Gut-to-brain regulation of Drosophila aging through neuropeptide F, insulin and juvenile hormone.

Dietary restriction slows aging in many animals, while in some cases the sensory signals from diet alone are sufficient to retard or accelerate lifespan. The digestive tract is a candidate location to sense nutrients, where neuropeptides secreted by enteroendocrine cells (EEC) produce systemic signals in response to food. Here we measure how Drosophila neuropeptide F (NPF) is secreted into adult circulation by enteroendocrine cells and find that specific enteroendocrine cells differentially respond to dietary sugar and yeast. Lifespan is increased when gut NPF is genetically depleted, and this manipulation is sufficient to blunt the longevity benefit conferred by dietary restriction. Depletion of NPF receptors at insulin producing neurons of the brain also increases lifespan, consistent with observations where loss of gut NPF decreases neuronal insulin secretion. The longevity conferred by repressing gut NPF and brain NPF receptors is reversed by treating adults with a juvenile hormone (JH) analog. JH is produced by the adult corpora allata, and inhibition of the insulin receptor at this tissue decreases JH titer and extends lifespan, while this longevity is restored to wild type by treating adults with a JH analog. Overall, enteroendocrine cells of the gut modulate Drosophila aging through interorgan communication mediated by a gut-brain-corpora allata axis, and insulin produced in the brain impacts lifespan through its control of JH titer. These data suggest that we should consider how human incretins and their analogs, which are used to treat obesity and diabetes, may impact aging.

Evolution of tissue-specific expression of ancestral genes across vertebrates and insects.

Regulation of gene expression is arguably the main mechanism underlying the phenotypic diversity of tissues within and between species. Here we assembled an extensive transcriptomic dataset covering 8 tissues across 20 bilaterian species and performed analyses using a symmetric phylogeny that allowed the combined and parallel investigation of gene expression evolution between vertebrates and insects. We specifically focused on widely conserved ancestral genes, identifying strong cores of pan-bilaterian tissue-specific genes and even larger groups that diverged to define vertebrate and insect tissues. Systematic inferences of tissue-specificity gains and losses show that nearly half of all ancestral genes have been recruited into tissue-specific transcriptomes. This occurred during both ancient and, especially, recent bilaterian evolution, with several gains being associated with the emergence of unique phenotypes (for example, novel cell types). Such pervasive evolution of tissue specificity was linked to gene duplication coupled with expression specialization of one of the copies, revealing an unappreciated prolonged effect of whole-genome duplications on recent vertebrate evolution.

The embryonic role of juvenile hormone in the firebrat, Thermobia domestica, reveals its function before its involvement in metamorphosis.

To gain insights into how juvenile hormone (JH) came to regulate insect metamorphosis, we studied its function in the ametabolous firebrat, Thermobia domestica. Highest levels of JH occur during late embryogenesis, with only low levels thereafter. Loss-of-function and gain-of-function experiments show that JH acts on embryonic tissues to suppress morphogenesis and cell determination and to promote their terminal differentiation. Similar embryonic actions of JH on hemimetabolous insects with short germ band embryos indicate that JH's embryonic role preceded its derived function as the postembryonic regulator of metamorphosis. The postembryonic expansion of JH function likely followed the evolution of flight. Archaic flying insects were considered to lack metamorphosis because tiny, movable wings were evident on the thoraces of young juveniles and their positive allometric growth eventually allowed them to support flight in late juveniles. Like in Thermobia, we assume that these juveniles lacked JH. However, a postembryonic reappearance of JH during wing morphogenesis in the young juvenile likely redirected wing development to make a wing pad rather than a wing. Maintenance of JH then allowed wing pad growth and its disappearance in the mature juvenile then allowed wing differentiation. Subsequent modification of JH action for hemi- and holometabolous lifestyles are discussed.

Juvenile hormones direct primordial germ cell migration to the embryonic gonad.

Germ cells are essential to sexual reproduction. Across the animal kingdom, extracellular signaling isoprenoids, such as retinoic acids (RAs) in vertebrates and juvenile hormones (JHs) in invertebrates, facilitate multiple processes in reproduction. Here we investigated the role of these potent signaling molecules in embryonic germ cell development, using JHs in Drosophila melanogaster as a model system. In contrast to their established endocrine roles during larval and adult germline development, we found that JH signaling acts locally during embryonic development. Using an in vivo biosensor, we observed active JH signaling first within and near primordial germ cells (PGCs) as they migrate to the developing gonad. Through in vivo and in vitro assays, we determined that JHs are both necessary and sufficient for PGC migration. Analysis into the mechanisms of this newly uncovered paracrine JH function revealed that PGC migration was compromised when JHs were decreased or increased, suggesting that specific titers or spatiotemporal JH dynamics are required for robust PGC colonization of the gonad. Compromised PGC migration can impair fertility and cause germ cell tumors in many species, including humans. In mammals, retinoids have many roles in development and reproduction. We found that like JHs in Drosophila, RA was sufficient to impact mouse PGC migration in vitro. Together, our study reveals a previously unanticipated role of isoprenoids as local effectors of pre-gonadal PGC development and suggests a broadly shared mechanism in PGC migration.

Differential gene expression and microRNA profile in corpora allata-corpora cardiaca of Aedes aegypti mosquitoes with weak juvenile hormone signalling.

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.

The embryonic role of juvenile hormone in the firebrat, Thermobia domestica, reveals its function before its involvement in metamorphosis.

To gain insights into how juvenile hormone (JH) came to regulate insect metamorphosis, we studied its function in the ametabolous firebrat, Thermobia domestica. Highest levels of JH occur during late embryogenesis, with only low levels thereafter. Loss-of-function and gain-of-function experiments show that JH acts on embryonic tissues to suppress morphogenesis and cell determination and to promote their terminal differentiation. Similar embryonic actions of JH on hemimetabolous insects with short germ band embryos indicate that JH's embryonic role preceded its derived function as the postembryonic regulator of metamorphosis. The postembryonic expansion of JH function likely followed the evolution of flight. Archaic flying insects were considered to lack metamorphosis because tiny, movable wings were evident on the thoraces of young juveniles and their positive allometric growth eventually allowed them to support flight in late juveniles. Like in Thermobia, we assume that these juveniles lacked JH. However, a postembryonic reappearance of JH during wing morphogenesis in the young juvenile likely redirected wing development to make a wing pad rather than a wing. Maintenance of JH then allowed wing pad growth and its disappearance in the mature juvenile then allowed wing differentiation. Subsequent modification of JH action for hemi- and holometabolous lifestyles are discussed.

Investigating the role of aae-miR-34-5p in the regulation of juvenile hormone biosynthesis genes in the mosquito Aedes aegypti.

Juvenile hormone (JH) controls the development and reproduction of insects. Therefore, a tight regulation of the expression of JH biosynthetic enzymes is critical. microRNAs (miRNAs) play significant roles in the post-transcriptional regulation of gene expression by interacting with complementary sequences in target genes. Previously, we reported that several miRNAs were differentially expressed during three developmental stages of Aedes aegypti mosquitoes with different JH levels (no JH, high JH, and low JH). One of these miRNAs was aae-miR-34-5p. In this study, we identified the presence of potential target sequences of aae-miR-34-5p in the transcripts of some genes encoding JH biosynthetic enzymes. We analysed the developmental expression patterns of aae-miR-34-5p and the predicted target genes involved in JH biogenesis. Increases in miRNA abundance were followed, with a delay, by decreases in transcript levels of target genes. Application of an inhibitor and a mimic of aae-miR-34-5p led respectively to increased and decreased levels of thiolase transcripts, which is one of the early genes of JH biosynthesis. Female adult mosquitoes injected with an aae-miR-34-5p inhibitor exhibited significantly increased transcript levels of three genes encoding JH biosynthetic enzymes, acetoacetyl-CoA thiolase (thiolase), farnesyl diphosphate phosphatase, and farnesal dehydrogenase. Overall, our results suggest a potential role of miRNAs in JH production by directly targeting genes involved in its biosynthesis.

Antibody Persistence Following Administration of a Hexavalent DTwP-IPV-HB-PRP~T Vaccine Versus Separate DTwP-HB-PRP~T and IPV Vaccines and Safety and Immunogenicity of a Booster Dose of DTwP-IPV-HB-PRP~T Administered With an MMR Vaccine in Healthy Infants in India.

BACKGROUND: Antibody persistence of a whole-cell pertussis-containing hexavalent vaccine (DTwP-IPV-HB-PRP~T) and its co- or sequential administration with measles, mumps, rubella (MMR) vaccine were evaluated. METHODS: Phase III, open-label, randomized, multicenter study in India. Healthy toddlers 12-24 months of age who had received DTwP-IPV-HB-PRP~T or separate DTwP-HB-PRP~T+IPV primary vaccination at 6-8, 10-12 and 14-16 weeks of age received a DTwP-IPV-HB-PRP~T booster concomitantly with MMR (N = 336) or 28 days before MMR (N = 340). Participants had received a first dose of measles vaccine. Immunogenicity assessment used validated assays and safety was by parental reports. All analyses were descriptive. RESULTS: All participants had prebooster anti-T ≥0.01 IU/mL and anti-polio 1 and 3 ≥8 1/dil, and ≥96.5% had anti-D ≥0.01 IU/mL, anti-HBs ≥10 mIU/mL, anti-polio 2 ≥8 1/dil and anti-PRP ≥0.15 µg/mL; for pertussis, antibody persistence was similar in each group. Postbooster immunogenicity for DTwP-IPV-HB-PRP~T was similar for each antigen in each group: ≥99.5% of participants had anti-D ≥0.01 IU/mL, anti-T ≥0.01 IU/mL, anti-polio 1, 2 and 3 >8 1/dil, anti-HBs ≥10 mIU/mL and anti-PRP ≥1 µg/mL; for pertussis, vaccine response was similar in each group [72.0%-75.9% (anti-PT), 80.8%-81.4% (anti-FIM), 77.6%-79.5% (anti-PRN), 78.2%-80.8% (anti-FHA)]. There was no difference in MMR immunogenicity between groups, and no difference in DTwP-IPV-HB-PRP~T booster immunogenicity based on the primary series. There were no safety concerns. CONCLUSIONS: DTwP-IPV-HB-PRP~T antibody persistence was similar to licensed comparators. Booster immunogenicity was robust after DTwP-IPV-HB-PRP~T with or without MMR, and MMR immunogenicity was not affected by coadministration with DTwP-IPV-HB-PRP~T. CLINICAL TRIALS REGISTRY INDIA NUMBER: CTRI/2020/04/024843.

Effects of mating on female reproductive physiology in the insect model, Rhodnius prolixus, a vector of the causative parasite of Chagas disease.

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.

Safety and immunogenicity of a new formulation of a pentavalent DTwP-HepB-Hib vaccine in healthy Indian infants-A randomized study.

BACKGROUND: Pentavalent vaccines (DTP-HepB-Hib) have been introduced in many countries in their routine public immunization programmes to protect against diphtheria (D), tetanus (T), pertussis (P), hepatitis B (Hep B) and Hemophilus influenzae type b (Hib) diseases. This study compared the safety and immunogenicity of a new formulation of a whole-cell Bordetella pertussis (wP) based pentavalent vaccine (DTwP-HepB-Hib). The new formulation was developed using well-characterized hepatitis B and pertussis whole cell vaccine components. METHODS: This was a phase III, observer-blind, randomized, non-inferiority, multi-center study conducted in India among 460 infants who were followed up for safety and immunogenicity for 28 days after administration of three doses of either investigational or licensed comparator formulations at 6-8, 10-12 and 14-16 weeks of age. RESULTS: The investigational formulation of DTwP-HepB-Hib vaccine was non-inferior to the licensed formulation in terms of hepatitis B seroprotection rate (% of subjects with HepB antibodies ≥10mIU/mL were 99.1% versus 99.0%, respectively, corresponding to a difference of 0.1% (95% CI, -2.47 to 2.68)) and pertussis immune responses (adjusted geometric mean concentrations of antibodies for anti-PT were 76.7 EU/mL versus 63.3 EU/mL, with a ratio of aGMTs of 1.21 (95% CI, 0.89-1.64), and for anti-FIM were 1079 EU/mL versus 1129 EU/mL, with a ratio of aGMTs of 0.95 (95% CI, 0.73-1.24), respectively). The immune responses to other valences (D, T, and Hib) in the two formulations were also similar. The safety profile of both formulations was found to be similar and were well tolerated. CONCLUSIONS: The investigational DTwP-HepB-Hib vaccine formulation was immunogenic and well-tolerated when administered as three dose primary series in infants. CLINICAL TRIAL REGISTRATION: Clinical Trials Registry India number: CTRI/2018/12/016692.

Female reproductive dormancy in Drosophila is regulated by DH31-producing neurons projecting into the corpus allatum.

Female insects can enter reproductive diapause, a state of suspended egg development, to conserve energy under adverse environments. In many insects, including the fruit fly, Drosophila melanogaster, reproductive diapause, also frequently called reproductive dormancy, is induced under low-temperature and short-day conditions by the downregulation of juvenile hormone (JH) biosynthesis in the corpus allatum (CA). In this study, we demonstrate that neuropeptide Diuretic hormone 31 (DH31) produced by brain neurons that project into the CA plays an essential role in regulating reproductive dormancy by suppressing JH biosynthesis in adult D. melanogaster. The CA expresses the gene encoding the DH31 receptor, which is required for DH31-triggered elevation of intracellular cAMP in the CA. Knocking down Dh31 in these CA-projecting neurons or DH31 receptor in the CA suppresses the decrease of JH titer, normally observed under dormancy-inducing conditions, leading to abnormal yolk accumulation in the ovaries. Our findings provide the first molecular genetic evidence demonstrating that CA-projecting peptidergic neurons play an essential role in regulating reproductive dormancy by suppressing JH biosynthesis.

Immunogenicity and Safety of a Hexavalent DTwP-IPV-HB-PRP~T Vaccine Versus Separate DTwP-HB-PRP~T, bOPV, and IPV Vaccines Administered at 2, 4, 6 Months of Age Concomitantly With Rotavirus and Pneumococcal Conjugate Vaccines in Healthy Infants in Thailand.

BACKGROUND: This study investigated the immunogenicity and safety of a fully liquid, hexavalent, diphtheria (D)-tetanus (T)-whole-cell pertussis (wP)-inactivated poliovirus (IPV)-hepatitis B (HB)- Haemophilus influenzae b (PRP-T) vaccine compared to licensed DTwP-HB-PRP~T, IPV, and bivalent oral poliovirus (bOPV) vaccines following co-administration with other pediatric vaccines [pneumococcal conjugate vaccine (PCV13) and rotavirus vaccine]. METHODS: Phase III, randomized, open-label study in Thailand. Healthy infants received DTwP-IPV-HB-PRP~T at 2, 4 and 6 months of age (N = 228), or DTwP-HB-PRP~T and bOPV (2, 4 and 6 months of age) and IPV (4 months of age) (N = 231). All participants received PCV13 (2, 4 and 6 months of age) and rotavirus vaccine (2 and 4 months of age). Immunogenicity for all antigens was assessed using validated assays, and noninferiority post-third dose was evaluated for anti-D, anti-T, anti-pertussis [anti-pertussis toxin (anti-PT) and anti-fimbriae 2/3 (anti-FIM)], anti-polio 1, 2, 3, anti-HB, and anti-PRP~T. Safety was assessed using parental reports. RESULTS: Noninferiority was demonstrated for each antigen, and overall noninferiority of DTwP-IPV-HB-PRP~T versus DTwP-HB-PRP~T+bOPV+IPV was concluded. Similarity in each group was observed for the GMC ratio for antirotavirus antibodies (20.9 and 17.3, respectively) and anti-PCV13 antibodies (range: 8.46-32.6 and 7.53-33.1, respectively). Two serious adverse events were related to DTwP-IPV-HB-PRP~T (febrile convulsion and acute febrile illness) and 1 was related to DTwP-HB-PRP~T+bOPV+IPV (febrile seizure), but overall there were no safety concerns with similar rates of participants experiencing solicited (99.1% and 98.3%) and unsolicited (19.3% and 19.5%) adverse events in each group. CONCLUSIONS: This study confirmed the suitability of DTwP-IPV-HB-PRP~T primary series vaccination in combination with rotavirus and PCV13 vaccines.

Genetics tools for corpora allata specific gene expression in Aedes aegypti mosquitoes.

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.

Role of Methoprene-tolerant in the regulation of oogenesis in Dipetalogaster maxima.

Juvenile hormone (JH) signalling, via its receptor Methoprene-tolerant (Met), controls metamorphosis and reproduction in insects. Met belongs to a superfamily of transcription factors containing the basic Helix Loop Helix (bHLH) and Per Arnt Sim (PAS) domains. Since its discovery in 1986, Met has been characterized in several insect species. However, in spite of the importance as vectors of Chagas disease, our knowledge on the role of Met in JH signalling in Triatominae is limited. In this study, we cloned and sequenced the Dipetalogaster maxima Met transcript (DmaxMet). Molecular modelling was used to build the structure of Met and identify the JH binding site. To further understand the role of the JH receptor during oogenesis, transcript levels were evaluated in two main target organs of JH, fat body and ovary. Functional studies using Met RNAi revealed significant decreases of transcripts for vitellogenin (Vg) and lipophorin (Lp), as well as their receptors. Lp and Vg protein amounts in fat body, as well as Vg in hemolymph were also decreased, and ovarian development was impaired. Overall, these studies provide additional molecular insights on the roles of JH signalling in oogenesis in Triatominae; and therefore are relevant for the epidemiology of Chagas´ disease.

Immunogenicity and safety of booster CYD-TDV dengue vaccine after alternative primary vaccination schedules in healthy individuals aged 9-50 years: a randomised, controlled, phase 2, non-inferiority study.

BACKGROUND: Dengue is endemic in many countries throughout the tropics and subtropics, and the disease causes substantial morbidity and health-care burdens in these regions. We previously compared antibody responses after one-dose, two-dose, or three-dose primary regimens with the only approved dengue vaccine CYD-TDV (Dengvaxia; Sanofi Pasteur, Lyon, France) in individuals aged 9 years and older with previous dengue exposure. In this study, we assessed the need for a CYD-TDV booster after these primary vaccination regimens. METHODS: In this randomised, controlled, phase 2, non-inferiority study, healthy individuals aged 9-50 years recruited from three sites in Colombia and three sites in the Philippines (excluding those with the usual contraindications to vaccinations) were randomly assigned 1:1:1 via a permuted block method with stratification by site and by age group using an independent voice response system to receive, at 6-month intervals, three doses of CYD-TDV (three-dose group), one dose of placebo followed by two doses of CYD-TDV (two-dose group), or two doses of placebo followed by one dose of CYD-TDV (one-dose group). Participants were also randomly assigned (1:1) to receive a CYD-TDV booster at 1 year or 2 years after the last primary dose. Each CYD-TDV dose was 0·5 mL and administered subcutaneously in the deltoid region of the upper arm. The investigators and sponsor, study staff interacting with the investigators, and participants and their parents or legally acceptable representatives were masked to group assignment. Neutralising antibodies were measured by 50% plaque reduction neutralisation testing, and geometric mean titres (GMTs) were calculated. Due to a change in study protocol, only participants who were dengue seropositive at baseline in the Colombian cohort received a booster vaccination. The primary outcome was to show non-inferiority of the booster dose administered at 1 year or 2 years after the two-dose and three-dose primary regimens; non-inferiority was shown if the lower limit of the two-sided adjusted 95% CI of the between-group (day 28 post-booster dose GMT from the three-dose or two-dose group vs day 28 GMT post-dose three of the three-dose primary regimen [three-dose group]) geometric mean ratio (GMR) was higher than 0·5 for each serotype. Non-inferiority of the 1-year or 2-year booster was shown if all four serotypes achieved non-inferiority. Safety was assessed among all participants who received the booster. This trial is registered with ClinicalTrials.gov, NCT02628444, and is closed to accrual. FINDINGS: Between May 2 and Sept 16, 2016, we recruited and enrolled 1050 individuals who received either vaccine or placebo. Of the 350, 348, and 352 individuals randomly assigned to three-dose, two-dose, and one-dose groups, respectively, 108, 115, and 115 from the Colombian cohort were dengue seropositive at baseline and received a booster; 55 and 53 in the three-dose group received a booster after 1 year and 2 years, respectively, as did 59 and 56 in the two-dose group, and 62 and 53 in the one-dose group. After the three-dose primary schedule, non-inferiority was shown for serotypes 2 (GMR 0·746; 95% CI 0·550-1·010) and 3 (1·040; 0·686-1·570) but not serotypes 1 (0·567; 0·399-0·805) and 4 (0·647; 0·434-0·963) for the 1-year booster, and again for serotypes 2 (0·871; 0·673-1·130) and 3 (1·150; 0·887-1·490) but not serotypes 1 (0·688; 0·479-0·989) and 4 (0·655; 0·471-0·911) for the 2-year booster. Similarly, after the two-dose primary schedule, non-inferiority was shown for serotypes 2 (0·809; 0·505-1·300) and 3 (1·19; 0·732-1·940) but not serotypes 1 (0·627; 0·342-1·150) and 4 (0·499; 0·331-0·754) for the 1-year booster, and for serotype 3 (0·911; 0·573-1·450) but not serotypes 1 (0·889; 0·462-1·710), 2 (0·677; 0·402-1·140), and 4 (0·702; 0·447-1·100) for the 2-year booster. Thus, non-inferiority of the 1-year or 2-year booster was not shown after the three-dose or two-dose primary vaccination regimen in dengue-seropositive participants. No safety concerns occurred with the 1-year or 2-year CYD-TDV booster. INTERPRETATION: CYD-TDV booster 1 year or 2 years after the two-dose or three-dose primary vaccination regimen does not elicit a consistent, meaningful booster effect against all dengue serotypes in participants who are seropositive for dengue at baseline. FUNDING: Sanofi Pasteur. TRANSLATION: For the Spanish translation of the abstract see Supplementary Materials section.

Coupling Stable Isotope Labeling and Liquid Chromatography-Trapped Ion Mobility Spectrometry-Time-of-Flight-Tandem Mass Spectrometry for De Novo Mosquito Ovarian Lipid Studies.

There is a need to better understand lipid metabolism during mosquito ovarian development. Lipids are the major source of energy supporting ovarian follicles development in mosquitoes. In this paper, we describe the complementary use of stable isotope labeling (SIL) and high-resolution mass spectrometry-based tools for the investigation of de novo triglycerides (TG) and diglycerides (DG) during the ovarian previtellogenic (PVG) stage (4-6 days posteclosion) of female adult Aedes aegypti. Liquid chromatography coupled to high-resolution trapped ion mobility spectrometry-parallel accumulation sequential fragmentation-time-of-flight tandem mass spectrometry (LC-TIMS-PASEF-TOF MS/MS) allowed the separation and quantification of nonlabeled and 2H/13C-labeled TG and DG species. Three SIL strategies were evaluated (H2O/2H2O with 50:50 and 95:5 mixtures, 13C-sucrose, and 13C-glucose). Results showed wide applicability with no signs of lipid ovarian impairment by SIL induced toxicity. The analytical workflow based on LC-TIMS-TOF MS/MS provided high confidence and high reproducibility for lipid DG and TG identification and SIL incorporation based on their separation by retention time (RT), collision cross section (CCS), and accurate m/z. In addition, the SIL fatty acid chain incorporation was evaluated using PASEF MS/MS. The 2H/13C incorporation into the mosquito diet provided information on how TG lipids are consumed, stored, and recycled during the PVG stage of ovarian development.

The insect somatostatin pathway gates vitellogenesis progression during reproductive maturation and the post-mating response.

Vitellogenesis (yolk accumulation) begins upon eclosion and continues through the process of sexual maturation. Upon reaching sexual maturity, vitellogenesis is placed on hold until it is induced again by mating. However, the mechanisms that gate vitellogenesis in response to developmental and reproductive signals remain unclear. Here, we have identified the neuropeptide allatostatin-C (AstC)-producing neurons that gate both the initiation of vitellogenesis that occurs post-eclosion and its re-initiation post-mating. During sexual maturation, the AstC neurons receive excitatory inputs from Sex Peptide Abdominal Ganglion (SAG) neurons. In mature virgin females, high sustained activity of SAG neurons shuts off vitellogenesis via continuous activation of the AstC neurons. Upon mating, however, Sex Peptide inhibits SAG neurons, leading to deactivation of the AstC neurons. As a result, this permits both JH biosynthesis and the progression of vitellogenesis in mated females. Our work has uncovered a central neural circuit that gates the progression of oogenesis.

Sexual dimorphism of diapause regulation in the hemipteran bug Pyrrhocoris apterus.

Diapause is one of the major strategies for insects to prepare for and survive harsh seasons. In females, the absence of juvenile hormone (JH) is a hallmark of adult reproductive diapause, a developmental arrest, which is much less characterized in males. Here we show that juvenile hormone III skipped bisepoxide (JHSB3) titers in hemolymph remarkably differ between reproductive males and females of the linden bug Pyrrhocoris apterus, whereas no JH was detected in diapausing adults of both sexes. Like in females, ectopic application of JH mimic effectively terminated male diapause through the canonical JH receptor components, Methoprene-tolerant and Taiman. In contrast to females, long photoperiod induced reproduction even in males with silenced JH reception or in males with removed corpus allatum (CA), the JH-producing gland. JHSB3 was detected in the accessory glands (MAG) of reproductive males, unexpectedly, even in males without CA. If there is a source of JHSB3 outside CA or a long-term storage of JHSB3 in MAGs remains to be elucidated. These sex-related idiosyncrasies are further manifested in different dynamics of diapause termination in P. apterus by low temperature. We would like to propose that this sexual dimorphism of diapause regulation might be explained by the different reproductive costs for each sex.

Crosstalk between Nutrition, Insulin, Juvenile Hormone, and Ecdysteroid Signaling in the Classical Insect Model, Rhodnius prolixus.

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.

Epoxidation of juvenile hormone was a key innovation improving insect reproductive fitness.

Methyl farnesoate (MF) plays hormonal regulatory roles in crustaceans. An epoxidated form of MF, known as juvenile hormone (JH), controls metamorphosis and stimulates reproduction in insects. To address the evolutionary significance of MF epoxidation, we generated mosquitoes completely lacking either of the two enzymes that catalyze the last steps of MF/JH biosynthesis and epoxidation, respectively: the JH acid methyltransferase (JHAMT) and the P450 epoxidase CYP15 (EPOX). jhamt-/- larvae lacking both MF and JH died at the onset of metamorphosis. Strikingly, epox-/- mutants, which synthesized MF but no JH, completed the entire life cycle. While epox-/- adults were fertile, the reproductive performance of both sexes was dramatically reduced. Our results suggest that although MF can substitute for the absence of JH in mosquitoes, it is with a significant fitness cost. We propose that MF can fulfill most roles of JH, but its epoxidation to JH was a key innovation providing insects with a reproductive advantage.