Molecular adjustment to a social niche: Brain transcriptomes reveal divergent influence of social environment on the two queen morphs of the ant Temnothorax rugatulus
Description
In this study, we experimentally disentangled the effect of queen morph and social organization by using a full-factorial design. We expected macrogynes to be well adapted to the situation of being the only queen in a colony, whereas for microgynes, this could be an unusual and potentially stressful social environment. Conversely, we predicted that macrogynes might respond to the lower level of care potentially provided in polygynous societies compared to microgynes, for whom polygyny is the typical social organization (Negroni et al., 2021). We focus here on the transcriptional activity in the brain, as the central nervous system is where information about the social environment is perceived, processed, and can lead to physiological or behavioral responses (Manfredini et al., 2022; Jernigan & Uy 2023). There is evidence that behavioral dynamics between workers and queens depend on queen morph, as trophallactic interactions with microgynes are much more frequent (Negroni et al., 2021). In addition to changes in the expression of behavioral genes, we were interested in whether the queens responded to their social environment by altering the expression of genes associated with stress, fertility, or lifespan functions. We also investigated the influence of queen form and behavioral task on brain transcriptome of workers to determine whether T. rugatulus workers from macrogynous colonies differ in brain activity and potentially behavior from those raised and living in microgynous societies.
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Ant collection, maintenance, and experimental manipulation T. rugatulus colonies were collected in rock crevices and under stones in oak-pine forests of the Chiricahua Mountains, Arizona, USA in August 2015. Collection permits for the Coronado National Forest were obtained through the Southwestern Research Station of the Museum of Natural History in Portal, Arizona. In the laboratory, ants were kept at 22°C with a 12:12 light: dark cycle and fed with crickets and honey twice weekly and provided with water ad libitum. Queens were classified into micro- or macrogyne based on the body size index (Rüppell et al., 1998; Choppin et al., 2021; Negroni et al., 2021) a measure that is closely linked to their dry weight. A total of 91 polygynous colonies (average colony size: 217.71 ± 169.33 workers), 44 with exclusively macrogyne queens, and 47 with only microgyne queens were selected for further experiments. To experimentally vary social structure, specifically queen number, we split 86 of the 91 colonies. Each experimental colony received 50 workers with a similar proportion of each behavioral type (18 nurses, 2 guards, 4 foragers, and 26 other in-nest workers for each experimental colony fragment), as well as 12 larvae, while all eggs were removed. Experimental colonies assigned to be monogynous contained one queen of the respective queen morph, while those assigned to be polygynous contained two queens of the respective queen morph. Overall, colonies remained under these experimental conditions for over four months, allowing queens and workers to adjust to the experimental conditions, i.e., colony size and queen number. During the course of the experiment, colony composition stayed consistent across treatments. Neither the number of workers nor queen number in the source colonies differed between the macrogyne and microgyne colonies (Mann-Whitney-U test: workers: z-value -0.15, p-value 0.88; queens: z-value 1.23, p-value 0.22), and the same holds true for colonies from the mono- and polygynous treatment (Mann-Whitney-U workers: z-value -0.46, p-value 0.65; queens: z-value -0.09, p-value 0.92). At the end of the experiment, the head of one queen from six colonies was decapitated and flash-frozen using liquid nitrogen and stored at -80°C until dissection. The same was done for one nurse and one forager for each of the polygynous colonies. RNA extraction, sequencing and gene expression analyses Brains were dissected into 50 µl of Trizol and RNA was extracted using the RNAeasy mini extraction kit (Qiagen) following the standard protocol. Samples were sent to Beijing Genomics Institute (BGI) Hongkong for sequencing on an Illumina HiSeq 4000, resulting in 20 to 100 million 100 bp long paired end reads per sample. The sequencing failed for one macro-polygynous queen.