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LARVICIDAL EFFECTS OF MOMORDICA CHARANTIA METHANOL LEAF EXTRACT ON TESSELLATUS

1Reema Sameem , 2 Koshala De Silva 1,2 BMS, School of Science [email protected]

ABSTRACT

Mosquito borne diseases contribute to the major disease burden around the world. The use of botanicals as an alternative to synthetic compounds have been explored due to its multifarious advantages. The present study investigated the larvicidal activity of Momordica charantia methanol leaf extracts against the second instar larvae of the laboratory reared, Anopheles tessellatus. Momordica charantia is a medical herb, belonging to the Cucurbitaceae family. The bioassay test was carried out by using the WHO procedure. The mean percentage mortality of Anopheles tessellatus was shown to increase with increasing concentration and increased time of exposure to the Momordica charantia methanol leaf extract. 100% larval mortality was observed at 48 hours following the exposure to plant extract at the highest concentration of 1000 ppm. The study revealed an LC50 value which decreased with time (251 ppm at 24 hours and 126 ppm at 48 hours). Additionally, behavioural changes during the larvicidal assay were observed in response to tactile stimuli. In order to identify histological changes in the cuticle and mid-gut upon exposure to Momordica charantia plant chemicals, the tissue was processed and sections were stained with Hematoxylin and Eosin. The most commonly observed characteristic changes in treated Anopheles tessellatus larvae include damage to cuticle and shrinkage of cells. Therefore, the present investigation revealed that Momordica charantia methanol extract demonstrated effective larvicidal properties against Anopheles tessellatus larvae which can be attributed to the phytochemicals present in the plant. Hence, the formulation of Momordica charantia methanol extracts may potentially be used as an effective and eco-friendly larvicide, which could be an alternative to malaria control.Keywords: , Anopheles tessellatus, Momordica charantia, larvicidal effect, mortality

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INTRODUCTION Vector control can be achieved by biological, physical and chemical Mosquitoes as vectors of diseases approaches. The strategy to combat these diseases primarily depends on disruption of Vector borne diseases are among the chief the disease transmission cycle by either causes of illness and decease in many targeting the mosquito larvae at breeding developing countries. Mosquitoes (Order: sites through spraying of stagnant water or Diptera) are undoubtedly the most by killing/repelling the adult mosquitoes medicinally significant which using insecticides (Bekele et al., 2014). are vectors for detrimental parasites and pathogens, including malaria, yellow fever, Control measures directed at the larval Japanese encephalitis, dengue, filariasis, stages are more effective especially in areas chikungunya and Zika virus disease where mosquito breeding sites are easily (Sharma et al., 2016). Malaria is a accessible. As the mosquito larvae are debilitating disease associated with high restricted within small aquatic habitats and morbidity and mortality, caused by the due to their low mobility, it is unlikely to Plasmodium parasite. Female mosquitoes escape control measures (Mallick et al., depends on a blood meal of vertebrate hosts 2016). Initially, mosquito control to initiate gametogenesis. These blood significantly relied on the application of meals mediate the interaction between broad-spectrum pesticides. Since the humans and the mosquito hosts in the discovery of the insecticide Dichloro– parasite life cycle (Nyamoita et al., 2013). diphenyl–trichloroethane (DDT), an organochlorine pesticide, mosquito control According to the World Malaria Report, approach has been almost completely based 214 million cases of malaria were reported on synthetic organic insecticides (Musau et globally in 2015 along with and 438 000 al., 2016). malaria deaths (World Malaria Report, 2016). In September 2016, was Nevertheless, the frequent use of chemical declared as a Malaria free country, with less insecticides has led to undesirable side than <0.04% prevalence of the malaria effects including, destabilization of the vector and no reported incidences of ecosystem, bioaccumulation, reduction in Malaria since 2012. In order to maintain biodiversity and unfavourable this status, vector control and surveillance biodegradability (Ollengo et al., 2016). measures have been implemented (Bulletin Moreover, the extensive use of synthetic of the World Health Organization, 2017). insecticides over the last five decades have resulted the development of physiological Methods of mosquito control resistance in vector species (Ali et al., Mosquito control plays a fundamental role, 2013). These factors have necessitated the in preventing the proliferation of mosquito urge for environmentally safe, cost- borne diseases and in improving quality of effective and target specific insecticides public health. Due to the absence of an against mosquito species. Accordingly, a effective vaccine or antiviral therapy, number of studies have emphasized the vector control is the only currently development of natural insecticides for available method to control mosquito borne controlling mosquitoes (Ileke et al., 2015; diseases (El-Sayed and El-Bassiony, 2016). Singh et al., 2014; Varun et al., 2013).

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Recent research have documented the use Momordica charantia and its of natural plant products against mosquito phytochemical constituents vectors (Haq et al., 2016; Kweka et al., 2016; Elumalai et al., 2015; Govindarajan, Momordica charantia Linn (Family: 2011). Although insecticides of plant origin Cucurbitaceae), commonly known as bitter have been extensively used on agricultural gourd/melon and balsam pear, is cultivated pests, a very limited extent has been used in tropical regions, including Asia, against mosquito vectors of public health Caribbean, East Africa and South America importance. Prospection for new larvicidal (Das et al., 2015). Momordica charantia molecules based on plant derivatives are contains suborbicular leaves, with 5-7 deep safer to use due to their innate blade palmate lobes and are variable in biodegradability (Govindarajan et al., their size. Bitter gourd is an important 2012; Kamaraj et al., 2010). functional food crop grown in low lands of Sri Lanka. This monoecious climber and LITERATURE REVIEW scrambling harbaceous vine is locally known as, ‘Karawila’ in Sinhala and Plant based chemicals compounds ‘Pavakka’ in Tamil (Giuliani et al., 2016).

Sri Lanka is a varietal emporium of Traditionally, the fruits, leaves, and roots of endemic and exotic flora enriched with bitter melon were believed to have phytochemicals having pharmacognostical medicinal importance. These medicinal and toxicological properties. The properties are attributed to the presence of phytochemicals derived from plant sources secondary metabolites such as alkaloids, can act as toxicant against the adult as well glycosides, tannins, triterpenes, protein, as larval stages of mosquitoes (Islam et al., flavonoids, saponins, minerals and 2011). Moreover, certain phytochemicals vitamins (Tan et al., 2014). interfere with growth, reproduction or act as a repellent against various species of Over the past few decades collection of mosquitoes (Noutcha et al., 2016). These research have been carried out on bitter plant based botanicals have a variety of melon and these studies have identified, advantages including, multifarious control antidiabetic, antiviral, antitumor, mechanisms against mosquitoes which antileukemic, antibacterial, anthelmintic, decreases the likelihood of developing antimutagenic, antimycobacterial, resistance and is target specific thereby non antioxidant, antiulcer, anti-inflammatory, harmful to humans (Paul and Choudhury, hypocholesterolemic, hypotriglyceridemic, 2016). hypotensive, immunostimulant, and insecticidal properties of Momordica In Sri Lanka, variety of plant species charantia (Subramaniam et al., 2017; including cinnamon, lemon grass, sweet Saengsai et al., 2015; Nagappan and flag, citronella, lemon, cardamom and Gomathinayagam, 2014). ginger have been tested for mosquito larvicidal activity against several species of Momordica charantia as a potential the subgenus Aedes, Culex and Anopheles larvicide (Ranaweera, 1996). The first reported insecticidal activity of Momordica charantia was against the

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Interntional Conference on Health and Medicine mustard saw and mustard aphid, Microscope slides, Aluminium ice box, Lipaphis erysimi (Mishra et al., 2006; Glass vials and Latex gloves. Kumar et al., 1979). More recently, the mosquito larvicidal property of Momordica Instrumentation charantia against three mosquito species of Analytical balance (SP 602 AM), Electrical the subgenus Anopheles stephensi, Culex grinder (LS750 W5), Forced Air Lab oven quinquefasciatus and Aedes aegypti have (DHG-9023A), Fume hood (FH1000), been reported (Kamaraj and Rahuman, Inverted Microscope (TCM 400), Light 2010; Maurya et al., 2009; Rahuman and Microscope (LX 300), Magnetic Venkatesan, 2008; Singh et al., 2006; stirrer (2ML), Magnetic stirrer bar (Q-19- Prabakar and Jebanesan, 2004). A), Paraffin Dispenser (TEC-2000), Rotary The objective of this study was to assess the Microtome (F163851404), Slide Dryer potential larvicidal effects of methanol (SD-2800), Vertical pressurized steam extracts of Momordica charantia against sterilizer (LS-B35L-I), Vortex Mixer (VM- the Anopheles tessellatus larvae. The aim 300), Water bath (WB 2800). of this study was to determine the mean METHODOLOGY percentage mortality of Anopheles tessellatus larvae following the exposure to Collection of plant material methanol extract of Momordica charantia leaf. Furthermore, the focus of this research Mature leaves of Momordica charantia was to identify histological changes of the were collected from a cultivation in organism upon exposure to methanol Colombo, Western Province, Sri Lanka extract of Momordica charantia leaf. (Figure 1). The geographical conditions of the region were 74 mm average MATERIALS precipitation, humidity 88% and mean annual temperature +27° C. Reagents

Methanol, Ethanol, Distilled water, De- chlorinated water, Xylene, 10% neutral buffered formalin, Hematoxylin, Eosin, Acid Alcohol, Canada Balsm, Paraffin wax, Dimethyl sulfoxide (DMSO).

Glassware and Consumables

Measuring cylinder (10ml and 100 ml), Beakers (50 ml and 500 ml), Micro- centrifuge tubes (1.5ml), Staining jars, Reagent bottles (50 ml), Aluminium foil, Whatman No.1 filter paper, Funnel, Petri dishes, Micropipettes (1000 µl), Steel spatula, Pipette tips, Pasteur pipette, Surgical forceps, Tissue paper, Air tight polythene sealer bags, Tissue Cassettes,

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Figure 1. Map of Sri Lanka showing under an inverted microscope (Model- location of sample collection, Colombo TCM 400) with an objective (×10 and x40). Western Province. Palagama, GP Using standard larval keys developed for coordinates 6.7645° N, 79.9654° E. Sri Lankan anophelines, the larvae were identified to the species level REARING OF MOSQUITO LARVAE (Amerasinghe, 1992). Further, larval species identification was reconfirmed The eggs of Anopheles species mosquito through adult identification. were cultured and hatched to larvae at the Department of Parasitology, Faculty of Morphological Species Identification – Medicine, University of Colombo, Sri Adult Mosquito Lanka. These larvae were brought to the BMS laboratory and kept in the container The adult mosquito was preserved in 70% for 2 hours. Thereafter, three larvae (n=3) ethanol and stored in the refrigerator were allocated into containers, each overnight. Thereafter, the mosquito was containing 10 ml of de-chlorinated water. dissected by using surgical forceps and the Physiological parameters of the de- parts were mounted by using Canada chlorinated water including temperature Balsm. Prepared permanent slides were and pH were measured. observed and identified under the light microscope (Model-LX 300). The The containers containing mosquito larvae mosquito was identified to species level by were covered by using a nylon net with using standard illustrated adult mosquito small pore size and allowed to adapt to the keys (Gunathilaka, 2017). environmental conditions for 24 hours, as shown in (Figure 2). During the Preparation of plant extract acclimatization period, the larvae were fed Plant leaves (Figure 3) were initially with brewer’s yeast. The humidity and light washed with tap water and allowed to air conditions were maintained in a dry for five days in a shaded environment homogenized manner. at room temperature.

Figure 2. A part of the experimental set up

Morphological Species Identification - Larvae

Stage II and III larvae were placed individually in petri dishes and observed

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Momordica charantia methanol extract in 100 µl of DMSO. Once the residue was Figure 3. Leaf of Momordica charantia completely mixed, it was dissolved by The dried leaves were powdered adding de-chlorinated water. The stock mechanically using an electric grinder solution was vigorously vortexed to obtain a homogenized mixture. (Model- LS750 W5). The powdered product was sealed in an air tight polythene Larvicidal Bioassay test bag wrapped in aluminium foil and stored in the refrigerator at +4°C prior to Stage II Instar larvae were sorted and extraction. chosen for the assay. Three sets of larval triplicates (n=3) were transferred into the The methanol extract of the plant was containers and treated with different prepared by dissolving 40g of dried powder concentrations of the Momordica charantia in 100 ml of solvent. The mixture was methanol extract (Table 1). The bioassay completely covered in aluminium foil left for the larvicidal activity was carried out by in the dark at room temperature for 24 using the WHO procedure with slight hours with shaking at regular intervals. modifications. Thereafter, the mixture was subjected stirring at 500 rpm for 20 minutes in a The control set was prepared by adding magnetic stirrer (Model-2ML). The 0.1% DMSO. The exposed larvae were solution was filtered using a glass funnel kept in a cool, low light area in the lab for and Whatman no. 1 filter paper. Filtrates 24 hours in an unfed state. All containers were dried in the fume hood. The covered with a net to reduce evaporation of evaporated plant extract was transferred the de-chlorinated water and to facilitate into an airtight container covered in oxygen dissolving. The mortality were aluminium foil and was refrigerated at +4o recorded following 24 hours and 48 hours C until further use. of exposure. Moreover, to analyze the behavioral changes, the response to tactile A 10 mg/ml (1%) stock solution was stimuli were assessed. prepared by dissolving 100 mg of Table 1. Group and treatment for larvicidal bioassay

Treatment Concentration of Momordica charantia (ppm)

C0 [control] 0

C1 25

C2 50

C3 80

C4 100

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Histological Studies statistical software SPSS, in which the significance was considered at a 95% For histological studies, live larvae were confidence level (P<0.05). Furthermore, obtained following 48 hours of exposure to individual mean significant difference was Momordica charantia methanol leaf calculated by using post hoc Tukey test by extract. The larvae were fixed in 5 ml of using SPSS software. Additionally, using 10% neutral buffered formalin and Microsoft Excel, the data were subjected to refrigerated. Following the dehydration in a log–probit regression analysis to calculate graded ethanol series of 70%, 80%, 90%, the median lethal concentrations, LC50 at and two washes in 100% ethanol, the larvae 95% confidence intervals. were bathed in two washes in xylene (Refer appendix). Thereafter, the larvae were RESULTS subjected to two paraffin wax baths at 58- 60o C, before infiltration. By using a wax Species Identification dispenser (Model-TEC-2000), the larvae was embedded in paraffin. Once solidified, Morphological Species Identification - the paraffin block was kept at +4ºC until Larvae use. The paraffin block was sectioned using The water dwelling larvae were observed to rotary microtome (Model-F163851404) at lie parallel to the water surface, which is a 5 µm. Sections were mounted on to glass characteristic feature of Anopheles genus. slides which were homogenizedly coated Three body regions, head, thorax and with egg albumin. The slides were placed abdomen were distinct as shown in Figure on the slide dryer (Model-SD-2800) at 38o 4 C for 30 minutes. Thereafter, the slides were observed under the light microscope (Model-LX300) at objective 40x to ensure the presence of larval tissue.

The larval tissues were stained using Hematoxylin and Eosin staining procedure (Refer appendix) and observed under the light microscope (Model-LX300). The histopathological effect on mid-gut epithelium of larvae and morphological aberration induced by the methanol extracts of Momordica charantia leaves were examined. Figure 4. Light Microscopic view of Statistical Analysis Anopheles stage II instar larvae (100x).

The experimental data were expressed as mean % mortality ± SE of three replicates at each concentration. The data were statistically analyzed using multiple factor ANOVA (Analysis of Variance) using the (A) (B)

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Figure 5. (A) Dorsal view of head (400x) (B) Ventral view of prothorax and thorax (400x)

Morphological features including, Seta 1-A Neomyzomyia series in Sri Lanka simple; 2-C inserted at least as far apart as (Amerasinghe, 2014). the distance between 2-C and 3-C on one side, in the dosal view of head as shown in Morphological characteristics observed in (Figure 5A) were observed. Thus the ventral view of prothorax, including confirming the subgenus Cellia. Seta 1-P weak, 2-5 branched; 1, 2-P arising (Amerasinghe, 1992). from separate basal tubercles and only tubercle of 2-P prominent and sclerotized, In the ventral view of the thorax, the as shown in (Figure 5B). Based on these following morphological features were features, the subgenus was identified to be observed, long thoracic pleural setae 9, 10, Anopheles Tessellatus. Species 12-P, 9, 10-M and 9, 10-T simple as shown identification was further confirmed in (Figure 5B). Hence the taxonomic series through adult identification (Amerasinghe, was identified as Neomyzomyia. 2014). Anopheles tessellatus and Anopheles elegans have been reported under Morphological Species Identification – Adult Mosquito

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(A)

(B)

Figure 6. (A). Mosquito Wing (200x) (B) Femur and tibia speckled (400x)

The morphological characteristics of the Four taxonomical series of Sri Lankan mosquito wing including the presence of anophelines belong to the subgenus Celia. four or more darks marks involving both Among which Anopheles elegans and costa and veins R-R1 accessory sector pale Anopheles tessellatus, members of the (ASP) spot present on costa and/or Neomyzomyia series, Anopheles subcostal, as shown in (Figure 6A) These Maculatus and Jamesii group, belonging to features aided in distinguishing subgenus Neocellia series exhibit speckled femur and Cellia from those of subgenus Anopheles tibia as shown in (Figure 6B). (Gunathilaka et al., 2014).

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(A)

(B)

Figure 7. (A) (B) Apical half of the proboscis pale scaled (400x)

Based on the characteristics of apical half leaf extract of Momordica charantia was of the proboscis pale scaled, as shown in observed. Table 2 shows the larval figure 7, the species was confirmed to be mortality of Anopheles tessellatus (II Anopheles tessellatus (Gunathilaka, 2017). instar) after treatment at different concentrations (250 – 1000 ppm). Larvicidal Bioassay Table 2. Larval mortality of the methanol The larval mortality of Anopheles extract of Momordica charantia leaf against tessellatus after treatment with methanol Anopheles tessellatus

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Treatment Momordica charantia Mean % Mortality ± SE methanol extract

Concentration (ppm)

24 hrs 48 hrs

N/C 0 0 ± 0 0 ± 0

C1 250 11.11 ± 11.00 55.56 ± 11.33

C2 500 22.22 ± 11.00 66.67 ± 19.34

C3 800 77.78 ± 22.33 100 ± 0

C4 1000 100 ± 0 100 ± 0

The larval mortality was recorded at 24 leaf. The results are presented in the graph hour and 48 hours after exposure to in (Figure 8). methanol extract of Momordica charantia

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Error Bars +/- 1 SE

Figure 8. Graphical representation of the mean percentage mortality of Anopheles tessellatus after 24 hr and 48 hr of exposure between five different concentrations of Momordica charantia methanol leaf extract. Mean % mortality at each exposure time at each concentration ± SE is presented.

The graph indicated that the percentage with the highest concentration of the mortality of Anopheles tessellatus extract, 1000 ppm, showed maximum % increased with increasing concentration mortality. The lowest concentration of 250 and increased time of exposure to the plant ppm showed lowest % mortality while the extract (P<0.05, Tukey’s pairwise tests control larvae showed 100% survival. At after one-way ANOVA). The replicates the highest concentration (1000 ppm), the

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% mortality of the larvae was significantly way ANOVA). The highest % mortality different with other concentrations was detected in every concentration apart including control (p=0.000), 250 ppm from control in 48 hours of exposure. (p=0.000) and 500 ppm (p=0.001) (P<0.05, Tukey’s pairwise tests after one-way Table 3. Summary of the two-way ANOVA ANOVA). balanced design between the Momordica charantia methanol leaf extract Moreover, statistical data clearly indicated concentrations and the time of exposure (Table 3) a significant increase in % upon the percentage mortality of mortality with exposure time period Anopheles tessellatus larvae. (P<0.05, Tukey’s pairwise tests after one-

Source Type III Sum df Mean Square F Sig. of Squares

Time 3740.833 1 3740.833 10.029 .005

Concentration 40580.800 4 10145.200 27.199 .000

2992.667 4 748.167 2.006 .132 Time * Concentration

Error 7460.000 20 373.000

Total 140001.000 30

Corrected Total 54774.300 29

Median Lethal Concentration (LC50)

Table 4. Log-Probit regression

2 Time (hrs) LC50 Regression Equation R

24 251 ppm y = 2.1769 x -0.1733 0.94

48 126 ppm y = 2.3398 x – 0.1475 0.95

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Behavioral changes in larvae and were alternating between the surface and the bottom of the container. At 250 and Physiological parameters of the de- 500 ppm, the larvae were moribund, chlorinated water including, temperature incapable of rising to the surface and not 0 and pH were recorded as 28 C and 6.6, showing characteristic diving action when respectively. Following 24 hours of the water was disturbed. At 800 and 1000 exposure, the control larvae were rapidly ppm, 100 % mortality and immobility were responding to tactile stimuli and were observed. The larvae did not induce alternating between the surface and the movement when probed in the abdomen bottom of the container. At 250, 500 and region. 800 ppm the larvae were lethargic and moderate response to stimuli were Histological Effects observed. At 1000 ppm, 100 % mortality and immobility were observed and thus The histological effects of Anopheles larvae remained at the bottom of the tessellatus larvae treated with Momordica container and were unresponsive to stimuli. charantia methanol extract were compared with untreated larvae (Figure 9 and 10). Following 48 hours of exposure, the control larvae rapidly responded to tactile stimuli

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Figure 9. (A) Cross section of untreated Anopheles tessellatus larvae (200x) (B) Cross section of Anopheles tessellatus larvae treated with 500 ppm methanol extract of Momordica charantia leaf, showing the effect after 48 hours of exposure (200x). 1. Midgut and peritrophic matrix; 2. Wing imaginal disc; 3. Gastric caeca; 4. Leg imaginal disc; 5. Thoracic ganglion

The results of the histological studies confirmed that Momordica charantia methanol extract caused distinguished damaged to the cuticle leading to completely destroyed and shrunken cells. When treated with Momordica charantia, the midgut epithelium and caeca of the larval tissue was affected, as indicated in (Figure 10).

Intact Cuticle (A) cuticle

Mid-gut

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(B) Damaged

Cuticle

cuticle Disintegrated mid-gut

Figure 10. (A) Longitudinal section of untreated Anopheles tessellatus larvae showing intact thick cuticle layer (200x) (B) Longitudinal section of Anopheles tessellatus larvae treated with 500 ppm of Momordica charantia methanol plant extract (200x) , showing the effect after 48 hours of exposure.

DISCUSSION significant mortality in Armyworm larvae (Wardhani, Abadi, and Himawan, 2015). In the emerging field of research, in Furthermore, bioactive compounds of mosquito control programmes, botanical Momordica charantia including, apigenin phytochemicals have been recognized as and tannins have established potential potential alternatives to synthetic anthelmintic effect (Vedamurthy et al., insecticides, due to their larvicidal 2015). properties (Zhao et al., 2017). The present study was undertaken to assess the The phytochemical components in larvicidal potential of the methanol extracts Momordica charantia could contain polar of Momordica charantia leaf against and non-polar compounds, which have to Anopheles tessellatus. Targeting the be dissolved in solvents in order to release stationary larvae phase with plant-derived its chemical constituents (Ghosh, larvicides is desirable as this will supress Chowdhury, and Chandra, 2012). Methanol the fecundity of mosquitoes. was selected as the solvent of choice, based on the polarity and the nature of A number of compounds, such as phytochemicals present in Momordica flavonoid, glycosides, alkaloid, and charantia. Since methanol has a polarity of saponin have been isolated from 5.1, it is capable of extracting various polar Momordica charantia and have reported to and certain non-polar components. exhibit larvicidal activity (Mala and Tulika, Therefore, the methanol extract of 2014). Additionally, momordicin I and Momordica charantia dissolved in DMSO momordicin II, have exhibited anti-feedant stock solution, revealed a significant and larvicidal activity in (Ling et al., difference (p<0.05) between percentage 2008; Li et al., 2015). Moreover, terpenoid mortality and concentration. Hence, the compounds including, momordicside-L bioassay showed potent larvicidal activity and momordicoside-K, have demonstrated (Edriss, Satti and Alabjar, 2013).

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The LC50 value of the present study is Histopathological alteration were seen in nearly similar to literature values. Rahuman the mid-gut of the treated larvae, including and Venkatesan have reported that the separation of the epithelial cells from the methanol extract of Momordica charantia basement membrane. However, in the were highly effective against the larvae of treated mid-gut the cells were not A. aegypti (LC50= 199.14±13.13 ppm), at pronounced and could not been identified 24 hours of exposure Similarly, the in some areas at the base of the epithelial larvicidal efficacies of methanol extracts of cells due to the sever destruction of the Momordica charantia, tested with LC50 epithelium. Epithelial cells of the treated values were 207.61±13.50 ppm, against the larvae were destroyed, large vacuoles were late third larval age group of C. present in the cells, irregular and disappear quinquefasciatus (Rahuman and cell boundaries were observed. Venkatesan, 2008). Thus, a significant Additionally, the cells appeared dislodged, number of studies demonstrate that the leaf sloughed and detached from each other. extracts of Momordica charantia are toxic The control larvae revealed an intact cuticle to larval species. Momordica charantia whereas the cuticle in treated larvae appear may, therefore, act as an effective bio- to be discontinuous (Farnesi et al., 2012). larvicide against mosquitoes in the future This is consistent with the histological (Singh, Dhiman and Mittal, 2006). changes observed when mosquito larvae were exposed to plant chemicals (Sarra et According to the results of the probit al., 2017; Yu et al., 2015). Several reports analysis at 95% confidence interval, the suggested that the larvicide substances lead LC50 had decreased over time. Hence an to morphological damage in epithelial cells increase in toxicity of the Momordica of the mid-gut which are likely where these charantia methanol extract was observed at substances are absorbed (Perumalsamy et longer exposure period compared to short al; 2013; Al-Mehmadi, and Al-Khalaf, exposure period. This implies that the 2010). chemical constituents in the Momordica charantia methanol extract are released at a The behavioral changes observed, are steady rate over a period of time suggestive that the exposure to Momordica (Ramalingum and Mahomoodally, 2014). charantia methanol extract impaired the As a consequence of exposure to the plant spontaneous motor activity of the larvae extract, induction of apoptosis in larval and provoked a change in propulsive tissue could occur during long exposure movement, which was assessed by the periods (Dusfour et al., 2015). A significant response to sensory stimuli. Hence, it is mean percentage mortality was observed at suggestive that at high concentrations of the the concentration with close proximity to Momordica charantia methanol extract, the the LC50, hence the larvae were evaluated larvae demonstrates neuro-behavioral for possible histological changes. effects, including restlessness, loss of equilibrium and death (Bailey, Oliveri, and The most affected tissue was the mid gut Levin, 2013). epithelium when compared with the untreated mid gut. The untreated mid gut A study conducted by Liu, evaluated the consists of a unicellular epithelium layer possible mechanism of action of resting upon a basement membrane. compounds that demonstrate larvicidal

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Interntional Conference on Health and Medicine properties. Alkaloids are nitrogenous for human health (Joseph and Jini, 2013; compounds present in Momordica Rawani et al., 2017). The larvicidal charantia, which are active molecules property of Momordica charantia can be against mosquito larvae. The most common attributed to the phytochemicals present in effect Liu and colleagues observed was the plant. Hence, the formulation of paralysis of the larval musculature, as Momordica charantia methanol extracts demonstrated by the lack of mobility may potentially be used as an effective and following exposure to Momordica eco-friendly larvicide, which could be an charantia methanol extract. This is possibly alternative to malaria control. due to inhibition of neuro-muscular transmission or disruption of enzymatic Several other aspects of this research can be activity involved in energy synthesis further studied. These include (Veerakumari, 2015). Larvae that suffered determination of the larvicidal activity of a spastic paralysis assumed a bent or different solvent extracts of Momordica twisted posture, while in flaccid paralysis, charantia plants on Anopheles species to the larvae were straight and relaxed (Islam identify the most efficient solvent for and Bloomquist, 2015). Another proposed extraction. Furthermore, isolation and mode of action is the acetylcholinestrase purification of compounds present in (AChE) or sodium channels, as inhibition Momordica charantia methanol extract of acetylcholinesterase activity is using column chromatography could be responsible for terminating the nerve performed in order to confirm the bioactive impulse transmission through synaptic compounds responsible for the larvicidal pathway. Alkaloids function by depressing property. Moreover, the larvicidal activity autonomic nervous system activity hence of the methanol extract of Momordica facilitating the plant extracts effectiveness charantia could be exploited for use in in killing mosquito larvae (Liu et al., 2012). potable waters against mosquito larvae. Generally the active toxic ingredients of Implementation of field trials could be plant extracts are secondary metabolites. performed to assess the efficacy and cost- The mosquito larvae feed on these effectiveness of this method of mosquito secondary metabolites potentially control. encountering toxic substances with a wide In conclusion, an attempt was made to range of molecular targets (Nagarani, evaluate the role of plant extracts in Abirami, and Siddhuraju, 2014). These in mosquito larvicidal activity. The results of turn, affects the larval physiology in the present study may encourage further various ways at numerous receptor sites, researches on using simple and inexpensive the principal of which is abnormality in the application methods for controlling nervous system (Ebadollahi et al., 2014). mosquitoes in their breeding sites The purpose of this study was successfully (Soleimani-Ahmadi et al., 2017). achieved and Momordica charantia ACKNOWLEDGEMENTS methanol leaf extracts have shown a potential larvicidal activity against I would like to extend to my sincere Anopheles tessellatus in laboratory gratitude to University of Northumbria and experiments. Toxicological studies have BMS for giving me the opportunity to revealed that Momordica charantia is safe conduct this research project. A special

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Interntional Conference on Health and Medicine gratitude to the Associate Dean of BMS, Amerasinghe, F. P (1992), “A Guide to the Dr.Sajani Dias and Deputy Dean Dr.Mathi identification of the Anopheline Kandiah, for providing us with quality mosquitoes (Diptera: Culicidae) of Sri itinerary throughout and all the other Lanka. II. Larvae”, Ceylon Journal of lecturers and staff of BMS for their continuous guidance. I would also like to Science (Biological Sciences), Vol. 22, express my deepest appreciation to my No. 1, pp. 1-16. project supervisor, Miss Koshala De Silva, Amerasinghe, F. P., Mukhtar, M. and Herrel, N. for her constructive suggestions, (2014), “Keys to the Anopheline encouragement, motivation and guidance Mosquitoes (Diptera: Culicidae) of throughout the duration of my project. Furthermore, I show my sincere Pakistan”, Journal of Medical appreciation to the Laboratory staff for Entomology, Vol. 39, No. 1, pp. 28-35. supporting me with the necessary materials Bailey, J., Oliveri, A. and Levin, E. (2013), upon request. I would also sincerely thank “Zebrafish model systems for Dr. Sisira Pathirana, of Department of developmental neurobehavioral Parasitology, Faculty of Medicine, toxicology”, Birth Defects Research University of Colombo, for providing us with specimens required for the project. Part C: Embryo Today: Reviews, Vol. 99, No. 1, pp. 14-23. A special thanks to all my batch mates for Bekele, D., Petros, B., Tekie, H. and Asfaw, Z. the continuous support and motivation all along. Finally, I am indebted to my parents (2014), “Larvicidal and Adulticidal and loved ones, whose immense assistance, Effects of Extracts from Some support and guidance have brought me this Indigenous Plants against the Malaria far. Vector, Anopheles Arabiensis (Diptera: Culicidae) in Ethiopia”, REFERENCES Journal of Biofertilizers & Ali, M. Y. S., Ravikumar, S. and Beula, J. M. Biopesticides, Vol. 5, No. 2, pp. 144- (2013), “Mosquito larvicidal activity 152. of seaweeds extracts against Bulletin of the World Health Anopheles stephensi, Aedes aegypti Organization (2017), “Sri Lankans and Culex quinquefasciatus”, Asian vigilant after bidding farewell to Pacific Journal of Tropical malaria”, Vol. 95, pp. 170-171. Disease, Vol. 3, No. 3, pp. 196-201. Available at: Al-Mehmadi, R. M. and Al-Khalaf, A .A. http://dx.doi.org/10.2471/BLT.17.020 (2010), “Larvicidal and histological 317 (Accessed: 30th December 2017). effects of Melia azedarach extract on Das, D. R., Sachan, A. K., Imtiyaz, M. and Culex quinquefasciatus Say larvae Shuaib, M. (2015), “Momordica (Diptera: Culicidae)”, Journal of King charantia as a Potential Medicinal Saud University-Science, Vol. 22, No. Herb: An Overview”, Journal of 2, pp. 77-85.

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World Malaria Report (2016), World Health (2-24 hours). The dehydration procedure Organization, Available at: was carried out in a graded ethanol series, http://apps.who.int/iris/bitstream/1066 70% Ethanol for 40 minutes, 80% Ethanol for 30 minutes, 90% Ethanol for 30 5/252038/1/9789241511711- minutes, 100% Ethanol for 30 minutes and eng.pdf?ua=1 (Accessed: 13 100% Ethanol for 1 hour. Thereafter, the December 2017). larvae were subjected to two washes in Yu, K. X., Wong, C. L., Ahmad, R. and Jantan, Xylene, for 7 and 15 minutes, respectively. I. (2015), “Larvicidal activity, Hematoxylin and Eosin staining procedure inhibition effect on development, (H & E Staining) histopathological alteration and In order to remove paraffin, slides morphological aberration induced by containing larvae sections were bathed in seaweed extracts in Aedes aegypti the following series of solutions: xylene (2 (Diptera: Culicidae)”, Asian Pacific washes of 5 minutes each), 100% ethanol journal of tropical medicine, Vol. 8, (two washes of 3 minutes each), 95% No. 12, pp.1006-1012. ethanol (3 minutes), 70% ethanol (3 minutes) and distilled water (30 seconds). Zhao M. P., Liu Q. Z., Liu Q. and Liu, Z. L. The HE staining was initiated with a bath in (2017) “Identification of Larvicidal hematoxylin solution (45 seconds), Constituents of the Essential Oil of followed by tap water, and distilled water Echinops grijsii Roots against the for 2 minutes. To remove excess Three Species of Mosquitoes”, hematoxylin the slides were bathed in 1% Molecules, Vol. 22, No. 2, pp. 205. acid alcohol for 1 minute and 30 seconds, followed by 70% ethanol (3 minutes), eosin

solution (6 seconds), 95% ethanol (30 APPENDIX seconds), 100% ethanol (2 washes of 3 minutes each) and xylene (2 washes of 3 Dehydration procedure for Histological minutes each). After the xylene bath, slides studies of larval tissues were mounted with cover slips, using Alive larvae were fixed in 5 ml of 10% Canada Balsm. neutral buffered formalin and refrigerated

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