Insect Hemocytes Development, formt, funetkm», und techniijues Edited by A. P. Gupta Profrxutr of b'.ntomoltnw Huturrx ( ntnTxity Cambridge University Press Cambhitfi,' Ismtltm \ru York MellMturtw Hemocyte types: their structures, synonymies, interrelationships, and taxonomie significance A. P. GUPTA Department of Entomology and EvommUv Zoolog, Hutuv \ru Bruusu'trk. .Veil Jrrtey ecoiues very difficult to compare hemocytes of one species with those of others. This has particularly hindered any phylogenetic consideration of the evolution of hemocyte types in various arthropod groups and the Onychuphora. Clearly, there is a need for a uniform lieinocyte classification for insects as well as other arthropml groups. The insect hemocyte classification that is generally used has evolved over more than half a century. According to Millara (1947), Cnenot (1896) was the first to classify insect hemocytes into four categories and was later followed in this attempt by Hollande (19()9, 1911) and others. Wigglesworth (1939) summarized most of the earlier classifications and presented a classification that was widely accepted. He modified it later (Wigglesworth, 1959). On the American side. Wager's (IMS) work stimulated considerable interest in the study of hemocytes. Jones (1962) revised and greatly improved Yeager's classification. In order to adopt a uniform hemocyte classification for discussing hemocytes and their physiological significance in various insects, it is necessary to homologi/e terminologies used by different authors on the bases of descriptions, observed functions, line drawings, and mi- 6- ii M i líi i is? Ifens feSfeä; i it i Si Iii I I a. I i 2 I s xxscxxxxa:- CEEEEEEECCEI h I OP 11 jl Jtjiill tlilli -I J i i c S i Bf I Í ä§|f I »sc ä«JE::ö!ZfJa 87 88 .A. P. Gupta erographs of hemocytes studied by those authors. A summary of the \e\en main hemocyte types in various insect orders is presented in Table 4.1. The three terms indicated hy the table's footnote were not used by the original authors, but have been adopted by me alter scrutinizing original descriptions and figures. Hemocytes categorized as ainoeboeytes and/or phagocytes by the original authors have l>een assigned mostly to the category of" plasntatocyte (PL), although they could Ih- included in granulocyte (OR), spherulocyte (SF), and/or adi-pohemocyte (AO), inasmuch as these last three (onus also are supposedly phagocytic in certain insects. 4.2. Main hemocyte types There is disagreement among insect hematologists about the number of hemocyte types in various insects. From one or a few to as many as nine or more types have been described, particularly by light microscopy. Ultrastructurally, however, only seven types have so far been identified in various insects: prohemocyte (PR), plasmatocyte (PL), granulocyte (GR), spherulocyte (SP), adipohemocyte (AD), oenocy-toid (OE), and eoagulocyte (CO). Of these seven, AD has been reported onlv by Devauchelle (1971) and CO by Goffinet and Gregoire (1975) and Ratclifle and Price (1974). Podocyte (PO) and vermicyte (VE) have not been recognized as distinct types in electron micro-scopic studies so tar. primarily because ultrastructurally they appear similar to PLs (Devauchelle, 1971). A general description of various hemocyte types, based on both light and electron microscopic studies, their synonymies, and interrelationships is presented below. I must emphasize that although I am including PO and VE in the following description, I do not regard them as distinct types. Furthermore, I believe that description of "new" hemocyte types, based on superficial dissimilarities, should be avoided (see also Chapters 8, 10). 4.2.1. Prohemocyte (PR) Structure PRs are small round, oval, or elliptical cells with variable sizes (6-10 ixiii wide and 6—14 jum long). The plasma membrane is generally smooth (Fig. 4.LA), but may show vesiculation (Fig. 4.3A). The nucleus is large, centrally located, and almost filling the cell; nuclear size is variable (3.6-12 /im) in various insects; several nuclei and nucleoli may be present. A thin or dense, homogeneous and intensely basophilic layer of cytoplasm surrounds the nucleus, the nucleocyto-plasmic ratio being 0.5-1.9 or more. The cytoplasm niav contain granules, droplets, or vacuoles (Fig. 4.3A). The laminar nature of the plasma and nuclear membranes may not Structure and taxonomic significance Fig. 4.1. A. Prohemocyte nf Prrivtanrta amrrirana. Ca x 8.0IX), B. Plasmatocyte of P. amrrinnui- Ca x 10,(100. C. Spherulocyte of\uuplu*-ta rinrrra. Ca. x 8.500. D. Granulocyte of/^«u*f« mitiratoria. E. Spherulmyte of .V rinrrra. Ca. x 25,000. (C MM E from Gupta and Sutherland. !967; D from CosHn. 1975) 90 A. P. Gupta In c\idcnt. The cytoplasm gciicralb contains a low concentration ot endoplasmic reticulum oung or small PLs. The\ may be numerous, rare, or absent, depending on the developmental and physiological state of the insect at the fljMB of observation. PRs are seldom seen in vivo. Synoin mies The term that has survived to date with little or no change since its adoption by llollandc (1911) is "prolcucocyte." Yeager (1945) used the term "proleucocytoid and Jones and Tauber (1954) "prohemocy-toid." I believe Arnold (1952) was the first to use the term "prohaemo-eyte." Other synonyms tor PR arc "macronucleocytc" (Paillot, 1919); " formative cell" (Midler, 1925); "jeune globule" (Bruntz, 1908); "smooth-contour chromophilic cell" (Yeager, 1945); "jeune leucocyte" (Millara, 1947); "plasmatocytelike cell" (Jones, 1959); "young plasmatocyte" (Gupta and Sutherland, 1966; Gupta, 1969); "young granulocyte" (Francois, 1974); and "proleucocyte" (many authors). Interrelationship with other types The controversial questions often raised regarding PRs are: (1) are they the stem cells that transform into other hemocytes r1 and (2) it tlu-y ar<-, are they the main postemhryonic source ot hemocytes;' Although there are substantiating reports that PRs do transform into at least a few other hemocyte t\ pes, evidence on their being tlx- main postembryonal' source is inconclusive. The term "prohemocyte" suggests that these cells give rise to other types, but it has not yet lieen demonstrated conclusively that all hemocyte types are derived from PRs. The most generallv accepted \ iew is that PRs transform into PLs (Yeager, 1945; Arnold, 1&52, 1970. 1974; Jones, 1954, 19.56, 1959; Shri-vastava and Richards, 1965; Mitsuhashi, 1966; Willc and Vecchi, 1966; Beaulaton, 1968; Devauchelle. 1971; Lai-Fook, 1973; Beaulaton and Monpeyssin, 1976, 1977). Several authors have suggested that PRs transform into other types as well (Muttkimski. 1924; Bogojav-lensky, 1932; Yeager, 1945; Arvy and Lhoste, 1946; Ashhurst and Richards, 1964). Arnold (1952) stated that "haemocytes, with the possible exception of the Oenocytoids, apparently develop originally from a common source, the prohaemocytes," but has now ehanged his mind. Wille and Vecchi (1966), however, suggested that PR can give rise to OK. Arnold (1970), in Diploptera punctata, stated that PRs are likely stem celts for PLs, GRs, and SPs, but the direction ot ditferentia- Stnicturc and taxonomic significance 91 Fig. 4.2. A. Oaaocytofd of L. migntoria, showing cytoplasmic filaments. B. <>ene\am helle (19711 reported that PKs, PLs, GRs, and ADs are derived from each other. Francois (1974) found that PRs transform into GRs. Recently, Sohi (1971) indicated by sulxiilturing that PRs are the germinal cells from which other categories develop, while Arnold and Sohi (1974) indicated two cell lines in subculture. Some authors (Gupta and Sutherland, 1966; Hoffmann et al., 1968; Altai, 1969; Gupta. 1969; Zachary and Hoffmann, 1973) did not recognize the existence of I'Rs (see also Chapters 2, 8). As I stated earlier, the evidence on whether FHs constitute the main postcmhryonic source of hemocytes is inconclusive. There is growing c\ idencc that FHs reside in the hemopoietic organs (Hoffmann et al., 1968; Arnold, 197«; Altai and Sato, 1973; Zachary and Hoffmann, 197.3; Francois, 1974; Hinlts anil Arnold, 1977) and differentiate into other hemocyte types. Hoffmann (1967), Arnold (1974), and Beaulaton and Monpeyssin (1976) stated that PRs are germinal cells. Earlier (197«), Arnold stilted that FRs appear in the hemolymph only intermittently and often in groups, suggestive of their release from hemopoietic tissue. Wille and Vecchi (1966) reported that PRs are abundant in newly emerged hees, hut rare in old ones. Gupta and Sutherland (1968) reported an increase in PLs, GRs, SPs, and COs (= CYs) in I'eriiilaneta umericunu following treatment with sublethal doses of chlonlane. 4,2.2. Plasnmtocute (PL) Structure PLs are small to large, polymorphic cells with variable sizes (3.3—5 lim wide and 3.3-40 mm long). The plasma membrane may have mi-cropapillae, filopodia, or other irregular processes, as well as pinocy-totic or vesicular invaginations (Figs. 4.18, 4.3B.C). The nucleus may be round or elongate and is generally centrally located. It may be lo-bate (Fig. 4.3C), vary in size (3-9 lim wide and 4-10 urn long) in various insects, and appear punctate. Scattered chromatin masses may be present along with the nucleolus (Fig. 4.3(7). Occasionally, binucleate PLs may be found. The laminar nature of the plasma and nuclear membranes may or may not be visible. The cytoplasm is generally abundant and may be granular or agranular; it is basophilic and rich in organelles. Gen-rr.ilk (hire is wcll-devclopeil and extensive RER (Fig. 4.4B), which may form greatly distended cistemae or a vacuolar system. Golgi bodies (= dictyosomes = golgiosomes or internal reticular apparatus) (Fig. 4.4A) and lysosomes (membrane-bounded, electron-dense bodies, 0.1-1.30 ytm in size) may be numerous; lysosomes can be 94 A. P. GiijiIm identified 1» the presence in them of the reaction products of the hy-drolytic marker enzymes, acid phosphatase and thiamine pyrophosphatase (Scharrer, 1972), and are often associated with the RER or the vacuolar svstcm. The Golgi bodies produce the electron-dens, gran-ulcs (generallv 0.5 um in diameter) that one observes in the PLs Ml-iroson.es and cistemae of the ER (- "crgastoplasmc" »1 French authors) mav he present. Free ribosomcs (polysomes or polyribosomes) Fi«. 4.4. A. Plasmas, of UM.mtha mftnhmtha. showing (iolgilCI a..d InMH» plasmic .llllll Iii*»' x 12.«« ■• P"*'"' Pl-smal'^e of ******{?• showing rough tndoplasnm reticulum (flf.H) and vacuoles (\ 1 Ca « 21.350. C. P"' In*, of plasmatocvto of Kpf.Mll« «i»WI». showiog manch......no., (.ml .«,.1 ...InKs- lnplasn.ii n.ks. Synonymies Veager and Miinson (1941) first introduced the term "plasm.it., cyte." Some of the commonly used synonyms of PL are "leucocste (Kolhnan. 1908; Metal..iltos. 1908); "inicniniicleiK-yte" (Paillot. 1919), "phagixyte," "amix-h.K ytc," and "lymphocyte" (many authors), "|x>-il.K\tc" (Dcsauchelle. 1971); and "vennifonn cell" (la'aalld Gilbert, 1966; Devauchelle, 1971). PLs also include the "lamelliK > te" of some authors and the "nemalixy te" of Hi/.ki (1953). Interrelationship with other types The first real problem one encounters with PLs is that ol ilistiu gnishing them, particularly the so-called young PLs, from the FRs This situation is further complicated b> the presence of many transitional forms between these two types. The distinction between FHs and PLs is generally based on the relative cell and nuclear si/, s. intensity of cytoplasmic basophilia, and the extent and development ol the intracellular organelles. The question that is often raised regarding PLs is whether tbe> are the primary cells that give rise to other forms by secondary transformation. Tavlor (1935) claimed that amocbocstcs (= mostly PLs), and not chromophils (= PRs), are the basic tyix-s. Gupta and Sutherland (1966) and Gupta {1969) supported Taylor's claim and considered PRs as young PLs. Direct transformation of PLs into GRs (Veager, 1945; Junes. 1956; Gupta and Sutherland 1966; Hoffmann. 1967; Devauchelle, 1971; Beaulaton and Monpeyssin. 1977), SPs (Devauchelle. 1971; Breugiion and L* Rerre, 1976; Beaulaton and Monpc> ssm. 1977), ADs (Yeager. 1945; Shrisast.ua and Richards, 1965; Gupta and Sutherland. 1966; Raina. 1976). COs (Gupta and Sutherland. 1966; Devauchelle, 1971). OEs (Gupta and Sutherland, 1966; Hoffmann. 1967; Beaulaton and Monpeyssin, 1977), VEs (Tuzet and Manier. 1959; Gupta and Sutherland. 1966; U-a and Gilbert. 1966; Devauchelle. 1971; Francois. 1974. 1975), and POs (Gupta and Sutherland. 1966; \appi. 1970; Devauchelle. 1971; Francois. 1974. 1975) has Ihcii reported, but not substantiated. Devauchelle (1971) considered VEs and POs ultrastmctiirally similar to PLs. That it is the PL which trans tonus into other t> |x-s is indicated also In the corresponding decrease of PLs and increase of other t>|xs in differential heiu.x sic 0......ts. For 96 A. P. Cui>tii Structure utul taxonomic vigni/irmu r 97 example, in Protlvnia, when PLs fall in number, spheroidocytes (= ADs) increase (Yeager, 1945), in Drosojthila mvlanoiwstvr. when POi increase. PLs decrease | Kizki. 1962); ami in P. americanu. within -4 hr of antemial hemorrhage. GRs increase, while PLs decrease (pcrs. obscrv .). !t has also been suggested (Gupta and Sutherland, 1966; Moran, 1971; Seharrer, 1972. Pnee and Katelilfe, 1974; Beaulaton and Mon-pe\ssin, 1976) that insects have only one basic type of hcinocyte and that the commonly recognized types of hemocytcs are merely different ph\siological manifestations of the same type, depending on the physiological needs of the insect at different times, Although the PL has been regarded as the primary type in insects, a survey of the he-mocyte types in other arthropod groups reveals that the GR, not the PL, is the basic type (Gupta. 1979) (see also Chapter 8). 4.2.3. C.runutovyte (GR) Structure GRs .ire small to large, spherical or oval cells (Figs. 4.1/), 4..*v\.o\ 4.6.A.B) with variable sizes (10-45 /im long and 4-32 fim wide, rarely larger). The plasma membrane may or may not have micropapillae, fil-opodia, or other irregular processes. The nucleus may be relatively small (compared with that in the PL), round or elongate, and is generally centrally located. Nuclear size is variable (2-8 pM long and 2-7 /.tin wide). The laminar nature of the plasma and nuclear membranes may not l>e visible. The cytoplasm is characteristically granular (Figs. 4.1/.), 4.J>\.B, 4.6A). Several types of" membrane-bounded granules have !>een described in the GRs of various insects (Figs. 4.6A.B, 4.7A-C; 4.HA.B). Recently, Goffinet and Cregoire (1975) summarized and syn-onymized various types of granules into three categories, based on their observations in Carausius morosus. The following summary and synonymy of granules are based on these and other works: 1. Structureless, electron-dense granules: = unstnictured inclusions (type 1) of Baerwald and Boush (1970); inelanosoine-like granules of Hagopian (1971); opacjue body of Moran (1971); type 2 bodies of Seharrer (1972); and electron-dense granules of Raiua (1976) and others 2. Structureless, thinly granular l>odies: type 3 of Seharrer (1972); and electron-lucent granules of Raina (1976) 3. Structured granules: = "glohules" or "granules tnultibullaires" of Beaulaton (1968); "grains denses stnictures" in the AD of Devauehelle (1971); and Landiireau and Grellet (1975); "corpus fihrillaires" of Hoffmann et al. (1968. 1970); cylinder inclusions (type 2), regular-packed inclusions (type 3), and inclusions with handlike units (type 4) of Baerwald and Boush (1970); "Granula init hihularer Binncnstniktur" of Stang-Voss 98 A. P (iufttu Fig. 4.6. A. Granulocvtc of P. umrrivana, showing structured (O and unstructured (u.») granules. Ga. x 16,00«. B. Portion of granulocyte of Isucophara madvntr. showing derivation of structured I = preiiielanosomc-like1 granule (.*) from Golgi «.'), structureless or unstructured ( = melanin-like) granule (to), ami nitnu ytoplasmic niitrohi-hules {m). M 3.000 (A reinterpreted from Baerwald and Boush. 1970; B reinterpreted from Ilagnpi.m. 1971) Fig. 4.5. A. Granulocyte of M. mehttnntha. \ = nucleus, x 6.300. B. Granulocyte of Thermohia ttomritica. x 9,400. (A courtesv of Dr. G. Devauehelle; B from Francois. 1975) Fig. 4.7. A. Structured granule Inirn granulocyte of /,. matlcrar. showing internal mi-crotuhules. M 10,000 B. An earlier stage of development of internal microtuhtiirs. * 50,000. C. Section of a structured granule showing concentric arrangement of in-tenial mit rotuhules. x 38.000. (A-C from Hagopian. 1971) Sinu tun' aiui taxonomie vigiii/irtino 101 Fig. 4.8. A. Section of stnu-turrťl líranulr of a gninnliKVtt' of /.. mmti'riu; sh(minti arrangement of inuTntnbnle* alaait 25 nm in diameter. M nVl.OOO. B. HiizliK mainlined view of mierotuhnles of structured granules. Note mteroMnicrotubules (5 nm in diameter, arrowt within microtubules anil limiting membrane (me) of grannie. * 240,000. (A and B from llagopian, [971) 100 102 A. P. <.'»|i<« (1974). M CRs. Gafloet ami Gregoire (1975) reported separate categories of CRa ami COs in CurmUtm MOWmn1. rVl a matter nl fail, the separate existence of the OK (not tube confused with PL, SI'. AD, ami CO) is now recognized by imi,'t authors, although Dm am hellc (19711 lias included both GR and CO in liis type 111. How arc CRs funned':1 An- thr> derived from PRl or PLa? both sources of origin have ban nrMwl (Arnold, 197-1 >. Cupta and Sutherland (1966) indicated that the derivation of GR from PL is a short step. Takada and Kitumi (1971) reported that CRs showed a trend to increase and PI.s to decrease with time. Are CRs capable of transform-on; into other type! of heinocytcs:'There arc reports that indicate that CRs do indeed gh c rise to SFs, AOs, and COs (Cnpta and Sutherland. 1966). Arnold (1974) has suggested that CRs "might b« considered hasic units from which more precisely structured and functioning classes ol cells have dc\ eloped." This is supported by my survc> ol hcniocyte types in Artlimpoda (CopfkV 1979). llinks and Arnold (1977), however, have suggested separate origins ol CRs and Si's. The presence of microtubules in the granules and in the cytoplasm of the CR also has caused debate. According to Crossley (1975), the microtubules of the granules tlo not have the "dimensions ol typical cytoplasmic microtubules (24-27 nm diameter), nor have been demonstrated to be sensitive to colchicine or vinblastine . and therefore they should not be called microtubules." According to him, only in Ia'UCo)iIuuu are the dimensions of the inclusion tubules (25 nm) comparable to those of the intracytoplasmic microtubules. The intracytoplasmic microtubules have been descrilied in several insects (Grimstone et al., 1967; Baerwald i.nd Boush, 1970. 1971; De-vauchelle, 1971; llagopian, 1971; Scharrer, 1972; to mention a few investigators) and are generally narrower in diameter than the microtubules of the granules. These intracytoplasmic microtubules may be arranged into marginal bundles (Hagopian, 1971) or may be randomly distributed in the cytoplasm (Devauchelle, 1971) and supposedly are found in all hemocyte types, except OEs (Devauchelle, 1971), although Raina (1976) has described them in OEs. 4.2.4. Sp/terulocufe (SP) Structure SPs are ovoid or round cells (Figs. 4.1C.K; 4.9A.B) with variable sizes (9-25 aa long and 5-10 u.m wide) and usually larger than CRs. The plasma membrane may or may not have micropapillac, filopodia, or other irregular processes. The nucleus is generally small (5-9 an long and 2.5-6 am wide), central or eccentric, rich in chromatin bodies, and generally obscured by the membrane-bounded, electron-dense, intracytoplasmic spherules that are characteristic of these cells. (1970) preinelaiiOMiiiie-likt- granules ol I ...cnpi.ih ilWTI), hilmle-eim-taiuuig IxhIhs oi T('H nl Monui tl971>. type 1 of Scharrer (1972). ami granules with a inu nituhulai stnutiin- uf li. it. hi It- autt 1'iui- (1974) The length ui the diameter nl (lie structureless granules varies Iron. 0.15 to 3 pin or more in various insects, while that of the structured granules varies from 0.5 to 2 pm. The shai*' of the granules may Im-spherical, ovoid, elongate, or irregularlv |>olygonal (Fins. 4..'i\,/f. -4.fi-\./i. 4.78Á'; 4.8A). The diameter of the microtubules within the structured grannies varies from 15 to 80 nm) in various inserts. Internally, the micmtubules may show mknwiucrotuhules alxiut 5 nm in diameter (Hagopian. 1971) (Fír. 4.HB): Akai and Sato (1973) also have described "suhunits of fibrils" in their so-called secretory vesicles. The iiiiiiiIht of microtubules per granule may vary from 9 to 80. From the accounts provided fay llagopian (1971), Scharrer (1972), Akai and Sato (1973), and Francois (1975), it appears that the granules are derived from the Colgi hodies (Fig. 4.6ÍJ), the microtubules developing during the later stages of morphogenesis. It is conceivable that the strm tureless, electron-dense granules represent the final stage of development of these granules in which the structured nature I >er of the spherules ma> van from lew to mam and the d.ameter from 1.5 to 5 „,„. The spherules eontaiu granular, fine-textured, filamentous, or Hocculent material (Kaina. 1976). The granules within the spherules niav van from 15 to 17 nm in diameter | Aka. and Sato. 9,3). In addition to the spherules, the cvtoplasm eont.uns polynbosomes (Fig. 4.10T). Colgi Uxlies fnoderatelv to well developed) (Fig. 4.KM). membrane-Umided vacuoles (= 'j...... J {Kig 4.90, numerous randomly distributed microtubules, ehmgated mito- RER , C Fi» 4.9. A. Sphemkxvte of .If. mrtolaMha. »h«m,n« eccnriric nuclru, (V) „d nu-TÄ™,:"1 ! yT'"'r V) " 9-45U »• Sph..n,l,«.yl(. „f B,„„lm ,„„rl. ~ I■ , C- 'f "Í" h"" "'ľ " '""Winn rramh ' 21.00(1. (A fr..,„ ft......ll.il, 1OTI; B from Akai and Sain. !973; C frnTn Raina. hm A. /■ (;»/■(« Slmctun' tnut Itivtmiuttii sii!fii/i,,,u,r H ň c hornina, .mil KKH (Figs. 4.9C, 4.1UI.C). Dcvaiichellc (1971) has ..K,> described ■ iiKMi- or less loose network of fibrils in the cytoplasm (Fík. 4.HIHI. Si's release tin- material in their spherules into the heino-Kinph by exix-ytosis. Histochcniicallv. the sphemles have lieen reporteil to eontain neu- * ' "1. Fig. 4.10. A. Portion of sphenilncyte oľf. K,i.vv,/pi,'/i",,, showing ronith endoplasmic-retic-ulitn, {RtlR) and (íolři (C) involved in formation of spherule I.Sji) Ca. * 15.750. B. Portion of sphemlocvte of M mAohmtha. shosvinif loose network of intrac-ytoplas-mie fihrils. C. Portion of sphenilnc-ytc of B. mori. showing spherule (,S;i) with fint' granules, rough endoplasmic reticulum containing rihrmis material in its cistern.,,-(arrows), and rionsomrs. x 90.400. (A from Rama. 1976; B from Devauchelle 1971 C from Akai and Sato. 1973) tral or acid mucnpolv saccharide- anil glycomucoprotcins by several authors (Vereauteren ami Acrts, 19.5«; Sittono, 1960; Ashhurst anil Richards. 1964; Gupta ami Sutherland. 1967; Costin. 1975; Beaulaton anil Monpeyssin. 1977). Much earlier. Ilollamle (1909) reported that the spherules eontain "lipoehrome" (a kind of earotenoid lipid). The presence of tyrosinase has Ihtii reported by Dennell (1947). Jones (1956). and Ri/ki and Hizki (1959). Most recently, Costin (1975) reported the presence ut nonsiillated sialoinucin. in addition to glycoproteins and neutral inucopolysaeehrides. Synonymies Ilollamle (1909) was the first to use the term "spherule cells." which is now generally used by most workers. Other terms that have been used by various authors are "cellules spherule-uses" or "cellules a sphemles" (Paillot, 1919; Paillot and Noel. 1928); "sphe-rocyte-s" (Bogojavlcnsky, 1932); "eruptive cells" (Ycager. 1945); "ix-noe vtoids" (Dennell. 1947); ' rlie-ginatix-ytcs" (rHHrdy, 1957); and "hyaline cells" (Whitten. 1964. her FiK. 1H). Harpaz et al. (1969) classified SPs .is ADs. Interrelationship with other types The main controversies about SPs concern the transformation of 111! •se cells into other tyjxs. formation of the sphemles. and the functions ol these cells. The transformation of SPs into other types is not well documented. Gupta and Sutherland (1966) suggested that SPs are capable of transforming into AOs and COs (= cystex-ytes) and that SPs are themselves derived from GRs. Miliars (1947) and Arnold and Salkeld (1967) also considered the SP as a phase in the life of a GR. Later, Arnold (1974) stated that "they seem to be another specialized cell within the granular homocyte complex." Beaulaton (1968) has suggested that SPs are degenerated OEs. Hinks and Arnold (1977) consider SPs as separate types with mitotic capabilities. Little information js available oil the formating of the spherules. According to Akai and Sato (1973), the material in the sphemles is first observed in enlarged cistemae of the RER, then transferred into the Colgi Complex, where it is packaged into the meinbrane-boundcd sphemles. The role of Sl\ is highly controversial. Hollande (1909) considered these cells respiratory in function because of the presence of the so-called lipoehrome. It has been demonstrated by Akesson (1945), Ash-hurst and Richards (1964), Arnold and Salkeld (1967), Gupta and Sutherland (1967), and Costin (1975) that the material contained in the sphemles is neutral or acid mucopolysaccharide, not a earotenoid lipid. Hollande (1909) stated also that these cells contained an oxidase. Dennell (1947). Jones (1959), and Rizki and Rizki (1959) re- A. P. Gupta ported tyrosinase in the sphemles of variant Diptera. but Gupta and Sutherland (1967) found no tyrosinase in the SPs of cockroaches. Gupta anil Sutherland I 1965) were supposedly the first to report SPs in cockroaches. Whitten (1964) suggested that SPs (= her hyaline cells) may pl.l> .1 role in the darkening ol the pliparilllll in s.....c c \- clorrhaphous Diptera. Perez (1910) reported that SPs took part in histolysis. Although this has been disputed by Akesson (1945). the histo-lytic role of SPs should not be surprising, considering the fact that before and after molting several SPs are observed to congregate..... histiilyzing tissue. Gupta (1970) has suggested the probable histolvtic nr phagocytic functions of SPs. It is probable that SPs both histulyze and phagocytize tissues in at least a lew insects The phagocytic function was reported by K0II111.111 (1908). Cameron (19.34). anil Akesson (1945). but further work is needed to demonstrate clearly the histobtic role of SPs. Raina (1976) found no evidence of their role in phagocytosis. Metalnikov and Chorine (1929) and Metalnikov (1934) found that the SPs in GaViVriu are related to bacterial Immunity. Nittono (I960) stated that strains of silkworm larvae that lacked SPs completely or incompletely tended to produce relatively smaller quantise* oi silk. Wigglesworth (1959) suggested that SPs are involved 111 the uptake and transport of other substances, such as hormones Akai and Sato (1973) suggested that SPs are sources of some blood proteins. 45.5. Adipohemocyle (AD) Structure ADs are small to large, spherical or oval cells (Fig. 4.2C) with variable sizes (7-45 (on in diameter). The plasma membrane may or may not have micropapillae. filopodia, or other irregular processes. The nucleus is relatively small (compared with that in a PL or SP). round or slightly elongate, and centrally or eeientrically located. Nuclear size is variable (4-10 am in diameter). The nucleus may appear con-case, biconvex, punctate, or lobate. The laminar nature of the plasma and nuclear membranes may not be visible. The cytoplasm contains characteristic small to very large refringent fat droplets (0.5-15 am in diameter) and other nonlipid granules (0.5-9 an in diameter) and vacuoles, which, according to Arnold (1974), become filled with lipids under certain conditions. In addition, the cytoplasm contains well-developed Golgj bodies, mitochondria, and polyrilxisomes. Histochemically, ADs are reported to contain PAS-positive substance in the granules (Ashhurst and Richards, 1964; l,ea and Gilbert, 1966). Costin (1975) did not recognize ADs as a type in her study. Structure 11111/ tiui-in»«i( siimnV nine- Synonymies ... Hollande (1911) first introduced the- term "adipolcncocyte, al-thiHigl. kolhnan (1908) had earlier used the- tern, "adipo-spherulc cell" lor some henioevtcs ol invertebrates. Other terms used for ADs an- "spheroid... v t.s (YeagW, 1945. Arnold. 1952. Hi/ki, 1953; Jones, 1959); "later stages ol sphemles (Whitten. 1964); and "adipocytes (Wigglesworth. 1965) (sec also Chapter8) Interrelationship with other Is pes The main controversy about ADs concerns their identity as a distinct category of hc.nncv tcs. Scmtins of the literature leads one to belu rve that tlicv are not a distinct type. Sove-ral authors have reported that it is difficult to distinguish then bum GHs (Jones. 1970; Arnold, 1974) and many others did not recognize the category of ADs in their studies (Wittig, 1968; Akai and Sato. 1973; Costin. 1975; Francois. 1975 Cofhm-t and Gn-gotrc. 1975; Boiteau and Perron. 1976; Raina, 1976^ Beaulaton and Monpe-vssin. 1977; to mention a lew recent ones). Raina (1976) noted a progressive accumulation of lipid drops in GR, and on that basis considered ADs as functional stages of GRs_ Gupta and Sutherland (1966) also have n-ix.rtcd the translonnatioii ol GRs into ADs. Only one of the ultrastnictural studies (IVvauchelle, 1971) of hemoevtes includes ADs as separate category. However, Ins micrographs (his Figs. 20, 21, 24) an- strikingly similar to the GRs (ct Fig. 4 11 with Fig. 4.6B) described by If—I fill (1971) and other au- ' On the basis of the histochemical nature of these cells also it is difficult to justify the term, and hence the category. »f ADs. According to Crosslev (1975). "ultrastnictural studies of so-called acli|>e>haenieK-ytes\ include cells which contain no re|>orteel lipid (Devaeichelle, 19,1, Fig. 18-22). lipid of doubtful authenticity (Pipa and Wexilever. 1965) or material believed to lx- mucoprotein or inuce.polysaccharidc (Beaulaton, 1968. Fig. 7)." Costin (1975) also did not n-cognize the AD on the basis of the histochemistry of hemexytes in Locuifu migraforiu The n-semblance of ADs to fat lxxiy cells is also confusing and adds to the difficulty of identifying ADs in fresh hemolymph samples^ For example, one mav find all gradations lx-tween small ADs and fat lxxiy cells (Wigglesworth, 1955). Jones (1965) suggested that hemcx-ytcs "with excentric nucleus and many brilliant fatlike droplets shemld he termed ADs onlv when they can lx- clearly distinguished from fat body cells." According to him (Jones. 1975). ADs are at least 10 times smaller than fat lxxiy cells. Gupta and Sutherland (1966) suggested that under certain conditions, such as chilling, starvation, and diapause (periexls of nemfceding and n-duced Metabolic rate), the PL, n-spond by changing into ADs 10« A. ř. GapM m .(Ii "lipid" droplets. This was based mi their observation thai mt-al-wonn larvae, when chilled lor 20-24 hr at 5 °C, showed numerous "lipid" droplets and ADs. other types of hemocytes being rare. Sueh larvae subsequently recovered. Ludwig and YVugineister (1953) noted an increased ainonnt of free fats in the heniolymph of the stars in« Japanese beetle. Popi/Iiii jrtpiiiiir«. and Clark and Chadhoume (1960) reported a greater number of ADs (= their spheroidocytes) in diapaus-ini! larvae of the pink bollwonn, Pirfiiitm/irirw govsi/iiie/iii. Thus, it Fit, 4.11. Supposed adipohcmocyte of M. melohmthv But note lhal II looks like granulocyte. « 41.300. (From Devauchelle. 1871) FÍH. 4.12. A. Ooimk ytoid ol H nmn. showintr i nm rntru .irraiiKi-lncnt of intracytn. plasniir fibrils (arrows) and fit oniric nucleus (\). x 1,000. B. Oolioeytoid of P. Krw-siil'irUa x ,6011 C. Portion of oenocytoid of H. morí. showing highly inaKliincd view ni entile mms of intrai > liipl.iMim (ilirils. Not,. a|M, uuoricutcd fibrils (arrows). Cm. x 30.000. Inaet (ca. x 2Í5.IKKK shims filtnls in cross section. D. Portion ol ih'iio-cytotd of P. Km yi< llu showinrí Inuiiitudinally arranged intracytnphismic microtubule! (imi*il. (.nt^i ((;). and nbosnnios (Ni), x .15.475. IA and C from Akai and Sato. 1071; B and D Ini.ii Haina. 197« 110 Structure ami taxonomie significana 109 seems very likely that the appearance (or transformation from PLs) of ADs in the hemolymph at certain times is governed by the physiological state of the insect. 4.2.6. Oenocytoid (OE) Structure OEs are small to large, thick, oval, spherical, or elongate cells (Figs. 4.2,4.8, 4.12A.B) with widely variable sizes (16-54 urn or more) and shapes. The plasma membrane is generally without micropapillae, fil-opodia, or other irregular processes. The nucleus is generally small, round or elongate, and generally eccentrically located (Figs. 4.28, 4.12A). Nuclear size may vary (3-15 urn). Occasionally, two nuclei may be present. The laminar nature ol the plasma and nuclear membranes may not lie visible. The cytoplasm is generally thick and homogeneous and has several kinds of plate-, rod-, or needlelike inclusions. According to Costin (1975), the OE is distinguished by an elaborate system of filaments that fills the cytoplasm (Figs. 4.2A, 4.12CJJ) and is visible under a phase-contrast microscope. Histochemically, the filaments resemble the cytoplasm and hence are not visible in stained preparations. Hoffmann (1966) and Hoffmann et a). (1968) also have reported such filaments. In addition to the above intracytoplasmic inclusions and filaments, a few electron-dense spherules may be present in the cell periphery (Devauchelle, 1971). With the exception of polyribosomes and the abundant, large mitochondria, which are conspicuous, other organelles, such as ER and Golgi, are poorly developed. Supposedly, lysosoines are absent. Histochemically, OEs are reported to contain tyrosinase (Dennell, 1947), protein (Akai and Sato, 1973), and PAS-positive-only granules, indicating the presence of glycoproteins or neutral mucopolysaccharides and sulfated, periodate-reactive sialomucin (Costin, 1975). One peculiarity of OEs seems to be their highly labile nature. They are particularly fragile in vitro and lyse quickly, ejecting material in the hemolymph. They are nonphagocytic. Synonymies No two terms have caused as much confusion as "oenocytes" and "oenocytoids." Oenocytes differ from oenocytoids in that they are ectodermal in origin, usually segmentally arranged, yellpw in color, and not hemocytes. Poyarkoff (1910) first introduced the term OE, followed by Hollande (1911). In order to avoid any confusion between oenocytes (proposed by Wielowiejsky, 1866) and oenocytoids. Hollande (1914) proposed to replace the term oenocyte by "cerodecytes." It is not surprising that several earlier authors mistook oenocytes for Structure and tuxtmomic significance 111 OEs. Even after Hollandc's (1920) detailed description of OEs. several authors (Metalnikov and Oaschen. 1922; Miiller, 1925; Tatoivva. 1928; Metalnikov and Chorine. 1929; logojav lensky, 1932; and Cameron. 1934) used the term ixnocytc instead of OE in their respective works. Other synonyms used for OEs are "ocnocytc-like cells" (Ycagcr. 1945); large "non-granular spindle cells" and "non-phagocytic giant .....cytes" (Wigglesworth. 1933, 1955; see discussion in Jones, 1965), "crystalloid" and "dark hyaline hemoevtes" (Selman, 1962); "crystal cells" (Hi/.ki. 1953, 1962; Nappi, 1970); and COs (Hoffmann and Stoekel, 1968). Interrelationship with other types The main controversy about OEs concerns their identity as a separate category, particularly their distinction from COs. The view that OEs are part ol the CO complex has received support owing to the observation by some authors (La and Gilbert. 1966) that OEs are unstable in v itro and that they undergo rapid and drastic transformation into hyaline cells (= COs). These authors report that in llyaloiihora cccro-piu, OEs begin to transform within 15-30 sec, and fully transformed OEs are found within 15 miu. Jones (1959) and Nittono (1960) also have reported such translijp'iatioii of OEs in Prodenia and Bomhyx, respectively. Coupled "'Toi these observations are the reports by many authors that OEs are either found in very small numbers or are absent. This may partly explain why several authors either have not reported OEs in their studies or do not recognize OEs as a distinct category. Crossley (1975), however, stated that ultrastructurally OEs and COs are different, and indeed some authors (Hoffmann et a!., 1968) have descril>ed both OEs and COs in their uitrastructur.il studies. The iiltrastrnctur.il identity of OEs is also supported by the fact that although these cells eject material into the hemolymph as COs do, this does not result in plasma gelation (Arnold, 1974). The origin or derivation of these cells is also controversial. Gupta and Sutherland (1966) and Heaulaton and Monpeyssin (1977) suggested that OEs are differentiated from PLs. Devauchelle (1971) has indicated that OEs might be derived from PRs. Arnold (1974) stated that "the cells seem to be allied with the complex of granular cells, but their origins and relationships are not understood." Hinks and Arnold (1977) found them originating in the hemopoietic tissue. 4.2.7. Coagulocyte (CO) Structure COs are generally small to large (3-30 /im long), spherical, hyaline, Iragilc, and unstable cells, combining the features of GRs and OEs 112 A. P. Gupta (Arnold, 1974). The plasma membrane is generally without any micro-papillae, hlopodia. or other in' gular processes. The nucleus is relatively small (5-11 /iin long), generally eccentric, oval, sharply outlined, and under phase-contrast may appear cartwheel-like owing to the arrangement of the chromatin in that lashion (Fig. 4.2D). According to Goffinet and Gregoire (1975), there is a pronounced perinuclear cistema (Fig. 4.13A), which, together with microniptures iu the plasma memhrane. supposedly distinguishes these cells from other types. The laminar nature of the plasma and nuclear membranes ma\ sot be visible. The plasma memhrane may show microniptures. The cytoplasm is hyaline and rich in polyribosomes, but has fewer mitochondria and moderately developed EH. In addition, the cytoplasm has some spherical or elongate granular inclusions, about 1 urn in diameter (Fig. 4.13A). Francois (1975) has described four types of such granules in Thernwhia dtimcstica: (1) electron-dense, homogeneous granules generally resembling those in GRs; (2) moderately electron-dense, homogeneous granules; (3) heterogeneous granules wi'.h a central or lateral dense zone, the remaining portion being homogeneously granular; and (4) structured granules, with internal microtubules (15 nm in diameter), arranged in a parallel fashion and 40 mn apart. Goffinet and Gregoire (1975) also descril>ed stnictural granules in the COs of (.'. monism. It is obvious that there is a very close resemblance between GRs and COs. Histochemically, COs are clearly distinguishable from PLs and GRs according to the periodic acid-Schiff (PAS) test (Costin, 1975). According to her, "compared with the cytoplasm of the other types of blood cells, that ol' coagulocyte has much reduced basophilia." It is very weakly PAS-positive. Synonymies Gregoire and Florkin (1950) for the first time introduced the term "coagulocyte" or "unstable hyaline hemocyte" in Gryllulux and Curausius. Earlier, Yeager (1945) for the first time used the term "cys-tocyte" for cells with cystlike inclusions. Jones (19.50) used that term for "coarsely granular haemocytes," and later (Jones. 1962) suggested that the "term coagulocyte for these cells may be prefened to cysto-cyte because these cells are only identified by their function." Wigg-lesworth (Chapter 11) synonymized "thrombocytoids" of Zachary and Hoffmann (1973) with COs. Intenelationship with other types The main controversies about COs concern their identity, function, and origin. It is still debatable whether the COs are ultrastructurally different from GRs. Devauchelle (1971) found them indistinguishable Structure tnitl taxonon 113 114 A. P. Gupta nymphs. More often th.,u not, radiate PLs "iih pseudopodia **e mis- taken lor POs. These hcniocytcs are very large (Fig. 4.2K). extremely flattened PL-like cells with sever il cytoplasmic extensions (Fig. 4.13B). The nucleus is generally large and centrally located and may apin-ar punctate. Fit 4.13. A. Cnwnkwylr. ihowtng perinuclear cistern**- (arrows) and those of endoplasmic reticulum ler). No* presenco-of electron-dense (sljurturrleM) **>»•*• MM* as an- found in granulocytes, x 20.000 B. P.xlocyte. showing pscudopo. la. Note re-semblance to prohemocyte or young plasniatocyte. .V - nucleus, x 9.500. (A and n courtesy Dr. G. Devauchelle) from CHs and synonymized them with the latter. Moran (1971) found a type ol cell in Hluhcrux tliscoulali* (frequently in newly molted, un-tanned adults) with meinbrane-bounded. tubule-containing bodies (TCB) filled with rows of 34-nm tubules, which are quite different from the intracytoplasmie microtubules. He suggested that these cells are equivalent to COs (cystoeytes). Rateliffe and Price (1974), however, have identified COs (their cystocyte) in their work. Most recently, Goffinet and Gregoire (1975) and Gregoire and Goffinet (Chapter 7) claimed a separate identity lor "them. According to them, the perinuclear cisteniae of the COs are .much more pronounced than those ol PRs, PLs. and (-Ms, and their plasma membrane is niptured during coagulation, whereas those in PRs, PLs, and GRs remain intact. The role ol the COs in hemolymph coagulation is generally accepted and has l>cen recently reconfinned by Gregoire (1974), Francois (1975), Goffinet and Gregoire (1975), and Gregoire and Goffinet (Chapter 7). Gupta and Sutherland (1966), however, have suggested that COs arc the effect rather than the cause of coagulation on the basis of their observation that as soon as coagulation starts, several PLs transform into COs. This view, however, is not accepted by Gregoire. Supposedly. COs also contain phenol-oxidizing enzymes (Crossley, 1975). It must be mentioned here that in several arthropod groups coagulation of the hemolymph is caused by the GR (Gupta, 1979), and evidence is accumulating that this is also tme iu some insects (Rowley and Rateliffe, 1976; Rowley, 1977). The origin of COs is still debatable. Gregoire and Goffinet (Chapter 7) and Hinks and Arnold (1977) have suggested that these cells originate in the hemopoietic organs. However, if we accept the premise that hemocytes respond to bodily injury in the insect, it is conceivable that either the injury itself would induce the production of COs or some other type of hemocyte would produce them bv transformation. For more details on the stnicture, functions, and origin of COs, the reader is referred to Chapter 7. 4.3. Other hemocyte types 4.3.1. Podocyle (PO) Structure These hemocytes have not been recognized as a separate category in any ultrastnietural study and are not ordinarily observed in the hemocyte samples under the light microscope. They should be regarded as a variant form of PL. According to Arnold (1974), they have been correctly identified only in Prodenia (Yeager, 1945; Jones, 1959). However, I (Gupta, 1969) have observed them in P. americana Structure and huumnmki ligntfnennj 115 Synonymies , , .„_.. Yeager (1945) introduced the tenn podocyte. (..rubers IWfiJ star-shaped an.ocbocytes" and Lutz's (1H95) radiate cells may have Included POs. Intenelationship with other types POs m derived from PLs. Gupta and Sutherland (1966) have suggested their transfonnation from PLs. This seems to l>e supported by Hizkis (1962) observation that in D. me/«m»g«v(er when POs increase in differential counts. PLs decrease. Hi/ki's (1953) POs appear to be IM.s. Whittcn (1984) questioned the concept "I POs, and DevaucheUe (1971) considered them variant forms ol PLs. 4.3.2. Vi-rimYl/lf (VE) Structure , This form is gcncralb called verniifonil cell and should not be regarded as a separate category. As the name suggests, these are ex-trc.uclv elongated cells with slightly granular or agranular cytoplasm. The nucleus may be located centrally or eccentrically (Fig. 4.2/- )■ Synonymies „ Yeager (1945) introduced the tern, "vermiform cell, but the term •Vermicyte" is more appropriate. Tuzet and Manier (1959) used the tenn "giant fusiform cells" lor \ Ks. Intenelationship with other types The origin ofVEs is unknown. However, it is conceivable that they arc derived from PLs, as has been suggested b) Gupta and Slither ami (1966) Lea and Gilbert (1966) considered them a variant tonn ot PLs. According to Arnold (1974), "they seem to occur mainly just pnor to pupation, but never in large numbers. 4.3.3. Additional miscellaneous hemocyte types In addition to the above nine heniocvte types, several authors have from time to time, reported hemocytes. nun) or all of which has e not been general!) accepted - lor example; "haemocytohlast ol Bogojav-lenskv (1932);' "leucoblast" of Any and Gabe (1946) and Arvy (1954); ••proleucocvtoid" and "prohaemocytoid" of Yeager (1945) and Jones (1950), respectively. Yeager also introduced the term "nemutocyte. Rizki (1962) in his works used the terms "lamellocyte and crystal cell " The latter was adopted also by Whitten (1964). According to Arnold (1974). crvstal cells and l'amellocytes are considered vanants oi 116 A. P. Gupta Stnuturr and taxonomii sinnißraim 117 OEl .titd PLt, respectively. Gupta (1969) also suggested lint (he crystal or)I is probably an OK. Terms such as "selenifonn tell" (Poyaxkoff, 1910); "atocytf" iTillyard, 1917); "splanchnot \ te" (Muttkowski, 1924). "teratocyte" (Hollande. 1920); "pycnonuclcocv tc" (Morgan-thalci. 195.3; Will*- and Vecchi, 1966), "micleocyte" and "rhcgniato-i v tc i Mrd\ , 1957) arc rare I > encountered in the literature, most likelv scv cral introdmed the term "grauuliK ytophagous" eell in his work on Hluxfnms pn . in the cockroach. Chotnphiulorliitm por-trnto^i. Zai hary and MotTmann (1973) deserihed the hemocyte tliiinnlteen studied. Kor example, as of that \<\u, among Apterygota. onlv Thysanura had lieen studied. Anions die orthopteroid groups, I sop t era and Emhioptera awaited studies. In the heiuipteroid complex, no account ol hentocytes v as available in /er ol species studied in various orders, Ix-pidop-ti-la. Il\ nienr)ptera. Coleoptera, and Diptera appear to l>c the most evil hmw-Iv Itodfced groups. In addition to the Heteroptera, Ilomoptera. and Odonata. ol which only a lew BpecJCl have so far l>een studied, Deiui.iptera. I'lccoptera. Trichoptcra. and Thysanoptera are the most poorly studied grnupf. Hemocvtcs of several insect orders are tin-know ii. With the exception of the GH and possibly also PLs, all other types nt lniniK \t.s are not present in all insect orders (see Tahle 4.1). Ac-i Ofdfog to Arnold 11974). all hemocyte types have heen reported only toPtOttrniii iVeagcr, 1945; Jones, 1959). Most insects seem to possess PR*, I'l.s, and GKs (see also Chapter 8). 1 5 | lesiomorphit- hemocyte and its differentiation into other types 1 have reported elsewhere (Gupta, '.979) that the granulocyte (GH) is tin pit■sioinorplnc hemocyte, and it is the only hemocyte type that has heen reported in all major arthropod groups, including all studied insect orders, and the Onvchophora. The following account of the presence ol GH in Insecta is based on Arnold's (1974) reinterpretation of various works, my own (Gupta, 1969) survey ol the hemocyte literatim- in main insects, and the most recent transmission electron microscopic (TKM) studies of heinocytes As far as is known, onl\ Hnmtz (1908), Millara(1947). Barra (1969), Gupta (1969), and Francois < 1974, 1975) have worked on the heinocytes of the Apterygota; and on the basis of these works, both Collembola (it is control ersial whether they should be included in ApteryK""*) and Thysanura possess GHs. All higher orders of insects possess GKs (see Table 4.1). Some of the most recent TKM studies (Hoffmann et at, 1968, 1970, Hacrwald and Bnush. 1970: Hagopian. 1971; Moran 1971;,Scharrer. 1972; Hatclitfeand Price. 1974; Onffinct and Gn-goire, 1975; Beaulaton and Monpeyssin, 1976; Brehelin et al.. 1976; Katclilfe et al.. 1976; Rowley and Ratclilfe. 1976; Row Icy. 1977; Schmit and Ratclifle, 1977) have reported (or can be interpreted to show) GHs in various insects. The most highly specialized ueuropteroid orders also possess GRs. Since it has been reported by several authors that one hemocyte type can and dot* differentiate into another type, it is conceivable that during evolution the plcsiomorphic GH differentiated into other hemocyte types. It can be postulated also that the GH originates from the so-called prohemocytc (PR) or stem cell and goes through the plasma-tocyte (PL) stage before becoming a distinct GR tyi>e. In taxa in which only GRs have Ireen observed (e.g., Xiphosura), the PR and PL are merely evanescent stages and have not achieved distinctness as types. In taxa that are reported to possess other types besides PH. PL. anil GR, the last perhaps further differentiated.jnto SP, AO, CO, and OE. not necessarily in that order. The post-CR differentiation is generally accompanied by distinct PRs and PLs Furthermore, in the more highly evolved taxa any of the types may lie suppressed. The main differentiation pathways as postulated aliove may lie represented as shown in the diagram. SP CO OK 4.6. Phylogenetic significance of hemocyte types The prospect of using variations in hemocyte types in various insert orders for phylogenetic considerations is severely limited owing to (1) I IK .1. P. Gupta lack n! .....form terminology, and hence the difficulty ol establishing comparisons, .mil i2> paucity of comprehensive studies ol hemocyte types in l.uge numbers ol species within various oiders to enable one to draw ine.uiinulul conclusions on the basis ol important variations. However, hemocyte tyjvcs and their numerical variations in many inserts oulcrs .uč known. Docs the number ol hemocyte types in various orders have any pliy Ingcuctic significance:1 Arnold 11972a.b. 1976) .nul Vrnold .mil Hinks ,1975) have used heinocytes in insect taxou-oinv , and several authors have suggested (see Gupta, 1979) that some pliv logenetic relationship among various arthropod groups can be tlcinmistralcd on the basis ol the occurrence of the GH and other he-inocv te Iv pes \ míru of tin- insert hemocyte literature indicates some phylogenetic trends m the diversity of tin' heniocvtc types in Insecta as a group as the evolutionary Udder is ascended i Fig. 1.14). This was observed also by Arnold (1974). although he attributed this diversity mure In "shifts in the emphasis on certain functions or in the assignment ol functions to different tissues" than to phy logeny. And al least in certain instances he inav be right. As far as is known, only Brunt/, t HXW). Millar., 11947). Barra (I960). Gupta 119691. and Francois (1971. 1975) have worked on the hemo-ivtcs ol the Apterygota; and on the basis of these works, Collcinliola possess onlv the plcsioiunrpliic (ML whereas the Thysanura (Lepis-matidae) seem to possess PL. GR SP. and CO. Assuming that the Thy-sainii.i originated from Sv tupli) la-like ancestors, and that the latter had heniocvtc tvpes comparable to those of Si utiucrctla (Gupta. 1968), we find that a reduction from sis (PR, PL, GR, SP. AO, and CO) in the symphylan ancestor to lour in the Thysanura has occurred. I have no evidence to suggest whether this is a secondary suppression and/or reduction at, as Arnold (1974) suggested, attributable to shifts in functions. It is also possible that future studies will reveal more types than are presently known. According to Carpenter (1976), the derivation of the Pterygota from the aptcrygotc Thysanura is almost universally accepted. It is also generally believed that the pterygotcs evolved along four evolutionary lines: palcopteroid. orthopteroid, heunpleroid. and ueuropteroid. It is interesting to note (Fig. 4.14) that in the Palaeoptera, although the number ol hemocyte types has not increased from the Bill nihil thy -sanuran number, the OK has already made its appearance in the very beginning ol the evolution of winged insects. In addition, the PR has achieved distinctness, and the CO is either suppressed or its function is taken over by the OR. which is generally the case in some other arthropods (aquatic Chelicerata and some Crustacea). It is also evident from Pig. 4.14 that Ivcyonďtho palcopteroid line the number of hemocyte types increased, and all the six or seven types Structure and taxonomie significance 119 were realized in the orthopteroid, hemipteroid, and ueuropteroid lilies. It seems that by the time the orthopteroid line evolved, the ple-siomnrphic GH had already differentiated into all the distinct types presently known in pterygote insects, and that no further evolution in the hemocyte types occurred beyond that point. It is doubtful whether within the orthopteroid group any phylogenetic significance of the he-inocyte types exists. The number of hemocyte types reported in various orders ol this group (see Gupta. 1969; Arnold, 1974) is so variable that it is very difficult to discern any phylogenetic trends. On the basis NEUROPTf ROID LINE MEMIP ER01D LINE ORTHOPTEROID LINE PALAEOPTEROID LINE ' >n... ..... Efirwflwoptaf* PleccplHi, Embiopwa DwMplM InpMn ThyMnooiwa PMiwapu.« i.f,l..iipte,a HmJUJUM ■ I > ODT*[. I .-p.lunlei. Oiplwa I PALAEODICTVOPTERA | COLLEMBOLA | j PBOTUHA I DIPLURA THYSANURA STMPHYLAN ANCESTOR Fig. 4.14. Diuxnini show inn distribution of various hei.xx.-yti- t> jx-* hi km evolutionary liii... (group*) and in >.ymph>laii ancestor ol lust-cta. Note that liatrd orders under each evolutionary- line are only some examples <>f representative orders, not nece*-sarily those in which hemocyte types designated under each evolutionary line are present. AIJ - adipohemocyte; CO -= coagulocytc; C.H - granulocyte, OE - oenncy-toul; ft = plasmattx-yte, FO = pod«K-yte, PR m prohemmvie, SP = sphemlocyte; VE x vermicyte. (Krom C^ipta. I979) 120 a. r. riipn Structure iMiii /uxoiiimiir \innijicum 121 <>l the uiti>>iii.tliiiii presently, available, the hctnocytc types vary from three tn i-ijjht nr nine liy lijrhl microscope, and seven (PH, PL, (JR. SP. 00, AD, OK) types have been demonstrated by TEM. Unfortunately, hemocytes oi several hemipteroid orders have not lieen studied (tmpta. 19f>9; Arnold. 19741. most of the work beinu confined to a lew species in the order Hemiptera (Poisson. 1924; Hamilton. 1931; Khanna. 1964; Witrojesv.orth, 1955, 1956; Jones, 1965; Uu-Kools. 1970; Za.li and Khan. 1975). Five (PK. PL, GH, AD, OE) types by litfht microscopy and four (PK, PL. GR, OE) by TEV1 studies have lieen identified. The apparent alisenee of SP and CO in the hemipteroid uroup as a whole is probably attrilintalile to lack ol'enough studied species. It is difficult to imagine that these two types have lieen suppressed in the hemipteroid orders. In the neuropteroid group, we find the seven major types (PR. PL, CR, SP, AD, CO, OE), although not all these types have lieen reported from each of the orders in this group (Gupta, 1969), and not all are recognized as types by all authors. As a matter of fact, the types of hemocytes reported vary widely even within an order in this group. For example, two to seven types have lieen reported in Lepidoptera, Coleoptera, and Diptera; five types in Hymenoptera. Neuroptera, and Mcgaloptera; and onlv three (PR. PL, GR) in Trichoptera (see Cupta, 1969. for various listings; Arnold, 1974). Since all the orders are highly evolved, it is quite conceivable that most or all major types would be found in all the orders as more studies become available. 4.7. Summary Among arthropods, hemocytes have l>een most extensively studied in insects. A uniform hemocyte classification is still lacking for insects as well as for other arthropod groups. There is disagreement among insect hematologists about the numl>er of hemocyte types in various insects. From one or a few to as many as nine or more types have been described, particularly by light microscopy. Ultrastmc-hirally, however, only seven types have so far been identified in various insects: prohemocyte (PR), plasmatocyte (PL), granulocyte (GR), sphemlocyte (SP), adipohemocyte (AD), oenocytoid (OE), and coagu-locyte (CO). Of these seven, AD and CO have been described by only a few authors. All seven types have been described under various other names, and these synonymies are mentioned. It is suggested that of the seven types of hemocytes, the GR is the plesiomorphic hemocyte type and has evolved into other hemocyte types. I have postulated that the GR originates from the PR and, goes through the PL stage before becoming a distinct GR type. In tax a in which onlv GRs have been found, the PR and PL are merely evanescent stages and have not achieved distinctness as types. In tax a that are reported to 122 A. P. Gupta iipoll- ( 111 / /'( tlflllt"l>\l\ . e l»Mh. Idc tititit aliou d.s .lustra HRtM I H |0V, Htt»i il'iiml i.O 757-«. Any. I... J. l.hosti' l*Wv l.:251 -hti. Matt.i I \. I WW I'tiinii.. ii des (aillcniholes. Prtsfim-(IIu'iihh .i «ranulr«. tlaiis Ic lii|iiitlc evuvial an coins tie la nun iltiseetes, Cnlleiuholes l. t". fl. Aetul. St i. Pari» MM) Nt-3. Bt-aiil atou. J IWirV Etude ultrastniclurale el * > to* himiquc des «landes pnithoracittucs tie \ rrs .1 sou- auv tniatricint- el tuii|iiunic ai!<-s lar\airrv I. Li tunua orofiria et srs relations a* it Its fibres i-nnjnnctivcs et Its hcinoc\tcs./ lltrastrurt fits. 2'J 474-*-«. Be.uilaton, J . and \I. Monpcsssiu. 197h\ lltrastnicturc et cvtocliimie des hemocytes tl'Anthrrara pernyi Guer. ilA-pidoptera. Attatidae.au tours tin einquieinc äjte lar-vaire. I. Prohenux-vtes. plasmat-w \ tt it tfranulot \ tes J. I'ltrastruct Rr\ i5(2):U'l -56. Beanlatnn. J., and M \Ioii|x\ssnt, 1977. I'ltrastnietnre et c>1»k-himit- des heimkyteN ii'.Xnthrrara prmyt Guer. (la-pttloptera, Attaeidae). II. Gellules a sphendes et m*uo-ot.mle^. Bio/. (Y/i. 88horhiar (Thomas) (Homopteni: Aphitlitlae). Can. J. 'lool 54i2):22M-M. Hrelulin. I). Zaehan. ami J. A. Hoffmann. I97f>. Fonetions des KraiudtKAtes typi- tmes dans la eit atrisation the/ lorthoptere hmista mifiratoriu L.J. A/it row . Biol. Cell. 25(2): 133-6. BreiiKiiou. St., and J. K. la- Bern*. 1976. Fluctuation ol the heiiKHvle fonnula and hcino-Kmph volume in the caterpillar Pirris hra-.su ar. Ann. 'tool. Fco/. An/m. fS(l):l-!2. (In French J Brunt/.. I. 190S. Nouvclles recherches stir l'excretion et la pha«i»c>tose che/ les Thv- saiMNires. .Vrt/i. Zoo/ Fr», lien. 'W.-47I-88. ("aim-ron. G. R. 1934. Inflammation in the caterpillars of lx-pitloptera. J. Pathol. Bar- trriol. 38.441-«6. Carpenter, F. M. 1976. Getdtwtal histors and evolution of the insect. PriK. /5t/i Int. Vonnr. F.ntomol. /976.63-70. ("lark, E. W., and I). S. ChadlMMirnt-. 196(1. The heintnytes of non-diapause antl diapause larvae and pupae of the pink Hoi]worm. Ann. Entiniml Soc. Atnrr. 53.682-5, CoNttu, \. M. 1975. HistiH'hemicat observations of the haemocytes of hicutta miura-toria. HistiH-hem. ]. 7.21-43. Ciossliv, A. C. 1975, The cvtophvsioltijrv of insect hltKxt. Ath . \n\ert Phyxiol. //.-U7-■221 ^ Cuenot, I. 1896. Etudes [>ln >iolouii|iics siir les < Irthopteres. .\rc/i Biol. I4:^l0n Dennell. R. 1947 A study of an insect cuticle. Prix. B So«. Lonil. (B) / 34.79-110. Des au« helle. (. 1971. Etude u 1 trastnichirale ties hetnocstes du Colcnpterc Mrlolontha mrlolontha iL.).J. I'ltraitnut. Bit. .34.492-516. FraiH,-ois, J. 1974. Etude ultrastnicturalc des hemtKytes du Thysatioure Thrrmohia dornest ir a (Insecte. Apterynote). Petlohioloiita 14:157-62. [kissi-ss ntlicr l\|iis IkmiIcs HH. PL, unci (;K. the last further ditU'renti-Mvs iutn SI'. AD. ami OK. lint mtvsvtrih in that urilcr This post-CJR (lifferentiatimi is KcucralK atxiimpanied liv distinct PKs anil PLs, Furthermore, in more liiithK evolved taxa. as well as in some lower oiit'v .in> of the types ma\ Ik- suppressed. A review of the insect heimx-yte literature indicates some phvlo-Kenetic trends in the diversity of the hemocyte t> pes as the evolutionär)' ladder is ascended in Insccta. Acknowledgments Tin- ui,ili-n.il |m-m-iit\ uiiim inr w ltd prints til ntn.itn i's .11 id .illosv inn inr to irpriKln..' their linlitisht'il illustrations. I am most indebted to l)rs. II Akai. H J Hwrwald. \ M ( tin, C Devaiiihflle. J. Fransiiis. C CofRnH. Ch. tircnolre. A. \'. lanid (for the late Dr. \l llailopian). A. k Haina. Salt, and S. Sato. I am Kratelnl to Dr. J. W. Arnold for his eomments and siiJUIestiiHis. The entire i-redlt and ni> dw'p appreiiation lor preparing all the illustrations of t'-is resievs o to Dr V T. Das and Mr. S. B. Hainas«anil. I sin-eerely appret late tile set retallal assislaiiee of Mrs. Joan (iross. References Akai. II. 1969. I'ltrastnietnre of liaenioeytes olisened on the fat-lmdv eells in f/li/o-siimirl dnriniE met.......rphosis. 7pn. 7 Anpf. Kntomol. Zoo/- ft. 17-21. Akai. II.. and S. Sato. 1973. I'ltrastnietnre of the larval lienineyles of the silkworm, flnni-Ityx muri L. tLepidoptera: Bombseidae). Int. J. lust-cl Worpfio/. Emhryol. 2(31:207-31. Akesson. B. 1945. Observations on the liaenioeytes during the metamorphosis of Cmt- lipnoro rrythrmrphala iMeig.i. Art Z<«rf 6(121:203-11. Arnold. J. W. 1952 The haemcKites of the Miditerranean flour moth, fcprie.lio toll- mi IIa Zell (Lepidoptera: Py ralididael. (oil ] Zoo/ 30.352-»4. Arnold. J W 1970. Ilaellloeyles of the Paeifie lieetle tiKkriweh. MulophTa imnttala Can. K11I01110/ /02i7l:830-5. Arnold. J. W. 1972a. Haenineyinlogy in insisi-t biosy steniaties: The prospisil. Can. Ento- mal. I0t.«55-9. Aniold. J. W. 1972b. A eolliparative study of the haemneylf s (IiIikxI eells) of CSTtkroaehes (lnM^-ta: Dietyoptera: Hlartaria). with a view ol their signifieanee ill taxonomy. Can. ITi i r I nrf MMiiM II Arnold, J. W 1974 The hellKK-ytes of Inseets. pp. 201-54 In M. HiH-kslein leil.l. The P/l|/.iio/n«o 0/ Insrcta. Vol. 5. 2nd ed. Aeademii 1'ress. New York. Aniold. J W. I97fi. Biiwysteinaties of the genus Kirxno (la'pidoptera: Notluidae). VIII. The heilUKytologieal (losition ofK. rocjt/iliruei in the "deelarata gn«ip."t.'on. Km/o-1110/ /fW. 1387-90. Arnold, J. W.. and C. F. Hinks. 1975. Biosysteinatu s of the genus Koxoo (l^epidoptera: Noctniilae). III. Hemmytologieal distinetions la-tueen twoelosely related species. E. camiH'ttris and S. ilctlarnld. Can. Enltmwl. HIT. 1095-1100. Arnold, J. W., and E. II. Salkeld. I9fi7. Morphology of the haemoeytes of the giant eoek-nvaeh, Blahrm\ gigooteni. with histochemieal tests. Can. Enlomitl. 99:1138-45. Arnold. J. W . and S. S. Sohl. 1974. Hemocytes of .Uo/orotomo (/iiilrio Hühner (Lepidoptera: 1-isioc.unpidae): Moriihology of the eells in fresh blood and after cultivation in cilro. Can). Zoo/ 52(4):481 j>. Stnulitn- antl laxtnunnif siHnifictlHcr 123 Kt.inyois. J 1975. Ileliuayte el organ. lH'inato|MiietU|ile lit f/ienno/.lo tttmratir* ll'a.k..idi (Thvsaiiura lainsiiMtioVl Inf I ln*rt M.nfM EmluytJ t(6l:477-94. (.olfiuet. (. ...id CI. C.regoire 1975 Coagilliaite alterations ill.toning liena.lvmpli ol Corousm. Miorom. L..\m/i Int P/n/.i.J Hior/irm. VM>707-22 Cralat.V 1871 t elar die Bliitkonarili.il tier Insiskten SU:/, it,../ Warn Am Will Hun 64 9-t4 (.regoiie. CI. 1971 He.....lymph . .kigul.itlon. |>1 HW-nO In M B.« ksteili l.sl.l Tlu Phavhifram "/ /mil to. Vol 5. 2nd ed. Ac.i leinic Press. Sew Villi. (.rigoir. Ch.....id M Kloikio 19VI Bl.««l oMgiilatlon 111 artlimraals I 11a- i.iagiila- tiou ol Insect bhaal. as st11di.1l with the lihase ...ntlast mUToscopc Pliu.io/ Comp ()..,.( J.2. 12B-.3». Crmislone A. \'., S. Holher.iin. alldti. Sail 19117. \n .•l.s1nm-liil.ios,ii,»- study ol eap- ..... lonu.il.oi. lo .......1 bl-«l eells. ( <>// Sri 2.281-92. Cupta. A P. I9H8 llenua-yfes ol S, ulig.r, IIa iiio.io. n/.i(o and the aluvslry ot Imerta. .tun t.iilomol S.o Amir 6/(41:1028-9. Cupta. A P 1989 Studies .4 the bhaal of Meloi.lae Ita.l.snpteral 1 The haenaa^le. of Kpi. onto .iiureo iforster). and a sy noiiyiny of ha.in.ay te tenninologie. ( nl.Jogio 34(21300-44. Cupia.A. P 197" Midgut lesions inEimanla rinrrra (Coleoptera Meloul.a-i Aim C.11- tnnnil So.. Amer. 63.1788-8. Cupta. A P 1979 Arthnaaal he. 11.« ites and |iliy hwny, pp. 669-715 In A P t.opl.i («1.1. .Arl/no/ioi/ P/ii//o«i-iiu. Van \nstraml Reillhold. New York. Cupta. A P.. and D. J Sutherland 196.5. Observations on the spherule nils Iii >.um- II!.ill.m.: |Ortho|nera). Rull. Entamol. im. .Aincr. //.-16I. Cupta. A P.. and D. J. Sutherland 1966 In film transformations of the insect plasmalo- cvte in ivrtain insisils / /11s. .1 Phutinl. 12 1369-75. Cupta, A. P.. and D. J Sutherland. 1967. Phase contrast and Ilisl.K heulical studies ,.r " spherule «flls in ciakroaches. Ann. EnUmal. Sue. .Amer. ««(.31:557-65. Cupta A P and D. J Sutherland. 1968. Kllects ofsublell.al doses ol chlord.ui......the hemocytes and midgut epithelium of /Vriu/oiirra omeriiona A.1.1. IM Sor. Amer. 61(4)910-18. Ilagopian. M 1971 l'nii|iu- structures in the insect granular henna lies. / t llraMrutl Acs 16 64fi-5H. llainilton. M A 1931. The morphology of the water scorpion. WmM riiurea Ijnn Pria Ziai/. So. lAmd 193:1067-1136. Ilarpar, F.. S. Kislev. ami A Zelcer. 1969. El.iln.n-mienisii.pic studies on bein.aytc. of the Egyptian iigtonwonn. Spm/onlem /illoro/ls (Boisduval) Inf.iKil with a nu-clear-|K.lyh.ilrosis vims, as compared to iinniiilectcd hemneytw. 1 Snninferted hemocytes. J. tnrerteor. Pathol. 14:175-85. ... Hinks. C. F . and ) W. Arnold. 1977 Haemopoiesis in Ia-pidoptera. II The role ol the haemo|Miietic organs. Can. J. Zoo/. W( 10): 1740-55. HolTllianil. J A. 1966. Elude des .a-iua-ytoides chez /a.riivrd mig.olorio (Orthopten).;. Wi< rim (Pari.).5.269-72. HoHinann. J. A. t9B7 Etude des hemocytes de bkMM migraloria I. (Orthoiiterel Arth Ztmt. Exp. Ceil. »«.251-91. Iloflinann. J. A . A Porte, ami P. Job 1971) On the localization »I phenolojiilase activity in coagulation of tlnlll inigroloria (L.) (Orthoptera) C H. Ht-M Sronrei .Arm/. Sri Pari. 270D 629-3L lloftuiai.il. J A . ami M. E Stoekel. 1968 Sur les modifications ultrastmcturalcs des coagulia vtes an iiairs dr la coagulation de Ihemolymphe chez un insert* Orthnp-ten.ide: Imk migroloria C ft Seonrer Sor. Bittl Slra.bourj: 162:2257-9. Hoftniann, J. A.. M. E Sna-kel. A. Porte, and P. Joly 1968 l'ltrastnii4ure des hemocytes dr tmmmt migrolorio Ittrthnptrfr). C A. HrM. Seonm Aran!. Sri. Pari« 266:503-5. Identification key for hemocyte types in hanging-drop preparations A. P. C U PTA /VjHiM mtrif n/ tiilumoliniy and Et oitomu Zoo/ncv. Huljtrn ' hu «ruf y. .Nr« Bru.iMj nl. Vi u /. rv.t/ rJMMJ. ( S ..A Contents 17.1. Introduction page 527 17.2. Selected species for examination 527 17.3. Procedure for hanging-drop preparation 529 17.4. Summary 529 Heferences 17.1. Introduction The identification key presented as Table 17.1 is for the novice who is studying insect-or for that matter any other arthropod-hemtK-ytes for the first time. It is often very frustrating for a beginner to try to identify \ anoiis types of hemoeytes in a hemol> inpli sample or film under light microscope. This key should be helpful in guiding the beginner to become acquainted with the seven main tyjx's of hemoeytes described in Chapter 4. Because not all hemocyte types are readily observed in any one species, at all developmental Stages, SUsd under all physiological conditions, it is important to examine hemolymph samples from different species at various developmental stages and under different physiological conditions. The method of study is no less important. The key is based on hemocyte observations under a phase-contrast microscope in hanging-drop preparations of fixed or unfixed hemo-Ivmpli irom various insects (Gupta and Sutherland, 1966, 1967; Gupta, 1968, 1969) 17.2. Selected species for examination Although the key can be used to identify hemoeytes from any species, I suggest the following insects !>e used in the beginning: adult Bla-ht-nis spp. for typical prohemocytes (PRs), plasmatocytes (PLs), gran-ulocytes (GRs), and spherulocytes (SPs); larvae of Gallcria meUoneUa and Porthetriu dispar for typical PRs. PLs, oenocytoids ILV I3.H) ................. PnJicrini* yh \ PR | I* Nucleus ii>>i uumpact, generally small in relation l<> it'll sue. mil m .nl\ filling the nil ... ..............2 2(1'' Nucleus with i hmmatin arranged in t artw hccl-hke I.isIiimii generally eccentric, oval, .nut sharply out lined, cytoplasm hvaliiic. general) v st-ant. may contain some spherical in fitmgate gi.miliar inclusions, it'll sometimes w it„ ev stlike lilehs in process of exoeytosis (Figs 4.2». 7 5. MJC, 13.17) ............CtxtuuhHVh- (Cl» (Beware! CO* mas he confused with OEs and with (•Ks in some insects' 2'. Nuclear t hromatin not arranged in cartwheel-like iaslnon. nucleus ei-centrii m central, cytoplasm not hyaline, abundant, homogeneous, and without any plate-, rod-, or needlelike inclusions and filaments ..........3 3(2'). Cytoplasm generally agranular or slight!) granular, nucleus round or elongate anil central, and may or ma) not appear punctate, cells poly morpliit and van- aide in size in various insects (Figs. 4.IB, 13.10) . . . Pla*matared with that in plasmatn-cyte) and compact, round or elongate, and gent rally central (Figs. 4.1». 13.14) ..............C.runuloct/tv iC.Ri iNote that CHs in lower orders are generally larger than in higher orders.) 5(4'). Crannies in cytoplasm considerably enlarged and appear as distinct spherules or droplets................5' 5'. Spherules nou re Inn gent, generally obscuring the nucleus, unmix r of spherules varying from few to many, nucleus rather small, central or eccentric; cells ovoid or round with variable tiles, usually larger Hi Ml granulocytes, and may l>e observed releasing material from spherules into hemolymph by cxtx-ytnsis (Figs. 4.1CJC. 13.19)....................SphrruhicyteiSPt 6(5'). S|>hemles or droplets refringeiit owing to presence of lipid; nucleus relatively small (compared with that in plasmatocytes or sphemlocytes). round or slightly elongate, central or eccentric, and may or may not appear concave, biconvex, punctate, or lohatc; cytoplasm may contain other nonlipid granules (Figs. 4.2C. 10.1C. 10.4 (larger cell) ...........AdiiHthemitcytr IAD) /e easier to focus them sharply near the periphery. 6. Make a fresh preparation for examination every 8-10 min because hemoeytes liegiu to deteriorate after bleeding. 7. For longer-lasting preparations, you may try hemolymph from an insect that has been heat-fixed in water at 60 °C for al>out 5 min. 17.4. Summary A beginner's identification key for seven main hemocyte types (prohe-mucytes. plasmatocytes, granulocytes, spherulocytes, adi|M>hemo-I \ tcs. coagulocytes, and oenocytoids) is presented. It is suggested that hanging-drop preparations of hemolymph be used to identify these hemoeytes under phase-contrast microscope. References Cupta, A. P. 1968. Hemoeytes of ScutiRerella Immaculata and the ancestry of Insecta. Anrt. Entomot. Sue. \..... 61 (4): 1028-9. Gupta, A. P, 1969. Studies of the blood of Meloidae (Coleoptera). 1. The haemocytes of Epicauta cinerea (Forster), and a synonymy of haemocyte terminologies. Cytologia 3^(2):30O-44. Cupta, A. P., and D. J. Sutherland. 1966. In vitro Iransfnnnations o) the insect plasmato- cyte in certain insects. J. Insect Physiol. 12:1369-75. Cupta, A. P., and D. J. Sutherland. 1967. Phase contrast and histochemical studies of spherule cells in cockroaches. Ann. EntonuA. Sor. Amer. 6Ö(3):557-65. 18 IV. Techniques [used hemocytes under light microscopy: technique* J \V AHNOU) AM) C f MINKS HtoiyWi mutti * hVsiiinh Jioflfuf* H< srun *i Nrum ii tn wifun ('(jMuWfi Ofidu a 1 i. K i \ "i '1 ( unit Ait (contents 18.1 Introduction 531 18.2. In sivn procedure 533 18J. In vitro procedure 534 18.4. Blood film preparation 534 18.4.1. hi tat ion and slid*- pn -p.ir.il..... 18.4J MainuiK Hap.d Cu-msa staining Full (tiemsa staining CouillM-llls Hematoxvliii-ensiu-ahtan blue staining Comments Vena aniline staining Comments At ndmm- orange staining Comments 18.5. sni mu.ni 537 References 18.1. Introduction In general, the technuiues used in vertebrate hematology must l>e modified for the study of insect hemocytes. The same principles of technique apply, and the insect hemocytes can be observed in vivo, in vitro, in living culture, and in blood films fixed and stained in a variety of wuvs. The procedures described below are a small sampling of these techniques. Each procedure provides a somewhat different view of the cells, and it is often useful to employ more than one method, if 'possible. Such a combination of methods at best includes an in vivo or in vitro technique as a basis for interpretation of fixed and stained cells. To some extent, all the methods are empirical l>ecause of variability in the character of both hemolymph and hemocytes in different species. Most of the following techniques have been found suitable for a wide \ ariety of insects, but there are preferred methods for certain species 531 M I W, AmoM ami ( h llinkx P- O jo Fins. 18.1-18.7 Hemoevtes of insects as seen with various techniques. Magnification as indicated, io iiiierons 18.1 Hemocytes of Rttdn-ru* giganfru-v {Dictyoptcra) in v i\ ii. mi a wmg vein. I8.2. Hemocytes ofEuxoa drrtarata ll.cpidnptera) in vitro, as a wet film I8.3. Hemoevtes of Aftf/rtfo.sowirt disxlria i I.epidopterat in \ itro. in siiUiil- /itsrcr Iuhuh utis under light miirov* o/ii/ techniques 533 and for tlillerent purposes, and the in v iv o procedure is obv louslv limited to species with transparent areas ol the ImmIv . IS. 2. In vivo procedure A relati\el> cleat and sometimes excellent view of living hemoevtes eau be obtained with transmitted light through transparent regions or appendages ol certain insects. The procedure is similar hi the one long used bv vertebrate hematologists to demonstrate living blood tells in thin tissues, such as the ear membrane of the rabbit 01 the toe membrane of the frog. Sirnplv, it involves immobilization of the insect so that the transparent structure can be manipulated under the compound microscojM'. Wings are most suitable lor this purpose, and for best results thev are sandwiched in glycerol or refined immersion oil between thin glass coverslips to reduce diffraction at the cuticle-air interlace (Arnold, 1959). The wings of orthopteroid insects are best bv far, as the blood circulates free I > there in thin veins and sinuses, and the licmocv tes are large- ('articular!) among the large hlal>crnid cockroaches, the hemoevtes can be obscived here with great claritv at high magnification {Kig. 18.1), circulating free I \ in the blood or moving amehnidally in regions out of the main How (Arnold, 1961). Not all dear-winged insects are so satisfactory, tor a variety of reasons (Arnold, 1964), including the small si/e of the hemocytes (e.g., among Hcmiptera), the extreme thickness of the vein walls (e.g., Odonata), the near occlusion of veins by tracheae (e.g., Lepidoptera), the poor circulation ol'blood in the wings (e.g., Dtptera), or the extremely rapid circulation of blood in the wings (e.g., Hymenoptcra). Nevertheless, w ith ingenuitv and patience main such insects can Ih> used to gain an impression of the real si/e and form of the living hemoevtes liefore resorting to techniques that involve injury or sacrifice of the insect or denaturation of the hemocytes. Similarly, with ingenuity and patience other appendages and clear regions of the insect body can serve as windows to the blood. The legs, prothoracic extensions, caudal eerci, and/or respiratory structures of some aquatic insects can lx- useful, and the dorsum of the abdomen sometimes provides a view of hemocytes in the dorsal sinus or heart. In these cases too, clarity is improved by covering the structure or area with saline (with a trace of wetting agent) or glycerol or immersion oil under a coverslip. Al- U1o(k1 from the insect to glass without or with little exposure to air. There are two common ones: hlood under oil and as a wet film under a coverglass. Perhaps the simplest technique is to sev er the antenna under mineral oil on a glass slide. BIikhI issues from the cut end and (onus a discrete glohule on the slide, protected from the ail h> the inert oil. I'uder oil immersion, or at lower magnification with an immersion adapter, the heinocytcs can lx- observed in their norma! form as they How from the antenna and in gradually changing form on the glass surface. The more common technique, the wet film, involves directly transferring a drop of hlood to the microscope slide and covering it immediately with a coverslip ringed with iH'troleum jelly to exclude air. The hemocytes are seen hen' clearly (Fig. 18.2), Ixith in susjieusion for a short time and attached to the glass. They slowly alter their form and size under these conditions and should be examined immedi-ately. It is here that the explosive so-called coagulocytcs may lx- identified best. The \ icw of tells m culture is somewhat similar to this (Arnold and Sohi. 1974), through the Hat surface of the standard tissue culture flask (Fig. 18.3), but the typical hemocyte forms are not maintained in subcultures. The technique lor wet films can be used effective!) also with treated hlixxl. Heat-fixed blood can Ik* used directly, or living blood can be diluted in fluids that suppress coagulation, such as Turk's diluting fluid (Fisher) or dilute solutions of sequestering agents such as tetlasodituu etln lenediaiiiinntetra.k ctatc. Here the form of the heino-cytes is retained for longer periods than in wet films of living hlotxl. 18.4. Blood film preparation Although vertebrate blood can be prepared lor microscopy by placing a drop directly on a microscope slide and drawing it out with a cover-slip to dry quickly in air, insect blood treated in this way will usually agglutinate before the film can lie made or else will show much cellular distortion. For this reason, it is best to fix insect blood before preparing the film. This is accomplished in two ways: by a suitable degree of heat fixation or by dilution of the blood in a fixative that does not uimiedlateb cause gcl.itmn ol the hciuolMiiph Heat fixal.....cau be lollowed In othei U|x-s ot tivation il desiie.l attei the film has Jilci on the slide Tile vertebrate teel.....|lie. lislng lne hhxxl dírce tl\ on the slide, lan serve in speciál prc|>aratlons where cell lom, is less iinpoit.uil lhán niicle.u uitcgrltv In such čase. a Ix-adnf tresh blond Is cx|xixcd......ic diatclv to starek .md v.i|»,i i,„ mmti fiv.in.on I Im nsiiueihia „ ,i, tailed Ix-lnw. is cllectne lot deinonstiating mitosis in h......a \ tes using miclt-ar stains 18.4.1. Fixation and slide preparation Heat can serve to fix hellnx Wis very rapidly within the nisei I. with out appreciable change to then sha|x- or si/e. and at the same time prevent coagulation of the hi-.....lymph. Coiiseqiicntb . after heal lixa .....,,M' can la- withdrawn limn the insect and spread e.isib on a microscope slide without clumping or distort.....of the cells At the same time, the hi.....f hlixxl ami its cells adheres evciilv to the glass without need for ailhesiv is Ileal fixation is accomplished In piling ing the insect into water laid at appm\im.,teh bO (' loi a short period. The teniixratiirc can lx- varied within 5 °C depending.....he size of the insect, anil the cvixisurc varied from I to III linn on the same basis Within these- limits, the teiii|k-rature and tune are not nli cal. but should Im- regulated lor particular sixt ics In empiru al lest ing. Slide preparation with heat-fixed hlood is ., simple process ,,f placing a drop on the slide and touching it with the edge ol a cnei slip, which is then drawn along the slide with the drop trailing lx--hind. The resulting hlixxl film is air-dried (preferabb on a slide wanner at alxiut 32 "(.') Ix-fore staining. Dilution of living blixal directly into a water-soluble eheiiiiial fiva-five also accomplishes rapid fixation of the hernias tes will, little coagulation of the heinolymph or cell distortion. It is the prefern d inethixl for some insects and for some puqxises, especially when a sin cession ot bhxxl samples is required from the same insect Dilute solutions of fonnalin f.OT ) or glutaraldelnde (0.4 M) are recommended The procedure involves the pooling of some of the fixative solution on the mi croscopc slide, immersing an appendage such as the antenna..... and severing the appendage so that the hlixxl Hows diiectb into the solution without contacting the air Bhxxl from other t> |xs ol wounds can also lx- drop|x-d from alxne directly into the fixatixi- so that expmure to air is very brief. In either case the mixture is stirred immediately to prevent clumping of the cells and can lx- spread on the slide if desired. The mixture is then air-dried completeb on the slide and rinsed to remove traces of fixative Ix-fore staining. 538 / » Aiuo/d um/ ( f IWatl 18.4.2. Staining For most purposes, henna vtcs arc stained in one ol the Konianow skv preparations, which depend on the iorination of azure and other oxidation pnxlucts ol inethvlcne bine, usually in combination with cosni ,H......ison. I!*>71 With these preparations, variation ol the biillcr level toward the acid side increases the precision ol nuclear staining and docnascs cytoplasmic basophilia, the reverse increases the ainonut of blue in various elements One can. therefore, alter tlx- cite, I ol the slam loi dlflcrcnt piiqxiscs We find lhal the Ciemsa prcpa-ration is most reliable and use it almost exclusively It call be used dm , tlx lor rapid staining oi with differentiation lor more elegant results .sec also Chapter 20). Rapid Ciemsa staining (Fig. W.4) Solution ol Cicnlxa (Fisher): 1 drop of concentrate pet milliliter distilled water in a Stender dish. Place air-dried slides directly in the solution or after 1 mill ill absolute methyl alcohol, Inspect slides lor depth ol staining alter 3 min (max. 5 mill). Rinse in distilled water lor 1 min Blot-dry using Kixlak lens-cleaning tissue. Mount pennanent slides in Canada balsam. Icinixir.uv slides in glycerol or immersion oil. Full Ciemsa staining (Fig. 18.5) Immerse air-dried films ol heat-fixed hcinnlyinph in Gienisa solution (1 drop of concentrate per millilitel distilled water) lor 20 mill to 2 hr. Rinse in distilled water; then immerse briefly in distilled water to which a lew drops ol 'lithium carbonate have been added (to differentiate red-staining structures). Rinse in distilled water, then immerse bricflv in distilled water to which a few drops of dilute hydnx-hloric acid have Ix-cn added ito dilfen-ntiate blue-staining stnicturcs). Rinse in distilled water and examine. Repeal dilTerentiation if staining is tixi dense. Blot dry using Kixlak lens-cleaning tissue. Mount ill Canada balsam. (nmiiii-iirv. Cells are latter differentiated than when rapid Ciemsa inethixl is used; excellent for photography. Hematoxylin-eosin-alcian blue staining (Fig. 18.6) Immerse air-dried films of heat-fixed heinolymph in LOT acetic acid in methanol for 20 min. Hydrate through graded alcohols to distilled water. Immerse in 1** alcian blue 8CX in 0.1 N HC1 for 20 mm. Rinse in distilled water and immerse in Harris's hematoxylin for 20 min. Rinse in distilled water and differentiate in acid alcohol; then blue in Scott's solution. Kxamine and repeat dilTerentiation if necessary. De- hiseet hemtteutes under light Wlicriiseopo fec/iiili/ues 537 hydrate to 9(r, alcohol and immerse in IS covin m 90S alcohol lor 3 min. Rinse oil excess stain in 90S alcohol, transfer to absolute alcohol, then to xylene, mount in Canada balsam. ('nnimeiifs Mitotic cells are easier to identity than in Cicnixa-st.uncil pre|*tratinus. Sphendia-ytes are verv distinct, retaining ixtlc bluc. whereas the cytoplasm ot all other cell l\ pes stains pink to pinkish purple. Acetocamiine staining (Fig. 18.7) Kxpress small lx-ad of fresh heiiioKuiph from a COf-narcotizcd insect onto the center ol a round coveiglass and invert osel a \ lal of glacial acetic acid for 90 min. Reverse coserglass with bcinolv inph tip-|x-nnost in a solid watch glass anil cover with accttx-annuic (Humason, 1967) for 90 mm. Blot off excess stain carefully and squash the preparation gently onto a microscope slide in a drop ol Venetian turix-ntme. Comments. Cell tyix-s are identifiable; mitotic cells very clear in all cell tytx-s, excellent for photographs. Acridine orange staining • Kxpress a small drop of fresh hemolymph from a COf-iian-otized insect onto a quartz inicroseoiK- slide. Mix will, an equal volume of acridine orange solution. 0.1 mg/nil in 0.9S N'aCl. Place covcrglass ovel hemiilymph preparation ami immediately examine with fluorescence microscope. Comments. The inclusions of sphendia-ytes an- rapidly and specifically stained to gist- an intense orange fluorescence. Nuclei ol all hc-mocytes give a yellowish green fluorosceiue. 18.5. Summary Techniques for light microscopy of insect heniiH-v ti-s are mcxiifica-tions of those used in vertebrate hematology and include in vivo ami in vitro pnxedures as well as methixls of fixation and staining. The in vivo pnxedures utilize- transparent regions of the insect lxxly, preferably the wings sandwiched in an inert solution under glass. The in vitni pnx-etlun-s include the examination ol bhxxl issuing Inxn a wound under oil, of the cells in culture, or mon* commonly of preparations of covered wet films of living bhxxl ringed with inert oil to exclude air. ľo 11.u.,11, u i of insect hlixxl for standard histological staining should lx* pn-teded by the killing of the insect in hot water to fix the cells and prevent heinolymph coagulation or In dilution of the living Techniques for total and differential hemocyte counts and blood volume, and mitotic index determinations M. SHAPIRO (-WW* Moth Mrthtxts /)ci e/ojimcnŕ laboratory. t'.S thiittittm-iit of Aurirulturr. Oft, Air hont ««»■. \la*\»irhu\,-tt\ OJWJ t s v Isaer 539 Contents 19.1. Introduction 19.2. Total hemocyte count 539 19.3. Differential hemocyte count 541 19.4. Blood volume 543 19.5. Reliability as influenced b> internal and external factors 544 19.5.1. Internal factors 19.5.2. External factors 19.6. Summary V4i> References 19.1. Introduction The three most common measurements made to descril>c the blood picture ol 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. 19.2. Total hemocyte count The first study of THCs in insects was made by Tauber and Yeager (1934). In 1935, they studied Orthoptera, Odonata, Hemiptera. ami 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 pi..leg. 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 (NaCI, 4.65 g; KC1, 0.15 g; CaCI„ 539 540 ■ Sfcapfca 0.11 g. gentian siolet. 0.005 g. and 0.125 ml acetic acid/UK) ml) was used als„ lis Ktsher (19351. Smith (1938), and Shapiro (1967. 1968). Other physiological saline solutions were utilized by Roxenlx*rgei and Jones (I960). Collin (1963), and Gupta and Sutherland (1968) Acetic acid, a component of the latiber-Yeager fluid, was used lor raWfria (Stephens, 196). Shapiro. 1966; Jones, 1967a) and for lit-Hutflii (Shapiro et al . 1969; Vinson. 197 1). Turk's solution (1-2'7i glacial acetic acid, slightly colored with gentian violet) was used forfYi -fmoji/ii.™ (Clark and Chadlxmme, 1960) and for Euxim (Arnold and Hmks, 1976) l'atton and Flint (1959) reported that Turk's solution did not present coagulation in hemolyniph samples of Periidanela. Ver-sene 11 -2' i tctrasodium EDTA) was superior to Turk's solution and oxalate and was routinely used. Wittig (1966), studying phagocytosis in Psrudaletia larvae, also found versene (2"? plus a trace of methylene blue) to la' superior to glacial acetic acid. Formalin (tM in 0.85'* NaCI) has also been employed as a diluting fluid (Jones. 1956). Physiological saline did not prevent cell agglutination, but the addition of acetic acid (lr/<) or formaldehyde reduced clumping. In making total counts from Pifri.v, the first and second drops of he-mob mph were utilized (Kitano, 1969). The first drop of hemolyniph in unfixed and unfed Rhodnius contained more hemocytes than the second drop (Jones, 1962). The number of hemocytes was also reduced from three successive drops of Bombljx hemolymph (Matsumoto and Sakurai, 1956). On the other hand, Wittig (1966) found no significant differences in DHCs taken from the first and second drops of Pavuda-li'lin hcmolsinph. Jones (1956) used the first drop of Setvophage hemolyniph for DHCs. The rest of the hemolymph was placed on a second slide, and a portion was used lor THCs. Some 3 or 1 drops of Prodenia hemolymph were allowed to flow on a glass slide. A portion of the blood was drawn into aThoma white blood cell pipette, diluted, and counted (Rosenberger and Jones, 1960). After the hemolymph was diluted in a pipette, the first 3 or 4 drops were discarded (Jones, 1967a; Shapiro. 1967, 1968). In many instances, hemolymph was drawn into a Thoma white I,I,.oil cell pipette, diluted, and counted in a hemacytometer. The he-nnib mph dilution ranged from 1:20 (Rosenberger and Jones, 1960; Wittig, 1966) to 1:50 (Clark and Chadbourne, 1960; Shapiro, 1967) to 1:100 (Fisher, 1935; Gupta and Sutherland, 1968). Wittig (1966) adjusted the dilution of hemolymph so that a suspension contained between 800 and 1,600 cells/mm2 area counted. This adjustment could not be made when the amount of blood available or the number of hemocytes per cubic millimeter was low. Fisher (1935) found that the standard white cell pipette required too much hemolymph from Periiiltmctti. A micropipette was made that re- llrwiociife ftrwnfs. hlthxt ndumt-.uttd itntuth im/cr 541 quired only 1.7 ,,i anil a final dilution of I 44. Partou anil Flint 11959) also used a s|x-cial micropi|x'ttc (or Ptripltuu-tti in which I id of hemolyniph could In drawn ami diluted 1: 100. THCs are counted in a standard hemacytometer according to the formula ijni.es, 1962): hemtx-ytes in v 1-mm squares x dilution x depth of ehamlxT number of I-mm squares counted kitano (1969) counted the millllxT of hcmix-ytes in the smallest square (0.00025 nun'), counted 80 squares, and multiplied b\ a factor 01 4,000 to give THC. Fisher (1935) counted hemocytes from three of the four white cell squares in each ol the two chamlx-rs. Wittig 11966) calculated the THC |xt cubic millimeter b\ i-ounting cells in four I-uini1 areas in each of the two chambers. Heimx-ytcs Irotn fi\e 1-inin1 squares (the lout cornel anil central squares' were counted li\ Rosenberger and Jones (1960), Jones (1967a), Shapiro (1966, 1967), and Gupta and Sutherland (1968). White cells m all nine 1-mnr squares were counted by Clark and Chadlxxirne (1960). In counting the hciiincytcs. a variation is to be expected. But when the distribution ol the cell count was uneven and clumping was oh-scrx ed. the counts were discarded (Rosenberger and Jones, I960; Shapiro, 1967; kitano. 1969). Stephens (1963) questioned whether it was reasonable to discard counts when the means of the two chambers differed greatly Wittig (1966) felt that such a rejection was justified if it was done on the basis ol percent of the mean instead of the number of cells per volume, "tor the weight of a luniilier is different for high and low means." 19.3. Differential hemocyte count In general, the method used by Shapiro (1966) may he considered typical, l^arvae were submerged in a hot-water bath (56-58 °C for I- 2 min), and a prolog on the sixth abdominal segment was cut with fine scissors. The hemoKuiph was allowed to fall on a clean, grease-free microscope slide, and a smear was mad" in the conventional manner. Ii> drawing a second slide across the first one at a 45 ° angle. The smear was allowed to air-dry and was stained by a mixlified Papjx-n-heim-panoptic method (Pappeiihcim, 1914). The details of the method are as follows; (1) Hoixl air-dried smear with Max-Gninwald solution and allow to remain on slide for 3 min; (2) add distilled water so that a layer is formed and allow to stand for 2 min; (3) discard the Ma\-(óunwald-water layer; (4) Hood slide with Cienca solution (1 part concentrated Cicmsa to40 parts distilled water) and allow to dry; (5) wash slide in running tap water and allow to dry. 542 \( .S/lfl-ll liii- smear is examined under oil inline ision. ami 2(KI cells per slide art- differentiated. B> this method ol staining, azurophilic material ap-|x*ars purple red, chromatin, reddish violet; and basic protoplasm, blue lPap|>cnhcun, 1914). I'sing the Ycager tl945i , l.issitication system as modified by Jones 119591. the following ty ]M's ol hemocytcs were counted: probemoi y tcs iPRs). plasmatoi v tcs iPl.vt, granulocytes (CRv). adipohdiitxy tcs (ADs), sphentlncytcs (Si's), potlocytes (POs). ami oenocytnids (OKs). Degenerating cells and cells in mitosis were also counted. In addition, heinocytes were lound that could not he placed in the preceding classes with certainty, these cells were designated as unclassified cells. Jones (1982) recommended that a minimum of five insects of a given stage anil pin siologica! status be used. Whenever possible, a ininimiiin ol 2tKI cells should be classified per insect. In Hhodnius. 1(H) heinocytes were classified Iroin each nl five insects as either PRs, Pl.s, GRs, or OEs. Mitotically dividing cells were also counted (Jones, 1987b). Vinson (1971) examined stained blood films from a minimum of five lh'lit>tlii\ larvae pet time |x*riod. A minimum ol 190 cells per larva was classified. A minimum ol 200 anil a maximum of 5,(XX) cells were counted in Tviwbho larvae (Join's and Taubcr. 1954). Jones (1987a) counted 200-1,000 cells in each Gulhriu larva. Whenever possible, 2(X) tells were counted in SarcopluiHti (Jones. 1956). in Pcriplaiwtu (Gupta ami Sutherland, 1968), and in OriMiip/u/u (N'appi ami Streams, 1969). Arnold anil Hinks (1976) examined 200 hemocytcs per smear of the uoctuid, Euxoa. Five smears were examined per instar. Frnm 15 to 20 sections of each face Hy larva, Ortlwllui. were examined, anil a minimum of 100 cells was counted per section (Nappi and Stnllolano, 1972). Wittig (1966) counted 350-400 hemocytcs lor each DHC from Pscudidctia. Veage. (1945) attempted to count at least 400 tells in each DHC from Prodcniti. but could not from young larvae, old pupae, and adults. Differential counts from Atui^usta larvae were obtained by classilying 500 cells prat smear. At least 10 larvae from each larval stage were used (Arnold, 1952a). In a subsequent study on the effects of fumigants on the heinocytes of Anwgd.v/ii, Arnold (1952b) made DHCs fror 20 larvae per time period; 500 cells were also counted in blood smears of Blabents (Arnold, 1969). 19.4. Blood volume BY is a little used but important value. It has been reported that an inv one relationship exists between the THC and BV in Bomhtix (\i(-tono, 1960), in adults of l^ttcusta (Webley, 1951), and in last-stage nymphs and adults of Periplunrta (Wheeler, 1962, 1963). In atldition. lltm,hVt, counts. /,/,««/ , olumi. ami mitotic ;,,,„., 543 Wheeler (19631 found that the absolute nuuiher „| circulating hemo-i vies per cubic inilliineter. obtained bv mtilliplving the BV and the IHC. was relatively constant, notwithstanding changes in both the n\ and the THC. Ill a few instances, BVx were detern......d bv weighing insects re moving as much hemolvniph as ,n>ssiblc. and reweighing the insects (Kichardson et al.. 1931; Arnold and Hinks. 1976, Tins cvs.mg,,,,,,-tioii method apiiears crude, as.....re sophisticated methods are available. Smith 119381 employed the tell dilution „,ctl„>d „I Vcagc „„| I auber 11932). The billowing lonnula was used: i, o ■ c, CO - ij where V„ = Intal Mood v ohiti.e;,/ = amount of dilution fluid in cubic millimeters; co = original tell count per cubic inilliineter e, - diluted blood cell count per cubit millimeter; and a = volume of blood drawn in making the original count. A dye solution method (Ycager ami Miinson. 1950) was used furS'ur-™gf 'J'""-- 195ßl* Tcm-brio (Jones, 19.57). and (;„//,ri« (Shapiro 1966; Jones, 1967a). An 0.2''; amaranth retl the in 0.85°, NaCI was in-letted into Sarcopluiuu larvae ami pique, H „( |KKlx weight (Jones 1956). Calbria larvae were injected with 10 „1 of |0j amara„th red in' sahne per «nun body weight. The dye » as allowed to circulate vv ill,,,, the hemocoel lor .3-5 min. Then hcniolytnph was drawn, ami the intensity ol color was compared to a series ol standards Hemolv mph volume percent were cunverted into microliters (Jones, 1967a) Shapiro (1966) investigated the BV of Galhria larvae The dye method employed was essentially that used l,v Ycager Mad Mm,so,, (1950) and modified by Lee (1961) with further modifications bv Mar-«gnoni and Milstcad (pcrs. comm.). Each larva was weighed .„„| i„. Idled will, ., 1'; aqueous amaranth solution. The volume line, led vv ,s equal to M ol the insect's laxly weight. Alter the amaranth was inlet ted. the Ian., was placed mashell vial (1.5 x 6.4 cm), .„„I tl„-tly, was allowed to circulate within the hemticoel. After 10 mi,, a proiea on the sixth abdominal segment was cut. and the blood was collected «1 capillars robe (Kitnax No. 34500) that had been Hooded with pure nttrogen to retartl molanization. The blunt end ol the tube was scald on an alcohol burner, ami the tube was refrigerated (4 °C) lor several minutes and centriftiged (3,100 rpm lor 10 min). The tube was re-cooled, and the portion of the tube containing seihincntcd henioevtcs was < „1 oil and discarded. The plasma was drawn into a disposable Driuiunontl nucropipette (I0-Ml capacity I and diluted in 1 „,| Arons-son s buffer (0.99.5 M). This solntion and the stantlard (Aronssnn's buffer) were placed in separate 1 ()-„,l cuvettes (10-mni light path) and 544 hi Sin the relative ahsnrbanccs were determined with a Bccknian DB spectrophotometer at 515 linn, the wavelength at which the maximum ab-sorh.mtc of amaranth occurred. Previously, the absorbanccs of known concentrations ol amaranth had been determined, and. in accordance with Beer's law. the absorbs*--* was proportional to the concentration. The absorbancc value ol the lest sample was platted against the concentrations ol known samples, and the tout filtration ol the test sample was thus obtained. < hue the concentration of amaranth in the test sample had been determined, the blood volume was calculated by the following lonnula: V ■)l (<") where V = blood volume in microliters; s-sible. ol the same stage, instar. age, size (Wittig, 1966), and possibly sex. Ill last-stage army worms, even among an apparently homogeneous group, individual THCs ranged from 75ecomes important not to add more variability to the system. It would lx* Ix'ncficial to the field ol insect hematology if worker* using the same species of test insect would use the same techniques, so that variations attributable to different techniques could be minimized. 19.6. Summary Techniques are available to investigate changes in hemocyte imputations regarding cell numbers (THCs), cell types (DHCs), and blood volumes. Moreover, these parameters can be combined to determine the absolute numbers of hemoeytes within an insect at a given time or through time. Unfortunately, many studies have involved a single parameter. The reliability of these techniques must be critically evaluated so that only the best available ones are used in hematological studies. We must answer the question: Does heat fixation present' the hemocyte population in situ or does it artifactually cause large numbers of hemoeytes to enter the circulation. Perhaps, each method J>e defined as to its lx*st usage, so that a mosaic of techniques iiiiutit tu^lWBBsi|-aU^>est advantage. Once a nu-thodi^sr^lBSBSsskLI" use. it must l>e followed precisely Variations in a method might prT5Wss|(^i|rjath>iis m results. Assuming that a given method is carried < !!re*ffr'.UIJW .lls[ be given to the test insect. The population should be defined as to age, stage, size, and sex in order to minimize inherent variations. If periodicity is a problem as far as reproducibility of data is concerned, then tests must be carried out at the same time. Is periodicity of hemocyte populations a problem? The growth and development of the insects should be optimal under laboratory conditions. The use of semisynthetic artificial diets and improved rearing techniques should improve the synchrony of growth and minimize the variability of food materials. References Aniohl, J. W. 1*152.1 Tin- haemoi \ ti - of the Mediterranean Hont moth. Ephentia kuh-nirlla /ell {Lepidoptera: Pyralididae). Can. J. tool. 30.352-64. Aniohl, J. W. 19521). Effects of certain fumigants on haernocytes ol the Mediterranean Hour moth, F.phestia kuhniella Zell. (Lepidoptera: Pyralididae). (ľan. }. Zintl 30.365-74. Arnold, J. W. 1966. An interpretation of the haemucyte complex in a rtonefly, Acrv-neuria arrmna (Pteeoptera: Perlidae) Can Entomol. 98 394-411. M N/mpifo Nhapiro. M I9h7 l'atholngit i hanges in the hlood ol dir iin.iln was mutli, (ialhna "M .'/.■ "i. IIa du ring ihe • mirsc ol uucleopolv hetlrosis and stan atlon I I otal heino- cyte tonnt / Imertrhr Palhol 9 111-13. Shapiro. \t l9hH Pathologu changes in the huxxl ol the greater was moth iiallrha mrtlnnella. diiriliK im» lenixilyhedrosis and starsatmu II DihVrcutial hemocyte coum / Imeetehr Pathot 10 230-4. Shapiro. \| . Ii I). Stink, and C. M IgunHo I9h9 llcmtxytf changes m larsae ol the Ixillstomi H-ii.'ihf :ea. inlected ssith a iiuclco|xilv hetlrosis sinis. f. Int rt Ii In Pathol 14 28-30. Smith. II W l'tis The hhxxl o| the i ixkroach Penplaneta amencana l, Cell stmeture and degeneratinn, aml eell eimiits Yen llampthirr Auru Elp Stn. Tech Rull Stephens. J. M 196.1 EHei ts ol ,u tu e imimini/atioii on total hemocyte counts ol larvae ol i lallt > i,. t„, !!..,„ II,i il.miiaens) 7 Innert Pathol "> 152-6. Tauhrf.n. E.and J E Yeager 1934 On the total Mond diemoly mph) cell count ol the tield encket, (.n///ie> mumi/n /x-rimi/ii «mein Hann, lou a Statt- ('oll. } Sei 9 13- 24. Tauher. () I and I I Yeatfer 1935 On total hemoh mph ihlotal) connts ol mseits I Orthoptcra. Odonata. Ilemiptera, and Momoptera. Ann Entomol Sur Amrr SA 229-411 I ml., i OK. aml 1 K Yeager !9itS On the total hemolvmph lilo.nl' all coiints ot in-sects II Neurnptcra. Colcoptera. I a-pidoptera. and Hy menopter.i Ann Entomol s« Asssr. Ist IM ss, \ ihm in, S B 1971 Defense reaction and heimxytic chaiiKes in //. liothi - i treuen* in response to tts hahitual paranoid Carttutehüet murircp\ f. Imertrhr. Pathai IH 94-100. "*-. Wehley. D P 1951 Iii eell counts in the Afncan imgratnry lociisl \l,ocu\ta mtnra ton** mipratunoutes Reiche and Eainnairel Pro« R Entomol. 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