in vitro acaricidal effect of guyabano (annona muricata

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IN VITRO ACARICIDAL EFFECT OF GUYABANO (Annona muricata) CRUDE

ETHANOLIC SEED EXTRACT AGAINST BROWN DOG TICKS

(Rhipicephalus sanguineus)

KIRSTEN ALEXANDRA P. MERRITT

NOELLE FIDELIS D. VILLACORTA

Submitted to the

Department of Biology

College of Arts and Sciences

University of the Philippines Manila

In partial fulfillment of the requirements

for the degree of

Bachelor of Science in Biology

June 2015

ii

Department of Biology College of Arts and Sciences

University of the Philippines – Manila Padre Faura, Manila

ENDORSEMENT

The thesis attached hereto, entitled In Vitro Acaricidal Effect of Guyabano (Annona

muricata) Crude Ethanolic Extract Against Brown Dog Ticks (Rhipicephalus

sanguineus) prepared and submitted by Kirsten Alexandra P. Merritt and Noelle Fidelis

D. Villacorta, in partial fulfillment of the requirements for the degree of Bachelor of

Science in Biology was successfully defended on May 13, 2015.

Anna Theresa Santiago, MPH Thesis Adviser

Fredeslinda Evangelista, PhD Janice Velasco Ng, MS Thesis Reader Thesis Reader

This undergraduate thesis is hereby officially accepted as partial fulfillment of the

requirements for the degree of Bachelor of Science in Biology.

Miriam De Vera, PhD Alex B. Gonzaga, PhD, DrEng Chair Dean Department of Biology College of Arts and Sciences

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Table of Contents

PAGE

Table of Contents................................................................................................................iii

List of Tables.......................................................................................................................v

List of Figures.....................................................................................................................vi

List of Appendices.............................................................................................................vii

Acknowledgements..........................................................................................................viii

Abstract.......……................................................................................................................ix

Introduction...........………...........................................…………………………................1

Background of the Study..................................................................................1

Statement of the Problem…………………………….………….…................2

Research Objectives………………………………….…………….................2

Significance of the Study………………………………..................….……...3

Scope and Limitations of the Study...………………..………….....................4

Review of Related Literature……….………………........................…………..................6

Conventional Chemical Acaricidal Agents.......................................................6

Plant Derived Products (PDPs) as Anti-Parasitic Treatment............................7

Pharmacological Benefits of Guyabano (Annona muricata)............................8

Guyabano Seed Extract as an Alternative Acaricidal Agent..........................10

The Brown Dog Tick as an Important Ectoparasite and Vector.....................12

iv

Methodology..........……………………...........................………………………….........15

Plant Extraction..............................................................................................15

Acquisition of Ticks………………………………………………...............16

Adult Immersion Test (AIT)………………………………….…………….16

Biosafety Measures……………………………………………………........18

Data Presentation and Analysis.....................................................................18

Results...........……………………...........……………………………………...…...........20

Discussion..........................................................................................................................23

Conclusion and Recommendations..............…………......................................................27

References............………...……………………..........……………………...........…......29

Appendix………….……………………………………...……………………................42

Curriculum Vitae...............................................................................................................56

v

List of Tables

Table

1 Daily cumulative percent mortalities per treatment for the first trial................................44

2 Daily cumulative percent mortalities per treatment for the second trial.................44

3 Daily cumulative percent mortalities per treatment for the third trial......................45

4 Raw data on tick mortality (Treated with 2.5% Guyabano Extract) .......................45

5 Raw data on tick mortality (Treated with 25% Guyabano Extract).........................46

6 Raw data on tick mortality (Treated with Cypermethrin).........................................47

7 Raw data on tick mortality (Treated with Water) ......................................................47

8 Relative cumulative frequencies of mortalities per treatment (Trial 1) ..................48



11 Relative cumulative frequencies of mortalities per treatment…………………...49

12 Summary for daily mortality (Box plot data)........................................................49

13 Oviposition and hatching rate of eggs of individual females - Trial 1....................50

14 Oviposition and Hatching rate of eggs of females per treatment - Trial 1………51

15 Oviposition and hatching rate of eggs of individual females - Trial 2…………..51

16 Oviposition and hatching rate of eggs of females per treatment - Trial 2.............51

17 Oviposition and hatching rate of eggs of individual females per treatment -

Cypermethrin, Trial 3.............................................................................................52

18 Oviposition and hatching rate of eggs of females per treatment - Trial 3…….....52

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19 Average Percent Hatching Per Treatment (All Trials)...............................................52

20 Line-Item Budget............................................................................................................54

List of Figures

Figure

1 Annona muricata plant (Blanco, 1883)....................................................................9

2 Molecular structure of annonaceous acetogenins, bioactive compound

of Annona species extracts (Champy,2011)..........................................................12

3 Rhipicephalus sanguineus developmental stages..................................................13

4 Rhipicephalus sanguineus life cycle (University of Florida, 2011)......................14

5 Crushed guyabano seeds in 95% ethanol (a), extracting solution in

rotary evaporator (b), treatment solutions: Cypermethrin, left; 2.5%

guyabano, right; 25% guyabano, middle (c). ........................................................15

6 Positive control (upper left),negative control (upper right), 25%

guyabano (lower left), and 2.5% guyabano (lower right) treatment groups..........17

7 Relative Cumulative Frequencies of Tick Mortalities Per Treatment...................20

8 Boxplot data of summarized daily tick mortalities per treatment..........................21

9 Comparison of percent hatching of female ticks according to treatment..............23

10 STATA 11 main input box....................................................................................43

11 STATA 11 options input box.................................................................................44

12 STATA 11 results..................................................................................................44

13 Descriptives of each treatment from ANOVA.......................................................51

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14 ANOVA of Average Percent Mortality in All Treatments....................................22

15 Kruskal-Wallis Test of Percent Hatching in All Treatments with Descriptives....22

16 Mosquito chamber.................................................................................................57

16 Adult Immersion Test for all treatment.................................................................57

viii

List of Appendices

Appendix

A Sample Size Calculation using STATA11.............................................................44

B Observed Results During Experimentation Period................................................46

C Line Item Budget...................................................................................................56

D Tools Used in the Study.........................................................................................57

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ACKNOWLEDGEMENT

First and foremost we offer our sincerest gratitude to our adviser, Anna Theresa

Santiago, who has supported us throughout our thesis with her patience and knowledge.

We attribute the completion of our undergraduate thesis to her encouragement, effort and

unbelievably fast feedback. We could not have asked for a better, more accommodating

and helpful adviser.

Our thanks goes out to sir Glenn Sia Su for providing DB with the mosquito

chamber, where majority of our work took place. Also to Kuya Mando of the Muntinlupa

City Pound who collected and provided the dog ticks that we used in our study.

We would also like to thank our readers, Ms. Fredeslinda Evangelista and Ms.

Janice Ng, for offering their advice and insights and giving us their precious time all for

the improvement of our study. Of course, we thank our parents for all their support,

especially in terms of funding our study. And to our friends who are always willing to

lend a helping hand when ours are tied up and who cheered us on from start to finish.

Lastly, we thank the Lord almighty for giving us the strength, knowledge and

perseverance to finish this journey with all the best that we could give. Without Him and

the people He surrounded us with, all this wouldn’t have been possible.

x

ABSTRACT

The in vitro acaricidal activity of two concentrations (2.5% and 25%) of Annona

muricata (guyabano) ethanolic seed extracts was tested against the dog tick

Rhipicephalus sanguineus. Engorged, female brown dog ticks were exposed to distilled

water (negative control), Cypermethrin (positive control), 2.5% and 25% guyabano seed

extracts using the adult immersion test and were observed daily for mortalities and

oviposition. The results show an increasing trend in mortality by the 25% and 2.5%

guyabano seed extracts, exhibiting 100% and 93% mortality on the 17th day, as compared

with controls, with 71% and 54% mortality, respectively. Despite the observed trend, the

25% and 2.5% guyabano seed extracts were not significantly different from the controls

(p=0.271). The calculated 10-day LC50 of guyabano seed extract was at 12%. Mean tick

hatching was lowest in the 25% guyabano seed extract group (23.62%) followed by the

2.5% guyabano seed extract group (24.64%) but differences across treatment groups were

not statistically significant (p=0.320). It can be concluded that ethanolic seed extracts

taken from Annona muricata have potential acaricidal activity. In vivo studies are

recommended to assess the acaricidal activity of the extract in relation to various factors

and its possible effects on the host.

Keywords: Acaricide, Annona muricata seed, Cypermethrin, Extract, Rhipicephalus

sanguineus

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Introduction

Background of the study

Ectoparasites are major causes of distress and disease in many animals (Ettinger

& Feldman, 2005) because they are considered as obligate haematophagous external

parasites in mammals, birds, and reptiles throughout the world (Habeeb, 2010).

Specifically in dogs, ectoparasites are common and an important cause of pruritic and

non-pruritic skin disorders and hypersensitivity reactions. Brown ticks are among the

etiological agents responsible for health problems in dogs, causing primary injury on

infested hosts and acting as biological vectors of viruses, bacteria, protozoa, and

helminthes (Chomel, 2011). Moreover, some of the main tick-borne pathogens are also

zoonoses of public health concern (Brianti et al, 2013).

Control of ticks is usually done by treatment with conventional chemicals, but this

causes two major problems, first being the periodical application of biocide molecules

such as organophosphates or pyrethroids on dogs and pen surfaces often create

nonstandard conditions for their elimination such as variable concentration and

frequencies of application, unpredictable residual effect in the environment and on dogs,

among others (Otranto and Wall, 2008). More importantly, toxicity for both personnel

and animals represents a major concern for this strategy (Brianti et al, 2013). Secondly,

the rapid development of resistance to the active ingredients and residues of pesticides in

animals has caused great concern in society (Fair et al, 2003). Misuse of chemicals, sub-

dosage, inadequate preparation, poor implementation of application and the like have

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caused the development of resistance among ticks. Each time that ticks survive an

application of acaricide, they transmit genetic information on resistance to the product on

to subsequent generations (Furlong and Silver, 2006).

Plant-based pesticides present promising alternatives to conventional chemical

pesticides on top of well-explored medicinal properties of plants which have long been

used in developing countries to cure common illnesses. In ectoparasite elimination, the

use of plant extracts as an alternative approach to biocides employ the bioactive attributes

of plant-derived products (PDPs). PDPs are beneficial due to low mammalian toxicity,

short environmental persistence, and complex chemistries that would limit development

of pest resistance against them (George et al., 2014).

Guyabano, being the common name of Annona muricata in the Philippines, has

long been subjected to in vitro and in vivo studies that investigate plant species

effectiveness as alternatives in the control of parasites (Slomp et al, 2009). In herbal

medicine, the guyabano plant has been used as a cancer treatment; its chemical

components being proposed as anti-tumor (Keinan et al., 1998). Further studies of the

medical and veterinary importance of guyabano had been conducted in the present study.

Statement of the problem

Is guyabano seed extract an effective acaricide against brown dog ticks?

Research Objectives

The study aims to determine the acaricidal effect of guyabano seed extract on

brown dog ticks, specifically:

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1. To determine toxicity of Annona muricata seed crude ethanolic extract against

Rhipicephalus sanguineus based on LC50

2. To measure percent mortality of ticks caused by guyabano seed extract,

3. To compare, based on percent mortality and percent hatching, the acaricidal

effects of two dilutions of guyabano seed extract (2.5% and 25%) in vitro.

Hypotheses

1. Ho: Guyabano seed extract does not have a statistically significant acaricidal effect

against brown dog tick

Ha: Guyabano seed extract has a statistically significant acaricidal effect against

brown dog tick

2. Ho: Varying concentrations of guyabano seed extract do not differ in acaricidal

effects

Ha: Varying concentrations of guyabano seed extract have a statistically significant

difference in acaricidal effects.

Significance of the Study

Tick bites and tick-borne diseases continue to be a serious public health problem

throughout the world. They transmit viral, rickettsial, bacterial, and protozoal diseases

affecting domestic animals, and in rare cases, humans. Being harmful to their hosts and

vectors of diseases, the development of a means of control for ticks is thus of significant

importance. Many acaricides like fipronil, amitraz, carbaryl and pyrethroids such as

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deltamethrin, permethrin and cypermethrin are some of the synthetic acaricides that were

developed and most employed for the control of dog ticks (WHO, 2006). However, in

recent studies, some authors report resistance of ticks to commercial formulations

containing compounds present in acaricides like in those stated above (Miller et al, 2001).

The use of synthetic products are becoming less viable in practical terms, since its

excessive use causes environmental pollution, toxicity to humans and the appearance of

chemical residues in products of animal origin.

When considering control of dog ticks, it should be noted that the entire

population of the parasite may not be found on dogs at a given period. The engorged

female, being fully fed is larger than the nymph and is gray in color, detaches itself from

the host and lays eggs on the surrounding environment. The eggs then, after hatching,

find their way to its previous host, or may otherwise find a new host to feed upon

(ESCCAP, 2012). Therefore, effective elimination of brown dog ticks goes beyond

acaricide application; it may also require an integrated control strategy, aimed at both

canine population and environment management.

It is thus important to look for natural, alternative treatments for dog ticks that is

safe for the environment, humans and their host. The guyabano plant, showing anti-

parasitic properties, could therefore be a potential acaricide that fits this criteria.

Scope and Limitations

The extracts were obtained only from the guyabano seeds. Concentrations of

2.5% and 25% of Guyabano ethanolic extract were prepared. Water and Cypermethrin

5

served as negative and positive controls, respectively. A total of four treatments were

applied separately only to engorged females of species Rhipicephalus sanguineus

Latreille in the Adult Immersion Test in three separate trials. Ticks were obtained from

the Muntinlupa City dog pound with the assistance of the pound care taker and

immediately transported to the laboratory. Experimentation was conducted at the

Arthropod chamber at the Department of Biology, University of the Philippines Manila.

6

Review of Related Literature

Conventional Chemical Acaricidal Agents

The more popular methods of tick control depend more on chemical acaricides

and repellents. A wide range of acaricides, including chlorinated hydrocarbons,

organophosphates, carbamates, formamidines and synthetic pyrethroids are being used

for controlling ticks (Arijo, 2006). Chlorinated hydrocarbons (CHs) are synthetic

chemicals used as insecticides. DDT ("dichlorodiphenyltrichloroethane") is the most

well-known among the chlorinated hydrocarbons. Its insecticidal properties were

discovered in 1939 and led to its widespread utilization. However, CHs remain in the

environment for a very long time and bio-accumulate. Due to toxicity and lifespan, the

use of CHs has been banned (USGS, 2013) and were replaced by organophosphates

(esters of phosphoric acid) and carbamates. Although organophosphates remain in the

environment for a shorter time compared to CHs, these chemicals have been discovered

to pose a higher risk of toxicity in livestock. Carbamates, which are chemically similar to

organophosphates, were also found to be toxic to mammals (Arijo, 2006). At present,

synthetic pyrethroids are the most used insecticide against ticks. Pyrethroids are not

persistent in the environment as these easily breakdown in sunlight. Although pyrethroids

are less toxic than the previously enumerated acaricides, these have been discovered to

cause urban runoff, resulting to harmful levels in water (Andrade, 2013).

Bayticol (Bayer) and Amitraz®, two of the most commonly used brands of

synthetic acaricides, may become obsolete because of overuse and misuse in different

cattle ranches across Australia and Northern America (Kearney, 2013). Amitraz®, has

7

already been shown to be ineffective in causing the immediate death of ticks and that

some surviving ticks have accomplished engorgement and ovipositon of viable eggs,

thereby forcing entomologists to classify Amitraz® as useful for tick control but not tick

eradication (Burridge et al, 2003). Generally, prolonged misuse of acaricides has been

found to be the primary reason for the development of tick resistance to these compounds

and also have negative effects on the environment. It is in this light that natural

alternative acaricides be given attention in scientific studies.

Plant Derived Products (PDPs) as Anti-Parasitic Treatment

Plant products may be an alternative source of antiparasitic agents since they

constitute a rich source of bioactive compounds that are environmentally safe and non-

toxic to humans (Madhumitha, 2012). Several pesticides based on plant-derived products

(PDPs) are already available, and in some cases widely utilized, in modern pest

management. According to George et al (2014), numerous proof of concept studies

support that additional PDPs could be of further use, including targeting numerous

veterinary and medical pests. Many more have a long and continuing history of use in

poorer areas of the globe where access to synthetic pesticides is often limited.

One of the PDPs that are used widespread around the globe is technical grade

pyrethrum. It is extracted from dried and ground flowers of the daisy Tanacetum

cinerariaefolium and typically contains 20-25% pyrethrins (I and II) as its main pesticidal

components (Isman, 2006). Pyrethrum is known to act in much the same way as DDT

(dichlorodiphenyltrichloroethane), an insecticide, attacking sodium channels and serving

as a neurotoxin. Knock-down is subsequently relatively fast particularly in flying insects

(Casida, 1980). Pyrethrum remains in widespread use to the present day, with its current

8

contribution to veterinary and medical pest management being primarily in the treatment

of premise pests, such as cockroaches and flies, which could serve as disease vectors

(George et al, 2014).

The triterpene azadirachtin is another PDP to which much of the pesticidal

activity of the neem tree (Azadirachta indica) is attributed to. Formed from seed extracts,

where azadirachtin is naturally occurring at relatively low levels (<1%), it is concentrated

to 10-50% to form technical grade material (Isman, 2006). Neem seed extract is known to

display activity to a vast range of pest invertebrates like ticks, ectoparasitic mites

(including D. gallinae), scabies mites (Sarcoptes scabiei) and head lice (Pediculus

humanus capitis) (Singh & Saxena, 1999). The effects exerted by neem are also well

known and primarily attributed to feeding deterrence and disruption to growth, though

oviposition deterrence, repellence, reduced fitness and sterility (Schmutterer, 1990). In

terms of its mode of action, neem has been shown to target the cholinergic system in

insects through inhibition of acetylcholinestrase (AChE), and is also reported to disrupt

hormonal balance (Rattan, 2010). Work with insect cell lines has further shown that

azadirachtin (the main chemical component of neem) may exert an anti-mitotic effect by

disrupting tubulin polymerisation (Salehzadeh et al, 2003).

Pharmacological Benefits of Guyabano (Annona muricata)

Annona muricata L. from family Annonaceae is a medium-sized fruit tree

commonly found in the tropics (Figure 1). Although members of the family Annonaceae

are generally consumed as fresh fruits, they are also widely used in folk medicine.

Several reports have characterized the pharmacological activity of these plants because of

9

their bioactive compounds, mainly acetogenins flavonoids and alkaloids which are found

in roots, leaves, bark, seeds and fruit (Carballo, 2010).

Annona muricata, sometimes called Graviola, is a small, upright evergreen tree,

5–6 m high, with large, glossy, dark green leaves. It produces a large, heart-shaped,

edible fruit that is 15–20 cm in diameter and green in color, with white flesh inside

(Taylor, 2005). A. muricata has been widely used in other countries for folk medicine as

anthelmintic, antipyretic, sedative, antispasmodic, anti-convulsant and as hypotensive

agent in humans.

Figure 1. Annona muricata plant (Blanco, 1883).

Investigations carried out with different species of the genus Annona showed that

aqueous extracts (Alawa et al, 2003; Ndjonka et al, 2011) and methanol extracts and ethyl

acetate extracts (Souza et al, 2008; Kamaraj & Abdul Rahuman, 2011) present in vitro

antihelminthic properties. The biological activities of Annona extracts have been

attributed to the occurrence of annonaceous acetogenins, which are a class of natural

compounds extracted from leaves and seeds (Chang and Wu, 2001) of species of the

Annonaceae family. In one of their studies, Souza et al. (2008) demonstrated the activity

of these compounds against parasites, specifically as an antihelminthic. In addition,

10

according to a study by Keinan et al. in 1998, these chemicals in general have been

documented with anti-tumorous, antiparasitic, insecticidal, and antimicrobial activities.

Further studies of the medical and veterinary importance of the guyabano have been

conducted more extensively in the past years.

Guyabano seed extract as an alternative acaricidal agent

Recent studies show the efficacy of different species of Annona extracts, such as

A. squamosa as an acaricidal against ectoparasites such as the cattle tick, Rhipicephalus

microplus (Madhumitha, 2012). In a study conducted by Broglio-Micheletti et al. in

2009, the extracts from the seeds of guyabano showed promising performance in vitro to

control cattle tick due to reduced oviposition of gravid females and reduced larval

hatchability by 100%. Also, in a study by Chungsamarnyart et al (1994), the seeds of

Annona muricta were tested as an acaricide to tropical cattle ticks (Boophilus microplus).

They also showed that a combination of plant extracts could also be a factor in acaricidal

activity, due to synergistic action of certain combined plant extracts. This emphasizes the

fact that there may be many factors that affect the acaricidal activity of extracts. Annona

squamosa, a plant which shares the same genera as Annona muricata, also exhibits

insecticidal properties from its seed extracts against numerous pests like the red flour

beetle (T. castaneum), leafhopper (N. lugen), hairy caterpillar, (S. obliqu), Brinjal spotted

leaf beetle (H. vigintioctopunctata) and the cotton boll worm, (D. koenigii)

(Khalequzzaman & Sultana, 2006). In a study conducted by Bagavan et al. (2009),

different crude solvent extracts were used on different plants and plant parts. This showed

the significance of different crude solvent extracts due to the difference in results on

different plant and plant part when tested on parasites.

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Several studies have used different forms of the extract from Annona species as

potential acaricide, for instance, aqueous and ethanolic. Between the two, ethanolic

extracts posed the highest efficacy, not to mention, ethanolic extracts at high

concentrations (Nahar et al, 2005). Ethanolic and aqueous extracts from different parts of

A. squamosa have been tested on a number pests. In the study of Breceda et. Al. (2012),

A. squamosa leaf and stem extracts were used as a larvicidal against the mexican fruit fly

(Anastrepha ludens). In another study by Lima et. al. (2011), A. squamosa leaf extracts

were again used as a larvicidal against the diamondback moth (Plutella xylostella), a pest

of cabbage. Larva of the diamondback moth (Plutella xylostella) exhibited a 100%

mortality when exposed to the leaf extracts.

Acetogenins are active secondary metabolites produced across almost all Annona

species, particularly in the seeds (thus, the common term annonaceous acetogenins).

These compounds are comprised of long carbon chains (about 30-40 carbon atoms)

originating from fatty acid group of biomolecules. Notable functional groups include an

alkyl chain bearing oxygen-containing tetrahydrofuranic (THF) rings and a terminal

butyrolactone (Figure 2). Acetogenins are found to be strong inhibitors of mitochondrial

complex I (NADH ubiquinone oxidoreductase), an integral component in the electron

transport chain phase of cellular respiration. These compounds are the subject of ongoing

studies, for instance, on Annona squamosa extracts as pest controls, especially in the

tropical regions. Furthermore, it was found to have abortifacient effects on pest females, a

very significant method of population control in insects and acarids (Khalequzzaman &

Sultana, 2006).

12

Figure 2. Molecular structure of annonaceous acetogenins, bioactive compound of

Annona species extracts (Champy, 2011).

The Brown Dog Tick as an Important Ectoparasite and Vector

The brown dog tick Rhipicephalus sanguineus (Figure 3) is the most widespread

tick of the ixodid species in the world. Ixodidae are the family of hard ticks and are

characterized by the presence of a scutum (hard shield) (Parolla and Raoult, 2001).

Rhipicephalus sanguineus in particular is characterized by its red-brown color, elongated

body shape, and hexagonal basis capituli, the latter being a diagnostic character of the

species (Lord, 2001). It is a parasite of dogs that can occasionally parasitize other hosts,

including humans (Torres, 2010). R. sanguineus is a three-host tick species, endophilic or

adapted to indoor living and monotropic meaning all developmental stages feed on the

same host species (Figure 3).

13

Figure 3. Rhipicephalus sanguineus developmental stages

Rhipicephalus sanguineus are carriers of numerous pathogens which can be

transferred to the host while it feeds. Though brown dog ticks seldom bite hosts other

than dogs, there are a few diseases which may be transmitted to humans. Rickettsia

conorii, bacteria which can be carried by R. sanguineus, affect dogs and is the cause of

Mediterranean spotted fever in humans (University of Florida, 2011). Rickettsia rickettsii,

another pathogen in R. sanguineus, causes Rocky Mountain Spotted Disease in humans.

This tick borne disease can be fatal to the human host (CDC, 2013). R. sanguineus can

also transmit the pathogens Babesia vogeli and Ehrlichia canis which cause babesiosis

and ehrlichiosis in dogs (Dantas-Torres et al., 2013).

Ticks in the family ixodidae require three hosts to complete its life cycle. Its

preferred hosts are domesticated dogs but will occasionally feed on other hosts (e.g.

humans) to survive (Dantas-Torres, 2010). An adult female will feed on the host for

around one to two weeks, then drop off the host. It has a pre-oviposition period of three

days to two weeks and then lays its eggs (Dantas-Torres, 2010). The engorged female

lays about 1000 to 4000 eggs, the oviposition period lasting for several weeks and the

number of eggs laid by each female directly correlated with the weight and the length of

14

the oviposition period (Koch, 1982). However, disturbed females can interrupt

oviposition although losses in egg production efficiency are usually minor (Dantas-

Torres, 2010). The larvae hatch two to five weeks later and feed for three to seven days,

then take about two weeks to develop into nymphs. The nymphs then feed for five to ten

days and again take about two weeks to develop into adults (Lord, 2001). As adults, both

males and females will attach to hosts and feed, although the males only feed for short

periods. The overall cycle can be completed in just over two months, but frequently will

take longer if there are few hosts available or in cold temperatures (Lord, 2001). Ticks

are notoriously long-lived, and can live as long as three to five months in each stage

without feeding. According to the European Medicines Agency (EMA) (2007), the earlier

stages of life (egg, larvae, nymph) of the R. sanguineus are more sensitive to drugs which

is why in laboratory testings for efficacy of acaricides, it is most convenient to test on an

engorged adult female.

Figure 4. Rhipicephalus sanguineus life cycle (University of Florida, 2011)

15

Methodology

Plant Extraction

The fruit of Annona muricata was obtained from Robinsons Supermarket in

Ermita, Manila, Philippines. Preparation of the crude ethanolic extract of guyabano

followed a protocol modified from Chungsamarnyart, (1994) and Broglio-Micheletti et

al., (2009). Approximately 100 grams of A. muricata seeds were dried under the sun for

48 hours. The dried seeds were powdered mechanically using commercial electrical

stainless steel blender until fine grains of sizes between 0.06-2.0 millimeters in diameter

were obtained. One part fine powder of approximately 100 grams was immersed in three

parts of approximately 300 milliliters 95% ethanol for 48 hours in a covered erlenmeyer

flask. The solution was then evaporated using a rotary evaporator to obtain the crude

extract. In separate erlenmeyer flasks, 0.5 ml or 5 ml of the crude ethanolic extract was

mixed with 20 mL of 95% ethanol to obtain the 2.5% and 25% concentrations,

respectively.

Figure 5. Crushed guyabano seeds in 95% ethanol (a), extracting solution in rotary

evaporator (b), treatment solutions: Cypermethrin, left; 2.5% guyabano, right; 25%

guyabano, middle (c).

(a) (b) (c)

16

Acquisition of Tick Subjects

A total of 104 engorged female ticks were obtained for all three (3) trials, a

minimum of 24 engorged female ticks per trial, weighing up to 350mg (Capinera, 2008),

of Rhipicephalus sanguineus were obtained from stray dogs found in the Muntinlupa City

Dog Pound in Tunasan, Muntinlupa City. The ticks were then randomly assigned to each

treatment group - 2.5% guyabano seed extract, 25% guyabano seed extract, distilled

water and Cypermethrin, such that each of the treatment groups will include at least 6

ticks which is the minimum calculated n=6 (Appendix A).

Adult Immersion Test (AIT)

Engorged female ticks were washed in distilled water to remove any eggs laid

during transport. Females who have already laid eggs were excluded from the set up. In

vitro testing was carried out by using the modified adult immersion test (AIT) based on

the protocols by Jonsson, Miller, & Robertson (2007) and Domingues et al. (2013). The

engorged female ticks were weighed and separated into groups with at least 6 ticks of

homogenous weights. The ticks were then immersed in Petri plates (5.5 cm diameter, 1.5

cm high) containing 15 mL of the respective treatment solutions, distilled water as

negative control and Cypermethrin, a chemical widely used as an insecticide, as positive

control for 5 minutes, with three trials for each test solution including the negative and

positive control (Figure 6). The ticks were then dried on absorbent paper and adhered to

masking tape strips in Petri plates and incubated at 27±1 ºC at relative humidity > 80%

and a 12:12 hour (L: D) photoperiod.

17

Figure 6. Positive control (a),negative control (b), 25% guyabano (c), and 2.5%

guyabano (d) treatment groups.

Mortality was observed and recorded daily until all treatment groups reached

100% mortality by counting dead ticks which were then transferred onto separate Petri

plates. Dead ticks were identified by cuticular darkness and absence of response to

stimuli (Aguilar et al, 2010). LC50 and percent mortality were computed using the

formulas found in Equation 1 and 2. The daily progress of the experiment was

documented through photographs and the number of females that have laid eggs

including number of eggs per oviposition were observed using a stereomicroscope. The

petri plates were then observed again under a stereomicroscope for the counting of the

number of hatched eggs and the percent hatched, computed using Equation 3 and

compared to the controls. The hatched eggs were determined by observing the apparent

change in color, from an opaque brown to a translucent grey, and by recognizing a

longitudinal fissure seen on the egg chorion (Dantas-Torres, 2010).

18

Equation 1. Median Lethal Concentration

LC50 = antilog {A + [(B/C)*D]}

where, A = log concentration below 50% mortality, B = 50 - mortality below 50%,

C = mortality above 50% - mortality below 50%, and

D = log concentration above 50% - log concentration below 50%

Equation 2. Percent Mortality

Percent Mortality = number of dead ticks x 100

total number of ticks

Equation 3. Percent Hatching

Percent Hatching = number of hatched eggs x 100

total number of eggs

Biosafety Measures

The investigators wore protective clothing including gloves and light-colored

clothing with long pants tucked into socks, to make ticks easier to detect, as well as

wearing closed shoes and keep the ticks on the outside of the clothes. Also, long hair was

tied at all times. Forceps were used at all times in handling ticks. The only reagent used,

95% Ethanol, was placed inside the mosquito chamber at room temperature, away from

acids or any oxidizing materials.

Data Presentation and Analysis

The percent mortality and percent hatching of ticks subjected to 2.5% guyabano,

25% guyabano, positive control (Cypermethrin) and negative control (Distilled water)

were summarized using Microsoft Excel (version 12.0) and PAST (Ver.3.x). Percent

19

mortality was compared through one way Analysis of Variance (ANOVA) while percent

hatching was evaluated through Kruskal-Wallis Test using GNU PSPP (Version 0.8.4)

both set at 0.05 level of significance.

20

Results

The results of the present study demonstrated the effect of the guyabano seed

extract in killing adult Rhipicephalus sanguineus in vitro in a shorter period of time as

compared with the controls. LC50 from adult immersion in guyabano seed extract was

computed using the modified Reed-Muench Method (Sanganuwan, 2011), the LC50 of the

guyabano seed extract was found to be 12.2% in the span of 10 days.

Based on Figure 7, engorged female dog ticks treated with 25% guyabano seed

extract reached 100% cumulative mortality within the 16th- 18th day, the earliest among

all treatments. This is followed by 2.5% guyabano seed extract having reached 100%

cumulative mortality on the 22nd- 24th day, then Cypermethrin on the 25th- 27th day, and

lastly, water on the 28th- 30th day. Although mortality in varying treatment groups showed

no statistically significant difference (p=0.271), an increasing trend in mortalities was

found in engorged female dog ticks exposed to 25% and 2.5% guyabano seed extract

Figure 7. Relative Cumulative Frequencies of Tick Mortalities Per Treatment (Based on

Table 12, Appendix B)

21

The two concentrations of the guyabano seed extracts used had a more immediate

acaricidal effect on the mortality of the dog ticks in relation to the negative control,

distilled water (Figure 8). Figure 8 below shows that 2.5% and 25% guyabano have the

earliest onset of mortality, both on the 2nd day, followed by Cypermethrin and the

negative control, having their first mortalities on the 9th day. First to reach 50% mortality

is 25% guyabano on the 8th day, followed by 2.5% guyabano on the 10th day,

Cypermethrin on the 13th day, and the negative control on the 16th day. The 25%

guyabano comes first in reaching 100% mortality on the 16th day, followed by 2.5%

guyabano on the 18th day, Cypermethrin and the negative control, on the 19th and 22nd

day, respectively.

Figure 8. Boxplot data of summarized daily tick mortalities per treatment (Based from

Table 13, Appendix B).

Figure 9 shows the average hatching rate for all three trials. The percent hatching

showed no significant difference (p=0.320), although both guyabano concentrations had

22

smaller percent hatching values than the negative control. Female dog ticks treated in

water exhibited the highest percent hatching at 49%, followed by Cypermethrin at 35%,

then by 25% guyabano at 25% and lastly, 2.5% guyabano with the lowest percent

hatching at 24%. The hatching rate of each treatment was computed by dividing the

number of hatched eggs by total number of eggs laid per individual female dog tick, and

then averaging all hatching rate values obtained from each female in their respective

group (Tables 15-19 in Appendix B).

Figure 9. Comparison of percent hatching of female ticks according to treatment (Based

on Table 20, Appendix B)

23

DISCUSSION

The study revealed a trend of greater mortality and lower percent hatching at both

concentrations of guyabano seed extract compared to the positive control. The 25%

guyabano seed extract showed a trend of having even greater effect compared to 2.5%

both in percent mortality and percent hatching as its concentration was increased tenfold

thus possibly increasing any effect it would have on the subjects. However, both

mortalities and percent hatching in different treatment groups showed no significant

difference.

Other studies on the Annona muricata seeds extracts reported its activity against

other ectoparasites in animals. In a similar study conducted by Broglio-Micheletti (2009),

various plant extracts were tested against Rhipicephalus microplus and it was found that

2% ethanolic seed extracts from Annona muricata also exhibited the highest mortality

(100%) within 21 days and reduced larvae hatchability by 100%. It was only the

guyabano extract that differed from the other treatments tested in relation to larval

hatching as there were none observed in the said treatment group. In a study by

Chungsamarnyart (1991) on tropical cattle ticks, Annona muricata exhibited 91-99%

mortality seven days after dipping.

The 10 day LC50 for the ethanolic extracts of Annona muricata seeds was found to

be 12.2%. This value is substantially higher compared to those found in Broglio-

Michelleti who reported that 2% crude ethanolic extracts of Annona muricata caused

100% mortality in Rhipicephalus microplus within 21 days. Variability in values may be

attributed to differences in sensitivity of test species used.

24

The death of adult ticks in this study may be attributed to the different chemical

components present in the seeds of guyabano, namely: annomuricatin A, annomuricatin

B, annomuricatin C, murihexol, donhexocin, and annonacin. In a study conducted by

Dahiya et al. (2009), annomuricatin B, a cycloheptapeptide, was synthesized and found

to exhibit cytotoxic activity against Ehrlich’s ascites carcinoma by apoptosis, antifungal

activity against Candida albicans, and bioactivity against dermatophytes. Murihexol,

donhexocin, and annonacin are acetogenins isolated from the seeds of Annona muricata.

Several studies show that the acaricidal property of Annona muricata is probably due to

its annonaceous acetogenins, specifically annonacin (Rieser, et al., 1993). Annonacin is

the most abundant acetogenin in A. muricata (McLaughlin, 2008) and are potent

inhibitors of NADH ubiquinone oxidoreductase, which is an essential enzyme in complex

I of the electron transport system (ETS) which eventually leads to oxidative

phosphorylation in mitochondria, the end result of which is ATP deprivation (Kedari, et

al., 2014).

The mortality rate in guyabano seed extract treated ticks may probably be

attributed to cuticular absorption of the different compounds present in the said extract.

The ticks’ integument is composed of the epicuticle, procuticle and hypodermis.

Annonacin being a lipophilic compound, can thus pass through the non-polar, lipophilic

integument of the ticks (Bermejo et al., 2005).

A possible reason for the larger number of mortalities in 2.5% and 25% guyabano

than the positive control, Cypermethrin, is the combined activity of the extract along

along with the solvent, 95% ethanol. In a study by Goncalves et al. (2006), the effect of

various solvents and detergents on Rhipicephalus microplus based on inhibition of

25

fecundity and percent mortality was evaluated. It was found that absolute ethanol had

14.2% mortality which was the lowest compared to Methanol (45.3% mortality) and

acetone (100% mortality). Also, no observations were made on the effect of the

solvents/detergents on hatching of the laid eggs by the treated ticks in that study. The

toxic effects of various solvents against Rhipicephalus annulatus were studied by

Ravindran et al. (2011) and they came up with similar results showing that ethanol had

higher mortality compared to the control.

The low number of mortalities of the positive control, Cypermethrin, may

possibly be due to resistance developed by ticks to the specific chemical. Numerous

studies have been conducted on resistance to certain commercial products that are

regularly used in households, farms, pounds and other places that warrant the elimination

of different species of ticks. A study by Sharma et al. (2012) on Rhipicephalus microplus

resistance to certain synthetic pyrethroids (SP) in farms noted that the overall prevalence

of SP-resistant ticks among the sampled farms was 66.6%. Cypermethrin, one of the SPs

investigated did not have as high a resistance compared to other SPs, although the results

showed a higher level of resistance in farms who utilize the said product extensively and

regularly. Also, according to Millar (1988) resistance to pyrethroids is usually described

in terms of family resistance and ticks resistant to one pyrethroid compound cannot be

controlled by another compound for a long time. In another study on resistance of cattle

ticks by Mendez et al. (2011), 82.6% of the populations studied were resistant to

Cypermethrin, levels of which varied from high to low.

The source of ticks for this study was from a dog pound where there is regular use

of acaricides to control tick levels. It has been reported that ever since its formulation

26

around the 1960s, Cypermethrin has been commercially available worldwide and has

been in use in the Philippines since 1970s (Heong, 1997). The continuous use of the

product ever since its availability is most likely the reason for resistance in ticks and thus

may explain why a delayed onset in mortality was observed in the results.

27

CONCLUSION AND RECOMMENDATIONS

The bioactivity of ethanolic guyabano seed extracts in two different

concentrations, 2.5% and 25%, against dog ticks or Rhipicephalus sanguineus was

investigated. The 10 day LC50 of the seed extracts was calculated as 12.2%. Based on the

results of the study, 25% guyabano showed the highest mortality rate followed by 2.5%

guyabano and then by Cypermethrin, the positive control, and then lastly, by the negative

control. The 25% guyabano also exhibited the lowest hatching rate among the four

treatments, closely followed by 2.5% guyabano and Cypermethrin, respectively. The

negative control, water, exhibited the highest hatching rate among all treatments. The

effects of guyabano seed extract on mortality and hatching were possibly brought about

by the annonacin found in the seeds of guyabano. The guyabano seed extracts were not

statistically different from the controls based on mortality (p=<0.271) and hatching

(p=0.320) values, although both have greater potential acaricidal effect compared to the

two controls.

The results of this study indicate that based on percent mortality and percent

hatching, the extracts from guyabano seeds may serve as a potential alternative to

chemical acaricides, exhibiting almost the same results as the latter. Besides causing

mortality of gravid females in the early days after treatment, they interfere with

replication which can be an alternative to chemical acaricides normally used. However,

an in vivo study is recommended to further assess its acaricidal activity in relation to

various factors and possible adverse reactions to the host animal. Also, identification and

28

further study of the active component and the mechanism through which it acts on the

ticks are recommended.

29

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42

Appendix A

Sample Size Calculation using STATA11

In Politi et al. (2012), it was found that 50.0 mg/mL concentration of T. patula

exhibited the best results against ticks. Using STATA 11, the minimum sample size was

determined by comparing the mortality of ticks in water and 50 mg/mL T.patula. Two-

sample comparison of proportions was chosen and the mortality of the ticks in water

(10.48%) and 50.0 mg/mL T.patula (99.78%) were inputted.

Figure 10. STATA 11 main input box

On the options tab, the significance level was set to 0.05. The significance level is

the probability of a Type I error (a false rejection of the null hypothesis). The power was

set to 0.80. The power of the statistical test is the likelihood of not committing a Type II

error (acceptance of a false null hypothesis.)

43

Figure 11. STATA 11 options input box

Below it is seen that the minimum sample size required for each treatment is 6.

Increasing the sample size, increases the significance as the sample better represents the

population.

Figure 12. STATA 11 results

44

Appendix B

Observed Results During Experimentation Period

Table 1. Daily cumulative percent mortalities per treatment for the first trial

Day

2.5 % Guyabano

(n=10)

25 % Guyabano

(n=10)a

(+) Cypermethrin

(n=10)

(-) Water

(n=10)

1 0 0 20 0

2 20 0 30 0

3 20 10 30 0

4 20 10 30 0

5 30 10 50 0

6 40 10 50 30

7 50 10 50 30

8 50 20 50 30

9 50 50 70 30

10 60 50 100 50

11 70 50 100 50

12 70 60 100 60

13 70 70 100 70

14 90 80 100 90

15 90 90 100 100

16 100 90 100 100 a100% cumulative percent mortality achieved on 17th day

Table 2. Daily cumulative percent mortalities per treatment for the second trial

Day

2.5 % Guyabano

(n=10)a

25 % Guyabano

(n=10)

Cypermethrin

(n=10)b

Water

(n=10)c

1 10 10 0 0

2 10 10 0 0

3 10 10 0 0

4 10 10 0 0

5 10 20 0 0

6 10 20 0 0

7 10 30 0 0

8 10 30 0 0

9 20 30 0 0

10 20 50 0 0

11 40 50 0 20

12 50 60 10 20

13 60 80 20 20

14 70 90 30 20

45

Table 2. (Continued) Daily cumulative percent mortalities per treatment for the second

trial

15 80 100 30 20

16 80 100 40 20

17 80 100 40 20

18 80 100 40 30 a100% cumulative percent mortality achieved on 22nd day b100% cumulative percent mortality achieved on 26th day c100% cumulative percent mortality achieved on 28th day

Table 3. Daily cumulative percent mortalities per treatment for the third trial

Day

2.5% Guyabano

(n=6)

25% Guyabano

(n=6)

Cypermethrin

(n=7)a

Water

(n=7)b

1 0 0 0 0

2 0 0 0 0

3 0 33 0 0

4 16.7 50 0 0

5 16.7 66.7 0 0

6 16.7 66.7 0 0

7 16.7 66.7 0 0

8 16.7 66.7 0 0

9 33 66.7 0 0

10 50 83 0 0

11 66.7 83 0 14.3

12 66.7 83 0 14.3

13 83 83 0 14.3

14 83 83 14.3 42.9

15 83 83 28.6 42.9

16 100 100 57.1 42.9

17 100 100 71.4 42.9

18 100 100 71.4 57.2 a100% cumulative percent mortality achieved on 21st day b100% cumulative percent mortality achieved on 22nd day

Table 4. Raw data on tick mortality (Treated with 2.5% Guyabano Extract)

Day

n=10 n=10 n=6

Trial 1 % Trial 2 % Trial 3 %

1 0 0 1 10 0 0.0

2 2 20 0 0 0 0.0

3 0 0 0 0 0 0.0

4 0 0 0 0 1 16.7

5 1 10 0 0 0 0.0

6 1 10 0 0 0 0.0

46

Table 4. (Continued) Raw data on tick mortality (Treated with 2.5% Guyabano Extract)

7 1 10 0 0 0 0.0

8 0 0 0 0 0 0.0

9 0 0 1 10 1 16.7

10 1 10 0 0 1 16.7

11 1 10 2 20 1 16.7

12 0 0 1 10 0 0.0

13 0 0 1 10 1 16.7

14 2 20 1 10 0 0.0

15 0 0 1 10 0 0.0

16 1 10 0 0 1 16.7

17 0 0 0 0 0 0.0

18 0 0 0 0 0 0

19 0 0 1 10 0 0

20 0 0 0 0 0 0

21 0 0 0 0 0 0

22 0 0 1 10 0 0

Table 5. Raw data on tick mortality (Treated with 25% Guyabano Extract)

Day

n=10 n=10 n=6


1 0 0 1 10 0 0.0

2 0 0 0 0 0 0.0

3 1 10 0 0 2 33.3

4 0 0 0 0 1 16.7

5 0 0 1 10 1 16.7

6 0 0 0 0 0 0.0

7 1 10 1 10 0 0.0

8 3 30 0 0 0 0.0

9 0 0 0 0 0 0.0

10 0 0 2 20 1 16.7

11 1 10 0 0 0 0.0

12 1 10 1 10 0 0.0

13 1 10 2 20 0 0.0

14 1 10 1 10 0 0.0

15 0 0 1 10 0 0.0

16 0 0 0 0 1 16.7

17 1 10 0 0 0 0

47

Table 6. Raw data on tick mortality (Treated with Cypermethrin)

Day

n=10 n=10 n=7


1 2 20 0 0 0 0.0

2 1 10 0 0 0 0.0

3 0 0 0 0 0 0.0

4 0 0 0 0 0 0.0

5 2 20 0 0 0 0.0

6 0 0 0 0 0 0.0

7 0 0 0 0 0 0.0

8 0 0 0 0 0 0.0

9 2 20 0 0 0 0.0

10 3 30 0 0 0 0.0

11 0 0 0 0 0 0.0

12 0 0 1 10 0 0.0

13 0 0 1 10 0 0.0

14 0 0 1 10 1 14.3

15 0 0 0 0 1 14.3

16 0 0 1 10 2 28.6

17 0 0 0 0 1 14.3

18 0 0 0 0 0 0.0

19 0 0 1 10 1 14.3

20 0 0 0 0 0 0

21 0 0 1 10 1 14.3

22 0 0 1 10 0 0

23 0 0 0 0 0 0

24 0 0 2 20 0 0

25 0 0 0 0 0 0

26 0 0 1 10 0 0

Table 7. Raw data on tick mortality (Treated with Water)

Day

n=10 n=10 n=7


1 0 0 0 0 0 0.0

2 0 0 0 0 0 0.0

3 0 0 0 0 0 0.0

4 0 0 0 0 0 0.0

5 0 0 0 0 0 0.0

6 3 30 0 0 0 0.0

7 0 0 0 0 0 0.0

8 0 0 0 0 0 0.0

9 0 0 0 0 0 0.0

10 2 20 0 0 0 0.0

48

Table 7. (Continued) Raw data on tick mortality (Treated with Water)

11 0 0 2 20 1 14.3

12 1 10 0 0 0 0.0

13 1 10 0 0 0 0.0

14 2 20 0 0 2 28.6

15 1 10 0 0 0 0.0

16 0 0 0 0 0 0.0

17 0 0 0 0 0 0.0

18 0 0 1 10 1 14.3

19 0 0 0 0 1 14.3

20 0 0 1 10 1 14.3

21 0 0 0 0 0 0

22 0 0 1 10 1 14.3

23 0 0 1 10 0 0

24 0 0 2 20 0 0

25 0 0 0 0 0 0

26 0 0 0 0 0 0

27 0 0 1 10 0 0

28 0 0 1 10 0 0

Table 8. Relative cumulative frequencies of mortalities per treatment (Trial 1)

Day

Treatment

2.5% Guyabano 25% Guyabano Cypermethrin (+) Negative Control

1st-3rd 20 10 30 0

4th-6th 40 10 50 30

7th-9th 50 50 70 30

10th-12th 70 60 100 60

13th-15th 90 90 100 100

16th-18th 100 100 100 100


Day

Treatment


1st-3rd 10 10 0 0

4th-6th 10 20 0 0

7th-9th 20 30 0 0

10th-12th 50 60 10 20

13th-15th 80 100 30 20

16th-18th 80 100 40 30

19th-21st 90 100 60 40

22nd-24th 100 100 90 80

25th-27th 100 100 100 90

28th-30th 100 100 100 100

49


Day

Treatment


1st-3rd 0.0 33.3 0.0 0

4th-6th 16.7 66.7 0.0 0

7th-9th 33.3 66.7 0.0 0

10th-12th 66.7 83.3 0.0 14.3

13th-15th 83.3 83.3 28.6 42.9

16th-18th 100.0 100.0 71.4 57.2

19th-21st 100.0 100.0 100.0 85.7

22nd-24th 100.0 100.0 100.0 71.4

Table 11. Relative cumulative frequencies of mortalities per treatment (Average of All

Trials)

Day

Treatment

2.5% Guyabano 25% Guyabano Positive control Negative control

1-3 10.00 17.76 10.00 0.00

4-6 22.23 32.23 16.67 10.00

7-9 34.47 48.90 23.33 10.00

10-12 62.27 71.13 36.67 31.43

13-15 84.50 91.13 52.87 54.30

16-18 93.40 100.00 70.50 62.40

19-21 96.73 100.00 86.70 75.27

22-24 100.00 100.00 96.70 93.37

25-27 100.00 100.00 100.00 96.70

28-30 100.00 100.00 100.00 100.00

Table 12. Summary for daily mortality (Box plot data)

2.5% 25% + -

Average Minimum Day of First

Mortalities 2.33 2.33 9.00 9.33

Average Day of 25% Mortalities 7.33 5.67 9.67 10.33




50

Figure 13. Descriptives of each treatment from ANOVA

Table 13. Oviposition and hatching rate of eggs of individual females - Trial 1

2.5% Guyabano

Female number Hatched total Hatching rate (%)

1 121 153 79.08

2 40 109 36.70

3 195 246 79.27

4 80 107 74.77

5 307 388 79.12

6 327 429 76.22

Total 1432 Average = 70.86

25% Guyabano

1 109 112 97.32

2 212 272 77.94

3 478 582 82.13

4 0 35 0.00

5 357 401 89.03

6 278 340 81.76

7 89 112 79.46

8 135 161 83.85

Total 1658 2015 Average = 73.94

Cypermethrin

1 101 230 43.91

2 76 160 47.50


Negative Control

1 61 72 84.72

2 382 491 77.80

3 62 119 52.10

4 338 391 86.45

51

Table 13.(Continued)Oviposition and hatching rate of eggs of individual females- Trial 1

5 312 355 87.89

6 161 206 78.16


Table 14. Total Oviposition and Hatching rate of eggs of females per treatment - Trial 1

2.5% Guyabano 25% guyabano Cypermethrin Water

Hatched 1070 1658 177 1316

Total Eggs 1432 2015 390 1634

Hatching rate 70.86 73.94 45.71 77.85

Table 15. Oviposition and hatching rate of eggs of individual females per treatment -

Trial 2

2.5% Guyabano


0 0 0

25% Guyabano

0 0 0

Cypermethrin

1 57 114 50.00

2 91 167 54.49

3 5 10 50.00

4 83 154 53.90

5 0 70 0.00

6 50 79 63.29

7 28 62 45.16

8 66 144 45.83


Negative Control

1 21 39 53.85

2 30 51 58.82

3 18 33 54.55

4 38 71 53.52

5 32 59 54.24

6 108 201 53.73

7 32 56 57.14

8 19 47 40.43

9 16 45 35.56


Table 16. Total oviposition and hatching rate of eggs of females per treatment - Trial 2


Hatched 0 0 380 314

Total Eggs 0 0 800 602

Hatching rate 0 0 45.33 51.31

52

Table 17. Oviposition and hatching rate of eggs of individual females per treatment -

Cypermethrin, Trial 3

2.5% Guyabano


0 0 0

25% Guyabano

0 0 0

Cypermethrin

1 128 228 56

2 0 109 0

3 0 122 0

4 0 80 0

5 0 57 0

6 11 56 20

Total 139 652 12.67

Negative Control

1 0 29 0.00

2 93 107 87

3 0 33 0.00

4 0 20 0.00

5 0 29 0.00

Total 93 218 17

Table 18. Total oviposition and hatching rate of eggs of females per treatment - Trial 3


Hatched 0 0 139 93

Total Eggs 0 0 652 218

Hatching rate 0 0 12.67 17.38

Table 19. Average Percent Hatching Per Treatment (All Trials)

Treatment

Trial 2.5% Guyabano 25% Guyabano Cypermethrin (+) Water (-)

1 70.86 73.94 45.71 77.85

2 0 0 45.33 51.13

3 0 0 12.67 17.38

Average 23.62 24.65 34.57 48.79

53

Figure 14. ANOVA of Average Percent Mortality in All Treatments

Figure 15. Kruskal-Wallis Test of Percent Hatching in All Treatments with Descriptives

54

Appendix C

Line Item Budget

Table 20. Line-Item Budget

CATEGORY ITEM UNIT PRICE

(Php)

QUANTITY TOTAL

(Php)

Specimen A. muricata fruit 80 8 640.00

Reagents

90% ethyl alcohol 416.67 3 1250.00

Tween 80 420.00 1 420.00

Other

materials

Electrical stainless

steel blender

3,000.00 1 3,000.00

Fabric chambers 50.00 5 250.00

Absorbent paper 100.00 1 100.00

Disposable Petri

plates

10.00 60 600.00

Glass vials 5.00 20 100.00

6360.00

55

Appendix D

Tools Used in the Study

Figure 16. Mosquito chamber

Figure 17. Adult Immersion Test of all treatments

in vitro acaricidal effect of guyabano (annona muricata

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