Reactive oxygen species-dependent cell signaling regulates the mosquito immune response to Plasmodium falciparum.
ABSTRACT Reactive oxygen species (ROS) have been implicated in direct killing of pathogens, increased tissue damage, and regulation of immune signaling pathways in mammalian cells. Available research suggests that analogous phenomena affect the establishment of Plasmodium infection in Anopheles mosquitoes. We have previously shown that provision of human insulin in a blood meal leads to increased ROS levels in Anopheles stephensi. Here, we demonstrate that provision of human insulin significantly increased parasite development in the same mosquito host in a manner that was not consistent with ROS-induced parasite killing or parasite escape through damaged tissue. Rather, our studies demonstrate that ROS are important mediators of both the mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt signaling branches of the mosquito insulin signaling cascade. Further, ROS alone can directly activate these signaling pathways and this activation is growth factor specific. Our data, therefore, highlight a novel role for ROS as signaling mediators in the mosquito innate immune response to Plasmodium parasites.
- SourceAvailable from: journalofdiabetology.org[Show abstract] [Hide abstract]
ABSTRACT: With increasing prevalence rates of diabetes mellitus in tropical countries, malaria and diabetes often coincide. The study was designed to investigate the effects of glucose and insulin upon in vitro proliferation of Plasmodium falciparum, the causative agent of malaria tropica. Plasmodium falciparum proliferation was determined via the Malstat™ assay, following incubation of the parasites at varying concentrations of glucose (0 -27.7mM) or insulin (10 pM -100µM) for 24 and 48 hrs. While Plasmodium falciparum proliferation was unaffected at concentrations of 5.5 – 27.7mM glucose, growth w as impaired below a threshold of 5.5mM. No effect was seen following incubation of the parasites in the presence of 10pM – 100µM insulin. Insulin levels did not affect parasite proliferation whilst glucose levels below 5.5mM reduced parasite growth. The Malstat™ assay was identified as a suitable screening assay to assess the effects of glycemic control upon Plasmodium falciparum growth.
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ABSTRACT: Insulin and insulin-like growth factor signaling (IIS) regulates cell death, repair, autophagy, and renewal in response to stress, damage, and pathogen challenge. Therefore, IIS is fundamental to lifespan and disease resistance. Previously, we showed that insulin-like growth factor 1 (IGF1) within a physiologically relevant range (0.013-0.13 µM) in human blood reduced development of the human parasite Plasmodium falciparum in the Indian malaria mosquito Anopheles stephensi. Low IGF1 (0.013 µM) induced FOXO and p70S6K activation in the midgut and extended mosquito lifespan, whereas high IGF1 (0.13 µM) did not. In this study the physiological effects of low and high IGF1 were examined in detail to infer mechanisms for their dichotomous effects on mosquito resistance and lifespan. Following ingestion, low IGF1 induced phosphorylation of midgut c-Jun-N-terminal kinase (JNK), a critical regulator of epithelial homeostasis, but high IGF1 did not. Low and high IGF1 induced midgut mitochondrial reactive oxygen species (ROS) synthesis and nitric oxide (NO) synthase gene expression, responses which were necessary and sufficient to mediate IGF1 inhibition of P. falciparum development. However, increased ROS and apoptosis-associated caspase-3 activity returned to baseline levels following low IGF1 treatment, but were sustained with high IGF1 treatment and accompanied by aberrant expression of biomarkers for mitophagy, stem cell division and proliferation. Low IGF1-induced ROS are likely moderated by JNK-induced epithelial cytoprotection as well as p70S6K-mediated growth and inhibition of apoptosis over the lifetime of A. stephensi to facilitate midgut homeostasis and enhanced survivorship. Hence, mitochondrial integrity and homeostasis in the midgut, a key signaling center for IIS, can be targeted to coordinately optimize mosquito fitness and anti-pathogen resistance for improved control strategies for malaria and other vector-borne diseases.PLoS Pathogens 06/2014; 10(6):e1004231. · 8.14 Impact Factor
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ABSTRACT: Susceptibility to Plasmodium infection in Anopheles gambiae has been proposed to result from naturally occurring polymorphisms that alter the strength of endogenous innate defenses. Despite the fact that some of these mutations are known to introduce non-synonymous substitutions in coding sequences, these mutations have largely been used to rationalize knockdown of associated target proteins to query the effects on parasite development in the mosquito host. Here, we assay the effects of engineered mutations on an immune signaling protein target that is known to control parasite sporogonic development. By this proof-of-principle work, we have established that naturally occurring mutations can be queried for their effects on mosquito protein function and on parasite development and that this important signaling pathway can be genetically manipulated to enhance mosquito resistance.Parasites & Vectors 06/2014; 7(1):287. · 3.25 Impact Factor
ORIGINAL RESEARCH COMMUNICATION
Reactive Oxygen Species-Dependent Cell Signaling
Regulates the Mosquito Immune Response
to Plasmodium falciparum
Win Surachetpong,* Nazzy Pakpour,* Kong Wai Cheung, and Shirley Luckhart
Reactive oxygen species (ROS) have been implicated in direct killing of pathogens, increased tissue damage, and
regulation of immune signaling pathways in mammalian cells. Available research suggests that analogous
phenomena affect the establishment of Plasmodium infection in Anopheles mosquitoes. We have previously shown
that provision of human insulin in a blood meal leads to increased ROS levels in Anopheles stephensi. Here, we
demonstrate that provision of human insulin significantly increased parasite development in the same mosquito
host in a manner that was not consistent with ROS-induced parasite killing or parasite escape through damaged
tissue. Rather, our studies demonstrate that ROS are important mediators of both the mitogen-activated protein
kinase and phosphatidylinositol 3-kinase=Akt signaling branches of the mosquito insulin signaling cascade.
Further, ROS alone can directly activate these signaling pathways and this activation is growth factor specific.
Our data, therefore, highlight a novel role for ROS as signaling mediators in the mosquito innate immune
response to Plasmodium parasites. Antioxid. Redox Signal. 14, 943–955.
over 1 million deaths annually. The absence of an effec-
tive vaccine along with increasing drug-resistant parasites
and pesticide-resistant mosquito vectors has resulted in a
surge of malaria cases in recent years, which highlights the
need for novel control strategies (40). One novel strategy is
based on the genetic modification of mosquitoes so that they
are unable to transmit malaria. Currently, only a few geneti-
cally engineered mosquito lines have been produced that are
refractory to malaria parasites, and none are close to being
field-tested (6, 28). This is due, in part, to our inability to
identify effective gene targets for transformation that render
the mosquito resistant to malaria infection without reducing
mosquito fitness. Plasmodium parasites undergo a series of
complex developmental transformations inside Anopheles
mosquitoes during which they experience significant losses
greatest reduction in parasite numbers generally occurs as
ookinetes cross the midgut epithelium to form oocysts (44).
During this stage of infection, parasites are eliminated by a
combination of anti-microbial peptides, nitric oxide, and
complement-like factors (5). Thus, mosquito immunity can
lasmodium falciparum infection is responsible for
directly impact parasite transmission and provides an excel-
lent target for genetic manipulation.
In the course of blood meal digestion, the mosquito midgut
epithelium is exposed to a variety of parasite-derived and
human blood-derived factors, such as human transforming
growth factor (TGF)-beta1 and insulin, which can affect
mosquito physiology and malaria parasite development (14,
24, 42). The signaling cascades that regulate these responses,
including the mitogen-activated protein kinase (MAPK)-
dependent cascades in general and the insulin=IGF-1 signal-
ing (IIS; 25) cascade in particular, are highly conserved. The
IIS cascade consists of two main signaling branches, an
(PI3K)=Akt-dependent pathway, both of which have been
shown to regulate a variety of cellular functions, including
innate immunity (Fig. 8; 25). We previously demonstrated
that both branches of the IIS cascade in the mosquito midgut
particular, both extracellular signal-regulated kinase (ERK)
and Akt phosphorylation are increased in the mosquito
midgut in response to ingested human insulin (14). Most re-
cently, we demonstrated that expression of constitutively
active Akt in the midguts of genetically engineered Anopheles
stephensi can completely inhibit P. falciparum infection in
Department of Medical Microbiology and Immunology, University of California, Davis, Davis, California.
*These two authors contributed equally to this work.
ANTIOXIDANTS & REDOX SIGNALING
Volume 14, Number 6, 2011
ª Mary Ann Liebert, Inc.
homozygous transgenic mosquitoes (6). While Akt is a central
regulator of IIS, the downstream mechanisms that underlie
Akt-mediated refractoriness are likely to be complex and
networked with multiple signaling pathways.
Provision of human insulin in the blood meal also signifi-
cantly decreases superoxide dismutase (SOD) activity in A.
stephensi and reduces mosquito lifespan (14), presumably due
toincreased levelsofreactive oxygen species (ROS).Increased
ROS can profoundly alter epithelial protein structure and
deleterious consequences for the vector. In particular, nu-
merous studies in mammals have linked ROS to the disrup-
tion of epithelial tight junctions and to increased permeability
orleakiness ofepithelial barriers toavarietyofpathogensand
toxins (34). In mosquitoes, the midgut epithelium serves both
a physiological role in the absorption of nutrients and an
immunological role as a barrier against pathogens. Greater
levels of insulin-induced ROS could result in the loss of
midgut barrier and integrity, allowing pathogens such as
malaria parasites to establish infection more easily.
Although excessive ROS levels can be damaging to host
cells, they can also be detrimental to infectious pathogens
such as malaria parasites (17, 26, 29, 33). In particular, dif-
ferences in systemic levels of ROS can result in differences in
the mosquito immune response to Plasmodium parasites (17)
and that provision of enzyme inhibitors or antioxidants in an
infectious blood meal can enhance parasite development (26,
29). High levels of ROS can be detrimental to the host and
invading organisms, whereas moderate levels of ROS can be
beneficial to a variety of cell signaling processes (1, 20, 27, 43).
For example, ROS can prime Drosophila hematopoietic cells
for differentiation (31) and, from more recent work, may be
directly involved in the catalytic cross-linking of proteins in a
protective mucin layer in the midgut of the African malaria
mammalian research has described a critical role for ROS in
facilitating normal signaling by insulin and a variety of other
hormones and growth factors (8). For example, hydrogen
peroxide (H2O2) has been shown to be a potent activator of
both ERK and PI3K signaling pathways in mammalian cells
(1, 43). Numerous studies have also shown that TGF-beta1
stimulation increases the synthesis of ROS, which can activate
signaling by the full complement of MAPKs—ERK, p38, and
JNK—in a variety of mammalian cell types (13, 15). Further,
the signaling effects of ROS are moderated by antioxidants,
including glutathione peroxidase, SODs, catalase, and the
peroxiredoxins (12). Collectively, these data suggest that ROS
may be functioning similarly in the mosquito in the regula-
tion of MAPK signaling in general and in the IIS cascade in
In this study, we found that the provision of human insulin
in an infectious blood meal to the Indian malaria mosquito A.
stephensi significantly increased development of P. falciparum
and that addition of the antioxidant Mn(III) tetrakis(4-benzoic
acid) porphyrin chloride (MnTBAP) significantly decreased
parasite numbers via an effect that was not attributable to
oxidative damage to the midgut. In the absence of an ROS-
damaged midgut, we hypothesized that insulin-induced ROS
may be altering immune signaling to regulate P. falciparum
development. In support of this hypothesis, we demonstrate
here that ROS can induce activation of mitogen activated
protein kinase kinase (MEK), ERK, and p38, known immune
modulatory MAPKs, as well as PI3K=Akt signaling in A. ste-
phensi cells. In addition, inhibition of insulin-induced ROS
resulted in decreased phosphorylation of downstream effec-
tors of the IIS. By comparison, ROS scavenging had little to no
effect on TGF-beta1-dependent MAPK activation. Our data
highlight an ROS-dependent signaling specificity in A.
stephensi that extends our appreciation of this biomedically
important species for the study of innate immune cell sig-
naling pathways that have applications for novel strategies
for malaria control.
Materials and Methods
Human insulin was purchased from Sigma-Aldrich and re-
combinant TGF-beta1 was purchased from R&D Systems.
Sigma-Aldrich and polyclonal anti-ERK1=2 antibodies were
purchased from Cell Signaling Technology. Anti-phospho-p38
MAPK antibody was obtained from Cayman Chemical, and
anti-GAPDH antibody from Abcam. Anti-phospho-forkhead
box O1 (FOXO) antibody and anti-phospho-p70S6K were pur-
chased from Millipore.
Horseradish peroxidase-conjugated polyclonal rabbit anti-
mouse IgG was purchased from Sigma-Aldrich. Horseradish
peroxidase-conjugated goat anti-rabbit F(ab’)2 fragment and
peroxidase-conjugated goat anti-rabbit IgG (HþL) were pur-
chased from Biosource International and Pierce, respectively.
The BCA assay kit and SuperSignal West Pico chemilumi-
nescent detection kit were purchased from Pierce. RPMI 1640
with HEPES was purchased from Gibco=Invitrogen. All other
chemicals and reagents were obtained from Sigma-Aldrich or
Fisher Scientific. Human serum and red blood cells (RBCs)
were obtained from Interstate Blood Bank. MnTBAP was
purchased from EMD Chemicals.
Mosquito cell culture, mosquito rearing,
and experimental treatments
Immortalized, A. stephensi embryo-derived (ASE), larva-
(gift from Hans-Michael Muller, EMBL) were maintained as
previously described (42). For in vivo studies, A. stephensi
278C and 75% humidity. All mosquito rearing and feeding
protocols were approved and in accordance with regulatory
guidelines and standards set by the Institutional Animal Care
and Use Committee of the University of California, Davis. For
experimental treatments, laboratory-reared 3–5-day-old fe-
male mosquitoes were maintained on water for 24–48h and
then allowed to feed for 30min on reconstituted human blood
meals provided through a Hemotek Insect Feeding System
(Discovery Workshops). Artificial blood meals contained wa-
shedhuman RBCs andsaline (10 mmol l?1NaHCO3, 15 mmol
insulin at 170 pM and with or without MnTBAP at 5mM.
Normal human blood insulin levels range from 17 pM at
fasting to 0.59 nM without fasting, indicating that 170 pM
insulin could be ingested by feeding mosquitoes (7). For
western blot analyses, midguts were dissected from 60 mos-
quitoes in each treatment group and processed as previously
described (42). Control mosquitoes were provided artificial
944SURACHETPONG ET AL.
blood meals supplemented with an equivalent volume of
diluent phosphate-buffered saline (PBS).
The details of our western blotting protocols have been
previously described (42). Protein lysates from cells or mos-
quito midguts weresubjectedto10%sodiumdodecylsulfate–
polyacrylamide gel electrophoresis polyacrylamide gels,
transferred to nitrocellulose membranes (BioRad), and pro-
bed for proteins of interest with various antibodies.
Quantitative reverse transcriptase–polymerase
To detect A. stephensi nitric oxide synthase (NOS) gene ex-
pression, total RNAs were isolated from cultured cells using
Trizol reagent (Invitrogen) at 6–48h post-treatment. Samples
were analyzed by quantitative reverse transcriptase–polymer-
ase chainreactionusing an ABI Prism 7300 Sequence Detection
System (Applied Biosystems). NOS expression levels were
and are represented as fold induction over control. Primers,
probes, and amplification conditions were described previ-
ously (42). Technical replicates consisting of triplicate reactions
with 100ng template RNA and no template controls were an-
alyzed simultaneously to assess amplification efficiency and
lack of genomic DNA cross-contamination, respectively. Bio-
cells were used for statistical analysis.
2-cys peroxiredoxin overexpression
ASE cells were transfected with pcDNA3.1=V5-His vector
(Invitrogen) expressing full-length A. stephensi 2-cys peroxir-
edoxin (AsPrx) referred to as ‘‘TLP-58’’ (33). Overexpressed
AsPrx protein in mosquito cells was detected by immunoblot-
ting with anti-V5 antibody as previously described in (33). The
copper-inducible pMT=V5-His, vector (Invitrogen) referred to
as ‘‘TLP-55’’ was used as the transfection control since in the
(33). ASE cells were plated in six-well plates and were trans-
fected with plasmid using Effectene Transfection Reagent
(Qiagen) according to the manufacturer’s protocol. After 48h,
with varying concentrations of insulin, TGF-beta1, or H2O2for
5min. After incubation, cell lysates were collected and pro-
cessed for western blotting analysis.
Malaria parasite culture and mosquito infection
twenty 3–5-day-old female A. stephensi were maintained on
water pads for 24h before blood feeding. Mosquitoes were
allowed to feed on P. falciparum NF54-infected RBCs for
30min. After 10 days, midguts from 50 mosquitoes with fully
group were dissected in PBS and stained with 0.1% mercur-
ochrome for direct counting of P. falciparum oocysts. Means of
oocysts per midgut in each treatment group were calculated
from all dissected mosquitoes, including zeros for mosquitoes
that contained no oocysts.
were grown in 10% heat-inactivated human serum and 6%
washed human RBCs in RPMI 1640 with HEPES (Gibco) and
hypoxanthine. At day 15, stage V gametocytes were evident
and exflagellation rates were evaluated on the day of feeding.
The 3–5-day-old A. stephensi were fed on mature gametocyte
cultures diluted with human RBCs and heat-inactivated
human serum with or without 170 pM human insulin and
with or without 5mM MnTBAP. All treatments were added to
the diluted P. falciparum culture immediately before blood
feeding. Protocols involving the culture and handling of
P. falciparum for mosquito feeding were approved and in
accordance with regulatory guidelines and standards set by
the Biological Safety Administrative Advisory Committee of
the University of California, Davis.
P. falciparum growth assays
Aliquots of P. falciparum NF54 culture were synchronized
48h before the assay as previously described (21) and were
then plated in 96-well flat-bottom plates in complete RPMI
1640 with HEPES, hypoxanthine, and 10% heat inactivated
human serum. Parasites were treated with the equivalent
volumes of PBS and human insulin at concentrations ranging
from 170 pM to 17mM or with PBS and MnTBAP at concen-
trations rangingfrom 50nM to50mM for 48hin acandle jarin
culture media with RPMI 1640 with 1% formalin. Ery-
throcytes were stained with 10mg=ml of propidium iodide
(Sigma) in PBS for 1h at room temperature. Infected RBCs
were counted with FACS Calibur flow cytometer, Becton
Dickinson (BD Biosciences). Relative levels of parasite growth
in response to treatment were normalized to PBS-treated
controls, which were set to 100%.
Quantification of mosquito midgut
protein carbonyl content
A total of 150 female A. stephensi (3–5 days old) from a
single cohort were transferred into four, 1-gallon containers
and were fed a single artificial blood meal supplemented with
either 170 pM human insulin, 5mM MnTBAP, insulin plus
MnTBAP, or an equivalent volume of PBS as a control. All
mosquitoes were allowed to feed for *1h and were then
provided with 10% sucrose-soaked cotton pads after blood
feeding. Thirty mosquito midguts were dissected daily as
described above and midgut protein carbonyl content (PCC)
was determined by using the Oxyblot kit western blotting
protocol (EMD chemicals) according to manufacturer’s rec-
ommendations. Film exposures of PCC membranes and
Coomassie-stained gels were scanned, and band densities
were determined using a GS-800-calibrated densitometer.
PCC levels were normalized to total protein levels in the same
samples as determined by Coomassie staining.
Three tests for normality were used: Kolmogorov–
Smirnov, D’Agostino–Pearson omnibus, and Shapiro–Wilk
(Graphpad Prism 5.02). Data that were normally distributed
were analyzed by analysis of variance for overall significance
and Student–Neuman–Keuls for pairwise comparisons or by
Student’s t-test to assess differences between paired con-
trols and treatments or between paired treatments. Data that
were not normally distributed were analyzed using the
ROS-DEPENDENT CELL SIGNALING IN MOSQUITOES945
Kruskal–Wallis test and Dunn’s test for pairwise compari-
sons. The prevalence of parasite infection was analyzed using
the chi-square test.
Insulin-dependent ROS alter P. falciparum development
in A. stephensi
Our previous work showed that human insulin treatment
increased H2O2levels and decreased SOD activity in mos-
quito cells in vitro and in vivo (14). Because higher levels of
ROS have been associated with decreased parasite develop-
ment in mosquitoes (17, 26, 29, 33), we sought to determine
the effects of insulin-dependent ROS production on P. falci-
parum oocyst development in A. stephensi. We expected that
provision of human insulin in the blood meal would increase
ROS production and decrease oocyst development. Further,
we predicted that the addition of antioxidants such as
MnTBAP, an SOD mimetic compound that has been utilized
in mosquito studies previously (14, 38), should reverse the
effects of insulin treatment on oocyst development. However,
we found that P. falciparum oocyst numbers in insulin-fed
MnTBAP reversed this effect. (A) Mosquitoes were fed with P. falciparum–infected red blood cells supplemented with PBS as
a control (Buffer), 5mM MnTBAP (MnT), 170 pM human insulin, or MnTBAP plus human insulin. Data from three inde-
pendent experiments with separate cohorts of mosquitoes were analyzed for the main effects of experiment and treatment.
No significant effects were noted for experiment, for treatment, or for the interaction of experiment and treatment, indicating
that these data could be combined for analysis. Horizontal lines indicate the means for three combined sets of 50 mosquitoes
(150 mosquitoes total) per treatment group. The data were not normally distributed and, therefore, analyzed using the
Kruskal–Wallis test and Dunn’s post-test. Significant differences between treatment groups are indicated. (B) Prevalence of
infection (Anopheles stephensi with at least one P. falciparum oocyst) from three independent experiments shown as percentages
of dissected mosquitoes. Data were analyzed by chi-square (a¼0.05) and no significant differences among treatments were
observed. Human insulin (C) and MnTBAP (D) did not affect growth of asexual-stage P. falciparum. Replicate cultures of P.
falciparum NF54 were incubated with increasing concentrations of human insulin or MnTBAP. Relative growth is compared
to the PBS control, which is set at 100%. Data from three independent experiments were analyzed by analysis of variance and
by Student–Neuman–Keuls (a¼0.05) for all pairwise comparisons. No significant differences among treatment groups and
controls were observed. MnTBAP, Mn(III) tetrakis(4-benzoic acid) porphyrin chloride; PBS, phosphate-buffered saline.
Provision of insulin significantly increased Plasmodium falciparum oocyst numbers, whereas the antioxidant
946 SURACHETPONG ET AL.
mosquitoes were significantly higher than oocyst numbers in
buffer-fed controls (Fig. 1A). Mosquitoes fed insulin in the
presence of MnTBAP had significantly fewer parasites than
did mosquitoes fed insulin alone. Additionally, mosquitoes
fed MnTBAP alone had significantly decreased oocyst num-
bers relative to insulin-fed mosquitoes, but these levels were
not significantly different from buffer-fed controls. Infection
prevalence was not affected by treatment (Fig. 1B). One ex-
planation for these results is that human insulin could en-
hance the growth of P. falciparum parasites directly in a
manner analogous to human insulin=IGF-1 growth induction
of Toxoplasma and Leishmania (10, 45). To test this possibility,
we examined the effects of increasing concentrations of
human insulin on the growth of synchronized asexual-stage
P. falciparum parasites in vitro. At all concentrations, including
those used for our in vitro assays (1.7mM) and our in vivo
assays (170 pM), human insulin had no significant effect on
parasite growth (Fig. 1C). We also examined the effects of
MnTBAP on P. falciparum growth and, although the highest
concentration (50mM) appeared to be toxic to parasites, the
concentration used in our feeding studies (5mM) had no sig-
nificant effect on parasite growth (Fig. 1D). These results in-
dicated that neither insulin nor MnTBAP had a direct positive
effect on parasite growth in the mosquito.
Human insulin does not alter the levels
of oxidative damage in the mosquito midgut
In previous studies, we demonstrated that blood meals
supplemented with human insulin significantly reduced A.
stephensi lifespan and that these effects were reversed by
provision of the antioxidant MnTBAP (14), suggesting that
ingested human insulin acts via the synthesis of ROS in
mosquito midguts. Based on these findings and the increased
parasite development observed in Figure 1A, we hypothe-
sized that higher levels of insulin-induced ROS may lead to
increased oxidative damage in the midgut that could facilitate
the establishment of Plasmodium infection. Such a finding
would be analogous to observations that increased damage of
the human intestinal barrier has been correlated with in-
creased bacterial translocation and disease progression in
other infections, such as HIV (4).
We quantified levels of oxidative damage in the midgut as
Midguts from A. stephensi were isolated at 0, 24, 48, and 72h
after a single blood meal supplemented with 170 pM human
insulin, with 5mM MnTBAP, with insulin plus MnTBAP, or
with an equivalent volume of buffer as a control. All groups
had increased PCC levels at 24h after blood feeding (Fig. 2),
an effect that was most likely due to the process of blood
digestion, which is known to increase ROS levels (17). How-
ever, none of the treatments were significantly different from
the buffer controls at any timepoint (Fig. 2). Further, any in-
crease in PCC in the controls was not reduced by provision of
the antioxidant MnTBAP (Fig. 2). As such it did not appear
more permissive to invading parasites. It also did not appear
that insulin-induced ROS were lethal to parasites, since we
observed significantly higher oocyst development in the
presence of insulin (Fig. 1A). Therefore, we hypothesized that
the increased parasite development observed in Figure 1A in
response to ingested human insulin resulted from the alter-
ation of immune signaling pathways that indirectly regulate
ROS are important mediators of the IIS cascade
In our system, insulin-induced ROS did not appear to be
directly deleterious to oocyst development, nor did they in-
crease oxidative protein damage in the mosquito midgut.
Therefore, we sought to determine whether insulin-induced
ROS could modify immune signaling through the IIS path-
way. Based on our previously published protocols (14), we
treated A. stephensi cells with 1.7mM human insulin in the
presence or absence of 5mM MnTBAP and examined the
phosphorylation of downstream signaling proteins of the IIS
pathway. Although immortalized mosquito cells provide a
useful model for studying cell signaling, they are not identical
to the highly specialized epithelial cells present in the midgut
of mosquitoes and as such require the use of a higher con-
centration of human insulin. We found that in accordance
with our previously published work (14, 24), insulin-treated
mosquito cells had higher MEK, ERK, and p38 phosphoryla-
tionrelativetoPBS-treatedcontrols (Fig.3A,C).Inaddition to
the effects on MEK, ERK, and p38, insulin significantly in-
duced p70S6K and FOXO phosphorylation, downstream
signaling targets of the PI3K=Akt signaling arm of the IIS
pathway (Fig. 3B, D). When cells were pretreated with the
midgut proteins through 72h after feeding. Mosquitoes
were fed on blood supplemented with PBS, 5mM MnTBAP
(MnT), 170 pM human insulin, or MnTBAP plus human
insulin. Midgut protein damage was quantified as total
protein carbonyl content (PCC) in midgut tissue pooled from
30 mosquitoes at 0, 24, 48, and 72h postblood feeding and
normalized to total protein content in each sample. Fold
changes in PCC levels relative to time 0 are shown. Data are
represented as means?SEMs from four independent ex-
periments. No significant differences among treatment
groups and controls were observed. ROS, reactive oxygen
species. SEM, standard error of the mean.
Insulin-induced ROS did not damage mosquito
ROS-DEPENDENT CELL SIGNALING IN MOSQUITOES947
reduced insulin-induced MAPK and PI3K=Akt signaling. ASE cells were treated with 5mM MnTBAP (MnT) for 40min before
stimulation with 1.7mM human insulin for 5min. Cell lysates were analyzed by western blotting with anti-phospho-specific
antibodies. The effects of MnTBAP pretreatment on MEK, ERK, and p38 phosphorylation (A, C), as well as FOXO and
p70S6K phosphorylation (B, D) are shown. Fold changes in phospho-specific proteins in pairwise comparisons of treatments
or of treatments and PBS-treated controls from three independent experiments were analyzed with Student’s t-test (a¼0.05).
GAPDH provided an assessment of protein loading and were used to normalize corresponding phospho-protein levels. Data
are represented as means?SEMs and significant differences are indicated. MAPK, mitogen-activated protein kinase; PI3K,
phosphatidylinositol 3-kinase; ERK, extracellular signal-regulated kinase; FOXO, forkhead box O1; MEK, mitogen activated
protein kinase kinase.
ROS mediate insulin signaling in A. stephensi cells in vitro. (A) Pretreatment with the antioxidant MnTBAP
948SURACHETPONG ET AL.
antioxidant MnTBAP, phosphorylation of MEK, ERK, and
p38 as well as FOXO and p70S6K was reduced relative to the
controls (Fig. 3), suggesting that ROS participate in the IIS
cascade in vitro.
A. stephensi mosquitoes were fed artificial blood meals con-
insulin plus 5mM MnTBAP, or MnTBAP alone. In accordance
withour previously publisheddata(14,24) andinvitro results
(Fig. 3), we found that ingested human insulin significantly
increased the phosphorylation of both ERK and FOXO in
mosquito midgut tissues at 30min after bloodfeeding (Fig. 4).
When mosquitoes were fed blood meals containing human
insulin in the presence of MnTBAP, phosphorylation of both
ERK and FOXO was significantly decreased relative to mos-
quitoes fed insulin alone (Fig. 4). Intriguingly, MnTBAP alone
significantly increased pERK relative to control levels, sug-
gesting that basal levels of ROS keep activation of this heavily
networked signaling protein in check. In general, these data
indicate that ROS are important mediators of both the MAPK
and PI3K=Akt branches of the IIS cascade in the mosquito
midgut epithelium, a key interface between malaria parasites
and the mosquito immune response.
ROS alone can activate MAPK and PI3K=Akt
signaling pathways in Anopheles cells
While the effects of MnTBAP indirectly implicated ROS in
IIS in A. stephensi (Figs. 3 and 4), it remained unclear whether
ROS could directly participate in MAPK and PI3K=Akt sig-
naling. To determine whether ROS had a direct effect on cell
were treated with increasing doses of H2O2and activation of
downstream signaling proteins was assessed by western
blotting. Although H2O2is the most stable ROS commonly
used to study cell signaling (41), our time–course analyses
revealed that maximum ERK phosphorylation occurred at
5min and then gradually declined after H2O2 treatment
(Supplementary Fig. S1; Supplementary Data are available
online at www.liebertonline.com=ars), suggesting that sig-
naling by H2O2peaks very rapidly in mosquito cells.
and p38 phosphorylation in two A. stephensi cell lines (Fig. 5)
and in A. gambiae cells (Supplementary Fig. S2). Notably,
pretreatment with the antioxidant enzyme catalase signifi-
cantly reduced H2O2-induced ERK and p38 phosphorylation
in A. stephensi ASE cells (Fig. 5A, B) and MSQ43 cells (Fig. 5C,
D), and showed a similar pattern in A. gambiae 4a3B cells
(Supplementary Fig. S2). Although MEK phosphorylation
levelsdidnot changesignificantly after catalasetreatment,we
have previously shown that small alterations in phosphory-
lated MEK levels, such as those observed here, can lead to
significant differences in ERK activation and are, therefore,
biologically significant (42). In a manner similar to MAPK
activation, H2O2also dose dependently induced FOXO and
p70S6K phosphorylation (Fig. 5E and Supplementary Fig. S3)
and this increase was reduced by catalase pretreatment (Fig.
5E and Supplementary Fig. S3). Collectively, these data
demonstrated that ROS alone can activate both the MAPK
and PI3K=Akt signaling pathways, suggesting that ROS play
a critical role in mosquito cell signaling.
In previous studies, we showed that insulin-induced acti-
vation of MAPK, but not PI3K=Akt signaling, was associated
with increased expression of A. stephensi NOS (24), an effector
gene that contributes to the control of malaria parasite de-
velopment (16, 18, 32). We have shown here that ROS alone
can activate the MAPK pathway. Therefore, we sought to
determine whether H2O2could mediate the expression of
induced NOS expression in A. stephensi cells, with the highest
induction occurring at 500mM H2O2although these trends
were not significant (Fig. 5F). H2O2also induced NOS ex-
pression in A. gambiae cells (Supplementary Fig. S4). Catalase
pretreatment reduced H2O2-dependent NOS expression in
S4), suggesting that ROS can regulate anti-parasite immune
responses as well as cell signaling in mosquito cells.
ROS are necessary for insulin signaling,
but not TGF-beta1 signaling, in mosquito cells
ROS have been shown to be necessary and important me-
diators of both the IIS and TGF-beta1 signaling pathways in a
variety of organisms. We have previously shown that both
human TGF-beta1 and human insulin can activate the MAPK
pathway and induce NOS gene expression in mosquito cells
(14, 24, 42). Therefore, we presumed that TGF-beta1 signaling
lin signaling in the A. ste-
phensi midgut epithelium in
vivo. Mosquitoes were fed
with PBS as a control, 170 pM
MnTBAP. (A) Representative
western blots of pERK and
pFOXO. (B) Fold changes in
phospho-specific proteins in
treatments or of treatments
and controls from three inde-
pendent experiments were analyzed with Student’s t-test (a¼0.05). GAPDH levels provided an assessment of protein
loading and were used to normalize corresponding phospho-protein levels. Data are represented as means?SEMs and
significant differences are indicated.
ROS mediate insu-
ROS-DEPENDENT CELL SIGNALING IN MOSQUITOES 949
induced MEK, ERK, and p38 phosphorylation in A. stephensi ASE cells (A, B) and MSQ43 cells (C, D). For catalase treatment,
cells were pretreated with 200 units=ml of catalase for 40min before treatment with increased doses of H2O2. Blots are
representative of three independent experiments. Fold changes of pMEK, pERK, and p-p38 treated with 100 and 500mM H2O2
in ASE cells (B) and 100mM H2O2in MSQ43 cells (D) are represented relative to paired PBS controls. Pairwise comparisons of
treatments were analyzed by Student’s t-test (a¼0.05). Data are represented as means?SEMs from three independent
experiments and significant differences within treatment pairs are indicated. (E) Increasing doses of H2O2activated FOXO
and p70S6K phosphorylation in ASE cells and pretreatment with catalase at 200 units=ml for 40min before H2O2treatment
reduced this phosphorylation. Blots are representative of three independent experiments. Fold changes of pFOXO and
p-p70S6K are shown in Supplementary Figure S3. (F) NOS gene expression was analyzed in ASE cells by quantitative reverse
transcriptase–polymerase chain reaction at 24h post-treatment. Fold inductions of NOS relative to paired controls from three
independent experiments, represented as means?SEMs, were analyzed by Student’s t-test (a¼0.05). No significant differ-
ences within treatment pairs were observed. NOS, nitric oxide synthase; H2O2, hydrogen peroxide.
ROS directly activated MAPK and PI3K=Akt signaling pathways in A. stephensi cells in vitro. Exogenous H2O2
950SURACHETPONG ET AL.
of ROS in these pathways. We found that pretreatment of cells
and p38 phosphorylation but had no impact on TGF-beta1
also reduced insulin-induced activation of FOXO and p70S6K
phosphorylation (Fig. 6B and Supplementary Fig. S6). Our
previous observations that ROS levels are increased in insulin-
treated mosquito cells (14) are consistent with the function of
did not induce significant ROS production in A. stephensi or in
A. gambiae cells (data not shown), consistent with that the fact
that ROS are not involved in TGF-beta1 signaling (Fig. 6 and
Supplementary Fig.S5).Takentogether,our findings indicated
signaling, in mosquito cells.
Overexpression of AsPrx reduced insulin
signaling and NOS expression
Thus far, we used exogenous MnTBAP and catalase to at-
tribute a function for ROS in mosquito cell signaling. We
previously showed that the endogenous AsPrx can protect
mosquito cells from oxidative damage (33). Therefore, we
ROS-mediated IIS in mosquito cells. To this end, we over-
expressed AsPrx and analyzed the subsequent effects on cell
signaling. The expression of full-length AsPrx protein was
naling response of A. stephensi
cells in vitro to human insulin
but not to human TGF-beta1.
(A) ASE cells were stimulated
with 6000pg=ml human TGF-
beta1 or 1.7mM human insulin
for 5min. To determine the effect
of ROS on signaling protein ac-
tivation, cells were pretreated
with 200 units=ml catalase for
40min before stimulation with
each treatment. Blots are repre-
sentative of three independent
experiments. (B) ASE cells were
treated with 200 units=ml cata-
lase for 40min before treatment
with 1.7mM of human insulin.
Representative blots of FOXO
from two experiments
shown. Fold changes of pFOXO
and p-p70S6K are shown in
Supplementary Figure S6. (C)
Relative fold changes of pMEK,
pERK, and p-p38 were normal-
ized to total ERK or GAPDH and
are shown relative to paired PBS
controls. Pairwise comparisons
of treatments were analyzed by
Student’s t-test (a¼0.05). Data
are represented as means?SEMs
from three independent experi-
within treatment pairs are indi-
cated. TGF, transforming growth
ROS mediated the sig-
ROS-DEPENDENT CELL SIGNALING IN MOSQUITOES951
vitro to human insulin but not to human TGF-beta1. ASE cells were transfected with plasmid TLP-58 encoding a constitutively
expressed AsPrx and were stimulated with 6000pg=ml human TGF- beta1, 1.7mM human insulin, or 500mM H2O2for 5min (32).
(A) Overexpressed 2-cys Prx was detected with anti-V5 antisera (upper panel) at 48h after transfection. Disulfide bonding
required for homodimerization and, hence, catalytic activity was confirmed (lower panel) under reducing conditions with
dithiothreitol (DTT). (B) Phosphorylation of MEK, ERK, and p38 by insulin and H2O2, but not by TGF-beta1, was decreased in
cells overexpressing AsPrx indicated as ‘‘Prxþ.’’ In the absence of copper stimulation, no AsPrx was expressed in cells transfected
with the copper-inducible plasmid TLP-55, so these controls are indicated as ‘‘Prx?.’’ Blots are representative of three independent
experiments. (C) Fold changes of pMEK, pERK, and p-p38 were normalized to total ERK or GAPDH and are shown relative to
paired PBS-treated control cells transfected with TLP-55. Pairwise comparisons of treatments were analyzed by Student’s t-test
(a¼0.05). Data are represented as means?SEMs from three independent experiments. Significant differences within treatment
pairs are indicated. (D) Overexpressed AsPrx reduced insulin induction of FOXO and p70S6K phosphorylation. Blots are rep-
resentative of two independent experiments. Fold changes of pFOXO and p-p70S6K are shown in Supplementary Figure S7. (E)
ASE cells were transfected with TLP-58 for 48h, and then stimulated with 1.7mM human insulin for 24 or 48h before RNA
isolation. NOS gene expression was analyzed with quantitative reverse transcriptase–polymerase chain reaction. Fold inductions
of NOS relative to paired TLP-55 transfected controls are shown from three independent experiments, represented as means?
SEMs, and were analyzed by Student’s t-test (a¼0.05). No significant differences within treatment pairs were observed.
Overexpression of A. stephensi 2-cys peroxiredoxin (AsPrx) reduced the signaling response of A. stephensi cells in
confirmed using antisera to the V5 tag protein (Fig. 7A).
Disulfide bonding required for AsPrx homodimerization and,
hence, catalytic activity was confirmed under reducing con-
ditions (Fig. 7A). Transfected cells were stimulated with
human TGF-beta1, human insulin, or H2O2. Similar to our
results with MnTBAP and catalase, cells stimulated with in-
sulin and H2O2, but not TGF-beta1 that also overexpressed
AsPrx exhibited reduced activation of MEK, ERK, and p38
relative to cells lacking AsPrx (Fig. 7B, C). Cells over-
expressing AsPrx also trended toward a reduction of insulin-
induced phosphorylation of FOXO and p70S6K (Fig. 7D and
Supplementary Fig. S7). Although not statistically significant,
overexpression of AsPrx also reduced NOS expression by 1.6-
fold at 24h and by 1.4-fold at 48h after insulin treatment
(Fig. 7E). Taken together, these data suggested that insulin-
induced ROS levels in mosquito cells can be regulated by
endogenous antioxidants, such as AsPrx.
We have shown that ingested insulin is beneficial for
P. falciparum oocyst development in A. stephensi and that this
this beneficial effect of insulin contrasts with the detrimental
effects of Akt overexpression on P. falciparum oocyst develop-
ment in A. stephensi (6), suggesting that the effects of insulin
activation of IIS are complex and distinct from those mediated
by overexpression of the single IIS protein Akt. The effects of
insulin-induced ROS do not appear to be mediated by in-
creased permeability ofthe midgut epithelialbarrier as a result
of ROS-induced damage but rather they are a consequence of
ROS-dependent signaling in Anophelesmosquitoes. In vitro, the
trend toward increased expression of the anti-parasite effector
gene NOS at 24h appeared to be partly dependent on insulin-
induced ROS. In vivo, we have shown previously that induc-
which P. falciparum oocyst formation is largely completed (3).
Thus, the timing of signaling events observed here in vitro and
in vivo would suggest that IIS-induced ROS regulate addi-
tional, earlier responses independent of NOS to govern oocyst
development. Indeed, given the negative effects of ROS scav-
enging on P. falciparum oocyst development, we propose that
early regulation by IIS-induced ROS may prime an anti-
inflammatory state in which parasite development is favored.
In support of our observations and hypotheses, the IIS
pathway and IIS-induced ROS-dependent signaling (2) have
been shown to broadly regulate the innate immune responses
ofa variety oforganisms.Inparticular, thebalance ofsignaling
among the two IIS branches can dictate the biological effects of
IIS.The PI3K=Akt branchoftheIISpathway hasbeenlinkedto
anti-inflammatory responses in mammals (11). In contrast, the
MAPK branch of the IIS pathway has been shown to have pro-
inflammatory effects (11). Elevated insulin levels and IIS have
been correlatedwith therelease ofpro-inflammatorycytokines
and chemokines (37). It is possible, therefore, that the MAPK
and PI3K=Akt branches of IIS have distinct and opposing ef-
fects on the mosquito innate immune response. Based on the
above observations and our in vivo results (Fig. 4), we are
currently analyzing the kinetics of IIS in our mosquito hosts to
fully elucidatethe resultanttemporalandimmunemodulatory
effects of human insulin on Plasmodium development.
We have demonstrated that the requirement for ROS is
specific to IIS since scavenging of ROS had no effect on TGF-
beta1-dependent MAPK activation (Fig. 6). In mammalian
cells, ROS have been shown to play a critical role in TGF-beta1
of highly conserved components to support TGF-beta1 sig-
naling (23), our data suggest thatdivergent, ROS-independent
mechanisms regulate TGF-beta1 signaling in mosquito cells.
Although the exact mechanism by which ROS regulate IIS in
mosquito cells remains to be determined, multiple mecha-
nisms for ROS facilitation of both the MAPK- and PI3K=Akt-
dependent signaling branches of the IIS cascade have been
activity of PTEN, a known inhibitor of the IIS pathway (22).
Alternatively, ROS can stimulate protein kinase C (PKC) ac-
tivity through the oxidative modification of PKC regulatory
domains (9). Several PKC isoforms have been shown to be
involved in the activation of the IIS cascade in mammalian
cells (30, 36). Mosquitoes possess many of the conserved
components of the IIS cascade, including PTEN and PKC
isoforms (35), so ROS-dependent IIS regulation may occur
through these signaling proteins in mosquito cells.
naling in mosquitoes. (1) Human insulin signals in the
mosquito midgut, (2) inducing the phosphorylation of MEK
and Akt. (3) Activated MEK and Akt phosphorylate down-
stream effectors such as ERK and FOXO. (4) This signaling
leads to increased ROS, which can positively feed back into
the insulin=IGF-1 signaling pathway, increasing the phos-
phorylation of downstream effectors such as ERK and
FOXO. (5) Ultimately, insulin=IGF-1 signaling, acting in part
through increased ROS levels, leads to a decrease in the
mosquito immune response and (6) a subsequent increase in
parasite development in the mosquito midgut epithelium.
Proposed model of ROS-mediated insulin sig-
ROS-DEPENDENT CELL SIGNALING IN MOSQUITOES953
The mosquito midgut is a key interface between the vector,
human host, and the malaria parasite and the sum of these
interactions determines the ability of mosquitoes to transmit
malaria. In this study we show that insulin-induced ROS are
critical mediators of the MAPK- and PI3K=Akt-dependent
signaling branches of the IIS cascade and of P. falciparum de-
velopment in A. stephensi (Fig. 8). Identifying the mechanisms
whereby IIS regulates innate immunity has obvious practical
value for understanding of mosquito physiology, but this
knowledge can also provide insights into the evolution of IIS
regulation in higher organisms. Further, selective targeting of
critical mediators of signaling pathways in the mosquito
midgut can facilitate discovery of novel suites of anti-parasite
effector genes that can be manipulated to develop mosquitoes
that are refractory to malaria infection.
We would like to thank Dr. Edwin E. Lewis for assistance
with statistical analyses and Hannah Smithers for critical re-
view ofthearticle.We would alsolike tothank Laura Dickson
of protein oxidation. Funding for these studies was provided
by NIH NIAID AI073745 and AI080799 and with facilities
improvement support by NIH NCRR C06 RR-12088–01.
Author Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Dr. Shirley Luckhart
Department of Medical Microbiology and Immunology
University of California, Davis
3437 Tupper Hall
One Shields Ave.
Davis, CA 95616
Date of first submission to ARS Central, June 17, 2010; date of
final revised submission, December 1, 2010; date of accep-
tance, December 2, 2010.
ASE¼A. stephensi embryo-derived
AsPrx¼A. stephensi 2-cys peroxiredoxin
ERK¼extracellular signal-regulated kinase
FOXO¼forkhead box O1
MAPK¼mitogen-activated protein kinase
MEK¼mitogen activated protein kinase kinase
MnTBAP¼Mn(III) tetrakis(4-benzoic acid)
NOS¼nitric oxide synthase
p38¼P38 mitogen-activated protein kinase
PCC¼protein carbonyl content
PKC¼protein kinase C
RBCs¼red blood cells
ROS¼reactive oxygen species
SEM¼standard error of the mean
TGF-beta1¼transforming growth factor-beta1
ROS-DEPENDENT CELL SIGNALING IN MOSQUITOES955