The cellular immune response of the pea aphid to foreign intrusion and symbiotic challenge.

Abstract

Recent studies suggest that the pea aphid (Acyrthosiphon pisum) has low immune defenses. However, its immune components are largely undescribed, and notably, extensive characterization of circulating cells has been missing. Here, we report characterization of five cell categories in hemolymph of adults of the LL01 pea aphid clone, devoid of secondary symbionts (SS): prohemocytes, plasmatocytes, granulocytes, spherulocytes and wax cells. Circulating lipid-filed wax cells are rare; they otherwise localize at the basis of the cornicles. Spherulocytes, that are likely sub-cuticular sessile cells, are involved in the coagulation process. Prohemocytes have features of precursor cells. Plasmatocytes and granulocytes, the only adherent cells, can form a layer in vivo around inserted foreign objects and phagocytize latex beads or Escherichia coli bacteria injected into aphid hemolymph. Using digital image analysis, we estimated that the hemolymph from one LL01 aphid contains about 600 adherent cells, 35% being granulocytes. Among aphid YR2 lines differing only in their SS content, similar results to LL01 were observed for YR2-Amp (without SS) and YR2-Ss (with Serratia symbiotica), while YR2-Hd (with Hamiltonella defensa) and YR2(Ri) (with Regiella insecticola) had strikingly lower adherent hemocyte numbers and granulocyte proportions. The effect of the presence of SS on A. pisum cellular immunity is thus symbiont-dependent. Interestingly, Buchnera aphidicola (the aphid primary symbiont) and all SS, whether naturally present, released during hemolymph collection, or artificially injected, were internalized by adherent hemocytes. Inside hemocytes, SS were observed in phagocytic vesicles, most often in phagolysosomes. Our results thus raise the question whether aphid symbionts in hemolymph are taken up and destroyed by hemocytes, or actively promote their own internalization, for instance as a way of being transmitted to the next generation. Altogether, we demonstrate here a strong interaction between aphid symbionts and immune cells, depending upon the symbiont, highlighting the link between immunity and symbiosis.

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The Cellular Immune Response of the Pea Aphid to
Foreign Intrusion and Symbiotic Challenge
Antonin Schmitz1,2,3, Caroline Anselme1,2,3¤, Marc Ravallec4, Christian Rebuf1,2,3, Jean-
Christophe Simon5, Jean-Luc Gatti1,2,3, Maryle `ne Poirie ´1,2,3*
1Institut National de la Recherche Agronomique (INRA), Unite ´ Mixte de Recherches 1355 ‘‘Institut Sophia Agrobiotech’’ (ISA), Sophia Antipolis, France, 2Centre National
de la Recherche Scientifique (CNRS), Unite ´ Mixte de Recherches 7254, Sophia Antipolis, France, 3Universite ´ Nice Sophia Antipolis, Nice, France, 4Institut National de la
Recherche Agronomique (INRA) - Universite ´ Montpellier 2, Unite ´ Mixte de Recherches 1333 ‘‘Diversite ´, Ge ´nomes et Interactions Microorganismes-Insectes’’, Montpellier,
France, 5Institut National de la Recherche Agronomique (INRA), UMR 1349, Institut de Ge ´ne ´tique, Environnement et Protection des Plantes, Le Rheu, France
Abstract
Recent studies suggest that the pea aphid (Acyrthosiphon pisum) has low immune defenses. However, its immune
components are largely undescribed, and notably, extensive characterization of circulating cells has been missing. Here, we
report characterization of five cell categories in hemolymph of adults of the LL01 pea aphid clone, devoid of secondary
symbionts (SS): prohemocytes, plasmatocytes, granulocytes, spherulocytes and wax cells. Circulating lipid-filed wax cells are
rare; they otherwise localize at the basis of the cornicles. Spherulocytes, that are likely sub-cuticular sessile cells, are involved
in the coagulation process. Prohemocytes have features of precursor cells. Plasmatocytes and granulocytes, the only
adherent cells, can form a layer in vivo around inserted foreign objects and phagocytize latex beads or Escherichia coli
bacteria injected into aphid hemolymph. Using digital image analysis, we estimated that the hemolymph from one LL01
aphid contains about 600 adherent cells, 35% being granulocytes. Among aphid YR2 lines differing only in their SS content,
similar results to LL01 were observed for YR2-Amp (without SS) and YR2-Ss (with Serratia symbiotica), while YR2-Hd (with
Hamiltonella defensa) and YR2(Ri) (with Regiella insecticola) had strikingly lower adherent hemocyte numbers and
granulocyte proportions. The effect of the presence of SS on A. pisum cellular immunity is thus symbiont-dependent.
Interestingly, Buchnera aphidicola (the aphid primary symbiont) and all SS, whether naturally present, released during
hemolymph collection, or artificially injected, were internalized by adherent hemocytes. Inside hemocytes, SS were
observed in phagocytic vesicles, most often in phagolysosomes. Our results thus raise the question whether aphid
symbionts in hemolymph are taken up and destroyed by hemocytes, or actively promote their own internalization, for
instance as a way of being transmitted to the next generation. Altogether, we demonstrate here a strong interaction
between aphid symbionts and immune cells, depending upon the symbiont, highlighting the link between immunity and
symbiosis.
Citation: Schmitz A, Anselme C, Ravallec M, Rebuf C, Simon J-C, et al. (2012) The Cellular Immune Response of the Pea Aphid to Foreign Intrusion and Symbiotic
Challenge. PLoS ONE 7(7): e42114. doi:10.1371/journal.pone.0042114
Editor: Kristin Michel, Kansas State University, United States of America
Received March 19, 2012; Accepted July 2, 2012; Published July 27, 2012
Copyright: ? 2012 Schmitz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a ‘‘Jeune Equipe’’ funding (2009–2011) from the National Institute for Agronomic Research (INRA). A. Schmitz PhD grant
was co-funded by the ‘‘Provence Alpes Co ˆte d’Azur (PACA)’’ region, and the INRA ‘‘Sante ´ des Plantes et Environnement (SPE)’’ department. C. Anselme received
post-doctoral support from the INRA SPE department. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of
the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: poirie@sophia.inra.fr
¤ Current address: Universite ´ de Picardie Jules Verne, Equipe d’accueil 4698 EDYSAN, Amiens, France
Introduction
Insect defense against pathogens relies on innate immune
mechanisms that have mainly been characterized in Diptera and
Lepidoptera [1–3], and show substantial conservation in Hyme-
noptera and Coleoptera [4,5]. In comparison, the immunity of
hemimetabolous insects has been fewly investigated, although they
comprise economically important species. The recent annotation
of the genome of the pea aphid Acyrthosiphon pisum [6] suggests that
many components central to immune functions in other insects are
missing. These include antimicrobial peptides (AMPs) such as
defensins, PeptidoGlycan Recognition Proteins (PGRPs), some of
the cellular Pattern Recognition Receptors (PRRs) and several
components of the Imd pathway [7]. Besides, all experiments
failed to identify significant changes in expression of immunity
genes in response to wounding, stress or pathogen challenges [7,8].
However, genomic approaches are only predictive and, for
instance, an extensive description of the nature and defense
functions of aphid hemocytes remained to be provided.
Another interest of aphids is their obligate association with a
primary symbiont, Buchnera aphidicola (Ba), and the large number of
facultative secondary symbionts they can host (e.g. Serratia
symbiotica (Ss), Hamiltonella defense (Hd), Regiella insecticola (Ri)).
Indeed, it has recently been demonstrated that symbionts can
significantly influence the host immune response [9–11]. For
instance, the presence of the alpha proteobacteria Wolbachia is
associated with increased susceptibility to parasitoid infection in
Drosophila simulans [12] or with alteration of immune components
in the woodlouse Armadillidium vulgare [13], and it mediates
protection against RNA virus infection in D. melanogaster [14].
The maintenance of symbiosis with regard to the host immunity
has also been investigated. Symbionts seem to be detected by the
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host immune system [15,16]; they can be observed inside host
hemocytes [13,16], possibly as a way of protecting themselves from
host defenses [17]. In addition, the host immune system can
control the division of symbionts and their localization, as
demonstrated recently [18].
In the pea aphid, some secondary symbionts are involved in
host resistance to fungal pathogens [19] and parasitoid wasps [20],
through mechanisms yet only partly identified. In addition, while
primary symbionts are usually confined to the cytoplasm of
specialized cells, the bacteriocytes, secondary symbionts are also
observed free in the hemolymph [21,22], raising the question of
how the immune system responds to the presence of symbionts
and whether symbionts may somehow affect aphid immunity. In
the predicted absence of strong humoral immunity, aphid immune
cells are the main components with which symbionts may interact.
However, there are few available descriptions of these cells, and
they only rely on light microscopy observations (see [23] as an
example). The first data on A. pisum were reported only recently,
with the description of three morphologically distinct types of
hemocytes: prohemocytes that may correspond to stem cells,
granulocytes that phagocytize bacteria, and oenocytoids that
exhibit melanotic activity [24].
Here, we deepen the knowledge of A. pisum hemocytes by
providing extensive morphological and functional characteriza-
tion, based on light and electron microscopy. We describe a new
cell category (the plasmatocytes) and decipher the role of the
different hemocytes in phagocytosis, coagulation, adhesion and
encapsulation. We also show that, besides being able to engulf
foreign bodies, plasmatocytes and granulocytes actively phagocy-
tize primary and secondary symbionts when present in the
hemolymph. Finally, we report a lower number of adherent cells
and a smaller granulocyte/plasmatocyte proportion in presence of
some, but not all, secondary symbionts. Aphid symbionts thus
strongly and diversely interact with the cellular immunity of their
host, a finding that opens new avenues of research to question the
link between symbiosis and immunity.
Results
Hemocyte characterization
Hemocyte characterization was primarily performed on a clone
devoid of secondary symbionts (LL01). Five main cell types were
defined using several morphological and functional criteria (e.g.
size, nucleus/cytoplasm ratio (N/C), morphology, adhesion
properties, staining properties). These cells categories were later
observed in all the aphid lines tested.
Prohemocytes are the smallest cells in hemolymph, often observed
in clusters of three or more cells (Fig. 1A). Their diameter ranges
from 5 to 8 mm; they have a spherical shape and a very high N/C
ratio (Fig. 1A inset and Fig. 2A). The central spherical nucleus
occupies most of the cell and is surrounded by a limited layer of
basophilic cytoplasm (May-Gru ¨nwald Giemsa (MGG) staining;
not shown). A small nucleolus can be observed by light microscopy
(Fig. 1A inset) and transmission electron microcopy (TEM)
(Fig. 2A). TEM observations showed a well-defined nuclear
envelope and an homogenous cytoplasm filled with very small
dense particles and also containing a few vesicles (Fig. 2A).
Organelles such as mitochondria, Golgi apparatus or endoplasmic
reticulum were barely distinguishable. Prohemocytes never
showed cytoplasmic extensions; they were never retrieved on
adherent hemocyte preparations (named thereafter AHPs; see
material and methods), nor adhering onto foreign material, and
were never observed phagocytizing injected particles.
Plasmatocytes are small round or occasionally spindle-shaped
cells, of 8 to 10 mm in diameter, with a high N/C ratio (Fig. 1B,
2B–C). Their eccentric nucleus is round to lobulated, with a well-
developed nucleolus. The basophilic cytoplasm (blue reaction with
MGG, Fig. 3E) is homogenous, but cytoplasmic vacuoles were
sometimes observed by phase contrast microscopy (Fig. 1B, inset).
By light microscopy, plasmatocytes differed from prohemocytes by
their extended filopodia (Fig. 1B). Under TEM observation
(Fig. 2B–C), the nucleus had a lobulated shape and the cytoplasm,
almost devoid of vesicles, contained clearly visible cytoplasmic
organelles (mitochondria, free ribosomes, rough endoplasmic
reticulum (RER)). Plasmatocytes have adhesive properties: they
attach to glass and can form clusters when entering in contact with
each other (inset of Fig. 3A, Fig. 3E). By F-actin staining on AHPs,
two types of adhesion profile were observed (Fig. 3A): filopodia
extension exclusively (inset of Fig. 3A), or lamellipodium extension
without apparent filopodia (Fig. 3A). Plasmatocytes were also
weakly positive for the presence of ROS (not shown).
Granulocytes are usually spherical or ovoid, with a 10 to 20 mm
diameter. They have a low N/C ratio and an eccentric, generally
lobulated nucleus, with a prominent nucleolus. Under light
microscopy, vacuoles and filamentous processes are observed as
well as granules that could be released by the cell (Fig. 1C). TEM
observation (Fig. 2D) showed numerous cytoplasmic organelles
(mitochondria, Golgi apparatus, free ribosomes, and RER),
suggesting they are highly active cells. In addition, large
cytoplasmic granules (up to 1 mm in diameter), filled with more
or less electron dense material, could be observed scattered at the
cell periphery. Granules’ variation in size and electron density may
be due to a maturation process. In addition to granules,
cytoplasmic clear membrane vesicles, as well as phagosome-like
Figure 1. Light microscopy pictures of Acyrthosiphon pisum
hemocytes (LL01 clone). (A) Three prohemocytes in cluster. Inset:
phase contrast showing the large central nucleus and the nucleolus
(Nu). Ba: B. aphidicola. (B) Plasmatocyte beginning to adhere, with
filopodia (Fp) extension. Inset: phase contrast showing large cytoplas-
mic vacuolar formation. (C) Adherent granulocyte containing cytoplas-
mic granules (G) and filopodia (Fp) extending from a lamellipodium
(Lm). (D) Spherulocyte with its large colored globular inclusions, small
yellow spherules (YS) and large green spherules (GS). (E) Wax cell
showing a large central vacuole (V) and colored globular inclusions that
differ from those of spherulocytes. Same magnification for all
micrographs; scale bar: 10 mm.
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structures surrounding ingested particles were observed. Granu-
locytes readily adhered to glass coverslips and were able to spread
more than four times their original size one hour after hemolymph
collection (Fig. 3A). Spreading occurred in a fan-like manner with
star-like filopodia extending from lamellipodia. Most granulocytes
spread symmetrically but some spread asymmetrically along one
axis (Fig. 3A, 3D). Granulocytes often clumped and spread when
they entered in contact with each other (Fig. 3B). The eosinophilic
cytoplasm was more or less filled with basophilic granular
inclusions (MGG staining, Fig. 3D), suggesting that granulocytes
Figure 2. TEM characterization of hemocytes. Cells from the LL01 clone (A–D) and the YR2-Hd line (E). (A) Prohemocyte characterized by a
round nucleus (N), a high N/C ratio, and homogenous cytoplasm devoid of apparent organelles. (B) Plasmatocyte with homogeneous cytoplasm, a
lobulated nucleus, a high N/C ratio and some cytoplasmic organelles clearly visible in box (C): Rough Endoplasmic Reticulum (RER), Mitochondria (M).
(D) Granulocyte with a lobulated nucleus, a low N/C ratio, and granules (G). The cytoplasm contains numerous organelles (see box). A phagosome is
observed that contains a large foreign particle (asterisk). (E) YR2-Hd spherulocyte with a round nucleus and a low N/C ratio. The large volume of
cytoplasm is filled with spherules (S) of different sizes, and numerous mitochondria are found in a small region (enlarged box). Ba: B. aphidicola; Hd: H.
defensa. Scale bar: 2 mm (A, B, D, E) and 0.5 mm (C).
doi:10.1371/journal.pone.0042114.g002
Figure 3. Histological characterization of adherent hemocytes (LL01 clone). (A) Fluorescent micrograph showing AHP after F-actin (green)
and nucleus (blue) staining. Plasmatocytes (Pl) are the smaller cells; they display two adhesion profiles (i) lamellipodia (Lm) extension without
apparent filopodia or (ii) filopodia extension (Fp) exclusively (inset). Granulocytes (Gr) have spread more than four times their original size; they
display lamellipodia with radiates filopodia. (B) Merger of DIC and fluorescent micrographs showing three clumped ROS-producing granulocytes
(green fluorescence of Rhodamine 123). (C) Intracellular PO staining on AHP using Dopamine hydrochloride showing a PO-positive hemocyte (brown
staining). (D–E) Adherent hemocytes stained with May Gru ¨nwald Giemsa. (D) Granulocyte with an eosinophilic cytoplasm (pink staining) that contains
basophilic granules (blue staining). (E) Clumped plasmatocytes with a limited layer of basophilic cytoplasm. Ba: B. aphidicola. Scale bars: 10 mm.
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might degranulate in the course of the experiment. Granulocytes
were strongly positive for the presence of ROS (Fig. 3B). About
2% (2.3961%) of adherent hemocytes on AHPs displayed a strong
intracellular PO staining (Fig. 3C). Since these cells had the same
size and shape, as well as similar lamellipodia/filopodia extensions
as granulocytes, they were considered as such.
Spherulocytes were observed both in freshly collected hemolymph
(Fig. 1D), and adherent under the cuticle (not shown), suggesting
they might be sessile cells that were released upon collection. They
are very large (from 20 to 100 mm in diameter), highly
polymorphic, with a spherical to undefined shape; they have a
low N/C ratio (Fig. 1D). Their round eccentric nucleus contains a
well-developed central nucleolus (intense blue coloration with
methyl blue, Fig. S1). Highly colored prominent vesicles (globules
or spherules) are found in their cytoplasm, with large green
spherules (.5 mm in diameter) and small yellow spherules (about
1–2 mm in diameter) (Fig. 1D). We observed that the color of the
spherules in different clones matched that of the aphid body
(Table 1), as previously reported [25].
Spherulocytes are fragile cells that broke easily during
collection, leaving free nuclei in suspension in the medium (Fig.
S1). Accordingly, within 30 min following collection, numerous
spherules were floating at the surface of the collected hemolymph.
This likely explains why we rarely observed these cells using TEM.
The only observation (Fig. 2E, from the clone YR2-Hd) showed
many empty vacuoles that matched in size and location with the
two types of spherules, as well as numerous mitochondria
concentrated in a restricted region of the cytoplasm. Spherulocytes
attached very poorly to glass and did not show filamentous
processes.
A main function of spherulocytes is their involvement in the
coagulation process that occurred rapidly following hemolymph
collection (Fig. 4A–D, movie S1). Spherulocytes underwent a
succession of modification steps that began with a loss of spherules
and formation of fibrils (Fig. 4A), followed by rapid expansion of
the cytoplasm, with the formation of a beam-like protuberance
from cytoplasmic swellings and blebs (Fig. 4B) that rapidly
changed into long strings of pearls (Fig. 4C). The process more
or less extended and it ended with the organization of a granular
coagulum into a delicate meshwork of fibrils (Fig. 4D). The
presence of numerous spherules and the high number of
mitochondria suggest that spherulocytes may also be involved in
energy storage.
Wax cells were sometimes observed in hemolymph preparations
(Fig. 1E) but they were mainly localized at the base and in the
canal of the aphid cornicles (Fig. 4E). These spherical to ovoid cells
have a 40–50 mm diameter and a low N/C ratio. They have a very
low density, floating at the surface of the collected hemolymph,
and their cytoplasm is more or less filled with large neutral lipid
inclusions (Nile blue sulfate staining, Fig. 4F), in agreement with
their involvement in wax production and lipid storage [26]. Wax
cells never showed cytoplasmic extensions nor adhesive properties.
They were never observed in TEM preparations, likely because
they were lost during the centrifugation steps. Because of their
restricted localization and putative functions, they were not
considered as true circulating hemocytes.
Adherent hemocyte number and granulocyte proportion
in different aphid lines
Due to the hemolymph contamination and spherulocytes’
leakage observed with all collection methods, the respective
abundance of hemocyte categories in aphid hemolymph could
not be accurately determined. However, spread cells were readily
recognizable after F-actin staining by their filamentous processes
(Fig. 3A). We then developed an image recognition method based
on AHPs (Methods S1, Fig. S2) to estimate the number of
adherent hemocytes and the proportion of granulocytes and
plasmatocytes in aphid lines (Fig. 5). The total adherent hemocyte
count (THC) (Fig. 5A) was about 31716634 for five pooled LL01
aphids (6346170 cells per individual), but it strongly differed
among aphid lines (ANOVA, p=7.0e-06). No significant differ-
ence was found between LL01 and YR2-Amp, the lines devoid of
secondary symbionts (SS) (TukeyHSD, p=1), and between YR2-
Amp and YR2-Ss (TukeyHSD, p=0.58). In contrast, THCs from
YR2(Ri) and YR2-Hd were on average 7.6 times and 13.4 times
lower than that of YR2-Amp, respectively (TukeyHSD, p=2.0e-
04 and p=1.0e-04). The proportion of granulocytes differed
among aphid lines similarly to the THC (Fig. 5B; ANOVA, p-
value=1.5e-05), being on average 3.7 times and 6.6 times lower in
YR2(Ri) and YR2-Hd compared to YR2-Amp, respectively
(TukeyHSD, p=1.6e-03 and p=3.5e-04). Contrastingly, there
was no difference between the lines devoid of SS, LL01 and YR2-
Amp (TukeyHSD, p=0.41), or between YR2-Amp and YR2-Ss
(TukeyHSD, p=0.81). In lines devoid of SS, granulocytes
(including PO-positive hemocytes) represented 35% (LL01) and
48% (YR2-Amp) of adherent hemocytes, others being plasmato-
cytes.
Hemocyte adhesion in vivo
Adhesion properties of hemocytes were tested in vivo by inserting
brush horsehair fragments in LL01 aphids. These fragments were
removed at different times and observed by microscopy. Adhesion
of some hemocytes was already observed 24 h post-insertion, and
their number increased 48 h post-insertion (data not shown). One
week after insertion, hairs were covered by adherent hemocytes
forming a more or less extended cell monolayer, but which never
happened to be complete (Fig. 6A ; 3D reconstruction in movie
S2). To question if this was due to the large size of the hairs, and
whether hemocytes could adhere to non-organic material, the
Table 1. Origin and characteristics of the aphid lines.
LinesColorPlant originLocationCollection dateSS composition (donor line)
LL01GreenAlfalfaLusignan (France)1987none
YR2(Ri)Pink Clover York (England)2002R. insecticola
YR2-Amp*Pink---none
YR2-Hd*Pink--- H. defensa (L1-22)
YR2-Ss*Green--- S. symbiotica (P136)
*: artificial lines.
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experiment was repeated with smaller epoxy resin and glass
capillary fragments. The same incomplete hemocyte layer was
observed, together with melanin deposits (data not shown) that
could not be observed on the black brush hairs. Interestingly, most
of the inserted objects covered with hemocytes were found
attached to aphid tissues such as the fat body, the trachea or the
digestive tract. At the ultrastructural level, the cell monolayer was
composed of both plasmatocytes and granulocytes, distinguishable
by their morphology (size, N/C ratio) and the presence of granules
(Fig. 6). Hemocytes occurred as either isolated cells (Fig. 6C–D) or
as multicellular aggregates containing both cell categories, without
any apparent organization (Fig. 6B). At places, the membranes of
neighbor hemocytes were in tight contact, with no intercellular
space (Fig. 6F), while in large cell aggregates intercellular spaces up
to 2 mm were observed (Fig. 6B). Those spaces contained cellular
debris (mitochondria, granules…) possibly originating from cell
lysis. An electron dense layer was always observed at the interface
between hemocytes and horsehairs (Fig. 6C–E), as well as on the
Figure 4. Functions of spherulocytes and wax cells. (A–C) Early alterations of spherulocytes after hemolymph collection: (A) loss of spherules
and fibrils’ formation (arrowhead). Ba: B. aphidicola. (B) Unstable cytoplasmic blebs (arrow) derive from spherulocytes while spherules remain intact
(arrowhead). (C) From blebs (arrow), long stable strands like strings of pearls extend (asterisk; see also movie 1). (D) Low magnification of a large
coagulum stained by neutral red showing a granular aspect and the presence of an intact spherulocyte (Sp). (E–F) Wax cells (Wx) are localized at the
base and inside the cornicles (Co), secretory appendices localized at the posterior end of aphids’ bodies (inset in (E)). (E) Red lipid staining showing
large accumulation of lipid-containing cells at the base of the cornicle. (F) Wax cell containing a large neutral lipidic inclusion (pink staining) that
almost fills entirely the cytoplasm. Scale bars: 15 mm (A), 10 mm (B, C, D, F) and 100 mm (E and inset in (E)).
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Figure 5. Total hemocyte counts (THC) and granulocyte proportions from AHPs of the different lines. Box-plot representations of the
THC (A) and the proportion of granulocytes (B), estimated on F-actin stained AHPs (pools of 5 adults per AHP), for aphid lines listed in Table 1. Box-
plots with the same letter have means that are not significantly different (TukeyHSD, alpha=5e-03, n=4).
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surface of the horsehair even in areas devoid of hemocytes. This
layer had a granular aspect when observed at high magnification
(thickness about 30 nm per granule) and its appearance varied
from a granular deposit of low electron density (grey color, Fig. 6C)
to a compact, less granular, electron dense matrix (black, Fig. 6D).
A second layer of material was sometimes observed between this
dense matrix and the hemocytes’ membrane (Fig. 6E) that had a
slightly granular aspect and contained cellular organelles (e.g.
mitochondria). Besides, a thin layer of electron-dense material was
observed at the surface of hemocytes facing the hemocoele cavity
(Fig. 6F).
Phagocytosis of latex beads and E. coli bacteria
The hemocyte capacity to internalize foreign particles was
tested by injecting latex beads or fluorescent E. coli DH5-alpha
bacteria inside the body cavity of LL01 aphids. Similar exper-
iments were performed with YR2-Amp aphids, with similar results
(data not shown).
The cellular uptake of injected particles was observed by
fluorescent microscopy, and intracellular localization was con-
firmed using confocal microscopy and TEM. Prohemocytes,
spherulocytes and wax cells did not contain any latex bead or
fluorescent bacteria whatever the time post-injection (data not
shown), thus being classified as non-phagocytic cells. In contrast,
both granulocytes and plasmatocytes mounted a strong and rapid
phagocytic response (as soon as 2 h post-injection, data not shown)
toward abiotic (latex beads, Fig. 7A–F) and biotic (E. coli, Fig. 7G–
J) particles. At all observation times (2 h, 5 h and 24 h), the
cytoplasm of many adherent cells was filled with latex beads or
bacteria, granulocytes being able to take up more particles than
plasmatocytes due to their bigger size. We estimated that 27% and
35% of adherent hemocytes had taken up three or more latex
beads 5 h and 24 h post-injection, respectively. 3D projections
generated from confocal optical sections demonstrated that beads
and bacteria are indeed inside the cells, and that a single hemocyte
could phagocytize more than 50 beads or bacteria (see movie S3
for latex beads engulfment in 3D projection). In addition, beads-
containing hemocytes frequently formed aggregates (Fig. 7E) and
some of them were totally melanized 24 h post injection (Fig. 7F).
Adherent hemocytes containing numerous particles had a
Figure 6. In vivo hemocyte adhesion assay (LL01 clone). (A) Observations of a brush horsehair (asterisk) seven days after insertion in the
hemocoele of an LL01 aphid: both plasmatocytes (Pl) and granulocytes (Gr) are observed (F-actin (green) and nuclei (blue) staining). Scale bar: 10 mm.
(B–F) TEM micrographs of the ‘‘encapsulation’’ process. (B) Part of a multilayered hemocytic capsule containing both plasmatocytes (Pl) and
granulocytes (Gr). The capsule is partly detached from the brush hair (asterisk). Intercellular spaces (IS) between hemocytes are relatively large (see
box magnified bottom left) and they often contain some cytoplasmic organelles (mitochondria, granules…). Scale bar: 5 mm; box: 1 mm. (C)
Plasmatocyte partly adhering to the brush hair (asterisk). An electron-dense matrix of granular aspect is observed at the interface between the hair
and the hemocyte (arrowhead). Scale bar: 2 mm. (D) Granulocyte largely spread onto the hair. Two characteristic layers are observed at the interface
between the horsehair and the hemocyte (box magnified in (E)). Scale bar: 5 mm. (E) The internal layer is highly electron-dense, compact and
homogenous (arrow). The external layer is heterogeneous and composed of cell debris and coagulated hemolymph (arrowhead). Scale bar: 1 mm.
This granulocyte is in tight contact with another ‘‘capsule’’-forming hemocyte (box magnified in (F); arrowheads). An electron-dense thin layer covers
the surface of the cell (arrow). Scale bar: 1 mm. N: Nucleus; M: Mitochondria; G: Granule; V: Vacuole.
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modified morphology, with less cytoplasmic extensions (lamellipo-
dia and filopodia) and spreading ability. The latex beads inside
hemocytes could not be visualized using TEM, likely because
beads were lost during sample processing, but empty prints of the
bead size were found in the cytoplasm (not shown). By contrast,
many hemocytes from aphids injected with E. coli had their
cytoplasm filled with bacteria (up to 45 bacteria observed by TEM
on one cell slice), all tightly enclosed in different types of
phagosomal structures (Fig. 7I–J). Phagosomes observed on
TEM were classified on a morphological basis according to [27]
(see also Figure legends). Some internalized bacteria showed
variable degrees of degradation as judged by the loss of internal
structure (Fig. 7I). Despite this intense phagocytosis, many bacteria
remained free in the hemolymph 24 h post-injection.
Phagocytosis of symbionts
Phagocytosis of secondary symbionts.
servation of AHPs from YR2(Ri), YR2-Hd and YR2-Ss lines,
whose hemolymph contains a high number of their respective
Surprisingly, ob-
secondary symbionts (SS), revealed the presence of SS inside the
hemocytes. A high proportion of plasmatocytes and granulocytes
had their cytoplasm full of bacteria, whose identity was confirmed
using specific FISH (data shown for YR2(Ri), Fig. 8B–C).
Occurrence of phagocytosis was confirmed by TEM, with up to
50 R. insecticola (YR2(Ri)), 100 S. symbiotica (YR2-Ss), and 180 H.
defensa (YR2-Hd) observed on a single slice of one granulocyte of
the corresponding line (Fig. 8D–F).
In YR2(Ri), phagolysosome-like structures were found that
contained Ri bacteria, but the bacteria did not present visible signs
of degradation. In YR2-Ss, numerous phagosomes with concentric
multiple membranes were observed, some but not all containing Ss
bacteria (Fig. 8E). The absence of Ss in part of these phagolyso-
some-like structures could possibly result from the complete
degradation of the bacteria. Finally, in the YR2-Hd line, Hd
symbionts were found in phagosomes but no phagolysosome-like
structure could be observed.
To determine if secondary symbionts freshly injected in a SS-
free line would be phagocytized in the same way as endogenous
Figure 7. In vivo phagocytosis of fluorescent latex beads and fluorescent E. coli 24 h post-injection in LL01 aphids. (A–D) Confocal
images of both granulocytes (A and B) and plasmatocytes (C and D) having ingested numerous latex beads (yellow-red fluorescence). (E) Merger of
DIC and fluorescent micrographs showing an aggregate of hemocytes actively phagocytizing beads (red fluorescence). (F) Melanization of ingested
latex beads (brown staining). (G–H) Granulocytes (in G) and plasmatocytes (in H) containing numerous red fluorescent bacteria (F-actin in green);
Scale bar: 10 mm (A–H). (I–J) TEM micrographs showing uptake and degradation of ingested bacteria. (I) E. coli bacteria inside the cytoplasm of a
granulocyte (arrowheads: bacteria at different degrees of degradation). (J) Granulocyte phagocytizing a bacterium in a zipper-like manner (P1 arrow).
Phagolysosome-like structures are seen that contain a single bacterium (magnified in box) or several bacteria in an electron-dense matrix (asterisk).
See also filopodial extensions (arrowhead). Scale bar: 5 mm (I–J) and 0.5 mm (magnifications box in (J)).
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symbionts, we injected Ri bacteria from the YR2(Ri) line into
LL01 and YR2-Amp aphids. 24 h and 48 h post-injection, the
hemolymph contained numerous free bacteria showing signs of
division. Ri cells were observed inside adherent hemocytes, using
DNA staining or specific FISH (Fig. 9C, 9E), and phagocytosis was
confirmed by TEM (plasmatocytes, Fig. 9F; granulocytes Fig. 9G).
We could observe phagocytosis of individual bacteria (Fig. 9F–G)
and of groups of bacteria with extracellular fluid (Fig. 9F), as well
as structures resembling phagolysosomes and containing Ri
(Fig. 9G, insert). Some of the Ri inside phagolysosomes of
granulocytes or large phagosomes of plasmatocytes showed more
or less advanced signs of degradation, notably an increase in
electron-density (Fig. 9F–G). Besides, hemocytes contained vacu-
oles of variable size as well as numerous membrane ruffles,
suggesting strong endocytosis and micropinocytosis activity
(Fig. 9F–G).
Phagocytosisofthe
aphidicola.
Buchnera bacteria can easily be identified by light
microscopy (Fig. 1A–B, 3B–D, 4A) and TEM (Fig. 2E, 7I, 8E–F)
primarysymbiont B.
as strongly basophilic cells (intense blue coloration with MGG,
Fig. 3D) with a characteristic spherical shape (2–4 mm of
diameter). They were observed in hemolymph samples whatever
the aphid line or the collection procedure. Although a few Buchnera
may naturally be present in the hemolymph, they are likely
released by the rupture of the bacteriocytes upon ventral dissection
in most of our experiments. A variable number of Buchnera were
observed on AHPs, some stuck on coverslips, but most of them at
the surface or inside adherent hemocytes (detection by DNA
staining in LL01 and YR2-Amp (Fig. 9A–B), by FISH in LL01 as
an example (Fig. 9D)). Unfortunately, as most hemolymph
preparations contained a low number of Buchnera, we were not
able to obtain TEM pictures of their internalization.
Discussion
Recent papers suggest that the aphid immune defense is
surprisingly low [7,8]. However, our knowledge remains incom-
plete and, for instance, the nature and functions of hemocytes were
Figure 8. In vivo phagocytosis of symbionts by hemocytes from SS-containing YR2 lines. (A–C) Fluorescent micrographs of YR2(Ri)
hemocytes. (A) F-actin (green) and (B) DNA staining (blue), of an adherent granulocyte. The cytoplasm is filled with R. insecticola secondary symbionts
as shown by bacterial DNA staining (arrowheads in B; arrow: cell nucleus), and (C) specific FISH detection (yellow, false color; DIC and confocal
micrographs overlay). Nucleus (N) location is detectable by the absence of yellow coloration. (D–F) TEM micrographs showing phagocytosis of
secondary symbionts by hemocytes of three YR2 lines. (D) YR2(Ri) granulocyte containing numerous R. insecticola inside the cytoplasm. Different
symbiont-containing phagosomal structures are observed (P1: zipper-like, P2: trigger-like, P3: macropinocytosis), as well as phagolysosomes
containing symbionts and membranous material (arrowheads and box). (E) YR2-Ss line granulocyte with numerous S. symbiotica inside cytoplasmic
membranous phagolysosomes (arrowheads). Insert box: detail of S. symbiotica engulfment by trigger-like phagocytosis. (F) YR2-Hd granulocyte
containing H. defensa symbionts in the three types of phagosomes. Inset: plasmatocyte with a large phagosome structure (macropinocytosis-like)
enclosing several symbionts and extracellular fluid. Scale bar: 5 mm (A–B), 10 mm (C), 5 mm (D), 2 mm (F) and 1 mm (magnifications boxes in (D) and
(E)).
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largely uncharacterized. Moreover, this lack of data also precluded
addressing the question of aphid hemocytes’ interaction with
primary and secondary symbionts. Our study was then set up to
extensively characterize immune cellular components in the pea
aphid, and to document the interaction between hemocytes and
symbionts in this insect model.
Characterization of aphid circulating cells
Prior studies of aphids’ hemocytes are scarce, and mainly based
on light microscopy [23,28–33]. The only study on A. pisum [24] is
recent and describes three cell categories, prohemocytes, granu-
locytes and oenocytoids, and part of their functions. Based on
morphological, histological, ultrastructural and functional criteria,
we have characterized five main cell types from A. pisum
hemolymph: prohemocytes, granulocytes, and the non previously
described plasmatocytes, as well as two additional categories,
spherulocytes and wax cells. Although they may not be ‘‘true’’
circulating cells, these last types were included since they are
involved in aphid defense. At last, non-adhering cells resembling
the previously described oenocytoids [24] were observed in a few
hemolymph samples. As they were rare and highly labile, we could
not characterize them further. Although cell characterization was
performed with LL01 aphids, similar types were observed in all
lines, whatever their genotype and the secondary symbiont they
host.
Wax cells were described in the hemolymph of other aphid
species, and they may have the same embryonic origin as
circulating hemocytes [28]. However, they mainly localize at the
base and inside the cornicles and are thus likely released in
hemolymph upon collection. These cells produce the wax that
sticks external aggressors [34], and their large lipid vesicle likely
contains alarm pheromones emitted by attacked aphids [35].
Spherulocytes are mainly located under the aphid cuticle, but they
were always observed in hemolymph preparations. Their location
suggests they may be involved in lipid synthesis and storage, as
oenocytes and oenocytoids of other insect species [36], or in
providing materials for cuticle renewal. In A pisum, we show that
spherulocytes are the main cells involved in the clotting reaction,
having the same function as coagulocytes in other species [37,38].
Spherules are also strongly reminiscent of the lipid-containing
particles described as pro-coagulants in other arthropods (review
in [39]), and may contain part of the coagulum-forming
compounds. Upon collection, cytoplasmic spherules are quickly
released and rapidly disintegrate to form fibrillar ‘‘fishnets’’ with
Figure 9. In vivo phagocytosis of primary symbionts and injected secondary symbionts in LL01 and YR2-Amp aphids (devoid of SS).
(A–B) Fluorescent images of AHP from LL01 (A) and YR2-Amp (B) unchallenged lines. Hemocytes have taken up B. aphidicola (Ba) cells (arrowheads);
F-actin in green, DNA in blue, arrows indicate cell nuclei. (C) Fluorescent images of AHP from the LL01 line, 24 h after injection of R. insecticola (Ri)
secondary symbionts. Ri are observed in the cytoplasm of a granulocyte (arrowhead); F-actin in green, DNA in blue, arrows indicate cell nuclei. (D)
Overlay of DIC and fluorescent images from AHP of unchallenged LL01 aphids, showing FISH detection of Ba cells (red fluorescence) in contact or
within adherent hemocytes. Nuclei are counterstained with DAPI (blue fluorescence). (E) Overlay of DIC and confocal images showing FISH detection
of Ri secondary symbionts (yellow fluorescence, false color) in a granulocyte of an LL01 aphid, 24 h post-injection. (F–G) TEM showing secondary
symbionts Ri within LL01 hemocytes 24 h post-injection. Notice the high vacuolization (V: Vacuoles), the presence of membrane ruffles (Mb) and the
phagosomes containing ingested Ri. (F) LL01 Plasmatocyte showing two zipper-like phagosomes containing individual bacteria (P1) and a
macropinocytosis-like phagosome (P3) containing several symbionts, some in advanced state of degradation (high darkening). (G) LL01 granulocytes
showing zipper-like phagosome (P1) and phagolysosomes containing symbionts (asterisk and box magnified). Scale bar: 10 mm (A–E), 1 mm (F) and
2 mm (G). N: Nucleus; M: Mitochondria; RER: Rough endoplasmic reticulum; G: Granule.
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the potential to sequester pathogens. Since spherulocytes are non-
adherent, their previous observation as groups of cells at the
surface of Aphidius ervi parasitoid eggs [25] may correspond to
attachment of a coagulum containing entrapped spherulocytes.
Among true circulating hemocytes, prohemocytes are likely
progenitor cells, able to differentiate into other hemocyte
categories. Plasmatocytes and granulocytes morphologically differ
from prohemocytes by the nucleus shape and the number of
cytoplasmic organelles, granulocytes being recognizable from
plasmatocytes by the presence of cytoplasmic granules and a
larger size distribution (see AHP digital analysis, Methods S1, Fig.
S2). Granulocytes and plasmatocytes also differ functionally from
prohemocytes in their capacity to adhere and spread, and to
phagocytize foreign bodies. Whether they correspond to different
lineages or to distinct maturation stages remains to be established;
characterization of cell markers will help solving this question as
well as locating the yet unidentified hematopoietic organ in aphids.
Estimation of the adherent hemocyte number
Our hemocyte counting was performed on adherent hemocytes
using an automatic method. On average, 0.4 ml of hemolymph
was collected per aphid and the hemocyte content was estimated
at about 1440 adherent hemocytes/ml in the LL01 line. This is
close to the estimate by Laughton et al. [24] of 1800 total
hemocytes/ml, suggesting that granulocytes and plasmatocytes
represent the majority of the circulating hemocytes. In compar-
ison, L3 Drosophila larvae contain about 2000 to 3000 hemocytes/
ml [40]. While the number of granulocytes was similar in the two
aphid studies, Laughton et al. [24] described prohemocytes as the
most abundant cells in hemolymph, which is very unlikely. Indeed,
our observations suggest that the prohemocyte proportion ranges
from 3% to 5%, and they are considered to be rare in insect
species [23,38,41]. This discrepancy is certainly due to the smear
technique used in [24], plasmatocytes differing mainly from
prohemocytes by their adhesion capacity.
The role of hemocytes in aphid defense
‘‘Encapsulation’’ and melanisation.
tion of parasitoids eggs has seldom been reported in aphids, it
seems to involve granule-containing cells [42] that likely corre-
spond to granulocytes. Accordingly, A. pisum granulocytes, but also
plasmatocytes, adhered in vivo to inserted foreign objects in a rather
similar way to hemocytes of most insect species [41,43]. However,
the encapsulation reaction was never complete, and the ‘‘patchy’’
cell monolayer observed clearly differed from the well-organized
capsules built by Diptera and Lepidoptera [43]. The incomplete-
ness of the encapsulation may result from a low number of
recruited hemocytes and/or a low speed of recruitment in A. pisum.
Accordingly, the number of cells on the foreign object was much
higher seven days post-insertion than after 48 h, a time at which
complete encapsulation is achieved in Drosophila. Encystment of
the ‘‘encapsulated’’ object in aphid tissues was often observed,
which may explain the ‘‘disappearance’’ of parasitoid eggs in
resistant strains [44].
As reported in numerous insects including other Hemiptera
[3,45], a deposit resembling melanin was observed at the surface of
the foreign object. This layer might also correspond to extracel-
lular matrix materiel deposition as suggested in Drosophila [46].
We also noticed numerous cells undergoing lysis, possibly in
relation with the release of melanization enzymes such as the PO.
PO activity has been described in almost all insect hemocyte types
[41]; in A. pisum, we observed PO-positive granulocyte-type cells,
as well as circulating granulocytes containing melanized latex
beads in the aphid hemolymph. These cells are thus likely involved
Although encapsula-
both in melanization and in the first steps of the ‘‘encapsulation’’
response.
Phagocytosis of pathogenic bacteria
Plasmatocytes and granulocytes were able to massively and
rapidly internalize latex beads or E. coli cells that had been injected
into the hemolymph. Although no PGRP-encoding genes have
been identified in the pea aphid genome, several genes are
predicted to encode proteins involved in opsonization, which can
lead to phagocytosis. They include for instance, the gram-negative
bacteria binding protein 1 (GNBP1) and C-type lectins, LPS-
binding proteins [7]. Dscam, the immunoglobulin superfamily
receptor Down syndrome cell adhesion molecule, which may act
both as a signaling receptor and an opsonin, is also present in the
genome. Moreover, numerous genes acting in the phagosomal and
lysosomal pathways have been identified (see KEGG pathways), as
well as genes encoding surface receptors involved in triggering
these pathways (e.g. integrins, multiple EGF-like-domains recep-
tors, low-density lipoprotein receptor, CD36 like protein). This is
in agreement with our observation that aphid plasmatocytes and
granulocytes display the full repertoire of phagocytosis types.
Finally, we observed evident signs of degradation of E. coli bacteria
in phagolysosome-like structures, showing that aphids can destroy
bacterial invaders.
Although aphid hemocytes are clearly able to perform classical
immune cellular functions, it remains to be determined whether they
play a significant role in aphid defense, notably against bacteria.
Aphids have a limited capacity to seal wounds by coagulation and
melanization [8]and werealsoreportedto besusceptible toinfection
by various strains of bacteria and resistant to others [47,48]. The
efficiency of phagocytosis in controlling bacterial infections will have
to be tested using various concentrations of injected or ingested
bacteria, as well as with naturally occurring pathogens.
Aphid cellular immunity and symbiosis
Phagocytosis of secondary symbionts.
adherent hemocytes in YR2(Ri), YR2-Ss and YR2-Hd lines led
to the striking observation that plasmatocytes and granulocytes
from each line contained a high number of the corresponding
secondary symbionts (SS) in their cytoplasm. A similar result was
observed following injection of R. insecticola symbionts into LL01 or
YR2-amp aphids. The presence of symbionts inside hemocytes has
previously been reported, notably in another Hemipteran species,
the triatomine bug [16]. TEM analyses confirmed the presence of
the SS in phagosome-like structures resembling those containing
injected E. coli. The pea aphid hemocytes thus have the capacity to
phagocytize secondary symbionts whether naturally present or
injected into the hemolymph. This phagocytic capacity depends
neither on the clone genotype nor on the nature of the SS (e.g.
Regiella, Hamiltonella, Serratia). However, quantitative differences
may occur, depending on these parameters.
The outcome of phagocytized SS was rather unclear compared
to that of E. coli bacteria since no clear pictures of degradation
were observed. However, the presence of many empty phagolyso-
somes in YR2(Ss) could suggest a rapid degradation of uptaken Ss
bacteria, while no such indication of degradation was found for Ri
and Hd. This observation and the persistence and proliferation of
Hd and Ri in Drosophila S2 cells [49] raises the question of their
possible survival inside hemocytes. These closely-related bacteria
carry active type III secretion systems, and produce putative
virulence factors [50,51], in contrast to Ss in which many genes
involved in pathogenesis seem to be inactivated [52]. Interestingly,
the type III secretion system was demonstrated to be required for
cell line infection and in vivo maintenance of the Sodalis-tsetse fly
The study of
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symbiont [53,54]. It is also well-known that bacterial pathogens,
notably other enterobacteria, use various strategies to survive host
cellular defenses, promoting their own internalization and/or
interfering with the phagosomal maturation processes in phagocytic
cells [55,56]. If Ri and/or Hd were demonstrated to use such
strategies, their survival inside aphid hemocytes might possibly
facilitate the symbionts’ colonization of host tissues (such as
bacteriome sheath cells and bacteriocytes), their intraspecific or
interspecific horizontal transfer, or their transmission to embryos of
subsequent generations. Interestingly, signs of degradation were
found forRibacteria injected into LL01 orYR2-amp aphidsbutnot
for bacteria naturally present in the YR2(Ri) line. This might result
either from the presenceof damaged Ri cells followingthe isolation/
injection procedure, or from the fact that injected Ri were
introduced into the hemolymph of aphids previously devoid of SS.
Adherent hemocyte number in presence of the different
secondary symbionts
The difference in the number of adherent hemocytes between
LL01 and YR2-amp lines, devoid of secondary symbionts, and
YR2(Ri) and YR2-Hd lines clearly demonstrates that the presence of
some secondary symbionts can affect aphid cellular immunity. This
raises the question of how this effect is mediated. The lower adherent
cell number in presence of Hd or Ri may originate from a lower
number of plasmatocytes and granulocytes in vivo, and/or a decrease
of their adhesion properties. A lower cell number may have several
possible origins, including a cost of the presence of the SS on the
aphid overall fitness, as previously reported [57,58]. Strikingly, the
presence of Ss did not affectthe adherent cellnumber, which suggests
that this effect is symbiont-dependent. Future studies will question
whether the effect of the presence of a given symbiont on aphid
circulating cells also depends on parameters such as the host
genotype, the bacterial strain, or the conditions of infection. Finally, it
is interesting to note that a reduction in immune competence has
been observed in the isopod A. vulgare infected by Wolbachia, this
bacteria being also detected host hemocytes [13,59].
Phagocytosis and primary symbionts
Buchnera (Ba) cells are usually enclosed inside primary bacter-
iocytes, surrounded by a membrane presumably derived from the
host cell (the M3 membrane), and thus protected from immune
cells. However, their transmission to A. pisum embryos is imperfect
[60], possibly because it occurs by specific exocytosis from the
maternal bacteriocyte [61], which may explain our sporadic
observation of Ba in the hemolymph. When released in high
number during hemolymph collection, Ba cells were readily taken
up by hemocytes, as were foreign bacteria or SS. Douglas et al.
[62] reported rapid lysis of Buchnera injected in the hemolymph of
aposymbiotic aphids, as well as their uptake and rapid subsequent
elimination by S2 Drosophila cells [63]. Although these bacteria
have drastically changed due to establishment of tight symbiosis,
surface molecular patterns that allow recognition by the insect
immune system, such as peptidoglycan, are still present [64];
accordingly, Drosophila S2 cells overexpress genes encoding AMPs
when infected with Ba [63]. In addition to the problem of the
recognition pattern of Ba by aphid hemocytes, our results raise
several questions such as whether Ba uptake by aphid hemocytes
occurs in natural conditions, and whether Ba are destroyed or may
be able to survive inside phagocytic hemocytes.
Conclusion
This work shows that aphids possess all classical components of
insect cellular immunity, and provides strong bases for future
studies of its role in aphid defense. It also opens new areas of
research in the challenging domain of the interaction between
immunity and symbiosis. Based on our observations, the aphid-
symbiont model may be considered as one of the most promising
to address such questions as: how symbionts are perceived by the
immune system, how the immune system may control their
location and proliferation, and whether symbionts may still show
pathogenic traits such as the ability to hide themselves in immune
cells. Here, we also demonstrate that the decrease in host
immunocompetence associated with some parasitic bacteria
[12,13,59] can also occur in a case of mutualism, thus highlighting
the continuum between parasitism and mutualism. Aphid
symbionts might be important players in function/homeostasis
of the immune system, as demonstrated for symbionts of the tsetse
fly [65]. An important feature brought by the aphid model is also
the evidence that different symbionts can differentially interfere
with the immunity of the same host. Finally, the presence of
symbionts inside aphid hemocytes might help understand the
remaining mysteries associated with transmission of aphid
symbionts.
Materials and Methods
Biological material
A. pisum individuals used were offspring of parthenogenetic
females from two field-originating lines, LL01 and YR2(Ri), and
three artificial lines (YR2-Amp, YR2-Hd, YR2-Ss) (Table 1). LL01
is devoid of secondary symbionts (SS) while YR2(Ri) (formally
named YR2) only hosts Regiella insecticola (formerly the U-type or
PAUS). YR2-Amp was produced from YR2(Ri) by elimination of
R. insecticola using ampicillin treatment, and is therefore artificially
free of SS [66]. YR2-Hd and YR2-Ss were obtained by injecting
Hamiltonella defensa (T-type or PABS) and Serratia symbiotica (R-type
or PASS), respectively, into the YR2-Amp line (Simon et al. [58]).
The four YR2 lines thus differ only in their SS composition status.
All strains were maintained in cages on fava bean, at 20uC, under
a 16:8 h light/dark cycle.
To obtain synchronized aphid individuals, 10 reproductive
mature females were left on a plant for 24 h. The produced
progeny was a synchronized cohort of aphids. All assays were
carried out on adults that began to reproduce 1–2 days prior to
experimentation (14–15 days-aged in our rearing conditions).
Hemolymphcollection
preparation (AHP).
Two protocols were used for hemolymph
collection. For hemolymph-clotting tests, we proceeded as
described by Fukatsu et al. [21]. Aphids were sterilized in 70%
ethanol, rinsed in water, and attached dorsally onto a Petri dish
using adhesive tape. The legs were removed, and the drops of
hemolymph collected using a capillary glass pre-filled or not with
fresh Schneider’s insect medium (SIM, Sigma). Since the collected
amounts of hemolymph (0.1 ml per aphid) and the hemocytes’
number were low, and the presence of cellular debris and
numerous free nuclei suggested hemocytes’ degradation (Fig. S1),
we set up a different collection protocol for all other experiments.
Briefly, aphids were sterilized and immerged into a drop (10 ml per
aphid) of SIM or Grace’s Insect Cell Culture Medium (Invitrogen),
on a Petri dish over ice. Following careful rupture of the ventral
cuticle under a stereomicroscope, the aphid-diluted hemolymph
was collected. We also estimated the quantity of undiluted
hemolymph recovered per aphid with this method at about
0.4 ml, using 0.5 ml glass capillaries (five repetitions). A higher
number of hemocytes could be obtained by dissecting successively
several aphids in the same drop of medium. For adherent
hemocyte preparations (AHPs), diluted hemolymph was trans-
and adherenthemocyte
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ferred to glass coverslips (Carl Roth, 2 cm diameter) in a multi-
well plate placed in a wet chamber. Hemocytes were allowed to
settle down and adhere for one hour at room temperature (15 min
for detection of ROS, see below), and coverslips were then washed
twice with the hemolymph collection medium.
F-actin and nuclei staining.
10 min with paraformaldehyde 4% (PFA 4% (Sigma) in 0.1 M
phosphate buffer, pH 7.4), and washed three times, 5 min each, in
phosphate-buffered saline (PBS, Sigma). To stain F-actin, AHPs
were permeabilized 10 min with PBS-0.1% triton 6100, washed
in PBS, and incubated one hour in the dark with Phalloidin-65-
FluoProbe 505 (Interchim; green fluorescence: Em=530 nm)
diluted 1:200 in PBS. After PBS washing, nuclei were counter-
stained in the dark 10 min with 1 mg/ml of 49,69-diamino-2-
phenylindole (DAPI, Sigma; blue fluorescence: Em=461 nm).
Next, AHPs were washed three times in PBS and one time in
deionized water. Coverslips were mounted on slides using aqueous
mounting medium containing an anti-fading reagent (RotiH-
Mount Fluorcare, Carl Roth) and observed under a fluorescent
microscope.
Detection ofintracellular
(ROS).
AHPs were incubated in the dark with non-fluorescent
dihydrorhodamine 123 (DHR 123, 50 ng/ml; Sigma) diluted in
SIM for 15 min RT. They were then washed once and the
generation of fluorescent rhodamine 123 (resulting from intracel-
lular oxidization of DHR 123 by ROS) was directly observed
under an epifluorescent microscope with DIC filter for green
fluorescence detection (Em=534 nm).
Hemocyte phenoloxidase (PO) activity.
hemocytes was detected according to Ling et al. [67], by incubating
AHPs for 30 min in freshly prepared dopamine hydrochloride
(1 mg/ml in 35% ethanol, Sigma). Reaction specificity was tested
using the following controls: 35% ethanol alone, and Dopamine
with phenylthiourea (PTU, 2 mg/ml) that specifically inhibits PO
activity. AHPs were then washed three times in PBS and coverslips
were mounted on slides (Faramount Aqueous Mounting medium,
Dakocytomation). PO activity was detected by the brown-dark
coloration. The proportion of adherent hemocytes having PO
activity was estimated by triplicate counting experiments on AHPs
(five aphids dissected in 50 ml SIM per AHP).
May-Gru ¨nwald Giemsa (MGG) staining.
with 1 ml of May-Gru ¨nwald solution (Carl Roth) for 3 min, and
1 ml of Sorensen’s phosphate buffer (33.9 mM KH2PO4;
32.8 mM Na2HPO4, 12 H2O; pH 6.8) was then gently added
for a 5 min incubation period. Coverslips were transferred in
Giemsa solution (Carl Roth) 1:20 in Sorensen’s buffer for 15 min,
washed in Sorensen’s buffer, mounted and observed by light
microscopy.
Nile blue sulfate staining (lipid detection).
used derives from the Nile Blue method for detecting phospho-
lipids, which stains neutral lipids pink while lipids with acidic
function are stained blue [68]. Whole adult aphids were fixed with
PFA 4% during one week at 4uC. After washing in PBS, they were
transferred into a saturated Nile Blue A sulfate (Carl Roth)
aqueous solution, and incubated at 60uC for 24 h. Aphids were
then washed twice 2 h in PBS and 1 h in distilled water. Finally,
they were transferred into a drop of distilled water and carefully
dissected. Staining was observed by light microscopy.
Hemolymph clotting.
The first steps of hemolymph clotting
were observed using freshly collected hemolymph. 15–20 ml drops
of diluted hemolymph (1:10 in SIM) were spotted on glass slides
(Carl Roth), covered with coverslips (2 cm diameter), and changes
in hemocytes/hemolymph appearance were followed during
30 min by light microscopy. Alternatively, undiluted hemolymph
Washed AHPs were fixed for
reactiveoxygen species
PO activity in
AHPs were fixed
The technique
collected from 50 aphids was spotted on glass slides. After a 1 hour
in a wet chamber, 10 ml of neutral red (1 mg/ml in SIM) were
gently added to the spotted drops, coverslips mounted, and the
coagulum observed by light microscopy.
In vivo hemocyte adhesion assay.
immobilized, the dorsal cuticle was punctured with the tip of a
glass needle and a brush horsehair fragment (about 500 mm long
and 80–150 mm diameter) was inserted into the hemocoele.
Aphids were then returned to plants under normal rearing
conditions. Fragments were removed 24 h, 48 h or 7 days after
insertion, by dissecting aphids in SIM. After fixation (PFA 4%,
2 h, 4uC), washing in PBS, and staining of F-actin and DNA,
fragments were examined for the presence of adhesive hemocytes.
Five to seven brush horsehair fragments were observed for each
time point. In addition, 20 fragments collected 7 days post-
insertion were treated for TEM. To determine if other foreign
objects could be ‘‘encapsulated’’ as well, we tested insertion of
small parts of stretched glass capillary and small fragments of resin
in the same manner.
In vivo phagocytosis assays.
erties were tested in vivo by microinjecting fluorescent latex beads,
fluorescent living bacteria or isolated secondary symbionts into
adult aphids (LL01 and YR2-Amp). Alternatively, we looked for
natural phagocytosis of SS by hemocytes of the YR2 lines using
fluorescent microscopy and FISH experiments (YR2(Ri) line), and
TEM (YR2(Ri), YR2-Ss and YR2-Hd). Microinjections were
performed with a Nano-injector (Nanoject, Drummond) on
immobilized aphids. A volume of 69 nl of sample was injected
into the body cavity through the dorsal cuticle of each aphid.
Injected aphids were then returned to rearing conditions until
hemolymph collection.
Fluorescent latex beads (2 mm-diameter carboxylate-modified
polystyrene latex beads, red fluorescent, Sigma) were washed twice
with SIM. Approximately 2000 beads (adjusted after counting on a
Thoma chamber) were injected per aphid. E. coli DH5-alpha
expressing the red fluorescent protein (DsRed) were cultured in LB
medium (Mo Bio Laboratories) and diluted in SIM. R. insecticola
samples were obtained from YR2(Ri) aphid hemolymph. Follow-
ing 5 min centrifugation at 200 g, the pellet (containing mainly
embryos and bacteriocytes) was discarded, and the supernatant
centrifuged at 2000 g for 5 min. The final pellet (that contained
mainly SS) was then re-suspended into an adequate volume of
SIM and approximately 1000 bacteria were injected per aphid. For
‘‘latex beads’’ and ‘‘fluorescent E. coli’’, hemolymph was collected
2 h, 5 h or 24 h post-injection from pools of 10 aphids, and either
directly observed or used for F-actin staining on AHPs prepara-
tion. The percentage of hemocytes phagocytizing fluorescent latex
beads was estimated by counting on AHPs the number of adherent
hemocytes containing three or more beads, 5 h (1029 hemocytes
analyzed) and 24 h (1023 hemocytes analyzed) post-injection. For
TEM, the hemolymph was collected 24 h post-injection (see
below). For ‘‘R. insecticola’’ injection, DNA staining and FISH
detection on AHPs were performed with hemolymph collected
24 h and 48 h post-injection, while TEM samples were prepared
from a collection 24 h post-injection.
Estimation of adherent hemocytes’ number.
number of hemocytes in hemolymph samples could not be directly
determined (see results), but we nevertheless estimated the number
and proportions of the different types of adherent hemocytes, i.e.
plasmatocytes and granulocytes. F-actin stained AHPs from five
aphids were photographed under a fluorescent microscope using a
video camera, and the digital images were processed and analyzed
using a specific routine developed using the ImageJ 1.42q software
(http://rsbweb.nih.gov/ij/; for details see Methods S1 and Fig.
Aphids (LL01 line) were
Hemocytes’ phagocytic prop-
The total
How Aphid Immune Cells Interfere with Symbionts
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Page 13

S2). Estimations were made in four replicates for each aphid line.
Results were analyzed with the one-way ANOVA and Tukey
multiple comparisons of means (95% family-wise confidence level)
test (TukeyHSD), after verification of assumptions (normality and
homogeneity of variances), using the R 2.12.1 software (www.r-
project.org/).
Fluorescent in situ hybridization (FISH).
performed according to Tsuchida et al. [69] on AHPs fixed for
10 min in 4% paraformaldehyde. The following probes were used
for detection of symbionts’ 16S rRNA: ApisP2a-Cy3 (59-Cy3-
CCTCTTTTGGGTAGATCC-39) for B. aphidicola, and U16-Cy5
(59-Cy5-GTAGCAAGC TACTCCCCGAT-39) for R. insecticola (U
type). Briefly, hybridization buffer (20 mM Tris-HCl, pH 8.0;
0.9 M NaCl; 0.01% sodium dodecyl sulfate; 30% formamide)
containing 10 pmol/ml of probe and 200 ng/ml of DAPI was
applied on fixed AHPs. Slides were incubated in a wet chamber at
46uC overnight, rinsed 10 min in 16SSC (0.15 M NaCl, 15 mM
sodium citrate) and 1 min in deionized water, mounted with
fluorescent mount medium and observed under epifluorescent or
confocal microscope to detect B. aphidicola and R. insecticola (red
fluorescence and far-red fluorescence respectively). No-probe
experiments were performed as a control.
FISH was
Transmission electron microscopy (TEM).
blocks were prepared from the pooled hemolymph of 100–200
aphids. Approximately ten samples of diluted hemolymph, each
from 20 aphids (dissected into 200 ml of SIM), were pooled into a
centrifuge vial, on ice. The same volume of fixative (4%
glutaraldehyde (Sigma) in 0.1 M sodium cacodylate buffer,
pH 7.2) was added to the vial that was next conserved 24 h at
4uC. Fixed hemolymph was then centrifuged (500 g, 10 min) to
pellet hemocytes and remove the fixative. The pellet was washed
and diluted in the same buffer, filtered through a 48 mm filter to
remove embryos and tissues debris, and centrifuged again (500 g,
10 min). Post-fixation was done by 2% osmium tetroxide in
cacodylate buffer. Following dehydration in graded series of ethanol
solutions, samples were embedded into Epon resin. The brush
horsehair fragments removed from aphid hemocoele 7 days after
insertion (see above) were put into fixative (2% glutaraldehyde in
0.1 M sodium cacodylate buffer, pH 7.2) for 24 h at 4uC. After
washing in the same buffer, the brush horsehair fragments were
post-fixed and embedded as described above.Samples sections were
cut with a diamond knife using a LKB ultramicrotome, mounted on
copper grids, stained with uranyl acetate and lead citrate, and
observed with a Zeiss EM10CR electron microscope at 80 kV.
Light and Fluorescent Microscopy.
ined using epifluorescent microscopes or a confocal microscope, all
fitted with differential interference contrast (DIC) optics. The
microscope ‘‘Axioplan 2’’ (ZEISS) with the objective ‘‘Plan
Neofluar 406/0.75’’ and the color camera ‘‘Axiocam color’’ was
used for acquisition of colored images without fluorescent staining.
The microscope ‘‘Imager.Z1’’ (ZEISS) with objectives ‘‘EC Plan-
Neofluar 406/0.75’’ and ‘‘Plan Apochromat 636/1.4 oil’’ and the
black and white camera ‘‘Axiocam MRm’’ was used for
acquisition of epifluorescent images and for black and white
DIC images. All confocal micrographs were acquired with the
microscope ‘‘LSM 510 Meta’’ (ZEISS) coupled with the ZEISS
software and using the objective ‘‘Plan-Neofluar 406/1.3 oil’’. All
captured images were exported to Adobe Photoshop for figure
assembly.
TEM sample
Samples were exam-
Supporting Information
Figure S1
from lysed spherulocytes. (A) Spherulocyte losing its spherules
In vivo observation of free nuclei originating
5 min after hemolymph collection. The nucleus and the central
highly stained blue nucleolus are visible. (B) Nucleus from a lysed
spherulocyte, 20 min after hemolymph collection. The nucleus is
associated with a few spherules and cellular debris. (C–E) Free
spherulocyte nucleus, 30 min after hemolymph collection. (C)
Methyl blue staining. (D) DNA staining using DAPI. E. Merge of
C and D. Legends: arrow: nucleus; arrowhead: nucleolus. Scale
bar: 10 mm.
(TIF)
Figure S2
by image processing and analysis. (A) Representative
examples of visually classified thumbnails of F-actin stained
adherent particles into four subgroups: undefined particles,
plasmatocytes, granulocytes and clusters of cells. (B) Outlines of
the particles presented in A after image processing and particle
analysis using ImageJ. (C) Plots of two measurements recorded
by ImageJ on the 244 visually pre-classified thumbnails. The
Feret’s diameter measurement did not accurately discriminate
the pre-established groups in contrast to the Area’s measure-
ment. Dotted lines represent the threshold values of the
hemocyte Area that allows
(40 mm2,Area,250 mm2) from granulocytes (250 mm2,Ar-
ea,1500 mm2). Undefined particles (Area,40 mm2) and part
of cell clusters (Area.1500 mm2) were removed from the
analysis. Scale bars: 20 mm for A and B.
(TIF)
Estimation of adherent hemocyte numbers
distinguishingplasmatocytes
Methods S1
(DOCX)
Adherent hemocyte counting.
Movie S1
first shows a degraded spherulocyte surrounded with numerous
fibrils of small size. Successive steps of the clotting process are
then observed, with isolated cytoplasmic blebs undergoing
vacuolization, and formation of long strands like strings of
pearls.
(AVI)
Observation of the clotting process. The movie
Movie S2
cytes partially covering a fragment of horsehair 7 days
after insertion in the body cavity of an LL01 adult aphid.
The horsehair is the same as in Fig. 6A. 3D-reconstitution was
generated from 20 confocal optical sections.
(MP4)
3D reconstitution of F-actin stained hemo-
Movie S3
optical sections of an adherent granulocyte having
engulfed numerous latex-beads. F-actin: green fluorescence;
latex-beads: red fluorescence.
(MP4)
3D reconstitution generated from confocal
Acknowledgments
We are very grateful to T. Fukatsu, R. Koga and T. Tsuchida for providing
YR2 lines with different secondary symbionts. We are also thankful to F.
Godiard from the ‘‘Service Commun de Microscopie E´lectronique’’ of the
University of Montpellier for support with Electronic Microscopy, to G.
Engler from the ‘‘Plate-forme de Biologie Cellulaire’’ of the Sophia
Agrobiotech Institute, to Hugo Mathe ´-Hubert for help with statistics, and
to anonymous reviewers for suggestions to improve the manuscript.
Author Contributions
Conceived and designed the experiments: AS JLG MP. Performed the
experiments: AS CA MR CR. Analyzed the data: AS CA JLG MP.
Contributed reagents/materials/analysis tools: CR JCS. Wrote the paper:
AS CA JCS JLG MP.
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Page 14

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