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Carbon dioxide (CO2) is a critical biological signal that is noxious to many animals at high concentrations. The earthwormDendrobaena venetalives in subterranean burrows containing high levels of CO2and respires through its skin. Despite the ecological and agricultural importance of earthworms, relatively little is known about how they make decisions in their environment, including their response to elevated levels of CO2. To examine CO2detection in this species, we designed the exudate assay, in which we placed an earthworm in a sealed container, exposed it to varying concentrations of CO2for one minute, and recorded the amount of exudate secreted. Because earthworms excrete exudate in response to noxious stimuli, we hypothesized that the amount of exudate produced was proportional to the amount of irritation. We repeated these experiments after treatment with several blockers for molecules with potential involvement in CO2detection, including carbonic anhydrases, guanylate cyclase, TRPA1 channels, ASICs, and OTOP channels. We also confirmed the presence of homologous transcripts for each of these gene families in an epithelial transcriptome forD. veneta.Additionally, since organisms often detect CO2levels indirectly by monitoring the conversion to carbonic acid (a weak acid), we used the exudate assay to evaluate aversion to additional weak acids (formic acid, acetic acid, and propionic acid). Earthworms excreted significantly more exudate in response to CO2in a dosage-dependent manner, and this response was muted by the general carbonic anhydrase inhibitor acetazolamide, the carbonic anhydrase IX/XII inhibitor indisulam, the calcium channel blocker ruthenium red, the sodium channel blocker amiloride, and the acid-sensing ion channel blocker diminazene aceturate. These data provide evidence of the role of carbonic anhydrase and ENaCs in earthworm CO2detection and contribute to our understanding of the mechanisms used by earthworms to detect and react to weak acids in their environment.Contribution to the field statement:Carbon dioxide (CO2) is a major byproduct of cellular respiration and an important biological signal. The metabolism of animals living underground in burrows can cause the concentration of CO2to increase above atmospheric CO2. Most animals have multiple molecular mechanisms that detect CO2and find high concentrations aversive or noxious; some subterranean animals have adaptations that make them more tolerant of concentrated CO2. Earthworms live in burrows, are a keystone species for subterranean ecosystems, and in their lightless environment must rely primarily on tactile and chemical signals. Despite the profound importance of earthworms to the ecosystems, few if any of the molecular mechanisms they use to detect these signals have been characterized. Here we use RNA sequencing to develop a list of candidate mechanisms that the European nightcrawler may use to respond to CO2. We also have developed an assay for comparing how noxious different concentrations of volatile compounds are to earthworms. In the present study, we utilize that assay with inhibitors to further narrow the possible multiple molecular mechanisms responsible for the detection of CO2. We suspect that our study is among the first attempts to combine RNA-sequencing technology and pharmacology to describe earthworm sensory biology. |
