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. 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
PupaBeetle
Egg
Larva
56
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 ml3 ml
30 ml2.2 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 EDCB
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 have been 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
D
E
f(x) = 0.0319x + 0.5848
R² = 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
OD765
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
) 0.587 0.587 0.587 0.587 0.586
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
) 0.586 0.586 0.586 0.586 0.586
Table 5.1.3 Mean, standard deviation, and p-value of optical densities of different color rice
𝑥̅ ± 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 0.585 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
Concentration(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
Weight(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 the control group, 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 the control group. Worms in the control group 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 loose connective tissues. Rupturing was observed endothelia. Cellular
adaptation was also observed in the abdominal tissue. Mucous membrane formation was
observed in columnar epithelia and accumulation was observed in dense connective tissues.
Both histopathological changes and cellular adaptation were determined using the criteria
in the 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
had the most accumulation in the tissue. Worms that ate white rice or black rice with
insecticide had the least accumulation. Worms that ate only white rice had no accumulation.
Worms that ate only black rice had 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 or
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, 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 from 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 those from 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 those from 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

Emilio Solomon Discussion / 58
carbosulfan may have avoided eating carbosulfan, an indication 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, rupturing in endothelia, and lysis in
dense connective tissue. 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 least 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: emilio.solomon@live.com

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