BACKGROUND: Juvenile hormone (JH) is synthesized by the corpora allata (CA) and controls development and reproduction in insects. We recently used CRISPR/Cas9 to establish a line lacking the enzyme that catalyzes the final step of JH biosynthesis in mosquitoes, a P450 epoxidase. The CA of the epox-/- mutants do not synthesize epoxidized JH III but methyl farneosate (MF), a weak agonist of the JH receptor. Female epox-/- mosquitoes have reduced JH signaling and show a substantial loss of reproductive fitness. To understand the molecular basis of this loss of fitness, we constructed ovarian mRNA libraries of Ae. aegypti of the Orlando strain wild-type (WT) and epoxidase null mutants (epox-/-) and investigated differential expression of reproductive genes. RESULTS: We performed triplicate RNA-seq analyses of female WT and epox-/- ovaries dissected at four critical stages of oogenesis: Ovaries from newly eclosed females (0h), sugar-fed females at 4 days post-eclosion (4d SF), females 16h (16h BF), and 48 h after a blood meal (48h BF). Silencing of epoxidase resulted in a drastic change in the expression of thousands of genes. CONCLUSIONS: Our results suggest that epoxidase deficiency leads to a reduction in JH signaling that has significant effects on Ae. aegypti ovarian transcriptome profiles. Ecdysteroid titers are dysregulated in the mutants, leading to a significant delay in the expression of vitelline membrane genes and other transcripts. We discovered changes in the expression of 230 long non-coding RNAs (lncRNAs) that may play an important role in the regulation of ovarian genes.

• Keywords
• Aedes aegypti, Ecdysteroids, Juvenile hormone, Ovaries, Transcriptome, lncRNA,
• MeSH
• Aedes * genetics   physiology   MeSH
• Juvenile Hormones * metabolism   MeSH
• Oogenesis * genetics   MeSH
• Ovary * metabolism   MeSH
• Signal Transduction * genetics   MeSH
• Gene Expression Profiling MeSH
• Transcriptome * MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Juvenile Hormones * MeSH

Sand flies infect more than 1 million people annually with Leishmania parasites and other bacterial and viral pathogens. Progress in understanding sand fly adaptations to xenobiotics has been hampered by the limited availability of genomic resources. To address this gap, we sequenced, assembled, and annotated the transcriptomes of 11 phlebotomine sand fly species. Subsequently, we leveraged these genomic resources to generate novel evolutionary insights pertaining to their adaptations to xenobiotics, including those contributing to insecticide resistance. Specifically, we annotated over 2,700 sand fly detoxification genes and conducted large-scale phylogenetic comparisons to uncover the evolutionary dynamics of the five major detoxification gene families: cytochrome P450s (CYPs), glutathione-S-transferases (GSTs), UDP-glycosyltransferases (UGTs), carboxyl/cholinesterases (CCEs), and ATP-binding cassette (ABC) transporters. Using this comparative approach, we show that sand flies have evolved diverse CYP and GST gene repertoires, with notable lineage-specific expansions in gene groups evolutionarily related to known xenobiotic metabolizers. Furthermore, we show that sand flies have conserved orthologs of (i) CYP4G genes involved in cuticular hydrocarbon biosynthesis, (ii) ABCB genes involved in xenobiotic toxicity, and (iii) two primary insecticide targets, acetylcholinesterase-1 (Ace1) and voltage gated sodium channel (VGSC). The biological insights and genomic resources produced in this study provide a foundation for generating and testing hypotheses regarding the molecular mechanisms underlying sand fly adaptations to xenobiotics.

• Keywords
• comparative genomics, cytochrome P450s, gene family evolution, phlebotomine sand flies, xenobiotic adaptation,
• MeSH
• Phylogeny * MeSH
• Genomics MeSH
• Glutathione Transferase genetics   metabolism   MeSH
• Insecticides * pharmacology   MeSH
• Inactivation, Metabolic genetics   MeSH
• Evolution, Molecular * MeSH
• Psychodidae * genetics   MeSH
• Insecticide Resistance * genetics   MeSH
• Cytochrome P-450 Enzyme System genetics   metabolism   MeSH
• Xenobiotics metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH
• Names of Substances
• Glutathione Transferase MeSH
• Insecticides * MeSH
• Cytochrome P-450 Enzyme System MeSH
• Xenobiotics MeSH

The corpora allata-corpora cardiaca (CA-CC) is an endocrine gland complex that regulates mosquito development and reproduction through the synthesis of juvenile hormone (JH). Epoxidase (Epox) is a key enzyme in the production of JH. We recently utilized CRISPR/Cas9 to establish an epoxidase-deficient (epox-/-) Aedes aegypti line. The CA from epox-/- mutants do not synthesize epoxidated JH III but methyl farneosate (MF), a weak agonist of the JH receptor, and therefore have reduced JH signalling. Illumina sequencing was used to examine the differences in gene expression between the CA-CC from wild type (WT) and epox-/- adult female mosquitoes. From 18,034 identified genes, 317 were significantly differentially expressed. These genes are involved in many biological processes, including the regulation of cell proliferation and apoptosis, energy metabolism, and nutritional uptake. In addition, the same CA-CC samples were also used to examine the microRNA (miRNA) profiles of epox-/- and WT mosquitoes. A total of 197 miRNAs were detected, 24 of which were differentially regulated in epox-/- mutants. miRNA binding sites for these particular miRNAs were identified using an in silico approach; they target a total of 101 differentially expressed genes. Our results suggest that a lack of epoxidase, besides affecting JH synthesis, results in the diminishing of JH signalling that have significant effects on Ae. aegypti CA-CC transcriptome profiles, as well as its miRNA repertoire.

• Keywords
• Aedes aegypti, Corpora allata, Epoxidase, Juvenile hormone, Mosquito, RNA-Seq, Transcriptome, microRNA,
• MeSH
• Aedes * genetics   metabolism   MeSH
• Corpora Allata metabolism   MeSH
• Gene Expression MeSH
• Juvenile Hormones metabolism   MeSH
• MicroRNAs * genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Juvenile Hormones MeSH
• MicroRNAs * MeSH

The blood-sucking hemipteran Rhodnius prolixus is one of the main vectors of Chagas disease, a neglected tropical disease that affects several million people worldwide. Consuming a blood meal and mating are events with a high epidemiological impact since after each meal, mated females can lay fertile eggs that result in hundreds of offspring. Thus, a better knowledge of the control of R. prolixus reproductive capacity may provide targets for developing novel strategies to control vector populations, thereby reducing vector-host contacts and disease transmission. Here, we have used a combination of gene transcript expression analysis, biochemical assays, hormone measurements and studies of locomotory activity to investigate how mating influences egg development and egg laying rates in R. prolixus females. The results demonstrate that a blood meal increases egg production capacity and leads to earlier egg laying in mated females compared to virgins. Virgin females, however, have increased survival rate over mated females. Circulating juvenile hormone (JH) and ecdysteroid titers are increased in mated females, a process mainly driven through an upregulation of the transcripts for their biosynthetic enzymes in the corpus allatum and ovaries, respectively. Mated females display weaker locomotory activity compared to virgin females, mainly during the photophase. In essence, this study shows how reproductive output and behaviour are profoundly influenced by mating, highlighting molecular, biochemical, endocrine and behavioral features differentially expressed in mated and virgin R. prolixus females.

• MeSH
• Chagas Disease * MeSH
• Oviposition physiology   MeSH
• Humans MeSH
• Parasites * MeSH
• Rhodnius * physiology   MeSH
• Reproduction MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH

Juvenile hormone (JH), a sesquiterpenoid produced by the insect corpus allatum gland (CA), is a key regulator of insect metamorphosis, reproduction, caste differentiation, and polyphenism. The first part of JH biosynthesis occurs via the universal eukaryotic mevalonate pathway. The final steps involve epoxidation and methylation. However, the sequence of these steps might not be conserved among all insects and Crustacea. Therefore, we used available genomic and transcriptomic data and identified JH acid methyltransferase (JHAMT), analyzed their genomic duplications in selected model organisms, and reconstructed their phylogeny. We have further reconstructed phylogeny of FAMeT proteins and show that evolution of this protein group is more complicated than originally appreciated. The analysis delineates important milestones in the evolution of several JH biosynthetic enzymes in arthropods, reviews major literature data on the last steps of JH synthesis, and defines questions and some hypotheses worth pursuing experimentally.

• Keywords
• Alternative splicing, Epoxidase, Evolution, Gene duplication, Juvenile hormone, Methyl transferase,
• MeSH
• Corpora Allata MeSH
• Insecta genetics   metabolism   MeSH
• Insect Proteins metabolism   MeSH
• Juvenile Hormones * metabolism   MeSH
• Sesquiterpenes * metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Insect Proteins MeSH
• Juvenile Hormones * MeSH
• Sesquiterpenes * MeSH

The rigorous balance of endocrine signals that control insect reproductive physiology is crucial for the success of egg production. Rhodnius prolixus, a blood-feeding insect and main vector of Chagas disease, has been used over the last century as a model to unravel aspects of insect metabolism and physiology. Our recent work has shown that nutrition, insulin signaling, and two main types of insect lipophilic hormones, juvenile hormone (JH) and ecdysteroids, are essential for successful reproduction in R. prolixus; however, the interplay behind these endocrine signals has not been established. We used a combination of hormone treatments, gene expression analyses, hormone measurements, and ex vivo experiments using the corpus allatum or the ovary, to investigate how the interaction of these endocrine signals might define the hormone environment for egg production. The results show that after a blood meal, circulating JH levels increase, a process mainly driven through insulin and allatoregulatory neuropeptides. In turn, JH feeds back to provide some control over its own biosynthesis by regulating the expression of critical biosynthetic enzymes in the corpus allatum. Interestingly, insulin also stimulates the synthesis and release of ecdysteroids from the ovary. This study highlights the complex network of endocrine signals that, together, coordinate a successful reproductive cycle.

• Keywords
• corpus allatum, endocrine signaling, hormone titers, insect, ovary,
• MeSH
• Ecdysteroids metabolism   MeSH
• Insect Hormones * metabolism   MeSH
• Insulin, Regular, Human MeSH
• Insulin metabolism   MeSH
• Juvenile Hormones metabolism   MeSH
• Rhodnius * metabolism   MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Ecdysteroids MeSH
• Insect Hormones * MeSH
• Insulin, Regular, Human MeSH
• Insulin MeSH
• Juvenile Hormones MeSH

Juvenile hormone (JH) is synthesized by the corpora allata (CA) and controls development and reproduction in insects. Therefore, achieving tissue-specific expression of transgenes in the CA would be beneficial for mosquito research and control. Different CA promoters have been used to drive transgene expression in Drosophila, but mosquito CA-specific promoters have not been identified. Using the CRISPR/Cas9 system, we integrated transgenes encoding the reporter green fluorescent protein (GFP) close to the transcription start site of juvenile hormone acid methyl transferase (JHAMT), a locus encoding a JH biosynthetic enzyme, specifically and highly expressed in the CA of Aedes aegypti mosquitoes. Transgenic individuals showed specific GFP expression in the CA but failed to reproduce the full pattern of jhamt spatiotemporal expression. In addition, we created GeneSwitch driver and responder mosquito lines expressing an inducible fluorescent marker, enabling the temporal regulation of the transgene via the presence or absence of an inducer drug. The use of the GeneSwitch system has not previously been reported in mosquitoes and provides a new inducible binary system that can control transgene expression in Aedes aegypti.

• MeSH
• Aedes * genetics   MeSH
• Corpora Allata * MeSH
• Drosophila MeSH
• Gene Expression MeSH
• Animals, Genetically Modified MeSH
• Juvenile Hormones MeSH
• Green Fluorescent Proteins genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Juvenile Hormones MeSH
• Green Fluorescent Proteins MeSH