Candidate molecules that circulate in human blood plasma and that stimulate different GPCRs were selected based on, a) their involved in the regulation of reproduction and nutrient metabolism in humans and b) the identification of orphan GPCRs in the mosquito genome that are sequence orthologues of human receptors .Human ligands for GPCRs of several receptor families were selected and included peptides that activate Class A GPCRs (a.k.a Rhodopsin family GPCRs): oxytocin, galanin (GAL), kisspeptin, neuropeptide Y (NPY) that are basic determinants of reproductive functions luteinizing hormone releasing hormone (LHRH), which stimulates the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and triggers ovulation in females and melatonin (MT) which regulates circadian rhythms of feeding and peaks in the blood at dawn (when mosquitoes are more actively searching a blood meal); Ligands of Class B GPCRs (a.k.a. Secretin-GPCRs) glucagon-like peptide 1 (GLP1), glucagon-like peptide 2 (GLP2) and vasoactive intestinal peptide (VIP) that regulate feeding behaviour, gut motility, glucose and insulin metabolism and parathyroid hormone (PTH), calcitonin (CT) and corticotrophin releasing hormone (CRH) which regulate ion metabolism and the stress response in humans and other vertebrates and also indirectly affects feeding behaviour and of Class C GPCRs (a.k.a Glutamate GPCRs) the Glutamate and ɣ-aminobutyric acid (or GABA) which are two important neurotransmitters that stimulate feeding. Two fish peptides (CT and LHRH) were also tested as they are potent activators of the human peptide receptors and have similar functions to their human orthologues. No potential activity or physiological effect of the selected human GPCR-ligands in mosquitoes is known.
To test the potential involvement of the selected peptides on mosquito reproduction and egg development, different diets were formulated. Peptides were added either to an initial liquid diet (i-liq_diet) providing amino acids, vitamins and carbohydrates (based on DMEM tissue culture medium ref. 12–604F from Lonza) or to a rich liquid diet (r-liq_diet), that consisted of i-liq_diet supplemented with: a) a phagostimulant (ATP), b) proteins (BSA) essential for egg maturation and c) cholesterol . Insects cannot synthesize cholesterol de novo and it is a precursor of the ecdysteroid hormone with a key role in yolk synthesis and egg maturation. Diets containing different peptides were supplied to female mosquitoes using a standard artificial feeding apparatus  and compared with fresh mouse blood. Oogenesis was assessed by changes in Vg expression 24h post-feeding. To confirm that Vg expression induced oogenesis progression, the number of females presenting eggs was evaluated.
Mosquito fitness is a determinant factor for the success of a mosquito colony when they are released into the wild. Colonies from each diet were maintained under standard insectary conditions, and the life cycle was followed for one generation. Larvae, pupae and adult mortality were recorded. Offspring from females fed on r-liq_diet + P2 (hGLP2), P3 (hPTH) or P6 (hGABA) showed better performances (eg. lower rates of dead larvae and dead adults/egg number/female) when compared to blood and other diets, however the total number of female progeny was slightly lower than in progeny of blood-fed females. A blood meal had the highest impact on larvae mortality, suggesting that stable highly nutritious artificial diets, without fresh blood, can reduce mortality improving mosquito rearing success. Variability (SE) was always higher in the blood group when compared with other diets and is probably a reflection of the variable composition of blood and emphasises the usefulness of fresh-blood-free diets.
The availability of a successful blood substitute diet will allow mass production of anautogenous mosquitoes without the need for costly animal care facilities or blood or plasma supply. The development of chemically well-defined artificial diet to provide a reliable and consistent nutrition to adult mosquitoes is a breakthrough for mass rearing of mosquitoes. Herein we described a formulated diet for anautogenous female Anopheles mosquitoes that mimicked a standardized vertebrate blood meal. The fresh-blood-free diet formulated stimulates oogenesis and egg production and has a similar or superior effect on mosquito fitness relative to a standard vertebrate blood in Anopheles coluzzii and we reveal it can also be used to feed other anopheline species. Supplementation of the blood-free diet with the vertebrate peptides that activate GPCRs regulating reproduction and metabolism reveals that they modify mosquito physiology. The putative mosquito GPCRs activated by the vertebrate peptides remain to be characterized. Of the tested human peptides, P2 (hGLP 2), had the most notable effect when it was introduced in the blood-free artificial diet and it significantly increased VTG expression, mosquito egg production and offspring fitness relative to blood-fed mosquitoes. P2 is a 33-amino acid peptide present in enteroendocrine L-cell and released in response to nutrient intake and it stimulates cell proliferation, inhibition of apoptosis and proteolysis in the small and large intestine in human [23,24].
Except otherwise indicated, all reactions were performed at room temperature (20°C), reagents were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA), and female mosquitoes of Anopheles coluzzii (former Anopheles gambiae M form) Yaoundé strain were used. The peptides calcitonin (from human), calcitonin (from salmon), glucagon-like peptide 1 [1-37aa] (human, bovine, guinea pig, mouse, rat) trifluoroacetate salt, and glucagon-like peptide 2 [1-33aa] (human) ammonium acetate salt were purchased from Bachem (Germany). The peptides parathyroid hormone (from human), oxytocin (from human), kisspeptin 10 (from human), melatonin, and neuropeptide Y (from human, rat) were purchased from Tocris (Biogene, Spain).
All animal experiments were carried out in strict accordance with the Portuguese law and guidelines for the use of laboratory animals. The protocols used were approved by Direção-Geral de Veterinária, Ministério da Agricultura do Desenvolvimento Rural e das Pescas, Portugal (id approvals: 023351 and 023355). Mosquitoes were obtained from a laboratory colony of A. coluzzii (Yaoundé line) and Anopheles stephensi. Anopheles aquasalis were obtained from a colony at the Entomology Department Insectary of the Fundação de Medicina Tropical Dr Heitor Vieira Dourado (FMT-HVD) (id approvals: CEUA 01/2013), that were derived from a colony established in 1995 . Mosquitoes were maintained under standard insectary conditions of 26 ± 1°C, 75% humidity and a 12h:12h light:dark cycle. Adult mosquitoes were fed on 10% glucose solution ad libitum until the day before feeding trials.
Female CD1 mice (Mus musculus), obtained from the IHMT Animal house, were intraperitoneally anesthetized with 20% Imalgène 1000 (Merial, Portugal) and 10% Rompun (Bayer, Portugal). Female mosquitoes fed directly on healthy female CD1 mice for 30–45 min, with regular monitoring to verify that mice were anesthetized. Unfed mosquitoes were removed and fully engorged mosquitoes were kept at 26 ± 1°C, 75% humidity. When needed, a cardiac puncture was performed on anesthetized mouse to collect 1 mL of blood for artificial blood feeding assays.
The ovaries of A. coluzzii female mosquitoes were collected twenty-four hours after feeding on blood or on supplemented liquid diet and fixed for 15 min with 4% v/v paraformaldehyde (Alfa Aesar, Massachusetts, USA) in PBS. Samples were washed twice with 0.5% Triton in PBS and incubated with 250 µM Nile Red (MP Biomedicals, USA) for one hour in the dark. Samples were washed twice with 0.5% Triton in PBS and the slides were mounted by using Vectashield with dapi (Vector Laboratories, USA) and analyzed using a Leica TCS SP5 laser scanning confocal microscope (20x, 40x, and 60x objectives).
Total RNA from female fatbodies was isolated using the TRI Reagent protocol and treated with 1 U DNase for 1 min according to the manufacturer’s instructions to eliminate genomic DNA contamination. DNase I treated total RNA (1,5 µg) was denatured at 70°C for 10 min, quenched on ice for 5 min and used for cDNA synthesis in a 20 µl final reaction volume containing 10 µL of 2 × RT buffer mix, 1 µL of 20 × RT enzyme mix (Thermofisher, Alfagene, Portugal), and nuclease-free water. cDNA was synthesized for 60 min at 37°C followed by 5 min at 95°C to stop the reaction and hold at 4°C. The quality and quantity of the cDNA produced was assessed by PCR amplification of the ribosomal protein S7 unit using the following protocol: 95°C for 3 min; 35 cycles of 95°C for 30 sec, 60°C for 30 sec, 72°C for 30 sec, followed by 72°C for 5 minutes . PCR reactions were carried out for a 10 µl final reaction volume containing 1.5 mM MgCl2 (Thermo Scientific, Alfagene, Portugal), 0.2 mM dNTPs (GE Healthcare, Spain), 0.25 µM of each gene specific primer pair and 0.5U of DreamTaq DNA Polymerase (5 U/µl, Thermo Scientific, Alfagene, Portugal) and the amplification products analysed on agarose electrophoresis gel.
Quantitative Real-time PCR (q-RT-PCR) analysis was used to quantify the expression of Vitellogenin-1 precursor (Vg), a protein biomarker of mosquito oogenesis initiation. Vg expression levels in the fatbodies were quantified using the ΔΔCT method a) after a blood meal, b) after microinjection with the different peptides, and c) after being fed on the various artificial diets. Primers used are described in Table S2 (Supplementary Information). Briefly, cDNA samples were diluted (1:10 or 1:5) with ultrapure water prior to use as a template in q-RT-PCR and reactions were performed in triplicate (<5% variation between replicates) using a CFX Connect Real-Time PCR Detection System (Bio-Rad, Portugal) for 96-well microplates (Bio-Rad, Portugal). Analyses were performed in 20 µl final reaction volume with 300 nM of forward and reverse primer, SsoFast EvaGreen supermix (Bio-Rad, Portugal) and 2 µl of the diluted cDNA template. Optimized cycling conditions consisted of 95°C for 30 sec, followed by 45 cycles of 95°C for 5 sec and the appropriate annealing temperature for each primer pair for 10 sec. PCR reactions included a standard curve, melting curves were performed to detect primer dimers and negative control reactions were included to assess for genomic contamination. PCR reaction efficiencies and r2 (coefficient of determination) were calculated for each target gene and transcript expression was normalized using ribosomal S7 subunit as reference gene.
Two-day-old female A. coluzzii mosquitoes were cold-anaesthetized and injected intrathoraxically with 69 nL of 10 µM peptide. For each experiment, a control group injected with PBS was included. Injections were performed using a microinjection system (Nanoject; Drummond Scientific). The complete list of the peptides used and respective abbreviations are presented on Supplementary Information, Table S1.
Mosquitoes (n = 30 approximately) were kept inside paper cups covered with a net. Each cup had a glass feeder on top connected to 2 plastic tubes for water inlet and outlet and temperature within the multiple cylindrical water-jacked plastic was kept at 37.5°C by a constant water flow supply. Parafilm® was stretched across the mouth of the feeder and 1 mL of a pre-warmed meal was pipetted into the glass feeder. Peptides were tested at a 10 µM final concentration. Mosquitoes were allowed to feed for 60 minutes. Unfed mosquitoes were removed and fully engorged female mosquitoes were kept at 26 ± 1°C under 75% humidity.
The i-liq_diet consisted on Dulbecco’s modified Eagle’s medium (high glucose with L-glutamine from Lonza, 12–604F). The formulation of the enriched artificial meal (r-liquid diet) is listed on Supplementary Information, Table S3. ATP, cholesterol and BSA were purchased from Sigma-Aldrich Corporation (A6419, C4951 and A7906 respectively). For each experiment, diet formulae were freshly prepared from stock solutions.
Fatbodies were collected from pools of 30–35 mosquitoes. Tissues were dissected, transferred to RNAlater (Ambion, Alfagene, Portugal) and stored at −20°C until RNA extraction. For the blood meal assays, mosquito fatbodies were collected at different time points (3, 6, 12, 24, 28, and 32 hours post blood meal). Fatbodies from females feed on 10% glucose at the same time points were used as controls. For the peptides screening experiment and artificial diet assays, mosquito fatbodies were dissected 24h post-injection and 24h post-feeding, respectively.
Experimental feeding of mosquitoes using blood, i-liquid diet, r-liquid diet (Supplementary Information, Table S3) or r-liquid diet supplemented with blood peptides (Supplementary Information, Table S1) was performed as described above. As a proxy of feeding success, the number of mosquitoes that were fully engorged was recorded and the percentage of fed mosquitoes determined. To determine the number of eggs per female, at 48 h post-feed, mosquitoes were dissected, the ovaries collected and the number of eggs per female determined under a hand held magnifying glass. For egg laying and mosquito development, 30 fully engorged females were placed in individual cages (20 × 20 × 20 cm) with humidified filter paper for egg laying (30 females/replicate, 3 replicates/diet). Eggs were counted 48 and 72h post-feed under a hand held magnifying glass. The total number of eggs/female was registered.
Data are presented as the mean ± standard deviation of at least three independent experiments (except where otherwise indicated), and the corresponding standard deviations in histograms are represented by error bars. The Student’s t test was used to compare independent groups when data followed a Gaussian distribution, and differences were considered significant when P ≤ 0.05. The Fisher’s exact test was used to compare the differences on proportions among distinct diet-fed groups. The statistical analysis was performed on GraphPad Prism6 software.
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