Hematopoietic organs of Manduca sexta and hemocyte lineages

Dev Genes Evol (2003) 213:477–491
DOI 10.1007/s00427-003-0352-6
ORIGINAL ARTICLE
James B. Nardi · Barbara Pilas · Elizabeth Ujhelyi ·
Karl Garsha · Michael R. Kanost
Hematopoietic organs of Manduca sexta and hemocyte lineages
Received: 2 April 2003 / Accepted: 8 July 2003 / Published online: 28 August 2003
Springer-Verlag 2003
Abstract Cells of the moth immune system are derived
from organs that loosely envelop the four wing imaginal
discs. The immune response in these insects is believed to
depend on the activities of two main classes of hemocytes:
plasmatocytes and granular cells. The fates of cells
that arise from these hematopoietic organs have been
followed by immunolabeling with plasmatocyte-specific
and granular-cell-specific antibodies. Cells within each
hematopoietic organ differ in their coherence and in their
expression of two plasmatocyte-specific surface proteins,
integrin and neuroglian. Within an organ there is no
overlap in the expression of these two surface proteins;
Edited by P. Simpson
J. B. Nardi () )
Department of Entomology and
Department of Natural Resources and Environmental Sciences,
University of Illinois,
320 Morrill Hall, 505 South Goodwin Avenue, Urbana,
IL, 61801, USA
e-mail: j-nardi@uiuc.edu
Tel.: +1-217-3336590
Fax: +1-217-2443499
B. Pilas
Flow Cytometry Facility, Biotechnology Center,
University of Illinois,
231 Edward R. Madigan Laboratory,
1201 West Gregory Drive, Urbana, IL, 61801, USA
E. Ujhelyi
Center for Microscopy and Imaging,
College of Veterinary Medicine,
University of Illinois,
2001 South Lincoln Avenue, Urbana, IL, 61801, USA
K. Garsha
Imaging Technology Group,
Beckman Institute for Advanced Science and Technology,
University of Illinois,
405 N Mathews Avenue, Urbana, IL, 61801, USA
M. R. Kanost
Department of Biochemistry,
Kansas State University,
104 Willard Hall, Manhattan, KS, 66506, USA
neuroglian is found on the surfaces of the coherent cells
while integrin is expressed on cells that are losing
coherence, rounding up, and dispersing. A granular-cellspecific
marker for the protein lacunin labels the basal
lamina that delimits each organ but only a small number
of granular cells that lie on or near the periphery of the
hematopoietic organ. When organs are cultured in the
absence of hemolymph, all cells derived from hematopoietic
organs turn out to immunolabel with the plasmatocyte-specific
antibody MS13. The circulating
plasmatocytes derived from hematopoietic organs have
higher ploidy levels than the granular cells and represent a
separate lineage of hemocytes.
Keywords Hemocytes · Plasmatocytes · Granular cells ·
Hematopoiesis · Insect immunity
Introduction
The immune response of caterpillars purportedly depends
on the activities of two main hemocyte populations,
granular cells and plasmatocytes, whose lineages have
remained obscure. In addition to the granular cells and
plasmatocytes that comprise approximately 85–95% of all
hemocytes in last instar larvae of Lepidoptera (Beetz et
al., submitted; Loret and Strand 1998), three other classes
of hemocytes have been usually recognized on the basis
of morphology: prohemocytes, spherule cells, and oenocytoids.
For other insect orders, the terminology applied
to hemocytes is likewise based on morphological features,
but these features often differ from order to order.
Morphological traits are also often a function of developmental
stage or the media in which hemocytes are
examined, frustrating attempts to compare hemocyte
classes from different insect orders.
A variety of lineages has been proposed for the
different classes of insect hemocytes. In some proposed
lineages, all classes of hemocytes arise from a single
population of pluripotent stem cells (Lanot et al. 2001;
Yamashita and Iwabuchi 2001; Beaulaton 1979; Gupta

478
and Sutherland 1966). In other postulated lineages, the
different classes of hemocytes are derived from at least
two different populations of precursors (Gardiner and
Strand 2000, 1999; Lebestky et al. 2000; Rizki and Rizki
1984; Shrestha and Gateff 1982; Hinks and Arnold 1977).
During differentiation, granular cells and plasmatocytes
of Lepidoptera can be distinguished by their
ultrastructure as well as their labeling patterns with
specific antibodies (Gardiner and Strand 1999; Willott et
al. 1994). These two classes of hemocytes represent two
antigenically distinct lineages. The sources of these two
different classes of circulating hemocytes in larval
Lepidoptera have been traced to (1) hematopoietic organs
and (2) the proliferation of other circulating hemocytes
(Ratcliffe et al. 1985). Immunolabeling of hematopoietic
organs from Spodoptera frugiperda with granular-cellspecific
and plasmatocyte-specific antibodies (Gardiner
and Strand 2000) revealed that about 90% of the
hemocytes in each organ are plasmatocytes.
Other findings have supported the proposal that
plasmatocytes, but not granular cells, originate from
these discrete hematopoietic organs associated with the
wing discs of Lepidoptera (Hinks and Arnold 1977; Akai
and Sato 1971). Examining populations of hemocytes in
situ following cauterization of Bombyx wing imaginal
discs and their associated hematopoietic organs, Nittono
(1964) observed a marked reduction in prohemocytes and
plasmatocytes. Nittono interpreted these results as implying
that the hematopoietic organs are the source of only
prohemocytes and plasmatocytes. Hinks and Arnold
(1977) infrequently observed the presence of granular
cells and spherule cells in hematopoietic organs of Euxoa
declarata caterpillars, but they concluded that these
particular cells were derived from the hemolymph and
did not arise intrinsically. The granular cells and spherule
cells were always found in those regions of the organ
from which other hemocytes had entered the hemolymph
by passing through openings in the organ’s basal lamina.
Although these findings support the view that hematopoietic
organs represent aggregations of stem cells that
populate the larval hemolymph with plasmatocytes (Gardiner
and Strand 2000; Hinks and Arnold 1977), other
authors have proposed that both granular cells and
plasmatocytes arise from hematopoietic organs (Yamashita
and Iwabuchi 2001; Beaulaton 1979).
For Manduca sexta, the cells of hematopoietic organs
have been characterized using a variety of approaches. At
the ultrastructural level a few cells with the distinctive
features of granular cells have been identified at the
surface of the organ. Immunolabeling the cells of
hematopoietic organs with plasmatocyte-specific and
granular-cell-specific markers has also been used to
establish the fate of these cells at a given stage. Both
electron microscopy and antibody labeling have shown
that granular cells are either (1) located on the outer
surface of the basal lamina that delimits cells within the
organ from the surrounding hemolymph or, (2) in some
instances, they are found just beneath this surface at
places where the basal lamina is disrupted. By culturing
hematopoietic organs in the absence of hemolymph, the
fate of individual cells derived from these organs can be
traced with specific antibody markers for plasmatocytes
and granular cells.
Materials and methods
Rearing and staging of larvae
All insects were reared on standard artificial diet under constant
temperature (26C) and photoperiod (18 h light:6 h ark). Each
larval stadium (L) is designated with a number (e.g., L5). Day 0
(d0) of a stadium marks the molt from the previous stadium. Each
subsequent day (n) of a stadium marks 24x(n) hours after the molt
from the previous stadium. Larvae were staged according to several
easily recognized developmental landmarks: the molt from the third
stadium (L3) to the fourth stadium (L4d0); the molt from the fourth
stadium (L4) to the fifth stadium (L5d0); and the initiation of the
wandering stage (L5d5) with its unique morphological and
behavioral features.
Sections for light and electron microscopy
Wing imaginal discs and surrounding hematopoietic organs were
removed with overlying larval integument from carefully staged
larvae and dissected in Grace’s tissue culture medium (Invitrogen).
Organs were separated from the adjacent discs with fine tungsten
needles and transferred to primary fixative of 2.5% glutaraldehyde
and 0.5% paraformaldehyde dissolved in a 0.1 M cacodylate buffer
containing 0.18 mM CaCl 2 and 0.58 mM sucrose (Tolbert and
Hildebrand 1981). After several rinses in the cacodylate buffer
containing only sucrose and CaCl 2 , tissues were post-fixed in the
same buffer containing 2% OsO 4 in place of aldehydes. Tissues
were once again rinsed in cacodylate buffer and dehydrated in a
series of ethanol concentrations (10–100%) before final infiltration
with propylene oxide and Medcast resin.
Sections (1–2 m) for light microscopy were cut with a Reichert
Ultracut E, arranged on glass slides, and stained with 1% toluidine
blue in a 1% borax solution. Sections for electron microscopy were
viewed with a Hitachi 600 at 75 kV.
Immunolabeling
Following a half-hour fixation with 4% paraformaldehyde dissolved
in phosphate-buffered saline (PBS, pH 7.4), tissues were
rinsed several times with PBS and then transferred to blocking
buffer (PBS +3% normal horse serum or 10% normal goat serum
+0.1% Triton X-100) for at least 30 min. Horse serum was added to
blocking buffer when labeling with horseradish peroxidase (HRP);
goat serum was added to blocking buffer when tissues were labeled
with Texas Red or fluorescein. Standard immunolabeling of tissues
has been described in earlier publications (Nardi et al. 1999; Nardi
and Miklasz 1989). A 1:10,000 dilution was used for each of the
following monoclonal antibodies (MAbs): MS13, MS34, MAb
3B11, and MAb 15D11. MS13 and MS34 are specific for
plasmatocytes and recognize the beta subunit of integrin (Levin
et al., in preparation). MAb 3B11 recognizes the cell adhesion
protein neuroglian; in addition to being expressed by a subpopulation
of plasmatocytes, neuroglian is expressed by a variety of
epithelial, neural and glial cells (Nardi 1994). MAb 15D11
recognizes the extracellular matrix protein lacunin that is expressed
by granular cells but not plasmatocytes (Nardi et al. 2001). As
controls, tissues were treated with normal mouse serum (1:1,000)
for 12 h in the cold prior to treatment with secondary antibodies.
Whole immunolabeled tissues were mounted in 30% 0.1 M Tris
(pH 9.0) in glycerol.

479
To double-label organs with two different mouse monoclonal
antibodies and yet ensure that secondary labeling of these mouse
monoclonal antibodies did not result in cross reactivity of the two
labels, one monoclonal antibody (MAb 3B11) was first labeled with
goat anti-mouse fluorescein according to the above procedure. The
second mouse primary antibody was biotinylated and secondarily
labeled with Texas Red-avidin D (Vector Laboratories). After the
fluorescein-labeled secondary antibody was rinsed from tissues, the
organs were incubated overnight at 4C with biotin-MS13 diluted
1:1,000 in blocking buffer. Unbound biotinylated antibody was
rinsed from organs at room temperature, and tissues then were
exposed overnight in the cold to Texas Red-avidin D diluted
1:1,000 in blocking buffer. Several final rinses with blocking buffer
at room temperature preceded mounting of tissues on glass slides.
Sections of tissues labeled with HRP were prepared from tissues
that had been refixed with the aldehydes and 2% OsO 4 as described
in the preceding section. After dehydration and infiltration with
Medcast resin, the tissues were sectioned at 1–2 m and mounted
on glass slides without staining.
Organ cultures and hemocyte cultures
Whole hematopoietic organs were dissected under sterile conditions
in Grace’s insect culture medium (Invitrogen) whose pH had
been adjusted to 6.5 and then filter sterilized. Organs adhered to
sterile cover glasses (22 mm 2 ) that were placed on the bottom of
small Falcon culture dishes (35”10 mm) containing Grace’s
medium (pH 6.5) plus 20% fetal calf serum (Sigma). Cultures
were maintained at 26C in large culture dishes lined with moist
filter paper.
Circulating hemocytes from last instar larvae were added to
culture dishes prepared as described above. As soon as a drop of
larval hemolymph touched the medium, the blood was swirled, and
cells were allowed to settle and adhere to the glass coverslip for 1 h.
After this culture period, the medium and unattached hemocytes
were removed and adherent cells were fixed with PBS containing
4% paraformaldehyde for 30 min at room temperature. These fixed
cells on cover glasses were then rinsed several times with PBS and
later processed for immunolabeling as described earlier.
Confocal microscopy
Whole organs that had been double-labeled with fluorescein and
Texas Red were observed using laser scanning confocal microscope
(LSCM) instrumentation housed and maintained by the Imaging
Technology Group at the University of Illinois’ Beckman Institute.
Imaging was performed using the Leica SP-2 spectral confocal
instrumentation equipped with a ”20 plan-apochromatic objective
and a ”63 plan-apochromatic oil immersion objective. The 488
laser line from an argon laser was selected for excitation of
fluorescein, while the 543 laser line from a helium-neon laser was
used to excite Texas Red. To minimize overlap of emission spectra
for the two fluorochromes, excitation was performed sequentially.
The emission detection ranges for fluorescein and Texas Red
labeling were tuned respectively to the following bandwidths: 500–
535 nm and 593–622 nm. Three-dimensional volumetric data sets
consisting of multiple optical sections were processed using Leica
Confocal Software to yield extended focus projections.
Flow cytometry
Larval mesothoracic wing discs along with the adjacent overlying
integument were dissected and transferred to Grace’s tissue culture
medium. Hematopoietic organs were removed intact from the
surfaces of these forewing imaginal discs using tungsten needles
and watchmakers’ forceps. Each organ was transferred to a separate
siliconized Eppendorf tube containing 500 l maceration solution
consisting of glycerin, glacial acetic acid, and water (1:1:13). This
mixture completely disaggregates tissues yet maintains the integrity
of individual cells (David 1973). Organs remained in this maceration
solution for at least 15 min and were thoroughly vortexed
prior to addition of 500 l 4% paraformaldehyde dissolved in PBS.
Cells of wing imaginal discs from L5d0 larvae were chosen as
examples of known diploid cells. At this larval stage, neither
tracheal cells nor hemocytes have colonized the extracellular space
between the two monolayers of the wing discs (Nardi et al. 1985).
The cells of these wing discs were disaggregated according to the
procedure above.
The disaggregated cells remained in fixative (2% paraformaldehyde)
for 30 min before being centrifuged at 200 g in a swinging
bucket rotor. The cell pellet was washed first with 1.0 ml PBS and
then with 1.0 ml blocking buffer (PBS +10% normal goat serum
+0.1% Triton X-100). The washed pellet was suspended in 100 l
blocking buffer containing the mitosis marker, rabbit anti-phosphohistone
H3 (Upstate), at a concentration of 10 g/ml. Controls were
exposed to 1:1,000 normal rabbit serum. The fixed cells remained
in the primary rabbit antibody for 3 h at room temperature and then
were washed twice with 1.0 ml blocking buffer before being
incubated with 15 g/ml fluorescein goat anti-rabbit IgG (Vector)
in 100 l blocking buffer. After the cells had been exposed to
secondary antibody for 2 h at room temperature, they were washed
once with 1.0 ml blocking buffer and 1.0 ml PBS and stored at 2C
in the dark until analyzed with flow cytometry no more than 2 days
later.
Both the total number of cells per hematopoietic organ as well
as the number of fluorescein-labeled mitotic cells per organ were
calculated by simultaneously adding a known number of 6-m
carmine beads (Molecular Probes, 620 nm emission) to suspensions
of macerated hematopoietic organs.
To establish the relationship between the class of circulating
hemocyte (granular cells or plasmatocytes) and their ploidy levels,
blood from larvae (L5d0, L5d4, L5d5) was collected in anticoagulant
buffer (AC buffer). To each tube containing 750 l AC buffer,
four drops of blood were added, mixed, and then fixed with 750 l
4% paraformaldehyde in PBS for 30 min at room temperature.
These fixed, circulating hemocytes were then immunolabeled with
plasmatocyte-specific MS13 and granular-cell-specific MAb
15D11 following the procedure outlined for immunolabeling of
macerated cells from hematopoietic organs. Propidium iodide
(1 mg/ml in distilled H 2 O) was added (8% by volume) to
suspensions of permeabilized cells in PBS to stain for DNA.
For cell counting and analysis of labeled cells, a Coulter EPICS
XL-MCL cytometer, equipped with an air-cooled,15-mW argon
laser operating at 488 nm was used. To separate fluorescence
emission of fluorescein isothiocyanate (FITC), carmine beads and
propidium iodide (PI), the following set of band pass filters was
used: 525, 620 and 675 nm, respectively. To discriminate doublets
when DNA distribution was measured, peak versus integral
fluorescence of PI was recorded at the same time.
Results
To establish the fate of cells in the hematopoietic organs
of M. sexta, a combination of approaches has been used:
(a) high-resolution imaging of cells that permits visualization
of features distinctive for granular cells; (b)
immunolabeling of well-permeabilized organs with antibodies
that are specific for particular classes of hemocytes;
(c) culture of isolated organs in vitro.
Morphology and fine structure of hematopoietic organs
Each hematopoietic organ is draped across a wing
imaginal disc. The inner surface of the organ lies adjacent
to the wing disc, and the outer surface of the organ faces

480
Figs. 1–4 In this series of four images, the position of the
hematopoietic organ is shown relative to the nearby wing disc,
consisting of a peripodial epithelium (p) that surrounds each wing
epithelium (W). Both groups of coherent cells and clusters of
noncoherent cells are found in each hematopoietic organ. Most of
the coherent cells are located near the inner surface of the organ
(i.e., adjacent to the wing disc). The magnification is the same for
each image. Tracheoles are indicated with arrows
Fig. 1 On L4d3, a day prior to the molt from the 4th instar to the
5th instar, this lobe of the hematopoietic organ lies between the
larval thoracic integument—epithelium (E) + cuticle (C)—and the
peripodial epithelium (p) of the wing disc
Fig. 2 Immediately after the molt from the 4th instar to the 5th
instar
t
(L5d0), an inner-outer polarity of the organ is eviden
Fig. 3 This section of an L5d1 hematopoietic organ was cut at the
periphery of the wing disc
Fig. 4 By the wandering stage of the 5th instar (L5d5), the
organization of the hematopoietic organ has changed with the basal
lamina of the organ becoming folded and convoluted (arrowheads)
the hemocoel (Figs. 1, 2, 3, 4). A basal lamina delimits
the organ with its surface intact on the inner surface of the
organ (facing the wing disc) but disrupted in places on its
outer surface that faces away from wing disc (Figs. 6,19,
21). Numerous thin sections of organs at different times
during the penultimate (4th) and last (5th) larval stadia
were cut and examined. The architecture of hematopoietic
organs as well as the fine structure of individual cells
within these organs and on their surfaces are presented in
a series of electron and light micrographs (Figs. 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14).
After the initiation of wandering (L5d5) the welldefined
differences between inner and outer surfaces of
organs become less evident as the delimiting basal lamina
becomes folded and convoluted. Many cells of the organ
lose their coherence and disaggregate. Some cells engulf
and phagocytose one another (Fig. 9). In this respect,
these cells resemble the phagocytic cells also observed in
Drosophila lymph glands at the wandering stage (Lanot et
al. 2001) or the reticular cells of Gryllus, Calliphora, and
Locusta hematopoietic organs that have dual functions of
hematopoiesis and phagocytosis (Hoffman et al. 1979).
As the hematopoietic organ degenerates at the end of
larval life, the remaining cells adopt interlocking, attenuated
forms and extend numerous fine processes from
their surfaces (Figs. 10, 11). Several days later at
Figs. 5–8 At higher resolution, most noncoherent and all coherent
cells of hematopoietic organs can be distinguished from morphologically
distinctive granular cells. These latter cells are always
located near the periphery of each organ
Fig. 5 Noncoherent cells from an L4d3 larva are interspersed with
thin strands of basal lamina (arrows)
Fig. 6 A thick basal lamina (arrow) separates coherent cells of an
L4d3 organ (lower right) from more peripheral cells, one of which
is a granular cell (G) with its characteristic inclusions. Note that this
granular cell lies within a break in the basal lamina (single
arrowheads)
Fig. 7 On L5d0 the thicker outer basal lamina (large arrows) can
be compared with thinner basal laminae (small arrows) that are
found in the interior of each organ. A granular cell (G) with
distinctive inclusions lies to the right on the periphery of the organ.
T Tracheole
Fig. 8 On L5d3 this coherent mass of cells is delimited by
relatively thick basal laminae (arrows) on both inner (top) and outer
(bottom) surfaces of the hematopoietic organ

481

482
Figs. 9–14 Views of hematopoietic organs between the wandering
stage of the 5 th instar (L5d5) and pupation 5 days later
Fig. 9 On L5d5 convoluted basal laminae (arrows) are found
throughout the hematopoietic organ. Basal laminae (b) as well as
cells (c) are being engulfed by phagocytic cells believed to be
granular cells (G)
Fig. 10 On L5d6 remaining cells of the organ interlock with long
processes and form large, spherical extracellular spaces (S). Cells
extend numerous fine processes (arrows) into these spaces
Fig. 11 The global architecture of the L5d6 organ and its large
number of spherical extracellular spaces are more clearly visualized
at lower magnification

pupation, the cells of the organ have lost most of their
conspicuous intercellular spaces, and their nuclei undergo
condensation and adopt highly ramified forms—classical
features of apoptotic cells (Figs. 11, 12, 13, 14).
Expression of surface proteins by cells
of the hematopoietic organ
Fig. 12 Four days later at pupation the conspicuous extracellular
spaces (S) are less numerous and cells are showing ultrastructural
features of programmed cell death
Fig. 13 A close up of a nucleus (N) from a cell of a hematopoietic
organ at pupation having the highly ramified form and condensation
typical of nuclei in apoptotic cells
Fig. 14 The global architecture of the organ at pupation showing
its tenuous connection to the pupal epidermis at E and the remnants
of the spherical extracellular spaces (arrows)
The cells of the organ’s inner surface are generally more
coherent and densely packed; on the outer surface, the
cells lose their coherence and disperse into the hemolymph
(Figs. 1, 2, 19, 21). Most of the closely coherent
cells are concentrated near the inner surface of the organ,
although a few clusters of coherent cells lie near the outer
surface.
All plasmatocytes that have dispersed from the hematopoietic
organ—circulating as well as adherent—immunolabel
with MS13 and MS34 (anti-integrins; Levin et
al., in preparation). Cells that express neuroglian are
always coherent cells that usually lie near the inner
surface of the organ, whereas cells that express integrin
are found on both inner and outer surfaces of hematopoietic
organs (Figs. 15, 16, 17, 18, 19). Upon becoming
noncoherent hemocytes, the surfaces of these particular
cells uniformly express integrin but lose their uniform
surface expression of neuroglian.
Cells of the organ form a coherent mass segregated
from granular cells of the hemolymph by a basal lamina.
Only a few cells of the hematopoietic organ immunolabel
with MAb 15D11, a specific marker for granular cells and
basal laminae. Granular cells are localized to the periphery
of the organ where they apparently can transverse
breaks in the basal lamina (Figs. 6, 7, 21). In crosssections
of hematopoietic organs labeled with MAb
15D11 the basal lamina clearly labels; however, cells
within the organ, with the exception of a few at the
periphery, are not immunoreactive (Figs. 20, 21). In
sections examined at high resolution, basal laminae are
found throughout the interior of the organs that are
thinner than the exterior, enveloping basal laminae
(Figs. 5, 7). None of these thinner basal laminae label
with MAb 15D11. The protein lacunin that is recognized
by this MAb (Nardi et al. 1999) is produced by granular
cells but not by plasmatocytes (Nardi et al. 2001).
Although all circulating plasmatocytes immunolabel
with the anti-integrins MS13 and MS34, only a small
fraction of the cells within the hematopoietic organ
immunolabel with anti-integrin. Those cells of hematopoietic
organs that label with anti-integrins, however, do
not label with anti-neuroglian (Figs. 22, 23).
Like developing T-cells in the mammalian thymus, the
plasmatocytes that develop within the moth hematopoietic
organs probably pass through phases marked by changes
in expression of surface proteins (Janeway et al. 2001).
Development within the lepidopteran hematopoietic organ
also seems to be compartmentalized, with differences
in surface protein expression and cell cohesion observed
along the inner-outer axis of the organ.
Culture and mitotic activity of cells
from hematopoietic organs
483
Hematopoietic organs of Lepidoptera are known to be
sources of dividing hemocytes (Gardiner and Strand
2000; Akai and Sato 1971). The fates of cells that arise
from isolated cultures of hematopoietic organs can be
tracked by marking these cells with antibodies that are
specific for particular classes of hemocytes. The markers
can establish the diversity of hemocyte classes that are
descended from the undifferentiated cells of a given
organ.
Cells derived from cultured organs in vitro either (1)
adhered and spread or (2) remained suspended in culture
medium after dispersing from the hematopoietic organ.
All cells that had dispersed from the organ immunolabeled
with plasmatocyte-specific MS13 and MS 34
(Figs. 24, 25), but not with granular-cell-specific MAb
15D11. Cells adhering to the glass and plastic substrata
within the culture dish as well as cells that were collected
from the medium were fixed and immunolabeled. The
specific immunolabeling of all these cultured cells
(adherent and nonadherent) with MS13 and MS34 (antiintegrins)
provides some of the best evidence that only
plasmatocytes are derived from the thoracic hematopoietic
organs.
Mitotic cells are abundant throughout each organ
(Figs. 26, 27). Some mitotic cells express the surface
protein neuroglian (Fig. 28); however, cells of the
hematopoietic organ that express integrin and are dispersing
were never observed to express the mitotic marker
in organs from each of the four L5d3 and four L5d5
larvae that were doubly immunolabeled (Fig. 29).
The number of cells in each organ clearly increases
during the first 5 days of the last larval stadium and then
decreases after the inception of wandering and the rise in
ecdysteroid levels in the hemolymph (Nardi et al.1985;
Riddiford et al. 1984). The fraction of mitotic cells within
the organ as a function of development parallels the time
course of change in ecdysteroid levels as well as total
number of cells within the organ (Figs. 30, 31). The
changes in size and form of whole hematopoietic organs
between day 0 and day 6 of the last larval stadium are
illustrated in Fig. 32. The corresponding growth of the
wing imaginal discs associated with each of these
hematopoietic organs are included for comparison.

484

Ploidy differences between circulating plasmatocytes and
granular cells
According to Arnold and Hinks (1976), circulating
plasmatocytes are a class of hemocytes that do not divide
in the noctuid moth Euxoa declarata. These authors did
observe, however, that plasmatocytes of the hemolymph
increase in size dramatically during the last two larval
stadia of E. declarata. Cell size is known to be
proportional to ploidy level. Although these authors did
not measure the DNA content of these plasmatocytes, the
observed increase in plasmatocyte size is consistent with
this class of hemocytes being endomitotic and polyploid.
A clear disparity in size exists between granular cells and
plasmatocytes in the hemolymph of last instar M. sexta
(Fig. 33).
The antibody, anti-phosphohistone H3, that was used
to label mitotic cells cannot distinguish between mitotic
cells destined to divide and endomitotic cells that do not
divide. The relative amount of DNA per cell, however, is
proportional to intensity of staining with propidium
iodide. Labeling nuclei of circulating plasmatocytes with
propidium iodide provided evidence for endomitotic
Figs. 15–21 Immunolabeling of hematopoietic organs (L5d4) with
two plasmatocyte-specific MAbs and one granular cell and basal
lamina-specific MAb. In all images HRP was used as a marker
Fig. 15 In this whole-mount of an organ labeled with antineuroglian
(MAb 3B11), groups of coherent cells on the inner
surface of the organ label on their cell surfaces. Epithelial cells of
tracheoles label with anti-neuroglian (arrows; Nardi 1994). The
inset shows cells on the outer surface of the same organ. Fewer
cells label with anti-neuroglian on the outer surface, and only
coherent cells label. Bar of inset 50 m
Fig. 16 A cross-section of an organ labeled with MAb 3B11 shows
the clusters of coherent cells that are labeled. The inner surface of
the organ faces down; it is on this surface that label is concentrated.
The surfaces of tracheal epithelial cells also label with MAb 3B11
(arrow)
Fig. 17 In this whole-mount of an organ viewed from its inner
surface, aggregates of cells as well as single cells label with antiintegrin
(MS13)
Fig. 18 An organ labeled with MS13 and viewed from its outer
surface again shows labeled cells that form loose aggregates
(arrow) rather than the closely coherent aggregates showing MAb
3B11 immunoreactivity
Fig. 19 A cross-section of an organ that was labeled with MS13.
Groups of labeled cells as well as labeled individual cells are
evident. The inner surface of the organ faces down
Fig. 20 The outer surface of an organ labeled with anti-lacunin
(MAb 15D11), an antibody that is specific for basal laminae as well
as granular cells. Folds and creases in the basal lamina (small
arrows) are darker than the smooth basal lamina. A few granular
cells label near the periphery of the organ (large arrows)
Fig. 21 A cross-section of an organ labeled with anti-lacunin. The
outer surface faces down and the inner surface lies adjacent to the
peripodial epithelium (p) and wing epithelium (w) of the wing disc.
Cells can be seen dispersing from breaks in the basal lamina of the
outer surface (arrows). The inset shows a section of another organ
whose inner surface faces up and is covered by a continuous basal
lamina; its outer surface is covered by a discontinuous basal lamina.
One labeled granular cell (arrow) lies near the tracheole (T). Bar of
inset 50 m
activity rather than mitotic proliferation of these cells in
M. sexta during the last larval stadium. Hemolymph as
well as cells of hematopoietic organs from four larvae on
each of three different days during this stadium (L5d0,
L5d4, L5d5) were doubly labeled with plasmatocytespecific
MS13 and propidium iodide (Fig. 34).
Differences in ploidy levels of different classes of
circulating hemocytes were noted when plasmatocytes
and granular cells were analyzed with flow cytometry.
Circulating hemocytes from last instar Manduca larvae
were doubly labeled with propidium iodide and FITCconjugated
MS13, the antibody marker specific for
plasmatocytes. The cells that are negative for MS13
labeling (Fig. 34A, R1) have the smallest amount of DNA
and presumably are diploid cells with mean relative
fluorescence peaks at 27 for G0/G1 and 51 for G2/M
(Fig. 34B; G1/G2 ratio =1.89). These cells are almost
entirely granular cells. The main population of cells that
are positive for MS13 (Fig. 34A, R2) presumably
represents a polyploid population with mean relative
fluorescence peaks at 102 for G1/G0 and 200 for G2/M
(Fig. 34C; G1/G2 ratio =1.96). Plasmatocytes and granular
cells of caterpillar hemolymph not only arise from
different lineages, but they also have distinct ploidy
levels.
Cells of wing imaginal discs are known to have diploid
nuclei and were used as a diploid marker for propidium
iodide staining. These wing epithelial cells were taken
from discs at a stage (L5d0) when the hemocoel of the
wing disc is free of hemocytes (Nardi et al. 1985). The
intensity of propidium iodide staining for granular cells as
well as cells of hematopoietic organs matches that for
diploid cells of the wing imaginal discs (Fig. 34D).
Endomitosis and additional DNA synthesis of plasmatocytes
apparently occur after cells have dispersed as
diploid cells from hematopoietic organs.
Discussion
Hemocyte cell lineages in Lepidoptera
485
In mammals hematopoietic stem cells are the precursors
of all blood cell lineages; in insects, the cells derived from
head mesoderm may likewise be the precursors for all
hemocyte lineages (Lebestky et al. 2000; Tepass et al.
1994). As in Drosophila, hemocytes of Manduca first
arise as involution of head mesoderm (Nardi, submitted).
Based on the surface marker(s) expressed by these cells
that are recognized by peanut agglutinin lectin (PNA), the
hemocytes that appear in early embryogenesis represent
granular cells. Moth granular cells not only are recognized
by specific lectins and antibodies, but they also
have characteristic granules within their cytoplasm that
are evident with both the light and/or electron microscope
(Figs. 6, 7).
A clear divergence in lineages of granular cells and
plasmatocytes occurs during embryogenesis (Nardi, submitted).
The plasmatocyte lineage that first appears late in

486
Figs. 22, 23 Laser scanning confocal images of hematopoietic
organs from L5d4 larvae that have been labeled with antineuroglian
(FITC) and anti-integrin (Texas Red). Note the absence
of co-expression of these two cell surface proteins by the cells of
these organs. Tracheal epithelial cells label with anti-neuroglian
(arrows)
Fig. 22 A low magnification view of an organ showing predominantly
neuroglian-positive (green) cells
Fig. 23 A higher magnification view of an organ showing the
integrin-positive (red) and neuroglian-positive (green) cell populations.
Tracheal epithelial cells label with anti-neuroglian (arrows)
embryogenesis either (1) loses the surface antigen(s)
recognized by PNA or (2) represents a lineage of
embryonic hemocytes that arises from precursors that
are distinct from the granular cell precursors of head
mesoderm. All the findings presented in this paper on the
postembryonic hematopoietic organs are consistent with
granular cells and plasmatocytes representing two different
lineages that diverged during embryogenesis.
1. In hematopoietic organs granular cells can be distinguished
from plasmatocytes at the ultrastructural level
as well as with specific immunolabels; granular cells
are confined to the surfaces of hematopoietic organs
facing the hemocoel.
2. The only adherent hemocytes derived from cultures of
isolated hematopoietic organs are plasmatocytes.
3. Nonadherent cells that morphologically match the
description of prohemocytes and express integrin are
derived from cultured hematopoietic organs and probably
represent precursors of differentiated plasmatocytes.
4. Circulating granular cells are diploid while circulating
plasmatocytes are polyploid.
Both single lineage and dual lineage models have been
offered to account for the origin of circulating hemocytes
and the fate of cells derived from hematopoietic organs of
Lepidoptera. With a panel of monoclonal antibodies
generated against hemocytes of Pseudoplusia includens,
Gardiner and Strand (1999) clearly showed that granular
cells and plasmatocytes represent two antigenically
distinct lineages. Their findings with immunolabeling
supported earlier claims (Hinks and Arnold 1977; Nittono
1964) that hematopoietic organs are sources of plasmatocytes
but not granular cells; the findings are consistent
with the two classes of hemocytes having separate
lineages.
Beaulaton (1979) noted that the hematopoietic organs
of the moths Bombyx and Antheraea are partitioned into
islets of cells, with compact islets of undifferentiated cells
occupying the inner surface of the organ closest to the
wing disc and with loose islets of cells on the outer
surface. Based at least in part on electron micrographs of
hematopoietic organs from several Lepidoptera (Monpeyssin
and Beaulaton 1978), the loose or heterogenic
islets were interpreted as representing hemocytes at
various stages of differentiation and with features of all
hemocyte classes. This interpretation that hemocytes of
all classes differentiate within the loose islets of lepidopteran
hematopoietic organs led Beaulaton (1979) to
postulate a single lineage for caterpillar hemocytes in
which plasmatocytes derived from prohemocytes serve as
pluripotent stem cells that give rise to granular cells,
oenocytoids, and spherule cells. The equally valid interpretation
that these hemocytes loosely associated with
hematopoietic organs on their outer surfaces had actually
differentiated as circulating cells of the hemolymph was
not considered.

487
Figs. 24, 25 Many cells that disperse from isolated, cultured
hematopoietic organs (L5d3) adhere and spread on a glass
substratum. Some cells do not adhere, but all cells immunolabel
with MS13 and MS34 (anti-integrins). Each scale bar represents
100 m
Fig. 24 Cells have been cultured for 24 h and labeled with MS13
Fig. 25 Cells have been cultured for 2 weeks and labeled with
MS13
Figs. 26, 27 Whole organs (L5d4) have been fixed and labeld with
the mitosis marker, anti-phospho-histone H3. A large percentage of
cells label with the marker, and only some of the labeled cells lie
within the plane of focus. Each scale bar equals 100 m
Fig. 26 An overview of mitotic labeling in two lobes of an organ
Fig. 27 A higher magnificaton view of another organ whose
mitotic cells have been labeled
Studies of hemocyte transformations in culture have
also been interpreted as support for granular cells and
plasmatocytes forming one lineage. Examining transformations
of hemocytes in vitro, Gupta and Sutherland
(1966) claimed that plasmatocytes are polymorphic as
well as pluripotent and can change either directly or
indirectly into all other types of hemocytes. By isolating
individual prohemocytes from hemolymph cultures of
Bombyx mori, Yamashita and Iwabuchi (2001) inferred
that prohemocytes can differentiate into either granular
cells or plasmatocytes. These latter authors concluded that
the prohemocytes released from hematopoietic organs are
the pluripotent cells of the hemolymph and can give rise
to both granular cells and plasmatocytes. These two
studies, however, were based strictly on subjective
morphological classifications of hemocyte types that have
often proved misleading (Gillespie et al. 1997).
Comparing Drosophila hemocytes with hemocytes
of Lepidoptera
Both single lineage and dual lineage models have
likewise been offered to account for the origin of

488
Figs. 28, 29 In each figure, a whole mount of a hematopoietic
organ (L5d4) has been fixed and doubly labeled with a marker for
mitosis (FITC) and another marker for a cell surface protein (Texas
Red). Each confocal image represents a 1.0 micron slice through
the hematopoietic organ
Fig. 28 The mitosis marker and anti-neuroglian label some of the
same cells
Fig. 29 The mitosis marker and anti-integrin are never localized to
the same cells
Figs. 30, 31 Flow cytometry established the total number of cells
in each hematopoietic organ at different days during the last larval
stadium (L5d0-L5d6) as well as the percentage of these cells
labeled with the mitosis marker. For each time point, cells from at
least six organs were counted. Bars represent standard errors
Fig. 30 The mean (€ SE) for day 0 is significantly different from
the mean ( € SE) for days 3 and 5 (p

489
Fig. 33 Hemocytes from an L5d4 larva that adhere to a cover glass
substratum after 1 h in culture. These cells were immunolabeled
with plasmatocyte-specific MS13 and photographed with differential
interference contrast optics. Note the disparity in sizes for
plasmatocytes (large arrows) and granular cells (small arrows).
One of the large neuroglian-positive plasmatocytes is indicated
with a double arrow. Scale 50 m
Drosophila hemocytes. In their investigation of hematopoietic
lymph glands in Drosophila larvae and prepupae,
Lanot et al. (2001) listed four cell types as being found
within the glands: (1) prohemocytes, (2) crystal cells,
plasmatocytes acting as (3) phagocytes and (4) secretory
cells. Lamellocytes were never observed within the lymph
glands, and these authors hypothesized that lamellocytes
arise not from plasmatocytes but from prohemocytes.
Prohemocytes of lymph glands serve as pluripotent stem
cells that differentiate into at least three hemocyte classes:
(1) lamellocytes, (2) plasmatocytes (phagocytes and
secretory cells), (3) crystal cells. This single lineage
model for differentiation of hemocyte types contrasts with
the dual lineage model for embryonic hematopoiesis
presented by Lebestky et al. (2000). A dual lineage model
for hemocyte lineages is also based on the extensive
observations of Rizki and Rizki (1984) of circulating
hemocytes as well as Shrestha and Gateff’s (1982)
examination of larval hematopoietic organs (lymph
glands).
The dual lineage model involves specification of two
hemocyte lineages in Drosophila: a plasmatocyte lineage
specified by transcription factor glial cell missing (gcm)
and a crystal cell lineage specified by transcription factor
lozenge (lz; Lebestky et al. 2000). Lamellocytes, the
hemocytes of Diptera that encapsulate foreign objects,
Fig. 34A–D Hemocytes from an L5d4 larva have been analyzed
according to their ploidy levels and their surface labeling with the
plasmatocyte-specific antibody MS13. Macerated and fixed cells of
wing imaginal discs from L5d0 larvae are known to be diploid. They
were labeled with propidium iodide in D and used as a diploid
marker. A The FITC fluorescence intensity of hemocytes labeled
with MS13. The population of plasmatocytes specifically labeled by
this antibody is represented by the peak in region 2 (R2). Region 1
(R1) represents the population of hemocytes not labeled with MS13
(mostly granular cells). B The DNA distribution in unlabeled cells
from R1. The first peak on the left represents the G0/G1 peak with a
mean fluorescence of 27. The second G2/M peak has a mean
fluorescence of 51. C The DNA distribution in the MS13-positive
cells from region 2 of A. The first peak on the left represents GO/G1
with a mean fluorescence of 102. The second peak is presumably the
G2/M peak with a mean fluorescence of 200. D The intensity of
propidium iodide staining for cells of wing imaginal disc epithelium
(unshaded peak) whose nuclei are known to be diploid. The labeling
peak for the wing disc cells (unshaded peak) aligns with the shaded
peak for cells of hematopoietic organs (between L5d0 and L5d5) as
well as with the peak for granular cells in B

490
were first postulated to arise from circulating plasmatocytes.
Rizki (1957) noted that lamellocyte numbers in
hemolymph increase as plasmatocyte numbers concurrently
decrease; this change in hemocyte populations
occurs without an accompanying increase in cell division
or apoptosis. By tracing the progression of cellular
phenotypes in the lymph glands of Drosophila, Shrestha
and Gateff (1982) hypothesized that plasmatocytes,
podocytes, and lamellocytes represent a single lineage
based respectively on their progressive increase in
number of (1) primary lysosomes, (2) phagocytic vacuoles,
and (3) cytoplasmic processes.
Whereas Drosophila lamellocytes and lepidopteran
plasmatocytes both function as encapsulating hemocytes
in response to foreign invasion, granular cells clearly are
the hemocytes of Lepidoptera that are involved in
secretion of basal laminae and phagocytosis (Nardi et
al. 2001; Nardi and Miklasz 1989). Lanot et al.(2001)
note that no counterpart of lepidopteran granular cells is
present in the hemolymph of flies (Diptera). In Lepidoptera
these granular cells degranulate as a first line of
defense in the presence of foreign objects (Schmit and
Ratcliffe 1977). The functions of moth granular cells–
secretion of extracellular matrix and phagocytosis–have
apparently been assumed in Drosophila by the plasmatocytes
(Lanot et al. 2001).
The relationship between plasmatocytes
and prohemocytes
Among the circulating hemocytes of Drosophila, mitotic
activity has been observed in prohemocytes and plasmatocytes
but not in crystal cells or in lamellocytes (Lanot et
al. 2001). In lepidopteran larvae, Arnold and Hinks (1976,
1983) rarely observed division of circulating plasmatocytes
but found that prohemocytes, granular cells, and
spherule cells were the only circulating hemocytes that
frequently divided.
Prohemocytes have been described in Lepidoptera as
oval or rounded cells with high nuclear to cytoplasm
ratios (Gardiner and Strand 1999, 2000). Such cells have
not been described for M. sexta (Beetz et al., submitted),
and only recently has a subpopulation of plasmatocytes in
P. includens that fit this morphological description been
identified by their special immunoreactivity (Gardiner
and Strand 2000). As these authors point out, Arnold and
Hinks (1976) had earlier suggested that prohemocytes are
actually precursors of plasmatocytes.
In the ultrastructural images of Manduca hematopoietic
organs (Figs. 5, 6, 7, 8, 9), the rounded cells clearly
have high nuclear to cytoplasmic ratios. As these cells
disperse from the hematopoietic organs and begin
expressing integrin (Figs. 18, 19), they have the same
morphological features used to describe prohemocytes
(Gardiner and Strand 1999; Jones 1962). In cultures of
Manduca hematopoietic organs, many plasmatocytes
adhere to the glass substrate (Figs. 24, 25); however,
numerous rounded cells remain in suspension. Both
adherent and nonadherent cells in these cultures stain
with anti-integrin. The nonadherent, integrin-positive
cells observed in cultures of Manduca hematopoietic
organs are derived from the same organ as the adherent,
integrin-positive plasmatocytes and probably represent
the class of hemocytes that other investigators have called
prohemocytes.
These observations of cells derived from cultured
hematopoietic organs of M. sexta are consistent with
Gardiner and Strand’s (2000) finding that two subpopulations
of plasmatocytes in P. includens can be distinguished
on the basis of their labeling with the particular
monoclonal antibody 43E9A10. They suggest that the
subpopulation of nonadherent, 43E9A10-negative plasmatocytes
is the equivalent of the prohemocytes described
by other researchers.
Following ligation of larvae into anterior and posterior
regions, granular cell and spherule cell populations of
hemocytes show only a slight increase in the anterior
region of the larva. However, prohemocytes and plasmatocytes
show a marked, several-fold increase in numbers
at the anterior end of the larva (Hinks and Arnold 1977).
While this phenomenon was evident in both Spodoptera
frugiperda and E. declarata, another noctuid caterpillar,
P. includens, showed no regional differences in hemocyte
densities following ligations. This latter species is known
to have greatly reduced hematopoietic organs and
presumably plasmatocyte populations in P. includens
are maintained by divisions of cells within the hemocoel
(Gardiner and Strand 2000). The BrdU labeling patterns
of circulating plasmatocytes in S. frugiperda and P.
includens also indicate that these cells are synthesizing
DNA and possibly proliferating as diploid cells.
Rather than finding evidence that circulating plasmatocytes
of the last larval instar of M. sexta proliferate as
diploid cells, however, their plasmatocytes were found to
undergo endomitosis and to have higher ploidy levels than
granular cells of the hemolymph. Differences in ploidy
levels for plasmatocytes and granular cells have not been
previously noted; however, both Arnold and Hicks (1976)
as well as Shrestha and Gateff (1982), respectively,
suggested that plasmatocytes increase in size within the
hemolymph of caterpillars and within the lymph glands of
fly larvae. This difference in ploidy levels between
granular cells and plasmatocytes is another structural
difference that distinguishes these two major classes of
hemocytes and that probably reflects the different functional
roles of granular cells and plasmatocytes in the
immune response.
Acknowledgements This research was supported by a grant from
the National Institutes of Health (1 R01 HL 64657). Charles Mark
Bee helped with the scanning and final preparation of the figures.
Andy Anderson and Stephanie Shockey carefully formatted the
final manuscript. Two anonymous reviewers provided helpful,
constructive suggestions for improving this manuscript.

491
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