Exposure to Batrachochytrium dendrobatidis metabolites altered ghost shrimp behavior and reduced mortality

Introduction

Batrachochytrium dendrobatidis, also referred to as Bd, is a pathogenic chytrid fungus implicated in the decline and extinction of hundreds of amphibian species worldwide (Scheele et al., 2019). This water borne fungus reproduces via sporangia, and is spread by the dispersion of infectious, flagellated zoospores (Longcore, Pessier & Nichols, 1999). Infections may be transmitted via direct contact with an infected host or with the free-swimming mobile zoospores. Indirect exposure is thought to occur through contact with contaminated environmental reservoirs (e.g., sediment (Kirshtein et al., 2007), boulders (Longcore, Pessier & Nichols, 1999; Wixson & Rogers, 2009), bromeliads (Cossel & Lindquist, 2009), aquatic birds (Garmyn et al., 2012; Burrowes & De la Riva, 2017) or snakes (Kilburn, Ibáñez & Green, 2011)), although transmission through this route has not been confirmed.

In amphibians, Bd infection can impact the keratinized layer of the epidermis and can cause pathological features such as skin degradation, sloughing, hyperkeratosis, hyperplasia, ulceration, erosions, and necrosis (Berger et al., 1998; Grogan et al., 2018). Infected amphibians may also show evidence of lethargy, loss of appetite, behavioral changes and in many instances, mortality (Gabor, Fisher & Bosch, 2015). The epidermis is critically important to physiological processes such as osmoregulation, thermoregulation, gas exchange and indirectly cardiac function. Bd infections have been directly implicated in the disruption of osmoregulation, including imbalances in sodium and potassium ion concentrations (Peterson et al., 2013; Voyles et al., 2009) and cardiac dysfunction, including decreased stroke volume, delayed cardiac cycles (Salla et al., 2018) and asystolic cardiac arrest (Voyles et al., 2009), any of which can lead to mortality.

Bd metabolites, water soluble chemicals produced by the chytrid fungus, have also been shown to cause pathological outcomes in amphibians. While the exact chemical composition of Bd metabolites is still unknown, we know that Bd produces proteolytic enzymes which have been implicated in skin disorders. Other metabolites have been shown to have immunomodulatory and inhibitory potential (e.g., methylthioadenosine, tryptophan, and polyamine spermidine; Rollins-Smith, Reinert & Burrowes, 2015; Rollins-Smith et al., 2019). Interestingly, Bd metabolites have also been shown to produce prophylactic effects, although the exact metabolites responsible have not been identified. This may be because the composition of metabolites Bd produces changes depending on the local environment (Rollins-Smith, Reinert & Burrowes, 2015). Specific Bd metabolites may have different impacts, some detrimental and others potentially prophylactic. Differences in the concentration of Bd metabolites an organism is exposed to may also account for the range of impacts, from toxic to pharmaceutically beneficial. While exposure to high doses of Bd metabolites may be detrimental to some freshwater species (McMahon et al., 2013; Nordheim, Grim & McMahon, 2021), studies have shown that exposure to low doses of Bd metabolites may actually produce beneficial effects. Indeed, amphibians exposed to Bd metabolites responded to this prophylactic treatment exposure, and had reduced subsequent Bd infection loads, and in some cases, there was even lower infection prevalence (McMahon et al., 2014, 2023, 2024; Barnett et al., 2021, 2023; Nordheim et al., 2022). Additionally, tadpoles exposed to low concentrations of Bd metabolites experienced higher developmental speeds, while maintaining equal growth and survival compared to control tadpoles (McMahon, Laggan & Hill, 2019). These findings suggest that while high concentrations of Bd metabolites may impact important physiological processes such as gas exchange and osmoregulation, lower concentrations may be neutral or may even present a prophylactic potential against Bd infection, including reduced pathogen load, increased tolerance or resistance and potentially reducing adverse outcomes such as disease severity and mortality.

The concept that Bd metabolites may be beneficial is not entirely novel. Other fungal species have been shown to produce metabolites with pharmaceutically beneficial effects for freshwater organisms. The fungal species Aspergillus niger, for example, has been shown to increase survival in white shrimp (Zhang et al., 2023) in aquaculture. In fact, A. niger has been shown to enhance growth, immunity and disease resistance (Cheng et al., 2023). Additionally, fungal polysaccharides have been found to yield immunostimulation, increased pathogen resistance and even improve growth in crustaceans (Mohan et al., 2019). Indeed, many fungal species have been shown to produce bioactive metabolites with antimicrobial, including antifungal properties, that have proved critical to the aquaculture industry (Xu et al., 2021; Du et al., 2022).

In this study, we examined the effects of Bd metabolites on the freshwater species, Palaemonetes paludosus, more commonly known as ghost shrimp. It is important to note that despite Bd’s prevalence within amphibian populations, it is not exclusive to amphibians as it has been identified on non-amphibian organisms, such as wading birds (Garmyn et al., 2012) and snakes (Kilburn, Ibáñez & Green, 2011), and confirmed on museum specimens of aquatic birds originating from the Bolivian Andes (Burrowes & De la Riva, 2017). In its non-amphibian hosts, specifically crayfish, Bd has been identified growing in the gastrointestinal tract and on the exoskeleton (McMahon et al., 2013; Brannelly et al., 2015; Román, O’Neil & James, 2016; Oficialdegui et al., 2019). Bd related mortality in these freshwater invertebrates is likely due to gill damage and the resulting impairment in gas exchange (McMahon et al., 2021; Nordheim, Grim & McMahon, 2021), and potentially osmoregulation, given the role the gills play in these processes. Similarly, Bd metabolites have been shown to cause pathological outcomes in non-amphibian organisms. Research has demonstrated that gill damage and mortality can occur in crayfish exposed to high concentrations of Bd metabolites, and that these detrimental impacts occur even in the absence of Bd infection (McMahon et al., 2013; Nordheim, Grim & McMahon, 2021). It should be noted that these detrimental effects were not seen when crayfish were exposed to low concentrations of Bd metabolites, suggesting that not only might specific metabolites confer prophylactic benefits, but that a concentration dependent relationship between the prophylactic and pathological effects of Bd metabolites may exist (McMahon et al., 2013; Nordheim, Grim & McMahon, 2021).

Ghost shrimp are native to the southeastern coastal plains of North America and reside in shallow regions of freshwater ponds, lakes, and streams. These small invertebrates are scavengers and bioturbators, and they co-occur with Bd and amphibians. Through borrowing activities, ghost shrimp can alter carbon and nutrient cycling, and influence microbiota interactions and dispersal. They are a critical freshwater species, making them an ideal model to understand the impact of Bd metabolites and indirect exposure to Bd on freshwater organisms (Wada, Urakawa & Tamaki, 2016; Adhikary, Klerks & Chistoserdov, 2024). Additionally, ghost shrimp are ideal for studying bioactive compounds because their characteristic behavioral responses to threats, e.g., hiding and escape responses, offer strong opportunities to understand the effects of Bd metabolites on well-studied critical behaviors. The biochemical pathways, including those that regulate these behaviors, have also been characterized extensively in crayfish, which share neurochemical homology to ghost shrimp (Sosa et al., 2004; Wongprasert et al., 2006; Fossat et al., 2014). Finally, ghost shrimp’s characteristic translucent carapace and bodies allow for unpresented physical assessment, including monitoring of heart rate and changes to the physical appearance of the carapace.

To date, we know little about the direct and indirect effects of Bd on non-amphibian organisms. Indeed, most research on Bd has focused on amphibian conservation, which is not surprising given the severe decline in this taxon. The goal of this study was to better understand the effects of Bd metabolites on non-amphibian, freshwater organisms, which is particularly critical if Bd metabolites are to be used as a prophylactic at a community level. Additionally, understanding the complex dynamics of indirect exposure to Bd on co-occurring freshwater invertebrates may provide important insight into the mechanisms through which Bd is affecting population dynamics, and will provide a more holistic view of how Bd impacts the freshwater ecosystems.

Materials and Methods

Statistical analysis

All analyses were conducted in R using R studio (version 4.2.2; R Core Team, 2022). A general linear model (package: stats, function: glm, family: gaussian; R Core Team, 2022) was used to verify that there was no difference in initial shrimp length between the treatment groups prior to the start of the experiment. A generalized linear model (package: glmmTMB, function: glmmTMB, family: gaussian; Brooks et al., 2017) was used to determine if there was an effect of treatment on whether ghost shrimp spent more time on the dark side of the enclosure post treatment exposure. Time spent on the dark side pre-exposure, number of observations post-exposure (number of times behavioral observations were made for each individual after exposure; this number varied due to mortality), and mortality status were accounted for as covariates in the model.

A general linear model (package: stats, function: glm, family: gaussian) was used to determine whether there was an effect of treatment or time after exposure on gill recession, whether there was a correlation between gill recession and activity level, whether there was an effect of treatment or number of post exposure observations on post exposure activity level, whether there was an effect of treatment or number of post exposure observations on height post exposure (taking into consideration average height pre-exposure), and whether there was an effect of treatment or number of post exposure observations on total number of molts post exposure. A general linear model (package: stats, function: glm, family: gaussian) was also used to determine if there was a difference in initial heart rate between the treatment groups prior to exposure to the treatments and to determine whether there was an effect of treatment on final heart rate (taking into consideration the initial heart rate of each shrimp). We used a general linear model (package: stats, function: glm, family: gaussian) to determine if treatment had an effect on whether shrimp exhibited an evasive movement in response to a stimulus. A survival analysis was performed and the data was fit to a cox proportional hazards regression (package: survival, function: coxph; Therneau, 2024) to determine if there was an effect of treatment on mortality.

Figures 1–3 were created in R (Fig. 1: package: survminer, function: ggsurvplot (Kassambara, Kosinski & Biecek, 2024)); Figs. 2 and 3: package: ggplot2, function: ggplot; Wickham, 2016, and Fig. 4 was created in Excel (Version 16.78.3).

Results

Discussion

This study found that exposure to Bd metabolites altered ghost shrimp behavior and mortality, but not necessarily in an expected manner. We found that ghost shrimp exposed to Bd metabolites experienced decreased mortality when compared to those exposed to ASW, suggesting that Bd metabolites may actually improve survival. The mechanism for increased survival in ghost shrimp was not studied here, but previous research found that Bd metabolites altered the host microbiome, shifting the microbiome towards more protective biota (Siomko et al., 2023). The microbiome, particularly the gut microbiome, has been shown to be intrinsically related to immune function (Holt et al., 2021). It is certainly possible that the ghost shrimp experienced a beneficial microbiome shift as well, though further research is needed to better understand this possible dynamic, the mechanism behind the increased survival conferred by Bd metabolite exposure, and its beneficial effects in other organisms.

Similar results were seen in studies in amphibians, with prophylactic exposure to Bd metabolites leading to reduced disease outcomes (Nordheim et al., 2022; Barnett et al., 2023), which would likely increase survival (McMahon et al., 2014). Indeed, tadpoles exposed to Bd metabolites experienced more rapid development while still maintaining strong growth and survival (McMahon, Laggan & Hill, 2019). This is unexpected, as typically when tadpoles develop faster due to local environmental conditions there is a negative trade off, including smaller size and higher mortality (see Newman, 1988; Bridges, 2002; Rohr et al., 2004). These results support observations in ghost shrimp, and further highlight possible beneficial impacts on survival of exposure to Bd metabolites.

We found that Bd metabolites did not induce gill recession in ghost shrimp. This finding was important given that previous studies in crayfish have reported gill recession which was associated with increased mortality (McMahon et al., 2013; Nordheim, Grim & McMahon, 2021). Importantly, in those previous studies, gill damage was associated with higher doses of Bd metabolites; in fact, low doses of Bd metabolites did not cause damage in crayfish either. The different responses in gill damage between crayfish and ghost shrimp may be associated with the effects of actual Bd infection vs exposure to just the Bd metabolites. It should be noted that Bd positive ghost shrimp have been found in the field (Rowley, Alford & Skerratt, 2006 but see Rowley et al., 2007, as well) and so, it is certainly possible that ghost shrimp are tolerant to Bd metabolites. The differential effects in gill damage may also be due to exposure to different specific metabolites as Bd produces different metabolites depending on the environment (Rollins-Smith, Reinert & Burrowes, 2015). Finally, previous research has found lower concentrations of Bd metabolites were not deleterious to other crustaceans (McMahon et al., 2013; Nordheim, Grim & McMahon, 2021), suggesting different concentrations of Bd metabolites may also confer differential effects.

Ghost shrimp exposed to Bd metabolites experienced impacts on the cardiac system and had a decreased final heart rate compared to those exposed to ASW. While this finding was based on a 3-s view of the heart rate pre- and post-exposure, the results are interesting, given our observations of increased survival and no gill recession. Some studies have reported cardiac disruptions in amphibians following active Bd infections, including decreased stroke volume and delayed cardiac cycles (Salla et al., 2018). Additionally, amphibians experiencing effects of chytridiomycosis have impaired epidermal osmoregulation, which was associated with asystolic cardiac arrest (Voyles et al., 2009). While we have no evidence that the decreased heart rate is detrimental in the ghost shrimp in our study, and the exact mechanism through which cardiac disruption is precipitated is not known, our findings indicate that the Bd metabolites may be associated with altered cardiac function. Further research is needed to fully understand the dynamics associated with Bd metabolite exposure on cardiac function because there are likely multiple physiological mechanisms at play, and the impact of Bd metabolites on cardiac function may point toward important mechanistic differences between direct exposure to Bd, and indirect exposure to Bd metabolites.

Ghost shrimp exposed to Bd metabolites were observed to spend less time on the dark side of their tank post treatment compared to ASW and were also less likely to exhibit escape behavior in response to an approaching foreign object. Both behaviors deviate from ghost shrimp’s expected natural behavioral profile, which include a preference for the dark or covering, and a tendency toward escape, behaviors aimed at avoiding predation. Reduced threat response behaviors, which are likely to increase ghost shrimps’ risk of predation, have been linked to disruption of the serotonergic pathway (Guler & Ford, 2010; Fossat et al., 2014, 2015). This connection has been supported through research in other decapods, with one study showing that animals exposed to antidepressants such as serotonin reuptake inhibitors, which target the serotonergic pathway, display reduced hiding and escape behavior (Guler & Ford, 2010). Indeed, tryptophan, which was identified as one of the most abundantly secreted metabolites in the Bd supernatant (Rollins-Smith, Reinert & Burrowes, 2015), is an essential amino acid and precursor to serotonin, a neurotransmitter critical in regulating behavior. Additionally, kynurenine, another Bd metabolite, is also central to the serotonergic pathway and immune regulation. Kynurenine, like serotonin, is a metabolite of tryptophan and represents a distinct pathway branchpoint to serotonin, making these two metabolic products key regulators of tryptophan and each other. Importantly, serotonin has been connected to Bd susceptibility in amphibians. One study in particular demonstrated correlations between skin serotonin levels and the susceptibility to Bd, and perhaps disease progression (Claytor et al., 2019). Serotonin was shown to inhibit the growth of Bd sporangia in vitro, and studies in Candida albicans showed that serotonin attenuated virulence in the fungus (Mayr et al., 2005). Altogether, this points toward a possible role for Bd metabolites, specifically tryptophan, in not only the pathogenesis and virulence of Bd, but as prophylactic agents.

This research found that exposure to Bd metabolites was not directly deleterious to ghost shrimp, but we did find that these metabolites had potentially adverse impacts that could be detrimental in the field. Given that researchers have been developing a Bd metabolite prophylaxis it is crucial that more research be done to understand the impact of these metabolites on critical freshwater organisms. Studies have been mixed in regards to the effects of Bd metabolites, with some showing evidence that exposure to high concentrations of Bd metabolites can increase mortality (e.g., see McMahon et al., 2013; Nordheim, Grim & McMahon, 2021) and others finding Bd metabolites confer prophylactic effects (e.g., see Barnett et al., 2023; McMahon et al., 2023, 2024). These discrepancies may be due to differences in concentration of metabolites, or the composition of metabolites organisms are exposed to, which can be dependent on the environment (Rollins-Smith, Reinert & Burrowes, 2015). It is also plausible that different strains of Bd may have differential impacts on organisms (e.g., see Barnett et al., 2021). Different organisms may also display differential responses to Bd metabolites, due to inherent physiological differences. It is important that we continue to examine the mechanisms through which Bd mediates its impact, including the role of specific metabolites. This is not only important in elucidating the mechanisms through which Bd induces deleterious impacts, but also potential targets for prophylactic and therapeutic intervention. The serotonergic pathway may be an important target given existing research showing a key role for serotonin in Bd virulence and animal susceptibility, and the identification of tryptophan and kynurenine as Bd metabolites. Addressing Bd’s impact on freshwater organisms may lie in our ability to modulate, at least in part, Bd’s effects on serotonin and its metabolites.

There have also been few studies focused on understanding the effects of Bd metabolites on non-amphibian organisms, despite evidence that they co-occur with amphibians and can be infected by and carry Bd. Many freshwater invertebrates, e.g., ghost shrimp and crayfish, could play an important role in understanding the impact of direct and indirect exposure to Bd because they are easy to maintain in the lab, their physiology and behavioral responses are well studied, many have transparent bodies and many are not experiencing dramatic declines in the wild. Instead of over utilizing amphibians in this type of work, freshwater invertebrates could be a crucial part of developing and implementing multilayered conservation efforts to protect critically endangered amphibian populations. Indeed, studying the impact of Bd and the metabolites it produces on freshwater invertebrates may provide much needed insight into the mechanisms associated with the pathogenicity and virulence of Bd as well as its impact on community ecology.

Conclusions

This study examined the effects of Bd metabolites in a ghost shrimp model system, focusing on the effects on behavior, cardiac function and mortality. We found that ghost shrimp exposed to Bd metabolites experienced reduced mortality when compared to those exposed to a water control and we observed decreased heart rate and reduced threat response behaviors. Previous studies reported increased mortality in crayfish following exposure to high concentrations of Bd metabolites, these disparate outcomes underscore the need for more research into understanding the impacts of Bd metabolites. Previous research has found that Bd metabolites can be used as a prophylaxis for amphibians, however, the use of Bd metabolites in a large-scale conservation effort in the field would likely yield exposure of both amphibian and non-amphibian organisms to these prophylactic agents. This work is promising, but it would only be feasible if we continue to do work to understand the impact of Bd metabolites on critical non-amphibian organisms.

Supplemental Information