Fast, ultra-trace detection of juvenile hormone III from mosquitoes using mass spectrometry

In the present work, a new protocol for fast separation and quantification of JH III from biological samples using liquid chromatography coupled to electrospray tandem mass spectrometry is described. In particular, the proposed protocol improves existing methodologies by combining a limited number of sample preparation steps with fast LC-MS/MS detection, providing lower limits of detection and demonstrated matrix effect control, together with high inter and intraday reproducibility. A limit of detection of 8 pg/mL (0.32 pg on column) was achieved, representing a 15-fold gain in sensitivity with respect to previous LC-MS based protocols. The performance of the LC-MS/MS protocol is comparable to previously described JH III quantitation protocol based on fluorescence detection, with the added advantage that quantification is independent of the availability of fluorescent tags that are often unavailable or show quite diverse responses on a batch-to-batch basis. Additionally, a detailed description of the JH III fragmentation pathway is provided for the first time, based on isolation of the molecular ion and their intermediate fragments using in-source MS/MS, MS/MSn and FT-ICR MS/MS measurements. The JH III workflow was evaluated as a function of developmental changes, sugar feeding and farnesoic acid stimulation in mosquitoes and can be applied to the detection of other juvenile hormones

Farnesyl Phosphatase, a Corpora allata Enzyme Involved in Juvenile Hormone Biosynthesis in Aedes aegypti

Background: The juvenile hormones (JHs) are sesquiterpenoid compounds that play a central role in insect reproduction, development and behavior. The late steps of JH III biosynthesis in the mosquito Aedes aegypti involve the hydrolysis of farnesyl pyrophosphate (FPP) to farnesol (FOL), which is then successively oxidized to farnesal and farnesoic acid, methylated to form methyl farnesoate and finally transformed to JH III by a P450 epoxidase. The only recognized FPP phosphatase (FPPase) expressed in the corpora allata (CA) of an insect was recently described in Drosophila melanogaster (DmFPPase). In the present study we sought to molecularly and biochemically characterize the FPP phosphatase responsible for the transformation of FPP into FOL in the CA of A. aegypti. Methods: A search for orthologs of the DmFPPase in Aedes aegypti led to the identification of 3 putative FPPase paralogs expressed in the CA of the mosquito (AaFPPases-1, -2, and -3). The activities of recombinant AaFPPases were tested against general phosphatase substrates and isoprenoid pyrophosphates. Using a newly developed assay utilizing fluorescent tags, we analyzed AaFPPase activities in CA of sugar and blood-fed females. Double-stranded RNA (dsRNA) was used to evaluate the effect of reduction of AaFPPase mRNAs on JH biosynthesis. Conclusions: AaFPPase-1 and AaFPPase-2 are members of the NagD family of the Class IIA C2 cap-containing haloalkanoic acid dehalogenase (HAD) super family and efficiently hydrolyzed FPP into FOL. AaFPPase activities were different in CA of sugar and blood-fed females. Injection of dsRNAs resulted in a significant reduction of AaFPPase-1 and AaFPPase-2 mRNAs, but only reduction of AaFPPase-1 caused a significant decrease of JH biosynthesis. These results suggest that AaFPPase-1 is predominantly involved in the catalysis of FPP into FOL in the CA of A. aegypti

Following de novo triglyceride dynamics in ovaries of Aedes aegypti during the previtellogenic stage

Understanding the molecular and biochemical basis of egg development is a central topic in mosquito reproductive biology. Lipids are a major source of energy and building blocks for the developing ovarian follicles. Ultra-High Resolution Mass Spectrometry (UHRMS) combined with in vivo metabolic labeling of follicle lipids with deuterated water (2H2O) can provide unequivocal identification of de novo lipid species during ovarian development. In the present study, we followed de novo triglyceride (TG) dynamics during the ovarian previtellogenic (PVG) stage (2–7 days post-eclosion) of female adult Aedes aegypti. The incorporation of stable isotopes from the diet was evaluated using liquid chromatography (LC) in tandem with the high accuracy (\u3c 0.3 ppm) and high mass resolution (over 1 M) of a 14.5 T Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (14.5 T FT-ICR MS) equipped with hexapolar detection. LC-UHRMS provides effective lipid class separation and chemical formula identification based on the isotopic fine structure. The monitoring of stable isotope incorporation into de novo incorporated TGs suggests that ovarian lipids are consumed or recycled during the PVG stage, with variable time dynamics. These results provide further evidence of the complexity of the molecular mechanism of follicular lipid dynamics during oogenesis in mosquitoes

A comparative analysis of corpora allata-corpora cardiaca microRNA repertories revealed significant changes during mosquito metamorphosis

The corpora allata (CA) are a pair of endocrine glands with neural connections to the brain and close association with another neuroendocrine organ, the corpora cardiaca (CC). The CA from adult female Aedes aegypti mosquitoes synthesize fluctuating levels of juvenile hormone (JH), which have been linked to the ovarian development and are influenced by nutritional signals. In this study, we investigated the potential involvement of microRNAs (miRNAs), a type of small non-coding RNAs, in the regulation of gene expression in CA-CC complexes during mosquito reproductive development, at stages with distinct JH biosynthesis patterns. We analyzed the miRNA repertoires expressed in the CA-CC of pupae, sugar-fed and blood-fed female Ae. aegypti. In total, 156 mature miRNAs were detected in the CA-CC, with 84 displaying significant differences in expression among the three CA-CC developmental stages. There were more miRNAs that were expressed in pupae, and decreased or were absent after adult emergence, when compared with changes between CA-CC of sugar and blood-fed females. Analysis of the genes identified as potential targets for the CA-CC miRNA repertoires classified them into the broad categories of metabolism, information storage and processing, and cellular processes and signaling; with genes involved in cellular processes and signaling representing the largest portion. Among them, the signal-transduction mechanisms and intracellular trafficking, secretion and vesicular transport contained almost 55% of the genes' targets. A substantial number of miRNAs were differentially abundant in the libraries of the three developmental stages, and those changes were much more notable when pupae and adult stages were compared. We detected putative binding sites for some of the most abundant miRNAs on genes encoding JH biosynthetic enzymes and CC neuropeptides. These studies should help us to gain a better understanding of the regulation of CA-CC activity mediated by miRNAs during major developmental stages in mosquitoes

Author Correction: Following de novo triglyceride dynamics in ovaries of Aedes aegypti during the previtellogenic stage (Scientific Reports, (2021), 11, 1, (9636), 10.1038/s41598-021-89025-6)

In the original version of this Article Veronika Michalkova was incorrectly affiliated with ‘Institute of Parasitology, Biology Centre CAS, Ceske Budejovice, Czech Republic’. The correct affiliations are listed below. Department of Biology, Florida International University, Miami, FL, USA. Institute of Zoology, Slovak Academy of Sciences, Dubravska cesta 9, 84506 Bratislava, Slovakia. The original Article and accompanying Supplementary Information file have been corrected

The juvenile hormone described in Rhodnius prolixus by Wigglesworth is juvenile hormone III skipped bisepoxide

Juvenile hormones (JHs) are sesquiterpenoids synthesized by the corpora allata (CA). They play critical roles during insect development and reproduction. The frst JH was described in 1934 as a “metamorphosis inhibitory hormone” in Rhodnius prolixus by Sir Vincent B. Wigglesworth. Remarkably, in spite of the importance of R. prolixus as vectors of Chagas disease and model organisms in insect physiology, the original JH that Wigglesworth described for the kissing-bug R. prolixus remained unidentifed. We employed liquid chromatography mass spectrometry to search for the JH homologs present in the hemolymph of fourth instar nymphs of R. prolixus. Wigglesworth’s original JH is the JH III skipped bisepoxide (JHSB3), a homolog identifed in other heteropteran species. Changes in the titer of JHSB3 were studied during the 10-day long molting cycle of 4th instar nymph, between a blood meal and the ecdysis to 5th instar. In addition we measured the changes of mRNA levels in the CA for the 13 enzymes of the JH biosynthetic pathway during the molting cycle of 4th instar. Almost 90 years after the frst descriptions of the role of JH in insects, this study fnally reveals that the specifc JH homolog responsible for Wigglesworth’s original observations is JHSB3.Fil: Villalobos Sambucaro, María José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Histología y Embriología Animal; ArgentinaFil: Nouzova, Marcela. Florida International University; Estados UnidosFil: Ramirez, Cesar E.. Florida International University; Estados UnidosFil: Alzugaray, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Histología y Embriología Animal; ArgentinaFil: Fernandez-Lima, Francisco. Florida International University; Estados UnidosFil: Ronderos, Jorge Rafael. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Cátedra de Histología y Embriología Animal; ArgentinaFil: Noriega, Fernando. Florida International University; Estados Unido

The juvenile hormone described in <i>Rhodnius prolixus</i> by Wigglesworth is juvenile hormone III skipped bisepoxide

Juvenile hormones (JHs) are sesquiterpenoids synthesized by the corpora allata (CA). They play critical roles during insect development and reproduction. The first JH was described in 1934 as a "metamorphosis inhibitory hormone" in Rhodnius prolixus by Sir Vincent B. Wigglesworth. Remarkably, in spite of the importance of R. prolixus as vectors of Chagas disease and model organisms in insect physiology, the original JH that Wigglesworth described for the kissing-bug R. prolixus remained unidentified. We employed liquid chromatography mass spectrometry to search for the JH homologs present in the hemolymph of fourth instar nymphs of R. prolixus. Wigglesworth's original JH is the JH III skipped bisepoxide (JHSB3), a homolog identified in other heteropteran species. Changes in the titer of JHSB3 were studied during the 10-day long molting cycle of 4th instar nymph, between a blood meal and the ecdysis to 5th instar. In addition we measured the changes of mRNA levels in the CA for the 13 enzymes of the JH biosynthetic pathway during the molting cycle of 4th instar. Almost 90 years after the first descriptions of the role of JH in insects, this study finally reveals that the specific JH homolog responsible for Wigglesworth's original observations is JHSB3.Facultad de Ciencias Naturales y MuseoConsejo Nacional de Investigaciones Científicas y Técnica

Approaches and Tools to Study the Roles of Juvenile Hormones in Controlling Insect Biology

The juvenile hormones (JHs) are a group of sesquiterpenoids synthesized by the corpora allata. They play critical roles during insect development and reproduction. To study processes that are controlled by JH, researchers need methods to identify and quantify endogenous JHs and tools that can be used to increase or decrease JH titers in vitro and in vivo. The lipophilic nature of JHs, coupled with the low endogenous titers, make handling and quantification challenging. JH titers in insects can easily be increased by the topical application of JH analogs, such as methoprene. On the other hand, experimentally reducing JH titers has been more difficult. New approaches to modulate JH homeostasis have been established based on advances in RNA interference and CRISPR/Cas9-based genome editing. This review will summarize current advances in: (1) the detection and quantification of JHs from insect samples; (2) approaches to manipulating JH titers; and (3) next-generation tools to modulate JH homeostasis

Allatostatin-C reversibly blocks the transport of citrate out of the mitochondria and inhibits juvenile hormone synthesis in mosquitoes.

Aedes aegypti allatostatin-C (AeaAST-C or PISCF-AST) is a strong and fast reversible inhibitor of juvenile hormone III (JH III) synthesis by the corpora allata (CA) of mosquitoes; however, its mechanism of action remains poorly understood. AeaAST-C showed no inhibitory activity in the presence of any of the intermediate precursors of JH III indicating that the AeaAST-C target is located before the entry of acetyl-CoA in the pathway. Stimulation experiments using different sources of carbon (glucose, pyruvate, acetate and citrate) suggest that AST-C acts after pyruvate is transformed to citrate in the mitochondria. In vitro inhibition of the citrate mitochondrial carrier (CIC) mimicked the effect of AeaAST-C, and was overridden by addition of citrate or acetate. Our results provide compelling evidence that AeaAST-C inhibits JH III synthesis by blocking the CIC carrier that transports citrate from the mitochondria to the cytosol, obstructing the production of cytoplasmic acetyl-CoA that sustains JH III synthesis in the CA of mosquitoes

A quantitative assay for the juvenile hormones and their precursors using fluorescent tags.

The juvenile hormones (JHs) are sesquiterpenoid compounds that play a central role in insect reproduction, development and behavior. The lipophilic nature of JHs and their precursors, in conjunction with their low concentration in tissues and susceptibility to degradation had made their quantification difficult. A variety of methods exist for JH quantification but few can quantify on the femtomole range. Currently applied methods are expensive and time consuming. In the present study we sought to develop a novel method for accurate detection and quantification of JHs and their precursors.A sensitive and robust method was developed to quantify the precursor, farnesoic acid (FA) and juvenile hormone III (JH III) in biological samples. The assay is based on the derivatization of analytes with fluorescent tags, with subsequent analysis by reverse phase high performance liquid chromatography coupled to a fluorescent detector (HPLC-FD). The carboxyl group of FA was derivatized with 4-Acetamido-7-mercapto-2,1,3-benzoxadiazole (AABD-SH). Tagging the epoxide group of JH III required a two-step reaction: the opening of the epoxide ring with sodium sulfide and derivatization with the fluorescent tag 4-(N,N-Dimethylaminosulfonyl)-7-(N-chloroformylmethyl-N-methylamino)-2,1,3-benzoxadiazole (DBD-COCl).The method developed in the present study showed high sensitivity, accuracy and reproducibility. Linear responses were obtained over the range of 10-20 to 1000 fmols. Recovery efficiencies were over 90% for JH III and 98% for FA with excellent reproducibility.The proposed method is applicable when sensitive detection and accurate quantification of limited amount of sample is needed. Examples include corpora allata, hemolymph and whole body of female adult Aedes aegypti and whole body Drosophila melanogaster. A variety of additional functional groups can be targeted to add fluorescent tags to the remaining JH III precursors