Published On: Thu, Mar 14th, 2019

Identification of possible hypoxia sensor for behavioral responses in a marine annelid, Capitella teleta [RESEARCH ARTICLE]


Hypoxia treatment induced escape behavior of C. teleta, and this behavior was suppressed by the administration of A-967079 (Fig. 1), while the locomotor activity was not suppressed by A-967079 (Fig. S1). The suppression of escape behavior in C. teleta was prominent at the beginning of the experiment, which suggests that A-967079 influenced a rapid response to hypoxia. In mice, TRPA1 is the key regulator of the hypoxia ventilatory response, i.e. of the increase in ventilation during hypoxia (Pokorski et al., 2014). When mice breathe hypoxic air, their respiratory rate increases rapidly, but that of TRPA1-inhibited mice does not. This is in accord with our results, which implied that TRPA1 was involved in the rapid behavioral response to hypoxia. At later time points, A-967079 also suppressed the hypoxia-escaping behavior. Hatano et al., 2012 showed that human TRPA1 was upregulated within several hours by activated hypoxia inducible factor 1 α, a hypoxia-responsive transcription factor. This suggests that the input of sensing hypoxia via the TRPA1 homologue could gradually increase as the time of exposure to hypoxia increased. This might explain our observation that the suppression of the escape behavior re-emerged in the late part of this experiment. In the middle part of this experiment, this suppression was not observed. This might be because other hypoxia-sensing mechanisms, such as soluble guanylyl cyclase (sGC) or hypoxia inducible factor pathways, overwhelmed the A-967079-induced TRPA1 inhibition.

Similar hypoxia-induced evacuation from sediment is observed in infaunal benthic species. The burrowing marine annelids Hediste diversicolor and Alitta virens expose themselves on the sediment in response to hypoxia (Vismann, 1990). Lomia medusa, a tubicolous marine annelid, escapes from its tubes in hypoxic conditions (Llansó and Diaz, 1994). The brittle stars Amphiura filiformis and A. chiajei, the deposit-feeding bivalves Abra alba and A. nitida, and the suspension-feeding bivalve Cerastoderma edule come out from sediment in response to hypoxic stimuli (Rosenberg et al., 1991). In this study, C. teleta climbed on the sides of glass vials with their mucous in response to hypoxia. We infer that when they are confronted by hypoxia in their natural habitat, they would crawl out of their burrow and migrate on the surface of the sediment to find more suitable conditions. Terrestrial model organisms also show similar hypoxia-induced behaviors. The larvae of Drosophila melanogaster evacuate from their food, yeast paste, in response to hypoxia (Wingrove and O’Farrell, 1999). Caenorhabditis elegans migrates to an environment with a preferred oxygen availability to avoid hypoxia and hyperoxia (Gray et al., 2004; Chang and Bargmann, 2008). These behavioral responses are thought to be regulated by the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signaling pathway. In this pathway, soluble guanylyl cyclases play a key role in sensing oxygen via NO production (Morton, 2004; Vermehren-Schmaedick et al., 2010). These cyclases produce cGMP in response to oxygen deprivation, and cGMP in turn activates cyclic nucleotide-gated ion channel (CNG). Activation of CNG allows calcium ions to pass through the plasma membrane, resulting in depolarization, which leads to the behavioral responses to hypoxia. Hypoxia-induced activation of TRPA1 homologue also increases calcium permeability. TRPA1 homologue as a hypoxia sensor may cooperate with sGC to promote escape from the sediment in hypoxic conditions.

The phylogenic analysis of the TRPA1 homologue gene suggested that the cloned TRPA gene from C. teleta was classified as a TRPAbasal gene, similar to nematode’s TRPA-1 homologues (Fig. 2). The known agonists of nematode’s TRPA-1 are cold (Xiao et al., 2013) and mechanical stimuli (Kindt et al., 2007). A starfish, Patiria pectinifera, also has two TRPAs, TRPA1 and TRPA basal (Saito et al., 2017). PpTRPA1 is thermosensitive and involved in thermotaxis, but PpTRPA basal is not activated by heat or several pungent chemicals. The ODD is important for activation of TRPA1 by hypoxia. In the normoxic condition, a proline residue in the ODD is hydroxylated by the PHD family, which are oxygen-dependent prolyl hydroxylases (Takahashi et al., 2011). Under hypoxic conditions, that proline is not hydroxylated, leading to activation of TRPA1. This oxygen-dependent hydroxylation was first found in the regulation of HIFα, which mediates the physiological response to hypoxia (Kaelin and Ratcliffe, 2008). Therefore, the ODD from HIF-1α was aligned with CtTRPAbasal to examine whether CtTRPAbasal possesses an ODD (Fig. 3). The results of alignment showed that CtTRPAbasal has an ODD in the N-terminal cytosolic region, like mTRPA1. This result suggested that CtTRPAbasal can be activated by hypoxia. On the other hand, online transcriptome data indicate the existence of a gene homologous to CtTRPAbasal, ELU03480.1, in C. teleta. This gene is also categorized into TRPAbasal (Fig. 2) but the proline residue in ODD is substituted by glutamine, which suggests that this gene is not involved in hypoxia detection (Fig. S2).

The whole-mount in situ hybridization analysis showed that CtTRPAbasal was transcribed precisely in the segment anterior to mouth called the prostomium (Fig. 4). In annelids, sensory cells for sensing environmental stimuli such as amino acids and pH are specifically localized in the prostomium (Lindsay et al., 2004; Laverack, 1960, 1961). Observation of the ventral view revealed that signals were observed in both sides of the prostomium. In the tip of the prostomium, nerve fibers are concentrated on both lateral sides (Meyer et al., 2015). Since the arrangement of nerve fibers is similar to the location of the CtTRPAbasal transcript signal there, CtTRPAbasal may function as a hypoxia sensor at the sensory cells in the prostomium. Therefore, C. teleta might sense DO in its direction of movement and effectively avoid hypoxic zones. To specify more precisely the localization of CtTRPAbasal protein, immunohistochemical analyses will be needed.

A-967079 antagonizes mammalian TRPA1 with high specificity. Nakatsuka et al. (2013) and Banzawa et al. (2014) showed that A-967079 does not antagonize, but instead activates, chicken and frog TRPA1. This difference of antagonistic or agonistic effects of A-967079 depends on several amino acids present in the 5th transmembrane region. The region containing these amino acids in CtTRPAbasal does not have high similarity with that in mammalian TRPA1. Therefore, there is a possibility that C. teleta has some other TRPA1 homologue with higher homology to mammalian TRPA1, and A-967079 has no antagonistic effect on CtTRPAbasal. On the other hand, CtTRPA1 contains an ODD, a key domain for hypoxia-activation, suggesting that CtTRPAbasal would regulate the hypoxia-induced response of C. teleta. The suppression of hypoxia-avoidance behavior of C. teleta by A-967079 supports the notion that A-967079 antagonizes CtTRPAbasal activation by hypoxia. Functional analyses of CtTRPAbasal itself will be needed to verify the hypoxia-sensing ability of CtTRPAbasal and the antagonistic effect of A-967079 on it.

To expand their habitats, it is important for organisms to sense the limit of their tolerance to physicochemical conditions for their survival. Organisms belonging to the C. capitata complex, including several capitellids of which C. teleta is one, can endure several days or more in severe hypoxic conditions (Warren, 1977). Capitella teleta is thought to decrease its aerobic metabolism to below approximately 1.5 mg/l to endure hypoxic conditions, based on the results of its oxygen uptake rate (Chareonpanich et al., 1994). These reports showed the high tolerance of C. teleta to hypoxia. In this study, C. teleta exhibited avoidance from severe hypoxia, possibly mediated by CtTRPAbasal, within 1 h, and hence they could migrate to an area with higher DO before the time limit for their survival in severely hypoxic conditions. Their behavioral response and tolerance to hypoxia led them to survive in niches in organically enriched sediments where hypoxia often occurs. However, whether CtTRPAbasal itself is activated by hypoxia needs to be clarified by loss-of-function methods or analysis of the ion channel function.

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