Journal of Cell Science 103, 1261-1267 (1992)
Printed in Great Britain © The Company of Biologists Limited 1992
1261
Haemocyte heterogeneity in the cockroach Periplaneta americana
analysed using monoclonal antibodies
BENJAMIN M. CHAIN, KAREN LEYSHON-SØRLAND* and MICHAEL T. SIVA-JOTHY†
Department of Biology, University College, London, Gower Street, London WC1E 6BT, UK
*Present address: Institute for Kirurgisk forskning, Rikshospitalet, 0027 Oslo 1, Norway;
†Present address: Department of Animal and Plant Sciences, University of Sheffield, Sheffield S10 2UQ, UK
Summary
A panel of monoclonal antibodies (mAbs) raised against
whole haemocytes from Periplaneta americana has
revealed two broad categories of immunogens.
Three mAbs showed reactivity with membrane-bound
antigens on the three major morphological classes of
haemocyte as well as the basement membrane of tissues
in contact with the haemocoel. Moreover, this reactivity showed heterogeneity within morphotypes: this heterogeneity was quantified using flow cytometry.
In contrast, two other mAbs did not show reactivity
with tissues other than haemocytes: both mAbs
appeared to label unique intracellular components of
the haemocytes.
We have demonstrated that mAbs can be used to
define haemocyte subpopulations in P. americana that
are not recognisable on morphological criteria alone.
We believe this heterogeneity underlies the mechanisms
responsible for the identification and neutralisation of
non-self in the ‘immune’ response of P. americana.
Introduction
their cellular activity during immunological responses,
remain unanswered. Even less is known about the molecular mechanisms (whether via soluble cytokines, or via
inter-cellular adhesion molecules) which govern these
responses.
In contrast to the situation in insects, our understanding
of vertebrate haematology, and the role of vertebrate blood
cells in immunity, has increased dramatically over the past
twenty years. In large part, this has been a result of a move
away from cellular morphology to the study of function of
individual cellular molecules, acting as well-defined markers of cellular development, differentiation and activation.
Thus vertebrate leukocytes, and their immune reactions, are
defined in terms of a large number of different molecules
on their surfaces, which are usually identified by specific
monoclonal antibodies (mAbs).
In a similar manner, advances in the analysis of insect
haemocyte function will require the identification of
specific molecules on haemocyte subpopulations.
With this goal in view we have developed mAbs directed
specifically against haemocytes of the cockroach Periplan eta americana. In this study we describe a number of the
reagents we have developed, and discuss the potential of
these antibodies in haemocyte classification, and in future
studies on haemopoesis and immunity in this, and other,
insects. Our antibodies reveal a great deal of heterogeneity
within the broad, morphologically identifiable cellular subpopulations, and begin to identify some of the molecular
Insects demonstrate complex immunological (defence)
responses to foreign material, including microorganisms,
metazoan parasites and xenogenic grafts. Much of the published work describing this immune system has been collated in a number of review articles (Lackie, 1988; Ratcliffe
et al., 1985; Gupta, 1986). Our present understanding is that
this system consists of both cellular and humoral components which interact during an immune response in a
manner analogous to the vertebrate system, although insects
do not possess the specific memory component which is
characteristic of vertebrate immunity.
Few of the molecular or cellular mechanisms which regulate and underpin insect immunity have been defined. In
terms of cellular immunity a major obstacle to progress has
been that studies have been almost entirely confined to
descriptive morphology, in terms of both cellular classification and function. This, and the variety of insect species
that have been studied, has led to considerable confusion.
The literature contains scores of different names for insect
blood cells describing what are probably only half-a-dozen
distinguishable cell morphotypes. In addition, while it is
generally accepted that the main haemocytes involved in
immunological responses are the plasmatocytes and granulocytes, even fundamental questions regarding the developmental relationship between these two cell types, heterogeneity within these populations, and the changes in
Key words: insect haemocytes, insect immunity, monoclonal
antibodies, Periplaneta americana.
1262
B. M. Chain and others
changes which accompany the best-studied insect immune
response: the formation of capsules in the haemocoel.
Materials and methods
Insects
Parasite-free cultures of P. americana were maintained at 27 (±
1)oC in plastic vats and fed ad libitum rat chow, cat food, fresh
fruit, vegetables and water.
Preparation of haemocyte monolayers
Individual cockroaches (adult male and female) were simultaneously anaesthetized and chilled by placing them in a sealed box
containing pellets of dry ice. 300 µl anti-coagulant (Mead et al.,
1986) (98 mM NaOH, 145 mM NaCl, 17 mM EDTA, 41 mM
citric acid, pH 4.5) was injected into the haemocoel between the
9th and 10th abdominal sclerites. The abdomen was palpated to
facilitate circulation and the insect was bled by severing a posterior leg at the femur-tibia joint. Haemocytes were washed once
with anti-coagulant solution by centrifugation (250 g for 3 min).
Cells were resuspended in cockroach saline (Treherne et al., 1986)
(157 mM NaCl; 3 mM KOH; 2 mM CaCl2; 2 mM MgCl2; 5 mM
trehalose; 8.6 mM HEPES buffer, pH 7.2) at a density of 4 × 105
cells ml-1. Monolayers were prepared from haemocyte suspension
by pipetting 100 µl aliquots onto poly-D-lysine coated multi-spot
slides (Hendley and Essex, Loughton, UK). The preparations were
left for 30 min at room temperature to allow haemocytes to spread
and adhere to the slides. In a control experiment this stage was
carried out on ice to allow cell adherence but inhibit cell spreading. In some experiments the nuclear stain DAPI (4,6-diamidino2-phenylindole, 1 µg ml -1) was added during the final ten minutes of adherence. After attachment the slides were placed on ice
and the cells were fixed with 3% paraformaldehyde in cockroach
saline for 30 min.
immunocytochemistry on haemocyte monolayers. Selected
hybridomas were sub-cloned by limiting dilution, and the cells
stored in liquid nitrogen.
Immunocytochemistry
Prior to the immunolabelling procedure, the capsule sections were
allowed to reach room temperature and washed in PBS. Cell
monolayers and capsule sections were then incubated for 30 min
in 50% horse serum in PBS to saturate non-specific protein binding sites. After washing in PBS the tissue was incubated in the
primary antibody (undiluted culture supernatant). Negative control treatments substituted a PBS/10% horse serum (PBS-HS)
wash instead of a primary antibody incubation. Following incubation in the primary antibody, the cells were washed with PBS
(3 × 5 min) and then incubated for 30 min in biotin-conjugated
goat anti-mouse IgG (Sigma, Poole, Dorset, UK) diluted 1:1000
in PBS-HS. After a further wash step the tissue was incubated in
fluorescein isothiocyanate (FITC) labelled Extravidin (Sigma),
diluted 1:2000 in PBS-HS for 30 min. Following the final wash,
the tissue was mounted in Citifluor anti-fade mounting medium
(Chemistry Department, City University, Northampton, UK) and
examined with a Zeiss Universal fluorescence microscope.
Flow cytometry
Haemocytes were collected in ice-cold anticoagulant as outlined
above and were subsequently fixed, in suspension, with ice-cold
3% p-formaldehyde for 5 min. They were washed with ice-cold
anticoagulant; the subsequent protocol was identical to the preparation of monolayers for immunocytochemistry (see above) except
that all stages were carried out in suspension, with centrifugal
washes (250 g for 3 min).
After staining, the cells were resuspended in filtered PBS at
approximately 1 × 106 cells ml -1 and analysed in a Beckton Dickinson FACScan.
Preparation of capsule
Results
Capsule formation was stimulated by injecting small polyacrylamide pieces into the haemocoel of adult cockroaches. A 10% acrylamide solution was prepared as for polyacrylamide gel electrophoresis and taken up before polymerisation in a 21 gauge
hypodermic needle. After polymerisation had occurred, the polyacrylamide was extruded through the needle: the strip so formed
was cut into 5 mm pieces, which were taken up in a 19 gauge
needle and injected into the cockroach haemocoel. The recipients
were killed 2, 24 and 48 h later and dissected under saline. The
encapsulated polyacrylamide pieces were recovered and fixed in
3% p-formaldehyde for 24 h. The capsules were then washed in
phosphate buffered saline (PBS), pH 7.4, and infused with 30%
sucrose and subsequently embedded in Tissue-tek embedding
medium (Ames Division, Miles Labs., Stoke Poges, UK) and
immersed in liquid nitrogen until solid. Sections (10 µm) were cut
at −30oC using a cryostat, collected on poly-D-lysine-coated multiwell slides and air dried at room temperature. The slides were
wrapped in aluminium foil and stored at −20oC until required.
Haemocyte monolayer immunocytochemistry
Five basic morphotypes of haemocytes have been described
from P. americana: prohaemocytes, plasmatocytes, granulocytes, coagulocytes and oenocytoids (Moran, 1971;
Rowley and Ratcliffe, 1981). In our preparations, in which
cells were allowed to adhere to poly-D-lysine-coated glass
slides at room temperature, we could identify four populations on the basis of morphology alone. Two were identifiable as plasmatocytes and granulocytes (Fig. 1A). A third
cell type was granular but more elongated than typical granular cells and showed distinctive filipodia after 30 min
adhesion (Fig. 1B). This cell may represent the oenocytoid
type described by other authors, and will be referred to as
a ‘putative’ oenocytoid in this paper. A fourth cell type, the
prohaemocyte, was very rarely observed; consequently we
do not refer to this cell type in our results or interpretations. When cells were allowed to adhere to the glass substrate at 4oC, the characteristic spreading reaction was
inhibited, and under these conditions it was difficult to
classify unambiguously haemocytes into any categories,
although differences in cell morphology were evident (Fig.
1C).
Our haemocyte-specific hybridomas produced two broad
categories of antibodies. In the largest category the antibodies stained the majority of haemocyte types, and
appeared to stain both the cell surface and structures within
Monclonal antibody production
Adult Balb-c mice (Imperial Cancer Research Fund breeding
colony, Potter’s Bar, UK) were immunised intraperitoneally with
2 × 106 p-formaldehyde fixed haemocytes, at day zero and day
14. After 4-12 weeks, mice received a further boost with the same
number of cells, and were killed 4 days later. Spleen cells were
fused with the NS1 myeloma (ratio 4-5:1) using polyethylene
glycol (Mr 1500) according to standard protocols (Harlow and
Lane, 1988). Cultures were screened for growth by eye, and supernatants from wells containing hybridomas were screened by
Haemocyte heterogeneity in P. americana
Fig. 1. Interference contrast appearance of P. americana
haemocytes (A) after 30 min at room temperature, showing
granulocytes (gr) and plasmatocytes (pl) and (B) showing
‘putative’ oenocytoids (oe); (C) after 30 min on ice the
haemocytes show heterogeneity, but with no correlation to the
morphologies seen in (A) and (B).
1263
the cell. Although many antibodies of this type were isolated, we have concentrated on three representative examples from this group, the monoclonal antibodies 9D, 2C2
and 489 (Fig. 2A, B and C respectively). All three antibodies stained the surface of almost all cells with a characteristic bright ring pattern, but we could not determine
whether this staining was due to molecules which were synthesised by the cell, or material adsorbed on the surface
from the haemocoel. However, some cells showed clear,
and very bright, intracellular staining, frequently perinuclear, and often concentrated at one pole of the cell (eg.
Fig. 2C). In some cells, individual granules of antibody
reactive material could be seen, but more often staining was
diffuse.
All three antibodies showed a great degree of heterogeneity in the qualitative staining of different haemocytes.
This heterogeneity was apparent even when cells adhered
to the substratum at 4oC (Fig. 2F), and did not therefore
result only from the differential spreading behaviour of different cell types.
The possibility that brightly staining cells corresponded
to non-specific accumulation of antibody by dead cells was
also ruled out by staining cultures prior to fixation with
DAPI, a fluorescent nuclear stain which is excluded by
viable cells. DAPI-stained cells were indeed brightly
stained by antibody, but not all brightly staining cells were
also DAPI-positive, thus confirming that antibody staining
heterogeneity cannot be attributed solely to cell death (not
shown).
Although some cells clearly stained much more brightly
than others, the relationship of this staining to cellular morphology was generally complex. Both 489 and 9D stained
plasmatocytes strongly, but revealed heterogeneity within
the plasmatocyte population which was not related to any
visible differences in morphology. An example of two
apparently identical cells showing completely different
degrees of 489 staining is illustrated in Fig. 2C. In contrast
to other mAbs in this category, antibody 2C2 usually
stained ‘putative’ oenocytoids (Fig. 2B), while plasmatocytes were weakly stained.
In addition to staining haemocytes, antibodies 9D and
489 stained material which was discharged from haemocytes during the adherence step of the monolayer preparation. The discharge of a ‘plume’ of antibody-reactive material from what appeared to be disintegrating cells was
clearly visualised by antibody 9D (Fig. 2A and D). DAPI
staining confirmed that cells releasing this plume of material were dead. Material within disintegrating cells seemed
to become concentrated into large granules (Fig. 2A), which
persisted after the cell membrane had broken down, and
only then were released into the medium and onto the surface of the slide. Antibody 489 stained strands of secreted
material which appeared to extend from one cell to another
(Fig. 2E). The release of antibody-reactive medium was an
active cellular process, since these characteristic staining
patterns were never observed in cells maintained at 4oC.
Antibodies 2C2, 489 and 9D shared one major feature.
In addition to staining haemocytes, all stained the basement
membrane surrounding other tissues which lie within the
haemocoel, including nerve, ovary, fat body and muscle
(Fig. 2J).
1264
B. M. Chain and others
In contrast to the set of antibodies described above, the
second category of antibody was represented by two antibodies with more selective staining patterns among haemocyte populations, and which did not react with any other
tissue tested (not shown). Antibody 2D11 gave a punctate
labelling of a restricted area within the cytoplasm of plasmatocytes (Fig. 2G). The characteristic polar perinuclear
distribution of fluorescent labelling suggested the antigenic
sites were associated with the Golgi apparatus. In addition,
this antibody showed weak membrane staining on the
majority of plasmatocytes. This antibody did not react with
‘putative’ oenocytoids.
Antibody 3D1 stained large granules within granulocytes
and ‘putative’ oenocytoids, but did not react with plasmatocytes (Fig. 2H). Intensely fluorescent granules were also
observed attached to fibrillar threads emanating from a
3D1-negative haemocyte (Fig. 2H, insert). These results
suggest the labelling is confined to discrete vesicles or granules which may be discharged by the cell upon activation.
The staining patterns described were consistent, and
repeatable, between the cells of different animals from one
culture, as well as between cultures.
Flow cytometry
In order to make a quantitative assessment of haemocyte
morphotypes and their relation to the staining patterns of
our mAbs, haemocyte populations were collected, fixed and
stained and subsequently analysed by flow cytometry.
Analysis of the granularity (side scatter) and size (forward scatter) of haemocytes revealed no distinct categories
of granular/agranular or large/small haemocytes (Fig. 3A).
The wide band of events in Fig. 3A suggests that there is
a trend for larger cells to be granular, whilst smaller cells
tend to be agranular, but shows no clear separation of the
population into granular cells (granulocytes) and agranular
cells (plasmatocytes).
Antibody 2C2 resolved the haemocyte population into
two distinct sub-populations: most haemocytes stained very
weakly or not at all (94% of the population), whilst a small
sub-set (5.5% of the population) showed a strong positive
reaction with the antibody. Analysis of the distribution of
these events by Taylor’s (1965) method reveals these distributions to be statistically distinct Gaussian populations.
Gated analysis of these populations showed that there was
no simple relationship between 2C2 labelling and either size
or granularity. Comparison with the monolayer staining
profile (Fig. 2B) suggests that the positive population are
‘putative’ oenocytoids: 2C2 probably detects an oenocytoid
specific marker.
Antibody 2D11 shows a trimodal staining pattern
(Taylor’s (1965) analysis) on P. americana haemocytes
(Fig. 3). There is a flat negative population (4% of total),
a flat intermediate positive population (7.8% of total) and
a large, very positive population (87% of total). Gated
analysis of the negative and very positive populations
showed no simple correlation with either size or granularity: however the intermediate population was 53% richer in
granular cells than the sample population. Comparison of
these data with the monolayer staining (Fig. 2G) reveals a
different profile, suggesting an important change in the distribution/configuration of the identified antigen during
adherence and spreading. However, monolayer preparations
indicate that 2D11 is oenocytoid negative and 4% of
FACs events are negative: this correlates with the FACs
data from 2C2 where 5.5% of events are positive and probably oenocytoids. ‘Putative’ oenocytoids may therefore
constitute 4-5.5% of the haemocyte population in P. amer icana.
Antibodies 489 and 9D both showed skewed unimodal
staining patterns, suggesting that all cells bear the antigen
marker, but in variable amounts. Some cells are clearly negative, but most cells bear antigen in sufficient quantity to
produce very bright fluorescence. The heterogeneity in the
monolayer staining profiles of these two antibodies is therefore the result of observing cells that are extremes of a
single Gaussian population. The value of these antibodies
for identifying functional sub-populations is therefore
restricted: the antigen they identify might, however, be an
important developmental/activation marker.
Antibody 3D1 consistently failed to produce stained cells
for flow cytometry so has not been included.
Capsule formation
Pieces of polymerised acrylamide induced a strong encapsulating response in the haemocoel; within 1 h the acrylamide implant was covered with a sheath of cells. The size
of capsule formed varied significantly. In some cases the
capsule thickness was only 1-3 cells deep while in others
the size ranged from 10-20 cells. Antibodies 9D, 489 and
2C2 strongly labelled cells of the early 1 h capsule. In areas
where the capsule consisted of only one cell layer the
labelling was seen as a fine fluorescent sheath (Fig. 4A). In
regions where the capsule was further developed the greatest intensity of labelling was found in the outermost cells,
while in the inner layer labelling was significantly reduced
and individual cells less easily discerned (Fig. 4B). In
addition, distribution of fluorescence at a single cell level
was not uniform, with fluorescence concentrated on the side
of the cell furthest from the acrylamide (Fig. 4B). However, it was difficult to determine conclusively whether this
fluorescence was intracellular or on the cell membrane.
After 24 h a clear zonation in antibody labelling was evident in the capsule (Fig. 4C). The inner layer displayed an
autofluorescence and a middle zone, devoid of fluorescence,
was now apparent. The third, outer zone revealed a compact structure, though the previously well-defined rounded
cells were now seen in more irregular and distorted conformations. Staining in these older capsules was weaker
than in younger capsules.
The 2D11 antibody labelled the 1 h and 24 h capsules
in a manner similar, though at a reduced intensity, to antibodies 9D, 489 and 2C2 (not shown). The gradation in antibody intensity through the capsule was not evident with this
antibody, but staining was quite heterogeneous, suggesting
that both the strongly and weakly staining plasmatocytes
participated in this reaction.
Antibody 3D1 did not label the 1 h or 24 h capsules (not
shown).
At longer time periods, the acrylamide implant became
ensheathed by a brown, melanised layer upon which closely
packed haemocytes were deposited. These capsules showed
strong autofluorescence (Fig. 4D), particularly of the inner
Haemocyte heterogeneity in P. americana
1265
Fig. 3. (A) Dot plot of 10,000 haemocytes
showing covariance of size (forward
scatter) and granularity (side scatter).
Clusters of events (indicative of discrete
categories of cell morphotypes) are not
evident. All other figures show frequency
distributions of fluorescently labelled
haemocytes: the labels associated with
each figure refer to the mAb used to stain
the sample. The abscissa is an arbitrary
(but constant across samples) log scale of
fluorescence intensity. The ordinate
indicates frequency of events. For an
explanation of the negative plot see
‘Immunocytochemistry’ in the Materials
and methods section. Antibodies 489 and
9D show strong unimodal staining; 2C2
shows clear bimodality whilst 2D11 shows
a trimodal staining pattern. (NB the model
used to analyse the modality of these
distributions does not generate a P-value,
but has been shown to generate the
expected distributions within accepted
statistical limits (Taylor, 1965)).
zone, perhaps due to the polyphenols produced during
melanisation (Dennell and Malek, 1955), and were therefore not analysed further by immunocytochemistry.
Discussion
This study describes the initial characterisation of a number
of monoclonal antibodies reacting with haemocytes of P.
americana. Most of the antibodies we have described
stained the haemocyte population non-uniformally, and thus
demonstrate the underlying molecular heterogeneity of the
haemocytes. Although the degree of staining sometimes
correlated to visible differences in morphology and/or granularity, more often the heterogeneity revealed by the antibody did not correlate with morphology.
We describe three antibodies (9D, 489, 2C2) which
reacted with haemocytes and with the basement membrane
of other tissues tested (including muscle, nerve and ovary).
Although we have not yet investigated the nature of any of
the molecules recognised by our antibodies, their presence
in basement membranes and in material secreted as
‘plumes’ or strands from haemocytes suggests that they
may be mucopolysaccharides or proteoglycans. These molecules have frequently been found (using histochemical
techniques) in insect haemocytes, and are likely to play an
important part in wound repair and basement membrane
formation in both invertebrates and vertebrates. The staining pattern obtained with these antibodies suggests that the
antigens in question are synthesised within the haemocytes
and released into the medium, probably as a result of contact with the foreign poly-D-lysine/glass substratum during
haemocyte adherence.
Antibody 2D11 has a very strong staining pattern on cells
that have not adhered (Fig. 3) but has very low intensity
staining on adhered and spread cells. The most parsimonious explanation is that the antigen has become unbound,
or released, during adherence and may therefore play a
communication/opsonisation role. The FACs data also
suggest a relationship between the antigen and a subset of
granular cells.
Our findings of cross-reactive staining between haemocytes and basement membrane also support the model
defended over a number of decades by Wigglesworth
(1956, 1973) and others (Shrivastava and Richards, 1965;
Ball et al., 1987) in which the basement membrane in
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B. M. Chain and others
Fig. 4. Micrographs showing the typical staining patterns of mAbs
489, 2C2 and 9D on capsules of different age formed within the
haemocoel of adult P. americana. (A) Capsule showing intensely
staining haemocyte monolayer; (B) a thicker region of capsule
showing an increase in intensity of staining towards the
haemocoelic face, as well as showing brighter staining on
individual cells towards the haemocoel; (C) after 24 h there is
clear zonation in the staining profile of these capsules; (D) after
longer periods the capsule shows a strongly autofluorescent region
close to the acrylamide tube. * denotes the interior of the
acrylamide tube.
insects, as opposed to vertebrates, is predominantly laid
down by circulating haemocytes, rather than by the underlying tissue itself. The antigens recognised by these antibodies are also found at the plasma membrane, although
we cannot as yet be certain whether they form an integral
part of this structure or, more likely in our view, whether
they are bound to the cell surface via specific receptors. In
either case, it is apparent that cells vary considerably in
membrane staining, just as they do in the degree of intracellular staining. Although we have no data on the functional significance of this heterogeneity, comparison with
vertebrate model systems would suggest that interaction
between surface receptors and various members of these
glycoprotein families may be an important determinant of
haemocyte adhesive behaviour. Thus we believe that further studies on the biosynthesis, secretion and adhesive
activity of the antigens recognised by these monoclonal
antibodies will yield considerable information on haemocyte function.
The formation of a multicellular capsule around a foreign object (analogous to the vertebrate granuloma) is one
of the best described forms of insect defence mechanism
(Ennesser and Nappi, 1984; Lackie et al., 1985). Staining
with monoclonal antibodies 9D, 2C2 and 489 of early capsules (one hour post-implantation) demonstrates that the
cells staining strongly with these antibodies are among the
first to adhere to the surface of the implant. This reaction
may reflect an initial attempt to lay down an extracellular
matrix around the implanted object or, alternatively, the
cells may be releasing molecules which trigger the adhesion of further layers of haemocytes. As the capsule thickness increases, synthesis of these antigens towards the
inside of the capsule is switched off, presumably in order
to restrict basement membrane formation to the outer layers
of the capsule. In contrast, we could detect no staining with
antibody 3D1, which preferentially stained granulocytes. It
is possible that 3D1-positive cells did initially stick to the
implant, but were rapidly degranulated and thus could not
be detected by our staining protocol. Alternatively, 3D1positive cells may represent a subset of granular cells not
involved in encapsulation. Characterisation of the molecular structure of the antigens recognised by these antibodies
will provide further information on their possible role in
haemocyte function and the defence system of this organism.
Two important points about haemocytes follow from this
study. Firstly, the four broad morphotypes of haemocyte
(prohaemocytes, plasmatocytes, granulocytes and oenocytoids) do not fall into quantifiable categories (at least by the
FACs). Fig. 1 clearly shows that cells in the haemocyte
population combine features of “classic” granulocytes and
“classic” plasmatocytes: the data in Fig. 3A strongly
suggest there are no discrete boundaries between cell morphotypes, which, in turn, questions the validity of interpreting insect haemocyte responses in terms of cell morphotype alone. Secondly, our mAbs provide a basis for
Haemocyte heterogeneity in P. americana
classifying haemocytes: we can, for example, fairly confidently identify ‘putative’ oenocytoids using mAb 2C2 (and
indirectly with 2D11). Morphotype and molecular character are probably hand in glove in this cell type, but other
data suggest that the two main cell morphotypes (granulocytes and plasmatocytes) show more complex, and as yet
unidentified, molecular bases. This underscores the fact that
invertebrate ‘immunologists’ need to identify molecular
markers before they can assess the role of different cell
types in the response to challenge. It is especially important to quantify haemocyte phenotypes (whether they be
anatomical or molecular) in order to understand the basis
of the observed variation: variation is, after all, an inherent
feature of all biological systems. Examination of relatively
small samples from unimodal Gaussian populations can
give the impression that there are distinct subpopulations
of phenotypes: this is particularly likely to occur in monolayer preparations where sampling is the product of
observer bias. Phenotypes with unimodal distributions are
just as likely to be as important in their consequences as
bi- or poly-modal ones, but the basis of the variation must
clearly be understood before experiments can be designed,
and results interpreted.
In conclusion, we have demonstrated that antibodies can
be used to define cellular sub-populations in P. americana,
on the basis of morphology, and probably also function and
lineage. Much further work will be required to define the
role and development of these different cell types in the
insectan defence system as well as how the different subpopulations interact with each other. Understanding the role
of these cell types will require the molecular characterisation of the antigens recognised by the antibodies we have
described.
This study represents the first stage in the molecular dissection of insect cellular immunity.
This work was supported by AFRC grant no. PG32/502, and
in part by AFRC grant no. AG50/561.
1267
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(Reveived 19 June 1992 - Accepted 4 September 1992)