High Hemocyte Load Is Associated with Increased
Resistance against Parasitoids in Drosophila suzukii,
a Relative of D. melanogaster
Balint Z. Kacsoh, Todd A. Schlenke*
Biology Department, Emory University, Atlanta, Georgia, United States of America
Abstract
Among the most common parasites of Drosophila in nature are parasitoid wasps, which lay their eggs in fly larvae and
pupae. D. melanogaster larvae can mount a cellular immune response against wasp eggs, but female wasps inject venom
along with their eggs to block this immune response. Genetic variation in flies for immune resistance against wasps and
genetic variation in wasps for virulence against flies largely determines the outcome of any fly-wasp interaction.
Interestingly, up to 90% of the variation in fly resistance against wasp parasitism has been linked to a very simple
mechanism: flies with increased constitutive blood cell (hemocyte) production are more resistant. However, this relationship
has not been tested for Drosophila hosts outside of the melanogaster subgroup, nor has it been tested across a diversity of
parasitoid wasp species and strains. We compared hemocyte levels in two fly species from different subgroups, D.
melanogaster and D. suzukii, and found that D. suzukii constitutively produces up to five times more hemocytes than D.
melanogaster. Using a panel of 24 parasitoid wasp strains representing fifteen species, four families, and multiple virulence
strategies, we found that D. suzukii was significantly more resistant to wasp parasitism than D. melanogaster. Thus, our data
suggest that the relationship between hemocyte production and wasp resistance is general. However, at least one
sympatric wasp species was a highly successful infector of D. suzukii, suggesting specialists can overcome the general
resistance afforded to hosts by excessive hemocyte production. Given that D. suzukii is an emerging agricultural pest,
identification of the few parasitoid wasps that successfully infect D. suzukii may have value for biocontrol.
Citation: Kacsoh BZ, Schlenke TA (2012) High Hemocyte Load Is Associated with Increased Resistance against Parasitoids in Drosophila suzukii, a Relative of D.
melanogaster. PLoS ONE 7(4): e34721. doi:10.1371/journal.pone.0034721
Editor: Robert DeSalle, American Museum of Natural History, United States of America
Received July 11, 2011; Accepted March 8, 2012; Published April 17, 2012
Copyright: ß 2012 Kacsoh, Schlenke. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by National Institutes of Health (NIH) grant AI081879 to TAS. The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: tschlen@emory.edu
Introduction
Fruitflies of the genus Drosophila are regularly attacked by
parasitoid wasps. In natural D. melanogaster populations, upwards of
50% of fly larvae are found to be infected by wasps, suggesting
they exert extremely strong selection pressures on Drosophila
populations in nature [1,2,3]. Once infected, fruitfly larvae mount
an immune response against wasp eggs, termed melanotic
encapsulation, that is thought to involve several steps [4,5]: The
response begins when circulating, constitutively produced plasmatocytes
recognize the wasp egg as foreign and signal to induce the
differentiation of larger lamellocytes from pro-hemocytes in the
lymph gland (the fly hematopoietic organ) and from other
circulating plasmatocytes (via the intermediate podocyte form)
[6,7]. These newly derived lamellocytes migrate towards, and
attach and spread around the wasp egg in a multi-layered capsule.
In the final step, the inner cells of the capsule surrounding the
wasp egg lyse and release reactive oxygen species and an
impermeable layer of melanin, resulting in death of the wasp
egg. However, parasitoid wasps can potentially evade host
immune responses by using a non-reactive coating on their eggs,
or suppress host immunity by injecting venom into hosts along
with their eggs. There is both between and within species genetic
variation in flies for resistance against wasps and among wasps for
virulence against flies [8,9,10,11,12,13,14,15,16].
In previous work, Drosophila species from the melanogaster
subgroup were found to have significantly different numbers of
constitutively produced plasmatocytes, and there was a significant
correlation (r2
= 0.90) between plasmatocyte counts and ability to
melanotically encapsulate the eggs of the immune-evasive
parasitoid wasp Asobara tabida [12]. It was also found that D.
melanogaster strains artificially selected for resistance against A. tabida
showed a significant increase in plasmatocyte numbers [17].
Furthermore, D. simulans, which makes significantly more
plasmatocytes than its sister species D. melanogaster, was significantly
more resistant against the more immune-suppressive wasp A. citri
[18]. Finally, D. melanogaster mutants producing a wide range of
hemocyte counts showed a significant correlation (r2
= 0.45)
between constitutive plasmatocyte numbers and encapsulation
ability against the wasp Leptopilina boulardi [19]. Altogether, this
work suggests that high constitutive production of hemocytes is an
effective and relatively simple mechanism by which hosts can
evolve resistance to one of their most common groups of parasites.
We were interested in whether the relationship between
Drosophila standing immune defense (hemocyte production) and
immune resistance against wasps is general across a large panel of
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diverse wasp lineages with unique infection strategies, and whether
the relationship extends beyond the melanogaster subgroup of the
genus Drosophila. Pilot data from a study aimed at characterizing
hemocyte lineages across the genus Drosophila (unpublished)
suggested D. suzukii, a member of the melanogaster group but not
the melanogaster subgroup, constitutively produces an extremely
large number of hemocytes compared to other Drosophila. Thus,
the goal of this study was to confirm whether D. suzukii
constitutively produces higher numbers of hemocytes than D.
melanogaster, and if so, to determine whether D. suzukii was also
more resistant against a large panel of parasitoid wasp species and
strains.
D. suzukii is native to east Asia but has recently gained
widespread attention due to its spread as an agricultural pest in
Europe and North America (Figure 1) [20,21,22,23]. Although
most of the ,1,500 described Drosophila species lay their eggs and
feed on decaying plant and fungal tissues, including rotting fruits
(like D. melanogaster), D. suzukii is one of a handful of species that live
on ripe fruits, using its serrated ovipositor to lay eggs in the flesh of
soft-skinned fruits (Figure 1C). Its larvae subsequently burrow
through the body of the fruit as they eat (Figure 1D), allowing
bacteria and other microorganisms access to the inside of the fruit,
which results in premature rotting. Because parasitoid wasps have
been successfully used as biocontrol agents against a wide range of
insect agricultural pests, including Coleopterans (e.g., weevils, bean
beetles), Hemipterans (e.g., scale insects, whiteflies, aphids,
leafhoppers, stinkbugs), Lepidopterans (e.g., various moth and
butterfly larvae), and Dipterans (e.g., Tephritid fruitflies, blackflies)
[24,25,26,27,28], study of D. suzukii resistance and susceptibility to
parasitoid wasps may have added applied value.
At least four families of parasitoid wasps are known to attack
Drosophila in nature [29]. These wasps use a variety of infection
strategies to defeat the fly immune response, including immune
suppressive and evasive tactics, and vary in their host ranges from
specialists of particular Drosophila species to generalist of the
genus. Members of the families Braconidae and Figitidae are larval
parasites – they lay single eggs in Drosophila larvae and, if not
killed, the hatched wasp larva begins to consume internal fly tissues
before eventually killing the fly and eclosing from the fly pupal
case. Members of the families Diapriidae and Pteromalidae are
pupal parasites - they lay single eggs inside Drosophila pupae, and
the hatched wasp larva consumes the fly pupal tissues, also
eventually killing the fly and eclosing from the fly pupal case. It is
unclear whether fly pupae can mount an immune response or
otherwise defend themselves once infected by pupal parasites.
Pupal parasites of the genus Trichopria (Family Diapriidae) lay
their eggs in the Drosophila hemocoel, like larval parasites, but
those of the genus Pachycrepoideus (Family Pteromalidae) lay
their eggs in the space between the Drosophila pupal case and the
pupa, and act as ectoparasites in the early stages of their life by
sucking fluids from the pupa externally [29]. A lack of pupal
immunity against wasps may explain in part why pupal parasitoid
wasps are thought to have more generalist host ranges than larval
parasitoid wasps [30,31].
The Drosophila-wasp system is ripe for study as a model for the
co-evolution of pathogen infection strategies and host immune
responses across lineages and communities of pathogens and hosts
[32]. We attempted to answer the following questions: Is the
melanotic encapsulation response observed in D. melanogaster
conserved in D. suzukii? Does D. suzukii have higher constitutive
hemocyte production than D. melanogaster? Is increased hemocyte
production by D. suzukii associated with stronger resistance against
a panel of parasitoid wasps with diverse life histories and infection
strategies? Do wasps make different oviposition choices depending
on host species? Do wasp phylogeny and biogeography play any
role in fly-wasp interactions? From an applied point of view, which
parasitoid wasp species show the most potential for use in D. suzukii
biocontrol in the field?
Materials and Methods
Insect Species
The D. melanogaster genome strain 14021-0231.36 was acquired
from the Drosophila Species Stock Center and was grown on
standard cornmeal/yeast/molasses Drosophila medium. The two
additional D. melanogaster strains originated from single wild-caught
females collected in Atlanta, GA in the summer of 2010. The
primary D. suzukii strain tested originated from four wild-caught
females collected in Atlanta, GA in the summer of 2010, while two
additional isofemale strains were collected in Atlanta, GA in the
summer of 2011. D. suzukii were maintained on standard
Drosophila medium supplemented with (thawed) frozen raspberries,
which were found to enhance egg-laying but were otherwise
unnecessary for fly development.
A total of 24 Drosophila parasitoid wasp strains collected from
around the world were used for infection trials on D. melanogaster
and D. suzukii (Figure 2). Strains LgG500 and LgG510 were
provided by R. Allemand, strain LbG486 was provided by D.
Hultmark, strains LcNet, AjJap, ApIndo, and AcIC were provided
by J. van Alphen, strain GxUg was provided by J. Pool, and strain
AtFr was provided by B. Wertheim. All other strains were
collected by the Schlenke lab. These wasp strains represent: (1) at
least 14 species, (2) representatives of all four Hymenopteran
families known to infect Drosophila, (3) larval and pupal parasites,
and (4) a worldwide range of collection localities (Figure 2).
Morphology and cytochrome oxidase I (COI) sequences from the two
Trichopria sp.1 strains suggested they were representatives of the
same species, perhaps Trichopria drosophila (Ashmead). Furthermore,
Figure 1. Fly morphology and behavior. (A) Female D. melanogaster;
(B) female D. suzukii; (C) serrated ovipositor from female D. suzukii;
(D) tunnel excavated by D. suzukii larva through agar food plate.
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morphology and COI sequences from the two Ganaspis sp.1 strains
suggest they are representatives of a single undescribed species. All
wasp species were maintained in the lab on D. melanogaster strain
Canton S, with the exception of L. clavipes, A. tabida, Aphaereta sp.1,
and Pachycrepoideus sp.1, which were maintained on D. virilis. To
grow wasps, adult flies were allowed to lay eggs in standard
Drosophila medium for several days before they were replaced by
adult wasps, which then attacked the developing fly larvae or
pupae. Wasp vials were supplemented with approximately 500 uL
of a 50% honey/water solution applied to the inside of the cotton
vial plugs. COI sequences for all wasp strains as well as ITS2
sequences for Figitid wasps have been deposited in Genbank under
accession numbers JQ808406–JQ808451. Wasp strains are available
upon request.
Hemocyte Counts
Fly-wasp development for all experiments took place in a 25uC
incubator on a 12:12 light:dark cycle. For hemocyte count
experiments, adult female D. melanogaster and D. suzukii were
allowed to lay eggs into fly food supplemented with yeast paste
(50:50 mix of baker’s yeast and water) or raspberries, respectively,
in 60 mm Petri dishes. After 72 hours, adult flies were removed
and developmental stage and size-matched second instar fly larvae
were collected for two independent experiments.
For hemocyte count experiments, D. melanogaster and D. suzukii
larvae were either uninfected or were infected by the wasp strain
LbG486, with three replicates per treatment. For parasitoid
infections, 50 fly larvae were moved into 35 mm diameter Petri
dishes filled with 1 mL of Drosophila medium. Ten female wasps
were immediately allowed to attack these fly larvae for 3 hours,
and five larvae per dish were later dissected to determine the
number of wasp eggs laid per fly larva. Fourteen of fifteen D.
melanogaster larvae across the three replicates were found to be
infected by single wasp eggs, as well as fourteen of fifteen D. suzukii
larvae, so we assumed the wasp infection rate was very similar
across the two host fly species. Hemocytes were counted at two
time-points, 12 and 24 hours post-infection, in which the induced
cellular immune response was expected to be highly activated.
Crystal cells, a distinct hemocyte type described below, were
counted independently 33 hours post-infection.
In an experiment to test hemocyte induction absent wasp
venom effects, D. melanogaster and D. suzukii larvae were either
untreated or were pierced with a sterile needle to simulate the
wounding associated with wasp oviposition. Such wounding has
been shown to induce the production of lamellocytes [33]. For
each of four replicates, 15 fly larvae were rinsed in 16PBS, dried
on Kimwipes, and immobilized on double sided tape. Their
posterior cuticles were then pierced with flame-sterilized 0.1 mm
diameter stainless steel dissecting pins (Fine Science Tools 26002-
10), with care taken to avoid harming internal organs. Fly larvae
were then removed from the tape with a wet paintbrush, and
allowed to recover in a moist chamber for one hour before being
moved to 35 mm diameter Petri dishes filled with 1 mL of
Drosophila medium. Control larvae were treated identically
Figure 2. Phylogenetic relationships and provenance of wasps used in this study. Tree topology is derived from previous phylogenetic
studies of Hymenopteran families [68], the family Figitidae [69,70], and the family Braconidae [71]. Branch lengths are approximated.
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except without piercing. Hemocytes were then counted 24 hours
post-infection, while crystal cells were counted independently
33 hours post-infection.
To count hemocytes, 5 third instar larvae from each treatment
replicate (including controls) were washed in Drosophila Ringer’s
solution, dried on a Kimwipe, and bled together into 20 mL of 16
PBS solution containing 0.01% phenylthiourea on a glass slide.
Dissection into buffer limits evaporation, and phenylthiourea
prevents the hemolymph from melanizing [34]. The bufferhemolymph
mixture was applied to a disposable hemocytometer
(Incyto C-Chip DHC-N01) and allowed to sit for 30 minutes to
allow hemocytes to settle. Hemocytes from each sample were
counted from sixteen 0.2560.2560.1 mm squares (e.g., Figure 3A,
3B), which make up a total volume of 0.1 mL. Thus, the number of
hemocytes from the whole 20 mL sample is expected to be ,200
times the number counted, or a per larva value of 40 times the
number counted.
The addition of hemolymph to the 20 mL of buffer is expected
to increase the total buffer-hemolymph volume to greater than
20 mL, leading to a downward bias in our absolute hemocyte
counts. However, the amount of hemolymph from five third instar
larvae is only approximately 2.5 mL, and in practice about this
much liquid evaporates before 20 mL of the buffer-hemolymph
mixture can be pipetted onto the hemocytometer. Our hemocyte
counts may also underestimate true hemocyte loads because a large
fraction of plasmatocytes are sessile (i.e., docked on host tissues)
[35], and may not detach from the larval tissues upon dissection.
D. melanogaster and D. suzukii adults and larvae are similar in size,
(Figure 1, 3), so we did not expect differences in species hemocyte
counts to result from fly size differences, but we were careful to use
larvae of the same size and developmental stage from both species
for all experiments. Hemocytes were classified as plasmatocytes
(small round cells with obvious nuclei), podocytes (activated
plasmatocytes that are larger and refract more light than
plasmatocytes), and lamellocytes (large, clear flattened cells) [7].
The fourth hemocyte cell type, crystal cells, are medium sized
cells containing cytoplasmic crystals made up of the substrate that
the phenoloxidase enzymatic cascade converts into melanin [36].
The crystals are rapidly lost upon dissection and the cells become
difficult to recognize, so a separate method was used to count
them. Crystal cells self-melanize when larvae are incubated at
60uC for 10 minutes [37]. Therefore, crystal cells were quantified
separately by counting dark spots from the dorsal side of incubated
whole larvae (e.g., Figure 3C, 3D) at 33 hours post-infection.
Crystal cells were counted and averaged from three larvae per
replicate. It is not yet known whether crystal cells play a role in the
melanotic encapsulation response [4].
Multivariable regression models assuming Poisson distributions
were specified to model hemocyte counts by fly species and
immune challenge (wasp infection, piercing). When hemocyte
counts were overdispersed, negative binomial distributions were
specified instead of Poisson distributions.
Resistance Trials
Each fly-wasp infection combination was replicated three times.
Adult female D. melanogaster and D. suzukii were allowed to lay eggs
into fly food supplemented with yeast paste (50:50 mix of baker’s
yeast and water) or raspberries, respectively, in 60 mm Petri
dishes. After 72 hours, adult flies were removed and size-matched
second instar fly larvae were collected for infections. For larval
parasitoid infections, 50 fly larvae were moved into 35 mm
diameter Petri dishes filled with 1 mL of Drosophila medium.
Three female wasps were immediately allowed to attack these fly
larvae for 72 hours. After attack, 10 of the 50 fly larvae were
dissected to determine the percent of larvae infected, the number
of wasp eggs laid per fly larva, and the proportion of fly larvae
bearing encapsulated wasp eggs in each sample. 30 of the 40
remaining larvae were then moved into Drosophila vials to
complete development. For pupal parasitoid infections, 40 fly
larvae were moved into vials containing Drosophila medium, and
were allowed to develop another 72 hours to the wandering third
instar stage, just before they began pupating on top of the medium
or on the sides of the vials. Three female wasps were then allowed
to attack the fly pupae for 72 hours, at which time the wasps were
removed and the fly pupae were left to complete development.
The infection conditions were chosen to be optimal for wasp
success. Control uninfected flies from both species were reared
under identical conditions and showed nearly 100% survival (data
not shown).
The total numbers of flies and wasps that eclosed from all wasp
treatments were determined 15 days and 30 days post-infection,
respectively, times by which all viable flies and wasps should have
emerged. Fly-wasp interactions may yield one of three outcomes,
which were compared between D. melanogaster and D. suzukii
infections: (1) a successful immune response by the fly, (2)
a successful parasitism by the wasp, or (3) death of the fly and
the wasp within it. Furthermore, for larval parasitoid infections,
Figure 3. D. suzukii hemocytes and encapsulation of wasp eggs.
(A) A 0.2560.2560.1 mm hemocytometer field from normal D. suzukii
larvae showing abundant plasmatocytes; (B) hemocytometer field from
D. suzukii larvae 12 hours after infection by wasp strain LbG486
showing increased podocyte and lamellocyte numbers; (C) control D.
melanogaster larva with melanized crystal cells; (D) control D. suzukii
larva with melanized crystal cells, showing color variation in inset; (E)
initiation of encapsulation of LbG486 egg by D. suzukii showing loose
hemocyte aggregation and melanization at anterior and posterior tips
of egg; (F) LbG486 egg melanotically encapsulated by D. suzukii,
showing surrounding layer of tightly spread hemocytes.
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the numbers of wasp eggs counted from dissected fly larvae were
assessed for evidence of under-dispersion, as wasps are known to
preferentially choose un-infected hosts for oviposition
[38,39,40,41]. If wasps layed eggs in fly larvae randomly, without
regard to host infection status, the number of wasp eggs per larva
would have been expected to follow a Poisson distribution, where
the average number of wasp eggs per fly larva and the variance in
the number of wasp eggs per fly larva should have been equal.
Figure 4. Hemocyte count comparison between D. melanogaster and D. suzukii. (A) 12 hours after infection by wasp strain LbG486; (B)
24 hours after infection by wasp strain LbG486; (C) 24 hours after piercing with a sterile needle. Average (+) standard deviation shown. Numbers are
approximately one fortieth of the number of cells per one fly larva (Methods).
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Thus, for each fly-wasp pair, we compared the average number of
wasp eggs laid per 10 dissected fly larvae to the variance in the
number wasp eggs laid per 10 dissected fly larvae across the three
replicates of each treatment, using one-tailed paired t-tests.
Although some figures show data for each wasp strain separately,
values for wasp strains of the same species were averaged into
single species values for all statistical analyses unless otherwise
noted.
Results
Hemocytes
D. suzukii hemocytes were morphologically similar to those of D.
melanogaster (Figure 3). In normal D. suzukii larvae, there were an
abundance of small round cells in the hemolymph that were
presumably homologous to plasmatocytes. In D. suzukii infected by
wasps, medium-sized round cells resembling podocytes became
much more numerous, as well as large irregular shaped cells that
resembled D. melanogaster lamellocytes. Heating D. suzukii larvae
resulted in the formation of darkened cells throughout the
hemocoel. In D. melanogaster, this phenomenon has been attributed
to the self-melanization of crystal cells, and suggested that D.
suzukii also possesses hemocytes responsible for carrying melanization
factors. Interestingly, while all self-melanized crystal cells in
D. melanogaster were dark black (Figure 3C), D. suzukii showed both
brown and black cells (Figure 3D, inset). Finally, D. suzukii larvae
encapsulated and melanized wasp eggs with hundreds of
hemocytes that flattened and spread over the wasp eggs to form
a tight capsule (Figure 3E, 3F). Thus, the stereotypic melanotic
Figure 5. Crystal cell count comparison between D. melanogaster and D. suzukii. (A) 33 hours after infection by wasp strain LbG486; (B)
33 hours after piercing with a sterile needle. Average (+) standard deviation shown.
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encapsulation response used by D. melanogaster against parasitoid
wasps appears to be conserved in its relative, D. suzukii.
Though hemocyte morphology was similar in the two fly
species, we found significant differences in constitutive and
induced hemocyte counts between D. melanogaster and D. suzukii.
We used two methods for inducing immune responses in these
flies. First, we infected flies with wasp strain LbG486, which is
relatively avirulent in D. melanogaster and has been shown to induce
production of hemocytes, and especially lamellocytes, in particular
infected D. melanogaster strains [42,43]. Second, in order to
stimulate lamellocyte production in the absence of any possible
immune inhibitory effects of wasp venoms, we pierced D.
melanogaster and D. suzukii larvae with sterile needles [33].
We tested the effects of fly species and immune challenge on fly
hemocyte counts using standard regression methods (Figure 4). We
found consistent, significant species effects on plasmatocyte,
podocyte, and lamellocyte numbers. Across time-points and
immune treatments, D. suzukii had significantly more plasmatoFigure
6. Hemocyte counts in other D. melanogaster and D. suzukii strains. (A) Constitutive plasmatocyte, podocyte, lamellocyte counts; (B)
constitutive crystal cell counts. Average (+) standard deviation shown.
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cytes, producing up to five times more plasmatocytes than D.
melanogaster. D. suzukii larvae also produced significantly more
podocytes than D. melanogaster, including constitutively produced
podocytes, which are not normally found in D. melanogaster larvae.
Furthermore, D. suzukii larvae produced significantly more
lamellocytes than D. melanogaster larvae. We found no effect of
immune challenge on plasmatocyte or podocyte numbers,
although as expected there were significantly more lamellocytes
in immune-challenged flies. Interestingly, the D. melanogaster
genome strain used in the present study was not resistant to
LbG486, unlike D. suzukii, (see below), and also showed no
significant increase in lamellocyte numbers at two time-points
post-infection when infected by LbG486 (Figure 4A, 4B). Finally,
there were significant species-by-immune challenge interaction
effects on podocyte and lamellocyte numbers in some experiments,
usually due to significantly greater induction of these cell types
after an immune challenge in D. suzukii. Thus, like D. melanogaster,
D. suzukii induces hematopoiesis and/or hemocyte differentiation
during a cellular immune response, although this induction is often
stronger than that observed in D. melanogaster.
We next tested the effects of fly species and immune challenge
on fly crystal cell counts using standard regression methods
(Figure 5). There was a significant effect of species on crystal cell
numbers in the piercing experiment, whereby D. suzukii had more
than three times the number of constitutively produced crystal cells
compared to D. melanogaster (Figure 5B). There was a similar, albeit
non-significant trend in the wasp-attack experiment (Figure 5A).
There were consistent, significant immune challenge effects of
crystal cell counts, whereby both species showed significant
reductions in crystal cell numbers following wasp infection or
piercing, suggesting either crystal cells or their crystals (which are
thought to contain the melanization precursors [36]) were spent
during the wound healing or immune responses. Significant
melanization was observed around the wound site in both species.
In order to confirm that hemocyte count differences between D.
melanogaster and D. suzukii are general, we conducted further
hemocyte counts experiments using two more strains of both fly
species (Figure 6). Once again, we found a significant effect of
species on constitutive numbers of plasmatocytes, podocoytes, and
crystal cells, with the D. suzukii strains having greater numbers of
these cell types in every case.
Fly Resistance
In the next experiment, both host species were infected with
a panel of parasitoid wasps. Since we did not observe the flies and
wasps for the duration of the infection period, it was important to
Figure 7. Numbers of eggs laid by each wasp strain in D. melanogaster (A) and D. suzukii (B). Average number of eggs per larva (+) standard
deviation shown. ANOVA results compare egglay numbers within fly species across wasp treatments. * = significant under-dispersion of wasp eggs in
fly larvae at p,0.05 using a one-tailed paired t-test (Methods).
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know whether wasp infection rates were similar across the two fly
species, so that any difference in fly eclosion could be attributed to
a successful encapsulation response rather than a lack of infection.
We compared the average number of eggs laid in the larvae of
both fly species by the panel of parasitoid wasps. Although
significant differences existed in the number of eggs laid by
different wasp strains within a fly species (D. melanogaster ANOVA
p,1024
, D. suzukii ANOVA p,1024
) (Figure 7), there was no
overall difference between fly species in the number of eggs laid by
the different wasp species (Figure 8), which averaged close to 1.25
eggs per fly larva in both fly hosts. Thus there was no evidence of
an overall infection preference by wasps for one fly species over the
other, and no evidence of differences in alternative mechanisms of
host defense, such as behavioral or physical immunity (e.g.,
a thickened cuticle) by the flies.
D. suzukii was able to melanotically encapsulate at least a small
proportion of eggs from all 21 larval parasitoid wasp strains tested,
whereas D. melanogaster was able to encapsulate some proportion of
eggs from only 8 of 21 wasp strains (LbFr, LbG486, LcNet, G1Fl,
G1Haw, AtFr, AtSw, and Aph1Atl) and only 5 of 12 wasp species
(Figure 9A). The difference in the proportion of wasp species that
the flies could melanotically encapsulate was statistically significant
(Fisher’s exact test p = 0.005). Qualitative melanotic encapsulation
differences between D. melanogaster and D. suzukii held across
additional strains tested of both species (Figure 9B). As expected,
the D. suzukii strains were able to encapsulate 3 of the 4 larval
parasites tested (Lb17, GxUg, ApIndo, but not AjJap), while D.
melanogaster was not able to encapsulate any of the parasites.
D. suzukii was also consistently more resistant to our panel of
parasitoid wasp species than D. melanogaster (Figure 10, 11). A
greater proportion of D. suzukii eclosed after wasp infection
compared to D. melanogaster for 20 of the 24 wasp strains tested, the
exceptions being D. suzukii infected by wasp strains G1Fl and
G1Haw (for which no flies of either species eclosed), AjJap, and
Tri1Cal. This corresponded to a significantly higher fly eclosion
rate for D. suzukii compared to D. melanogaster across wasp species
(Figure 11A). Furthermore, a lesser proportion of wasps eclosed
from infected D. suzukii larvae compared to D. melanogaster for 19 of
the 24 wasp strains tested, the exceptions being D. suzukii infected
by wasp strains G1Fl, G1Haw, AjJap, Tri1Cal, and Pac1Atl. This
corresponded to a significantly lower wasp eclosion rate in D.
suzukii compared to D. melanogaster across wasp species (Figure 11B).
The proportion of attacks that led to death of both the fly and the
wasp growing within the fly was also lower in D. suzukii, with D.
suzukii showing a lower proportion of death than D. melanogaster for
17 of the 24 wasp strains tested. However, this difference was not
significant across wasp species (Figure 11C). When we tested
additional strains of both D. suzukii and D. melanogaster, we found
qualitatively similar eclosion results (Figure 12). As expected,
a greater proportion of D. suzukii eclosed following infection
compared to D. melanogaster for 3 wasp strains (Lb17, GxUg,
ApIndo) D. suzukii was previously successful against, but not for
two wasp strains D. suzukii previously did poorly against (AjJap,
Tri1Cal).
Given our understanding of the Drosophila immune response
against wasp parasitism, we expect that flies that successfully
encapsulate particular wasp species will also have greater eclosion
success against those same wasp species. To test this expectation,
we assayed for correlations between encapsulation success and fly
eclosion for both flies species infected by the panel of wasp species.
Although we found a trend in the expected direction for both fly
species, there was no significant correlation in either fly species
(Figure 13).
Wasp Choice
Previous work using D. melanogaster has shown that wasps can
differentiate between infected and un-infected flies, and that they
preferentially lay eggs in fly hosts that have not already been
infected [38,39,40,41,44,45]. This preference is presumably
adaptive because it limits competition between juvenile wasps
that require the resources from an entire fly to complete
development. Such preference should lead to an under-dispersion
of wasp eggs in any group of infected fly larvae, i.e., a more even
distribution of eggs per larvae than expected by chance. We found
significant under-dispersion of wasp eggs in D. melanogaster larvae
for 15 of the 21 larval parasite wasp strains (Figure 7). The wasp
strains that laid the most eggs in D. melanogaster tended to show the
least under-dispersion, suggesting that the wasps could not
differentiate between infected flies once they were infected with
more than one wasp egg [38,39]. Only 4 of 21 wasp strains showed
a significant under-dispersion of eggs across D. suzukii larvae. This
suggests that whatever cue the wasps use to identify infected D.
melanogaster larvae, whether it is a tag left by the previous wasp or
some aspect of the D. melanogaster response to infection [40], is
generally missing in D. suzukii larvae. In no fly-wasp interaction
was there a significant over-dispersion of wasp eggs.
Figure 8. Parallel plot comparing average egglay numbers for
each wasp species between hosts. There was no overall difference
between fly species in numbers of eggs laid by wasps, nor was there
a correlation between the number of eggs laid in D. melanogaster and
the number of eggs laid in D. suzukii across the panel of wasp species
(as indicated by the non-parallel connecting lines).
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Drosophila parasitoid wasps can also distinguish between fly
host species, and preferentially lay eggs in host species in which
their offspring have a higher chance of survival [29,46,47]. We
tested whether larval parasitoid wasps tended to lay more eggs in
the fly hosts that their offspring more successfully eclosed from in
our trials (note that in our trials the wasps did not have a choice
between host species, only whether or not to lay eggs in a single
given host) (Figure 14). There was no relationship between wasp
species success and the number of eggs laid per larva with D.
melanogaster as host (r2
= 0.033, ANOVA p = 0.574). For D. suzukii,
however, there was a highly significant relationship (r2
= 0.585,
ANOVA p = 0.004) that was due in large part to the wasp species
A. japonica (strain AjJap) and Ganaspis sp.1 (combined strains G1Fl
and G1Haw). AjJap in particular laid the highest number of eggs
in D. suzukii in our infection trials, and also had the highest
eclosion success.
Specificity In Fly-Wasp Interactions
As described above, we found significant differences in the
number of eggs laid by different wasp strains within fly species but
not between fly species. This could mean that wasps that lay
higher numbers of eggs in D. melanogaster also lay higher numbers
of eggs in D. suzukii, i.e., some wasps could have generally higher
egglay rates than others. However, there was no correlation
between the number of eggs laid in D. melanogaster and the number
of eggs laid in D. suzukii for the panel of wasp species (r2
= 0.016,
ANOVA p = 0.696), suggesting that egglay rate is a plastic wasp
trait that wasps tailor to the host species they encounter.
There were significant differences across the panel of wasp
strains in the infection outcomes within fly species (ANOVA
p,1024
for all six comparisons: fly survival, wasp survival, death
in D. melanogaster, D. suzukii) (Figure 10). Although these differences
in infection outcomes were due to significant variation both
between wasp species and within wasp species (variation amongst
strains), the largest differences in infection outcomes are seen
between wasp species rather than wasp strains. For each fly host,
some wasp species were very successful infectors, some were very
susceptible to the fly immune responses, and some induced a large
amount of death. As described above there were also significant
differences in the infection outcomes between fly species. Despite
the superior wasp resistance of D. suzukii, it is possible that wasps
that were more successful in D. melanogaster were also more
successful in D. suzukii, i.e., some wasps are generally more virulent
than others. However, there was no correlation in the proportions
of any of the three infection outcomes between D. melanogaster and
D. suzukii (fly success r2
= 0.102, ANOVA p = 0.265; wasp success
r2
= 0.001, ANOVA p = 0.908; death r2
= 0.041, ANOVA
p = 0.489). This indicates there was specificity in the outcome of
wasp infections depending on the particular host fly species,
Figure 9. Encapsulation success of wasp-infected fly larvae. (A) Average proportion of fly larvae that encapsulated a wasp egg; (B) average
proportion of fly larvae from additional fly strains that encapsulated a wasp egg.
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despite D. melanogaster and D. suzukii being part of the same
Drosophila species group.
There is a strong influence of wasp phylogeny on D. melanogaster
infection outcomes. Members of the Leptopilina clade that
includes L. boulardi and L. heterotoma are very successful against D.
melanogaster, showing an average of 69% wasp eclosion. Infections
by L. clavipes and members of the genus Ganaspis, which are
likewise members of the family Figitidae, did not result in high
eclosion rates in D. melanogaster, but instead caused an average of
79% death of D. melanogaster larvae (Figure 8). Thus, D. melanogaster
appears to lack an immune mechanism to counter shared
virulence strategies of Figitid parasitoids. There appeared to be
little influence of wasp phylogeny on the ultimate outcome of D.
suzukii–wasp interactions, as D. suzukii was resistant to the majority
of wasps tested. However, the larval parasitoid that eclosed from
D. suzukii at the greatest rate (79%), A. japonica, is endemic to Japan
where it is sympatric with D. suzukii.
Discussion
Previous studies have shown that fly species and strains with
a greater constitutive production of hemocytes are more resistant
against and/or are better able to encapsulate parasitoid wasp eggs
[12,18,19,48]. Although a correlation does not necessarily imply
causation, these data suggest that evolution of higher constitutive
production of hemocytes is a relatively simple way for hosts to
defeat one of their most common classes of parasites. However, the
previous studies were limited to flies in the melanogaster subgroup
and to a few wasp species/strains that represent only a small
fraction of the diverse virulence strategies used by Drosophila
parasitoid wasps. To determine if increased hemocyte production
by flies is a panacea against wasp infection, we first compared
hemocyte numbers between D. melanogaster and D. suzukii, a relative
of D. melanogaster outside the melanogaster subgroup.
We found that third instar D. suzukii larvae made constitutively
greater numbers of plasmatocytes, podocytes, and crystal cells than
D. melanogaster larvae, and also induce greater production of
podocytes and lamellocytes (Figure 4, 5, 6). Compared to our
recently wild-derived D. suzukii strains, the D. melanogaster genome
strain we used may have had relatively poor genetic immune
ability for its species due to its homozyosity and its long-term
selection in a lab environment. However, hemocyte counts from
the two additional wild-caught D. melanogaster strains we assayed
were very similar to those from the genome strain. The hemocyte
numbers we observed in our D. melanogaster strains were also similar
to those seen in a variety of other studies where the unit of
measurement was cells per larva [33,35,42], and also appeared
similar to numbers found in studies that counted cells per volume
of hemolymph (using a rough conversion factor of approximately
0.5 uL hemolymph per third instar larva) [12,19,43,49,50]. Thus,
we have no reason to believe that differences we observe between
our D. melanogaster and D. suzukii strains were due to a biased
sampling of strains rather than actual species differences. In
comparison with hemocyte numbers from other studies, D. suzukii
Figure 10. Infection outcomes for host larvae infected by each wasp strain. Average (+) standard deviation shown for D. melanogaster (A)
and D. suzukii (B). ANOVA results compare fly eclosion, wasp eclosion, or death proportions within fly species across wasp treatments.
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appears to have somewhat greater constitutive hemocyte counts
than D. simulans, which has the highest counts of any member of
the melanogaster subgroup [12].
Using a diverse panel of parasitoid wasp strains and species, we
found that infection rates in D. melanogaster and D. suzukii were
similar (Figure 7, 8), but that D. suzukii was significantly better at
melanotically encapsulating, and surviving infection by, the wasps
(Figure 9, 10, 11,12). The panel of wasps included relatively
specialist and generalist wasp species, such as L. boulardi and L.
heterotoma, respectively [15], as well as relatively immune evasive
versus immune suppressive wasp species, such as A. tabida and G.
xanthopoda, respectively [51,52]. Our data suggest that a general
protection against parasitoid wasps is afforded to fly species that
have higher constitutive hemocyte loads. The association between
hemocyte load and encapsulation ability reported previously [12]
also appears to extend beyond the melanogaster subgroup of fly
hosts, as D. suzukii is part of the melanogaster group but not the
melanogaster subgroup. Future infection trials using the same
panel of parasitoid wasps, but a much wider range of fly species,
will be needed for determining the true extent of the relationship
between hemocyte load and resistance against parasitoid wasps.
The current model for the melanotic encapsulation process is
that plasmatocytes act as sentinels of wasp infection and signal to
activate other circulating plasmatocytes as well as the lymph gland
once infection is recognized [4,5]. The activated plasmatocytes
develop cytoskeletal projections and become known as podocytes,
which may be an intermediate form between the smaller
plasmatocytes and larger lamellocytes [6,7]. Lamellocytes are also
induced via differentiation of pro-hemocytes in the lymph gland.
The lamellocytes then migrate towards and surround the wasp
egg, forming a tight capsule. The capsule becomes melanized, but
it is not yet known whether melanin precursors stored in crystal
cells are used in this process. Thus, any or all of the hemocyte cell
types that D. suzukii produced in excess may have been responsible
for the relatively high resistance of D. suzukii against wasp eggs.
Flies with more hemocytes may suffer fewer effects of wasp
venom for a variety of reasons, enabling them to mount a quicker
and/or stronger encapsulation reaction against wasps. For
example, venoms often alter hemocyte structure and function
[50,53], and thus an increased number of hemocytes could
potentially dilute the effects of a standard dose of venom.
Alternatively, hemocytes may be responsible for destroying venom
components found in the hemolymph, via endocytosis or some
other mechanism, preventing the venom from exerting its effects
on other tissues. It is unclear whether an excess of constitutively
produced hemocytes (plasmatocytes, crystal cells) or the increased
induced production of podocytes and lamellocytes drives the
relationship between hemocyte counts and wasp resistance, but the
distinction may be unimportant given that constitutively produced
cells can differentiate into induced cell types [6,7]. However, in
support of the idea that constitutive production of hemocytes alone
is not sufficient for wasp resistance, Drosophila species of the
obscura group that make relatively high numbers of plasmatocytes,
but apparently do not produce a lamellocyte class of cells, are
unable to encapsulate foreign objects and are highly susceptible to
wasp infection [54,55].
Figure 11. Parallel plot comparing outcomes between host larvae infected by each wasp species. (A) fly eclosion; (B) wasp eclosion; (C)
death. There were significant overall differences between fly species in fly eclosion and wasp eclosion proportions, but not in proportion dead. There
is no correlation between fly eclosion, wasp eclosion, or death proportions between D. melanogaster and D. suzukii across the panel of wasp species
(as indicated by the non-parallel connecting lines).
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Unlike for D. melanogaster, larval parasitoid wasps rarely underdispersed
their eggs across D. suzukii larvae. Wasps are thought to
discriminate naı¨ve host larvae from previously infected larvae
either by recognizing a mark left by the previous wasp, or by
recognizing the host response to infection [40]. Given that D.
suzukii has a significantly more robust immune response against
wasp infection than D. melanogaster, it seems unlikely that these
wasps use host immune cues to avoid superparasitism. If fly
Figure 12. Infection outcomes for host larvae of other strains. (A, B) D. melanogaster extra strain 1 and 2; (C, D) D. suzukii extra strain 1 and 2.
Average (+) standard deviation shown.
doi:10.1371/journal.pone.0034721.g012
Figure 13. Relationship between encapsulation rate and fly eclosion. Average proportion of fly larvae that encapsulated a wasp egg for D.
melanogaster (A) and D. suzukii (B).
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hemocytes are responsible for clearing wasp venom components
from the hemolymph, wasp ‘‘possession marks’’ might also be lost
in fly hosts that make abundant hemocytes, leading to more
random dispersal of wasp eggs across host larvae.
We expected to find a correlation between encapsulation ability
and fly success in both D. melanogaster and D. suzukii, but although
there was a trend in this direction, the correlations were not
significant (Figure 13). Three factors likely contribute to this lack of
correlation. First, we counted fly larvae as having successful
encapsulations if any encapsulation was seen, even if flies were
super-parasitized and hadn’t encapsulated all wasp eggs they were
infected by. Thus, flies scored as showing encapsulation could still
succumb to infection. Second, some fly-wasp combinations that
yielded encapsulations culminated in neither fly nor wasp eclosion,
but high rates of death of by both fly and wasp. Third, wasp
parasites sometimes die inside their fly hosts even if the fly has not
encapsulated them by the time-point we assayed.
Interestingly, D. suzukii does not have a clear survival advantage
over D. melanogaster when infected by the two pupal parasite species
(three strains) in our panel of wasps. Very little is known about the
determinants of infection outcomes with regards to pupal parasites
of flies, or even whether venom plays an important role. Although
Trichopria acts as a pupal endoparasitoid, the Drosophila pupal
stage does not appear able to mount melanotic encapsulation
responses against them. Furthermore, Pachycrepoideus lays its
eggs in the space between the pupal case and the pupa, and acts as
an ectoparasite for most of its development [29], which could
negate any ability the flies have to mount an internal, physiological
immune response. In other systems, pupal parasitoid wasps are
known to have more generalist host ranges than larval parasites
[30,31], but they do not have unlimited host ranges either, so some
specificity in their utilization of host resources is inherent.
Although our data suggests increased hemocyte load has little
effect on fly resistance against pupal parasites, a definitive
statement will require data from a greater range of pupal parasite
species.
Still, if increased hemocyte load provides general protection
against larval parasitoids, why do some fly species, such as D.
melanogaster, produce such low numbers compared to their close
relatives? Hosts face an evolutionary tradeoff between investing in
immune responses against parasites versus investing in other
aspects of fitness [56,57,58,59]. The constitutive production and
maintenance of hemocytes must obviously impart an energetic cost
on the host, diverting resources from other aspects of host fitness.
Thus, if hosts are rarely infected by wasps in nature, or are
commonly infected by specialist wasps that can overcome
hemocyte-based immunity, it may make evolutionary sense to
invest in fecundity rather than immunity, or in other aspects of
immunity, such as behavioral immunity. On the other hand,
investment in high constitutive hemocyte levels might be selected
in host species that are commonly infected by non-specialist
parasites.
Although D. suzukii is generally more resistant against larval
wasp parasites than D. melanogaster, there were a small number of
obvious exceptions. A. japonica is sympatric with D. suzukii in its
native east Asian range, and was significantly more successful at
infecting D. suzukii than D. melanogaster. Previous studies showed A.
japonica successfully parasitizes D. suzukii both in the field and in the
lab [60,61]. A japonica also laid approximately three times more
eggs in D. suzukii than in D. melanogaster, and laid the highest
number of eggs in D. suzukii of any larval parasitoid wasp.
Altogether, these data suggest A. japonica may have co-evolved
a specialized virulence strategy able to overcome the high
hemocyte load of D. suzukii, and may have evolved an infection
preference for D. suzukii as well. The only other larval parasite able
to eclose from D. suzukii hosts at any appreciable rate is Ganaspis
sp.1, an undescribed species collected in Florida and Hawaii.
Although G. xanthopoda was found to emerge from D. suzukii pupae
collected in the field in Japan [61], the two G. xanthopoda strains
used in this study, from Hawaii and Uganda, were very poor
infectors of D. suzukii, suggesting populations of this wasp species
may have locally adapted to D. suzukii host use in Japan.
Figure 14. Relationship between wasp eclosion success and number of eggs wasps choose to lay in a host. There was no significant
relationship for the panel of wasp species attacking D. melanogaster (A), but there was a significant relationship for the panel of wasp species
attacking D. suzukii (B).
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D. suzukii has recently spread into Europe and North America as
a pest species [20,21,22,23]. It was first documented in the United
States in California in 2008, from where it quickly spread to
Oregon and Washington. In these west coast states, D. suzukii was
responsible for up to 80% yield losses in berry and cherry crops
depending on location, and is estimated to be causing yearly
monetary losses in the range of 500 million dollars [62,63]. In
2009, D. suzukii became established in Florida, and in 2010 reports
of collections were made from a handful of new states [23].
However, experimental studies testing the efficacy of various
management strategies for D. suzukii are as yet lacking [64,65].
One common pest management strategy is the use of biocontrol
agents such as natural enemies (parasites, predators) [66], and
parasitoid wasps have successfully controlled numerous other
arthropod pests in the past [24,25,26,27,28]. Furthermore,
Drosophila parasitoid wasps often infect a large proportion of fly
larvae in natural populations [1,2,3], and the potential for an
endemic Figitid species (L. boulardi) to control native Drosophila
populations in California was previously considered [67]. It
appears the wasp species with the highest potential for use in
biocontrol of D. suzukii are the larval parasites A. japonica and
Ganaspis sp.1, and the pupal parasite Trichopria sp.1. A. citri might
also be considered a potential biocontrol agent for D. suzukii
because of the high death rates it caused in D. suzukii, but this wasp
had much higher eclosion rates using D. melanogaster as host than D.
suzukii. Because infection trial conditions in this study were
designed to be ideal for success of the wasps, and such conditions
(easy access to hosts, no competition with other parasites,
controlled temperature, abundant resources, etc) are unlikely to
be replicated in the field, extensive field experiments will be
required to assess the efficacy of the use of parasitoid wasps in D.
suzukii biocontrol in practice.
Acknowledgments
We thank R. Allemand, D. Hultmark, J. van Alphen, J. Pool, and B.
Wertheim for providing wasp strains, Kate Ellingson for help with
statistical analyses, and members of the Schlenke lab for helpful comments
on the manuscript.
Author Contributions
Conceived and designed the experiments: BZK TAS. Performed the
experiments: BZK TAS. Analyzed the data: BZK TAS. Contributed
reagents/materials/analysis tools: TAS. Wrote the paper: BZK TAS.
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