Scientific Poster

1
Antibacterial Properties in Helix aspersa
Jessica Lau Biology Department, Skyline College,
San Bruno CA

• Methods
• Extract preparation
• Gather snails and collect mucus and blood.
• Separate mantle, crop, stomach, intestine, and
  lung. Homogenize each part in distilled water or
  80 ethanol (1.0 g/mL).
• Disk diffusion assay
• Aseptically inoculate nutrient agar plates with
  Escherichia coli, Staphylococcus aureus,
  Pseudomonas aeruginosa, and Bacillus subtilis.
• Saturate sterile filter paper disks with each
  extract. Controls filter paper disks saturated
  with distilled water or 80 ethanol.
• Place filter paper disks on the inoculated agar.
• Incubate plates at 37C for 24-48 hr.
• Measure zones of inhibition.
• Bacterial growth rate
• Aseptically inoculate 4.5 mL nutrient broth with
  0.5 mL of E. coli.
• Add a piece (0.15 g) of mantle to one tube,.
• Incubate at 37C and measure the absorbance at
  540 nm at 30 min. intervals.
• Lysozyme activity
• Prepare a 110 dilution of mantle extract in
  lysozyme buffer.
• Inoculate with Micrococcus luteus.
• Record the absorbance at 540 nm for 15 min.
• Repeat with egg-white lysozyme.

• Results
• Mantle (Figure 2) and blood (Figure 3) inhibited
  growth of bacteria.
• E. coli and S. aureus were inhibited by the
  ethanolic mantle extract (2.0 g/mL) (Table 1).
• The growth rate of E. coli in the presence of the
  whole mantle is 33.8 (range 17.1-50.5) slower
  compared to the control (Figures 4 and 5).
• Lysozyme analysis (Figure 6) shows that the
  mantle does not produce lysozyme.

Abstract Overuse and misuse of antibiotics over
the past 50 years has led to an increase in
antibiotic resistant bacteria. Therefore, there
is a need to develop new antibacterial agents,
which includes those that are obtained naturally.
Many animals produce bactericidal factors,
however, information on mollusk defenses is
scare. To date, antimicrobial activity in
mollusks has been studied only in the sea hare
(Aplysia californica) and African land snail
(Achatina fulica). The terrestrial gastropod,
Helix aspersa, crawls on its ventral foot
exposing its mucous membranes to potentially
pathogenic bacteria, suggesting they produce
antimicrobial compounds. My hypothesis is that
the mucus, digestive gland, salivary gland,
mantle, and crop of H. aspersa have antibacterial
properties. Ethanolic and aqueous extracts of
each structure were tested against Escherichia
coli, Staphylococcus aureus, Pseudomonas
aeruginosa, and Bacillus subtilis bacteria using
the disk diffusion assay. Bacteria were incubated
on solid nutrient media followed by assessment of
inhibition by direct observation of zones of
inhibition. Only the ethanolic extract of the
mantle (2000 mg/mL) inhibited Escherichia coli
and Staphylococcus aureus and further tests were
done to determine the nature of antibacterial
properties. The mantle is not bactericidal but
does inhibit growth it does not produce
lysozyme. The growth rate of E. coli in the
presence of the mantle is 50 slower compared to
the control. Antibacterial properties found in
snails may be useful for developing antibacterial
agents for use in veterinary and human medicine.
Figure 3. Blood inhibited E. coli. Size bar 1
cm.

• Discussion Conclusion
• The mantle is not bactericidal but does inhibit
  growth. Extraction with ethanol increased
  inhibition suggesting the active compound is
  inside cells and is a lipid or protein.
• Antibacterial activity from the mantle is not due
  to lysozyme.
• Antibacterial properties found in snails may be
  useful for developing antibacterial agents for
  use in veterinary and human medicine.
• Further study concentrate mantle/blood, separate
  using HPLC, and test fractions for antibacterial
  activity.
• Identify potential pathogens that may be used as
  biocontrols against H. aspersa.

Figure 1. Helix aspersa, the brown garden
snail, is a pulmonate mollusk native to the
Mediterranean coasts of Spain and France. It was
introduced into California and is known to cause
considerable damage to crops and competition for
native snails. It is also found in most
southeastern states and along the east coast
north to New Jersey.
Hypothesis The mucus, digestive gland, salivary
gland, mantle, blood, and crop of Helix aspersa
have antibacterial properties.
Figure 4. The growth rate of E. coli was slower
in the presence of mantle.

• Literature Cited
• Bellows, T.S. and T.W. Fisher. Handbook of
  Biological Control. New York Academic Press,
  1999.
• Campion, M. The structure and function of the
  cutaneous glands in Helix aspersa. Q J Microsc
  Sci 102195216, 1961.
• Fiolka J. M. and A. Witkowski. Lysozyme-like
  activity in eggs and in some tissues of land
  snails Helix aspersa maxima and Achatina
  achatina. Folio Biologica (Krakow)
  52(3-4)233-7, 2004.
• Glinski, Z, and J. Jarosz. Molluscan immune
  defenses. Arch Immunol Ther Exp (Warsz)
  45(2-3)149-55, 1997.
• Kamio, M., K.C. Ko, S. Zheng, B. Wang, S. L.
  Collins, G. Gadda, P. C. Tai, C. D. Derby. The
  chemistry of escapin identification and
  quantification of the components in the complex
  mixture generated by an L-amino acid oxidase in
  the defensive secretion of the sea snail Aplysia
  californica. Chemistry 15(7)1597-603, 2009.
• Teague, E. Slime Time We've set foot on the
  moon, but we still haven't stamped out the snail.
  The best we can do for now is crowd control. The
  Fresno Bee (Fresno, CA) Jan 10, 2002, p. 6.
• http//elicinanz.com/index.html (Downloaded 12
  August 2009).

• Background
• The brown garden snail, Helix aspersa, is native
  to the shores of the Mediterranean Sea (Figure
  1).
• Introduced to California in the 1850s by French
  immigrants to be raised as a food source, H.
  aspersa is well adapted for the California
  climate. It has become an agricultural pest,
  feeding on leaves, twigs, and fruits. H. aspersa
  is also threatening native California snails by
  competing for food.
• Much of the 10 million annually spent by the
  U.S. citrus industry on pest control is used
  against H. aspersa. Attempts to develop
  biological control began with the introduction of
  predaceous snails and beetles in the 1950s and
  early 1960s (1, 6).
• As H. aspersa crawls around on its ventral foot,
  it is exposed to snail pathogens that may enter
  via the mucous membrane, suggesting there are
  some protective chemicals in the mucus (2).
• Antimicrobial peptides have been identified in a
  wide variety of multicellular eukaryotes
  including insects and vertebrates (3, 4).
• Elicina, a cosmetological cream made from
  secretions of H. aspersa Müller, is claimed to
  alleviate a number of skin conditions including
  acne (7).
• The sea hare, Aplysia californica, secretes ink
  containing escapin, an L-amino acid oxidase that
  oxidizes L-lysine, producing a mixture of
  chemicals that is antipredatory and antimicrobial
  (5).

Figure 2. A section of mantle inhibited
E.coli. Size bar 1 cm.

• Acknowledgements
• Dr. Christine Case, Skyline College Biology
  Professor
• Patricia Carter, Skyline College Biology Lab
  Technician
• Stephen Fredricks, Skyline College MESA Director
• Tiffany Reardon, California MESA Assistant
  Director

Figure 6. Egg white lysozyme (control) lysed
Micrococcus luteus. The mantle (test) did not
lyse the bacteria.
Figure 5. The generation time for E. coli
bacteria was longer in the presence of mantle.
Error bars 1 standard deviation.