1. A Characterization of Toxin
Properties in the Venom of the
Wandering Spider Ctenus hibernalis
By Adam Krahn
Mentor: Dr. Brad C. Bennett

2. Spider Venom Characteristics
• Functions: Paralysis, Lethality, Digestion, and
Bacterial Protection
• Composition: Proteins/peptides, salts, and other
organic components
Ctenus hibernalis
Dr. Hataway

3. Study Questions and Objectives
Objectives:
• Characterize C. hibernalis venom
• Assess its biological activity
• Express and analyze individual venom proteins
Question:
• Why is C. hibnernalis venom
likely less toxic than
Phoneutria nigriventer venom?
Phoneutria nigriventer
https://en.wikipedia.org/wiki/Brazilian_wandering_spider

4. Methodological Overview
• Spider Collection and Venom Extraction
• Characterization via SDS-PAGE and BCA Assays
• Growth Inhibition Assays for Antimicrobial Activity
• Mammalian Cell Assays for Toxicity and Viability
• Recombinant Expression of C. hibernalis Proteins

5. Spider Collection and Venom Extraction
Collection:
• Site: Homewood Preserve
• Method: By hand at night
Extraction:
• Anesthetized with CO2
• Milked via electrostimulation
of the venom gland
Venom Extraction Apparatus

6. SDS-PAGE Showed Most Components
Between 50 and 75 kDa and
less than 15 kDa
Average Venom
Concentration:
47.7 mg/mL

7. Growth Inhibition Assays Revealed No
Antimicrobial Effect on 8 Species of Bacteria

8. Time Course Growth Inhibition Assays Showed
No Evidence of Immediate yet Temporary
Antimicrobial Activity

9. C. hibernalis Venom Markedly Reduces
C2C12 Myocyte Viability
Average Median Lethal Dose
Concentration: 0.035 ug/uL
Median Lethal Dose Concentrations:
1) 0.030 ug/uL
2) 0.039 ug/uL
MTT Assay
96-well plate

10. Bright Field Microscopy Shows Dose
Dependent Morphological Changes
Gewies. 2003

11. Recombinant Expression Was Unsuccessful,
Likely Due to Primer Imprecision
6000bp
2072bp
600bp
100bp
PCR Amplification
(genes 1-5)
Vector Digest PCR Test for DNA
Template Viability

12. Conclusions
• Venom potency appears selective toward
eukaryotic cells
• Toxin mechanism of action is likely not purely
cytolytic (appears to be necrotic)
• A transcriptome of the venom gland is needed
to express recombinant venom components

13. Acknowledgements
• Dr. Bennett
• Dr. Hataway and Dr. Sahawneh
• Dr. D. Johnson and Antonio Bradley
• Jonathan Berkuta
• Shannon Gilstrap and Kris Martinez
• Marissa Garrett
• REU Students who assisted with spider catching
• Samford University
• NSF

14. References
• Casewell, N. R. et al., (2013)
• De Paula Le Sueur, L. et al., (2005)
• Diniz, M. R. V. et al., (2006)
• Haeberli, S. et al., (2000)
• Herzig, V. et al., (2002)
• Kang, C. et al., (2009)
• Kim, M. et al., (2014)
• Mattiello-Sverzut, A. C. et al., (1998)
• Silva, A. O. et al., (2012)
• Vassilevski, A. A. et al., (2009)
• Zhong, Y. et al., (2014)

15. Agar Plate Assays Confirmed the Absence of
Growth Inhibition