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Parasitoid wasp venom SERCA regulates Drosophila calcium levels and inhibits cellular immunity


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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 fly Drosophila melanogaster is a model system for understanding 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 immune response 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 encapsulation. To gain insight into the mechanisms underlying wasp virulence 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/endoplasmic reticulum calcium ATPase (SERCA) pump. Accordingly, we found that fly immune cells termed plasmatocytes normally undergo a cytoplasmic calcium burst following infection, and that this 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 parasitoid species. Our characterization of parasitoid wasp venom proteins led us to identify plasmatocyte cytoplasmic calcium bursts as an important aspect of fly cellular immunity.
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Parasitoid wasp venom SERCA regulates Drosophila
calcium levels and inhibits cellular immunity
Nathan T. Mortimer
, Jeremy Goecks
, Balint Z. Kacsoh
, James A. Mobley
, Gregory J. Bowersock
James Taylor
, and Todd A. Schlenke
Departments of
Biology and
Mathematics and Computer Science, Emory University, Atlanta, GA 30322; and
Department of Surgery and
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,
identication and functional characterization of these factors can
provide insight into poorly understood host immune mechanisms.
The fruit yDrosophila melanogaster is a model system for under-
standing humoral innate immunity, but Drosophila cellular innate
immune responses remain incompletely characterized. Fruit ies
are regularly infected by parasitoid wasps in nature and, following
infection, ies 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 y 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 y 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 y 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 y cellular immunity.
The outcome of a parasitic infection is largely determined by the
interaction between parasite virulence factors and host immune
defenses (1). Therefore, identication of virulence factors can
provide insight into host immune mechanisms and, in the case of
medically relevant parasites, suggest potential treatments. The fruit
yDrosophila 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
deciency (Imd) signaling pathways in antimicrobial immunity
(2, 46). 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 (1113).The study of antimicrobial immunity in ies has
therefore allowed for a detailed understanding of these important
mammalian immune pathways.
Although these y 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 ies in natural
populations (14). Larval parasitoids attack y larvae, simultaneously
injecting both an egg and a complex mixture of venom proteins di-
rectly into the larval hemocoel. In response to wasp infection, ies
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 y and mammalian hematopoeisis and other aspects of
cellular immunity (2022).
Parasitoid wasps use virulence factors in their venom to short
circuit the yencapsulationresponsetoprotecttheirdeveloping
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 specicaspectsoftheencapsulationresponse.For
instance, whereas venom from the parasitoid Leptopilina heterotoma
causes lamellocyte cell death (23), the venom of its sister species,
Leptopilina victoriae,specically 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 y 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.
In laboratory trials, we found that G1 readily attacks D. mela-
nogaster larvae (Table S1), laying eggs that attach to internal y
tissues within 12 h post attack (PA) (Fig. 1A). G1 can efciently
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 conict 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 rst version, GAIW01000000. G1 COI and ITS2 sequences have been deposited in
GenBank (accession nos. JQ808430 and JQ808406, respectively).
To whom correspondence should be addressed. E-mail:
This article contains supporting information online at
1073/pnas.1222351110/-/DCSupplemental. PNAS Early Edition
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 signicantly rescued by G1 attack (Fig. 1 DF), 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 signicant 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 sufcient
number of hemocytes for encapsulation of the wasp egg (Fig. S1).
These data suggest that G1 venom suppresses the y immune
response by disabling, rather than destroying, host hemocytes.
To understand how G1 disables y hemocytes, we used our
recently described sequencing/bioinformatic approach to identify
G1 venom genes (30). We rst 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 ltered by RSEM to remove low quality and
low abundance sequences (30), resulting in a nal assembly of
27,354 transcripts. We then used mass spectrometry to identify
the proteins in puried 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-ltered
assembly (Table S2), meaning that the identied venoms repre-
sent a specic 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 conrm the specicity 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 specic 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 identied venom
genes (Table 1), showing broad overlap across venom identica-
tion methods. Furthermore, we found that the most highly repre-
sented venom transcript by SSH was also one of the most abundant
venom proteins identied 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 afnity for calcium
ions (32). These protein isoforms are conserved in insects; both the
D. melanogaster homolog Ca-P60A and the G1 homolog identied
in our transcriptome data (comp1045) encode two protein isoforms
Fig. 1. (A) G1 egg (circled) attached to ygut(whitearrow)andfatbody
(black arrow) 24 h PA. (B) Hatched G1 larvae dissected from y 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.
| Mortimer et al.
(referred to as SERCA
according to size).
Interestingly, only SERCA
was specically identied 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
represents a venom-specic isoform, we
performed transcript-specic PCR on cDNA samples from venom
glands and carcasses. We found SERCA
transcript in both
carcass and venom gland samples, whereas SERCA
was spe-
cically found in venom glands (Fig. 2A). The identication of
a single SERCA isoform by venom mass spectrometry despite the
coexpression of both isoforms in the venom gland is consistent
with SERCA
being a venom-specic isoform and supports the
specicity of our results. To further conrm that SERCA is found
in G1 venom, Western blots were performed on puried 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
puried venom sample (Fig. 2B). These ndings 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 purication.
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 y cells. Puried G1 venom (or PBS control) was
pretreated with either thapsigargin (TG), an irreversible and
specic 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 uo-
rescent calcium sensor (GCaMP3; ref. 34) bled from third instar
larvae. The intensity of GCaMP3 uorescence is proportional to
intracellular calcium levels (34), and we assayed the ability of each
sample to alter calcium levels by measuring GCaMP3 uorescence
during the incubation period. We found that incubation with the
PBS samples had no effect on GCaMP3 uorescence (Fig. 2C,blue
and green lines). However, incubation with G1 venom/DMSO
resulted in a signicant decrease in GCaMP3 uorescence (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), conrming 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
Identied transcripts are listed according to number of SSH clones, annotation, and peptide hits from venom
Fig. 2. (A) Isoform-specic PCR of wasp carcass and
venom gland cDNAs. (B) Western blot of wasp pro-
tein extracts and puried venom with anti-SERCA. (C)
Corrected total cell uorescence 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-specic PCR on cDNAs from
D. melanogaster third instar larvae and hemocytes.
(Eand F) Corrected total cell uorescenceof 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
levels suggests that it is functionally distinct from the endogenous
y plasmatocyte SERCA. Isoform-specicPCRshowsthatincon-
trast to the SERCA
isoform found in G1 venom, yhemocytes
specically express the SERCA
isoform (Fig. 2D). Data from
mammalian systems show that the two isoforms have different af-
nities 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
yhemocytes.Thesendings 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 signicant increase in
GCaMP3 uorescence at 6 h PA, but no change in GFP uo-
rescence at this time (Fig. 2E), indicating a specic elevation of
intracellular calcium levels following wasp attack. Following
L. clavipes attack, we did not detect a change in GCaMP3 uo-
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
specic to plasmatocytes and is part of an immediate response to
wasp attack. Conversely, plasmatocytes from G1-attacked larvae
showed a signicant decrease in GCaMP3 uorescence at 6 h PA,
whereas GFP levels remained constant (Fig. 2E). There was no
alteration in GCaMP3 uorescence 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 uorescence in lamello-
cytes following attack by either wasp, conrming that the calcium
burst is specic 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
specically targets the wasp-induced calcium burst in yplas-
matocytes that occurs immediately following attack.
The calcium burst seen in y 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 y 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-specic 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 y larvae attacked by the normally avirulent
wasp L. clavipes (Fig. 3A), suggesting that He-GAL4driven PV
expression prevented capsule initiation by y 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-specic knockdown of Rya-r44F, but not Itp-r83A, also
resulted in a signicant decrease in the proportion of L. clavipes
eggs encapsulated by larval hemocytes (Fig. 3A). Ryr is also re-
quired for phagocytosis by y 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 ylarvalhemocytestoencapsulate
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
,theD. melanogaster homolog of the
Orai calcium release-activated calcium channel, and Ca-P60A
(43, 44). We found that hemocytes from both olf186-F
mutant larvae were able to encapsulate G1 eggs at
a signicantly higher rate than their genetic background controls
[yellow,white (y,w) and Canton S (CS), respectively] (Fig. 3Band
Table 2). Hemocyte-specic knockdown of Ca-P60A also con-
ferred larvae with increased encapsulation ability against G1 (Fig.
3C), conrming the cell specicity of the calcium signaling phe-
notype. These results suggest that elevated calcium levels either
block G1 virulence specically or make y larvae generally more
wasp resistant. To distinguish between these possibilities, we at-
tacked these same ies 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 specically 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
Encapsulation rate
of G1 eggs, % n
Encapsulation rate
of L. boulardi eggs, % n
y,w 0 82 0 73
21.7 92 0 69
CS 0 90 0 67
61.3 80 0 76
| Mortimer et al.
The identication 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 yimmune
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 yhemocytesfortheactivationofImdpathway
signaling in response to infection with Gram-negative bacteria (46),
suggesting that the activation of diverse immune responses likely has
highly conserved plasmatocyte calcium burst and prevent the initi-
ation of the encapsulation response. This nding 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 nding 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 identied 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
(29), Ca-P60A
(43), and olf186-F
(44); the
transgenic constructs Cg-GAL4 (36), He-GAL4 (37), UAS-GCaMP3 (34), UAS-GFP
{AH2},UAS-PV (40), Ca-P60A
and the control lines y,w,Canton S (CS), Oregon R (OR), a nd w
. For the host
range experiment, the y species used were as follows: Drosophila simulans,
Drosophila mauritiana,Drosophila sechellia,Drosophila yakuba,Drosophila
erecta,Drosophila cusphila,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). Briey, three
female wasps were allowed to attack 40 second instar y larvae in 35-mm
Petri dishes lled 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 y larvae were recovered from each plate and allowed to
eclose at 25 °C. The total number of ies and wasps that eclosed were de-
termined 15 d and 30 d after infection, respectively, times by which all viable
ies and wasps should have emerged. All experiments were performed
in triplicate.
tu(1)Sz Phenotype Suppression. The temperature-sensitive tu(1)Sz
encapsulation mutant was used to assay wasp virulence strategy (29). The
ies 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, ve larvae from each replicate
were washed in Drosophila Ringers 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-
ed by using the Dynabeads mRNA Direct kit (Invitrogen) according to
manufacturer specications. 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 ltered 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 puried 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)
identied peptides were mapped back to the transcriptome data by using
SEQUEST software. See ref. 30 for more detailed protocols.
SERCA Isoform-Specic 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 specic 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 amplication.
PCR products were cloned were the Strataclone PCR cloning kit (Stratagene)
and sequenced.
Western Blotting. G1 venom was puried as described above. Protein extracts
from puried 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 specicandirreversibleinhibitorof
SERCA (33). G1 venom was puried 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
and incubated at room temperature. During the incubation period, cells
were imaged by using an Olympus BX51 microscope with a FITC lter and
Olympus DP2-BSW software. Corrected total cell uorescence (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. Fishers 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 Tukeys HSD test was used for
pairwise comparisons. Differences in in vivo GCaMP3 and GFP uorescence
levels were compared by two-way ANOVA at 6 h and 24 h PA to test effects
for of ygenotypeandwaspattack.TukeysHSDtestwasusedforpairwise
comparisons within each time point. To test the effect of venom and PBS on
GCaMP3 uorescence throughout the incubation period in the ex vivo assay
we used repeated measures ANOVA (from the car R library) and pairwise
ttests to compare uorescence 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 les.
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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| Mortimer et al.
... The inner cells of the capsule produce melanin and release free radicals into the capsule, killing the wasp (Russo, 1996;Carton, Poirié & Nappi, 2008). Wasps have evolved a diverse array of strategies that counter the fly-encoded defense (Schlenke et al., 2007;Mortimer et al., 2013). Whether or not Spiroplasma contributes to enhancing the fly-encoded defense against wasps has not been extensively investigated. ...
... The strains of wasps used were: the Spiroplasma-susceptible Leptopilina heterotoma strain Lh14 (Schlenke et al., 2007;voucher USNMENT01557081;hereafter "Lh"); and the all-female Spiroplasma-resistant Ganaspis sp. strain G1FL (Mortimer et al., 2013;voucher USNMENT01557080; also known as "drop_ Gan_sp53" in the Drosophila parasitoid database; Lue et al., 2021;hereafter "Gh"). Wasps were reared using second instar Canton S Spiroplasma-free larvae. ...
... The two wasp species used in this study, L. heterotoma and Ganaspis sp., belong to the same family (Figitidae), but their parasitism strategies are quite different (Schlenke et al., 2007;Mortimer et al., 2013). Our fly transcriptome analysis also revealed differences in the effects of these wasps. ...
Full-text available
Background Several facultative bacterial symbionts of insects protect their hosts against natural enemies. Spiroplasma poulsonii strain s Mel (hereafter Spiroplasma ), a male-killing heritable symbiont of Drosophila melanogaster , confers protection against some species of parasitic wasps. Several lines of evidence suggest that Spiroplasma -encoded ribosome inactivating proteins (RIPs) are involved in the protection mechanism, but the potential contribution of the fly-encoded functions (e.g., immune response), has not been deeply explored. Methods Here we used RNA-seq to evaluate the response of D. melanogaster to infection by Spiroplasma and parasitism by the Spiroplasma -susceptible wasp Leptopilina heterotoma , and the Spiroplasma -resistant wasp Ganaspis sp. In addition, we used quantitative (q)PCR to evaluate the transcript levels of the Spiroplasma -encoded Ribosomal inactivation protein (RIP) genes. Results In the absence of Spiroplasma infection, we found evidence of Drosophila immune activation by Ganaspis sp., but not by L. heterotoma , which in turn negatively influenced functions associated with male gonad development. As expected for a symbiont that kills males, we detected extensive downregulation in the Spiroplasma -infected treatments of genes known to have male-biased expression. We detected very few genes whose expression patterns appeared to be influenced by the Spiroplasma-L. heterotoma interaction, and these genes are not known to be associated with immune response. For most of these genes, parasitism by L. heterotoma (in the absence of Spiroplasma ) caused an expression change that was at least partly reversed when both L. heterotoma and Spiroplasma were present. It is unclear whether such genes are involved in the Spiroplasma -mediated mechanism that leads to wasp death and/or fly rescue. Nonetheless, the expression pattern of some of these genes, which reportedly undergo expression shifts during the larva-to-pupa transition, is suggestive of an influence of Spiroplasma on the development time of L. heterotoma -parasitized flies. One of the five RIP genes (RIP2) was consistently highly expressed independently of wasp parasitism, in two substrains of s Mel. Finally, the RNAseq data revealed evidence consistent with RIP-induced damage in the ribosomal (r)RNA of the Spiroplasma -susceptible, but not the Spiroplasma -resistant, wasp. Acknowledging the caveat that we lacked adequate power to detect the majority of DE genes with fold-changes lower than 3, we conclude that immune priming is unlikely to contribute to the Spiroplasma -mediated protection against wasps, and that the mechanism by which Ganaspis sp . resists/tolerates Spiroplasma does not involve inhibition of RIP transcription.
... The encapsulation response in Drosophila melanogaster is mediated by hemocytes (immune cells), including circulating macrophage-like cells known as plasmatocytes, as well as lamellocytes, a highly specialized infection-induced immune cell subtype [13]. Plasmatocytes are physiologically activated by parasitoid wasp infection and, following activation, they migrate and adhere to the surface of the parasitoid egg [14,15]. Immune stimulation also triggers the production of lamellocytes [16,17], which adhere to the plasmatocyte cell layer and form a melanized capsule around the egg, killing the developing parasitoid [15,18]. ...
... Because of the importance of hemocyte number for resistance, many of these parasitoid virulence mechanisms target host hemocytes. This includes venom virulence proteins that act on host hemocytes in a variety of ways including inducing hemocyte lysis [28], promoting death of hemocyte precursor cells [29,30], and inhibition of hemocyte function leading to immunodeficiency [14,18,[31][32][33][34]. Many of these venom proteins specifically target lamellocytes [17,18,28,34,35], reinforcing the vital role that this hemocyte subtype plays in the encapsulation response. ...
... Along with these active immune suppression mechanisms, parasitoids can also use passive immune evasive mechanisms to escape encapsulation [36,37]. Several passive mechanisms have been proposed including the binding of parasitoid eggs to host tissues as a form of camouflage from the immune response [14,36,38]; an increase in parasitoid egg size following infection [39,40]; and superparasitism, where a single host is multiply infected by conspecific parasitoids and has been suggested to increase parasitoid infection success [40][41][42][43]. ...
Full-text available
The interactions between Drosophila melanogaster and the parasitoid wasps that infect Drosophila species provide an important model for understanding host–parasite relationships. Following parasitoid infection, D. melanogaster larvae mount a response in which immune cells (hemocytes) form a capsule around the wasp egg, which then melanizes, leading to death of the parasitoid. Previous studies have found that host hemocyte load; the number of hemocytes available for the encapsulation response; and the production of lamellocytes, an infection induced hemocyte type, are major determinants of host resistance. Parasitoids have evolved various virulence mechanisms to overcome the immune response of the D. melanogaster host, including both active immune suppression by venom proteins and passive immune evasive mechanisms. We identified a previously undescribed parasitoid species, Asobara sp. AsDen, which utilizes an active virulence mechanism to infect D. melanogaster hosts. Asobara sp. AsDen infection inhibits host hemocyte expression of msn, a member of the JNK signaling pathway, which plays a role in lamellocyte production. Asobara sp. AsDen infection restricts the production of lamellocytes as assayed by hemocyte cell morphology and altered msn expression. Our findings suggest that Asobara sp. AsDen infection alters host signaling to suppress immunity.
... In M. sexta, plasmatocytes require Ca 2+ to facilitate spreading (47). Indeed, an endoparasitoid wasp against D. melanogaster encodes a Ca 2+ blocker mimicking SERCA to shut down Ca 2+ bursts, which results in the host immunosuppression (48). ...
Full-text available
Innate immune responses are effective for insect survival to defend against entomopathogens including a fungal pathogen, Metarhizium rileyi , that infects a lepidopteran Spodoptera exigua . In particular, the fungal virulence was attenuated by cellular immune responses, in which the conidia were phagocytosed by hemocytes (insect blood cells) and hyphal growth was inhibited by hemocyte encapsulation. However, the chemokine signal to drive hemocytes to the infection foci was little understood. The hemocyte behaviors appeared to be guided by a Ca ²⁺ signal stimulating cell aggregation to the infection foci. The induction of the Ca ²⁺ signal was significantly inhibited by the cyclooxygenase (COX) inhibitor. Under the inhibitory condition, the addition of thromboxane A 2 or B 2 (TXA 2 or TXB 2 ) among COX products was the most effective to recover the Ca ²⁺ signal and hemocyte aggregation. TXB 2 alone induced a microaggregation behavior of hemocytes under in vitro conditions. Indeed, TXB 2 titer was significantly increased in the plasma of the infected larvae. The elevated TXB 2 level was further supported by the induction of phospholipase A 2 (PLA 2 ) activity in the hemocytes and subsequent up-regulation of COX-like peroxinectins ( SePOX-F and SePOX-H ) in response to the fungal infection. Finally, the expression of a thromboxane synthase ( Se-TXAS ) gene was highly expressed in the hemocytes. RNA interference (RNAi) of Se-TXAS expression inhibited the Ca ²⁺ signal and hemocyte aggregation around fungal hyphae, which were rescued by the addition of TXB 2 . Without any ortholog to mammalian thromboxane receptors, a prostaglandin receptor was essential to mediate TXB 2 signal to elevate the Ca ²⁺ signal and mediate hemocyte aggregation behavior. Specific inhibitor assays suggest that the downstream signal after binding TXB 2 to the receptor follows the Ca ²⁺ -induced Ca ²⁺ release pathway from the endoplasmic reticulum of the hemocytes. These results suggest that hemocyte aggregation induced by the fungal infection is triggered by TXB 2 via a Ca ²⁺ signal through a PG receptor.
... Drosophila larvae and pupae are regularly infected by parasitoid wasps in nature (43)(44)(45), and their blood cells attack the wasp eggs (46,47). In this encapsulation response, plasmatocytes become activated and adhere to the surface of the parasitoid egg (48,49). Immune stimulation also triggers the production of specialized flattened immune cells known as lamellocytes (50,51), which adhere to the plasmatocyte cell layer to form a multicellular, multilayered capsule around the parasitoid egg. ...
Full-text available
In order to respond to infection, hosts must distinguish pathogens from their own tissues. This allows for the precise targeting of immune responses against pathogens and also ensures self-tolerance, the ability of the host to protect self tissues from immune damage. One way to maintain self-tolerance is to evolve a self signal and suppress any immune response directed at tissues that carry this signal. Here, we characterize the Drosophila tuSz 1 mutant strain, which mounts an aberrant immune response against its own fat body. We demonstrate that this autoimmunity is the result of two mutations: 1) a mutation in the GCS1 gene that disrupts N-glycosylation of extracellular matrix proteins covering the fat body, and 2) a mutation in the Drosophila Janus Kinase ortholog that causes precocious activation of hemocytes. Our data indicate that N-glycans attached to extracellular matrix proteins serve as a self signal and that activated hemocytes attack tissues lacking this signal. The simplicity of this invertebrate self-recognition system and the ubiquity of its constituent parts suggests it may have functional homologs across animals.
... Despite long being of interest and the topic of debate, the detailed mechanism underlying superparasitism avoidance remains largely underexplored, which limits further addressing its enigmatic ecological context. Drosophila parasitoids have been widely utilised as both experimental and theoretical models to study the mechanisms underpinning the host response to parasite infection [28][29][30][31][32][33][34] . ...
Full-text available
Intraspecific competition is a major force in mediating population dynamics, fuelling adaptation, and potentially leading to evolutionary diversification. Among the evolutionary arms races between parasites, one of the most fundamental and intriguing behavioural adaptations and counter-adaptations are superparasitism and superparasitism avoidance. However, the underlying mechanisms and ecological contexts of these phenomena remain underexplored. Here, we apply the Drosophila parasite Leptopilina boulardi as a study system and find that this solitary endoparasitic wasp provokes a host escape response for superparasitism avoidance. We combine multi-omics and in vivo functional studies to characterize a small set of RhoGAP domain-containing genes that mediate the parasite’s manipulation of host escape behaviour by inducing reactive oxygen species in the host central nervous system. We further uncover an evolutionary scenario in which neofunctionalization and specialization gave rise to the novel role of RhoGAP domain in avoiding superparasitism, with an ancestral origin prior to the divergence between Leptopilina specialist and generalist species. Our study suggests that superparasitism avoidance is adaptive for a parasite and adds to our understanding of how the molecular manipulation of host behaviour has evolved in this system. Evolutionary arms races can drive adaptations in hosts and parasites as well as among competing parasites. A combination of multi-omics and functional tests identifies a set of genes that allow a parasitic wasp to minimize intraspecific competition by inducing hosts to escape before more wasps can parasitize them.
... Caterpillar hemocytes undergo depolarization during immune stimulation [42], although the mechanism(s) and significance of this phenomenon are unknown. Drosophila melanogaster hemocyte calcium transients are required for orientation and migration, and disruption by a parasitoid venom component reduces cellular immunity [43]. While circumstances suggest that the Vinnexin-induced depolarization is important to hemocyte pathology, the mechanisms underlying it are currently unknown [21], and both mechanism and implications are under study. ...
Full-text available
Polydnaviruses are dsDNA viruses associated with endoparasitoid wasps. Delivery of the virus during parasitization of a caterpillar and subsequent virus gene expression is required for production of an amenable environment for parasitoid offspring development. Consequently, understanding of Polydnavirus gene function provides insight into mechanisms of host susceptibility and parasitoid wasp host range. Polydnavirus genes predominantly are arranged in multimember gene families, one of which is the vinnexins, which are virus homologues of insect gap junction genes, the innexins. Previous studies of Campoletis sonorensis Ichnovirus Vinnexins using various heterologous systems have suggested the four encoded members may provide different functionality in the infected caterpillar host. Here, we expressed two of the members, vnxG and vnxQ2, using recombinant baculoviruses in susceptible host, the caterpillar Heliothis virescens. Following intrahemocoelic injections, we observed that >90% of hemocytes (blood cells) were infected, producing recombinant protein. Larvae infected with a vinnexin-recombinant baculovirus exhibited significantly reduced molting rates relative to larvae infected with a control recombinant baculovirus and mock-infected larvae. Similarly, larvae infected with vinnexin-recombinant baculoviruses were less likely to survive relative to controls and showed reduced ability to encapsulate chromatography beads in an immune assay. In most assays, the VnxG protein was associated with more severe pathology than VnxQ2. Our findings support a role for Vinnexins in CsIV and more broadly Ichnovirus pathology in infected lepidopteran hosts, particularly in disrupting multicellular developmental and immune physiology.
... Host-parasite evolutionary interactions are predicted to be particularly intense for insect parasitoids, because the successful development of a parasitoid depends on the death of its host. Insect hosts have evolved a suite of molecular mechanisms for resisting parasitoids [9], often involving the innate immune system [10,11], and natural selection is expected to shape these mechanisms in response to pressure from parasites. However, little is known about the molecular basis of variation in the mechanisms that insects use to recognize and resist parasitoids. ...
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Evolutionary interactions between parasitoid wasps and insect hosts have been well studied at the organismal level, but little is known about the molecular mechanisms that insects use to resist wasp parasitism. Here we study the interaction between a braconid wasp ( Aphidius ervi ) and its pea aphid host ( Acyrthosiphon pisum ). We first identify variation in resistance to wasp parasitism that can be attributed to aphid genotype. We then use transcriptome sequencing to identify genes in the aphid genome that are differentially expressed at an early stage of parasitism, and we compare these patterns in highly resistant and susceptible aphid host lines. We find that resistant genotypes are upregulating genes involved in carbohydrate metabolism and several key innate immune system genes in response to parasitism, but that this response seems to be weaker in susceptible aphid genotypes. Together, our results provide a first look into the complex molecular mechanisms that underlie aphid resistance to wasp parasitism and contribute to a broader understanding of how resistance mechanisms evolve in natural populations.
In the lengthy co-evolution between insects and their animal or plant hosts, insects have evolved a wide range of salivary strategies to help evade host defenses. Although there is a very large literature on saliva of herbivorous and hematophagous insects, little attention has been focused on the saliva of parasitoid wasps. Some parasitoid species are natural enemies that effectively regulate insect population sizes in nature that they are applied for biological control of agricultural pests. Here, we demonstrate the influence of the endoparasitoid, Pteromalus puparum, larval saliva on the cellular and humoral immunity of its host. Larval saliva increases mortality of hemocytes, and inhibits hemocyte spreading, a specific cellular immune action. We report that high saliva concentrations inhibit host cellular encapsulation of foreign invaders. The larval saliva also inhibits melanization in host hemolymph. The saliva inhibits the growth of some bacterial species, Bacillus subtilis, Staphylococcus aureus and Pseudomonas aeruginosa in vitro. This may promote larvae fitness by protecting them from infections. Insight into such functions of parasitic wasp saliva provides a new insight into host-parasitoid relationships and possibly leads to new agricultural pest management technologies.
Both hosts and parasitoids evolved a diverse array of traits and strategies for their antagonistic interactions, affecting their chances of encounter, attack and survival after parasitoid attack. This review summarizes the recent progress that has been made in elucidating the molecular mechanisms of these adaptations and counter-adaptations in various Drosophila host-parasitoid interactions. For the hosts, it focuses on the neurobiological and genetic control of strategies in Drosophila adults and larvae of avoidance or escape behaviours upon sensing the parasitoids, and the immunological defences involving diverse classes of haemocytes. For the parasitoids, it highlights their behavioural strategies in host finding, as well as the rich variety of venom components that evolved and were partially acquired through horizontal gene transfer. Recent studies revealed the mechanisms by which these venom components manipulate their parasitized hosts in exhibiting escape behaviour to avoid superparasitism, and their counter-strategies to evade or obstruct the hosts' immunological defences.
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Several facultative bacterial symbionts of insects protect their hosts against natural enemies. Spiroplasma poulsonii strain sMel, a male-killing heritable symbiont of Drosophila melanogaster, confers protection against some species of parasitic wasps. Several lines of evidence suggest that Spiroplasma-encoded ribosome inactivating proteins (RIPs) are involved in the protection mechanism, but the potential contribution of the fly-encoded functions has not been deeply explored. Here we used RNA-seq to evaluate the response of D. melanogaster to infection by Spiroplasma and parasitism by the Spiroplasma-susceptible wasp Leptopilina heterotoma, and the Spiroplasma-resistant wasp Ganaspis hookeri. In the absence of Spiroplasma infection, we found evidence of Drosophila immune activation by G. hookeri, but not by L. heterotoma, which in turn negatively influenced functions associated with male gonad development. As expected for a symbiont that kills males, we detected extensive downregulation in the Spiroplasma-infected treatments of genes known to have male-biased expression. We detected very few genes whose expression was influenced by the Spiroplasma-L. heterotoma interaction, and they do not appear to be related to immune response. For most of them, parasitism by L. heterotoma (in the absence of Spiroplasma) caused an expression change that was at least partly reversed when Spiroplasma was also present. It is unclear whether such genes are involved in the Spiroplasma-mediated mechanism that leads to wasp death or fly rescue. Nonetheless, the expression pattern of some of these genes, which reportedly undergo expression shifts during the larva-to-pupa transition, is suggestive of an influence of Spiroplasma on the development time of L. heterotoma-parasitized flies. In addition, we used the RNAseq data and quantitative (q)PCR to evaluate the transcript levels of the Spiroplasma-encoded RIP genes. One of the five RIP genes (RIP2) was consistently highly expressed independently of wasp parasitism, in two substrains of sMel. Finally, the RNAseq data revealed evidence consistent with RIP-induced damage in the ribosomal (r)RNA of the Spiroplasma-susceptible, but not the Spiroplasma-resistant, wasp. We conclude that immune priming is unlikely to contribute to the Spiroplasma-mediated protection against wasps, and that the mechanism by which G. hookeri resists/tolerates Spiroplasma does not involve inhibition of RIP transcription.
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In larvae of Drosophila paramelanica, eggs and larvae of the endoparasitic wasp Leptopilina heterotoma succumb to an effective host reaction that does not involve blood cell–mediated melanotic encapsulation, a response that characterizes cellular immunity in various species of Drosophila and in many insects and other arthropods. A significant increase occurs, however, in the number of lamellocytes, a type of blood cell that functions in encapsulation reactions. The appearance of activated lamellocytes in D. paramelanica is viewed as an early response to infection, one most likely initiated by non–self-recognition processes that similarly function in other wasp-infected Drosophila. However, ensuing cytotoxic responses, about which little is presently known, are not accompanied by melanotic encapsulation in D. paramelanica. Concurrent analyses of the cell-signaling molecule nitric oxide (.NO) revealed significant alterations in the levels of this free radical during the early stages of infection, most notably a dramatic increase immediately upon infection, and precipitous decreases occurring at times when parasites were killed. Injections of a specific inhibitor of nitric oxide synthase (NOS) into the host’s body cavity prior to infection significantly increased parasite survival. These observations suggest some involvement of .NO in the host immune response, either in ecruiting hemocytes to sites of infection or as a component of the insect’s cytotoxic arsenal, given the capacity of the radical to generate toxic molecules through interactions with various intermediates of oxygen and nitrogen.
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Insects respond to microbial infection by the rapid and transient expression of several genes encoding potent antimicrobial peptides. Herein we demonstrate that this antimicrobial response of Drosophila is not aspecific but can discriminate between various classes of microorganisms. We first observe that the genes encoding antibacterial and antifungal peptides are differentially expressed after injection of distinct microorganisms. More strikingly, Drosophila that are naturally infected by entomopathogenic fungi exhibit an adapted response by producing only peptides with antifungal activities. This response is mediated through the selective activation of the Toll pathway.
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The fruit fly Drosophila melanogaster and its endoparasitoid wasps are a developing model system for interactions between host immune responses and parasite virulence mechanisms. In this system, wasps use diverse venom cocktails to suppress the conserved fly cellular encapsulation response. Although numerous genetic tools allow detailed characterization of fly immune genes, lack of wasp genomic information has hindered characterization of the parasite side of the interaction. Here, we use high-throughput nucleic acid and amino acid sequencing methods to describe the venoms of two related Drosophila endoparasitoids with distinct infection strategies, Leptopilina boulardi and L. heterotoma. Using RNA-seq, we assembled and quantified libraries of transcript sequences from female wasp abdomens. Next, we used mass spectrometry to sequence peptides derived from dissected venom gland lumens. We then mapped the peptide spectral data against the abdomen transcriptomes to identify a set of putative venom genes for each wasp species. Our approach captured the three venom genes previously characterized in L. boulardi by traditional cDNA cloning methods as well as numerous new venom genes that were subsequently validated by a combination of RT-PCR, blast comparisons, and secretion signal sequence search. Overall, 129 proteins were found to comprise L. boulardi venom and 176 proteins were found to comprise L. heterotoma venom. We found significant overlap in L. boulardi and L. heterotoma venom composition but also distinct differences that may underlie their unique infection strategies. Our joint transcriptomic-proteomic approach for endoparasitoid wasp venoms is generally applicable to identification of functional protein subsets from any non-genome sequenced organism.
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Although Drosophila possesses potent immune responses, little is known about the microbial pathogens that infect Drosophila. We have identified members of the bacterial genus Erwinia that induce the systemic expression of genes encoding antimicrobial peptides in Drosophila larvae after ingestion. These Erwinia strains are phytopathogens and use flies as vectors; our data suggest that these strains have also evolved mechanisms for exploiting their insect vectors as hosts. Erwinia infections induce an antimicrobial response in Drosophila larvae with a preferential expression of antibacterial versus antifungal peptide-encoding genes. Antibacterial peptide gene expression after Erwinia infection is reduced in two Drosophila mutants that have reduced numbers of hemocytes, suggesting that blood cells play a role in regulating Drosophila antimicrobial responses and also illustrating that this Drosophila–Erwinia interaction provides a powerful model for dissecting host–pathogen relationships.
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In nature, larvae of the fruitfly Drosophila melanogaster are commonly infected by parasitoid wasps, and so have evolved a robust immune response to counter wasp infection. In this response, fly immune cells form a multilayered capsule surrounding the wasp egg, leading to death of the parasite. Many of the molecular mechanisms underlying this encapsulation response are conserved with human immune responses. Our findings suggest that protein N-glycosylation, a common protein post-translational modification of human immune proteins, may be one such conserved mechanism. We found that membrane proteins on Drosophila immune cells are N-glycosylated in a temporally specific manner following wasp infection. Furthermore we have identified mutations in eight genes encoding enzymes of the N-glycosylation pathway that decrease fly resistance to wasp infection. More specifically, loss of protein N-glycosylation in immune cells following wasp infection led to the formation of defective capsules, which disintegrated over time and were thereby unsuccessful at preventing wasp development. Interestingly, we also found that one species of Drosophila parasitoid wasp, Leptopilina victoriae, targets protein N-glycosylation as part of its virulence mechanism, and that overexpression of an N-glycosylation enzyme could confer resistance against this wasp species to otherwise susceptible flies. Taken together, these findings demonstrate that protein N-glycosylation is a key player in Drosophila cellular encapsulation and suggest that this response may provide a novel model to study conserved roles of protein glycosylation in immunity.
Insects counteract infection by a variety of reactions, partly humoral but principally cellular. This monograph considers their cellular reactions, especially the phagocytosis of micro-organisms and the encapsulation of larger parasites, from two main points of view: parasitological and cytologica. The first aspect involves description of the reactions and of their effects on parasites. This part of the subject is basic to the biological control of insect pests, because a better understanding of cellular defence reactions could lead to improved methods of using insect parasites to human advantage. The second aspect involves analysis of the stimuli that evoke cellular reactions. This part of the monograph attempts to relate the defensive activities of insect blood cells to general problems of cytology, such as the recognition of foreign bodies, the aggregation of cells and their adhesion to foreign surfaces and their extreme flattening on each other as they form capsules. Two final chapters discuss the efficiency and specificity of insect defence mechanisms and compare them with the immunity reactions of vertebrates.
A sex-linked, temperature-sensitive melanotic tumor mutation inDrosophila melanogaster, tu (1) Sz ts, was mapped at 34.3and localized to bands 10A10-11 of the polytene chromosomes. At 26Ctu-Sz ts larvae develop melanotic tumors whereas 18C is non-permissive for tumor formation. Tumorigenesis at 26C involves the encapsulation of abnormal caudal fat body regions by precociously differentiated hemocytes. Low temperature blocks the development of the abnormal adipose cells and the overlying aberrant tissue surfaces but does not inhibit precocious differentiation of the hemocytes to the lamellocytic form. This phenotypic difference at the two temperatures indicates that lamellocyte encapsulation to form melanotic tumors is directed against abnormal tissue surfaces. On the basis of these observations and an earlier study (Rizki and Rizki 1979) we propose that hereditary melanotic tumors inD. melanogaster are a calss of autoimmune disorders in which affected tissue surfaces arouse the body''s cellmediated defense response.