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PROJECT TOPIC AND MATERIAL ON STUDIES ON ANTIMICROBIAL AND HAEMOLYTIC ACTIVITIES, PROTEIN PROFILE AND TRANSCRIPTOMES OF AGELENOPSIS NAEVIA WALCKENAER, 1842 (GRASS SPIDER)VENOM

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  • Name: STUDIES ON ANTIMICROBIAL AND HAEMOLYTIC ACTIVITIES, PROTEIN PROFILE AND TRANSCRIPTOMES OF AGELENOPSIS NAEVIA WALCKENAER, 1842 (GRASS SPIDER)VENOM
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ABSTRACT

The study was carried out to investigate the antimicrobial and haemolytic activities,profile protein component of Agelenopsis naevia venom as well as transcript coding for protein in A. naevia venom. Venom was collected from the spiders by microdissection after homogenization of the venom gland and the concentration of venom was determined in a nanodrop spectrophotometer. Antimicrobial activity of venom againstBacilus subtilis, Candida albicans, and Salmonella typhi was carried out by disc diffusion and well diffusion assay. Haemolytic activity was carried out using purified 1% human erythrocyte. Crude venom was subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) using a precasted 4-20% gel. Gel was stained with commassie blue and destained to reveal the protein bands. Two dimension gel electrophoresis was also carried out in 13-cmimmobilized pH gradient(IPG) strip with a linear range of ƿH 3 to 10. Electrofocusing was carried out and the gel was rehydrated and subjected to a second dimension electrophoresis using a 15% SDS gel. The spots were visualized using commasie blue and detected using dynamic spot detector software. The messenger RNAs (mRNA) were isolated from venom gland and the first and second complimentary DNA (cDNA) strand were synthesized using ThermoScientific kit following manufacturer’s instructions. The cDNA library was constructed and purified. Pair-end sequencing was carried out using illumina NextSeq 500. The transcriptomes derived were searched against public databases using BLASTx alogrithm. Multiple sequence alignment andphylogenetic analysis was achieved using ClustalX and MEGA softwares respectively. Antimicrobial activity data were subjected to ANOVA to compare means, where significant, Duncan multiple range test was used to separate means. Effective concentration of crude venom was calculated using probit analysis. The crude venom of A. naevia showed significant activity against Bacillus subtilis(23.5±0.5) when compared to the Controls and no activity against Candida albicans and Salmonella typhi on the disc diffusion assay. However, the venom did not show activity against the three micro-organisms using well diffusion assay. The crude venom also showed haemolytic activity on human erythrocytes with activity within the first 1hr (42.40%) for all the three concentrations (0.579mg/ml, 2.843mg/ml and 4.044mg/ml) used. Percentage haemolysis ranged between 42.40%-52.52% within 1-6hr using three different concentrations. The venom has an EC50 of 2.07mg/ml. Six bands were evident on the SDS-PAGE gel with molecular weights ranging from below 6-64kDa. The crude venom showed both protein (>10kDa) and peptide (<10kDa) resolutions. Over 300 spots were detected on the 2D gel with molecular weight ranging from below 14kDa to 94kDa. Twelve transcriptomes homologous to sequences in databases were identified. Transcript from Agelenopsis naeviavenom gland clustered with that of Stegodyphus mimosarum. It was concluded that A. naevia venom have both antimicrobial and heamolytic activities. Its venom is made up of both proteins and peptides that are both cationic and anionicwhich could be harnessed as a potential bioinsecticide and antimicrobial agent.

TABLE OF CONTENTS

Title Page…………………………………………………………………………………i Approval page…………………………………………………………………………….ii Declaration ………………………………………………………………………………iii Certification………………………………………………………………………………iv Dedication………………………………………………………………………………..v Aknowledgements………………………………………………………………………..vi Abstract…………………………………………………………………….…………..viii
Table of Contents………………………………………………………………………..ix
List of Figures………………………………..………………………………………..xiii
List of Table………………………………………………………………..…………..xiv
List of Plates………………………………………..……………………………………xv
List of Appendices……………………………………………………………………..xvi
List of Abbreviations………………………………………………………………….xvii
CHAPTER ONE…………………………………………………………………………………………………1
1.0 INTRODUCTION…………………………………………………………………………………………1
1.1 Background Information……………………………………………………………1
1.2 Biologyof Agelenopsis naevia………..…………………………………………….4
1.3 Statement of Research Problem………………………………………………………5
1.4 Justification……………………………………………………………………….….6
1.5 Aim of Research……………………………………………………………………..7
1.6 Objectives ofthe Research…………………………………….……………………..7
1.7 Research Questions…………………………………………………………………8
CHAPTER TWO……………………………………………………………………….9
2.0 LITERATURE REVIEW…………………………………………………………….9
2.1 Spiders…………………………………………………………………………………………………………9
2.2 Spider Venom…………………………………………………………..…………..10
x
2.3 Components of Spider Venom………………………………….…………………11
2.4 Neurotoxins…………………………………………………………………………12
2.4.1 Proteins and peptides………………………………………………………………12
2.4.2 Acylpolyamines……………………………………………………………………14
2.5 Peptide Nomenclature……………………………………………………………….16
2.6 Sex and Geographical Location as Factors for Intra-specific Variation in Spider Venom…………………………………………………………….………..17
2.7 Spider Peptides as Potential Bioinsecticides……………………………………….18
2.7.1 Spider venom peptides targeting sodium (Nav) channels…………………………….20
2.7.2 Spider venom peptides targeting calcium (Cav) channels…………………………….22
2.7.3 Selection criteria for bioinsecticide leads…………………………………………….23
2.8 Spider Peptides as Potential Therapeutics…………………………………………………….26
2.8.1 Spider venom toxins as antiarrhythmic drugs………………………..………………26
2.8.2 Erectile dysfunction treatment using spider toxin………………….……………..27
2.8.3 Spider toxin as an antimicrobial agent…………………………….…………………27
2.8.4 Antimalarial spider toxins…………………………………………………………28
2.8.5 Spider toxin in treating cancer……………………………………………………..28
2.8.6 Spider toxin as an anticonvulsant………………………………………………….29
2.9 Potential Therapeutics from other Venomous Animals……..………………….30
CHAPTER THREE…………………………………………….……………………….31
3.0 MATERIALS AND METHODS………………………………………………….31
3.1 Description of Site of Spider Collection ………………………………………….31
3.2Collection of Agelenopsis naevia………………….……………………………….31
3.3 Determination of Sex of Agelenopsis naevia………………………………………………….33
3.4 Extraction of Venom……………………………………………….……………….33
3.5 Antimicrobial Assay of Agelenopsis naevia Venom……………………………………….36
3.6 Haemolytic Assay of Agelenopsis naevia Venom……………………………………………36
xi
3.7 Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS- PAGE) Analysis of Crude Venom …………………………..…………….37
3.7.1Staining and destaining……………………………………………………………38
3.8 Two Dimension Gel Electrophoresis……………………………………………..38
3.9 Messenger RNA Isolation……………………………………………….…………39
3.9.1 Complementary DNA (cDNA) synthesis and sequencing………………………..39
3.9.2 Sequence analysis……….………………………………….……………………….39
3.9.3 Phylogenetic analysis of toxins……………………………………………………40
3.10 Data Analysis………………………………………………..………………………41
CHAPTER FOUR…………………………………………………………………………………………….42
4.0 RESULTS……………………………………………………………..……………42
4.1 Antimicrobial Activity of Agelenopsis naeviaCrude Venom……………………42
4.2 Haemolytic Activity of Agelenopsis naevia Venom…………………………………………42
4.3 Protein Profile of Agelenopsis naevia Venom…………………………………………42
4.4 Transcriptomes of Agelenopsis naevia Venom……………………………………..48
4.5 Phylogenetic Relationship of Agelenopsis naevia Toxins…………………………48
CHAPTER FIVE…………………………………..…………………………………..54
5.0 DISCUSSION……………………………………………………………………………………………..54
5.1 Antimicrobial Activity of Agelenopsis naevia Venom……………………………54
5.2 Haemolytic Activity of Agelenopsis naevia Venom………………………………………..55
5.3 Protein Profile of Agelenopsis naevia Venom……………………………………56
5.4 Transcriptomes of Agelenopsis naevia Venom…………………………………..57
5.5 Phylogenetic Relationship of Agelenopsis naevia Toxins……………………….60
CHAPTER SIX…………………………………………………………………………..62
6.0 SUMMARY, CONCLUSION AND RECOMMENDATIONS.………………….62
6.1 Summary………………………………………………………………..…………….61
6.2 Conclusions…………………………………………………………………………………………………64
xii
6.3 Recommendations……………………………………………………………………………………….65
REFERENCES…………………………………………………..………………………66
APPENDICES………………………………………………………………………….85

CHAPTER ONE

INTRODUCTION
1.1 Background Information
Among various venomous animals, spidersare the mostsuccessful, the most
geographically distributed and consumed the most diverse prey with an estimated
120,000 species (Agnarsson et al., 2013). Majority of spiders employ a fatal mixture
of toxins that they use to subdue their preys, which are, often, larger than their size.
Even though these creatures have fearsome reputation, only about a handful of these
arthropodsare harmful to humans (Isbister and White, 2004; King, 2004).
Nonetheless, the few medically important species prompted scientists, more than half
a century ago to begin exploring the pharmacological complexity of spider venoms.
Though various other animals, such as scorpions, snakes, bees and ants amongst
others employ venom for prey capture as well as defence, spiders are the most
successful. Most spiders feed on other arthropods while few species feed on other
animals like small fish, reptiles, amphibians, birds, and mammals (Rash and Hodgson,
2002). Consequently, spider venoms contain a diverse amount of toxins which targets
vertebrate and invertebrate species (Escoubas et al., 2004).
Spider body is divided into two main parts; the cephalothorax (prosoma) and the
abdomen (ophistosoma) joined by a pedicel. Extending from the rear end of the
prosoma are the chelicerae whilefrom the rear end of the ophistosoma are the
spinnerets (Foelix, 2011). Anterior to the cheliceral basal segmentlies the venom
glands. The well-developed venom glands are still found in the basal segment of the
chelicerae in Mygalomorph spider which produce venom that is injected via the
cheliceral fangs into the victim. However, in Araneomorphae, the venom glands have
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become much longer and reach into the prosoma where they take up considerable
proportion of their body part (Sutter and Stratton, 2013).
Despite the key role that venom gland plays in the survival of spiders, some spiders
have significantly reduced or lost their venom glands. The Uloboridae evolved a
unique technique to compact the living prey with extensive wrapping and by wetting
the entire prey with a fluid containing digestive enzymes that kill insects quickly
(Weng et al., 2006). Forster and Platnick, (1977) reported that the only two species of
the genus Holarchaea have very long and slender cheliceral fangs without a venom
gland opening on the cheliceral top. In spitting spiders (Sycotidae), the venom glands
are highly modified to produce a gluey fibrous glycoprotein which is used to fix their
prey to the ground from a distance (Kovoor, 1987).
Spider venom consists of components with different types of biological activities. On
a conservative estimate of 100,000 species, there are 200 peptide toxins per venom
with more than 10 million bioactive peptides in the spider venoms (King et al., 2008).
Some spider toxins have been used pharmacologically to probe the structure and
function of ion channels and receptors (Liang, 2004; Siemens et al., 2006; Bohlen et
al., 2010). Roerig and Howse (1996) have reported on the effect of ω-agatoxin IVA, a
toxin isolated from venom of American funnel web spider (Agelenopsis aperta),
against thermal stimulation in the tail flick test, when co-administered with morphine
intrathecally. Also, intrathecal injection of ω-agatoxin IVA (0.2nmol/kg) in mice was
reported to decreased the licking time in both the early and late response phases in a
dose-dependent manner in formalin test (Murakami et. al., 2004). This peptide could
be of particular benefit if used as an analgesic in patients tolerant or opioid-dependent,
since this compound exhibits selectivity for the Ca2+ion channels (Rajendra et al.,
2004). Other spider toxins are being developed as environmentally-friendly
3
bioinsecticides (Windley et al., 2012; King and Hardy, 2013) andas potential drug
leads (Escoubas and King, 2009; Saez et al., 2010). Proteomicsand peptidomics have
become very powerful tools for exploring the complexity of the spider venom (Liao et
al., 2007; Yuan et al., 2007; Liang, 2008; Tang et al., 2010). In particular,
transcriptomics which is the study of complete collection of messenger RNA in a cell
at a particular time (Jeremy et al., 2012) has been widely used to discover the
molecular diversity of venom toxins from spider venom glands (Chen et al., 2008;
Jiang et al., 2008;Diego-Garcia et al., 2010; Tang et al., 2010; Zhang et al., 2010).
Despite various reports on spider toxin, only 916 peptide toxins from 85 spider
species have been characterized and deposited in the database of ArachnoServer 2.0
(Herzig et al., 2011).
In most studies, RNA extracted from the venom glands of multiple spiders were used
to generate the pooled cDNA library (Chen et al., 2008; Jiang et al., 2008;Gremski et
al., 2010; Zhang et al., 2010). However, Escoubas et al.(1999) reported that sex was a
substantial factor in the intraspecific variation of spider venom using reversed-phase
chromatography and matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry (MALDI-TOF-MS). In Korea-sourced spider,Araneus ventricosus,
Avtox-1 and Avtox-2 toxins were identified from the cDNA library of the venom
gland. However, these were not found from the venom gland cDNA library of the
China-sourced spider A. ventricosus (Duan et al., 2013). Thus, spiders of the same
species from different natural environment may show variation in venom
composition.
Steiner et al.(1981) identified the first antibacterial peptides, cecropins, from the
Hyalophora cecropia moth. This led to a search for antimicrobial agents in animals
peptides presenting antibacterial activity were isolated throughout the entire animal
4
kingdom, ranging from different insect orders to amphibia, humans and other
mammals (Boman, 1991; Haeberlia et al., 2000). Gomesin was the first peptide
isolated from a spider exhibiting antimicrobial activities. This highly cationic peptide
is composed of 18 amino acid residues including four cysteines that form two
disulfide linkages (Mandard et al., 2002). Similarly, Kuhn-Nentwig et al. (2002) have
investigated the antimicrobial activity of the venom ofCupiennius salei. They isolated
several antimicrobial peptides from it, synthesized their analogs and reported that
these peptides had lytic activity on human red blood cells and also insecticidal effect
on Drosophila melanogaster. In another study, Corzo et al. (2002) reported five
amphipathic peptides – called oxyopinins which exhibit antimicrobial, haemolytic and
insecticidal activities which were isolated from the crude venom of the wolf spider
(Oxyopes kitabensis).
1.2 Biologyof Agelenopsis naevia
Agelenopsis naevia belongs to the Family Agelenidae which is a large family of
Araneomorph spiders commonly referred to as grass spiders (Platnick, 2007).
A.naeviashare the same family with the hobo spider (Tegenaria agrestis) whose
venom may or may not be harmful to human (Binford, 2001; Gaver-Wainwright et
al., 2011). The number and arrangement of spider’s eyes are often helpful in
determining to which family it belongs. Agelenids (like spiders in many other
families) have eight eyes and in most genera, these are arranged in two procurved
transverse rows, i.e., two rows of four, each of which has lateral eyes set farther
forward than the median eyes. Many Agelenids have light brown bodies with paired
darker longitudinal stripes on the cephalothorax (and sometimes on the abdomen as
well). They have long and conspicuous posterior spinnerets that extend well beyond
the tip of the abdomen. The extension of spinnerets beyond the tip of the abdomen is a
5
distinguishing feature of Agelenids. Their legs are relatively long and they are able to
run very quickly (Bennett, 1987).
Agelenids build a flat sheet of web that has a funnel-shaped retreat in the middle.
They usually stay in their retreat awaiting prey to blunder by.Agelenid spiders
typically trap their prey on the sheet-like portion of their webs, often with the aid of
barrier of silk spun a few centimeters above the sheet. Flying insects are trapped to
this sheet, where their legs become ensnared in the lattice works of silk. Within an eye
blink, the grass spider shoots out of its funnel, grabs the insect, envenomates it and
brings it back to the safety of the funnel where it feeds. The spider does not become
caught in its own web because it places its tarsi in an almost vertical tip-toe poise that
avoids entanglement as it runs across the sheet. In contrast, insects generally place
their tarsi in a flat splayed fashion that is easily caught in silken. While some other
spiders in the Agelenidae family produce stick silk to wrap insects in their sheet web,
Agelenopsis naevia always use purely mechanical ensnarement (Bennett, 1987).
1.3Statement of Research Problem
Over, 10,000 arthropod species are currently considered to be pest organisms. They
are estimated to contribute to the destruction of approximately 14% of the world’s
annual crop production and transmit many pathogens. Presently, arthropod pests of
agriculture and health significance are controlled by chemical insecticides (Windley
et. al., 2012). However,insects are becoming more resistant to existing pesticides and
other pesticides are deregistered due to established ecological and human health risks
associated with their use. Thus, there is an urgent need for new insecticides that can
be used in insect control such as bioinsecticides. Bioinsecticides are natural organisms
or their metabolic products that can be employed to control insect pests (King and
6
Hardy, 2013).Various microorganisms have developed resistance against antibiotics
(Ang et al., 2004). Widespread misuse of broad-spectrum antibiotics has led to the
emergence of drug-resistant pathogens, both in medicine and in agriculture (Benli and
Yigit, 2008). The first case of envenomation by Agelenopsis aperta was reported in
Southern California (Vetter 1998). Victims showed several envenomation symptoms
such as severe lesion, acute pain and itching among others at the site of
bite.Agelenopsis bite in defense when they feel threatened without an option to escape
(Whitman-Zaiet al., 2015).The effect of the venom of A. naevia has not been
characterized.
Spider venom peptides are produced in a combinatorial fashion, which leads to an
estimated total of about1.5 million spider venom peptides (Escoubas et al., 2006).
Consequently, spider venoms are a rich source of interesting novel compounds that
have received increased attention from pharmacologists and biochemists recently
which could be applied in pharmacological and agro-allied industries. However,
during the past decades, only a few spider venoms have been studied in sufficient
details, and therefore less than 0.01% of spider venom peptides have been identified
so far (Escoubas and Rash, 2004; Tedford et al., 2004; Sollod et al., 2005).
1.4Justification
The potential medicinal uses of spider venom are largely due to their selectivity and
affinity for ion channels and other receptors. This makes them suitable for studying
cell function and for designing new therapeutics (King and Hardy, 2013).
Antimicrobialpeptides are present in both vertebrates (Nicolas and Mor, 1995; Fathi,
et al., 2010) and invertebrates (Liuet al., 2009; Umairet al., 2012) and have broad
spectrum activity against awide range of micro-organisms, including viruses, Gram
7
positive and Gram-negative bacteria, protozoa, yeasts and fungi, and may also be
haemolytic and cytotoxic to cancer cells (Larrick and Wright, 1996; Hancock and
Lehrer, 1998).Gomesin was the first peptide isolated from a spider exhibiting
antimicrobial activities. This highly cationic peptide is composed of 18 amino acid
residues including four cysteines that form two disulfide linkages (Mandard et al.,
2002). Since then, several antimicrobial peptides have been isolated from spider
venom.Protein profiling, antimicrobial and haemolytic activity as well as
transcriptomic studies provide information which contributes to the discovery of
novel potential drugs and bioinsecticides from spider venom and a better
understanding of evolutionary relationship of spider toxin (Liang, 2008; Liu et al.,
2009;Jiang et al., 2013). Due to their immense importance, spider venoms have
recently garnered most attention from several research groups worldwide. Despite the
uses of spiders’ venom and their abundance, there is dearth of information on spiders’
especially their venom in different geographical regions like the Tropics. This study
serves as baseline information on A. naevia venom transcriptomes, proteins,
antimicrobial and haemolytic activities which could be engineered towards
pharmacological and/or agro-allied applications.
1.5Aim of Research
The aim of this research is tostudy theantimicrobial andhaemolytic activities, protein
profile and transcriptomes coding for protein/peptides ofA. naevia venom.
1.6Objectives of the Research
The specific objectives include to determine;
i) Antimicrobial activities of A. naevia venom onBacillussubtilis, Salmonella typhi
and Candida albicans.
8
ii) Haemolytic activity of A. naevia venom on human erythrocytes.
iii) Molecular weight of proteins/peptides ofA. naevia venom.
iv) Transcriptomes encoding proteins/peptides ofA. naevia venom.
v) Phylogenetic relationship of toxins in A. naevia venom.
1.7Research Questions
i) Does A. naevia venom have antimicrobial activity on Bacillus subtilis,
Salmonella typhi and Candida albicans?
ii) What is the effect of A. naeviavenom on human erythrocytes?
iii) What are the molecular weights of proteins/peptides found in A. naevia
venom?
iv) What are the transcripts that codes for A. naevia venom proteins/peptides?
v) What is the phylogenetic relationship of toxins in A. naevia venom?

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