Extensive geographical variation in testes size and ejaculate traits in a terrestrial-breeding frog.

Ejaculate traits vary extensively among individuals and species, but little is known about their variation among populations of the same species. Here, we investigated patterns of intraspecific variation in male reproductive investment in the terrestrial-breeding frog Pseudophryne guentheri. Like most anurans, breeding activity in P. guentheri is cued by precipitation, and therefore the timing and duration of breeding seasons differ among geographically separated populations, potentially leading to differences in the level of sperm competition. We, therefore, anticipated local adaptation in sperm traits that reflect these phenological differences among populations. Our analysis of six natural populations across a rainfall gradient revealed significant divergence in testes and ejaculate traits that correspond with annual rainfall and rainfall seasonality; males from the northern and drier edge of the species range had significantly smaller testes containing fewer, smaller and less motile sperm compared with those from mesic central populations. These findings may reflect spatial variation in the strength of postcopulatory sexual selection, likely driven by local patterns of precipitation.

Environmental Stress Increases the Magnitude of Nonadditive Genetic Variation in Offspring Fitness in the Frog Crinia georgiana.

When organisms encounter heterogeneous environments, selection may favor the ability of individuals to tailor their phenotypes to suit the prevailing conditions. Understanding the genetic basis of plastic responses is therefore vital for predicting whether susceptible populations can adapt and persist under new selection pressures. Here, we investigated whether there is potential for adaptive plasticity in development time in the quacking frog Crinia georgiana, a species experiencing a drying climate. Using a North Carolina II breeding design, we exposed 90 family groups to two water depth treatments (baseline and low water) late in larval development. We then estimated the contribution of additive and nonadditive sources of genetic variation to early offspring fitness under both environments. Our results revealed a marked decline in larval fitness under the stressful (low water) rearing environment but also that additive genetic variation was negligible for all traits. However, in most cases, we found significant sire-by-dam interactions, indicating the importance of nonadditive genetic variation for offspring fitness. Moreover, sire-by-dam interactions were modified by the treatment, indicating that patterns of nonadditive genetic variance depend on environmental context. For all traits, we found higher levels of nonadditive genetic variation (relative to total phenotypic variation) when larvae were reared under stressful conditions, suggesting that the fitness costs associated with incompatible parental crosses (e.g., homozygous deleterious recessive alleles) will only be expressed when water availability is low. Taken together, our results highlight the need to consider patterns of nonadditive genetic variation under contrasting selective regimes when considering the resilience of species to environmental change.

Differences in the timing of cardio-respiratory development determine whether marine gastropod embryos survive or die in hypoxia.

Physiological plasticity of early developmental stages is a key way by which organisms can survive and adapt to environmental change. We investigated developmental plasticity of aspects of the cardio-respiratory physiology of encapsulated embryos of a marine gastropod, Littorina obtusata, surviving exposure to moderate hypoxia (PO2 =8 kPa) and compared the development of these survivors with that of individuals that died before hatching. Individuals surviving hypoxia exhibited a slower rate of development and altered ontogeny of cardio-respiratory structure and function compared with normoxic controls (PO2 >20 kPa). The onset and development of the larval and adult hearts were delayed in chronological time in hypoxia, but both organs appeared earlier in developmental time and cardiac activity rates were greater. The velum, a transient, 'larval' organ thought to play a role in gas exchange, was larger in hypoxia but developed more slowly (in chronological time), and velar cilia-driven, rotational activity was lower. Despite these effects of hypoxia, 38% of individuals survived to hatching. Compared with those embryos that died during development, these surviving embryos had advanced expression of adult structures, i.e. a significantly earlier occurrence and greater activity of their adult heart and larger shells. In contrast, embryos that died retained larval cardio-respiratory features (the velum and larval heart) for longer in chronological time. Surviving embryos came from eggs with significantly higher albumen provisioning than those that died, suggesting an energetic component for advanced development of adult traits.

Fitness consequences of targeted gene flow to counter impacts of drying climates on terrestrial-breeding frogs.

Targeted gene flow (TGF) could bolster the adaptive potential of isolated populations threatened by climate change, but could also lead to outbreeding depression. Here, we explore these possibilities by creating mixed- and within-population crosses in a terrestrial-breeding frog species threatened by a drying climate. We reared embryos of the crawling frog (Pseudophryne guentheri) on wet and dry soils and quantified fitness-related traits upon hatching. TGF produced mixed outcomes in hybrids, which depended on crossing direction (origin of gametes from each sex). North-south crosses led to low embryonic survival if eggs were of a southern origin, and high malformation rates when eggs were from a northern population. Conversely, east-west crosses led to one instance of hybrid vigour, evident by increased fitness and desiccation tolerance of hybrid offspring relative to offspring produced from within-population crosses. These contrasting results highlight the need to experimentally evaluate the outcomes of TGF for focal species across generations prior to implementing management actions.

Combining motion analysis and microfluidics--a novel approach for detecting whole-animal responses to test substances.

Small, early life stages, such as zebrafish embryos are increasingly used to assess the biological effects of chemical compounds in vivo. However, behavioural screens of such organisms are challenging in terms of both data collection (culture techniques, drug delivery and imaging) and data evaluation (very large data sets), restricting the use of high throughput systems compared to in vitro assays. Here, we combine the use of a microfluidic flow-through culture system, or BioWell plate, with a novel motion analysis technique, (sparse optic flow - SOF) followed by spectral analysis (discrete Fourier transformation - DFT), as a first step towards automating data extraction and analysis for such screenings. Replicate zebrafish embryos housed in a BioWell plate within a custom-built imaging system were subject to a chemical exposure (1.5% ethanol). Embryo movement was videoed before (30 min), during (60 min) and after (60 min) exposure and SOF was then used to extract data on movement (angles of rotation and angular changes to the centre of mass of embryos). DFT was subsequently used to quantify the movement patterns exhibited during these periods and Multidimensional Scaling and ANOSIM were used to test for differences. Motion analysis revealed that zebrafish had significantly altered movements during both the second half of the alcohol exposure period and also the second half of the recovery period compared to their pre-treatment movements. Manual quantification of tail flicking revealed the same differences between exposure-periods as detected using the automated approach. However, the automated approach also incorporates other movements visible in the organism such as blood flow and heart beat, and has greater power to discern environmentally-driven changes in the behaviour and physiology of organisms. We suggest that combining these technologies could provide a highly efficient, high throughput assay, for assessing whole embryo responses to various drugs and chemicals.