Abscisic acid induces a transient shift in signaling that enhances NF-κB-mediated parasite killing in the midgut of Anopheles stephensi without reducing lifespan or fecundity
© The Author(s). 2017
Received: 23 March 2017
Accepted: 6 July 2017
Published: 13 July 2017
Abscisic acid (ABA) is naturally present in mammalian blood and circulating levels can be increased by oral supplementation. We showed previously that oral ABA supplementation in a mouse model of Plasmodium yoelii 17XNL infection reduced parasitemia and gametocytemia, spleen and liver pathology, and parasite transmission to the mosquito Anopheles stephensi fed on these mice. Treatment of cultured Plasmodium falciparum with ABA at levels detected in our model had no effects on asexual growth or gametocyte formation in vitro. However, ABA treatment of cultured P. falciparum immediately prior to mosquito feeding significantly reduced oocyst development in A. stephensi via ABA-dependent synthesis of nitric oxide (NO) in the mosquito midgut.
Here we describe the mechanisms of effects of ABA on mosquito physiology, which are dependent on phosphorylation of TGF-β-activated kinase 1 (TAK1) and associated with changes in homeostatic gene expression and activity of kinases that are central to metabolic regulation in the midgut epithelium. Collectively, the timing of these effects suggests a transient physiological shift that enhances NF-κB-dependent innate immunity without significantly altering mosquito lifespan or fecundity.
ABA is a highly conserved regulator of immune and metabolic homeostasis within the malaria vector A. stephensi with potential as a transmission-blocking supplemental treatment.
We showed previously that oral supplementation of mice with the isoprenoid abscisic acid (ABA) increased circulating levels of ABA and reduced parasitemia, gametocytemia, and disease pathology as well as transmission of Plasmodium yoelii 17XNL to Anopheles stephensi . In parallel studies, ABA had no direct effects on growth of cultured P. falciparum asexual stages or gametocyte development in vitro. In A. stephensi, ABA ingestion modestly induced expression of two antimicrobial peptide genes and markedly (13-fold) induced nitric oxide synthase (nos) midgut transcript levels in mosquitoes fed on supplemented P. falciparum culture relative to controls at 4–6 h post-feeding. Addition of the NOS inhibitor L-NAME confirmed that ABA-dependent NO synthesis in the mosquito midgut eliminated parasites prior to oocyst formation . Given that A. stephensi NO synthesis is regulated by multiple signaling pathways with broad effects on mosquito physiology [2–4], the timing and mechanism(s) whereby ABA induces NO synthesis could provide significant insights into the potential effects of ABA on other life history traits that are important to vectorial capacity, including lifespan and egg production .
ABA is an ancient signaling molecule, with known biology in plants, invertebrates, and mammals, likely acting through pathways that have been conserved over evolutionary time. In mammalian studies, ABA has been shown to stimulate insulin release from pancreatic beta cells . In A. stephensi, the insulin- and insulin-like growth factor (IIS) pathway can coordinately regulate anti-parasite defenses as well as mosquito lifespan and reproduction [7–13]. These effects are due, in part, to IIS-dependent changes in midgut intermediary metabolism, mitochondrial function, and epithelial homeostasis [10, 11, 13, 14]. ABA signaling in plants has been associated with activation of mitogen-activated protein kinases (MAPKs), which regulate pathogen sensing and developmental processes as well as senescence [15, 16]. Notably, nearly 25% of the 1500 ABA-regulated genes in plants derive from ABA-dependent activation of a single MAP2K known as MKK3 . ABA signaling also impacts intermediary metabolism in plants, with increasing evidence for anterograde and retrograde signaling as well as changes in mitochondrial function that can impact plant defenses [17–19]. In A. stephensi, p38 MAPK and extracellular signal-regulated kinase (ERK MAPK) are involved in pathogen sensing and NOS activation [20, 21]. Both ERK and p38 MAPK are known to be regulated by transforming growth factor-β-associated kinase 1 (TAK1), a MAP3K that also regulates NOS activation in A. stephensi [22–25]. Given that IIS and MAPK signaling control metabolism and mitochondrial function in A. stephensi, ABA signaling through these pathways could coordinately regulate defense, lifespan, and reproduction in the mosquito host.
Lifespan is a determinant of the total number of eggs laid by a female mosquito and the probability that she will survive the extrinsic incubation period, or the time required for development of infectious sporozoites, following a blood meal. Changes in mosquito lifespan have a fourth order effect on vectorial capacity, with senescence notably increasing this effect size [5, 26, 27]. Reduced egg production yields fewer adults in the next generation, which impacts biting rate and, therefore, parasite transmission. Accordingly, we sought to characterize ABA signaling in A. stephensi and the life history traits that are connected by these cellular pathways to better understand the effects of ABA on this important vector species.
Reagents and chemicals
Anti-phospho-AMPKα Thr172 (#2535), phospho-TAK1 Thr184 (#4537), phospho-GSK-3α/β Ser21/9 (#9331), and phospho-Akt Ser473 (#9271) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-phospho-p70S6K Thr412 (#07–018) and phospho-FOXO Thr32 (#07–694) were purchased from Millipore (Billerica, MA, USA). Anti-phospho-ERK (#M8159) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-phospho-JNK (#44-682G) was purchased from Biosource (Carlsbad, CA, USA). Anti-phospho-p38 MAPK Thr180/Tyr182 (#10009177) was purchased from Cayman Chemical. Anti-GAPDH (#ab36840) was purchased from Abcam (Cambridge, MA, USA). Goat anti-rabbit secondary antibody (#ALI4404) was purchased from Biosource. Rabbit anti-mouse secondary antibody (#A9044) was purchased from Sigma-Aldrich. Morpholinos were purchased from Gene Tools, LLC (Philomath, OR, USA).
Plasmodium falciparum infections
Three- to five-day-old A. stephensi were provided with P. falciparum-infected blood containing 100 nM ABA or a diluent control as previously described . Non-engorged individuals were removed immediately after feeding. Midguts were dissected at various time points for gene expression and protein analysis. Oocysts were counted on dissected midguts stained with 0.5% mercurochrome at 10 days post-feeding.
For TAK1-knockdown experiments mosquitoes were fed a saline-ATP meal with 10 μM A. stephensi TAK1-targeted (5′-GAT CCT TAT TAC GTT TCG CTT CGT A-3′) or control human beta-globin-targeted (5′-CCT CTT ACC TCA GTT ACA ATT TAT A-3′) vivo morpholinos as previously described 3 days before being provided with a P. falciparum-infected blood meal .
Transcript levels of P. falciparum a18s rDNA, pfs16 and pfs25 in infected mosquito midguts were determined by qRT-PCR . Data were normalized to transcript levels for A. stephensi ribosomal s7 protein, a18s rDNA, and control levels as previously described . nos, defensin, apl1, tep1, and lrim transcript levels were determined by qRT-PCR and analyzed as described previously .
Western blotting assays
Western blots were prepared and analyzed as described . Briefly, proteins were extracted from 10 pooled midguts, separated by gel electrophoresis on a 10% sodium dodecyl sulfate-polyacrylamide gel, transferred onto a nitrocellulose membrane (BioRad, Hercules, CA, USA) and blocked in 5% dry milk in Tris-buffered saline-0.1% Tween 20 (TBST) for 1 h at room temperature. Membranes were incubated overnight at 4 °C in primary (1°) and secondary (2°) antibodies diluted in 5% non-fat dry milk/TBST as follows: 1:1000 phospho-TAK1 and 1:10,000 goat anti-rabbit IgG; 1:1000 phospho-GSK-3 and 1;10,000 goat anti-rabbit IgG; 1:1000 phospho-p70S6K and 1:2000 goat anti-rabbit IgG; 1:1000 phospho-FOXO and 1:2000 goat anti-rabbit IgG; 1:1000 phospho-Akt and 1:5000 goat-anti-rabbit IgG; 1:10,000 phospho-ERK and 1:20,000 rabbit anti-mouse IgG; 1:1250 phospho-JNK and 1:20,000 goat anti-rabbit IgG; 1:1250 phospho-p38 MAPK and 1:20,000 goat-anti-rabbit IgG; 1:10,000 anti-GAPDH and 1:20,000 goat-anti-rabbit IgG. For detection of phospho-AMPK, membranes were incubated in 1:1000 goat anti-rabbit IgG in 5% milk (TBST) for 2 h at room temperature before imaging. All data were normalized to GAPDH levels in the same samples and to target protein levels in matched control midguts.
Lifespan and fecundity measurements
Uninfected female A. stephensi were given the opportunity to bloodfeed once per week on a 1:1 vol: vol mixture of washed human red blood cells (RBCs) and phosphate-buffered saline (PBS) supplemented with 100 nM ABA or with an equivalent volume of diluent control. Mosquitoes were maintained on 10% sucrose solution and allowed to oviposit once per week. Dead individuals were removed and counted three times per week. For analyses of lifespan under nutrient stress, mosquitoes were treated as above but maintained on 3% sucrose solution between weekly blood meals.
To analyze the effects of ABA on infected lifespan, 3–5 day old mosquitoes were provided with a P. falciparum-infected blood meal supplemented with 100 nM ABA or with an equivalent volume of diluent control. Mosquitoes that did not fully engorge were removed from the cartons. For the remainder of the infected lifespan study, mosquitoes were given the opportunity to feed once per week on uninfected blood with or without ABA. Mosquitoes were allowed to oviposit once per week and maintained on 10% sucrose solution between weekly blood meals. Dead individuals were counted every 24 h and each dead individual was placed in Trizol for subsequent RNA isolation. Infection status of each mosquito was determined by detection of transcript levels of P. falciparum mitochondrial cytochrome c oxidase subunit 1 (Pfcox1) gene (forward: 5′-TGC CAG GAT TAT TCG GAG GA-3′; reverse: 5′-CCA TCC AGT TCC ACC ACC AA-3′) by qRT-PCR .
For fecundity studies, 3–5 day old female A. stephensi were fed on uninfected RBCs in PBS (1:1 vol: vol) supplemented with or without 100 nM ABA. Fully engorged females were housed individually and provided oviposition cups at 2 days post-feeding. Two days after the provision of oviposition cups, eggs were counted and prepared for hatching in individual water cups. First instar larvae were provided with a 2% solution of 2:1 Sera Micron® powdered fish food (Sera North America, Montgomeryville, PA, USA) and baker’s yeast. First instar larvae were counted at 2 days after hatching.
Levels of phosphorylated proteins were analyzed by Student’s t-test. Nos transcript levels were analyzed by Wilcoxon matched-pairs signed rank test. All other qRT-PCR data were analyzed by Student’s t-test. Infection prevalence, egg laying rate, and egg hatch rate were analyzed using Fisher’s exact test. Median survival rate and clutch size were analyzed by Wilcoxon matched-pairs signed rank test and Student’s t-test, respectively. Lifespan data were analyzed using log rank test and Gehan-Breslow-Wilcoxon test. Differences were considered significant at P < 0.05.
Sexual stage commitment of P. falciparum is restricted by ABA treatment within 24 h of mosquito infection
ABA reduced Plasmodium infection in the mosquito by increasing TAK1-regulated host immunity
To determine whether TAK1 phosphorylation mediated ABA-dependent immune gene expression and the effects of ABA on P. falciparum infection, we used a knockdown strategy based on supplementation of an infected blood meal with TAK1-targeted morpholino. Although phosphorylated TAK1 is expected to constitute a relatively small proportion of total TAK1 protein, TAK1 morpholino-treated, infected A. stephensi had significantly reduced midgut phospho-TAK1 levels in response to ABA relative to infected mosquitoes treated with control morpholino (Mann-Whitney test, U = 1, n 1 = 4, n 2 = 4, P = 0.029) (Fig. 4b), confirming TAK1 knockdown. In TAK1 morpholino-treated, infected mosquitoes, ABA-dependent nos expression trended downward (Wilcoxon test, W = -10, P = 0.06) (Fig. 4c) and expression levels of apl1 (t = 4.074, df = 4, P = 0.008), lrim (t = 2.23, df = 4, P = 0.023), and tep1 (t = 3.02, df = 4, P = 0.039) were significantly reduced relative to controls by 6–8 h post-infection (Fig. 4d). defensin expression, which was only modestly induced by ABA in earlier studies, was not altered by TAK1 knockdown (Fig. 4d).
ABA transiently altered metabolism and homeostasis in the mosquito midgut
GSK-3 functions as a monitor of cellular energy status and under energy-rich conditions promotes dephosphorylation of AMP kinase (AMPK) at Thr172 , which inhibits AMPK activity. Inhibition of AMPK prevents the activation of catabolic pathways that generate ATP, the induction of mitochondrial biogenesis, and the inhibition of biosynthetic pathways that consume ATP. Accordingly, we examined AMPK in the midgut and found that Thr172 phosphorylation was significantly reduced in ABA-treated A. stephensi relative to controls at both 30 min (t = 3.145, df = 5, P = 0.0175) and 4 h post-feeding (t = 3.852, df = 2, P = 0.03) (Fig. 6, Additional file 1: Figure S1). Sustained inactivation or deficiency of AMPK promotes mitochondrial dysfunction, which can lead to elevated levels of mitochondrial reactive oxygen species (ROS) and inflammatory levels of NO [41, 42]. ABA-TAK1 signaling (Fig. 4) and AMPK inhibition (Fig. 6), therefore, could additively generate high levels of NO that, based on our previous observations , would be damaging to cell health and autophagic repair in the midgut epithelium.
ABA does not alter mosquito lifespan or fecundity
ABA supplementation did not alter A. stephensi lifespan. Median survival times of control and ABA-treated mosquitoes are shown from five lifespans in which mosquitoes received a weekly uninfected blood meal with or without ABA supplementation. Differences in lifespan between treatment groups were analyzed by log-rank test and Gehan-Breslow-Wilcoxon test
Median survival (days)
Extension of infected lifespan can increase parasite transmission, but the relationship between age-dependent mortality and infection is complex . Accordingly, we conducted a lifespan study in which the first blood meal contained P. falciparum-infected RBCs with and without 100 nM ABA. Subsequent weekly blood meals were uninfected with and without ABA. As individual mosquitoes died they were collected and tested for parasite infection by detection of Pfcox1 by qRT-PCR. Mosquitoes that did not have detectable levels of Pfcox1 were considered uninfected and not included in the analysis. Even in the presence of parasite infection, however, ABA supplementation had no significant effect on the lifespan of A. stephensi (Fig. 9b).
Within 30 min to 1 h post-feeding, ABA induced changes in phosphorylation and activity of major immune and metabolic regulators in the mosquito midgut, including TAK1, AMPK, and GSK-3. ABA supplementation increased levels of phosphorylated TAK1, a positive regulator of NF-κB signaling, and TAK1 knockdown in the mosquito midgut abrogated the effects of ABA on immune gene expression and P. falciparum infection prevalence. These data support the hypothesis that ABA enhances anti-parasite defenses by increasing TAK1-dependent expression of NF-κB-regulated immune genes, including nos. These data are also consistent with findings in mammalian cells, where ABA treatment can enhance NF-κB nuclear translocation and transcriptional activity in multiple cell types [53–55].
The changes in metabolic protein activity and gene expression in response to ABA suggest a temporary shift in bioenergetic regulation within the midgut that facilitates and perhaps enhances parasite clearance. Specifically, a reduction in AMPK phosphorylation within 30 min post-ingestion of ABA can promote mitochondrial NO production [41, 42], an effective anti-parasite defense in A. stephensi . Further, ABA-dependent activation of GSK3 by 1 h post-feeding would be predicted to sustain dephosphorylation of AMPK, enhancing NF-κB activity and NO production through alternate signaling pathways [40, 56–61]. Combined, these predicted synergistic effects on NO synthesis would support effective killing of P. falciparum sexual stages prior to invasion of the midgut epithelium. The repression of AMPK activity is also consistent with the repression of autophagy  observed 24–72 h post-feeding (Fig. 7). Interestingly, inhibition of autophagy can increase caspase-dependent apoptotic cell death [63, 64]. Together with high levels of NO synthesis, an increase in apoptosis in the midgut epithelium during ookinete invasion would be predicted to additively reduce oocyst formation in the A. stephensi midgut .
We have observed that inflammatory NO synthesis can also damage the A. stephensi midgut epithelium, with concomitant reductions in stem cell proliferation and differentiation (escargot, prospero). Increased NF-κB activity has also been linked with decreased expression of autophagy-related genes as well as decreased cell renewal and differentiation [66, 67], suggesting multiple mechanisms whereby ABA could alter midgut homeostasis. In A. stephensi engineered to overexpress Akt under a midgut-specific promoter , damage to midgut mitochondrial function and epithelial homeostasis was profound and sustained, resulting in a significant reduction in lifespan. Here we saw no effect of ingested ABA on lifespan, even under the stresses of nutrient limitation and P. falciparum infection, most likely because the effects of ABA on signaling and gene expression were transient. Similarly, ABA had no effect on egg laying or egg viability within the first gonotrophic cycle, providing further evidence that transient enhancement of host defenses by ABA does not significantly alter fitness. Importantly, since lifespan and reproduction in the first gonotrophic are also not increased, the use of ABA as a therapeutic with transmission blocking activity would not be expected to increase vectorial capacity in mosquitoes that might feed on treated individuals.
Overall, our studies have demonstrated that the effects of ABA on mosquito immunity and key metabolic regulatory kinases are consistent with effects that have been reported in plants and in mammals. Interestingly, in plants ABA can activate or inhibit AMPK signaling in a tissue dependent manner , suggesting that the effects of ABA in the mosquito could vary across tissues. Further, pro- and anti-inflammatory effects of ABA have been reported for acute infection and chronic diseases, respectively, in mammals  suggesting that the effects of ABA in mosquitoes could vary depending on the nature of the infecting pathogen or in response to changes in associated microbiota.
ABA is a highly conserved regulator of immune and metabolic homeostasis within the malaria vector A. stephensi with potential as a transmission-blocking supplemental treatment. Further investigation of ABA signaling will help determine its potential efficacy as a transmission-blocking antimalarial therapeutic and increase our understanding of a highly conserved mediator of immune and metabolic homeostasis.
We thank Kong Wai Cheung for P. falciparum culture maintenance.
Funding for this study was provided by the UC Davis T32 training grant “Animal Models of Infectious Diseases” and by the Floyd and Mary Schwall Dissertation Year Fellowship in Medical Research, awarded to EKKG.
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
BKT and SFM processed mosquito midgut samples for gene expression and western blot analysis. JME conducted the uninfected lifespans and fecundity studies. EKKG performed all other experiments. EKKG and SL designed experiments and wrote the manuscript. All authors read and approved the final manuscript.
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SL and EKKG are authors on a patent. All other authors have no competing interests.
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