Insect Biochemistry and Molecular Biology 32 (2002) 1295–1309
www.elsevier.com/locate/ibmb
Insect hemocytes and their role in immunity
M.D. Lavine, M.R. Strand ∗
Department of Entomology, University of Georgia, Athens, GA 30602, USA
Received 21 December 2001; received in revised form 14 March 2002; accepted 2 April 2002
Abstract
The innate immune system of insects is divided into humoral and cellular defense responses. Humoral defenses include antimicrobial
peptides, the cascades that regulate coagulation and melanization of hemolymph, and the production of reactive intermediates
of oxygen and nitrogen. Cellular defenses refer to hemocyte-mediated responses like phagocytosis and encapsulation. In this review,
we discuss the cellular immune responses of insects with emphasis on studies in Lepidoptera and Diptera. Insect hemocytes originate
from mesodermally derived stem cells that differentiate into specific lineages identified by morphology, function, and molecular
markers. In Lepidoptera, most cellular defense responses involve granular cells and plasmatocytes, whereas in Drosophila they
involve primarily plasmatocytes and lamellocytes. Insect hemocytes recognize a variety of foreign targets as well as alterations to
self. Both humoral and cell surface receptors are involved in these recognition events. Once a target is recognized as foreign,
hemocyte-mediated defense responses are regulated by signaling factors and effector molecules that control cell adhesion and
cytotoxicity. Several lines of evidence indicate that humoral and cellular defense responses are well-coordinated with one another.
Cross-talk between the immune and nervous system may also play a role in regulating inflammation-like responses in insects during
infection.  2002 Elsevier Science Ltd. All rights reserved.
Keywords: Hemocytes; Encapsulation; Nodulation; Phagocytosis; Clotting; Immunity; Antimicrobial peptides; Cytokines; Lepidoptera; Diptera;
Drosophila; Insects
1. Introduction
Multicellular animals defend themselves against
infectious organisms by two systems known as innate
and acquired immunity. The innate immune system
relies on germline encoded factors for recognition and
killing of foreign invaders, whereas the acquired immune
system produces receptors by somatic gene rearrangement
that recognize specific antigens and that allow
organisms to develop an immunological memory
(Fearon, 1997). Insects lack an acquired immune system
but have a well-developed innate response. Initial defenses
include the physical barriers of the integument or gut,
clotting responses by hemolymph, and the production of
various cytotoxic molecules at the site of wounding.
Foreign entities that pass these barriers and enter the
∗
Corresponding author. Tel.: +1-706-583-8237; fax: +1-706-542-
2279.
E-mail address: mrstrand@bugs.ent.uga.edu (M.R. Strand).
0965-1748/02/$ - see front matter  2002 Elsevier Science Ltd. All rights reserved.
PII: S0965-1748(02)00092-9
hemocoel must contend with additional cytotoxic molecules
as well as an array of different hemocytes.
The insect immune system is further subdivided into
humoral and cellular defense responses. Humoral defenses
include the production of antimicrobial peptides
(Meister et al., 2000; Lowenberger, 2001), reactive intermediates
of oxygen or nitrogen (Bogdan et al., 2000;
Vass and Nappi, 2001), and the complex enzymatic cascades
that regulate coagulation or melanization of hemolymph
(Muta and Iwanaga, 1996; Gillespie et al., 1997).
In contrast, cellular defense refers to hemocyte-mediated
immune responses like phagocytosis, nodulation and
encapsulation (Strand and Pech, 1995; Schmidt et al.,
2001). While great progress has been made over the last
several years in identifying antimicrobial peptides and
the signaling pathways that regulate their synthesis,
much less is known about control of cellular defense
responses. This is due in large part to the small size of
many insects, which makes collection of hemocytes and
identification of hemocyte-produced effector molecules
difficult. It is also often difficult to conduct manipulative
experiments on hemocyte-mediated defense responses in
1296 M.D. Lavine, M.R. Strand / Insect Biochemistry and Molecular Biology 32 (2002) 1295–1309
vivo or to isolate defined populations of hemocytes for
use in experiments conducted in vitro.
Dividing the insect immune system into cellular and
humoral responses is somewhat arbitrary, as many
humoral factors affect hemocyte function and hemocytes
are an important source of many humoral molecules.
There also is considerable overlap between humoral and
cellular defenses in processes like the recognition of
foreign intruders. Hemocytes recognize foreignness
either by direct interaction of surface receptors on hemocytes
with molecules on the invading organism, or
indirectly by recognition of humoral receptors that bind
to and opsonize the surface of the invader. Inter- and
intracellular signaling events must then coordinate effector
responses like phagocytosis or encapsulation. The
purpose of this review is to summarize our understanding
of cellular immune responses. Emphasis is placed on
studies with insects in the order Lepidoptera and dipterans
like Drosophila melanogaster, as the majority of
studies on insect hemocytes have been conducted in
these taxa.
2. Hemocyte types and hematopoiesis
Insects produce several types of hemocytes that are
traditionally identified using morphological, histochemical
and functional characteristics (Gupta, 1985; Brehelin
and Zachary, 1986). More recently, antibody and genetic
markers have been characterized in selected species that
more reliably distinguish different hemocyte types from
one another (Chain et al., 1992; Mullet et al., 1993; Willott
et al., 1994; Strand and Johnson, 1996; Gardiner and
Strand, 1999; Lebestky et al., 2000). The most common
types of hemocytes reported in the literature are prohemocytes,
granular cells ( ϭ granulocytes), plasmatocytes,
spherule cells ( ϭ spherulocytes), and oenocytoids (Fig.
1). These hemocyte types have been described from
species in diverse orders including Lepidoptera, Diptera,
Orthoptera, Blattaria, Coleoptera, Hymenoptera, Hemiptera,
and Collembola (Ahmad, 1988; Azambuja et al.,
1991; Ahmad, 1992; Luckhart et al., 1992; Fenoglio et
al., 1993; Sonawane and More, 1993; Butt and Shields,
1996; Joshi and Lambdin, 1996; Hernandez et al., 1999;
de Silva et al., 2000a).
In larval stage Lepidoptera, granular cells and plasmatocytes
are the only hemocyte types capable of adhering
to foreign surfaces, and together usually comprise more
than 50% of the hemocytes in circulation (Lackie, 1988;
Ratcliffe, 1993; Strand and Pech, 1995). In the noctuid
moth Pseudoplusia includens, two distinct subpopulations
of plasmatocytes are distinguished by antibody
markers while no antigenic differences are detected
among circulating granular cells (Gardiner and Strand,
1999). The other hemocytes described from Lepidoptera
are non-adhesive spherule cells, oenocytoids and prohemocytes.
Spherule cells have been suggested to transport
cuticular components (Sass et al., 1994), while oenocytoids
contain cytoplasmic phenoloxidase precursors
that likely play a role in melanization of hemolymph
(Ashida and Dohke, 1980; Iwama and Ashida, 1986;
Jiang et al., 1997). Prohemocytes are hypothesized to
be stem cells that differentiate into one or more of the
aforementioned hemocyte types. The hemocytes
described from Drosophila are named somewhat differently
from those of most other insects, including lower
dipterans such as culicids and simuliids (Lanot et al.,
2001). During embryogenesis, the only hemocytes
present are called macrophages or plasmatocytes. In larvae,
plasmatocytes remain the most abundant hemocyte
type in circulation but two other cell types, called lamellocytes
and crystal cells, are also present. Like plasmatocytes,
lamellocytes are able to attach to foreign surfaces
while crystal cells are non-adhesive hemocytes that contain
phenoloxidase precursors and appear morphologically
similar to what are called oenocytoids in other
insects.
In mammals, all blood cells derive from hematopoietic
stem cells that differentiate into different lineages under
control of transcription factors like GATA 1–3 and
AML1 (Lebestky et al., 2000). Hemocytes first develop
in insects during embryogenesis from head or dorsal
mesoderm (Hoffmann et al., 1979; Ratcliffe et al., 1985;
Tepass et al., 1994). Insects also continue to produce
hemocytes during larval or nymphal stages via division
of stem cells in mesodermally derived hematopoietic
organs and/or by continued division of hemocytes
already in circulation (Jones, 1970; Akai and Sato, 1971;
Feir, 1979; Ratcliffe et al., 1985). Numerous scenarios
have been proposed for the lineage relationships among
different hemocyte types. In Lepidoptera, Beaulaton
(1979) proposed a linear maturation process in Bombyx
mori whereby prohemocytes in hematopoietic organs
differentiate into plasmatocytes that are released into circulation.
Plasmatocytes in circulation then differentiate
into granular cells, spherule cells and oenocytoids. The
short-term culture experiments of Yamashita and Iwabuchi
(2001), however, suggest that B. mori prohemocytes
are able to differentiate into plasmatocytes, granular
cells, or spherule cells. Studies with the noctuid moths
Euxoa declarata and Spodoptera frugiperda suggest a
dual origin for hemocytes during larval development.
Hematopoietic organs contain almost exclusively putative
stem cells and plasmatocytes, while granular cells,
spherule cells and oenocytoids are only observed in circulation
(Arnold and Hinks, 1976; Hinks and Arnold,
1977; Gardiner and Strand, 2000). Using bromodeoxyuridine
(BrdU) incorporation and hemocyte-specific antibody
markers, Gardiner and Strand (2000) determined
that prohemocytes/plasmatocytes in hematopoietic
organs and all cell types in circulation, with the exception
of oenocytoids, actively divide. These results also
1297M.D. Lavine, M.R. Strand / Insect Biochemistry and Molecular Biology 32 (2002) 1295–1309
Fig. 1. Nomarski images of the five types of hemocytes found in lepidopterans like Pseudoplusia includens. (A) Prohemocytes, (B) spherule cell,
(C) oenocytoid, (D) granular cells, and (E) plasmatocytes. Scale bar ϭ 50 µm.
suggested that: (1) stem cells ( ϭ prohemocytes) in hematopoietic
organs differentiate primarily into plasmatocytes,
whereas other hemocyte types differentiate after
release into circulation, and (2) maintenance of hemocyte
populations in circulation depends heavily on continued
division of each cell type after differentiation.
In Drosophila, the GATA homolog Serpent (Srp) is
expressed in hematopoietic stem cells (Rehorn et al.,
1996). Two additional transcription factors, glial cell
missing (Gcm) and the AML1-like protein Lozenge (Lz),
function downstream of Srp and are required for plasmatocyte
and crystal cell development, respectively
(Lebestky et al., 2000). Misexpression of Gcm in crystal
cells appears to cause transformation of these cells into
plasmatocytes based on changes in morphology and
expression of the plasmatocyte-specific receptor Croquemort.
However, expression of Lz in plasmatocytes does
not convert them into crystal cells. A small population
of Lz-expressing precursor cells also appear to develop
into plasmatocytes instead of crystal cells. Upon maturation,
these plasmatocytes no longer express Lz but do
express Gcm. Whether these plasmatocytes represent a
functionally distinct plasmatocyte sub-population is
unknown. Other genes implicated in proliferation and
differentiation of hemocytes include the Janus kinase
(JAK)/signal transducer and activator transcription
(STAT) (Dearolf, 1999; Myrick and Dearolf, 2000) and
Toll signaling pathways (Govind, 1999). Identification
of homologs for Srp, Lz, and Gcm have not yet been
reported from other insects, but expression patterns of
these factors in combination with other markers could
greatly help in clarifying the relationships among hemocyte
types in different insect taxa.
3. Hemocyte types involved in cellular immune
responses
As noted in the Introduction, the main defense
responses involving hemocytes are phagocytosis, nodulation
and encapsulation. Hemocytes also respond to
external wounding by participating in clot formation.
Phagocytosis refers to the engulfment of entities by an
individual cell. Hemocytes phagocytose both biotic tar-
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gets like bacteria, yeast, and apoptotic bodies as well as
small abiotic targets like synthetic beads or particles of
India ink (Yokoo et al., 1995; Hernandez et al., 1999; de
Silva et al., 2000b). The particular hemocytes reported to
be phagocytic varies among insect taxa, and in some
cases discrepancies even exist in the literature among
studies on the same species (Anggraeni and Ratcliffe,
1991; Tojo et al., 2000). These differences are likely due
in part to difficulties with identifying hemocytes in some
insects. In Lepidoptera, granular cells and plasmatocytes
are the only hemocyte types reported to be phagocytic,
while in Drosophila plasmatocytes are the main phagocytic
hemocyte (Elrod-Erickson et al., 2000).
Nodulation refers to multiple hemocytes binding to
aggregations of bacteria while encapsulation refers to the
binding of hemocytes to larger targets like parasitoids,
nematodes and chromatography beads. Nodule and capsule
formation look nearly identical at the ultrastructural
level (Ratcliffe and Gagen, 1976, 1977) which suggests
they are essentially the same process albeit against different
targets. Unlike phagocytosis, nodulation and
encapsulation result in the formation of an overlapping
sheath of hemocytes around a target. Again, the two
types of hemocytes most often observed in capsules are
granular cells and plasmatocytes in Lepidoptera, and
lamellocytes in Drosophila (Schmidt et al., 2001; Vass
and Nappi, 2001). Nodules and capsules melanize in
some insect species but not others (Strand and Pech,
1995). For those species in which melanization occurs,
oenocytoids or in Drosophila crystal cells may also play
a role in capsule formation.
The distribution of granular cells and plasmatocytes in
capsules formed by some lepidopterans appears random
(Wiegand et al., 2000) while in others it is highly
organized, with granular cells being the first cells to bind
to the target and plasmatocytes attaching thereafter (Sato
et al., 1976; Schmit and Ratcliffe, 1977, 1978; Lackie
et al., 1985; Pech and Strand, 1996). For example,
encapsulation of Dowex 1X-2 chromatography beads by
hemocytes from the moth P. includens begins when individual
granular cells attach to the beads. Thereafter, plasmatocytes
attach to the target and one another, forming
a multicellular sheath. Capsule formation then ends
when a monolayer of granular cells attach and apoptose
on the capsule periphery (Pech and Strand, 1996, 2000)
(Fig. 2). Neither granular cells nor plasmatocytes form
capsules alone, but plasmatocytes readily encapsulate
this target following attachment of granular cells. While
Dowex 1X-2 beads are encapsulated more rapidly in
vivo, capsule architecture is virtually identical to capsules
formed in vitro (Pech and Strand, 1996; Loret and
Strand, 1998). Lavine and Strand (2001a) elaborated on
these studies by finding that many other abiotic targets
are also only encapsulated by plasmatocytes following
attachment of granular cells. However, some targets can
be encapsulated by plasmatocytes in the absence of
Fig. 2. Encapsulation of a foreign target (Dowex 1X-2 chromatography
bead) by hemocytes from Pseudoplusia includens. Fluorescent
images of hemocytes labeled with the granular-cell-specific antibody
marker 48F2F5 and plasmatocyte-specific marker 52F3A5 or
49B11C6. (A) Light microscopy image 30 min after presentation of
the target to hemocytes. Some hemocytes have already bound to the
target’s surface. Scale bar ϭ 20 µm. (B) Fluorescent image of the
same target after double-labeling for granular cells (green) and plasmatocytes
(red). Only granular cells (G) are attached to the target. (C)
Light microscopy image 12 h after presentation of the target to hemocytes.
Overlapping layers of hemocytes have now attached to the target.
Scale bar ϭ 120 µm. (D) Fluorescent image of the same target
after double-labeling granular cells (red) and plasmatocytes (green).
The overlapping layers of hemocytes attached to the target are almost
exclusively plasmatocytes (P). (E) Fluorescent image of a 16 h old
capsule. The core of the capsule is comprised primarily of plasmatocytes
(P) (green) while rounded granular cells (G) (red) are attached
to the capsule periphery. (F) Fluorescent image of a 30 h old capsule.
Granular cells (G) around the capsule periphery have apoptosed, leaving
a flocculent outer layer labeled by monoclonal antibody (mAb)
48F2D5 (G, arrow). Scale bar for A–D is 30 µm. Figure adapted from
Pech and Strand (1996, 2000).
1299M.D. Lavine, M.R. Strand / Insect Biochemistry and Molecular Biology 32 (2002) 1295–1309
granular cells if they are preincubated in plasma. These
results indicate that both humoral and cellular receptors
are involved in recognition of encapsulation targets.
After granular cells or humoral opsonins bind to the target,
they induce plasmatocytes to change from nonadhesive
to strongly adhesive cells that form the majority
of the capsule. Encapsulation is then terminated by the
attachment of granular cells to the capsule periphery.
The literature offers little insight into the hemocytes
involved in clot formation, although we would speculate
that granular cells and plasmatocytes are likely key participants
in Lepidoptera. As with melanizing capsules,
oenocytoids or crystal cells may also play an important
role in clotting given that external wounds usually melanize.
However, epidermal tissue and plasma are other
sources of phenoloxidases that could play an equally
important role in melanization. In summary, all of the
hemocyte-mediated defense responses discussed above
involve the following sequence of events. The foreign
target or wound site first has to be recognized as foreign
and then one or more hemocytes must be activated to
perform a particular effector response. The biochemical
and molecular factors involved in regulating these events
are discussed below.
4. Recognition of foreignness
In the absence of an acquired response, the biggest
challenge to the innate immune system of insects is how
to recognize pathogens and other entities from self. This
has been addressed in vertebrates by the evolution of
pattern-recognition receptors (PRRs) that bind conserved
pathogen-associated molecular pattern (PAMP) molecules
produced by microorganisms like bacteria and
fungi (Fearon, 1997). Most identified PAMPs are cellwall
components like lipopolysaccharide (LPS), peptidoglycan,
and β-1,3-glucans that are recognized by either
humoral or cellular PRRs. Humoral PRRs like mannosebinding
protein bind to the PAMP, which results in
opsonization of the infectious organism. The bound PRR
is then recognized by a cell-surface protein like the C1q
receptor expressed by macrophages. In contrast, cellular
PRRs are expressed on the surface of immune cells and
bind PAMPs directly (Aderem and Underhill, 1999).
4.1. Humoral PRRs involved in recognition of
microorganisms
Several factors including lectins, hemolin, LPS-binding
protein, gram-negative bacteria recognition protein,
peptidoglycan recognition protein, and the thioester-containing
protein αTEP1 have been identified from insects
as candidate PRRs (Bulet et al., 1999; Schmidt et al.,
2001). While the receptors on hemocytes or other tissues
that recognize these PRRs are unknown, studies do indicate
that some of these factors enhance phagocytosis or
nodulation responses by hemocytes after binding to
microorganisms. For example, the mosquito Anopheles
gambiae produces αTEP1 which is related to vertebrate
complement factors and α2-macroglobulins (Levashina
et al., 2001). αTEP1 binds to gram-negative bacteria and
suppression of this factor by RNA interference reduces
phagocytosis by a hemocyte-like A. gambiae cell line.
LPS-binding protein from B. mori (BmLBP) binds to
rough strains of Escherichia coli, which in turn are nodulated
more quickly than smooth strains of E. coli that
are not recognized by BmLBP (Koizumi et al., 1999).
A polyclonal antibody generated against BmLBP also
appears to block the opsonizing activity of B. mori
plasma since it reduces nodulation of rough strain E. coli
in in vitro assays. The immunoglobulin (Ig)-superfamily
protein, hemolin, binds LPS but whether this facilitates
any kind of effector responses by hemocytes is unclear
(Zhao and Kanost, 1995; Faye and Kanost, 1998). A
number of lectins have been purified from insects and
suggested to function as humoral PRRs (Pendland et al.,
1988; Yu and Kanost, 2000). However, in most
instances, the ligands for these lectins are unknown.
4.2. Cellular PRRs involved in recognition of
microorganisms
Less is known about the cellular PRRs on hemocytes
involved in recognition of microorganisms. Mammalian
immune cells express several Toll-like receptors that are
considered cellular PRRs, because they directly recognize
LPS and other microbial products. These receptors
in turn activate signal transduction by way of NF-␬B
that results in production of pro-inflammatory cytokines
(Akira, 2001). The Drosophila genome contains Toll,
seven additional Toll-related genes (Toll-3-8, 18wheeler)
and the gene immune deficiency (imd) (Imler
and Hoffmann, 2000). Toll-like genes have also been
identified in other insects including mosquitoes and the
Tsetse fly, Glossina papalis papalis (Luo and Zheng,
2000). In Drosophila, genetic studies indicate that Toll-,
18w- and imd-mediated signaling pathways activate different
suites of antimicrobial genes. However, Toll does
not appear to be a legitimate PRR, because a protealytically
cleaved form of the spatzle (spz) gene product is
its only known extracellular ligand (Levashina et al.,
1999). One potential humoral PRR involved in initiating
the proteolytic cascade that results in cleavage of Spatzle
and activation of Toll is a peptidoglycan recognition protein
that may recognize gram-positive bacteria (Michel
et al., 2001). Analysis of the Drosophila genome database
further reveals the presence of at least 12 genes with
sequence homology to known peptidoglycan recognition
proteins (Werner et al., 2000). Several of these genes
have transmembrane domains that suggest they are cellsurface
receptors, but to our knowledge no functional
1300 M.D. Lavine, M.R. Strand / Insect Biochemistry and Molecular Biology 32 (2002) 1295–1309
experiments have been conducted on any of these factors.
The humoral PRR hemolin lacks a transmembrane
region but membrane extracts from hemocytes suggest
this factor may exist in a membrane-associated form in
Lepidoptera (Bettencourt et al., 1997).
Another potential class of cellular PRRs involved in
recognition of microbes is the integrins. This large family
of (α,β)-heterodimeric transmembrane proteins are
produced by metazoans in a variety of tissues and cells.
Ligand specificity is determined by the specific α- and
β-chain and can vary greatly among family members. In
mammals, most integrins bind extracellular matrix
(ECM) proteins like laminin, collagen IV, and fibronectin
that contain the recognition sequence Arg–Gly–Asp
(RGD) (Gonzalez-Amaro and Sanchez-Madrid, 1999).
However, some integrins, like CD11b/(CD18), bind bacterial
cell wall components like LPS. The Drosophila
genome contains five α- and two β-subunits, and three
complete integrins have been functionally characterized
(Hynes and Zhao, 2000). Each of these integrins is
essential for developmental processes but none have
been implicated in recognition of microorganisms. However,
studies with the Mediterranean fruit fly, Ceratitis
capitata, indicate that soluble RGD decreases phagocytosis
of E. coli by hemocytes, as does a monoclonal
antibody directed against a murine integrin β-subunit
(Foukas et al., 1998).
4.3. Recognition of other foreign targets and altered
self
It is not surprising that insects have evolved PRRs that
recognize bacteria and fungi, because these microorganisms
have surface proteins that are biochemically
distinct from the hosts they infect. However, insect hemocytes
also recognize many other targets that are less
molecularly distinct from themselves. These range from
other biological entities like protozoans, nematodes,
insect parasitoids and xenobiotic transplants of tissues
from other insect species (Lackie, 1988; Strand and
Pech, 1995; Gillespie et al., 1997) to self or conspecific
tissues that have a damaged basement membrane (BM)
(Rizki and Rizki, 1980a, 1980b; Karacali et al., 2000).
Hemocytes also recognize abiotic targets like nylon,
latex, and chromatography beads that are made of
materials insects are unlikely to ever have encountered
during evolutionary time. These observations emphatically
underscore that the insect immune system recognizes
a much broader suite of molecules than just the
PAMPs on microorganisms. The question is, how insects
are able to do this in the absence of an acquired immune
system? Few answers are currently available, but we
think it highly unlikely that insects express receptors
individually tailored to recognize the eclectic assemblage
of targets hemocytes are able to phagocytose or
encapsulate. Instead we would hypothesize that insects
more likely rely on receptors with promiscuous binding
properties that interact with a variety of pattern molecules
normally not encountered in the hemocoel.
Consider first the recognition of altered self tissues.
While still poorly defined, the BM of insects and vertebrates
contains many components including proteoglycans,
insoluble fibrous proteins, like Type IV collagen,
and soluble multiadhesive matrix proteins. BM lines the
hemocoel and all internal organs of insects, and hemocytes
normally do not attach or attach only weakly to
this substrate. However, several lines of evidence indicate
that hemocytes are sensitive to changes in this
matrix. First, hemocytes are known to be involved in
remodeling of the BM which is broken down and rebuilt
during metamorphosis (Wigglesworth, 1973; Ball et al.,
1987; Nardi and Miklasz, 1989; Nardi et al., 2001).
Granular cells appear to be the main hemocyte involved
in secretion of BM components in Lepidoptera (Nardi
and Miklasz, 1989; Nardi et al., 2001), and, in the dipteran
Sarcophaga peregrina, a 200 kDa surface protein
has been implicated in binding of granular cells to basal
lamina during metamorphic reprogramming (Kurata et
al., 1991, 1992). Second, a number of studies report that
hemocytes aggregate at external wound sites or encapsulate
tissue transplants from conspecifics if they have a
damaged BM (Rizki and Rizki, 1980b; Lackie, 1988).
The Drosophila melanotic tumor mutants tu-W and
tu(l)Szts
similarly exhibit an autoimmune response whereby
hemocytes encapsulate their own fat body. Studies
with these mutants suggest that encapsulation is due to
recognition of an altered BM (Rizki and Rizki, 1974,
1980a).
Taken together, these trends argue that detection of
altered self by hemocytes involves changes to the BM.
The lack of response by hemocytes to undamaged BM
could be due to either the absence of surface receptors
on hemocytes that recognize BM components, or to the
presence of molecules on BM that inhibit hemocyte
binding. In vertebrates, inhibitors of cell adhesion such
as thrombospondin, tenascin, SPARC, and various mucopolysaccharides
localize to BM (Greenwood et al.,
1998; Iozzo, 1998). Hemocytes from P. includens and
Manduca sexta likewise adhere poorly or not at all to
commercially available BM preparations like Matrigel
or highly purified polysaccharides like reagent-grade
agarose (Pech et al., 1995; Lovallo and Cox-Foster,
1999; Strand and Clark, 1999). In contrast, hemocytes
may bind to sites where the BM is altered, because of
structural modifications to glycoconjugants or because
novel ligands are presented. An example of the former
occurs in the wax moth, Galleria mellonella, which normally
does not encapsulate self tissues but rapidly encapsulates
self tissues pretreated with neuraminidase that
removes sialic acid residues (Karacali et al., 2000).
Removal of sialic acid could either unmask antigens
recognized as foreign or removal of these residues alters
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the glycoprotein in a way that allows soluble or cellsurface
receptors to bind.
A possible example of hemocytes recognizing altered
self by novel molecules is phagocytosis of apoptotic
bodies. In Drosophila, the surface receptor Croquemort
is specifically expressed on plasmatocytes, and Croquemort
null mutants are unable to phagocytose apoptotic
cells (Franc et al., 1996, 1999). Although the ligand on
apoptotic cells recognized by Croquemort is unknown,
apoptotic and necrotic cells present novel surface molecules
like phosphatidyl serine (PS) that are absent on
healthy cells. Croquemort also shows some sequence
similarity to CD36, which is a type of scavenger receptor
(SR) found on mammalian macrophages. SRs are
defined primarily by their promiscuous binding of
characteristic, though not invariant, polyanionic ligands
like modified lipoproteins, anionic polysaccharides, and
anionic phospholipids. Some SRs are also able to bind
prokaryotic PAMPs like LPS and lipoteichoic acid,
nucleic acids, and even abiotic surfaces like glass (Platt
and Gordon, 1998). In vertebrates, such ligand promiscuity
implicates SRs in recognition of molecules associated
with both altered self and foreign invaders. However,
Croquemort does not appear to be a typical SR, because
it does not bind the standard ligands recognized by other
family members (Franc et al., 1996). Plasmatocytes from
Croquemort null mutants also retain the ability to phagocytose
bacteria (Franc et al., 1999), suggesting that this
receptor may function primarily in recognition of apoptotic
cells. A novel scavenger receptor (dSRC-I) has
been identified from Drosophila that does bind polyanionic
ligands (Pearson et al., 1995). However, dSRCI
does not as yet have a known function in immunity.
The molecular mechanisms by which hemocytes
recognize altered self may also be involved in recognition
of foreign targets, like xenotransplants and parasitoids,
that are also of insect origin. Differences in BM
and other surface components are likely to increase with
phylogenetic distance between species. Consistent with
this assumption, hemocytes tend to encapsulate xenobiotic
transplants from phylogenetically more distant species
of insects more readily than tissues from closely
related species (Lackie, 1988). The similarity of the surface
components of parasitoids to the insect host’s own
BM may help determine the ability of the host to mount
a hemocytic defense response. For example, hemocytes
from obligate hosts usually do not bind to the eggs or
larvae of parasitoids. However, mechanical or enzymatic
disruption of surface factors or membranes from the
parasitoid egg or larva almost always results in rapid
encapsulation (Schmidt et al., 2001). Unfortunately, the
specific surface features and receptors involved in these
interactions are unknown.
Unlike the complexities of BM and other biological
surfaces, chromatography beads are made of welldefined
materials that could provide insight about the
types of surfaces hemocytes recognize (Vinson, 1974;
Lackie, 1983; Wiesner, 1992; Zahedi et al., 1992; Lavine
and Strand, 2001a). Yet, comparisons among species
identify few unifying themes as to the types of beads
hemocytes most avidly encapsulate. For example, Lavine
and Strand (2001a) used 19 different bead types as
encapsulation targets in P. includens. Encapsulation
assays revealed that some beads are recognized by
granular cells and then encapsulated by plasmatocytes,
others are encapsulated directly by plasmatocytes if
opsonized by humoral molecules in plasma, and some
beads are never encapsulated. Beads with certain functional
groups (sulfonic, diethylaminoethyl, and quaternary
amine) are encapsulated more readily than other
functional groups tested, and positively charged beads
are better encapsulated than negatively or neutrally
charged beads. In contrast, targets with carbohydrate
moieties on their surface (neutral dextran, neutral agarose,
N-acetyl-glucosamine, N-acetyl-galactosamine) are
the most poorly encapsulated targets. These results indicate
that: (1) both humoral and cellular receptors are
involved in recognition of abiotic targets just as they are
with microorganisms, and (2) the importance of humoral
and cellular receptors differs as a function of bead
matrix, charge and functional group. However, when
these beads were tested in other noctuid moths, some
responses were similar to P. includens while others differed
greatly. For example, CM-Sephadex is rarely
encapsulated by P. includens but is always encapsulated
by Spodoptera frugiperda, and DEAE-Sepharose is
strongly encapsulated by P. includens but is rarely
encapsulated by S. frugiperda. Thus, while we would
expect that insects in the same family use the same types
of molecules as recognition receptors, affinities for specific
ligands can still greatly differ.
One factor potentially involved in recognition of chromatography
beads is a glutamine-rich protein purified
from the beetle Tenebrio molitor (Cho et al., 1999). This
protein exists as two isoforms, with molecular masses
of 56 kDa and 48 kDa. Neutralization experiments using
antibodies against each isoform suggest that both are
required for opsonization of targets. Studies with P.
includens indicate that, in the absence of plasma,
addition of the tripeptide RGD to culture medium dosedependently
inhibits binding of P. includens plasmatocytes
(strongly) and granular cells (weakly) to polystyrene.
This indicates that, similar to some microorganisms
(see above), recognition of some abiotic
targets also involves an RGD-dependent mechanism
(Pech and Strand, 1996). Recent studies further indicate
that P. includens granular cells and plasmatocytes
express three alpha and one beta integrin subunit (Lavine
and Strand, 2001b). Functional studies are still in progress
but expression of these subunits by hemocytes
strongly suggests that integrins play a role in recognition
of both microbial and abiotic targets.
1302 M.D. Lavine, M.R. Strand / Insect Biochemistry and Molecular Biology 32 (2002) 1295–1309
5. Hemocytic-mediated effector responses
5.1. Phagocytosis
Phagocytosis is a widely conserved cellular response
that occurs in many Protozoa and all metazoan phyla. It
has been most extensively studied in mammalian leukocytes
like macrophages (Aderem and Underhill, 1999).
An extensive review of the mechanisms controlling formation
and maturation of the phagosome in macrophages
is beyond the scope of this article. However, the
process of phagocytosis begins when a target binds to
its cognate receptor. This activates signaling cascades
that regulate formation of a phagosome and target ingestion
via an actin polymerization-dependent mechanism.
The phagosome matures into a phagolysosome when
effector molecules are introduced by vesicle fusion
events. These effector molecules then kill and/or degrade
the target. Phagocytosis has not been extensively studied
in insect hemocytes. However, antibodies to some mammalian
signaling molecules that regulate phagocytosis
cross-react with proteins in hemocyte lysates from the
fruit fly, C. capitata. This has led to the suggestion that
the signal transduction pathways regulating phagocytosis
are conserved among insects and mammals (Foukas et
al., 1998; Metheniti et al., 2001).
Mammalian phagocytes are well known to generate
reactive oxygen intermediates (ROI) and reactive nitrogen
intermediates (RNI) which are released into the
phagosome or extracellularly, and which are toxic to a
variety of microorganisms (Nathan and Hibbs, 1991;
Robinson and Badwey, 1994). ROI or RNI have also
been detected in the hemolymph and/or hemocytes of
many insects. For example, increased superoxide levels
occur in the hemolymph and hemocytes of the West
Indian leaf cockroach Blaberus discoidalis after
exposure to heat-killed E. coli, LPS, and laminarin
(Whitten and Ratcliffe, 1999). E. coli growth is also
enhanced when ROI scavengers or inhibitors of the
mammalian respiratory burst are added to B. discoidalis
hemolymph, suggesting that ROI is an important killing
mechanism (Whitten and Ratcliffe, 1999). Although not
directly related to phagocytosis, infection of the mosquito
Anopheles stephensi by Plasmodium berghei and
Plasmodium falciparum increases nitrite/nitrate levels
which are end products of the RNI generating conversion
of l-arginine to l-citrulline by nitric oxide synthase in
hemolymph (Luckhart et al., 1998). Plasmodium infection
also induces increased expression of an A. stephensi
nitric oxide synthase gene (Luckhart et al., 1998; Luckhart
and Rosenberg, 1999). Addition of l-arginine to A.
stephensi’s diet decreased the mean number of Plasmodium
ookinetes in infected mosquitoes, while addition of
an l-arginine analog to the diet resulted in increased
numbers of ookinetes relative to controls (Luckhart et
al., 1998).
Some of the effects of ROI and RNI may be due to
their roles in immune-related signal transduction pathways,
rather than to any direct cytotoxic effects on parasites
or pathogens. In mammals, ROI such as H2O2, and
RNI such as NO, function as second messengers in signal
transduction pathways that include activation of NF␬B
(Kaul and Forman, 1996). These types of activities
have been poorly studied in insects, although exogenous
H2O2 is reported to increase expression of the antimicrobial
peptide attacin in the giant silkmoth, Hyalophora
cecropia (Sun and Faye, 1995). Superoxide anion, H2O2
and NO have also been detected during cellular immune
responses in Drosophila (Nappi et al., 2000), but
whether these factors are related to NF-␬B activation
is unknown.
Although the fat body is usually considered the most
important source of antimicrobial peptides, hemocytes
also produce these molecules in some insects
(Lowenberger, 2001). To our knowledge, antimicrobial
peptides from insect hemocytes have not been implicated
in killing of phagocytosed bacteria. However, studies
with a mollusc indicate that hemocytes release the antibacterial
peptide mytilin into both the medium and phagolysosomes
when phagocytosing bacteria (Mitta, 2000).
5.2. Encapsulation and nodulation
Once a target is recognized, formation of a capsule or
nodule requires that circulating hemocytes change from
non-adhesive to adhesive cells that are able to bind to
the target and one another. Changes in the adhesive state
of mammalian immunocytes are regulated by signaling
molecules (cytokines), cell adhesion molecules, and their
cognate receptors. For example, specific chemokines
activate monocyte adhesion by inducing the rapid export
of adhesion molecules from cytoplasmic granules to the
cell surface (Baggiolini et al., 1997). In lepidopterans
like P. includens, binding of granular cells or humoral
PRRs to a foreign surface similarly induces a rapid
change in the adhesive state of plasmatocytes which, as
previously discussed, are the main capsule-forming cells.
The most potent plasmatocyte activator identified to
date is a 23-amino-acid cytokine called plasmatocyte
spreading peptide (PSP) (Clark et al., 1997) (Fig. 3).
Both purified and synthetic PSP induce plasmatocytes to
aggregate or adhere to foreign surfaces within seconds
at concentrations Ն100 pM. PSP is expressed as a propeptide
of 142 residues with the PSP sequence located
at the C-terminus (Clark et al., 1998). This biologically
inactive precursor is then cleaved by an unknown protease
to release the mature peptide. The structure of PSP
consists of a disordered N-terminus (residues 1–6), and
a well-structured core (residues 7–22) stabilized by a disulfide
bond, hydrophobic interactions, and a short β-hairpin
turn (Volkman et al., 1999). PSP homologs have
been identified from a number of other lepidopterans,
1303M.D. Lavine, M.R. Strand / Insect Biochemistry and Molecular Biology 32 (2002) 1295–1309
Fig. 3. Processing and activity of the cytokine plasmatocyte spreading
peptide (PSP). The 142-amino-acid PSP precursor protein consists
of a strongly hydrophobic signal sequence domain at its N-terminus
(red), a central domain (green), and a C-terminal domain that encodes
PSP (blue). Two basic residues (Lys117
and Arg119
) N-terminal from
Glu120
serve as potential recognition sties for endoproteolytic cleavage
of the precursor protein to yield the mature peptide. Dose–response
studies indicate that PSP dose-dependently induces the attachment and
spreading of plasmatocytes to foreign surfaces and one another at concentrations
Ն100 pM. At concentrations of Ն10 nM, PSP induces plasmatocytes
to aggregate or adhere in less than 30 s.
and in some cases these homologs have also been shown
to be plasmatocyte activators (Wang and Jiang, 1999;
Strand et al., 2000). Based on the consensus sequence
of their N-termini (Glu–Asn–Phe–X–X–Gly), these molecules
are referred to as the ENF-peptide family (Strand
et al., 2000). Comparison with other proteins reveals
that, despite sequence identity at only four positions, the
core region of PSP adopts a very similar structure to the
C-terminal subdomain of human epidermal growth factor
(hEGF) and the fifth domain of the anticoagulant protein
thrombomodulin (hTM5). In contrast, the N-terminus
of PSP is similar only to other members of the
ENF-peptide family (Volkman et al., 1999). Mutational
analysis indicates that specific residues in both the disordered
and ordered domains of the molecule are
required for biological activity (Clark et al., 2001a,
2001b). Several other partially purified factors have been
reported to induce changes in hemocyte adhesion (Chain
and Anderson, 1982; Davies et al., 1988). Injection of
certain inhibitors of eicosanoid biosynthesis into M.
sexta reduces nodulation of bacteria, leading to the
suggestion that cyclo- and lipoxygenase products may
also regulate hemocyte adhesion responses (Miller et al.,
1994). However, whether any of these factors are signaling
molecules or possible homologs of the ENF-peptide
family is unknown.
The next question of interest is how these extracellular
signaling molecules induce a change in the adhesive
state of plasmatocytes. Studies with PSP suggest that
binding to its cognate receptor causes plasmatocytes to
export cytoplasmically stored adhesion molecules to
their surface (Strand and Clark, 1999). In vertebrates,
four main families of receptors mediate cell adhesion
events: cadherins, Ig-superfamily members like NCAMs,
selectins and integrins. The identification of multiple
integrin subunits and the fact that soluble RGD
inhibits plasmatocyte adhesion circumstantially suggest
that integrins are involved in plasmatocyte binding during
encapsulation (Pech et al., 1995; Lavine and Strand,
2001b). Integrins have also been implicated in regulating
adhesion of crustacean and molluscan hemocytes
(Johansson, 1999). Neutralization experiments with the
antibody M13 further suggests that an unidentified protein
on the surface of plasmatocytes from M. sexta is also
required for aggregation and binding to foreign surfaces
(Wiegand et al., 2000).
Examination of capsules from a number of species
indicates that termination of an encapsulation response
occurs when a BM-like layer appears on the capsule’s
periphery (Grimstone et al., 1967; Pech and Strand,
1996; Liu et al., 1998). In P. includens, this BM-like
layer is produced by a monolayer of granular cells that
attaches to the periphery of the capsule at the end of an
encapsulation response (Pech and Strand, 1996, 2000).
The signal(s) from plasmatocytes that recruits granular
cells to the periphery of the capsule is unknown, but the
role of granular cells in terminating capsule formation
is notable as granular cells are also the main hemocyte
type involved in secretion of the BM components that
line the hemocoel (see Section 4.3). In effect, the periphery
of the capsule likely assumes the characteristics of
intact BM, which creates a self surface to which plasmatocytes
no longer bind. In H. cecropia and M. sexta, the
Ig-related protein hemolin inhibits hemocyte aggregation
(Ladendorff and Kanost, 1991; Bettencourt et al., 1997)
but whether this activity has any role in capsule termination
is unknown.
Once a capsule has formed, the encapsulated organism
almost always dies. Several factors including asphyxiation,
production of toxic quinones or hydroquinones via
the prophenoloxidase (PPO) cascade, ROI and RNI, and
antibacterial peptides have been suggested to function as
killing agents (Salt, 1970; Gillespie et al., 1997; Nappi
et al., 2000). However, as with phagocytic responses by
insect hemocytes, there is little direct experimental evidence
to support a role for any of these factors in killing
encapsulated targets. For example, resistant strains of
Drosophila that encapsulate the parasitoid Leptopilina
boulardi produce increased levels of superoxide anion,
H2O2 and nitrite compared with susceptible strains
(Nappi et al., 1995; Nappi and Vass, 1998; Nappi et al.,
2000). Yet, superoxide anion and H2O2 do not appear to
1304 M.D. Lavine, M.R. Strand / Insect Biochemistry and Molecular Biology 32 (2002) 1295–1309
be toxic to L. boulardi, and it is equally unclear whether
elevated levels of these factors actually exist in the capsule
itself. Although some insects produce capsules that
never melanize, capsules formed by Drosophila and
many other species often do. This has led to the suggestion
that cytotoxic quinoid intermediates produced during
melanization reactions may serve as killing agents
(Nappi et al., 2000). Again, though, no studies we are
aware of support this experimentally.
5.3. The role of hemocytes in wounding and clotting
PAMPs like LPS activate a complicated proteolytic
cascade that results in rapid clotting of hemolymph in
crustaceans and in chelicerates. Elegant studies in the
horseshoe crab, Limulus polyphemus, have identified
several important humoral- and hemocyte-produced
components of this clotting cascade that ultimately result
in precipitation of the protein coagulogen into insoluble
coagulen (Muta and Iwanaga, 1996; Iwanaga et al.,
1998). The hemolymph of some insects also coagulates
upon external wounding but little is known about these
clotting systems save their dependence on both humoral
factors and hemocytes. A lectin and the serine protease
scolexin have been implicated in clotting of hemolymph
in M. sexta (Minnick et al., 1986; Finnerty et al., 1999),
while a multidomain protein with some similarity to the
vertebrate clotting protein von Willibrand factor has
been suggested to play a role in clotting in B. mori
(Kotani et al., 1995). Hemocytes also contribute to clot
formation in insects by aggregating at wound sites
(Gregoire, 1974). Damaged epidermal cells release a
partially purified protein named hemokinin that induces
rapid aggregation of hemocytes in H. cecropia (Cherbas,
1973). Whether hemokinin is a signaling molecule or an
adhesion protein is unknown.
6. Coordination of the insect immune system
The preceding discussion makes clear that the humoral
and cellular arms of the insect immune system both
contribute to defense against pathogens. This is well
illustrated in Drosophila, where disrupting phagocytosis
does not reduce survival of wild-type flies injected with
E. coli, but does reduce survival of imd flies that are
unable to synthesize antibacterial peptides in response
to gram-negative bacteria (Elrod-Erickson et al., 2000).
Other Drosophila mutants, like domino, reduce the number
of circulating hemocytes while bc inhibits hemolymph
phenoloxidase activity. Imd, domino, bc, and
bc/imd double mutant flies all show similar survival rates
to wild-type flies when injected with bacteria. In contrast,
domino/imd and domino/bc double mutants, and
domino/imd/bc triple mutants all show greatly increased
mortality (Braun et al., 1998). While uncharacterized
pleiotropic effects could clearly contribute to these outcomes,
these results overall suggest that both humoral
and cellular immunity are essential for survival.
Few pathways linking humoral and cellular defense
responses have been identified, although several lines of
evidence suggest such linkages exist. For example, hemocytes
do not appear to be an important source of antimicrobial
peptides in Drosophila as domino mutants
show wild-type levels of synthesis after injection with
bacteria or fungi (Braun et al., 1998). However, domino
and other hematopoietic mutants like l(3)hem show
decreased levels of the antimicrobial peptide diptericin
when infected per os, which suggests that hemocytes
may coordinate antimicrobial peptide responses in the
absence of physical injury (Basset et al., 2000).
Migration of hemocytes to sites of injury or infection
may also contribute to humoral defense responses. As
previously noted, bacterial PAMPs stimulate hemocytes
from Limulus and molluscs to rapidly discharge clotting
factors and/or antimicrobial peptides (Iwanaga et al.,
1998; Mitta, 2000), while hemocytes from insects likely
release PPOs, proteases and other immune-related molecules
(Jiang et al., 1997; Wittwer and Wiesner, 1998;
Muller et al., 1999). A role for the PPO cascade in pattern
recognition and defense is also suggested from studies
with certain mosquitoes that form melanotic as
opposed to cellular capsules around foreign targets
(Richman and Kafatos, 1996; Gorman and Paskewitz,
2001).
A final point of interest is the potential cross-talk that
may occur between the immune and nervous system.
Studies with vertebrates indicate that several neuropeptides
and biogenic amines produced in the central nervous
system (CNS) are also produced and have biological
effects on immunocytes (Roitt et al., 1993). For
example, neural signaling molecules like the opioid peptide
Met-enkephalin stimulate lymphocyte proliferation
as well as migration and release of pro-inflammatory
cytokines by macrophages (Salzet et al., 2000). The biological
effects of Met-enkephalin are unlikely indirect as
vertebrate immune cells have opioid receptors and produce
the appropriate proteases to process opioid precursor
proteins, like proenkephalin, to their mature peptides.
Interestingly, opioid precursor proteins also encode antibacterial
peptides, like enkelytin, that are released along
with opioid peptides after processing. This arrangement
of signaling and killing factors could result in both an
immediate antibacterial response as well as recruitment
of immune cells and signaling of danger to the CNS.
Proenkephalins containing opioid peptide sequences
and enkelytin have been identified in molluscs and
leeches, but not in insects (Stefano et al., 1998). However,
a partially analogous situation exists in the case of
ENF peptides, like PSP, that are processed from precursor
proteins and are expressed in the CNS, fat body, and
immune cells (Clark et al., 1998; Hayakawa and Nogu-
1305M.D. Lavine, M.R. Strand / Insect Biochemistry and Molecular Biology 32 (2002) 1295–1309
chi, 1998). Besides being potent activators of plasmatocytes,
ENF peptides also have important effects on larval
growth (Hayakawa, 1990; Strand et al., 2000). The titers
of ENF-peptide family members like growth-blocking
peptide (GBP) fluctuate in concert with the larval molting
cycle, which circumstantially suggests a role for this
molecule in maintenance of normal homeostatic activity.
However, stress factors like parasitism or low rearing
temperatures induce a rapid and significant elevation in
hemolymph GBP titer that correlates with reductions in
larval growth rates and delays in molting (Hayakawa,
1990; Ohnishi et al., 1995). Given that insects often
exhibit delays in growth after wounding or infection
(Lackie, 1988; Strand and Pech, 1995), it is tempting to
speculate that, in addition to maintenance functions,
ENF peptides also serve as inflammatory mediators following
noxious perturbations. Localized release from
hemocytes and/or rapid processing of propeptide already
in circulation would clearly promote rapid adhesion of
plasmatocytes to sites of injury or infection. However,
decreased larval mobility and delays in growth due to a
systemic elevation in peptide titer could also facilitate
the healing process before the cuticle is next shed.
7. Conclusions
Considerable strides have been made in the last few
years to develop new tools for identifying hemocytes and
understanding their differentiation. Progress has also
been made in developing methods for collecting cells
and conducting manipulative experiments on isolated
sub-populations. These kinds of studies have enhanced
our understanding of the role different hemocytes play
in specific defense responses. The concept of insects
recognizing microorganisms using PRRs that bind conserved
microbe-specific surface factors logically extends
ideas originally conceived with the innate immune system
of vertebrates in mind. In the absence of an acquired
immune response, however, recognition of more closely
related foreign targets, like parasitoids, or alterations to
self are more difficult to explain, as these targets lack
surface molecules distinctly different from self tissues.
Understanding how the insect immune system is able to
distinguish such entities from self is an important question
for the future. Our understanding of the signaling
factors that regulate hemocyte-mediated defense
responses like encapsulation is also at an early stage.
Genetic screens in model species like Drosophila offer
one approach to identification of these signaling factors,
as might expressed sequence tag (EST) or other genomic
screens targeted at specific defense responses like encapsulation.
However, such molecular approaches will still
have to be complemented by bioassay-driven, biochemical
approaches since many studies already indicate that
proteolytic cascades and other post-translational processing
events are critical to production of biologically
active molecules. Lastly, ROI and other factors that hemocytes
might produce to kill phagocytosed or encapsulated
organisms have been repeatedly discussed in
reviews, but scant experimental progress has actually
been made on the subject in the past 10 years. The availability
of hemocyte markers, along with in vitro methods
for manipulating encapsulation responses, opens the
door for developing appropriate bioassays to identify
hemocyte factors involved in killing protozoan and metazoan
parasites.
Acknowledgements
Some work discussed here was funded by NIH grants
AI32617 and AI38917, and USDA NRI grant 2002-
35302-1554 to M.R.S. M.D.L. is a recipient of a predoctoral
traineeship from NIH AI107414.
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