Changes In Incubation Temperature Research Articles

Incubation as a driver of maternal effects: Temperature influences levels of yolk maternally derived 5α-dihydrotestosterone

In birds, maternal hormones deposited into eggs in response to environmental stimuli can impact offspring phenotype. Although less studied, environmental conditions can also influence females’ incubation behavior, which might play a role in regulating embryo exposure to maternal hormones through changes in incubation temperature that affect the activity of the enzymes responsible for converting testosterone (T) to 5α-dihydrotestosterone (DHT) or estradiol. Here, we tested the hypothesis that the initial T content of the yolk and incubation temperature determine exposure to T metabolites during early embryo development. In the Japanese quail (Coturnix japonica), we experimentally manipulated yolk T and incubation temperature (38° C versus 36° C) and analyzed DHT and estradiol titers on day four of incubation. We found that eggs with experimentally increased T and those incubated at 36° C showed higher DHT concentration in egg yolk (with no synergistic effect of the two treatments). Estradiol titers were not affected by T manipulation or incubation temperature. Our study suggests that incubation temperature influences DHT titers and may act as an understudied source of maternal influence on offspring phenotype.

Identification and functional studies of microbial volatile organic compounds produced by Arctic flower yeasts.

Microbial volatile organic compounds (mVOCs) can serve as a communication channel among microorganisms, insects and plants, making them important in ecosystem. In order to understand the possible role of mVOCs in Arctic ecology, the microbes in Arctic flowers and their mVOCs and effects on plants were investigated. This study aims to isolate different yeast species from the flowers of five Arctic plant species and further to explore the function of mVOCs emitted by these microbes to plant. It was found that the composition and amount of mVOCs produced by the isolated yeasts were considerably affected by changes in incubation temperature. When the incubation temperature rose, the species of alcohols, aldehydes, esters, organic acids, and ketones increased, but substances specific to low temperature decreased or disappeared. When yeasts were co-cultured with Arabidopsis thaliana without any direct contact, mVOCs produced by the isolated yeasts inhibited the seed germination of A. thaliana at low temperatures; however, the mVOCs promoted the chlorophyll content, fresh weight, root weight and flowering rate of Arabidopsis plants. Although the overall growth-promoting effect of yeast mVOCs was higher at 20°C than at 10°C, the growth-promoting effect on roots, flowers and chlorophyll was highest at 10°C. When cultured at 10°C, the mVOCs produced by Cystofilobasidium capitatum A37, Cryptococcus sp. D41, and Sporidiobolus salmonicolor D27 had the highest growth-promoting effects on the root, flowering rate and chlorophyll content of Arabidopsis, respectively. In the co-culture system, some new mVOCs were detected, such as hendecane, tetradecane, and 1-hexanol that have been proven to promote plant growth. In addition, mVOCs of the isolated Arctic yeasts could inhibit the growth of several microorganisms, especially filamentous fungi. It was the first time to prove that mVOCs produced by the isolated yeasts had varying effects on plant growth at different incubating temperatures, providing a reference for the interactions between microorganisms and plants and their possible responses to climate change in the Arctic area. Moreover, the characteristics of promoting plant growth and inhibiting microbial growth by mVOCs of Arctic yeasts would lay a foundation for potential applications in the future.

Robustness Testing of the xMAP Food Allergen Detection Assay: A Multiplex Assay for the Simultaneous Detection of Food Allergens

The xMAP food allergen detection assay (xMAP FADA) can simultaneously detect 15 analytes (14 food allergens plus gluten) in one analysis. The xMAP FADA typically employs two antibody bead sets per analyte, providing built-in confirmation that is not available with other antibody-based assays. Before an analytical method can be used, its reliability must be assessed when conditions of the assay procedure are altered. This study was conducted to determine the effects on assay performance associated with changes in incubation temperature, amounts of the antibody bead cocktail, and concentrations of detection antibody and β-mercaptoethanol in the reduced-denatured extraction buffer. The analysis of buffered-detergent extracts revealed lower responses at 22°C than at 37°C, but temperature had no effect on the analysis of reduced-denatured extracts. Changes in β-mercaptoethanol and detection antibody concentrations had an effect on the detection of only milk in the reduced-denatured extracts. A slight change in the measured bead count was observed when one-fourth of the bead cocktail was used, and a large decrease in the bead count was noted when one-eighth of the recommended amount was used, but this number (≥25) was still sufficient to provide reliable results. Overall, the xMAP FADA was very robust to changes in the assay procedure, which may inadvertently occur.

Progressive Myofibril Reorganization of Human Cardiomyocytes on a Dynamic Nanotopographic Substrate.

Cardiomyocyte (CM) alignment with striated myofibril organization is developed during early cardiac organogenesis. Previous work has successfully achieved in vitro CM alignment using a variety of biomaterial scaffolds and substrates with static topographic features. However, the cellular processes that occur during the response of CMs to dynamic surface topographic changes, which may provide a model of in vivo developmental progress of CM alignment within embryonic myocardium, remains poorly understood. To gain insights into these cellular processes involved in the response of CMs to dynamic topographic changes, we developed a dynamic topographic substrate that employs a shape memory polymer coated with polyelectrolyte multilayers to produce a flat-to-wrinkle surface transition when triggered by a change in incubation temperature. Using this system, we investigated cellular morphological alignment and intracellular myofibril reorganization in response to the dynamic wrinkle formation. Hence, we identified the progressive cellular processes of human-induced pluripotent stem cell-CMs in a time-dependent manner, which could provide a foundation for a mechanistic model of cardiac myofibril reorganization in response to extracellular microenvironment changes.

Limited Support for Thyroid Hormone or Corticosterone Related Gene Expression as a Proximate Mechanism of Incubation Temperature-Dependent Phenotypes in Birds.

The conditions that animals experience during early development can have profound consequences for health and fitness. In birds, one of the most important aspects of development is egg incubation temperature. A small decrease in average temperature leads to various impacts on offspring phenotype, such as smaller body sizes, slower growth rates, and less efficient metabolic activity. Little is known, however, about the proximate mechanisms underlying these incubation temperature-induced phenotypic changes. Two important hormones which could play a proximate role are thyroid hormone and corticosterone, which mobilize stored energy reserves and coordinate the normal growth of tissues, particularly in the brain. Previous research shows that circulating blood concentrations of both hormones are influenced by incubation temperature, but the mechanism by which incubation temperature may lead to these changes is unknown. We hypothesized that incubation temperature induces changes in thyroid hormone and corticosterone regulation, leading to changes in expression of hormone-sensitive genes in the brain. To test this, we incubated wood duck (Aix sponsa) eggs at three different temperatures within the natural range (35.0, 35.8, and 37.0°C). We measured mRNA expression of thyroid hormone-related neuroendocrine endpoints (deiodinase 2/3, thyroid hormone receptor α/β, neural regeneration related protein, and Krueppel-like factor 9) in newly hatched ducklings and corticosterone-related neuroendocrine endpoints (mineralocorticoid receptor, glucocorticoid receptor, cholecystokinin, and brain-derived neurotrophic factor) in 15 day-old ducklings using qPCR on brain tissue from the hippocampus and hypothalamus. Contrary to our predictions, we found that mRNA expression of thyroid hormone-related endpoints in both brain areas were largely unaffected by incubation temperature, although there was a trend for an inverse relationship between mRNA expression and incubation temperature for several genes in the hypothalamus. We also found that mineralocorticoid receptor mRNA expression in the hypothalamus was lower in ducklings incubated at the low relative to the high temperatures. This study is the first to evaluate the effects of incubation temperature on mRNA expression of neuroendocrine endpoints in the developing avian brain and suggests that these particular endpoints may be largely resistant to changes in incubation temperature. Thus, further research into the proximate mechanisms for incubation temperature-induced developmental plasticity is needed.

Impact of a controlled temperature gradient from 35.0°- 37.5°C on mouse embryo development and morphokinetics

To examine the impact of different in vitro culture temperatures on mouse embryo development and morphokinetic timings. Prospective study An embryo culture incubator with 6 individual chambers and time-lapse imaging (ESCO MIRI TLTM) was set to vary the temperature between chambers by 0.5°C to give a gradient that ranged from 35.0-37.5°C. Gas concentration was consistent between chambers (6% CO2, 5% O2, 89% N2). Frozen-thawed 1-cell mouse embryos (B6C3F1xB6D2F1, EmbryotechTM) were thawed, pooled and randomly distributed between 6 culture dishes in Global culture medium + 10% SPS. One dish containing individually cultured embryos was placed into each culture chamber/temperature for simultaneous culture and assessment. Embryos were cultured over 96h, recording blastocyst formation as well as morphokinetic timings for time to 2-cell, 3-cell, 4-cell, compaction and blastocoel formation. Experiments were repeated 3-times. Data were analyzed using ANOVA and Fisher Multiple-Comparison Test and are presented as the mean ± SEM. Statistically significance was measured at p<0.05. Changes in temperature during mouse embryo culture significantly impacted blastocyst formation and morphokinetic timings. Blastocyst formation rates were highest at 37.0°C and decreased at other temperatures examined. Cell division timings became more rapid as temperature increased and significantly differed between temperature treatments for most endpoints examined.Tabled 1Mouse morphokinetic timings and blastocyst development at different culture temperaturesTemperatureCell Division Timings (hrs)% Blastocystt-2cellt-3cellt-4cellt-Compactiont-Blastocyst35.0° C8.7±0.633.6±1.0a34.9±1.1a61.2±1.6a80.5±5.1a31.2%a35.5° C9.1±0.5a34.1±0.8a35.4±0.8a61.4±0.8a81.5±1.1a52.7%b36.0° C8.6±0.431.7±0.9ab33.4±0.7a57.9±1.3b77.5±1.6a55.5%b36.5° C7.7±0.430.3±0.7bc31.1±0.6b53.1±0.8c69.3±1.2b52.7%b37.0° C7.3±0.428.6±0.4c30.5±0.7bc53.5±1.3c70.7±3.0bc65.5%c37.5° C6.8±0.4b28.0±0.c28.6±0.7c49.2±0.9d62.1±0.6c56.7%bcDifferent superscripts within a column represent statistically significant differences between tempertaures p<0.05. Open table in a new tab Different superscripts within a column represent statistically significant differences between tempertaures p<0.05. New incubator technology offers the opportunity to examine the impact of temperature on embryo development while controlling for other culture variables. Changes in incubation temperature as little as 0.5°C have a significant impact on mouse embryo development, affecting blastocyst formation and morphokinetic timings. This data has potential implications for application of morphokinetic selection algorithms between facilities, as temperature between labs must be tightly controlled.

Isolation and characterization of pectolytic bacterial pathogens infecting potatoes in Nakuru County, Kenya.

Isolation and characterization of pectolytic bacteria associated with soft rot disease of potatoes in Nakuru, Kenya, to provide the basis for the development of disease control measures. Potato tubers showing symptoms of soft rot were collected from different farms in Molo and Mau Narok regions within Nakuru county. Isolation was done using crystal violet pectate medium (CVPM). Out of the 71 isolates that showed growth on CVPM, pathogenicity tests revealed that 36 of them had the ability to macerate tissues of potato tubers. All the isolates yielded a fragment of approximately 1500bp after 16S rDNA amplification. Using the BIOLOG microbial identification system, 20 bacterial isolates were identified as Pectobacterium carotovorum subsp. carotovorum, 7 were Pseudomonas fluorescens B while 9 were Ps. fluorescens A. Y1/Y2 primers successfully amplified pectate lyase-encoding (pel) gene, approximately 434bp, in all the 20 P. carotovorum species. The virulence of the isolated strains to cause disease, according to pectinolytic tests, varied with change in incubation temperature of the test samples. Pectobacterium carotovorum strains were the most virulent at 30°C while disease severity due to infection by Ps. fluorescens A strains was high at 20°C compared to the other isolates. This study reveals the identity of pectolytic bacterial species from two genera, Pectobacterium and Pseudomonas, as causative agents of potato soft rot in Nakuru, Kenya. Research findings from this study will aid in developing suitable risk mitigation methods for adoption by farmers to prevent losses due to soft rot.

Incubation temperature impacts nestling growth and survival in an open-cup nesting passerine.

For oviparous species such as birds, conditions experienced while in the egg can have long‐lasting effects on the individual. The impact of subtle changes in incubation temperature on nestling development, however, remains poorly understood, especially for open‐cup nesting species with altricial young. To investigate how incubation temperature affects nestling development and survival in such species, we artificially incubated American robin (Turdus migratorius) eggs at 36.1°C (“Low” treatment) and 37.8°C (“High” treatment). Chicks were fostered to same‐age nests upon hatching, and we measured mass, tarsus, and wing length of experimental nestlings and one randomly selected, naturally incubated (“Natural”), foster nest‐mate on days 7 and 10 posthatch. We found significant effects of incubation temperature on incubation duration, growth, and survival, in which experimentally incubated nestlings had shorter incubation periods (10.22, 11.50, and 11.95 days for High, Low, and Natural eggs, respectively), and nestlings from the Low treatment were smaller and had reduced survival compared to High and Natural nestlings. These results highlight the importance of incubation conditions during embryonic development for incubation duration, somatic development, and survival. Moreover, these findings indicate that differences in incubation temperature within the natural range of variation can have important carryover effects on growth and survival in species with altricial young.

Interdependence of Primary Metabolism and Xenobiotic Mitigation Characterizes the Proteome of Bjerkandera adusta during Wood Decomposition.

ABSTRACTThe aim of the current work was to identify key features of the fungal proteome involved in the active decay of beechwood blocks by the white rot fungus Bjerkandera adusta at 20°C and 24°C. A combination of protein and domain analyses ensured a high level of annotation, which revealed that while the variation in the proteins identified was high between replicates, there was a considerable degree of functional conservation between the two temperatures. Further analysis revealed differences in the pathways and processes employed by the fungus at the different temperatures, particularly in relation to nutrient acquisition and xenobiotic mitigation. Key features showing temperature-dependent variation in mechanisms for both lignocellulose decomposition and sugar utilization were found, alongside differences in the enzymes involved in mitigation against damage caused by toxic phenolic compounds and oxidative stress.IMPORTANCE This work was conducted using the wood decay fungus B. adusta, grown on solid wood blocks to closely mimic the natural environment, and gives greater insight into the proteome of an important environmental fungus during active decay. We show that a change in incubation temperature from 20°C to 24°C altered the protein profile. Proteomic studies in the field of white-rotting basidiomycetes have thus far been hampered by poor annotation of protein databases, with a large proportion of proteins simply with unknown function. This study was enhanced by extensive protein domain analysis, enabling a higher level of functional assignment and greater understanding of the proteome composition. This work revealed a strong interdependence of the primary process of nutrient acquisition and specialized metabolic processes for the detoxification of plant extractives and the phenolic breakdown products of lignocellulose.

Confocal laser scanning microscopy to estimate nanoparticles' human skin penetration in vitro.

ObjectiveWith rapid development of nanotechnology, there is increasing interest in nanoparticle (NP) application and its safety and efficacy on human skin. In this study, we utilized confocal laser scanning microscopy to estimate NP skin penetration.MethodsThree different-sized polystyrene NPs marked with red fluorescence were applied to human skin, and Calcium Green 5N was used as a counterstain. Dimethyl sulfoxide (DMSO) and ethanol were used as alternative vehicles for NPs. Tape stripping was utilized as a barrier-damaged skin model. Skin biopsies dosed with NPs were incubated at 4°C or 37°C for 24 hours and imaged using confocal laser scanning microscopy.ResultsNPs were localized in the stratum corneum (SC) and hair follicles without penetrating the epidermis/dermis. Barrier alteration with tape stripping and change in incubation temperature did not induce deeper penetration. DMSO enhanced NP SC penetration but ethanol did not.ConclusionExcept with DMSO vehicle, these hydrolyzed polystyrene NPs did not penetrate intact or barrier-damaged human “viable” epidermis. For further clinical relevance, in vivo human skin studies and more sensitive analytic chemical methodology are suggested.

Incubation Temperature, But Not Pequi Oil Supplementation, Affects Methane Production, and the Ruminal Microbiota in a Rumen Simulation Technique (Rusitec) System.

Lipid supplementation is a promising strategy for methane mitigation in cattle and has been evaluated using several different lipid sources. However, limited studies have assessed the effect of temperature on methane emissions from cattle and changes in incubation temperature have also not been extensively evaluated. The aim of this study was to evaluate the combined effect of pequi oil (high in unsaturated fatty acids) and incubation temperature on fermentation characteristics and microbial communities using the rumen simulation technique. A completely randomized experiment was conducted over a 28-day period using a Rusitec system. The experiment was divided into four periods of 7 days each, the first of which was a 7-day adaptation period followed by three experimental periods. The two treatments consisted of a control diet (no pequi oil inclusion) and a diet supplemented with pequi oil (1.5 mL/day) which increased the dietary fat content to 6% (dry matter, DM-basis). Three fermenter vessels (i.e., replicates) were allocated to each treatment. In the first experimental period, the incubation temperature was maintained at 39°C, decreased to 35°C in the second experimental period and then increased again to 39°C in the third. Pequi oil was continuously supplemented during the experiment. Microbial communities were assessed using high-throughput sequencing of the archaeal and bacterial 16S rRNA gene. Methane production was reduced by 57% following a 4°C decrease in incubation temperature. Supplementation with pequi oil increased the dietary fat content to 6% (DM-basis) but did not affect methane production. Analysis of the microbiota revealed that decreasing incubation temperature to 35°C affected the archaeal and bacterial diversity and richness of liquid-associated microbes, but lipid supplementation did not change microbial diversity.

PERANCANGAN SISTEM TELEMETRI SUHU PADA INKUBATOR BAYI

Pengukuran suhu pada system telemetri dimaksudkan untuk mendapatkan data suhu didalam inkubator secara lengkap, sehingga sejumlah data yang diperoleh akan dapat memberikan petunjuk terhadap kemungkinan terjadinya perubahan temperatur pada inkubator. Data tersebut selanjutnya diinformasikan kepada operator. Dengan cara sistem Telemetri pengontrolan suhu dapat dilakukan ditempat yang berbeda. Perangkat telematri terdiri dari hardware dan software, dimana perangkat terdapat dibagian pengirim dan penerima. Dibagian pengirim terdapat sensor suhu yang akan terintegrasi dengan mikrokontroler, kemudian dihubungkan ke PC. Pengontrolan suhu dilakukan dibagian penerima. Bagian penerima membutuhkan suatu transduser lain untuk mengubah sinyal elektronis tersebut kembali kebentuk asalnya, dan juga bagian penerima harus ada alat penguat sinyal berupa penguat Kata Kunci : Telemetri, Telemetri Digital, Transmisi data, Inkubator

Identification of Pathogenicity-Related Genes in Biofilm-Defective Acidovorax citrulli by Transposon Tn5 Mutagenesis.

Biofilm formation is important for virulence of a large number of plant pathogenic bacteria. Indeed, some virulence genes have been found to be involved in the formation of biofilm in bacterial fruit blotch pathogen Acidovorax citrulli. However, some virulent strains of A. citrulli were unable to format biofilm, indicating the complexity between biofilm formation and virulence. In this study, virulence-related genes were identified in the biofilm-defective strain A1 of A. citrulli by using Tn5 insertion, pathogenicity test, and high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR). Results from this study indicated that 22 out of the obtained 301 mutants significantly decreased the virulence of strain A1 compared to the wild-type. Furthermore, sequence analysis indicated that the obtained 22 mutants were due to the insertion of Tn5 into eight genes, including Aave 4244 (cation diffusion facilitator family transporter), Aave 4286 (hypothetical protein), Aave 4189 (alpha/beta hydrolase fold), Aave 1911 (IMP dehydrogenase/GMP reductase domain), Aave 4383 (bacterial export proteins, family 1), Aave 4256 (Hsp70 protein), Aave 0003 (histidine kinase, DNA gyrase B, and HSP90-like ATPase), and Aave 2428 (pyridoxal-phosphate dependent enzyme). Furthermore, the growth of mutant Aave 2428 was unaffected and even increased by the change in incubation temperature, NaCl concentration and the pH of the LB broth, indicating that this gene may be directly involved in the bacterial virulence. Overall, the determination of the eight pathogenicity-related genes in strain A1 will be helpful to elucidate the pathogenesis of biofilm-defective A. citrulli.

Evidence in duck for supporting alteration of incubation temperature may have influence on methylation of genomic DNA

Incubation temperature has an immediate and long-term influence on the embryonic development in birds. DNA methylation as an important environment-induced mechanism could serve as a potential link between embryos’ phenotypic variability and temperature variation, which reprogrammed by DNA (cytosine-5)-methyltransferases (DNMTS) and Methyl-CpG binding domain proteins (MBPS) 3&5 (MBD3&5). Five genes in DNMTS and MBPS gene families were selected as target genes, given their important role in epigenetic modification. In this study, we aimed to test whether raising incubation temperature from 37.8°C to 38.8°C between embryonic days (ED) 1–10, ED10–20 and ED20–27 have effect on DNA methylation and whether DNMTS, MBPS play roles in thermal epigenetic regulation of early development in duck. Real-time quantitative PCR analysis showed that increased incubation temperature by 1°C has remarkably dynamic effect on gene expression levels of DNMTS and MBPS. Slight changes in incubation temperature significantly increased mRNA levels of target genes in breast muscle tissue during ED1–10, especially for DNMT1, DNMT3A and MBD5. In addition, higher temperature significantly increased enzyme activities of DNMT1 in leg muscle during ED10–20, liver tissue during ED1–10, ED20–27 and DNMT3A in leg muscle and breast muscle tissue during ED10–20. These results suggest that incubation temperature has an extended effect on gene expression levels and enzyme activities of DNMTS and MBPS, which provides evidence that incubation temperature may influence DNA methylation in duck during early developmental stages. Our data indicated that DNMTS and MBPS may involved in thermal epigenetice regulation of embryos during the early development in duck. The potential links between embryonic temperature and epigenetic modification need further investigation

Isolating the effects of storm events on arctic aquatic bacteria: temperature, nutrients, and community composition as controls on bacterial productivity.

Storm events can pulse nutrients and carbon from soils and provide an important subsidy to food webs in oligotrophic streams and lakes. Bacterial nutrient limitation and the potential response of stream aquatic bacteria to storm events was investigated in arctic tundra environments by manipulating both water temperature and inorganic nutrient concentrations in short (up to 4 days) and long duration (up to 2 weeks) laboratory mesocosm experiments. Inorganic N and P additions increased bacterial production (14C-labeled leucine uptake) up to seven times over controls, and warmer incubation temperatures increased the speed of this response to added nutrients. Bacterial cell numbers also increased in response to temperature and nutrient additions with cell-specific carbon uptake initially increasing and then declining after 2 days. Bacterial community composition (BCC; determined by means of 16S denaturing gradient gel electrophoresis fingerprinting) shifted rapidly in response to changes in incubation temperature and the addition of nutrients, within 2 days in some cases. While the bacteria in these habitats responded to nutrient additions with rapid changes in productivity and community composition, water temperature controlled the speed of the metabolic response and affected the resultant change in bacterial community structure, constraining the potential responses to pulsed nutrient subsidies associated with storm events. In all cases, at higher nutrient levels and temperatures the effect of initial BCC on bacterial activity was muted, suggesting a consistent, robust interaction of temperature, and nutrients controlling activity in these aquatic systems.

Local variation in weather conditions influences incubation behavior and temperature in a passerine bird

Incubation is an important component of avian parental care and slight changes in incubation temperature can affect offspring phenotype. Although many extrinsic and intrinsic factors may generate variation in incubation temperature, they remain underexplored under natural conditions. Using a robust data set encompassing 55 nests, 22 816 behavioral observations, and &gt; 1 million paired ambient and egg temperatures, we describe the relationships among abiotic factors, female incubation behavior, incubation temperature, and incubation period for tree swallows Tachycineta bicolor. We report a large amount of individual variation in incubation behaviors and average incubation temperatures for our study population. The average on‐bout incubation temperature was 34.1°C, with daily egg temperatures ranging from 18.0–39.2°C. Females modulated the number of times they left the nest and the amount of time they stayed off the nest according to interactions between precipitation and temperature patterns. Models generated from our observations predicted that the number of female off‐bouts was the lowest under warm and dry conditions while more off‐bouts were taken under cold and dry or warm and wet conditions. During cold and dry conditions, females stayed off their nest ∼4 times longer than under warm and dry conditions. However, this pattern was reversed under periods of rainfall; females tended to take shorter off‐bouts when it was rainy and cold compared to longer off‐bouts during warmer rain events. Furthermore, variation in female behavior was associated with differences in overall incubation temperature such that females that maintained greater incubation constancy produced higher incubation temperatures at a given ambient temperature than those that displayed lower incubation constancy. Our results provide perspective on the timing of breeding, as some of the advantages of breeding early may be countered by cooler, early season temperatures and precipitation that cause reproducing females to favor self‐maintenance at a potential cost to optimal incubation temperatures for offspring development.

Embryonic critical windows: changes in incubation temperature alter survival, hatchling phenotype, and cost of development in lake whitefish (Coregonus clupeaformis).

The timing, success and energetics of fish embryonic development are strongly influenced by temperature. However, it is unclear if there are developmental periods, or critical windows, when oxygen use, survival and hatchling phenotypic characteristics are particularly influenced by changes in the thermal environment. Therefore, we examined the effects of constant incubation temperature and thermal shifts on survival, hatchling phenotype, and cost of development in lake whitefish (Coregonus clupeaformis) embryos. We incubated whitefish embryos at control temperatures of 2, 5, or 8 °C, and shifted embryos across these three temperatures at the end of gastrulation or organogenesis. We assessed hatch timing, mass at hatch, and yolk conversion efficiency (YCE). We determined cost of development, the amount of oxygen required to build a unit of mass, for the periods from fertilization-organogenesis, organogenesis-fin flutter, fin flutter-hatch, and for total development. An increase in incubation temperature decreased time to 50 % hatch (164 days at 2 °C, 104 days at 5 °C, and 63 days at 8 °C), survival decreased from 55 % at 2 °C, to 38 % at 5 °C, and 17 % at 8 °C, and hatchling yolk-free dry mass decreased from 1.27 mg at 2 °C to 0.61 mg at 8 °C. Thermal shifts altered time to 50 % hatch and hatchling yolk-free dry mass and revealed a critical window during gastrulation in which a temperature change reduced survival. YCE decreased and cost of development increased with increased incubation temperature, but embryos that hatched at 8 °C and were incubated at colder temperatures during fertilization-organogenesis had reduced cost. The relationship between cost of development and temperature was altered during fin flutter-hatch, indicating it may be a critical window during which temperature has the greatest impact on energetic processes. The increase in cost of development with an increase in temperature has not been documented in other fishes and suggests whitefish embryos are more energy efficient at colder temperatures.

The relationship between anammox and denitrification in the sediment of an inland river

This study measured the microbial processes of anaerobic ammonium oxidation (anammox) and denitrification in sediment sampled from two sites in the estuary of an inland river (Koisegawa River, Ibaragi prefecture, Japan) using a nitrogen isotope pairing technique (IPT). The responses of anammox and denitrification activities to temperature and nitrate concentration were also evaluated. Further, to elucidate the correlation between anammox and denitrification processes, an inhibition experiment was conducted, using chlorate to inhibit the first step of denitrification.Denitrification activity was much higher than anammox activity, and it reached a maximum at the surface layer in February 2012. Denitrification activity decreased as sediment depth increased, and a similar phenomenon was observed for anammox activity in the sediment of site A, where aquatic plants were absent from the surroundings. The activities of both denitrification and anammox were temperature-dependent, but they responded differently to changes in incubation temperature. Compared to a linear increase in denitrification as temperature rose to 35°C, the optimal temperature for anammox was 25°C, after which the activity decreased sharply.At the same time, both anammox and denitrification activities increased with NO3− concentration. The Michaelis–Menten kinetic constants (Vmax and Km) of denitrification were significantly higher than those of the anammox process. Furthermore, anammox activity decreased accordingly when the first step of denitrification was inhibited, which probably reduced the amount of the intermediate NO2−. Our study provides the first direct exploration of the denitrification-dependent correlation of anammox activity in the sediment of inland river.

Development and Characterization of an In Vitro Release Assay for Liposomal Ciprofloxacin for Inhalation

A novel method was developed and optimized to measure the in vitro release (IVR) of two liposomal ciprofloxacin formulations under development to treat lung infection. The release agent, bovine serum, has components that interact with liposomes to cause the encapsulated drug to be released. The precision and accuracy of the method were characterized. The method has a nearly linear release phase initially, which then approaches a plateau value after 2–4h. The robustness of the method was verified over a range of release agent and liposomal concentrations, and in response to changes in incubation temperature, buffer pH, and storage containers of serum. For this “sample and separate” IVR method, there is less than 2% release at the T = 0 time point, indicating negligible artifactual release before analysis. For both inhaled liposomal ciprofloxacin products, the plateau value represents 100% release of the encapsulated drug. The key elements of this IVR method may prove useful for characterization of other liposomal products as well. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:314–327, 2014

Incubation temperature influences locomotor performance in young wood ducks (Aix sponsa)

Incubation temperature is an important maternal effect in birds that can influence numerous offspring traits. For example, ducklings from eggs incubated at lower temperatures have lower growth rates, protein content, and are in poorer body condition than ducklings from eggs incubated at higher temperatures. Based on these observations, we predicted that incubation temperature would indirectly influence performance through its direct effects on body size. Wood duck (Aix sponsa) eggs were incubated at three ecologically relevant temperatures (35, 35.9, 37°C). After hatching, all ducklings were housed under identical conditions and were subjected to aquatic and terrestrial racing trials at 15 and 20 days posthatch (dph). Contrary to our prediction, incubation temperature did not influence most duckling body size parameters at 15 or 20 dph. However, incubation temperature did have a strong influence on locomotor performance independent of body size and body condition. Ducklings hatched from eggs incubated at the lowest temperature had significantly reduced maximum aquatic swim velocity than ducklings from higher temperatures. Maximum terrestrial sprint velocity followed a similar pattern, but did not differ statistically among incubation treatments. To our knowledge, this is the first study to demonstrate that slight changes in incubation temperature can directly affect locomotor performance in avian offspring and thus provide a significant source of phenotypic variation in natural wood duck populations.