One of the major goals of evolutionary biology is to link phenotypic variation with specific genetic variation, yet for behavioral phenotypes in non-model species, this task remains daunting and generally elusive. Although behaviors are heritable and clearly acted upon by evolutionary forces, they are generally polygenic, flexibly expressed, and context-dependent. Two recent papers, however, accomplished this very thing, in white-throated sparrows (Zonotrichia albicolis; Merritt et al. 2020) and in a species of jumping spider from southeastern Asia (Portia labiata; Chang et al. 2020)!
Bottom left: White-striped and tan-striped morphs of the white-throated sparrow, Photo Credit Jennifer Merritt
Bottom right: White-mustached Portia Jumping Spider, Photo Credit Richard Ong on Project Noah
White-throated sparrows are an excellent model for investigating genotype-phenotype links because a chromosomal re-arrangement on chromosome two has created a supergene, or a group of genes that are inherited together and regulate a system of discrete phenotypes. White-throated sparrows with this supergene exhibit a white-striped plumage morph and are more aggressive than those without the supergene, also known as the tan-striped plumage morph (see this link for more information).
https://www.nature.com/news/the-sparrow-with-four-sexes-1.21018
This supergene captures ~1000 genes which are approximately 1% diverged from the gene sequences on non-rearranged version chromosome (Sun et al. 2018). Previous work by this lab group has correlated the expression of estrogen receptor alpha (ESR1) in areas of the brain related to social behavior like TnA (e.g., the bird versions of the medial amygdala) with aggression and parenting behavior in both males and females of each morph, but this work was only correlative (Horton et al. 2014). In this recent paper by Jennifer Merritt and colleagues (including yours truly; Merritt et al. 2020), the authors knocked down the expression of ESR1 mRNA in the brains of wild-caught sparrows and showed that decreasing the amount of ESR1 mRNA resulted in decreased aggression by the aggressive white-striped morph!
Figure 2 from Merritt et al. 2020
ESR1 expression mediates the morph difference in aggression. (A) ESR1 expression was reduced by ESR1 knockdown (ESR1-KD) in TnA. (B) Depiction of the set-up for behavioral testing, in which birds with and without ESR1 knockdown were dosed with either estrogen (E2) to stimulate ESR1 or with a control (CON) vehicle. (C–H) The y-axes depict the changes in behavior between the CON and E2 trials. ESR1 knockdown significantly reduced the degree to which E2 increased the number of attacks (C) and the time spent near the opponent (in the light blue area in B) in WS birds only (F). Both behaviors were correlated with the level of ESR1 expression (D and G) but not with the level of ESR2 expression (E and H) (n = 20). Residuals from a partial correlation controlling for morph are plotted in D, E, G, and H. All birds were laboratory-housed.
Then the authors explored the possible mechanisms that might underpin the differences in ESR1 expression between the two morphs. The supergene and wildtype alleles are significantly differentially expressed within the brains of the white-striped morph (Figure 3) and the amount of allelic imbalance in TnA correlates with the level of aggression exhibited by adult birds. This imbalance might be mediated by a number of different mechanisms, including differences in transcription factor binding at the promoters and differences in CpG methylation of ESR1.
Allelic imbalance in ESR1 expression. We quantified allelic imbalance in three brain regions in heterozygous (WS) adults (A, D, and G) and nestlings (C, F, and I) sampled from a free-living population (adults, n = 15 to 18; nestlings, n = 26 to 27). In the bar graphs, each column represents the relative expression of ZAL2 (blue) and ZAL2m (red) in a single bird. The dashed line represents a null ratio of 0.5. Behavioral responses of adult males to STI (Methods) were not predicted by the degree of allelic imbalance in HYP (B) or POM (E); however, they were significantly correlated with allelic imbalance in TnA (H) (n = 10)
Chang and colleagues examined gene expression differences in the brains of aggressive and docile jumping spiders to identify 58 genes that were differentially expressed – from these 58, they chose several target genes for further study. Of these, differential expression of serotonin receptor 1A (5-HT1A) and BTB/POZ domain containing protein 17 (BTBDH) explained nearly 10% of the variation in aggressive behavior, with expression of both genes being significantly higher in docile spiders compared to aggressive spiders. BTBDH is potentially responsible for immune response to viruses and the authors actually found differences between aggression phenotypes in viral loads of three different viral RNAs; Xinzhou spider virus RNAs were more abundant in docile females, while both the Duwamo virus RNA and Hubei picorna-like virus 69 RNA were more abundant in aggressive females (Figure 2). Several possible explanations exist for this finding – viral infection could cause changes in the aggression levels and BTBDH expression of the spiders. Or spiders with differing levels of BTBDH expression and aggression vary in their probability of infection.
Female spider aggression varying with viral loads and associated with the putative BTB/POZ domain-containing protein 17 (BTBDH) gene. (a) Significant differences in virus RNAs (Hubei picorna-like virus 69, Duwamo virus, Xinzhou Spider virus) detected by RNA-seq between aggressive and docile spiders. FPKM stands for fragments per kilobase of transcript per million mapped reads (average ± SE). (b) RNA-seq showed differential expression of the putative BTBDH gene between aggressive and docile spiders. The gene expression (FPKM) in aggressive spiders was zero.
Because the authors also found that differences in 5-HT1A expression, involved in the serotonin signaling pathway, were associated with differences in aggression, the authors administered serotonin and several serotonin antagonists and measured aggressive behavior. Serotonin significantly reduced aggression 3 hours after administration, as did high doses of the serotonin antagonist, methiothepin (Figure 3).
Putative serotonin receptor (5-HT1A) gene associated with female aggressive behaviour. (a) RNA-seq showed differential expression of the putative 5-HT1A gene between aggressive and docile spiders (FPKM ranging > 2 to = 5 is omitted, the values more than 5 are 5.71 and 6.95, respectively). (b) Changes in aggressive behavior (distance to the mirror) between before and after treatments (1 hr, 3 hr and 24 hr after the treatment). The spider with shorter distance to the mirror was considered more aggressive. Negative values indicate spiders become more aggressive (pink) after the treatment, while positive values indicate spiders become less aggressive (blue) after the treatment. DMSO represents the DMSO solvent control. Changes in distance to the mirror were compared between each treatment and the DMSO control for each time point. ns = nonsignificant difference between the treatment and the DMSO control. Ketanserin is a selective serotonin receptor antagonist, and methiothepin is a nonselective serotonin receptor antagonist. High = high dosage and low = low dosage.
Unlike the white-throated sparrow, the white-mustached jumping spider does not have a supergene that has been accumulating nucleotide divergences; however, the authors used targeted genotyping strategies to find one SNP within BTBDH and two SNPs within 5-HTR1A that are associated with aggression (Figure 2c & 3c).
The serotonin and steroid hormone signaling pathways have long been known to play an important role in the regulation of behavior, particularly aggression. Similarly, a classic hypothesis in evolutionary biology revolves around the trade-off between immune function and survival in the form of aggressive behavior and reproduction. Although aggression is a complex behavior, these studies provide a blueprint for linking genotype to phenotype when studying the evolution of animal behavior.
References
Chang, C., Connahs, H., Tan, E. C. Y., Norma-Rashid, Y., Mrinalina, Li, Daiqin L., Chew, F. T., (2020). Female spider aggression is associated with genetic underpinnings of the nervous system and immune response to pathogens. Molecular Ecology, 29, 2626-2638.
Horton, B. M., Hudson, W. H., Ortlund, E. A., Shirk, S., Thomas, J. W., Young, E. R., Zinzow-Kramer, W. M., and Maney, D. L. (2014). Estrogen receptor α polymorphism in a species with alternative behavioral phenotypes. Proceedings of the National Academy of Sciences, 111, 1443-1448.
Merritt, J. R., Grogan, K. E., Zinzow-Kramer, W. M., Sun, D., Ortlund, E. A., Yi, S. V., and Maney, D. L. (2020). A supergene-linked estrogen receptor drives alternative phenotypes in a polymorphic songbird. Proceedings of the National Academy of Sciences, 117 (35), 21673-2168.
Sun, D., Huh, I., Zinzow-Kramer, W. M., Maney, D. L., and Yi, S. V. (2018). Rapid regulatory evolution of a non-recombining autosome linked to divergent behavioral phenotypes. Proceedings of the National Academy of Sciences, 115, 2794-2799.