Novel applications of thermocyclers for phenotyping invertebrate thermal responses

Abstract

1 High-throughput genomic methods are increasingly used to investigate invertebrate thermal responses with greater dimensionality and resolution than previously achieved. However, corresponding methods for characterizing invertebrate phenotypes are still lacking. To scale up the characterization of invertebrate thermal responses, we propose a novel use of thermocyclers as temperature-controlled incubators.2 Here, we tested the performance of thermocyclers as incubators and demonstrated the application of this method to efficiently characterize the thermal responses of model and non-model invertebrates.3 We found the thermocyclers performed with high precision, accuracy and resolution under various and fluctuating ambient conditions. We were able to successfully characterize the temperature-dependent development of grasshopper eggs (Warramaba virgo), as well as the effects of fluctuating temperature cycles on the survival of mosquito eggs (Aedes aegypti) and developmental success of Drosophila simulans larvae, all with similar survival rates to conventional methods. 4 Thermocyclers are a general and transferrable means to scale up current methods of incubating small invertebrates. They permit rapid characterization of high-dimensional physiological responses to natural thermal regimes. When combined with existing approaches in thermal and evolutionary biology, these methods will advance our understanding of, and ability to predict, biological adaptations and responses to environmental changes.

Loss of cytoplasmic incompatibility in Wolbachia-infected Aedes aegypti under field conditions

“…aegypti eggs and the density of Wolbachia under a range of temperature conditions. We followed methods described in Kong et al [45] with some modifications. Eggs from uninfected, w Mel, w AlbB and w MelPop colonies were collected on sandpaper strips which were then partially dried, wrapped in paper towel and held in sealed zip-lock bags.…”

“…Batches of 15 to 39 eggs (mean 25.7) were added to each tube. Tubes were closed and then tapped on the bench to ensure that eggs sank to the bottom of the tube where temperature control in the thermocycler is the most accurate [45]. Tubes were then placed in heat blocks of Biometra TProfessional TRIO 48 thermocyclers with tubes from each population arranged randomly in each block.…”

Summer egg diapause in a matchstick grasshopper synchronizes the life cycle and buffers thermal extremes

“…a result of poor handling techniques). Subsequent and ongoing work has dramatically improved this hatch rate and produced the same developmental responses (Kong et al 2016). The narrow developmental temperature range of approximately 10 °C of W. virgo contrasts with the wider range of temperatures reported for other grasshoppers (Acrididae) in both temperate and semi-arid regions, typically between 15 and 20 °C (Birch 1942;Hunter-Jones 1970;Wingerden et al 1991;Ando 1993;Gehrken & Doumbia 1996;Hao & Kang 2004;Fielding & Defoliart 2010).…”

Molecular Ecological Basis of Grasshopper (Oedaleus asiaticus) Phenotypic Plasticity under Environmental Selection

“…These findings coincide with previous studies that the environmental microstructures—including vegetation structure, fine scale distribution of food plants, and warm spots—can influence the behavior and spatial distribution of grasshopper (Bouaichi et al, 1996 ; Baythavong, 2011 ; Vendrami et al, 2017 ). Inasmuch as grasshoppers tend to be thermomaximizers (Kong et al, 2016 ), where shade limits solar-heating, thereby decreasing grasshopper body temperatures, this subsequently decreases metabolic rate as well, which is positively related to body temperature. Dense vegetation also produces high humidity, which can benefit grasshopper pathogens.…”