Abstract
Domoic acid (DA) is a neurotoxin produced by certain species ofPseudo-nitzschia (PSN ) that can cause damage to neural tissues and can be fatal to marine animals. Copepods, direct consumers of PSN , exhibit remarkable resistance to DA. Given that gut microbiota facilitate various detoxification processes in copepods, we hypothesize that gut microbiota may play a crucial role in aiding copepods in DA detoxification. In this study, we investigated the detoxification capability of copepod gut microbiota by feeding both wild-type and gut-microbiota-free Acartia erythraea toxicPSN . Our results demonstrated that, although DA suppressed the growth of A. erythraea , the presence of gut microbiota enhanced the survival of copepods exposed to a DA diet. We subsequently feedA. erythraea both toxic and non-toxic PSN, and explored the potential mechanisms of DA detoxification through amplicon and metatranscriptome approaches. We identified both anaerobic and aerobic DA detoxification pathways in copepod gut bacteria, mediated by the genera Aureispira , Tenacibaculum ,Pseudoalteromonas , Shewanella , and Vibrio . In the anaerobic pathway, DA could be biotransformed into detoxification products through a series of main degradation steps, including decarboxylation, dehydrogenation, carboxylation, and multiple β-oxidation processes. In the aerobic pathway, DA undergoes reactions including hydration, dehydrogenation, hydrolysis, hydroxylation, and oxidation, resulting in the formation of terminal detoxification products. Overall, our findings elucidate the mechanisms by which copepod gut microbiota detoxify DA, thereby advancing our understanding of copepod resilience in the face of a toxic diet.
Keywords: Pseudo-nitzschia , Domoic acid, Gut microbiota, Detoxification, Copepods, Metatranscriptome
Introduction
The genus Pseudo-nitzschia (PSN ) comprises a group of pennate, chain-forming diatoms that are prevalent in marine environments (Bates et al. 2018). In China’s coastal waters, PSN frequently appears in phytoplankton communities (Chen et al. 2005; Lü et al. 2012), and can trigger harmful algal blooms. Notably, four such blooms occurred in Daya Bay between 1990 and 2005 (Chen et al. 2005; Liu et al. 2016). The Agriculture, Fisheries and Conservation Department (http://www.afcd.gov.hk) has documented over 20 PSN blooms in Hong Kong waters since 2009, with the largest bloom affecting an area of 150 km², leading to significant ecological and fishery damage. SomePSN species produce a neurotoxin known as domoic acid (DA), with 29 out of 63 species known to produce this toxin (Lundholm 2024; Niu et al. 2024). In China, 11 of 31 PSN species have been identified as DA producers (Li et al. 2017; Dong et al. 2020). Interestingly, severalPSN species previously deemed non-toxic have been found to produce DA in Chinese coastal waters (Li et al. 2017; Huang et al. 2019; Dong et al. 2020). For instance, five species isolated from Victoria Harbour and Mirs Bay were newly reported to produce DA, with concentrations ranging from 0.012 to 40.3 fg cell-1(Dong et al. 2020). Bates et al. (2018) proposed that under suitable conditions, all PSNs could potentially produce DA.
DA can travel through the food web, affecting higher trophic levels such as seabirds, whales, and seals (Leandro et al. 2010; Tammilehto et al. 2012; D’Agostino et al. 2017). It also causes amnesic shellfish poisoning in humans (Wright et al. 1990). The presence of toxic DA has led to considerable marine animal mortality and economic losses in North American and European coastal waters (Bates et al. 2018). More countries are reporting issues related to this diatom genus (Bates, Garrison, and Horner 1998; Bates et al. 2018). Recent research indicates that DA can also hinder denitrification and Anammox processes, which are vital for nitrogen removal in sediments, thus affecting nitrogen cycling (Li et al. 2023).
While DA is linked to widespread poisoning in marine mammals and birds, copepods show some resistance to this toxin. Research suggests that copepod species like Calanus sp. and Acartia sp. consume toxic PSNs without significant deterrence and do not preferentially select non-toxic prey (Tester et al. 2000; Maneiro et al. 2005; Leandro et al. 2010; Harðardóttir et al. 2019). Moreover, studies have shown no significant difference in mortality rates, egg production and hatching rates, between zooplankton consuming toxic and non-toxicPSNs (Lincoln et al. 2001; Miesner et al. 2016). However, other studies indicates that DA does affect copepods. Although grazing rates may not decrease significantly, physiological stress is observed in copepods consuming DA-producing Pseudo-nitzschia seriata , as shown by gene expression profiles (Harðardóttir et al. 2019). Additionally, Arctic copepods like Calanus hyperboreus andC. glacialis exhibit reduced escape responses after feeding on DA-producing diatoms (Harðardóttir et al. 2018). Compared to higher trophic level organisms, copepods and other zooplankton accumulate relatively low levels of DA (Liefer et al. 2013), which might explain their reduced sensitivity to this toxin.
Some researchers suggest that the gut microbiota of zooplankton may help detoxify this toxin, reducing its harmful effects (Li et al. 2019; Gorokhova et al. 2021; Yang et al. 2024). The zooplankton gut provides a unique microhabitat for microorganisms, offering specific environmental conditions and sources of carbon and nutrients. Gut microbiota play a vital role by supplying essential nutrients and vitamins that enhance the host’s digestive capacity (Harris 1993). While bacteria can enter the gut through detritus or planktonic prey, studies show that copepod gut communities differ from those in the surrounding seawater, indicating specialized, long-term microbial communities (Shoemaker and Moisander 2017). Recent finding reveal that variations in gut microbiota, influenced by environmental factors and host genotype, can mediate Daphnia’s tolerance to toxic cyanobacteria, highlighting the microbiota’s role in adapting to toxic diets (Macke et al. 2017). Moreover, zooplankton neonates from mothers zooplankton fed toxic algae showed improved growth and reproduction compared to those from mothers consuming non-toxic algae, suggesting a possible maternal transfer of beneficial gut microbiota (Lyu et al. 2016; Macke et al. 2017). Prior research in our laboratory has demonstrated that Daphnia gut microbiota can detoxify silver nanoparticles (AgNPs) by converting silver ions (Ag+) to less harmful forms and neutralizing them with flagellin protein (Li et al. 2019). Additionally, the maternal transfer of gut microbiota may enable younger generations to adapt to toxicity more swiftly (Li, Wang, and Liu 2022). Consequently, the gastrointestinal microorganisms of zooplankton are increasingly recognized as crucial factors influencing host physiological condition and fitness, especially regarding toxin exposure. However, there remains limited knowledge about the detoxification processes and molecular mechanisms of zooplankton gut microbiota.
In this study, we explored the effects of toxic (P. cuspidata ) and non-toxic PSNs (P. brasiliana ) on Acartia erythraea , a dominant copepod species in Hong Kong coastal waters, using physiological assessments and multi-omics techniques. Our goal was to test the hypothesis that copepod gut microbiota aids in detoxifying DA. Specifically, we sought to identify which gut microbiota taxa inA. erythraea are involved in DA detoxification and to uncover molecular evidence of potential detoxification pathways.
Materials and methods