Antioxidant Gene Expression in Vocal Hindbrain of a Teleost Fish

Abstract

Plainfin midshipman fish (Porichthys notatus) have a remarkable capacity to generate long duration advertisement calls known as hums, each of which may last for close to two hours and be repeated throughout a night of courtship activity during the breeding season. The midshipmans striking sound production capabilities provide a unique opportunity to investigate the mechanisms that motor neurons require for withstanding high-endurance activity. The temporal properties of midshipman vocal behaviors are largely controlled by a hindbrain central pattern generator that includes vocal motor neurons (VMN) that directly determine the activity pattern of target sonic muscles and, in turn, a sounds pulse repetition rate, duration and pattern of amplitude modulation. Of the two adult midshipman male reproductive phenotypes -- types I and II-- only type I males acoustically court females with hums from nests that they build and guard, while type II males do not produce courtship hums but instead sneak or satellite spawn to steal fertilizations from type I males. A prior study using next generation RNA sequencing showed increased expression of a number of cellular respiration and antioxidant genes in the VMN of type I males during the breeding season, suggesting they help to combat potentially high levels of oxidative stress linked to this extreme behavior. This led to the question of whether the expression of these genes in the VMN would vary between actively humming versus non-humming states as well as between male morphs. Here, we tested the hypothesis that to combat oxidative stress, the VMN of reproductively active type I males would exhibit higher mRNA transcript levels for two superoxide dismutases (sod1, sod2) compared to the VMN of type II males and females that do not hum and in general both of which have a more limited vocal repertoire than type I males. The results showed no significant difference in sod1 transcript expression across reproductive morphs in the VMN and the surrounding hindbrain, and no difference of sod2 across the two male morphs and females in the SH. However, we observed a surprising, significantly lower expression of sod2 transcripts in the VMN of type I males as compared to type II males. We also found no significant difference in sod1 and sod2 expression between actively humming and non-humming type I males in both the VMN and surrounding hindbrain. These findings overall lead us to conclude that increased transcription of sod1 and sod2 is not necessary for combatting oxidative stress from the demands of the midshipman high-endurance vocalizations, but warrant future studies to assess protein levels, enzyme activity levels, as well as the expression of other antioxidant genes. These results also eliminate one of the proposed mechanisms that male midshipman use to combat potentially high levels of oxidative stress incurred by motor neurons driving long duration vocalization and provide more insight into how motor neurons are adapted to the performance of extreme behaviors.