Signaling between mammalian adiponectin and a mosquito adiponectin receptor reduces Plasmodium transmission

ABSTRACT When a female mosquito engorges on a mammalian host, components of the blood meal can affect mosquito fitness and indirectly influence pathogen infectivity. We demonstrate that mammalian adiponectin, ingested during Anopheles gambiae blood feeding, co-localizes within mosquito midguts and decreases Plasmodium infection in the vector. Transcriptomic and RNAi studies reveal that the A. gambiae adiponectin receptor is involved in downregulating lipophorin, a lipid transporter that is important for egg development and Plasmodium infection in mosquitoes. These studies characterize a cross-phyla interaction between the mammalian host and A. gambiae that negatively impacts Plasmodium survival in its arthropod vector. IMPORTANCE When a female mosquito takes a blood meal from a mammalian host, components of the blood meal can affect mosquito fitness and indirectly influence pathogen infectivity. We identified a pathway involving an Anopheles gambiae adiponectin receptor, which, triggered by adiponectin from an incoming blood meal, decreases Plasmodium infection in the mosquito. Activation of this pathway negatively regulates lipophorin expression, an important lipid transporter that both enhances egg development and Plasmodium infection. This is an unrecognized cross-phyla interaction between a mosquito and its vertebrate host. These processes are critical to understanding the complex life cycle of mosquitoes and Plasmodium following a blood meal and may be applicable to other hematophagous arthropods and vector-borne infectious agents.

lipophorin (Lp) and vitellogenin (Vg), serve as important effector genes for lipid transport and metabolism after the blood meal and regulate vitellogenesis and oogenesis (2,9,(11)(12)(13)(14)(15)(16).Silencing of lipophorin or Vg leads to fewer Plasmodium in the mosquitoes (13,17).Based on all this information, lipid transport and lipid utilization serve as an important link between oogenesis and Plasmodium development inside the mosquito.
Adiponectin, the adipocyte complement-related protein of 30 kDa (or Acrp30), regulates metabolism, alters insulin sensitivity, and affects inflammation in different species (18)(19)(20).The effects of adiponectin depend, in part, on binding to adiponectin receptors via a globular C1q domain (19,21).There are two mammalian adiponectin receptors, AdipoR1 and AdipoR2 (22).Adiponectin receptors have seven transmem brane domains with an internal N terminus and an external C terminus (22).AdipoR1and AdipoR2-deficient mice have glucose intolerance and increased triglyceride levels, demonstrating that the adiponectin pathway regulates glucose and lipid homeostasis (18,23).An adiponectin receptor homologue in Drosophila melanogaster modulates insulin secretion and also controls both glucose and lipid metabolism, despite the absence of an identified adiponectin homologue in fruit flies (24).Recently, our group demonstrated that Ixodes scapularis ticks have an adiponectin receptor-like gene (ISARL) and ISARL both regulates phospholipid metabolism and facilitates Borrelia burgdorferi colonization of the tick (25).These interactions are mediated through an I. scapularis complement C1q-like protein (25).
In the present study, we characterize an adiponectin receptor (AdpR, AGAP004486) from Anopheles gambiae and its role when mosquitoes take a blood meal.Transcrip tomic analysis and RNA interference (RNAi) studies demonstrate that activation of the AdpR is associated with an inhibition of mosquito lipophorin expression.Interest ingly, lipophorin-mediated pathways enhance Plasmodium survival in mosquitoes (17), and AdpR-deficient mosquitoes are more readily colonized by Plasmodium.This study identifies a mosquito pathway triggered by a component of blood meal, adiponectin, which negatively impacts Plasmodium infection in its vector, mosquitoes.This is an example of cross-talk between the mosquito and the vertebrate host on which it feeds, which is detrimental to Plasmodium infection in the arthropod.

Characterization of an Anopheles adiponectin receptor protein
When mosquitoes take a blood meal, components of mammalian blood interact with the arthropod (26)(27)(28)(29).These relationships have the potential to influence metabolic pathways in the mosquito and modulate Plasmodium infection in its vector (30,31).A systematic gene search demonstrates that AGAP004486 encodes a putative adiponec tin receptor (AdpR) in A. gambiae, which has substantial homology with the AdpRs in humans, mice, and rats (Table S1).The full-length AdpR encodes a protein with 425 amino acid residues and 65% amino acid sequence similarity to both human and murine AdpR 1 and 2. Compared with human and murine AdpR 1 and 2 (32), the homologous seven transmembrane domains and zinc-binding sites, the 3× His and Asp residues and a Ser residue, are well-conserved between mosquito AdpR and mammalian AdpRs (Table S1).In contrast, the A. gambiae genome does not contain a gene with homology to adiponectin.To evaluate the expression levels of AdpR in different tissues, we collected midguts, ovaries, and fat bodies from mosquitoes after feeding.By reverse transcription quantitative polymerase chain reaction (RT-qPCR), the expression level of AdpR in the midgut, ovaries, and fat bodies did not show any statistically significant differences (Fig. 1A).In addition, there was no significant change after a blood meal (Fig. 1A).As A. gambiae has a putative AdpR but lacks adiponectin, we determined whether mamma lian adiponectin can be identified on the midgut surface.The midgut was collected and incubated with murine adiponectin.Recombinant murine adiponectin could be detected on the surface of some midgut cells, compared with controls (Fig. 1B).

Plasmodium berghei infection of mosquitoes is altered by the lack of adipo nectin
As Plasmodium are acquired by mosquitoes from the vertebrate host during a blood meal, we postulated that interactions between mosquito AdpR and mammalian blood could potentially affect Plasmodium infection of mosquitoes.We collected Plasmodiuminfected red blood cells (RBCs) from adiponectin-knockout (KO, Adipoq -/-) mice and then mixed the cells with plasma from wild-type or adiponectin-KO infected blood.Mosqui toes were then fed on this blood by membrane feeding.The expression of AdpR in the midgut was similar, regardless of the presence of adiponectin in the blood meal (Fig. 2A).There was a significantly higher P. berghei burden in the mosquito midgut, 2 and 14 days after taking the blood meal with plasma lacking adiponectin (Fig. 2B and C, P < 0.05).This suggests that adiponectin indirectly impedes Plasmodium infection of mosquitoes.

Silencing mosquito AdpR enhances P. berghei infection
Since mammalian adiponectin may interact with the A. gambiae, AdpR and the absence of adiponectin in a blood meal enhanced Plasmodium infection of mosquitoes, we hypothesized that RNAi-mediated silencing of the A. gambiae AdpR would increase Plasmodium transmission to mosquitoes.To silence AdpR, AdpR or Gaussia luciferase (Glu, control) dsRNA was injected into the thorax of naïve mosquitoes.Two days later, the mosquitoes were allowed to feed on the same P. berghei-infected mouse.RT-qPCR showed a significant decrease of AdpR expression in the midguts of dsAdpR-injected mosquitoes when compared to that in the midguts of control dsGluc-injected mosqui toes (Fig. 2D, P < 0.001), indicating that the expression of AdpR was successfully decreased.Two or 14 days after feeding, AdpR-silenced mosquitoes showed a marked increase in the P. berghei burden in the midguts when compared to that in control mosquitoes (Fig. 2E and F, P < 0.05).To further demonstrate that an interaction between mammal adiponectin and mosquito AdpR affects the transmission of Plasmodium to mosquitoes, we let control (dsGluc) and AdpR-silenced (dsAdpR) mosquitoes fed on P. berghei-infected adiponectin-knockout (Adipoq -/-) mice.There was no difference in the Plasmodium burden 2 days after blood meal (Fig. S1A).These data suggest that an association between murine adiponectin and the mosquito adiponectin receptor alters the establishment of Plasmodium infection in mosquitoes.

AdpR-related pathways associated with P. berghei infection of mosquitoes
To investigate the mechanisms underlying the association of AdpR and P. berghei infection, we assessed the presence or absence of AdpR on mosquito physiology by comparing transcriptomes of AdpR-silenced and control A. gambiae 2 days after feeding on P. berghei-infected mice.After taking a blood meal on P. berghei-infected mice, 94 genes were significantly differentially expressed with 86 upregulated and 8 downregula ted genes in the midguts of AdpR-silenced mosquitoes when compared to that in control mosquitoes (Fig. 3A and B; Table S2).The transcriptome analysis also demonstrated that the AdpR gene was successfully silenced by RNAi, consistent with our RT-qPCR results.One of the most significantly upregulated genes was lipophorin (adjusted P value < 10 −4 ), which has been reported to play an important role in lipid transport and mosquito egg development (15,16).In addition, several lipid homeostasis-associated genes, including fatty acyl-CoA reductase 2 (AGAP005985) or elongation of very long chain fatty acids protein (AGAP013094), were also upregulated in the absence of AdpR after feeding on P. berghei-infected mice, further suggesting that AdpR may have a role in mosquito metabolism after a blood meal.Transcriptomic data also demonstrated that AdpR silencing resulted in the upregulation of CLIP domain serine proteases (Table S1).collected from Adipoq -/-mice, and then equal amounts of plasma from Adipoq -/-mice or wild-type mice were mixed with the infected RBCs.Mosquitoes were fed on blood lacking adiponectin or wild-type plasma by membrane feeding.Two days after the blood meal, the midguts were collected, and the expression levels of the adiponectin receptor (A) and the burden of Plasmodium were determined by RT-qPCR (B).(C) Fourteen days after the blood meal, the midguts of mosquitoes were collected and the burden of Plasmodium was determined by RT-qPCR.(D) Mosquitoes were injected with dsAdpR (AdpR knockdown, AdpR KD), or dsGluc (control).Two days after dsRNA injection, the mosquitoes were fed on P. berghei-infected mice.Two days after the blood meal, the midguts were collected and the expression levels of adiponectin receptor (D) and the burden of Plasmodium were determined by RT-PCR (E).(F) Fourteen days after the blood meal, the midguts of mosquitoes were collected, and the burden of Plasmodium was determined by RT-PCR.Each dot represents one mosquito.(Median ± interquartile range, P < 0.05 using the Mann-Whitney U-test.)

The interaction between mammalian adiponectin and A. gambiae AdpR mediates the expression of lipophorin
To further investigate the downstream effects of the interaction of mammalian adiponectin and mosquito AdpR, we selected three downregulated and six upregulated genes to validate the accuracy and reproducibility of the transcriptome bioinformatic analyses by RT-qPCR (Fig. 3C; Fig. S1).The RT-qPCR samples were collected from the midguts of AdpR-silenced or control mosquitoes, which were not part of the initial sequencing samples, 2 days after a blood meal.The RT-qPCR results indicate that AGAP008338 and AGAP012532 were significantly downregulated after silencing of AdpR, and that lipophorin and AGAP013092 were significantly upregulated (Fig. 3C, P < 0.05).At the same time, the difference between the other four genes has a similar trend as the RNAseq data, but without statistical significance (Fig. S2).Next, we determined whether the expression of these four genes was affected when mosquitoes fed on a blood meal that lacked adiponectin.Among the four genes, lipophorin was also significantly upregulated 2 days after mosquitoes fed on P. berghei-infected adiponectin-knockout mice (Fig. 3D, P < 0.05).Based on these transcriptomic analyses, lipophorin is negatively regulated by signals associated with the adiponectin receptor and mouse adiponectin.
Since Plasmodium infection can be enhanced by lipophorin (13,15) and lipophorin is negatively regulated by adiponectin receptor, we next determined whether the enhancement of Plasmodium infection in mosquitoes due to silencing AdpR was mediated by lipophorin (Lp).We silenced AdpR and Lp together to investigate their effects on P. berghei infection.By RT-qPCR, we confirmed that co-injection of dsAdpR and dsLp significantly decreased the expression level of both AdpR and Lp (Fig. 4A and B).Silencing of Lp did not affect the expression of the adiponectin receptor (Fig. 4A), which indicated that it is unlikely that AdpR functions downstream of Lp.The decreased synthesis of lipophorin protein in dsLp group was also confirmed by SDS-PAGE (Fig. 4C).After feeding on P. berghei-infected mice, mosquitoes that had both AdpR and lipophorin silenced had a similar Plasmodium burden as the control group and a significantly lower Plasmodium infection level compared to the AdpR-silenced group (Fig. 4D).When Lp was silenced, the Plasmodium infection level was significantly decreased compared to the control (dsGluc) and AdpR silencing group (Fig. 4D).There was no difference between the dsLp, and the AdpR and Lp silencing groups (Fig. 4D).Based on our studies, the effects of silencing AdpR on Plasmodium infection in mosquitoes depend on the lipophorin pathway.

AdpR affects Plasmodium development in the mosquito midgut
To further understand the of the adiponectin receptor on the development of Plasmodium in the midgut, we examined the number of oocysts in the midguts in AdpR-silenced mosquitoes.Seven days after taking a blood meal from GFP-expressing P. berghei-infected mice, the midguts from control mosquitoes (both untreated and dsGluc-treated) and AdpR-silenced mosquitoes were collected to determine the number and size of oocysts.By counting the oocysts, there was a significant higher prevalence of infection in AdpR-silenced mosquitoes (75%) compared to control (46.5%) or dsGluc-trea ted mosquitoes (48.8%) (P = 0.02 by the Chi-square test, Fig. 5A).There was a significant increase in the oocyst number in AdpR-silenced mosquitoes compared to untreated mosquitoes (Fig. 5A, P < 0.05).There was no difference between untreated and dsGluctreated or dsGluc-treated and AdpR-silenced mosquitoes.In contrast to the increased number of oocysts, the diameters of oocysts were slightly smaller in AdpR-silenced mosquitoes compared to untreated mosquitoes or the dsGluc-treated mosquitoes (Fig. 5B, P < 0.05).Furthermore, we let control (dsGluc) and AdpR-silenced (dsAdpR) mosqui toes fed on P. berghei-infected adiponectin-knockout (Adipoq -/-) mice.There was no difference in the prevalence and oocyst number between these two groups when there was a lack of adiponectin in the blood meal (Fig. S1B).All these results demonstrate that silencing of adiponectin receptor affects the development of oocysts in the midgut.

DISCUSSION
Hematophagous arthropods acquire blood from other animals to obtain nutrients for physiological and reproductive needs.Nutrients in the blood directly influence fitness (26).However, other factors in blood can indirectly influence both fitness and Plas modium survival (3).A human miRNA, hsa-miR-150-5p, released from blood, can be disseminated into the mosquito hemocoel and suppress antiviral genes, an example of cross-species RNAi-mediated gene regulation (33).Insulin in a blood meal can bind to an insect insulin-like peptide receptor in mosquitoes and tsetse flies (34)(35)(36).In Anopheles mosquitoes, administration of insulin can increase the Plasmodium burden (35)(36)(37).All these results demonstrate the intimate interaction between the vertebrate host, Plasmodium, and mosquitoes.
Female mosquitoes modulate lipid metabolism after a blood meal to complete vitellogenesis .Following engorgement, the secretion of 20E leads to an upregulation of lipophorin and vitellogenin, a prerequisite for egg development in the ovary.Lipophorin is important for egg maturation, and Plasmodium utilize mosquito lipids through lipophorin (13,15,17).Silencing lipophorin led to fewer ookinetes and oocysts in the midgut of mosquitoes (13,17).In our study, we demonstrate that silencing the adiponec tin receptor led to upregulation of lipophorin and that there were increased number of oocysts in the AdipR-silenced mosquitoes, which is consistent with the impact of lipophorin on oocyst development (9,13).The oocysts were slightly smaller in size in the AdipR-silenced mosquitoes, while oocyst size usually increases when lipophorin is upregulated (9,13).This suggests that other factors may influence oocyst size.Further studies will elucidate the diverse factors related to how AdpR interactions alter various aspects of oocyst development.
The transcriptomic data also demonstrated that AdpR silencing resulted in the modest upregulation of CLIP domain serine proteases (Table S2).Silencing of CLIPA9 in mosquitoes leads to a lower P. falciparum oocyst intensity in the midguts of mosquitoes (38), and knockdown of either CLIPA2/CLIPA5 induces mosquito melanization and directly kills ookinetes (39).Taking together, the increased expression of CLIP domain serine proteases following AdpR silencing may also contribute to alterations in Plasmodium infection.Further studies will be needed to clarify the impact of CLIP domain serine proteases on Plasmodium development after AdpR silencing.
In contrast to the role of the adiponectin receptor during Plasmodium infection in mosquitoes, ISARL, an adiponectin-like receptor in ticks also serves as a metabolic regulator, but it is activated by a C1q-like protein in ticks and not by mammalian adiponectin (25).When ISARL is silenced in ticks, a critical phospholipid metabolism enzyme, PTDSS1, is affected (25).Silencing of ISARL significantly reduced the Borrelia burgdorferi burden in the tick (25).Unlike ISARL, we found that silencing of mosquito AdpR and the absence of adiponectin in the murine blood meal led to a similar effect on the acquisition of Plasmodium.There was also a similar effect on the expression of lipophorin, all of which suggest that the mosquito adiponectin receptor may be modulated by an incoming blood meal which contains mammalian adiponectin.Since different species of insects have homologues of adiponectin receptor proteins (40), it is possible that other ligands, such as C1q-like proteins, can activate the adiponectin receptor.While homology searches do not uncover any C1q-like protein in mosquitoes, we cannot exclude the possibility that there are additional unknown ligands for the adiponectin receptor in mosquitoes.In contrast to the positive effect of ISARL on Borrelia burgdorferi burden (25), there is a negative effect on the Plasmodium burden after activation of the mosquito adiponectin receptor.Therefore, different downstream effects in various arthropods can lead to the distinct effects in specific vector-borne pathogens.
In summary, we identified a novel pathway involving an A. gambiae adiponectin receptor, which triggered by adiponectin from an incoming blood meal, decreases Plasmodium infection in the mosquito.Activation of this pathway negatively regulates lipophorin expression, an important lipid transporter that both enhances egg develop ment and Plasmodium infection.The relative cost involved in altering egg development and Plasmodium infection may depend on the overall fitness of the mosquito.This is an unrecognized cross-phyla interaction between a mosquito and a mammal on which it feeds.These processes are critical to understanding the complex life cycle of mosquitoes and Plasmodium following a blood meal and may be applicable to other hematophagous arthropods and vector-borne infectious agents.

Animals
A. gambiae (4arr strain) mosquitoes were raised at 27°C, 80% humidity, under a 12/12hour light/dark cycle and maintained with 10% sucrose under standard laboratory conditions in the insectary at Yale University.Swiss Webster mice were purchased from Charles River Laboratories.C57BL/6 J mice wild type (WT) and C57BL/6 J mice deficient in adiponectin (Adipoq -/-) were purchased from the Jackson Laboratory.

Hemolymph collection
Hemolymph was collected as previously described (41,42).Briefly, mosquitoes were placed on ice and the proboscis was clipped.The lateral aspects of the thorax were gently pressed using fine forceps to push out a drop of hemolymph from the cut site.Hemolymph droplets from 10 dsRNA knockdown or control mosquitoes were collec ted into Laemmli sample buffer using a pipette tip.Proteins in the hemolymph were separated by SDS-PAGE and analyzed by Coomassie staining.

dsRNA synthesis and microinjection
The adiponectin receptor gene (AGAP004486) fragment between base pairs 37 and 564 of the coding sequence and the lipophorin gene (AGAP001826) fragment between base pairs 9370 and 9936 of the coding sequence were amplified from cDNA collected from midguts using PCR.Adiponectin receptor (AdpR) and lipophorin dsRNAs were further synthesized from the PCR-amplified fragments using a T7 RiboMAX kit (Promega).The primer sequences are listed in Table S3 in the supplemental material.Gaussia lucifer ase dsRNA was used as the control for dsRNA injection.Two days before the blood meal, 100 ng of dsRNA was injected into the thoraces of female mosquitoes under ice anesthesia.The injection was performed using a nanoinjector (Nanoject II; Drummond) with a glass capillary needle, as previously described (43).

Gene expression and Plasmodium load
The RNeasy Mini Kit (QIAGEN, CA) was used to extract RNA from the midguts, ovaries, and fat bodies of the mosquitoes.All extractions followed the manufacturer's protocols.The iScript RT-qPCR kit (Bio-Rad, CA) was used to generate cDNA from RNA.Using iTaq SYBR Green Supermix (Bio-Rad, CA), real-time PCR was performed on a CFX96 real-time platform (Bio-Rad).PCR involved an initial denaturation at 95°C for 2 min, 50 cycles of 15 sec at 95°C, 15 sec at 60°C, and 20 sec at 72°C.Fluorescence readings were taken at 72°C after each cycle.At the end of each reaction, a melting curve (60-95°C) was checked to confirm the identity of the PCR product.The burden of Plasmodium in midguts after blood meals was determined by assessing the expression level of P. berghei 18S rRNA, normalized to A. gambiae actin mRNA.These data were presented as copy number of the target gene per 10,000 copies of the housekeeping gene, actin.The primers used for the expression of sporozoite genes are listed in Table S3.

Transcriptome analyses
dsRNA (dsAdpR and dsGluc) microinjected A. gambiae mosquitoes were fed on P. berghei-infected Swiss Webster mice.Two days after a blood meal, the mosquitoes were collected for midgut dissection.Total RNA was purified as described above.RNA samples were then submitted for library preparation using TruSeq (Illumina, San Diego, CA, USA) and sequenced using Illumina HiSeq 2500 by paired-end sequencing at the Yale Centre for Genome Analysis (YCGA).The transcript data were mapped with the A. gambiae PEST genome (AgamP4.13)downloaded from VectorBase (https://vectorbase.org/vector base/app/) (44) and indexed using the kallisto-index (45).The reads from the sequencer were pseudo-aligned with the index reference transcriptome using kallisto (45).The counts generated were processed by DESeq2 (46) in RStudio (https://rstudio.com).The transcriptomic data are available in the Gene Expression Omnibus repository at the National Center for Biotechnology Information under the accession number: GSE198929.

Binding assays of murine adiponectin in mosquito midgut
The midgut was dissected from the mosquitoes and then incubated with 1 µg/µL of mouse adiponectin (SinoBiological, #50636-M08H), or BSA for 1 hour.The tissues were washed with PBS and fixed in 4% PFA for 15 min at room temperature.Then, the cells were blocked in 1% BSA in PBS for 1 hour, and subsequently immunolabeled with an Alexa Fluor 555 anti-His antibody (1:100, ThermoFisher, # MA1-21315-A555) for 1 hour.Nuclei were stained with DAPI (Invitrogen, #D9542).After staining, the fluorescence signals were examined with a Leica SP5 confocal microscope.

P. berghei infection
P. berghei (NK65 RedStar or ANKA GFP, ATCC) were maintained by serial passage in 6 to 8-week-old female Swiss Webster or C57BL/6 mice as previously described (47).Briefly, Swiss Webster or C57BL/6 mice were challenged with P. berghei-infected RBCs by intraperitoneal injection.A. gambiae mosquitoes then took a blood meal from the infected mice, when the parasitemia was approximately 5%.Seventeen to 24 days after P. berghei infection, the mosquitoes were sorted using the fluorescent signal of the salivary glands.
For artificial membrane feeding, mosquitoes were starved for at least 17 hours before exposure to blood.Blood was collected from Adipoq -/-or wild-type mice infected with P. berghei and treated with EDTA to prevent coagulation.Blood samples were centrifuged at 2,000 g for 5 min.The RBCs from Adipoq -/-mice were mixed with an equal amount of plasma from Adipoq -/-or wild-type mice.The blood was then transferred into a Hemotek blood reservoir unit.Mosquitoes were then fed at a constant temperature of 37°C using the Hemotek blood feeding system with stretched Parafilm as a membrane.After 30 min of blood feeding, mosquitoes were kept in paper cups covered with net and maintained as described above.

Oocyst numbers and sizes
Seven days post-feeding on P. berghei (ANKA GFP) infected mice, the midguts of mosquitoes that had completed a blood meal were dissected, and the midguts were evaluated with a EVOS fluorescence microscope.The number and size of oocysts in the midguts were determined using GFP as a marker (48).

Statistical analysis
All experiments were repeated at least three times.Data from at least three biological replicates were used to calculate medians for graphing purposes.Statistical analyses employed the Mann-Whitney test to determine the difference, and the data were presented as the median with interquartile range (IQR) or mean with standard deviation (SD).A P-value of <0.05 was considered statistically significant.The analysis, graphs, and statistics of all data were performed using Prism 9.0 software (GraphPad Software).

FIG 1
FIG 1 Mammalian adiponectin co-localizes with the mosquito midgut.(A) Following a sugar meal or blood meal, mRNA from the mosquito midgut, ovary, or fat body was collected.The expression level of the adiponectin receptor was determined by RT-PCR.Each dot in the midgut and ovary groups represents a pooled sample from two mosquitoes.Each dot in the fat body group represents a pooled sample from four mosquitoes.(Mean ± S.D.) (B) Murine adiponectin bound to mosquito midguts.His-tag murine adiponectin or control protein (BSA) was incubated with the mosquito midgut, and the presence of adiponectin in the midgut was determined by confocal microscopy.

FIG 2
FIG 2 Lack of adiponectin in the incoming blood meal increases infection of Anopheles gambiae with Plasmodium berghei.(A) P. berghei-infected RBCs were

FIG 3
FIG 3 Gene expression analysis reveals that lipophorin (AGAP001826) is regulated by the adiponec tin receptor.Mosquitoes were injected with dsAdpR (AdpR knockdown, KD) or dsGluc (control, Ctrl).Two days after dsRNA injection, the mosquitoes were fed on P. berghei-infected mice.Two days after the blood meal, each midgut was collected, and RNA from each midgut was isolated for RNA-seq analysis.(A) A hierarchical clustering of differentially expressed genes was generated by comparing data from AdpR knockdown and control mosquitoes.(B) Volcano plots showing significant changes in gene expression between AdpR knockdown and control mosquitoes.Dispersion graph of -10*log10(P value) (y-axis) against the log2(fold change) (x-axis) corresponding to the genes by their differential expression.(C) RT-qPCR validation of gene expression in the mosquito midgut after injection with dsAdpR (Adiponectin knockdown, AdpR KD) or dsGluc (control).Two days after dsRNA injection, mosquitoes were fed on P. berghei-infected mice.Two days after the blood meal, the midguts were collected for RT-qPCR.(D) RT-qPCR validation of expression in mosquito midguts 2 days after feeding on adiponectin knockout (Adipoq -/-) or wild-type P. berghei-infected mice.Each dot represents one mosquito.(Median ± interquartile range, P < 0.05 using the Mann-Whitney U-test.)

FIG 4
FIG 4 Adiponectin signaling affects Plasmodium infection in mosquitoes through the lipophorin pathway.Mosquitoes were injected with dsAdpR (AdpR knockdown), dsLp (lipophorin knockdown), both dsAdpR and dsLp, or dsGluc (control).Two days after injection, the mosquitoes were fed on P. berghei-infected mice.Two days after the blood meal, the midguts were collected, and the expression levels of adiponectin receptor (A) and lipophorin (B) were determined by RT-qPCR.(C) Hemolymph from 10 knockdown or control mosquitoes was collected, and the levels of lipophorin (Lp) and vitellogenin (Vg) were determined by SDS-PAGE gel.(D) The Plasmodium burden was determined by RT-qPCR.Each dot represents one mosquito.(Median ± interquartile range, P < 0.05 using the Mann-Whitney U-test.)

FIG 5
FIG 5 Silencing of adiponectin signaling affects the oocyst number and size in mosquitoes.Mosquitoes were injected with dsAdpR (AdpR knockdown) or dsGluc (control).Two days after injection, the mosquitoes were fed on GFP-expressing P. berghei-infected mice.(A) Seven days after the blood meal, the midguts were collected.The number of oocysts per midgut was determined by the GFP signals using EVOS fluorescence microscopy.The dot blots represent the number of oocysts from each group of infected midguts.(Median ± interquartile range (IQR), P < 0.05 using the Mann-Whitney U-test.)The pie charts represent the percentage of infected mosquitoes in each group (P < 0.05 using the Chi-square test).(B) The size of oocysts in each group was determined by GFP signals using EVOS fluorescence microscopy.(Median ± IQR, P < 0.05 using the Mann-Whitney U-test.) These studies were supported by the National Institute of Allergy and Infectious Disease (RO1 grant AI158615-01, R21 grant AI142708, and R41 grant AI142708 to E. F.) and the Howard Hughes Medical Institute Emerging Pathogens Program.Conceptualization: Y-M.C., H.S., and E.F.Methodology, Data curation, Project administration, Formal analysis: Y-M.C., H.S., S.A., and X.T. Funding acquisition: E.F.Writing -original draft: Y-M.C. Writing -review and editing: Y-M.C., X.T., and E.F.