Monoclonal antibodies bind distinct classes of hemocytes in the moth Pseudoplusia includens

Journal of Insect Physiology 45 (1999) 113–126

Monoclonal antibodies bind distinct classes of hemocytes in the moth Pseudoplusia includens Elisabeth M.M. Gardiner, Michael R. Strand

*

Department of Entomology, University of Wisconsin-Madison, Madison, WI 53706, USA Received 25 February 1998; accepted 25 April 1998

Abstract Insect hemocytes have historically been identified on the basis of morphology, ultrastructure and hypothesized function. Among insects in the order Lepidoptera, five hemocyte classes are usually recognized: granular cells, plasmatocytes, spherule cells, oenocytoids and prohemocytes. We have generated a panel of monoclonal antibodies (mAbs) against hemocytes of the moth Pseudoplusia includens. In this study, hemocyte identification using 16 different mAbs was compared to identification methods using morphological characters. Three main categories of mAb binding activity were identified: (1) mAbs that specifically labeled only one morphological class of hemocytes, (2) mAbs that labeled granular cells and spherule cells, and (3) mAbs that labeled plasmatocytes and oenocytoids. With one exception, none of the antibodies bound to other tissues in P. includens. However, certain mAbs that specifically labeled granular cells and/or spherule cells in separated hemocyte populations also labeled plasmatocytes co-cultured with granular cells or cultured in granular cell conditioned medium. Overall, our results suggest that granular cells are antigenically related to spherule cells, and that plasmatocytes are antigenically related to oenocytoids. The use of mAbs as hemocyte markers are discussed.  1999 Elsevier Science Ltd. All rights reserved. Keywords: Insect immunity; Hemocyte; Lineage; Ontogeny; Encapsulation

1. Introduction Accurate identification of hemocytes is essential to understanding cell-mediated immune responses in insects. Hemocyte identification has historically been based upon morphology, ultrastructure, and hypothesized function (Jones, 1962; Gupta, 1986; Brehelin and Zachary, 1986; Lackie, 1988; Ratcliffe, 1993). Unfortunately, these characteristics often vary with taxa, life stage, and how hemocytes are collected or maintained in culture. As a result, confusion still exists in the literature on how hemocytes should be classified, the ontogeny of different hemocyte classes during postembryonic development, and the function of different hemocytes in immunity. The hemocytes of Lepidoptera are usually divided into five classes on the basis of morphology: granular cells, plasmatocytes, spherule cells, oenocytoids and prohemo* Corresponding author. Fax: ⫹ 1-608-262-3322; E-mail: [email protected]

cytes (Ratcliffe et al., 1985; Brehelin and Zachary, 1986; Lackie, 1988; Strand and Pech, 1995). In the moth Pseudoplusia includens (Lepidoptera: Noctuidae), granular cells and plasmatocytes together account for approximately 85% of the hemocytes in circulation in last stadium larvae (Strand and Noda, 1991; Pech et al., 1994). Both cell types adhere to and spread on the surface of untreated culture plates, are phagocytic, and participate in capsule and clot formation (Strand, 1994; Pech et al., 1994; Pech and Strand, 1996; Clark et al., 1997). Plasmatocytes and granular cells are distinguished from one another by differences in size, ultrastructure and gross morphology when spread on a planer surface. Granular cells usually spread uniformly with each axis of the cell being approximately equal in length, whereas plasmatocytes spread asymmetrically with one axis of the cell almost always longer than the other. Spherule cells, oenocytoids, and prohemocytes are nonadhesive cell types. Spherule cells account for approximately 8% of the circulating hemocyte population and are identifiable by their large, cytoplasmic inclusions. Oenocytoids account for less than 5% of the circulating

0022–1910/99/$ - see front matter  1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 1 9 1 0 ( 9 8 ) 0 0 0 9 2 - 4

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hemocyte population and are distinguished ultrastructurally by their homogeneous cytoplasm and lack of inclusions. These cells also exhibit high levels of endogenous phenoloxidase activity; rapidly turning black when fixed in methanol and incubated in l-dihydroxyphenolalanine (Pech et al., 1994). Prohemocytes are described in Lepidoptera as rounded cells with a small nuclear to cytoplasmic ratio (Jones, 1962; Brehelin and Zachary, 1986), yet we rarely observe hemocytes in circulation that unambiguously match this description in P. includens larvae. With the exception of prohemocytes, each of the other hemocyte classes can be isolated by density gradient centrifugation (Pech et al., 1994; Pech and Strand, 1996), and maintained in vitro for extended periods of time (Pech and Strand, 1995, 1996). While the aforementioned characters are very useful, P. includens hemocytes, like those of most insects, are sufficiently pleomorphic that some ambiguity always exists when identifying individual cells by morphology. In particular, it is very difficult to identify cells by light microscopy when granular cells and plasmatocytes have not yet assumed their spread morphology. One approach to improving hemocyte identification methods is to classify cells on the basis of antigenic differences using antibody markers. Toward this end, we generated a panel of monoclonal antibodies (mAbs) against P. includens hemocytes (Strand and Johnson, 1996). Some of these antibodies have been used as markers to identify plasmatocytes and granular cells during hemocyte-mediated defense responses like encapsulation (Pech and Strand, 1996; Clark et al., 1997; Loret and Strand, 1998). The goal of this study was to carefully compare the binding patterns of 16 different mAbs in relation to: (1) other criteria used to identify P. includens hemocytes, and (2) culture conditions that affect hemocyte morphology and behavior. Here we report that each antibody specifically labeled one or more classes of hemocytes, and with one exception did not cross-react with other tissues in P. includens. However, granular cells do release proteins during in vitro culture that appear to bind to plasmatocytes. Collectively, our results indicate that granular cells share several antigens with spherule cells, and plasmatocytes share several antigens with oenocytoids. The implication of these results for understanding hemocyte ontogeny during postembryonic development are discussed.

2. Materials and methods 2.1. Insects P. includens was reared and physiologically staged as outlined by Strand (1990). Larvae were reared in 30 ml plastic cups at 27 ⫾ 1°C and a 16L:8D photoperiod while moths were fed a 10% sucrose solution. P.

includens larvae undergo five instars. The timing of events within an instar was recorded in hours post-ecdysis to the instar. Briefly, the duration of the first-fourth stadia is approximately 2 days each. Larvae feed until critical period (release of prothoracicotropic hormone (PTTH)) at 22–24 h, initiate apolysis (head capsule slippage phase) at 26–28 h, and ecdyse to the next instar at 44–48 h. The fifth stadium is approximately 4 days in duration with critical period occurring at 50 h, the wandering phase at 60–66 h and initiation of cocoon spinning at 72–82 h. Pupation occurs between 86 and 92 h. 2.2. Hemocyte collection, separation, and culture Hemocytes were collected from larvae at selected stages of development by the procedure of Pech et al. (1994). Larvae were anaesthetized with CO2, surface sterilized with 95% ethanol, and bled from a proleg into 500 ␮l of anticoagulant buffer (98 mM NaOH, 186 mM NaCl, 1.7 mM EDTA and 41 mM citric acid, buffer pH 4.5) (Mead et al., 1986). Cells collected in this manner contain all hemocyte morphotypes and are referred to as unseparated hemocytes. Specific experiments were conducted using either unseparated hemocytes or fractions of hemocytes separated on Percoll gradients. Gradient separations of hemocyte types were performed as outlined by Pech and Strand (1996) with slight modification. Hemocytes (ca. 2 ⫻ 106 hemocytes total) were collected from four larvae (36–48 h fifth stadium) into anticoagulant, rinsed once in Ex-cell 400 medium (JRH Biosciences, Lenexa, KS), and layered onto a Percoll (Sigma, St Louis, MO) step gradient formed in sterile, 12 ⫻ 75 mm round-bottom polystyrene tubes (Becton Dickinson, Lincoln Park, NJ). Isotonic Percoll (100%) was made by adding 10 ⫻ physiological saline (1.54 M NaCl, 2.6 mM KCl, 1.3 mM CaCl, 116 mM Dextrose) to Percoll (9:1 v/v) (Pech et al., 1994). The gradient consisted of 2 ml of 47.5% Percoll (Sigma, St Louis, MO) in Ex-cell 400 medium layered over 2 ml of 62.5% Percoll, and 0.5 ml of 90% Percoll. Gradients were centrifuged for 15 min at 480 g in a Beckman J221 centrifuge with a JS-7.5 swinging bucket rotor. The band at the top of the gradient contained on average 1 ⫻ 106 hemocytes (50% of the starting population); the majority of which were granular cells as identified using morphological characters. The cells at the interface of the 47.5% and 62.5% Percoll were primarily plasmatocytes (6 ⫻ 105 cells, 30% of the starting population), and the cells at the interface of the 62.5% and 90% Percoll were spherule cells and oenocytoids (5.5 ⫻ 104 cells, 3% of the starting population). These hemocyte fractions are referred to throughout the paper as the G, P and S–O fractions respectively. In P. includens, we are unable to isolate any cell fraction identifiable as prohemocytes.

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Cell fractions were removed from gradients, washed twice in Ex-cell medium and pelleted at 400 g using a Lourdes clinical centrifuge. The final pellet for each fraction was resuspended in 50 ␮l of Ex-cell medium, counted, and aliquots of 1 ⫻ 104 cells were placed in individual wells of a 96-well tissue culture plate (Corning). Each well of the culture plate contained 50 ␮l of TC-100 medium plus 10% fetal bovine serum (FBS) (Hyclone). Note that hemocytes collected from gradients are rounded, unattached to one another, and are morphologically indistinguishable from hemocytes collected directly from P. includens (Strand, 1994; Pech et al., 1994). Cells were maintained in vitro at 27 ⫾ 1°C. Under these conditions, > 90% of granular cells, whether in unseparated populations or purified on gradients, attach and spread on the bottom of culture wells after 1 h in vitro. The majority ( > 70%) of plasmatocytes in unseparated populations also spread in 1 h, whereas gradient-purified populations spread 3–4 h after placement into culture (Pech et al., 1994, 1995). 2.3. Antibodies The mAbs used in this study were generated from the hybridoma screen of Strand and Johnson (1996). Briefly, BALB/c mice were immunized with unseparated hemocytes collected from 36–48 h fifth stadium P. includens. Spleen cells were fused with P3/NSI/1–Ag401 myeloma cells (NS-1; ATCC; Rockville, MD) using polyethylene glycol 1500. For the current study, selected hybridomas were cloned by limiting dilution in 96-well plates at 0.3 and 0.1 cells per well. Clones were considered statistically valid if fewer than nine wells per plate had colonies and if only a single colony was observed per well. The resulting mAbs were assigned names on the basis of the culture plate and cloning well from which a given antibody-secreting cell line was isolated. Monoclonal antibodies used in experiments were tissue culture supernatants. 2.4. Antibody binding to hemocytes Hemocytes were fixed in 5% formalin for 10 min and rinsed in PBS (13.7 mM NaCl, 0.27 mM KCl, 0.43 mM Na2HPO4, 0.14 mM KH2PO4, pH 7.3) Cells were left either unpermeabilized or were permeabilized for 15 min in PBT (PBS plus 0.1% Triton X-100). After blocking for 1 h with 3% bovine serum albumin (BSA; fraction V, Boehringer Mannheim) in PBS (blocking solution), cells were incubated with primary antibody diluted 1:1– 1:10. After rinsing 4 ⫻ in PBS, hemocytes were incubated with fluorescein isothiocyanate (FITC) or rhodamine (Rh)-conjugated goat anti-mouse IgG ⫹ M secondary antibody (Jackson Labs) diluted 1:100 in blocking solution. In some cases, the secondary antibody was Texas Red (TR)-conjugated goat anti-mouse IgG ⫹ M

115

(Jackson Labs) diluted 1:150. Double labeling experiments were conducted with selected combinations of antibodies as described by Pech and Strand (1996). Samples were examined using a Nikon Diaphot epifluorescence microscope with Hoffman optics. Each antibody was tested against 5–10 independently collected samples of hemocytes. For each sample, 200 hemocytes were randomly selected in three culture wells and scored for being labeled or unlabeled by a given mAb. As controls, hemocytes from the same samples were processed identically with the exception that the primary or secondary antibody was omitted from the reaction. Following the completion of assays on separated populations of hemocytes, the same assays were repeated using unseparated hemocytes collected directly from P. includens or by combining and culturing different fractions of hemocytes separated on Percoll gradients. 2.5. Tissue specificity of anti-hemocyte antibodies To test whether any anti-hemocyte mAb bound to other tissues, whole mount analyses were performed using P. includens second instar larvae. Larvae were sagitally opened along their dorsal or ventral midline using microdissection scissors. Larvae were fixed in 10% formalin for 3 h, rinsed in PBS, and permeablized in PBT. Antibody reactions were carried out as described by Grbic et al. (1996) with slight modification. MAb 49B8B10 was diluted 1:10 in PBT, whereas the other primary antibodies were diluted 1:1 in PBT. Tissues were incubated with gentle agitation in 1.5 ml Eppendorf tubes at room temperature for 4 h. Secondary antibody (alkaline phosphatase conjugated goat anti-mouse IgG ⫹ M; Jackson) was preabsorbed against P. includens larvae for 3 h and diluted 1:2000 in PBT before use. Primary antibody binding was detected with NBT and BCIP (Harlow and Lane, 1988). Larvae were examined using a compound or stereomicroscope. As a positive control, each primary antibody was incubated with hemocytes and visualized identically to the procedure outlined above for whole mounts. As a negative control, either the primary or secondary antibody was omitted during processing. 2.6. Immunoblotting Hemocytes or hemocyte conditioned medium were collected at selected intervals from primary cultures incubated in Ex-cell 400 medium. Protein concentrations were assessed by Bradford assay (Bio-Rad), using bovine plasmatic gamma globulin (Bio-Rad) as a standard. Ten ␮l of conditioned medium (5 ␮g protein) was collected from hemocyte cultures at selected intervals, diluted 1:1 with loading buffer and boiled for 5 min. Samples were then separated on 12.0% polyacrylamide gels under reducing conditions (SDS–PAGE). Proteins

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were transferred to Nitrobind nitrocellulose paper (Hoeffer, San Francisco, CA) using a semi-dry transfer unit (Hoeffer). Filters were blocked for 30 min using 0.2% Tween 20 (Sigma) and 5% casein in PBS, and then incubated with the primary antibody diluted 1:10–100 in blocking buffer. After rinsing 3 ⫻ in PBS, blotted proteins were incubated for 1 h with peroxidase-conjugated goat anti-mouse IgG ⫹ M (Kirkegaard and Perry) diluted 1:2000 in blocking buffer. After rinsing 3 ⫻ in PBS, proteins were visualized using 0.06% diaminobenzamine tetrahydrochloride (DAB) plus 0.3% NiCl and H2O2 (Harlow and Lane, 1988). Control experiments included examination of unconditioned medium or omission of the primary or secondary antibody. 2.7. Image processing Microscope images were captured on film or as electronic images using Metamorph software (Metamorph 1.0) interfaced with a Photometrics high resolution camera. Western blots were scanned at a resulution of 300 dpi using an Eagle Eye documentation station (Stratagene). Files were printed from Adobe Photoshop 3.0 using a Tektronix Phaser IISDX dye sublimation printer.

3. Results 3.1. MAb screening using gradient-separated hemocytes In a previous study (Pech et al., 1994), we reported that P. includens granular cells, plasmatocytes, spherule cells and oenocytoids differ sufficiently in density that they can be separated from one another on Percoll gradients. The purity of fractions obtained from these gradients was determined by classifying cells using morphological characters. Since then, we have attempted to improve our separation methods by using different gradient schemes and Percoll concentrations (Pech and Strand, 1996; Loret and Strand, 1998). In preliminary experiments for this study, we assessed the purity of fractions produced using these different gradient schemes by identifying hemocytes using morphological characters and mAbs. We found that the approach outlined in the Section 2 was the simplest and yielded fractions of granular cells, plasmatocytes and spherule cells/oenocytoids least contaminated by other cell types. These results are discussed below. To characterize the binding characteristics of specific mAbs, we first used each antibody against gradient separated hemocytes collected from 36–48 h old fifth stadium larvae. This approach had two advantages over screening mAbs using unseparated hemocytes. First, it allowed us to examine each mAb against fractions of

hemocytes enriched for a particular morphological class of cells. This was especially important in the case of oenocytoids and spherule cells which are at low abundance in unseparated hemocyte populations and are difficult to distinguish from other rounded, unspread hemocytes after processing for immunofluorescence microscopy. Second, using gradient purified hemocytes reduced the possibility of antigens from one hemocyte type binding to other hemocytes (see below). We initially examined hemocytes immediately after collection from gradients. At this time all hemocytes are rounded and nonadhesive. The binding characteristics of each mAb toward permeablized and unpermeablized hemocytes are summarized in Table 1. Three main categories of binding activity were identified: (1) mAbs that specifically labeled only one morphological class of hemocytes, (2) mAbs that labeled granular cells and spherule cells, and (3) mAbs that labeled plasmatocytes and oenocytoids. One mAb, 44H6H10, labeled hemocytes of all classes. While some antibodies labeled only permeablized hemocytes, most also labeled the periphery of unpermeablized cells indicating that they recognized antigens on the surface of hemocytes. To determine the number and size of the antigens recognized by each mAb, hemocyte lysates were prepared and subjected to SDS–PAGE electrophoresis. Western blot analysis revealed that most of the mAbs recognized one or more proteins of different molecular mass [Fig. 1(A)]. Some mAbs like 56G7E9 and 46D11A1 bound to a single protein, others labeled two or more proteins, and a few recognized no proteins on blots. Immunoblots using selected mAbs are presented in Fig. 1(B). 3.2. MAbs exhibit labeling patterns that correlate with identification of hemocytes by morphology To determine whether selected mAbs labeled all hemocytes of a given morphological class or subpopulations thereof, we placed hemocytes from each Percoll fraction into culture and first classified them using morphological criteria. The cells were then incubated with a given antibody and the number of labeled cells determined by immmunofluorescence microscopy. On the basis of morphology, the G fraction from Percoll gradients contained on average 82% granular cells and was contaminated primarily by plasmatocytes (Tables 2 and 3). The P fraction was 84% plasmatocytes, contaminated by granular cells, and the S–O fraction was 90% spherule cells and 7% oenocytoids (Tables 2 and 3). The antibody 41A2G4 labeled the same percentage of cells in the G fraction as identified by morphology to be granular cells. Since 41A2G4 also labeled the same percentage of cells in the P and S–O fractions identified by morphology as contaminating granular cells, we concluded that this mAb specifically bound to most if not all hemocytes that we identify as granular cells (Table

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Table 1 Summary of binding affinities for anti-hemocyte mAbs using gradient-purified P. includens hemocytes mAb

Dilution Granular cells

Spherule cells

Plasmatocytes

Oenocytoids

permeablized unpermeablized permeablized unpermeablized permeablized unpermeablized permeablized unpermeablized 41A2G4 48F2D5 49B8B10 46E12F4 56G7E9 45E7B9 42C3G12 49B11C6 43E9A10 52F3A5 49E4C11 49G3A3 53A1A1 46D11A1 50C7B11 44H6H10

1:10 1:3 1:3 1:5 1:10 1:10 1:3 1:5 1:3 1:3 1:1 1:1 1:5 1:3 1:10

⫹ ⫹ ⫹ ⫹ ⫹ ⫹ – – – – – – – – – ⫹

⫹ – – ⫹ ⫹ ⫹ – – – – – – – – – ⫹

– ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ – – – – – – – – ⫹

– – – ⫹ ⫹ ⫹ ⫹ – – – – – – – – ⫹

2). The antibodies 48F2D5, 49B8B10 and 45E7B9 also labeled the same percentage of cells in the G fraction identified by morphology as granular cells (Table 2) [Fig. 2(A, B)]. However, these antibodies also labeled most cells in the S–O fraction which by morphology we identified as spherule cells (Table 2). Each of the aforementioned mAbs labeled both unspread and spread granular cells. After 12 h in culture, the proportion of cells labeled by each mAb did not change (data not presented). The intensity of labeling remained constant for some mAbs (48F2D5, 42C3G12) but noticeably declined with others (41A2G4, 49B8B10, 45E7B9). MAb 42C3G12 labeled almost no cells in the G and P fractions, but bound to greater than 80% of the hemocytes present in the S–O fraction (Table 2) [Fig. 2(C, D)]. Hemocytes labeled by this antibody were identified by morphology and density to be exclusively spherule cells. The mAbs 49B11C6 and 46D11A1 labeled the same percentage of cells in the P fraction as identified by morphology to be plasmatocytes (Table 3). Since 49B11C6 and 46D11A1 also labeled the same percentage of cells in the G fraction identified by morphology to be contaminating plasmatocytes, we concluded that these mAbs labeled all plasmatocytes. The antibody 43E9A10 also labeled a large number of hemocytes in the P fraction [Fig. 2(E, F)], but the mean percentage of labeled cells was significantly smaller than the percentage of cells identified to be plasmatocytes by morphology (Table 3). The mAbs 52F3A5, 49G3A3 and 53A1A1 labeled all cells in the P fraction identified by morphology to be plasmatocytes, as well as the same percentage of cells in the S–O fraction identified by morphology as oenocytoids (Table 3) [Fig. 2(G, H)]. As discussed previously,

– – – – – – – ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

– – – – – – – ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ – ⫹

– – – – – – – – – – – ⫹ ⫹ ⫹ ⫹ ⫹

– – – – – – – – – – – ⫹ ⫹ ⫹ – ⫹

plasmatocytes are initially rounded and unspread when first placed into culture, but after 6 h most plasmatocytes spread on the surface of culture plates or form aggregations. Each of the anti-plasmatocyte mAbs described here labeled unspread plasmatocytes that had been in culture for less than 1 h. However, when used against cells in culture for more than 6 h, mAb 43E9A10 labeled spread plasmatocytes almost exclusively. In contrast, the other anti-plasmatocyte mAbs bound to plasmatocytes that had spread as well as those that had not. The intensity with which plasmatocytes were labeled by mAb 49G3A3 declined with increasing culture time, whereas the intensity of labeling by 43E9A10 appeared to increase. Labeling patterns using the other anti-plasmatocyte mAbs remained constant (data not presented). 3.3. Most mAbs are hemocyte specific All of the antibodies listed in Table 1 were tested against whole mounts of second instar larvae. None of the antibodies labeled other tissues with the exception of mAb 49B8B10 (data not presented) which consistently bound to all tissues. Each tissue was uniformly stained around its periphery suggesting that 49B8B10 bound to antigens associated with the extracellular matrix that lines the hemocoel and surrounds all tissues. Close inspection of individual cells within tissues indicated that 49B8B10 did not label other structures. In control experiments, each antibody visualized using NBT and BCIP exhibited the same hemocyte specificities as described above using fluorochrome-conjugated secondary antibodies. No labeling signal was observed in hemocytes or other tissues when any of the primary antibodies or the secondary antibody was omitted.

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3.4. The labeling specificities of certain mAbs change when used against unseparated hemocytes

Fig. 1. Anti-hemocyte mAbs recognize antigens of different size classes. (A) Summarization of immunoblot data for each antibody examined in the study. The antibody tested is noted on the left and the molecular masses (kDa) for the proteins detected are presented on the right. (B) Specific immunoblots using the antibodies 56G7E9 (lane 1), 49B8B10 (lane 2), 52F3A5 (lane 3), 48F2F2 (lane 4), and 46D11E8 (lane 5). Molecular mass standards (in kDa) are indicated on the left. Hemocyte lysates were prepared from 2 ⫻ 107 cells (unseparated hemocytes). After determining the total amount of protein present, lysates were subjected to SDS–polyacrylamide gel electrophoresis (6.5 ␮g of protein per lane) and immunoblot analysis using each antibody. Each antibody was tested at least three times against different hemocyte samples. *In addition to ca. 50.0 and 60.0 kDa proteins, 48F2D5 also sometimes detects proteins of 23 and 37 kDa. This is not a repeatable result and may be due to breakdown products. The 20 and 27 kDa proteins detected by mAb 52F3A5 are doublets.

To determine whether the presence of different hemocyte morphotypes affected mAb labeling patterns, unseparated populations of hemocytes were maintained in vitro and labeled individually with the antibodies listed in Table 1. Screening revealed that all of the antibodies that recognized plasmatocytes and oenocytoids exhibited the same labeling specificities against unseparated populations of hemocytes as documented using gradient purified cells [Fig. 3(A–D)]. The anti-granular cell/spherule cell mAbs 48F2D5 and 49B8B10 similarly maintained their specificities when used against unseparated populations of hemocytes [Fig. 3(A, B)]. However, the antigranular cell mAb 41A2G4 and anti-granular cell/spherule cell mAbs 46E12F4, 56G7E9, and 45E7B9 also labeled plasmatocytes when used on cultures of unseparated hemocytes. This alteration in labeling specificity was clearly time dependent and correlated with spreading of granular cells. For example, mAb 45E7B9 labeled only granular cells and spherule cells 15 min after hemocytes were placed into culture [Fig. 3(E, F)], but after 1 h also labeled plasmatocytes [Fig. 3(G, H)]. Granular cells and spherule cells were labeled by 45E7B9 in a distinctly punctate pattern, whereas plasmatocytes were labeled by 45E7B9 on their surface. The other anti-granular cell mAbs that bound plasmatocytes in unseparated cultures (41A2G4, 46E12F4, 56G7E9) also labeled the surface of plasmatocytes. To further examine why certain anti-granular cell mAbs bound to plasmatocytes in unseparated cultures, we conducted immunoblotting experiments using mAb 45E7B9 and gradient purified populations of granular cells and plasmatocytes. When medium from granular cell cultures was examined, proteins recognized by 45E7B9 were detected in medium within 15 min of placing granular cells into culture [Fig. 4(A)]. These proteins were also detected in granular cell lysates, even after granular cells had been in culture for 4 h [Fig. 4(B)]. In contrast, 45E7B9 did not detect any proteins in gradient purified plasmatocytes that had been in culture for 4 h [Fig. 4(B)]. To determine whether granular cells were the source of the antigens detected on plasmatocytes in unseparated cultures, we gradient purified 2 ⫻ 105 granular cells and placed them into culture for 4 h. The conditioned medium was then collected and used to culture gradient purified plasmatocytes. Purified plasmatocytes cultured in unconditioned medium served as controls. After 15 min, all plasmatocytes in granular cell conditioned medium were labeled by 45E7B9, whereas plasmatocytes in unconditioned medium were not (Fig. 5). Since P. includens granular cells and plasmatocytes endocytose an array of particles and proteins (Pech et al., 1994), we also compared labeling patterns with 45E7B9 by culturing plasmatocytes in granular cell con-

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Table 2 Mean percentage ( ⫾ SD) of each hemocyte class in each Percoll fractions as identified by morphology or labeling with selected anti-granular cell or spherule cell mAbs Percoll fraction

Mean % ⫾ SD

Morphology

41A2G4 G fraction Granular cells Plasmatocytes Spherule cells Oenocytoids P fraction Granular cells Plasmatocytes Spherule cells Oenocytoids S–O fraction Granular cells Plasmatocytes Spherule cells Oenocytoids

48F2D5

49B8B10

45E7B9

42C3G12

81.7 14.9 0.5 1.0

⫾ ⫾ ⫾ ⫾

4.6 82.8 ⫾ 8.6 4.8 0.5 0.5

89.6 ⫾ 4.8

85.2 ⫾ 2.3

81.1 ⫾ 8.4

0.3 ⫾ 0.4*

12.6 83.5 1.6 1.0

⫾ ⫾ ⫾ ⫾

4.5 11.6 ⫾ 6.1 5.5 1.1 0.5

9.6 ⫾ 5.4

6.4 ⫾ 3.5

15.8 ⫾ 4.9

1.6 ⫾ 1.5*

0.4 2.2 90.4 7.1

⫾ ⫾ ⫾ ⫾

0.6 2.0 4.5 2.3 ⫾ 1.9* 2.8

87.3 ⫾ 7.4

87.7 ⫾ 5.8

84.2 ⫾ 14.7

80.9 ⫾ 12.1

All experiments were conducted using at minimum 5 independent collections of hemocytes separated on different gradients. For each fraction, the column summarizing identification of hemocytes by morphology does not add up to 100%, because a percentage of cells could not be identified as belonging to a specific class. Within each fraction, classification of hemocytes by morphology and antibody labeling was analysed by 1-way ANOVA using arcsin transformed data. Means followed by an asterisk (*) were significantly different from other values in the row (Fisher PLSD test). G fraction: F5, 33 ⫽ 189.2; P ⬍ 0.0001. P fraction: F5, 32 ⫽ 135.3; P ⬍ 0.0001. S–O fraction: F5, 26 ⫽ 95.7; P ⬍ 0.0001.

Table 3 Mean percentage ( ⫾ SD) of each hemocyte class in each Percoll fractions as identified by morphology or labeling with selected anti-plasmatocyte or oenocytoid mAbs Percoll fraction

Morphology

Mean % ⫾ SD 49B11C6

G fraction Granular cells Plasmatocytes Spherule cells Oenocytoids P fraction Granular cells Plasmatocytes Spherule cells Oenocytoids S–O fraction Granular cells Plasmatocytes Spherule cells Oenocytoids

81.7 14.9 0.5 1.0

⫾ ⫾ ⫾ ⫾

4.6 4.8 12.1 ⫾ 5.2 0.5 0.5

12.6 83.5 1.6 1.0

⫾ ⫾ ⫾ ⫾

4.5 5.5 85.2 ⫾ 10.3 1.1 0.5

0.4 2.2 90.4 7.1

⫾ ⫾ ⫾ ⫾

0.6 2.0 4.5 2.8 1.3 ⫾ 0.6*

43E9A10

46D11A1

52F3A5

49G3A3

53A1A1

8.4 ⫾ 5.8

11.9 ⫾ 5.3

13.0 ⫾ 1.4

13.2 ⫾ 6.8

8.6 ⫾ 4.9

68.6 ⫾ 3.6*

85.5 ⫾ 6.4

79.2 ⫾ 3.8

85.3 ⫾ 5.2

84.9 ⫾ 13.9

2.8 ⫾ 1.2*

7.1 ⫾ 6.5

6.4 ⫾ 4.3

5.1 ⫾ 3.8

12.2 ⫾ 5.0

All experiments were conducted using at minimum 5 independent collections of hemocytes separated on different gradients. For each fraction, the column summarizing identification of hemocytes by morphology does not add up to 100%, because a percentage of cells could not be identified as belonging to a specific class. Within each fraction, classification of hemocytes by morphology and antibody labeling was analysed by 1-way ANOVA using arcsin transformed data. Means followed by an asterisk (*) were significantly different from other values in the row (Fisher PLSD test). G fraction: F6, 34 ⫽ 1.4; P > 0.2. P fraction: F6, 34 ⫽ 4.1; P ⬍ 0.03. S–O fraction: F6, 26 ⫽ 40.1; P ⬍ 0.05.

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Fig. 2. Anti-hemocyte mAbs bind to specific classes of P. includens hemocytes. (A) Hoffman and (B) fluorescent images of gradient purified granular cells stained by the antibody 49B8B10. Granular cells had attached and spread to the surface of culture plates before fixation. This antibody stains permeablized granular cells in a punctate pattern. (C) Hoffman and (D) fluorescent images of unseparated hemocytes stained by the antibody 42C3G12. Hemocytes were fixed after granular cells had spread on the culture plate. This antibody labels permeablized spherule cells (S) in a punctate pattern but does not label granular cells (Gr) or other hemocyte types. (E) Hoffman and (F) fluorescent images of gradient purified plasmatocytes labeled by antibody 43E9A10. This antbody labels the cytoplasm of spread plasmatocytes (P–sp) but does not label most unspread plasmatocytes (P–usp). Granular cells (Gr) that contaminate the plasmatocyte fraction are also not labeled by this antibody. (G) Hoffman and (H) fluorescent images of gradient purified oenocytoids stained by the antibody 49G3A3. This antibody uniformly labels the surface of unpermeablized oenocytoids (O) but does not label spherule cells also present in the S–O fraction. The secondary antibody for all images is Texas Red–goat antimouse IgG ⫹ M. Scale bars, 150 ␮m.

ditioned medium at 2° and 27°C. Plasmatocytes incubated in medium containing Texas Red-conjugated secondary antibody served as a control. After 30 min, the surface of unpermeablized plasmatocytes cultured at 2°C

were labeled by 45E7B9 identically to cells cultured at 27°C (see [Fig. 5(B)]). In contrast, no plasmatocytes cultured at 2°C in medium containing secondary antibody were labeled above background.

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Fig. 3. Anti-hemocyte mAbs vary in their binding specificities when used against primary cultures of unseparated hemocytes. (A) Hoffman and (B) fluorescent images of unseparated hemocytes stained by the antibodies 49B8B10 and 43E9A10. Hemocytes had been in culture for 3 h before fixation. Most granular cells (Gr) and plasmatocytes (P) are spread. The antibody 49B8B10 (green) labels only granular cells, whereas 43E9A10 labels only plasmatocytes (red). (C) Hoffman and (D) fluorescent images of plasmatocytes in unseparated cultures double labeled by the antibodies 49G3A3 and 43E9A10. Hemocytes had been in culture for 3 h before fixation, and most granular cells (Gr) and plasmatocytes (P) are spread. The antibody 49G3A3 (red) uniformly labels the cytoplasm of permeablized plasmatocytes,whereas 43E9A10 (green) labels the cytoplasm of spread plasmatocytes in a dense, punctate pattern. No other hemocyte types in unseparated cultures are labeled by these antibodies. Scale bar 150 ␮m for A–D. (E) Hoffman and (F) flourescent images of unseparated hemocytes stained with the antibody 45E7B9. Hemocytes had been in culture for 15 min before fixation. Some granular cells (Gr) and plasmatocytes (P) have begun to spread but most remain unspread. Only granular cells (red) are labeled. Scale bar 200 ␮m for E and F. (G) Hoffman and (H) flourescent images of unseparated hemocytes stained by 45E7B9 after 1 h in culture. Both granular cells (Gr) and plasmatocytes (P) are labeled. Scale bar 150 ␮m for G and H.

3.5. MAbs maintain their labeling specificities throughout larval development of P. includens To determine whether our anti-hemocyte mAbs maintained their binding specificities during the course of lar-

val development, each antibody in Table 1 was tested against hemocytes collected from second-fifth instar larvae. All of the antibodies labeled the same classes of hemocytes documented for the fifth instar in Tables 1– 3. Results obtained using the anti-granular cell/spherule

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Fig. 4. Granular cells release antigens during primary culture that are recognized by mAb 45E7B9. (A) For each sample, gradient purified granular cells (1 ⫻ 105 cells) were placed into total of 150 ␮l of Excell 400 medium and cultured for 1 min, 15 min, 45 min, 4 h and 6 h. The conditioned medium was then subjected to SDS–polyacrylamide gel electrophoresis (1 ␮g of protein per lane) and immunoblot analysis using 45E7B9. (B) Hemocyte lysates were prepared from gradient purified plasmatocytes (2 ⫻ 105 cells) or granular cells (2 ⫻ 105 cells) that had been in culture for 4 h. Plasmatocyte (P) and granular cell lysates (Gr) were subjected to SDS–polyacrylamide gel electrophoresis (3 ␮g of protein/lane) and subjected to immunoblot analysis using 45E7B9.

Fig. 5. Granular cell antigens recognized by mAb 45E7B9 bind to plasmatocytes. (A) Hoffman and (B) fluorescent images of gradient purified plasmatocytes cultured in granular cell conditioned medium. Conditioned medium was prepared by culturing 2 ⫻ 105 granular cells for 4 h in 50 ␮l of Excell 400 medium. The medium was then collected, placed into a new well to which 1 ⫻ 104 plasmatocytes was added. After 15 min in conditioned medium, the plasmatocytes were fixed and stained by the antibody 45E7B9. Note that plasmatocytes (P) are still rounded but have begun to aggregate. All cells are also labeled by 45E7B9. (C) Hoffman and (D) fluorescent images of gradient purified plasmatocytes (1 ⫻ 104 cells/well) cultured for 15 min in 50 ␮l of unconditioned Excell 400 medium. After 15 min, the cells were fixed and stained by 45E7B9. All plasmatocytes are rounded, non-aggregated, and unlabeled by 45E7B9. Scale bar 100 ␮m.

mAb 49B8B10, the anti-plasmatocyte/oenocytoid mAb 49G3A3, and the plasmatocyte specific mAb 43E9A10 are presented in Fig. 6. Total hemocyte counts indicated that the number of hemocytes in circulation increased greatly between the second and fifth instar [Fig. 6(A)]. Differential hemocyte counts using morphological characters and antibodies yielded very similar results [Fig. 6(B,C)]. Plasmatocytes accounted for approximately 50% of the circulating hemocyte population dur-

ing the second and third stadium, but thereafter declined to approximately 30% of the total hemocyte population during the fourth and fifth stadium [Fig. 6(B)]. In contrast, granular cells increased from 22% to greater than 50% of the hemocyte population between the second and fifth instar [Fig. 6(C)]. The proportion of hemocytes identified as spherule cells remained relatively constant [Fig. 6(C)]. As noted previously, 49G3A3 recognized all plasmatocytes, whereas 43E9A10 labels plasmatocytes

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Fig. 6. Total and differential hemocyte counts from second-fifth stadium P. includens larvae. (A) Mean number of hemocytes ( ⫾ SD) present per ␮l of hemolymph. Total hemocyte counts were obtained by bleeding ten larvae per datum point. (B) Differential hemocyte counts using morphology (solid bar) or the antibody 49G3A3 (open bar) to identify plasmatocytes. (C) Differential hemocyte counts using morphology to identify granular cells (solid bar) and spherule cells (hatched bar), or the antibody 49B8B10 to identify granular cells plus spherule cells (open bar). For (B) and (C), hemocyte samples were collected from ten larvae per datum point. The percentage of plasmatocytes, granular cells or spherule cells ( ⫾ SD) at each time point was determined for each sample by dividing the number of each cell type present by the total number of hemocytes present. Oenocytoids are not presented in these graphs because they represented such a small percentage of the total hemocyte population. (D) The mean percentage of hemocytes ( ⫾ SD) in second-fifth stadium larvae labeled by the antibodies 49G3A3 (solid bar) and 43E9A10 (open bar). A total of fifteen larvae were bled per time point. The percentage of labeled hemocytes was determined for each sample by dividing the number of cells labeled by each antibody by the total number of hemocytes present.

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capable of spreading on foreign surfaces. While the overall percentage of plasmatocytes in circulation declined during larval development, the percentage of plasmatocytes labeled by 43E9A10 increased [Fig. 6(D)].

4. Discussion Antibodies have long served as essential probes for identification of mammalian immune cells, yet only recently have they begun to be used for identification of insect hemocytes (Chain et al., 1992; Mullet et al., 1993; Willott et al., 1994; Strand and Johnson, 1996). While mAb markers unquestionably have great potential as immunological tools, caution must also be exercised when evaluating these reagents as probes for cell identification. Some antibodies may label specific cell types, but others may recognize antigens present on multiple cell types, antigens that change in distribution during development or after immmunological challenge (i.e. activation markers), or antigens that vary with maturation state of the cell. The purpose of this study, therefore, was to: (1) compare hemocyte identification using mAbs with hemocyte identification using morphology and sedimentation density, (2) determine whether culture conditions that affect the morphology and adhesive state of certain hemocyte types also affect mAb binding properties, (3) determine whether mAb binding of hemocytes varies during larval development, and (4) assess whether anti-hemocyte mAbs bind to other cells or tissues. By comparing antibody labeling patterns to identification of hemocytes by morphology, we verified during the current study that one mAb (41A2G4) labels only granular cells, five mAbs (49B11C6, 43E9A10, 52F3A5, 46D11E8, and 49E4C11) label only plasmatocytes, one mAb (42C3G12) labels only spherule cells, and one mAb (44H6H10) labels all classes of hemocytes. The other antibodies labeled either granular cells and spherule cells or plasmatocytes and oenocytoids. Western blot analysis indicated that each of these antibodies bound to proteins of different molecular mass and whole mount studies revealed that none of the antibodies with the exception of 49B8B10 cross-reacted with other tissues. We conclude from these experiments that these antibodies recognize different antigens and that most of these antigens are associated specifically with hemocytes. Different hemocytes, including granular cells and spherule cells, have been implicated as sources of extracellular matrix molecules including laminin, collagin IV and tiggrin (Fessler and Fessler, 1989; Chain et al., 1992; Fogerty et al., 1994). The cross-reactivity of 49B8B10 with granular cells, spherule cells and the extracellular matrix of P. includens is consistent with these observations.

All of the antibodies that labeled granular cells and/or spherule cells recognized the same proportion of hemocytes in Percoll fractions that we identified to be granular cells and spherule cells on the basis of morphology. Our anti-granular cell mAbs also labeled granular cells when unspread or spread on the surface of culture plates. These results indicate that none of the these antibodies recognize unique subpopulations of granular cells or spherule cells, and suggest that the granular cells and spherule cells we isolate on Percoll gradients are homogeneous. Most of our anti-plasmatocyte/oenocytoid mAbs also did not recognize unique subpopulations of these hemocyte classes. The notable exception is mAb 43E9A10. Consistent with previous observations (Clark et al., 1997; Loret and Strand, 1998), results of this study indicate that at least two plasmatocyte subpopulations exist in P. includens: one recognized by 43E9A10 that readily spreads on foreign surfaces and another that is recognized by our other anti-plasmatocyte mAbs but that is not labeled by 43E9A10. We view 43E9A10 as a particularly important marker given that spread morphology is often the most important character for identifying plasmatocytes in Lepidoptera (Horohov and Dunn, 1982; Mead et al., 1986; Davies et al., 1987; Strand and Noda, 1991). Indeed, without 43E9A10, we did not recognize that any plasmatocyte subpopulations existed in P. includens. Plasmatocytes labeled by 43E9A10 do not differ from 43E9A10 negative plasmatocytes in sedimentation density, size or morphology. Previously, we had observed that some cells in the P fraction of Percoll gradients never spread in vitro and had assumed that the failure of these cells to spread was due to culture conditions or differences in cell viability (Pech et al., 1994; Pech and Strand, 1995). We now suggest that 43E9A10 negative plasmatocytes may be functionally distinct from 43E9A10 positive plasmatocytes. Supporting this conclusion, we recently identified plasmatocyte spreading peptide (PSP1): a 2500 Da peptide isolated from P. includens plasma that stimulates spreading and changes in the adhesive state of plasmatocytes (Clark et al., 1997). Bioassays revealed that only 43E9A10 positive plasmatocytes spread in response to PSP1. Recently, we reported that both plasmatocytes and granular cells from P. includens release proteins during short term culture that are recognized by some of our anti-hemocyte mAbs (Loret and Strand, 1998). Here, we found that all of our anti-plasmatocyte/oenocytoid antibodies maintained their binding specificities when used against hemocytes in unseparated populations. In contrast, certain mAbs that label only granular cells (41A2G4) or granular cells and spherule cells (49B8B10, 45E7B9) in gradient-fractionated populations also labeled plasmatocytes in cultures of unseparated hemocytes. These changes in labeling specificities are most likely due to the release of antigens from granular cells that

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then bind to plasmatocytes. This conclusion is most strongly supported by the observation that plasmatocytes cultured in granular cell conditioned medium were labeled within minutes by anti-granular cell mAbs like 45E7B9, whereas plasmatocytes in unconditioned medium were not. It is also unlikely that labeling of plasmatocytes is due to nonspecific endocytosis of granular cell antigens given that 45E7B9 also labeled plasmatocytes in granular cell conditioned medium when cells were cultured at 2°C. From the perspective of using mAbs as markers, our results underscore the importance of relating antibody binding patterns to other methods of cell identification. First and foremost, we found that characterization of mAb binding properties was greatly aided by comparing labeling patterns using both gradient fractionated and unseparated populations of hemocytes. Second, our results indicate that culture conditions and the presence of different hemocyte classes influence the binding properties of certain antibodies. When first screening the hybridomas that yielded the mAbs described here (Strand and Johnson, 1996), several errors were made about the binding properties of certain lines largely because we screened antibodies using hemocytes in unseparated populations. For example, the line yielding mAb 49G3A3 was recorded as labeling plasmatocytes when in all liklihood it also labeled oenocytoids. Our error arose because oenocytoids account for approximately 5% of the total hemocyte population in P. includens, are often similar in diameter to unspread plasmatocytes, and these nonadhesive cells are easily lost when being processed for immunofluorescence microscopy. Confusion similarly arose in regard to spherule cells which also comprise a small proportion of the total hemocyte population in P. includens. These nonadhesive cells are also difficult to identify by light microscopy after fixation and permeablization, because the cytoplasmic inclusions that are so easily seen in living cells are not apparent. However, by producing fractions enriched for these cell types, the labeling of oenocytoids and spherule cells by different antibodies became clear. The influence of culture conditions is well illustrated by our finding that some granular cell antigens are detected on plasmatocytes when these cell types are cocultured. This is perhaps not unexpected given that granular cells from several species have been observed to release factors when spreading on foreign surfaces that appear to affect the behavior of plasmatocytes (Schmit and Ratcliffe, 1977; Ratner and Vinson, 1983; Anggraeni and Ratcliffe, 1991; Wiesner and Gotz, 1993). Our own studies indicate that granular cell conditioned medium from P. includens greatly accelerates the spreading of plasmatocytes on planar surfaces (Pech and Strand, 1995) and the ability of plasmatocytes to encapsulate foreign targets (Pech and Strand, 1996). A final issue is whether the mAb binding patterns

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reported here offer any insight about hemocyte ontogeny and differentiation. While most authors agree that hemocytes arise during embryogenesis from median mesoderm (Mori, 1979; Tepass et al., 1994), it remains unclear how hemocyte populations are maintained during postembryonic development. In Lepidoptera, both hematopoietic tissues and mitosis of hemocytes in circulation have been implicated in maintainance of hemocyte populations (Akai and Sato, 1971; Gupta, 1986; Ratcliffe et al., 1985). Beaulaton (1979) proposed in Bombyx mori that prohemocytes give rise to plasmatocytes which are pluripotent and capable of producing granular cells, spherule cells and oenocytoids. In contrast, studies with Galleria mellonella suggested that prohemocytes, granular cells and plasmatocytes form one lineage, whereas spherule cells and oenocytoids arise from different lineages of unknown origin (Shrivastava and Richards, 1965). Finally, Arnold and Hinks (Arnold and Hinks, 1976; Hinks and Arnold, 1977) conducted a series experiments in the noctuid Euxoa declarata and concluded that hemocytes have a dual origin. Granular cells and spherule cells are maintained by mitotic division of these cells in circulation, whereas prohemocytes, plasmatocytes and oenocytoids arise from hematopoietic organs. Prohemocytes are solely plasmatocyte stem cells and oenocytoids arise from a different population of stem cells in hematopoietic organs. Interestingly, several of our antibodies crossreact with granular cells and spherule cells or plasmatocytes and oenocytoids. At minimum, these results suggest that granular cells and spherule cells are antigenically more related to one another than they are to plasmatocytes and oenocytoids. Of potentially greater importance is the possibility that some of these antigens may be characteristic of different lineages or stages of maturation. If so, our observations are broadly most consistent with those of Arnold and Hinks who, as noted above, concluded that plasmatocytes and oenocytoids arise from a similar source, and that granular cells and spherule cells are maintained from hemocytes in circulation. Future comparative studies with these and other species will undoubtedly help clarify these issues and the role of different hemocyte classes in immunity.

Acknowledgements

We would like to thank J. Johnson for assistance in maintaining hybridoma lines. This work was supported by grants from the National Institutes of Health (AI32917), the United States Department of Agriculture (95-37302-1811) and the USDA Hatch Program to MRS.

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References Akai, H., Sato, S., 1971. An ultrastructural study of the haemopoietic organs of the silkworm Bombyx mori. Journal of Insect Physiology 17, 1665–1676. Anggraeni, T., Ratcliffe, N.A., 1991. Studies on cell–cell cooperation during phagocytosis by purified haemocyte populations of the wax moth Galleria mellonella. Journal of Insect Physiology 37, 453– 460. Arnold, J.W., Hinks, C.F., 1976. Haemopoiesis in Lepidoptera. I. The multiplication of circulating haemocytes. Canadian Journal of Zoology 54, 1003–1012. Beaulaton, J., 1979. Hemocytes and hemocytopoiesis in silkworms. Biochemie 61, 157–164. Brehelin, M., Zachary, D., 1986. Insect haemocytes: a new classification to rule out the controversy. In: Brehelin, M. (Ed.), Immunity in Invertebrates. Springer-Verlag, Berlin, pp. 36–48. Chain, B.M., Leyshon-Sorland, K., Siva-Jothy, M.T., 1992. Haemocyte heterogeneity in the cockroach Periplaneta americana analysed using monoclonal antibodies. Journal of Cell Science 103, 1261– 1267. Clark, K., Pech, L.L., Strand, M.R., 1997. Isolation and identification of a plasmatocyte spreading peptide from hemolymph of the lepidopteran insect Pseudoplusia includens. Journal of Biological Chemistry 272, 23440–23447. Davies, D.H., Strand, M.R., Vinson, S.B., 1987. Changes in differential haemocyte count and in vitro behaviour of plasmatocytes from host Heliothis virescens caused by Campoletis sonorensis polydnavirus. Journal of Insect Physiology 33, 143–153. Fessler, J.H., Fessler, L.I., 1989. Drosophila extracellular matrix. Annual Review of Cell Biology 5, 309–339. Fogerty, F.J., Fessler, L.I., Bunch, T.A., Yaron, Y., Parker, C.G., Nelson, R.E., Brower, D.L., Gullgerg, D., Fessler, J.H., 1994. Tiggrin, a novel Drosophila extracellular matrix protein that functions as a ligand for Drosophila ␣PS2␤PS integrins. Development 120, 1747–1758. Grbic, M., Nagy, L.M., Carroll, S.B., Strand, M.R., 1996. Polyembryonic development: insect pattern formation in a cellularized environment. Development 122, 795–804. Gupta, A.P., 1986. Hemocytic and Humoral Immunity in Insects. Wiley, New York. Harlow, E., Lane, D., 1988. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Hinks, C.F., Arnold, J.W., 1977. Haemopoiesis in Lepidoptera: the role of the haemopoietic organs. Canadian Journal of Zoology 55, 1740–1755. Horohov, D.W., Dunn, P.E., 1982. Changes in the circulating hemocyte population of Manduca sexta larvae following injection of bacteria. Journal of Invertebrate Pathology 40, 327–339. Jones, J.C., 1962. Current concepts concerning insect hemocytes. American Zoologist 2, 209–246. Lackie, A.M., 1988. Haemocyte behaviour. Advances in Insect Physiology 21, 85–177. Loret, S.M., Strand, M.R., 1998. Followup of protein release from Pseudoplusia includens hemocytes: a first step toward identification of factors mediating encapsulation in insects. European Journal of Cell Biology, in press. Mead, G.P., Ratcliffe, N.A., Renwrantz, L.R., 1986. The separation of insect hemocyte types on Percoll gradients; methodology and problems. Journal of Insect Physiology 32, 167–177.

Mori, H., 1979. Embryonic hemocytes: origin and development. In: Gupta, A.P. (Ed.), Insect Hemocytes. Cambridge University Press, pp. 4–27. Mullet, H., Ratcliffe, N.A., Rowley, A.F., 1993. The generation and characterisation of anti-insect blood cell monoclonal antibodies. Journal of Cell Science 105, 93–100. Pech, L.L., Strand, M.R., 1995. Encapsulation of foreign targets by hemocytes of the moth Pseudoplusia includens involves an RGDdependent mechanism. Journal of Insect Physiology 41, 481–488. Pech, L.L., Strand, M.R., 1996. Granular cells are required for encapsulation of foreign targets by insect haemocytes. Journal of Cell Science 109, 2053–2060. Pech, L.L., Trudeau, D., Strand, M.R., 1994. Separation and behavior in vitro of hemocytes from the moth Pseudoplusia includens. Cell and Tissue Research 277, 159–167. Pech, L.L., Trudeau, D., Strand, M.R., 1995. Effects of basement membranes on the behavior of hemocytes from Pseudoplusia includens: development of an in vitro encapsulation assay. Journal of Insect Physiology 41, 801–807. Ratcliffe, N.A., 1993. Cellular defense responses of insects: unresolved problems. In: Beckage, N.E., Thompson, S.N., Federici, B.A. (Eds.), Parasites and Pathogens of Insects, vol. 1. Academic, San Diego, pp. 267–304. Ratcliffe, N.A., Rowley, A.F., Fitzgerald, S.W., Rhodes, C.P., 1985. Invertebrate immunity: basic concepts and recent advances. International Review of Cytology 97, 186–350. Ratner, S., Vinson, S.B., 1983. Encapsulation reactions in vitro by haemocytes of Heliothis virescens. Journal of Insect Physiology 29, 855–863. Schmit, A.R., Ratcliffe, N.A., 1977. The encapsulation of foreign tissue implants in Galleria mellonella larvae. Journal of Insect Physiology 23, 175–184. Shrivastava, S.C., Richards, A.G., 1965. An autoradiographic study of the relation between hemocytes and connective tissue in the wax moth Galleria mellonella L. Biological Bulletin 128, 337–345. Strand, M.R., 1990. Characterization of larval development in Pseudoplusia includens (Lepidoptera: Noctuidae). Annals of the Entomological Society of America 83, 538–544. Strand, M.R., 1994. Microplitis demolitor polydnavirus infects and expresses in specific morphotypes of Pseudoplusia includens haemocytes. Journal of General Virology 75, 3007–3020. Strand, M.R., Noda, T., 1991. Alterations in the haemocytes of Pseudoplusia includens after parasitism by Microplitis demolitor. Journal of Insect Physiology 37, 839–850. Strand, M.R., Pech, L.L., 1995. Immunological basis for compatibility in parasitoid–host relationships. Annual Review of Entomology 40, 31–56. Strand, M.R., Johnson, J.A., 1996. Characterization of monoclonal antibodies to hemocytes of Pseudoplusia includens. Journal of Insect Physiology 42, 21–31. Tepass, U., Fessler, L.I., Aziz, A., Hartenstein, V., 1994. Embryonic origin of hemocytes and their relationship to cell death in Drosophila. Development 120, 1829–1837. Wiesner, A., Gotz, P., 1993. Silica beads induce cellular and humoral immune responses in Galleria mellonella larvae and in isolated plasmatocytes, obtained by a newly adapted nylon wool separation method. Journal of Insect Physiology 39, 865–876. Willott, E., Trenczeck, T., Thrower, L.W., Kanost, M.R., 1994. Immunochemical identification of insect hemocyte populations: monoclonal antibodies distinguish four major hemocyte types in Manduca sexta. European Journal of Cell Biology 65, 417–423.