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Parasitoid wasp venom SERCA regulates Drosophila
calcium levels and inhibits cellular immunity
Nathan T. Mortimer
a,1
, Jeremy Goecks
a,b
, Balint Z. Kacsoh
a
, James A. Mobley
c,d
, Gregory J. Bowersock
c,d
,
James Taylor
a,b
, and Todd A. Schlenke
a
Departments of
a
Biology and
b
Mathematics and Computer Science, Emory University, Atlanta, GA 30322; and
c
Department of Surgery and
d
Comprehensive
Cancer Center, University of Alabama at Birmingham, Birmingham, AL 35294
Edited by Ruth Lehmann, New York University Medical Center, New York, NY, and approved April 16, 2013 (received for review December 26, 2012)
Because parasite virulence factors target host immune responses,
identification and functional characterization of these factors can
provide insight into poorly understood host immune mechanisms.
The fruit flyDrosophila melanogaster is a model system for under-
standing humoral innate immunity, but Drosophila cellular innate
immune responses remain incompletely characterized. Fruit flies
are regularly infected by parasitoid wasps in nature and, following
infection, flies mount a cellular immuneresponse culminating in the
cellular encapsulation of the wasp egg. The mechanistic basis of this
response is largely unknown, but wasps use a mixture of virulence
proteins derived from the venom gland to suppress cellular encap-
sulation. To gain insight into the mechanisms underlying wasp viru-
lence and fly cellular immunity, we used a joint transcriptomic/
proteomic approach to identify venom genes from Ganaspis sp.1
(G1), a previously uncharacterized Drosophila parasitoid species,
and found that G1 venom contains a highly abundant sarco/endo-
plasmic reticulum calcium ATPase (SERCA) pump. Accordingly, we
found that fly immune cellstermed plasmatocytes normally undergo
a cytoplasmic calcium burst following infection, and thatthis calcium
burst is required for activation of the cellular immune response. We
further found that the plasmatocyte calcium burst is suppressed by
G1 venom in a SERCA-dependent manner, leading to the failure of
plasmatocytes to become activated and migrate toward G1 eggs.
Finally, by genetically manipulating plasmatocyte calcium levels,
we were able to alter fly immune success against G1 and other par-
asitoid species. Our characterization of parasitoid wasp venom pro-
teins led us to identify plasmatocyte cytoplasmic calcium bursts as
an important aspect of fly cellular immunity.
The outcome of a parasitic infection is largely determined by the
interaction between parasite virulence factors and host immune
defenses (1). Therefore, identification of virulence factors can
provide insight into host immune mechanisms and, in the case of
medically relevant parasites, suggest potential treatments. The fruit
flyDrosophila melanogaster is used as a model of humoral innate
immune responses in both mammals and insect vectors of human
disease (2, 3). Studies of humoral immunity in D. melanogaster have
largely focused on the roles of the closely related Toll and Immune
deficiency (Imd) signaling pathways in antimicrobial immunity
(2, 4–6). Both Toll and Imd signaling culminate in the activation of
NF-κB homologs that are required for the induction of humoral
immunity (7, 8). Subsequently, a role in mammalian innate im-
munity has been found for the family of homologous Toll-like
receptors (9, 10), and their downstream mechanisms are strikingly
conserved (11–13).The study of antimicrobial immunity in flies has
therefore allowed for a detailed understanding of these important
mammalian immune pathways.
Although these fly responses to microbial pathogens are well
characterized, the response to macroparasites is only partially un-
derstood. Among the most common macroparasites of Drosophila
are parasitoid wasps, which can infect up to 80% of flies in natural
populations (14). Larval parasitoids attack fly larvae, simultaneously
injecting both an egg and a complex mixture of venom proteins di-
rectly into the larval hemocoel. In response to wasp infection, flies
mount a cellular immune response against wasp eggs termed mel-
anotic encapsulation (15). Following recognition of the wasp egg,
circulating plasmatocytes are activated via an unknown molecular
mechanism. Activated plasmatocytes migrate toward and bind to
the wasp egg, leading to the formation of a continuous plasmatocyte
layer (16, 17). Recognition of the wasp egg also induces the pro-
duction of specialized immune cells termed lamellocytes that are
present at high numbers in the hemolymph within 24 h following
infection. Lamellocytes bind to the primary plasmatocyte layer
surrounding the wasp egg, forming a dissociation-resistant outer
layer (17, 18). The wasp egg is then melanized inside the hemocyte
capsule, leading to the death of the developing wasp. This encap-
sulation response is conserved across arthropods (19). The molec-
ular mechanisms underlying encapsulation are largely unknown,but
recent work has revealed a high degree of genetic conservation
between fly and mammalian hematopoeisis and other aspects of
cellular immunity (20–22).
Parasitoid wasps use virulence factors in their venom to short
circuit the flyencapsulationresponsetoprotecttheirdeveloping
offspring (17, 23, 24). In nature, Drosophila are targeted by parasitoids
from at least four Hymenopteran families with numerous virulence
strategies (14, 25, 26), suggesting that wasp venom proteins have
evolved to target specificaspectsoftheencapsulationresponse.For
instance, whereas venom from the parasitoid Leptopilina heterotoma
causes lamellocyte cell death (23), the venom of its sister species,
Leptopilina victoriae,specifically inhibits protein N-glycosylation of
lamellocyte surface proteins (17). The study of naturally coevolving
pathogens has allowed for a better understanding of Drosophila hu-
moral immunity (27), and we hypothesize that the examination of
coevolved virulence strategies from multiple parasitoid species will
similarly provide insight into the mechanisms of fly cellular im-
munity. Here, we identify the virulence strategy of a previously
uncharacterized Figitid larval parasitoid of Drosophila,Ganaspis
sp.1 (G1), as a window into Drosophila cellular immunity.
Results
In laboratory trials, we found that G1 readily attacks D. mela-
nogaster larvae (Table S1), laying eggs that attach to internal fly
tissues within 12 h post attack (PA) (Fig. 1A). G1 can efficiently
escape encapsulation in D. melanogaster (Table S1), and following
host pupation, the wasp eggs hatch into larvae that begin to con-
sume host tissues (Fig. 1B), resulting in successful parasitization,
with adult wasps emerging from nearly 100% of attacked
D. melanogaster hosts (Fig. 1C). Drosophila parasitoid wasp species
have widely differing host ranges (26), varying from specialists that
target a narrow range of phylogenetically related species to gen-
eralists that can successfully infect a wide range of Drosophila spe-
cies. To determine the host range of G1, we repeated our attack
Author contributions: N.T.M. and T.A.S. designed research; N.T.M. and B.Z.K. performed
research; J.G. and J.T. contributed new reagents/analytic tools; N.T.M., J.G., J.A.M., G.J.B.,
J.T., and T.A.S. analyzed data; and N.T.M. and T.A.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The G1 Transcriptome Shotgun Assembly project has been deposited at
DDBJ/EMBL/GenBank (accession no. GAIW00000000). The version described in this paper
is the first version, GAIW01000000. G1 COI and ITS2 sequences have been deposited in
GenBank (accession nos. JQ808430 and JQ808406, respectively).
1
To whom correspondence should be addressed. E-mail: nathantmortimer@gmail.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1222351110/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1222351110 PNAS Early Edition
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IMMUNOLOGY
trials on a diverse subset of Drosophila species. We found that G1
is a generalist, successfully parasitizing 13 of the 15 species tested
(Fig. 1C), including 7 of 8 species from the melanogaster group
and representative species of an additional six species groups.
The melanica group member Drosophila paramelanica is G1 re-
sistant and mounts an encapsulation-independent anti-wasp im-
mune response (28), suggesting that G1 uses an antiencapsulation
virulence strategy.
Parasitoid wasps may be characterized as immune evasive, in
which case the egg is not detected by the immune response, or im-
mune suppressive, in which case the immune response is inhibited.
To differentiate between these strategies, we tested the ability of
G1 to suppress the self-encapsulation phenotype of tu(1)Sz mutant
larvae. tu(1)Sz mutants encapsulate their own posterior fat body
tissue in a manner analogous to wasp egg encapsulation (29). We
predict that an immune evasive wasp would have no effect on the
progression of self-encapsulation in tu(1)Sz mutants, whereas an
immune suppressive wasp would be expected to inhibit the self-
encapsulation phenotype. We found that the tu(1)Sz phenotype
is significantly rescued by G1 attack (Fig. 1 D–F), demonstrating
that G1 venom has immune suppressive properties.
Because immune suppression has been linked to the destruction
of hemocytes, and in particular lamellocytes (25, 26), we assayed
the total hemocyte count (THC) and the number of lamellocytes
in G1 attacked larvae at 48 h PA. There is a significant increase in
both THC (Fig. S1A) and lamellocyte number (Fig. S1B) in G1
attacked larvae compared with unattacked controls, and there was
no observable hemocyte death in G1-attacked larvae at 24, 48, or
72 h PA. Furthermore, G1-attacked larvae show increased THC
and lamellocyte numbers relative to larvae attacked by the aviru-
lent wasp Leptopilina clavipes (which is encapsulated by D. mela-
nogaster), demonstrating that G1-attacked larvae have a sufficient
number of hemocytes for encapsulation of the wasp egg (Fig. S1).
These data suggest that G1 venom suppresses the fly immune
response by disabling, rather than destroying, host hemocytes.
To understand how G1 disables fly hemocytes, we used our
recently described sequencing/bioinformatic approach to identify
G1 venom genes (30). We first performed RNA-Seq on mRNAs
isolated from dissected female wasp abdomens, and the sequence
data were assembled into 234,516 transcripts (Table S2). The
transcriptome was filtered by RSEM to remove low quality and
low abundance sequences (30), resulting in a final assembly of
27,354 transcripts. We then used mass spectrometry to identify
the proteins in purified venom, and the resulting 2,891 peptides
were mapped against the transcriptome sequences. The peptides
mapped to the predicted ORFs of 166 different transcripts
(Datasets S1 and S2) for an average of 17.4 peptides per ORF,
with an average protein coverage of 23.5% (Table S3). These
venom genes accounted for just 0.61% of the expression-filtered
assembly (Table S2), meaning that the identified venoms repre-
sent a specific subset of abdomen transcripts. We also found that
the venom proteinsare more likely to contain secretion signals than
nonvenoms (28% of venom genes vs. 6% of nonvenoms; P<0.01),
consistent with the hypothesis that many venom genes encode
small, classically secreted proteins (Table S4). Similar to Leptopilina
boulardi and Leptopilina heterotoma venoms, Gene Ontology (GO)
term enrichment suggests that G1 venom may regulate host phys-
iology (via carbohydrate and nucleotide metabolism and antioxi-
dant activity; Table S4) (30), but provides few clues to potential
virulence mechanisms.
To further confirm the specificity of our approach, we per-
formed suppression subtractive hybridization (SSH) (31) on
cDNA samples made from G1 venom glands and carcasses (whole
wasps with venom glands removed) to select for the most abundant
transcripts that are specific to, or overrepresented in, the venom
gland sample. Of the 71 SSH clones sequenced, 56 aligned to G1
transcripts and 14 of these clones matched our identified venom
genes (Table 1), showing broad overlap across venom identifica-
tion methods. Furthermore, we found that the most highly repre-
sented venom transcript by SSH was also one of the most abundant
venom proteins identified by mass spectrometry (Table S5) and
showed strong homology to the sarco/endoplasmic reticulum cal-
cium ATPase (SERCA). SERCA plays a conserved role in calcium
homeostasis, inhibiting intracellular calcium levels by pumping
calcium ions from the cytoplasm into SR/ER stores and is there-
fore a negative regulator of calcium-mediated signaling pathways.
Based on this interesting putative function, and abundance of
SERCA in both the proteomic and SSH sequencing projects, we
decided to focus on characterizing its role in G1 venom.
Mammalian genomes have multiple SERCA genes, each en-
coding at least two protein isoforms. These isoforms differ only in
the extreme C terminus with the longer isoform having an addi-
tional transmembrane domain and a higher affinity for calcium
ions (32). These protein isoforms are conserved in insects; both the
D. melanogaster homolog Ca-P60A and the G1 homolog identified
in our transcriptome data (comp1045) encode two protein isoforms
Fig. 1. (A) G1 egg (circled) attached to flygut(whitearrow)andfatbody
(black arrow) 24 h PA. (B) Hatched G1 larvae dissected from fly pupae, feeding
on host fat body (white arrow) or hemolymph (white arrowhead). (C)Wasp
eclosion success (by percent) across the genus Drosophila. G1-resistant species
shown in red. Branch lengths are approximated. (D) Penetrance of tu(1)Sz
phenotype in control and attacked larvae, *P<0.01 relative to control, error
bars indicate SE, n=6 independent replicates for each treatment. Control (E)
and attacked tu(1)Sz pupae (F). Scale bars as indicated.
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www.pnas.org/cgi/doi/10.1073/pnas.1222351110 Mortimer et al.
(referred to as SERCA
1020
and SERCA
1002
according to size).
Interestingly, only SERCA
1002
was specifically identified by both
venom mass spectrometry and SSH sequencing (Fig. S2), despite
the presence of both transcripts in wasp abdomens. To test the
idea that SERCA
1002
represents a venom-specific isoform, we
performed transcript-specific PCR on cDNA samples from venom
glands and carcasses. We found SERCA
1020
transcript in both
carcass and venom gland samples, whereas SERCA
1002
was spe-
cifically found in venom glands (Fig. 2A). The identification of
a single SERCA isoform by venom mass spectrometry despite the
coexpression of both isoforms in the venom gland is consistent
with SERCA
1002
being a venom-specific isoform and supports the
specificity of our results. To further confirm that SERCA is found
in G1 venom, Western blots were performed on purified venom
and total protein extract with anti-SERCA antiserum and we ob-
served distinct bands in the two samples: smaller bands of ∼150
kDa and a larger band of ∼200 kDa that is excluded from the
purified venom sample (Fig. 2B). These findings demonstrate that
the SERCA found in our sequencing projects represents a true
venom protein rather than a contaminant released from un-
intentionally lysed cells during venom purification.
To test whether venom SERCA is active, we designed an ex vivo
assay to measure the ability of G1 venom to regulate intracellular
calcium levels in fly cells. Purified G1 venom (or PBS control) was
pretreated with either thapsigargin (TG), an irreversible and
specific inhibitor of SERCA (33), or vehicle control (DMSO) and
then dialyzed to remove excess TG. These samples were then in-
cubated with plasmatocytes expressing a genetically encoded fluo-
rescent calcium sensor (GCaMP3; ref. 34) bled from third instar
larvae. The intensity of GCaMP3 fluorescence is proportional to
intracellular calcium levels (34), and we assayed the ability of each
sample to alter calcium levels by measuring GCaMP3 fluorescence
during the incubation period. We found that incubation with the
PBS samples had no effect on GCaMP3 fluorescence (Fig. 2C,blue
and green lines). However, incubation with G1 venom/DMSO
resulted in a significant decrease in GCaMP3 fluorescence (Fig. 2C,
yellow line), showing that G1 venom is able to manipulate host
intracellular calcium levels. This effect of G1 venom was com-
pletely blocked by pretreatment with the SERCA inhibitor TG
(Fig. 2C, red line), confirming that venom SERCA actively removes
calcium ions from the plasmatocyte cytoplasm. The ability of
venom SERCA to profoundly affect host plasmatocyte calcium
Table 1. Results of SSH sequencing
Transcript ID Annotation No. of SSH hits Venom peptide hits
comp1045_seq1 SERCA 5 79
comp845_seq1 Troponin C 2 7
comp755_seq1 Arginine kinase 1 129
comp181_seq1 —1 69
comp844_seq5 Neprilysin-2 1 11
comp630_seq1 Myosin LC alkali 1 11
comp171_seq2 Cysteine protease 1 6
comp317_seq1 —15
comp636_seq2 Erythrose-4-P dehydrogenase 1 4
comp1_seq1 28S rRNA 30 —
comp9199_seq1 —4—
comp0_seq1 18S rRNA 2 —
comp696_seq1 Torso 2 —
comp3562_seq1 Titan 1 —
comp25221_seq1 —1—
comp111_seq1 —1—
comp1032_seq1 CG31997 1 —
Identified transcripts are listed according to number of SSH clones, annotation, and peptide hits from venom
proteomics.
Fig. 2. (A) Isoform-specific PCR of wasp carcass and
venom gland cDNAs. (B) Western blot of wasp pro-
tein extracts and purified venom with anti-SERCA. (C)
Corrected total cell fluorescence plotted over time
during ex vivo incubation of GCaMP3-expressing
hemocytes with the indicated treatment. *P<0.05
compared with PBS/DMSO control, error bars in-
dicate SE, n=3 independent replicates per treat-
ment. (D) Isoform-specific PCR on cDNAs from
D. melanogaster third instar larvae and hemocytes.
(Eand F) Corrected total cell fluorescenceof GCaMP3
and GFP expressing hemocytes,6 h PA (E)and24hPA
(F) with the indicated wasps. *P<0.01 compared
with respective unattacked controls, error bars in-
dicate SE, n=3 independent replicates for each time
point per treatment.
Mortimer et al. PNAS Early Edition
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IMMUNOLOGY
levels suggests that it is functionally distinct from the endogenous
fly plasmatocyte SERCA. Isoform-specificPCRshowsthatincon-
trast to the SERCA
1002
isoform found in G1 venom, flyhemocytes
specifically express the SERCA
1020
isoform (Fig. 2D). Data from
mammalian systems show that the two isoforms have different af-
finities for calcium and are subject to different modes of regulation,
resulting in a higher maximal pumping rate for the shorter SERCA
isoform (32, 35). If this difference is conserved in insect SERCA
isoforms, it could account for the effect of G1 venom SERCA
1002
in
flyhemocytes.Thesefindings demonstrate that G1 venom antago-
nizes host hemocyte calcium levels in a SERCA-dependent manner.
The presence of active SERCA in G1 venom suggests that reg-
ulation of intracellular calcium concentration might be important
for wasp virulence and, in turn, host resistance. To assay in-
tracellular calcium levels in vivo, we expressed GCaMP3 in larval
immune tissues with the Cg-GAL4 driver, which expresses in the
plasmatocytes and fat body (36). We attacked these larvae with the
avirulent wasp L. clavipes to assay calcium levels in a successful
immune response and with G1 to assay the effect of G1 venom on
host calcium levels in vivo. We simultaneously attacked larvae
expressing calcium-independent GFP under the control of the
Cg-GAL4 driver to control for differences in GAL4 expression
following wasp attack. We found that plasmatocytes from
L. clavipes-attacked larvae showed a significant increase in
GCaMP3 fluorescence at 6 h PA, but no change in GFP fluo-
rescence at this time (Fig. 2E), indicating a specific elevation of
intracellular calcium levels following wasp attack. Following
L. clavipes attack, we did not detect a change in GCaMP3 fluo-
rescence either in plasmatocytes at 24 h PA (Fig. 2F) or in the fat
body at either time point, demonstrating that the calcium burst is
specific to plasmatocytes and is part of an immediate response to
wasp attack. Conversely, plasmatocytes from G1-attacked larvae
showed a significant decrease in GCaMP3 fluorescence at 6 h PA,
whereas GFP levels remained constant (Fig. 2E). There was no
alteration in GCaMP3 fluorescence in plasmatocytes at 24 h fol-
lowing G1 attack (Fig. 2F), or in the fat body at either time point.
We also expressed GCaMP3 with the pan-hemocyte driver He-
GAL4 (37) and did not detect GCaMP3 fluorescence in lamello-
cytes following attack by either wasp, confirming that the calcium
burst is specific to plasmatocytes. These data show that the calcium
regulatory activity of G1 venom demonstrated in our ex vivo assay
is also functional in vivo following wasp attack and that G1 venom
specifically targets the wasp-induced calcium burst in flyplas-
matocytes that occurs immediately following attack.
The calcium burst seen in fly plasmatocytes following attack
with the avirulent wasp L. clavipes suggests that calcium signaling
may be required to activate hemocytes for encapsulation. A similar
calcium burst is seen in both fly and mammalian immune cells in
response to diverse pathogen stimuli (38, 39). To test for a role of
the calcium burst in wasp egg encapsulation, we used He-GAL4 to
express parvalbumin (PV), a vertebrate-specific calcium binding
protein that negatively regulates calcium levels in D. melanogaster
cells (40). At 72 h PA, there was no evidence of capsuleformation in
a large proportion of fly larvae attacked by the normally avirulent
wasp L. clavipes (Fig. 3A), suggesting that He-GAL4–driven PV
expression prevented capsule initiation by fly plasmatocytes. Fur-
thermore, increased intracellular calcium is typically mediated by
the release of calcium from ER stores by either the IP3 receptor
(IP3R) or Ryanodine receptor (Ryr), the major ER calcium release
channels in eukaryotic cells (41). Both of these calcium channels
have homologs in D. melanogaster [encoded by the Inositol 1,4,
5,-tris-phosphate receptor (Itp-r83A)andRyanodine receptor 44F
(Rya-r44F) genes, respectively] (41, 42), and we found that he-
mocyte-specific knockdown of Rya-r44F, but not Itp-r83A, also
resulted in a significant decrease in the proportion of L. clavipes
eggs encapsulated by larval hemocytes (Fig. 3A). Ryr is also re-
quired for phagocytosis by fly hemocytes (38) and is important for
the calcium burst in human B and T cells (39). These data show
that the hemocyte calcium burst is important for hemocyte acti-
vation during the D. melanogaster encapsulation response and is
conserved between mammalian and insect immune responses.
If the ability of G1 venom to antagonize the hemocyte calcium
burst is important for G1 virulence, ectopically raising hemocyte
calcium levels should allow flylarvalhemocytestoencapsulate
G1 eggs. To test this hypothesis, we used G1 wasps to attack larvae
from two mutant lines with a demonstrated elevation of intracellular
calcium levels (olf186-F
EY01467
,theD. melanogaster homolog of the
Orai calcium release-activated calcium channel, and Ca-P60A
Kum170
)
(43, 44). We found that hemocytes from both olf186-F
EY01467
and
Ca-P60A
Kum170
mutant larvae were able to encapsulate G1 eggs at
a significantly higher rate than their genetic background controls
[yellow,white (y,w) and Canton S (CS), respectively] (Fig. 3Band
Table 2). Hemocyte-specific knockdown of Ca-P60A also con-
ferred larvae with increased encapsulation ability against G1 (Fig.
3C), confirming the cell specificity of the calcium signaling phe-
notype. These results suggest that elevated calcium levels either
block G1 virulence specifically or make fly larvae generally more
wasp resistant. To distinguish between these possibilities, we at-
tacked these same flies with the melanogaster subgroup specialist
L. boulardi, a wasp whose venom does not contain homologs of
any known calcium regulators (30). We found that L. boulardi eggs
were not encapsulated by any of these genotypes regardless of
calcium level (Table 2), showing that increased hemocyte in-
tracellular calcium specifically affects G1 virulence.
Fig. 3. (A) Proportion of L. clavipes eggs encapsulated in the indicated
genotypes. *P<0.01 compared with control, error bars indicate SE in all
graphs, within each graph, n=3 independent replicates per genotype. (B)
Proportion of G1 eggs encapsulated in the indicated genotypes. *P<0.01
compared with genetic background controls. (C) Proportion of G1 eggs
encapsulated in the indicated genotypes. *P<0.01 compared with control.
Table 2. Encapsulation rates of G1 and L. boulardi eggs in the indicated genotypes
Genotype
Encapsulation rate
of G1 eggs, % n
Encapsulation rate
of L. boulardi eggs, % n
y,w 0 82 0 73
olf186-F
EY01467
21.7 92 0 69
CS 0 90 0 67
Ca-P60A
Kum170
61.3 80 0 76
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Discussion
The identification of waspvenom proteins led us touncover a unique
and important aspect of the Drosophila innate cellular immune re-
sponse against macroparasites. Flyplasmatocytesundergoacyto-
plasmic calcium burst within 6 h of parasitoid wasp infection, which is
required for activation of the antiwasp immune response. A similar
calcium burst is observed in plasmatocytes before phagocytosis of
bacteria or apoptoticcells (38). Whenthecalciumburstisgenetically
blocked by knockout or knockdown of Rya-r44F, plasmatocytes fail
to initiate capsule formation in response to wasp infection (Fig. 3)
and are unable to phagocytize invading bacteria (38), demonstrating
that calcium signaling is a conserved mechanism among flyimmune
responses to various pathogens.
Calcium signaling also plays an important role in mammalian
immunity. Increased cytoplasmic levels of calcium are observed in
lymphocytes following activation of antigen receptors (39), and this
increase activates the calcium-dependent phosphatase calcineurin,
which is required for the activation of mammalian immune responses
via regulation of the nuclear factor of activated T-cells (NFAT)
family of transcriptional activators (45). Interestingly, calcineurin is
also required in flyhemocytesfortheactivationofImdpathway
signaling in response to infection with Gram-negative bacteria (46),
suggesting that the activation of diverse immune responses likely has
asharedgeneticbasis.G1waspsusevenomSERCAtotargetthis
highly conserved plasmatocyte calcium burst and prevent the initi-
ation of the encapsulation response. This finding demonstrates the
advantage of studying naturally coevolving host-pathogen/parasite
interactions to gain insight into conserved immune mechanisms.
Identifying parasitic wasp venom proteins can enhance our un-
derstanding of the delivery and function of immunomodulatory
proteins in general. Wasp venom genes were proposed to encode
small, classically secreted proteins (47), and this idea appears to be
somewhat true of G1 venom proteins; 28% contained a predicted
secretion signal, and these proteins had an average size of 44 kDa.
However, venom SERCA is a large (110-kDa) transmembrane pro-
tein with no classical secretion signal sequence, and this finding was
not unusual across G1 and other Hymenopteran venoms; G1 venom
proteins range in size from 9 kDa to 379 kDa, and bioinformatic
analyses using transmembrane domain prediction software (48)
reveals that 7% (12/166) of G1 venom proteins and 20% (176/864) of
the previously identified Hymenopteran venoms in GenBank (30)
contain predicted transmembrane domains. How a parasite might
secrete and deliver transmembrane proteins into hosts is unknown.
The venoms of wasps closely related to G1 have been shown to
contain “virus-like particles,”thought to act as venom delivery vehi-
cles, that enter host hemocytes to mediate wasp virulence (49), and we
hypothesize that G1 SERCA may use a similar mechanism. Un-
derstanding the packaging of SERCA in G1 venom, and its delivery to
host hemocytes, represents an interesting subject for future study.
Materials and Methods
Insect Strains. The followingD. melanogasterstrains were usedin this study: the
mutant alleles tu(1)Sz
1
(29), Ca-P60A
Kum170
(43), and olf186-F
EY01467
(44); the
transgenic constructs Cg-GAL4 (36), He-GAL4 (37), UAS-GCaMP3 (34), UAS-GFP
{AH2},UAS-PV (40), Ca-P60A
TRiP.JF01948
,Itp-r83A
TRiP.JF01957
,andRya-r44F
TRiP.JF03381
;
and the control lines y,w,Canton S (CS), Oregon R (OR), a nd w
1118
. For the host
range experiment, the fly species used were as follows: Drosophila simulans,
Drosophila mauritiana,Drosophila sechellia,Drosophila yakuba,Drosophila
erecta,Drosophila ficusphila,Drosophila ananassae,Drosophila pseudoobscura,
Drosophila willistoni,Drosophila funebris,Drosophila immigrans,Drosophila
mojavensis,Drosophila paramelanica,andDrosophila virilis.
This study also used the following wasps: G1, L. clavipes (strain LcNet), and
L. boulardi (strain Lb17). The G1 wasps used in this study were caught in
Homestead, FL, in 2008. G1 COI and ITS2 sequences have been deposited in
GenBank (accession nos. JQ808430 and JQ808406, respectively). LcNet was
provided by J. van Alphen (University of Amsterdam, Amsterdam) and Lb17
has been described(26). Laboratory cultures of G1 and Lb17 aremaintained on
D. melanogaster and LcNet is maintained on D. virilis.
Wasp Attack. Wasp attacks were performed as described (17). Briefly, three
female wasps were allowed to attack 40 second instar fly larvae in 35-mm
Petri dishes filled with Drosophila medium for a 72-h period at 25 °C. To
assay attack and encapsulation rates, larvae were dissected and scored for
the presence of an encapsulated wasp egg or live wasp larva. To assay
eclosion rates, 30 fly larvae were recovered from each plate and allowed to
eclose at 25 °C. The total number of flies and wasps that eclosed were de-
termined 15 d and 30 d after infection, respectively, times by which all viable
flies and wasps should have emerged. All experiments were performed
in triplicate.
tu(1)Sz Phenotype Suppression. The temperature-sensitive tu(1)Sz
1
self-
encapsulation mutant was used to assay wasp virulence strategy (29). The
flies were raised at 28 °C and to assay the ability of wasp venom to suppress
the phenotype, 40 second instar larvae were attacked by three female wasps
for 3 h at 28 °C. The attacked larvae were raised for a further 96 h at 28 °C.
Attacked pupae and age-matched controls were then scored for the tu(1)Sz
phenotype and dissected to ensure attack status; unattacked pupae were
discarded from analysis.
Imaging. Images were acquired by using a Leica stereo-dissecting scope with
a Moticam MIP 2.0 and Multi-Focus Pro software. Figures were compiled by
using Adobe Photoshop.
Hemocyte Counts. Hemocyte counts were performed in triplicate according
to ref. 17. After a 72-h wasp attack period, five larvae from each replicate
were washed in Drosophila Ringer’s solution and bled into PBS containing
0.01% phenylthiourea to prevent melanization. Hemocytes were applied to
a disposable hemocytometer (Incyto C-Chip DHC-N01) and allowed to ad-
here for 30 min. Hemocytes of each replicate were counted from 16 0.25 ×
0.25 ×0.1 mm squares, and the counts were normalized to a per larva value.
Wasp Transcriptomes. RNA was extracted from ∼200 female wasp abdomens
by using the standard TRIzol (Invitrogen) protocol. Poly(A)RNAs were puri-
fied by using the Dynabeads mRNA Direct kit (Invitrogen) according to
manufacturer specifications. Double-stranded cDNAs were synthesized by
using the SuperScript II ds cDNA Synthesis kit (Invitrogen). The cDNAs were
then sequenced by using an Illumina HiSeq 2000. Transcripts were de novo
assembled by using Trinity (version r2011-10-29) (50) and filtered by RSEM
(51) using a cutoff of one transcript per million. See ref. 30 for more detailed
protocols. Tools for secretion signal analysis and GO term annotation and
enrichment are described in ref. 30.
Mass Spectrometry. Venom proteins were purified from venom glands dis-
sected into PBS supplemented with 0.5 mM EDTA and Complete Protease
Inhibitor Mixture (Roche). Venom glands were homogenized under nonlysing
conditions, and gland cells were pelleted by centrifugation. Venom proteins
were run on SDS/PAGE, trypsinized, and subjected to Nano LC-MS(MS)
2
.The
identified peptides were mapped back to the transcriptome data by using
SEQUEST software. See ref. 30 for more detailed protocols.
SERCA Isoform-Specific PCRs. To assay expression of SERCA isoform transcripts,
total RNA was made from wasp venom glands or wasp carcasses (wasps with
venom gland removed), and whole third instar larvae or dissected larval
hemocytes, by standard TRIzol preparations as described above. RNA was
reverse transcribed to cDNA by using the QuantiTect reverse transcriptase kit
(Qiagen). Isoform specific primers were used to amplify SERCA from each
cDNA sample (primer sequences available upon request).
SSH. SSH was performed essentially as described (31) by using venom gland
cDNAs as the tester library and wasp carcass cDNAs as the driver library and
with the following alterations: libraries were hybridized at a 30:1 (driver:
tester) ratio and then used undiluted for two rounds of PCR amplification.
PCR products were cloned were the Strataclone PCR cloning kit (Stratagene)
and sequenced.
Western Blotting. G1 venom was purified as described above. Protein extracts
from purified venom and wasp carcasses were run on 9% (vol/vol) poly-
acrylamide SDS/PAGE gels and transferred to PVDF membranes. Blots were
probed with the 809-27 SERCA antibody at a concentration of 1:100,000 (52).
GCaMP3 Calcium Assays. Ex vivo assay. We used thapsigargin, a plant-derived
sesquiterpene lactone that acts as a specificandirreversibleinhibitorof
SERCA (33). G1 venom was purified as described above and incubated with
1μM thapsigargin in DMSO or DMSO control at room temperature for 15 min.
Samples were then twice dialyzed against PBS at room temperature (15 min,
1 h), added to dissected GCaMP3-expressing third instar larval hemocytes,
Mortimer et al. PNAS Early Edition
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IMMUNOLOGY
and incubated at room temperature. During the incubation period, cells
were imaged by using an Olympus BX51 microscope with a FITC filter and
Olympus DP2-BSW software. Corrected total cell fluorescence (CTCF) was
calculated as described (53).
In vivo assay. Larvae expressing GCaMP3 or GFP were attacked by wasps as
described and dissected at 6 or 24 h PA. Cells were imaged, and CTCFs were
calculated as for the ex vivo assay.
Statistics. All analyses were performed in R version 2.15.0. Fisher’s exact
test was used to compare tu(1)Sz phenotype penetrance between G1
attacked and control samples and to compare the results of Signal P analysis
on G1 venom and nonvenom genes. Total hemocyte counts and lamellocyte
numbers were compared by ANOVA, and Tukey’s HSD test was used for
pairwise comparisons. Differences in in vivo GCaMP3 and GFP fluorescence
levels were compared by two-way ANOVA at 6 h and 24 h PA to test effects
for of flygenotypeandwaspattack.Tukey’sHSDtestwasusedforpairwise
comparisons within each time point. To test the effect of venom and PBS on
GCaMP3 fluorescence throughout the incubation period in the ex vivo assay
we used repeated measures ANOVA (from the car R library) and pairwise
ttests to compare fluorescence at each time point. Finally, generalized linear
models with binomial errors and logit link functions were used to examine
differences in encapsulation rate between genotypes. Bonferroni correction
was used for multiple comparisons.
Data Access. RNA-seq is available at the DNA Data Bank of Japan Sequence
Read Archive under accession number SRP020527; single-end data under SRA
accession number SRX259389 and paired-end data are under SRX259413.
All G1 venom transcript and venom sequences are attached as Fasta
formatted files.
ACKNOWLEDGMENTS. We thank Jesper Moller, Loren Looger, Subhabrata
Sanyal, the Bloomington Drosophila Stock Center, and the Drosophila
Species Stock Center at the University of California San Diego, for stocks and
reagents, the Emory Georgia Research Alliance Genome Center, and Erin
Keebaugh for technical assistance. This work was supported by the National
Institutes of Health Grants R01 AI081879 (to T.A.S.) and R21 HG005133 (to J.T.).
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