Interntional Conference on Health and Medicine

LARVICIDAL EFFECTS OF LANTANA CAMARA ETHANOL LEAF EXTRACT ON TESSELLATUS

1 Thimali Rathnayaka , 2 Koshala De Silva [email protected]

ABSTRACT

Mosquitoes transmit thoughtful human diseases, causing millions of deaths every year and the development of resistance to chemical insecticides resultant in recovering vectorial capacity. Plants can alternative sources of control agents. The present study assessed the role of natural products of plant origin with insecticidal properties for control of vectors. The résistance to chemical insecticides among mosquito species have been considered as a setback in vector control. Botanical larvicides are including conspicuously as alternative to synthetic chemical insecticides which are less degradable and toxic to non-target organisms. The aim of this study was larvicidal effects of Lantana camara plant extract on Anopheles tessellatus. The larvicidal activity and phytochemical showing of ethanol extract of leaves of Lantana camara were investigated in the laboratory against third instar larvae of Anopheles tessellatus for 24 and 48 hours and Lantana camara extracted were prepared and four different concentrations. Statistical analysis showed significant differences between the higher mortality for larvae. The highest (100%) mortality in the larvae occurred on treatment with 2000 ppm extract of L.camara. The maximum adult mortality was detected in the leaf extract (LC50 1000 ppm for 24 hours and LC50 199.526 ppm for 48 hours) while no mortality was noticed in the control groups. The L.camara plant extracts effect on larvae with introduction of apoptosis or effect of larvae DNA. Therefore, done in a DNA fragment analysis and observed histological changes in tissue of larvae. The extracts of Lantana camara plant showed potent larvicidal efficacy. This study suggests that, the leaf extracts of the Lantana camara can considered as promising larvicide against Anopheles tessellates third stage larvae. Keywords: Anopheles tessellatus, Lantana camara, Larvae, Mosquitoes, Malaria

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INTRODUCTION used as an alternative to synthetic insecticides (Jirakanjanakit et al., 2007). Vector-borne diseases Malaria A vector is a living organism that transfers an infectious agent from an infected Malaria is caused by one of four species of to a human or another animal. Vector-borne the Plasmodiumnspecies parasite diseases area transmitted by the bite of transmitted by female Anopheles spp. infected species, such as mosquitoes (Kuhn, Campbell-Lendrum & mosquitoes, ticks, triatomine bugs, Davies, 2002). Historically malaria was sandflies, and blackflies (Confalonieri et endemic in Europe, but it was finally al., 2007). Arthropod vectors are eliminated in 1975 through a number of ectothermic and sensitive to climatic factors connected to socioeconomic factors. However, climate is only one of development. Any role that climate played several factors influencing vector in malaria reduction would have been distribution, such as habitat destruction, small. However, malaria transmission is using land, pesticide application, and host intricately connected to meteorological density (Roberts, Schmidt, and John, conditions such as temperature and 2008). precipitation (Guerra et al., 2008).

Mosquitoes are best known vector for Epidemiology of Malaria disease and major public healthqproblem The World HealthcOrganization (WHO) throughout the world (World Health has publishedxglobal estimates ofathe Organization, 2016). Among the 3000 number of people thatqdie from malaria. In species of mosquitoes noted worldwide, these 15 yearsqthe global deathqtoll has more than a hundred species are been cut in half: from1839, 000 deaths 1in accomplished of transmitting various 2000 to 438,000 in 2015. Among these diseases in humans and other vertebrates. diseases, malariaqremains theqmost Vector-borne infectious diseases are thoughtful vector-borneqdisease malaria, denguebfever, yellow fever, and affectingqsome 300-500 millionvpeople plague, causeca significant fraction of the and 1.4 to 2.6 million deathsqyearly worldwide infectiousbdisease burden; throughout the world. More than 40% indeed, half of theaworld’s populationais ofqthe world populationqlives in areas infected with at least one type of vector- proneqto malaria (Figure 01) (Moss et al., bornevpathogen (Ricci et al., 2012). 2015). The World Health Organizationqhas Over the years, synthetic insecticides were certified thatqSri Lanka is a malaria-free introduced. Even though, these are nation, inqwhat it called a truly effective; insect tends to develop resistance extraordinaryqachievement. But, the to such products. These problems have WHOqsaid, the countryqhad begun an anti- required the essential for search and malaria campaignqthat progress of environmentally safe, successfullyqtargeted the mosquito-borne biodegradable, low-cost, native methods parasiteqthat causes the disease (WHO, for malaria vector controlling which can be 2015).

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Figure 01. Malaria incidence rates by countries 2000-2015 (World Health Organization, 2015)

ANOPHELES MOSQUITOES

Anopheles species mosquitoes occur almost worldwide, with the four species of human pathogenic Plasmodium spp. are transmitted significantly in nature by only some 30 species of Anopheles (Figure 02). Of the vectorial species, a handful is important in stable malaria, while others only become involved in epidemic spread of unstable malaria (Vinayagam, Senthilkumar and Umamaheswari, 2008).

Sri Lanka is a Tropical Island located close to the southern tip of . The most common species of Anopheles in such as A. culicifacies, A. fluviatilis, A. stephensi and A. tessellates (Kannathasan et al., 2008). Anopheles life cycle is showed in Figure 03.

Figure 02. The life cycle of mosquitoes. Stages within 24 to 48 hours, the eggs will ANOPHELES LIFE CYCLE hatch into larvae. The larvae will grow approximately 5mm in length within a span of seven to 10 days; the larvae will enter the stage of a pupa. When a mosquito is fully developed, it will emerge from its pupal

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Interntional Conference on Health and Medicine case and will become a mosquito. The new Methods to controlled mosquito a adult, at this time, will stand upon the water necessary component of disease prevention to dry its wings and prepare for its flight programs in developing countries. (Eckhoff, 2011). Mosquitoes of Anopheles in theqlarval stage are attractive target for THE MALARIA PARASITE LIFE controlqprocess due to theirslow mobility CYCLE in theqbreeding habitatscand the ease to control in thesexhabitats (Howard, Zhou & Omlin, 2007). According to study of Lima et al., (2006), focusedcinterest on alternative compounds for mosquito control due tovincreasing resistance of mosquitoesvto the insecticides. Plants, being a natural source of various compounds are known to contain larvicidal agents (Govindarajan et al., 2011).

Natural products are best option because they are less harmful to environment and

non-target organisms. Several compounds from different plants families have been

evaluated for new and promising larvicides Figure 03. The malaria parasite life cycle (Hemalath et al., 2015). In recent years, the involves two hosts. During a blood meal, a top priority in finding a nature based malaria-infected female Anopheles insecticide is that, they must be of plant mosquito inoculates sporozoites into the origin and should not have any ill effects on human host. After this initial replication in the ecosystem. Isolated compounds of the liver, the parasites undergo asexual plants provided alternative source of multiplication in the erythrocytes. The mosquito repellents agents (Yang et al., gametocytes, male and female are ingested 2004). Lantana camara an important by an Anopheles mosquito during a blood medicinal plant with several medicinal uses meal. The parasites’ multiplication in the in traditional medication system. mosquito is known as the sporogonic cycle. The zygotes in turn become motile and Lantana camara elongated which invade the midgut wall of Lantana camara is a species of flowering the mosquito, which make their way to the plant within the family Verbenacea. It is a mosquito's salivary glands. Inoculation of hefty extensive evergreen shrub which can the sporozoites into a new human host grow up to 3 m in height and has a strong perpetuates the malaria life cycle (Centers scent. It is a perpetual shrub found growing for disease Control and Prevention, 2016) up to 2000m altitude in tropical, subtropical parts of the world. All parts of this plant MALARIA CONTROL BY USING have been used conventionally for NATURAL PRODUCTS numerous illnesses all through the world (Vardien, et al., 2012).

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Lantana camara also known as Ganda- Studies showed that L. camara has Pana or Rata Hinguru in Sri Lanka. An antimicrobial, fungicidal band insecticidal invasive plant is introduced to Sri Lanka in activity against stored grain insectbpests, 1926 and currently has spread across the vegetable crops pest, mosquito larvae, and island to a significant extent (Gunatnillake other biological activities. L. camara has and Ranasinghe, 2001). Morphologybof L. also been used in traditional herbal camara is showed in Figure 04. medicines for treatment of ailments, including cancer, skin itches,qrabies, asthma and ulcers (Sousa and Costa, 2012).

This study will assumed to assess in the present communication, an attempt has been made to evaluate the larvicidal efficacy of the ethanol extracts from the Lantana camara leaves against medically important species of Anopheles spp.

METHODOLOGY

Collection of Lantana camara plant

The mature leaves of Lantana camara were collected from Homagama area in western province, Sri Lanka. The area around 6° 59' 36" N, 79° 58' 29" showed Figure 05. The Lantana camara plant and leaves showed

Figure 06. Figure 04. Lantana camara plant with its pink-yellow flower

Whole plant parts have been thoroughly studies for their chemical constituents. The major constitutes of leaf and flower essential oils are showed in Table 01.

Table 01. Major compounds of Lantana camara leafs (Tesch et al., 2011)

Chemical compounds Terpenoids bicyclogermacrene steroids germacrene D alkaloids carvone phellandrene β-caryophyllene limonene zingiberene cineole δ-humulene bisabolene

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Figure 05. Lantana camara plant leaves collecting from Homagama area in Sri Lanka

A B

Figure 06. Lantana camara plants

Fresh leaves (Figure 07 A) of 300.0g were washed tap water and air dried on polythene layer and it covered from polythene layer in 7 days in a shaded environment at room temperature. After 7 days, the dried leaves were grinder and prepared the 200g powder. Then powder was packed in a polythene bag (Figure 07 B) and it was covered in aluminum foil. The powder was refrigerated at 4o C.

A B

Figure 07. A ) Lantana camara fresh mature leaf B) Lantana camara leaf powder packet

REARING MOSQUITO LARVAE dishes, each containing 10.0 ml of de- chlorinated water (Figure 08). Larvae The mosquito larvae were obtained from feeding with appropriate provide a food. the Department of Parasitology, Faculty of Physiological parameters of the de- medicine, University of Colombo. Three chlorinated water including temperature larvae (n=3) were allocated into a petri and pH were measured. Container were

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Interntional Conference on Health and Medicine covered by using nylon net with small pore using standard adult mosquito keys sizes and allowed to adapt to the developed for Sri Lankan Anophelines. environmental conditions for 24 hours. PLANT EXTRACTS PREPARATION

The ethanol extract of the plant wasxprepared by dissolving 50.0g of driedspowder in 150.0 ml of solvent. The mixture was left in the dark at room temperature for 24 hours with shaking at regular intervals. Thereafter, the mixture was subjected to stirring at 500 rpm for 20 minutes in a magnetic stire. The solution was filtered using a glass funnel and Whatman no. 1 filter paper. Filtrates were dried in the fume hood. The evaporated Lantarna camera extract was collected to an air tight container. Covered with Figure 08. Three larvae were allocated into aluminum foil and stored at 4o C petri dishes. refrigerated until further use.

LARVAL IDENTIFICATION A 20 000 ppm stock solution was prepared by dissolving a measured 160.0 mg of Stage III larvae were placed individually in Lantana camera extract in 100.0µL petri dishes and observed under an inverted DMSO. Once the residue was completely light microscope (LABOMED - TCM 400) dissolved, de-chlorinated water until the with an objective (100x and 400x). Using 8.0 ml. (The ultimate concentration of standard larval keys developed for Sri DMSO in the stock solution was 0.1%). Lankan anophelines. They were identified The stock solution was vigorously vortex to to the species level using Gunarathna, 2017 obtain a homogenized mixture. Anopheles larvae key. Key larval characters were recorded for each species. LARVICIDAL BIOASSAY TEST Further, larval species identification was reconfirmed through adult identification. Four batches of concentrations were prepared each containing three sets of ADULT MOSQUITO larvae triplicates (n=3) treated with IDENTIFICATION different concentration Lantana camara plant extract. The larvicidal activity was The adult mosquito immersed in 70% assessed by the procedure (World Health ethanol and refrigerated overnight. Organization, 2005). Each concentration Thereafter, the mosquito was dissected and was prepared by replacing, an appropriate parts were mounted by using Canada volume of de-chlorinated water with the balsam. Prepared permanent slides were stock solution. The control set was observed and identified under the light prepared by adding 0.1% DMSO. The microscope (CXL) with objective 400x larval behavior was noted at regular time intervals and the mortality was recorded

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Interntional Conference on Health and Medicine following 24 and 48 hours of exposure chamber until it covered the gel. Then (Table 03). 8.0µL ethidium bromide was added to the chamber. Then 10 µL of DNA extract were Table 03. Concentration with stock and mixed with 5µL of gel loading buffer and deionized water loaded into the wells. The lid of the chamber was closed and gel was let to run Treatment Concentration at 100V for 1 hour till the dye. The gel was (ppm) placed in the UV Trans-illuminator and C0 [control] 0 C1 100 bands were observed using the software C2 500 and the monitor. C3 1000 HISTOLOGICAL PROCEDURE C4 2000

For histological studies, 2000 ppm live DNA FRAGMENTATION ANALYSIS larvae after 24 and 48 hours exposure to the Lantarna camera extract were fixed in 5 ml The concentration of 2000 ppm larvae of 10% neutral buffered formalin and obtained 24 hours post-exposure was in 5 refrigerated. After dehydration (see ml of ice cold PBS. For the DNA extraction Appendix 02) in a graded ethanol series, the procedure, PBS buffer was removed from larvae were subjected to two washes in the micro centrifuge tube. Thereafter, the xylene. Thereafter, larvae were immersed larvae was homogenized in Liffton’s buffer in two paraffin wax baths at 58-60o C and kept for 30 minutes. Subsequently, before infiltration by using a paraffin Proteinase K was added to the dispenser (TEC 9000). For the paraffin homogenized sample and vortex. The block preparation, the mould was filled mixture was incubated for 1 hour at 55°C. with wax in to which the larvae were oriented and the cassette was placed. Once This was followed by the addition of solidified, paraffin block was kept at 4oC phenol: choloroform: Isoamyl alcoholcto until use. The paraffin block was sectioned the sample. The sample was vortex and using rotary microtome (SHANDON centrifuged at 12 000crpm for 15 minutes. FINESSE 325) at 7 µm. Sections were The aqueous layer of the sample was mounted on to glass slides which were transferred to a new micro centrifuge tube homogenized coated with egg albumin. The and equal volume of Ice cold Isopropanol slides were placed on the slide dryer (SD was added. The extract was stored in -20°C 2800) at 38o C for 30 minutes. The slides for 30 minutes. Thereafter, the extract was were observed under the light microscope centrifuged at 12 000 rpm for 15 minutes. (CXL) at objective 100x to ensure the The supernatant was removed from the presence of larval tissue. Eppendorf and the pellet was dried. Subsequently, nucleus free water was The larval tissues were stained using added to micro centrifuge tube. Hematoxyllin and Eosin staining procedure (See Appendix 02) and observed under the 1.0% Agarose gel was placed on the light microscope (CXL) at objective 400x electrophoresis chamber in a horizontal to identify histological changes in the position and TBE buffer was added to the

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Interntional Conference on Health and Medicine cuticle after the exposure to the plant were calculated using the SPSS 11.5 extract. (Statistical Package of Social Sciences) software and Excel. Results with P < 0.05 It was noted the death of larvae exposed to were considered to be statistically high concentrations. Therefore, to study significant used two-way ANOVA. The histological changes in Anopheles larvae, mean mortality percentage of larvae the experiment was extended. A new set of mortality data was subjected to probit second instar larvae were exposed to C3 analysis for calculatingcLC50. C4, control (n=3) in triplicates.

RESULTS STATISTICAL ANALYSIS Larvae Identification Statistics at 95% confidence limits of upper confidence limit, lower confidence limit Morphological features of head, thorax, and abdomen three body regions were noted.

A B

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1 2 9 10 3 9 P 10 M 9 10 T 9

C

Figure 09. A) Microscopic of view of mosquito larvae (100x) B) Figure 2: Seta 1-A simple; 2- C inserted at least as far apart as the distance between 2-C and 3-C on one side C) Long thoracic pleural setae 9, 10, 12-P, 9, 10-M and 9, 10-T simple. Seta 1-P weak, 2-5 branched; 1, 2-P arising from separate basal tubercles, only tubercle of 2-P prominent and sclerotized (400x)

According to the morphological features of Based on the characteristics observed in the the dosal view of head (Figure 7 A), the dosal view of prothorax (Figure 09) subgenus Cellia was disguised from the distinguished the spices as A. tessellatus subgenus Anopheles (Amarasinghe, 1997). from A. elegans. The species identification The Sri Lankan anophelines of this was further confirmed by observing the subgenus belong to four taxonomic series: morphological features of the adult the Myzomyia, Pyretophorous,qNeocellia, mosquito. andcNeomyzomyia series. The subgenus Cellia includes several species; ADULT MOSQUITO culicifacies, karwari, subpictus, tessellatus, IDENTIFICATION vagus, varuna (Gunathilaka, 2017). In the adult mosquito, following The features observed ventraleview (Figure morphological features distinguished 09, C) distinguished the taxonomic series species of subgenus Cellia frombthose as Neomyzomyia. Two species, A. of the subgenus Anopheles: wing with 4 tessellatus and A. elegans have been or more darks marks involving both reportedvunder Neomyzomyia series in Sri costa and veins R-R1 accessory sector Lanka. pale (ASP) spot present on costa. The subgenuscCellia includes several

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Interntional Conference on Health and Medicine species; culicifacies, pseudojamesi,qsubpictus, tessellatus, vagus, varuna (Gunathilaka, 2017).

A B

Figure 10. Features of adult mosquito A) Femur and tibia speckled B) Apical half of the proboscis pale scale

According to these features such as 11.11 55.56 speckled femur and tibia as shown in C1 100   Figure 08. Based on the following 11.11 11.11 characteristics distinguished the A. 22.22 tessellatus (Gunathilaka, 2017). 33.33 C2 500   0 Mortality rate of Anopheles tessellatus 11.11

44.44 88.89 C3 1000   The following table showed in a mortality 11.11 22.22 rate of Anopheles tessellatus within 24 and 48 hours. 77.78 100  C4 2000  Table 04. Mean percentage mortality of 0 11.11 Anopheles tessellatus caused Lantana camara

Mortality Samp Concentrat MeanSE (%) le ion (ppm) The highest rate of mortality in larvae was 24hrs 48hrs detected where the highest concentration of extraction was exposed (P<0.05, Tukey’s N/C 0 0 0 pairwise tests after one way ANOVA). Highest mortality rate was observed in the highest concentration of the extract. According to statistical test results, it was

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Interntional Conference on Health and Medicine evident that the mortality of larvae exposed 370 1 370 11. .00 to 2000ppm was significantly different Time 3.63 3.63 11 3 from control, 100ppm, 500 ppm and 0 0 0 1000ppm in 24h exposure(P<0.05, Tukey’s pairwise tests after one way ANOVA). And conce 185 4 463. 1.3 .27 also the mortality rate was increased with ntratio 2.03 009 89 4 the exposure period (P<0.05, Turkey’s n * 7 pairwise tests after one way ANOVA). Time

666 20 333. Error 7.11 356 1

933 30 Total 33.3 34

Corre 398 29 cted 53.2 Total 59 Figure 11. Mean percentage mortality rates MEDIUM LETHAL of A. tessellatus larvae at different concentrations of different time periods of CONCENTRATION LC50 Lantana camara (SE) According to results of the probit analysis Table 05. Summary of the two-way at a 95% confidence level showed that the

ANOVA (balanced design) between the LC50 values gradually decreased with the Lantana camara extract concentrations and exposure time from 24 to 48 hours. the time of exposure upon the percentage Table 06. Probit regression line mortality of Anopheles tessellatus larvae parameters response of Anopheles tessellatus larvae to Lantana camara plant extracts. Sourc Typ df Mea F Sig Tim LC50 Regression R e e III n . e equation value Sum Squ peri of are od Squ 24 1000ppm y=1.66x+0. 0.982 hour 072 61 ares s 48 199.526p y=2.17x+0. 0.909 conce 276 4 690 20. .00 hour pm 036 39 ntratio 30.4 7.62 72 0 s n 82 0 1

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Behavioral changes of A. tessellatus Metamorphosis was not occurred in larvae larvae, after exposing to the ethanol leaf during the 48 hours. extract Lantana camara DNA FRAGMENTATION ANALYSIS Physical characteristics of water used for the study, medium temperature 280C and The quality of genomic DNAqusing pH 6.6 were within the permissible limits phenol:chloroform extraction methods was throughout the study period. visualized on 1% Agarose gel (Figure 12). Clear bands were observed control and Mosquito larvae exposed to the L.camara sample (2000 ppm concentration), leaf extract showed significant behavioral indicating no degradation of genomic changes during 30 minutes of exposure. DNA. The most clear behavioral change observed in A. tessellatus was inability to come on the surface. According to the 1 hour exposure results, all the larvae of control showed movements. All the larvae of highest concentration (2000 ppm) came to the surface of the solution. Also, 55.56% larvae in 1000 ppm concentration came to the surface and others were showed some movements. All the larvae of 500ppm and 1000 bp 100ppm concentration showed lethargic.

After the exposing 24 hours, all the larvae of control showed less movement. 55.56% larvae were dead and 22.22% were in lethargic and 2 were in showed fewer movements in 2000ppm concentration. 33.33% larvae were dead and 33.33% were in lethargic and 33.33% were showed some movements in 1000ppm concentration. 11.11% larvae dead and 33.33% larvae were lethargic and others were showed some movements in 500 ppm. 22.22% larvae were lethargic and others were showed movements of 100 ppm concentration. No such behavioral changes were obtained in control group.

Figure 12. Gel image of DNA from larvae Anopheles tessellatus larvae treated with obtain agarose gel electrophoresis at 1 Lantana camara extracts hour. Lane M contains molecular weight marker (50 bp DNA ladder). Lane 1 for HISTOLOGY control and Lane 2 for extracted DNA in

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Anopheles tessellates larvae treated with damaged to the cuticle leading to Lantana camara extract were compared complementary destroyed. When treated with untreated larvae. The results of the with Lantana camara, the mid gut histological studies confirmed that Lantana epithelium and caeca of the larval tissue camara extract caused distinguished was affected, as showed in figure 13.

Damaged cuticle Intact Distinguished mid Cuticle gut Mid Gut

A B

Figure 13. Histological alterations on Anopheles tessellatus larvae. (A) Larvae exposed to the Lantana camara extract. The larvae tissue was showed degradated tissue B). Control larvae stained with HE. The close association among rope-like cuticle. Cuticle was interact and showed mid gut

DISCUSSION degradable, and are readilyfavailable in many areasqof the world (Sivagnaname Mosquitoqlarval controlqusing and Kalyanasundaram, 2004). Many plant larvicidalaagents is a majorqcomponent in extractsqare known to be toxicqto different the control of vectorwborne diseases. specieswof mosquitoes and could be Plantqas larvicide is consideredqas usedqto control the diseasesethey transmit practical and chosen alternative in (Willcox et al., 2004). theqcontrol of the mosquitoqspecies at theqcommunity level (Hemalatha et al., A large number of plant extracts have been 2015). Therefore, alternativeccontrol reported to shave mosquitocidal or methods arevin useful; plant repellent activities against mosquito sourcesbpossess a widebrange of vectors, but few plant products have shown pharmaceutical andfinsecticidal assets. practical utility for mosquito control (Sun Botanicalbinsecticides serve as appropriate et al., 2006). In the present study ehanolic alternatives tobsynthetic insecticides in extract of Lantana camara showed 100% futurebas they are relativelynsafe, larvicidal activity against the third instar larvae of Anopheles tessellatus.

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A pointqto be highlightedwhere is that yields 100% mortality rate on the syntheticrpesticides have been usedqto concentration achieving the same results killqmosquito larvae. Similarly, the present with ethanolic extractqof the flower. study purposeqof this work was larvicidal effects of Lantana camara plant extract on The effects of extract were studied win a Anopheles species. The larvicidal dose dependent manner (Yuan and Hu, properties of Lantana camara have 2010). The ethanol extract of L. camara beenvinvestigated in a numberbof studies was found to have a higher rate of mortality (Remia and Logaswamy 2010). Kumar et on Anopheles tessellatus. The third-instar al., (2013) foundwthat the ethanol extract stage of A. tessellatus larvae was treated of L. camara leaveseshowed with different concentrations. And also, the maximumbmortality towards Aedes mortality rate increased with increase in aegypytinand Culex quinquefasciatus concentration and exposure of time. mosquito larvae. The present studybwas Furthermore, ethanol is safe to man, not as carried outmto study the effectstof the highly toxic as methanol which is known to L.camara leaf extractbon the mortality of produce skin irritations and even blindness Anopheles larvaewwhich may be helpful in when ingested (Alothman et al., 2009; the controlfof Anopheles mosquitoes. Durling et al., 2007). Then again, in a study Lantana camara is reported to possess conducted byvRajasekaran & Duraikannan insecticidal activity against stored (2012), ethanol extract Lantana camara grainqpest, vegetable crops pest, mosquito showed 100%qmortalitydafter 24hrs of larvae and antifungal, repellent, and other incubation. biological activities (Barreto et al., 2010). Also, the results of the study showed that In the present study the powder extracted there were significant differences between from L. camara leaves showed adulticidal the higher concentrations of each plant activityqagainst various mosquitoes against extract and the larvicide against An. A. tessellatus using 100% tessellatus larvae. The highest rate of extractsnrespectively. This is in agreement mortality in larvae was detected where the with Dua et al., (2010) who found the highest concentration of extraction was adulticidalqactivity of the L. camara to be exposed. Therefore done by a statistical mostqtoxic to An. fluviatilis followed by A. analysis to these data according to culicifacies and A. tessellatus and ANOVA, Kolmogorov smirnov test. This (Kemabonta et al., 2014). Senthilnathan, showed significance level less than the 0.05 (2010) studied the ethanolqextract of L. that mortalities increased with camara which shows high larvicidal concentration (0.002 < 0.05), these results activity againstqAe. aegypti and Culex confirm the report of (Pelah et al, 2005) quinquefasciatus. that thereawas a positive correlation As opposed to the comparative study of between concentration and the percentage Sathish and Maneemegalai (2008) wherein of the larval mortality (Rahuman et al., they utilized different parts of L. camara, 2009). The data confirmed in normally the present study only bused the leaf part distributed and post hoc tests equal which is the abundant part of the plant. In variance assumed by Tukey and it was their study, methanolicrflower extract showed results in significance level then done by a Two-way ANOVA test.

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The present study also shows that there is The mosquito larvae exposed under significant difference among the treatments L.camara plant extract showed significant and the concentrations of all yields L. behavioral changes observed withinq30 camara ethanolic leaf extract as an minutes, 1 hour and 24 hours of exposure. ineffectivedlarvicide as different to the The most obvious sign of behavioral study made by Sathish and Maneemegalai changes observed in A. tessellatus was (2008). The extraction solvent and the part inability to come on the surface. The larvae of the plant utilized greatly affect the result also showed restlessness, loss of of the experiment recently done. equilibrium and finally led to death. These behavioral effects were more pronounced However, on the individual basis, L. in case of L.camaranextracts after camara exhibited a higher larvicidal effect exposure. These effects may be due the and observed with their LC50. Ethanol leaf presence of neurotoxic compounds inn extract of L. camara showed leastqLC50 plant (Remia and Logaswamy, 2010). value of 1000 ppm for 24 hours and These larvae showed different levels of 199.526 ppm for 48 hours at 95% susceptibility demonstrated by the time confidence interval confirming the required to larvae lose their agility after presence of bioactive compounds in the exposed toppling extract. This was extract. These results show the drastically probably because larvae lost their capacity decreased LC50 values with the exposure to maintain at the water surface to get to the time from 24 to 48 hours. Low surface fornbreathing. concentrations (1000 ppm) which makes them preferable to synthetic insecticides. Results of the experiment conducted form However, combined effects or synergistic evaluating the larvicidal efficacy of effects of various control agents have Lantana camara showed that was toxic to proved very advantageous in the control of the A. tessellatus. According to the study of various pests (Seyoum et al., 2002) park et al., (2002) determined the LC50 and reviewed different mosquito larvicidal observed behavioral changes and mortality plant species with growth retarding, in the larvae. Similar observations were reproduction inhibiting, ovicides, noticed in the present study. synergistic, additive and antagonistic action of botanical mixtures (Shaalan et al., According to all these results, L.camara 2002). plant extract may be effect on larvae tissues of the organism with induction of apoptosis The results of lantana leaves extract of or effect of larvae DNA. Therefore, done in ethanol proved that they have larvicidal a DNA fragmentation analysis and properties againstnA. tessellatus larvae. observed histological changes in tissue of These findings agreed with Nath et al., larvae. (2006) who found that leaf extract of L. Based on our results of DNA analysis, had camara showed larvicidal (LC50) activity against Aedes albopictus. Another study by no Lantana camarabethanol extract effect Innocent et al., (2008) showed the effect of in A. tessellatus DNA. It can be due to the the root barks extracts of Lantana camara time period and exposed concentration of against late third instar larvae of Anopheles the plant. These all larvae were exposed in gambiae. 24 hours therefore, the time period may not enough to effect larvae DNA. Although, L.

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Interntional Conference on Health and Medicine camara plant compounds were slowly The recent study is guided by the following broken down enzymatically (Juen and objectives all throughout the research, to Traugott, 2005). This could be either due to determine the larvicidal effects of Lantana the fact that plant compounds were harder camara leaf in ethanol extract against to digest or caused by thebphysiology Anopheles sp. Larvae of mosquitoes found andbmetabolic rates of these larvae. within the Sri Lanka, to assess the effectiveness of the treatments in a dose- Phenol chloroform extraction is used a dependent manner which were used lysed cells or homogenised tissue and this different concentrations of Lntana camara procedure was often performed multiple extract and to figure out if there is a times to increase the purity of the DNA. significant difference in the mortality This procedure takes advantage of the fact among them treatments used. that deproteinization is more efficient when two different organic solvents fare used The recent study is guided by them instead of one. Furthermore, phenol following objectives all throughout the denatures proteins efficiently; it does not research, to determine the larvicidal completely inhibit RNasenactivity. The potential of L. camara leaf extract against phenol chloroform isolation method is larvae of common species of mosquitoes, to notbefficient, because many samples can be assess the effectiveness of the treatments in processed in parallel and hazardous a dose-dependent manner which are 100, chemicals are used (Sambrook and Russell, 500, 1000 and 2000 ppm and to figure out 2017). However, DNA generated by the if there was a significant difference in the phenolechloroform isolation method is mortality among the treatments used. The sheared band often contains a huge amount L. camara ethanolic leaf extracts was found of contaminating compounds, which make to have larvicidal potential against A. this DNA unsuitable for requests tessellatus mosquitoes. Results showed that susceptible to sheared or contaminated it had 100% percentage of mortality on the DNA. larvae tested for 48 hours.

In the histological study the whole larva FUTURE WORK processing method can be easily and quickly performed in a Histology For future work, to consider using bother laboratory, with only minimal precautions parts of the Lantana camara such as the to achieve an optimal fixation and flowers, because it has-been proven to have inclusion. However, the exposed A. highermmortality rate than then leaves that tessellatus larvae were degreagated had used in this experiment. Utilize a compared to the control. This can be due to higher treatment dosage to check the some errors of tissue fixing and sectioning. potency of then extract. Also, extend the Fixative fixation protocol may depend on observation of larvae form about 72 hours the additional processing steps and final after pouring the extract because the analyses that are planned. In this case, efficacy of the extract takes longer hours. quick fix method using cold formalin for Last, to identify the A. tessellatus mosquito around 24nhours is typically mused and larval and the exact instar they are going to that time can vary depending on the utilize to know if the Lantana camara has biological material.

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Interntional Conference on Health and Medicine a different effect based on a specific A. opportunity they gave me to conduct this tessellatus and instar. study.

Recently, abnovel method has become REFERENCES available for identifying the A. tessellatus of mosquito larvae. This method compares DNA sequences from field-collected, Alothman, M., Bhat, R. & Karim, A.A. (2009), unidentified larval samples with those of “Antioxidant capacity and phenolic identified adult samples. Using this content of selected tropical fruits from molecular species identification technique, studies have revealed previously unknown , extracted with different morphological and ecologicalmtraits of solvents”, Food Chemistry, Vol. 115, many A. tessellatus during larval stages. No. 3, pp. 785–788. Amarasinghe, F.P. (1992), “A Guide to the The Lantana camara leaf extract effect of the A. tessellatus larvae, DNA extraction identification of the Anolheline or can be used directly for molecular analyses Mosquitoes (Diptheria:Culicidae) of such as PCR. This is an added advantage, Sri Lanka, Larvae”, Ceylon Journal of especially where small initial samples have Science, Vol. 22, No. 1, pp: 1-13. DNA of low concentration due to the loss Barreto, F., Sousa, E., Rodrigues, F., Costa, J. of some of the DNA in the process of extraction and elution. & Campos, A. (2017), “Antibacterial Activity Of Lantana Camara Linn A partially purified toxin fraction and Lantana Montevidensis Brig Extracts lantadene can be obtained from Lantana From Cariri-Ceara, Brazil”, Journal of camara leaves by batch extraction, column chromatography and fractional Young Pharmacists, Vol. 2, No. 1, pp: crystallization. Further scientific 42-44. investigation for the development of Confalonieri, U., Menne, B., Akhtar. R., Ebi, effective therapeutic compounds will be K.L., Hauengue, M., Kovats, R.S., found in Lantana camara. Revich, B. & Woodward, A. (2007), ACKNOWLEDGEMENT Human Health. In: Climate Change 2007: Impacts, Adaptation and I would like to grant my sincere gratitude to Vulnerability. Contribution of my supervisor, Ms. Koshala de Silva, the Working Group II to the Fourth lecturer of BMS for her guidance, advices and extreme support throughout this Assessment Report of the project. I would also like to thank all the Intergovernmental Panel on Climate staff of Parasitology Department, Faculty Change, Cambridge, U.K. of medicine, University of Colombo and Durling, N. E., Catchpole, O. J., Grey, J. B., also thank Ms. Vajirapani de Silva and the Webby, R. F., Mitchell, K. A., Foo, L. team of Genetech for their support. I am also obliged to BMS, School of science, Y. & Perry, N. B. (2007), “Extraction University of Northumbria for this of phenolics and essential oil from dried sage (Salvia officinalis) using

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ethanol-water mixtures”, Food Symposium 2001. At: Chemistry, Vol. 101, No. 4, pp: 1417- http://journals.sjp.ac.lk/index.php/fes 1424. ympo/article/viewFile/1625/792, Eckhoff, P.A. (2011), “A Malaria Visited: 01 May 2017 Transmission-Directed Model Of Hemalatha, P., Elumalai, D., Janaki, A., Babu, Mosquito Life Cycle And Ecology”, M., Velu, K., Velayutham, K. & Malaria Journal, Vol. 10, pp: 303. Kaleena, P.K (2015), Larvicidal Govindarajan, M., Mathivanan, T., Elumalai, activity of Lantana camara aculeate K., Krishnappa, K. & Anandan, A. against three important mosquito (2011), “Mosquito Larvicidal, species”, Journal of Entomology and Ovicidal, and Repellent Properties Of Zoology Studies, Vol. 3, No. 1, pp: Botanical Extracts Against Anopheles 174-181. Stephensi, Aedes Aegypti, And Culex Howard, A. F., Zhou, G. & Omlin, F. X. (2007), Quinquefasciatus (Diptera: “Malaria Mosquito Control Using Culicidae)”, Parasitology Research, Edible Fish In Western Kenya: Vol. 109, No. 2, pp: 353-367. Preliminary Findings of a Controlled Green, M. R. & Sambrook, J. (2017), “Isolation Study”, BMC Public Health, Vol. 7, of High-Molecular-Weight DNA No. 9, pp: 199. Using Organic Solvents”, Cold Spring Innocent, E., Cosam, J., Gikonyo, N., Nkunya, Harbor Protocols, Vol. 4, pp: M. & Hassanali, A. (2009), pdb.prot093450. “Larvicidal Properties Of Some Guerra, C.A., Gikandi, P.W., Tatem, A.J., Tanzanian Plant Species Against Noor, A.M., Smith, D.L., Hay, S.I. & Anopheles Gambiae S.S. Gile Snow, R.W. (2008), “The limits and (Diptera:Culicidae) Mosquitoes”, intensity of Plasmodium falciparum International Journal of Biological and transmission: implications for malaria Chemical Sciences, Vol. 3, N0. 2, pp: control and elimination worldwide”, 266-270. PLoS Med, Vol. 5, No. 2, pp:e38 Jirakanjanakit, N., Saengtharatip, S., Gunathilaka, N. (2016), “Illustrated Key to The Rongnoparut, P., Duchon, S., Bellec, Adult Female Anopheles (Diptera: C. & Yoksan, S. (2007), “Trend of Culicidae) Mosquitoes Of Sri Lanka”, temephos resistance in Aedes Applied Entomology and Zoology, (Stegomyia) mosquitoes in Vol. 52, No. 1, pp: 69-77. during 2003-2005”, Environmental Gunatnillake, N. & Ranasinghe, D.M.S.H.K. entomology, Vol. 36, No. 3, pp:506- (2001), “Habitat utilization pattern of 11. Lantana camara in udwalawa national Juen, A. & M. Traugott, M. (2005)Detecting park”, Proceedings of the Seventh predation and scavenging by DNA Annu'll Forestry and Environment gut-content analysis: a case study

19

Interntional Conference on Health and Medicine

using a soil insect predator-prey Lima, E.P., Oliveira, Filho, A.M., Lima, system, Oecologia, Vol. 142, No. 3, J.W.O., Ramos-Junior, A.N., pp. 344–352. Cavalcanti, L.P.G. & Pontes, R.J.S. Kalita, S., Kumar, G., Karthik, L., Venkata, K. (2006), “Resistencia do Aedes aegypti & Rao, B (2012), “A Review on ao temefos em municipios do Estado Medicinal Properties of Lantana do ceara”, Journal of the Brazilian camara Linn”, Research Journal of Society of Tropical Medicine, Vol. 39, pharmacology and technology, Vol. 5, No. 3, pp:259-263. No. 6, pp:711-715. Mayes, M.A. &.Barron, M.G. (1991), Aquatic Kannathasan, S., Antonyrajan, A., Toxicology and Risk Assessment, Karunaweera, N., Anno, S. & ASTM international. Surendran, S. (2008), “Identification Moss, W. J., Dorsey, G., Mueller, I., Laufer, M. of Potential Malaria Risk Areas Of K., Krogstad, D. J., Vinetz, J. M., The Jaffna District Of Northern Sri Guzman, M., Rosas-Aguirre, A. M., Lanka: A GIS Approach”, Journal of Herrera, S., Arevalo-Herrera, M., the National Science Foundation of Sri Chery, L., Kumar, A., Mohapatra, P. Lanka, Vol. 37, No. 3, pp: 231-239. K., Ramanathapuram, L., Srivastava, Kemabonta, K. A., Anikwe, J. C. & H. C., Cui, L., Zhou, G., Parker, D. M., Adaezeobiora, I. (2013), “Bioefficacy Nankabirwa, J. & Kazura, J. W. of skeetar in Anopheles gambiae and (2015), “Malaria Epidemiology And Aedes aegypti mosquitoes from Control Within The International insecticides resistance areas in Lagos Centers Of Excellence For Malaria and Oyo State”, Biology Agriculture Research”, American Journal of HealthCare, Vol. 3, No. 2, pp: 122– Tropical Medicine and Hygiene, Vol. 135. 93, No. 3, pp: 5-15. Kuhn, K.G., Campbell-Lendrum, D.H. & Nath, D., Bhuyan, M. and Goswami, S. (2006), Davies, C.R. (2002), “A continental “Botanicals as Mosquito Larvicides”, risk map for malaria mosquito Defence Science Journal, Vol. 56, No. (Diptera: Culicidae) vectors in 4, pp: 507-511. Europe”, Journal of Medicine Park, D. C., Lautenschlager, G., Hedden, T., Entomology, Vol. 39, No. 4, pp:621- Davidson, N. S., Smith, A. D. and 30. Smith, P. K. (2002), “Models Of Kumar, S., Bajwa, B.S., Kuldeep, S. & Kalia, Visuospatial And Verbal Memory A.N. (2013), “Anti-inflammatory Across The Adult Life Span”, activity of herbal plants: A review”, Psychology and Aging, Vol. 17, No. 2, International Journal of advances in pp: 299-320. Pharmacy, Biology and Chemistry, Pelah, D., Abromovich, Z., Markus, A . and Vol. 2, No. 2, pp: 272-281. Wiesman, Z. (2005), “The use of

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commercial saponin from Quallaja Biological Research, Vol. 8, No. 2, pp: saponaria bark as a natural larvicidal 39-43 agent against Aedes aegypti and Culex Senthilnathan S. (2007), “The use of Eucalyptus pipiens”, Journal of tereticornis Sm. (Myrtaceae) oil (Leaf Ethnopharmacology, Vol. 81, No. 3, extract) as a natural larvicidal agent pp: 407-409. against the malaria vector Anopheles Rahuman, A. A., Bagavan, A., Kamaraj, C., stephensi Liston (Diptera Culicidae)”, Saravanan, E., Zahir A. A. and Elango Bioresource Technology, Vol. 98, No. G. (2009), “Efficacy of larvicidal 9, pp:1856-1860. botanical extracts against Culex Seyoun, A., Palsson, K., Kng’a, S., Karbiru, quinquefasciatus Say (Diptera: E.W., Lwande, W., Kllen, G.F., Culicidae)”, Parasitology Research, Hassanali, A. and Knols, B.G.J., Vol. 104, No. 6, pp: 1365-1372. (2002), “Traditional use of mosquito Rajasekaraan, A. and Duraikannan, G. (2012), repellent plants in western Kenya and “Larvicidal activity of plants extracts their evaluation in semi-field an Aedes aegypti”, Asian Pacific experimental huts against Anopheles Journal tropical biomedicine, Vol. 2, gambiae: ethnobotanical studies and pp: S1578-S1582. application by thermal expulsion and Remia, K.M. and Logaswamy, S.L. (2010), direct burning”, Transactional Royal “Larvicidal efficacy of two botanicals Society Tropical Medicine &. against mosquito vector Aedes Hygiene, Vol. 96, No. 3, pp: 225-231. ageptyi”, Indian Journal of Natural Shaalan, E.H.S., Canyonb, D., Younese, products and Recourses, Vol. 1, No. 2, M.W.F., Wahab, A. and Mansoura, pp: 208-212. A.H., (2005), “A review on botanical Ricci, I., Valzano, M., Ulissi, U., Epis, S., phytochemicals with mosquitocidal Cappelli, A. and Favia, G. (2012), potential”, Environment International, “Symbiotic Control Of Mosquito Vol. 3, No. 8, pp: 1149-1166. Borne Disease”, Pathogens and Global Sinka, M.E. (2013), “Global Distribution Of Health, Vol. 106, No. 7, pp: 380-385. The Dominant Vector Species Of Roberts, L., S., Schmidt, G.D. and John, J. Malaria”, Oxford, UK (2008), Foundations of Parasitology. Sivagnaname, N. and Kalyanasundaram, M. 8th edn. McGraw Hill. (2014), “Laboratory Evaluation Of Sathish, M.K. and Maneemegalai, S. (2008), Methanolic Extract Of Atlantia “Evaluation of larvicidal effect of Monophylla (Family: Rutaceae) Lantana Camara Linn againstmosquito Against Immature Stages Of species Aedes aegypti and Mosquitoes And Non-Target Culexquinquefasciatus”, Advances in Organisms”, Memórias do Instituto

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Interntional Conference on Health and Medicine

Oswaldo Cruz, Vol. 99, No. 1, pp: Human Reproduction, Vol. 19, No. 7, 115-118. pp: 1539-1543. Sousa, E. O. and Costa, J. G. (2012), “Genus Yang, Y.C., Le, E.H., Lee, H.S., Lee, D.K. and Lantana: Chemical Aspects And Ahn, Y.J. (2004), “Repellency of Biological Activities”, Revista aromatic medicinal plant extracts Brasileira de Farmacognosia, Vol. 22, Aedes aegypti”, Journal of American No. 5. Mosquito Control Association, Vol. Sun. R., Sacalis, J.N., Chin, C.K. and Still, C.C. 20, No. 2, pp: 146-149. (2001), “Bioactive aromatic Yuan, Z. & Hu, X.P. (2012), “Repellent, compounds from leaves and stems of antifeedant, and toxic activities of Vanilla fragrans”, Journal of Lantana camara leaf extract against Agricultural and Food Chemistry, Vol. Reticulitermes flavipes (Isoptera: 49, No. 11, pp: 51- 61. Rhinotermitidae)”, Journal of Econ Tesch, N.R., Mora, F., Rojas, L., Diaz, T., Entomology, Vol. 105, No. 6, pp: Velasco, J., Yanez, C., Rios, N., 2115-21. Carmona, J. and Pasquale, S. (2011), “Chemical composition and APPENDIX 01 – HISTOLOGY antibacterial activity of essential oil of PROCEDURE Latana camara var. moritziana”, Natural Product Communications, Hematoxylin and Eosin staining Vol. 6, pp: 1031-1034. procedure (H & E Staining) Vardien, W., Richardson, D., Foxcroft, L., The mounted larval tissue slides were Thompson, G., Wilson, J. and Le stained with using following H & E Roux, J. (2012), “Invasion Dynamics Staining Procedure. Of Lantana Camara L. (Sensu Lato) In Xylene I - 5 minutes South Africa”, South African Journal of Botany, Vol. 81, pp: 81-94. Xylene II - 5 minutes Vinayagam, A., Senthilkumar, N. and 100% Ethanol I - 3 minutes Umamaheswari, A. (2008), “Larvicidal Activity of Some 100% Ethanol II - 3 minutes

Medicinal Plant Extracts against 95% Ethanol - 3 minutes Malaria Vector Anopheles stephensi”, 70% Ethanol -3 minutes Research Journal of Parasitology, Vol. 3, pp: 50-58. Distilled Water - 30 Seconds Wilcox, A.J. (2002), “On the Frequency Of Hematoxylin- 45 Seconds Intercourse Around Ovulation: Evidence For Biological Influences”, Tap water - 2 -3 minutes

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Distilled water - 1-2 minutes 100% Ethanol II - 3 minutes

Acid Alcohol - 15 Seconds Xylene I - 3 minutes

70% Ethanol -3 minutes Xylene II -3 minutes

Eosin - 5-10 Seconds After the staining procedure, a drop of Canada balsam was put on the section and 95% Ethanol - 30 Seconds placed a cover slip, press to remove air 100% Ethanol I - 3 minutes bubbles. Let it dry for 5-10 minutes.

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