Cnidaria

The phylum Cnidaria or Coelenterates includes sea anemones, jellyfish, hydras, sea fans, and, of course, the corals. With few exceptions they are all marine organisms and most are inhabitants of shallow water. In spite of the great variation in shape, size, and mode of life, they all possess the same basic metazoan structural features: an internal space for digestion (gastrovascular cavity or coelenteran), a mouth, and a circle of tentacles, which are really just an extension of the body wall. The body wall in turn is composed of three layers: an outer layer of epidermis, an inner layer of cells lining the gastrovascular cavity, and, sandwiched between them, a so-called mesoglea (Barnes 1980). All these features are present in both of the basic structural types: the sessile polyp and the free-swiming medusa. During their life cycle, some cnidarians exhibit one or the other structural type whereas others pass through both. Most Cnidaria have no mineralized deposits. The ones that, to date, are known to have mineralized deposits are listed in Table 5.1. They are found in both the free-swimming medusae and the sessile polyps. Not surprisingly, these have very different types of mineralized deposits. In the medusae they are located exclusively within the statocyst where they constitute an important part of the organism’s gravity perception apparatus. Interestingly the statoconia of the Hydrozoa, examined to date for their major elemental compositions only, are all composed of amorphous Mg-Ca-phosphate, whereas those of the Scyphozoa and Cubozoa are composed of calcium sulfate. Calcium sulfate minerals (presumably gypsum) are not commonly formed by organisms and the only other known occurrence is in the Gamophyta among the Protoctista. Spangenberg (1976) and her colleagues have expertly documented this phenomenon in the Cnidaria. (For a more detailed discussion of mineralization and gravity perception see Chapter 11.) The predominant mineralized hard part associated with the sessile polyps is skeletal. These can take the form of skeletons composed of individual spicules, spicule aggregates, or massive skeletons. They are composed of aragonite, calcite, or both.

Biomineralization Processes

The large number of different minerals formed by organisms from almost 50 different phyla described in Chapter 2 should in itself discourage anyone from searching for the mechanism of biomineralization. On the other hand, the survey of macromolecules used by many organisms to control mineralization (Chapter 2), even though limited primarily to carbonate- and phosphate-bearing mineralized hard parts, shows that similar and rather unusual acidic glycoproteins and proteoglycans are widely utilized in biomineralization. This raises the possibility that many organisms may have adopted common approaches or strategies for regulating mineral formation. We do not know whether this arose as a result of divergence from a common ancestor or is a product of convergent evolution in which many different phyla independently began utilizing similar macromolecules for controlling mineralization (see Chapter 12). Either way we view the diversity in biomineralization as the product of a very broad and almost continuous spectrum of processes that organisms use to control mineralization. This ranges from no apparent control at one end to, it seems, control over every detail at the other. However, this is achieved by a fairly limited number of different basic processes used in various combinations and ways to produce a unique final product. This last statement is, we readily admit at this point in time, more an act of faith than an established fact. In this chapter we will try to identify and/or speculate about some of these basic processes. We will draw upon material from many different sources, and, in particular, we will refer whenever possible to the more detailed descriptions of mineralization processes given in the chapters that follow. As a consequence, this chapter may also be used by the reader as a guide toward more discriminating reading on selected topics in the remainder of the book. The spectrum of biomineralization processes can in principle be easily divided into cases in which control is exercised in some way over mineralization and those in which it is not. In practice the differentiation is not that simple as all organisms do exercise some control at one level or another, even if it simply involves, for example, removing from the cell some undesirable metabolic end-product or ion that combines with another ion in the external medium and precipitates.

Echinodermata

The Echinodermata are certainly one of the most unusual and interesting phyla from the biomineralization point of view. They all live in the marine environment. The five major taxonomic classes (Asteroidea or sea stars, Ophiuroidea or brittle stars, Echinoidea or sea urchins, Crinoidea or sea lilies, and Holothuroidea or sea cucumbers) have quite different anatomical shapes and are characterized by fivefold symmetry. Each group forms mineralized hard parts. In the Echinoidea the skeletal elements are fused together to form a rigid test, whereas in the Asteroidea, Ophiuroidea and Crinoidea the skeletal elements or ossicles are articulated with one another. In the Holothuroidea the skeleton is usually reduced to microscopic ossicles or spicules, and, in some cases, mineralized granules as well. The hard parts of echinoderms vary enormously in shape and function and include not only the diverse skeletal elements, but also spines and teeth. Remarkably, with very few exceptions, the mineralized hard parts are formed from the same mineral, magnesium-bearing calcite [usually 5–15% as magnesium carbonate (Chave 1952, 1954; Raup 1966)], which has some unique and interesting properties. The ultrastructure of many of the macroscopic skeletal hard parts has a characteristic spongy or fenestrate structure (called the stereom) and is riddled with labyrinthine cavities (collectively called the stereom space). In echinoid spines the stereom spaces are secondarily filled in to form areas of solid mineral. The surfaces of the mineral phase are very smooth, even when examined a high magnification in the SEM (Towe 1967; Millonig 1970). Furthermore, the broken surfaces show no characteristic ultrastructural motif, which is observed in almost all other mineralized tissues in which the individual crystals are enveloped by layers of organic material. The fracture surfaces of echinoderm calcite actually have a conchoidal cleavage (Towe 1967), which is characteristic of glassy or amorphous materials. It is, therefore, most surprising that when individual skeletal plates, spines, spicules, ossicles, and even whole teeth are examined in polarized light or by X-ray diffraction, they behave as if they are single crystals! (Towe 1967; Donnay and Pawon 1969).

Mollusca

Mollusks have a well-deserved reputation for being expert mineralizers based only on their much-admired shell-making abilities. Table 6.1 shows that the reputation is deserved 10-fold as shell formation is just one of many different processes that these animals perform in which biogenic minerals are utilized. The table lists no less than 21 different minerals and about 17 different functions! The list contains both amorphous minerals (amorphous fluorite, calcium carbonate, calcium phosphate, calcium pyrophosphate, and silica) and many crystalline ones, including rather uncommon ones such as weddelite, calcium fluorite, barite, magnetite, lepidocrocite, and goethite. Weddelite, for example, is a calcium oxalate mineral frequently formed pathologically in vertebrates. Certain gastropods use the rather soft weddelite nonpathologically to cap pestlelike objects (gizzard plates) in their stomachs (Lowenstam 1968), which they use for crushing shelled prey. One mollusk, the chambered Nautilus, forms no less than five different minerals. An individual tooth of a chiton contains three different mature minerals that are products of two other transient minerals. In addition to the more familiar functions of mineralized tissues, mollusks use biogenic minerals as buoyancy devices, trap doors, egg shells, and love darts. The varieties of crystal shapes, sizes, organizational arrays, and tissue sites present a picture of overwhelming diversity all within one phylum. It is illustrative to compare the mollusks with the echinoderms. The echinoderms also use minerals for a wide variety of functions, but in contrast to the mollusks they use essentially the same “building material” for many different purposes. Thus, understanding how one echinoderm mineralized tissue forms provides insight into how most of the others form. This is not so with mollusks. It seems futile to expect that they too have adapted one basic process to form all their mineralized tissues. It seems just as futile to look for a different explanation for each type of mineralized product. The mollusks force us to seek a level of understanding of mineralization that identifies common approaches, strategies, and principles and, at the same time, appears to dispel any “dreams” about discovering the mechanism of mineralization. The mollusk phylum contains seven different taxonomic classes.

Evolution of Biomineralization

Biomineralization among living organisms is widespread, occurring in both prokaryotes and eukaryotes. It is diverse with some 60 or so minerals known to be formed by organisms under a wide variety of conditions. They are deposited at many different locations both inside and outside cells. Biomineralization occurs on such an enormous scale that it influences processes in the biosphere itself. It is, therefore, of interest to determine how this all came about—the evolution of biomineralization. The evolutionary history of biomineralization is a fascinating subject in its own right, which is the primary reason for including it in this book. However, a well-substantiated understanding of this subject is also of crucial importance to the interpretation of many aspects of research into the mechanisms of biomineralization in living organisms. An example is the observation by Veis et al. (1986) that antibodies raised against the rat incisor acidic proteins, phosphophoryns, crossreact with proteins extracted from a sea urchin test. The proteins presumably share some similar molecular structures. Did they inherit them from a common ancestor or did they evolve independently from each other to fulfill similar functions? This type of question can be asked about many comparative studies in biomineralization between phyla or even within lower taxa of the same phyla. As long as we do not have answers to these questions, the powerful tool of comparative biology in biomineralization is compromised. Studying the evolution of biomineralization has one enormous advantage over many other topics in evolutionary biology; the very material that we are interested in has the best chance of surviving the vagaries of time and being preserved in the fossil record. The fossil record at least during the last 600 million years or so is, for the most part, a documentation of remnants of the history of mineralized hard part formation by organisms. Thus, the evolution of biomineralization is one topic that can, and that should be based on the direct documentation of the fossil record. This is the way it is presented in this chapter. The corollary of this statement is also worth considering. The fossil record should be interpreted bearing in mind the evolution of biomineralization.

Environmental Influences on Biomineralization

For the most part, the minerals that organisms form do not form abiologically in the environment in which the organism lives. In fact, some of them do not form abiologically anywhere in the biosphere. A striking example is the crystalline strontium sulfate test of the Acantharia, a group of planktonic proctoctists that is found in most of the world’s oceans. Seawater is undersaturated with respect to strontium sulfate. Thus, the biological environment of mineral formation is isolated from the surroundings. This is, in fact, a generally observed phenomenon (Chapter 3) and it is, therefore, surprising to find that in a wide variety of organisms, the environment does still in some ways influence these biogenic minerals. This can manifest itself in the particular mineral deposited (for example, the deposition of the calcium carbonate polymorph calcite as opposed to aragonite or vice versa), the concentration of minor and trace elements, the stable isotopic composition of the mineral, and, at the ultrastructural level of the tissue, the distribution of growth lines. The degree to which the environment can affect biomineralization is a function of how isolated the process is from the outside world. Poorly controlled mineralization processes are expected to be affected more than well-controlled processes, although as we show in this chapter each case must be examined individually. There are examples of a clear environmental effect occurring in one species of a particular genus, whereas another species or even subspecies of the same genus appears to be unaffected (Lowenstam 1954c). The environmental effect can and very often is filtered out by the physiological processes of the organism, which either completely or partially determine the solution characteristics of the microenvironment in which the crystals grow. A lack of appreciation of this fact has caused considerable confusion in the literature, with some investigators concluding that an environmental influence on biogenic minerals does not exist in a whole taxonomic group, because they found that it was absent in one or a number of species (Bornhold and Milliman 1973; Taylor et al. 1969). The first published documentation of this phenomenon (Lowenstam 1954a) clearly showed that closely related organisms can differ in this respect and that extrapolations and sweeping conclusions are not justified.

Chordata

In the field of biomineralization the phylum Chordata is the most intensively studied, as one of its subphyla, the Craniata, includes our own species. The Craniata are often referred to as the vertebrates, a term that alludes to the importance of the endoskeleton in denning the essential character of these animals. The phylum Chordata also contains three other subphyla, only one of which has members that form mineralized hard parts. They belong to the Urochordata or tunicates. In fact, mineralization is confined to several families of a single class of urochordates, the Ascidiacea. Table 9.1 is a compilation of the known biogenic minerals formed by members of the Chordata, together with the sites at which they form and their presumed functions. The table includes no less than 17 different minerals, which should dispel any notion that mineralization in the chordates is synonymous with "calcium phosphate" deposition. It is, of course, true that the mineralized skeletal hard parts of most of the Craniata or vertebrates contain a calcium phosphate mineral, usually in the carbonated form called dahllite. However, the vertebrates also form four different carbonate minerals that are most commonly found in the vestibulary apparatus (see Chapter 10). They form three different iron minerals, which includes magnetite found in the navigation system of various vertebrate genera. The Ascidiacea also form a diverse array of minerals. Interestingly, however, their diversity is essentially confined to one class, the Pyuridae, which form no less than six different minerals, including two phosphate minerals. In this chapter we first describe biomineralization processes in the Ascidiacea followed by detailed discussions of mineralization processes in the Chordata or vertebrates. For convenience, the section on vertebrate mineralization is divided according to the major mineralized tissues: bone (dentin), cartilage, and tooth enamel. Mineralization in the vestibulary apparatus is discussed in Chapter 10.

Arthropoda

The arthropods are distinguished by having segmented bodies and appendages, as well as hardened external skeletons. Growth is achieved by shedding the exoskeleton and then regenerating a new and larger one. The hardening of the exoskeleton usually occurs by chemical cross-linking (sclerotization) of the macromolecular constituents, mostly proteins and the polysaccharide, α-chitin. The major exception is the class Crustacea. The members of this group harden their skeleton not only by sclerotization, but also by the addition of inorganic minerals. After each molting, the new exoskeleton is remineralized. The result is that many Crustacea, particularly those that live in freshwater or on land where the availability of calcium is limited, have evolved novel and diverse temporary storage sites for mineral (reviewed by Greenaway 1985). From the perspective of biomineralization processes, this adaptation is certainly one of the “highlights” of the Arthropod phylum. Interestingly one taxonomic order within the Crustacea, the Cirripedia or barnacles, does not moult their heavily mineralized cuticles, even though their “organic” exoskeleton does go through periodic molting cycles (Darwin 1854). Table 7.1 lists many of the known reports of biomineralization processes in the Arthropoda. The table is already impressively long. However, as this phylum is by far the largest in the animal kingdom, we have no doubt whatsoever that the true extent of mineralization processes in the Arthropoda is far from having been ascertained. In the insects alone nearly half a million species have been described, and our list comprises just a few documented cases of insects that mineralize. Interestingly, the list of minerals formed by insects includes a number of so-called “organic minerals,” for example, uric acid, crystalline wax, and long chain paraffins. We strongly suspect that many more “organic minerals” have yet to be discovered among the insects.

Minerals and Macromolecules

Biomineralization is a diverse, widespread, and common phenomenon. This statement is based on our current knowledge of the known diversity of biogenic mineral types, the taxonomic affinities of the organisms that form these minerals, and their abundance in the biosphere. In this chapter, we present an updated compilation of biogenic mineral types and the organisms that form them. We also briefly discuss aspects of their impact on the environment. In addition, we list the basic types of macromolecules that are often, but by no means always, associated with biogenic minerals. Information of this type is invaluable for gaining an overall perspective of the subject, for beginning to identify any common trends and strategies, and eventually for determining whether or not different organisms use similar underlying principles for forming their minerals. It is also one of the only means available for roughly assessing what proportion of mineralizing organisms has been discovered to date and what proportion still remains to be discovered. Table 2.1 lists the known biogenic mineral types and the taxonomic affinities of the organisms that form them at the phylum level. This compilation differs from earlier published versions in that the extensive key to the table allows the reader to identify the literature sources upon which the data are based. Table 2.2 lists the common names and chemical formulas of known biogenic minerals. Table 2.1 lists almost 60 different biogenic minerals! In 1963 only 10 different mineral types had been identified (Lowenstam 1963); this increased to 19 mineral types by 1974 (Lowenstam 1974), 30 by 1981 (Lowenstam 1981), and 39 by 1983 (Lowenstam and Weiner 1983).

Some Nonskeletal Functions in Biomineralization

The functions of mineralized hard parts are often self-evident. In many of the tables throughout the book we note the assigned or very often assumed functions of many different mineralized bodies. Often, however, assumed functions do not stand up to closer examination. A good example is the study of the cells of the hepatopancreas of gastropods (Howard et al. 1981). These glands have numerous cells containing intracellular mineralized granules. It was generally assumed that they all functioned as transient storage sites for calcium ions, until it was found that a subpopulation forms granules of a different type, which are used for heavy metal detoxification. Granules can be used in other ways as well. Certain polychaete worms, for example, strengthen their muscles by packing them with granules (Gibbs and Bryan 1984). Spicules are also commonly formed by many organisms and their functions are often not understood. They tend to have elaborate morphologies and mineralogies that are species specific, implying that they do perform specialized functions. These are just a few of many examples in which the functions of mineralized bodies still need to be determined. In this chapter we describe four different cases in which the functions are fairly well established. They have been investigated in some detail and, thus, provide good guidelines as to the various approaches by which function can be investigated. Some gravity receptors have been closely examined with respect to neuroanatomy and function, but not with respect to the specific adaptations of structure and mineralogy of the ubiquitous “heavy bodies.” Studies of biologic magnetic field receptors, in contrast, have focused on the mineral, and virtually nothing is known about the neuroanatomy. The molecular structure of the iron storage molecule ferritin is known with a resolution of a few Angstroms. Ferritin provides us with a glimpse of the insights that can be gained into function from such detailed structural information. Finally, some studies on the control of proteins on ice crystal formation represent the first application of the powerful techniques of molecular biology to determining function in biomineralization. These are undoubtedly the forerunners of many function-oriented studies using molecular biological techniques.