Maternal Deposition Research Articles

Improving the suppressive power of homing gene drive by co-targeting a distant-site female fertility gene

Gene drive technology has the potential to address major biological challenges. Well-studied homing suppression drives have been shown to be highly efficient in Anopheles mosquitoes, but for other organisms, lower rates of drive conversion prevent elimination of the target population. To tackle this issue, we propose a gene drive design that has two targets: a drive homing site where drive conversion takes place, and a distant site where cleavage induces population suppression. We model this design and find that the two-target system allows suppression to occur over a much wider range of drive conversion efficiency. Specifically, the cutting efficiency now determines the suppressive power of the drive, rather than the conversion efficiency as in standard suppression drives. We construct a two-target drive in Drosophila melanogaster and show that both components of the gene drive function successfully. However, cleavage in the embryo from maternal deposition as well as fitness costs in female drive heterozygotes both remain significant challenges for both two-target and standard suppression drives. Overall, our improved gene drive design has the potential to ease problems associated with homing suppression gene drives for many species where drive conversion is less efficient.

Zebrafish Suppressor of Cytokine Signaling 4b (Socs4b) Is Dispensable for Development but May Regulate Epidermal Growth Factor Receptor Signaling.

The suppressor of cytokine signaling (SOCS) family of proteins were named after their defining role as negative feedback regulators of signaling initiated by numerous cytokine receptors. However, multiple members of the SOCS family likely function outside of this paradigm, including SOCS4. Zebrafish possess two SOCS4 paralogues, with socs4a previously shown to participate in central nervous system development and function. This study examined the role of the other paralogue, socs4b, through expression analysis and functional investigations in vivo and in vitro. This revealed maternal deposition of socs4b mRNA, specific zygotic expression during late embryogenesis, including in the brain, eye and intestine, and broad adult expression that was highest in the brain. A mutant allele, socs4bΔ18, was generated by genome editing, in which the start codon was deleted. Fish homozygous for this likely hypomorphic allele showed no overt developmental phenotypes. However, in vitro studies suggested the Socs4b protein may be able to regulate EGFR signaling.

Inconsistent Transcriptomic Responses to Hexabromocyclododecane in Japanese Quail: A Comparative Analysis of Results From Four Different Study Designs.

Efforts to use transcriptomics for toxicity testing have classically relied on the assumption that chemicals consistently produce characteristic transcriptomic signatures that are reflective of their mechanism of action. However, the degree to which transcriptomic responses are conserved across different test methodologies has seldom been explored. With increasing regulatory demand for New Approach Methods (NAMs) that use alternatives to animal models and high-content approaches such as transcriptomics, this type of comparative analysis is needed. We examined whether common genes are dysregulated in Japanese quail (Coturnix japonica) liver following sublethal exposure to the flame retardant hexabromocyclododecane (HBCD), when life stage and test methodologies differ. The four exposure scenarios included one NAM: Study 1-early-life stage (ELS) exposure via a single egg injection, and three more traditional approaches; Study 2-adult exposure using a single oral gavage; Study 3-ELS exposure via maternal deposition after adults were exposed through their diet for 7 weeks; and Study 4-ELS exposure via maternal deposition and re-exposure of nestlings through their diet for 17 weeks. The total number of differentially expressed genes (DEGs) detected in each study was variable (Study 1, 550; Study 2, 192; Study 3, 1; Study 4, 3) with only 19 DEGs shared between Studies 1 and 2. Factors contributing to this lack of concordance are discussed and include differences in dose, but also quail strain, exposure route, sampling time, and HBCD stereoisomer composition. The results provide a detailed overview of the transcriptomic responses to HBCD at different life stages and routes of exposure in a model avian species and highlight certain challenges and limits of comparing transcriptomics across different test methodologies. Environ Toxicol Chem 2024;00:1-11. © 2024 The Author(s). Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Fertility decline in Aedes aegypti (Diptera: Culicidae) mosquitoes is associated with reduced maternal transcript deposition and does not depend on female age.

Female mosquitoes undergo multiple rounds of reproduction known as gonotrophic cycles (GC). A gonotrophic cycle spans the period from blood meal intake to egg laying. Nutrients from vertebrate host blood are necessary for completing egg development. During oogenesis, a female prepackages mRNA into her oocytes, and these maternal transcripts drive the first 2 h of embryonic development prior to zygotic genome activation. In this study, we profiled transcriptional changes in 1-2 h of Aedes aegypti (Diptera: Culicidae) embryos across 2 GC. We found that homeotic genes which are regulators of embryogenesis are downregulated in embryos from the second gonotrophic cycle. Interestingly, embryos produced by Ae. aegypti females progressively reduced their ability to hatch as the number of GC increased. We show that this fertility decline is due to increased reproductive output and not the mosquitoes' age. Moreover, we found a similar decline in fertility and fecundity across 3 GC in Aedes albopictus. Our results are useful for predicting mosquito population dynamics to inform vector control efforts.

Pentadecanoic acid (C15:0, PA) induces mild maternal glucose intolerance and promotes the growth of the offspring partly through up-regulating liver PPARα and MAPK signaling pathways.

Gestational diabetes mellitus (GDM) is one of the most common metabolic disturbances during pregnancy, which poses a serious threat to both maternal and offspring health. Pentadecanoic acid (C15:0, PA) is one of the most common odd-chain saturated fatty acids (OCS-FAs). However, its safety and nutritional value are yet to be verified. Herein, we provide a systematic assessment of the effects of PA on maternal and progeny health and insulin sensitivity for the first time. Our results showed that consumption of 1% PA during pregnancy could increase the contents of PA and heptadecanoic acid (C17:0) in maternal plasma, fetal tissue and offspring plasma, but it had no effect on embryonic development. During pregnancy, PA treatment caused mild insulin resistance, while it had little effect on the maternal body composition. During lactation, PA treatment caused mild insulin resistance and oxidative stress. Maternal body fat deposition was also reduced, but the growth rate of the offspring was faster. It is worth noting that PA treatment decreased plasma and liver TG content and increased the antioxidant capacity of the offspring. The effect of PA on the transcription and expression genes in the liver of pregnant mice was investigated using RNA-seq. PPARα and MAPK signaling pathways, both closely related to lipolysis, inflammation, oxidative stress, and insulin resistance were significantly increased. The expression of c-JUN, ERK, JNK and P65 proteins was also significantly up-regulated. In conclusion, our results suggest that 1% PA can induce a mild decrease in the maternal glucose tolerance and lipolysis mainly by activated MAPK and PPARα signaling. Moreover, low concentrations of PA may be an effective nutrient to alleviate the oxidative stress and reduce blood lipid levels of offspring.

From maternal glucocorticoid and thyroid hormones to epigenetic regulation of offspring gene expression: An experimental study in a wild bird species.

Offspring phenotype at birth is determined by its genotype and the prenatal environment including exposure to maternal hormones. Variation in both maternal glucocorticoids and thyroid hormones can affect offspring phenotype, but the underlying molecular mechanisms, especially those contributing to long-lasting effects, remain unclear. Epigenetic changes (such as DNA methylation) have been postulated as mediators of long-lasting effects of early-life environment. In this study, we determined the effects of elevated prenatal glucocorticoid and thyroid hormones on handling stress response (breath rate) as well as DNA methylation and gene expression of glucocorticoid receptor (GR) and thyroid hormone receptor (THR) in great tits (Parus major). Eggs were injected before incubation onset with corticosterone (the main avian glucocorticoid) and/or thyroid hormones (thyroxine and triiodothyronine) to simulate variation in maternal hormone deposition. Breath rate during handling and gene expression of GR and THR were evaluated 14 days after hatching. Methylation status of GR and THR genes was analyzed from the longitudinal blood cells sampled 7 and 14 days after hatching, as well as the following autumn. Elevated prenatal corticosterone level significantly increased the breath rate during handling, indicating an enhanced metabolic stress response. Prenatal corticosterone manipulation had CpG-site-specific effects on DNA methylation at the GR putative promoter region, while it did not significantly affect GR gene expression. GR expression was negatively associated with earlier hatching date and chick size. THR methylation or expression did not exhibit any significant relationship with the hormonal treatments or the examined covariates, suggesting that TH signaling may be more robust due to its crucial role in development. This study provides some support to the hypothesis suggesting that maternal corticosterone may influence offspring metabolic stress response via epigenetic alterations, yet their possible adaptive role in optimizing offspring phenotype to the prevailing conditions, context-dependency, and the underlying molecular interplay needs further research.

Making waves: Comparative analysis of gene drive spread characteristics in a continuous space model.

With their ability to rapidly increase in frequency, gene drives can be used to modify or suppress target populations after an initial release of drive individuals. Recent advances have revealed many possibilities for different types of drives, and several of these have been realized in experiments. These drives have advantages and disadvantages related to their ease of construction, confinement and capacity to be used for modification or suppression. Though characteristics of these drives have been explored in modelling studies, assessment in continuous space environments has been limited, often focusing on outcomes rather than fundamental properties. Here, we conduct a comparative analysis of many different gene drive types that have the capacity to form a wave of advance in continuous space using individual-based simulations in continuous space. We evaluate the drive wave speed as a function of drive performance and ecological parameters, which reveals substantial differences between drive performance in panmictic versus spatial environments. In particular, we find that suppression drive waves are uniquely vulnerable to fitness costs and undesired CRISPR cleavage activity in embryos by maternal deposition. Some drives, however, retain robust performance even with widely varying efficiency parameters. To gain a better understanding of drive waves, we compare their panmictic performance and find that the rate of wild-type allele removal is correlated with drive wave speed, though this is also affected by other factors. Overall, our results provide a useful resource for understanding the performance of drives in spatially continuous environments, which may be most representative of potential drive deployment in many relevant scenarios.

Intergenerational plasticity to cycling high temperature and hypoxia affects offspring stress responsiveness and tolerance in zebrafish.

Predicted climate change-induced increases in heat waves and hypoxic events will have profound effects on fishes, yet the capacity of parents to alter offspring phenotype via non-genetic inheritance and buffer against these combined stressors is not clear. This study tested how prolonged adult zebrafish exposure to combined diel cycles of thermal stress and hypoxia affect offspring early survival and development, parental investment of cortisol and heat shock proteins (HSPs), larval offspring stress responses, and both parental and offspring heat and hypoxia tolerance. Parental exposure to the combined stressor did not affect fecundity, but increased mortality, produced smaller embryos and delayed hatching. The combined treatment also reduced maternal deposition of cortisol and increased embryo hsf1, hsp70a, HSP70, hsp90aa and HSP90 levels. In larvae, basal cortisol levels did not differ between treatments, but acute exposure to combined heat stress and hypoxia increased cortisol levels in control larvae with no effect on larvae from exposed parents. In contrast, whereas larval basal hsf1, hsp70a and hsp90aa levels differed between parental treatments, the combined acute stressor elicited similar transcriptional responses across treatments. Moreover, the combined acute stressor only induced a marked increase in HSP47 levels in the larvae derived from exposed parents. Finally, combined hypoxia and elevated temperatures increased both thermal and hypoxia tolerance in adults and conferred an increase in offspring thermal but not hypoxia tolerance. These results demonstrate that intergenerational acclimation to combined thermal stress and hypoxia elicit complex carryover effects on stress responsiveness and offspring tolerance with potential consequences for resilience.

Optimising the zebrafish Cre/Lox toolbox. Codon improved iCre, new gateway tools, Cre protein and guidelines.

We recently introduced the Cre/Lox technology in our laboratory for both transient (mRNA injections) and stable/transgenic experiments. We experienced significant issues such as silencing, mosaicism, and partial recombination using both approaches. Reviewing the literature gave us the impression that these issues are common among the zebrafish community using the Cre/Lox system. While some researchers took advantage of these problems for specific applications, such as cell and lineage tracing using the Zebrabow construct, we tried here to improve the efficiency and reliability of this system by constituting and testing a new set of tools for zebrafish genetics. First, we implemented a codon-improved Cre version (iCre) designed for rodent studies to counteract some of the aforementioned problems. This eukaryotic-like iCre version was engineered to i) reduce silencing, ii) increase mRNA stability, iii) enhance translational efficiency, and iv) improve nuclear translocation. Second, we established a new set of tol2-kit compatible vectors to facilitate the generation of either iCre-mRNA or iCre-transgenes for transient and transgenic experiments, respectively. We then validated the use of this material and are providing tips for users. Interestingly, during the validation steps, we found that maternal iCRE-mRNA and/or protein deposition from female transgenics systematically led to complete/homogeneous conversion of all tested Lox-responder-transgenes, as opposed to some residual imperfect conversion when using males-drivers or mRNA injections. Considering that we did not find any evidence of Cre-protein soaking and injections in the literature as it is usually conducted with cells, we tested these approaches. While soaking of cell-permeant CRE-protein did not lead to any detectable Lox-conversion, 1ng-10ng protein injections led to robust and homogeneous Lox-recombination, suggesting that the use of protein could be a robust option for exogenous delivery. This approach may be particularly useful to manipulate housekeeping genes involved in development, sex determination and reproduction which are difficult to investigate with traditional knockout approaches. All in all, we are providing here a new set of tools that should be useful in the field.

Does contaminant exposure disrupt maternal hormones deposition? A study on per- and polyfluoroalkyl substances in an Arctic seabird

Maternal effects are thought to be essential tools for females to modulate offspring development. The selective deposition of avian maternal hormones could therefore allow females to strategically adjust the phenotype of their offspring to the environmental situation encountered. However, at the time of egg formation, several contaminants are also transferred to the egg, including per- and polyfluoroalkyl substances (PFAS) which are ubiquitous organic contaminants with endocrine disrupting properties. It is, however, unknown if they can disrupt maternal hormone deposition. In this study we explored relationships between female PFAS burden and maternal deposition in the eggs of steroids (dihydrotestosterone, androstenedione and testosterone), glucocorticoids (corticosterone) and thyroid hormones (triiodothyronine and thyroxine) in a population of the Arctic-breeding black-legged kittiwake (Rissa tridactyla). Egg yolk hormone levels were unrelated to female hormone plasma levels. Second-laid eggs had significantly lower concentrations of androstenedione than first-laid eggs. Triiodothyronine yolk levels were decreasing with increasing egg mass but increasing with increasing females' body condition. Testosterone was the only transferred yolk hormone correlated to maternal PFAS burden: specifically, we found a positive correlation between testosterone in yolks and circulating maternal perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDcA) and perfluoroundecanoic acid (PFUnA) in first-laid eggs. This correlative study provides a first insight into the potential of some long-chain perfluoroalkyl acids to disrupt maternal hormones deposition in eggs and raises the question about the consequences of increased testosterone deposition on the developing embryo.

Daisy-chain gene drives: The role of low cut-rate, resistance mutations, and maternal deposition.

The introgression of genetic traits through gene drive may serve as a powerful and widely applicable method of biological control. However, for many applications, a self-perpetuating gene drive that can spread beyond the specific target population may be undesirable and preclude use. Daisy-chain gene drives have been proposed as a means of tuning the invasiveness of a gene drive, allowing it to spread efficiently into the target population, but be self-limiting beyond that. Daisy-chain gene drives are made up of multiple independent drive elements, where each element, except one, biases the inheritance of another, forming a chain. Under ideal inheritance biasing conditions, the released drive elements remain linked in the same configuration, generating copies of most of their elements except for the last remaining link in the chain. Through mathematical modelling of populations connected by migration, we have evaluated the effect of resistance alleles, different fitness costs, reduction in the cut-rate, and maternal deposition on two alternative daisy-chain gene drive designs. We find that the self-limiting nature of daisy-chain gene drives makes their spread highly dependent on the efficiency and fidelity of the inheritance biasing mechanism. In particular, reductions in the cut-rate and the formation of non-lethal resistance alleles can cause drive elements to lose their linked configuration. This severely reduces the invasiveness of the drives and allows for phantom cutting, where an upstream drive element cuts a downstream target locus despite the corresponding drive element being absent, creating and biasing the inheritance of additional resistance alleles. This phantom cutting can be mitigated by an alternative indirect daisy-chain design. We further find that while dominant fitness costs and maternal deposition reduce daisy-chain invasiveness, if overcome with an increased release frequency, they can reduce the spread of the drive into a neighbouring population.

Effects of Dietary Fiber, Crude Protein Level, and Gestation Stage on the Nitrogen Utilization of Multiparous Gestating Sows

Simple SummaryTwo major concerns for sustainable swine production are the shortage of protein feed resources and the excretion of nitrogen and phosphorus into the environment. Those concerns can be partly alleviated by increasing nitrogen utilization by improving nutrition for pigs. This study observed that a low crude protein and dietary fiber supplemented diet can improve nitrogen utilization and reduce urinary nitrogen excretion. Additionally, late gestating sows have greater nitrogen utilization than those of early gestation, characterized by reduced nitrogen in urine and lower amino acids in serum. These findings offer nutritional solutions for improving nitrogen utilization.To investigate the effects of dietary fiber (DF), crude protein (CP) level, and gestation stage on nitrogen utilization, 28 Landrace-Yorkshire cross gestating sows at parity two were randomly divided into four dietary treatments with seven duplicates of one pig with a repeated-measures design. The diets comprised one with normal crude protein (CP) of 13.3%, one with a low CP diet of 10.1%, and two diets, one with dietary fiber (DF) supplementation of inulin and cellulose at the ratio of 1:1 and one without DF. The total litter size, litter size alive, and newborn birthweight of piglets did not differ between treatment groups. Sows that received high DF levels had greater nitrogen output in feces, lower urinary nitrogen, and increased nitrogen retention. Sows that received a low CP diet had reduced nitrogen excretion in feces and urine, lower nitrogen retention, and an unchanged nitrogen retention ratio. Sows at the late stage of gestation on days 95 to 98 had lower nitrogen excretion in urine and greater nitrogen retention than in the early stage of gestation on days 35 to 38, associated with a significant decrease in serum amino acids in late gestation. Maternal protein deposition was increased by high DF, decreased by low CP, and lower in late gestation compared with early gestation. Collectively, DF improved nitrogen utilization by decreasing urine nitrogen output, and nitrogen utilization increased as gestation advanced.

USP16-mediated histone H2A lysine-119 deubiquitination during oocyte maturation is a prerequisite for zygotic genome activation.

Maternal-to-zygotic transition (MZT) is the first and key step in the control of animal development and intimately related to changes in chromatin structure and histone modifications. H2AK119ub1, an important epigenetic modification in regulating chromatin configuration and function, is primarily catalyzed by PRC1 and contributes to resistance to transcriptional reprogramming in mouse embryos. In this study, the genome-wide dynamic distribution of H2AK119ub1 during MZT in mice was investigated using chromosome immunoprecipitation and sequencing. The results indicated that H2AK119ub1 accumulated in fully grown oocytes and was enriched at the TSSs of maternal genes, but was promptly declined after meiotic resumption at genome-wide including the TSSs of early zygotic genes, by a previously unidentified mechanism. Genetic evidences indicated that ubiquitin-specific peptidase 16 (USP16) is the major deubiquitinase for H2AK119ub1 in mouse oocytes. Conditional knockout of Usp16 in oocytes did not impair their survival, growth, or meiotic maturation. However, oocytes lacking USP16 have defects when undergoing zygotic genome activation or gaining developmental competence after fertilization, potentially associated with high levels of maternal H2AK119ub1 deposition on the zygotic genomes. Taken together, H2AK119ub1 level is declined during oocyte maturation by an USP16-dependent mechanism, which ensures zygotic genome reprogramming and transcriptional activation of essential early zygotic genes.

Loss of zebrafish dzip1 results in inappropriate recruitment of periocular mesenchyme to the optic fissure and ocular coloboma.

Cilia are essential for the development and function of many different tissues. Although cilia machinery is crucial in the eye for photoreceptor development and function, a role for cilia in early eye development and morphogenesis is still somewhat unclear: many zebrafish cilia mutants retain cilia at early stages due to maternal deposition of cilia components. An eye phenotype has been described in the mouse Arl13 mutant, however, zebrafish arl13b is maternally deposited, and an early role for cilia proteins has not been tested in zebrafish eye development. Here we use the zebrafish dzip1 mutant, which exhibits a loss of cilia throughout stages of early eye development, to examine eye development and morphogenesis. We find that in dzip1 mutants, initial formation of the optic cup proceeds normally, however, the optic fissure subsequently fails to close and embryos develop the structural eye malformation ocular coloboma. Further, neural crest cells, which are implicated in optic fissure closure, do not populate the optic fissure correctly, suggesting that their inappropriate localization may be the underlying cause of coloboma. Overall, our results indicate a role for dzip1 in proper neural crest localization in the optic fissure and optic fissure closure.

Toward a CRISPR-Cas9-Based Gene Drive in the Diamondback Moth Plutella xylostella.

Promising to provide powerful genetic control tools, gene drives have been constructed in multiple dipteran insects, yeast, and mice for the purposes of population elimination or modification. However, it remains unclear whether these techniques can be applied to lepidopterans. Here, we used endogenous regulatory elements to drive Cas9 and single guide RNA (sgRNA) expression in the diamondback moth (DBM), Plutella xylostella, and test the first split gene drive system in a lepidopteran. The DBM is an economically important global agriculture pest of cruciferous crops and has developed severe resistance to various insecticides, making it a prime candidate for such novel control strategy development. A very high level of somatic editing was observed in Cas9/sgRNA transheterozygotes, although no significant homing was revealed in the subsequent generation. Although heritable Cas9-medated germline cleavage as well as maternal and paternal Cas9 deposition were observed, rates were far lower than for somatic cleavage events, indicating robust somatic but limited germline activity of Cas9/sgRNA under the control of selected regulatory elements. Our results provide valuable experience, paving the way for future construction of gene drives or other Cas9-based genetic control strategies in DBM and other lepidopterans.

Cardiac physiology and metabolic gene expression during late organogenesis among F. heteroclitus embryo families from crosses between pollution-sensitive and -resistant parents

BackgroundThe teleost fish Fundulus heteroclitus inhabit estuaries heavily polluted with persistent and bioaccumulative chemicals. While embryos of parents from polluted sites are remarkably resistant to toxic sediment and develop normally, embryos of parents from relatively clean estuaries, when treated with polluted sediment extracts, are developmentally delayed, displaying deformities characteristic of pollution-induced embryotoxicity. To gain insight into parental effects on sensitive and resistant phenotypes during late organogenesis, we established sensitive, resistant, and crossed embryo families using five female and five male parents from relatively clean and predominantly PAH-polluted estuaries each, measured heart rates, and quantified individual embryo expression of 179 metabolic genes.ResultsPollution-induced embryotoxicity manifested as morphological deformities, significant developmental delays, and altered cardiac physiology was evident among sensitive embryos resulting from crosses between females and males from relatively clean estuaries. Significantly different heart rates among several geographically unrelated populations of sensitive, resistant, and crossed embryo families during late organogenesis and pre-hatching suggest site-specific adaptive cardiac physiology phenotypes relative to pollution exposure. Metabolic gene expression patterns (32 genes, 17.9%, at p < 0.05; 11 genes, 6.1%, at p < 0.01) among the embryo families indicate maternal pollutant deposition in the eggs and parental effects on gene expression and metabolic alterations.ConclusionHeart rate differences among sensitive, resistant, and crossed embryos is a reliable phenotype for further explorations of adaptive mechanisms. While metabolic gene expression patterns among embryo families are suggestive of parental effects on several differentially expressed genes, a definitive adaptive signature and metabolic cost of resistant phenotypes is unclear and shows unexpected sensitive-resistant crossed embryo expression profiles. Our study highlights physiological and metabolic gene expression differences during a critical embryonic stage among pollution sensitive, resistant, and crossed embryo families, which may contribute to underlying resistance mechanisms observed in natural F. heteroclitus populations living in heavily contaminated estuaries.

Is maternal thyroid hormone deposition subject to a trade-off between self and egg because of iodine? An experimental study in rock pigeon.

ABSTRACTMaternal hormones constitute a key signalling pathway for mothers to shape offspring phenotype and fitness. Thyroid hormones (THs; triiodothyronine, T3; and thyroxine, T4) are metabolic hormones known to play crucial roles in embryonic development and survival in all vertebrates. During early developmental stages, embryos exclusively rely on exposure to maternal THs, and maternal hypothyroidism can cause severe embryonic maldevelopment. The TH molecule includes iodine, an element that cannot be synthesised by the organism. Therefore, TH production may become costly when environmental iodine availability is low. This may yield a trade-off for breeding females between allocating the hormones to self or to their eggs, potentially to the extent that it even influences the number of laid eggs. In this study, we investigated whether low dietary iodine may limit TH production and transfer to the eggs in a captive population of rock pigeons (Columba livia). We provided breeding females with an iodine-restricted (I−) diet or iodine-supplemented (I+) diet and measured the resulting circulating and yolk iodine and TH concentrations and the number of eggs laid. Our iodine-restricted diet successfully decreased both circulating and yolk iodine concentrations compared with the supplemented diet, but not circulating or yolk THs. This indicates that mothers may not be able to independently regulate hormone exposure for self and their embryos. However, egg production was clearly reduced in the I− group, with fewer females laying eggs. This result shows that restricted availability of iodine does induce a cost in terms of egg production. Whether females reduced egg production to preserve THs for themselves or to prevent embryos from exposure to low iodine and/or THs is as yet unclear.

Ecotoxicology assessments in avian species using cell-based models: A review

Cell-based models in avian species have historically focused on virology due to the demands of animal agriculture and vaccine production industries. Recent years have witnessed a gradual rise in the use of these models ( in ovo, cell lines, primary cell cultures, organ slices, and organ-on-a-chip) in ecotoxicological studies as scientists and governments begin the shift to new approach methodologies, a shift validated by the recent memo by the Environmental Protection Agency announcing the end of mammalian testing in the next two decades. This rise has been hindered by the limited standards available for avian species and the unknowns surrounding cell-based assay applicability in extrapolation to in vivo. Toxicologists have incorporated these models in many different studies, including maternal deposition, mechanistic, metabolic, and non-target analysis methods, demonstrating the broad utility of cell-based assays. In ovo methods are ideal for reproductive and early life stage development studies, primary cell cultures for metabolic analysis, cell lines for long term studies requiring culture, organ slices for metabolic research, and organ-on-a-chip models for predictive analysis. These models all have their limitations that researchers need to consider when choosing which is most appropriate for the intended research, however. The current indications are that future avian cell-based model testing would benefit from expanding the species diversity available in cell lines and increasing metabolic conservation in full replacement methods. In ovo and primary cell culture methods should also be examined to increase efficiency and further reduce animal usage. This review examines the use, limitations, and published applications of these models in an ecotoxicological context to understand the current state of avian cell-based models to explain what future directions should be taken and how best to apply the methods available to current problems that avian researchers are approaching.

Mosquito metallomics reveal copper and iron as critical factors for Plasmodium infection.

Iron and copper chelation restricts Plasmodium growth in vitro and in mammalian hosts. The parasite alters metal homeostasis in red blood cells to its favor, for example metabolizing hemoglobin to hemozoin. Metal interactions with the mosquito have not, however, been studied. Here, we describe the metallomes of Anopheles albimanus and Aedes aegypti throughout their life cycle and following a blood meal. Consistent with previous reports, we found evidence of maternal iron deposition in embryos of Ae. aegypti, but less so in An. albimanus. Sodium, potassium, iron, and copper are present at higher concentrations during larval developmental stages. Two An. albimanus phenotypes that differ in their susceptibility to Plasmodium berghei infection were studied. The susceptible white stripe (ws) phenotype was named after a dorsal white stripe apparent during larval stages 3, 4, and pupae. During larval stage 3, ws larvae accumulate more iron and copper than the resistant brown stripe (bs) phenotype counterparts. A similar increase in copper and iron accumulation was also observed in the susceptible ws, but not in the resistant bs phenotype following P. berghei infection. Feeding ws mosquitoes with extracellular iron and copper chelators before and after receiving Plasmodium-infected blood protected from infection and simultaneously affected follicular development in the case of iron chelation. Unexpectedly, the application of the iron chelator to the bs strain reverted resistance to infection. Besides a drop in iron, iron-chelated bs mosquitoes experienced a concomitant loss of copper. Thus, the effect of metal chelation on P. berghei infectivity was strain-specific.

Modeling standardized ileal digestible lysine requirements during gestation on gilts and sows

The objective of this study was to model daily standardized ileal digestible (SID) Lys-requirements in gestating sows. Data from 877 females (459 gilts, 418 sows; Camborough®, PIC, Hendersonville, TN) was collected and modeled using Dourmad et al. (2008) equations. Individual feed intake and body weight (BW) were recorded daily throughout gestation. Dietary treatments included 11, 13.5, 16, and 18.5 g/d SID Lys-intake throughout gestation. Data were divided into 2 parity groups: gilts and sows, and gestation was divided into 3 stages: d 5 to 39 (early), 40 to 74 (mid), and 75 to 108 (late). The model follows the principle that energy is partitioned between maintenance, growth of conceptus (fetus, placenta, and fluids), and maternal protein and lipid deposition. Requirements for SID Lys-were estimated based on predicted whole body protein deposition. After dividing energy requirements into tissue pools for maintenance, products of conceptus, and maternal reserves, the greatest portion of the energy requirement was for maintenance. Estimated protein retention of the conceptus increased in each sequential stage of gestation (P < 0.05) but there were no differences (P > 0.05) among treatments. Regardless of parity or stage of gestation, increasing SID Lys-increased estimated maternal protein deposition (linear, P < 0.001), but it decreased in each sequential stage of gestation (P < 0.001). Estimated maternal lipid deposition increased with increasing SID Lys (linear, P = 0.076) and decreased in each sequential stage of gestation (P < 0.05), with gilts having to mobilize maternal lipid in late gestation. For gilts and sows, estimated SID Lys-requirements increased in each sequential stage of gestation (P < 0.05). Estimated SID Lys-balance (SID Lys-intake - SID Lys-requirement) increased with increasing SID Lys-in the diet (quadratic, P < 0.054), and decreased (P < 0.05) in each sequential stage of gestation. Overall, the model shows how changes in protein retention of the conceptus and maternal protein deposition differ by parity and stage of gestation for a single gestation phase. Based on predicted changes in protein deposition, and SID Lys-balance, providing females with 11.0 g/d SID Lys-throughout gestation adequately meets Lys-requirements for most of gestation; however, gilts and sows were in a negative SID Lys-balance for the last 5- to 10 d of gestation and this can be prevented by providing gilts and sows with 13.5 g/d SID Lys.