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