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biol 200 - leech thesis proposal (draft)

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zen banaag

on 30 September 2013

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Transcript of biol 200 - leech thesis proposal (draft)

why Land Leeches??
hypotheses
overview
Experimental Methods
SUSHMITA ZEN A. BANAAG
ARIANNE LYRA P. GAMBOA
FATIMA NYLA S. MADDALORA

1 October 2013


THE SALIVARY ANALYSIS AND POPULATION DENSITY OF
LAND LEECHES IN POBLACION, ATOK, BENGUET

Rationale
Hypothesis
Experimental methods and procedures

Terrestrial leeches are less studied compared to aquatic leeches

used for a variety of medical purposes

Exhibit useful compounds (antithrombin, anticoagulant and vasodilators)



Terrestrial leech salivary contents will exhibit a high anticoagulant property

Soil pH influences proliferation of leeches in Poblacion



Study Period
The present study will be carried out for a period of four months from November 2013 to February 2014

Experimental methods


Direct searching
Adult, juvenile leeches and soil collection
Morphometrics
Soil acidity and alkalinity
(Poblacion, Paoay and Caliking)


A. Collecting/Sampling Method


quadrat method (0.5m x 0.5m)
Relative abundance = number of animals / m2



B. Population Density/Abundance of land leeches


Ethanol solution as preservatives
Relative body size


C. Morphometric Study and
Species Identification

-Starvation (3-4 weeks)
-12h: 12h light and dark cycle at the room temperature (25±1°C)
-phagostimulatory solution
-keep in ice containers for 15-20 minutes
-centrifuged at 3000 rpm
-Filtered fluids as the crude saliva extract


D. Saliva Analysis
1.a. leech saliva extraction

-sodium chloride crystals for 15 minutes
-macerate in the blender
-centrifuge at 1500 rpm at 4°C for 60 mins
-crude saliva extract store at 4°C for 24 hrs
-0.5% of copper sulfate until no precipitation
-filtration of precipitate on a cheese cloth


D. Saliva Analysis
1.b. leech saliva extraction

-Bradford assay procedures with BSA
-phagostimulatory solution as a blank
-absorbance reading at A595



2. Protein Concentration in Leech Saliva Extract
D. Saliva Analysis

-dialysis into visking tubing
-shaking machine for 8 hours at 100 rpm
-store at 4°C for 24 hours
-anticoagulant power activity

D. Saliva Analysis
3. Anticoagulant capacity of LSE on blood
Figure 1. Site Map of Poblacion, Atok, Benguet taken from Google Earth.

Figure 2. Moist Pine Forest of Poblacion, Atok, Benguet

The use of European medicinal leeches (H. medicinalis)
for the treatment of obesity, ca. 1750.
Leech while sucking the solution
through the membrane.
Full transcript