116 CIRCULATORY SYSTEM, BLOOD AND IMMUNE SYSTEMS 0 12 3 4 hemotymph volume ((il.mg dry wt~1) Fig. 5.24. Osmotic effectors in the hemolymph of Onymachus at different levels of hydration. The quantity of each component is indicated by the area between successive lines. The top line shows the total concentration of all the solutes combined. Arrow indicates the normal level of hydration (after Machin, 1981). 5.2.3.2 pH The pH of insect hemolymph is usually between 6.4 and 6.8, although slightly alkaline values have been recorded in a dragonfly larva and in the larva of the midge, Chironomus. During normal activity there is a tendency for the blood to become more acid due to the liberation of acid metabolites, including carbon dioxide. The buffering capacity of insect blood (that is, its ability to prevent change in pH) is low in the normal physiological range, but increases sharply above and below this range. Within the normal range, bicarbonates and phosphates are the most important buffers. On the acid side of the range, carboxyl groups of organic acids are important, while on the alkaline side the amino groups of amino acids are most significant. Proteins buffer over a wide range of pH. Review: Mullins, 1985 5.2.4 Hemocytes Suspended in the blood plasma are blood cells or hemocytes. Many different types of hemocyte have been described, but a comprehensive classification is difficult because individual cells can have very different appear- ances under different conditions and a variety of niques have been used in their study. Rowley & Ratcli (1981) and Gupta (1979a, 1979b, 1985, 1991) attempt synonymize them across the different orders and redu( them to six main types (Fig. 5.25). They are; p, hemocytes, plasmatocytes, granulocytes (which are prol bly the same as cystocytes or coagulocytes), spherule (spherulocytes), oenocytoids and adipohemocytes. Prohemocytes are characterized by a high nuclear: t plasmic ratio and a general lack of organelles involved synthesis. They rarely comprise more than 5% of thetoi hemocyte population. They are the stem cells from whii most other hfsmocyte types are formed. Plasmatocytes are very variable in shape. They con moderate amounts of rough endoplasmic reticulum Golgi complexes and may contain membrane-bound g] ules. They are amongst the most abundant hemocytes usually account for more than 30% of the total hemoc count. Plasmatocytes are involved in phagocytosis encapsulation of foreign organisms invading the hemocoej Granulocytes contain large amounts of endoplasmic reticulum which is often extensively dilated. Golgi coi plexes are also abundant and the cells contain numbers of membrane-bound granules. They comprise considerable proportion, usually more than 30% of thi hemocyte population. They discharge their contem (degranulate) on the surfaces of intruding organisms an early part of the defense response. Granulocytes probably derived from plasmatocytes and intermediate! between the two types of cell occur (see Chain, Leyshon-I Sorland & Siva-Jothy, 1992). Cystocytes are probably granulocytes in which the synthesis of granular contents is complete. They contain abundant granules, but usually; contain smaller amounts of Golgi complexes and roughs endoplasmic reticulum than granulocytes. They have i relatively high nucleus: cytoplasm ratio. They are ofterf common, but have not been recognized in Diptera,; Lepidoptera and Hymenoptera. Fig. 5.25. Different types of hemocyte (a) after Chiang, Gupta & Han, 1988; others after Rowley andRatcliffe, 1981): (a) prohemocyte of B/attella; (b) plasmatocyte of larval Galleria; (c) granulocyte of larval Galleria; (d) granulocyte (cystocyte) of Clitumnus. Arrowheads indicate swollen perinuclear cisterna; (e) spherule cell of larval Galkria. The large open areas, looking like vacuoles (and labelled V), are probably caused by extraction of spherules during preparation; (f) oenocytoid from larval Galleria. Inset shows size of nucleus relative to whole cell. Abbreviations; G, granules; GO, Golgi complex; IG, developing granules; M, mitochondria; MT, microtubules; MVB, multivesicular body; N, nucleus; PE, protoplasmic" extensions; PO, ribosomes; PV, pinocytotic vesicles; R, ribosomes; RER, distended cisternae of rough endoplasmic reticulum; SP, spherules; V, vacuole. 118 CIRCULATORY SYSTEM, BLOOD AND IMMUNE SYSTEMS Fig. 5.26. Monocyte production during the larval stages of Euxoa (data from Arnold & Hinks, 1976). (a) Mitotic activity in different types of hemocytes, expressed as percentage of each type. Data from a subsequent paper (Arnold & Hinks, 1983) indicates that the values obtained in the original work, and which are used in this diagram, were probably too low, but the pattern of change is probably not affected, (b) Hemocyte counts per microliter of blood, (c) Hemocyte profile - relative frequency of different types of hemocyte, expressed as percentage of total number of blood cells. 60 50 40 30-20 -10-0 2 3 4 larval stage c) hemocyte profile Spherule cells are characterized by the large, retractile spherules which may occupy 90% of the cytoplasm. They are not usually very common although they are found in most of the species studied. Their function is unknown. Oenocytoids occur mainly in Lepidoptera where thev are amongst the largest of the hemocytes. These cells exhibit little development of rough endoplasmic reticulum or Golgi complexes, but they have a complex array of microtubules and sometimes also crystalline inclusions. Their function is unknown. Adipohemocytes characteristically contain lipid droplets. The nucleus: cytoplasm ratio is low, and they contain well-developed endoplasmic reticulum and Golgi complexes. Reviews: Brehelin & Zachary, 1986; Gupta, 1979a, b, 1985,1991; Rowley &Ratcliffe, 1981 5.2.4.1 Origin of hemocytes Hemocytes are derived from the embryonic mesoderm. Subsequently, new hemocytes are produced by mitotic division of existing, circulating hemocytes, or from previously undifferentiated cells in structures known as hemopoietic organs. Mitotic division of hemocytes The production of new hemocytes by mitosis of existing blood cells is a Widespread phenomenon. In adult holometabolous insects that lack hemopoietic organs, new hemocytes can only he produced in this way. This appears also to be the case during the larval stages of the milkweed bug, Oncopeltus. " Elsewhere, hemocyte production from existing cells! appears to complement production in hemopoietic organs**! but where the hemopoietic organs persist in adult insects^ as in Blattodea and Orthoptera, mitotic division of existing! hemocytes is relatively rare. Not all types of cell divide and the rates of division varyl even amongst those that do. Between 0.2 and 0.4% of pro- I hemocytes were found in division in blood samples taken I during the first four stages of larval development of the moth, Euxoa, but this level declined in the final larval stage J {Fig. 5.26a). Mitotic activity was similar in granulocytes, but amongst spherule cells it increased from zero in the first two stages to about 0.25% in the final stage. Plasmatocytes only rarely divide in at least a majority of insects. Despite this, the number of plasmatocytes per unit volume of hemolymph increases throughout larval development (Fig.5.26b). They are probably derived from the prohemocytes which remain constant in relative abundance despite their high mitotic rate. Much of the literature suggests that the mitotic rate for all the cells only rarely exceeds 1%, but some work indicates much higher rates. Arnold & Hinks (1983) suggest that in the final larval stage of Euxoa, the mitotic index of ^HEMOLYMPH cell* may exceed 10% (sec caption to Fig. 5.26) the final larval stage of the milkweed bug, Wjtus a mitotic index of 4% was recorded. ^e kasjs 0f the mitotic activity of the cells, it is sug-•^d that tjic vvhole population of granulocytes in the last sta(re of Euxoa turns over in about 5 days; the ule ce|| population would turn over in less than one (Arnold & Hinks, 1983). Other estimates of hemocyte -■ gyjtv jn Gallena, suggest that plasmatocytes survive at least nine days. Hemopoietic organs Blood is formed in structures called hemopoietic organs. Since, in insects, only the blood cells, ^not the plasma, are produced in these structures, they should strictly be called hemocytopoietic organs, but the -general term is more usual. Hemopoietic organs have been described in some Orthoptera, a blattid and a few larval ^Lepidoptera, Diptera and Coleoptera. They persist in .'adult Orthoptera, but not in adults of holometabolous .species. No hemopoietic organs are present at any stage of "me milkweed bug, Oncopeltus. I The positions of hemopoietic organs vary from species 5[0 species, but in most cases they are associated with, Sough not necessarily connected with, the heart. In the ^cricket, Gryllus, and the mole cricket, Gryllotalpa, they are ^paired, segmental structures on either side of the heart and opening into it (Fig. 5.2). In Locusta, Periplaneta, and larvae of cyclorrhaphous flies and of the beetle, Melolontha, they ■consist of irregular accumulations of cells close to the heart, but not connected with it (Fig. 5.27a,b). By contrast, in caterpillars they are groups of ceils around the developing imaginal wing discs (Fig. 5.27c,d). Only in the grylloids is the hemopoietic organ a discrete structure bounded by a cell layer and with an ill-defined lumen opening into the heart. Even here, the bounding layer of cells is incomplete. Within this boundary are irregularly shaped reticular cells apparently embedded in a connective tissue matrix. These cells undergo mitotic divisions and give rise to hemocyte stem cells. By further division, the stem cells form clusters of cells which differentiate synchronously to form hemocytes. Granulocytes and plasmatocytes are formed in this way. They separate from the cortical region and enter the circulation, presumably via the heart. The reticular cells are also phagocytic, taking up foreign material from the hemolymph. Because of this the hemopoietic organs in these insects were originally called phagocytic organs. The 119 process of hemopoiesis appears essentially similar in other insects although the reticular cells exist as aggregations with no bounding layer and, in Lepidoptera, reticular cells are absent. Reviews: Feir, 1979 - mitosis; Hoffmann et «./., 1979 -hemopoietic organs 5.2.4.2 Numbers of hemocytes Estimates of the total number of hemocytes in an insect show that small insects have many fewer hemocytes than large insects. Adult female mosquitoes have a total of less than 10000 hemocytes, whereas adult Periplaneta have more than 9 000 000. Similar trends occur within a species. Second stage caterpillars of Euxoa have>about 4000 hemocytes; sixth stage larvae have about 2 400 000. The number may also vary cyclically. For example, in the last stage larva of the wax moth, Galleria, the'lotal number of cells is at first constant at about 2.2 million and then increased to almost 4 million before the insect molts (Fig. 5.28a). An even bigger relative increase occurs during the postfeeding stages of the final stage larva of Sarcophaga, but at the time of pupariation, when the larva becomes immobile, there is a sudden rapid decline (Fig. 5.29). Increases in numbers of circulating cells may result from the production of new cells or, possibly, by the recruitment of cells adhering to other tissues. Reduction in hemocyte number may result from cell death or from an increase in the numbers adhering to the tissues. Counts of the number of cells per unit volume of hemolymph (usually called the 'total hemocyte count') may not reflect the total number of hemocytes in circulation because the blood volume varies. For example, in the last stage larva of Galleria, when the total number of cells is constant (Fig 5.28, weight less than 200mg) the number per unit volume decreases because the blood volume is increasing. From a functional standpoint, such as wound healing or combatting invaders, the number per unit volume may be more important than the total number. _ ,( , The number of hemocytes per unit volume of blood tends to increase throughout larval development, but with additional variation within each developmental stage (Fig. 5.30). It reaches a maximum at the time of each ecdysis, except the pupa/adult ecdysis. The lack of a peak at this time may reflect the fact that major restructuring of the tissues occurs earlier in the pupal period.. In hemimeta-bolous insects, the numbers are generally similar in larval 120 CIRCULATORY SYSTEM, BLOOD AND IMMUNE SYSTEMS IK I I' Fig. 5.27. Hemopoietic organs in different insects. Stippling indicates hemopoietic tissue (see also Fig. 5.2). (a) Locusta; (b) Calliphora larva; (c), (d) Caterpillar, (c) Showing the positions of the organs (arrows), (d) section through one wing disc (after Monpeyssin & Beaulaton, 1978). c) brain heart brain heart - heart d) -'-j.^v- developing—^s^Wf^<_.?.Jft.- ' hemocytes . ■ "?- -«£s*5r connective tissue hemocoel peripodial membranes developing wing and adult insects, but in holometabolous species it is usual for larvae to have more cells per unit volume of blood than adults. In general, adult females have a higher number of hemocytes than males. Hemocyte profile The relative abundance of different types of hemocytes (called the hemocyte profile or a differential hemocyte count) is not constant. Plasmatocytes and granulocytes are usually the most abundant, often comprising more than 80% of the total hemocyte population (Figs. 5.26c, 5.29b). The relative abundance of plasmatocytes tends to decline, and that of granulocytes to increase, through the larval period, but a sharp reversal occurs at pupariation in Sarcophaga when the total hemocyte count drops. The relative numbers of other cell types also change; spherule cells virtualW*dis-appear from the blood of Sarcophaga at pupariation. In Rhodnius, changes in relative abundance occur in relation to feeding and molting. Review: Shapiro, 1979 5.2.4.3 Functions of hemocytes Hemocytes perform a variety of functions. Among the; more obvious are wound repair and defense against; parasites and pathogens (see below), but they have roles t many aspects of the normal functioning of the insect. Granulocytes and spherule cells of larval Calpodes syn-j thesize polypeptides which are secreted into the hcmo-1 lymph and subsequently incorporated into the cuticle." Other peptides produced by hemocytes are probably I added to the basal lamina (Sass, Kiss & Locke, 1994). The hemocytes contain many proteases some of which appear to be involved in the breakdown of tissues at metamorphosis. For example, some hemocytes of Sarcophaga IMMUNITY I a) total hemocytes 4l 121 m 2 0> b) hemolymph volume 100- -0) 75- £ o > 50- -a -E o 25- £ ■sz c) hemocyte count 50,000 -i 25,000 f 150 175 200 225 weight (mg) Fig. 5.28. Changes in the hemolymph during the last larval stage Galkria (data From Shapiro, 1979): (a) total number of nocytes; (b) blood volume; (c) hemocyte count per microliter Sofblood. have a 200 kDa protein in the cell membrane. These cells .increase in number at the time of pupation and the 200 kDa {protein binds to sites on the basal lamina of the fat body Here the cells release a cathepsin-type protease which dis-| sociates the fat body (Kurata, Saito & Natori, 1993). If the epidermis is damaged, a blood clot forms beneath the wound. Formation of the clot involves components from both the hemocytes and the plasma. Granulocytes a) total hemocytes 2.5 t 2 0 1 is