Maternal GABAergic and GnRH/corazonin pathway modulates egg diapause phenotype of the silkworm Bombyx mori

Diapause represents a major developmental switch in insects and is a seasonal adaptation that evolved as a specific subtype of dormancy in most insect species to ensure survival under unfavorable environmental conditions and synchronize populations. However, the hierarchical relationship of the molecular mechanisms involved in the perception of environmental signals to integration in morphological, physiological, behavioral, and reproductive responses remains unclear. In the bivoltine strain of the silkworm Bombyx mori, embryonic diapause is induced transgenerationally as a maternal effect. Progeny diapause is determined by the environmental temperature during embryonic development of the mother. Here, we show that the hierarchical pathway consists of a γ-aminobutyric acid (GABA)ergic and corazonin signaling system modulating progeny diapause induction via diapause hormone release, which may be finely tuned by the temperature-dependent expression of plasma membrane GABA transporter. Furthermore, this signaling pathway possesses similar features to the gonadotropin-releasing hormone (GnRH) signaling system for seasonal reproductive plasticity in vertebrates.

To ensure survival under unfavorable environmental conditions and synchronize populations, most insect species enter diapause, which is a seasonal adaptation that evolved as a specific subtype of dormancy (1, 2). Diapause is not a passive response to changing conditions but rather an actively induced state that precedes adverse natural situations. Therefore, this diapause phenotype is accompanied by changes in energy metabolism or storage to improve cold/stress tolerance in later life stages, or progeny via reproductive switch (3). Although it has been generally suggested that brain/neuroendocrine systems are associated with this seasonal reproductive plasticity in both vertebrates and invertebrates (3, 4), the hierarchical relationship of the molecular mechanisms involved in the perception of environmental signals to integration into morphological, physiological, behavioral, and reproductive responses, known as the diapause syndrome, remains unclear (3).The silkworm Bombyx mori is a typical insect that arrests normal development during early embryogenesis, which is accompanied by metabolic changes in diapause (5, 6). The development of diapause-destined embryos is arrested during the G2 cell cycle stage immediately after the formation of the cephalic lobe and telson and sequential segmentation of the mesoderm (7). The bivoltine strain of B. mori has two generations per year, and progeny diapause is transgenerationally induced as a maternal effect and is determined by the environmental temperature, photoperiod, and nutrient conditions during embryonic and larval development of the mother (5, 6). The temperature signal during the mother’s embryonic development predominantly affects diapause determination, even if silkworms of the bivoltine Kosetsu strain are exposed to all cases of photoperiods during embryonic and larval development. In the Kosetsu strain, when eggs are incubated at 25 °C under continuous darkness, the resultant female moths (25DD) lay diapause eggs in almost all cases. In contrast, incubation of eggs at 15 °C in dark condition results in moths (15DD) that lay nondiapause eggs in almost all cases (6).Embryonic diapause is induced by the diapause hormone (DH) signaling pathway, which consists of highly sensitive and specific interactions between a neuropeptide, DH, and DH receptor (DHR) (6, 8). DH is exclusively synthesized in seven pairs of neurosecretory cells (DH-PBAN–producing neurosecretory cells [DHPCs]) located within the subesophageal ganglion (SG) in the mother’s generation (6). DH is released into the hemolymph during pupal–adult development and acts on the DHR, which belongs to the G protein-coupled receptors (GPCRs) (9). DH levels in the hemolymph are higher in the 25DD than 15DD pupae in the middle of pupal–adult development when the developing ovaries are sensitive to DH (6). Furthermore, the embryonic Bombyx TRPA1 ortholog (BmTRPA1) acts as a thermosensitive channel that is activated at temperatures above ∼21 °C and affects diapause induction through DH release (10). However, there remain questions about the thermal information that is received by BmTRPA1 and linked to DH signaling to induce diapause.From the 1950s, it has been suggested that the DH release was controlled by signals derived from certain region(s) in the brain based on surgical experiments, such as midsagittal bisection or transection (11–13). Especially, the operation in nondiapause producers changed them to diapause producers while transection of the protocerebrum had no effect on the diapause producers. These surgical results suggested the involvement of the protocerebrum in the inhibitory control of DH secretion (12, 14). Furthermore, the accumulation of the ovarian 3-hydroxykynurenine (3-OHK) pigment that accompanies the diapause syndrome was affected by injection with γ-aminobutyric acid (GABA) and the plant alkaloid picrotoxin (PTX), which is a widely used ionotropic GABA and glycine receptor antagonist (15, 16), and the selective ionotropic GABA receptor (GABAR) antagonist bicuculline. This suggests that a GABAergic neurotransmission via ionotropic GABAR is involved in DH secretion, which may be active in nondiapause producers but inactive in diapause producers throughout the pupal–adult development (14, 17). In general, ionotropic GABAR is composed of homo- or hetero-pentameric subunits. All known GABAR subunits display a similar structural scheme, with a large N-terminal extracellular domain involved in the formation of a ligand-binding pocket and a pore domain made of four transmembrane alpha-helices (TM1–TM4) (16, 18). Four homologous sequences of the ionotropic GABAR subunit genes were identified as RDL, LCCH3, GRD, and a GRD-like sequence named 8916 in various insects (19). However, the in vivo physiological roles of both signals derived from the brain and the GABAergic pathway in diapause induction have not been previously investigated.Corazonin (Crz) is an undecapeptide neurohormone sharing a highly conserved amino acid (a.a.) sequence across insect lineages and is involved in different physiological functions, such as heart contraction (20), stress response (21, 22), various metabolic activities (23–25), female fecundity (26), melanization of locust cuticles (27), regulation of ecdysis (28, 29), and control of caste identity (30). Moreover, Crz belongs to the gonadotropin-releasing hormone (GnRH) superfamily alongside adipokinetic hormone (AKH) and AKH/Crz-related peptide (ACP). Duplicates of an ancestral GnRH/Crz signaling system occurred in a common ancestor of protostomes and deuterostomes through coevolution of the ligand receptor (31, 32).Herein, we demonstrated that the hierarchical pathway consists of a GABAergic and Crz signaling system modulating progeny diapause induction by acting on DH release. We propose that the PTX-sensitive GABAergic signal may act to chronically suppress Crz release in dorsolateral Crz neurons (under nondiapause conditions) and that diapause conditions (or PTX injection) inhibits GABAergic signaling, resulting in accelerated Crz release, which in turn induces DH release. GABA signaling may be finely tuned by the temperature-dependent expression of the plasma membrane GABA transporter (GAT), which differs between the 25DD and 15DD conditions. Furthermore, this signaling pathway possesses similar features to the GnRH signaling system with respect to seasonal reproductive plasticity in vertebrates.     

Nintendo® Wii Fit based sleepiness tester detects impairment of postural steadiness due to 24 h of wakefulness

A field-usable sleepiness tester could reduce sleepiness related accidents. 15 subjects’ postural steadiness was measured with a Nintendo^® Wii Fit balance board every hour for 24 h. Body sway was quantified with complexity index, C_I, and the correlation between C_I and alertness predicted by a three-process model of sleepiness was calculated. The C_I group average was 8.9 ± 1.3 for alert and 7.9 ± 1.4 for sleep deprived subjects (p < 0.001, ρ = 0.94). The Wii Fit board detects the impairment of postural steadiness. This may allow large scale sleepiness testing outside the laboratory setting.     

Can nurses exclude middle-ear effusion without otoscopy in young asymptomatic children in primary care?

Objective. Scandinavian guidelines recommend controlling middle-ear effusion (MEE) after acute otitis media. The study aim was to determine whether nurses without otoscopic experience can reliably exclude MEE with tympanometry or spectral gradient acoustic reflectometry (SG-AR) at asymptomatic visits. Design. Three nurses were taught to perform examinations with tympanometry and SG-AR. Pneumatic otoscopy by the study physician served as the diagnostic standard. Setting. Study clinic at primary health care level. Patients. A total of 156 children aged 6–35 months. Main outcome measures. Predictive values (with 95% confidence interval) for tympanometry and SG-AR, and the clinical usefulness, i.e. the proportion of visits where nurses obtained the exclusive test result from both ears of the child. Results. At 196 visits, the negative predictive value of type A and C1 tympanograms (tympanometric peak pressure > −200 daPa) was 95% (91–97%). Based on type A and C1 tympanograms, the nurse could exclude MEE at 81/196 (41%) of visits. The negative predictive value of SG-AR level 1 result was 86% (79–91%). Based on SG-AR level 1 results, the nurse could exclude MEE at 29/196 (15%) of visits. Conclusion. Tympanograms with tympanometric peak pressure > −200 daPa (types A and C1) obtained by nurses are reliable test results in excluding MEE. However, these test results were obtained at less than half of the asymptomatic visits and, thus, the usefulness of excluding MEE by nurses depends on the clinical setting.     

Characterization of the rapamycin-sensitive phosphoproteome reveals that Sch9 is a central coordinator of protein synthesis

The target of rapamycin complex 1 (TORC1) is an essential multiprotein complex conserved from yeast to humans. Under favorable growth conditions, and in the absence of the macrolide rapamycin, TORC1 is active, and influences virtually all aspects of cell growth. Although two direct effectors of yeast TORC1 have been reported (Tap42, a regulator of PP2A phosphatases and Sch9, an AGC family kinase), the signaling pathways that couple TORC1 to its distal effectors were not well understood. To elucidate these pathways we developed and employed a quantitative, label-free mass spectrometry approach. Analyses of the rapamycin-sensitive phosphoproteomes in various genetic backgrounds revealed both documented and novel TORC1 effectors and allowed us to partition phosphorylation events between Tap42 and Sch9. Follow-up detailed characterization shows that Sch9 regulates RNA polymerases I and III, the latter via Maf1, in addition to translation initiation and the expression of ribosomal protein and ribosome biogenesis genes. This demonstrates that Sch9 is a master regulator of protein synthesis.     

Experimental Chlamydia pneumoniae infection in NIH/S mice: effect of reinoculation with chlamydial or cell preparation on culture,PCR and histological findings of lung tissue

The cellular components present in chlamydial preparations may contribute to the course of the experimental infection. NIH/S mice were inoculated and reinoculated intranasally with Chlamydia pneumoniae or a cellular preparation. The mock inoculation induced only mild histological changes in the lungs, which possibly induced partial protection against subsequent C. pneumoniae infection and, when given as reinoculation, possibly reactivated the culture-negative infection as culture-positive. In addition, serum antibodies against mouse heat shock protein 60 (Hsp60) were found in a few mice. In conclusion, the main immunopathogenic factors in a C. pneumoniae mouse model are chlamydial components. However, a cellular preparation may participate in an inflammatory reaction. Autoimmunity against Hsp60 may also play a role in the pathogenesis of C. pneumoniae infection.     

Markers of collagen synthesis and degradation in urogenital tissue from women with and without stress urinary incontinence

AIMS: Multiparity and obesity are risk factors for stress urinary incontinence (SUI), but collagen synthesis and metabolism in the urogenital tissue itself may also affect its function and control of micturition. Whether changes in synthesis or degradation of collagen are part of the etiology of SUI is not known and published studies show diverging results. The aims of the present study was to investigate collagen turnover in urogenital tissue in women with SUI (n=71) and in urologically healthy women (n=31). METHODS: Markers of collagen synthesis and breakdown, the carboxy-terminal propeptide of type I procollagen (PICP), the carboxy-terminal telopeptide of type I collagen (ICTP), and the amino-terminal propeptide of procollagen III (PIIINP) were assayed in urogenital tissue homogenates and peripheral serum. RESULTS: In the total clinical material SUI patients were significantly older, had a significantly higher body mass index (BMI) and significantly lower serum PICP and tissue ICTP levels than the controls. When healthy controls were compared with SUI patients matched for age, BMI, parity, and hormonal/menopausal status (31 women in each group), the SUI patients had significantly lower serum concentrations of PICP and significantly lower tissue concentrations of PIIINP and ICTP than the controls. Within the total material of SUI patients, post-menopausal women with weak and strong HRT and pre-menopausal women had significantly lower S-ICTP concentrations than untreated post-menopausal patients. Significant negative correlations to parity were found for T-PIIINP and T-PICP and to BMI for T-ICTP. CONCLUSIONS: The low tissue collagen marker levels in women with SUI suggest a reduced collagen turnover, which may negatively affect tissue strength and elasticity.