Egg Water Research Articles

A Toolbox for Efficient Proximity-Dependent Biotinylation in Zebrafish Embryos

Understanding how proteins are organized in compartments is essential to elucidating their function. While proximity-dependent approaches such as BioID have enabled a massive increase in information about organelles, protein complexes, and other structures in cell culture, to date there have been only a few studies on living vertebrates. Here, we adapted proximity labeling for protein discovery in vivo in the vertebrate model organism, zebrafish. Using lamin A (LMNA) as bait and green fluorescent protein (GFP) as a negative control, we developed, optimized, and benchmarked in vivo TurboID and miniTurbo labeling in early zebrafish embryos. We developed both an mRNA injection protocol and a transgenic system in which transgene expression is controlled by a heat shock promoter. In both cases, biotin is provided directly in the egg water, and we demonstrate that 12 h of labeling are sufficient for biotinylation of prey proteins, which should permit time-resolved analysis of development. After statistical scoring, we found that the proximal partners of LMNA detected in each system were enriched for nuclear envelope and nuclear membrane proteins and included many orthologs of human proteins identified as proximity partners of lamin A in mammalian cell culture. The tools and protocols developed here will allow zebrafish researchers to complement genetic tools with powerful proteomics approaches.

Amino acid cues emanating from Pacific salmon eggs and ovarian fluid.

The eggs of salmonid fishes are an important food source for many aquatic predators that detect eggs using olfaction. Moreover, chemicals from eggs and ovarian fluid aid sperm cells in detecting and locating eggs for fertilization, and ovarian fluid is attractive to conspecific males. Thus chemicals from eggs and ovarian fluid may facilitate reproduction but may also attract egg predators. The authors sampled mature females of three Pacific salmon species - Chinook (Oncorhynchus tshawytscha), coho (Oncorhynchus kisutch) and sockeye (Oncorhynchus nerka) - and determined the proportional representation of amino acids, potent fish odorants, from their eggs and ovarian fluid (Chinook and coho salmon only). They then tested juvenile coho salmon, an egg predator, for responses to ovarian fluid and egg odours using the electro-olfactogram (EOG) recording technique. The amino acid compositions of the salmon species were significantly and positively correlated with each other, and the interspecific differences were comparable to those between individuals of the same species. The egg water samples were, on average, dominated by lysine, alanine and glutamine (12.6%, 12.4% and 10.9%, respectively). The ovarian fluid samples were dominated by lysine (20.5%), followed by threonine (9.7%), glycine (9.2%) and arginine (8.8%). EOG recordings demonstrated the ability of juvenile coho salmon to detect the chemical traces of eggs and ovarian fluid. It is concluded that salmon eggs are a potent source of odours for potential predators but likely not highly differentiated among salmon species.

Experimental analysis of basalt fiber reinforced egg shell/water hyacinth loaded epoxy composites

In this research work the basalt fiber have been considered as the prime reinforcement agent, the commonly available epoxy resin has been chosen as the matrix material based on the rule of mixture. In order to increase the physical strength of the basalt reinforced composites, as well, in the view of to fabricate the eco friendly fiber reinforced composite, the exact proportionate of egg shell and water hyacinth has been selected as the particulate matter upon the epoxy matrix. The chosen combinations are 0%, 3%, 6% of Egg shell and Water hyacinth powders separately, as well as the combinations of both the particulates have also been fabricated, to validate the hybrid as the superior than the individual particulate matter mixture. Moreover to validate the mechanical strength of the fabricated composites, the tests like tensile, flexural and impact have been conducted as per the ASTM standards. The results evidenced that the significant improvement upon loading of the particulates (egg shell/water hyacinth) in to the epoxy matrix in all tests, whereas the hybrid composites have shown the (combination of eggshell and water hyacinth) superior behavior as compared with the individual particulate loading into the matrix in all tests.

Role of formula and rice varieties on the characteristics of rice-based spring roll wrappers

The used formula in food processing could affect the product characteristics, including on rice-based spring roll wrappers. This research was aimed to find out an appropriate formula to get cohesive and firm mass by using four kinds of rice-based formulas on two different varieties of rice. Mentik Wangi and Ciherang varieties were used as the two preferred local rice varieties. The research used the randomized block design of nested experiment. The ingredients used for each formulation were rice flour and water (F1); rice flour, tapioca starch and water (F2); rice flour, egg white albumin and water (F3) and rice flour, tapioca starch, egg white albumin and water (F4). The weakness of rice flour is the difficulty to form cohesive mass and firm network because of its lack of gluten. The combination of egg white albumin and tapioca starch along with sufficient mechanical energy intended to carry out viscosity increasing and create an appropriate texture. The study indicated that best formula was F4 from both of Mentik Wangi and Ciherang varieties which produced cohesive and firm mass. There was no significant difference between the two rice varieties (P > 0.05). The characteristics of rice-based spring roll wrapper from Mentik Wangi and Ciherang varieties were; firmness: 0.5532±0.0051 N and 0.8131±0.0057 N; elongation at break: 15.98±0.47% and 16.28±0.29% and moisture retention capacity: 38.29±0.12% and 42.60±0.29% respectively.

Influence of restoration age on egg bank richness and composition: an ex situ experiment

AbstractZooplankton community assembly after flooding in temporary ponds depends mostly on abundance and diversity of the dormant propagule bank. However, our understanding of the hatching patterns of zooplankton is imperfect. We performed an ex situ experiment to study the species composition and temporal sequence of zooplankton emergence from sediments in three ponds that were restored at different times: 1998, 2003 and 2007. The patterns of species hatching observed in our microcosms were compared with the emergent zooplankton community of their equivalent, synchronically inundated ponds. An escalation in species richness and a delay in hatching time were detected with increasing restoration age. In addition, some discrepancies between the egg bank and active populations were observed, especially in the oldest pond. This study illustrates the use of hatching experiments in comparing ponds with different characteristics. Nevertheless, egg bank hatching and water column sampling should complement each other in order to afford a full reflection of past diversity and contribute to a better understanding of the efficiency of restoration practices.

Abstract 6128: Evaluation of therapeutic efficacy of combination of 4HPR and SAHA for inhibition of growth of human glioblastoma T98G xenografts in zebrafish

Abstract Glioblastoma is the most aggressive and deadly primary brain cancer in humans. Despite the current treatments using surgery, radiotherapy, and chemotherapy, glioblastoma reappears due to drug resistance. New approaches for treatment of glioblastoma suggest that combination of two drugs may overcome the drug resistance. In this study, we have evaluated the efficacy of the combination of N-(4-hydroxphenyl) retinamide (4HPR) and suberoylanilide hydroxamic acid (SAHA) for inhibition of human glioblastoma T98G cells in a zebrafish xenograft model in our laboratory. Both 4HPR and SAHA have been approved by the United States Food and Drug Administration for treatment of cancers in the clinics. The synthetic retinoid 4HPR works via retinoic acid receptor signaling pathways to inhibit cancer cell proliferation and induce apoptosis, while SAHA is a histone deacetylase (HDAC) inhibitor that targets class I, II, and IV HDACs for controlling growth of cancer cells. Although rodent models have provided valuable insights into the biology and pathology of glioblastoma, these models are frankly unsuitable and increasingly unaffordable for high-throughput screening and profiling of novel therapeutic agents for rapid translation to the clinics. The optical transparency of zebrafish embryos and larvae provides the unique opportunity to track tumors in a way that is impossible in rodent models. Small size of zebrafish embryos allows for study design in small plates, making them a more useful laboratory system than other cancer models. Zebrafish lack a fully functioning adaptive immune response until 4 days post-fertilization, allowing rejection-free xenografts of human tumors during this period. Recently, zebrafish xenograft models have emerged as a new powerful tool for studying different types of human tumors including glioblastoma and for evaluating therapeutic efficacy of different drugs alone and in combination. Organ systems and cell types are common in human and zebrafish. Tumors that occur in the same organs in human and zebrafish look and behave alike, often share common genetic makeups, and employ basic mechanisms for tumor formation, expansion, and spread. Zebrafish xenograft model of human glioblastoma offers us a major opportunity for discovering key pathways of this tumor growth and for evaluating efficacy of a novel combination therapy. We loaded exponentially grown T98G cells labeled with CellTrace™ Red into the glass needle (0.75 mm internal diameter without filament, World Precision Instruments, FL) at a density of about 1 × 106 cells/200 µl. Using a micro-injector, each anesthetized zebrafish larva was microinjected with about 100 T98G cells into the duct of Cuvier at 1.5 - 3.0 psi. After injection, larvae were transferred to fresh egg water for recovery during 1 h at 28 ± 0.5°C and then incubated at 33°C for the rest of the experiment. After 2 days of tumor growth, we treated zebrafish larvae with 4 µM 4HPR and 8 µM SAHA alone and in combination and incubated for 3 days. Results showed that 4HPR and SAHA alone inhibited growth of T98G xenografts in zebrafish, but the combination of the two drugs most significantly reduced T98G cell proliferation inside of the zebrafish. Our work suggested that the zebrafish xenograft model could be useful to identify the therapeutic efficacy of combination of drugs in the treatment of glioblastoma. Citation Format: Firas Khathayer, Katie L. Kathrein, Mitzi Nagarkatti, Swapan K. Ray. Evaluation of therapeutic efficacy of combination of 4HPR and SAHA for inhibition of growth of human glioblastoma T98G xenografts in zebrafish [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6128.

Egg size does not universally predict embryonic resources and hatchling size across annual killifish species

Egg size has a crucial impact on the reproductive success of a mother and the performance of her offspring. It is therefore reasonable to employ egg size as a proxy for egg content when studying variation in offspring performance. Here, we tested species differences in allometries of several egg content parameters with egg area. We measured individual eggs in five species of annual killifish (Cyprinodontiformes), a group of fish where egg banks permit population survival over dry season. Apart from comparing allometric scaling exponents, amounts and compositions of egg components across the different species, we assessed the explanatory power of egg area for egg wet and dry weight and for hatchling size. We found notable species-specific allometries between egg area and the other egg parameters (egg dry weight and water content, elemental composition and triglyceride content). Across species, egg area predicted egg wet weight with highest power. Within species, coefficients of determination were largest in A. elongatus, a large piscivorous species with large eggs. Our study shows that systematically using egg area as a proxy of egg content between different species can ignore relevant species-specific differences and mask within-species variability in egg content.

Oophila is monophyletic within a three-taxon eukaryotic microbiome in egg masses of the salamander Ambystoma maculatum

Tests of hypotheses of vertical transmission and competitive exclusion for particular resource-exchange mutualisms require assessments of the number and identity of partners. Unicellular green algae form a symbiosis with embryos of the northeastern yellow spotted salamander Ambystoma maculatum. The extent to which the composition of egg mass-associated taxa depends on temporal or spatial factors is unknown and the discovery of an endosymbiotic component to this symbiosis has increased the importance of knowing the specificity of the association. We assessed the diversity and relative abundance of algal and other unicellular eukaryotes in egg masses by subjecting DNA harvested from cells in egg capsules or water from breeding habitat to 18S amplicon sequencing. A nested sampling regime for two breeding seasons allowed comparisons among capsules within egg masses, among egg masses in ponds, between breeding locales, between pond water and capsule fluid, and between years. Based upon 4.33 X106 reads, we found no evidence inside fifty-eight egg capsules of algal lineages outside the Oophila clade and report that, whereas non-algal eukaryotic sequence diversity was low, cercozoan protists and chytridomycete fungi were sometimes abundant. There were weak or inconsistent egg mass, year or locale effects, but there was a very strong localization effect, indicating that capsules are rarefied spaces for eukaryotic aquatic taxa. This work provides a foundation for comparing microbial community structure within and among egg masses across the geographic range of the host.

EFFECT OF EGG STORAGE PERIOD ON THE IMMUNITY AND THE HATCHED DUCKLING'S PERFORMANCE

This study was conducted at the French Group Company at Al-Sadat City in Menoufia Governorate. The hundred forty-eight fertile eggs were collected from 200 females of Pekin ducks (Anas platyrhynchos) at the age of (26 weeks of production), which mated by artificial insemination with 30 male of Muscovy ducks (Cairina moschata) to produce Molar ducks. Eggs were divided into four equal treatments (each of 96 eggs) and subjected to different storage periods (0, 4, 8 and 12 days) to determine the effects of different storage periods on egg weight loss, hatchability, hatch window, duckling quality, duckling immunity, and its body weight were measured for the different treatments. The results revealed that there were insignificantly differences (p ≤ 0.05) in egg weight loss by the different preincubation egg storage period. Storage eggs for 0 days had the highest hatchability percentage (92.2 %) compared to other experimental groups. Data demonstrated that storage eggs for 12 days achieved the largest hatch window (17:30 hours) when compared with 0, 4 and 8 days of storage. The relationship between storage period and duckling quality revealed that storage eggs for 0 days achieved the highest quality compared to the other groups. Immunity also, was affected by storage period (0 days was the highest immunity) compared with the other groups. The effect of storage period on duckling body weight at age of one day was also studied. Data indicated that third group (storage periods 8 days) achieved significantly (P ≤ 0.01) highest body weight (76 g). It could be concluded that storage of fertile duck eggs should not exceed 8 days to avoid excessive loss of egg water that impair the albumen contents which needed by the developing embryo for growth during incubation. The storage also, affected the transfer of maternal immunity to the embryos and the hatched ducklings.

Communal egg‐laying behaviour and the consequences of egg aggregation in the brown anole (Anolis sagrei)

AbstractCommunal nesting is a behaviour that involves multiple females laying eggs in the same nest or nesting site. This behaviour may be a consequence of a shortage of preferable nest sites (constraint hypothesis) or an adaptation generated by fitness benefits associated with egg aggregation (adaptive hypothesis). Experimental tests of these hypotheses require information about maternal nest site choice and its fitness consequences. To address these, we studied a lizard (brown anole; Anolis sagrei) that produces single‐egg clutches, but often aggregates eggs in nest sites. In a lab study, females were given the option of nesting in (a) soil previously used as nest substrate vs. fresh soil and (b) soil with eggshells vs. without eggshells. We also experimentally examined the effects of egg aggregation by incubating eggs singly, in groups of four, and in groups of nine. We recorded egg surface temperature, water uptake, and hatchling morphology. Females were more likely to nest in pots with used soil and with eggshells than in pots with fresh soil or without eggshells. We observed no effects of egg aggregation on egg survival, egg temperature, or most measures of hatchling morphology. However, singly‐incubated eggs absorbed more water than eggs incubated in the four and nine egg aggregations and this resulted in offspring with greater body condition (i.e. heavier for their length) at hatching. The behavioural experiment demonstrates that females actively choose nest sites that have been used previously (as expected under the adaptive hypothesis), but the egg‐aggregation experiment shows no benefits to offspring based on the variables measured. Thus, results of the behaviour study support the adaptive hypothesis; however, results from our egg‐incubation study do not. Likely, the adaptive and constraint hypotheses are not mutually exclusive, and a diversity of factors influence the evolution of communal nesting behaviour.

Mitochondrial response and resilience to anthropogenic chemicals during embryonic development

Mitochondria are integral to maintaining cellular homeostasis. Optimum mitochondrial function is critical during embryonic development, as they play a key role in early signaling cascades and epigenetic programming, in addition to sustaining an adequate energy production. Mitochondria are sensitive targets of environmental toxins, potentially even at levels considered safe under current regulatory limits. Most mitochondrial analyses have focused only on chemical exposure effects in vitro or in isolated mitochondria. However, comparatively little is known about mitochondrial effects of chemical exposure during vertebrate embryogenesis, especially during the recovery phase following a chemical insult. Here, we used the zebrafish (Danio rerio), in a 96-well plate system, to examine mitochondrial effects of 24 chemicals including pharmaceuticals, industrial chemicals, and agrochemicals. We used oxygen consumption rate (OCR) during embryogenesis as a proxy for mitochondrial function. Embryonic OCR (eOCR) was measured in clean egg water immediately following 24 h of chemical exposure and subsequently for an additional 8 h. Each chemical, dependent upon the concentration, resulted in a unique eOCR response profile. While some eOCR effects were persistent or recoverable over time, some effects were only detected several hours after being removed from the exposure. Non-monotonic dose response effects as well as mitochondrial hormesis were also detected following exposure to some chemicals. Collectively, our study shows that mitochondrial response to chemicals are highly dynamic and warrant careful consideration when determining mitochondrial toxicity of a given chemical.

QUALITY AND MYCOBIOTA COMPOSITION OF STORED EGGS

The aim of this study was to monitor the quality and mycobiota composition of table eggs during storage period. The most significant changes in the egg weight and water activity were observed on Day 7. To identify the mycobiota present on the eggshell by PCR method, a newly designed procedure for the extraction of fungal DNA based on a combination of commercial isolation kit, proteinase K and ultrasound was implemented. Identified mold genera included Penicillium spp., Cladosporium spp., Fusarium spp. and Alternaria alternata group. Their ratio varied considerably during storage with the dominance of Penicillium spp. on Day 14.

Egg-induced changes to sperm phenotypes shape patterns of multivariate selection on ejaculates.

Sperm cells exhibit extraordinary phenotypic diversity and rapid rates of evolution, yet the adaptive value of most sperm traits remains equivocal. Recent findings suggest that to understand how selection targets ejaculates, we must recognize that female-imposed physiological conditions often alter sperm phenotypes. These phenotypic changes may influence the relationships among sperm traits and their association with fitness. Here, we show that chemical substances released by eggs (known to modify sperm physiology and behaviour) alter patterns of selection on a suite of sperm traits in the mussel Mytilus galloprovincialis. We use multivariate selection analyses to characterize linear and nonlinear selection acting on sperm traits in (a) seawater alone and (b) seawater containing egg-derived chemicals (egg water). Our analyses revealed that nonlinear selection on canonical axes of multiple traits (notably sperm velocity, sperm linearity and percentage of motile sperm) was the most important form of selection overall, but importantly these patterns were only evident when sperm phenotypes were measured in egg water. These findings reveal the subtle way that females can alter patterns of selection, with the implication that overlooking environmentally moderated changes to sperm, may result in erroneous interpretations of how selection targets phenotypic (co)variation in sperm traits.

Embryonic Exposure to 2,2',3,5',6-pentachlorobiphenyl (PCB-95) Causes Developmental Malformations in Zebrafish.

2,2',3,5',6-Pentachlorobiphenyl (PCB-95) is an environmental neurotoxicant. There is accumulated evidence that some neurotoxic effects of PCB-95 are caused by increased spontaneous Ca2+ oscillations in neurons resulting from modifying ryanodine receptors (RyR) in calcium-releasing channels. However, there are large gaps in explaining brain and other developmental malformations on embryonic PCB-95 exposure. In the present study, we address those deficiencies by studying the toxic effects of PCB-95 using zebrafish as an ontogenetic model. To characterize these effects, zebrafish embryos with intact chorions were exposed to 4 different concentrations of PCB-95 (0.25, 0.5, 0.75, and 1 ppm) for 3 consecutive days. The controls were maintained in 0.5 × E2 medium or egg water and in 0.1% (v/v) dimethyl sulfoxide (DMSO)/0.5 × E2 medium or egg water. PCB-95-treated groups showed dose-dependent decreases in survival and hatching rates, with increased rates of developmental malformations when compared to controls. These include morphological malformations, brain cell necrosis, and smaller eye sizes at 5 d post fertilization. These data suggest potential mechanisms underlying the abnormal behavior observed in a visual stimulus assay. The present study provides insight into PCB-95-induced developmental toxicity and supports the use of the zebrafish model in understanding the effects of PCB-95 exposure. Environ Toxicol Chem 2019;39:162-170. © 2019 SETAC.

Fluorometric determination of fipronil by integrating the advantages of molecularly imprinted silica and carbon quantum dots.

A fluorometric method is described for the determination of fipronil, a frequently-used insecticide. It exploits the blue fluorescence of carbon quantum dots (CQDs) and the selectivity of molecularly imprinted silica (MIS). The MIS was prepared via the sol-gel method by using fipronil as the template, 3-aminopropyltriethoxysilane as functional monomer, and tetraethoxysilane as cross-linker in the presence of CQDs. The blue fluorescence of the CQD@MIS, with excitation/emission peaks at 340/422nm, is quenched by fipronil. The assay works in the 0. 70 pM to 47μM fipronil concentration range, and the limit of detection is 19 pM. The method was successfully applied to the quantitation of fipronil in spiked eggs, milk, and tap water. Recoveries between 83.8 and 114.0% were achieved. The corresponding relative standard deviations (RSD) are less than 6.67%. Graphical abstractSchematic representation of a high sensitivite and selectivite fluorescence nanoprobe constructed by combining the excellent fluorescence property of carbon quantum dots and the predicted selectivity of molecularly imprinted silica. It was applied to analyze fipronil in egg, milk and tap water, respectively.

Water Footprint Assessment of Eggs in a Parent-Stock Layer Breeder Farm

Egg production and consumption of eggs in China account for about 40% of the global total, and this constitutes a significant demand for water resources. The shortage of water resources in China means egg production in this country has serious water challenges. However, there are few studies concerning water use in egg production. In this study, the water footprint network (WFN) methodology was applied to analyze the water footprint (WF) of intensive egg production using a typical parent-stock layer breeder farm in North China as a model, which raises 208,663 layer breeders and produces about 2791.39 t eggs per year. The feed and water consumption over 353 days were collected for analysis, and the water footprint of chicken eggs was estimated at the farm level. The water footprint of eggs in a parent-stock layer breeder farm in China was therefore assessed in detail for the first time, and suggestions are put forward to reduce the egg water footprint from the perspective of the production chain and improving water use efficiency on the farm. The results show that (1) the green WF of eggs (water volume/egg weight) ranged from 1.917 to 2.114 m3/kg, the blue WF was 0.584 to 0.644 m3/kg and the grey WF was 0.488 to 0.538 m3/kg; (2) the indirect WF generated by feed contributed over 99.8% of the total; (3) eggs laid by Hy-line Brown hens have a lower WF than those from Hy-line Sonia hens, and the studied layer breeder farm had a higher WF than the global average based on the literature. In this paper, the variation of the WF was also analyzed, and some advice on water management for layer farms in China is provided.

Improvement of Storage Stability of Meatballs Using Propolis

We investigated the food storage stability of propolis extracts using homemade meatballs. Propolis extracts were prepared by using the ethanol extraction method. After making the meatballs, propolis was mixed into the meatball materials and egg water with a final concentration of 1% v/v. Microbial cultivation results showed that the cultivation of Staphylococcus aureus, which is one of the food poisoning bacteria, was reduced by more than 90% and the period of storage was 1.5 times longer. Also, it was observed to be effective when propolis was added to meatballs, it was even more effective when propolis was mixed with egg water. Thus, propolis extracts may be a useful ingredient for food storage stability due to its anti-microbacterial function.

ASSESSMENT OF A MICROPLATE SYSTEM FOR MEASURING INDIVIDUAL REAL-TIME RESPIRATION IN SMALL MODEL ORGANISMS OF AGING

The ability to measure oxygen consumption rates of a living organism in real-time provides an indirect method of monitoring dynamic changes in metabolism reflecting organismal level mitochondrial function. In this study, we assessed the Loligo Systems microplate system for measuring individual respiration in small organisms. This included adult nematodes (Caenorhabditis elegans, N2), zebrafish embryos (Danio rerio, AB), and adult fruit flies (Drosophila melanogaster, w1118). Organisms were placed inside 80 µL glass chambers on a 24-well microplate atop a 24-channel optical fluorescence oxygen reading device. Adult nematodes and zebrafish embryos were in liquid culture, M9 buffer and egg water respectively, and the adult flies were in room air. The microplate and reader were placed inside an incubator for temperature control. A silicone gasket with a thin liner was used to seal the chambers. Reference standard oxygen consumption (respiration) of single and multiple adult nematodes (n=1–4 animals/well), zebrafish embryos (n=1–4 animals/well), and adult flies (n=1–2 animals/well) in the microplate system were achieved. Significant differences across numbers of animals/well and by sex were observed. Validation experiments of the oxygen consumption rates measured in C. elegans in parallel with Seahorse extracellular flux (XF) experiments are underway. The Loligo Systems microplate system offers a non-invasive, non-destructive method to measure real-time respiration in smaller organisms. These data provide preliminary evidence for utility of the system for a variety of biomedical applications that relate to organismal and mitochondrial function/dysfunction, including research in the basic biology of aging in these highly-utilized, pre-clinical, genetic model organisms.

Pipefish embryo oxygenation, survival, and development: egg size, male size, and temperature effects.

In animals with uniparental care, the quality of care provided by one sex can deeply impact the reproductive success of both sexes. Studying variation in parental care quality within a species and which factors may affect it can, therefore, shed important light on patterns of mate choice and other reproductive decisions observed in nature. Using Syngnathus typhle, a pipefish species with extensive uniparental male care, with embryos developing inside a brood pouch during a lengthy pregnancy, we assessed how egg size (which correlates positively with female size), male size, and water temperature affect brooding traits that relate to male care quality, all measured on day 18, approximately 1/3, of the brooding period. We found that larger males brooded eggs at lower densities, and their embryos were heavier than those of small males independent of initial egg size. However, large males had lower embryo survival relative to small males. We found no effect of egg size or of paternal size on within-pouch oxygen levels, but oxygen levels were significantly higher in the bottom than the middle section of the pouch. Males that brooded at higher temperatures had lower pouch oxygen levels presumably because of higher embryo developmental rates, as more developed embryos consume more oxygen. Together, our results suggest that small and large males follow distinct paternal strategies: large males positively affect embryo size whereas small males favor embryo survival. As females prefer large mates, offspring size at independence may be more important to female fitness than offspring survival during development.

Subtle temperature increase can interact with individual size and social context in shaping phenotypic traits of a coldwater fish.

Temperature and individual egg size have been long studied in the development of fishes because of their direct effects on individual fitness. Here we studied the combined effects of three important factors for fish development, i.e. egg size, social environment and water temperature. Arctic charr (Salvelinus alpinus), a coldwater fish known to be phenotypically plastic, was used to investigate how these factors may affect growth and foraging behaviour of juvenile fish in a benign environment. We accounted for the social environment during early development by comparing fish raised in groups and in isolation. We examined the effect of egg size and a 2 °C difference on foraging behaviour, activity and growth a few weeks after first feeding. Growth trajectories of fish originating from large and small eggs were similar within each temperature: larger fish coming large eggs were at all time larger than smaller fish. There was no indication that small fish raised at a higher temperature grew faster than larger fish raised at a lower temperature. A 2 °C difference in temperature affected the behaviour of fish differently according to body size and/or social context. The foraging probability difference between fish raised in groups and fish briefly isolated was higher at 4.5 °C than at 6.5 °C for both size fish. Finally, there was no repeatability in foraging behaviour and mobility for isolated individuals. These results highlight the importance of small changes in temperature when evaluating growth and behaviour of fishes, and reveal the importance of considering the interaction of temperature with other factors, e.g. individual size and social environment, especially at early stages of development in fishes. We discuss these findings in the context of rapid changes in temperature and how temperature and its interaction with other factors may affect the phenotypes, ecology and evolution of coldwater fishes.