California State University, Northridge the Effects Of

CALIFORNIA STATE UNIVERSITY, NORTHRIDGE

THE EFFECTS OF LECTINS IN SEA URCHIN LYTECHINUS PICTUS DURING GASTRULATION IN LOW CALCIUM SEA WATER

A thesis submitted in partial fulfillment of the requirements

For the degree of Master of Science

in Biology

Siavash Nikkhou

December 2013

The thesis of Siavash Nikkhou is approved by:

------

Dr. Aida Metzenberg Date

------

Dr. Stan Metzenberg Date

------

Dr. Steven B. Oppenheimer, Chair Date

California State University, Northridge

Acknowledgements

I would like to sincerely thank Dr. Steven B. Oppenheimer for believing in me and being the best mentor and an advisor a graduate can ask for and with his well rounded knowledge in the field assisted me throughout the research.

I would like to thank the entire Biology faculty more specifically I would like to thank Dr Karels, Dr. Aida Metzenberg and Dr. Stan Metzenberg for their support and encouragement and answering every questions.

I would like to thank my colleagues in Dr. Oppenheimer’s lab for helping me throughout the project.

I would like to express gratitude towards my family for their never ending support and especially would like to thank my girlfriend and my best friend, Shadi Asadabadi for extensive support and patience she has showed in my journey throughout the past two years through thick and thin and sticking by my side to achieve this goal.

iii

Table of Contents

Signature Page ii

Acknowledgement Page iii

List of Tables v

List of Figures vi

Abstract x

Introduction 1

Materials and Methods 14

Table of lectins 23

Results 24

Discussion 78

References 84

List of Tables

Table 1: Lectins and their Specific Carbohydrate 23

Table 2: Embryo count for morphologies exhibited in

control and experimental concentrations for lectins tested 25

Table 3: Summary of effects in control and experimental concentrations in embryos 68

Table 4: Comparison of t-test for mean percentage of morphologies

for control and various concentrations for each lectin using two- tailed test 71

Table 5: Major Outcomes of Lectins 76

List of Figures

Figure 1A Control Lytechinus pictus embryos

48 hours after fertilization. 28

Figure 1B Control Lytechinus pictus embryos

48 hours after fertilization. 28

Figure 1C Control Lytechinus pictus embryos

48 hours after fertilization. 29

Figure 2A Lytechinus pictus embryos

treated with 0.1 mg/ml of Artocarpus integrifolia 30

Figure 2B Lytechinus pictus embryos

treated with 0.1 mg/ml of Artocarpus integrifolia 30

Figure 2C Lytechinus pictus embryos

treated with 0.1 mg/ml of Artocarpus integrifolia 31

Figure 3A Lytechinus pictus embryos

treated with 0.01 mg/ml of Artocarpus integrifolia 32

Figure 3B Lytechinus pictus embryos

treated with 0.01 mg/ml of Artocarpus integrifolia 32

Figure 3C Lytechinus pictus embryos

treated with 0.01 mg/ml of Artocarpus integrifolia 33

Figure 4A Lytechinus pictus embryos

treated with 0.001 mg/ml of Artocarpus integrifolia 34

Figure 4B Lytechinus pictus embryos

treated with 0.001 mg/ml of Artocarpus integrifolia 34

Figure 4C Lytechinus pictus embryos

treated with 0.001 mg/ml of Artocarpus integrifolia 35

Figure 5A Lytechinus pictus embryos

treated with 0.0001 mg/ml of Artocarpus integrifolia 36

Figure 5B Lytechinus pictus embryos

treated with 0.0001 mg/ml of Artocarpus integrifolia 36

Figure 5C Lytechinus pictus embryos

treated with 0.0001 mg/ml of Artocarpus integrifolia 37

Figure 6A Lytechinus pictus embryos

treated with 0.00001 mg/ml of Artocarpus integrifolia 38

Figure 6B Lytechinus pictus embryos

treated with 0.00001 mg/ml of Artocarpus integrifolia 39

Figure 6C Lytechinus pictus embryos

treated with 0.00001 mg/ml of Artocarpus integrifolia 39

Figure 7A Control Lytechinus pictus embryos

48 hours after fertilization. 41

Figure 7B Control Lytechinus pictus embryos

48 hours after fertilization. 42

Figure 7C Control Lytechinus pictus embryos

48 hours after fertilization. 42

Figure 8A Lytechinus pictus embryos

treated with 0.1 mg/ml of Triticum vulgaris 43

Figure 8B Lytechinus pictus embryos

treated with 0.1 mg/ml of Triticum vulgaris 44

Figure 8C Lytechinus pictus embryos

treated with 0.1 mg/ml of Triticum vulgaris 44

Figure 9A Lytechinus pictus embryos

treated with 0.01 mg/ml of Triticum vulgaris 45

Figure 9B Lytechinus pictus embryos

treated with 0.01 mg/ml of Triticum vulgaris 46

Figure 9C Lytechinus pictus embryos

treated with 0.01 mg/ml of Triticum vulgaris 46

Figure 10A Lytechinus pictus embryos

treated with 0.001 mg/ml of Triticum vulgaris 47

Figure 10B Lytechinus pictus embryos

treated with 0.001 mg/ml of Triticum vulgaris 48

Figure 10C Lytechinus pictus embryos

treated with 0.001 mg/ml of Triticum vulgaris 48

vii

Figure 11A Lytechinus pictus embryos

treated with 0.0001 mg/ml of Triticum vulgaris 49

Figure 11B Lytechinus pictus embryos

treated with 0.0001 mg/ml of Triticum vulgaris 50

Figure 11C Lytechinus pictus embryos

treated with 0.0001 mg/ml of Triticum vulgaris 50

Figure 12A Lytechinus pictus embryos

treated with 0.00001 mg/ml of Triticum vulgaris 51

Figure 12B Lytechinus pictus embryos

treated with 0.00001 mg/ml of Triticum vulgaris 51

Figure 12C Lytechinus pictus embryos

treated with 0.00001 mg/ml of Triticum vulgaris 52

Figure 13A Control Lytechinus pictus embryos

48 hours after fertilization. 55

Figure 13B Control Lytechinus pictus embryos

48 hours after fertilization. 55

Figure 13C Control Lytechinus pictus embryos

48 hours after fertilization. 56

Figure 14A Lytechinus pictus embryos

treated with 0.1 mg/ml of Phaseolus vulgaris PHA-L 57

Figure 14B Lytechinus pictus embryos

treated with 0.1 mg/ml of Phaseolus vulgaris PHA-L 57

Figure 14C Lytechinus pictus embryos

treated with 0.1 mg/ml of Phaseolus vulgaris PHA-L 58

Figure 15A Lytechinus pictus embryos

treated with 0.01 mg/ml of Phaseolus vulgaris PHA-L 59

Figure 15B Lytechinus pictus embryos

treated with 0.01 mg/ml of Phaseolus vulgaris PHA-L 59

Figure 15C Lytechinus pictus embryos

treated with 0.01 mg/ml of Phaseolus vulgaris PHA-L 60

Figure 16A Lytechinus pictus embryos

viii

treated with 0.001 mg/ml of Phaseolus vulgaris PHA-L 61

Figure 16B Lytechinus pictus embryos

treated with 0.001 mg/ml of Phaseolus vulgaris PHA-L 62

Figure 16C Lytechinus pictus embryos

treated with 0.001 mg/ml of Phaseolus vulgaris PHA-L 62

Figure 17A Lytechinus pictus embryos

treated with 0.0001 mg/ml of Phaseolus vulgaris PHA-L 63

Figure 17B Lytechinus pictus embryos

treated with 0.0001 mg/ml of Phaseolus vulgaris PHA-L 63

Figure 17C Lytechinus pictus embryos

treated with 0.0001 mg/ml of Phaseolus vulgaris PHA-L 64

Figure 18A Lytechinus pictus embryos

treated with 0.00001 mg/ml of Phaseolus vulgaris PHA-L 65

Figure 18B Lytechinus pictus embryos

treated with 0.00001 mg/ml of Phaseolus vulgaris PHA-L 65

Figure 18C Lytechinus pictus embryos

treated with 0.00001 mg/ml of Phaseolus vulgaris PHA-L 66

Figure 19 Percentage of embryos exhibiting different morphologies

for treated and control embryos using lectin Artocarpus

integrifolia 73

Figure 20 Percentage of embryos exhibiting different morphologies for

for treated and control embryos using lectin Triticum vulgaris 74

Figure 21 Percentage of embryos exhibiting different morphologies for

for treated and control embryos using lectin Phaseolus

vulgaris (PHA-L) 75

ABSTRACT

THE EFFECTS OF LECTINS IN SEA URCHIN LYTECHINUS PICTUS DURING GASTRULATION IN LOW CALCIUM SEA WATER

Siavash Nikkhou

Master of Science in Biology

In order to help learn about the possible role of carbohydrate – containing molecules in gastrulation in the model sea urchin embryo (Lytechinus pictus), 24 hr sea urchin embryos were incubated with 3 lectins (carbohydrate binding proteins) Triticum vulgaris (wheat germ agglutinin), Artocarpus integrifolia agglutinin and Phaseolus vulgaris PHA-L agglutinin at 0.1-0.00001 mg/ml. Triticum is a specific binder of N-acetyl-D-glucosamine- like residues, as is Phaseolus, while Artocarpus is a specific binder of D-galactose-like residues. The embryos were treated with and without these lectins at all 5 concentrations for an additional 24 hrs at 15 ⁰C in lower calcium artificial seawater (that speeds entry of molecules into the interior of the embryos) in 96 wells well flat bottom microplates. The wells were treated with 10% formaldehyde to fix the embryos at the 48 hr stage (late gastrula). All embryos in each well were scored as to their morphologies: complete archenteron, incomplete archenteron, non-invaginated, exogastrulated or dead. Thousands of embryos were scored. Means of percentages of each morphology for each concentration of each lectin was plotted with standard error bars and a t-test was used to determine if any differences in the experimentals vs. controls were statistically significant (P<0.05). The results indicated statistically significant concentration dependent effects of all 3 lectins on altering the morphologies of the embryos. These preliminary results suggest that N-acetyl –D- glucosamine and D-galactose groups maybe involved in archenteron elongation and organization. The microplate assay is an effective means of quantitatively determining the precise effects of reagents on sea urchin morphologies, an NIH designated model for higher organisms including humans.

INTRODUCTION

Sea urchins are model organisms used to provide insight into how organisms function and develop. Sea urchins are simple organisms that are translucent during the larval stage, allowing for observation of cell division and reproduction. Sea urchins are used so widely as a model organism because they provide an excellent model for the study of cell differentiation, reproduction, development, and cell adhesions (Gustafson et al., 1963).

Sea urchins have been used as a model system for over a century. Their popularity as a research subjects comes from their widespread availability, the ease with which they can be stored, and the large numbers of gametes that can be harvested in a very short period of time. Although sea urchins are simple, development of their embryos and larva involves transcription factors and genetic components that are very similar to much more complex organisms. Sea urchins have been around for more than 500 million years and share common ancestors with many complex organisms of today (Hardin and Cheng, 1986).

Lytechinus pictus is a species of sea urchin that is native to California. They are inexpensive and easy to capture, and as such they have been used for research purposes since at least the 1800’s. In addition to the developmental similarities that sea urchins share with more complex organisms, sea urchin genes have high levels of homology with vertebrate immune system related genes (Hardin and Cheng, 1986).

Lytechinus pictus exhibits five-fold radial symmetry known as pentamerism. The larvae forms of sea urchins have bilateral symmetry. The adult organisms move by generating hydraulic pressure with their vascular system, which is used to move hundreds of transparent tube feet. The adults range in size from 6 to

12 cm across, and they are found in the intertidal ocean at depths of up to 5000 meters.

This background information and complete understanding of sea urchins allow us to look at the similarities the sea urchins have in common with humans. The genetic and biological similarities sea urchins have to humans makes them ideal model organisms to study cellular interactions that mirror those found in human cells.

Development of Sea Urchins

The simplicity and easy use of sea urchins have been the main reasons for their use in today’s studies. The injection of the male and female sea urchins with a simple potassium chloride solution produces large amounts of gametes that can be used for a variety of research projects (Miller, 1985). The artificial seawater used as a medium can be cheaply and easily produced. The sea urchin embryos are visible throughout their development, and can easily be viewed and manipulated using simple microscopic techniques (Balinsky, 1959; Campbell & Reece 2002). The interaction among sea urchin sperm and egg shows specific cell-cell interactions also observed in other cnidarians, mollusks and echinoderms. This process is due to the release of chemicals and chemotactic factors which attract the sperm to the appropriate eggs, allowing for fertilization to take place (Gilbert, 1988).

Production of eggs and sperm allows the sea urchins to sexually reproduce (Bowers 2006). The success of spawning which is the release of eggs and sperm into artificial seawater in this case which led to successful fertilization is critical to outcome of our research.

The sea urchin egg has a chemotaxic mechanism which attracts the sperm to the egg in a species- specific manner. This chemical recognition by the egg allows for the sperm to fertilize the egg properly

(Yoshida and Yoshida, 2011). This chemical mechanism is important for maximizing the probability that each egg is fertilized by a single sperm. However with lots of sperms and eggs available, millions of embryos are produced which can be used. Among the sea urchins that have been studied, the species

Arabacia punctulata showed that they are surrounded by a jellycoat containing 14 amino acid proteins known as resact. The sperm of this species have receptors on their surfaces that recognize the amino acids and bind to it. In the research done by Ward et al, 1985 once resact binds to the receptors on the sperm cells it causes activation of movement in the sperm’s tail causing it to become motile and propel towards the jelly coat of the egg (Ward et al, 1985). The flagellum of the sperm cells assists the sperm in moving towards the egg as a successful fertilization depends on being fully prepared to be functional. The motility of the flagellum is very important since the sperm cells need to travel long distances through the medium to arrive at the egg and break through the walls of the egg cells and fertilize them. In order for the sperm to have the ability to penetrate and fertilize the egg, it needs to have an efficient highly specialized membrane

2 used as a storage compartment at the tip of the sperm. The compartment is called the acrosome which contains enzymes which aid in breaking and digesting proteins and sugars. The moment sperm cells touch the outer coating of the egg cell the acrosome ruptures, releasing its contents in a process known as the acrosomal reaction (Summers and Hylander 1974).

The initiation of the process of acrosomal reaction begins with a sperm cell touching the jelly coat of the egg cell on the exterior surface making a union of the acrosomal membrane with the sperm cell membrane. The substance found in the egg jelly that causes the acrosomal reaction to take place is very specific in every species of sea urchins showing the species-specific mechanism allowing for the correct sperm uniting and fertilizing the correct egg (Vacquier et al., 1978; Hylander and Summers, 1981).

The egg and sperm fuse using the protein bindin which is closely linked with acrosomal substances. Bindin is found in the head of the sperm and only released at the end of the acrosomal reaction.

The isolation of bindin from the acrosome is detected when it is competent to bind to the eggs of the species of sea urchins that are the same; hence it is species- specific. This specificity works like lock and key as the receptors are the lock on the egg cell surface that solely identify the bindin protein that is the correct key from the sperm of the same species leading to successful fusion of the cells (Summers and

Hylander, 1975). Bindin protein that is recognized is the beginning process of both egg and sperm syngamy as the membrane of both cells fuse allowing for the next stage of fertilization and cell division to take place

(Vacquier, 1978).

One important phenomenon that takes place in sea urchins that ensures one sperm fertilizes one egg is the concept of blockage to polyspermy. If polyspermy takes places, it results in a non-viable zygote, but due to this phenomenon this does not take place. This method prevents multiple sperms penetrating the eggs cell and fertilizing it. This mechanism is a rapid blockage of multiple sperm fertilizing an egg cell.

The process happens as one sperm is binding to the egg cell and penetrating it. It takes about one to three seconds, and is the result of changes in the calcium ions found in the egg cell extending all over the cell membrane while electrical activity takes place. The blockage of polyspermy is the result of calcium ions and electrical activity that is short term prevention so that no more sperm cell can enter the egg. Another process known as the cortical reaction is result of events and reactions which result in alteration of the

3 surface protein coat. This process takes place in addition to the quick block response of the sperm and egg cells (Colwin and Colwin, 1963). This mechanism is called the slow block to polyspermy. Using a compound microscope it is feasible to see the elevation of the fertilization envelope although the rapid blockage takes place in seconds making it hard to observe it with naked eye. As soon as the fertilization envelope is lifted, additional sperm cannot fertilize the egg (Bleil and Wassarman, 1980a).

Once the egg has been fertilized, the zygote begins dividing, eventually forming a structure called the blastula, a spherical structure composed of approximately 128 cells. Within the blastula there is a fluid filled cavity called the blastocoel. It is in this stage of cellular division that there are contacts between the blastula cells and the hyaline layer (Gilbert, 1988). During the blastula stage there is appearance of tight junctions which allow for the cells in the surrounding blastomeres to bind tightly so there is no interaction and communication with the outside environment (Dan-Sohkawa and Fujisawa, 1980). The morphology and the shape of the embryo are due to adhesive properties between the cells and the hyaline layer (Wolpert and Gustafson, 1961).

Gastrulation is composed of multiple stages of cellular invagination and rearrangement, resulting in the creation of three germ layers. The endoderm creates the inner linings of the respiratory and digestive tracts. The ectoderm makes up the nervous system, mouth and nose. The mesoderm becomes the circulatory, muscular, and excretory systems.

The well documented and studied stages of gastrulation in sea urchins include (1) ingression in primary or skeletogenic mesenchyme (2) invagination process in the vegetal plate producing the early archenteron (3) elongation in the archenteron which creates the secondary mesenchyme cells and the last phase (4) touching the tip of the archenteron at the animal pole close to the apical plate where the area is thickened ectodermally (Gustafson and Wolpert, 1961). There are different types of proteins that aid with movements of the primary mesenchymal cells during gastrulation (Katoh and Hayashi, 1985).

A vast amount of research has been done on sea urchin gastrulation, and photomicrography of each event has been documented (Gustafson and Wolpert 1967; Gustafson and Toneby 1971). Once the

4 primary mesenchyme cells derived from the micromeres located in the center of vegetal pole region travel into the blastocoel, gastrulation begins. This cell movement is called ingression as it takes place once the hatched blastula is completed. This is a result of flattening of the hatched blastula starting gastrulation. The embryo at this early stage of gastrulation represents the mesenchyme blastula. The location of primary mesenchyme cells (PMCs) is in the vegetal plate that turns into the mesoderm (Shook, 2003). Loss of affinity of the PMCs to the outside of the embryo cells called the hyaline layer occurs. PMCs increased affinity for basal the lamina is essential and critical for the cells to start their movement within the blastocoel (Fink and McClay, 1985). The basal lamina is important as it is the site where PMCs move and interact with cells on the blastocoel wall. At the initiation of ingression process, there are changes in the shapes and morphologies caused by microtubules that are observed and detected on the axis of the PMCs

(Tiley and Gibbins, 1969; Katow and Solursh, 1986).

The process of invagination has two phases; primary and secondary invagination (Dan and

Okazaki, 1956; Gustafson and Kinnander, 1956). This process of invagination results in sheets of epithelial cells curving inwardly that creates an inpocketing. As a result of inpocketing, there is a tube created once the invagination at the vegetal region proceeds. The tube is called the archenteron or the primitive gut that has one opening at one end while the opposite end is closed. This process of elongation of the archenteron takes place through the blastopore (Ettensohn, 1988). The process is a result of constant rearrangement and shifting of epithelial cells on the vegetal plate (Hardin and Shying, 1986).

Elongation continues during the secondary invagination as the projections in the cytoplasm extend from secondary cells at the tip of the archenteron. The cytoplasmic projections are long filopodia that start stretching toward the animal pole of the embryo until exact and precise contacts and attachments are created with the ectoderm (Hardin and Cheng, 1986). Eventually the archenteron tip that is composed of secondary mesenchymal cells and coelomic mesodermal cells make contact with the opposite side of blastopore on the ectodermal region. By the end of gastrulation, secondary and coelomic cells will depart from the tip of the archenteron and get separated (Davidson et al., 1989).

The proper cytoplasmic extension of the filopodia is critical for the complete development and formation of archenteron and process of gastrulation. Pancreatin destroys the function and action of

5 filopodia hence the archenteron fails to develop fully and elongation is halted once the toxic agent is added

(Dan Okazaki, 1956). This is critical as studies by Hardin and Cheng, 1986 showed that filopodia are essential for primary and secondary mesenchymal cells since they can enter the basal lamina and get in the middle of ectodermal cells where the cells can attach and the process can be completed fully (Hardin and

Cheng, 1986). The phase in which the cells are contacting one another is critical since adhesive reactions among cells take place and further developmental stages will proceed. The archenteron near the roof of the blastocoel is created as the secondary mesenchyme cells start to disappear. The mouth of the embryo cell is developed at the site where the archenteron contacts the roof of the blastocoel. The archenteron which has an elongated tube looking shape makes up the digestive tube and lastly the anus of the embryo will be created from the blastopore (Davidson et al., 1998).

The vegetal region takes approximately 20-24 hours after fertilization to start showing invagination. The process of gastrulation to completion takes approximately 48 hours. At this point the embryo is called a larva which proceeds to pluteus stage where post fertilization mouth, gut and anus are now evident. Once this stage ends, the larva makes components resembling adult sea urchin (Davidson et al., 1998).

Lectins

Lectin is derived from the Latin word, legere which means to choose or to select which covers all non-immune carbohydrate-specific agglutinins in regards to specificity for blood types (Sharon and Lis,

2004). In the cells, membranes, and secretomes of living being are sites where lectins are found having a binding pocket or binding site that allows ligands such as specific carbohydrates to bind (McMahon et al.,

2005). Some of the functions of lectins include extension and expansion of cell walls, stimulating mutation causing agents, protein storage, immune and defensive mechanisms, carbohydrate transportations and shipping, packaging, and movement of stored material (Boyd et al., 1963). The process of induction can take place among lectins by making the animal cells divide and differentiate. The binding of lectins to the carbohydrates on the surface of various cells are extremely specific interactions. Lectins are proteins that

6 bind to oligosaccharides/glycans (Minko, 2004). The earliest descriptions of lectins and study of lectins are found in works of Hermann Stillmark dating back to 1888. In his studies, he describes the binding of ricin

(Bies et al., 2004; Sharon and Lis, 2004). Originally, lectins were detected in plants, but in recent years a variety of lectins have been collected from different microorganisms and even animals (Sharon and Lis,

2004). Structurally, lectins isolated from plants and animals show no homology, although they show similar characteristics in binding to similar carbohydrates. Plants and animals’ lectin genes may have evolved together hence making the interaction of lectins and their specific carbohydrates they bind to of more importance (Gorelik et al., 2001). The most important function of lectins that has been studied is the defensive role and mechanisms related to how they protect and defend the plants (Etzler, et al., 1985). The lectins attack microorganisms via processes in enzymes that are inhibited and/or hydrolyzed. In a research done by Pueppke et al., 1983, it was shown that lectins also can bind to nitrogen fixing bacteria seen in roots of legume plants.

Diversity among structures of lectins has been studied for years. In a study done by Sumner et al.,

1919, Concanvalin A isolated from jack beans was crystallized, and by using X-ray diffraction the structure of the lectin was identified. This process shows the structure of lectin which evidently aids in understanding of how the lectin binds specifically to specific carbohydrate allowing researchers understand the 3-D conformational shape and morphology of concanvalin A (Sumner et al., 1919). Studies on the seeds of the

Dolichos biflorus plants showed that lectin is found specifically at the terminal non- reducing α-linked N- acetylgalactosamine residues (Etzler et al., 1984). Furthermore the study showed the lectin is found in the cotyledons localized in protein bodies when the seed is maturing (Etzler et al., 1977). Studies from Talbot et al., 1978, showed that the lectin started breaking down while the seed of Dolichos biflorus was germinating. This was coincidental with the growth of the seedling at the same rate as the protein was stored in the cotyledon. The lectin was not found in any other parts of the plant (Talbot et al., 1978).

Background Information on Triticum vulgaris

Triticum vulgaris is isolated from many organisms and structures including seeds, plant roots and bark, fungi, bacteria, seaweed, sponges, mollusks, fish eggs, bodily fluids of invertebrates and lower vertebrates and cell membranes from the mammalian organisms. The physiological and cellular function of the lectin is not clear but it is important in vitro and can be used in blood grouping and polyagglutination in erythrocyte studies, mutation generating agents (mitogenic) that stimulate lymphocytes and histochemical studies on normal and pathogenic cells.

Lectin Triticum vulgaris extracted from wheat germ and also called wheat germ agglutinin (WGA) is purified by the process of affinity chromatography. The lectin binds specifically to N-acetyl-D- glucosamine- like residues to WGA (Liener et al., 1988). This lectin contains two subunits and has a molecular weight of thirty- six kilodaltons. This lectin is known to bind to N-acetyl-β-D- glucosamine

(GlcNAc) and to N-acetylneuraminic acid known as the sialic acid residues of glycoproteins and glycolipids (Johnson, 1992). Triticum vulgaris lectin causes agglutination of the erythrocytes (red blood cells) and malignant cells in which the cells are different from normal tissues. In small concentrations (Less than 4 μg/ml), the lectin agglutinates human erythrocytes. It is used in conjunction with treatment of trypsin of the cells. When using low amount such as 200mM N-acetyl-D- glucosamine added to Triticum vulgaris, a full inhibitory effect was observed. Triticum vulgaris inhibits C5a receptor interaction (Johnson, 1992).

Triticum vulgaris is used commonly with Con A to study glycoproteins and cell surface proteins by analyzing the results and the outcomes of such binding. Triticum vulgaris is a white to pale-yellow lyophilized form from 10M CH3COONH4 with no additional preservatives added. The purity of this lectin is determined using SDS-PAGE that creates three bands which contribute to the bands of isolectins of

Triticum vulgaris.

Background Information on Phaseolus vulgaris (PHA-L)

Phytohemagglutinin- L is isolated from Phaseolus vulgaris that is a leucoagglutinating seed lectin from a family of lentil (legumes). PHA-L is a tetramer and is different from the well studied lectin

Concanvalin A. PHA-L consists of two canonical legume lectins, dimeric in structure, packed together and closely in contact with one another (Shade et al., 1994). This family of lectin is glycoprotein meaning that each subunit is N-glycosylated at two loci (Sturm and Chrispeels, 1986). PHA-L binds to minimum of five saccharides Galβ14GlcNAcβ12[Galβ1– 4GlcNAcβ1– 6] Mannose that is found in the complex found from the mammalian origin (Hammerstrom et al., 1982). The lectin binds covalently to oligosaccharides but specifically to GlcNAc (N-Acetyl-β-D-glucosamine). PHA-L is extracted mainly from red kidney bean and is used in cellular signaling and recognizing carbohydrates. PHA-L is also important in effects in glycobiology, immunity signaling, lectin antibodies and modifications in post translational proteins. It is found in white and yellow powder (Rueben, 1977).

Phaseolus vulgaris leucoagglutinin (PHA-L) contains a high mutagenic agent (mitogenic) and agglutinates leukocytes with four subunits and five isolectins. This lectin is extracted from plants and is transported anterogradely by neurons in central nervous system. This lectin selectively is taken up at iontophoretic injection sites. These sites include the axons, dendrites and terminal boutons. PHA-L is used as a biogenic marker (Gerfen, 1985). Once PHA-L is injected, it takes about 4-6 mm/day for the transport rate. It is visualized in the neural cells using light microscopy when the antibody binds to lectin (Rueben,

1977).

Background Information on Artocarpus integrifolia

Lectin Artocarpus integrifolia (Jacalin) is isolated from seeds of Jackfruit and is purified using the affinity chromatography. Artocarpus integrifolia belongs to the family of T-antigen and is D-galactose binding (Liener, 1988). Artocarpus integrifolia has a tetrameric two chain structure with molecular weight of 66 kilodaltons. This lectin specifically binds to carbohydrate structures on the surface of the cell and is used in hematology, immunology and as a specific marker for glycoproteins on the membrane structures

(Sankaranarayanan, 1996). Furthermore, this lectin is used to isolate IgA from human serum, isolation of human plasma glycoproteins and industry in histochemistry. Modifications in the post translational proteolytic process of the lectin give a new carbohydrate binding site which interacts with the N-terminus of the protein. The structure of protein identifies the relative affinities of the lectin to its specific carbohydrate, galactose derivatives and structurally to Thomsen-Friedenreich (T)-antigen

(Sankaranarayanan, 1996). Artocarpus integrifolia is found as a white to light yellow powder and the purity is measured using SDS-PAGE. The SDS-PAGE reveals two homogenous bands at two sites of 12 kDa and

16 kDa. This lectin is useful in research related to AIDS.

The diversity among all kinds of lectins is seen as they bind specifically to specific carbohydrates.

The lectin Artocarpus integrifolia is extracted from jack fruit and binds specifically to disaccharide Gal-

β13-GalNac and binds to IgA on the proteins of human cells. It shows specificity for T-antigen and contains alpha and beta chains with different three dimensional structures to other plant lectins (Pilatte,

1995). Artocarpus integrifolia is composed of an unusual quaternary configuration which binds the alpha chains to beta chains in a unique pattern. Artocarpus integrifolia does not come from Moraceae family of lectins and does not have any homology with legume or other plant lectins due to its unique orientation and patterns described (Lee et al., 1989). Jacalin attaches and binds to the heme binding protein of hemopexin and receptors of C4β-binding proteins in human bloods. The binding of the hemopexin and Jacalin shows that there is a single O- linked oligosaccharide which is essential and necessary to allow for the binding of protein and lectin to occur (Pilatte, 1995). Jacalin can be used as an instrument to identify proteins that are linked to O-linked glycans (Hortin, 1990). In a study by Monestier et al., they were able to show how immobilized Jacalin was used to mark IgA1 class of immunoglobin in nephropathy (Monestier et al.,

1994).

The capacity of lectins to cause agglutination with erythrocytes has been previously mentioned.

This is a distinctive characteristic which makes lectins more useful tools in medicine than other ligands that bind to carbohydrates. The type and structure of carbohydrates determine the lectin that will bind to these sites making them generally very specific like the lock and key. If there are any modifications or alterations exerted on the carbohydrate sites, the appropriate and particular lectin generally cannot bind hence the binding sites are highly conserved for specific lectin (Sharon et al., 1986). Different lectins may be different in structures and functions however they all have a binding site for the ligand which is specific carbohydrate. The specific carbohydrate on the cell surface normally a monosaccharide binds the lectin to the surface of the cell. The differences among the families of lectins are driven from the type of carbohydrate they bind and what conformational changes may be caused when binding is completed

(Liener et al., 1988). The lectins that bind to same carbohydrates are classified together in the same family hence usually have similar effects on the structural components not necessarily functional aspects (Sharon et al., 1996). The lectins that bind to erythrocytes and leukocytes cause agglutination via cross bridging.

Agglutination shows that the correct binding took place between the lectin for specific carbohydrate on the surface of the cell. If such agglutination does not take place either the lectin is not correctly oriented or it is not specific for the carbohydrate to bind properly (Liener, 1988).

Cellular interactions and communications are essential while the lectin is securely and properly attached to the specific carbohydrate binding site. Lectins are considered to be multivalent molecules that can result in linking several receptor sites on the surface of the cells explaining lectin agglutination once the lectin binds to the specific carbohydrate. The interaction and recognition pattern allows for cellular communication and cellular trafficking created from sperm and egg cells to implantation, fertilization, gastrulation and neurulation and further developments in embryogenesis (Halomori et al., 2000).

Research performed by Stillmark in the 19th century noticed that when extracts were taken from castor bean seeds they caused agglutination of human erythrocytes. Other studies showed that extracts from beans of 800 species of plants, mostly legumes, affects cell properties.

Research done by Khurrum et al., 2004 suggested that carbohydrates are essential in how the cells interact in sea urchins embryos and their presence allows cascades of events to continue. In their study they used glycoprotein/proteoglycan inhibitors such as tunicamycin and sodium selenate and β-amylase, α- glucosidase and α-mannosidase and they found that no complete archenteron was observed. On the cellular levels, the development was halted as there was no elongation of the cells and consequently cells did not reach the roof of the blastocoel to make the mouth of the embryos in these sea urchins (Khurrum et al.,

2004) When calcium levels are low proper cell adhesion and attachment properties with other cells are lost

(Oppenheimer and Carroll, 2004; Oppenheimer, 2006).

For a long time the only way that macromolecules including lipids, proteins and carbohydrates were inserted into the blastocoels of embryo was through microinjection (Kiehart, 1982). There are downsides associated with microinjection as only few embryos can be used at a time not allowing large

11 numbers of embryos to be studied. The technique of microinjection has limitations that can be insidious. In the research by Latham et al., 1998, the colleagues showed that instead of using microinjection, incubation of a flurochrome-labeled lectins and bovine albumin which were the selected macromolecules were able to go in the blastocoels of sea urchin embryos. The processes of macromolecules entering the blastocoels of the embryos were documented by using a laser scanning confocal microscopy. The entrance of the macromolecules occurred at faster rate when the medium used was low calcium seawater (Latham et al.,

1998). This study by Latham et al., 1998 was important to research going on since it can be done on huge populations of sea urchin embryos at a time, as it will save time and save resources for more trials to take place. In addition this process is less invasive than microinjection. The low calcium seawater allows the epithelium of blastula stage to turn highly permeable to macromolecules and substances added (Itza et al.,

2005). During the incubation period in low calcium seawater the macromolecules can enter through the process of simple diffusion (Itza et al., 2005).

In this study, Itza et al., 2005 showed that the blastula in sea urchins show septate junctions that are looser than tight junctions seen in chordate epithelium (Itza et al., 2005). Using low calcium seawater treatments allow experimenting on millions of embryos at once if necessary. The results can be measured while the technique used in non invasive and causes no harm or damage is done to embryos.

The study performed by Latham et al., 1999 showed the effect of various lectins at different post fertilization hours during gastrulation stages in Strongylocentrotus purpuratus sea urchin embryos. They found important results with lectins Concanvalin A, succinylconcanavalin A, Lens culinaris agglutinin, wheat germ agglutinin, Pisum sativum and Artocarpus integrifolia with concentrations of 0.01-100 µg per ml in artificial seawater at 15-28 hour incubation after fertilization. They determined that Pisum sativum and Lens culinaris attached and agglutinated by binding to SMCs that was important in anchoring of archenteron during gastrulation. The results showed that lectins caused exogastrulation in the embryos. On the other hand the results from wheat germ agglutinin showed atypical and unusual skeletogenesis. These results from Latham et al., 1999 demonstrated that Pisum sativum and Lens culinaris that bind D-mannose residues that seem critical in development of the archenteron and anchoring the cells. Furthermore, the results from their study suggested that N-acetyl-D- glucosamine like residues cause primary mesenchymal

12 cells to be utilized (Latham et al., 1999). Based on the study by Latham et al., 1998, if a presented lectin inserted into the blastocoel of the embryo cells, it may hinder the cellular mechanism and communications between the cells (Latham et al., 1998).

In the present study, three lectins were chosen including: Artocarpus integrifolia, Triticum vulgaris , Phaseolus vulgaris PHA- L. Lectin from Artocarpus integrifolia binds to D-galactose –like residues, Triticum vulgaris and Phaseolus vulgaris PHA- L attach to N-Acetyl- D- glucosamine- like residues. These lectins were examined under five different experimental concentrations of 0.1, 0.01, 0.001,

0.0001 and 0.00001 mg of lectins per ml of low calcium seawater. The experimental concentrations are compared to the Control which is made from low calcium seawater only. The purpose of this study is to determine possible roles and interactions of carbohydrate of containing molecules in gastrulation as the cells are rearranging and developing. By using a light inverted microscope, the process of adhesion and cellular movements during gastrulation in the sea urchin embryos from L. pictus. I hypothesized that the introduction of each lectin named can interfere with the actions of the specific carbohydrates present on the outer surface of embryo cells and block and obstruct events from occurring. Based on previous studies already mentioned, there are specific carbohydrates that bind to lectins. If lectin binding takes place, this may result in abnormal development among the sea urchin embryos and can suggest that these specific carbohydrates are present on the surface of the cells and maybe involved in gastrulation cellular interactions. This way we can learn more about the mechanism of what functions and tasks these known carbohydrates and their receptors have on the sea urchin embryo cells during the communication between the cells and their development during gastrulation.

MATERIALS AND METHOD

Materials: Lytechinus pictus were used for the experiments performed. The sea urchins were bought from

Marinus Scientific located in Garden Grove, California. After purchase, the sea urchins were stored in refrigerated seawater aquaria at temperature of 10-14 ⁰C.

Preparation of Artificial Seawater (ASW)

Artificial Seawater (ASW) was made using the protocol from the Marine Biological Laboratory

Formula (Bidwell and Spotte, 1985). The optimal pH for the ASW was 8.0, and was achieved by titration with HCl or TRIS (Fisher Scientific, USA) as needed. The chemical components were purchased from

Sigma Chemical Co, St. Louis, Missouri. The protocol that was used from Marine Biological Laboratory

Formula 130 was followed to produce four liters of ASW. Four liters (4000 ml) measured with large graduated cylinders of distilled Arrowhead water. Water was poured into an autoclaved and sterile 4000ml

Erlenmeyer flask.

The following components were measured out and added to 4L of distilled water in a sterile

Erlenmeyer flask: 98.88 grams NaCl, 2.68 grams KCl, 5.44 grams CaCl2*2H2O (0.009M), 18.64 grams

MgCl2*6H2O, and 25.16 grams MgSO4*7H2O. These measurements were obtained using an electric balance. The components as described were allowed to stir for 15-20 minutes at 300-500 rpm until all components were dissolved. After 15-20 minutes, 0.72 grams of NaHCO3 was measured and added to the stirred solution to balance the pH. The mixed solution was allowed to stir for another 15 minutes until all the NaHCO3 was dissolved. Next the pH of the solution was measured and was adjusted to pH of 8.0 by use of a pH meter obtained from Beckham Coulter (Brea CA). When the optimal pH of 8.0 was achieved, the ASW in the Erlenmeyer flask was sealed using Para-film M (American Can, Chicago, IL), dated, labeled and stored in the incubator at 15 ⁰C for up to one week.

Preparation of Low Calcium Artificial Seawater

Low calcium artificial seawater (LcASW) was made using the protocol from the Marine

Biological Laboratory Formula (Bidwell and spotte, 1985) same as ASW but at lower calcium

14 concentrations. The optimal pH for the LcASW is 8.0 that is the ideal pH for sea urchins which is obtained using acid or base depending on pH detected. The chemical components were purchased from Sigma

Chemical Co, St. Louis, Missouri. The protocol used from Marine Biological Laboratory Formula was followed to produce one liter of LcASW. One liter (1000 ml) measured with large graduated cylinders of distilled Arrowhead was poured into an autoclaved and sterile Erlenmeyer Flask that measures 4000 ml

(4L).

The components that were added into the Erlenmeyer flask started first by putting in 24.72 grams of NaCl, followed by 0.67 grams KCl, then 0.113 grams CaCl2*2H2O followed by 4.66 grams

MgCl2*6H2O then added 6.29 grams of MgSO4*7H2O were all measured and in order were added. These measurements were obtained using an electric scale and all were added accordingly to the distilled

Arrowhead water. The components as described were allowed to stir for 15-20 minutes at 300-500 rpm until all components were dissolved. After 15-20 minutes, 0.18 grams of NaHCO3 was measured and added to the stirred solution to balance the pH. The mixed solution was allowed to stir for another 15 minutes until all of NaHCO3 was dissolved. Next the pH of the solution was measured and was adjusted to pH of

8.0. By using a pH meter obtained from Beckham Co. initial pH for the LcASW was obtained.

After calibration was done, optimal pH of 8.0 was made using same procedures as ASW. When the optimal pH of 8.0 was achieved, the LcASW in the Erlenmeyer flask (Pyrex, USA) was sealed using

Para-film M (American Can, Chicago, IL), dated, labeled and was put into the incubator at 15 ⁰C for future uses. This LcASW was good for use for up to one week.

Preparation of 0.55M Potassium Chloride (KCl)

To prepare 0.55 M potassium chloride, 4.125 grams of KCl that was purchased from Sigma (St.

Louis, Missouri) was placed into a 200 ml Erlenmeyer flask. To the flask, 100 ml distilled Arrowhead water was added. All of the measurements were done accurately and precisely by using an electric scale to eliminate any errors. When the KCl and distilled Arrowhead water were added, they were allowed to be mixed on the magnetic stirrer using a stir bar for approximately 10-15 minutes until all KCl was dissolved.

This mixture produced 100 ml of 0.55M KCl, and was sealed using a para-film M (Chicago, IL, 60631),

15 labeled and was put into the incubator at 15⁰C to be used during the experiments. This solution can be used up to one month from the day it was prepared.

Extraction of Gametes

To extract the gametes either sperm or egg from the male or female sea urchins, 1 ml (1cc) of 0.55

M KCl (refer to section to make 0.55M KCl) was injected within the coelom. (intracoelomically) into three separate areas around the mouth of Lytechinus pictus. The injection of KCl was done using a 5 ml syringe.

The multiple injections into the Lytechinus pictus allowed for the gametes to be released. It took approximately 1-3 minutes for the gametes to be released when the injection was completed. The eggs had a yellow-brownish coloration and viscous and aqueous texture and the sperm was white with thicker texture.

Collection of Gametes

When the sea urchins were taken out of aquaria and taken to the lab, they were injected with approximately 3 ml of potassium chloride (KCl). If there was release of eggs (yellow gametes), the sea urchin was female and if they released sperm (white gametes) they were considered males. The eggs from the female sea urchins were washed with artificial seawater (ASW) at pH 8.0 by using transfer pipettes

(Fisher Scientific, USA). The ASW and lcASW were poured in two separate 50 ml beaker and to protect it from contamination; they were covered with para-film M (American National Can, Chicago, IL). When the gamete was determined to be female by sea urchin excreting yellow viscous eggs, the sea urchin was put onto a small beaker filled with ASW and was covered with Para-film M with a small opening made by razor or scissors. The sea urchin was put upside down so the eggs could be released in the ASW. The sea urchin was put on the beaker while para-film protected it from falling into the ASW. The egg releasing continued until no more eggs could be extracted from the sea urchin which usually took 20-30 minutes.

When all the eggs were released, the eggs were washed three times using fresh ASW. This technique ensured that no contamination in the eggs caused any changes to results and the outcomes. The washings were done when all of the eggs settled to the bottom of the beaker; the liquid portion was drawn out by a pipette that was attached to a hose that was attached to air suction. When the valve was open, the

16 air took out the aqueous portion of the beaker into a discarded flask. Then more ASW was added to the beaker and the procedure was done twice more. This process took up an hour and half to achieve best results. When the washing was done, the egg samples from each sea urchin were tested for fertility, viability and high-quality morphology. This process was done by taking a transfer pipette, cutting the tip so the narrow tip did not damage the eggs and placed few drops of the eggs into a petri dish. The petri dishes used were Falcon Petri Dish, 60x15 mm to test for good eggs. The egg samples were observed using a

Fisher Scientific Compound Microscope. The good, viable and fertile eggs were those that had a full round circle with no damages to the egg. The good egg contained a full round pronucleus. If the eggs were lysed or damaged, they were not used and were discarded. The viable eggs from female sea urchin were put into a larger Pyrex dish tray that was filled with more ASW. The larger Pyrex dish increased the surface area for the eggs and sperm to be fertilized.

After the sperm was recovered from the male sea urchins, the sea urchins were placed upside down onto a petri dish over ice which stimulated the release of sperm. When all of the sperm was recovered and extracted from the sea urchin, the petri dish was diluted with ASW. The sperm was checked for viability by taking a transfer pipette and putting few drops on a slide and checked if the sperm was active, motile and functional. The sperm was tested and checked for viability using a Fisher Scientific Compound

Microscope. The viable and active sperms were determined when more than 90% of the sample showed movement. Using a plastic transfer pipette the rest of the sperm were collected from the petri dish and stored into a 15-ml falcon tube (Polystyrene Conical Tube 17x120 mm style; Becton Dickinson, Franklin

Lakes, New Jersey) incubated over ice until it was time to be used for fertilization.

Preparation of Fertilization Sample

To check for viability and fertility of sperm and eggs extracted from the Lytechinus pictus, a sample of eggs and sperms was fertilized to detect any abnormalities in proper development. Using a transfer pipette with the tip cut so it does not damage the eggs while transferring, few drops of eggs were put into a Falcon petri dish (60x15 mm) and with a different transfer pipette few drops of sperm were added to the Falcon petri dish to cause fertilization. Using a Fisher Scientific Compound Microscope the fertilization processes was observed to detect any abnormalities. A successful fertilization was achieved

17 when there was a fertilization membrane enclosing an egg as a result of syngamy between sperm and egg.

Furthermore, eggs and sperms that showed over 90% success with fertilization membranes were used and any low fertilization of eggs was discarded. This ensured that the development in the embryos continued and better results were achieved.

Preparation of Fertilization of the Gametes

The Pyrex dish (8” x 10” or 8” x 8”, New York, USA) was first autoclaved to avoid any contaminants while fertilizing the eggs and sperms. After these dishes were autoclaved they were also rinsed using 95% Ethanol and rinse with ASW pH of 8.0. The good eggs recovered from each female sea urchin were decanted into each dish. This process was done slowly and carefully so they are not lysed or damaged. After all the eggs were deposited into a glass Pyrex dish, additional 250-300 ml of ASW was added. Next the sperm were diluted by adding 8-10 ml of ASW at pH 8.0 to 1 ml sperm placed into 15 ml

Falcon tube (Polystyrene Conical Tube 17 x 120 mm style, Becton Dickinson, Franklin Lakes, New

Jersey). The diluted sperm was stored in a falcon tube, mixed gently to create a homogeneous solution and left on ice to slow down the sperm motility until time to be used. When the glass Pyrex dish with eggs and diluted sperm was prepared, 3 ml at a time of the diluted sperm was added to the Pyrex dish with the eggs evenly throughout. The addition of diluted sperm was done uniformly and evenly using a transfer pipette

(Fisher Scientific, USA). To make sure the fertilization was taking place the Pyrex dish was gently agitated.

The Pyrex dish was lightly rocked backed and forth a few times to ensure that all eggs had proper sperm content in the vicinity. The Pyrex dish was allowed to incubate at 15 ⁰C for about 30-45 minutes then a sample was taken out and was examined under Fisher Scientific Compound Microscope to make sure successful fertilization has taken place. An additional 1000ml (1L) of the prepared ASW was added to the

Pyrex dish, covered partially with aluminum foil so air can get into it. After partial coverage of the dish, it was labeled with date and time of fertilization. The embryos were incubated for 24 hours at 15 ⁰C.

Preparation of Microassay

After the embryos were incubated for 24 hours at 15 ⁰C, a few ml of the embryos now 24-hour blastulas were examined under a Fisher Scientific Compound Microscope for viability. The embryos were

18 examined and were checked by pipetting few drops onto a petri dish (Falcon petri Dish, 60 x 15 mm). The embryos that did not show any progress in development or were damaged or lysed were discarded and if such a problem was consistent after checking a few, the embryos were discarded. The samples were taken and examined from the middle and top surface of the Pyrex dish to ensure they were swimming blastulas.

For the further studies, only free swimming blastulas were used.

To test the lectins of interest, 96 well microassay flat bottom plates (Falcon, Becton Dickinson,

Franklin Lakes, New Jersey) were used. Each plate was labeled with type of lectin being tested with different concentrations starting from the highest to lowest concentrations. One column for each trial was dedicated to Control to compare the results. On each plate, there were duplicate results of five different lectin concentrations and two duplicates of Control for low calcium seawater.

Preparation of 10% Formaldehyde

To produce 10% formaldehyde solution, 7.0 ml of freshly made ASW at pH 8.0 (refer to section on Preparation of artificial sea water) was added into a 15ml falcon tube (Polystyrene Conical Tube 17x20 mm style, Becton Dickinson, Franklin Lakes, New Jersey) using a transfer pipette (Fisher Scientific, USA).

To obtain 10% formaldehyde dilution, 3.0 ml of 37% formaldehyde (Ted Pella, Redding, California) was mixed in to the ASW under a fume hood. The solution was allowed to be mixed using a magnetic stirrer for about 10-15 minutes to achieve a homogeneous mixture. When the solution was made, it was capped, labeled and stored in the fume hood until it was needed. This mixture would be good to use for up to one week.

Preparation of Lectins

The prepared lectins used in these experiments came from Sigma Chemical Co. St. Louis,

Missouri and were in powder form. The lectins that were used included: Triticum vulgaris , Phaseolus vulgaris, and Artocarpus integrifolia. For each lectin, 1 milligram was used and mixed in with 9 milliliter of Low Calcium Artificial Seawater (LcASW) which created a final concentrations of 0.1 milligram/milliliter (30µl of 1mg/ml lectin concentration was added to 270µl of the embryos fertilized at 24 hours. This gives the final concentrations of 0.1 mg/ml that was the highest concentration for our trials

19 using each lectin. As previously stated Low calcium artificial seawater increases the rate and speed at which the lectin can enter into the embryos (Latham et al., 1998; Itza and Mozingo, 2005). Therefore, for our experimental purposes the lectins were diluted in Low Calcium Artificial Seawater instead of regular

ASW. By using the process of serial dilutions, four more concentrations were also tested for each lectin.

The concentrations used were: 0.1 mg/ml, 0.01 mg/ml, 0.001 mg/ml, 0.0001 mg/ml and 0.00001 mg/ml. To do the transferring of the lectins, embryo cells, and Low Calcium Artificial Seawater appropriate pipettes and pipette tips were used. The tips were changed all the time when different concentrations of lectins were used.

Treatment of Embryo Cells with Lectin

The embryos incubated in a 15 ⁰C incubator for 24 hours removed and since the live embryos were on top of the Pyrex dish, they were collected in a larger beaker. To assure that the collected embryos were alive; a sample was placed in a small petri dish and was observed using the Fisher Scientific

Compound Microscope to assess if development was normal. According to the calculations to make a 0.1 mg/ml lectin, 30 µl of each lectin at different concentrations were added in 270 µl of embryos into clear

Microplate Fisher brand Polystyrene Flatwells untreated and sterile that contains 96 wells. For the experimental embryos, each plate ran 10 wells running horizontally and 8 wells running vertically for total of 80 wells. Control embryos without lectin were also inoculated in the microplate. There were 2 columns of 8 per plate dedicated to Control embryos. The total Control was 2 columns times 8 wells for total of 16

Control wells per plate. The Control columns on each plate contained 30 µl of Low Calcium Artificial

Seawater and 270 µl of 24 hour fertilized embryo cells. 30µl of each lectin concentration were added to

270µl of 24 hour fertilized embryo that were added to each well using a wide-bore pipette. The wide-bore pipette was used so the embryos were not damaged when transferring from the beaker into the each microplate wells. The microplates were incubated for another 24 hours in a 15 ⁰C incubator.

Fixation of Embryos

After the embryos were treated with proper lectin concentrations, they were observed under Fisher

Scientific Compound Microscope. The Control embryos were examined to make sure high levels of full

20 development has taken place. When it was observed that the Control embryos in low calcium seawater have shown complete archenterons, all embryos were fixed using 10 µL of the 10% formaldehyde.

Observation of Embryos

In order to determine the effects of lectins on the embryos, the number of each embryo with a particular morphology was recorded and photos were taken. Since the sea urchin embryos were translucent, the archenteron was apparent and as a result the morphology and effect of lectins on cell-cell interactions at different concentrations could be recorded. The embryos fixed with 10% formaldehyde earlier were examined using an Inverted Fisher Scientific Compound Microscope and morphologies recorded were as follows: complete archenteron, incomplete archenteron, no invagination, exogastrulation and dead.

Photography of Embryos

Using a Samsung WB100 Digital Camera that features 6.0x optical zoom with 22-580 nm super- zoom lenses and 16.2 Megapixels resolution sensor embryos were photographed. The total magnification included the ocular lens magnification (10x) and the magnification of objective lenses (10x or 20x). The magnification of the camera was 3.5x making the total magnification of each photograph either x350 or x700.

Statistical Analysis of Embryos

To determine the validity and significance of the results a t-test was performed comparing the results of the means of the control to the treated embryos. The mean of each category of morphology such as complete archenteron, no invagination, exogastrulation, incomplete archenteron or dead were calculated.

The mean percentage of each morphology from the experimental concentrations and the Control were calculated using unpaired t-test calculator from graphpad software, InSTAT and compared exactly two groups of controls and experimentals and the results were imported onto excel

Upon determining the number of the embryos for each concentration of lectins, the percentages of embryos showing each kind of morphology were calculated using the total of embryos observed for those specific concentrations. For each lectin there were twelve trials performed at five different concentrations

21 therefore five different sets of percentages were calculated and t-tests were conducted to see if results were significant.

Most of the lectins observed and numbers calculated showed specific morphological changes among the embryos. Furthermore, one concentration may have showed different changes in morphologies using different lectins. Using the data, the standard deviation for each mean also was calculated and on the graphs, error bars were shown. The significance of the error bars is that if they are non-overlapping, they suggest that the results are significant. This goes parallel to the results that are found when doing the P- values. Normally a P-value of less than 0.05 (P<0.05) shows significant differences among the mean values of control and the experiments at different concentrations and morphologies.

Table 1

Lectins and Their Specific Carbohydrate

Lectin Carbohydrate

Artocarpus integrifolia D-galactose

Triticum vulgaris N-Acetyl-β-D-glucosamine

Phaseolus vulgaris PHA- L N-Acetyl-β-D-glucosamine

RESULTS

Artocarpus integrifolia: The sea urchin embryos were treated after 24 hours fertilization using concentrations of 0.1 mg/ml, 0.01mg/ml, 0.001 mg/ml, 0.0001 mg/ml and 0.00001 mg/ml in sea urchin embryos of Lytechinus pictus in low calcium seawater (Table 2). The results and effects of the lectin were observed and photographed after an extra 24 hours making it 48 hours after fertilization (Figures 1(A-C)- 6(A-C)). In addition to taking pictures of morphologies of the sea urchin embryos, using In STAT, p-values for mean percentages of embryos displaying complete archenteron (CA), no invagination (NI), incomplete archenteron(IA), exogastrulation (EXO) and dead (D) morphologies were measured for their validity and significance. This showed how significant or not the results were comparing to the Control that only contained the low calcium seawater (Table 4a).

Triticum vulgaris: The sea urchin embryos were treated after 24 hours fertilization using concentrations of 0.1 mg/ml, 0.01mg/ml, 0.001 mg/ml, 0.0001 mg/ml and 0.00001 mg/ml in sea urchin embryos of Lytechinus pictus in low calcium seawater (Table 2). The results and effects of the lectin were observed and photographed after an extra 24 hours making it 48 hours after fertilization (Figures 7(A-C)-12(A-C)). In addition to taking pictures of morphologies of the sea urchin embryos, using In STAT, p-values for mean percentages of embryos displaying complete archenteron (CA), incomplete archenteron(IA), no invagination (NI), exogastrulation (EXO) and dead (D) morphologies were measured for their validity and significance. This showed how significant or not the results were comparing to the Control that only contained the low calcium seawater (Table 4b).

Phaseolus vulgaris PHA-L The sea urchin embryos were treated after 24 hours fertilization using concentrations of 0.1 mg/ml, 0.01mg/ml, 0.001 mg/ml, 0.0001 mg/ml and 0.00001 mg/ml in sea urchin embryos of Lytechinus pictus in low calcium seawater (Table 2). The results and effects of the lectin were observed and photographed after an extra 24 hours making it 48 hours after fertilization (Figures 13(A-C)- 18(A-C)). In addition to taking pictures of morphologies of the sea urchin embryos, using In STAT, p- values for mean percentages of embryos displaying complete archenteron (CA), incomplete archenteron (IA), no invagination (NI), exogastrulation (EXO) and dead morphologies were measured for their validity and significance. This showed how significant or not the results were comparing to the Control that only contained the low calcium seawater (Table 4c).

Table 2

Embryo count for morphologies exhibited in Control and experimental concentrations for lectins tested

Effects of Control and various lectins on morphology of Lytechinus pictus embryos. The values represent embryos of Control and treated with different concentrations for each lectin and total numbers of morphologies observed.

Lectin and Concentrations Embryo Count and Morphology

Artocarpus integrifolia Complete archenteron- 1839 (69.47%) Incomplete archenteron- 255(9.63%) CONTROL No invagination-260 (9.82%) Exogastrulation-280 (10.58%) Dead- 13 (0.49%)

Artocarpus integrifolia Complete archenteron- 1010 (40.14%) Incomplete archenteron- 25.40 (16.7%) 0.1mg/ml lcASW No invagination -608 (24.16%) Exogastrulation- 242 (9.62%) Dead-17 (0.68%)

Artocarpus integrifolia Complete archenteron- 1041 (43.74%) Incomplete archenteron- 534 (22.43%) 0.01mg/ml lcASW No invagination -563 (23.66%) Exogastrulation- 218 (9.16%) Dead-24 (1.01%)

Artocarpus integrifolia Complete archenteron- 1341 (49.26%) Incomplete archenteron- 531 (19.51%) 0.001mg/ml lcASW No invagination -509 (18.7%) Exogastrulation- 318 (11.68%) Dead- 23 (0.84%)

Artocarpus integrifolia Complete archenteron- 1707 (58.58%) Incomplete archenteron- 455 (15.61%) 0.0001mg/ml lcASW No invagination -450 (15.44%) Exogastrulation- 276 (9.47%) Dead- 26 (0.99%)

Artocarpus integrifolia Complete Archenteron- 1975 (63.87%) Incomplete Archenteron- 410 (13.26%) 0.00001mg/ml lcASW No invagination-383 (12.39%) Exogastrulation- 298 (9.64%) Dead- 26 (0.84% )

Table 2A- Shows number of Control and embryos induced by various concentrations of Artocarpus integrifolia and morphologies they exhibited

Lectin and Concentrations Embryo Count and Morphology

Complete archenteron- 2041 (68.35%) Triticum vulgaris Incomplete archenteron- 281(9.41%) No invagination -400 (13.40%) CONTROL Exogastrulation-253 (8.47%) Dead- 11 (0.37%)

Triticum vulgaris Complete archenteron- 765 (33.12%) Incomplete archenteron- 538 (23.29%) 0.1mg/ml lcASW No invagination -434 (18.79%) Exogastrulation- 549 (23.77%) Dead-24 (1.04%)

Triticum vulgaris Complete archenteron- 1264 (44.26%) Incomplete archenteron- 558 (19.54%) 0.01mg/ml lcASW No invagination -674 (23.60%) Exogastrulation- 344 (12.04%) Dead-16 (0.56%)

Triticum vulgaris Complete archenteron- 1573 (49.20%) Incomplete archenteron- 567 (17.73%) 0.001mg/ml lcASW No invagination -709 (22.18%) Exogastrulation- 336 (10.51%) Dead- 12 (0.37%)

Triticum vulgaris Complete archenteron- 1812 (55.31%) Incomplete archenteron- 460 (14.04%) 0.0001mg/ml lcASW No invagination -667 (20.36%) Exogastrulation- 323 (9.86%) Dead- 14 (0.43%)

Triticum vulgaris Complete archenteron- 2247 (62.17%) Incomplete archenteron- 392 (10.85%) 0.00001mg/ml lcASW No invagination -585 (16.19%) Exogastrulation- 382 (10.57%) Dead- 8 (0.22% )

Table 2B- Shows Control and embryos induced by various concentrations of Triticum vulgaris and morphologies they exhibited

Lectin and Concentrations Embryo Count and Morphology

Phaseolus vulgaris PHA-L Complete archenteron- 2278 (74.32%) CONTROL Incomplete archenteron- 290(9.46%) No invagination -360 (11.74%) Exogastrulation-45 (1.47%) Dead- 92 (3.00%)

Phaseolus vulgaris PHA-L Complete archenteron- 1274 (49.59%) Incomplete archenteron- 576 (22.42%) 0.1mg/ml lcASW No invagination -582 (22.65%) Exogastrulation- 85 (3.31%) Dead-52 (2.02%)

Phaseolus vulgaris PHA-L Complete archenteron- 1367 (49.55%) Incomplete archenteron- 607 (22.00%) 0.01mg/ml lcASW No invagination -652 (23.63%) Exogastrulation- 67 (2.43%) Dead-66 (2.39%)

Phaseolus vulgaris PHA-L Complete archenteron- 1448 (47.26%) Incomplete archenteron- 689 (22.49%) 0.001mg/ml lcASW No invagination -772 (25.20%) Exogastrulation- 63 (2.06%) Dead- 92 (3.00%)

Phaseolus vulgaris PHA-L Complete archenteron- 1787 (55.51%) Incomplete archenteron- 566 (17.58%) 0.0001mg/ml lcASW No invagination -713 (22.15%) Exogastrulation- 59 (1.83%) Dead- 94 (2.92%)

Phaseolus vulgaris PHA-L Complete archenteron- 2420 (72.63%) Incomplete archenteron- 288 (8.64%) 0.00001mg/ml lcASW No invagination-445 (13.36%) Exogastrulation- 49 (1.47%) Dead- 130 (3.90% )

Table 2C- Shows Control and embryos induced by various concentrations of Phaseoulus vulgaris PHA-L and morphologies they exhibited.

100 µm NI µm A

Figure 1A- Shows Control Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and high numbers of the embryos show complete archenteron development (CA). There are some embryos exhibiting no invagination (NI) and incomplete archenteron (IA). Bar scale represents 100 µm. (x350)

100 µm µm B

Figure 1B- Shows Control Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulated state and high numbers of the embryos show complete archenteron development (CA). There are some embryos exhibiting no invagination (NI). Bar scale represents 100 µm. (x350)

100 µm C µm Figure 1C- Shows Control Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in gastrulated state and high number of the embryos exhibit complete archenteron development (CA). There are some embryos exhibiting no invagination (NI) and incomplete archenteron (IA) as well. Bar scale represents 100 µm. (x350)

EXO

100 µm µm

a A

Figure 2 A- Shows 0.1 mg/ml Artocarpus integrifolia in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in gastrulated state and embryos exhibited incomplete archenteron (IA), exogastrulation (EXO) and complete archenteron (CA). Bar scale represents 100 µm. (x350

IA NI

CA B 100 µm µm Figure 2 B- Shows 0.1 mg/ml Artocarpus integrifolia in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in gastrulated state. Embryos show no invagination (NI) and incomplete archenteron (IA). Fewer embryos exhibit complete archenteron (CA). Bar scale represents 100 µm. (x350)

IA EXO

100 µm C µm

Figure 2 C- Shows 0.1 mg/ml Artocarpus integrifolia in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulated state with high number of the embryos exhibiting incomplete archenteron (IA) and exogastrulation (EXO). Bar scale represents 100 µm. (x700)

NI NI

IA NI

EXO A 100 µm µm

Figure 3 A- Shows 0.01 mg/ml Artocarpus integrifolia in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulated state with high number of the embryos showing no invagination (NI), incomplete archenteron (IA) and exogastrulation (EXO). Bar scale represents 100 µm. (x350)

CA IA EXO

B 100 µm µm Figure 3 B- Shows 0.01 mg/ml Artocarpus integrifolia in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and many of the embryos show no invagination (NI) and incomplete archenteron (IA) while less embryos exhibit complete archenteron development (CA) and exogastrulation (EXO). Bar scale represents 100 µm. (x350)

EXO

100 µm µm

Figure 3 C- Shows 0.01 mg/ml Artocarpus integrifolia in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state exhibiting incomplete archenteron (IA) while some embryos displayed exogastrulation (EXO). Bar scale represents 100 µm. (x700)

IA CA CA

100 µm µm A

Figure 4 A- Shows 0.001 mg/ml Artocarpus integrifolia in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state with embryos exhibiting slight increase in complete archenteron (CA) but high number display incomplete archenteron (IA) as shown. Bar scale represents 100 µm. (x350)

100 µm µm

CA B

Figure 4 B- Shows 0.001 mg/ml Artocarpus integrifolia in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state. There is a slight increase in complete archenteron (CA). Bar scale represents 100 µm. (x700)

EXO

100 µm IA µm

Figure 4 C- Shows 0.001 mg/ml Artocarpus integrifolia in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state with embryos exhibiting slight increase in complete archenteron (CA) but high number display incomplete archenteron (IA). Minor number of embryos showed exogastrulation (EXO). Bar scale represents 100 µm. (x700)

CA IA

EXO

100 µm CA µm A

Figure 5 A- Shows 0.0001 mg.ml Artocarpus integrifolia in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and increase in embryos demonstrating complete archenteron (CA) is seen while number of embryos displaying incomplete archenteron (IA) and exogastrulation (EXO) has fallen. Bar scale represents 100 µm. (x350)

B 100 µm µm Figure 5 B- Shows 0.0001 mg.ml Artocarpus integrifolia in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state. The embryos display more complete archenteron (CA) development while less embryos display no invagination (NI) and incomplete archenteron (IA). Bar scale represents 100 µm. (x350)

C CA CA

100 µm µm Figure 5 C- Shows 0.0001 mg.ml Artocarpus integrifolia in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state. Embryos exhibit increase in complete archenteron (CA) development while less embryos exhibit incomplete archenteron (IA).Bar scale represents 100 µm. (x700)

CA CA

100 µm µm A

Figure 6 A- Shows 0.00001 mg/ml Artocarpus integrifolia in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and high numbers of the embryos display complete archenteron (CA) development. Fewer embryos with incomplete archenteron (IA) were also observed. Bar scale represents 100 µm. (x350)

IA 100 µm µm

Figure 6 B- Shows 0.00001 mg/ml Artocarpus integrifolia in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and high numbers of the embryos display complete archenteron (CA) and fewer embryos with no invagination (NI) were also noted. Bar scale represents 100 µm. (x350)

100 µm µm

Figure 6 C- Shows 0.00001 mg/ml Artocarpus integrifolia in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and high numbers of the embryos display complete archenteron (CA). Bar scale represents 100 µm. (x700)

Effect of Artocarpus integrifolia at Control and various concentrations

Summary of results:

Control: The embryos show high percentage of complete archenteron (69.47%) development.

0.1mg lectin/ml LcASW: High percentage of incomplete archenteron shows an increase in action of lectin with the embryos (25.40% vs. 9.63% in control). There is an increase in number of no invagination development as well (24.16% vs. Control 9.82%) which was significant.

0.01mg lectin/ml LcASW: Slightly lower in number of incomplete archenteron development and no invagination than shown comparing to 0.1 mg concentration (22.43% and 23.66% respectively). Also there is a slight increase in number of complete archenteron development as well (43.74% versus 40.14% in latter concentration). Comparing the percentage of exogastrulation in 0.1 and 0.01 mg lectin, the values were similar hence not significant (9.16% vs. 9.62%).

0.001 mg lectin/ml LcASW: There is higher number of embryos exhibiting complete archenteron (49.26%) comparing to 0.01 mg concentration (43.74%). The number of no invaginated embryos dropped significantly in this concentration (18.70% vs. 22.43%). In addition, number of incomplete archenteron embryos was lowered to 19.51% versus 22.43% in 0.01 mg lectin as well. There was a slight increase in percentage of Exogastrulation observed in 0.001 mg (11.68% vs. 9.16%).

0.0001 mg lectin/ ml LcASW: At this concentration there was an increase in number of complete archenteron development (58.58% vs. 49.26% in 0.001 mg lectin) which shows increase of approximately 9.4%. The number of incomplete archenteron was lowered to 15.61% from observed 19.51% in 0.001 mg lectin which showed about 4 % decrease as well. The number of exogastrulation embryos was also lowered by about 2.3% from the previous concentration (9.47% versus 11.68%). The number of No invagination dropped from 15.44% in 0.0001 mg from 18.70% seen in 0.001 mg.

0.00001 mg lectin/ml LcASW: This concentration projected the lowest concentration used for the experiments. The embryos showed complete archenteron of 63.87% which was the highest among the concentrations tested. This percentage was very close to the Control (69.47%). The number of no invagination did not change when compared to the Control and was very insignificantly higher by 0.77%. The number of incomplete archenterons increased very slightly by 1.7%. This value is not significant taking into account a large sampled tested. The number of exogastrulation embryos in the lowest concentration of 0.00001 mg lectin also dropped by 2.34%.

*** In all of the concentrations there was very minimal % of dead embryos found that was not significant in the overall results. The table shows the number of dead embryos.

A CA CA

CA 100 µm µm CA IA

Figure 7A- Shows Control in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and high numbers of the embryos show complete archenteron (CA) development and there are some embryos exhibit incomplete archenteron (IA).Bar scale represents 100 µm. (x350)

B CA

100 µm µm IA

Figure 7B- Shows Control in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and high numbers of the embryos show Complete Archenteron (CA) development. There are some embryos exhibit incomplete archenteron (IA). Bar scale represents 100 µm. (x700)

IA C CA

100 µm µm

Figure 7C- Shows Control in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and high numbers of the embryos show complete archenteron (CA) development and there are some embryos exhibit incomplete archenteron (IA). Bar scale represents 100 µm. (x350)

IA NI

NI O

EXO

IA IA 100 µm µm A EXO

Figure 8A- Shows 0.1 mg/ml Triticum vulgaris in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and high numbers of the embryos show exogastrulation (EXO) and incomplete archenteron (IA). Embryo exhibiting no invagination (NI) also seen. Bar scale represents 100 µm. (x350)

B IA

NI IA

100 µm NI µm

Figure 8B- Shows 0.1 mg/ml Triticum vulgaris in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and high numbers of the embryos show no invagination (NI). Some embryos show incomplete archenteron (IA) and some exhibit complete archenteron (CA) development. Bar scale represents 100 µm. (x350)

100 µm µm EXO C

Figure 8C- Shows 0.1 mg/ml Triticum vulgaris in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and these embryos exhibiting both exogastrulation (EXO) and no invagination (NI). Bar scale represents 100 µm. (x700)

A NI EXO CA IA

EXO NI

100 µm µm

Figure 9A- Shows 0.01 mg/ml Triticum vulgaris in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and high number of the embryos exhibit no invagination (NI) and incomplete archenteron (IA). There are some embryos that show exogastrulation (EXO) and few embryos exhibit Complete Archenteron (CA) development. Bar scale represents 100 µm. (x350)

IA CA IA

EXO

100 µm µm EXO B

Figure 9B- Shows 0.01 mg/ml Triticum vulgaris in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and high number of the embryos exhibit no invagination (NI) and incomplete archenteron (IA). There are some embryos that show exogastrulation (EXO) and some embryos exhibit complete archenteron (CA) development. Bar scale represents 100 µm. (x350)

NI 100 µm µm C

Figure 9C- Shows 0.01 mg/ml Triticum vulgaris in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and the embryos show no invagination (NI) observed for high number of morphology. Bar scale represents 100 µm. (x350)

CA EXO IA NI

CA EXO 100 µm A

Figure 10A- Shows 0.001 mg/ml Triticum vulgaris in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and high number of embryos showing no invagination (NI) and incomplete Archenteron (IA) while fewer of them display complete archenteron (CA) development and exogastrulation (EXO). Bar scale represents 100 µm. (x350)

B IA IA CA EXO

CA 100 µm

Figure 10B- Shows 0.001 mg/ml Triticum vulgaris in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state. Many embryos show no invagination (NI) and incomplete archenteron (IA). There are some embryos that exhibit complete archenteron (CA) development. There are embryos observed in exogastrulated (EXO) as well. Bar scale represents 100 µm. (x350)

EXO

100 µm IA NI CA C

Figure 10C- Shows 0.001 mg/ml Triticum vulgaris in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and high number of the embryos shows in no invagination (NI) and incomplete archenteron (IA) stage. There are some of embryos that display complete archenteron (CA) development and exogastrulation (EXO). Bar scale represents 100 µm. (x350)

IA NI

NI 100 µm A

Figure 11A- Shows 0.0001 mg/ml Triticum vulgaris in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and high numbers of the embryos show Complete Archenteron (CA). There are some embryos exhibit Incomplete Archenteron (IA) and No Invagination (NI) . There number of No Invagination (NI) exceeded the number of embryos presented displaying Incomplete Archenteron (IA) development .Bar scale represents 100 µm.

100 µm

CA CA

CA NI B

Figure 11B- Shows 0.0001 mg/ml Triticum vulgaris in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and high numbers of embryos show complete archenteron (CA). Many embryos also exhibit no Invagination (NI) morphology. Bar scale represents 100 µm. (x350)

100 µm C IA

Figure 11C- Shows 0.0001 mg/ml Triticum vulgaris in l Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation and high numbers of embryos show complete archenteron (CA). Many embryos also exhibit no invagination (NI) morphology and incomplete archenteron (IA). Bar scale represents 100 µm. (x350)

A CA

CA CA 100 µm

Figure 12A- Shows 0.00001 mg/ml Triticum vulgaris in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and high numbers of the embryos show complete archenteron (CA) development. Bar scale represents 100 µm. (x350)

100 µm

CA IA B

Figure 12B- Shows 0.00001 mg/ml Triticum vulgaris in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and high numbers of the embryos show complete archenteron (CA) development. Some embryos exhibit incomplete archenteron (IA). Bar scale represents 100 µm. (x350)

100 µm C CA

Figure 12C- Shows 0.00001 mg/ml Triticum vulgaris in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and high numbers of the embryos show complete archenteron (CA) development. Some embryos exhibit display incomplete archenteron (IA). Bar scale represents 100 µm. (x350)

Effects of Triticum vulgaris at Control and Various Concentrations

Summary of results:

Control: The embryos show high levels of full development, Complete Archenteron (68.35%). A number of embryos exhibit No invagination as well (13.40%).

0.1mg lectin/ml LcASW: Comparing this highest concentration of lectin introduced in embryo cells, the percentage and number of embryos displaying Complete Archenteron; 33.12% versus 68.35% respectively. Furthermore, the percentages of embryos exhibiting incomplete archenteron also increased nearly three times (9.41% in Control versus 23.29% in 0.1 mg lectin). The embryos also showing No invagination increased from 13.40% seen in Control to 18.79% for 0.1 mg lectin. The number of embryos displaying Exogastrulation increased by more than three times as well when compared to the Control (8.47% Control and 23.77% in 0.1 mg lectin). These display changes shown to be significant when comparing highest lectin used to the Control.

0.01mg lectin/ml LcASW: The embryos exhibiting Complete Archenteron increased by about one and half times from previous concentration however the percentage was still way below the Complete Archenteron seen in Control (44.26% in 0.01 mg lectin, 33.12% in 0.1 mg and 68.35% Control). This was significant changes observed in the embryos displaying this type of morphology. Comparing the Incomplete archenteron and No invagination showed similar results to 0.1 mg lectin however they were way above the Control results. The embryos demonstrating incomplete archenteron was 19.54% in 0.01 mg whereas Control showed 9.41%. The embryos exhibiting No invagination in 0.01 mg showed 23.60% and the Control displayed 13.40%. These values comparing to the Control showed to be significant. The embryos showing Exogastrulation were similar to Control (12.04% in 0.01 mg versus 8.47% in Control) however the percentage was significantly lower than what was seen in 0.1 mg (23.77%).

0.001 mg lectin/ml LcASW: Comparing the percentages of Complete Archenteron displayed were little above what was observed in 0.01 mg lectin (49.20% versus 44.29% seen in 0.01 mg) however still the number showed below the Control full development in Complete Archenteron (68.35%). The embryos exhibiting incomplete archenteron were within what was observed in 0.01 mg lectin (17.73% in 0.001 mg versus 19.54% in 0.01 mg lectin). However the embryos exhibiting this morphology to the Control showed almost two times as much observed (Control; 9.41%). The embryos displaying No invagination were significantly higher than Control (22.18% in 0.001 versus 13.40% in Control) but the percentage was similar to what was observed in the previous concentration of 0.01 mg (23.60%). The embryos showing Exogastrulation in this concentration (10.51%) showed to be similar and not significantly different than observed in Control (8.47%) and 0.1 mg lectin concentration (12.04%).

0.0001mg lectin/ml LcASW: The embryos displayed higher percentage of Complete Archenteron (55.31%) but still notably below observed in Control (68.35%) however higher than 0.1 mg (49.20%). The embryos presenting incomplete archenteron (14.04%) were still notably higher than found in Control (9.41%) but less than the embryos exhibiting this morphology in 0.001 mg (17.73%). The embryos demonstrating No invagination (20.36%) showed to be remarkably higher than observed in Control (13.40%) but within the same percentage comparing to 0.001 mg (22.18%). The percentages for Exogastrulation for this concentration were within the same range comparing to Control and latter concentration (9.86%).

0.00001mg lectin/ml LcASW: This concentration was used as the lowest concentration for the experiments. The embryos displayed 62.17% for Complete Archenteron in 0.00001 mg lectin. This value was reasonable

53 closer to observed percentage seen in the Control (68.35%). The Incomplete archenteron showed to be within same vicinity percentage of the Control (10.85% in 0.0001 and 9.41% in Control). This percentage was considerably lower seen in latter concentration of 0.0001 mg (14.04%). The embryos displaying No invagination dropped notably in this concentration (16.19%) from latter concentration (20.36%) however this morphology was displayed slightly higher than in Control (13.40%). The Exogastrulation was within the similar range in this concentration (10.57%) comparing to the Control (8.47%) and latter concentration of 0.0001 mg (9.86%)

*** In all of the concentrations there was very minimal % of dead embryos found that was not significant in the overall results. The table shows the number of dead embryos.

CA A

100 µm

CA CA NI

Figure 13A- Shows Control Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and high number of the embryos exhibit complete archenteron (CA) development and some embryos present no invagination (NI). Bar scale represents 100 µm. (x350)

B 100 µm

Figure 13B- Shows Control Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and high number of the embryos exhibit complete archenteron (CA) development. Bar scale represents 100 µm. (x350)

100µm

Figure 13C- Shows Control Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and high number of the embryos exhibit Complete Archenteron (CA) development. Bar scale represents 100 µm. (x700)

A NI

NI IA

100 µm

Figure 14A- Shows 0.1 mg/ml Phaseolus vulgaris PHA-L in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and high numbers of embryos shows incomplete archenteron (IA) and no invagination (NI). Bar scale represents 100µm. (x350)

NI IA

100 µm IA B

Figure 14B- Shows 0.1 mg/ml Phaseolus vulgaris PHA-L in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and high numbers of embryos shows incomplete archenteron (IA) and no invagination (NI). Bar scale represents 100 µm. (x700)

EXO

NI NI

IA IA

100 µm

C NI

Figure 14C- Shows 0.1 mg/ml Phaseolus vulgaris PHA-L in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and high numbers of embryos shows incomplete archenteron (IA) and no invagination (NI). There are some embryos exhibiting exogastrulation (EXO). Bar scale represents 100 µm. (x350)

A IA

100 µm

Figure 15A- Shows 0.01 mg/ml Phaseolus vulgaris PHA-L in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and high numbers of embryos shows incomplete archenteron (IA) and no invagination (NI). Bar scale represents 100 µm. (x350)

100 µm IA

NI B

Figure 15B- Shows 0.01 mg/ml Phaseolus vulgaris PHA-L in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and high numbers of embryos show incomplete archenteron (IA) and no invagination (NI). Bar scale represents 100 µm. (x350)

100 µm

Figure 15C- Shows 0.01 mg/ml Phaseolus vulgaris PHA-L in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and high numbers of embryos show incomplete archenteron (IA) and no invagination (NI). Bar scale represents 100 µm. (x350)

IA IA

NI A NI 100 µm

Figure 16A- Shows 0.001 mg/ml Phaseolus vulgaris PHA-L in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and high numbers of the embryos exhibit no invagination (NI) and incomplete archenteron (IA). Bar scale represents 100 µm. (x350)

100 µm

Figure 16B- Shows 0.001 mg/ml Phaseolus vulgaris PHA-L in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and high numbers of the embryos exhibit no invagination (NI) and incomplete archenteron (IA). Bar scale represents 100 µm. (x350) C NI NI

100 µm

Figure 16C- Shows 0.001 mg/ml Phaseolus vulgaris PHA-L in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and high numbers of the embryos exhibit no invagination (NI). Bar scale represents 100 µm. (x350)

NI A CA

CA NI

IA 100 µm

Figure 17A- Shows 0.0001 mg/ml Phaseolus vulgaris PHA-L in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and high numbers of the embryos exhibit no invagination (NI) and incomplete archenteron (IA) embryos also seen but there are many embryos displaying complete archenteron (CA) development. Bar scale represents 100 µm. (x350))

NI B

100 µm

Figure 17B- Shows 0.0001 mg/ml Phaseolus vulgaris PHA-L in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and high numbers of the embryos exhibit no invagination (NI) and incomplete archenteron (IA) embryos also seen but there are many embryos displaying complete archenteron (CA) development. Bar scale represents 100 µm. (x350)

100 µm

Figure 17C- Shows 0.0001 mg/ml Phaseolus vulgaris PHA-L in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and high numbers of the incomplete archenteron (IA) embryos were seen but there are many embryos displaying complete archenteron (CA) development. Bar scale represents 100 µm. (x350).

CA A

100 µm

CA CA

Figure 18A- Shows 0.00001 mg/ml Phaseolus vulgaris PHA-L in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and very high numbers of the embryos display complete archenteron (CA) development. Bar scale represents 100 µm. (x 350)

CA B

100 µm

Figure 18B- Shows 0.00001 mg/ml Phaseolus vulgaris PHA-L in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and very high numbers of the embryos display complete archenteron (CA) development. Bar scale represents 100 µm. (x 700)

CA C

100 µm

Figure 18C- Shows 0.00001 mg/ml Phaseolus vulgaris PHA-L in Lytechinus pictus embryos at 48 hours after fertilization. All of the embryos are in the gastrulation state and very high numbers of embryos display complete archenteron (CA) development. Bar scale represents 100 µm. (x 700)

Effects of Phaseolus vulgaris Agglutinin PHA-L at Control and Various Concentrations

Summary of results:

Control: The embryos show high percentage of complete archenteron development (74.32%) and very low levels in the other morphologies recorded for incomplete archenteron (9.46%), No attached archenteron (11.74%), exogastrulation (1.47%) and dead embryos (3.00%).

0.1mg lectin/ml LcASW: Significantly lower number of complete archenteron development as the percentage dropped to 49.59%. There is a significant increase in the morphology with incomplete archenteron and no attached archenteron was observed (22.42% and 22.65% respectively). These percentages are much higher than observed for the control. The percentages of incomplete archenteron and no attached archenteron for the Control showed to be 9.46% and 11.74% indicating significant changes in the morphologies. However the percentages in exogastrulation and dead embryos were not different significantly although very slight increase in exogastrulation among the embryos for 0.1 mg/ml LcASW was observed.

0.01mg lectin/ml LcASW: There was no change in embryos with complete archenteron morphology comparing to 0.1 mg concentration as similar result was observed (49.55% vs. 49.59%). In addition, comparing the percentage in no attached archenteron, incomplete archenteron, exogastrulation and dead embryos did not show no significant differences in embryo percentages as similar numbers were achieved when comparing to 0.1 mg lectin per ml lcASW.

0.001 mg lectin/ml LcASW: Very slight decrease in complete archenteron percentages observed when comparing to 0.01 mg concentration while incomplete archenteron showed to be very similar in numbers. Also, the embryos exhibiting no attached archenteron morphology increased slightly than seen in latter concentration (25.20% versus 23.63%). There were no significant changes in the embryos exhibiting both exogastrulation and dead morphologies.

0.0001mg lectin/ml LcASW: At this concentration the number of embryos showing complete archenteron increased by more than 8% (from 47.26% in 0.001 mg to 55.51%). Furthermore, the percentage of embryos displaying incomplete archenteron dropped significantly from 22.49% from latter concentration to 17.58%. There was a slight decrease in number of embryos displaying no attached archenteron as well. The percentages dropped from 25.20% in the latter concentration to 22.15% for 0.0001mg lectin concentration. The percentage of exogastrulation and dead embryos were not significantly different from the latter concentration when comparing the results.

0.00001mg lectin/ml LcASW: This concentration anticipated to be the lowest concentration that was used for the experiments. The embryos showed similar percentage for complete archenteron as seen in the Control embryos. The percentage of complete archenteron for this lowest concentration was 72.63% showing similar result as 74.32% for the control. This also showed the highest percent complete archenteron than any other concentrations measured. There was a slight increase for embryos exhibiting no attached archenteron comparing to Control but not significant enough. In addition, lower percentage of embryos exhibited incomplete archenteron (8.64% vs. 9.46%) however this was not significant either as there was a slight difference. The embryos exhibiting exogastrulation were very similar in both Control and 0.00001 mg lectin concentration and not much difference was seen when looking at the dead embryos for both concentrations (3% Control vs. 3.9 % for 0.00001 mg lectin).

*** In all of the concentrations there was very minimal % of dead embryos found that was not significant in the overall results. The table shows the number of dead embryos.

Table #3

Summary effects of Control and experimental concentrations using Artocarpus integrifolia in embryos

Concentration of Control 0.1 mg/ml LcASW 0.01 mg/ml LcASW Artocarpus integrifolia

Hours Added 24 24 24 After Fertilization

Time Photo was 48 48 48 Taken

Figures numbers 1A-C 2A-C 3A-C

Effects of Most embryos exhibit Most embryos show High number of the embryos Artocarpus complete archenteron no invagination (NI) show no invagination (NI) and integrifolia (CA) and incomplete incomplete archenteron (IA); archenteron (IA); fewer embryos exhibit Less embryos display complete archenteron (CA) complete archenteron (CA)

Table 3 A- Shows Control and all the concentrations of Artocarpus integrifolia used in lcASW, time added after fertilization, time photos were taken and the corresponding figures and the effects that were seen in sea urchin embryos.

Concentration of 0.001 mg/ml LcASW 0.0001 mg/ml 0.00001 mg/ml LcASW Artocarpus LcASW integrifolia

Hours Added 24 24 24 After Fertilization

Time Photo was 48 48 48 Taken

Figures numbers 4A-C 5A-C 6A-C

Effects of The embryos exhibit Many embryos Most but not all embryos shows Artocarpus low in complete exhibited higher complete archenteron and some integrifolia archenteron (CA) but complete archenteron displayed no invagination (NI) and showed higher no (CA); Less embryos incomplete archenteron (IA). invagination (NI) and displayed both No incomplete invagination (NI) and archenteron (IA) incomplete archenteron (IA) Table 3 A- Shows Control and all the concentrations of Artocarpus integrifolia used in lcASW, time added after fertilization, time photos were taken and the corresponding figures and the effects that were seen in sea urchin embryos.

Summary effects of Control and experimental concentrations using Triticum vulgaris in embryos

Concentration of Control 0.1 mg/ml LcASW 0.01 mg/ml LcASW Triticum vulgaris

Hours Added 24 24 24 After Fertilization

Time Photo was 48 48 48 Taken

Figures numbers 7A-C 8A-C 9A-C

Effects of Most embryos exhibit High number of the Embryos exhibiting lower complete Triticum vulgaris complete archenteron embryos exhibit both archenteron (CA). Many embryos (CA). Some embryos exogastrulation (EXO) show incomplete archenteron (IA) showing incomplete and incomplete and no invagination (NI) while there archenteron (IA). Archenteron (IA). were fewer embryos exhibited Fewer embryos exogastrulation (EXO). displayed no invagination (NI). Number of complete archenteron (CA) fell considerably. Table 3 B- Shows Control and all the concentrations of Triticum vulgaris used in lcASW, time added after fertilization, time photos were taken and the corresponding figures and the effects that were seen in sea urchin embryos.

Concentration of 0.001 mg/ml LcASW 0.0001 mg/ml 0.00001 mg/ml LcASW Triticum vulgaris LcASW

Hours Added 24 24 24 After Fertilization

Time Photo was 48 48 48 Taken

Figures numbers 10A-C 11A-C 12A-C

Effects of Embryos exhibit lower More embryos High number of the embryos displayed Triticum vulgaris complete archenteron exhibit complete complete archenteron (CA) development. (CA). High number of archenteron (CA). embryos exhibit The incomplete incomplete archenteron archenteron (IA) (IA) and no and no invagination (NI). invagination (NI) Fewer embryo display were exhibited in exogastrulation (EXO). fewer numbers. Table 3 B- Shows Control and all the concentrations of Triticum vulgaris used in lcASW, time added after fertilization, time photos were taken and the corresponding figures and the effects that were seen in sea urchin embryos.

Summary effects of Control and experimental concentrations using Phaseolus vulgaris PHA-L in embryos

Concentration of Control 0.1 mg/ml LcASW 0.01 mg/ml LcASW Phaseolus vulgaris PHA-L

Hours Added After 24 24 24 Fertilization

Time Photo was 48 48 48 Taken

Figures numbers 13A-C 14A-C 15A-C

Effects of Most embryos exhibit Embryos show significant Embryos show significant Phaseolus vulgaris complete archenteron decrease in complete decrease in complete PHA-L (CA). archenteron (CA). Embryos archenteron (CA). Embryos display high incomplete display high incomplete archenteron (IA) and no archenteron (IA) and no invagination (NI). invagination (NI). Table 3C- Shows Control and all the concentrations of Phaseoulus vulgaris PHA-L used in lcASW, time added after fertilization, time photos were taken and the corresponding figures and the effects that were seen in sea urchin embryos.

Concentration of 0.001 mg/ml LcASW 0.0001 mg/ml LcASW 0.00001 mg/ml LcASW Phaseolus vulgaris PHA-L

Hours Added After 24 24 24 Fertilization

Time Photo was 48 48 48 Taken

Figures numbers 16A-C 17A-C 18A-C

Effects of Embryos display lower Embryos slightly show High number of the embryos Phaseolus vulgaris complete archenteron decrease in complete showed complete archenteron (CA). PHA-L (CA). Many embryos archenteron (CA). Many display no invagination embryos exhibit no (NI) and many embryos invagination (NI). A exhibit incomplete number of embryos exhibit archenteron (IA). incomplete archenteron (IA).

Table 3C- Shows Control and all the concentrations of Phaseoulus vulgaris PHA-L used in lcASW, time added after fertilization, time photos were taken and the corresponding figures and the effects that were seen in sea urchin embryos.

Table #4

Summary of comparison of p-value for mean percentage for morphologies of Control and various concentrations

Lectin Artocarpus Artocarpus Artocarpus Artocarpus Artocarpus integrifolia integrifolia integrifolia integrifolia integrifolia

Compared complete incomplete no invagination exogastrulation dead morphology archenteron archenteron

Two- tailed 0.0033 0.0024 0.0042 0.1769 0.0002 P- value

Result Very Very Very statistically Not statistically Extremely statistically statistically significant significant statistically significant significant significant Table 4a- Shows p-value using two tailed t-test and results for different morphologies in control and concentrations of 0.1, 0.01, 0.001, 0.0001 and 0.00001 mg Artocarpus integrifolia used.

Lectin Triticum Triticum Triticum Triticum Triticum vulgaris vulgaris vulgaris vulgaris vulgaris

Compared complete incomplete no invagination exogastrulation dead morphology archenteron archenteron

Two- tailed 0.0042 0.0074 0.0008 0.1005 0.3036 P- value

Very Very Extremely Not statistically Not Result statistically statistically statistically significant statistically significant significant significant significant

Table 4b- Shows p-value using two tailed t-test and results for different morphologies in control and concentrations of 0.1, 0.01, 0.001, 0.0001 and 0.00001 mg Triticum vulgaris used.

Lectin Phaseolus Phaseolus Phaseolus Phaseolus Phaseolus vulgaris vulgaris vulgaris vulgaris vulgaris PHA-L PHA-L PHA-L PHA-L PHA-L

Compared complete incomplete no invagination exogastrulation dead morphology archenteron archenteron

Two- tailed 0.0031 0.0087 0.0016 0.0440 0.6418 P- value

Very Very Very Statistically Not statistically Result statistically statistically statistically significant significant significant significant significant

Table 4c- Shows p-value using two tailed t-test and results for different morphologies in control and concentrations of 0.1, 0.01, 0.001, 0.0001 and 0.00001 mg of Phaseolus vulgaris PHA-L used.

Statistical Analyses- Percentage of morphologies for control and various concentrations for each lectin

100

Exogastrulated 60

No 50 invagination

Percentage (%)Percentage Incomplete 40 archenteron Dead 30 Complete 20 archenteron

0 0 0.1 0.01 0.001 0.0001 0.00001 Concentration of Artocarpus integrifolia (mg/ml)

Figure 19- Shows different concentrations for lectin Artocarpus integrifolia treatment (0.1, 0.01, 0.001, 0.0001, 0.00001 mg/ml) and control (0 treatment). The Y-axis shows the percentage of embryos exhibiting morphologies that were observed. The red line indicates the percentage of complete archenteron in each treatment; the purple shows percentage of embryos exhibiting incomplete archenteron; the blue line shows the embryos showing no invagination while the orange line indicates embryos exhibiting exogastrulation. The dead embryos are shown with green line.

100

60 Exogastrulated

50 No invagination

40 Incomplete archenteron Dead percentage (%) 30 Complete 20 archenteron

0 0 0.1 0.01 0.001 0.0001 0.00001 Concentration of Triticum vulgaris (mg/ml)

Figure 20- Shows different concentrations for lectin Triticum vulgaris treatment (0.1, 0.01, 0.001, 0.0001, 0.00001 mg/ml) and control (0 treatment). The Y-axis shows the percentage of embryos exhibiting morphologies that were observed. The red line indicates the percentage of complete archenteron in each treatment; the purple shows percentage of embryos exhibiting incomplete archenteron; the blue line shows the embryos showing no invagination while the orange line indicates embryos exhibiting exogastrulation. The dead embryos are shown with green line.

100

60 Exogastrulated

50 No invagination 40 Incomplete archenteron Dead

30 Percentage (%)Percentage Complete 20 archenteron

0 0 0.1 0.01 0.001 0.0001 0.00001 Concentration of Phaseolus vulgaris PHA-L (mg/ml)

Figure 21- Shows different concentrations for lectin Phaseolus vulgaris PHA-L treatment (0.1, 0.01, 0.001, 0.0001, 0.00001 mg/ml) and control (0 treatment). The Y-axis shows the percentage of embryos exhibiting morphologies that were observed. The red line indicates the percentage of complete archenteron in each treatment; the purple shows percentage of embryos exhibiting incomplete archenteron; the blue line shows the embryos showing no invagination while the orange line indicates embryos exhibiting exogastrulation. The dead embryos are shown with green line.

TABLE 5

Major Effects of Lectins (Summary)

LECTIN CARBOHYDRATE MAJOR OUTCOME SPECIFICITY

Embryos exhibited lower complete archenteron development in 0.1 mg/ml, 0.01 mg/ml, 0.001 and 0.0001 mg/ml. In 0.1 mg/ml incomplete archenteron and no invagination was almost three times compared to control. In 0.01 mg/ml the number of embryos exhibiting complete archenteron, no invagination and Artocarpus D-galactose exogastrulation remained similar to 0.1 mg/ml. However, integrifolia incomplete archenteron increased drastically. In 0.001 mg/ml the embryos exhibiting complete archenteron development increased although the incomplete archenteron and no invagination still showed about double what was observed in control. Exogastrulated embryos slightly increased in this concentration. In 0.0001 mg/ml higher number of embryos showed complete archenteron development than observed in 0.001 mg/ml. However number of embryos exhibited incomplete archenteron, no invagination and exogastrulation decreased. In 0.00001 mg/ml highest number of embryos showed complete archenteron development similar to the control. The significance of the results was tested using p-value analyses using InSTAT.

At concentration of 0.1 mg/ml some complete archenteron development occurred but no invagination and incomplete archenteron and exogastrulation morphologies showed to be the highest. At 0.01 mg/ml embryos showed some complete development in archenteron but the levels of incomplete archenteron, exogastrulation and no invagination showed to be at Triticum N-Acetyl-β-D- high levels. In 0.001 mg/ml complete archenteron development vulgaris glucosamine increased however no invagination and incomplete archenteron were also seen to be high. Embryos exhibiting 0.0001 mg/ml showed steady increase in complete archenteron levels and number of embryos exhibiting no invagination also increased. In 0.00001 mg/ml high number of embryos showed complete archenteron development. The significance of the results was tested using p- value analyses using InSTAT.

In 0.1 mg/ml, 0.01 mg/ml, 0.001 mg/ml and 0.0001 mg/ml the complete archenteron development dropped significantly from observed in control. In all of these concentrations (0.1, 0.01, 0.001 and 0.0001 mg/ml) the incomplete archenteron and no invagination Phaseolus N-acetyl- β D- were also increased by more than double for each concentration and vulgaris PHA-L glucosamine morphology comparing to the control. Exogastrulated embryos were higher in the 0.1 mg/ml showing more than twice as many. In 0.00001 mg/ml high number of embryos exhibited complete archenteron development and levels are incomplete archenteron, no invagination and exogastrulation showed similar numbers to the control. The significance of the results was tested using p-value analyses using InSTAT.

DISCUSSION

Lectins have helped researchers for many years to determine the roles of carbohydrate containing receptors in sea urchin embryos (Lallier, 1972; Neri et al., 1975; Robertson et al., 1975; McCaig and

Robertson, 1982; Contini et al., 1992; Latham et al., 1995a). Previous research showed that macromolecules enter the blastocoel of starfish embryos that were incubated in hypertonic media showing the entrance was due to junctions that occur among cells that were not strongly sealed (Dan-Sohkawa et al.,

1995). Sea urchin embryos were treated with low calcium seawater to increase the speed in which lectins entered into the blastocoel. This media did not alter development of the embryo (Latham et al., 1998).

This study investigated the effects of three lectins on sea urchin embryonic development. Studies have shown that a lectin is composed of two or more carbohydrate binding sites. The linkage between the lectin and the carbohydrate is a weak non-covalent interaction. Itza and Mozingo, 2005 showed that when a divalent cation such as calcium is not used this results in the sea urchin blastula/gastrula epithelium becoming more permeable allowing it to be less rigid and stiff allowing molecules to penetrate (Itza and

Mozingo, 2005).

The aim of this research was to study the effects of five different concentrations of three lectins in low calcium seawater. The various concentrations selected had different impacts on the development and morphologies and resulted in changes in elongation of the archenteron and attachment of archenteron. The findings from this research can help to understand specific roles for specific receptors during sea urchin cellular interactions.

Studies by Khurrum et al., 2004 examined glycoprotein/proteoglycan synthesis inhibitors that caused abnormalities in archenteron organization, elongation and attachment at the tip of the blastocoel roof in sea urchin embryos (Khurrum et al., 2004). This research also demonstrated that supernatant taken from 24-32 hour sea urchin embryos that resulted in exogastrulation contained macromolecules that blocked archenteron elongation.

Treatment of Artocarpus integrifolia in low calcium seawater caused most embryos to exhibit incomplete archenteron development and no attachment of the archenteron for 0.1 mg/ml, 0.01 mg/ml,

0.001mg/ml and 0.0001 mg/ml concentrations (Figures 2(A-C)- 5(A-C)). At the lowest concentration of

0.00001 mg/ml high numbers of the embryos showed complete archenteron development although some exhibited incomplete archenterons and no invagination (Figure 6(A-C)). To confirm the validity and significance of the results the p-values were calculated for each morphology and concentrations and compared to the control. A p-value of less than 0.05 is considered to be significant. The unpaired t-test comparing the mean percentage of complete archenterons in controls to all of the concentrations tested (0.1 mg/ml, 0.01 mg/ml, 0.001 mg/ml, 0.0001 mg/ml and 0.00001 mg/ml) showed a p value of 0.0033 which showed the results were statistically significant (Table 4a). The unpaired t-test comparing the mean percentage of incomplete archenterons from the experimental concentrations to the controls showed a p- value of 0.0024 indicating statistical significance (Table 4a). The unpaired t-test comparing the mean percentage of no invagination of the control to various concentrations tested also showed to be statistically significant as the p-value was 0.0042 (Table 4a). The unpaired t-test comparing the mean percentage of exogastrulation of Control compared to the other concentrations showed to have a p-value of 0.1769, showing no statistical significance (Table 4a). The previous studies performed by Latham et al., 1999 showed that exogastrulation may result due to the lectin wheat germ agglutinin preventing the archenteron from properly developing and instead dropping outward creating exogastrulated morphology (Latham et al., 1999). On the other hand, the unpaired t-test for comparing the mean percentage of dead embryos in

Control to the other concentrations showed a p-value of 0.0002 making the results statistically significant

(Table 4a). Artocarpus integrifolia is a lectin that binds to D-galactose- like residues. The binding of lectin

Artocarpus integrifolia may result in halting and prevention of the filopodia of secondary mesenchyme cells attaching and binding to blastula roof properly. The filopodia of the secondary mesenchymal cells find the binding site and the proper attachment is performed.

In the lowest concentration used, 0.00001 mg/ml, the embryos showed more complete archenteron development when compared to the control making the effects of this lectin concentration dependent. This suggests that at lower concentration of 0.00001 mg/ml not all of the D-galactose residues were obstructed hence cellular interactions did take place and more embryos exhibited complete archenteron development

(Figure 6 (A-C)). At the highest concentration used, 0.1 mg/ml (Figure 2(A-C)), the embryos showed the least amount of development. The results from morphologies obtained from Artocarpus integrifolia show that it is possible that these cellular adhesive interactions are dependent on the binding of D-galactose residues.

Treatment of the sea urchin embryos with Triticum vulgaris in low calcium seawater showed different results using five different concentrations compared to untreated Control embryos. The lectin

Triticum vulgaris binds to N-Acetyl-β-D-glucosamine residues. At the highest concentration (0.1 mg/ml) of Triticum vulgaris it was noticed that number of complete archenteron development dropped significantly by more than fifty percent and this number only started to increase when the concentration dropped to 0.01 mg/ml (Figure 7(A-C) – 8(A-C)). Exogastrulated embryos were found to be at the highest level at the highest concentration of 0.1 mg/ml concentration dependent results were found in disruption in the organization and cellular elongation at the tip of blastocoel roof in the archenteron (Figure 8(A-C)). In 0.1 mg/ml, there are high numbers of embryos exhibited exogastrulation compared to other concentrations tested. This morphology implicates that the development occurred towards the outside of the embryos

(envagination) and no invagination occurred into the embryos (Hardin and Cheng, 1986). As observed mostly in the embryos with 0.1 mg/ml concentration (Figure 8(A-C)), the primitive gut either is dropped completely or it is partially falls from the interior of the embryo (Hardin and Cheng, 1986). On the other hand the concentrations 0.01 mg/ml, 0.001 mg/ml, 0.0001 mg/ml and 0.00001 mg/ml all show lower numbers of exogastrulated embryos (Figure 9(A-C)- 12(A-C)). When the 24 hour Lytechinus pictus embryos were incubated with 0.1 mg/ml, 0.01 mg/ml and 0.001 mg/ml high number of the embryos showed disorganization and disaggregation of tip of the archenteron leading to improper and failure of full elongation to take place. In response high number of embryos showed incomplete archenteron and no invagination (Figure 8(A-C) - 11(A-C)). The p-value for the unpaired t-test for comparing the mean of incomplete archenteron and no invagination (table 4b) showed to be statistically significant. The p-value for unpaired t test for mean percentage of incomplete archenteron showed to be 0.0074 making it statistically significant and the p-value for mean percentage for no attached archenteron was 0.0008 also showing statistical significance (figure 4b). As the concentration of Triticum vulgaris was decreased from

0.1 mg/ml to 0.00001 mg/ml, the number of embryos displaying complete archenteron also increased

80 showing that proper organization and elongation took place in the archenteron at the animal pole (Figure

12(A-C)). The increase in the complete archenteron may be a result of low Triticum vulgaris concentration that did not attach to N-Acetyl-β-D-glucosamine residues and the secondary mesenchymal cells were able to reach the animal pole by elongation and attachment to the blastocoel roof via filopodia (Khurrum et al.,

2004). In a similar study by Latham et al in 1999, wheat germ agglutinin (WGA)-Triticum vulgaris - which has the specificity for N-acetyl-D-glucosamine-like residues, obstructed the activity of the primary mesenchymal cells from migrating hence the skeletal formation was arrested This novel study by Latham et al was used under conditions of low calcium seawater to make the entrance of the macromolecules into the embryo cells faster than normal artificial seawater (Latham et al., 1999). There was a concentration dependent effect between the sea urchin carbohydrate residues and lectin Triticum vulgaris.

When treating the embryos from Lytechinus pictus sea urchins with Phaseolus vulgaris PHA-L which binds to N-Acetyl-β-D-glucosamine residues different morphologies were found. Phaseolus vulgaris

PHA-L exhibited concentration dependent effects. The concentration of Phaseolus vulgaris PHA-L decreased more embryos exhibited complete archenteron development. The development involves the proper organization and attachment of the archenteron tip at the blastocoel roof. In the lowest concentration of Phaseolus vulgaris PHA-L, it is apparent that least number of blockages possibly occurred with N-

Acetyl-β-D-glucosamine residues. With the results from the t-test from mean percentage of complete archenteron between the control (untreated embryos) and the mean of embryos that were treated at various concentrations and the result show a p-value of 0.0031 for all of the concentrations that is considered to be statistically significant and showing concentration dependent effects (Table 4C). This suggests that more

N-Acetyl-β-D-glucosamine residues and receptors are important in the cellular and developmental interactions. The differences showed that there are statistically significant effects at the lowest concentration (0.0001 mg/ml) and highest concentration (0.1 mg/ml) (Figure 13(A-C) and 18(A-C)). There were no significant differences observed in other treated concentrations. The p-values of the mean percentages of incomplete archenteron and no invagination between the control and all other concentrations of Phaseolus vulgaris PHA-L were 0.0087 and 0.0016 respectively indicating statistical significance in both morphologies when compared to control in all of the treated concentrations (Table 4C). Incomplete archenteron and no invagination morphologies demonstrated a concentration dependent as the embryos

81 displaying these morphologies dropped gradually from 0.1 mg/ml (highest concentration) to 0.00001 mg/ml (lowest concentration) in the experiments (Figure 14(A-C) – 18(A-C)).

The results suggest that secondary mesenchymal cells that are driven by the filopodia use N-

Acetyl-β-D-glucosamine or similar ligands and receptors to attach to the blastocoel roof although in all of these structures we do not known exactly how the lectins affect the embryos.

The current study was one of few on investigating, analyzing and interpreting the effects of three lectins, Artocarpus integrifolia, Triticum vulgaris, Phaseolus vulgaris PHA-L with five concentrations of

0.1 mg/ml, 0.01mg/ml, 0.001 mg/ml, 0.0001 mg/ml and 0.00001 mg/ml in Lytechinus pictus sea urchin embryos using low calcium seawater. From the results of the selected lectins, the highest concentration of each lectin caused lower numbers of complete archenteron development in the sea urchins. The results would indicate that the lectins mainly at the highest concentrations bind to the specific carbohydrates on the surface of the embryo cells and create failure in the cellular and developmental interactions and communications. These results suggest entry of lectins into the sea urchins at faster speed using low calcium seawater. Low calcium seawater allowed for studying more embryos without the process of microinjection. In addition to studying three lectins, various concentrations were used to detect morphological differences among the sea urchin embryos that were involved in the process of gastrulation.

Sea urchins are used as model systems due to their simplicity, transparency, cost effectiveness with handling and media in which they survive as well as amount of offspring that can be obtained from fertilization. These embryos exhibit some cellular and developmental attributes similar to higher organisms.

Helpful information can be gathered about carbohydrate presence and specificity on the surface of cells.

The sea urchins can be used to look at the embryological, cytological as well as molecular levels to better understand cellular interactions and communications during the process of gastrulation. Furthermore using additional lectins with different properties can help in identification and investigation of the role of carbohydrate receptors in interactions and communications in which they are involved. The current study looked at the effect of three lectins, Artocarpus integrifolia, Triticum vulgaris, Phaseolus vulgaris PHA-L at five concentrations of 0.1 mg/ml, 0.01mg/ml, 0.001 mg/ml, 0.0001 mg/ml and 0.00001 mg/ml but future studies and researches can focus on other lectins extracted from various organisms such as plants and

82 animals to detect differences in their effects at various concentrations. Future studies can use different lectin concentrations to detect for specificity of ligands and receptors at the cellular and molecular levels to determine the adhesive interactions of the secondary mesenchymal cells to the blastocoel roof in sea urchin embryos. To better demonstrate the specificity of lectin effects, future experiments should use Hapten inhibitors such as N-Acetyl-β-D-glucosamine versus a non-Hapten sugar incubated with the lectins before treating the embryos with the lectins. It is likely however, that the lectins used in this study are binding to their specific sugar residues in the embryos. This is the likely because two lectins are specific for N-Acetyl-

β-D-glucosamine and had similar effects. Also, effects were generally concentration dependent suggesting that the lectins were active based on carbohydrate specificity. This is a preliminary study that suggests a role for N-Acetyl-β-D-glucosamine and possibly D-galactose residues in archenteron development in the sea urchin gastrula.

It should be noted that in order to better determine which molecules are involved in the actual binding of the tip of the archenteron to the blastocoel roof, this laboratory has microdissected the archenteron and the blastocoel roof out of the embryos and treated them with specific enzymes. In this way it is possible to determine which ligands may be directly responsible for the adhesive interaction (Singh et al., 2013). This laboratory approaches the molecular basis of sea urchin gastrulation using many tools including lectins (as done here), enzymes, sugars and various assays such as the quantitative microplate method used here and in other studies and the microdissection method (Singh et al., 2013). While the microplate method can not identify exactly how reagents are acting in the embryo, its ability to test thousands of embryos at a time is one effective approach in helping in the understanding of sea urchin morphogenesis.

REFERENCES

Balinsky, BI (1959). "An electromicroscopic investigation of the mechanisms of adhesion of the cells in a sea urchin blastula and gastrula". Experimental cell research16 (2): 429–33.

Bleil, J. D., and P. M. Wassarman. 1980a. Structure and function of the zona pelucida: identification and characterization of the proteins of the meuse oocyte's zona pelucida. Dev. Biol. 76:185-202.

Bower S. (1996) Disease risks associated with transplantation of abalone, geoducks , sea urchins, and spot prawns in British Columbia. Aquaculture Update, 76, 1-4.

Colwin A. L. and Colwin L.H. Role of the gamete membranes in fertilization in Saccoglossus kowalevskii (Enteropneusta). I. The Acrosomal region and its changes in early stages of fertilization. J.Cell Biol. 1963 December 1; 19(3): 477-500.

Contini A, Falugi C, and Fasulo S (1992). Developmental regulation of lectin-binding patterns in Paracentrotus lividus gonads, gametes, and early embryos. Acta histochem. 92: 179-189

Dan K, Okazaki K. Cyto-embryological studies of sea urchins III. Role of secondary mesenchyme cells in the formation of the primitive gut in sea urchin larvae. Biol. Bull. 1956;110:29–42

Dan-Sohkawa M, Kaneko H, and Noda K (1995) Paracellular, transepithelial permeation of macromolecules in the body wall epithelium of starfish embryos. J Exp Zool 271: 264-272

Davidson E H. Lineage-specific gene expression and the regulative capacities of the sea urchin embryo: A proposed mechanism. Development. 1989;105:421–445

Ettensohn, C.A. and D.R. McClay (1988). Cell lineage conversion in the sea urchin embryo. Dev. Biol 125 (2): 396-408

Ettensohn, C.A. and S.W. Ruffins (1993). Mesodermal cell interactions in the sea urchin embryo: Properties of skeletogenic secondary mesenchyme cells. Development 117 (4): 1275-85.

Etzler ME, Macmillan S, Scates S, Gibson DM, James DW, Cole D, Thayer S. Subcellular localizations of two Dolichos biflorus lectins. Plant Physiol. 1984 Dec;76(4):871–878

Etzler ME, Talbot CF, Ziaya PR. NH2-terminal sequences of the subunits of Dolichos biflorus lectin. FEBS Lett. 1977 Oct 1; 82(1):39–41

Gerfen CR, Sawehenko PE (1985). A method for anterograde axonal tracing of chemically specified circuits in the central nervous system: combined Phaseolus vulgaris leueoagglutinin (PHA-L) tract tracing and immunohistoehemistry. Brain Res 1985;343:133-150.

Gustafson, T. (1963). Cellular mechanisms in the morphogenesis of sea urchin embryo. Cell contacts within the ectoderm and between mesenchyme and ectoderm cells. Exp Cell Res 32:570-89

Gustafson, T. and L.Wolpert (1961). Studies on the cellular basis of morphogenesis in the sea urchin embryo. Directed movements of primary mesenchyme cells in normal and vegetalized larvae. Exp Cell Res 24:64-79

Gustafson, T. and L.Wolpert (1963). Cellular movement and contact in sea urchin morphogenesis development. Int Rev Cytol 15: 139-214.

Hakomori S.I.(2000). Cell adhesion/recognition and signal transduction through glycosphingolipid microdomain. Glycoconj. J. Mar-Apr;17(3-4):143-51.

Halverson Larry J. and Stacey Gary(1984). Host recognition in the rhizobium-soybean symbiosis. Evidence for the involvement of lectin in nodulation. Department of Microbiology and graduate program of ecology, University of Tennessee.

Hankins C N and L M Shannon (1978). The physical and enzymatic properties of a phytohemagglutinin from mung beans. J. Biol. Chem. (21):7791-7797.

Hardin, J. (1986). The mechanisms and mechanics of archenteron elongation during sea urchin gastrulation. Dev Biol 115:490-501.

Hardin J. and D.R. McClay (1990). Target recognition by the archenteron during sea urchin gastrulation. Dev Biol 142 (1): 86-102. .

Hylander B. L. and Summers R. G. (1981). The effect of local anesthetics and ammonia on cortical granule-plasma membrane attachment in the sea urchin egg. Developmental Biology; Volume 86, issue 1, August 1981, Pages 1-11.

Itza, E.M. and N.M. Mozingo (2005). Septate junctions mediate the barrier to paracellular permeability in sea urchin embryos. Zygote 13 (3):255-64.

Johnson R. J., Simpson S., Van Epps D. E., Chenoweth D. E. (1992) Wheat germ agglutinin inhibits the C5a receptor interaction: implications for receptor microheterogeneity and ligand binding site. Journal of Leukocyte Biology Vol 52, Issue 1, 3–10.

Khurrum, M.A. Hernandez, et al. (2004). Carbohydrate involvement in cellular interactions in sea urchin gastrulation. Acta Histochem 106 (2): 97-106.

Kiehart DP (1982) Microinjection of echinoderm eggs: apparatus and procedures. In methods in cell biology, vol 25. Academic Press, New York, pp 13-31

Kominami, T., Takata, H. (2004). Gastrulation in the sea urchin embryo: a model system for analyzing the morphogenesis of a monolayered epithelium. Development, growth & differentiation 46 (4): 309– 26. doi:10.1111/j.1440-169x.2004.00755.x. Lee, X., Johnson, R.A.Z., Rose, D.R. and Young, N.M. (1989) Crystallization and preliminary X-ray diffraction studies of the complex of Maclura pomifera agglutinin with the disaccharide Gal beta 1- 3GalNAc.J. Mol. Biol. 210, 685-686.

Lallier R (1972). Effects of concanavalin A on the development of sea urchin egg. Exp Cell Res 72: 157- 163

Latham VH, DucutJ, Rostamiani K, Chun HH, Lopez ME, Herrera S, and Oppenheimer SB (1995 a) A rapid lectin receptor binding assay: comparative evaluation of sea urchin embryo cell surface lectin receptors. Acta histochem 97: 89-97

Latham, V.H., M.J. Tully, et al. (1999). A putative role for carbohydrates in sea urchin gastrulation. Acta Histochem 278(34):32439-47.

Liener I. E., Sharon N., Goldstein I. J., (1988). The lectins – properties, functions and applications in Biology and Medicine. Volume 32, issue 2, pages 212-213.

McCaig CD, and Robinson KR (1982): The distribution of lectin receptors on the plasma membrane of the fertilized sea urchin during first and second cleavage. Dev BioI 92: 197-202

McMahon SA, Miller JL, Lawton JA, Kerkow DE, Hodes A, Marti-Renom MA, Doulatov S, Narayanan E, Sali A, Miller JF, Ghosh P (2005). The C-type lectin fold as an evolutionary solution for massive sequence variation. Nat Struct Mol Biol 12:886–892.

Monestier M, Fasy TM, Novick KE, Losman MJ, Rigal D, Wong GY, Terzidis-Trabelsi H, Pilatte Y, Rostoker G. (1994). Histone-reactive IgA antibodies in adult IgA nephropathy and other primary glomerulonephritis. Nephron. 1994;68(1):25-31.

Oppenheimer, S.B (1989). Introduction to embryonic development. Allyn and Bacon.: 314-318

Pilatte Y, Rostoker G, Violleau K, Basta M, Hammer CH. Further identification of human plasma glycoproteins interacting with the galactose-specific lectin Jacalin. J Chromatogr B Biomed Appl. 1995 Jun 9; 668(1):1-11. Pueppke, S.G. (1983). Soybean lectin: does it have an essential role in the rhizobium soybean symbiosis? Prog Clin Biol Res 138:225-36.

Rueben, L., et al., Activities of lectins and their immobilized derivatives in detergent solutions. Implications on the use of lectin affinity chromatography for the purification of membrane glycoproteins. Biochemistry, 16, 1787-1794 (1977).

Sankaranarayanan R., Sekar K., Banerjee R., Sharma V., Surolia A., Vijayan M, (1996) A novel mode of carbohydrate recognition in jacalin, a Moraceae plant lectin with a b-prism fold. Nature Structural Biology 3, 596–603.

Sharon, C., M.C. Tirindeli, et al. (1986). Homozygous protein C deficiency with moderately severe clinical symptoms. Thromb Res 41 (4): 483-8

Sharon, N. and H. Lis (1990). Legume lectins—a large family of homologous proteins.” FASEB J 4(14):3198-208.

Shook, D; Keller, R (2003). Mechanisms, mechanics and function of epithelial-mesenchymal transitions in early development. Mechanisms of development 120 (11): 1351–83.

Singh, S. Karabidian, E. Kandel , A. , Metzenberg, S., Carroll, E.J., Oppenheimer, S.B., (2013) a role for polyglycans in a model sea urchin cellular interaction, Zygote, March 27: 1-11.

Solursh, M. (1986). Migration of sea urchin primary mesenchyme cells. Dev Biol. NY 2: 391-431.

Talbot CF, Etzler ME. (1978). Development and distribution of Dolichos biflorus Lectin as measured by radioimmunoassay. Plant Physiol. 1978 May; 61(5):847–850.

Varki, A. (1994). Selectin ligands.” Proc Natl Acad Sci USA 91(16): 7390-7.

Yoshida M.,and Yoshida K. (2011). Sperm chemotaxis and regulation of ﬂagellar movement by Ca+2. Molecular Human Reproduction, volumr.17, No.8 pp. 457–465.