Early developmental carry-over effects on exploratory behaviour and DNA methylation in wild great tits (Parus major).

Adverse, postnatal conditions experienced during development are known to induce lingering effects on morphology, behaviour, reproduction and survival. Despite the importance of early developmental stress for shaping the adult phenotype, it is largely unknown which molecular mechanisms allow for the induction and maintenance of such phenotypic effects once the early environmental conditions are released. Here we aimed to investigate whether lasting early developmental phenotypic changes are associated with post-developmental DNA methylation changes. We used a cross-foster and brood size experiment in great tit (Parus major) nestlings, which induced post-fledging effects on biometric measures and exploratory behaviour, a validated personality trait. We investigated whether these post-fledging effects are associated with DNA methylation levels of CpG sites in erythrocyte DNA. Individuals raised in enlarged broods caught up on their developmental delay after reaching independence and became more explorative as days since fledging passed, while the exploratory scores of individuals that were raised in reduced broods remained stable. Although we previously found that brood enlargement hardly affected the pre-fledging methylation levels, we found 420 CpG sites that were differentially methylated between fledged individuals that were raised in small versus large sized broods. A considerable number of the affected CpG sites were located in or near genes involved in metabolism, growth, behaviour and cognition. Since the biological functions of these genes line up with the observed post-fledging phenotypic effects of brood size, our results suggest that DNA methylation provides organisms the opportunity to modulate their condition once the environmental conditions allow it. In conclusion, this study shows that nutritional stress imposed by enlarged brood size during early development associates with variation in DNA methylation later in life. We propose that treatment-associated DNA methylation differences may arise in relation to pre- or post-fledging phenotypic changes, rather than that they are directly induced by the environment during early development.

How much multiple paternity should we expect? A study of birds and contrast with mammals.

Parentage analyses via molecular markers have revealed multiple paternity within the broods of polytocous species, reshaping our understanding of animal behavior, ecology, and evolution. In a meta-analysis of multiple paternity in bird and mammal species, we conducted a literature search and found 138 bird and 64 mammal populations with microsatellite DNA paternity results. Bird populations averaged 19.5% multiple paternity and mammals more than twice that level (46.1%). We used a Bayesian approach to construct a null model for how multiple paternity should behave at random among species, under the assumption that all mated males have equal likelihood of siring success, given mean brood size and mean number of sires. We compared the differences between the null model and the actual probabilities of multiple paternity. While a few bird populations fell close to the null model, most did not, averaging 34.0-percentage points below null model predictions; mammals had an average probability of multiple paternity 13.6-percentage points below the null model. Differences between bird and mammal species were also subjected to comparative phylogenetic analyses that generally confirmed our analyses that did not adjust for estimated historical relationships. Birds exhibited extremely low probabilities of multiple paternity, not only compared to mammals but also relative to other major animal taxa. The generally low probability of multiple paternity in birds might be produced by a variety of factors, including behaviors that reflect sexual selection (extreme mate guarding or unifocal female choice) and sperm competition (e.g., precedence effects favoring fertilization by early or late matings).

Early-life environmental effects on mitochondrial aerobic metabolism: a brood size manipulation in wild great tits.

In avian species, the number of chicks in the nest and subsequent sibling competition for food are major components of the offspring's early-life environment. A large brood size is known to affect chick growth, leading in some cases to long-lasting effects for the offspring, such as a decrease in size at fledgling and in survival after fledging. An important pathway underlying different growth patterns could be the variation in offspring mitochondrial metabolism through its central role in converting energy. Here, we performed a brood size manipulation in great tits (Parus major) to unravel its impact on offspring mitochondrial metabolism and reactive oxygen species (ROS) production in red blood cells. We investigated the effects of brood size on chick growth and survival, and tested for long-lasting effects on juvenile mitochondrial metabolism and phenotype. As expected, chicks raised in reduced broods had a higher body mass compared with enlarged and control groups. However, mitochondrial metabolism and ROS production were not significantly affected by the treatment at either chick or juvenile stages. Interestingly, chicks raised in very small broods were smaller in size and had higher mitochondrial metabolic rates. The nest of rearing had a significant effect on nestling mitochondrial metabolism. The contribution of the rearing environment in determining offspring mitochondrial metabolism emphasizes the plasticity of mitochondrial metabolism in relation to the nest environment. This study opens new avenues regarding the effect of postnatal environmental conditions in shaping offspring early-life mitochondrial metabolism.

Reproductive performance of Peregrine falcons relative to the use of organochlorine pesticides, 1946-2021.

Populations of some fish- and meat-eating birds suffered dramatic declines globally following the introduction of organochlorine pesticides during the late 1940s and 1950s. It has been hypothesised that these population declines during the 1950s-1970s were largely driven by a combination of reproductive failure due to eggshell-thinning, egg breakage and embryonic death attributable to DDT and its metabolites, and to enhanced mortality attributable to the more toxic cyclodiene compounds such as aldrin and dieldrin. Using 75 years (1946-2021) of Peregrine falcon (Falco peregrinus) monitoring data (315 unique nest-sites monitored for 6110 nest-years), we studied the breeding performance of a resident Peregrine population in southern Scotland relative to the spatiotemporal pattern of organochlorine pesticide use. We show that (i) Peregrine breeding success and measures of breeding performance increased substantially following the reduction in, and subsequently a complete ban on, the use of organochlorine pesticides; (ii) improvements in Peregrine breeding performance were more dramatic in southeastern Scotland where agriculture was the predominant land use than in southwestern Scotland where there was less arable and more forested land; (iii) Peregrines nesting closer to the coast generally had higher fledging success (that is, a higher proportion of clutches that produced at least one fledgeling) than those nesting inland farther away from the coast; (iv) low temperatures and excessive rain in May negatively affected Peregrine fledging success; and (v) Peregrine abundance increased in parallel with improvements in reproductive performance following the reduction and then complete ban on the use of organochlorine pesticides in the UK. However, recovery was gradual and occurred over four decades, and rate of recovery varied among measures of reproductive performance (egg, nestling and fledgeling production). Our results suggest that the temporal pattern of organochlorine pesticide use strongly influenced Peregrine reproductive parameters but that the pattern of influence differed regionally. Overall results are consistent with the hypothesis that reproductive failure caused by organochlorine pesticides was an important driver of the decline in the south Scottish Peregrine population, and that improvements in all measures of breeding performance following a reduction and eventual ban on organochlorine use facilitated the observed increase in this population.

Early life conditions influence fledging success and subsequent local recruitment rates in a declining migratory songbird, the Whinchat Saxicola rubetra.

Life history traits and environmental conditions influence reproductive success in animals, and consequences of these can influence subsequent survival and recruitment into breeding populations. Understanding influences on demographic rates is required to determine the causes of decline. Migratory species experience spatially and temporally variable conditions across their annual cycle, making identifying where the factors influencing demographic rates operate challenging. Here, we use the Whinchat Saxicola rubetra as a model declining long-distance migrant bird. We analyse 10 years of data from 247 nesting attempts and 2519 post-fledging observations of 1193 uniquely marked nestlings to examine the influence of life history traits, habitat characteristics and weather on survival of young from the nestling stage to local recruitment into the natal population. We detected potential silver spoon effects where conditions during the breeding stage influence subsequent apparent local recruitment rates, with higher recruitment for fledglings from larger broods, and recruitment rate negatively related to rainfall that chicks experienced in-nest. Additionally, extreme temperatures experienced pre- and post-fledging increased fledging success and recruitment rate. However, we could not determine whether this was driven by temperature influencing mortality during the post-fledging period or later in the annual cycle. Brood size declined with hatching date. In-nest survival increased with brood size and was highest at local temperature extremes. Furthermore, nest survival was highest at nests surrounded with 40%-60% vegetation cover of Bracken Pteridium aquilinum within 50 m of the nest. Our results show that breeding phenology and environmental factors may influence fledging success and recruitment in songbird populations, with conditions experienced during the nestling stage influencing local recruitment rates in Whinchats (i.e. silver spoon effect). Recruitment rates are key drivers of songbird population dynamics. Our results help identify some of the likely breeding season mechanisms that could be important population drivers.

Variation in oxidative status, but not structural and physiological development, associated with changing ontogenetic environments.

Despite the potential for temporally dependent relationships between trait values and fitness (e.g. as juveniles approach life-stage transitions such as fledging), how developmental stage affects canalization (a measure of robustness to environmental variation) of morphological and physiological traits is rarely considered. To test the sensitivity of morphological and physiological traits to environmental variation in two developmental stages, we manipulated brood size at hatch in European starlings (Sturnus vulgaris) and cross-fostered chicks between enlarged and reduced broods approaching fledging. We measured body size (mass, tarsus, wing length) and physiological state (aerobic capacity, oxidative status) at asymptotic mass on day 15, then cross-fostered chicks between 'high' and 'low' quality environments and assessed the same traits again on day 20, after 5 days of pre-fledging mass recession. Chicks in reduced broods were heavier at asymptotic mass and had lower reactive oxygen metabolites than enlarged broods, whereas structural size, aerobic capacity and antioxidant capacity were unaffected by experimental brood size. The observed canalization of structural and physiological traits during early development was maintained after cross-fostering, during late development. However, in contrast to early development, antioxidant capacity approaching fledging appeared sensitive to environmental conditions, as trajectories varied by cross-fostering treatment. Elevated reactive oxygen metabolites observed after early development in enlarged brood chicks were maintained after cross-fostering, suggesting that canalized development in low-quality environments could produce oxidative costs that carry over between life stages, even when conditions improve. These data reveal trait-specific relationships between environmental conditions and development, and highlight how natal environment effects may vary by developmental stage.

Environmental Drivers of Growth and Oxidative Status during Early Life in a Long-Lived Antarctic Seabird, the Adélie Penguin.

AbstractIn vertebrates, developmental conditions can have long-term effects on individual performance. It is increasingly recognized that oxidative stress could be one physiological mechanism connecting early-life experience to adult phenotype. Accordingly, markers of oxidative status could be useful for assessing the developmental constraints encountered by offspring. Although some studies have demonstrated that developmental constraints are associated with high levels of oxidative stress in offspring, it remains unclear how growth, parental behavior, and brood competition may altogether affect oxidative stress in long-lived species in the wild. Here, we investigated this question in a long-lived Antarctic bird species by testing the impact of brood competition (e.g., brood size and hatching order) on body mass and on two markers of oxidative damage in Adélie penguin chicks. We also examined the influence of parental effort (i.e., foraging trip duration) and parental body condition on chick body mass and oxidative damage. First, we found that brood competition and parental traits had significant impacts on chick body mass. Second, we found that chick age and, to a lesser extent, chick body mass were two strong determinants of the levels of oxidative damage in Adélie penguin chicks. Finally, and importantly, we also found that brood competition significantly increased the levels of one marker of oxidative damage and was associated with a lower survival probability. However, parental effort and parental condition were not significantly linked to chick levels of oxidative damage. Overall, our study demonstrates that sibling competition can generate an oxidative cost even for this long-lived Antarctic species with a limited brood size (maximum of two chicks).

Brood size in an uncertain world.

Reproduction in an uncertain world is fraught. The consequences of investing in too many offspring in a resource poor season can be disastrous but so too is missing the opportunity of a resource rich year. We consider a simple population and individual growth model and use Lyapunov exponents to find analytical results for the optimum brood size under stochastic environmental conditions. We show that if the environment shows dramatic changes between breeding seasons choosing a smaller brood size is more likely to be successful but the best strategy is to synchronize your reproduction to the food availability. Finally, we show that if the cost of having offspring is high it can be better to live in a highly varying world with a plastic strategy that synchronizes to the environment than to live in a deterministic world with a constant strategy, a finding with implications for invasive species and climate change.

Developmental stress does not induce genome-wide DNA methylation changes in wild great tit (Parus major) nestlings.

The environment experienced during early life is a crucial factor in the life of many organisms. This early life environment has been shown to have profound effects on morphology, physiology and fitness. However, the molecular mechanisms that mediate these effects are largely unknown, even though they are essential for our understanding of the processes that induce phenotypic variation in natural populations. DNA methylation is an epigenetic mechanism that has been suggested to explain such environmentally induced phenotypic changes early in life. To investigate whether DNA methylation changes are associated with experimentally induced early developmental effects, we cross-fostered great tit (Parus major) nestlings and manipulated their brood sizes in a natural study population. We assessed experimental brood size effects on pre-fledging biometry and behaviour. We linked this to genome-wide DNA methylation levels of CpG sites in erythrocyte DNA, using 122 individuals and an improved epiGBS2 laboratory protocol. Brood enlargement caused developmental stress and negatively affected nestling condition, predominantly during the second half of the breeding season, when conditions are harsher. Brood enlargement, however, affected nestling DNA methylation in only one CpG site and only if the hatch date was taken into account. In conclusion, this study shows that nutritional stress in enlarged broods does not associate with direct effects on genome-wide DNA methylation. Future studies should assess whether genome-wide DNA methylation variation may arise later in life as a consequence of phenotypic changes during early development.

No evidence for associations between brood size, gut microbiome diversity and survival in great tit (Parus major) nestlings.

BACKGROUND: The gut microbiome forms at an early stage, yet data on the environmental factors influencing the development of wild avian microbiomes is limited. As the gut microbiome is a vital part of organismal health, it is important to understand how it may connect to host performance. The early studies with wild gut microbiome have shown that the rearing environment may be of importance in gut microbiome formation, yet the results vary across taxa, and the effects of specific environmental factors have not been characterized. Here, wild great tit (Parus major) broods were manipulated to either reduce or enlarge the original brood soon after hatching. We investigated if brood size was associated with nestling bacterial gut microbiome, and whether gut microbiome diversity predicted survival. Fecal samples were collected at mid-nestling stage and sequenced with the 16S rRNA gene amplicon sequencing, and nestling growth and survival were measured. RESULTS: Gut microbiome diversity showed high variation between individuals, but this variation was not significantly explained by brood size or body mass. Additionally, we did not find a significant effect of brood size on body mass or gut microbiome composition. We also demonstrated that early handling had no impact on nestling performance or gut microbiome. Furthermore, we found no significant association between gut microbiome diversity and short-term (survival to fledging) or mid-term (apparent juvenile) survival. CONCLUSIONS: We found no clear association between early-life environment, offspring condition and gut microbiome. This suggests that brood size is not a significantly contributing factor to great tit nestling condition, and that other environmental and genetic factors may be more strongly linked to offspring condition and gut microbiome. Future studies should expand into other early-life environmental factors e.g., diet composition and quality, and parental influences.

Insulin-like growth factor 1 and the hormonal mediation of sibling rivalry.

In altricial animals, young are completely dependent on parents for provisioning. The ability to outcompete siblings to receive parental provisioning has clear fitness benefits, and may be mediated by hormones that influence growth. We analyzed the effects of insulin-like growth factor 1 (IGF-1) on body size, growth, and sibling rivalry in eastern bluebirds (Sialia sialis). To determine whether IGF-1 is upregulated in response to the competitive environment, we manipulated brood sizes and examined the effect on IGF-1 levels, nestling body size, growth rate, and behavior. In a separate experiment, we injected nestlings with exogenous IGF-1 to study its impacts on body size, growth rate, and sibling competition. Brood size manipulation did not influence endogenous IGF-1 levels, but male nestlings with higher IGF-1 levels early in the nestling period tended to have greater mass gain than males with lower IGF-1 levels. Nestlings with higher IGF-1 levels also tended to be fed more frequently by parents. In the injection experiment, IGF-1 injected individuals tended to be heavier than vehicle injected young by the end of the nestling period, which suggests that IGF-1 can influence mass gain in bluebirds. IGF-1 has been proposed to be a mediator of life-history strategies and post-hatching behavior. Our results suggest that although bluebird nestlings do not adaptively elevate IGF-1 in response to the presence or number of siblings, IGF-1 may influence growth during the nestling period. These findings shed light on sibling competition, life history strategies, and the hormones that underlie them.

Weather and nest cavity characteristics influence fecundity in mountain chickadees.

Background: Examining direct and indirect effects on reproduction at multiple scales allows for a broad understanding of species' resilience to environmental change. We examine how the fecundity of the mountain chickadee (Poecile gambeli), a secondary cavity-nesting, insectivorous bird, varied in relation to factors at three scales: regional weather conditions, regional- and site-level food availability, site-level community dynamics, and nest-level cavity characteristics. We hypothesized that earlier laying dates and higher fecundity (clutch size, nest survival, brood size) would be associated with milder climatic conditions, increased food from insect outbreaks, lower densities of conspecifics and nest predators (red squirrel; Tamiasciurus hudsonicus), and safer (smaller, higher) cavities. Methods: We collected data on laying date, clutch size, brood size, nest fate (success/failure), and cavity characteristics from 513 mountain chickadee nests in tree cavities in temperate mixed coniferous-broadleaf forest in interior British Columbia, Canada, from 2000 to 2011. We surveyed annual abundances of mountain chickadees and squirrels using repeated point counts, and mountain pine beetle (Dendroctonus ponderosae) and lepidopteran defoliators by monitoring host trees and by using regional-scale aerial overview forest insect survey data. We used weather data (temperature, rain, snow) from a local Environment and Climate Change Canada weather station. We modeled laying date, clutch size, daily nest survival, and brood size as a function of predictors at regional-, site-, and nest-scales. Results and Conclusions: Measures of fecundity varied dramatically across years and spatial scales. At the regional (study-wide) scale, chickadees laid earlier and larger first clutches in warmer springs with minimal storms, and daily nest survival (DSR) increased with a 2-year lag in growing season temperature. Despite a doubling of mountain chickadee density that roughly accompanied the outbreaks of mountain pine beetle and lepidopteran defoliators, we found little evidence at the site scale that fecundity was influenced by insect availability, conspecific density, or predator density. At the nest scale, DSR and brood size increased with clutch size but DSR declined with nest cavity size indicating a positive reproductive effect of small-bodied cavity excavators. Double-brooding, rare in chickadees, occurred frequently in 2005 and 2007, coinciding with early breeding, high food availability from insect outbreaks, and warm spring temperatures with 0-1 spring storms. Our results support the idea that fecundity in secondary cavity-nesting species is impacted directly and indirectly by weather, and indirectly through changes in community dynamics (via cavity resource supply). We stress the importance of adopting holistic, community-level study frameworks to refine our understanding of fecundity in opportunistic and climate-sensitive species in future.

Reproductive Span of Caenorhabditis Elegans is Extended by Microbacterium Sp.

The reproductive span (RS) of organisms could be affected by different factors during their lifetime. In the model nematode, Caenorhabditis elegans, RS is affected by both genetic and environmental factors. However, none of the factors identified so far were related to environmental bacteria, which may incidentally appear anywhere in the habitats of C. elegans. We aimed to find environmental bacteria that could affect the RS of C. elegans and related species. We tested 109 bacterial isolates and found that Microbacterium sp. CFBb37 increased the RS and lifespan of C. elegans but reduced its brood size. We studied the effect of M. sp. CFBb37 on the RS of Caenorhabditis briggsae, Caenorhabditis tropicalis, and another Rhabditidae family species, Protorhabditis sp., and found similar trends of RS extension in all three cases, suggesting that this bacterial species may induce the extension of RS broadly among Caenorhabditis species and possibly for many other Rhabditidae. This work will facilitate future research on the mechanism underlying the bacterial extension of RS of nematodes and possibly other animals.

Early social environment affects attention to social cues in juvenile common ravens, Corvus corax.

Social competence, i.e. defined as the ability to adjust the expression of social behaviour to the available social information, is known to be influenced by early-life conditions. Brood size might be one of the factors determining such early conditions, particularly in species with extended parental care. We here tested in ravens whether growing up in families of different sizes affects the chicks' responsiveness to social information. We experimentally manipulated the brood size of 13 captive raven families, creating either small or large families. Simulating dispersal, juveniles were separated from their parents and temporarily housed in one of two captive non-breeder groups. After five weeks of socialization, each raven was individually tested in a playback setting with food-associated calls from three social categories: sibling, familiar unrelated raven they were housed with, and unfamiliar unrelated raven from the other non-breeder aviary. We found that individuals reared in small families were more attentive than birds from large families, in particular towards the familiar unrelated peer. These results indicate that variation in family size during upbringing can affect how juvenile ravens value social information. Whether the observed attention patterns translate into behavioural preferences under daily life conditions remains to be tested in future studies.

Experimentally manipulated food availability affects offspring quality but not quantity in zebra finch meso-populations.

Food availability modulates survival, reproduction and thereby population size. In addition to direct effects, food availability has indirect effects through density of conspecifics and predators. We tested the prediction that food availability in isolation affects reproductive success by experimentally manipulating food availability continuously for 3 years in zebra finches (Taeniopygia guttata) housed in outdoor aviaries. To this end, we applied a technique that mimics natural variation in food availability: increasing the effort required per food reward without affecting diet. Lower food availability resulted in a slight delay of start of laying and fewer clutches per season, but did not affect clutch size or number of offspring reared per annum. However, increasing foraging costs substantially reduced offspring growth. Thus, food availability in isolation did not impact the quantity of offspring reared, at the expense of offspring quality. Growth declined strongly with brood size, and we interpret the lack of response with respect to offspring number as an adaptation to environments with low predictability, at the time of egg laying, of food availability during the period of peak food demand, typically weeks later. Manipulated natal brood size of the parents did not affect reproductive success. Individuals that were more successful reproducers were more likely to survive to the next breeding season, as frequently found in natural populations. We conclude that the causal mechanisms underlying associations between food availability and reproductive success in natural conditions may be more complex than usually assumed. Experiments in semi-natural meso-populations can contribute to further unravelling these mechanisms.

Using Caenorhabditis elegans as a Model for Mechanistic Insights of Craniofacial Development.

Caenorhabditis elegans has served as a powerful model for understanding the molecular and cell biology of clinically important human proteins due to the conservation of genes that are associated with human disorders. It is well established that evolution has conserved critical domains of proteins and their cellular functions even though the phenotypic output for analogous mutations can be distinct among organisms. To that end, the genes that are associated with human craniosynostosis such as TWIST1, TCF12, and FGFR2 have homologs in C. elegans hlh-8, hlh-2, and egl-15, respectively. Whereas mutations in these human genes lead to bone defects in the skull, mutations in the C. elegans genes lead to defects primarily in nonstriated muscles that are responsible for laying eggs and controlling defecation. Even though the phenotypes are distinct in nature, the ability to quantify them in C. elegans can give a sense of the severity to provide a genotype-phenotype correlation. With the advent of CRISPR/Cas-9 genome editing in C. elegans, it is possible to model specific patient mutations that affect conserved amino acids in C. elegans proteins. These mutant strains can then be evaluated for their phenotypes in both homozygous and heterozygous animals. The assays that can be used to measure these phenotypes are described in this chapter.

Natural variation in reproductive timing and X-chromosome nondisjunction in Caenorhabditis elegans.

Caenorhabditis elegans hermaphrodites first produce a limited number of sperm cells, before their germline switches to oogenesis. Production of progeny then ensues until sperm is depleted. Male production in the self-progeny of hermaphrodites occurs following X-chromosome nondisjunction during gametogenesis, and in the reference strain increases with age of the hermaphrodite parent. To enhance our understanding of the reproductive timecourse in C. elegans, we measured and compared progeny production and male proportion during the early and late reproductive periods of hermaphrodites for 96 wild C. elegans strains. We found that the two traits exhibited natural phenotypic variation with few outliers and a similar reproductive timing pattern as previous reports. Progeny number and male proportion were not correlated in the wild strains, implying that wild strains with a large brood size did not produce males at a higher rate. We also identified loci and candidate genetic variants significantly associated with male-production rate in the late and total reproductive periods. Our results provide an insight into life-history traits in wild C. elegans strains.

Variation in reproductive investment increases body temperature amplitude in a temperate passerine.

Many birds and mammals show substantial circadian variation in body temperature, which has been attributed to fluctuations in ambient temperature and energy reserves. However, to fully understand the variation in body temperature over the course of the day, we also need to consider effects of variation in work rate. We made use of a dataset on body temperature during the resting and active periods in female marsh tits (Poecile palustris) that bred in a temperate area and were subjected to experimental changes in reproductive investment through brood size manipulations. Furthermore, the amplitude increased with daytime, but were unaffected by nighttime, ambient temperature. Amplitudes in females with manipulated broods were 44% above predictions based on inter-specific allometric relationships. In extreme cases, amplitudes were > 100% above predicted values. However, no individual female realised the maximum potential amplitude (8.5 °C, i.e. the difference between the highest and lowest body temperature within the population) but seemed to prioritise either a reduction in body temperature at night or an increase in body temperature in the day. This suggests that body temperature amplitude might be constrained by costs that preclude extensive use of both low nighttime and high daytime body temperatures within the same individual. Amplitudes in the range found here (0.5-6.7 °C) have previously mostly been reported from sub-tropical and/or arid habitats. We show that comparable values can also be found amongst birds in relatively cool, temperate regions, partly due to a pronounced increase in body temperature during periods with high work rate.

Plastic adjustments of biparental care behavior across embryonic development under elevated temperature in a marine ectotherm.

Phenotypic plasticity in parental care investment allows organisms to promptly respond to rapid environmental changes by potentially benefiting offspring survival and thus parental fitness. To date, a knowledge gap exists on whether plasticity in parental care behaviors can mediate responses to climate change in marine ectotherms. Here, we assessed the plasticity of parental care investment under elevated temperatures in a gonochoric marine annelid with biparental care, Ophryotrocha labronica, and investigated its role in maintaining the reproductive success of this species in a warming ocean. We measured the time individuals spent carrying out parental care activities across three phases of embryonic development, as well as the hatching success of the offspring as a proxy for reproductive success, at control (24℃) and elevated (27℃) temperature conditions. Under elevated temperature, we observed: (a) a significant decrease in total parental care activity, underpinned by a decreased in male and simultaneous parental care activity, in the late stage of embryonic development; and (b) a reduction in hatching success that was however not significantly related to changes in parental care activity levels. These findings, along with the observed unaltered somatic growth of parents and decreased brood size, suggest that potential cost-benefit trade-offs between offspring survival (i.e., immediate fitness) and parents' somatic condition (i.e., longer-term fitness potential) may occur under ongoing ocean warming. Finally, our results suggest that plasticity in parental care behavior is a mechanism able to partially mitigate the negative effects of temperature-dependent impacts.

High-Throughput Measurement for Toxic Effects of Metal Mixtures in Caenorhabditis elegans.

The contamination of heavy metals, a class of naturally occurring and persistent toxicants, has become a major public health concern due to increasing industrial and anthropogenic activities. The use of COPAS Biosort, a flow cytometer capable of measuring thousands of nematodes in minutes via high-throughput assays, has been widely applied in C. elegans studies for assessing toxicity of individual metals; however, such application yet to be seen for metals or other chemical mixtures. In the present protocol, we investigated toxic effects of individual metals, Cd, Pb, and Mn, as well as their binary and ternary mixtures, using nematode C. elegans. The toxic outcomes, including effects on growth, reproduction, and feeding behavior, were measured using high-throughput platform analysis (COAPS Biosort).