UGAL strain Aedes aegypti were used for all experiments. Mosquito colonies were maintained at 27°C on a 16:8h L:D cycle. Larvae were fed Cichlid Gold fish pellets (Hikari, USA, Hayward, CA), and adult mosquitoes were fed an 8% sucrose solution until 2 days post-emergence. Adult females were fed defibrinated rabbit blood (Hemostat Laboratories, Dixon, CA, USA) by an artificial feeding apparatus warmed to 37°C.

Putative AAEL003647 and AAEL019988 orthologs were identified using OrthoDB (44). Taxa were chosen to represent all possible insect orders with available genome sequences ( Tables S1 , S2 ). Protein sequences were aligned using hmmalign as implemented in HMMER (45) with the –trim option. Gaps in alignments were manually removed, and trimmed alignments were used to construct maximum likelihood phylogenies using PhyML (46) using the options “-d aa -m LG -f e -o tl -b -2”. FigTree version 1.4.4 was used for visualization of trees and trees were rooted on the midpoint. Accessions of included sequences are given in file S1.

Eight to ten-day old, non-blood fed mated females were collected and dissected into head, gut, fat body, abdominal carcass (“pelt”), and clean ovaries without bursa or accessory glands in sterile, nuclease-free, Aedes saline. Additional ovary samples were collected from females at 2-hour intervals post-feeding (pbf) until 12 hours, then at 24, 48, and 72 hours pbf. Four or more tissue samples were collected for each tissue and time point. After collection, tissue samples were stored at -80°C prior to RNA extraction. Tissue samples were thawed on ice and homogenized with a rotor pestle. Total RNA was isolated from homogenized tissues using the RNeasy Mini kit (Qiagen, Venlo, The Netherlands) according to manufacturer instructions. DNA was removed from each RNA sample using the Turbo DNA-free kit (Ambion, Austin, TX, USA). One hundred nanograms of RNA was used as input to synthesize cDNA using the iScript cDNA synthesis kit (BioRad, Hercules, CA, USA). cDNA templates were used for quantitative real-time PCR, with the Quantifast SYBR Green PCR kit (Qiagen) and gene specific primers ( Table S3 ). Standard curves for each gene were generated by cloning qPCR products into the pSCA vector with the Strataclone PCR cloning kit (Agilent, Santa Clara, CA, USA), isolating plasmid DNA using the GeneJET Plasmid Miniprep Kit (Thermo Scientific, Vilnius, Lithuania), and preparing plasmid standards to a known copy number. Expression levels of ribosomal protein S7 were used as a housekeeping gene to normalize transcript abundance.

A 400-500 bp region of each gene was chosen as a target for dsRNA synthesis for AAEL003647 and AAEL019988, subsequently referred to as ds3647 and ds19988, respectively. Primers including the T7 promoter sequence were used to amplify each target using cDNA synthesized from RNA isolated from whole body, non-blood fed females ( Table S3 ). PCR products were cloned into the pSCA vector and plasmid DNA was extracted using methods listed above. Plasmid DNA from each target and an EGPF control were used as the templates for dsRNA synthesis. dsRNA was synthesized using the MEGAscript RNAi kit (Ambion, Vilnius, Lithuania), according to manufacturer instructions. Following dsRNA synthesis, dsRNA was precipitated in ethanol and resuspended in Aedes saline to a concentration of 2µg/µL.

Newly emerged (≤ 1d post eclosion) mated females were injected with 2 µg ds3647, ds19988, or dsEGFP. To validate receptor knockdown, whole body females were collected 7 days post-injection. qPCR was used to validate knockdown of each gene using the methods detailed above. Females were blood fed three days post-injection and separated into individual egg laying chambers consisting of a damp paper towel in a plastic cup with a lid and a dental wick with 8% sucrose solution, for yolk deposition and fecundity bioassays. For yolk deposition bioassays, females were collected at 24, 48, and 72 hours PBF. Ovaries were dissected and yolk deposition per oocyte was measured along the anterior-posterior axis using an ocular micrometer. Five oocytes were measured and averaged per female, and 5 females were used per time point and treatment. Egg laying was measured by providing females with a wet paper towel at 72 h post blood feeding to stimulate egg deposition. Females were given 48 h to deposit eggs. After 48 h hours, the number of eggs laid per individual female was counted. Another cohort of knockdown females were allowed to lay eggs then dissected and the number of retained, mature oocytes were counted. Eggs that were laid were separated by parent and allowed to hatch, and the proportion of hatched versus unhatched eggs was recorded for each treatment.

Our phylogenetic analysis included diverse insect species to identify the closest receptor relatives across both holometabolous and hemimetabolous insects. Our results for AAEL003647 indicate this receptor groups in a strongly supported clade of receptors that are distinct from, but sister to, the SIFamide receptors ( Figure 1 ). These receptors are found in the genomes of culicids as well as cockroaches (Periplaneta americana and Blattella germanica), termites (Zootermopsis nevadensis). This robustly supported sister clade to SIFamide receptors suggest an ancient split between SIFamide receptors and the orthologs of AAEL003647 which predates the split of hemi- and holometabolous insects. Orthologs of AAEL003647 appear to have been lost in many lineages. No orthologs were found in lepidopteran, coleopteran or hymenopteran genomes. In contrast, most sequenced hemipteran genomes contained orthologs, several of which have subsequently duplicated. In the order Diptera, AAEL003647 orthologs were found in most nematoceran genomes, but absent from many available brachyceran genomes, including sequences from all members of the genus Drosophila. This loss was not complete in Brachycera, as Rhagoletis zephyria and Hermetia illucens both encode AAEL003647 orthologs in their genomes. Within the Culicidae, each species examined has a single ortholog of AAEL003647 with the notable exception of Anopheles maculatus, which has five orthologous sequences in OrthoDB (44). Two sequences were identified as orthologs of the SIFamide receptor (AMAM023590 and AMAM011260), and two orthologs identified as orthologs of AAEL003647 (AMAM023042 and AMAM009506). All of these sequences are lacking the complete 7 transmembrane region of canonical GPCRs and it seems likely that these sequences do not reflect true orthologs but rather annotation artefacts, potentially fragments of a single ortholog to the SIFamide receptor and AAEL003647. An additional duplication in Anopheles maculatus groups with the SIFamide-like receptor of Thrips palmi. Further investigation of this ortholog suggests that it is unique to An. maculatus, and that its grouping with non-mosquito sequences is likely an artifact of the alignment. Improved sequencing of the An. maculatus genome will likely resolve this in the future.

Our analysis identified AAEL019988 as an ortholog of the D. melanogaster trapped in endoderm (tre1) GPCR with strong support ( Figure 2 ). Tre1 appears to be highly conserved among holometabolous insects but is absent from many hemimetabolous lineages. Only the orders Blattodea, Odonata, Thysanoptera, and Grylloblattidae encode orthologs. The sister group to this clade includes both the GPCRs Moody and Moody-like, which are known to be important to blood-brain barrier in Drosophila melanogaster ( 47).

We investigated expression patterns of AAEL003647 and AAEL019988 among life stages, sexes, and tissues. Expression of AAEL003647 was highest in females relative to males and immature stages (one-way ANOVA, p < 0.0001) ( Figure 3A ). Expression of AAEL019988 was higher in adult females relative to 1^st, 3^rd, 5^th instar larval, and pupal stage mosquitoes (one-way ANOVA, p < 0.05). There was no significant difference in expression between females and males ( Figure 3B ). We next examined tissue tropism of the receptors in females. The highest expression of AAEL003647 and AAEL019988 was observed in the ovaries ( Figures 4A, B ). We next measured receptor expression across a time series following a blood meal. Our results demonstrate that expression of AAEL003647 was highest in non-blood fed, 2h, 4h, and 6h pbf female ovaries ( Figure 4C ). Expression of AAEL019988 was highest in NBF ovaries ( Figure 4D ).

The peaks of expression prior to feeding and nearing the time of oviposition informed our hypothesis that AAEL003647 and AAEL019988 may be important in regulation of egg production and/or oviposition. To understand the effects of both orphan GPCRs on oviposition, we injected newly eclosed female mosquitoes with 2 µg of ds3647, ds19988, or dsEGFP. For each receptor, we were able to achieve an 85% whole body transcript knockdown (one-way ANOVA, p < 0.0163, p < 0.0163, respectively; Figures 5A, B ). Following dsRNA injection, females were allowed to mate and were fed 3 days post-injection. After feeding, females were separated into individual enclosures for oviposition assays. We found that ds3647 and ds19988 injected females laid significantly fewer eggs than dsEGFP injected females (one-way ANOVA, p = 0.0184, p = 0.0393, respectively; Figure 5C ).

The observed reduction in egg laying by mosquitoes treated with ds3647 or ds19988 could be due to a disruption of egg maturation or egg laying. To disentangle this, we examined whether yolk deposition of ds3647 and ds19988 injected females was impaired, which would suggest that the receptors are important in post-vitellogenic egg development. We injected newly eclosed females with dsEGFP, ds3647 or ds19988, fed females a blood meal at 3 days post injection, and dissected ovaries from blood fed females at 24, 48, and 72h pbf. Following dissection, we measured the packaged yolk in per individual oocyte with an ocular micrometer. We found no significant difference among oocyte yolk lengths in ds3647, ds19988, or dsEGFP injected females (one-way ANOVA, p > 0.05; Figure 5D ), suggesting that the receptors mediate physiological events after egg maturation. We then examined the effect of receptor knockdown on the egg retention and egg hatching. Knockdown of AAEL003647 did not result in retained eggs in females, but AAEL019988 knockdown mosquitoes retained more mature oocytes than dsEGFP controls ( Figure 6A , p = 0.0476; Wilcoxon rank-sum test). Of eggs that were laid, there was no difference in the proportion of eggs that hatched, suggesting that knockdown of the receptors does not interfere with fertilization ( Figure 6B ).