Altered Sex Ratio Research Articles

Sex Ratios, Single Motherhood, and Gendered Structural Relations: Examining Female-Headed Families Across Racial-Ethnic Populations

ABSTRACTSex ratio research often ignores how structural inequalities influence sex ratio effects. Employing decomposition analysis and using standardized U.S. Census tract data from 1970 to 2000, this research investigates how adult sex ratio effects predict the proportion of single parenting women among White, Black, and Hispanic/Latino populations, examining both main and conditional effects of sex ratios on female-headed families. Analyses reveal that main sex ratio effects consistently predict the proportion of single mothers across all racial-ethnic groups, but the conditional effect of gender-distributed labor force participation differentially influences sex ratio effects across race-ethnicities. Specifically, relative labor force participation consistently weakens the sex ratio effects on female headship among Whites. However, the relative female employment alters sex ratio effects among Blacks only when examining the same area over time, and it does not alter sex ratio effects among Hispanic/Latino populations. Findings underscore the importance of understanding family as a sociopolitical phenomenon.

Correlation of Infant Gender with Postpartum Maternal and Paternal Depression and Exclusive Breastfeeding Rates.

Male bias in India has resulted in an altered sex ratio. It also results in maternal postpartum depression (PPD) and lower breastfeeding rates. We studied depression among fathers in this context. Edinburgh postnatal depression scale (EPDS) questionnaire was administered to 479 parents separately. Breastfeeding on day 7 was determined. Depression was significantly higher in fathers of girls. Mean EPDS score was 5.86 ± 4.98 versus 2.5 ± 2.64 (p < 0.001). Concordance between parents was significant (r = 0.95, p < 0.001). Boys were more likely to be exclusively breastfed (64.46% versus 35.54%, p < 0.001). In second-order births, if the first born was a girl and second baby was again a girl, 23% were exclusively breastfed compared with 86% if it was boy after a girl (p < 0.001). Among parents who had EPDS scores ≥11, no babies were exclusively breastfed. Among the 25 babies who received no breast milk, 21 were girls and 4 were boys (p < 0.042). The multiple logistic regression analysis indicated that birth of girls (OR 0.269, 95% CI 0.076-0.953), high EPDS score in mothers (OR = 0.080, 95% CI 0.026-0.249), and high EPDS score in fathers (OR = 0.096, 95% CI 0.031-0.299) were associated with lower breastfeeding rates. Paternal depression correlates closely with maternal PPD and low exclusive breastfeeding of girls. Breastfeeding has implications for survival of girls. Efforts are needed to support the parents of girl children with PPD and such support must extend to fathers to improve survival.

Effect of triclosan on anuran development and growth in a larval amphibian growth and development assay.

A larval amphibian growth and development assay was performed to evaluate the potential effects of environmentally-relevant concentrations of triclosan (TCS) on amphibian development and growth. Xenopus laevis were exposed to TCS 0.0 (control), 6.3, 12.5 and 25.0 μg l-1 (estimated maximum tolerable concentration) until 10 weeks post-metamorphosis. At median metamorphosis time (Nieuwkoop and Faber stage 62), five larvae per replicate were collected for snout-vent length, hind limb length and body weight measurements, and histopathological examination of thyroid glands. Endpoints evaluated at test termination were based on draft guidance (USEPA, ) and included: survival; snout-vent length; body weight; gender; nuptial pad development (males); and liver, kidney, gonad and gonadal ducts histopathology. Exposure to TCS did not decrease survival, induce general signs of toxicity, affect median metamorphosis time or alter sex ratios. Exposure to TCS 12.5 and 25 μg l-1 increased growth during the metamorphic stages relative to the control, but did not influence growth during the post-metamorphic phase. Overall, several statistically significant findings were found in larvae exposed to TCS, such as a decrease in the prevalence of stage 3 Müllerian ducts in the anterior trunk sections of TCS 25.0 μg l-1 dose group females as compared to controls; most were not considered toxicologically relevant. Copyright © 2017 John Wiley & Sons, Ltd.

Sea turtle climate change biologists should do more measuring and less modeling

Anthropogenic climate change poses many threats to threatened and endangered species, including sea turtles. These threats include the erosion of nesting beach habitat, altered sex ratios of hatchlings, and latitudinal shifts in preferred nesting conditions as temperatures warm globally. Researchers and conservation practitioners often model the potential consequences of climate change to help guide management practices, but greater focus should be placed on measuring the responses of species to climate change through long-term field studies. Here, we review studies undertaken to assess, simulate, or project the impacts of climate change on sea turtles. We placed 53 recently published (2003–2015) studies into one of three categories (Historical Assessment Only, Current Conditions and Projections, Historical Assessment and Projections). The first and most common category (58% of surveyed publications) includes short-term studies of current environmental needs and models of future climate changes to determine if those conditions will be met in the future. The second category (32%) includes studies of historical responses of sea turtles to climate change derived from long-term (&gt;10 year) datasets, without projections into the future. The least common approach (9%) included studies that used both long-term datasets on species’ responses to observed climate change and quantitative models of future climate scenarios. We synthesize the relevant literature on this topic and argue for new studies that integrate long-term historical datasets for species responses to climate change, rather than models extrapolated from current conditions.

Do mothers bias offspring sex ratios in carotenoid-rich environments?

If environmental or maternal factors favour the fitness of one sex over the other, theory predicts that mothers should produce more offspring of the sex most likely to benefit from prevailing conditions. For species where males depend on carotenoid-based colourful ornaments to secure territory or attract mates, carotenoid availability in the environment could be one such component: mothers experiencing high availability of carotenoids should produce more sons. Here, we test this hypothesis by providing carotenoids to a wild population of a sexually dimorphic passerine, the hihi (stitchbird: Notiomystis cincta). Access to carotenoids during early life influences the colour of male hihi plumage, which improves territory acquisition as adults. Therefore, carotenoid availability when young may influence male fitness. However, we found no evidence of sex ratio bias in treated or untreated groups, either before or after hatching. First-laid eggs, where carotenoid concentrations are usually highest, were also unbiased. For hihi, access to carotenoids during egg-laying does not appear to encourage mothers to alter sex ratios of offspring. Alternatively, the fitness of daughters may also benefit from increased carotenoids during development. Disentangling these alternatives requires further work.

Altered sex ratios in response to climate change-Who will fall into the (epigenetic) trap? (Comment on DOI 10.1002/bies.201600058).

BioEssaysVolume 38, Issue 10 p. 939-939 Thoughts & Opinion Altered sex ratios in response to climate change—Who will fall into the (epigenetic) trap? (Comment on DOI 10.1002/bies.201600058) Francesc Piferrer, Corresponding Author Francesc Piferrer piferrer@icm.csic.es Institut de Ciències del Mar, Consejo Superior de Investigaciones Científicas (CSIC), Barcelona, Spain Corresponding author: Francesc Piferrer E-mail: piferrer@icm.csic.esSearch for more papers by this author Francesc Piferrer, Corresponding Author Francesc Piferrer piferrer@icm.csic.es Institut de Ciències del Mar, Consejo Superior de Investigaciones Científicas (CSIC), Barcelona, Spain Corresponding author: Francesc Piferrer E-mail: piferrer@icm.csic.esSearch for more papers by this author First published: 31 August 2016 https://doi.org/10.1002/bies.201600172Citations: 10Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume38, Issue10October 2016Pages 939-939 RelatedInformation

Lifetime reproductive effort is equal between the sexes in seed beetles (Callosobruchus maculatus): dispelling the myth of the cheap male

In the evolution of sexual reproduction, the development of asymmetrical gametes (anisogamy) often leads to the characterisation that males invest less in reproduction than females. This view is founded on the fact that male investment in individual gametes (sperm) and in gonadal tissue is often less than that of females. While it may be true that within a single mating interaction the relative reproductive investment by males and females differs, over their respective lifetimes an evolutionary view predicts that total lifetime reproductive investment should be the same for both sexes. We tested this prediction by measuring total reproductive investment in reproducing adult male and female seed beetles. Since seed beetle adults are facultatively aphagous, not needing to feed or drink as adults, we could track the daily loss of biomass in the reproducing adults as a proxy for total lifetime investment in maintenance and reproduction. By the end of their lifetime there was not a significant difference in total reproductive effort between males and females. Both sexes invested 70 ± 1% SE of their initial body mass in maintenance and reproduction. Altering sex ratios, creating male-biased or female-biased treatments, significantly influenced total investment between the sexes, but not in the manner predicted. Ours is one of the few studies that have measured total adult lifetime reproductive investment in an organism, and our results support the theoretical expectation of equal total reproductive investment between the sexes.

Epigenetic-induced alterations in sex-ratios in response to climate change: An epigenetic trap?

We hypothesize that under the predicted scenario of climate change epigenetically mediated environmental sex determination could become an epigenetic trap. Epigenetically regulated environmental sex determination is a mechanism by which species can modulate their breeding strategies to accommodate environmental change. Growing evidence suggests that epigenetic mechanisms may play a key role in phenotypic plasticity and in the rapid adaptation of species to environmental change, through the capacity of organisms to maintain a non-genetic plastic memory of the environmental and ecological conditions experienced by their parents. However, inherited epigenetic variation could also be maladaptive, becoming an epigenetic trap. This is because environmental sex determination can alter sex ratios by increasing the survival of one of the sexes at the expense of negative fitness consequences for the other, which could lead not only to the collapse of natural populations, but also have an impact in farmed animal and plant species.

Changing sex ratios

Climate Change Climate-warming temperatures might be expected to affect the sex ratio of species if sex determination is temperature-dependent. Petry et al. show that indirect climate effects could also alter sex ratios in species in which sex is genetically determined and damage reproductive fitness (see the Perspective by Etterson and Mazer). Over four decades, sex ratios in populations of a dioecious alpine plant have shifted toward females as a result of the different water needs of the male and female plants. The lack of males has reduced the reproductive success and fitness of the females. Similar subtle differences between sexes in environmental sensitivities could eventually lead to population declines. Science , this issue p. [69][1]; see also p. [32][2] [1]: /lookup/doi/10.1126/science.aaf2588 [2]: /lookup/doi/10.1126/science.aag1624

Sex-specific responses to climate change in plants alter population sex ratio and performance.

Males and females are ecologically distinct in many species, but whether responses to climate change are sex-specific is unknown. We document sex-specific responses to climate change in the plant Valeriana edulis (valerian) over four decades and across its 1800-meter elevation range. Increased elevation was associated with increased water availability and female frequency, likely owing to sex-specific water use efficiency and survival. Recent aridification caused male frequency to move upslope at 175 meters per decade, a rate of trait shift outpacing reported species' range shifts by an order of magnitude. This increase in male frequency reduced pollen limitation and increased seedset. Coupled with previous studies reporting sex-specific arthropod communities, these results underscore the importance of ecological differences between the sexes in mediating biological responses to climate change.

Glyphosate and adverse pregnancy outcomes, a systematic review of observational studies.

BackgroundA study in frog and chicken embryos, and reports of a high incidence of birth defects in regions of intensive GM-soy planting have raised concerns on the teratogenic potential of glyphosate-based herbicides. These public concerns prompted us to conduct a systematic review of the epidemiological studies testing hypotheses of associations between glyphosate exposure and adverse pregnancy outcomes including birth defects.MethodsA systematic and comprehensive literature search was performed in MEDLINE, TOXLINE, Bireme-BVS and SCOPUS databases using different combinations of exposure and outcome terms. A case–control study on the association between pesticides and congenital malformations in areas of extensive GM soy crops in South America, and reports on the occurrence of birth defects in these regions were reviewed as well.ResultsThe search found ten studies testing associations between glyphosate and birth defects, abortions, pre-term deliveries, small for gestational date births, childhood diseases or altered sex ratios. Two additional studies examined changes of time-to-pregnancy in glyphosate-exposed populations. Except for an excess of Attention Deficit Hyperactivity Disorder - ADHD (OR = 3.6, 1.3-9.6) among children born to glyphosate appliers, no significant associations between this herbicide and adverse pregnancy outcomes were described. Evidence that in South American regions of intensive GM-soy planting incidence of birth defects is high remains elusive.ConclusionsCurrent epidemiological evidence, albeit limited to a few studies using non-quantitative and indirect estimates and dichotomous analysis of exposures, does not lend support to public concerns that glyphosate-based pesticides might pose developmental risks to the unborn child. Nonetheless, owing to methodological limitations of existing analytical observational studies, and particularly to a lack of a direct measurement (urine and/or blood levels), or an indirect estimation of exposure that has proven valid, these negative findings cannot be taken as definitive evidence that GLY, at current levels of occupational and environmental exposures, brings no risk for human development and reproduction.

Zeta Sperm Selection Improves Pregnancy Rate and Alters Sex Ratio in Male Factor Infertility Patients: A Double-Blind, Randomized Clinical Trial.

Selection of sperm for intra-cytoplasmic sperm injection (ICSI) is usually considered as the ultimate technique to alleviate male-factor infertility. In routine ICSI, selection is based on morphology and viability which does not necessarily preclude the chance injection of DNA-damaged or apoptotic sperm into the oocyte. Sperm with high negative surface electrical charge, named "Zeta potential", are mature and more likely to have intact chromatin. In addition, X-bearing spermatozoa carry more negative charge. Therefore, we aimed to compare the clinical outcomes of Zeta procedure with routine sperm selection in infertile men candidate for ICSI. From a total of 203 ICSI cycles studied, 101 cycles were allocated to density gradient centrifugation (DGC)/Zeta group and the remaining 102 were included in the DGC group in this prospective study. Clinical outcomes were com- pared between the two groups. The ratios of Xand Y bearing sperm were assessed by fluorescence in situ hybridization (FISH) and quantitative polymerase chain reaction (qPCR) methods in 17 independent semen samples. In the present double-blind randomized clinical trial, a significant increase in top quality embryos and pregnancy rate were observed in DGC/Zeta group compared to DGC group. Moreover, sex ratio (XY/XX) at birth significantly was lower in the DGC/Zeta group compared to DGC group despite similar ratio of X/Y bearings sper- matozoa following Zeta selection. Zeta method not only improves the percentage of top embryo quality and pregnancy outcome but also alters the sex ratio compared to the conventional DGC method, despite no significant change in the ratio of Xand Ybearing sperm population (Registration number: IRCT201108047223N1).

TBBPA chronic exposure produces sex-specific neurobehavioral and social interaction changes in adult zebrafish

The toxicity of tetrabromobisphenol A (TBBPA) has been extensively studied because of its high production volume. TBBPA is toxic to aquatic fish based on acute high concentration exposure tests, and few studies have assessed the behavioral effects of low concentration chronic TBBPA exposures in aquatic organisms. The present study defined the developmental and neurobehavioral effects associated with exposure of zebrafish to 0, 5 and 50nM TBBPA during 1–120days post-fertilization (dpf) following by detoxification for four months before the behaviors assessment. These low concentration TBBPA exposures were not associated with malformations and did not alter sex ratio, but resulted in reduced zebrafish body weight and length. Adult behavioral assays indicated that TBBPA exposed males had significantly higher average swim speeds and spent significantly more time in high speed darting mode and less time in medium cruising mode compared to control males. In an adult photomotor response assay, TBBPA exposure was associated with hyperactivity in male fish. Female zebrafish responses in these assays followed a similar trend, but the magnitude of TBBPA effects was generally smaller than in males. Social interaction evaluated using a mirror attack test showed that 50nM TBBPA exposed males had heightened aggression. Females exposed to 50nM TBBPA spent more time in the vicinity of the mirror, but did not show increased aggression toward the mirror compared to unexposed control fish. Overall, the hyperactivity and social behavior deficits ascribed here to chronic TBBPA exposure was most profound in males. Our findings indicate that TBBPA can cause developmental and neurobehavioral deficits, and may pose significant health risk to humans.

Incubation temperature causes skewed sex ratios in a precocial bird

Many animals with genetic sex determination are nonetheless capable of manipulating sex ratios via behavioral and physiological means, which can sometimes result in fitness benefits to the parent. Sex ratio manipulation in birds is not widely documented, and revealing the mechanisms for altered sex ratios in vertebrates remains a compelling area of research. Incubation temperature is a key component of the developmental environment for birds, but despite its well-documented effects on offspring phenotype it has rarely been considered as a factor in avian sex ratios. Using ecologically relevant manipulations of incubation temperature within the range 35.0-37.0°C, we found greater mortality of female embryos during incubation than males regardless of incubation temperature, and evidence that more female than male embryos die at the lowest incubation temperature (35.0°C). Our findings in conjunction with previous work in brush turkeys suggest incubation temperature is an important determinant of avian secondary sex ratios that requires additional study, and should be considered when estimating the impact of climate change on avian populations.

Detoxification Efforts in Longnose Dace (&amp;lt;i&amp;gt;Rhinichthys cataractae&amp;lt;/i&amp;gt;) Exposed to Municipal and Agricultural Inputs

Ecological impacts of contaminants on population patterns in wild fish are impacted by many contaminants that readily enter aquatic systems. Responses to toxicants by individuals in lab studies generally do not predict population level consequences in natural systems. Trace levels of contaminants are present in all major rivers in southern Alberta, Canada, with concentrations higher down-stream of anthropogenic inputs like agricultural land-use and inputs of municipal wastewater effluents. Longnose dace (Rhinichthys cataractae) were used as a sentinel species to study field-based population-level responses to contaminants. We hypothesized that biomarker activity, triggered by contaminant exposure, should increase downstream of anthropogenic inputs in two southern Alberta rivers, with corresponding relations between biomarker activity and sex ratios, after accounting for age structure. Liver detoxification (ethoxyresorufin-O-deethylase activity = EROD) measured at reference and exposed sites on each river differed significantly in only the Bow River system. Sex ratios varied more downstream of anthropogenic inputs than upstream, but the direction of sex ratio bias was inconsistent and temporally dynamic. Sex ratios correlated with liver detoxification in only the Bow River. Taken together, these results suggest that contaminants alter sex ratios in long-nose dace, but that there is variation in anthropogenic stressors among rivers.

Should we be promoting embryo transfer at blastocyst stage?

Improved laboratory standards and better culture media have made extended culture to blastocyst stage a reality to identify embryos with maximum implantation potential. The strategy of extended culture has become more popular across the world at a time when regulatory bodies have emphasized the need to increase the uptake of elective single embryo transfer, minimize complications associated with multiple births and aim for a healthy singleton live-birth as the preferred outcome in IVF. New data on perinatal outcomes suggest that pregnancies after embryo transfer at blastocyst stage are associated with a higher risk of preterm delivery, large for gestational age babies, monozygotic twins and altered sex ratio compared with those following embryo transfers at cleavage stage. In addition, concerns have been raised of increased congenital anomalies and epigenetic modifications with embryo transfer at blastocyst stage. Twenty-four years on from the first embryo transfer at blastocyst stage, we examine the reasons for extended embryo culture, evaluate the risks and benefits of this strategy and suggest the need to reconsider this policy in the interests of fetal safety.

Ameliorative Effect of Olive Leaf Extract on the Fetal Lung Tissue of Diabetic Pregnant Rats

Aim of the work- Diabetes mellitus is a common metabolic disease not only affecting the individual, but also imposes adverse effects on the offsprings. Besides increasing congenital malformations, maternal diabetes is suggested to be associated with early pregnancy loss, altered sex ratio in the offspring and long-term postnatal diseases. Antidiabetic plants are used as supportive therapy in the treatment of diabetes during pregnancy, so the present study aims to investigate the protective effect of olive leaf extract on the fetal lung of the diabetic pregnant rats. Material and methods - Forty pregnant albino rats were used and categorized after mating into four groups; group 1: control group(C), group 2: rats treated with olive leaf extract during the period of pregnancy (O) (1 ml/100gm. b. wt), group 3: streptozotocin induced diabetic rats (D)(STZ 35 mg/kg b.wt), group 4: diabetic rats treated with olive leaf extract (D+O) (as in groups 2&3). The pregnant females of different groups were dissected during the 19 th day of pregnancy. Lung samples of fetuses were taken for the histological and histochemical studies. Results- Histopathological and histochemical observations of fetal lung tissue showed that the olive leaf extract succeeded to minimize the drastic changes which were observed in the fetal lung of diabetic rats. Conclusion- It is recommended that the use of the olive leaf extract has the ability to minimize the adverse effects in the fetal lung tissue of diabetic rats.

Protective Effect of Olive Oil against Teratogenecity Induced by Mancozeb in Albino Rats

Aim: this study aimed to investigate the protective effect of extra-virgin olive oil (EVOO) against teratogenicity of the fungicide mancozeb. Methods: after pregnancy confirmation, 32 pregnant rats were divided into 4-groups (n=8). The 1st group orally administered tap water (-ve control), the 2nd group (+ve control) was administered EVOO (0.5ml/dam) from the1st to 20th day of pregnancy. The 3rd and the 4th groups were administered 200 mg/kg mancozeb during the period of organogenesis, from the 6th to 15th day of pregnancy. The 4th group received the mentioned dose of EVOO prior to the pesticide administration. Cesarean section was performed on day 20 of pregnancy and the maternal and fetal parameters were recorded. Results: mancozeb induced maternal toxicity manifested as lower body weight gain of dams, increased number of late resorption sites/litter in comparison with the control group and mancozeb group pretreated with EVOO. Mancozeb evoked a decrease in fetal body weight, altered sex ratio (M/F) as well as increased incidence of fetal external, visceral and skeletal abnormalities. Treatment with virgin oil reduced the congenital malformations. Conclusively, the present study elucidates the protective role of EVOO as a result of antioxidant activity which scavenges the reactive oxygen species which induced cytotoxicity and increased prenatal mortalities.

Journey of a Girl Child in India During Health and Disease.

Gender inequality can express as inequalities in several dimensions – survival, natality, facilities, ownership, sharing of household benefits and chores, and domestic violence. Inequality in gender of one type may lead to gender inequality of many other types. Gender inequality in health is evident as asymmetry in mortality, sex-selective abortions, and differential care-seeking [1]. Sex selection before birth and neglect of the female child after birth leads to an altered sex ratio. There are 1076 women per 1000 men in Europe and 1029 women per 1000 men in North America, but there are only 914 women per 1000 men in India [2]. This is a further decline from the figure of 927 females per 1000 males in 2001 Indian census [3]. During the last decade, the number of female children to male children in the younger age group dropped from 945 per 1000 males to 927 per 1000 males. There are now 48 fewer girls per 1000 boys than there were in 1981 [4]. This is despite the fact that the survival rate for girls is often more than that of boys in the younger age groups. These numbers reflect neglect and mistreatment of the girl child in India.

Life cycle exposure of the frog Silurana tropicalis to arsenate: Steroid- and thyroid hormone-related genes are differently altered throughout development

Arsenic contaminates water surface and groundwater worldwide. Several studies have suggested that arsenic acts as an endocrine disruptor in mammalian and non-mammalian species, although its chronic effect during development remains largely unknown. To address this question, life cycle exposures to 0, 0.3 and 0.8ppm of arsenate (pentavalent arsenic; AsV) were performed in the Western clawed frog (Silurana tropicalis) from the gastrulae stage (developmental stage Nieuwkoop-Faber; NF12) until metamorphosis (NF66). Tissue samples were collected at the beginning of feeding (NF46; whole body), sexual development (NF56; liver), and at metamorphosis completion (NF66; liver and gonadal mesonephros complex). Real-time RT-PCR analysis quantified decreases in mRNA levels of genes related to estrogen- (estrogen receptor alpha and aromatase), androgen- (androgen receptor and steroid 5-alpha-reductase type 2), and cholesterol metabolism- (steroidogenic acute regulatory protein) at stage NF46. Similarly, arsenate decreased steroid 5-alpha-reductase type 2 expression in stage NF56 livers, but transcript increases were observed for both estrogen receptor alpha and steroidogenic acute regulatory protein at this stage. Given the changes observed in the expression of genes essential for proper sexual development, gonadal histological analysis was carried out in stage NF66 animals. Arsenate treatments did not alter sex ratio or produce testicular oocytes. On the other hand, arsenate interfered with thyroid hormone-related transcripts at NF66. Specifically, thyroid hormone receptor beta and deiodinase type 2 mRNA levels were significantly reduced after arsenate treatment in the gonadal mesonephros complex. This reduction in thyroid hormone-related gene expression, however, was not accompanied by any morphological changes measured. In summary, environmentally relevant concentrations of AsV altered steroidogenesis-, sex steroid signaling- and thyroid hormone-related gene expression, although transcriptional changes varied among tissues and developmental stages.