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Maternal lipid mobilization is essential for embryonic development in the malaria vector Anopheles gambiae [1]

['Maurice A. Itoe', 'Department Of Immunology', 'Infectious Diseases', 'Harvard T.H. Chan School Of Public Health', 'Boston', 'Massachusetts', 'United States Of America', 'W. Robert Shaw', 'Howard Hughes Medical Institute', 'Iryna Stryapunina']

Date: 2025-01 

Abstract Lipid metabolism is an essential component in reproductive physiology. While lipid mobilization has been implicated in the growth of Plasmodium falciparum malaria parasites in their Anopheles vectors, the role of this process in the reproductive biology of these mosquitoes remains elusive. Here, we show that impairing lipolysis in Anopheles gambiae, the major malaria vector, leads to embryonic lethality. Embryos derived from females in which we silenced the triglyceride lipase AgTL2 or the lipid storage droplet AgLSD1 develop normally during early embryogenesis but fail to hatch due to severely impaired metabolism. Embryonic lethality is efficiently recapitulated by exposing adult females to broad-spectrum lipase inhibitors prior to blood feeding, unveiling lipolysis as a potential target for inducing mosquito sterility. Our findings provide mechanistic insights into the importance of maternal lipid mobilization in embryonic health that may inform studies on human reproduction.

Citation: Itoe MA, Shaw WR, Stryapunina I, Vidoudez C, Peng D, Du EW, et al. (2024) Maternal lipid mobilization is essential for embryonic development in the malaria vector Anopheles gambiae. PLoS Biol 22(12): e3002960. https://doi.org/10.1371/journal.pbio.3002960 Academic Editor: Louis Lambrechts, Institut Pasteur, FRANCE Received: April 29, 2024; Accepted: November 29, 2024; Published: December 17, 2024 Copyright: © 2024 Itoe et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. The numerical data are accessible from the Harvard Dataverse online repository using the link https://doi.org/10.7910/DVN/ULTW1K. Metabolomics and Lipidomics data are accessible from the EMBL-EBI MetaboLights online database using the link https://www.ebi.ac.uk/metabolights/MTBLS9881. The RNAseq data are accessible from the Gene Expression Omnibus (GEO) online database using the link https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE263712. Funding: F.C. is funded by Howard Hughes Medical Institute (HHMI, www.hhmi.org) as an HHMI Investigator and by the National Institute of Health (NIH) grants R01AI148646, R01AI153404. M.A.I. was funded by the Charles A. King Trust postdoctoral research fellowship in Basic Sciences from Health Resources in Action (HRiA, www.hria.org). I.S. was funded by Natural Sciences and Engineering Research Council of Canada (NSERC, www.nserc-crsng.gc.ca) as a scholarship recipient (PGSD3 - 545866 – 2020). The funders were not involved in study design, data collection, analysis, or interpretation. Competing interests: A patent application covering the concept of the application of lipase inhibitors to induce sterility in mosquitoes is being processed on behalf of F.C. and M.A.I. by the President and Fellows of Harvard University. The authors state that they have no other competing interests. Abbreviations: ACTH, adrenocorticotropic hormone; AKH, adipokinetic hormone; ATGL, adipose triglyceride lipase; BSA, bovine serum albumin; cAMP, cyclic adenosine monophosphate; CE, cholesterol ester; DAG, diglyceride; FDR, false discovery rate; HSL, hormone-sensitive lipase; LD, lipid droplet; LPE, lysophosphatidylethanolamine; MB, maleate buffer; MPA, mobile phase A; MPB, mobile phase B; PBM, post blood meal; PBS, phosphate-buffered saline; PFA, paraformaldehyde; PKA, protein kinase A; TAG, triglyceride; TCA, tricarboxylic acid; TEM, transmission electron microscopy

Introduction Reproduction is a highly energy-demanding process that requires tight metabolic control coordinated by hormones, metabolites, and immune factors [1,2]. In mammals, lipid stores in the form of triglycerides (TAGs) are deposited in the cells of adipose tissue, where they serve as a dynamic energy reservoir. As TAGs are unable to cross cell membranes, for transport to other tissues, they need to be either incorporated into vesicles or remobilized. Regulation of these processes is critically important during gestation as they coordinate the necessary energy supply to the growing fetus, and their perturbations can have negative effects on pregnancy outcomes [3,4]. In reproductive organs, lipid mobilization allows essential reproductive functions such as the development of ovarian follicles and the synthesis of steroid hormones, which prepare the endometrium for implantation [3]. In simplified terms (reviewed in [5]), TAG remobilization follows a hydrolytic process requiring their breakdown into free fatty acids (FFAs) and glycerol, regulated by hormonal stimuli that activate lipolytic enzymes like hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) [6–8]. The canonical pathway for lipolysis activation in adipocytes is driven by catecholamines, adrenocorticotropic hormone (ACTH), and secretin, which upon binding to β-adrenergic G-protein-coupled receptors stimulate cyclic adenosine monophosphate (cAMP) synthesis, activating protein kinase A (PKA). PKA activation causes the phosphorylation of HSL, which induces its translocation onto the lipid droplet (LD) surface aided by PKA-mediated phosphorylation of perilipin-1 [9,10]. Perilipin-1 phosphorylation also results in the dissociation of the co-activator comparative gene identification-58 (CGI-58) [11,12], which can then bind to ATGL and activate lipolysis [13]. Upon completion of hydrolysis, FFAs will bind to carrier proteins that transport them to target tissues in the body for their use in energy production through ß-oxidation, hormone synthesis, or other cellular metabolic needs [14]. Although the last common ancestor of insects and mammals is estimated to have lived over 500 million years ago, insects possess a similar metabolic cascade to mobilize lipids. Lipolysis of TAGs is hormonally stimulated by a neuropeptide hormone, adipokinetic hormone (AKH), that has an analogous role to glucagon in mammals and is released from the corpora cardiaca [15,16]. Once released, AKH binds to its G protein-coupled receptor (AKHR) triggering an increase in intracellular calcium levels and cAMP synthesis, followed by PKA activation [17]. Activated PKA in turn phosphorylates lipid storage droplet protein 1 (LSD1), a homologue of perilipin 1-like protein, on the surface of LDs, ultimately inducing the activation of TAG lipases to catalyze the rate-limiting step in the hydrolysis of fatty acids from TAGs to generate FFA and diglycerides (DAGs) [18–22]. In hematophagous insects, after blood feeding, complex lipids such as TAGs and phospholipids are broken down into FFAs in the midgut lumen for transport across the luminal membranes [23], where they are reutilized to synthesize DAGs, TAGs, esters, and phospholipids [24]. These lipids are then loaded—in concert with lipid transfer proteins—onto circulating lipoproteins, principally lipophorin (Lp), on the basal side of the midgut epithelium for delivery to key tissues such as flight muscles and ovaries, or directed to the fat body for storage or further mobilization [19,20,25,26]. Dynamic changes occur in the lipid profile of the fat body during a reproductive cycle [27], with substantial proportions of stored lipids being mobilized for egg production [28,29]. Lp-transported lipids, as well as the yolk protein vitellogenin (Vg), are incorporated into developing oocytes [30,31] whereupon lipid is organized into LDs and Vg crystallizes into vitellin yolk granules [32] to be degraded after oviposition to nourish the developing embryo [33]. Coordinated lipolysis regulation is mediated by multiple conserved signaling pathways, including 20-hydroxyecdysone [34,35], Hedgehog [36], Decapentaplegic [37], NF-κB [38], as well as the neuropeptides FMRFamide [39] and AKH [40]. The components of the lipolytic machinery are shared by important insect vectors of human diseases including malaria, Chagas disease, and African Trypanosomiasis [23,41–44], and could potentially be targeted to reduce the size of vector populations and aid in disease control efforts. Orthologues of TAG lipase and AKHR are significantly increased after blood feeding in the fat body of Aedes aegypti mosquitoes [34,40], and TAG stores in the fat body, which are sharply reduced after blood feeding due to their mobilization and incorporation into developing eggs, are not mobilized when the AKH receptor AKHR is silenced by RNAi [40]. Similarly, disruption of lipid mobilization through targeting AKH signaling, ATGL or Perilipin/LSD1 in several insects decreases fecundity [43,45] and causes embryonic lethality [21,46]. However, information regarding lipid mobilization in Anopheles is relatively scarce. In one of the most important vectors of human malaria, the Anopheles gambiae mosquito, lipid transport by Lp has been shown to impact the development of Plasmodium falciparum and Plasmodium berghei, respectively, a human and rodent malaria parasite. Indeed, silencing Lp before an infectious blood meal reduces oocyst numbers in both parasite species [47–49], while it impairs oocyst growth and sporozoite infectivity in P. berghei but not P. falciparum [48–50]. These studies also revealed that Lp, and to a lesser extent Vg, which transports approximately 5% of lipids within oocytes [51], are essential for egg development as their silencing drastically reduces the number of developing ovarian oocytes [31,47,48,50]. A role of upstream lipolytic pathways in the reproduction of this or other human malaria vectors has yet to be determined, and their function has only previously been studied in the context of pathogen infections. Silencing of the TAG lipase AgTL2 (a pancreatic lipase that is the functional homologue of ATGL and predominantly induced in the fat body after blood feeding [52]) by RNAi leads to accelerated P. falciparum oocyst growth, possibly due to the accumulation of TAGs, DAGs, and phosphatidylcholine observed in midguts and fat bodies [48], while depleting AKH1 and AKHR leads to reduced number of sporozoites in the salivary glands [53]. Whether impairing lipolysis impacts reproduction in this anopheline is unknown, and addressing this question could uncover key reproductive targets to reduce mosquito populations and curb malaria incidence in disease endemic countries. Here, we demonstrate a key role of the An. gambiae maternal lipolytic machinery in shaping the development and survival of progeny. Targeted depletion of AgTL2 in mothers induces considerable perturbations to energy metabolism in the progeny, preventing embryos from hatching and causing embryonic lethality. Treating adult females with orlistat, a broad-spectrum hydrolase inhibitor, mimics the effects of AgTL2 silencing, causing dose-dependent embryonic mortality without, however, accelerating P. falciparum development. These data provide a proof of principle that targeting lipolysis can aid mosquito control and increase our understanding of the importance of lipid metabolism during pregnancy and embryonic development.

Discussion Our data reveal an unappreciated role for maternal TAG remobilization in shaping the development of An. gambiae embryos. We show that impairing lipolysis via AgTL2 depletion in the fat body, the fat storage tissue, leads to a substantial decrease in TAGs in the ovaries of adult females, the destination of lipids shuttled via the function of lipoproteins. In turn, we demonstrate that inheriting vastly reduced TAG levels triggers a cascade of nutritional dysregulation that prevents embryos from hatching and leads to their death. The almost complete penetrance of this phenotype led us to test the possibility of targeting lipolysis in mosquitoes to reduce the size of field populations. We were pleasantly surprised by the efficacy in proof of principle experiments of the lipase inhibitor orlistat, which induced significant embryonic lethality when applied to females. Orlistat is a drug used in humans to counter obesity and therefore it would not be suitable for a mosquito-targeting strategy. However, in the future, it may be feasible to generate Anopheles-specific inhibitors that target the function of AgTL2, AgLSD1, or other factors critical to mosquito lipolysis, for incorporation in bed nets (or other vector control strategies like indoor residual sprays [64]). Developing these inhibitors for mosquito control may prove challenging given the close homology between mosquito and human lipases [21], although it is possible that allosteric inhibitors may show higher specificity against mosquito enzymes [65]. Potential impacts on nontarget organisms would need to be evaluated through toxicological testing in mammals and insect surveys during efficacy trials of the intervention. If necessary, effects could be mitigated through usage methods, dosages, or formulations that reduce exposure of other insects to these inhibitors, such as physical incorporation into bed nets, which generally only target night-biting species contacting the net. Importantly, as mentioned above, compounds that interfere with mosquito reproduction are already incorporated into bed nets utilized in malaria control strategies. Indeed, dual-ingredient nets including the insect growth regulator pyriproxyfen show promise in malaria endemic countries [59–61], as they act as secondary active agents in the event of mosquito resistance to the primary insecticide. This is particularly relevant at a time when insecticide-treated nets, after contributing to averting millions of malaria cases worldwide [66], are showing worrying signs of inefficacy [67], representing one of the principal causes behind the increase in malaria cases after years of decline [68,69]. Once developed, Anopheles-specific inhibitors of the lipolytic machinery could therefore contribute to feed the pipeline for the next generation of dual-ingredient bed net products. Besides Lp-transported lipids, the yolk protein Vg conveys approximately 5% of total oocyte lipids [51], and so yolk deposition could potentially be affected in AgTL2 embryos and contribute to their abnormal development. Interestingly, embryonic lethality was reported when maternally deposited enzymes required for yolk usage in the embryo were silenced in Rhodnius prolixus embryos [70], suggesting that such phenotypes are due to an inability to mobilize yolk effectively. Our studies in An. gambiae have however shown that embryonic lethality occurs much earlier when Vg is silenced. Indeed, Vg depletion in mothers, which led to reduced yolk protein deposition into the oocyte, arrested embryonic development within the first few hours of oviposition [31], suggesting the late mortality observed in dsAgTL2 embryos is not due to defects in yolk deposition. The effects of AgTL2 silencing on Anopheles reproduction are in line with phenotypes observed in insects after manipulation of other components of the lipolytic machinery. In Drosophila, knockout of brummer (bmm), the orthologue of the mammalian adipose TAG lipase ATGL, in both parents leads to excessive fat accumulation and impaired lipid mobilization from fat bodies, and severely affects hatching of mutant embryos by unknown mechanisms [21], and similar results were obtained in recent studies in Bombyx mori [46] and R. prolixus [45]. Although these studies across insects did not determine the mechanisms triggered by inhibition of lipolysis, they highlight a degree of conservation in the importance of TAG mobilization in insect reproduction that could well be exploited to target populations of insect vectors of human and animal pathogens, as well as agricultural pests. Our multi-omics analyses revealed that embryos from AgTL2-depleted mothers have considerable deficits in key metabolic processes. Carbohydrate metabolism and ß-oxidation are shut down in early developmental phases, followed by disruption of the TCA cycle. While these metabolic defects are not apparent in the histological samples and are not reflected by changes in transcriptomic profiles at the early RNAseq time points, it is likely that their accumulation leads eventually to the mis-regulation of several genes observed at the last time point analyzed (38 h post oviposition), a time when control embryos are close to hatching. Up-regulated genes are enriched in stress response and protein folding genes, revealing embryos that are under severe stress. This is paralleled with a strong down-regulation in key sensory signaling receptors and neuropeptides, eclosion hormone [54,55], and cuticular proteins that form the second cuticle needed before hatching [58]. LDs are known to be multifunctional storage compartments in the developing embryo [71], providing a source of not only TAGs and phospholipids but other lipids classes as well, such as the waxy saturated hydrocarbons required for cuticle synthesis [72]. Along with a significant decrease in expression of 26 annotated cuticle-related genes (S3 Data), a lack of LD breakdown in dsAgTL2 embryos may sequester these structural lipids away from cuticle synthesis enzymes, preventing the production of adequate cuticle for hatching [58]. Lipids stored within LDs may also serve as the source of several key signaling molecules, such as eicosanoids, hormones derived from polyunsaturated FFAs with roles in egg and eggshell formation [73]. Limited eicosanoid signaling may also explain embryonic lethality as eggshell defects and low hatching rates were recently reported in R. prolixus after bmm silencing [45], and eggshell defects have been observed after inhibition of eicosanoid signaling in Aedes mosquitoes [74]. We also observed a highly significant GO term enrichment for neural functions in down-regulated genes at 38 h post oviposition (top 4 terms), including neuropeptide and GPCR signaling pathways. CCHamide1-related proteins, induced in control but not dsAgTL2 embryos, are arthropod neuropeptides secreted by enteroendocrine cells in the gut upon sensing of different dietary conditions in the intestinal lumen. They regulate activity of dopaminergic neurons in the brain to control gut motility and other physiological behaviors [75–77], although interestingly they have not previously been implicated in embryogenesis and hatching. Lipids and their derivatives are required for neuronal signaling through their roles in synapse function and axonal wrapping [78,79]. Indeed, we observed increased levels of LPE and decreased ceramide phosphoethanolamine (CerPE) in dsAgTL2 embryos at 38 h post oviposition, changes that resemble Drosophila glial mutants unable to ensheathe axons [80], again suggestive of neuronal defects. In mammals, deficiencies in lipid metabolism and lipid signaling have been associated with developmental and cognitive problems as well as with neurodegenerative diseases. Imbalances in fatty acids during gestation causes altered brain development in the fetus, while severe fatty acid deprivation can impair cognitive functions (reviewed in [78]). Although we could not pinpoint the precise mechanisms behind embryonic lethality, our results show how reduced energy stored caused by impaired lipid remobilization and low TAG levels leads to the collapse of normal metabolic functions, eventually causing severe stress and cellular death. Finally, our data confirm that P. falciparum parasites are affected by the mosquito lipolytic machinery. Depletion of AgTL2 (previous studies [48]) and AgLSD1 (this study) leads to accelerated oocyst growth rates and faster sporozoite invasion of salivary glands. Interestingly, however, such acceleration is not recapitulated by orlistat treatment, an observation that implicates fat body (rather than midgut) lipids in parasite growth. Indeed, while both AgTL2 depletion and orlistat treatment induce glyceride accumulation in the midgut (this study and [48]), differences in glyceride levels in the fat body are only detected after lipase silencing. This difference suggests that orlistat, while closely phenocopying the effects of AgTL2 depletion in terms of oogenesis and embryonic development, may inhibit different or additional aspects of the female lipolytic machinery. Regardless of the mechanism, when combined with the observed effects on fertility, these data support the idea that if Anopheles-specific lipase inhibitors were to be generated, they may represent promising tools for field interventions aimed at reducing the size of field populations of malaria vectors, aiding efforts to control this devastating disease.

Materials and methods Rearing of An. gambiae mosquitoes Anopheles gambiae (the standard laboratory wild-type strain G3) were reared in cages at 27°C, 80% humidity, and on a 12 h light-dark cycle. Adults were fed 10% glucose ad libitum and weekly on purchased human blood (Research Blood Components) for maintenance. Culturing of P. falciparum parasites Plasmodium falciparum (NF54 strain) parasites were maintained as asexual stages in human erythrocytes and gametocytes were induced as described in [48]. Embryo collection Glass bowls containing circular Whatman filter papers submerged in deionized water were provided for 2 h to mated control and AgTL2-knockdown females at 4 days (d) PBM. Embryos were aged outside cages and collected for an assay of interest at 4 to 6 h, 22 to 24 h, and 36 to 38 h after oviposition. Measurement of triglyceride (TAG) levels Non-blood fed and blood fed control and AgTL2-silenced adult female An. gambiae mosquitoes were cooled on ice and decapitated in 1× phosphate-buffered saline (PBS). Ovaries, midgut, and fat body were dissected and transferred into separate tubes containing two 2 mm beads, diluted NP-40 substitute assay buffer (Triglyceride Assay Colorimetric kit, Cayman Chemicals), and 1× cocktail of protease inhibitor (cOmplete Protease Inhibitor Cocktail, EDTA-Free, Roche). For orlistat experiments, tissues were dissected from control or orlistat-treated mated wild-type blood fed mosquitoes at 48 h PBM. Each tube contained tissues pooled from 3 animals. For TAG determination in embryos, 200 embryos laid by control and AgTL2-silenced females were collected at 6, 24, and 30 h after oviposition. TAG quantification in tissues and embryos was performed according to the manufacturer’s instructions in the Triglyceride Assay Colorimetric kit (Cayman Chemicals). The assay quantifies glycerol content derived after release of fatty acids by lipoprotein lipase hydrolytic activity. Protein content in each pool was determined by Bradford assay and used for normalization of TAG levels. Embryo fixation, immunofluorescence assays, and transmission electron microscopy (TEM) Embryos were fixed as described by [81]. Briefly, embryos were rinsed with distilled water onto a filter paper at the indicated time point and bleached for 75 s with 25% bleach (2% sodium hypochlorite). After rinsing 6 to 8 times with distilled water, they were transferred into scintillation vials with 1:1 solution of 9% paraformaldehyde (PFA) and heptane and rotated on a roller shaker for 25 min at 70 rpm at room temperature. The water phase was replaced once and then washed with distilled water for 30 min on a shaker. Following incubation with boiled and iced-cold distilled water for 30 s and 15 s, respectively, the water phase was removed, and heptane was replaced. To remove the endochorion, an equal volume of methanol was added, and the vials were strongly swirled once and allowed to stand for 15 min at room temperature. The heptane-methanol mixture was removed, and embryos were washed 3 to 4 times with methanol and stored in fresh methanol at −20°C until the day of removal of eggshell. To remove eggshells, embryos were transferred onto a double-sided tape and peeled according to [82]. For immunofluorescence assays, embryos were permeabilized and blocked with a 2% bovine serum albumin (BSA) in 0.1% Tween-20 in 1× PBS solution for 30 min at room temperature. For TEM, peeled embryos were collected in 200 μl of fixative (2.5% PFA, 5% glutaraldehyde, 0.06% picric acid in 0.2 M cacodylate buffer) and submitted to the Harvard Medical School Electron Microscopy Core. There, they were washed once with 0.1 M cacodylate buffer, twice with water, and then postfixed with 1% osmium tetroxide and 1.5% potassium ferrocyanide in water for 1 h at room temperature. Samples were then washed twice in water followed by once in 50 mM maleate buffer (pH 5.15) (MB). Next, the samples were incubated for 1 h in 1% uranyl acetate in MB, followed by 1 wash in MB, and 2 washes in water. Samples were then dehydrated via an ethanol gradient: increasing concentrations of ethanol were used with samples placed successively in 50%, 70%, 90%, 100%, 100% ethanol for 10 min each. After dehydration, samples were placed in propylene oxide for 1 h and then infiltrated overnight in a 1:1 mixture of propylene oxide and TAAB 812 Resin (www.taab.co.uk, #T022). The following day, samples were embedded in TAAB 812 Resin and polymerized at 60°C for 48 h. Ultrathin sections (roughly 80 nm) were cut on a Reichert Ultracut-S microtome, sections were picked up onto copper grids, stained with lead citrate, and imaged in a JEOL 1200EX transmission electron microscope equipped with an AMT 2K CCD camera. Staining of lipids with LD540 or Nile Red Ovaries, midguts, and fat body tissues were dissected from control dsGFP, TL2-silenced, control DMSO-, or orlistat-injected females at 48 h PBM and fixed in 4% PFA for 45 min at room temperature. Tissues were washed twice with 1× PBS for 15 min and incubated in permeabilization and blocking solution (1% BSA, 0.1% Tween 20, 1× PBS) for 30 min with shaking at room temperature. Samples were stained with LD540 in 1× PBS (0.5 μg/ml) according to [83]. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) in 1× PBS (1 μg/ml) for 5 min and mounted on microscope slides with Vectorshield (Vector laboratories). Peeled embryos were fixed, permeabilized, and blocked as above but stained with Nile Red (10 μg/ml) for 30 min with shaking at room temperature. Images were captured on an Inverted Zeiss microscope Observer Z1. Lipidomic and metabolomic analyses All solvents were HPLC-MS grade from Sigma-Aldrich. For lipidomic and metabolomic analyses of An. gambiae embryos, 200 embryos oviposited by control and AgTL2-knockdown blood fed mosquitoes in 3 biological replicates were collected at 4 to 6 h, 22 to 24 h, and 36 to 38 h after laying with 1 ml of methanol into a vial containing five 2 mm steel beads. Embryos were homogenized in a bead-beating homogenizer (Mini-Beadbeater 96, BioSpec Products) for 5 min at 2,400 rpm in a cold block and transferred to 8 ml glass vials (Duran Wheaton Kimble Life Sciences). After rinsing the bead-beater tubes with an additional 1 ml of methanol, 4 ml of cold chloroform was added to each glass vial and vortexed for 1 min, and 2 ml of ultrapure water was added to each vial and vortexed for another 1 min. Glass vials were centrifuged at 3,000 g for 10 min at 4°C, and 3 ml of the upper aqueous phase and 3 ml of the lower chloroform phase were transferred into separate glass vials for metabolomic and lipidomic analyses, respectively. For metabolomics, samples were dried under nitrogen flow and resuspended in 50 μl of acetonitrile 25% in water. A pool was created by combining 10 μl of each sample. Samples were analyzed by LC-MS on a Vanquish LC coupled to an ID-X MS (Thermo Fisher Scientific); 5 μl of sample was injected on a ZIC-pHILIC PEEK-coated column (150 mm × 2.1 mm, 5 μm particles, maintained at 40°C, Sigma-Aldrich). Buffer A was 20 mM ammonium carbonate, 0.1% ammonium hydroxide in water and Buffer B was acetonitrile 97% in water. The LC program was as follow: starting at 93% B, to 40% B in 19 min, then to 0% B in 9 min, maintained at 0% B for 5 min, then back to 93% B in 3 min and re-equilibrated at 93% B for 9 min. The flow rate was maintained at 0.15 ml min-1, except for the first 30 s where the flow rate was uniformly ramped from 0.05 to 0.15 ml min-1. Data was acquired on the ID-X in switching polarities at 120,000 resolution, with an automatic gain control target of 1e5, and an m/z range of 65 to 1,000. MS1 data was acquired in switching polarities for all samples. MS2 and MS3 data was acquired on the pool sample using the AquirX DeepScan function, with 5 reinjections, separately in positive and negative ion mode. Data was analyzed in Compound Discoverer 3.2 (Thermo Fisher Scientific). Identification was based on MS2/MS3 matching with a local mzvault library and corresponding retention time built with pure standards (level 1) or on mzcloud match (level 2). Each match was manually inspected. For lipidomics, samples were dried under nitrogen flow and resuspended in 60 μl of chloroform. Each sample was split into 2 equal aliquots, one for each polarity analysis. LC-MS analyses were modified from [84] and were performed on an Orbitrap Exactive plus MS (Thermo Fisher Scientific) in line with an Ultimate 3000 LC (Thermo Fisher Scientific). Each sample was analyzed in positive and negative modes, in top 5 automatic data-dependent MS/MS mode. Column hardware consisted of a Biobond C4 column (4.6 × 50 mm, 5 μm, Dikma Technologies). Flow rate was set to 100 μl min−1 for 5 min with 0% mobile phase B (MPB), then switched to 400 μl min−1 for 50 min, with a linear gradient of MPB from 20% to 100%. The column was then washed at 500 μl min−1 for 8 min at 100% MPB before being re-equilibrated for 7 min at 0% MPB and 500 μl min−1. For positive mode runs, buffers consisted for mobile phase A (MPA) of 5 mM ammonium formate, 0.1% formic acid, and 5% methanol in water, and for MPB of 5 mM ammonium formate, 0.1% formic acid, 5% water, 35% methanol in isopropanol. For negative runs, buffers consisted for MPA of 0.03% ammonium hydroxide, 5% methanol in water, and for MPB of 0.03% ammonium hydroxide, 5% water, 35% methanol in isopropanol. Lipids were identified and their signal integrated using the Lipidsearch software (version 4.2.27, Mitsui Knowledge Industry, University of Tokyo). Integrations and peak quality were curated manually before exporting and analyzing the data in Microsoft Excel. Heatmaps and principal component analysis plot were generated using MetaboAnalyst 5.0 [85]. Egg development and fertility assays To determine the number of eggs laid and fertility after dsRNA injections or orlistat treatment, treated females were put into individual oviposition cups containing distilled water and Whatman filter paper at 48 to 72 h PBM. One week after oviposition, the number of eggs laid and the fertility was determined using a Nikon SMZ1000 dissecting microscope. Specifically, fertility was assessed by the degree of melanization of laid eggs (black for fertile and brown for infertile), followed by needle pricking of unhatched eggs to assess the presence (or absence) of fully developed larvae. Knockdown of gene expression using dsRNA Creation of templates for dsRNA production. cDNA from female whole bodies without heads was used for RT-qPCR amplification of fragments of interest. AgTL2 (AGAP000211, 406 bp) and AgLSD1 (AGAP002890, 485 bp) fragments were amplified using the following primers with the T7 promoter sequence added to their 5′ ends: AgTL2-Fwd: 5′-taatacgactcactatagggCTTTAGTCGGGTGGAGGACA-3′, AgTL2-Rev: 5′-taatacgactcactatagggGATGATCGGTGTTCGGCTT-3′; AgLSD1-Fwd: 5′-taatacgactcactatagggCGCAGATGGAGTCGCTGT-3′, AgLSD1-Rev: 5′-taatacgactcactatagggGCACGAGTCTGGCTGATCTT-3′. GFP fragments were amplified from plasmid pCR2.1-eGFP plasmid as in [48]. The PCR products were confirmed by gel electrophoresis and dsRNA was transcribed using the Megascript T7 transcription kit (Thermo Fisher Scientific) previously described [86,87]. dsRNA injections. For single knockdown experiments, 690 ng of dsRNA (dsGFP, dsAgTL2, dsAgLSD1) was injected (Nanoject III, Drummond) into 1-day-old females at a concentration of 10 ng/nl. Gene knockdown levels were determined in at least 3 biological replicates by quantitative real-time PCR (RT-qPCR). RNA extraction, cDNA synthesis, and RT-qPCR. For RNA extraction, 7 to 10 female mosquitoes were decapitated in 1× PBS and transferred to Tri-reagent (Thermo Fisher Scientific). Samples were stored at −80°C until the day of extraction. RNA was extracted according to the manufacturer’s instructions, with minor modifications. RNA pellet was washed 3 times with 75% ethanol in RNase-free water and air-dried for 7 to 8 min. Pellets were resuspended in heated water for 10 min and DNase-treated using Turbo DNase according to manufacturer’s instructions (Thermo Fisher Scientific). Purified RNA was quantified using a NanoDrop Spectrophotometer 2000c (Thermo Fisher Scientific) and cDNA synthesis was performed as in [87] using 2 μg of RNA in 100 μl cDNA reactions. For quantification of target genes, RT-qPCR was performed in duplicate either in 7.5 or 15 μl reactions containing 1× PowerUp SYBR Green Master mix for RT-qPCR (Thermo Fisher Scientific), primers, and 2.5 or 5 μl of 10-fold diluted cDNA. Reactions were run on a QuantStudio 6 Pro real time-PCR thermocycler system (Thermo Fisher Scientific). Primers used for RT-qPCR had been previously used or designed spanning exon-exon junctions using Primer-BLAST [88]. Relative quantities of each gene in 3 or more biological replicates were determined using a standard curve with normalization against the ribosomal gene Rpl19 (AGAP004422). With the exception of RT-qPCR primers for AgTL2 that have previously been published [48], the following additional primer sets were used: AgLSD1: Fwd: 5′-CATTCAGCATACCTACGAC-3′, Rev: 5′-CACTGGTGGTGATCTTTT-3′; AgFAS: Fwd: 5′-GAGCTTGCGTAGAGATAGAT-3′, Rev: 5′-GCTAAGATTCTGTTGGAATG-3′; AGAP012959: Fwd: 5′-CTACTCTGTTCTTCCTGGTC-3′, Rev: 5′-TACCCTGTACTTCGGACTT-3′; AGAP013072: Fwd: 5′-TGAAGGCTACTCTGTTCTTC-3′, Rev: 5′-CCTGTACTTCGGACTTTC-3′; Hsp70 (AGAP004581): Fwd: 5′-CGTACTGTTTCAGTCTCAAG-3′, Rev: 5′-ATCGTACTCTTCCTTCTCTG-3′. RNA sequencing and differential gene expression analysis For RNAseq, RNA was extracted from embryos collected at different time points after oviposition from control and AgTL2-depleted blood fed An. gambiae females in 4 biological replicates as above, and then purified using PureLink RNA purification mini kit according to manufacturer’s instructions (Invitrogen). Libraries were prepared using a SciClone G3 NGSx workstation (Perkin Elmer) using the Kapa mRNA HyperPrep kit (Roche Applied Science). Polyadenylated mRNAs were captured using oligo-dT-conjugated magnetic beads (Kapa mRNA HyperPrep kit, Roche Sequencing) from 300 ng of total RNA on a Perkin Elmer SciClone G3 NGSx automated workstation. Poly-adenylated mRNA samples were immediately fragmented to 300 to 400 bp using heat and magnesium. First-strand synthesis was completed using random priming followed by second-strand synthesis and A-tailing. A dUTP was incorporated into the second strand to allow strand-specific sequencing of the library. Libraries were enriched and indexed using 11 cycles of amplification (Kapa mRNA HyperPrep kit, Roche Sequencing) with PCR primers, which included dual 8 bp index sequences to allow for multiplexing (IDT for Illumina unique dual 8 bp indexes). Excess PCR reagents were removed through magnetic bead-based cleanup using Kapa Pure magnetic beads on a SciClone G3 NGSx workstation (Perkin Elmer). The resulting libraries were assessed using a 4200 TapeStation (Agilent Technologies) and quantified by qPCR (Roche Sequencing). Libraries were pooled and sequenced on one lane of an Illumina NovaSeq S4 flow cell using paired-end, 100 bp reads. Sequencing reads were aligned to the An. gambiae genome (PEST strain, version 4.14, VectorBase release 62) using HISAT2 (version 2.2.1) with the default parameters. Reads with mapping quality scores <30 were removed using Samtools (version 1.17). The numbers of reads mapped to genes were counted using htseq-count (version 2.0.2) with the default parameters. Calculation of normalized read counts and analysis of differential gene expression was performed using the DESeq2 package (version 1.40.2) in R (version 4.3.1). Orlistat treatment Approximately 5.0 to 6.7 mg orlistat (Sigma-Aldrich) was dissolved in 50 μl pure dimethyl sulfoxide (DMSO) to give a 200 to 270 mM stock solution, which was then diluted as required with 1× PBS to a 10 mM working solution, resulting in 3.7% to 5.0% DMSO in 1× PBS. The 5 mM and 2.5 mM orlistat solutions were prepared by 2-fold serial dilution with 1× PBS. Mated wild-type females were cooled on ice and pre-injected with control or diluted amounts of orlistat (1.0, 0.5, 0.25 μg/mosquito). The amount of orlistat injected was achieved by injecting 207 nl of either the 10, 5, or 2.5 mM solutions, respectively. The control group was injected with the same DMSO concentration as the 10 mM orlistat group (i.e., 3.7% to 5.0% DMSO in 1× PBS). After 3 to 4 h of recovery at 27°C, some mosquitoes were blood fed and ovaries, midgut, and fat body tissues were dissected from non-blood fed and blood fed females at 24 and 48 h after feeding for TAG analysis. For neutral lipid staining, tissues dissected at 48 h PBM were fixed and stained with the neutral lipid dye LD540. At 3 d PBM, the remaining blood fed females were put into individual oviposition cups to assess the number of eggs developed and fertility. For tarsal delivery, mated wild-type females were allowed to rest on their tarsi for 1 h on glass plates previously coated with orlistat at 1 or 2 mmol/m2 or control plates treated with the acetone vehicle alone. After 30 min of recovery, mosquitoes were blood fed and egg development, and fertility were determined after 7 d of oviposition. P. falciparum infections of An. gambiae mosquitoes Treated mosquitoes aged 4 to 6 d were housed in small cages and provided about 400 μl of P. falciparum gametocyte cultures for 1 h through heated membrane feeders. After blood feeding, poorly fed mosquitoes were removed and cages were maintained for the duration of the experiment in a glove box: 7 to 8 d post infectious feed for oocyst quantification or 13 d for sporozoite quantification. While in the glove box, mosquitoes were provided 10% glucose solution ad libitum. Statistical analysis Data were analyzed using GraphPad Prism 10 and JMP 16 Pro statistical software. GraphPad Prism 10 was used to calculate Fisher’s exact test, Student’s t tests, and 1- or 2-way analysis of variance (ANOVA) with Dunnett’s or Šídák’s multiple comparisons correction. Prior to ANOVA, data were first assessed for normal distribution and were transformed if found not be normally distributed. JMP Pro 16 was used to generate Least Square Means models incorporating treatment, time, and their interactions as fixed effects and replicate as a random effect. A post hoc t test between groups was done to assess significance and p-values corrected using a false discovery rate (FDR) of 0.05.

Acknowledgments We are particularly grateful to Aaron Stanton, Emily Selland, and Elizabeth Nelson for mosquito rearing and all the members of the Catteruccia laboratory for helpful suggestions. We are also grateful to The Bauer Core Facility at Harvard University for RNA sequencing and the Harvard Medical School Electron Microscopy Facility for TEM. The findings and conclusions within this publication are those of the authors and do not reflect the policies or positions of the HHMI or the NIH.

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