senior project- bsc biological sciences

EVALUATION OF ANTIOXIDANT PROPERTIES IN

DIFFERENT COLORS OF ASIAN RICE (ORYZA SATIVA)

AGAINST INSECTICIDE CARBOSULFAN USING

MEALWORM (TENEBRIO MOLITOR) ASSAY

EMILIO SOLOMON

A SENIOR PROJECT SUBMITTED IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF

BACHELOR OF SCIENCE (BIOLOGICAL SCIENCE)

MAHIDOL UNIVERSITY INTERNATIONAL COLLEGE

MAHIDOL UNIVERSITY

2015

COPYRIGHT OF MAHIDOL UNIVERSITY

Senior Project

entitled

EVALUATION OF ANTIOXIDANT PROPERTIES IN

DIFFERENT COLORS OF ASIAN RICE (ORYZA SATIVA)

AGAINST INSECTICIDE CARBOSULFAN USING

MEALWORM (TENEBRIO MOLITOR) ASSAY

was submitted to the Mahidol University International College, Mahidol University

for the degree of Bachelor of Science (Biological Science)

on

December 3, 2015

……………….………….…..………

Emilio Solomon

Candidate

………………….…..………….……

Asst. Prof. Wannee Jiraungkoorskul

Ph.D.

Advisor

……………………….….…..………

Aram Tangboondouangjit

Ph.D.

Chair of Science Division

Mahidol University International College

Mahidol University

……………………….….…..………

Laird B. Allan

Program Director

Bachelor of Science in Biological Science

Mahidol University International College

Mahidol University

iii

ACKNOWLEDGEMENTS

This research project would not have been possible without the knowledge

gained from my undergraduate professors from Mahidol University International

College (MUIC). Thank you Dr. Chayanant Hongfa (PhD) and Dr. Pakorn

Bovonsombat (PhD) for teaching me various essential chemistry concepts. Thank you

Dr. Saovanee Chancharoensin (PhD) for helping me understand biochemistry well. I

never would have thought that what I learned from your classes, became extremely

useful until I started doing my research project. Last but not least, a big thank you to

my Pathology teacher and my awesome senior project advisor, Dr. Wannee

Jiraungkoorskul (PhD). Thank you for giving me the essential knowledge, required to

perform my research investigation and thank you for helping me and guiding me

through the whole research project; from the proposal, to the experiment, and to the

final paper.

Emilio Solomon

Emilio Solomon Senior Project / iv

EVALUATION OF ANTIOXIDANT PROPERTIES IN DIFFERENT COLORS OF

ASIAN RICE (ORYZA SATIVA) AGAINST INSECTICIDE CARBOSULFAN

USING MEALWORM (TENEBRIO MOLITOR) ASSAY

EMILIO SOLOMON 5580132

B.Sc. (BIOLOGICAL SCIENCE)

SENIOR PROJECT ADVISOR: ASST. PROF. WANNEE JIRAUNGKOORSKUL,

Ph.D.

ABSTRACT

Asian rice (Oryza sativa) in white, brown, and black colors were used to

evaluate its efficiency in protecting mealworms (Tenebrio molitor) against insecticide,

carbosulfan. Three tests were performed including a total phenolic measurement test,

an acute toxicity test, and a histopathological process. The optical densities and total

phenolic content of white, brown, and black rice were obtained in the total phenolic

measurement test, with black rice having the highest total phenolic content, followed by

brown and white rice (p < 0.05). Furthermore, the characteristics and weight

differences of worms were determined in the acute toxicity test. Worms fed with both

rice and carbosulfan displayed abnormal characteristics and minimal weight gain.

Finally, histopathological changes and cellular adaptation were observed in the

histopathological process. Worms that were exposed with carbosulfan and fed with

white rice showed the most histopathological changes, followed by brown rice, and

black rice. Worms fed with black rice had the best cellular adaptation, followed by

brown rice and white rice. Data analysis demonstrated that the antioxidant properties of

O. sativa helped protect mealworms against insecticide, carbosulfan.

KEYWORDS: ANTIOXIDANT / CARBOSULFAN / INSECTICIDE /

MEALWORM / ORYZA SATIVA / RICE / TENEBRIO MOLITOR

68 pages

v

CONTENTS

Page

ACKNOWLEDGEMENTS iii

ABSTRACT (ENGLISH) iv

LIST OF TABLES vi

LIST OF FIGURES vii

CHAPTER I INTRODUCTION 1

CHAPTER II OBJECTIVES 4

CHAPTER III LITERATURE REVIEW 5

3.1 Oryza sativa 5-8

3.2 Carbosulfan 8-10

3.3 Tenebrio molitor 10-14

CHAPTER IV MATERIALS AND METHODS 15

4.1 Chemicals/Solution 15

4.2 Devices/Machines 15

4.3 Miscellaneous materials 16

4.4 Extraction 16

4.5 Total phenolic content 17

4.6 Acute toxicity test 20

4.7 Abdomen collection 22

4.8 Histopathological process 22

CHAPTER V RESULTS 27

5.1 Total phenolic measurement 27-30

5.2 Acute toxicity test 31-34

5.3 Histopathological process 34-56

CHAPTER VI DISCUSSION 57

CHAPTER VI CONCLUSION 60

REFERENCES 62

APPENDICES 67

BIOGRAPHY 68

vi

LIST OF TABLES

Table Page

4.6.1 Experimental groups 21

4.8.1 Schedule for histopathological process 25

4.8.2 Schedule for staining process 26

5.1.1 Total phenolic content standard curve 27

5.1.2 Optical densities of different color rice 29

5.1.3 Mean, standard deviation, and p-value of optical densities of

different color rice

29

5.1.4 Total phenolic content of different color rice at various times 30

5.2.1 Experimental groups 31

5.2.2 Weights of mealworms before and after feeding 32

5.2.3 Worm characteristics after feeding 33

5.3.1.1 Mealworm tissue under 40x magnification 35

5.3.4.1 Cuticle 38-41

5.3.4.2 Columnar epithelia (pseudo-stratified) 41-43

5.3.4.3 Endothelia 44-46

5.3.4.4 Dense connective tissue 46-49

5.3.4.5 Loose connective tissue 49-51

5.3.4.6 Skeletal muscle 52-54

5.3.5.1 Criteria for histopathological changes and cellular adaptation in

abdominal tissue

55

5.3.5.2 Histopathological changes and cellular adaptation in abdominal

tissue

55

vii

LIST OF FIGURES

Figure Page

3.1.1 Oryza sativa plant (left) and white, brown, and black rice (right) 5

3.1.2.1 White (top), brown (bottom left), and black rice (bottom right) 7

3.1.2.2 Oryza sativa morphology (Drawings by Polato, 2013). 7

3.2.1 Carbosulfan 8

3.2.1.1 Chemical structure of carbosulfan 9

3.3.1 Tenebrio molitor 11

3.3.2.1 Morphology of T. molitor 13

3.3.3.1 Life cycle of T. molitor 14

4.4.1 Rice extracts on shaker (left) and rice extracts after 1 hour of

shaking (right)

16

4.4.2 Centrifuge (left) and supernatant of white, black, and brown rice at

3 hours (right)

17

4.5.1 Gallic acid (left) and spectrophotometer (right) 17

4.5.1.1 Preparation of 10% Folin-Ciocalteu phenol reagent 18

4.5.2.1 Preparation of 0.7 M sodium carbonate 18

4.5.3.1 Preparation of gallic acid standard solution 19

4.5.3.2 Serial dilution of Gallic acid standard solution (left) and Gallic acid

standard solutions (right)

19

4.6.1 Mealworms before sorting (left) and after sorting (right) 20

4.6.2 Mealworms after feeding 21

4.6.3 Surgical blades and handle stainless (Left) and abdomen collection

(right)

21

4.7.1 Ethyl-3-aminobenzoate methane sulfonate (left) and ethyl-3-

aminobenzoate methane sulfonate with distilled water (right)

22

4.8.1 Mealworms fixed in formalin 22

viii

LIST OF FIGURES (cont.)

Figure Page

4.8.2 Order during histopathological process 23

4.8.3 Paraffin dispenser (left) and rotary microtome (right) 23

4.8.4 Chemicals used for staining process (Top and left) and histological

slides (right)

23

4.8.5 Permount 24

4.8.6 Microscopy apparatus 25

5.1.1 Total phenolic content standard curve 28

5.1.2 Total phenolic content of different color rice at various times 30

5.2.1 Weight of mealworms before and after feeding 32

5.2.2 Appearance of mealworms after feeding 33

5.3.2.1 Cuticle 36

5.3.2.2 Columnar epithelia (pseudo-stratified) 36

5.3.2.3 Endothelia 36

5.3.2.4 Dense connective tissue 37

5.3.2.5 Loose connective tissue 37

5.3.2.6 Skeletal muscle 38

Mahidol University International College B.Sc. (Biological Science) / 1

CHAPTER I

INTRODUCTION

Many of the foods we eat contain antioxidants. Antioxidants are

substances that help protect the cells in our body from oxidative stress (Carunchia et

al., 2015). They can be found in a variety of foods, including fruits, vegetables, and

seeds (Carunchia et al., 2015). Other sources of antioxidants are whole-grained

products, nuts, herbs and spices and even chocolate and dietary supplements

(Embuscado, 2015). Antioxidants are known to be beneficial to our health, as they can

help prevent the risk of cardiovascular diseases and cancer and improve our immune

system (Hercberq et al., 1998).

Oryza sativa, or Asian rice is a commodity that is widely consumed by

people around the world. In Asia, O. sativa is a major component in many people’s

diets. O. sativa has two major subspecies, including O. sativa L. ssp. japonica, which

is primarily consumed in Southeast Asia, Japan, Europe and the U.S., and O. sativa L.

ssp. indica, which is primarily consumed in India and Southern China (Bordiga et al.,

2014). O. sativa comes in different colors such as red, white, brown, and black. The

color of rice, is a result of the different molecules found within the pericarp of the rice.

For example, anthocyanin contributes black color to the rice (Bordiga et al., 2014).

This commodity is important just like any other crops, in spite of its nutritional

significance. O. sativa contains antioxidants, which help destroy free radicals (Walter

et al., 2013). O. sativa is also low in fat and high in starchy carbohydrates, making it a

good source for energy (Renuka et al., 2016). This staple food is also packed with

vitamins such as vitamin B (thiamine, niacin) & vitamin E and minerals like

potassium, all of which can help improve vitamins and minerals deficiency-related

symptoms (Renuka et al., 2016). The consumption of O. sativa can help ward off

diseases such as heart disease and cancer (Chung and Shin, 2007), as well as help

combat type II diabetes and obesity (Kang et al., 2010).

O. sativa can either be processed or be left as a whole grain. For example,

white rice, the most widely consumed rice is processed through a series of mechanized

Emilio Solomon Introduction / 2

steps, including hulling and milling (Rohman et al., 2014). This can influence the

rice’ nutritional value, by altering its glycemic index. For example, the glycemic index

of white rice (processed) is higher than its non-processed brown and whole grain

counterparts. The mean glycemic index for white rice is 64, whereas the mean

glycemic index for brown and black rice are 55 and 25, respectively (Hu et al., 2012).

Besides rice processing, the rice’ sugar content can also affect its nutritional value.

According to the USDA (2015a, 2015b, and 2015c), white rice has 29 grams of

carbohydrates per 100 grams of rice, whereas brown rice has 24 grams of

carbohydrates per 100 grams of rice. Studies have shown that people who ate less

whole grain rice (white) were more susceptible to weight gain, as opposed to people

who ate more whole grain rice (brown) consistently (Rohman et al., 2014). Apart from

the negative effects of rice processing and sugar content on its nutritional value, the

use of insecticides on rice has become a growing concern to people today.

Insecticides kill, harm, or repel any invading insects. This helps in

increasing food production, decreasing the cost of food, and improving consumer

benefits. In the Pearl River delta, China, insecticides have been increasingly used to

improve agricultural output (Wei et al., 2015). Insecticides are also used to help

prevent the outbreak of pests in rice, making them a viable option for food security

and non-targeted species safety (Xu et al., 2015). Although much has been said about

the benefits of insecticides, insecticides in fact, pose many disadvantages. Studies have

shown that insecticides increase the risk of suicide among farmers (Freire and

Koifman, 2013) and can harm an unborn child in pregnant women (Lewis et al. 2015).

Other disadvantages are associated with the environment such as air and water

pollution, where fish can experience metabolic disturbances and stunted growth when

exposed (Wei et al., 2015). The consumption of foods, treated with insecticides can

affect the nervous system, endocrine system, and skin or eyes in humans, thus leading

to many adverse health effects in humans (FAO, 2003).

There have been many studies on O. sativa, most of which mainly focused

on the phenolic characterization and antioxidant activity of O. sativa only. However,

there have not been a lot of the findings of the efficiency of O. sativa in reducing the

toxicity of insecticides. In order to measure this efficiency of O. sativa, an organism is

required. Insecticides can enter an organism through various routes. For humans,


insecticides can enter through the lungs by inhalation, skin by penetration, or mouth

by ingestion (Fishel, 2014). This can lead to various kinds of symptoms, ranging from

mild symptoms to severe symptoms. The acute toxicity test will be used to examine

the severity of symptoms in mealworms (Tenebrio molitor). The body weight of

mealworms will also be determined before and after feeding in the acute toxicity test.

Similar to other studies, the phenolic content of O. sativa will be tested as well as a

measure of the rice’ antioxidant property. In order to test for the phenolic content in O.

sativa, a bioassay will be performed using different reagents and a spectrophotometer.

The first purpose of this research is to determine the antioxidant properties

of Asian rice, O. sativa in different color rice. The second purpose is to evaluate the

mealworms’ (Tenebrio molitor) protection against insecticide, carbosulfan. O. sativa

will first be measured for the total phenolic content through a bioassay using different

reagents and a spectrophotometer. After measuring the total phenolic content in

different colors of O. sativa, mealworms will eventually be exposed to rice with or

without insecticide for 7 days. Weights of mealworms will be accounted before and

after feeding. Mealworms will then be dissected in order to obtain the abdomen for the

histopathological process. During the histopathological process, the abdomen of

mealworms in each group will be fixed in 10% formalin for at least 24 hours, followed

by a series of other steps, including dehydration, clearing, embedding, and sectioning.

The histological slides of each mealworm’s abdomen will determine whether O. sativa

has the efficiency in reducing the effect of the insecticide. Such findings can hopefully

be used by prospective researchers or food scientists to investigate alternatives for O.

sativa in reducing the toxicity of insecticides in various foods.

Emilio Solomon Objectives / 4

CHAPTER II

OBJECTIVES

2.1 To compare the antioxidant properties of Oryza sativa in different colors and

different time extractions.

2.2 To analyze the efficiency of Oryza sativa in different colors in reducing the

effect of carbosulfan insecticide using Tenebrio molitor histopathological

analysis.


CHAPTER III

LITERATURE REVIEW

3.1 Oryza sativa

O. sativa, or commonly known as Asian rice contains two major sub-

species. O. sativa L. spp. japonica, which is the sticky, short grained rice and O. sativa

L. spp. indica, which is the non-sticky, long grained rice (Bordiga et al., 2014). O.

sativa comes in a variety of colors such as white, brown, black, purple, and red. O.

sativa can either be pigmented or non-pigmented (Kim et al., 2014). The non-

pigmented rice is consumed by 85% of the world’s population, while the white

pigmented rice is consumed as a specialty in East Asia (China, Japan, and Korea) for

its flavor and health benefits (Kim et al., 2014). O. sativa accounts for 95% of global

rice production (Kim et al., 2014) and consumption and is the second highest

commodity produced, after Zea mays. It is commonly consumed as polished white rice

with the husk, bran, and germ fractions removed (Bordiga et al., 2014). More than

three billion people in the world consume O. sativa (Sarkar et al., 2009).

Figure 3.1.1 Oryza sativa plant (left) and white, brown, and black rice (right)

Emilio Solomon Literature review / 6

3.1.1 Taxonomy

Kingdom: Plantae

Subkingdom: Tracheobionta

Superdivision: Spermatophyta

Division: Magnoliophyta

Class: Liliopsida

Subclass: Commelinidae

Order: Cyperales

Family: Poaceae

Genus: Oryza

Species: Oryza sativa

3.1.2 Morphological description

O. sativa has three major parts, including the husk, bran, and germ.

Several studies focused on the husk part of rice (Figure 3.1.2.2). According to a study

from Phytochemistry Letters, the husk provides a valuable source of nutrients,

including diterpenes, phenols, steroids, long-chain fatty acids, and flavonoids (Li et

al., 2014). The researchers of this study, also state that the husk accounts for 20% of

the rice grain (Li et al., 2014). Unlike this study, researchers from the Journal of

Cereal Science, focused more on the phenolic substances found within the husk part of

rice. The researchers found that the husk is concentrated with vanillic and p-coumaric

acids (Shao et al., 2014). Furthermore, other studies focused on the bran part of rice.

According to a study from Food Research International, the bran accounts for 70 to

90% of phenolic acids in light brown pericarp color grains, and around 85% of

anthocyanin in black pericarp color grains (Walter et al., 2013). Another study from

Food Research International similarly addressed the presence of phenolic acids in rice

bran. The researchers state that rice bran contains high amounts of fiber and bioactive

molecules, such as vitamin B complex, tocopherols, tocotrienols, oryzanols and other

phenolic compounds (Bordiga et al., 2014). Researchers from International Food

Research Journal have shown that Vitamin B in O. sativa can improve vitamin B

deficiency-related symptoms such as muscle weakness and neuritis (Rohman et al.,

2014). The researchers have also discussed the uses of rice bran, stating that rice bran


is often times extracted for producing oil (Hamada et al., 2012). There have not been

a lot of studies done on the germ, however several studies have confirmed that there is

less antioxidant activity in the germ, in comparison to the bran and husk parts of O.

sativa (Shao et al., 2014). Other studies also show that proteins, fats, vitamins, and

minerals are present in greater quantities in the germ than in the endosperm (Renuka,

Mathure, Zanan, Thengane, & Nadaf, 2015)

Figure 3.1.2.1 White (top), brown (bottom left), and black rice (bottom right)

Figure 3.1.2.2 Oryza sativa morphology (Drawings by Polato, 2013).


3.1.3 Phytochemical substances

O. sativa is well-known for having potent antioxidant properties. Studies

have shown that the antioxidant properties of O. sativa are found in the rice’ phenolic

compounds. A study from Food Research International has shown that the phenolic

compounds exist in both soluble and insoluble forms, with the soluble form

representing 38% to 60% of the total polyphenols content in light brown rice grains,

and around 81% in red and black pericarp color grains (Walter et al., 2013). Other

studies elaborate on the significance of phenolic compounds. Researchers from

Molecules found that O. sativa contains phenolic compounds, such as anthocyanin and

proanthocyanidin (Kim et al., 2014). They believe that anthocyanin possess

antioxidant, anti-inflammatory, and hypoglycemic effects (Kim et al., 2014). They

also state that the proanthocyanidin, found in some cereals, legume seeds, and various

fruits have superior antioxidant activities (Kim et al., 2014).

3.2 Carbosulfan

Carbosulfan is an insecticide that can be used in killing or repelling

invading insects. It is available as emulsifier concentrates, dusts and granular

formulations for the control of insects, mites and nematodes; mainly on potatoes, sugar

beet, rice, maize, and citrus (Altinok et al., 2012) . It can be used against certain insect

pests not controlled by organochlorine or organophosphorus insecticides (Nwami et

al., 2010) and can also be used for the control of pyrethroid-resistant mosquitoes (Giri

et al., 2002). Carbosulfan is primarily used to improve crop productivity (Nwami et

al., 2010)

Figure 3.2.1 Carbosulfan


3.2.1 Chemical structure

Carbosulfan, is a benzofuranyl methylcarbamate insecticide, with a

molecular formula of C20H32N2O3S (Giri et al., 2002).

Figure 3.2.1.1 Chemical structure of carbosulfan (ChemService Inc. (2015).

3.2.2 Properties

Carbosulfan primarily affects the nervous system of both aquatic and

terrestrial organisms. A study from Food and Chemical Toxicology shows that

carbosulfan acts as a neurotoxicant by affecting synaptic transmission in cholinergic

parts of the nervous system of aquatic organisms (Nwami et al., 2010). A study from

Food and Chemical Toxicology elaborates on this mechanism of action. Researchers

from this study state that its toxicity is mediated by the inhibition of

acetylcholinesterase, an enzyme that cleaves acetylcholine in the nervous system of

various organisms, including aquatic organisms (Giri et al., 2002). Other studies also

mention the inhibition of acetylcholinesterase by discussing the effects. According to

the United Nation’s Food and Agriculture Organization, Carbosulfan can act as a

dermal sensitizer (FAO, 2003). Carbosulfan can cause slight skin irritation in rabbits

(FAO, 2003)

3.2.3 Environmental contamination

Carbosulfan is known for its environmental contamination. When used,

Carbosulfan residues are capable of dispersing into the environment, exerting harmful

consequences for humans and animals (Banji et al., 2014). According to a study from

Food and Chemical Toxicology, Carbosulfan is widely used in rural areas, making it

easier to contaminate the aquatic environment (Nwami et al., 2010). Another study


from Pesticide Biochemistry and Toxicology describes the route of contamination of

carbosulfan. The researchers from the study state that the insecticide can enter water

through surface runoff, leaching, and/or erosion after insecticide residues enter the

atmosphere and precipitate (Altinok et al., 2012). Carbosulfan concentrations in

environment can range between 0.64 µg L-1 and 29 µg L-1 (Altinok et al., 2012).

3.2.4 Toxicity of carbosulfan in living organism

Carbosulfan is known for causing toxicity in various organisms, including

aquatic organisms such as fish, and terrestrial organisms such as rats and humans. In

fish, carbosulfan can cause liver cells to undergo edema and necrosis, as well as

oxidative stress, where the production of reactive oxygen species is induced (Capkin

and Altinok, 2013). In rats, carbosulfan is capable of inhibiting acetylcholinesterase,

the enzyme that cleaves acetylcholine (Banji et al. 2014). The inhibition of

acetylcholinesterase can lead to alterations in sensorimotor tasks, motor function, and

elevated anxiety in rats (Banji et al., 2014). Researchers from Genetic Toxicology and

Environmental Mutagenesis found additional effects of carbosulfan in rats. They

found that carbosulfan can lead to a decrease in mitotic index in bone marrow cells of

rats (Giri et al., 2002). In humans, carbosulfan is capable of inducing chromosome

aberrations in peripheral lymphocytes (Giri et al., 2002). Carbosulfan is also capable

of causing mental disturbances in humans. For example, it has been found that farmers

who worked with organochlorine insecticides were 90 percent more likely to have

been diagnosed with depression than those who did not work with them (Bienkowski,

2014).

3.3 Tenebrio molitor

T. molitor, or mealworm, the darkling beetle, or the stink bug belongs to

the family Tenebrionidae. This insect is native to Europe and is now distributed

worldwide (Gnaedinger et al., 2013). It is usually found under decaying trees and bark

in nature. It can also be found in flour mills or barns where livestock feed are stored

(Gnaedinger et al., 2013). T. molitor can be produced industrially as feed for pets and

zoo animals, including birds, small reptiles, mammals, and fish (Gnaedinger et al.,

2013). It can be fed alive or eaten canned or dried (Gnaedinger et al., 2013). It is


omnivorous; it can eat both plants and animals (Gnaedinger et al., 2013). It feeds on

cereal bran or flour (wheat, oats, maize) supplemented with fresh fruits and vegetables

(carrots, potatoes, lettuce) for moisture (Gnaedinger et al., 2013). It is known to be an

international and a serious pest of stored products (Dastranj et al., 2013). Both adult

and larval forms can destroy flour, grain, pasta, bread, bran, and insect collections

(Gnaedinger et al., 2013).

Figure 3.3.1 Tenebrio molitor

3.3.1 Taxonomy

Kingdom: Animalia

Phylum: Arthropoda

Class: Insecta

Order: Coleoptera

Family: Tenebrionidae

Genus: Tenebrio

Species: Tenebrio molitor

3.3.2 Properties

T. molitor reproduces at an optimal temperature of 25-27.5° C (Park et al.,

2014). It is nocturnal; when exposed with light, it hides in grains (Park et al., 2014). It

is high in protein and fat and is rich in oleic acid (Park et al., 2014). It is capable of

using small amounts of water contained in dry feeds, however, water-deprived

mealworms will exhibit low productivity (Gnaedinger et al., 2013).


3.3.2 Morphological description

T. molitor can be divided into three parts in order, including the head,

thorax, and abdomen (Figure 3.3.2.1). The worm’s thorax can be subdivided in order

into the prothorax, mesothorax, and metathorax. Worms have thirteen segments as

well; one for the head, and three and nine segments for the thorax and abdomen,

respectively. A study from Moscow University Biological Sciences Bulletin focused on

the mealworm’s segments. The researchers state that the three thoracic segments

include one strong chitinized basal segement, a longer pedicel, a large flagellum

beginning from the dorsolateral side of the antennal pedicle (Farazmand and Chaika,

2007). They also state that second segment is the longest, whereas the third segment is

the shortest (Farazmand and Chaika, 2007). The first segment has a length in between

of the second and third segments (Farazmand and Chaika, 2007). Another study from

Cell and Tissue Research focused on the mealworm’s legs. The researchers state that

mealworms have legs at the pro and mesothorax that project hair sensilla, or hair

sensory receptors (Breidbach, 1990). They found that the mealworm’s hair sensilla are

innervated by sensory neurons (Breidbach, 1990). Other studies focused on the

mealworm’s abdomen and its role in digestion. Researchers from Comparative

Biochemistry and Physiology state that different enzymes are found within the

mealworm’s midgut, including trypsin and cysteine proteinases (Vinokurov et al.,

2006). Researchers from Insect Biochemistry and Molecular Biology state that

cysteine peptidases are specifically found within the anterior midgut, whereas trypsin-

like and chymotrypsin-like serine peptidases are found within the posterior midgut

(Goptar et al., 2013). They believe that these enzymes play an important role in the

mealworm’s digestion (Goptar et al., 2013).


Figure 3.3.2.1 Morphology of T. molitor (Feitl, 2010)

3.3.3 Life cycle

According to Feedipedia.org, the life cycle of T. molitor lasts between 280

to 630 days, depending on the worm. The larva hatches after 10 to 12 days, and

undergo a variable number of stages (8- 20) until maturation by molting (Gnaedinger

et al., 2013). The mature larva is of light yellow-brown color with a length of 20-32

mm and a weight of 130 to 160 mg (Gnaedinger et al., 2013). The mealworm can live

for two to three months (Gnaedinger et al., 2013) before it begins its life cycle again.

A study from International Journal of Industrial Entomology describes the molting

process in terms of its mechanism and its role in the mealworm’s life cycle studies, as

well as examined the mealworm’s life cycle. Researchers found that the molting

process is facilitated by the worm’s molting hormones (Park et al., 2014). They also

found that as the worm molts, the worm’s cuticle is shed (Park et al., 2014). With

regards to the mealworm’s life cycle, the researchers found that molting occurs until

the worm becomes a pupa (Park et al., 2014). They state that the pupa stage usually

occurs after approximately 14 days and after approximately 20 days, the worm reaches

maturity (Park et al., 2014). The researchers believe that the development of

mealworms is overall, influenced by the age of parents (Park et al., 2014). For

example, they found that young parents are associated with the highest egg hatching

rates (Park et al., 2014).


Figure 3.3.3.1 Life cycle of T. molitor

Molted

worm

Large

worm

Pupa Beetle

Egg

Larva

5 6

2

1

3

4


CHAPTER IV

MATERIALS AND METHODS

4.1 Chemicals/Solutions

- Eosin (881, Sigma-Aldrich)

- Absolute ethanol (459844, Sigma-Aldrich)

- Folin-Ciocalteu’s phenol reagent (F9252-, Sigma-Aldrich)

- Formaldehyde 37wt. % (252549, Sigma-Aldrich)

- Gallic acid (G7384, Sigma-Aldrich)

- Hematoxylin (H3136, Sigma-Aldrich)

- Paraplast X-TRA Paraffin (Oxford Labware, USA)

- Sodium Carbonate (6392,Merck)

- Sodium Chloride (K2101, Lab Scan)

- Xylene (247642, Sigma-Aldrich)

- Distilled water

4.2 Devices/Machines

Digital balance (Zepper EPS-302, People's Republic of China)

Digital camera (Canon EOS 1100D, Japan)

Centrifuge

Embedding machine (Axel Johnson Lab system, U.S.A)

Hot plate and magnetic stirrer (Lab-Line PyroMagnestir, USA)

Laminar airflow cabinet

Light microscope (Olympus CX-31, Japan)

Microtome (Histo STAT, Reichert, USA)

Refrigerator

Shaker (Germmy Orbit Shaker model VRN-480, Taiwan)

Spectrometer (Model 340, Sequoia Turner Corp., Taiwan)

Emilio Solomon Materials and Methods / 16

4.3 Miscellaneous materials

Plastic centrifuge tubes

Laboratory film (parafilm)

Erlenmeyer’s flask

Funnel

Gloves

Glass bottles

Gauze

Filter paper

Test tubes

Test tube rack

4.4 Extraction

O. sativa in white, brown and black color, were made in power and extracted

in distilled water. Approximately 2.50 gm of O. sativa and 50 ml of distilled water were

used. Once extracted, O. sativa were shaken at 180 rpm for 0.5, 1, 3, 5, and 24 hours

(Figure 4.4.1). After extraction, O. sativa were centrifuged at 4000xg for 10 minutes

(Figure 4.4.2). The supernatant was then be collected and used for measuring the total

phenolic content of O. sativa (Figure 4.4.2).

Figure 4.4.1 Rice extracts on shaker (left) and rice extracts after 1 hour of shaking (right)


Figure 4.4.2 Centrifuge (left) and supernatant of white, black, and brown rice at 3 hours

(right)

4.5 Total phenolic content

The total phenolic content was determined according to the method of

McDonald et al. (2001) with some minor modifications. The 50 µl of 2.5 mg ml-1 extract

was mixed with 250 µl of 10% Folin-Ciocalteu phenol reagent (Figure 4.5.1) with

distilled water and 200 µl of 0.7 M sodium carbonate, then added with 4.5 ml of distilled

water. The mixture was then incubated at room temperature for 2 hours in the dark, then

measured at 765 nm using a spectrophotometer (Figure 4.5.1). The total phenolic

content was measured five times using ethanol solution of Gallic acid. A standard curve

was plotted and expressed as mg of Gallic acid equivalent (GAE) g-1 of dried crude

extract. The equation from the standard curve was then used to calculate the total

phenolic content of all rice at different times (Calculations 4.5.1).

Figure 4.5.1 Gallic acid (left) and spectrophotometer (right)


Calculations 4.5

1. Total phenolic content

Total phenolic content = (OD765 (avg))

4.5.1 Preparation 30 ml of 10% Folin-Ciocalteu phenol reagent

3 ml of 100% Folin-Ciocalteu phenol-reagent was obtained and diluted with 27

ml of distilled water, making it 10% in concentration. 250 µl of the prepared reagent

was used for 100 tubes (Figure 4.5.1.1)

Figure 4.5.1.1 Preparation of 10% Folin-Ciocalteu phenol reagent

4.5.2 Preparation 30 ml of 0.7 M sodium carbonate

2.2 gm of sodium carbonate was dissolved into 30 ml of distilled water, making

0.7 M in concentration. 200 µl of sodium carbonate was used for 100 tubes (Figure

4.5.2.1).

Figure 4.5.2.1 Preparation of 0.7 M sodium carbonate

27 ml 3 ml

30 ml 2.2 gm


4.5.3 Preparation Gallic acid standard solution

Five standard solutions of Gallic acid; A, B, C, D, E were prepared (Figures

4.5.3.1 & 4.5.3.2). 5 ml of ethanol was added to each solution, except for A, where 10

ml of ethanol was added. For solution A, 0.1 gm of Gallic acid was dissolved into 10 ml

of ethanol. After solution A was made, a serial dilution was performed in order to make

solutions B, C, D, and E (Figure 4.5.3.1). Solution B was made by using 5 ml of solution

A with 5 ml of ethanol. Solution C was made by using 5 ml of solution B with 5 ml of

ethanol. Solution D was made by using 5 ml of solution C with 5 ml of ethanol. Solution

E was made by using 5 ml of Solution D with 5 ml of ethanol.

Solution A = 0.1 g Gallic â + Ethanol 10 ml concentration 10 mg ml -1

Solution B = 5 ml of solution A + Ethanol 5 ml concentration 5 mg ml -1

Solution C = 5 ml of solution B + Ethanol 5 ml concentration 2.5 mg ml -1

Solution D = 5 ml of solution C + Ethanol 5 ml concentration 1.25 mg ml -1

Solution E = 5 ml of solution D + Ethanol 5 ml concentration 0.625 mg ml -1

Figure 4.5.3.1 Preparation of Gallic acid standard solution

Figure 4.5.3.2 Serial dilution of Gallic acid standard solution (left) and Gallic acid

standard solutions (right)

A E D C B

5 ml 5 ml 5 ml 5 ml


4.6 Acute toxicity test

T. molitor (n=80) in similar sizes were randomly divided into 8 groups as

shown in Figure 4.6.1. They were fed with an average of 21.7% (Calculations 4.6.1)

body weight of food each day (Figure 4.6.2). Rice bran acted as the control and

carbosulfan was used as an insecticide. The worms in treated groups were fed with fifty

percent of O. sativa in different colors (white, brown, black) and fifty percent of rice

bran with or without 10,204 ppm carbosulfan (Calculations 4.6.2). After 7 days of

exposure, worms in each group were dissected for abdomen collection (Figure 4.6.3).

Calculations 4.6

1. The percentage of food per body weight

% Food per body weight = Food

Time×

100

BWbefore

1.40 g

7 days×

100

0.90 g= 21.74 %

2. Concentration of carbosulfan

Concentration of carbosulfan (ppm) =Amount of carbosulfan (mg)

Amount of food (g)÷ 0.001 mg/g÷7

100 mg

1.40 g÷0.001

mg

g÷7 = 10,204 ppm

Figure 4.6.1 Mealworms before sorting (left) and after sorting (right)


Figure 4.6.2 Mealworms after feeding

Table 4.6.1 Experimental groups

Part

Worm (n=10)

Control White rice Brown rice Black rice

1 2 3 4 5 6 7 8

Rice bran (gm) 1.40 1.40 0.70 0.70 0.70 0.70 0.70 0.70

White rice (Wh)

(gm) - - 0.70 0.70 - - - -

Brown rice (Br) (gm) - - - - 0.70 0.70 - -

Black rice (Bl) (gm) - - - - - - 0.70 0.70

Carbosulfan (gm) - 0.10 - 0.10 - 0.10 - 0.10

Figure 4.6.3 Surgical blades and handle stainless (Left) and abdomen collection (right)


4.7 Abdomen collection

The 0.2 g/L of ethyl-3-aminobenzoate methane sulfonate solution was

prepared. Mealworms were then anesthetized in the solution (Figure 4.7.1). Once

mealworms were anesthetized, mealworms were sliced at the thorax (Figure 4.6.3).

The remaining mealworms that were still moving, were euthanized with a more

concentrated ethyl-3-aminobenzoate methane sulfonate solution and sliced at the

thorax.

Figure 4.7.1 Ethyl-3-aminobenzoate methane sulfonate (left) and ethyl-3-

aminobenzoate methane sulfonate & distilled water (right)

4.8 Histopathological process

The purpose of the histopathological process was to observe for the

abnormalities in the abdominal tissue of T. molitor. In order to observe for such

abnormalities, histological slides were first made following the schedule in Tables

4.8.1 & 4.8.2. The abdomen of T. molitor was fixed in 10% formalin for at least 24

hours.

Figure 4.8.1 Mealworms fixed in formalin


This helped preserve the tissue. After fixation with formalin, the abdomen

was dehydrated with a series of alcohols from 70%, 80%, 95%, and absolute alcohol

(100%) for 1 hour each in order to remove water from the tissues. The alcohol from the

tissue was then cleared and removed with xylene, a substance miscible with the

embedding medium for 2 hours (Figures 4.8.2).

Figure 4.8.2 Order during histopathological process

The tissue was then infiltrated with paraffin, the embedding agent for

another 2 hours inside a paraffin dispenser (Figure 4.8.3). Paraffin, which is of candle-

wax material was used to mold in the tissue. The tissue was then, embedded for proper

alignment and orientation in the block of paraffin, and sectioned with a rotary microtome

(Figure 4.8.3).

Figure 4.8.3 Paraffin dispenser (left) and rotary microtome (right)


Before staining occurred, the embedded tissue was deparaffinized and run

in the reverse order from xylene to alcohol to water as shown in Figure 4.8.4. The slide

was then stained with hematoxylin and eosin (H & E) (Figure 4.8.4) for enhancing image

clarity when viewing the slide under the microscope. Hematoxylin reacted with the

basophilic structures of the abdominal tissue, while eosin reacted with the acidophilic

structures. Finally, the slide was mounted using Permount (Figure 4.8.5).

Figure 4.8.4 Chemicals used for staining process (Top and left) and histological slides

(right)

Figure 4.8.5 Permount

1. Xylene I, II, III 2. Absolute Alcohol I, II, III 3. 95% Alcohol I, II, III 4. 80% & 70% Alcohol

8. Xylene I, II, III 7. Absolute Alcohol I, II, III 6. 95% Alcohol I, II, III 5. Eosin, Hematoxylin


The completed histological slide was examined under the microscope for

tissue abnormalities in T. molitor using a light microscope (Figure 4.8.6). Images were

taken as well using a digital camera, hooked to the microscope (Figure 4.8.6). This was

done for tissues in all groups.

Figure 4.8.6 Microscopy apparatus

Table 4.8.1 Schedule for histopathological process

No. Process Time (hr)

1 10% buffered formalin 24

2 70% alcohol 1

3 80% alcohol 1

4 95% alcohol I 1

5 95% alcohol II 1

6 Absolute alcohol I 1

7 Absolute alcohol II 1

8 Xylene I 1

9 Xylene II 1

10 Paraffin I 1

11 Paraffin II 1

12 Embedding

13 Sectioning


Table 4.8.2 Schedule for staining process

No. Process Time (min)

1 Deparaffin slide in 60-70 ⁰ C hot air oven 30- 60

2 Xylene I 5

3 Xylene II 5

4 Xylene III 5




8 95% alcohol 5

9 80% alcohol 5

10 70% alcohol 5

11 Running water 7

12 Hematoxylin 7

13 Running water 7

14 Eosin 5

15 95% alcohol I 5

16 95% alcohol II 5

17 95% alcohol III 5



20 Absolute alcohol III 5

21 Xylene I 5

22 Xylene II 5

23 Xylene III 5

24 Mounting

25 Observation by microscope


CHAPTER V

RESULTS

5.1 Total phenolic measurement

The total phenolic measurement was determined according to the method of

McDonald et al. (2001) with some minor modifications. The 50 µl of 2.5 mg ml-1 extract

was mixed with 250 µl of 10% Folin-Ciocalteu phenol reagent with distilled water and 200

µl of 0.7 M sodium carbonate, then added with an additional 4.5 ml of distilled water. The

mixture was then incubated at room temperature for 2 hours in the dark, then measured at

765 nm using a spectrophotometer. The total phenolic content was measured five times

using ethanol solution of Gallic acid. A standard curve was plotted and expressed as mg of

Gallic acid equivalent (GAE) g-1 of dried crude extract. The standard curve was then, used

to calculate the concentration of white, brown, and black rice at 0.5, 1, 3, 5, and 24 hours

(Calculations 4.5.1).

Table 5.1.1 Total phenolic content standard curve

Tube A B C D E

Concentration (mg ml-1) 10.000 5.000 2.500 1.250 0.625

OD765 0.860 0.834 0.676 0.610 0.561

Figure 5.1.1 presents the total phenolic standard curve. According to the figure,

the total phenolic content of Gallic acid solution was directly proportional to the optical

density. It was shown that as the concentration of Gallic acid solution increased, the optical

density increased. For example, 10 mg ml-1 of solution A had the most optical density,

whereas 0.625 mg ml-1 of solution E had the least optical density. In other words, 10 mg

ml-1 of solution A had the most total phenolic content, whereas 0.625 mg ml-1 of solution E

had the least total phenolic content. The optical densities of solution A and solution E were

0.860 and 0.561, respectively. The optical densities of solutions B, C, and D were 0.834,

0.676, and 0.610, respectively.

Emilio Solomon Results / 28

Figure 5.1.1 Total phenolic content standard curve

The R2 value was equal to 0.8273, indicating that the concentration of Gallic

acid solution and the total phenolic content were indeed, positively correlated. P-value

results showed that the optical densities of different color rice were statistically significant

(Table 5.1.3).

Table 5.1.2 presents the optical densities of different color rice at different

times. The optical densities for white, brown, and black rice were averaged. The average

optical densities of white, brown, and black rice, along with the linear equation from the

total phenolic standard curve were then used to calculate the total phenolic content of

different color rice at 0.5, 1, 3, 5, and 24 hours.

A

B

C

DE

f(x) = 0.0319x + 0.5848R² = 0.8273

0.500

0.550

0.600

0.650

0.700

0.750

0.800

0.850

0.900

0.950

0.000 2.000 4.000 6.000 8.000 10.000 12.000

OD

765

Concentration (mg ml-1)

Total Phenolic Content Standard Curve


Table 5.1.2 Optical densities of different color rice

White

Tube 0.5 1 3 5 24

1 0.007 0.010 0.011 0.013 0.013

2 0.014 0.008 0.010 0.022 0.015

3 0.008 0.011 0.014 0.012 0.020

4 0.011 0.007 0.011 0.011 0.019

5 0.010 0.009 0.014 0.026 0.012

OD765 (avg) 0.010 0.009 0.012 0.017 0.016

Total Phenolic Content (mg g-1) 0.585 0.585 0.585 0.585 0.585

Black

Tube 0.5 1 3 5 24

1 0.060 0.058 0.074 0.063 0.049

2 0.051 0.051 0.055 0.059 0.052

3 0.058 0.059 0.055 0.061 0.048

4 0.058 0.065 0.054 0.061 0.051

5 0.058 0.053 0.061 0.064 0.051

OD765 (avg) 0.057 0.057 0.060 0.062 0.050


Brown

Tube 0.5 1 3 5 24

1 0.033 0.024 0.029 0.026 0.037

2 0.043 0.029 0.033 0.031 0.033

3 0.044 0.034 0.026 0.027 0.036

4 0.051 0.026 0.046 0.028 0.037

5 0.036 0.025 0.027 0.028 0.059

OD765 (avg) 0.041 0.028 0.032 0.028 0.040


Table 5.1.3 Mean, standard deviation, and p-value of optical densities of different color rice

𝑥 ̅ ± 𝜎 1 2 3 4 5

White 0.010±0.003 0.009±0.002 0.012±0.002 0.017±0.007 0.016±0.004

Brown 0.041±0.007 0.028±0.004 0.032±0.008 0.028±0.002 0.040±0.011

Black 0.057±0.003 0.057±0.005 0.060±0.008 0.062±0.002 0.050±0.002

P-value 1 2 3 4 5

White 0.013

Brown 0.010

Black 0.016


Table 5.1.4 Total phenolic content of different color rice at various times

Rice

Time (hrs)

0.5 1 3 5 24

White 0.585 0.585 0.585 0.585 0.585

Black 0.587 0.587 0.587 0.587 0.586

Brown 0.586 0.586 0.586 0.586 0.586

Figure 5.1.2 Total phenolic content of different color rice at various times

Figure 5.1.2 summarizes the antioxidant activities of white, black, and brown

rice over 0.5, 1, 3, 5, and 24 hours. According to the figure, black had the highest

antioxidant activity, followed by brown and white rice at 0.5, 1, 3, and 5 hours. The total

phenolic content of black rice was 0.587 mg g-1 at 0.5, 1, 3, and 5 hours. The total phenolic

content of brown rice was 0.586 mg g-1 at 0.5, 1, 3, and 5 hours. The total phenolic content

of white rice was 0.585 mg g-1 at 0.5, 1, 3, and 5 hours. At 24 hours, the antioxidant activities

of black and brown rice were the same. The total phenolic content of both black and brown

rice were both 0.586 mg g-1. The total phenolic content of white rice was still the lowest at

24 hours. The total phenolic content of white rice at 24 hours was 0.585 mg g-1.

0.583

0.584

0.585

0.586

0.587

0.588

0.5 1 3 5 24

Co

nce

ntr

atio

n (

mg/

g)

Time (hr)

Total Phenolic Content of Different Color Rice

White Black Brown


5.2 Acute toxicity test

T. molitor (n=80) in similar sizes were randomly divided into 8 groups as shown

in Table 5.2.1. Worms were fed with an average of 21.7% (Calculations 4.6.1) body weight

of food each day. Rice bran acted as the control and carbosulfan was used as an insecticide.

The worms in each group were fed with fifty percent of O. sativa in different colors (white,

black, brown) and fifty percent of rice bran with or without 10,204 ppm (Calculations 4.6.2)

carbosulfan. After 7 days of exposure, worms in each group were dissected for abdomen

collection.

Table 5.2.1 Experimental groups

% Food per body weight (n= 80): 21.7%

Part

Worm (n=10)

Control White rice Brown rice Black rice

1 2 3 4 5 6 7 8

Rice bran (gm) 1.40 1.40 0.70 0.70 0.70 0.70 0.70 0.70

White rice (Wh) (gm) - - 0.70 0.70 - - - -

Brown rice (Br) (gm) - - - - 0.70 0.70 - -

Black rice (Bl) (gm) - - - - - - 0.70 0.70

Carbosulfan (gm) - 0.10 - 0.10 - 0.10 - 0.10

Body weight before

(gm) 0.90 0.88 0.93 0.88 0.98 0.88 0.98 0.91

Body weight after

(gm) 1.20 0.97 1.21 0.85 1.15 0.77 1.24 1.02

% food per body

weight 21.74 22.72 21.51 22.72 20.41 22.72 20.41 21.98

# of worms alive after

feeding 10 9 10 8 10 8 10 10

# of worms dead after

feeding 0 1 0 2 0 2 0 0


Table 5.2.1 summarizes the data recorded during the acute toxicity test.

According to the table, a majority of the worms survived after the feeding process. Worms

in groups 1, 3, 5, 7, and 8 all survived. All worms in these groups were fed with rice bran

and/or rice, with the exception of worms in group 8. Worms in group 8 were fed with rice

bran, black rice, and carbosulfan. Unfortunately, several worms in groups 2, 4, and 6 died

after the feeding process. In group 2, 9 worms survived, while 1 worm died. In groups 4

and 6, 8 worms survived, while 2 worms died. Worms in groups 2, 4, and 6 were fed with

rice bran and/or rice and carbosulfan.

Table 5.2.2 Weights of worms before and after feeding

Group

Before

(gm) After (gm) Difference (gm) % Difference

1 0.090 0.120 0.030 25

2 0.088 0.108 0.020 19

3 0.093 0.121 0.028 23

4 0.088 0.106 0.018 17

5 0.098 0.115 0.017 15

6 0.088 0.096 0.008 8

7 0.098 0.124 0.026 21

8 0.091 0.102 0.011 11

Figure 5.2.1 Weight of worms before and after feeding

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

1 2 3 4 5 6 7 8

Wei

ght

(gm

)

Group

Weight of worms before and after feeding

Before After Difference


Table 5.2.2 and Figure 5.2.1 summarize the weight of worms before and after

feeding. The figure also indicates the difference between before and after body weights.

After 7 days of feeding, worms that were treated with only rice, as shown from groups 3,

5, and 7 experienced significant changes in body weight. Worms in group 3 gained weight

by 23%, whereas worms in groups 5 and 7, gained weight by 15% and 21% respectively.

These are opposed to worms that were treated with both rice and insecticide, carbosulfan.

Worms that were treated with both rice and carbosulfan, as shown from groups 4, 6, and 8

had minimal changes in body weight. Worms in group 4 gained weight by 17%. Worms in

group 6 gained weight by 8% and worms in group 8 gained weight by 11%. Worms that

only ate white rice gained the most weight. Overall, worms that only ate white rice

experienced the most weight gain whereas worms that ate brown rice and carbosulfan

experienced the least weight gain.

Table 5.2.3 Worm characteristics after feeding

Group Molting Black abdomen

1 X -

2 X X

3 - -

4 X X

5 X -

6 X X

7 X -

8 X X

Figure 5.2.2 Appearance of worms after feeding

5 6 7 8

1 2 3 4


Table 5.2.3 presents the characteristics observed in mealworms after feeding. It

was shown that worms exhibited molting and/or a black abdomen. Almost all worms

demonstrated molting, with the exception of worms in group 3. Worms in group 3 were fed

with rice bran and white rice. Furthermore, worms in groups 2, 4, and 6 exhibited a black

abdomen. Worms in groups 2, 4, and 6 were fed with rice bran and rice along with

insecticide, carbosulfan. Worms in group 1, as well as groups 3, 5, and 7 had unaffected

abdomens. These worms were treated with only rice bran and rice, with the exception of

worms in group 1. Worms in group 1 were treated with only rice bran. Figure 5.2.2 shows

the appearance of worms in all groups after feeding.

5.3 Histopathological analysis

The purpose of the histopathological process was to observe for the

abnormalities in the abdominal tissue of T. molitor. In order to observe for such

abnormalities, a histological slide was first made following the schedule in Table 4.8.1. The

abdomen of T. molitor was fixed in 10% formalin for at least 24 hours. This helped preserve

the abdominal tissue. After fixation with formalin, the abdomen was dehydrated with a

series of alcohols from 70%, 80%, 95%, and absolute alcohol (100%) for 1 hour each in

order to remove water from the tissues. The alcohol from the tissue was then cleared and

removed with xylene, a substance miscible with the embedding medium for 2 hours. Tissue

was then infiltrated with paraffin, the embedding agent for another 2 hours. Paraffin, which

is of candle-wax material was used to mold in the tissue. The tissue was then, embedded

for proper alignment and orientation in the block of paraffin, and sectioned with a rotary

microtome. Before staining occurred, the embedded tissues were deparaffinized and run in

the reverse order from xylene to alcohol to water as shown in Table 4.8.2. This formed the

stains in the histological slide and enhanced image clarity when viewing the slide under the

microscope. Finally, the slide was stained with hematoxylin and eosin (H & E). The

hematoxylin reacted with the basophilic structures of the abdominal tissue, while eosin

reacted with the acidophilic structures. The stained glass slides were examined under a light

microscope for tissue abnormalities in T. molitor. Tissues were viewed under 40x, 400x,

and 1000x magnifications. Images were taken as well using a Canon EOS 1100D DSLR

camera, hooked to the microscope.


5.3.1 Image Analysis under 40x magnification

Various structures were observed for all groups under 40x magnification,

including the cuticle, loose connective tissue, dense connective tissue, endothelia, columnar

epithelia (pseudo-stratified), and skeletal muscle.

Table 5.3.1.1 Mealworm tissue under 40x magnification

1

(Rice bran)

2

(Rice bran +

carbosulfan)

3

(White rice)

4

(White rice +

carbosulfan)

5

(Brown rice)

6

(Brown rice +

carbosulfan)

7

(Black rice)

8

(Black rice +

carbosulfan)

5.3.2 Histology of structures in mealworm tissue under physiological

conditions

Figures 5.3.2.1 to 5.3.2.5 presents the histology of structures in mealworm

tissue under physiological conditions. Figures 5.3.2.1 to 5.8.2.5 were used as a guide in

identifying tissue abnormalities in T. molitor.


Figure 5.3.2.1 Cuticle

Figure 5.3.2.2 Columnar epithelia (pseudo-stratified)

Figure 5.3.2.3 Endothelia

Basement membrane

Cuticle

Epithelial cell nuclei

Epithelia

Pericyte


Figure 5.3.2.4 Dense connective tissue

Figure 5.3.2.5 Loose connective tissue

Elastic fibers

Fibroblast nuclei

Fibroblast nuclei

Collagen fibers


Figure 5.3.2.6 Skeletal muscle

5.3.4 Image analysis under 1000x magnification

The cuticle, loose connective tissue, dense connective tissue, endothelia,

columnar epithelia, and skeletal muscle were observed closely under 1000x magnification.

Tables 5.3.3 to 5.3.8 show the images taken for each structure under 1000x magnification

Table 5.3.4.1 Cuticle

Group Image (1000x)

1 Rice bran

Skeletal muscle nuclei

Sarcoplasm

Artefact


2 Rice bran + carbosulfan

3 White rice

4 White rice + carbosulfan


5 Brown rice

6 Brown rice + carbosulfan

7 Black rice


8 Black rice + carbosulfan

Table 5.3.4.2 Columnar epithelia (pseudo-stratified)

Group Image (1000x)

1 Rice bran



3 White rice


5 Brown rice



7 Black rice



Table 5.3.4.3 Endothelia

Group Image (1000x)

1 Rice bran


3 White rice



5 Brown rice



7 Black rice


Table 5.3.4.4 Dense connective tissue

Group Image (1000x)

1 Rice bran



3 White rice



5 Brown rice


7 Black rice



Table 5.3.4.5 Loose connective tissue

Group Image (1000x)

1 Rice bran



3 White rice


5 Brown rice



7 Black rice



Table 5.3.4.6 Skeletal muscle

Group Image (1000x)

1 Rice bran


3 White rice



5 Brown rice



7 Black rice


5.3.5 Histopathological changes in various structures of abdominal tissue

Various histopathological changes were observed in the structures found in the

abdominal tissue of mealworms. Lysis was observed in the basement membrane of the

cuticle, as well as in columnar epithelia and loose connective tissues. Rupturing was

observed in endothelia. Cellular adaptation was also observed in the abdominal tissue.

Mucous membrane formation was observed in columnar epithelia and accumulation was

observed in loose and dense connective tissues. Both histopathological changes and cellular

adaptation were determined using the criteria in Table 5.3.5.1.


Table 5.3.5.1 Criteria for histopathological changes and cellular adaptation in abdominal

tissue

Structure - + +++

Cuticle1 No basement

membrane lysis

Mild basement

membrane lysis

Severe basement

membrane lysis

Columnar epithelia2 No mucous

membrane

Thin mucous

membrane

Thick mucous

membrane

Endothelia1 No rupturing Slightly ruptured Severely ruptured

Dense connective

tissue2

No accumulation Slight accumulation High accumulation

Loose connective

tissue2

No accumulation Slight accumulation High accumulation

Muscle No lesion Mild inflammation Severe

inflammation

1= Histopathological changes, 2= Cellular adaptation

Table 5.3.5.2 Histopathological changes and cellular adaptation in abdominal tissue

Structure

Worms (n=80)

1 2 3 4 5 6 7 8

Cuticle1 - - - + - - - -

Columnar

epithelia2 + + + -* + + - +

Endothelia1 - - - + - + - -

Dense connective

tissue2 - +++ - + + +++ + +

Loose connective

tissue2 - + - -* - + - +

Muscle - - - - - - - -

*=Lysis, 1= Histopathological changes, 2= Cellular adaptation

Table 5.3.5.2 presents histopathological changes and cellular adaptation

observed in the abdominal tissue of T. molitor. Basement membrane lysis was observed in

only worms that ate white rice. Unlike in worms that ate white rice, the basement membrane

remained intact in worms that ate rice bran, brown rice, or black rice. In columnar epithelia,


mucous membrane thickening was observed. Worms that ate black rice had mucous

membranes that became thicker. Worms that ate rice bran or brown rice had mucous

membranes that remained unchanged. Worms that ate white rice had mucous membranes

that became thinner. In endothelia, rupturing was observed. Worms that ate white rice or

brown rice had endothelia that ruptured. Worms that ate black rice or rice bran on the other

hand, had endothelia that remained intact. In dense connective tissues, accumulation was

observed. Accumulation was observed in the dense connective tissues of all worms,

however the degree of accumulation was different. Worms that ate brown rice or rice bran

only had the most accumulation in the tissue. Worms that ate black rice had less

accumulation. Worms that ate only white rice had no accumulation. Accumulation was also

observed in loose connective tissues. Accumulation was observed in worms that ate rice

bran, brown rice, or black rice. All of these worms had the same degree of accumulation in

the loose connective tissues. Worms that ate white rice on the other hand, showed no signs

of accumulation. There was however, lysis in worms that ate white rice. No

histopathological changes nor cellular adaptation were observed in the skeletal muscle of

T. molitor.


CHAPTER VI

DISCUSSION

It was initially hypothesized that O. sativa had the ability to reduce insecticide

toxicity in T. molitor, depending on its color. After three processes, including the total

phenolic measurement test, acute toxicity test, and histopathological process, it was

confirmed that O. sativa, indeed had the ability to reduce insecticide toxicity in T. molitor.

According to many studies, including Kim et al. (2014), it was known that the

total phenolic content of all rice represented its antioxidant property. In the total phenolic

measurement test, it was observed that black rice had the best antioxidant property,

followed by brown rice and white rice. This was shown in Table 5.1.3 and Figure 5.1.2.

Specific studies, such as Walter et al. (2013) and Chung and Shin (2007) supported the fact

that black rice had better antioxidant properties than brown rice and white rice in total

phenolic measurement test. The antioxidant properties of these rice were most likely,

affected by the extent of rice processing (Rohman et al., 2014).

In the acute toxicity test, it was observed that worms fed with rice and

insecticide, carbosulfan experienced minimal weight gain compared to worms fed with only

rice. According to Table 5.2.2, worms that were fed with only rice experienced significant

weight gain. Worms that experienced the most weight gain were fed with only white rice,

as compared to worms that ate brown rice or black rice. Worms that ate only brown rice or

black rice gained less weight than worms that ate only white rice. The significant weight

gain, observed in worms fed with only rice was most likely associated with the rice’s

nutritional value. Several studies, including Rohman et al. (2014) and Abbas et al. (2011)

and factsheets, such as those from the USDA (2015a, 2015b, and 2015c), have explained

the nutritional value of different rice. According to the USDA (2015a, 2015b, and 2015c),

white rice contains the most carbohydrates, per 100 grams, whereas brown rice and black

rice contain less carbohydrates per 100 grams. Unlike the significant weight gain observed

in those worms, the minimal weight gain observed in worms fed with rice and insecticide

was most likely associated with the eating behavior and control of T. molitor. Worms that

were fed with both rice and carbosulfan may have avoided eating carbosulfan, an indication

Emilio Solomon Discussion / 58

that the control with insecticide works. Researchers from Dastranj et al. (2013) agree with

this. Besides weight changes, worm characteristics were also observed in the acute toxicity

test. Black abdomens were observed in only worms fed with both rice and carbosulfan. It

is known that the black abdomen is an indication of a dying mealworm. Furthermore

molting, the process where worms shed their skin (Gnaedinger et al., 2013) was observed

in all worms, except for worms that only ate white rice.

In the histopathological process, various histopathological changes were

observed, including lysis in the basement membrane of the cuticle and loose connective

tissue, mucous membrane lysis in columnar epithelia, and rupturing in endothelia. It was

observed that worms fed with white rice, underwent the most histopathological changes.

Worms fed with white rice had histopathological changes in almost all structures, including

the cuticle, columnar epithelia, endothelia, and loose connective tissues. Worms fed with

black rice on the other hand, showed no signs of histopathological changes. Worms fed

with brown rice experienced more histopathological changes than worms fed with black

rice, but less than worms that ate white rice. These worms had histopathological changes in

only endothelia. Besides the various histopathological changes observed, cellular

adaptation changes were also observed.

These changes included mucous membrane formation in columnar epithelia and

accumulation in loose and dense connective tissues. It was observed that worms fed with

black rice or brown rice had the best cellular adaptation. Worms fed with black rice or

brown rice had mucous membrane formation, and loose and dense connective tissue

accumulation. Worms fed with white rice had the worst cellular adaptation. Worms fed with

white rice displayed only dense connective tissue accumulation. For columnar epithelia and

loose connective tissues, lysis was rather observed.

Overall, the histopathological process demonstrated that worms fed with black

rice had the best protection against carbosulfan, the insecticide. Worms fed with brown rice

had less protection against the insecticide than worms fed with black rice, but more

protection than worms fed with white rice. Worms fed with white rice, had the least

protection. The protection of worms against carbosulfan was most likely influenced by the

rice’s antioxidant properties, as presented in the total phenolic measurement test. According

to Table 5.1.3 and Figure 5.1.2, black rice, which had the best protection, had the highest

total phenolic content. Brown rice, which had the second best protection, had the second


highest total phenolic content. White rice, which had the worst protection, had the lowest

total phenolic content. Although there have not been many studies on the protection of

worms against carbosulfan, there have been studies conducted on the effects of carbosulfan

in animals such as fish and rats, and in humans. The effects mentioned in the studies

included acetylcholinesterase inhibition in fish (Capkin and Altinok, 2013), decrease in

mitotic index in bone marrow cells in rats (Giri et al., 2002), and mental disturbances in

humans (Bienkowski, 2014).

Emilio Solomon Conclusion / 60

CHAPTER VII

CONCLUSION

The research investigation aimed to determine whether Oryza sativa had

the ability to help protect mealworms (Tenebrio molitor) against insecticide,

carobsulfan. In order to determine this, various lab procedures were performed,

including procedures for the total phenolic measurement test, acute toxicity test, and

histopathological process. The total phenolic measurement test helped determine the

antioxidant property of different color rice. In the total phenolic measurement test, O.

sativa was extracted at various times, including 0.5, 1, 3, 5, and 24 hours. Rice

extracts were eventually used to create a standard curve. The best-fit line equation of

the standard curve along with the optical densities of white, brown, and black rice

were then used to calculate the total phenolic content of white, black, and brown rice

at various times. The acute toxicity test on the other hand, helped in the observation of

worm characteristics and weight changes. The histopathological process helped

determine the ability of O. sativa in protecting the mealworms against carbosulfan.

Results from all tests together, confirmed that the antioxidant properties of rice (O.

sativa), indeed helped protect mealworms (T. molitor) against insecticide, carbosulfan.

Results from the histopathological process supported the initial hypothesis, that black

rice was best in protecting worms against the insecticide. Worms that ate black rice

with carbosulfan experienced least histopathological changes, but the most cellular

adaptation whereas worms that ate white rice experienced the most histopathological

changes, but the least cellular adaptation. Worms that ate brown rice had more

histopathological changes than worms fed with black rice, but less than worms fed

with white rice. Worms that ate brown rice also had more cellular adaptation than

worms fed with white rice, but less than worms fed with black rice. Other tests,

including the total phenolic measurement test and acute toxicity test, strengthened the

findings from the histopathological process. In the total phenolic measurement test, it

was observed that black rice had the highest total phenolic content, followed by brown

rice and white rice. In the acute toxicity test, it was observed that worms fed with both


rice and carbosulfan had a black abdomen, an indication of a dying worm under

stressful conditions. It was also observed that worms fed with both rice and

carbosulfan experienced minimal weight gain, an indication of the worm’s eating

behavior under stressful conditions. For further research, the activity of various

enzymes found in the abdominal tissue of T. molitor should be investigated. Various

studies have already confirmed that digestive enzymes, such as cysteine proteinases

and trypsin are found in the digestive of T. molitor and help aid in the worm’s

digestion (Vinokurov et al., 2006). Deoxyribonucleic acid and ribonucleic acid found

in the cells of various structures of T. molitor should also be investigated for

transcription and translation activities. The structures found in T. molitor including the

cuticle, columnar epithelia, endothelia, loose connective tissue, dense connective

tissue, and skeletal muscle, have already been observed in the histopathological

process of this research investigation. Lastly, other insecticides and food should be

tested against T. molitor. Sufficient understanding of enzymes and genes found in the

abdominal tissue of T. molitor, as well as of other insecticides and food will hopefully

help prospective researchers, food scientists, as well as nutritionists and entomologists

in investigating better alternatives to insecticides, including carbosulfan. This

knowledge will also hopefully help the general public in understanding the dangers of

insecticides in terms of its uses and routes of exposure.

Emilio Solomon References / 62

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APPENDICES

Calculations

4.5.1 Total phenolic content

Total phenolic content = (OD765 (avg))

4.6.1 The percentage of food per body weight

4.6.2 Concentration of carbosulfan

Emilio Solomon Biography / 68

BIOGRAPHY

NAME Mr. Emilio Solomon

DATE OF BIRTH November 10, 1993

PLACE OF BIRTH Bangkok, Thailand

INSTITUTIONS ATTENDED Mahidol University International College

Bangkok, Thailand

(2012-2016)

International School Bangkok

Bangkok, Thailand

(1999-2012)

HOME ADDRESS Bangkok, Thailand

Tel: 02-967-9588

Email: [email protected]

senior project- bsc biological sciences

Health & Medicine