Wednesday, March 26, 2014

LARVICIDAL EFFECT OF MADRE DE CACAO (GLIRICIDIA SEPIUM) LEAF EXTRACT ON HOUSEHOLD MOSQUITO (CULEX PIPIENS)”

         
                                                




Approval Sheet
             This undergraduate thesis entitled: “LARVICIDAL EFFECT OF MADRE DE CACAO (GLIRICIDIA SEPIUM) LEAF EXTRACT ON HOUSEHOLD MOSQUITO (CULEX PIPIENS)” submitted by MA. CHZARMYN T. ANTONIO, AINA RIE M. ANTOY, AND JANCES M. LUMOGDA in partial fulfillment of the requirements for the degree BACHELOR OF SECONDARY EDUCATION (Major in Biological Sciences) is hereby approved.

Prof. ZENAIDA T. JUADA
                                                Adviser

Mr. LEMUEL I.GENTAPANAN                     Mr. DENNIS M. CARDENAL
   Subject Instructor                                             Panel Member               
            
                                                                  
Mr. RICHELLE L.  SANCHEZ
     Panel Member


            
             Accepted in partial fulfillment of the requirements for the degree BACHELOR OF SECONDARY EDUCATION (Major in Biological Sciences).

                                                          JELYN O. ALENTAJAN, PH.D
Dean, College of Teacher Education

March 2014                                      



Acknowledgement

The researchers would like to extend their sincere thanks, deep gratitude, and appreciation to all those who, in a way or another, contributed to the realization of this study;                  
First, to Mr. Lemuel I. Gentapanan our subject instructor for his guidance, encouragement, and support and for approving our research study;
To Prof. Zenaida T. Juada our thesis adviser for her unending patient and effort in checking and improving our work, for directing us what to do, and for generous sharing of her expertise;   
To Dr. Jelyn O. Alentajan, Mr. Richelle L. Sanchez, and Mr. Dennis M. Cardenal for extending their help to us in making this work a success;
To our parents, who offered their assistance and guidance for the completion of this study for their understanding, prayers, and financial support;                                                                          
To our friends, for the moral support to help us overcome the challenges; and           



Most of all, thanks to Almighty God who gave us infinite guidance and support to help us accomplish this research.

M. C. ANTONIO
A. R. ANTOY
J. LUMOGDA
 March 2014











Abstract
Larvicidal Effect of Madre de Cacao Leaf Extract (Gliricidia sepium) on Mosquito Larvae (Culex pipiens).
By
Ma. Chzarmyn T. Antonio
Aina Rie M. Antoy
Jances M. Lumogda


Prof. Zenaida T. Juada
Adviser       
             This experimental research was conducted to determine the level of effectiveness of Madre de Cacao (Gliricidia sepium) leaf extract as a larvicide for household mosquitoes (Culex pipiens). It further looked into whether there is no significant difference in the level of effectiveness of Madre de cacao leaf extract as larvicide at different concentrations.
              A total of 555 household mosquito larvae (Culex pipiens) were cultured and treated with Madre de Cacao leaf extract. There were five treatments used which include 0%, 25%, 50%, 75% and 100 % of Madre de Cacao leaf extract. The Mean was used  to determine the level of effectiveness of Madre de Cacao leaf extract as larvicide described as less effective, moderately effective, effective and very effective. The ANOVA

was used to determine the significant difference in the level of effectiveness of different concentrations of Madre de Cacao on household mosquito larvae (Culex pipiens) set at 0.01 level of significance.
             The study showed that 50%, 75% and 100% concentration of extract were found to be very effective in killing household mosquito larvae (Culex pipiens) while 25% concentration of the extract came out only as effective. There was a significant difference in the level of effectiveness of Madre de Cacao (Gliricidia sepium) leaf extract as larvicide to household mosquitoes between 25% concentration and 50%, 75%, and 100% concentration while there was no significant difference in the level of effectiveness of Madre de Cacao leaf extract as larvicide when 50%, 75%, and 100% concentration were used.
             From the results of the study, Madre de cacao leaf extract can be a potential natural larvicide on mosquitoes and can be an alternative substitute to commercial mosquito pesticide. The researchers recommend the use of Madre de cacao leaf extract as an alternative natural larvicide on household mosquitoes to reduce the side effect of using commercial pesticides especially on places where the plant grows abundantly and to wet places prone to mosquitoes. Further research is


recommended on other potential use of Madre de Cacao aside from its larvicidal effect.






Table of Contents
                                                                   Page Number
Title Page                                                                                 i        
Approval Sheet                                                                        ii
Acknowledgement                                                                             iii
Abstract                                                                                   v
List of Tables                                                                           x
Appendices                                                                              xi
Chapter
I.     Introduction to the Study                                          1
Background of the Study                                           1
Statement of the Problem                                          2
Hypothesis                                                                 3
Scope and Limitation of the Study                                      3
Significance of the Study                                           4
Definition of Terms                                                    5
II.   Review of Related Literature                                                8
Morphological description of Madre de Cacao      8
      (Gliricidia sepium)
Chemical Components of Madre de Cacao           9

                        
Uses and Efficacy of Madre de Cacao                         10
(Gliricidia sepium)
Medicinal Value of Madre de Cacao Leaf Extract        14
Related Studies about the potentiality of                   15
Madre de Cacao (Gliricidia sepium)
III. Research Methodology                                               18
Experimental Design and Treatment                         19
Research Paradigm                                                    20
Procedure                                                                   21
Instrumentation                                                         23
Statistical Tools                                                          24
IV. Results and Discussion                                              25     
V.   Summary, Findings, Conclusions and
Recommendations                                                      32
           Summary                                                             33
Findings                                                               34
 Conclusions                                                                   35        Recommendations                                                35     
References                                                                               37
Appendices                                                                              40                                           

List of Tables
Table                                                                                                               Page Number
 1                                 Treatment, Level of Concentration of                                 19       
Madre de Cacao Leaf Extract
and Replicates.

 2                                 Results showing the Treatment,                                       26                                            Number of Mosquito Larvae Died in each                                                                                     Replicate and the Level of Effectiveness.
3                                  Mean Standard Deviation and Standard                          29                                            Error at Different Concentration of Madre                                                                                    de Cacao Leaf Extract.
4                           Results of the ANOVA                                         30
 5                          Multiple Comparisons                                          31
















List of Appendices


Appendices                                                                    Page Number

A                           Pictures in Preparation of Extract               41     
C                          Results in 0 % Concentration                     43
D                          Results in 25 % Concentration                            44
E                           Results in 50 % Concentration                            45
F                           Results in 75 % Concentration                            46
G                          Results in 100 % Concentration                 47









 CHAPTER   I
Introduction

This chapter presents the background of the study, statement of the problem, hypothesis, scope and limitation, significance of the study, and definition of terms.

                          Background of the Study                                                  Mosquitoes are among the common insects that endanger human health by spreading human diseases through bites. Millions of death worldwide every year is attributed to mosquitoes. By bloodsucking, female mosquitoes need blood to nourish their eggs, multiply and transmit diseases. Dengue or hemorrhagic fever is a dreadful disease caused by a virus and transmitted by mosquitoes. To prevent proliferation of mosquito bites diseases and to improve the quality of environment and public health, mosquito control is essential. (Gosh, Chowdhury and Chandra 2012).There are many methods of control that can be implemented to reduce the number of mosquitoes. Local councils may use commercial larvicides (pesticides that kill the larvae) which prevent mosquitoes from maturing to adults.
In an effort to help the government combat the disease and eradicate mosquito carrier, the researchers determined the effect of Madre de Cacao (Gliricidia sepium) leaf extract on mosquito larvae. Studies have shown that the plant Madre de Cacao is an insect repellant and has the ability to kill many insects which can become potential carrier of many diseases. At present there is still a wide use of commercial and synthetic chemicals to destroy pests which are potential carriers and are expensive. The researchers are aware of the risk of high toxic chemical compounds and become interested in using natural pesticides derive from plants as a means of destroying pests hence the conduct of this study.                                                                                                                                                                                                                                                Statement of the Problem                                    
The main objective of the study was to determine the larvicidal effectiveness of Madre de Cacao (Gliricidia sepium) leaves extract on mosquitoes.                                                                                        Specifically, this study aimed to answer the following questions:
1. What is the level of effectiveness of Madre de Cacao leaf extract as a mosquito larvicide?                                                                                 2.  Is there significant difference in the level of effectiveness of Madre de Cacao leaf extract as a mosquito larvicide at different concentrations?                                                                                                                                                                                                                                               Hypothesis                                                                
     Based on the problem cited this hypothesis is advanced:            
No: There is no significant difference in the level of effectiveness of Madre de cacao leaf extract in killing mosquito larvae at different concentrations.                                                                                                                                                                                                    Scope and Limitations of the Study                             
  This study determined the larvicidal effect of Madre de Cacao leaf extract on household mosquitoes (Culex pipiens). The study was conducted at University of Antique Science Building Laboratory room. The experimental period lasted for 24 hours. Five treatments with different concentrations of Madre de Cacao leaf extract were used and each treatment containing three replicates with thirty seven larvae arranged in Parallel Group Design.                                                                 
To determine the level of effectiveness of Madre de Cacao leaves as a larvicide, the Mean was used and is described as less effective, moderately effective, effective ad very effective.                                         
The One Way Analysis of Variance (ANOVA) was used to determine the significant difference in the level of effectiveness of Madre de Cacao leaf extract at different concentration and tested at .01 level of significance.                                                                                                                                                Significance of the Study                                            
    This study aimed to discover the potential use of Madre de Cacao leaf extract in eradicating mosquitoes and as a substitute for commercial pesticides. The researchers believed that this study will be beneficial to the following:                                                                                                 
   “Medical Practitioners” Results of the study will provide these people with basic knowledge and information as to the potential use of natural pesticide specifically Madre de cacao in eradicating mosquito larvae as well as its usefulness in eradicating other similar pests that may cause different kinds of diseases brought by them.         
Government” The results of this study will help lessen government expenses intended in eradicating mosquitoes.                                  
“Community” This study will benefit the community for it would provide them cheaper and effective mosquito larvae repellent and improve their awareness of the important value of Madre de Cacao as an alternative larvicide.
Researchers.” The result of this study will serve as baseline information for those who are interested to conduct further investigation related to the problem.                                                                                                                                                                                                                  Definition of Terms                                         
    The following terms are defined in order to give a clearer understanding on how the words are used in the study.

“Concentration” It refers to the percentage by volume of Madre de cacao leaf extract in a solution and is described as 0%, 25%, 50%, 75% and 100%.                                                                            
   “Effectiveness” It refers to the potential level of Madre de cacao in killing mosquito larvae and is described as less effective, moderately effective, effective and very effective.                                                  
   “Larvicide” It refers to a pesticide that kills mosquito larvae.                  
“Leaf Extract” It refers to the liquid substance obtained from pounded and blended Madre de cacao leaves.                                  
   “Madre de Cacao (Gliricidia sepium)” A medium sized leguminous tree which can grow from 10 to 12 meters high belonging to the family Fabaceae.                                                                                    
    “Mosquito Larvae” They refer to the wrigglers obtained from the culture of household mosquito (culex pipiens) in duration of five to seven days.                                                                                                
“Replicate” This term refers to every treatment that appears more than once in the experiment to provide a means for estimating experimental error. In this study, it refers to the number of trials in each treatment.                                                                                  
       “Treatment” This term refers to the different concentration of Madre de Cacao leaf extract in the experimental group and is described as Treatment 1 (control), Treatment 2, Treatment 3, Treatment 4 and Treatment 5.                       




                                 CHAPTER II                                                                                    Review of Related Literature and Studies                                                                                                                                         
         This chapter gives the description of the morphology and characteristics of Madre de Cacao, its uses and efficacy, medicinal value and related studies as to its larvicidal effect.                                                                                                                            Morphological description of Madre de Cacao (Gliricidia sepium)                

            Madre de Cacao (Gliricidia sepium) is a small to medium-sized, thorn less, leguminous tree up to 10-12 m high.  It is branching frequently from the base with basal diameters reaching 50-70 cm.  Bark is smooth, varying in color from whitish grey to deep red-brown. The trees display spreading crowns.  Leaves are odd pinnate, usually alternate, sub-opposite or opposite, and approximately 30 cm long. The leaflets are 5-20, ovate or elliptic, 2-7 cm long, and 1-3 cm wide.  The leaflet midrib and rachis are occasionally striped red.  Inflorescences appear as clustered racemes on distal parts on new and old wood, 5-15 cm long. Flowers borne singly with 20-40 per raceme with colors that vary from bright pink to lilac, tinged with white, usually with a diffuse pale yellow spot at the base of the standardpetal, and the calyxglabrous, green, often tinged red.  Standardpetal  is round and nearly erect, approximately 20 mm long keel petals 15-20 mm long, 4-7 mm wide.  Fruit green, sometimes tinged reddish-purple when unripe, light yellow-brown when mature, narrow, 10-18 cm long, 2 cm wide, valves twisting in dehiscence;  seeds 4-10, yellow-brown to brown, nearly round. (http://www.tropicalforages.info/key/Forages/Media/Html/Gliricidia_sepium.html)

Chemical Components of Madre de Cacao (Gliricidia sepium)                
    There are many compounds found in Gliricidia sepium. The ones most researched are the tannins. In one study, Gliricidia was found to contain 40.7g of condensed tannins/kg dry matter. Tannins bind to protein and can make plants with high levels have an astringent dry mouth taste. The exact quantity of tannins varies with the location of the tree. The active medicinal compounds may be the tannins or other compounds such as afrormosin, medicarpin, or some isoflavins. Most of the research with Gliricidia and its compounds have focused on its nutritive quality. However, some studies have focused on the ability of the plant and/or roots to decrease soil nematode populations, and control insects or fungi. Some of the compounds in Gliricidia sepium are: Afrormosin (an isoflavan), reported to be an antitumor promoting agent; Formononetin(an isoflavan); Gliricidin-6a–wood; Gliricidol-9A–wood; Medicarpin (a pterocarpan), reported to be antifungal; 7,4'-dihydroxy-3'-Methoxyisoflavin; 2'-O-Methylsepiol–plant; Tannin, reported to have potential antidiarrheic, antidysenteric, antimutagenic, antinephritic, antioxidant, antiradicular, antiviral, bactericide, cancer-preventive, hepatoprotective, pesticide, psychotropic, and viricide activities; and 7,3',4'-Trihydroxyflavanone – plant. (http://www.ansci.cornell.edu/plants/medicinal/gliricid.html)

Uses and Efficacy of Madre de Cacao (Gliricidia sepium)                      
       Gliricidia is used by farmers in some Latin American countries to repel insects. The leaves are ground up and combined with water. The animal is then bathed with the resulting paste. According to some of the farmers, if this is repeated every 7-14 days, the number of torsalo (tropical warble fly) infections is decreased. No published studies could be found to substantiate this claim. However, when interviewing farmers and checking goats in Honduras, It was found out that the goats who the farmers claimed to have bathed with Gliricidia had only 2-3 torsalos, while others had 10 or more. Also, one study did indicate that the heartwood of Gliricidia contains compounds that attract and are toxic to certain insects (e.g., southern army worm, cabbage looper, yellow woolly bear, and Glyptotermes dilatatus, a termite).                                    
    Other study, Gliricidia was found to inhibit the growth of various strains of Neisseria gonorrhoea in vitro tests. Tinctures made from the leaves were used for these tests. (http://www.ansci.cornell.edu/plants/medicinal/gliricid.html)         
          According to the National Academy of Sciences (1980a), the leaves contain over 20% crude protein and are nutritious for cattle though toxic to most other animals including horses. The tree is widely planted as shade for chocolate, coffee, tea, and vanilla. There are few "living fence" species that strike root from cuttings more readily, also widely planted as a hedge and/or windbreak. Tilth and fertility of the soil beneath the trees are greatly improved from the leaf- and flower-fall. The timber is said to finish smoothly and be used for furniture, agricultural instruments, posts, railroad ties, and heavy construction. Flowers are a good source of forage for bees. Flowers are consumed by Mexican rural inhabitants who use the pods for rat poison. In the Philippines, the foetid leaves are crushed and rubbed onto cattle. In Indonesia, the tree is planted as a firebreak. This and other fast-growing leguminous trees have the vigor to outgrow or compete with the Imperata grass. In the shade of Gliricida, the grass finally dies, leaving nothing that can sustain a grass fire.                 
  Gliricidia sepium may be the most common living fence species in the tropics. Fence posts are established from large stakes. They may be planted at 1 to 2 m spacing and joined with barbed-wire or bamboo. Alternatively, they may be planted 10 to 20 cm apart as a stockade and their branches interwoven (Stewart 1996). Fuel wood, stakes, fodder and green manure are harvested from fences. The wood has a specific gravity of 0.5 to 0.8. It makes a good fuel, burning with little smoke and no sparks, and has a calorific value of 4,900 kcal/kg. Natural stands, secondary forests and woodlots of Gliricidia have been managed for commercial fuel production (Glover 1989, Stewart 1996). The wood is also used for poles, timber, furniture and agricultural implements.              
      Responding well to frequent cutting, Gliricidia produces abundant amounts of nutritious fodder containing 18 to 30% crude protein. Livestock respond well to the fodder. Some animals are reluctant to eat gliricidia, but training may overcome this problem. Once Gliricidia is accepted, subsequent offspring readily consume it. Toxicity problems are reported with non-ruminants. Pruning trees before the dry season enables coppice growth to be retained for use as dry season feed. Fodder plantings vary from hedgerows with 10 to 50 cm in-row spacing and 1 to 4 meter between row spacing, to block plantings of 50 x 50 cm to 1 x 3 m. Production varies from 2 to 20 t/ha (Glover 1989, Stewart 1996, Allison and Simons 1996). Pod peels are eaten by livestock as a dry season fodder in Bali.                        
   When used as mulch or green manure in farming systems, the nitrogen-rich foliage improves crop production through the addition of nutrients, weed control, conservation of moisture and reduction of soil temperature. Leaf biomass is usually produced from hedgerows or fences around or in the cropping area. Companion crops include paddy and upland rice, corn, cassava and coconuts. Hedgerows are used on sloping farmland for erosion control and passive terrace formation. Hedgerow management should minimize competition with crops. Hedgerow systems can be labor intensive, which may limit their adoption. Gliricidia is used as a shade for tea, coffee and cacao; and as a support for cassava, yams, vanilla, pepper, and passion fruit. These crops also benefit from the soil improvement characteristic of Gliricidia. The presence of Gliricidia in fields reduces incidence of some fungal and insect attacks (Glover 1989, Stewart 1996).

 Medicinal Value of Madre de Cacao Leaf Extract                              
         In the Philippines, Gliricidia is washed and pounded to extract the juice from the leaves. It is then applied to the area affected by external parasites once to twice a day for one week. In Guatemala, the bark and leaves of Gliricidia are used to treat human skin diseases. (http://www.ansci.cornell.edu/plants/medicinal/gliricid.html)
Madre de Cacao was reported to be expectorant, insecticidal, rodenticidal, sedative, suppurative. Madre de Cacao is a folk remedy for alopecia, boils, bruises, burns, colds, cough, debility, eruptions, erysipelas, fever, fractures, gangrene, head-ache, itch, prickly heat, rheumatism, skin, sore, tumors, ulcers, urticaria, and wounds (Duke and Wain, 1981 in http://www.customessaymeister.com/ customessays/Botany/16649.html)

Related Studies about the potentiality of Madre de Cacao (Gliricidia sepium)                                                                                         
  Research has been conducted on both the antifungal and antimicrobial properties of Gliricidia extracts. In a brine shrimp toxicity test, a general screening method indicative of cytotoxicity and pesticidal activity, the LC50 was 454ug.ml (CI 328-608). , medicarpin, one of the compounds in the leaves and heartwood of gliricidia, is supposed to be antifungal. In an antifungal study, gliricidia extracts inhibited the germination of Drechsleraoryzae only 6%. However, in another study, 50ug of stem chloroform extracts inhibited the growth of Cladosporium cucumerinum and slightly inhibited the growth of Candida albicans. In contrast, in another study, the antimicrobial properties of extracts from the bark of gliricidia were tested. It was effective against bacteria causing dermatitis. However, it was not effective against enterobacteria or Candida albicans. The discrepancy in the effectiveness against Candida albicans could be because of the quantity of plant extract used or the types of extracts used. In another study, leaf extracts were found to be effective against the dermatophytes, microsporumcanis, Trichophytonmenta grophytes ,varalgodonosa, and T. rubrum. It was not effective against Epidermophyton floccosum, M. gypseum or T.mentagrophytes vargranulare. (http://www.ansci.cornell.edu/plants/medicinal/gliricid.html)           
    A study done by Alfredo Rabena, a full-time professor at the University of Northern Philippines in Vigan City, found that kakawati leaves are good source of coumarins, a toxic substance that can kill almost all types of pests and insects. Rabena said one kilogram of kakawati leaves, soaked in water overnight, can produce seven gallons of “botanopesticides” (botanical pesticides) as an effective pest control. (http://www.philstar.com/Article.aspx?articleid=401291.)                  Another study conducted by Zamboanga National High School Science Lab (2008), about the larvicidal property of crude aqueous Madre de cacao extract against larvae. Extracts were taken from fresh young stem’s scrapings and leaves mixed with equal parts of water. 20, 15 and15 mL of the two extracts were independently treated on the 10 mosquito wrigglers suspended in 5.0 mL rain water in separate Petri dishes. Five trials were done for all the setups. All the groups were undisturbed for 24 hours. The greatest mortality was observed in 20 and 15 mL leaf extract with both means of 9.4 (94% mortality, with n=5).                                  In the study Larvicidal Activity of Gliricidia sepium Against Mosquito Larvae of Anopheles stephansi, Aedes aegypti and Culex quinquefasciatus by Sharma, N., et al., (1998), Crude ethanol extracts (CEE) of dried leaves (CEE1), fresh leaves (CEE2), dried petioles (CEE3) and stem bark (CEE4) of Gliricidia sepium were investigated for their toxic properties on the late third instar larvae of Anopheles stephansi, Aedes aegypti and Culex quinquefasciatus. The results indicate that all the CEEs are toxic to the larvae of the three species, causing 100% mortality at or below a dosage of 16,000 ppm. Although the mortality rate is dose-dependent, the larvae of different species exhibit genetic variability with respect to their tolerance at lower doses of CEE. It is likely that the toxicity, at least in some cases, could be due to more than one toxic principle. The findings also suggest that toxic principles vary quantitatively and/or qualitatively in different organs of the plant. Overall, CEE2 shows a higher toxicity and CEE3 shows a weaker toxicity compared to CEEs from fresh leaves and bark.                               
         The above related literature and studies gave more information and insights to the researchers in conducting this research study.






 Chapter III                                                  
Methodology             

This chapter presents the procedure, experimental design and treatment and the materials used in conducting the study.

                                  Experimental Design and Treatments                                   
          This study made use of the experimental method of research. The experiment consisted of five treatments with three replicates arranged in a Parallel Group Design shown below.


Table 1.                                                                                                              Experimental Design showing treatment, level of concentration of Madre de Cacao leaf extract and replicates.
Madre de Cacao Leaf Extract Treatment
Replicates                       indicating number of larvae used
Total number of larvae

1
2
3
111
T1 (0%)
37
37
37
111
T2 (25%)
37
37
37
111
T3(50%)
37
37
37
111
T4(75%)
37
37
37
111
T5(100%)
37
37
37
111
                                                                                      Total: 555

The target population was 125 mosquito larvae in each treatment. This was computed using the formula  Ss = NV + [ Se2(1- p ) ]NSe + [ V2 (p) (1- p ) ]
Since there were five treatment and replicated three times, fifteen petri dishes and a total of 111 mosquito larvae were used for each treatment. Each replicates for the five treatments contained thirty seven mosquito larvae placed on a petri dish.



Research Paradigm:

Independent Variables                                Dependent Variables


 Concentration of Madre de Cacao Leaf Extract (0%, 25%, 50%, 75% and 100%)

       Level of Effectiveness of Madre de Cacao Leaf Extract as Natural Larvicide


 










                                                                Procedure                                                  
                          Culturing of Mosquito Larvae                                                                                            The mosquito larvae were cultured for duration of five to seven days. The bucket was filled with water and handfuls of manure were added for the mosquitoes to be feed on. The mosquito eggs were checked frequently. If there were already small eggs on the surface of water, the eggs were transferred from the bucket to a jar at room-temperature using fresh water and provided with a fish net for easier catch until they reach their larval stage of development. At this stage, the larvae looked like long insects that wiggle when disturbed. After seven days, the larvae were taken from the jar using a fish net and used as a subject in the study.                                
Collection and Processing of Madre de Cacao Plant                                                     
    The  leaves of Madre de Cacao were gathered from its planting areas in Ilaya, Bugasong, Antique, Philippines. The leaves were washed and pounded using amortar and pestle then blended and the extracts were collected using a strainer. The extract was diluted by adding distilled water as to different concentrations.
Treatment                                                                                                                         
     The concentration was varied to twenty five percent (25%), fifty percent (50%), seventy five percent (75 %) and one hundred percent (100%) by volume respectively. There were five treatments used. Treatment (1) is the control with zero percent (0 %) concentration of the extract. Treatment (2) used 25 % concentration containing 10 ml of extract and 30 ml of distilled water. Treatment (3) used 50% concentration containing 20 ml of extract and 20 ml of distilled water. Treatment (4) used 75% concentration containing 30 ml of the extract and 10 ml of distilled water and lastly is  Treatment (5)  100% containing 40 ml of extract with no distilled water.                                 The prepared Madre de Cacao extract at varying concentrations were placed in a petri dish with three (3) replicates for each treatment, a total of fifteen (15) petri dishes. Thirty seven (37) larvae were added on each petri dish and were observed by the researchers for duration of twenty four (24) hours.  The number of dead larvae in each petri dish were then counted and recorded by the researchers.                 

                                                    Instrumentation                                          
                The materials and equipments used were:                                                                   Blender– an electrical appliance with whirling blades for chopping, mixing or liquefying foods. In the study, it is used for further grinding and mixing the pounded Madre de Cacao leaves.                                                                                          
Graduated Cylinder - used to measure the amount and percentage concentration of the extract.                                                                                                   
       Madre de Cacao leaves- the leaves were chopped pounded and extracted using a blender and a strainer.                                                                                     
Mortar and Pestle – used in pounding the Madre de Cacao leaves to obtain the   extract.                                                                                                                 
Mosquito larvae – a wormlike feeding form that hatch from the egg of household mosquito.                                                                                                              
        Petri Dish- used as a container for the set up and treatments.                                                            Strainer- used in separating the extract from the blended Madre de cacao leaves.
                                          Statistical Tools                                                                         
  To determine the level of effectiveness of Madre de Cacao leaf extract as larvicide for mosquitoes, the Mean was used. The Mean was obtained by adding the number of mosquito larvae that died in three replicates divided by three. The following descriptions were used:



Descriptions
Mean Ratings
(Number of mosquito larvae died)
Less effective
0 -9.24
Moderately effective
9.25-18.49
Effective
18.50-27.74
Very effective
27.75-37

           






To determine whether there is a significant difference in the level of effectiveness of Madre de Cacao leaf extract in killing mosquito larvae as to different concentrations, the One Way Analysis of Variance (ANOVA) was used using 0.01 level of significance.



                                                                 CHAPTER IV
Results and Discussion
                                                                                                                                                          
  This chapter presents the analysis, discussion and findings of the results of the study. This study investigated the level of effectiveness of Madre de Cacao leaf extract as a larvicide to mosquitoes. It also looked into whether there is a significant difference in the level of effectiveness of Madre de Cacao leaf extract as larvicide using varied concentration.

A.  Level of Effectiveness of Madre de Cacao leaf Extract on mosquito larvae.            
  To determine the level of effectiveness of Madre de Cacao leaf extract as larvicide on household mosquitoes, the mean was used. The description of the level of effectiveness is based on the following mean rating scale.                                                 A Mean Rating Scale of 0-9.24 is described as less effective, 9.25-18.49 as moderately effective, 18.50-27.74 as effective and 27.75-37 as very effective.  The results of the study are shown in Table 2.


Table 2.                                                                                                                       Results showing the treatment, number of mosquito larvae died in each replicate and the level of effectiveness.

Treatment

Number of Mosquito larvae died

Level of effectiveness
(Description)


Replicate 1
Replicate 2
Replicate 3
    Mean


1 (0%)
        0
1
1
     0.7


2 (25%)
      28
        28
     26
27.33
Effective

3 (50%)
       35
        37
    35
   35.67
Very effective

4 (75%)
       37    
       37
     37
     37
Very effective

5 (100%)
       37
       37
    37
     37
Very effective

Results on the treatment of Madre de Cacao leaf extract on mosquito larvae showed that in the duration of twenty four hours, in 25% concentration, 28 mosquito larvae died in Trial 1, 28 mosquito larvae died in Trial 2 and 26 mosquito larvae died in Trial 3 with the Mean of 27.33. This indicated that at this concentration, the Madre de cacao leaf extract is an effective larvicide on mosquitoes.                                              As regards, 50% concentration, 35 mosquito larvae died in Trial 1, 37 in Trial 2 and 35 died in Trial 3 with the Mean of 35.67. The results indicated that at this concentration Madre de Cacao leaf extract is considered as a very effective treatment for killing mosquito larvae.
When 75% and 100% concentration were used, all the 37 mosquito larvae died in Trials 1, 2 and 3 with the Mean of 37. This indicates that at this concentration Madre de Cacao becomes very effective in killing mosquito larvae.                                As to the control group, no mosquito larvae died in trial, and only 1 mosquito larvae died in trial 2 and trial 3 with a mean of 0.7.
B. Difference in the level of Effectiveness of Madre de Cacao leaf extract as a larvicide at varied concentration.
            To determine whether there is a significant difference in the level of effectiveness of  Madre de Cacao leaf extract in killing household mosquito larvae at different concentration, the One Way Analysis of Variance (ANOVA) was used and tested at 0.01 level of significance.                                                                          
  Results of the ANOVA computation showed that there were significant differences in the level of effectiveness of Madre de Cacao (Gliricidiasepium) leaf extract  between 0% concentration and 25% concentration, 0% concentration and 50% concentration, 0% concentration and 75% concentration, 0 and 100%, 25% concentration and 50% concentration, 25% concentration and 50% concentration, 25%oncentration and 75% concentration, and 25% concentration and 100% concentration since the obtained values were lower than .01. Therefore the null hypothesis was rejected. The data were shown in Table 5.
On the other hand when a test of significant difference between 50% concentration and 75% concentration, 50% concentration and 100% concentration, 75% concentration and 100% concentration were computed.  Results revealed that there is no significant difference between these concentrations since the obtained value is higher than 0.01. Therefore the null hypothesis was accepted
Table 3.                                                                                                                         Mean Standard Deviation and Standard Error at Different Concentration of Madre de Cacao Leaf Extract.

N
Mean
Std. Deviation
Std. Error
95% Confidence Interval for Mean
Minimum
Maximum
Lower Bound
Upper Bound
Control
3
.6667
.57735
.33333
-.7676
2.1009
.00
1.00
25%
3
27.3333
1.15470
.66667
24.4649
30.2018
26.00
28.00
50%
3
35.6667
1.15470
.66667
32.7982
38.5351
35.00
37.00
75%
3
37.0000
.00000
.00000
37.0000
37.0000
37.00
37.00
100%
3
37.0000
.00000
.00000
37.0000
37.0000
37.00
37.00
Total
15
27.5333
14.41164
3.72107
19.5524
35.5142
.00
37.00




Table 4.                                                                                      Results of the ANOVA computations showing the Significant Differences in the Level of Effectiveness of Madre de Cacao at Different Concentrations

No. of dead larvae
Sum of Squares
df
Mean Square
F
Sig.
Between Groups
2901.733
4
725.433
1209.056
.000
Within Groups
6.000
10
.600


Total
2907.733
14























Table. 5
Mulltiple Comparisons
Dependent Variable:   No. of Dead Larvae
LSD 
(I) Treatment
(J) Treatment
Mean Difference (I-J)
Std. Error
Sig.
99% Confidence Interval
Lower Bound
Upper Bound
Control-
25%
-26.66667*
.63246
.000
-28.6711
-24.6622
-32.9956
50%
-35.00000*
.63246
.000
-37.0044
75%
-36.33333*
.63246
.000
-38.3378
-34.3289
100%
-36.33333*
.63246
.000
-38.3378
-34.3289
25%
Control-
26.66667*
.63246
.000
24.6622
28.6711
-6.3289
50%
-8.33333*
.63246
.000
-10.3378
75%
-9.66667*
.63246
.000
-11.6711
-7.6622
100%
-9.66667*
.63246
.000
-11.6711
-7.6622
50%
Control-
35.00000*
.63246
.000
32.9956
37.0044
10.3378
25%
8.33333*
.63246
.000
6.3289
75%
-1.33333
.63246
.061
-3.3378
.6711
.6711
100%
-1.33333
.63246
.061
-3.3378
75%
Control-
36.33333*
.63246
.000
34.3289
38.3378
25%
9.66667*
.63246
.000
7.6622
11.6711
50%
1.33333
.63246
.061
-.6711
3.3378
100%
.00000
.63246
1.000
-2.0044
2.0044
38.3378
100%
Control-
36.33333*
.63246
.000
34.3289
25%
9.66667*
.63246
.000
7.6622
11.6711
50%
1.33333
.63246
.061
-.6711
3.3378
75%
.00000
.63246
1.000
-2.0044
2.0044






                                     CHAPTER V                                      
      Summary, Findings, Conclusions and Recommendations                    
This chapter presents the summary of the study, findings, conclusions and recommendations.

                                   Summary of the Study                                  
                    The primary objective of this study was to determine the larvicidal effectiveness of Madre de Cacao (Gliricidia sepium) leaf extract on household mosquito larvae (Culex pipiens).                                  
  Specifically, it sought to answer the following questions:                     
  1. What is the level of effectiveness of Madre de Cacao leaf extract as mosquito larvicide?                                                                           
2.Is there a significant difference in the level of effectiveness of Madre de Cacao leaf extract as a mo0squito larvicide at different concentrations?                                                                                              
                  The null hypothesis tested was there was no significant difference in the level of effectiveness of Madre de Cacao leaf extract in killing household mosquito larvae at different concentration.                                                                                    This study made use of the experimental method of research using the Parallel Group Design.                                                                
                There were 5 treatments used. Treatment 1 (0 %), Treatment 2 (25%), Treatment 3 (50%), Treatment 4 (75%), and Treatment 5 (100%). A total of 555 larvae were used. Each treatment with 111 larvae was divided into three replicates containing 37 larvae each. The mosquito larvae were cultured within 5-7 days and the experimental period lasted for 24 hours.                                                                                   
   The data obtained were analyzed using the mean to determine the level of effectiveness of Madre de Cacao leaf extract at different concentration in killing mosquitoes.                                                          
     The One Way Analysis of Variance (ANOVA) was used to determine the significant difference in the level of effectiveness at different concentrations of Madre de Cacao leaf extract using the 0.01 level of significance.



Findings of the Study
             The study revealed the following findings:                           
         1. The level of effectiveness of Madre de Cacao leaf extract at 25 % concentration was effective when treated with household mosquito larvae. The succeeding concentration of 50 %, 75 % and 100 % were found to be very effective in killing mosquito larvae.                          
        2. There was a significant difference in the level of effectiveness of Madre de Cacao leaf extract in killing household mosquito larvae between 0%  and extract at higher concentrations.                           
       Likewise, a significant difference was also noted in the level of effectiveness of Madre de cacao between  25% concentration and 50%, 25% and 75%, and 25% and 100%  concentration.                         
However, there was no significant difference in the level of effectiveness between 50% and75%, 50 % and 100% concentration.                                                                                                                                                                                                               
                               Conclusions                                 
    From the findings of the study, these conclusions were made: 1.Madre de Cacao Leaf extract is an effective household mosquito larvicide.
             Since Madre de Cacao leaf extract is an effective household mosquito larvicide, it is a potent substitute for commercial mosquito larvicide.                                                                                 
                   2.The effectiveness of Madre de cacao leaf extract in killing household mosquito larvae increases with higher concentrations.

                                     Recommendations                       
                         Based from the findings and conclusion of the study, the following recommendations were made:                                                            
1. Since Madre de Cacao is a potent larvicide, its use in eradicating and killing household mosquitoes is highly recommended especially on wet places prone to mosquitoes.   `                                  
    2. To reduce the side effects of using commercial pesticides in killing mosquitoes, the use of Madre de Cacao as a natural larvicide is recommended.                                                                                  
     3. A similar study should be conducted using a bigger number of larvae and other parts of Madre de Cacao to test its effectiveness as well as to verify the validity of the results.









REFERENCES



References
Alingayao, C. S. and Parado, M. M. (2008). Retrieved from sibugay-research.blogspot.com .
Anupam G., Nandita C. andGoutam C. (2012).Plant Extracts as Potential Mosquito Larvicides. Department of Zoology.The University of Burdwan. India.
Cornell University. (2004). Retrieved from http://www.ansci.cornell.edu/plants/medicinal/ gliricid.html.
Crans,W. J. and  Mahmood, F. (2008). Retrieved from http://www-rci.rutgers.edu/~insects/house.htm.
Glover, N.(1989). Gliricidia. Its Name Tells its Story: Nitrogen Fixing Tree Highlights. Winrock International, Arkansas, USA.
Mannetje, L. and Jones, R.M. (1996).Plant Resources of South-East Asia No. 4.Forages.(Pudoc Scientific Publishers, Wageningen, Netherlands.

Prakash, A. (2008). Retrieved from www.jbiopest.com/users/LW8/efiles/Anand%20Prakash_4_1.pdf.
Rahila, N. et al. (2008).Insecticidal, Nematicidal and Antibacterial Activities of GliricidiaSepium.Food and Marine Resources Research Center. Pakistan
Stewart, J.L., Allison, G.E. and Simons, A.J. (1996) Gliricidiasepium: Genetic resources for farmers. Oxford Forestry Institute, University of Oxford, UK.
Wiersum, K.F. and Nitis, I.M. (1996). Gliricidia sepium. Plant Resources of South-East Asia No. 4.Forages.(Pudoc Scientific Publishers, Wageningen, Netherlands.










                                      APPENDICES







Appendix A
Preparation of Extract
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Appendix B
Results in 0 % Concentration

Replicate
No. of Larvae Used
No. of Mosquito Larvae Died
1
37
0
2
37
1
3
37
1






Appendix C
Results in 25 % Concentration

Replicate
No. of Larvae Used
No. of Mosquito Larvae Died
1
37
28
2
37
28
3
37
26








Appendix D
Results in 50 % Concentration

Replicate
No. of Larvae Used
No. of Mosquito Larvae Died
1
37
35
2
37
37
3
37
35







Appendix E
Results in 75 % Concentration

Replicate
No. of Larvae Used
No. of Mosquito Larvae Died
1
37
37
2
37
37
3
37
37






Appendix F
Results in 100 % Concentration

Replicate
No. of Larvae Used
No. of Mosquito Larvae Died
1
37
37
2
37
37
3
37
37





4 comments:

  1. in what grams does madre de cacao leaves applied in this study? badly needed :-(

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