Stage-specific expression of an odorant receptor underlies olfactory behavioral plasticity in Spodoptera littoralis larvae.

BACKGROUND: The detection of environmental cues and signals via the sensory system directs behavioral choices in diverse organisms. Insect larvae rely on input from the chemosensory system, mainly olfaction, for locating food sources. In several lepidopteran species, foraging behavior and food preferences change across larval instars; however, the molecular mechanisms underlying such behavioral plasticity during larval development are not fully understood. Here, we hypothesize that expression patterns of odorant receptors (ORs) change during development, as a possible mechanism influencing instar-specific olfactory-guided behavior and food preferences. RESULTS: We investigated the expression patterns of ORs in larvae of the cotton leafworm Spodoptera littoralis between the first and fourth instar and revealed that some of the ORs show instar-specific expression. We functionally characterized one OR expressed in the first instar, SlitOR40, as responding to the plant volatile, ß-caryophyllene and its isomer α-humulene. In agreement with the proposed hypothesis, we showed that first but not fourth instar larvae responded behaviorally to ß-caryophyllene and α-humulene. Moreover, knocking out this odorant receptor via CRISPR-Cas9, we confirmed that instar-specific responses towards its cognate ligands rely on the expression of SlitOR40. CONCLUSION: Our results provide evidence that larvae of S. littoralis change their peripheral olfactory system during development. Furthermore, our data demonstrate an unprecedented instar-specific behavioral plasticity mediated by an OR, and knocking out this OR disrupts larval behavioral plasticity. The ecological relevance of such behavioral plasticity for S. littoralis remains to be elucidated, but our results demonstrate an olfactory mechanism underlying this plasticity in foraging behavior during larval development.

An Efficient Solid-Phase Microextraction-Gas Chromatography-Mass Spectrometry Method for the Analysis of Methyl Farnesoate Released in Growth Medium by Daphnia pulex.

Methyl farnesoate (MF), a juvenile hormone, can influence phenotypic traits and stimulates male production in daphnids. MF is produced endogenously in response to stressful conditions, but it is not known whether this hormone can also be released into the environment to mediate stress signaling. In the present study, for the first time, a reliable solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) method was developed and validated for the ultra-trace analysis of MF released in growth medium by Daphnia pulex maintained in presence of crowding w/o MK801, a putative upstream inhibitor of MF endogenous production. Two different clonal lineages, I and S clones, which differ in the sensitivity to the stimuli leading to male production, were also compared. A detection limit of 1.3 ng/L was achieved, along with good precision and trueness, thus enabling the quantitation of MF at ultra-trace level. The achieved results demonstrated the release of MF by both clones at the 20 ng/L level in control conditions, whereas a significant decrease in the presence of crowding was assessed. As expected, a further reduction was obtained in the presence of MK801. These findings strengthen the link between environmental stimuli and the MF signaling pathway. Daphnia pulex, by releasing the juvenile hormone MF in the medium, could regulate population dynamics by means of an autoregulatory feedback loop that controls the intra- and extra-individual-level release of MF produced by endogenous biosynthesis.

Correction: Prevention of obesity in toddlers (PROBIT): a randomised clinical trial of responsive feeding promotion from birth to 24 months.

In the original Article, Dr. Laura Fontana's name was missing from the author list. This has been corrected (Dr. Fontana's name and details have been added to the HTML, PDF and XML version of this Article).

Prevention of obesity in toddlers (PROBIT): a randomised clinical trial of responsive feeding promotion from birth to 24 months.

INTRODUCTION: The aims of the PROBIT trial (clinicaltrials.gov: NCT03131284) were to prevent overweight or obesity occurring at two years of life, and improve feeding patterns during infancy. METHODS: The trial compared 252 northern Italian newborns whose paediatricians offered their parents an educational programme from the child's birth to the age of two years (intervention arm) with 216 newborns whose parents did not undergo the programme (control arm). This sample size was 80% powerful to detect, with a 0.05 α error, a 40% lower prevalence of overweight/obesity and a 57% lower prevalence of obesity in the intervention arm. At each well visit, the parents of the children in the intervention arm were given oral and written information about protective behaviours, with particular emphasis on responsive feeding. Overweight and obesity at two years of age were, respectively, defined as a body mass index of more than the 85th and the 95th percentile in accordance with the WHO growth charts. The sample size had 80% power to detect a 40% lower prevalence of overweight/obesity and a 57% lower prevalence of obesity in the intervention arm. RESULTS: At the age of two years, the prevalence of obesity in the intervention arm was 35% lower than among the controls, but the difference was not statistically significant (8.7% vs. 13.4%; p = 0.10) There was no difference in the prevalence of overweight/obesity between the groups (26.8% vs. 28.3%; p = 0.49). At the age of three months, a higher proportion of the infants in the intervention group were fed on demand (93% vs. 80%, p < 0.001). CONCLUSIONS: The PROBIT trial failed to detect a significantly lower prevalence of obesity in the intervention arm, but did improve early feeding patterns. More powerful trials and meta-analyses are required to establish whether educating newborns' parents can decrease the prevalence of early obesity.

Methy-sens Comet assay and DNMTs transcriptional analysis as a combined approach in epigenotoxicology.

Epigenotoxicology needs simple and fast tools to assess xenobiotic epigenetic load. This work proposes a comet assay modification designed to detect global methylation changes (Methy-sens Comet) through enzymatic digestion with two restriction enzymes (HpaII, MspI). In the methylation-sensitive protocol tested for repeatability on A549 cells, nickel chloride induced hypermethylation and decitabine-induced hypomethylation. A concomitant assessment of DNA methyltransferases (DNMTs) genes transcriptional levels has been performed, to implement a multifunctional approach to epigenotoxicology. Methy-sens Comet showed a general good repeatability and sensitivity to methylation changes while DNMTs transcriptional levels granted additional proof of xenobiotic-induced impairment of methylome maintenance.

Lifetime response of contemporary versus resurrected Daphnia galeata Sars (Crustacea, Cladocera) to Cu(II) chronic exposure.

Resurrecting legacy lineages of organisms from sediment cores of known geological age allows us to understand how environmental change can cause selection pressures that constrain the variation of populations over time. We quantified the lifetime tolerance and effects of Cu(II) exposure on Daphnia galeata in a polluted subalpine lake by comparing extant individuals with those resurrected from ephippia extracted from ca. 30-years-old sediments. Laboratory experiments were conducted using two Cu(II) concentrations, 40 and 10 µg L(-1), corresponding to the levels recorded in the lake, during chemical recovery, when Daphnia first re-appeared and succeeded. Contemporary Daphnia were unable to survive after the 10th day at either of the Cu(II) concentrations, and were unable to successfully reproduce. Daphnia cohorts from the past performed better in low Cu(II) concentrations than in copper-free, control conditions. The copper-adapted, tolerant Daphnia strains grew faster under non-toxic conditions, but were unable to survive new pollution events.

To sleep or not to sleep: Dormancy and life history traits in Eucypris virens (Crustacea, Ostracoda).

Dormancy represents an investment with its own costs and benefit. Besides the advantage obtained from the avoidance of harsh environments and from the synchronization of life cycles with seasonal changes, an organism could benefit from a temporary stop in growth and reproduction. To test this hypothesis a transgenerational experiment was carried out comparing the life history traits of clonal females of Eucypris virens from resting and non-resting eggs at two different photoperiods: short day length (6:18 L:D), proxy of favorable but unpredictable late winter-spring hydroperiod, and long day length (16:8 L:D) proxy of dry predictable unfavorable season, inducing resting egg production and within-generation plasticity (WGP). Clonal females that were dormancy deprived showed the highest age at first deposition and the lowest fecundity. Dormancy seems to work as a resetting mechanism of reproduction. Transgenerational plasticity (TGP) had a bounce back pattern: the phenotype of F1 generation was influenced by cues experienced in the F0 generation but the effects of F0 exposure were not evident in the F2. TGP might be adaptive when a mother experiences some kind of seasonality or stochasticity producing both resting and nonresting eggs. A positive relationship between the number of resting eggs and the total number of eggs per females suggested the absence of trade-off between dormancy and reproduction. Both WGP and TGP increase the mother long term fitness with important consequences on population dynamics, on the way a species spread throughout space and time and might respond to climate change.

Acoustic features as a tool to visualize and explore marine soundscapes: Applications illustrated using marine mammal passive acoustic monitoring datasets.

Passive Acoustic Monitoring (PAM) is emerging as a solution for monitoring species and environmental change over large spatial and temporal scales. However, drawing rigorous conclusions based on acoustic recordings is challenging, as there is no consensus over which approaches are best suited for characterizing marine acoustic environments. Here, we describe the application of multiple machine-learning techniques to the analysis of two PAM datasets. We combine pre-trained acoustic classification models (VGGish, NOAA and Google Humpback Whale Detector), dimensionality reduction (UMAP), and balanced random forest algorithms to demonstrate how machine-learned acoustic features capture different aspects of the marine acoustic environment. The UMAP dimensions derived from VGGish acoustic features exhibited good performance in separating marine mammal vocalizations according to species and locations. RF models trained on the acoustic features performed well for labeled sounds in the 8 kHz range; however, low- and high-frequency sounds could not be classified using this approach. The workflow presented here shows how acoustic feature extraction, visualization, and analysis allow establishing a link between ecologically relevant information and PAM recordings at multiple scales, ranging from large-scale changes in the environment (i.e., changes in wind speed) to the identification of marine mammal species.

Unsupervised machine learning and geometric morphometrics as tools for the identification of inter and intraspecific variations in the Anopheles Maculipennis complex.

Geometric morphometric analysis was combined with two different unsupervised machine learning algorithms, UMAP and HDBSCAN, to visualize morphological differences in wing shape among and within four Anopheles sibling species (An. atroparvus, An. melanoon, An. maculipennis s.s. and An. daciae sp. inq.) of the Maculipennis complex in Northern Italy. Specifically, we evaluated: (1) wing shape variation among and within species; (2) the consistencies between groups of An. maculipennis s.s. and An. daciae sp. inq. identified based on COI sequences and wing shape variability; and (3) the spatial and temporal distribution of different morphotypes. UMAP detected at least 13 main patterns of variation in wing shape among the four analyzed species and mapped intraspecific morphological variations. The relationship between the most abundant COI haplotypes of An. daciae sp. inq. and shape ordination/variation was not significant. However, morphological variation within haplotypes was reported. HDBSCAN also recognized different clusters of morphotypes within An. daciae sp. inq. (12) and An. maculipennis s.s. (4). All morphotypes shared a similar pattern of variation in the subcostal vein, in the anal vein and in the radio-medial cross-vein of the wing. On the contrary, the marginal part of the wings remained unchanged in all clusters of both species. Any spatial-temporal significant difference was observed in the frequency of the identified morphotypes.  Our study demonstrated that machine learning algorithms are a useful tool combined with geometric morphometrics and suggest to deepen the analysis of inter and intra specific shape variability to evaluate evolutionary constrains related to wing functionality.

The longin SNARE VAMP7/TI-VAMP adopts a closed conformation.

SNARE protein complexes are key mediators of exocytosis by juxtaposing opposing membranes, leading to membrane fusion. SNAREs generally consist of one or two core domains that can form a four-helix bundle with other SNARE core domains. Some SNAREs, such as syntaxin target-SNAREs and longin vesicular-SNAREs, have independent, folded N-terminal domains that can interact with their respective SNARE core domains and thereby affect the kinetics of SNARE complex formation. This autoinhibition mechanism is believed to regulate the role of the longin VAMP7/TI-VAMP in neuronal morphogenesis. Here we use nuclear magnetic resonance spectroscopy to study the longin-SNARE core domain interaction for VAMP7. Using complete backbone resonance assignments, chemical shift perturbations analysis, and hydrogen/deuterium exchange experiments, we conclusively show that VAMP7 adopts a preferentially closed conformation in solution. Taken together, the closed conformation of longins is conserved, in contrast to the syntaxin family of SNAREs for which mixtures of open and closed states have been observed. This may indicate different regulatory mechanisms for SNARE complexes containing syntaxins and longins, respectively.

Alternative splicing of the human gene SYBL1 modulates protein domain architecture of Longin VAMP7/TI-VAMP, showing both non-SNARE and synaptobrevin-like isoforms.

BACKGROUND: The control of intracellular vesicle trafficking is an ideal target to weigh the role of alternative splicing in shaping genomes to make cells. Alternative splicing has been reported for several Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptors of the vesicle (v-SNAREs) or of the target membrane (t-SNARES), which are crucial to intracellular membrane fusion and protein and lipid traffic in Eukaryotes. However, splicing has not yet been investigated in Longins, i.e. the most widespread v-SNAREs. Longins are essential in Eukaryotes and prototyped by VAMP7, Sec22b and Ykt6, sharing a conserved N-terminal Longin domain which regulates membrane fusion and subcellular targeting. Human VAMP7/TI-VAMP, encoded by gene SYBL1, is involved in multiple cell pathways, including control of neurite outgrowth. RESULTS: Alternative splicing of SYBL1 by exon skipping events results in the production of a number of VAMP7 isoforms. In-frame or frameshift coding sequence modifications modulate domain architecture of VAMP7 isoforms, which can lack whole domains or domain fragments and show variant or extra domains. Intriguingly, two main types of VAMP7 isoforms either share the inhibitory Longin domain and lack the fusion-promoting SNARE motif, or vice versa. Expression analysis in different tissues and cell lines, quantitative real time RT-PCR and confocal microscopy analysis of fluorescent protein-tagged isoforms demonstrate that VAMP7 variants have different tissue specificities and subcellular localizations. Moreover, design and use of isoform-specific antibodies provided preliminary evidence for the existence of splice variants at the protein level. CONCLUSIONS: Previous evidence on VAMP7 suggests inhibitory functions for the Longin domain and fusion/growth promoting activity for the Δ-longin molecule. Thus, non-SNARE isoforms with Longin domain and non-longin SNARE isoforms might have somehow opposite regulatory functions. When considering splice variants as "natural mutants", evidence on modulation of subcellular localization by variation in domain combination can shed further light on targeting determinants. Although further work will be needed to characterize identified variants, our data might open the route to unravel novel molecular partners and mechanisms, accounting for the multiplicity of functions carried out by the different members of the Longin proteins family.

Geometric morphometrics and machine learning as tools for the identification of sibling mosquito species of the Maculipennis complex (Anopheles).

Geometric morphometrics allows researchers to use the specific software to quantify and to visualize morphological differences between taxa from insect wings. Our objective was to assess wing geometry to distinguish four Anopheles sibling species of the Maculipennis complex, An. maculipennis s. s., An. daciae sp. inq., An. atroparvus and An. melanoon, found in Northern Italy. We combined the geometric morphometric approach with different machine learning alghorithms: support vector machine (SVM), random forest (RF), artificial neural network (ANN) and an ensemble model (EN). Centroid size was smaller in An. atroparvus than in An. maculipennis s. s. and An. daciae sp. inq. Principal component analysis (PCA) explained only 33% of the total variance and appeared not very useful to discriminate among species, and in particular between An. maculipennis s. s. and An. daciae sp. inq. The performance of four different machine learning alghorithms using procrustes coordinates of wing shape as predictors was evaluated. All models showed ROC-AUC and PRC-AUC values that were higher than the random classifier but the SVM algorithm maximized the most metrics on the test set. The SVM algorithm with radial basis function allowed the correct classification of 83% of An. maculipennis s. s. and 79% of An. daciae sp. inq. ROC-AUC analysis showed that three landmarks, 11, 16 and 15, were the most important procrustes coordinates in mean wing shape comparison between An. maculipennis s. s. and An. daciae sp. inq. The pattern in the three-dimensional space of the most important procrustes coordinates showed a clearer differentiation between the two species than the PCA. Our study demonstrated that machine learning algorithms could be a useful tool combined with the wing geometric morphometric approach.

Roundup causes embryonic development failure and alters metabolic pathways and gut microbiota functionality in non-target species.

BACKGROUND: Research around the weedkiller Roundup is among the most contentious of the twenty-first century. Scientists have provided inconclusive evidence that the weedkiller causes cancer and other life-threatening diseases, while industry-paid research reports that the weedkiller has no adverse effect on humans or animals. Much of the controversial evidence on Roundup is rooted in the approach used to determine safe use of chemicals, defined by outdated toxicity tests. We apply a system biology approach to the biomedical and ecological model species Daphnia to quantify the impact of glyphosate and of its commercial formula, Roundup, on fitness, genome-wide transcription and gut microbiota, taking full advantage of clonal reproduction in Daphnia. We then apply machine learning-based statistical analysis to identify and prioritize correlations between genome-wide transcriptional and microbiota changes. RESULTS: We demonstrate that chronic exposure to ecologically relevant concentrations of glyphosate and Roundup at the approved regulatory threshold for drinking water in the US induce embryonic developmental failure, induce significant DNA damage (genotoxicity), and interfere with signaling. Furthermore, chronic exposure to the weedkiller alters the gut microbiota functionality and composition interfering with carbon and fat metabolism, as well as homeostasis. Using the "Reactome," we identify conserved pathways across the Tree of Life, which are potential targets for Roundup in other species, including liver metabolism, inflammation pathways, and collagen degradation, responsible for the repair of wounds and tissue remodeling. CONCLUSIONS: Our results show that chronic exposure to concentrations of Roundup and glyphosate at the approved regulatory threshold for drinking water causes embryonic development failure and alteration of key metabolic functions via direct effect on the host molecular processes and indirect effect on the gut microbiota. The ecological model species Daphnia occupies a central position in the food web of aquatic ecosystems, being the preferred food of small vertebrates and invertebrates as well as a grazer of algae and bacteria. The impact of the weedkiller on this keystone species has cascading effects on aquatic food webs, affecting their ability to deliver critical ecosystem services. Video Abstract.

Early visual and neuro-development in preterm infants with and without retinopathy.

BACKGROUND: Retinopathy of prematurity (ROP) is often associated with visual impairment and multiple developmental disabilities. AIMS: As most of the previous studies include infants with brain lesions, that can determine visual impairment per se, a cohort of low neurological risk preterm infants without ROP and with various degree of severity of ROP was assessed in order to establish visual and neurodevelopmental outcome. STUDY DESIGN: Preterm infants born at <31 weeks gestation, without major brain lesions, underwent visual function assessment at 1 year corrected age and neurodevelopmental assessment at 2 years corrected age. SUBJECTS: One hundred and five infants were included in the study: 42 infants did not develop ROP, 7 reached stage 1 in zone 2 ROP, 37 reached prethreshold (untreated) type 2 ROP. The remaining 19 infants were classified as type 1 ROP. OUTCOME MEASURES: Visual function (including fixing, tracking, visual acuity, visual field, attention at distance and nystagmus) were assessed at 12 months corrected age and Griffiths Scales at 2 years corrected age. RESULTS: The severity of ROP was strongly correlated (p < 0.001) with both visual function at 1 year and neurodevelopment at 2 years. Similarly, the presence of nystagmus was also strongly correlated with visual and neurodevelopmental sequelae. CONCLUSIONS: Infants with no or milder retinopathy showed normal visual function at 1 year and neurodevelopment at 2 years. Infants who underwent treatment more frequently showed abnormal results on several aspects of visual function. Presence of nystagmus appeared to increase the risk for abnormal visual function and neurodevelopmental outcome.

Comparative analysis of plant genomes allows the definition of the "Phytolongins": a novel non-SNARE longin domain protein family.

BACKGROUND: Subcellular trafficking is a hallmark of eukaryotic cells. Because of their pivotal role in the process, a great deal of attention has been paid to the SNARE proteins. Most R-SNAREs, or "longins", however, also possess a highly conserved, N-terminal fold. This "longin domain" is known to play multiple roles in regulating SNARE activity and targeting via interaction with other trafficking proteins. However, the diversity and complement of longins in eukaryotes is poorly understood. RESULTS: Our comparative genome survey identified a novel family of longin-related proteins, dubbed the "Phytolongins" because they are specific to land plants. Phytolongins share with longins the N-terminal longin domain and the C-terminal transmembrane domain; however, in the central region, the SNARE motif is replaced by a novel region. Phylogenetic analysis pinpoints the Phytolongins as a derivative of the plant specific VAMP72 longin sub-family and allows elucidation of Phytolongin evolution. CONCLUSION: "Longins" have been defined as R-SNAREs composed of both a longin domain and a SNARE motif. However, expressed gene isoforms and splice variants of longins are examples of non-SNARE motif containing longins. The discovery of Phytolongins, a family of non-SNARE longin domain proteins, together with recent evidence on the conservation of the longin-like fold in proteins involved in both vesicle fusion (e.g. the Trs20 tether) and vesicle formation (e.g. sigma and mu adaptin) highlight the importance of the longin-like domain in protein trafficking and suggest that it was one of the primordial building blocks of the eukaryotic membrane-trafficking machinery.

Longins and their longin domains: regulated SNAREs and multifunctional SNARE regulators.

Longins are the only R-SNAREs that are common to all eukaryotes and are characterized by a conserved N-terminal domain with a profilin-like fold called a longin domain (LD). These domains seem to be essential for regulating membrane trafficking and they mediate unexpected biochemical functions via a range of protein-protein and intramolecular binding specificities. In addition to the longins, proteins involved in the regulation of intracellular trafficking, such as subunits of the adaptor and transport protein particle complexes, also have LD-like folds. The functions and cellular localization of longins are regulated at several levels and the longin prototypes TI-VAMP, Sec22 and Ykt6 show different distributions among eukaryotes, reflecting their modular and functional diversity. In mammals, TI-VAMP and Ykt6 are crucial for neuronal function, and defects in longin structure or function might underlie some human neurological pathologies.

Transgenerational response to early spring warming in Daphnia.

Temperature and photoperiod regulate key fitness traits in plants and animals. However, with temperature increase due to global warming, temperature cue thresholds are experienced at shorter photoperiods, disrupting the optimal seasonal timing of physiological, developmental and reproductive events in many species. Understanding the mechanisms of adaptation to the asynchrony between temperature and photoperiod is key to inform our understanding of how species will respond to global warming. Here, we studied the transgenerational mechanisms of responses of the cyclical parthenogen Daphnia magna to different photoperiod lengths co-occurring with warm temperature thereby assessing the impact of earlier spring warming on its fitness. Daphnia uses temperature and photoperiod cues to time dormancy, and to switch between sexual and asexual reproduction. Daphnia life cycle offers the opportunity to measure the relative contribution of plastic and genetic responses to environmental change across generations and over evolutionary time. We use transgenerational common garden experiments on three populations 'resurrected' from a biological archive experiencing temperature increase over five decades. Our results suggest that response to early spring warming evolved underpinned by a complex interaction between plastic and genetic mechanisms while a positive maternal contribution at matching environments between parental and offspring generation was also observed.

Antiviral and Antioxidant Activity of a Hydroalcoholic Extract from Humulus lupulus L.

A hydroalcoholic extract from female inflorescences of Humulus lupulus L. (HOP extract) was evaluated for its anti-influenza activity. The ability of the extract to interfere with different phases of viral replication was assessed, as well as its effect on the intracellular redox state, being unbalanced versus the oxidative state in infected cells. The radical scavenging power, inhibition of lipoperoxidation, and ferric reducing activity were assayed as antioxidant mechanisms. A phytochemical characterization of the extract was also performed. We found that HOP extract significantly inhibited replication of various viral strains, at different time from infection. Viral replication was partly inhibited when virus was incubated with extract before infection, suggesting a direct effect on the virions. Since HOP extract was able to restore the reducing conditions of infected cells, by increasing glutathione content, its antiviral activity might be also due to an interference with redox-sensitive pathways required for viral replication. Accordingly, the extract exerted radical scavenging and reducing effects and inhibited lipoperoxidation and the tBOOH-induced cytotoxicity. At phytochemical analysis, different phenolics were identified, which altogether might contribute to HOP antiviral effect. In conclusion, our results highlighted anti-influenza and antioxidant properties of HOP extract, which encourage further in vivo studies to evaluate its possible application.

Mother's Age and Hatching Phenology Strategy of Heterocypris incongruens (Crustacea: Ostracoda) in Unpredictable Environment.

We report experimental evidence of egg polyphenism in clonal lineages of Heterocypris incongruens from an ephemeral pond on a Mediterranean Island (Lampedusa, Italy). In controlled laboratory conditions, clonal females produced three different kinds of eggs: (i) resting and desiccation-resistant eggs, (ii) nonresting eggs that hatched gradually within 2 month hydroperiod, and (iii) nonresting delayed development eggs that hatched synchronously. Clonal females showed a diversified bet-hedging strategy to spread risk of reproduction in ponds with unpredictable hydroperiod. They adjusted proportion of different egg phenotypes in response to experimental temperature and photoperiod that are proxies for different hydroperiod unpredictability. The proportion of resting eggs is not affected by maternal age at deposition, but mother's age has a key role in defining the development time and the hatching phenology of nonresting eggs. Genetically identical eggs kept at the same controlled laboratory conditions showed a U-shaped distribution of development time. Development time variance decreased with mother's age at deposition. The resulting wedge-shaped relationship between development time and mother's age at deposition may be interpreted by considering mother's age as a proxy for the probability of drought onset. The older the mother, the shorter the latency to drought and the shorter is the time for nonresting eggs to hatch. Considering only the delayed development of eggs that hatch synchronously, development time is inversely related to the mother's age at deposition: this relationship generates the observed hatching peak.