AESTIVATION INDUCTION AND EVIDENCE OF CONFORMATIONAL DIFFERENCES BETWEEN OXY-HAEMOCYANIN AND DEOXY- HAEMOCYANININ AESTIVATING AND NON-AESTIVATING SNAILS

ABSTRACT

Haemocyanin is a high molecular weight, dioxygen, transport, copper-glycoprotein with a di- copper active site  found  in the  haemolymph of several marine and  terrestrial invertebrates belonging  to  the  phyla  Mollusca  and  Arthropoda.  Haemocyanin  exists  in  two  distinct conformers:  the  T-conformer  (Tense)  and  the  R-conformer  (Relaxed).Knowledge  of  the molecular architecture around the copper atoms in the active site of haemocyanin is important in understanding how  these  proteins reversibly bind  oxygen. Induction of aestivation and  the evidence of conformational differences between oxy-haemocyanin and deoxy-haemocyanin in aestivating and non-aestivating snails was studied.Aestivation induction was studied by treating five groups 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 oxy- haemocyanin and deoxy-haemocyaninwas also studied by treating the haemolymph of two snail samples (Snail 1  and  Snail 2)  with  nitrogen gas.After the  induction of aestivation,  it  was observed that the snails in groups A, B and C administered with the respective volumes of

haemolymph extracted from aestivating snails began to synthesize epiphragm layer on the 4th day

after  injection, on the 5th  day after  injection, the epiphragm layer was completely formed. Whereas the snails in groups D and E began to synthesize epiphragm layer on the 5th day, on the

6th  day, the epiphragm layer was completely formed. It was also observed that the snails in groups A, B and C that were injected with haemolymph extracted from non-aestivating snails beganto synthesize epiphragm layer on the 4th  day, on the 5th  day, the epiphragm layer was completely formed. It was also observed that the snails in groups D and E that were injected with different volumes of water, began to synthesize epiphragm layer on the 3rd day, at about 4 days and 8hours after injection, the epiphragm layer was completely formed. Whereas the snails in groups A, B and C began to synthesize epiphragm layer on the 4th  day, at about 5 days after injection, the epiphragm layer was completely formed. Results from the UV-Visible scanning showed that oxyhaemocyanin exhibited spectral activity both in the near-UV region and in the mid-UV  region,  whereas deoxyhaemocyanin only  showed  spectral activity  in  the  near-UV region.

CHAPTER ONE

1.1 INTRODUCTION

Mollusca are the second largest phylum of the animal kingdom, forming a major part of theworld fauna. The Gastropoda is the only class of molluscs which have successfully invaded 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 almost35,000 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 plantsincluding 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 whiletraveling across a surface (Denny, 1983). The unique  mechanical properties of snail pedalmucus enable the animal’s locomotion. The mucus trail performs a number of other functions, including the provision of mechanisms for re-tracing a path and for finding a mate of thesame species by following a trail (Al-Sanabani, 2008). An understanding of thefunctionality of trail mucus, including its interactions with water vapour, can thereforelead to a means of controlling the reproduction of snails and thereby limiting their impacton the environment, especially vegetable crops. When freshly deposited by terrestrialsnails, trails of pedal mucus are reported to be in the range of 10 –

20 mm thick(Denny, 1989). Since the mucus typically consists of between 90 and 99.7% water by weight(Denny, 1983), the trails dry to leave a much thinner solid film. It is generally believed thatthe fundamental structure of mucus gels consists of giant protein– polysaccharidecomplexes. This complex is usually classified into the broad categories ofmucopolysaccharides and glycoproteins (Denny, 1983; Davies and  Hawkins, 1998); mucussecretions can  function as effective adhesives due to their viscoelasticity (Grenon and Walker, 1980; Daoud, 2004; Abd El- wakeil,2005).

Snails (escargot) have a long history of being used as food by humans and are still much sought after in many countries in the world as a delicacy.The demand for snail meat became prominent

during the rule of the Roman Empire. At the Imperial Court in Rome, snail meat was thought to contain aphrodisiac properties and was often served to visiting dignitaries in the late evening (Taylor, 1900). In West Africa, snail meat popularly called “Congo”meathas traditionally been a major ingredient in the diet of people living in the tropical rain forest. In Nigeria the rearing of snails (Heliculture) has been on an increase because the animal is highly nutritious and has a lot of medicinal and cultural/social values. Hence snail meat provides alternative sources of animal protein complementing that from chicken, cattle, sheep, goats swine and pigs(Cobbinahet al.,

2008; Agbogidi and Okonta, 2011; Kalioand Etela, 2011).

Achatinaglutinosa,commonly called  the  African  land  snailis  a  large  specie  of  pulmonate gastropods of the family Achatinidae. They are indigenous to Africa and are found mainly in sub-Saharan Africa, ranging from the Gambia in the West to the Lake Chad region in the East. Their distribution extends southwards to the Orange River in South Africa (Hodasi, 1995). Achatina glutinosa belong to two main genera namely Achatina(Lamarck) and Archachatina(Albers). The former occurs all over Africa whilst the latter is restricted to the West African sub-region. In Nigeria and Ghana, three species of the Achatinagenus, namely Achatinaachatina, Achatina glutinosaand Achatinafulica, and then two species of the Archachatinagenus, i.e.,  Archachatinadegneriand Archachatinamarginataare known to occur (Monney, 1994). Both genera are known to be snails of the humid tropical environment.Achatinaglutinosa are of much use to humans in that they (i) provide cheap protein- rich food for consumption, (ii) are used for varied medicinal purposes by local people in different parts of west tropical Africa and (iii) are of paramount importance in studying the various adaptation mechanisms exhibited during adverse environmental conditions such as prolonged unavailability of food and longer periods of desiccation and during altered man-made environmental conditions.

The farming potentials of the African snails, more specifically the species Achatina glutinosa, Achatina achatina, Achatinafulicaand Achatina marginata have boosted international trade on snails in Europe, South East Asia and the Americas. In Ghana, Nigeria and Côte d’Ivoire, where snail meat is particularly popular, snails are gathered from the forest during the wet season (Cobbinahet  al.,  2008).  In  recent  years,  however,  wild  snail  populations  have  declined

considerably, primarily because of the impact of such human activities as deforestation, pesticide use, slash and-burn agriculture, spontaneous bush fires, and the collection of immature snails(Cobbinahet al., 2008). It is therefore important to encourage snail farming in order to conserve this important resource (Cobbinahet al., 2008). One of the disadvantages of heliculture is the expensive artificial means of climate control. Snails do not like hot and dry conditions. When temperature and/or humidity are not in favour of snails, their survival is usually through the phenomenon known as aestivation. Aestivation has restricted snail farming to the humid tropical  forest  zone,  which  offers  a  constant  temperature  and  a  high  relative  humidity (Cobbinahet al., 2008).

Terrestrial moluscs such as snails are noted for their ability to aestivate during dry seasons. These snails occur in the tropics where they bury themselves deeply in the grounds or under rocks during dry seasons. During aestivation, A. glutinosa and other pulmonate land snails become inactive by burying themselves deep in the ground, under rocks or leaves, withdrawing into their shells and secreting layers of a thin film and calcified mucous membrane called epiphragm across their  apertures (Herreid,  1977; Hodasi,  1979).  During this  period,  snails depend on their reserves at much reduced rate which implies an imminent reduction in body weight and loss of valuable growing time, as growth during aestivation is reduced. They can survive in this state for months and possibly years. Aestivation is an important survival stage of the snail’s life cycle. Aestivation plays a significant role in the snails’ ability to breed, grow and reproduce (Okafor, 2001). Other benefits range from agility, fertility, guts clearance, healing and rejuvenation. Due to these benefits snails therefore, have adapted a wide range of behavioral, biochemical, morphological and physiological mechanisms that may maximize their chances of successfully  surviving  under  adverse  environmental conditions  such  as  during  aestivation. During aestivation in land snails, gaseous exchange is discontinuous and depends on the environmental temperature (Barnhart and McMahon, 1987).

Haemocyanin, a copper-containing protein, is the respiratory pigment in snails as well as some arthropods (Van Holde and Miller, 1982).Haemocyanins is the respiratory pigments of snails, other mollusc as well as some arthropods. They are copper proteins; the most prevalent after haemoglobin (vanHolde and Miller, 1982, 1995). During aestivation one of the biochemical

adaptations of snails is a regulation in protein metabolism (Somero, 1995; Terwilliger, 1998; Decker and Foll, 2000; Bridges, 2001; Chaussonet al., 2004; Lukong and Onwubiko, 2004a;

2004b) and the duration of aestivation may be limited by the accumulation of noxious end- products of protein catabolism. It is therefore of importance to evaluate some of the structural and functional adaptations of haemocyanin from Achatina glutinosa asa means to unveil some of the biochemical and physiological mechanisms that enable the snail to survive months and even years of aestivation in the arid tropical regions of Africa. For haemocyanin to be biologically active, it must be folded into a unique three-dimensional structure. This structure is distorted by a number of osmolytes and extremes of temperature. During aestivation, snails accumulate a number of osmolytes (Horne, 1971; Rees and Hand,1993) and are exposed to high environmental temperatures. These factors could affect the structural and biological properties of haemocyanin by facilitating its unfolding into anunnatural state.

1.1.1Achatinaglutinosa(A. glutinosa)

Achatinaglutinosapopularly called the African land snail, is species of air-breathing land snails, a terrestrial pulmonate gastropod mollusc in the family Achatinidae, with shells that can reach a length of about 10-15 cm (Ramzy, 2009).

1.1.2 Taxonomy of Achatinaglutinosa

A. glutinosa belong to the phylum Mollusca, the second largest animal phylum in the world, with an estimated total species diversity ranging from less than 50,000 to as much as 200,000 and inhabiting freshwater, marine and terrestrial habitats (Van Bruggen, 1995). Although members of this phylum exhibit diversity in form as exemplified by snails, clams, octopods, squids, chitons and the tusk shells, this group possesses three unique hallmarks that distinguish its members from other animal phyla. These hallmarks are: (1) a muscular foot for locomotion; (2) a fleshy skin fold called mantle that secretes a calcareous shell; and (3) a feeding organ called a radula. Other characteristics of molluscs are the lack of true segmentation, the reduction of the body cavity, and the presence of spiral cleavage during early development (Van Bruggen, 1995).The family Achatinidae to which A. glutinosabelong are confined to Sub-Saharan Africa; however, they are now referred to as the travelling species in that they have moved or been moved all over the world (Mead and Palcy, 1992;Raut and Barker, 2002). Commerce and intentional spread by

man appear to be the most likely pathways for introduction of these snails to the U.S. (Lambert and Tillier, 1993).

Shells of Achatinidae are mostly dextral, higher than wide and are fusiform, ovoid or pillar- shaped (Schileyko, 1999). Classification within the family is based on conchological features (Bequaert, 1950)  and  the  highly variable reproductive tract  (Mead, 1991). There are  three subfamilies within the Achatinidae: (1) the Callistopeplinae in which members have medium sized shells with a truncated columellar margin and a vas deferens that is not attached to the penis sheath (Mead, 1994); (2) the Limicolariinae in which members also have medium sized shells but with a non-truncated columellar margin and a vas deferens that is attached to the penis sheath (Schileyko, 1999); and (3) the Achatininae with medium to large shells with truncated columellar margin and a vas deferens that is also attached to the penis sheath (Mead, 1994), (Figure 1).   The African land snails which are the Achatininae, belong to two main genera namely Achatina(Lamarck) and Archachatina(Albers). The former occurs all over Africa whilst the latter is restricted to the West African sub-region (Monney, 1994).

Figure 1: Anatomy of Achatinaglutinosa(Microsoft Encarta Premium, 2009 edition)

Variation also exists within the genera. In the Achatina, for instance, the West and Central African Achatina(Lamarck) to which A. glutinosa belongs, has a sculptured nepionic whorl (Bequaert, 1950), a plateaued apex (Mead, 1995) and no muscular bulboid enlargement in the

basal vagina (Mead, 1991; 1995) while the East African Achatina(Lissachatina), to which A. fulicabelongs, has a smooth nepionic whorl (Bequaert, 1950) and a half-dome apex (Mead, 1995) as well as a muscular bulboid enlargement of the basal vagina (Mead, 1991; 1995).

It is classified as follows: Kingdom:                   Animalia Phylum:                      Molusca Class:              Gastropoda

(Unranked)                 clade Heterobranchia clade Euthyneura clade Eupulmonata

clade Stylommatophora

Superfamily:               Achatinoidea Family:                       Achatinidae Subfamily:                  Achatininae Genus:                        Achatina

Specie:                        glutinosa (Pfeiffer, 1854)

1.2METABOLIC DEPRESSION IN SNAILS

An extended period of inactivity and reduced metabolic rate of many animals and plants, as well as unicellular organisms, has long been recognized by natural historians, e.g. Aristotle and Pliny. Biologists have studied this phenomenon since the 1550s (Gessner) and 1700s (Van Leeuwenhoek, Buffon). A confusing array of different terms has been used to describe these periods of inactivity. The period of inactivity can be less than a day, a few consecutive days or weeks, an entire season, or even many years. It can involve very different physiological states in response to a variety of environmental stimuli, such as extreme temperatures or unavailability of food or water. These periods of inactivity have been described and classified according to the group of organisms in question, extent and duration of the metabolic depression, ambient and body temperatures (low or high), the physical state of body water (frozen or hyperosmotic), or availability of oxygen.  Two  different  states of  inactivity and  metabolic  depression can  be discerned, dormancy and cryptobiosis (Keilin, 1959).

1.2.1Dormancy

Dormancy is a central feature of phenomena characterized by a state of hypometabolism or lethargy, which include: aestivation, hibernation, torpor, dauer, diapause (Storey and Storey,

1990; Storey and Storey, 2004). Dormancy can be a response to a circannual rhythm, or a variety of environmental factors, including temperature or the availability of food or water. It can be a short-term event (<24 hour) or it can occur for a few consecutive days, an entire season, or even many years. Dormancy can also involve seasonal arrest (diapause) or opportunistic inactivity (quiescence) of a developmental stage (Heldmaier et al., 2004; Storey and Storey, 1990; 2004;

2007).   Unlike cryptobiosis, dormancy does not involve marked changes in the physiological state of the animal (e.g. osmotic or hydration state). While cryptobiosis is a reactive response to an environmental stress (e.g. freezing at low ambient temperature; desiccation at low ambient humidity; hypoxia at  low ambient partial pressure of oxygen), dormancy is preemptive and anticipates stressful physiological conditions to avoid dramatic changes in the internal environment. Dormant animals are inactive but can usually be aroused by disturbance without requiring any major change in their physiological state, e.g. level of hydration or oxygen level (Storey and Storey, 2010). Mechanisms for dormancy are more likely to be intrinsic metabolic controls rather than molecular adaptations to an altered state of intracellular water or oxygen levels.Most major groups of animals as well as plants have some representatives that can become dormant. Dormancy vary in the degree of metabolic reduction, ranging from only slightly lower metabolism during the periodic, short-duration dormancy of deep sleep to more extreme reductions for extended periods of time (Schmidt-Nielsen, 1991).Principles of all dormant states include (a) an overall strong suppression of metabolic rate (hypometabolism), typically at least a

70–80% reduction as compared with normal resting metabolic rate. (b) differential control over the rates of various metabolic processes so that energy use is reprioritized to favor core vital cell functions (e.g., membrane potential difference) while largely shutting off “optional” activities (e.g., protein synthesis, cell division), and (c) implementation of actions that protect cells and preserve viability over what could be many months of dormancy (Storey, 1997; 2002; 2003; Storey and Storey, 1990; 2004; 2007; Guppy et al., 1994).

1.2.1.1Aestivation

Aestivation is a survival strategy used by many vertebrates and invertebrates to endure arid environmental conditions. Key features of aestivation include severe metabolic rate suppression, strategies to retain body water, conservation of energy and body fuel reserves, altered nitrogen metabolism, and mechanisms to preserve and stabilize organs, cells and macromolecules over many weeks or months of dormancy. Cell signaling is crucial to achieving both a hypometabolic state and reorganizing multiple metabolic pathways to optimize long-term viability during aestivation (Storey and Storey, 2012).

Aestivation is typically defined as a summer or dry season dormancy. The word is derived from the Latin for summer (aestas) or heat (aestus). Arid conditions that restrict water and food availability are the common trigger for aestivation, often but not always accompanied by hot summer temperatures. Aestivation is an ancient trait. Spurred on by ‘selfish genes’, all organisms are driven to grow, develop and reproduce. The primary inputs needed for this  are water, nutrients (both building blocks for biosynthesis and fuels for energy production) and energy (mainly  ATP  and  reducing  equivalents,  mostly  derived  from  oxygen-based  respiration  in animals. Physiological and biochemical adaptations supporting aestivation have been studied in many species and are particularly well-researched in lungfish (e.g. Protopterus spp.) water- holding frogs (e.g. Cyclorana sp. and Neobatracus spp.) spadefoot toads (Scaphiopus spp.) and several species of land  snails  (e.g.  Helix species and  Otala  lactea). Current  knowledge is excellently summarized in a recent book (Navas and Carvalho, 2010). Strong metabolic rate depression during aestivation minimizes energy use to prolong total survival time, but this also means that the normal turnover of macromolecules (synthesis and degradation) is much reduced so that preservation strategies are needed to extend their functional lifespans. This is provided by mechanisms including enhanced antioxidant defenses and elevated chaperone proteins, strategies that are well-known components of the stress response (Kültz, 2005) but are also widely used across all forms of natural hypometabolism to support viability and life extension (Storey and Storey, 2007; Storey and Storey, 2010; Storey and Storey, 2011).

1.2.1.2 Hibernation

Hibernation (winter dormancy) is long-term torpor in response to cold and scarcity of food. The above-ground structures of plants  may die  during unfavorable winter  conditions, but  some develop protective scales around the stem tips  so that the buds survive. Some plants have dormant underground bulbs, rhizomes, tubers, or corms, which are buffered from environmental extremes by the soil. For ectothermic animals, hibernation is primarily an inactive state accompanying a low body temperature, with many physiological sequelae including a lowered metabolic rate (Storey and Storey, 2010).

1.2.1.3   Diapause

Diapause involves the cessation of development of a sub-adult life stage, often for avoidance of harsh environmental conditions. It is often a programmed and obligate part of the life cycle, in response to regular and predictable environmental extremes, and synchronises the next stages of the life cycle with appropriate environmental conditions. Diapause is especially common in insects but is also observed in a variety of other invertebrate animals, as well as many plants (e.g. buds,  bulbs,  rhizomes, and  seeds),  but  is  rare  amongst  vertebrate animals.  The  embryonic diapause of a variety of mammals (e.g., macropod marsupials, mustelids, and deer) is a reproductivestrategy for delayed implantation and development of embryos rather than a strategy for metabolic depression, but presumably the diapaused embryo metabolically depresses during this period of arrested development. Diapaused eggs of annual killifish (Astrofundulus limnaeus) form a vitreous-like egg envelope and have a high resistance to desiccation; the embryonic tissues do not become dehydrated during diapause (Podrabsky et al., 2001).

1.2.1.4Torpor

Torpor is inactivity and a reduction in metabolic rate below the normal resting value, often in response to extreme ambient conditions. It characteristically occurs in adults and is usually a response to daily or seasonal environmental changes (hibernation in winter or aestivation in summer). Torpor by endothermic mammals and birds involves daily or seasonal changes in their thermoregulation set point and use of thermogenic metabolism so it is physiologically different from ectothermic torpor, which can occur at constant ambient and body temperatures (Storey and Storey, 2010).

1.2.1.5Dauer

A state of stasis in Caenorhabditis elegans nematodes, triggered by poor environmental conditions in larval stages L1 and L2 and leading to exit into the dauer instead of molt into L3. Dauer larvae are highly resistant to environmental stress (Storey and Storey, 2012).

1.2.2   CRYPTOBIOSIS

The study of cryptobiosis (or abiosis) started with observations by van Leeuwenhoek, of certain “animalcules” (wheel animals, rotifers) that he found in dry sediment in roof gutters, in an apparently lifeless state but which resumed normal activity when rehydrated (Schmidt, 1948; Keilin, 1959; Tunnacliffe and Lapinski, 2003).Cryptobiosis which means “hidden life,” is an extreme behavioral and physiological state characterized by “suspended animation” which is associated  with  complete  inactivity  and  almost  a  100%metabolic  depression.  Dormancy describes a state of reduced (but not complete lack of) metabolism, i.e., hypometabolism. Thus, there would appear to  be a continuum in the  potential metabolic state of organisms, from complete absence of metabolism, through hypometabolism, to the normal resting metabolic rate. Cryptobiosis is most commonly observed for invertebrates (Keilin, 1959) and is often a strategy to survive seasonal cold or desiccation. The similar term anabiosis, or “return to life” (Preyer,

1891), describes the resurrection of apparently completely lifeless organisms. Van Leeuwenhoek did not, however, suggest that these “lifeless” animals were completely desiccated, nor did he describe this state in terms of latent life or resurrection. His observations were followed about a half century later by descriptions of microscopic nematodes (eelworms, Anguillulina tritici) in an apparently lifeless state; these “lifeless” white fibres in grain quickly resumed activity when rehydrated, although in their desiccated state they crumbled to powder if disturbed (Needham,

1743). The extremely depressed metabolic state of cryptobiosis is often related to a dramatic change   in  the   state  of  cell  water  through  desiccation  (anhydrobiosis),  osmotic  stress (osmobiosis), or freezing (cryobiosis), although metabolic depressiondue to a lack of oxygen (anoxybiosis/anaerobiosis) occurs under conditions of normal cellwater. Cryptobiosis has independently evolved several times, within bacteria and protists as well as many multicellular plants (mosses, lichens, liverworts, higher plants) and animals (nematodes, rotifers, tardigrades,

crustaceans, insects; Clegg, 2001; Rebecchi et al.,2006). A few remarkable organisms are able to survive all of these forms of cryptobiosis, e.g. diapaused cysts of brine shrimp (e.g. Clegg et al.,1986; Clegg, 1997) and tardigrades (Nelson, 2002).

1.2.2.1  Anhydrobiosis

Anhydrobiosis is a response to desiccation (loss of cell water). It has been observed for a variety of invertebrate animals and plants during extreme desiccating conditions (Spallanzani, 1776; Keilin,  1959;  and  Rebecchi et  al.,2007),  but  not  vertebrates.  Spallanzani (1776)  described “resurrection” by cryptobiotic rotifers and tardigrades, which had a remarkable tolerance of high temperatures and  could survive  in a  vacuum.  Also,  many species of soil-dwelling animals (especially nematodes) are capable of surviving total dehydration if their initial rate of water loss is not too great. The slowly desiccated animals said to be in a state of anhydrobiosis (Crowe et al., 1992),which appear shrunken and lifeless, rapidly rehydrate and assume active life when they come in contact with water. In contrast, animals dried rapidly are killed by the dehydration process. Metabolic depression is anhydrobiosis is almost 100%.

1.2.2.2 Cryobiosis

Cryobiosis is a form of cryptobiosis that also involve an altered state of cell water. Cryobiosis involve perturbation of the  physical state  of water  in the  intracellular  environment, which presumably results in the extreme metabolic depression. Many animals (and plants) can survive freezing temperatures (Storey and Storey, 1996). Some organisms supercool or have antifreeze solutes to avoid freezing. A super-cooled state would be associated with asubstantial reduction in metabolic rate. However, some mhhanimals and plants tolerate actual freezing of their extracellularfluids. In 1663, it  was observed that vinegar eelworms (probably the nematode Turbatrixaceti) survived freezing (Wharton, 2002). Intertidal invertebrates such as gastropods, mussels, and barnacles routinely freeze when exposed at low tide (Aarset, 1982; Loomis, 1987). Some nematodes, slugs, and centipedes also tolerate freezing.Amongst vertebrates, some amphibians (Rana, Pseudacris, Hyla spp) and reptiles (turtles, lizards and snakes) can survive freezing  (Schmid,  1982;  Costanzo  et  al.,  1988;  Storey and  storey,  1988;  Claussen  et  al.,

1990;Costanzo et al.,1995; Dinkelacker et al., 2005). Freeze-tolerant animals can only withstand freezing of their extracellular fluids as any ice formed within cells disrupts cell membranes and

destroys intracellular integrity (Storey and Storey, 1996). As ice forms, solutes are excluded from the  ice-crystal structure and  this  increases  the  osmotic concentration of the  unfrozen extracellular fluids, thereby lowering its freezing point. Intracellular fluid remains in osmotic equilibrium with the extracellular fluid, which consequently also becomes osmo-concentrated. Some freeze-tolerant animals have specific ice-nucleating agents in their extracellular fluids, to promote freezing there rather than inside cells. Metabolic rate is reduced for frozen insects and frogs; tissue ATP declines during freezing, and anaerobic end-products accumulate (Storey and Storey, 1986, 1988).

1.2.2.3  Anoxybiosis /Anaerobiosis

Anoxybiosis (Keilin, 1959) involves survival of lack of oxygen, but at a normalhydration level (in contrast to the other forms of cryptobiosis). It occurs in someinvertebrate and vertebrate animals. For example, cysts of brine shrimp (Artemia)can survive extended periods of anoxia. During anoxybiosis, the metabolic rate ofhydrated Artemia cysts is so low as to be unmeasurable,

but  it  returns to  prediapauselevels (approximately 0.007 ml O2g-1  h−1) when the cyst  is  in

oxygenated water(Clegg, 1997; Glasheen and Hand 1988; Clegg et al.,1995). Anoxybiosis israre amongst vertebrate animals. Hatchling turtles survive in nitrogen at 4°C for 17(Malaclemys terrapin) to 50 days (Graptemys geographica; Dinkelacker et al.2005). Some adult turtles are extremely tolerant of anoxia, especially at reducedtemperatures, e.g. painted turtles Chrysemys picta survive up to 5 months of anoxiaat 3°C (Jackson, 2002); they have a coordinated reduction of both ATPconsumingand ATP-producing pathways involving various metabolic adaptations(Storey, 1997; Hochachka and Lutz, 2001).

1.2.2.4 Osmobiosis

Osmobiosis requires that animals are tolerant to a high ambient osmotic concentration(Keilin,

1959). For example, the metabolic rate of hydrated Artemia cystsduring osmobiosis (exposed to

5 mol l−1 NaCl) is only 0.00009 ml O2  h−1  (Glasheenand Hand, 1988). The obligate parasitic nematode Steinernema carpocapsae hassimilar tolerance of osmotic stress, at least for nonionic solutes such as glycerol andpolyethylene glycol (Glazer and Salame, 2000). Soil nematodes tolerate  osmoticconcentrations  of  up  to  1  M  (Van  Gundy,  1965).  Many  freshwater  and

terrestrialtardigrades form a resistant tun when exposed to concentrated salt solutions(Wright et al., 1992). The hyperosmotic tolerance of osmobiotic animals is similarto that of cryobiotic animals,  and  extracellular  and  intracellular  compatible  cryoprotectantsare also  important  in osmobiosis.

1.3AESTIVATION IN PULMONATE SNAILS (A. glutinosa)

A.  glutinosacan stay active  (eating,  reproducing) throughout  the  year  or  have  a  period  of aestivation depending on the environmental conditions (Figure 3). The main factors that determine the activity of snails are temperature, the amount of rain and the availability of food, with the first two as most important (Hodasi, 1979; 1982). Light intensity or photoperiod has shown to be of little or no importance. During aestivation, snails have absolutely no intake of food and water, and hence produce minimal or no urine and faecal materials for an extended period thereby, slowing down the biological time in relation to the clock time.

Aestivation   in   snails   comprises  three   phases   (Herreid,   1977,Riddle,   1983):   induction, maintenance and arousal. During the induction phase, animals detect environmental cues and turn  them  into  some  sort  of  internal  signals  that  would  instil  the  necessary  behavioural, structural, physiological and biochemical changes in preparation for aestivation. After entering the maintenance phase, they have to preserve their biological structures and sustain a slow rate of waste production to avoid pollution of the internal environment. Upon return of favourable environmental conditions, they must arouse from aestivation, excrete the accumulated waste products, and feed for repair and growth. Completion of aestivation occurs only if arousal is successful; if not, the animal would have had apparently succumbed to certain factors during the maintenance phase.

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