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Parasitoid wasp virulence: A window into fly immunity


Abstract and Figures

In nature, larvae of the fruit fly Drosophila melanogaster are commonly infected by parasitoid wasps. Following infection, flies mount an immune response termed cellular encapsulation in which fly immune cells form a multilayered capsule that covers and kills the wasp egg. Parasitoids have thus evolved virulence factors to suppress cellular encapsulation. To uncover the molecular mechanisms underlying the anti-wasp response, we and others have begun identifying and functionally characterizing these virulence factors. Our recent work on the Drosophila parasitoid Ganaspis sp1 has demonstrated that a virulence factor encoding a SERCA-type calcium pump plays an important role in Ganaspis sp1 virulence. The SERCA venom antagonizes fly immune cell calcium signaling and thereby prevents the activation of the encapsulation response. In this way, the study of wasp virulence factors has revealed a novel aspect of fly immunity, namely a role for calcium signaling in fly immune cell activation, which is conserved with human immunity, again illustrating the marked conservation between fly and mammalian immune responses. Our findings demonstrate that the cellular encapsulation response can serve as a model of immune cell function and can also provide valuable insight into basic cell biological processes.
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Fly 7:4, 242–248; October/November/December 2013; © 2013 Landes Bioscience
In nature, larvae of the fruit fly
Drosophila melanogaster are
commonly infected by parasitoid
wasps. Following infection, f lies mount
an immune response termed cellular
encapsulation in which fly immune cells
form a multilayered capsule that covers
and kills the wasp egg. Parasitoids
have thus evolved virulence factors to
suppress cellular encapsulation. To
uncover the molecular mechanisms
underlying the antiwasp response, we
and others have begun identifying
and functionally characterizing these
virulence factors. Our recent work
on the Drosophila parasitoid Ganaspis
sp.1 has demonstrated that a virulence
factor encoding a SERCA-type calcium
pump plays an important role in
Ganaspis sp.1 virulence. This venom
SERCA antagonizes fly immune cell
calcium signaling and thereby prevents
the activation of the encapsulation
response. In this way, the study of wasp
virulence factors has revealed a novel
aspect of f ly immunity, namely a role
for calcium signaling in f ly immune
cell activation, which is conserved with
human immunity, again illustrating the
marked conservation between fly and
mammalian immune responses. Our
findings demonstrate that the cellular
encapsulation response can serve as a
model of immune cell function and can
also provide valuable insight into basic
cell biological processes.
Drosophila melanogaster and
Drosophila Parasitoid Wasps: A
Model Host-Parasite Interaction
The fruit fly Drosophila melanogaster
has proven to be an excellent system for
identifying highly conserved immune
response pathways in both mammals and
other insects, including pollinators and
vectors of human disease.1-4 Fly immune
responses to microbial infection have
been especially well described5,6 and
are characterized by the production of
antimicrobial peptides downstream of
the canonical Toll and Imd pathways,7-9
and by the phagocytosis of microbes by
circulating immune cells.10,11 However, in
addition to microbial infection, flies can
be infected by macroparasites, though
the immune responses mounted against
parasite infection are only beginning
to be understood.12 Among the most
common macroparasites of flies are
Hymenopteran parasitoid wasps, with
infection rates reaching up to 80% in
natural fly populations.13 Because the
study of natural host-parasite interactions
can be particularly useful for discovering
specialized immune mechanisms,14 we
have chosen to study the interaction
between fruit flies and their parasitoid
wasps in order to further characterize the
antiparasite response.
Upon infection, female larval
endoparasitoid wasps inject an egg
directly into the hemocoel of a developing
fly larva. The fly’s immune cells then
recognize the egg as foreign and mount a
cellular immune response directed against
the invading egg (summarized in Fig. 1).15
Parasitoid wasp virulence
A window into fly immunity
Nathan T Mortimer
School of Life Sciences; Gibbet Hill Campus ; University of Warwick; Coventry, UK
Keywords: Drosophila, Ganaspis,
parasitoid, immunity, SERCA,
calcium, encapsulation
Correspondence to: Nathan T Mortimer;
Submitted: 08/15/2013; Revised:
09 /0 6/2 013; A ccept ed: 09/13 /2013
Extra View to:Mortimer NT, Goecks J, Kacsoh
BZ, Mobley JA, Bowersock GJ, Taylor J, Schlenke
TA. Parasitoid wasp venom SERCA regulates
Drosophila calcium levels and inhibits cel-
lular immunity. Proc Natl Acad Sci U S A 2013;
PMID:23690612; 110:9427-32; http://dx.doi.
org /10 .1073/pn as .1222351110
243 Fly Volume 7 Issue 4
In this response, circulating immune cells
(known as plasmatocytes) are activated
and then migrate toward and adhere
to the wasp egg.16-18 The plasmatocytes
spread across the surface of the egg and
form stable intercellular adhesions via the
development of septate junctions, resulting
in an uninterrupted plasmatocyte layer
surrounding the wasp egg.16,17,19,20 While
the plasmatocyte layer is forming, wasp
infection also induces the differentiation
of specialized immune cells called
lamellocytes.21-23 These lamellocytes are
large f lattened cells that migrate toward
the egg and adhere to the plasmatocyte
layer.16,17 The capsule then melanizes and
the lamellocyte layers undergo a protein
glycosylation-dependent process known
as consolidation.17, 2 4 During consolidation
the lamellocytes form novel cell-cell
adhesions to produce a single continuous,
dissociation resistant layer,17 leading to
death of the wa sp egg. This c ellular im mune
response clearly provides a good model for
the functional study of basic cell biological
processes including cell differentiation,
cell migration, cell-cell adhesion, signal
transduction, and the regulation and
function of post-translational protein
modif ications.16-20,25,26
Not surprisingly, parasitoid wasps
have evolved virulence mechanisms to
overcome the fly cellular encapsulation
response. Wasp virulence factors are
expressed in the venom gland, secreted
into the venom, and are co-injected with
the egg during infection. If the virulence
factors are sufficient to prevent the egg
from being encapsulated, it will hatch
into a larva that will consume the fly
host’s tissues before eventually eclosing as
an adult wasp from the fly’s pupal case.
The interactions between wasp virulence
factors and f ly immune defenses therefore
determine the outcome of an infection.
Virulence Activity of the
Parasitoid Wasp Ganaspis sp.1
There are several distinct aspects to
virulence in host-parasite interactions.
Along with anti-immune factors,
parasites also use factors that modulate
host physiology and behavior to ensure
successful parasitization.27, 28 For our
studies, we have chosen to specifically
focus on the anti-immune mechanisms
employed by the larval endoparasitoid
wasp Ganaspis sp.1 to overcome the fly
cellular encapsulation response.18
We found that Ganaspis sp.1 was highly
virulent and successfully parasitized nearly
100% of larvae from several wild type
strains of D. melanogaster.18 Additionally,
Ganaspis sp.1 had a high parasitization
success rate against several species within
the melanogaster species subgroup, and
against more distantly related f lies across
multiple species groups.18 This suggests
that Ganaspis sp.1 can be classified as
a generalist (broadly infects f ly species
of the genus Drosophila) rather than
a specialist (infects a narrow range of
Drosophila species) parasitoid wasp.13,18, 29
Interestingly, one of the few Ganaspis
sp.1 resistant species is D. paramelanica,
a fly species that has previously been
demonstrated to use a non-cellular
encapsulation mediated antiwasp
response.30 This finding suggests that
Ganaspis sp.1 virulence may be targeted to
a cellular encapsulation-specific aspect of
fly immunity.
Parasitoid virulence activities are
generally characterized as being either
immune suppressive, in which the wasp’s
virulence factors globally inhibit fly
immunity, or immune evasive, in which
the wasp’s egg is rendered virtually
invisible to the fly’s immune response,
without inhibiting other immune
responses.31-33 We found that within 12 h
following infection, Ganaspis sp.1 eggs
begin to adhere to host tissues, namely
the fat body and gut, and that during
the next 12 h (12–24 h post infection)
Ganaspis sp.1 eggs were completely
covered by host tissue and likely hidden
from immune cells circulating in the
hemolymph.18 This is similar to the
observed immune evasive virulence
strategy of the Drosophila parasitoid
species Asobara tabida.34 ,35 However,
whereas in A. tabida infected larvae, the
egg-host tissue adhesion occurs prior to
the onset of the cellular encapsulation
response,36 in Ganaspis sp.1 infected
larvae, egg-host tissue adhesion begins
after the plasmatocyte layer should
begin to form.18 We never observed
plasmatocytes binding to Ganaspis sp.1
eggs prior to tissue adhesion, suggesting
that Ganaspis sp.1 venom must be
suppressing the encapsulation response.18
Figure1. Fly immune respons e to parasitoid wasp infection . Progression of the cellular enca psulation response following w asp infection, with the majo r
milestones and approximate times (post-infection) of completion indicated. Fly 244
These findings demonstrate that there
is not always a clear distinction between
immune suppressive and evasive strategies,
so we have proposed to define wasp
virulence using a functional assay. In this
method, we used tu (1) Sz mutant larvae,
which mount a self-directed cellular
encapsulation response that mimics the
process of wa sp egg encapsulation.37 tu(1) Sz
mutant larvae were infected by parasitoid
wasps, and the activity of a wasp’s venom
was quantified by measuring the degree to
which wasp infection could suppress this
self encapsu lation phenotype. We predicted
that a wasp w ith immune suppressive venom
would efficiently suppress the tu(1) Sz
phenotype, while an immune evasive
wasp would escape encapsulation without
altering the degree of self-encapsulation.
We found that Ganaspis sp.1 significantly
suppressed the development of the tu(1)Sz
phenotype, suggesting that its venom has
immune suppressive properties.18
Mechanisms of
Parasitoid Virulence
In nature, f ly larvae are infected by a
wide variety of parasitoid wasp species,13
which have an equally diverse array of
virulence activities. Interestingly, while
several parasitoid wasp species display
immune suppressive properties, distinct
virulence strategies have evolved among
these wasps (summarized in Fig. 2).
These strategies include inhibiting
immune cell activity, either by altering
lamellocyte morphology, as seen in larvae
infected by Leptopilina boulardi38,39 and L.
heterotoma,29,40,41 or by altering cell surface
properties, as described in larvae infected
by L. victoriae,42 whose venom acts by
inhibiting the glycosylation of lamellocyte
surface proteins.17 The immune
suppressive parasitoids L. heterotoma, A.
citri, and A. japonica act by decreasing
the total number of immune cells, either
by causing cell death or by blocking cell
differentiation.32,40,4 3,44 The parasitoids L.
boulardi, L. heterotoma, and A. citri have
also been shown to inhibit the fly phenol
oxidase pat hway, which plays an important
role in capsule melanization.32,45,46 These
studies illustrate the diversity of virulence
activities found across parasitoid wasp
species and also demonstrate that a
single parasitoid species can employ
multiple activities to overcome the cellular
encapsulation response.
Our recent study has found that the
immune suppressive activity of Ganaspis
sp.1 venom also inhibits immune cell
activity, but does so in a novel way
(Fig. 2). We found that Ganaspis
sp.1 infected larvae have comparable
immune cell numbers to larvae infected
by an avirulent wasp.18 This suggests
that immune cell differentiation is not
inhibited by Ganaspis sp.1 venom, nor did
we observe evidence of immune cell death
Figure2. Virulence strategies of immune suppressive parasitoid wasps. Immune suppressive wasps target a wide range of stages of the cellular encap-
sulation respons e, and a single parasitoid wasp spec ies can target multiple stag es as part of its virulence s trategy. Note: Ganaspis sp.1 is unique in target-
ing the plasmatocyte activation stage. This stage requires an increase in intracellular calcium levels, which is blocked by Ganaspis sp.1 venom and the
activity of vSERCA.
245 Fly Volume 7 Issue 4
in infected larvae.18 On the other hand, we
found that plasmatocytes in Ganaspis sp.1
infected larvae failed to migrate toward
or adhere to the wasp egg.18 These cells
have an entirely normal morphology,
suggesting that Ganaspis sp.1 venom may
suppress the encapsulation response by
inhibiting the activation of plasmatocytes.
Activation of an immune cell refers to the
signal transduction events that take place
within the cell that promotes its function
as part of the host defense response. Our
observation of Ganaspis sp.1 virulence
activity suggests that Ganaspis sp.1 venom
may therefore act to block intracellular
signaling in fly plasmatocytes.
Exploiting Parasitoid
Virulence Activities to Study
Host Immune Responses
The study of parasite virulence
mechanisms also provides an opportunity
to characterize the immune defenses
of their natural hosts.14 In particular,
parasitoid wasp virulence factors have
evolved to target specific aspects of host
immune defenses,33 and by identifying
these factors and their activities, we
can gain insight into cellular immune
mechanisms. Accordingly, several labs
have attempted to identify venom proteins
from Drosophila parasitoid wasps using
both single gene and high-throughput
sequencing methods.45 -51 Most notable
are recent attempts at understanding
Drosophila parasitoid wasp virulence
using approaches that combine high-
throughput transcriptomics with venom
proteomics.18,50,51 These studies have
identified a large number of venom
proteins from 3 Drosophila parasitoid
species (Ganaspis sp.1, L. boulardi, and L.
heterotoma) and have revealed that these
venoms contain unique putative virulence
factors, in keeping with their distinct
virulence mechanisms.
To try to uncover the molecular basis
for Ganaspis sp.1 virulence, we used RNA-
Seq together with proteomic analysis
of purified venom to identify all of the
putative Ganaspis sp.1 virulence factors
found in the venom (for additional
methodological details please see,50 full
transcriptome sequencing results are
available from GenBank, Accession #
GAIW00000000). Using this approach,
we identified 166 Ganaspis sp.1 venom
proteins.18 To narrow down this list
of candidates, we decided to begin by
investigating the most abundant venom
proteins. We supplemented our proteomic
data with a non-normalized subtractive
suppressive hybridization method to
reveal abundant venom gland specific
transcripts. Both of these approaches
identified a venom protein with high
homology to a sarco/endoplasmic
reticulum calcium ATPase (SERCA)-
type calcium pump as among the most
abundant venom proteins.18
Ganaspis sp.1 Venom
SERCA Antagonizes Fly
Intracellular Calcium Levels
SERCA calcium pumps are highly
conserved in all metazoans and function
to antagonize cellular calcium signaling by
the active transport of calcium ions from
the cytoplasm into the sarco/endoplasmic
reticulum stores. Considering this
conserved function, we hypothesized that
Ganaspis sp.1 venom SERCA (vSERCA)
may act to regulate calcium levels in f ly
immune cells. To test this hypothesis, we
used the genetically encoded fluorescent
calcium sensor GCaMP3.52 This sensor
encodes a fusion protein that fluoresces
in the presence of calcium, and thus,
fluorescence intensity can be used
as a measure of intracellular calcium
To assay the effect of Ganaspis sp.1
venom on fly intracellular calcium
levels, we isolated GCaMP3 expressing
plasmatocytes from f ly larvae. These
plasmatocytes were then incubated with
purif ied Ganaspis sp.1 venom or PBS
control, and we measured the change in
fluorescence intensity over time. Using
this ex vivo assay, we found that while
there was no effect of PBS control on
GCaMP3 fluorescence, the f luorescence
intensity of plasmatocytes incubated
with Ganaspis sp.1 venom decreased
significantly over the course of the 45
min incubation period.18 These findings
demonstrate that Ganaspis sp.1 venom has
calcium regulatory activity.
To test the role of vSERCA in the
calcium modulatory activity of Ganaspis
sp.1 venom, we pretreated Ganaspis sp.1
venom (and PBS control) with th apsigargin
(TG), a highly specif ic and irreversible
inhibitor of SERCA,53 and repeated the
ex vivo GCaMP3 fluorescence assay.
Following pretreatment, the samples were
dialyzed to remove unbound TG and
prevent free TG from directly effecting
plasmatocyte calcium levels. Accordingly,
the TG-treated PBS control sample had
no effect on GCaMP3 f luorescence.
However, TG pretreatment abolished the
calcium modulatory activity of Ganaspis
sp.1 venom, demonstrating that vSERCA
is required for this function.18
Calcium Signaling Plays a
Conserved Role in Immune
Cell Activation in the Fly
Antiwasp Immune Response
Based on the ability of Ganaspis
sp.1 venom to regulate fly intracellular
calcium levels, we hypothesized that
calcium signaling may play an important
role in the cellular encapsulation
response. We predicted that if calcium
signaling is important for the antiwasp
response, intracellular calcium levels
would increase following parasitoid
infection. To test this prediction, we used
the parasitoid L. clavipes, a specialist of
fungivorous Drosophila species,54 which is
efficiently encapsulated by wild-type D.
melanogaster.17 Thus, L. clavipes is a useful
tool to investigate the D. melanogaster
antiwasp immune response. We infected
fly larvae expressing GCaMP3 in a
variety of immune cell types with
L. clavipes and assayed GCaMP3
fluorescence. We observed a significant
increase in GCaMP3 fluorescence
specifically in plasmatocytes within
6 h following infection.18 Interestingly,
fluorescence intensities return to control
levels within the next 18 h, although the
encapsulation response remains active
throughout this period. Furthermore,
this increase in intracellular calcium
levels is specific to plasmatocytes, and
was not seen in other tested immune
tissues, including lamellocytes.18 These
findings demonstrate that calcium levels,
and likely calcium signaling, specifically
increase in plasmatocytes at the time of
the onset of the encapsulation response, Fly 246
and suggest that calcium signaling may
be required for plasmatocyte activation.
To test whether this increase in
plasmatocyte calcium levels is required
for the cellular encapsulation response,
we used L. clavipes to infect flies in
which calcium levels were genetically
lowered using 2 distinct approaches.
In the first approach, we ectopically
expressed Parvalbumin (PV), a vertebrate-
specific calcium binding protein that
acts as a calcium buffer when expressed
in D. melanogaster cells.55 We found
that expression of PV specifically in
immune cells significantly decreased
the encapsulation rate.18 For the second
approach, we expressed RNAi knockdown
constructs against either the Itp-r83A or
Rya-r44F calcium release channels in
fly immune cells. These channels act to
release calcium from sarco/endoplasmic
reticulum stores to raise intracellular
calcium levels in response to specific
stimuli.56 Larvae with either of these
channel s knocked down were infecte d with
L. clavipes, and we found that Rya-r44F
RNAi specifically lead to a decrease in fly
encapsulation ability.18 Taken together,
these findings suggest that increased
calcium levels in plasmatocytes following
infection are required for the cellular
encapsulation response. Interestingly,
calcium signaling is also required
for the activation of B and T cells in
human acquired immune responses,57
and plays a role in plasmatocyte-
mediated phagocytosis of microbes in
D. melanogaster.58 This again illustrates
the marked conservation between f ly
and mammalian immune responses, and
suggests that the D. melanogaster cellular
encapsulation response may provide a
good model for the study of immune cell
Regulation of Plasmatocyte
Calcium Levels is Important
for Ganaspis sp.1 Virulence
To test the properties of Ganaspis sp.1
venom in vivo, we allowed Ganaspis sp.1
wasps to infect D. melanogaster larvae
expressing GCaMP3, and measured
fluorescence intensity in immune cells at
various time points following infection.
We found that GCaMP3 f luorescence
following Ganaspis sp.1 infection mirrored
our findings of GCaMP3 f luorescence
in L. clavipes infected larvae. Namely,
GCaMP3 fluorescence was decreased
specifically in plasmatocytes, and only
within the first 6 h following infection.18
This suggests that the vSERCA activity
observed in ex vivo experiments also
functions to regulate plasmatocyte
calcium levels in vivo.
Since the timing of this Ganaspis
sp.1 venom-induced calcium decrease is
coincident with that of the plasmatocyte
calcium increase required for the cellular
encapsulation response, we hypothesized
that this function is important for
Ganaspis sp.1 virulence. To test this, we
infected Ca-P60AKum170 and olf186-FEY 0146 7
mutant larvae with Ganaspis sp.1. Both
of these mutations have ectopically
increased intracellular calcium levels,59,60
and we found that they confer larvae with
resistance to Ganaspis sp.1 infection.18
This could suggest either that increased
intracellular calcium makes flies generally
more resistant to wasp infection, or that
it specifically counteracts the ability of
Ganaspis sp.1 venom to decrease calcium
levels. To distinguish between these
possibilities, we infected the mutant larvae
with a second virulent wasp, L. boulardi,
that does not have any recognizable
calcium modulatory venom proteins.50
Neither of the mutations conferred
resistance to L. boulardi infection,18
suggesting that the increased intracellular
calcium levels specifically counteract
Ganaspis sp.1 virulence. This further
suggests that suppressing the plasmatocyte
calcium increase is an important part
of the Ganaspis sp.1 virulence strategy.
These findings are consistent with the
observation that Ganaspis sp.1 venom acts
to suppress plasmatocyte function prior to
Ganaspis sp.1 egg-host tissue adhesion.
Our work has illustrated the utility
of studying parasitoid virulence factors
to uncover fly immune mechanisms.
The cellular encapsulation response is
widely conserved across insect species61
and shows a high degree of genetic
conservation with mammalian immune
cell mechanisms.17-19,2 6, 62 Our studies of
Ganaspis sp.1 virulence have uncovered a
role for calcium signaling in plasmatocyte
activation in the encapsulation response.18
This role is highly conserved, both
among other immune responses of D.
melanogaster and with immune cell
activation in human immunity.57,58 Thus,
the interaction between Ganaspis sp.1
and D. melanogaster may provide a novel
model for the study of calcium-mediated
immune cell activation.
The ability of Ganaspis sp.1 venom
to alter calcium levels in host cells raises
several interesting questions. First, how
does vSERCA so efficiently antagonize
intracellular calcium levels when D.
melanogaster plasmatocytes endogenously
express SERCA protein? This suggests
that the activities of vSERCA and
endogenous D. melanogaster SERCA are
differentially regulated in plasmatocytes.
In most metazoan species, the SERCA
locus encodes 2 distinct protein isoforms,
with the longer isoform having an
additional transmembrane domain. This
long isoform (SERCA102 0 in insects) has
a higher affinity for calcium, and thus,
a slower maximal calcium translocation
rate than the short isoform (SERCA1002
in insects).63,64 We found that vSERCA
encodes the SERCA100 2 isoform, whereas
D. melanogaster plasmatocytes specifically
express SERCA1020, and the predicted
difference in translocation kinetics
could potentially account for the activity
difference.18 Alternatively, despite the
high degree of homology between D.
melanogaster and Ganaspis sp.1 SERCA
proteins, there are several amino acid
differences, which could be evidence for
the evolution of differential regulation or
function between the proteins.
Second, how is vSERCA, a multi-
pass transmembrane protein, transferred
from Ganaspis sp.1 venom gland cells
into the venom fluid and finally into
fly plasmatocytes? Considering the
hydrophobicity of the transmembrane
domains, it is unlikely that vSERCA is
secreted as a n individual protein, but rather
may be packaged into a proteinaceous or
membrane bound structure for transport.
It has been demonstrated that other
Drosophila parasitoid wasp species package
their venom proteins into struc tures known
as virus-like particles (VLPs) for delivery
247 Fly Volume 7 Issue 4
into the host during infection.41,42,65,66 It
is not yet known whether Ganaspis sp.1
venom contains VLPs; however, we would
hypothesize that vSERCA is packaged
within some type of larger structure to be
delivered to host cells.
Third, what makes vSERCA function
plasmatocyte specific? GCaMP3
fluorescence imaging revealed that
other than in plasmatocytes, calcium
levels appear to be unchanged in cells
of Ganaspis sp.1 infected larvae.18 This
specificity is likely to be an important
property of Ganaspis sp.1 venom;
since calcium signaling is essential for
the functioning of many cell types,
deregulated vSERCA activity could lead
to premature death of the host, which, in
turn, would kill the developing parasitoid.
Perhaps the mechanism underlying
vSERCA transport to host cells allows
for plasmatocyte specif ic delivery. This
hypothesis is supported by findings from
other Drosophila parasitoid species, where
VLPs and other venom proteins have
been shown to specif ically interact with
fly immune cells.39,41,65 Gaining insight
into the processes by which parasitoids
package virulence proteins for delivery to
specific host cell types would be of broad
biological interest.
In summary, the D. melanogaster
antiwasp cellular encapsulation response
provides a good system in which to
study conserved aspects of the genetics
and cell biology of cellular immune
responses. Cellular encapsulation requires
strictly regulated cell differentiation
and migration, the formation of cell-cell
adhesions, and signaling through a variety
of conserved pathways. In particular,
our work has shown that parasitoid
virulence factor activity can be studied
to understand host immunity and gain
insight into the underlying cell biological
mechanisms, making the D. melanogaster-
Drosophila parasitoid wasp interaction an
exciting model system for future research.
Disclosure of Potential Conflicts of Interest
No potential conf licts of interest were
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... In general, parasitoid female wasps seek out fruit fly larvae and use a modified ovipositor to pierce the cuticle, and then inject an egg into host. There are a series of host-parasitoid interactions between the host immune response and the invading wasp egg such as encapsulation to and virulence defense mechanism (Mortimer, 2013). The immune response from the host exhibits defensive action to kill the egg so that the fruit fly can complete its development; the wasp egg invader, attempts to develop into a larva quickly enough to evade the immune response, consume the host from the inside-out and develop into an adult wasp. ...
... Hosts may defend themselves against parasitism through a wide variety of defense mechanisms (behavioral, physical, or physiological responses), but due to finite resources, investment in one defense mechanism may trade-off with investment in another mechanism. Multiple pioneering biological observations and cytogenetic research have been performed and reported on host-parasitoid interaction in the laboratory condition, especially on studying foraging, oviposition behavior, development, and reproduction are still considered to be of profound importance today (Mortimer, 2013;Flanders, 1950;Doutt, 1959;Lawrence, 2005;Paladino et al., 2013). ...
The oriental fruit fly, Bactrocera dorsalis, is an important agricultural pest and biological control is one of the most effective control methodologies. We conducted an investigation on the molecular response of the fruit fly to parasitism by the larval parasitoid, Diachasmimorpha longicaudata using two-dimensional gel electrophoresis and mass spectroscopy. We identified 285 differentially expressed protein spots (109 proteins) during parasitism. The molecular processes affected by parasitism varied at different time point during development. Transferrin and muscle specific protein 20 are the only two proteins differentially expressed that play a role in host immunity 24 h after parasitism. Developmental and metabolic proteins from parasitoids (transferrin and enolase) were up-regulated to ensure establishment and early development of parasitoids 48 h post parasitism. 72 h after parasitism, larval cuticle proteins, transferrin and CREG1 were overexpressed to support the survival of para-sitoids while host metabolism proteins and parasitoid regulatory proteins were down-regulated. Host development slowed down while parasitoid development went up at 96 h after parasitism. All developmental, regulatory , structural, and metabolic proteins were expressed at their optimum at 120 h post parasitism. Host development was reduced, metabolism and regulatory proteins were strongly involved in the activities. The development deteriorated further at 144 h after parasitism. Enolase and CREG1 were indicators of parasitoid survival. Hexamerin and transferrin from the parasitoid was peaked at 168-216 h after parasitism, strongly indicating that parasitoid would survive. This study represents the first report that reveals the molecular players involved in the interaction between the host and parasitoid.
... Cellular immune responses are an important aspect of innate host defense against infection and are broadly conserved from insects to mammals. The model organism Drosophila melanogaster uses the cellular encapsulation response to protect against macroparasite infection (Carton et al., 2008;Mortimer, 2013). This response shows genetic conservation with human immune responses (Howell et al., 2012), and may serve as a useful model to better understand human immune cell functions. ...
Full-text available
In nature, Drosophila melanogaster larvae are infected by parasitoid wasps and mount a cellular immune response to this infection. Several conserved signaling pathways have been implicated in coordinating this response, however our understanding of the integration and regulation of these pathways is incomplete. Members of the S1A serine protease family have been previously linked to immune functions, and our findings suggest roles for two S1A family members, CG10764 and CG4793 in the cellular immune response to parasitoid infection.
... Parasitoid wasps that infect Drosophila are a valuable model for understanding parasite behavior and have provided important ecological and molecular insights into host-parasite interactions [1][2][3]. In this system, parasitoids infect larval Drosophila, and following infection, Drosophila mount a cellular encapsulation response to overcome the invader [4]. ...
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.
... Little is known, however, on Ganaspis biology, apart from the fact that at least some Ganaspis species parasitize Drosophila larvae (such as G. brasiliensis and the uncharacterized G. sp used in this study). Some Ganaspsis species have been investigated in terms of immune interaction with the Drosophila (Ferrarese et al. 2009;Mortimer et al. 2013), revealing important differences with Leptopilina species (Mortimer 2013). ...
Full-text available
Some species of parasitic wasps have domesticated viral machineries to deliver immunosuppressive factors to their hosts. Up to now, all described cases fall into the Ichneumonoidea superfamily, which only represents around 10% of hymenoptera diversity, raising the question of whether such domestication occurred outside this clade. Furthermore, the biology of the ancestral donor viruses is completely unknown. Since the 1980’s, we know that Drosophila parasitoids belonging to the Leptopilina genus, which diverged from the Ichneumonoidea superfamily 225My ago, do produce immuno-suppressive virus-like structure in their reproductive apparatus. However, the viral origin of these structures has been the subject of debate. In this paper, we provide genomic and experimental evidence that those structures do derive from an ancestral virus endogenization event. Interestingly, its close relatives induce a behaviour manipulation in present-day wasps. Thus, we conclude that virus domestication is more prevalent than previously thought and that behaviour manipulation may have been instrumental in the birth of such associations.
... Little is known, however, on Ganaspis biology, apart from the fact that at least some Ganaspis species parasitize Drosophila larvae (such as G. brasiliensis and the uncharacterized G. sp used in this study). Some Ganaspsis species have been investigated in terms of immune interaction with the Drosophila (Ferrarese et al. 2009;Mortimer et al. 2013), revealing important differences with Leptopilina species (Mortimer 2013). ...
... GTPase activating protein (LbGAP), which is the major venom component of L. boulardi, consists of RhoGAP domain derived from cellular RhoGAP, and is known to activate Rho-like GTPases (Colinet et al., 2007a(Colinet et al., , 2007b. The cis-regulated variation in expression of LbGAP was found to be responsible for the difference in virulence (the ability to supress the host immune system) of the two strains (Colinet et al., 2010;Mortimer, 2013). Moreover, several RhoGAP domain containing proteins were also identified in the venoms of L. heterotoma and L. boulardi, which were speculated to have originated via extensive gene duplication, followed by subcellular relocalisation and the acquisition of venom-gland specific signal peptides (Colinet et al., 2013;Poirié et al., 2014). ...
Comprising of over a million described species of highly diverse invertebrates, Arthropoda is amongst the most successful animal lineages to have colonized aerial, terrestrial, and aquatic domains. Venom, one of the many fascinating traits to have evolved in various members of this phylum, has underpinned their adaptation to diverse habitats. Over millions of years of evolution, arthropods have evolved ingenious ways of delivering venom in their targets for self-defence and predation. The morphological diversity of venom delivery apparatus in arthropods is astounding, and includes extensively modified pedipalps, tail (telson), mouth parts (hypostome), fangs, appendages (maxillulae), proboscis, ovipositor (stinger), and hair (urticating bristles). Recent investigations have also unravelled an astonishing venom biocomplexity with molecular scaffolds being recruited from a multitude of protein families. Venoms are a remarkable bioresource for discovering lead compounds in targeted therapeutics. Several components with prospective applications in the development of advanced lifesaving drugs and environment friendly bio-insecticides have been discovered from arthropod venoms. Despite these fascinating features, the composition, bioactivity, and molecular evolution of venom in several arthropod lineages remains largely understudied. This review highlights the prevalence of venom, its mode of toxic action, and the evolutionary dynamics of venom in Arthropoda, the most speciose phylum in the animal kingdom.
... In addition to the humoral immune response to infection, the Drosophila immune system can mount a cellular response to eliminate pathogens by encapsulation. It has become increasingly apparent that the mechanisms that control this cellular response and hostpathogen recognition rely on conserved proteins that are also important for human immunity (Howell et al., 2012;Mortimer, 2013;Mortimer et al., 2012). ...
Full-text available
Innate immune responses depend on the action of multiple conserved signaling pathways. Pathways important for activation of humoral immune responses following microbial infection are well-characterized in the genetic model species Drosophila melanogaster, but our understanding of fly cellular immunity, and how parasites suppress it, is relatively fragmentary. Fly larvae mount a coordinated cellular immune response following infection by endoparasitoid wasps, characterized by the production of specialized blood cells called lamellocytes that form a tight capsule around wasp eggs in their hemolymph. The conserved JAK/Stat signaling pathway has been implicated in lamellocyte proliferation and is required for successful encapsulation of wasp eggs. Here we show that activity of Stat92E, the D. melanogaster Stat ortholog, is induced in the fat body and hemocytes following wasp infection. Virulent wasp species are able to suppress activation of a Stat92E activity reporter during infection, suggesting they target upstream activation of this pathway as part of their virulence strategies. Furthermore, two wasp species (Leptopilina guineaensis and Ganaspis xanthopoda) suppress phenotypes associated with gain-of-function mutations in hopscotch, the D. melanogaster JAK ortholog, showing that they inhibit JAK/Stat activity downstream of JAK. Our data demonstrate that endoparasitoid wasp virulence factors block JAK/Stat signaling to overcome fly immune defenses.
... parasitization (Lee et al., 2009). The composition of these particles and their effect on the host differ between species and strains (Dupas, Brehelin, Frey & Carton, 1996;Lee et al., 2009;Mortimer, 2013;Poirié, Carton & Dubuffet, 2009), which therefore might play a role in the observed intraspecific variation in D. suzukii -parasitoid outcome. ...
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The development of biological control methods for exotic invasive pest species has become more challenging during the last decade. Compared to indigenous natural enemies, species from the pest area of origin are often more efficient due to their long coevolutionary history with the pest. The import of these well‐adapted exotic species, however, has become restricted under the Nagoya Protocol on Access and Benefit Sharing, reducing the number of available biocontrol candidates. Finding new agents and ways to improve important traits for control agents (“biocontrol traits”) is therefore of crucial importance. Here, we demonstrate the potential of a surprisingly under‐rated method for improvement of biocontrol: the exploitation of intraspecific variation in biocontrol traits, for example, by selective breeding. We propose a four‐step approach to investigate the potential of this method: investigation of the amount of (a) inter‐ and (b) intraspecific variation for biocontrol traits, (c) determination of the environmental and genetic factors shaping this variation, and (d) exploitation of this variation in breeding programs. We illustrate this approach with a case study on parasitoids of Drosophila suzukii, a highly invasive pest species in Europe and North America. We review all known parasitoids of D. suzukii and find large variation among and within species in their ability to kill this fly. We then consider which genetic and environmental factors shape the interaction between D. suzukii and its parasitoids to explain this variation. Insight into the causes of variation informs us on how and to what extent candidate agents can be improved. Moreover, it aids in predicting the effectiveness of the agent upon release and provides insight into the selective forces that are limiting the adaptation of indigenous species to the new pest. We use this knowledge to give future research directions for the development of selective breeding methods for biocontrol agents.
Dipteran endoparasitoids avoid host immune response; however, antidefense components from the Dipterans are unknown. Infestation of commercial silkworm Bombyx mori Linnaeus (Lepidoptera: Bombycidae) by endoparasitoid Exorista bombycis Louis (Diptera: Tachinidae) induced immune reactions, cytotoxicity, granulation, degranulation, and augmented release of cytotoxic marker enzyme lactate dehydrogenase (LDH), and degranulation-mediator enzyme β-hexosaminidase in hemocytes. In this study, by reverse phase high-performance liquid chromatography, fractions of E. bombycis larval tissue protein with antihemocytic activity are separated. From the fraction, peptides of hemocyte aggregation inhibitor protein (HAIP) and pyridoxamine phosphate oxidase (PNPO) are identified by mass spectrometry. Interacting partners of HAIP and PNPO are retrieved that further enhance the virulence of the parasitoid. PNPO and HAIP genes showed a four- to seven fold increase in expression in the integument of the parasitoid larva. Together, the dipteran endoparasitoid E. bombycis exploit antihemocyte activity to inhibit host defense reactions in addition to defense evasion contemplated.
Hymenopteran parasitoid wasps are a diverse collection of species that infect arthropod hosts and use factors found in their venoms to manipulate host immune responses, physiology, and behaviour. Whole parasitoid venoms have been profiled using proteomic approaches, and here we present a bioinformatic characterization of the venom protein content from Ganaspis sp. 1, a parasitoid that infects flies of the genus Drosophila. We find evidence that diverse evolutionary processes including multifunctionalization, co-option, gene duplication, and horizontal gene transfer may be acting in concert to drive venom gene evolution in Ganaspis sp.1. One major role of parasitoid wasp venom is host immune evasion. We previously demonstrated that Ganaspis sp. 1 venom inhibits immune cell activation in infected Drosophila melanogaster hosts, and our current analysis has uncovered additional predicted virulence functions. Overall, this analysis represents an important step towards understanding the composition and activity of parasitoid wasp venoms.
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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 hymenopteran Asobara tabida Nees (Braconidae: Alysiinae) parasitizes the larval stages of Drosophila species. In D. melanogaster, 90% of A. tabida eggs avoid encapsulation and are found embedded within the tissues (digestive tube, fat body, tracheae, etc.) of their larval host. Electron microscopy was used to investigate if the embedment of the parasitic egg within the tissues of its host may prevent encapsulation. Study revealed that the outer layer of the A. tabida egg consists of a fibrous exochorion that exhibits adhesive properties within the tissues of D. melanogaster. Three hours after oviposition, any basement membrane surrounding the host tissues that is in contact with the parasitic egg is stuck to the exochorion. Attachment rapidly increases. Nine hours post oviposition, the A. tabida egg is almost totally surrounded by a sheath of host tissue, with only limited areas of the surface remaining uncovered and exposed to attack by the host hemocytes. It is therefore concluded that the adhesion of A. tabida eggs to the host tissues is likely to contribute to their protection from encapsulation during embryogenesis. Also, electron microscopy reveals that no virus-like particles occur in A. tabida, unlike any other braconid parasitoid studied. The absence of virus-like particles is discussed with regard to the importance of host regulation of the particles in other braconid species.
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Immune responses against opportunistic pathogens have been extensively studied in Drosophila, leading to a detailed map of the genetics behind innate immunity networks including the Toll, Imd, Jak-Stat, and JNK pathways. However, immune mechanisms of other organisms, particularly plants, have primarily been investigated using natural pathogens. It was the use of natural pathogens in plant research that revealed the plant R/Avr system, a specialized immune response derived from antagonistic coevolution between plant immune proteins and their natural pathogen's virulence proteins. Thus, we recommend that researchers begin to use natural Drosophila pathogens to identify novel immune mechanisms that may have arisen through antagonistic coevolution with common natural pathogens. In this review, we address the benefits of using natural pathogens in research, describe the known natural pathogens of Drosophila, and discuss exciting prospects for future research on select natural pathogens of Drosophila.
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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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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.
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.
Analysis of natural host-parasite relationships reveals the evolutionary forces that shape the delicate and unique specificity characteristic of such interactions. The accessory long gland-reservoir complex of the wasp Leptopilina heterotoma (Figitidae) produces venom with virus-like particles. Upon delivery, venom components delay host larval development and completely block host immune responses. The host range of this Drosophila endoparasitoid notably includes the highly-studied model organism, Drosophila melanogaster. Categorization of 827 unigenes, using similarity as an indicator of putative homology, reveals that approximately 25% are novel or classified as hypothetical proteins. Most of the remaining unigenes are related to processes involved in signaling, cell cycle, and cell physiology including detoxification, protein biogenesis, and hormone production. Analysis of L. heterotoma's predicted venom gland proteins demonstrates conservation among endo- and ectoparasitoids within the Apocrita (e.g., this wasp and the jewel wasp Nasonia vitripennis) and stinging aculeates (e.g., the honey bee and ants). Enzyme and KEGG pathway profiling predicts that kinases, esterases, and hydrolases may contribute to venom activity in this unique wasp. To our knowledge, this investigation marks the first functional genomic study for a natural parasitic wasp of Drosophila. Our findings will help explain how L. heterotoma shuts down its hosts' immunity and shed light on the molecular basis of a natural arms race between these insects.