Studies on Antimicrobial And Haemolytic Activities, Protein Profile and Transcriptomes of agelenopsis naevia walckenaer, 1842 (Grass Spider)Venom

Studies on Antimicrobial and Haemolytic Activities, Protein Profile and Transcriptomes of agelenopsis naevia walckenaer, 1842 (Grass Spider)Venom.

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 micro- dissection 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.

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TABLE OF CONTENTS

Title Page……. i

Approval page….ii

Declaration…….. iii

Certification……… iv

Dedication…………… v

Acknowledgements….. 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

• INTRODUCTION……….. 1
• Background Information………. 1
• Biologyof Agelenopsis naevia……. 4
• Statement of Research Problem. 5
• Justification…… 6
• Aim of Research….. 7
• Objectives ofthe Research……. 7
• Research Questions……. 8

Chapter Two

LITERATURE REVIEW………. 9

Spiders… 9

Spider Venom  10

Components of Spider Venom…….. 11

Neurotoxins……. 12

Proteins and peptides…….. 12

Acylpolyamines… 14

Peptide Nomenclature……….. 16

Sex and Geographical Location as Factors for Intra-specific Variation in Spider Venom 17

Spider Peptides as Potential Bioinsecticides…… 18

Spider venom peptides targeting sodium (Na_v) channels….. 20

Spider venom peptides targeting calcium (Ca_v) channels…… 22

Selection criteria for bioinsecticide leads……. 23

Spider Peptides as Potential Therapeutics.. 26

Spider venom toxins as antiarrhythmic drugs… 26

Erectile dysfunction treatment using spider toxin…. 27

Spider toxin as an antimicrobial agent…… 27

Antimalarial spider toxins……….. 28

Spider toxin in treating cancer…….. 28

Spider toxin as an anticonvulsant……… 29

Potential Therapeutics from other Venomous Animals…….. 30

Chapter Three

• MATERIALS AND METHODS……………. 31
• Description of Site of Spider Collection……… 31
• Collection of Agelenopsis naevia………… 31
• Determination of Sex of Agelenopsis naevia…….. 33
• Extraction of Venom………………… 33
• Antimicrobial Assay of Agelenopsis naevia Venom…….. 36
• Haemolytic Assay of Agelenopsis naevia Venom……….. 36
• Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis

(SDS- PAGE) Analysis of Crude Venom…… 37

• Staining and destaining…….. 38

• Two Dimension Gel Electrophoresis……… 38
• Messenger RNA Isolation….. 39
  • Complementary DNA (cDNA) synthesis and sequencing.. 39
  • Sequence analysis…………. 39
  • Phylogenetic analysis of toxins….. 40
• Data Analysis…. 41

Chapter Four

• RESULTS………. 42
• Antimicrobial Activity of Agelenopsis naeviaCrude Venom…..42
• Haemolytic Activity of Agelenopsis naevia Venom…….. 42
• Protein Profile of Agelenopsis naevia Venom…………. 42
• Transcriptomes of Agelenopsis naevia Venom……….. 48
• Phylogenetic Relationship of Agelenopsis naevia Toxins…….. 48

Chapter Five

• DISCUSSION………. 54
• Antimicrobial Activity of Agelenopsis naevia Venom.. 54
• Haemolytic Activity of Agelenopsis naevia Venom…. 55
• Protein Profile of Agelenopsis naevia Venom……. 56
• Transcriptomes of Agelenopsis naevia Venom………. 57
• Phylogenetic Relationship of Agelenopsis naevia Toxins…… 60

Chapter Six

• SUMMARY, CONCLUSION AND … 62
• Summary………. 61
• Conclusions……. 64

• Recommendations……….. 65

REFERENCES……….. 66

APPENDICES  85

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INTRODUCTION

1.1 Background of the Study

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 become much longer and reach into the prosoma where they take up considerable proportion of their body part (Sutter and Stratton, 2013).

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REFERENCES

Agnarsson, I., Coddington, J. A., and Kuntner, M. (2013).Systematics – progress in the study of spider diversity and evolution. In: Penney, D., editor, Spider Research in the 21st Century. 58–111p.
Ali, S. A.,Stoeva, S., Grossmann, J.G., Abbasi, A. and Voelter, W. (2001).Purification, characterization and primary structure of four depressant insect-selective neurotoxin analogs from scorpion (Buthus sindicus) venom.Archives of Biochemistry and Biophysic, 15:197–206.
Fu, T. J., Abbott, U. R. andHatzos, C. (2002). Digestibility of food allergens and non- allergenic proteins in simulated gastric fluid and simulated intestinal fluid—a comparative study. Journal of Agricultural Food Chemistry, 50:7154–7160.
Gao, L., Shan, B.E., Chen, J., Liu, J.H., Song, D.X. and Zhu, B. C. (2005). Effects of spiderMacrothele ravenvenom on cell proliferation and cytotoxicity in HeLa cells. Acta Pharmacologica Sinica, 26:369–376.
Gao, L., Yu, S., Wu, Y. and Shan, B.(2007). Effect of spider venom on cell apoptosis and necrosis rates in MCF-7 cells.DNA Cell Biology. 26:485–489.
Pukala, T. L., Boland,  M. P.,  Gehman,  J. D.,  Kuhn-Nentwig,  L.,  Separovic,  F.  and Bowie,  J.H. (2007).  Solution  structure  and  interaction  of  cupiennin  1a, a spider venom peptide, with phospholipid bilayers. Biochemistry, 46:3576– 3585.
Quistad, G. B., Reuter, C. C., Skinner, W. S., Dennis, P. A., Suwanrumpha, S. and Fu,