ABSTRACT
is a high molecular weight, dioxygen, transport, copper-glycoprotein with a dicopper active site found in the haemolymph of several marines and terrestrial invertebrates belonging to the phyla Mollusca and Arthropoda.
Haemocyanin exists in two conformers: The T-conformer (Tense) and the R-conformer (Relaxed). Knowledge of the molecular architecture around the copper atoms in the site of haemocyanin is important in understanding how these proteins reversibly bind oxygen.
of aestivation and the evidence of conformational differences between oxy-haemocyanin and deoxy-haemocyanin in aestivating and non-aestivating snails were studied.
Aestivation induction was studied by treating five of snails (groups A, B, C, D and E) with respective volumes of oxy-haemocyanin from aestivating snails, respective volumes of oxy-haemocyanin from non-aestivating snails and respective volumes of distilled water.
Evidence of conformational differences between oxyhaemocyanin and deoxy-haemocyanin was also studied by treating the haemolymph of two snail (Snail 1 and Snail 2) with nitrogen gas.
TABLE OF CONTENTS
Title page …..…………………………………………………………………….. i
Approval page …………………………………………………………………….. ii
Dedication ….……………………………………………………………………… iii
Acknowledgement ……………………………………………………………………. iv
Abstract …………………………………………………………………………….. v
Table of contents…………………………………………………………………. vi
List of figures…………………………………………………………………….. x
List of tables ………………………………………………………………………. xiii
CHAPTER ONE:
1.1 General introduction ..………………………………………………………….1
1.1.1Achatinaglutinosa(A. glutinosa) ………………………………….……….. 4
1.1.2Taxonomy of A. glutinosa………………………………………………….. 4
1.2 Metabolic depression in snails…………………………………………………. 6
1.2.1 Dormancy……………………………………………………………………. 7
1.2.1.1Aestivation ………………………………………………………………… 8
1.2.1.2 Hibernation ……………………………………………………………….. 8
1.2.1.3Diapause …………………………………………………………………..9
1.2.1.4Topor ……………………………………………………………………… 9
1.2.1.5 Dauer…………………………………………………………………….. 10
1.2.2Cryptobiosis………………………………………………………………… 10
1.2.2.1 Anhydrobiosis…………………………………………………………… 11
1.2.2.2Cryobiosis…………………………………………………………….….. 11
1.2.2.3Anoxybiosis /Anaerobiosis……………………………………………… 12
1.2.2.4Osmobiosis……………………………………………………………… 13
1.3 Aestivation in pulmonatesnails (A.glutinosa) ………………………………. 13
1.3.1 Entry into aestivation ….……………………………………………………. 15
1.3.2 Physiological changes in snails during aestivation ………..………………. 15
1.3.2.1Body temperature and water loss during aestivation ……………………… 15
1.3.2.2 Respiration during aestivation …………………………………………… 16
1.3.2.3 Metabolic rate during aestivation ……………………………………….. 16
1.3.3 Arousal from Aestivation ………………………………………………….. 18
1.4Regulation and gene expression during aestivation…………………………. 19
1.4.1 Metabolic control by reversible phosphorylation in aestivation …………… 19
1.4.1.1Glucose-6-phosphate dehydrogenase…………………………………….. 21
1.4.1.2 Ion motiveATPasesduring aestivation …………………………………… 22
1.4.1.3 Protein synthesis during aestivation ……………… …………………….. 22
1.4.1.4 Protein degradation during aestivation …………………………………………. 23
1.5Dioxygen transport proteins during aestivation ……………………………… 24
1.6Haemocyanins ………………………………………………………………… 26
1.6.1 Active site structure and oxygenation of haemocyanins …………………… 27
1.6.2 Molluscan and arthropodanhaemocyanins ………………………………… 28
1.6.3 Haemocyanin as a respiratory pigment……………………………………. 36
1.6.4 Haemocyanin as a phenoloxidase…………………………………………. 38
1.6.5 Medical, industrial and agricultural importance of haemocyanins………… 40
1.7 Rationale of study…………………………………………………………….. 41
1.8 Aim and objectives of research.……………………………….……………… 41
CHAPTER TWO: MATERIALS AND METHODS
2.1 Materials ………………………………………………………………………. 43
2.1.1 Reagents / Chemicals……………………………………………………….. 43
2.1.2 Equipment / Instrument……………………………………………………… 43
2.1.3 Animal Procurement …………………………………………………………. 43
2.2 Methods……………………………………………………………………….. 43
2.2.1 Feeding of snails…………………………………………………………….. 43
2.2.2 Induction of aestivation in snails……………………………………………43
2.2.3 Collection of haemolymph……………………………………………………44
2.2.4 Injection of aestivating snail haemolymph into snails.……………………… 44
2.2.5 Injection of non-aestivating snail haemolymph into snails …………………. 45
2.2.6 Injection of distilled water into snails……………………………………………… 45
2.2.7Deoxygenation of snail haemolymph……………………………………… 46
2.2.8 UV- Visible scanning of haemocyanin………………………………………….. 46
2.2.8.1 UV- Visible scanning of oxy-haemocyanin………………………………. 47
2.2.8.1.1 UV- Visible scanning of oxy-haemocyanin containing different
volumes of aestivating haemocyanin…………………………………. 47
2.2.8.1.2 UV- Visible scanning of oxy-haemocyanin containing different
volumes of non- aestivating haemocyanin………………………….….. 47
2.2.8.2 UV- Visible scanning of haemocyanin contained in haemolymph injected
with different volumes of water………………………………………… 47
2.2.8.3 UV- Visible scanning of deoxy-haemocyanin…………………………… 47
CHAPTER THREE: RESULTS
3.1 Induction of aestivation ………………………………………………………… 48
3.1.1 Induction of aestivation in snails injected with aestivating haemolymph…… 48
3.1.2 Induction of aestivation in snails injected with non- aestivating haemolymph. 48
3.1.3 Induction of aestivation in snails injected with distilled water…………..……48
3.2 UV Spectroscopy…………………………………………………………….. 52
3.2.1.UV- Visible spectra of oxy-haemocyanin in haemolymph extracted
from snails injected with different volumes of non-aestivating
haemolymph……………………………………………………………… 52
3.2.2 UV- Visible spectra of oxy-haemocyanin in haemolymph extracted
from snails Injected with different volumes of non-aestivating
haemolymph………………………………………………………….…. 57
3.2.3UV spectra of haemocyanin in haemolymph extracted from snails injected
with different volumes of distilled water…………………………………… 62
3.2.4UV- Visible Spectra of Deoxy-Haemocyanin and the Corresponding
Oxyhaemocyanin from Snail I and II……………………………………… 68
CHAPTER FOUR: DISCUSSION AND CONCLUSION
4.1 Discussion …………………………………………………………………….. 71
4.2 Conclusion …………………………………………………………………… 74
Recommendations for further studies……………………………………………. 75
References…………………………………………………………………………. 76
INTRODUCTION
Mollusca is the second largest phylum of the animal kingdom, forming a major part of the world fauna. The Gastropoda is the only class of molluscs that have successfully invaded the land.
They are one of the most diverse groups of animals, both in shape and habit. Among gastropods, land snails (subclass: Pulmonata) are one of the most numerous with almost 35,000 described species of the world.
The Phylum Mollusca is probably the third most important animal group after the arthropods and vertebrates (South, 1992). Snails and slugs belong to the class Gastropoda.
They are molluscs, a group of invertebrates with soft unsegmented bodies. Slugs are often described as snails without a shell, while snail bodies are enclosed in calcareous shells (Barker, 2001; Ramzy, 2009).
The terrestrial Mollusca including snails and slugs are destructive agricultural pests causing economic damage to a wide variety of plants including horticulture, field crops, and forestry.
In addition, they are of importance in medical and veterinary practice. Gastropods such as slugs and snails secrete a trail of mucus from their pedal gland while ravelling across a surface (Denny, 1983).
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