Introduction

The blood cells of many higher animals are formed in specialized tissues called hemocytopoietic tissues (blood-cell-making tissues; poiesis = “making”) or hemopoietic tissues. Although less accurate, the latter term is generally used to mean the same thing as hemocytopoietic tissues and will be preferred in this chapter.

Hemopoietic tissues are concerned with producing different lines of blood cells and delivering them into the blood; in some animals they may also function to remove dying or dead cells and histolytic debris from circulation. This function is assumed by phagocytic cells that originate from a particular cell lineage, generally called reticular or reticuloendothelial cells; it is this cell type that gives to hemopoietic tissues a characteristic reticular arrangement. It must be stressed that a third major function of hemopoietic tissues in higher animals may be immunologic, involving specialized cells such as lymphocytes and plasmocytes.

That in at least some insects definite hemopoietic tissues are present was reported near the end of the last century (Kowalevsky, 1889). However, discrepancies obviously exist between the organizational level of hemopoietic tissues (or their equivalents) in different insect orders; in addition, this organization may change during the development in the same species, as will be explained for Calliphora.

These differences are the major source of the controversies about the postembryonic origin of hemocytes that existed between Kowalevsky (1889) and Cuénot (1896). Another source is the lack of quantitative data.

Jones (1970) published an exhaustive report on these controversies (250 references), and it is unnecessary to duplicate such a review here (see also Chapter 3).

Introduction

The purpose of this chapter is to review the contribution of the hemocytes to the processes, and particularly to the control, of insect growth and metamorphosis. But there are certain ancillary matters that must be briefly considered.

Hemocytes as a source of “embryonic cells”

Hemocytes arise as residual mesodermal cells at the conclusion of embryonic growth. Berlese (1900, 1901) regarded their differentiation to form muscles and other mesodermal tissues as being their main function during postembryonic growth in higher Diptera. Likewise, in Galerucella (Col.: Chrysomelidae), Poyarkoff (1910) included the imaginal myoblasts among the hemocytes. Nowadays, although embryonic cells (notably myoblasts, derived from imaginal histoblasts) may be moving freely in the hemocoel at certain stages in development, it is usual not to include them among the hemocytes (Crossley, 1964, 1975). However, Penzlin (1963), studying regeneration of limbs in Periplaneta, concluded that the new muscles are formed by blastema cells derived from the hemocytes, not from the epidermis. And there are many claims for fat body formation during postembryonic development from “mesodermal leucocytes” (see Wigglesworth, 1959, and Chapter 1).

Hemocyte types

There is a vast descriptive literature on the varied types of hemocytes in different groups of insects (Wigglesworth, 1959; Jones, 1962; Gupta, 1969; Crossley, 1975). In recent years the tendency has been to simplify these descriptions and to put the emphasis on a limited number of more or less well-defined types. It is hoped that this procedure will lead to the recognition of true homologies between blood cells of different insects, with the allocation of definite functions to common types of cells.

Introduction

The postembryonic origin and multiplication of insect hemocytes continue to provide food for thought and material for research projects for many entomologists, comparative physiologists, and comparative pathologists. Review articles discussing insect hemocytes, total and differential cell counts, and hemopoiesis include Jones (1962, 1970), Wigglesworth (1959), Arnold (1974), and Crossley (1975). One should consult these articles for the historical development of the topic as well as for an extensive review of the literature.

Because the terms “hemopoiesis,” “hematopoiesis,” and “hemocytopoiesis” seemed to be used interchangeably in the literature, I decided to do a little checking before using one term or the other. According to Webster's Third New International Dictionary (1965) “hemopoiesis” and “hematopoiesis” are equivalent in meaning and in derivation and therefore can be used interchangeably. Both mean formation of blood or blood cells within the body. Webster does not list “hemocytopoiesis,” but does list “hemocytogenesis” as being the part of hematopoiesis concerned with the formation of blood cells. I will use “hemopoiesis” because it is the shortest of the terms and includes the formation of blood cells in its definition (see also Chapter 10).

The process of hemopoiesis includes both cell proliferation and cell differentiation. As cell differentiation proceeds, the capacity for cell proliferation decreases. Mature cells are incapable of proliferation (Best and Taylor, 1966). Even though I will be principally concerned with cell proliferation and multiplication in this chapter, I will also cover some aspects of cell differentiation because it bears an important relationship to the amount of cell proliferation in the normal animal.

Insect hematology is a difficult and controversial subject. Developing suitable techniques to study hemocytes – their origin, development, differentiation, multiplication, and functions – has presented many problems; and the existing differences of opinion among insect hematologists on almost all aspects of insect hemocytes surely indicate the controversial nature of the subject. As a consequence, the study of insect hemocytes has not been popular, and this in turn has hindered progress in this area. Disagreement over the exact number of basic hemocyte types still remains, and I doubt if a consensus on this will ever be reached because this is largely a matter of opinion. Fortunately, however, insect hematologists recognize the existence of certain morphologically distinct hemocyte types, and there is a more or less general agreement on the names of these types. This has facilitated, to some degree, comparisons of various descriptions and has made it possible to define more accurately the physiological roles of at least some hemocyte types. Much of the physiological significance of hemocytes, however, remains to be understood, particularly their role in the insect endocrine system.

The book seeks no consensus on any aspect of hemocytes, but attempts to present a critical and, I hope, a balanced account of the subject as it is presently perceived by the contributors. Each contributor has had complete freedom to develop, interpret, and present his/her views on the subject. Portions of the chapters by Drs. Arnold, J. A. Hoffmann, Jones, Shapiro, and Sohi were presented at the XV International Congress of Entomology (1976) in a Special Interest Conference on Hemocytes.

Introduction

There has been much speculation in the past about the possible role of hemocytes in the production of the connective tissues in insects. At the time hemocytes were first implicated (Lazarenko, 1925; Wermel, 1938), our knowledge of insect connective tissues was rudimentary. It is only since 1955 that information about the biochemistry and fine structure of these tissues has accumulated.

Before insect connective tissues and the hemocytes are discussed in more detail, it is pertinent to consider current concepts about connective tissues in other animals, especially those of mammals and birds. Any notion that these are irrelevant to this discussion must be immediately dispelled. Recent work on insect tissues has provided much evidence of their biochemical, structural, and functional similarities to the tissues of higher animals.

The term “connective tissue” is currently used to include all the material found between the cells, whether it is that filling the large spaces in matrixes such as bone or cartilage or the small narrow spaces between cells. The constituents of the matrix are collagenous proteins, other glycoproteins, and glycosaminoglycans (formally termed acid mucopolysaccharides). The detailed structure of collagen molecules is now almost fully known (Miller, 1976); at least four molecular species of collagen exist in vertebrates. Type I collagen is found in bone, skin, and tendon. Type II collagen occurs in cartilage. Type III collagen forms the thin fibrils associated with the loose networks of connective tissue supporting organs such as the spleen, and in embryonic skin and Type IV is the nonfibrous basement membrane collagen. The other glycoproteins are not yet well characterized.

Introduction

The three most common measurements made to describe the blood picture of a given insect at a given time or from one time to another are total hemocyte count (THC), differential hemocyte count (DHC), and blood volume (BV). The purpose of this chapter is to examine the methods used to obtain these values and their reliability.

Total hemocyte count

The first study of THCs in insects was made by Tauber and Yeager (1934). In 1935, they studied Orthoptera, Odonata, Hemiptera, and Homoptera. A year later, these same authors extended their study to include Neuroptera, Coleoptera, Lepidoptera, and Hymenoptera. The insects were heat-fixed (60 °C for 5–10 min) and bled from a proleg, after which the blood sample was diluted with physiological saline. The THC (i.e., the number of circulating hemocytes per cubic millimeter) was determined by the method employed for mammalian blood counts. This work represents an outstanding contribution and has served as a model for subsequent investigations.

Tauber-Yeager (1935) fluid (NaCl, 4.65 g; KC1, 0.15 g; CaCl2, 0.11 g; gentian violet, 0.005 g; and 0.125 ml acetic acid/100 ml) was used also by Fisher (1935), Smith (1938), and Shapiro (1967, 1968). Other physiological saline solutions were utilized by Rosenberger and Jones (1960), Collin (1963), and Gupta and Sutherland (1968). Acetic acid, a component of the Tauber-Yeager fluid, was used for Galleria (Stephens, 1963; Shapiro, 1966; Jones, 1967a) and for Heliothis (Shapiro et al., 1969; Vinson, 1971). Turk's solution (1–2% glacial acetic acid, slightly colored with gentian violet) was used for Pectinophora (Clark and Chadbourne, 1960) and for Euxoa (Arnold and Hinks, 1976).

Introduction

Insect hemocytes may be studied in several ways: (1) examination of hemocytes within the living insect; (2) examination of unfixed hemolymph, stained or unstained; (3) examination of fixed hemolymph, stained or unstained (Arnold, 1974); (4) examination of sections, fixed or unfixed. It is the intent of this chapter to examine each method and to analyze its advantages and disadvantages.

Examination of insect hemocytes

Hemocytes in vivo

The ameboid motion of hemocytes of the giant cockroach, Blaberus giganteus, the course of blood circulation within the mature embryo, as well as the morphology of hemocytes and blood circulation in insect wings were studied within living insects by Arnold (1959a, b, 1960, 1961, 1964, 1966) and by Arnold and Salkeld (1967). Circulation of blood was studied in insects belonging to 14 orders. Much valuable information was obtained, but the method has limited use and has not found widespread advocacy. Jones and Tauber (1954) attempted to differential counts of hemocytes (DHCs) in the wing membrane of the mealworm, Tenebrio molitor. Counts could not be made because the cells could not be clearly seen under high magnification.

Clark and Harvey (1965) studied cellular membrane formation in diapausing Cecropia pupae by utilizing a modified telobiotic preparation. In this method, pupae were attached to a Plexiglas chamber, so that living hemocytes could be observed directly using phase-contrast optics. Attachment of plasmatocytes (PLs) and migration of cells were also studied. As the hemocyte population in the blood chamber increased, the proportion of fusiform PLs increased. These cells became rounded, however, when they made contact with the hemacytometer surfaces.

Introduction

Insects are exposed to the toxins and poisons of their predators as well as to all sorts of chemical poisons. There has been relatively little written on the specific responses of insect hemocytes or hemolymph to poisons. Perry and Agosin (1974) reviewed the physiological responses of insects, resistance in particular, to insecticides. Arnold (1974) has a very short paragraph on detoxication of poisons as a function of hemocytes and concludes that the hemocytes do not function in direct defense against the toxicants. Smith (1962) discussed detoxication mechanisms in insects, but the hemocytes and hemolymph were not included among the mechanisms.

The available literature on this topic is small. I have confined this discussion to responses of hemocytes and hemolymph and have included no other detoxication mechanisms.

Venoms and toxins of predators

Kamon (1965a–d) has done a number of studies on the responses of locusts to scorpion venom. In one experiment (1965a), he injected scorpion venom into locusts, removed the hemolymph, and stored it at 4 °C. After 24 hr of storage there were considerable sediment and opacity in the hemolymph from the venom-treated locusts, but none in the hemolymph of the controls. He found that the increased sediment and opacity were associated with an increase in the specific gravity of the hemolymph and that these phenomena occurred only in treated females. The specific gravity remained high for 14 days, which was the maximum length of the testing period. There was no increase in specific gravity in treated male hemolymph. Kamon could give no explanation for these findings.

Introduction

Like other cells, hemocytes are generally differentiated anatomically by differences in size and shape of the soma and nucleus; by type, number, size, and staining affinities of their inclusions; and by the general appearance and staining of the cytoplasm. They can also be distinguished by inherent behavioral differences in their ability to divide, in speed of degranulation (or vacuolization) of their inclusions, in their general cytoplasmic fragility, by the development (and types) of pseudopodia (or surface extensions), and by their tendencies to stick to each other or to different surfaces. They may also be classed according to their relative abundance at specific periods during the life span of a given species. Finally, hemocytes may be identified with some definite activity, such as phagocytosis, coagulation of the hemoplasma, or trephocytosis (Jones, 1962, 1964, 1977).

Hemocytopoiesis

The pitfalls in this area of insect hematology are particularly numerous. They begin with the acceptance of an imprecise vertebrate terminology. Because the words “hemopoiesis,” “hematopoiesis,” and “hematogenesis” all refer to the production or formation of blood (Dorland, 1974), they can refer to the formation of both the liquid fraction (plasma and lymph) and the formed elements (erythrocytes and leucocytes). Because insects have a combination of blood and lymph (hemolymph) and because they do not have red blood cells, it is better to use the word “hemocytopoiesis” to refer specifically to the formation of hemocytes (Jones, 1970) and to coin some other word, like “hemoplasmopoiesis,” to refer only to the formation of the liquid fraction of the hemolymph – hemoplasma. Hemolymph includes the hemoplasma and its indigenous cells, the hemocytes.

Introduction

The life of an insect may be uneventful, progressing from the immature to the adult with no trauma. On the other hand, the insect may face the trauma of injury, the lack of food, or the challenge of natural enemies. In this chapter I hope to discuss quantitative and qualitative changes in hemocyte populations during the life of an insect undergoing growth and development or exposed to injury or insult. Population changes will be determined primarily by changes in numbers (total hemocyte counts), in types of hemocytes (differential hemocyte counts), and in blood volume. Some emphasis will be placed on the wax moth, Galleria mellonella (L.) (Lepidoptera), on which much information has been obtained. In vivo studies will be emphasized.

Changes during development

Salt (1970), in his excellent review, The Cellular Defense Reactions of Insects, cautioned about the total hemocyte count (THC). He felt the THC must be used with caution because of inherent changes in the ratio of circulating to sedentary hemocytes and the changes owing to sampling procedures.

Orthoptera

Tauber and Yeager (1935) studied the THC in Orthoptera, Odonata, Hemiptera, and Homoptera. THCs from Blatta and Periplaneta were higher in females carrying oothecae than in “normal” females. Adults of Udeopsylla and Melanoplus (crickets and grasshoppers), Plathermis (a dragonfly), and Euschistus (a stink bug) had higher cell populations than nymphs. The authors felt that a gradual increase in hemocytes occurred during development.

In Periplaneta, the variation in cell counts was great (Smith, 1938). The lowest average count was 34,000/mm3 and the highest was 158,000/mm3.

Introduction

Mesodermal stem cells (i.e., cells migrating inward at the gastral groove during gastrulation of the embryo) differentiate into a host of specialized tissues including hemocytes (Anderson, 1972a,b) (see also Chapter 1). Specialization is recognized by particular synthetic abilities, and ultimately differentiation might be regarded as derepression of certain genes leading to synthesis of particular proteins. A particular gene may be derepressed briefly or more permanently, when we come to be able to recognize the cell as differentiated using biochemical or ultrastructural criteria. The wide variety of possible protein syntheses makes it difficult not only to classify differentiated hemocytes, but also to distinguish hemocytes from other mesodermal cells, for if hemocytes are considered simply as a mobilization or muster of mesodermal cells, then this muster at times includes transient migrations of mesodermal cells such as myoblasts (Crossley, 1965, 1972a). Conversely, if circulation is made an exclusive parameter, then hemocytes in hemocytopoietic organs and in hemocytic reservoirs (Jones, 1970) are excluded artificially. The importance of sessile hemocytic reservoirs, noted by Wigglesworth (1965), is emphasized by comparison of hemocyte counts before and after heat treatment, which prevents clumping and attachment of hemocytes (Wheeler, 1963).

The activities and hence appearance of sessile hemocytes may not differ greatly from those of connective tissue cells such as fibroblasts. Indeed, the embryological origin of fibroblasts is not clear, and they may, like hemocytes, arise from median mesoderm. Their relation to hemocytes is considered below, under “Hemocytes, Fibroblasts, and Collagen Secretion.” Cells believed to arise from lateral mesodermal somites are excluded from this review; this excludes myoblasts, cardioblasts, fat body cells, and pericardial cells.

Introduction

The importance of insect tissue and cell cultures has long been recognized by entomologists because cells, tissues, and organs grown in vitro in controlled physical environment and nutrient media provide ideal systems for studying insect pathology, developmental biology, hemocytology, physiology, and cell biology.

Insect hemocyte cultures are of much value in the study of the origin and differentiation of hemocyte types. They can help elucidate the origin and function of the hemopoietic organs in insects and the interrelationships among various hemocytes, and their use could settle the controversies about insect hemocyte types.

Unfortunately, however, in vitro cultivation of insect tissues and cells has been very difficult (Vaughn, 1968; Brooks and Kurtti, 1971; Feir and Pantle, 1971). Although the first known attempt to grow insect tissue cultures was made in 1915 (Goldschmidt, 1915), it was not until almost 50 years later that the first continuous insect cell line was obtained by Grace (1962). Culturing insect hemocytes in vitro has been even more difficult than culturing other insect tissues (Arnold, 1974; Mazzone, 1976). But considerable progress has been made in recent years in growing insect tissue cultures. Over 100 continuous lines of insect cells, including six of hemocytes, have been developed since 1962 (Hink, 1976).

Although six continuous cell lines of insect hemocytes have been obtained, it is not always possible to start a successful primary culture of hemocytes at will, let alone develop a new continuous cell line. In recent years insect tissue culture has been reviewed by Brooks and Kurtti (1971), Hink (1972), Stanley (1972), and Granados (1976).