Nonbiologists may be excused for questioning whether microbial diversity is really under threat. At a superficial level, micro-organisms seem to be tolerant of almost any set of conditions thrown at them. Also, they appear to have reproductive capacities able to generate populations of truly astronomic numbers in very little time. However, that is a superficial understanding and any belief that microbial species are not threatened is simply wrong.

James T. Staley of the University of Washington gave his answer to the challenge ‘Microbiologists are not concerned with endangered species, are we?’ in a commentary published in 1997 (Staley, 1997). His simple answer to this question is ‘Yes, some microbial species are threatened’, but the argument Staley develops is interesting and has some valuable points for mycologists to ponder. Even though the commentary was written largely from the bacteriologist's point of view, Staley mentions lichens and fungi so it is clear that he does include even the mushrooms and toadstools within his definition of micro-organisms. This is useful for us as we attempt in this brief introductory chapter to highlight and provide cross-references to the wide variety of aspects of fungal conservation that are included in this book. We are not alone in the belief that such topics are important!

Indeed, Staley puts the level of importance very high. Micro-organisms produced the original biosphere of Earth.

Introduction

The Royal Society for the Protection of Birds (RSPB) is the largest wildlife conservation charity in Europe with over one million members. It manages over 150 nature reserves throughout the UK, covering more than 108 000 hectares. Away from the reserves the Society safeguards sites and species of conservation importance through research, lobbying and education, whilst outside the UK the RSPB is working with Birdlife International Partners on major conservation projects in 18 countries in Europe, Africa and Asia. The RSPB, together with voluntary organisations, has produced a practical guide to conserving the UK's biodiversity that has been adopted at Government level.

The Society is committed to the management of nonavian taxa on its reserves, and is UK champion for nonbird species such as the medicinal leech, Hirudo medicinalis. The Society's interest in other biotic groups has been somewhat biased towards invertebrates and lower plants, for the most part to the exclusion of the fungi. This latter fact is understandable given the difficulties with identification and the complex taxonomy within mycology. In recent years an interest in the fungi on RSPB reserves has been encouraged by one or two active field mycologists, and has gradually led to the development of an in-house strategy for their conservation.

Threatened in Europe

A rapid decline of fungi across northern Europe has been reported since the 1980s. Before discussing habitats and species on RSPB reserves the European situation needs to be understood.

Introduction

Conservation of fungi, particularly populations of rare and endangered fungal species, is only possible when their habitats are protected. Consequently, efforts in fungal conservation will be successful only when cooperation with other environmentalists interested in nature conservation is established. Joint, well-motivated proposals to create new protected areas or to regulate a conservation regime in existing reserves will then accepted more easily than otherwise. The role of mycologists is not only to study the mycota of nature reserves, but also to take the initiative in creating new reserves.

Protection of primeval and old-growth forests

Primeval forests are very rare in Europe. Some kind of forest management has been practised almost everywhere except in those forest stands that are low in productivity and poor in species composition growing on oligotrophic Sphagnum bogs in northern areas. Seminatural old-growth forests with minimal human impact have survived mainly in nature reserves.

In Estonia, of the 138 hemerophobic vascular plant species found, 90 are growing only or mainly in forests (Kukk, 1999; Trass, Vellak & Ingerpuu, 1999); there are 79 hemerophobic bryophytes and 88 lichens in the Estonian forests. In the boreal zone, old unmanaged forest is the main refugium for rare and endangered species, including fungi. Protection of different forest site types is effective for conservation of species as well as of communities.

In the European Union, the main target of nature protection has been different groups of higher organisms (primarily birds) and a few selected habitat types (wetlands, Mediterranean communities).

This division comprises the entire continent from the Panamanian border southwards to Tierra del Fuego, together with the Falkland (Malvinas) Islands. Associated islands in the eastern Pacific Ocean come within Region 01, while those in the South Atlantic form part of Region 03. The chain of islands off the Caribbean coast, which encompasses Trinidad (with Tobago), the islands of the Venezuelan state of Nueva Esparta, and thence westwards to Aruba – once, with the nearby mainland, known as the ‘Spanish Main’ – is here included with the West Indies (as Regions 28 and 29).

Nervous systems

One of the characteristics of higher animals is their possession of a more or less elaborate system for the rapid transfer of information through the body in the form of electrical signals, or nervous impulses. At the bottom of the evolutionary scale, the nervous system of some primitive invertebrates consists simply of an interconnected network of undifferentiated nerve cells. The next step in complexity is the division of the system into sensory nerves responsible for gathering incoming information, and motor nerves responsible for bringing about an appropriate response. The nerve cell bodies are grouped together to form ganglia. Specialized receptor organs are developed to detect every kind of change in the external and internal environment; and likewise there are various types of effector organ formed by muscles and glands, to which the outgoing instructions are channelled. In invertebrates, the ganglia which serve to link the inputs and outputs remain to some extent anatomically separate, but in vertebrates the bulk of the nerve cell bodies are collected together in the central nervous system. The peripheral nervous system thus consists of afferent sensory nerves conveying information to the central nervous system, and efferent motor nerves conveying instructions from it. Within the central nervous system, the different pathways are connected up by large numbers of interneurons which have an integrative function.

In the previous chapter we examined some of the properties of skeletal muscles without giving much consideration to the mechanisms of the contraction process. It is as if we had investigated the properties of a motor vehicle by measuring its top speed, its fuel consumption, and so on, without finding out how the engine works. Now it is time to look under the bonnet.

Excitation–contraction coupling

The way in which the muscle cell is excited has been described in Chapter 7: an all-or-nothing action potential sweeps along the whole length of the fibre. This is followed by contraction, and the process linking the two events is called the excitation–contraction coupling process. The question we have to answer is: how does the action potential cause contraction?

Depolarization of the cell membrane

When muscle fibres are immersed in a solution containing a high concentration of potassium ions, they undergo a relatively prolonged contraction called a potassium contracture. The tension produced is related to the potassium concentration in a sigmoidal way as is shown in Fig. 10.1. The membrane potential is of course reduced under these conditions (see Chapter 3), so it seems that depolarization is an adequate stimulus for contraction. Normally this depolarization occurs during the propagated action potential.

Skeletal muscles are the engines of the body. They account for over a quarter of its weight and the major part of its energy expenditure. They are attached to the bones of the skeleton and so serve to produce movements or exert forces. Hence they are used in such activities as locomotion, maintenance of posture, breathing, eating, directing the gaze and producing gestures and facial expressions.

Skeletal muscles are activated by motoneurons as we have seen in previous chapters. Their cells are elongate and multinuclear and the contractile material within them shows cross-striations, hence skeletal muscle is a form of striated muscle. In contrast, cardiac and smooth muscles have cells with single nuclei, and smooth muscles are not striated; we shall examine their properties in a later chapter.

Anatomy

Skeletal muscle fibres are multinucleate cells (Fig. 9.1) formed by the fusion of numbers of elongated uninucleate cells called myoblasts. Mature fibres may be as long as the muscle of which they form part, and 10 to 100 μm in diameter. The nuclei are arranged around the edge of the fibre. Most of the interior of the fibre consists of the protein filaments which constitute the contractile apparatus, grouped together in bundles called myofibrils. The myofibrils are surrounded by cytoplasm (or sarcoplasm), which also contains mitochondria, the internal membrane systems of the sarcoplasmic reticulum and the T system, and a fuel store in the form of glycogen granules and sometimes a few fat droplets.

Both voltage-gated and ligand-gated ion channels are large protein molecules, as is the sodium pump Na,K-ATPase. In recent years the primary structure of a number of them has been determined, and by combining this information with the biophysical evidence major advances have been made in our understanding of how they work at the molecular and sub-molecular levels.

cDNA sequencing studies

A protein consists of a long chain built up of twenty different amino acids (Table 5.1), folded on itself in a rather complicated way. Its properties depend critically on the arrangement of the folds, which is determined by the exact order in which its constituent amino acids are strung together. This in turn is specified by the sequence of the nucleotide bases that make up the DNA molecules which constitute the genetic material of the cell. There are only four different bases, and each of the twenty amino acids corresponds according to a universally obeyed triplet code to a specific group of three of them. The information embodied in the base sequence of a DNA molecule is transcribed on to an intermediary messenger RNA, and is then translated during the synthesis of the protein to yield the correct sequence of amino acids. Rapid sequencing methods for nucleotides were perfected by Sanger and his colleagues, and modern recombinant DNA technology makes possible the cloning of DNA so that the quantity required for the determination can be prepared from a single gene.

Skeletal muscles are innervated by motor nerves. Excitation of the motor nerve is followed by excitation and contraction of the muscle. Thus excitation of one cell, the nerve axon, produces excitation of another cell which it contacts, the muscle fibre. The region of contact between the two cells is called the neuromuscular junction. The process of the transmission of excitation from the nerve cell to the muscle cell is called neuromuscular transmission. This chapter is concerned with how this process occurs.

Regions at which transfer of electrical information between a nerve cell and another cell (which may or may not be another nerve cell) occurs are known as synapses, and the process of information transfer is called synaptic transmission. Neuromuscular transmission is just one form of synaptic transmission; we shall examine the properties of some other synapses in the following chapter.

The neuromuscular junction

Each motor axon branches so as to supply an appreciable number of muscle fibres. Fig. 7.1 shows the arrangement in most of the muscle fibres in the frog. Each axon branch loses its myelin sheath where it contacts the muscle cell and splits up into a number of fine terminals which run for a short distance along its surface. The region of the muscle fibre with which the terminals make contact is known as the end-plate. Structures and events occurring in the axon are called presynaptic whereas those occurring in the muscle cell are called post-synaptic.

Dr David Aidley died suddenly but peacefully at home on August 24th 2000.

David was a gifted teacher: generations of students at the University of East Anglia benefited from his broad knowledge and relaxed style. A much wider audience knew David through his books, which over a thirty-year period have provided information, guidance and inspiration to students and their teachers in many parts of the world. The Physiology of Excitable Cells was first published in 1971 and is currently available in a fourth edition. Nerve and Muscle (with Richard Keynes) first appeared in 1981 and the third edition was to appear in proof the week he died. Ion Channels: molecules in action (with Peter Stanfield) was published in 1996. Each book represents an exemplary example of lucid prose and a clear grasp of the subject matter.

David was the perfect author; his books were delivered on time, in good order and each found a ready audience. He was, for these and many other reasons, a delight to work with, and we join his many friends in lamenting his untimely death and extending our condolences to his wife Jessica and their family.

Muscle cells have become adapted to a variety of different functions during their evolution, so that the details of the contractile process and its control are not always identical with those in vertebrate skeletal muscles. In this chapter we examine the properties of mammalian heart and smooth muscles.

Cardiac muscle

Mammalian heart muscle consists of a large number of branching uninucleate cells connected to each other at their ends by intercalated discs (Fig. 11.1). Electron micrography shows that the intercalated discs consist largely of accumulations of dense material on the insides of the two cell membranes; these apparently serve to fix the cells together and allow the filaments of the contractile apparatus in one cell to pull on those of the next one in the line. Gap junction channels (p. 116) are also present in the intercalated discs and these allow electrical currents to flow from one cell to another.

The contractile apparatus is much the same as in skeletal muscles, with thick myosin and thin actin filaments aligned transversely so that the muscle cells as a whole are cross-striated in appearance. As in a skeletal muscle fibre, the interior of the cell also contains mitochondria, sarcoplasmic reticulum and the transverse tubules of the T system.

The cardiac action potential

Intracellular recordings from heart muscle fibres were first made using isolated bundles of Purkinje fibres from dogs. The Purkinje fibres form a specialized conducting system which serves to carry excitation through the ventricle.

The functioning of the nervous system depends largely on the interactions between its constituent nerve cells, and these interactions take place at synapses. In most cases synaptic transmission is chemical in nature, so that, as in neuromuscular transmission, the presynaptic cell releases a chemical transmitter substance which produces a response in the postsynaptic cell. There are a few examples of electrically transmitting synapses, which we shall consider briefly at the end of this chapter.

Acetylcholine is only one of a range of different neurotransmitters. Fig. 8.1 shows some of the variety found in the central nervous system. For a long time it was thought that any one cell would only release one neurotransmitter, but several cases where two of them are released at the same time are now known.

Different chemically transmitting synapses differ in the details of their anatomy, but some features are common to all of them. In the presynaptic terminal the transmitter substance is packaged in synaptic vesicles. The pre- and postsynaptic cells are separated by a synaptic cleft into which the contents of the vesicles are discharged. There are specific receptors for the neurotransmitter on the postsynaptic membrane.

Just as with the neuromuscular junction, our knowledge of how synapses work was greatly affected by the invention of the intracellular microelectrode. Much of the fundamental work with this technique was performed by J. C. Eccles and his colleagues on the spinal motoneurons of the cat, so it is with these that we shall begin our account of synapses between neurons.

The impedance change during the spike

An important landmark in the development of theories about the mechanism of conduction was the demonstration by Cole and Curtis in 1939 that the passage of an impulse in the squid giant axon was accompanied by a substantial drop in the electrical impedance of its membrane. The axon was mounted in a trough between two plate electrodes connected in one arm of a Wheatstone bridge circuit (Fig. 4.1) for the measurement of resistance and capacitance in parallel. The output of the bridge was displayed on a cathoderay oscilloscope, and Rv and Cv were adjusted to give a balance, and therefore zero output, with the axon at rest. When the axon was stimulated at one end, the bridge went briefly out of balance (Fig. 4.2) with a time course very similar to that of the action potential. The change was shown to be due entirely to a reduction in the resistance of the membrane from a resting value of about 1000 ohm cm2 to an active one in the neighbourhood of 25 ohm cm2. The membrane capacitance of about 1 μF/cm2 did not alter measurably.

The sodium hypothesis

Cole and Curtis's results were not wholly unexpected, because it had long been supposed that there was some kind of collapse in the selectivity of the membrane towards K+ ions during the impulse.