The Journal of Basic and Applied Zoology (2020) 81:6 Page 10 of 12 supplementary energy to combat the chemical stress. gut/digestive enzymes of the M. domestica larvae. Value are mean ± SE Hence, treatment with EOs might have interrupted this (Standard error). Data were analyzed by One-Way ANOVA, followed by Tukey’s test and Significance at *p < 0.001 compared with control. process and resulted in larval mortality. The biochemical and metabolic disruption by the EO Abbreviations treatment was also superficially revealed by the FESEM AChE: Acetylcholinesterase; ANOVA: Analysis of variance; CPD: Critical point analysis where a complete distortion of the body was ob- drying; DDT: Dichlorodiphenyl trichloroethane; DNSA: 3,5-Dinitrosalicylic acid; served. Our inferences were supported by the conclusion DTNB: 5,5′-Dithiobis, 2-nitrobenzoic acid; EO: Essential oil; FESEM: Field emission scanning electron microscopy; GABA: Gamma amino butyric acid; of Insun et al. (1999) who demonstrated the damage to GC-MS/MS: Gas chromatography-mass spectrometry/mass spectrometry; the surface morphology of mosquito larvae by Kaemp- HMDS: Hexamethylenedisilazane; LC: Lethal concentration; LD: Lethal dose; feria galangal extract. Moreover, the effect of monoter- NaOH: Sodium hydroxide; NCL: National chemical laboratory; PBS: Phosphate-buffered saline; PIR: Percent inhibition rate; RH: Relative penes on insect morphology was also investigated by humidity; SEM: Scanning electron microscope; SEM: Standard error means; Sukontason et al. (2004) who studied the structural USFDA: United States Food and Drug Administration changes induced in housefly larvae through SEM ana- Acknowledgements lysis after the application of eucalyptol oil. The authors JC is highly indebted to the authorities of the S.P. Pune University, Pune, reported significant deformation in the integument, inter India, for providing the research stipend. MD is grateful to the University segmental spines, and bleb formation of the treated lar- Grants Commission (UGC), New Delhi, India, for senior research fellowship under the Maulana Azad National fellowship scheme. RSP acknowledges the vae. The FESEM analysis of the treated larvae revealed UPE-II (nanobiotechnology), UoP-BCUD grant (15-SCI-001422), and DRDP and extreme dehydration and surface distortion compared DST-PURSE schemes provided to the laboratory. with the undisturbed, free, and smooth surface of con- Authors’ contributions trol larvae, therefore confiraffirming the effect of tested JC and RSP conceived and designed the experiments. JC and MD performed EO on housefly larvae. The above all results of the pre- the experiments. JC, MD, and RSP wrote the paper. All authors read and sented research indicate that EOs from medicinal plants approved the final manuscript. such as L. nobilis could be a potential alternative for use Funding as housefly fumigants, provoking death of the insect The work was supported by Savitribai Phule Pune University, Pune, under the within a short period of time. project code UoP-BCUD grant (15-SCI-001422), as well as DRDP and DST-PURSE schemes provided to the RSP. Conclusions Availability of data and materials The housefly is a cosmopolitan home invader linked to All the data generated from this study is included in the paper and can be accessed from the corresponding author upon request approval. poor hygiene and low socioeconomic status. The search for new, highly selective, biodegradable insecticides is Ethics approval and consent to participate mandatory to solve the problem of residual toxicity to the Not applicable. environment, fauna or flora. Nature provides many prod- Consent for publication ucts acting as an excellent alternative; among these are the Not applicable. botanical insecticides. With the recent rise of the green in- Competing interests secticide concept and increased public awareness, several The authors declare that they have no competing interests. attempts to use indigenous plants extracts as potential alternatives have been done, but only few plant-based con- Received: 12 June 2019 Accepted: 13 January 2020 trol products appear in the market. This study recom- mends the usage of bay-based products in domestic References botanical anti-fly insecticides, as it is ethnobotanical, en- Andronikashvili, M., & Reichmuth, C. H. (2002). Repellency and toxicity of essential demic, safe, cheap, and available all year. 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The larval midgut of the housefly (Musca domestica): Ultrastructure, fluid fluxes and ion secretion in relation to the organization of digestion. Journal of Insect Physiology., 34, 463–472 https://doi.org/10.1016/0022-1910(88)90187-4. Ultee, A., Kets, E. P., Alberda, M., Hoekstra, F. A., & Smid, E. J. (2000). Adaptation of the food-borne pathogen Bacillus cereus to corvacrol. Archives of Microbiology, 174, 233–238 PMID: 11081791. Van Handel, E., & Day, J. F. (1988). Assay of lipids, glycogen and sugars in individual mosquitoes: correlations with wing length in field-collected Aedes vexans. Journal of the American Mosquito Control Association, 4, 549–550 PMID: 3225576. Wanaratana, S., Amonsin, A., Chaisingh, A., Panyim, S., Sasipreeyajan, J., & Pakpinyo, S. (2013). Experimental assessment of houseflies as vectors in avian influenza subtype H5N1 transmission in chickens. Avian Diseases, 57, 266–272 https://doi.org/10.1637/10347-090412-Reg.1. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Basic and Applied Zoology Springer Journals

Biocontrol efficacy of bay essential oil against housefly, Musca domestica (Diptera: Muscidae)

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10.1186/s41936-020-0138-7
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Abstract

Background: The synanthropic housefly, Musca domestica, augments the transmission of several detrimental diseases like cholera and avian flu. Consequently, during the last century, many physico-chemical methods including synthetic compounds have been applied for its control. But these methods have proven to be prohibitive due to their side effects and serious issues like resistance development, environmental contamination, and detrimental effects on non-target fauna. Therefore, in view of these objectives, we investigated the effects of bay essential oil (EO) against M. domestica. Methods: The attractant/repellent assays were conducted by double choice technique. Different enzyme assays evaluating the effect of LC concentration of the tested essential oil on larval gut were taken into consideration. To determine the composition, the tested oil was subjected to GC-MS/MS analysis. Further, the morphological alterations caused by EO treatment to third instar larvae were observed in a Nova Nano SEM machine. Data was statistically analyzed by one-way ANOVA using Tukey’stest (p < 0.001). The LC and LC values were calculated by probit 50 90 analysis. Results: The adulticidal bioassay revealed significant effects with LC concentration as 43.03 mg/dm against the newly emerged adult flies while in larvicidal assay mortality was dose dependent showing maximum effect at LC 0.0629 μg/cm . The pupicidal activity was more effective at a dose of LD 64.09 μl/0.25 L of air which either killed the pupae or caused deformity in the emerged adults. Likewise total sugar, protein, glycogen, and lipid contents of larvae were reduced after treatment with EO when compared with the normal larvae along with some gut enzymes. The EO reduced the acetylcholinesterase activity from 0.013 U/mg protein in normal larvae to 0.0093 U/mg protein after EO treatment. The GC-MS/MS analysis of the bay EO showed the abundance of myrcene, linalool, eugenol, chavicol, and anethole along with diterpenoid, geranylgeraniol. However, the insecticidal activity of tested EO might be majorly imparted by eugenol content. The FESEM analysis showed shrinkage of integument and distortion to intersegmental regions caused by the tested compound. Conclusion: The present study concludes the significant efficacy of bay EO against M. domestica which could be employed to breakdown its population below threshold levels to prevent the menace of vector-borne diseases. Keywords: Biocontrol, Essential oil, Laurus nobilis, Musca domestica,FESEM, GC-MS * Correspondence: mudasir.dar@unipune.ac.in; panditrao499@gmail.com Department of Zoology, Savitribai Phule Pune University, Pune, Maharashtra 411007, India © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Chintalchere et al. The Journal of Basic and Applied Zoology (2020) 81:6 Page 2 of 12 Background Materials and methodology The housefly, Musca domestica (Diptera: Muscidae), is a Chemicals and reagents well-known pest of livestock and human health import- The EO of the Laurus nobilis was supplied by Sigma Al- ance. It constitutes a worldwide problem wherever poor drich, USA. The oil doses were prepared freshly by dilu- sanitation and bad hygienic conditions exist (Khan et al., tion in acetone solvent and immediately used for tests. 2013). Moreover, the biology and ecology of Musca All other chemicals/reagents used in this study were of domestica makes it an ideal organism to carry and dis- highest purity and molecular or analytical grade unless seminate human and animal pathogens, such as hel- defined. minth parasites, protozoan cysts, viruses, and bacteria (Fotedar et al., 1992; Greenberg, 1973; Kobayashi et al., Rearing of housefly colony 1999). Recently, it has been inculpated to transmit avian The laboratory-reared colonies of the model organism, flu (Wanaratana et al., 2013) also. Therefore, hitherto, a i.e., M. domestica, were generously provided by the En- large variety of synthetic compounds such as DDT and tomology Section, National Chemical Laboratory (NCL), cyromazine have been used against this vector to prevent Pune (M.S.), India, which were free from insecticides the epidemics. However, indiscriminate use of these and pathogens. The culture of housefly was maintained chemical insecticides has resulted in many serious prob- in vitro at a temperature of 28 ± 2 °C and 60–70% rela- lems like insect resistance, persistence of chemicals in tive humidity (RH) in plastic jars (35 × 15 cm), covered the environment, and biomagnifications through trophic with cheese cloth for several generations. A cotton swab levels leading to detrimental effects on human beings soaked in milk (10% w/v) was offered as a food to adult (Tabashnik & Johnson, 1999). Therefore, researchers are flies which also served as a substratum for oviposition. continuously prospecting for active natural products of The eggs were transferred to another set of jars contain- plant origin as potential alternatives to conventional ing animal feed and water or cotton swab soaked in milk insecticides. for hatching and larval development. Similarly, pupae The eco-toxicological property of plants such as lower were collected and kept in another container for adult toxicity to humans, cheap, easy cultivation and degrad- emergence. All stages of the fly such as eggs, larvae, ation along with reduced environmental impact makes pupae, and adults were continuously available for the them promising candidates for the management of in- experiments. sect pests (Kumar et al., 2011). Additionally, aromatic plants have proved effective insecticides and their essen- Larvicidal and adulticidal bioassays tial oils (EOs) often constitute the bioactive fraction The larvicidal and adulticidal bioassays were carried out (Regnault-Roger, 1997). Plant metabolites act by either by following the methods of Busvine (Busvine, 1971) and exerting their effect on octopaminergic nervous system Palacios et al. (2009), respectively, with few modifica- of insect pests (El-Zayyat et al., 2017) or interfering with tions. To determine the effect of EO, 10 individuals of GABA-gated chloride channels (Khater, 2012). Further the third instar larvae were exposed to different concen- effects can be seen in behavioral modifications (attrac- trations of EO in each test. Primarily, a range of desir- tion/repellency) and contact toxicity for different life able doses were tested to determine the LC and LC 50 90 stages of the targeted organisms. Similarly, some natural values. For residual film method, 1 ml each of different oils are complexes of many biologically active constitu- concentrations of EOs was applied on filter paper discs ents including terpenes, acyclic monoterpene alcohols, kept inside the glass petri dish of 90 mm diameter. The monocyclic alcohols, aliphatic aldehydes, aromatic phe- EO doses were applied uniformly in order to make a uni- nols, monocyclic ketones, bicyclic monoterpenic ke- form film over the filter paper. Initially, the treated petri tones, acids, and esters (Koul, 2008). dishes were air dried for few minutes to allow the solvent In the past few years, there is a strong impetus to develop evaporation, followed by release of larvae (n = 10) and EOs of botanical origin as potential agents for pest control then incubating the plates under laboratory conditions for strategies. Since aromatic angiosperm, Laurus nobilis L., is 24 h. widely exploited for treatment of gastrointestinal disorders In case of adulticidal assay, 10 adult flies were placed (Lorenzi & Matos, 2008), the aqueous extracts of this plant in plastic jars (1.2 dm ) containing a 7-cm-long cotton are also used for the treatment of many open wounds from yarn suspended from the cap of the jar. Different dos- ancient times (Nayak et al., 2006). In view of these proper- ages of EO ranging from 10 to 100 mg/dm were used ties, the present study aimed to determine the chemical for the tests. Each dose was applied after being dissolved composition of Laural nobilis essential oil and evaluate its in 10 μl of acetone then applied to a cotton yarn. In the efficiency against Musca domestica.Further,weinvesti- control jar, the cotton yarn was treated with 10 μlof gated the larvicidal effect and oviposition deterrent activities acetone only. The jars were then sealed tightly and kept posedbythisoil to thedifferent lifestagesof the fly. at 28 ± 2 °C for 30 min. All tests were replicated five Chintalchere et al. The Journal of Basic and Applied Zoology (2020) 81:6 Page 3 of 12 times with acetone solution being used as a control The percent inhibition rate (% IR) on pupal emergence treatment. The mortality rate of the treated larvae and was calculated by using the below given formula no. 4. flies was calculated after 24 h of treatment by using below mentioned formula 1. Where X denotes the per- %inhibition rate ¼ Cn−Tn=Cn  100 ð4Þ centage of larvae that survived in the control test and Y represents the percentage survival of larvae in the tested doses. where Cn represents the number of newly emerged in- sects in the control set and Tn depicts the number of in- Mortalityð%Þ¼½X−Y =X100 ð1Þ sects emerged after treatment. The data were pooled and analyzed by standard probit analysis to obtain LC and LC values. However, the 50 90 Effect of essential oil on biochemical aspects of housefly actual dose of EO present in 1 ml mixture was calculated larvae by using the formula 2. In larvicidal bioassays, 10 individuals of third instar larvae Dose=cm ¼ value present in 1 ml=area of petridish of the fly were exposed to LC concentrations of essential oil for 24 h. After the treatment, the exposed larvae were ð2Þ collected and subjected to biochemical analyses such as determination of total sugars, glycogen, lipids, and pro- Attractant/repellant bioassays teins by following the Anthrone method specified by The attractant and repellant bioassays were carried out Plummer (Plummer, 1988). However, proteins and lipids by using the double choice method (Campbell, 1983) were estimated by the methods of Bradford (Bradford, where 20 newly emerged adults of mixed sexes were re- 1976) and Van Handel and Day (Van Handel & Day, leased in a cage (size 20 × 12 × 8 cm) with two conical 1988), respectively. Results obtained were analyzed with flasks. One flask contained 0.1% test oil in 5 ml of milk, one-way ANOVA, followed by Tukey’s test (mean com- while the other contained acetone as a solvent and 5 ml parison) using GraphPad software version 3.6. of milk to serve as control. The conical flasks were fitted with funnel (10 cm in diameter) to avoid the escape of the flies. The assays were replicated five times, and the Enzyme assays numbers of flies attracted towards the tested oil flask Different enzyme assays were carried out by treating the and in the control flask were counted after 24 h to deter- larvae with LC concentration approximately as de- mine the percentage of repellency. The results were scribed in the larvicidal bioassay. The whole guts of the expressed in terms of percentage (%) attraction whereas treated larvae were suspended in lysis buffer and homog- percentage repellency (% R) was calculated by the fol- enized on ice using a glass homogenizer. The homogen- lowing formula. ate was centrifuged at 10,000 rpm for 10 min at 4 °C. The supernatant obtained was treated as enzyme extract %R ¼½ 100ðÞ C  T =C ð3Þ and used for different enzyme assays. The α-amylase ac- where C is the number of flies trapped in the control tivity was determined by dinitrosalicylic acid (DNSA) flask and T indicates the number of flies trapped in the procedure described by (Bigham et al. 2010), using 1% treated flask. (w/v) starch as substrate. Briefly, 20 μl of the enzyme ex- tract was mixed with 50 μl of PBS (pH 7.4) and 25 μlof Pupicidal bioassay 1% starch solution (w/v). The mixture was incubated at Pupicidal bioassays were carried out by following the 35 °C for 30 min to allow the reaction to occur which method described by Kumar et al. (Kumar et al., 2011) was subsequently terminated by adding 85 μl DNSA re- with slight modifications. The bioassays were performed agent. The terminated reactions were heated in a boiling with 20 pupae (3 days old) in a 250-ml conical flask. The water bath for 10 min. Similarly, α- and β-glucosidase pupae were exposed to different doses in the range of activities were determined by measuring the amount of 20 μl/0.25 l to 100 μl/0.25 l of air to assess the effect of p-nitrophenol released from 5 mM p-nitrophenyl–α–D- fumigation through impregnation on a cotton swab. In glucopyranoside and p-nitrophenyl–β–D-glucopyrano- control treatment, the pupae were exposed to acetone side, respectively. Here the assay mixtures were incu- only. These pupicidal assays were performed at 28 ± bated at 25 °C for 30 min followed by termination with 2 °C and RH 65 ± 5%. The observation was recorded till 100 μl of NaOH (0.1 M) solution. The absorbance was th the 6 day after exposure to EOs during which emer- read spectrophotometrically at 540 nm, and the reducing gence into adults was documented. These assays were sugars released were estimated by using glucose as carried out with five or more independent replicates. standard. Chintalchere et al. The Journal of Basic and Applied Zoology (2020) 81:6 Page 4 of 12 Inhibition of acetylcholinesterase (AChE) activity drying (CPD) in hexamethylenedisilazane (HMDS). The To determine the acetylcholinesterase (AChE) activ- specimens were mildly coated with gold (100 Å) followed ity caused by the EO treatment, the sample preparation by observation at a voltage of 5.0 kV in a FEI NOVA was carried out as described above except in place of the Nano SEM (NPEP303, FEI USA). whole gut only head regions were taken for inhibition ana- lysis. The activity was assayed by the modified method of Statistical analysis Ellman and colleagues (Ellman et al., 1961). Briefly, 10 μl Data obtained was subjected to statistical analysis. Re- of enzyme extract was mixed with 10 μl deionized water, sults were reported as mean ± standard error means incubated at room temperature for 15 min followed by (SEM) of five or more independent replicates which addition of 20 mM acetylthiocholine iodide and 10 μlof were subjected to one-way ANOVA using Tukey’s test 0.5 M DTNB [5,5′-dithiobis (2-nitrobenzoic acid)]. The (p < 0.001). The LC and LC values with their 95% 50 90 control was treated by taking acetone in place of the en- confidence limits were determined by probit analysis zyme extract. The absorbances were recorded at 412 nm, which helped to analyze the dose–mortality response and enzyme activity was measured in U/min/mg of (Finney, 1952). Analysis of data was carried out in SPSS protein. software version 19 as well as Microsoft Excel (Microsoft office suite, 2013). Gas chromatography-mass spectrometric (GC-MS) analysis of essential oil Results The chemical composition of tested EO was analyzed by Larvicidal and adulticidal efficacy gas chromatography-mass spectrometric (GC-MS) The larvicidal assay carried out by the residual film method. The liquid phase used in the chromatographic method showed that LC concentration of the bay EO procedures helps to characterize the oil constituents re- shows higher larval mortality at 4 mg/ml with an actual sponsible for the insecticidal property of the oils. For dose 0.0629 μg/cm which is a very low concentration. GC-MS, analysis of bay EO was carried out by the The results observed with the larvicidal assay are method of Peris and Blazquez (Peris & Blázquez, 2015). depicted in Table 1. However, the LC concentration of The column temperature in the program was 60 °C dur- the EO was observed to be 9.231 mg/ml with an actual ing 5 min, with 6 °C/min increase up to 180 °C, followed dose of 0.144 μg/cm (Fig. 1a). As the dose concentra- by a 20 °C/min increase up to 280 °C, which was main- tion of EO increases, the percent (%) mortality of the tained for 10 min. The carrier gas used was helium at a treated larvae also increased. The efficacy of the tested flow rate of 1 ml/min and a split ratio of 1:30. The ana- oil was very significant when compared with the control lysis was performed using a GCMS-TQ8030 apparatus, tests indicating short-term exposure of larvae to lethal equipped with RTX 5MS column. Finally, the compo- doses can markedly increase their mortality over time, nent identification was elucidated based on the compari- thereby reducing the number of viable adults, leading to son of their relative retention time and mass spectra possible significant diminution in overall population of analysis with those of NIST11 library data (GC-MS sys- M. domestica. The effect of tested doses of the EO on tem), and available literature. adult insects showed significant mortality (Fig. 1b) vary- ing with the dose range. However, the LC and LC 50 90 Field emission scanning electron microscopy (FESEM) concentrations for adult mortality by bay EO were found 3 3 In order to have a clear and discernible observation of to be 43.03 mg/dm and 84.42 mg/dm , respectively the effects of oil treatment on larval body, 10 larvae of (Table 1). the third instar stage were treated with LC concentra- tion of tested EO. The sacrificed (treated) larvae and Attractant/repellant bioassay control larvae were prepared for FESEM visualization by The EOs derived from plants are volatile, natural, and using the protocol of Kumar et al. (Kumar et al., 2011) complex organic compounds characterized by a strong with few modifications to compare the structural odor formed as secondary metabolites. The repellency changes caused by the oil treatment. The specimens potential of the EO from L. nobilis was evaluated against were primarily fixed in 2.5% glutaraldehyde (v/v) in dis- housefly in glass chambers. In this assay, EO showed tilled water for 15–17 h. Thereafter, sample specimens 27.58% repellency at a concentration of 0.1% (Table 2). were washed with distilled water for 20 min, followed by Among the tested essential oil concentrations, 0.5% was fixation in 4% osmium tetroxide for about 2 h. The sam- found to be the most effective with 100% repellency ples were washed with deionized water for 20 min and whereas no repellency was observed on a dose of 0.1% then dehydrated serially with different grades (30, 50, 70, after 1 h of continuous observations. The repellency po- 90, and 100% v/v) of alcohol each for 10 min. After de- tential of EO doses followed the pattern 0.1% > 0.5% > hydration, samples were subjected to critical point 1.0%. The tested laurel oil elicited a strong and abrupt Chintalchere et al. The Journal of Basic and Applied Zoology (2020) 81:6 Page 5 of 12 Table 1 Larvicidal and adulticidal activities of bay essential oil on M. domestica # # Essential LC (mg/ml) 95% confidential limit (LC ) Regression LC (mg/ml) Chi 50 50 90 3 ## 3 ## oil (mg/dm ) equation (mg/dm ) square LCL UCL Larvicidal 4 3.48 4.45 y = 0.245 − 0.98x 9.231 4.37*(5) Adulticidal 43.03 34.833 50.866 Y = 0.31 − 1.33X 84.42 8.051*(5) Each value represents the mean of five replicates. LC and LC are lethal concentrations at which 50% and 90% population dies, respectively. *Significant at p < 0.001. 50 90 # ## Unit used for larval mortality; Unit used for adulticidal assay. The mortality rate of larvae or adult flies in control sets was negligible when compared with the treatment assays effect against the adults of M. domestica at concentra- content of the treated larvae as compared with the con- tions higher than 0.5% indicating 0.5% sufficient for trol set. Further, the biochemical assays revealed that 100% mortality of the flies. bay EO showed notable changes in total sugar, glycogen, lipid, and protein contents of third instars of M. domes- Pupicidal bioassays tica (Table 3). The tested oil remarkably reduced the The pupicidal assay carried out by fumigation and con- total sugar content from 3.169 ± 0.191 μg/larva in the tact toxicity protocols against the tested organism exhib- control to 0.448 ± 0.036 μg/larva in the treated larva. ited significant variation in growth inhibition with Not only sugars, the treatment also reduced total glyco- different doses of the bay EO (Table 2). The results of gen content, i.e., 0.117 ± 0.007 μg/larva after treatment the fumigation assay revealed that a LD dose of bay comparable to normal larva (7.302 ± 0.131 μg/larva). A EO for the pupal death was 64.09 μl/0.25 l of air (Fig. 2) similar trend was observed with lipid content of the in- whereas LD concentration was found to be over sect where its amount was decreased from 1400.2 ± 104.35 μl/0.25 l of air. In case of higher doses, a complete 38.87 μg/larva in normal individual to 474.62 ± 57.35 μg/ inhibition of pupal emergence was noted (Additional file larva after treatment. In case of total protein content of 1: Figure S1). Further, the increase in the dose concen- the treated and control larvae, the proteins were signifi- tration caused increased percent inhibition rate (PIR) cantly reduced from 1000.6 ± 11.47 μg/larva to 636.72 ± which eventually lead to the deformities in adults in case 26.98 μg/larva due to EO treatment. of survived pupae. Similarly, in contact toxicity, we found that not a single pupa can transform into adult Effect of EO on gut enzymes successfully at all tested doses indicating 100% mortality Since digestive tract is the main interface between insect of the pupae. and the environment, given that nutrition is a decisive factor in the evolutionary process of these organisms, Effect of EO on biochemistry of housefly the main aspect of the pest control is the selective inhib- The secondary metabolites of the plant origin cause a di- ition of digestive enzymes secreted by the alimentary verse effect on the metabolism of the insects. Therefore, canal of pests. Keeping that in mind, we checked the ef- we tested the effect of different concentrations of the fect of tested EO on gut enzymes such as α-amylase, bay EO on the larvae of housefly. The inferences of the α and β-glucosidases. There was a significant difference present study elucidate that LC dose of the bay EO in- (Fig. 3a–c) in enzyme activities between treated and con- duced marked reduction in the level of total protein trol larvae in case of both the enzymes under Fig. 1 Bioefficacy of bay essential oil against M. domestica. a Percent mortality of larvae after a treatment of 24 h. b Adulticidal activity of the tested EO against newly emerged M. domestica after 24-h treatment. LC and LC are lethal concentrations at which 50% and 90% population 50 90 dies, respectively. Each value represents the mean of five replicates. Asterisk indicates significance at p < 0.05 Chintalchere et al. The Journal of Basic and Applied Zoology (2020) 81:6 Page 6 of 12 Table 2 Percent (%) inhibition repellency of the adult houseflies by different doses of the EO using the fumigation method Dose (μl/ % LD (μl/ 95% confidential limit (LC ) Regression LD (μl/ Chi 50 50 90 0.25 l of air) IR 0.25 l of air) equation 0.25 l of air) square LCL UCL 20 14 40 22 60 44 64.09 55.79 73.58 Y = 0.032x − 2.040 104.35 9.44*(5) 80 68 100 88 Each value represents the mean of five replicates. LC and LC are lethal concentrations at which 50% and 90% population dies, respectively. df degree of 50 90 freedom. *Significant at p < 0.001. Negligible repellency or inhibition rate was observed for the control set consideration showing a p < 0.001 only. The bay essen- GC-MS analysis of essential oil tial oil significantly decreased α-amylase activity (0.092 ± The GC-MS analysis of the bay EO revealed an abun- 0.002 μg/larva) when compared with control test (0.214 dance of seven major components (Fig. 4). The compo- ± 0.004 μg/larva). We observed a substantial decrease of nents observed (Table 4) were terpenes such as 57.1% in the activity of α-amylase indicating the effect of myrcene, linalool (3, 7-dimethyl-1, 6-octadien-3-ol), and bay EO on M. domestica. Similarly, the effect of EO on diterpenoids (E,E,E)-3,7,11,15-tetramethylhexadeca-1,3,6. α-glucosidase showed considerable reduction (1.878 ± Some of the phenolic compounds observed were eu- 0.043 μg/larva) in its secretion by fly larvae as compared genol, chavicol (phenol,4- (2-propenyl)) and anethole. with control set (l2.375 ± 0.074 μg/larva) marking a dif- The highest amount found was of the eugenol (58.95%) ference of 21%. It also showed 1.3-fold reduction in β- which proved to be the main component followed by glucosidase activity from 3.066 ± 0.071 μg/larva to 2.19 phenol (13.96%), tetra methyl hexadeca (10.57%), and β- ± 0.024 μg/larva after treatment with the EO (Additional myrcene (6.03%). The insecticidal property of this plant file 1: Table S1). The tested EO significantly affected the EO could be attributed majorly to eugenol content and α-amylase of the gut extract upon treatment showing a to some extent linalool compound. However, the com- decrease of over 51% activity (Fig. 3a). pounds like β-myrcene, eugenol, and linalool comprise only about 3% of the total chemical compounds present in this plant. Acetylcholinesterase (AchE) activity As shown in Additional file 1: Figure S2, the bay EO was found to inhibit the AchE activity of M. domestica larvae Field emission scanning electron microscopy (FESEM) after 24 h of treatment. The AchE activity in treated lar- In order to clearly visualize the effect of EO treatment on vae was 0.0093 U/mg of protein compared with control the larval body surface, we employed FESEM technique to larvae where it was much higher (0.013 U/mg of protein) compare the ultra-structural changes in larval integument indicating significant reductions caused by the exposure due to EO treatment with normal larvae. Upon analysis, a to EO. clear observation showed the toxic effect of bay oil in the form of integument shrinkage particularly at spinose rings with distorted intersegmental regions. However, control/ normal larvae of M. domestica showed normal appearance with a smooth textured integument and well-shaped inter- segment spines (Fig. 5a–c). Therefore, it can be concluded Table 3 Effect of bay EO on nutritional reserves of M. domestica larvae Sr. no. Analysis Control larvae (μg/larva) Treated larvae (μg/larva) 1 Sugar 3.169 ± 0.191 0.448 ± 0.036* 2 Glycogen 7.302 ± 0.131 0.117 ± 0.007* 3 Lipid 1400.2 ± 38.87 474.62 ± 57.35* Fig. 2 Pupicidal assay showing percent (%) mortality of M. domestica 4 Protein 1000.6 ± 11.47 636.72 ± 26.98* larvae by fumigation method with bay EO. Significant *p < 0.001 Values are mean ± SE (standard error). Data were analyzed by one-way when compared with the control. Each value represents the mean ANOVA, followed by Tukey’s test and significance at *p < 0.001 compared with of five replicates the control Chintalchere et al. The Journal of Basic and Applied Zoology (2020) 81:6 Page 7 of 12 Fig. 3 Effect of bay essential oil on the gut enzymes of M. domestica larvae. a α-Amylase, b α-glucosidase, and c β-glucosidase activities of M. domestica. Values are means ± SE (standard error). Data analyzed by one-way ANOVA, followed by Tukey’s test and significance at *p < 0.001 compared with the control from the observed results that fly larvae upon contact with quite higher than the concentration used in this study. EO show superficial alterations to the integument. Similarly, Asid and co-workers stated that Citrullus colo- cynthis extracts exhibit potential efficacy against all Discussion stages of M. domestica at 50% concentration (Asid et al., Pesticides derived from plants are organic in nature, 2015). Previously, many authors have reported the anti- considered to be biodegradable, non-mutagenic or non- bacterial (Evrendilek, 2015), antifungal (Gumus et al., toxic to animals, and more particularly target specific 2010), antioxidant (Inan et al., 2012), analgesic and anti- than their chemically synthesized counterparts. Some of inflammatory properties (Sayyah et al., 2003), apart from them also inhibit or reduce the insecticide detoxifying some acaricidal (Senfi et al., 2014), larvicidal (Pavela, enzymes of the pests thereby could be important from 2008), and insecticidal (Sertkaya et al., 2010) activities by the agriculture viewpoint (Wang et al.,2016). Therefore, laurel EO. keeping in mind the importance of plant-based EOs, we In order to ascertain the potential of bioactive com- tested the efficacy of EO from L. nobilis against the dif- pounds present in the bay EO, we used the GC-MS ana- ferent stages of the nuisance-causing housefly, M. lysis that revealed dominance of eugenol and phenolic domestica. We observed higher larval mortalities at very content which was in congruence with previous reports low doses of the compound which was only 0.0629 μg/ that have stated the chemical composition of the EO cm . The efficacy of Laurus EO against M. domestica as from the leaves of L. nobilis (Senfi et al., 2014; Sertkaya observed in this study is much higher than the insecti- et al., 2010). Further insecticidal property of many com- cidal property of EOs derived from various other plants pounds present in EO has been conducted topically by (Pavela, 2008). Recently, the efficiency of the EOs from Rozman and co-workers (2007) wherein eugenol was L. nobilis has been tested on several other insects such found highly toxic to all tested beetles. Eugenol is also as stored grain pests, where authors have suggested known to show higher repellency against beetles (Andro- species-specific dose requirements for the control of nikashvili & Reichmuth, 2002; Huang et al., 2002; Roz- pests (Drapeau et al., 2009; Pavela, 2008; Sertkaya et al., man et al., 2007) and other stored grain pests 2010). Ours is the pioneering study to report the effects (Cosimi et al., 2009; Lee et al., 2003). Further, these au- of L. nobilis EO on the adults of M. domestica. Recently, thors concluded the complete destruction of the eggs Pavela and his colleagues (Pavela, 2008) tested more and immature stages of T. castaneum by eugenol. Our than 30 plant extracts against M. domestica and con- results were in line with Nehir et al. (2014) who found cluded that EO from Pogostemon cablin is the most effi- eugenol; 1,8-cineole; linalool; methyl eugenol; α-terpinyl cient against fly at a concentration of 3 μg/fly which is acetate; α-pinene; and β-pinene as major components of Fig. 4 Chromatogram obtained from GC-MS/MS analysis shows the chemical composition and abundance of some compounds of the bay EO Chintalchere et al. The Journal of Basic and Applied Zoology (2020) 81:6 Page 8 of 12 Table 4 GC-MS analysis of bay essential oil revealing the abundance of certain compounds Peak R. time Area Area % Height Height % Name 1 9.048 8,758,593 6.03 2,457,962 10.47 Beta myrcene 2 12.304 5,117,500 3.52 1,400,514 5.97 1,6-Octadien-3-ol,3,7-dimethyl 3 16.540 20,281,918 13.96 2,876,306 12.26 Phenol,4-(2-propenyl) 4 19.233 85,635,465 58.95 6,319,156 26.93 Eugenol 5 29.826 15,347,881 10.57 5,460,173 23.27 (E,E,E)-3,7,11,15-Tetramethylhexadeca-1,3,6 6 30.114 5,874,016 4.04 3,065,184 13.06 (E,E,E)-3,7,11,15-Tetramethylhexadeca-1,3,6 7 33.403 4,246,485 2.92 1,887,660 8.04 Anethole laurel EO. Most of the terpenoids and phenols of plant monoterpenes alter the physiological functions by competi- origin have minimal vertebrate toxicity and are regarded tive inhibition of acetylcholine esterase in houseflies and as safe by United States Food and Drug Administration cockroaches (Grundy & Still, 1985; Ryan & Byrne, 1988). (USFDA). These compounds apparently cause hyperactiv- The toxic effects exerted by EO of L. nobilis could be attrib- ity, tremors, and convulsions followed by knockdown of uted to the toxic constituent like eugenol, linalool, myrcene pests similar to the symptoms by insecticides such as or- and other phenolic compounds present therein. Besides the ganophosphates and carbamates. However, previously simi- fumigant and repellent activities of bay EO against M. lar signs have been described by insects upon exposure to domestica, it has also proven insecticidal against red flour pure monoterpenes (Coats et al., 1991). Some beetle, T. castaneum (Cosimi et al., 2009; Lee et al., 2003). Fig. 5 The ultra-structural and superficial changes induced by bay EO to the larvae of M. domestica after a treatment for 24 h. a, c, and e show the anterior, posterior, and abdominal segments respectively of the control larva exhibiting normal appearance with smooth integument and well-defined or swollen intersegment spines, whereas b, d, and f show the FESEM micrograph anterior, posterior, and abdominal segments depicting the ultra-structural changes like shrinkage, rupture of body fluids, and corrugation of the integument induced by bay EO during treatment Chintalchere et al. The Journal of Basic and Applied Zoology (2020) 81:6 Page 9 of 12 Further, monoterpenoids abundantly distributed in EOs of whereas β-glucosidases catalyze the hydrolysis of β- aromatic plants are lipophilic in nature thereby inter- glycosides into respective monosaccharides (Sezginturk vene with metabolic, biochemical, physiological, or be- & Dinckaya, 2008). Many insects are known to show β- havioral functions of insects (Brattsten et al., 1983). glucosidase activities required for metabolism of Similar to our study, Palacios et al. (2009b) examined ingested food such as Diatraea saccharalis (Azevedo et al. the efficacy of EOs from several medicinal and edible 2003) and Parnassiusapollos sp. (Nakonieczny, Michalc- plants against housefly and stated that orange peel and zyk, & Kedziorski, 2006). The tested EO depicted neuro- eucalyptus leaves are the most toxic to flies which had toxic effects by inhibiting AchE activity of the M. limonene (92.5%) and 1,8-cineole (56.9%), as the princi- domestica larvae after 24 h of treatment with LC doses pal compounds. However, Kumar and co-workers only. Many secondary metabolites such as EOs and (Kumar et al., 2011) have observed that EOs of Mentha monoterpenes of aromatic plants are known to inhibit piperita and Eucalyptus globules are the most effective the AchE activity of insects (Senthilm et al., 2008). plant extracts against M. domestica showing both Our observations were in accordance with Rajashekar repellent and insecticidal properties. From time imme- and co-workers (2014) who concluded the inhibition morial, plant-derived substances have been used to repel of AchE in M. domestica by Coumaran, extracted or kill mosquitoes before the advent of synthetic chemi- from L. camara. Therefore, for designing a biocontrol cals (Curtis et al., 1990). The tested EO also showed strategy, it becomes imperative to understand the higher repellency against adult flies preventing them interaction of pesticide with digestive enzymes and from oviposition in the nearby vicinity which is also AchE of thetargetorganism. Hencewedetermined stated by other researchers against rust-red flour beetle the effect of EO on gut enzymes of M. domestica Tribolium castaneum (Andronikashvili & Reichmuth, where drastic changes in expression levels were ob- 2002), Sitophilus zeamais, Cryptolestes ferrugineus, served. The treatment with EO reduced the activities Tenebriomolitor (Cosimi et al., 2009), and adult females of the α-amylase and α-and β-glucosidases by 57.1, of Culex pipiens (Erler et al.2006). The repellency of 21, and over 30%, respectively, in treated larvae as these compounds appears due to the presence of volatile compared with larvae without treatment (control). monoterpenoids (Buescher et al., 1982; Curtis et al., The reduction in enzyme activity could be a conse- 1990;Rutledge et al.,1983). Besides, Papachristos and Stam- quence of the cytotoxic effect of plant compounds on poulos (2002) showed that EO from bay plant presented a epithelial cells of the gut that are responsible for the syn- repellent activity against Acanthoscelides obtectus. thesis of α-amylase (Hichri et al.t, 2019). Cytotoxicity ap- The pupicidal effect exerted by the tested EO was also pears to include membrane damage by causing significant for the bio control of housefly. Besides vari- coagulation of the cytoplasm (Gustafson et al., 1998) ous fumigant and repellent activities of bay leaf EO thereby damaging its lipid and protein contents against stored grain pest (Cosimi et al., 2009; Lee et al., (Ultee et al., 2000) or disrupting cell membrane leading 2003), there are few reports available about the insecti- to leakage of macro molecules and ultimately cell lysis cidal potential of bay leaf oil and its fractions against T. (Oussalah et al., 2007). Some plant EOs are also known castaneum. In the present study, significant mortality or to form stable complexes with digestive enzymes, mak- inhibition of pupal emergence was observed by both ing dissociation difficult. In case of biochemical con- contact and fumigation pupicidal bioassays. In contrast, tents, decreased levels of proteins, lipids, and glycogen Kumar et al. (2011) observed 100% mortality of the same were observed due to treatment of EO which could be insect by contact toxicity method only. primarily due to blocking of gut hydrolases, which leads Since most insects feed on carbohydrate-rich food to poor nutrient utilization, retarded growth, and conse- products such as seed, fruits, crops, and stored grains, quent death by starvation (Jongsma & Bolter, 1997). their guts are efficient natural biochemical reactors se- However, the reduction in body protein content may be creting a suite of hydrolytic enzymes such as amylase attributed to inhibition of DNA and RNA synthesis at and α- and β-glucosidases. These enzymes hydrolyze α- molecular levels which then reflects the decrease in en- D-1, 4-glucan linkages of starch, glycogen, and some zymatic activities. Our results were in congruence with other carbohydrates (Ferreira & Terra, 1989; Strobl earlier reports (Nathan et al., 2005) where authors have et al., 1998). The amylase enzyme transforms starch into stated similar reduction in protein and metabolite con- maltose, which is then converted to glucose by α- tents in moth, Cnaphalocrocis medinalis, after treatment glucosidase and thus utilized as an energy source by the with plant compounds. The changes in total lipid con- insect. In insects, only α-amylases have been found to tents were also caused by the treatment of EOs. Some hydrolyze long α-1, 4-glucan chains of starch or glyco- authors have suggested that decreased lipid content may gen (Terra et al., 1988). Likewise, α-glucosidases break be a result of transformation of lipids into proteins to non-reducing 1, 4-linked α-D-glucose ends of glycogen substitute the reduction in protein content or produce Chintalchere et al. The Journal of Basic and Applied Zoology (2020) 81:6 Page 10 of 12 supplementary energy to combat the chemical stress. gut/digestive enzymes of the M. domestica larvae. Value are mean ± SE Hence, treatment with EOs might have interrupted this (Standard error). Data were analyzed by One-Way ANOVA, followed by Tukey’s test and Significance at *p < 0.001 compared with control. process and resulted in larval mortality. The biochemical and metabolic disruption by the EO Abbreviations treatment was also superficially revealed by the FESEM AChE: Acetylcholinesterase; ANOVA: Analysis of variance; CPD: Critical point analysis where a complete distortion of the body was ob- drying; DDT: Dichlorodiphenyl trichloroethane; DNSA: 3,5-Dinitrosalicylic acid; served. Our inferences were supported by the conclusion DTNB: 5,5′-Dithiobis, 2-nitrobenzoic acid; EO: Essential oil; FESEM: Field emission scanning electron microscopy; GABA: Gamma amino butyric acid; of Insun et al. (1999) who demonstrated the damage to GC-MS/MS: Gas chromatography-mass spectrometry/mass spectrometry; the surface morphology of mosquito larvae by Kaemp- HMDS: Hexamethylenedisilazane; LC: Lethal concentration; LD: Lethal dose; feria galangal extract. Moreover, the effect of monoter- NaOH: Sodium hydroxide; NCL: National chemical laboratory; PBS: Phosphate-buffered saline; PIR: Percent inhibition rate; RH: Relative penes on insect morphology was also investigated by humidity; SEM: Scanning electron microscope; SEM: Standard error means; Sukontason et al. (2004) who studied the structural USFDA: United States Food and Drug Administration changes induced in housefly larvae through SEM ana- Acknowledgements lysis after the application of eucalyptol oil. The authors JC is highly indebted to the authorities of the S.P. Pune University, Pune, reported significant deformation in the integument, inter India, for providing the research stipend. MD is grateful to the University segmental spines, and bleb formation of the treated lar- Grants Commission (UGC), New Delhi, India, for senior research fellowship under the Maulana Azad National fellowship scheme. RSP acknowledges the vae. The FESEM analysis of the treated larvae revealed UPE-II (nanobiotechnology), UoP-BCUD grant (15-SCI-001422), and DRDP and extreme dehydration and surface distortion compared DST-PURSE schemes provided to the laboratory. with the undisturbed, free, and smooth surface of con- Authors’ contributions trol larvae, therefore confiraffirming the effect of tested JC and RSP conceived and designed the experiments. JC and MD performed EO on housefly larvae. The above all results of the pre- the experiments. JC, MD, and RSP wrote the paper. All authors read and sented research indicate that EOs from medicinal plants approved the final manuscript. such as L. nobilis could be a potential alternative for use Funding as housefly fumigants, provoking death of the insect The work was supported by Savitribai Phule Pune University, Pune, under the within a short period of time. project code UoP-BCUD grant (15-SCI-001422), as well as DRDP and DST-PURSE schemes provided to the RSP. Conclusions Availability of data and materials The housefly is a cosmopolitan home invader linked to All the data generated from this study is included in the paper and can be accessed from the corresponding author upon request approval. poor hygiene and low socioeconomic status. The search for new, highly selective, biodegradable insecticides is Ethics approval and consent to participate mandatory to solve the problem of residual toxicity to the Not applicable. environment, fauna or flora. Nature provides many prod- Consent for publication ucts acting as an excellent alternative; among these are the Not applicable. botanical insecticides. With the recent rise of the green in- Competing interests secticide concept and increased public awareness, several The authors declare that they have no competing interests. attempts to use indigenous plants extracts as potential alternatives have been done, but only few plant-based con- Received: 12 June 2019 Accepted: 13 January 2020 trol products appear in the market. This study recom- mends the usage of bay-based products in domestic References botanical anti-fly insecticides, as it is ethnobotanical, en- Andronikashvili, M., & Reichmuth, C. H. (2002). Repellency and toxicity of essential demic, safe, cheap, and available all year. 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