Scoring thermal limits in small insects using open-source, computer-assisted motion detection.

Scoring thermal tolerance traits live or with recorded video can be time consuming and susceptible to observer bias, and as with many physiological measurements, there can be trade-offs between accuracy and throughput. Recent studies show that automated particle tracking is a viable alternative to manually scoring videos, although some of the software options are proprietary and costly. In this study, we present a novel strategy for automated scoring of thermal tolerance videos by inferring motor activity with motion detection using an open-source Python command line application called DIME (detector of insect motion endpoint). We apply our strategy to both dynamic and static thermal tolerance assays, and our results indicate that DIME can accurately measure thermal acclimation responses, generally agrees with visual estimates of thermal limits, and can significantly increase throughput over manual methods.

Increased pupal temperature has reversible effects on thermal performance and irreversible effects on immune system and fecundity in adult ladybirds.

The environmental conditions an organism encounters during development vary in their lasting impact on adult phenotypes. In the context of ongoing climate change, it is particularly relevant to understand how high developmental temperatures can impact adult traits, and whether these effects persist or diminish during adulthood. Here, we assessed the effects of pupal temperature (17 °C - normal temperature, 26 °C - increased temperature, or 35 °C - heat wave) on adult Harmonia axyridis thermal stress tolerance, immune function, starvation resistance, and fecundity. The temperature during pupation significantly affected all investigated traits in fresh adults. Heat acclimation decreased adult haemocyte concentration, cold tolerance, and total egg production, and had a positive effect on heat tolerance and starvation resistance. The negative effects of heat acclimation on cold tolerance diminished after seven days. In contrast, heat acclimation had a lasting positive effect on adult heat tolerance. Our results provide a broad assessment of the effects of developmental thermal acclimation on H. axyridis adult phenotypes. The relative plasticity of several adult traits after thermal acclimation may be consequential for the future geographic distribution and local performance of various insect species.

Vitellogenin expression corresponds with reproductive status and caste in a primitively eusocial bee.

Vitellogenin (vg) expression is consistently associated with variation in insect phenotypes, particularly egg-laying. Primitively eusocial species, such as eusocial sweat bees, have behaviourally totipotent castes, in which each female is capable of high levels of ovarian development. Few studies have investigated vg expression patterns in primitively eusocial insects, and only one study has focused on a primitively eusocial bee. Here we use a primitively eusocial sweat bee, Lasioglossum laevissimum, and Real Time quantitative PCR (RT-qPCR) to investigate the relationship between vg expression, castes, and variation in phenotypes associated with castes differences. These assays showed that females with high ovarian development had the highest levels of vg expression, and that vg expression levels reflected the reproductive status of females first and caste second. This is in contrast to vg expression patterns observed in advanced eusocial queens and workers, which differ in vg expression based on caste and have caste-specific vg expression patterns. Furthermore, future queens (gynes) do not have ovarian development and had similar vg expression levels to early spring foundresses, which do have ovarian development, supporting Vg's function as a transporter of lipids and amino acids before diapause.

Integrating GWAS and Transcriptomics to Identify the Molecular Underpinnings of Thermal Stress Responses in Drosophila melanogaster.

Thermal tolerance of an organism depends on both the ability to dynamically adjust to a thermal stress and preparatory developmental processes that enhance thermal resistance. However, the extent to which standing genetic variation in thermal tolerance alleles influence dynamic stress responses vs. preparatory processes is unknown. Here, using the model species Drosophila melanogaster, we used a combination of Genome Wide Association mapping (GWAS) and transcriptomic profiling to characterize whether genes associated with thermal tolerance are primarily involved in dynamic stress responses or preparatory processes that influence physiological condition at the time of thermal stress. To test our hypotheses, we measured the critical thermal minimum (CTmin) and critical thermal maximum (CTmax) of 100 lines of the Drosophila Genetic Reference Panel (DGRP) and used GWAS to identify loci that explain variation in thermal limits. We observed greater variation in lower thermal limits, with CTmin ranging from 1.81 to 8.60°C, while CTmax ranged from 38.74 to 40.64°C. We identified 151 and 99 distinct genes associated with CTmin and CTmax, respectively, and there was strong support that these genes are involved in both dynamic responses to thermal stress and preparatory processes that increase thermal resistance. Many of the genes identified by GWAS were involved in the direct transcriptional response to thermal stress (72/151 for cold; 59/99 for heat), and overall GWAS candidates were more likely to be differentially expressed than other genes. Further, several GWAS candidates were regulatory genes that may participate in the regulation of stress responses, and gene ontologies related to development and morphogenesis were enriched, suggesting many of these genes influence thermal tolerance through effects on development and physiological status. Overall, our results suggest that thermal tolerance alleles can influence both dynamic plastic responses to thermal stress and preparatory processes that improve thermal resistance. These results also have utility for directly comparing GWAS and transcriptomic approaches for identifying candidate genes associated with thermal tolerance.

High-Throughput Assays of Critical Thermal Limits in Insects.

Upper and lower thermal limits of plants and animals are important predictors of their performance, survival, and geographic distributions, and are essential for predicting responses to climate change. This work describes two high-throughput protocols for measuring insect thermal limits: one for assessing critical thermal minima (CTmin), and the other for assessing heat knock down time (KDT) in response to a static heat stressor. In the CTmin assay, individuals are placed in an acrylic-jacketed column, subjected to a decreasing temperature ramp, and counted as they fall from their perches using an infrared sensor. In the heat KDT assay, individuals are contained in a 96 well plate, placed in an incubator set to a stressful, hot temperature, and video recorded to determine the time at which they can no longer remain upright and move. These protocols offer advantages over commonly used techniques. Both assays are low cost and can be completed relatively quickly (~2 h). The CTmin assay reduces experimenter error and can measure a large number of individuals at once. The heat KDT protocol generates a video record of each assay and thus removes experimenter bias and the need to continuously monitor individuals in real time.

How flies respond to honey bee pheromone: the role of the foraging gene on reproductive response to queen mandibular pheromone.

In this study we test one central prediction from sociogenomic theory--that social and non-social taxa share common genetic toolkits that regulate reproduction in response to environmental cues. We exposed Drosophila females of rover (for(R)) and sitter (for(s)) genotypes to an ovary-suppressing pheromone derived from the honeybee Apis mellifera. Surprisingly, queen mandibular pheromone (QMP) affected several measures of fitness in flies, and in a manner comparable to the pheromone's normal effect on bee workers. QMP-treated sitter flies had smaller ovaries that contained fewer eggs than did untreated controls. QMP-treated rover flies, by contrast, showed a more variable pattern that only sometimes resulted in ovary inhibition, while a third strain of fly that contains a sitter mutant allele in a rover background (for(s2)) showed no ovarian response to QMP. Taken together, our results suggest that distinctly non-social insects have some capacity to respond to social cues, but that this response varies with fly genotype. In general, the interspecific response is consistent with a conserved gene set affecting reproductive physiology. The differential response among strains in particular suggests that for is itself important for modulating the fly's pheromonal response.