Timing of increased temperature sensitivity coincides with nervous system development in winter moth embryos

Climate change is rapidly altering the environment and many species will need to genetically adapt their seasonal timing to keep up with these changes. Insect development rate is largely influenced by temperature, but we know little about the mechanisms underlying the temperature sensitivity of development. Here, we investigate seasonal timing of egg hatching in the winter moth, one of the few species which has been found to genetically adapt to climate change, likely through selection on temperature sensitivity of egg development rate. To study when during development winter moth embryos are most sensitive to changes in ambient temperature, we gave eggs an increase or decrease in temperature at different moments during their development. We measured their developmental progression and time of egg hatching, and used fluorescence microscopy to construct a timeline of embryonic development for the winter moth. We found that egg development rate responded more strongly to temperature once embryos were in the fully extended germband stage. This is the phylotypic stage at which all insect embryos have developed a rudimentary nervous system. Furthermore, at this stage, timing of ecdysone signaling determines developmental progression, which could act as an environment dependent gateway. Intriguingly, this may suggest that, from the phylotypic stage onward, insect embryos can start to integrate internal and environmental stimuli to actively regulate important developmental processes. As we found evidence that there is genetic variation for temperature sensitivity of egg development rate in our study population, such regulation could be a target of selection imposed by climate change

Phenological mismatch affects individual fitness and population growth in the winter moth

Climate change can severely impact species that depend on temporary resources by inducing phenological mismatches between consumer and resource seasonal timing. In the winter moth, warmer winters caused eggs to hatch before their food source, young oak leaves, became available. This phenological mismatch changed the selection on the temperature sensitivity of egg development rate. However, we know little about the fine-scale fitness consequences of phenological mismatch at the individual level and how this mismatch affects population dynamics in the winter moth. To determine the fitness consequences of mistimed egg hatching relative to timing of oak budburst, we quantified survival and pupation weight in a feeding experiment. We found that mismatch greatly increased mortality rates of freshly hatched caterpillars, as well as affecting caterpillar growth and development time. We then investigated whether these individual fitness consequences have population-level impacts by estimating the effect of phenological mismatch on population dynamics, using our long-term data (1994-2021) on relative winter moth population densities at four locations in The Netherlands. We found a significant effect of mismatch on population density with higher population growth rates in years with a smaller phenological mismatch. Our results indicate that climate change-induced phenological mismatch can incur severe individual fitness consequences that can impact population density in the wild.</p

Color of artificial light at night affects incubation behavior in the great tit, Parus major

Artificial light at night (ALAN) has been recognized as a biodiversity threat due to the drastic effects it can have on many organisms. In wild birds, artificial illumination alters many natural behaviors that are important for fitness, including chick provisioning. Although incubation is a key determinant of the early developmental environment, studies into the effects of ALAN on bird incubation behavior are lacking. We measured nest temperature in nest boxes of great tits during the incubation period in two consecutive years. Nest boxes were located in eight previously dark field sites that have been experimentally illuminated since 2012 with white, green, or red light, or were left dark. We tested if light treatment affected mean nest temperature, number of times birds leave the nest (off-bout frequency), and off-bout duration during the incubation period. Subsequently, we investigated if incubation behavior is related to fitness. We found that birds incubating in the white light during a cold, early spring had lower mean nest temperatures at the end of incubation, both during the day and during the night, compared to birds in the green light. Moreover, birds incubating in white light took fewer off-bouts, but off-bouts were on average longer. The opposite was true for birds breeding in the green light. Low incubation temperatures and few but long off-bouts can have severe consequences for developing embryos. In our study, eggs from birds that took on average few off-bouts needed more incubation days to hatch compared to eggs from birds that took many off-bouts. Nevertheless, we found no clear fitness effects of light treatment or incubation behavior on the number of hatchlings or hatchling weight. Our results add to the growing body of literature that shows that effects of ALAN can be subtle, can differ due to the spectral composition of light, and can be year-dependent. These subtle alterations of natural behaviors might not have severe fitness consequences in the short-term. However, in the long term they could add up, negatively affecting parent condition and survival as well as offspring recruitment, especially in urban environments where more environmental pollutants are present

No effect of artificial light of different colors on commuting Daubenton's bats (Myotis daubentonii) in a choice experiment

Progressive illumination at night poses an increasing threat to species worldwide. Light at night is particularly problematic for bats as most species are nocturnal and often cross relatively large distances when commuting between roosts and foraging grounds. Earlier studies have shown that illumination of linear structures in the landscape disturbs commuting bats, and that the response of bats to light may strongly depend on the light spectrum. Here, we studied the impact of white, green, and red light on commuting Daubenton's bats (Myotis daubentonii). We used a unique location where commuting bats cross a road by flying through two identical, parallel culverts underneath. We illuminated the culverts with white, red, and green light, with an intensity of 5 lux at the water surface. Bats had to choose between the two culverts, each with a different lighting condition every night. We presented all paired combinations of white, green, and red light and dark control in a factorial design. Contrary to our expectations, the number of bat passes through a culvert was unaffected by the presence of light. Furthermore, bats did not show any preference for light color. These results show that the response of commuting Daubenton's bats to different colors of light at night with a realistic intensity may be limited when passing through culverts.</p

Transcriptional regulation underlying the temperature response of embryonic development rate in the winter moth

Climate change will strongly affect the developmental timing of insects, as their development rate depends largely on ambient temperature. However, we know little about the genetic mechanisms underlying the temperature sensitivity of embryonic development in insects. We investigated embryonic development rate in the winter moth (Operophtera brumata), a species with egg dormancy which has been under selection due to climate change. We used RNA sequencing to investigate which genes are involved in the regulation of winter moth embryonic development rate in response to temperature. Over the course of development, we sampled eggs before and after an experimental change in ambient temperature, including two early development weeks when the temperature sensitivity of eggs is low and two late development weeks when temperature sensitivity is high. We found temperature-responsive genes that responded in a similar way across development, as well as genes with a temperature response specific to a particular development week. Moreover, we identified genes whose temperature effect size changed around the switch in temperature sensitivity of development rate. Interesting candidate genes for regulating the temperature sensitivity of egg development rate included genes involved in histone modification, hormonal signalling, nervous system development and circadian clock genes. The diverse sets of temperature-responsive genes we found here indicate that there are many potential targets of selection to change the temperature sensitivity of embryonic development rate. Identifying for which of these genes there is genetic variation in wild insect populations will give insight into their adaptive potential in the face of climate change

Artificial Light at Night Reduces Daily Energy Expenditure in Breeding Great Tits (Parus major)

The ecological impact of artificial light at night (ALAN) is an increasingly recognized process that accompanies expanding urbanization. Yet, we have limited knowledge on the impact of ALAN on wild species, and on the potential to mitigate any negative effects by using different light sources and colors. In birds, effects of ALAN on activity levels are reported for several species and, hence, their daily energy expenditure (DEE) may be affected. DEE is a potent mediator of life-history trade-offs and fitness and thus an important aspect to consider when examining the potential long-term ecological effects of ALAN. Previous work has suggested that birds exposed to ALAN show higher levelsof provisioning and nocturnal activity, suggesting that white ALAN increases DEE. Other factors regulating DEE, such as provisioning behavior and food availability, might also respond to ALAN and thus indirectly affect DEE. We tested the hypothesis that ALAN increases DEE using an experimental setup where four previously unlit transects were illuminated with either white, green, or red LED light, or left dark as a control treatment.This setup was replicated in eight locations across the Netherlands.Wemeasured DEE of our focal species, the great tit (Parusmajor), using a novel doubly labeled water technique that uses breath rather than blood samples. Contrary to our expectations, birds feeding their offspring under white and green ALAN showed lower DEE compared to birds in the control dark treatment. Differences in chick provisioning activity did not explain this result, as neither visit rates nor daily activity timing was affected by light treatment. However, food availability under white and green light was much higher compared to red light and the dark control. This difference strongly suggests that the lower DEE under white andgreen ALAN sites is a consequence of higher food availability in these treatments. This result shows that there can be positive, indirect effects of ALAN for breeding song birds which may balance against the negative direct effects shown in previous studies

Transcriptional regulation underlying the temperature response of embryonic development rate in the winter moth

Climate change will strongly affect the developmental timing of insects, as their development rate largely depends on ambient temperature. However, we know little about the genetic mechanisms underlying the temperature sensitivity of embryonic development in insects. We investigated embryonic development rate in the winter moth (Operophtera brumata), a species with egg dormancy that has been under selection due to climate change. We used RNAseq to investigate which genes are involved in the regulation of winter moth embryonic development rate in response to temperature. Over the course of development, we sampled eggs before and after an experimental change in ambient temperature, including two early development weeks when the temperature sensitivity of eggs is low and two late development weeks when temperature sensitivity is high. We found temperature-responsive genes that responded in a similar way across development, as well as genes with a temperature response specific to a particular development week. Moreover, we identified genes whose temperature effect size changed around the switch in temperature sensitivity of development rate. Interesting candidate genes for regulating the temperature sensitivity of egg development rate included genes involved in histone modification, hormonal signalling, nervous system development, and circadian clock genes. In conclusion, the diverse sets of temperature-responsive genes we found here indicate that there are many potential targets of selection to change the temperature sensitivity of embryonic development rate. Identifying for which of these genes there is genetic variation in wild insect populations will give insight into their adaptive potential in the face of climate change.,This dataset consists of all processed data and phenotypic data needed to reproduce the analysis of RNAseq data from winter moth embryos, published in Molecular Ecology under the same title. Data included here: the final transcriptome incl. functional annotation, GO annotation table, final gene counts matrix, and phenotypic data. The raw RNAseq reads can be found on the European Nucleotide Archive (ENA) under accession no. PRJEB55675. All the scripts needed to process the data and reproduce the analysis can be found on GitHub at https://github.com/NEvanDis/WM_RNAseq. Briefly: raw RNAseq reads were quality screened and processed according to the 'new Tuxedo' pipeline (see Pertea et al. 2016 Nature Protocols 11(9). This pipeline includes transcript assembly and quantification with StringTie, guided by the winter moth reference genome v1 with annotation v2. StringTie produces a transcriptome including only transcripts in the dataset with each transcript coordinated to the winter moth reference genome. We functionally annotated this transcriptome e.g. with BLAST, which included producing a GO annotation table. StringTie then uses this transcriptome to do transcript quantification at the gene level, of which the product is the final gene counts matrix deposited here. This final gene counts matric and the phenotypic data were used for statistical analysis and the production of the final figures in the Mol Ecol publication. Please see the scripts on GitHub for pipeline and analysis details.,File extensions of the deposited data files include .gff, .txt, and .csv. These are all text-based file formats that can be opened with any text processor

STING-Activating Adjuvants Elicit a Th17 Immune Response and Protect against Mycobacterium tuberculosis Infection

Summary: There are a limited number of adjuvants that elicit effective cell-based immunity required for protection against intracellular bacterial pathogens. Here, we report that STING-activating cyclic dinucleotides (CDNs) formulated in a protein subunit vaccine elicit long-lasting protective immunity to Mycobacterium tuberculosis in the mouse model. Subcutaneous administration of this vaccine provides equivalent protection to that of the live attenuated vaccine strain Bacille Calmette-Guérin (BCG). Protection is STING dependent but type I IFN independent and correlates with an increased frequency of a recently described subset of CXCR3-expressing T cells that localize to the lung parenchyma. Intranasal delivery results in superior protection compared with BCG, significantly boosts BCG-based immunity, and elicits both Th1 and Th17 immune responses, the latter of which correlates with enhanced protection. Thus, a CDN-adjuvanted protein subunit vaccine has the capability of eliciting a multi-faceted immune response that results in protection from infection by an intracellular pathogen. : Van Dis et al. demonstrate that STING-activating cyclic dinucleotides provide significant protection when used as adjuvants in a protein subunit vaccine against Mycobacterium tuberculosis and show that mucosal administration of this vaccine elicits a Th17 immune response that correlates with enhanced protection. Keywords: Mycobacterium tuberculosis, vaccine adjuvant, cyclic dinucleotides, Th1