The alternative oxidase (AOX) increases sulphide tolerance in the highly invasive marine invertebrate Ciona intestinalis

Ecological communities and biodiversity are shaped by both abiotic and biotic factors. This is well illustrated by extreme environments and invasive species. Besides naturally occurring sulphide-rich environments, global change can lead to an increase in hydrogen sulphide episodes that threaten many multicellular organisms. With the increase in the formation, size, and abundance of oxygen minimum zones and hypoxic environments, bacterial-associated sulphide production is favoured and as such hydrogen sulphide-rich environments increase subsequently. Many species are challenged by the inhibiting effect of sulphide on aerobic energy production via cytochrome c oxidase, ultimately causing the death of the organism. Interestingly, many protist, yeast, plant, and also animal species possess a sulphide-resistant alternative oxidase (AOX). In this study, we investigated whether AOX is functionally involved in the sulphide stress response of the highly invasive marine tunicate, Ciona intestinalis. At the LC50, the sulphide-induced reduction of developmental success was three times stronger in AOX knock-down embryos than in control embryos. Further, AOX mRNA levels were higher under sulphide than control conditions and this effect increased during embryonic development. Together, we found that AOX is indeed functionally involved in the sulphide tolerance of Ciona embryos, hence, very likely contributing to its invasive potential; and that the response of AOX to sulphide seems to be controlled at the transcriptional level. We suggest that AOX-possessing species play an important role in shaping marine ecological communities, and this importance may increase under ongoing global change. Jo ur na l o f E xp er im en ta l B io lo gy • A cc ep te d m an us cr ip t


Introduction
Ecological communities and biodiversity are shaped by two main factors, climate change and invasive species (Mainka and Howard, 2010). With the currently ongoing climate change, many environments have already experienced alterations in their characteristics, and will continue to do so, while extremes increase in abundance and severity, consequently affecting ecological communities (Bellard et al., 2012;Mainka and Howard, 2010;Worm and Lotze, 2021). Invasive species play another important role in shaping ecological communities and biodiversity (Bax et al., 2003;Molnar et al., 2008). By nature, invasive species have to be well prepared to cope with diverse stressors, and this in turn may be one key feature that enables them to compete in a new environment. Therefore, understanding the underlying mechanisms of how organisms in general and invasive species in particular respond to extreme environments is crucial to improve predictions for ecological communities and biodiversity, especially in the context of climate change.
The oceans, covering about 70% of the Earth's surface, play a central role in climate change.
With increased carbon dioxide levels in the atmosphere, the oceans are greatly affected by changing temperature (becoming warmer) and pH (becoming more acidic). The consequences of those changes are manifold, ranging from less oxygenated oceans, rises in sea levels, changes in ocean currents and increasing weather extremes (Diaz and Rosenberg, 2008). One additional consequence with considerable impact on the survival of multicellular organisms has rarely been mentioned in the climate change debate: the increase in hydrogen sulphide (H 2 S) in the oceans. In general, high sulphide concentrations are due to geothermal and biological processes (Bagarinao, 1992). Interestingly, biological processes relate to oxygen levels: with depleted oxygen levels, bacterial sulphate reduction takes place and H 2 S is produced (Jørgensen et al., 1982;Schunck et al., 2013), and eventually moves up the water column (Bakun and Weeks, 2004;Brüchert et al., Journal of Experimental Biology • Accepted manuscript cultures, sterilized with a 0.22 µm filter) and 1% agarose-coated petri dishes ( 55 mm, Gosselin, Hazebrouck, France) (Sardet et al., 2011). In all experiments we used unrelated families as biological replicates, i.e. sperm and eggs for each family were only used once. On the day of the experiment, eggs and sperm for each family were freshly sampled and eggs were dechorionated and fertilized according to (Sardet et al., 2011

Statistical analyses
We analysed the dose-response data using a generalized linear mixed model with logit-link function and binomial error distribution under Laplace approximation to the likelihood as implemented in the R-function glmer of the R-package lme4 (Bates et al., 2015). The proportions of individuals that survived were modelled with a fixed covariate for sulphide concentration (in μM; representing a logit regression), and random terms for family, family-by-concentration, and family-by-concentration-by-replicate (accounting for detected overdispersion). We determined the LC 50 relative to the survival at sulphide absence based on the estimated model intercept = 1.689 ± 0.163 (estimate ± se) and regression slope = -0.132 ± 0.009 as 15.2 μM sulphide, which we rounded to 15 μM for use in the AOX knock-down experiment.
We analysed the AOX knock-down experiment data using a generalized linear mixed model with logit link function like for the dose-response data. The developmental success binaries (embryos that developed to tailbud stage relative to all embryos) were modelled with fixed terms for the sulphide treatment (15 μM or no sulphide), the MO groups, and their interaction, and random terms for family, family-by-treatment, and family-by-MO. We performed pairwise comparisons of predicted means for each MO group within each treatment and between treatments within each MO group using t-tests and adjusted the p-values for the false discovery rate (Benjamini and Hochberg, 1995).
We analysed the qPCR data using linear mixed models. We first fitted a model to test for a constant log2 Cq across development for each of the two housekeeping genes and a constant difference between them, which held for the latter but not the former. Next, we averaged the Cq across housekeeping genes per family, treatment, and developmental stage. We then standardised

Elevated hydrogen sulphide levels reduce Ciona developmental success
The dose-response experiment indicated that Ciona larval development success depended strongly on the sulphide concentration (Fig. 1). The average maximum developmental success in the absence of sulphide was 84%, whereas at sulphide concentrations around 50 µM, absolute developmental success was approaching zero. Based on the logistic mixed model, we determined a sulphide LC 50 of ~15 µM compared to embryos not exposed to sulphide. We used this concentration in the following experiments.

Knockdown of AOX reduces the tolerance to sulphide
In the absence of sulphide, we did not detect any significant difference in developmental success among the four MO groups (Fig. 2 . 2). These sulphide effects can be translated into odds ratios, i.e. the odds for an embryo to survive in control conditions relative to sulphidic conditions within each MO group. For the uninjected group, the odds ratio was 3.9 (95% CI 2.1-7.1), for the MO Control group, it was 3.5 (95% CI 1.9-6.7) and for the rescue group the odds ratio was 3.5 (95% CI 1.7-7.6). For the AOX KD group, the odds to survive under control conditions were 11.6 (95% CI 5.9-23.0) times higher than to survive under sulphidic conditions. Further, to evaluate the relative effect of AOX, we contrasted the odds ratio of the AOX KD group with each of the two control MO groups. In other words, we quantified the ratio of developmental success of control embryos (uninjected and MO Ctrl -injected) and AOX KD embryos under sulphide conditions to control conditions. We found the odds to survive under LC 50 sulphide conditions were higher for control embryos than for AOX KD embryos. Specifically, we found the odds to survive under LC 50 sulphide conditions for the uninjected control group to be 3 times higher (95% CI 1.7-5.3) and for the MO Ctrl group 3.3 times higher (95% CI 1.8-6.2) compared to the AOX KD group.

AOX transcript levels at different developmental stages under sulphide exposure
We detected differences in the effects on housekeeping gene transcript levels at different developmental stages (Gene-by-Stage; Table 3), i.e. the housekeeping gene transcript levels change with development asynchronously, which hinders an interpretation of the results as changes across development. However, the developmental stage effect on transcript levels between housekeeping genes did not differ between the 15 μM and 0 μM sulphide treatments (Gene-by-Stage-by-Treatment; Table 3). This absence of stage-by-treatment interaction for the housekeeping genes (used for the AOX Cq standardisation) allows for valid AOX transcript level contrasts between sulphide treatments. Therefore, we performed treatment contrasts, i.e. the difference in AOX mRNA levels (relative to housekeeping gene levels) between sulphide and control treatments for each developmental stage; but we did not perform treatment contrasts across development.

Journal of Experimental Biology • Accepted manuscript
Relative AOX transcript levels between the 15 µM and 0 µM sulphide exposed embryos were very similar at 32-cell stage while their difference increased significantly at the neurula and tailbud stages (Fig. 3). Further, the AOX mRNA level differences between the control and sulphide treatment for the 32-cell stage, gastrula and neurula were similar, while the AOX mRNA level difference was significantly higher for the tailbud stage (Table 4, Table 5).
Because of the changing housekeeping gene mRNAs levels across developmental stages, the underlying cause of this increase cannot be fully pinpointed (either an increase in AOX transcript levels or a decrease in the average housekeeping gene transcript levels under sulphide treatment relative to the control without sulphide).

Discussion
In this study, we investigated whether AOX is crucial for the development and survival of the highly invasive marine tunicate Ciona intestinalis under sulphide stress. To do so, we combined a MO-based knock-down approach and studied the transcriptional response of AOX. First, we demonstrated that early developmental success decreases with increasing sulphide concentrations. Second, we provided evidence that AOX is required for sulphide tolerance and, finally, showed that sulphide elicits an increased relative amount of AOX transcripts. Together, these results provide support for the idea that the presence of AOX in C. intestinalis increases its sulphide tolerance during development and may thereby contribute to its invasive potential.
The determined LC 50 of 15.2 μM sulphide and the fully stalled development at a concentration of 50 μM (Fig. 1)  Ciona to sulphide is two orders of magnitude higher than that of eggs, fry and juveniles of non-

Journal of Experimental Biology • Accepted manuscript
AOX hosting fish species such as walleye, northern pike, sucker, and rainbow trout (Adelman and Smith, 1970;Smith and Oseid, 1972;Smith and Oseid, 1974). While the maximum possible safe level of sulphide for fish eggs lies between 0.41 and 0.53 μM, it is between 0.11 and 0.18 μM for yolk-sac fry. Our results also align with the suggestions that developmental stages of many marine invertebrates are more sensitive to stress compared to their adult stages (Pineda et al., 2012;Ringwood, 1992). sulphide-rich events have been observed off the coast of Peru with up 6 μM sulphide (Schunck et al., 2013) and Namibia (up to 30 μM sulphide) which leads to regular mass mortalities of fish (Copenhagen, 1953;Lavik et al., 2009). Furthermore, it is expected that areas of toxic H 2 S concentrations will increase in abundance, size and severity in many marine environments due to the increase in anoxic waters (Schobben et al., 2015), which can be expected to affect ecosystems via large animal kills. Interestingly, and as one of the worst case scenarios, the end-Permian marine mass extinction has been linked to anoxic and sulphide-rich oceanic conditions (Schobben et al., 2015).
To our current and best knowledge, this is the first functional study of AOX in an animal species.
This, however, also impedes a direct comparison of our results to other studies. Nonetheless, the importance of AOX in the sulphide response was suggested previously. A study on mitochondria of a polychaete, the lugworm (Arenicola marina), found strong indication that the alternative pathway via AOX is involved in the oxidation of sulphide (Hildebrandt and Grieshaber, 2008).
Specifically, under elevated sulphide levels, oxygen consumption was high while no ATP was produced; and this reaction was completely blocked when AOX was inhibited (Hildebrandt and Grieshaber, 2008). These latter results may also partially explain why A. marina can survive quite high sulphide concentrations of up to 10 mM (Groenendaal, 1980). A study on another polychaete, the echiuran worm (Urechis unicinctus), found AOX mRNA levels in the body wall and hindgut tissue to increase with both, sulphide concentration and sulphide exposure time both, the Pacific oyster and the freshwater mussel are also considered invasive species, which supports our suggestion that AOX may indeed be an additional mechanism in slow or sessile marine organisms to cope with a variety of environmental factors and that it may give those species an advantage under future ocean conditions.
Among species lacking the AOX gene, sulphide tolerance varies enormously but can also be quite high (Bagarinao, 1992;Grieshaber and Völkel, 1998). Cellular mechanisms, other than AOX, that explain sulphide tolerance include the sulphide-quinone oxidoreductase (SQR)-related sulphide oxidation as present in most domains of life (Theissen et al., 2003), the H 2 S-resistant COX such as found in the shortfin molly (Poecilia mexicana), and the H 2 S-binding haemoglobins of the giant tube worm (Riftia pachyptila) and the California killifish (Fundulus parvipinnis) (Bagarinao and Vetter, 1992;Zal et al., 1998). Using H 2 S-binding haemoglobins, the California killifish for example, has an 8-h LC 50 of 300 μM H 2 S (Bagarinao, 1992).
To better understand the control of the AOX response to sulphide, we also investigated the transcriptional response of AOX to sulphide exposure at different developmental stages.
Unfortunately, changes of the two "housekeeping" gene mRNA levels across developmental stages prevented us from being able to make clearer statements on the change of AOX mRNA across developmental stages. This exemplifies the crucial need of a careful analysis of "housekeeping" gene responses, particularly during dynamic biological processes, such as development, to allow for more reliable conclusions on a target gene response. However, we were able to draw conclusions about the difference of AOX mRNA levels between control and sulphide conditions at each developmental stage. We found no difference in AOX mRNA levels between sulphide and control conditions at the 32-cell stage, whereas the AOX mRNA levels were increasingly higher under LC 50 sulphide exposure compared to no sulphide exposure at the later stages (Fig. 3). Further, the sulphide effect was clearly higher for the last studied, the tailbud