Impacts of a newly identified behaviour-altering trematode on its host amphipod: from the level of gene expression to population.

Changes to host behaviour induced by some trematode species, as a means of increased trophic transmission, represents one of the seminal examples of host manipulation by a parasite. The amphipod Echinogammarus marinus (Leach, 1815) is infected with a previously undescribed parasite, with infected individuals displaying positive phototaxic and negative geotaxic behaviour. This study reveals that the unknown parasite encysts in the brain, nerve cord and the body cavity of E. marinus, and belongs to the Microphallidae family. An 18 month population study revealed that host abundance significantly and negatively correlated with parasite prevalence. Investigation of the trematode's influence at the transcriptomic level revealed genes with putative neurological functions, such as serotonin receptor 1A, an inebriated-like neurotransmitter, tryptophan hydroxylase and amino acid decarboxylase, present consistent altered expression in infected animals. Therefore, this study provides one of the first transcriptomic insights into the neuronal gene pathways altered in amphipods infected with a trematode parasite associated with changes to its host's behaviour and population structure.

Crustacean intersexuality is feminization without demasculinization: implications for environmental toxicology.

The dysfunction associated with intersexuality in vertebrates and molluscs is often a serious threat to ecosystems. Although poorly understood, crustacean intersexuality is associated with contamination and includes forms linked to increased sex-ratio distorting parasites at polluted sites. Despite the importance of crustaceans for monitoring vulnerable aquatic habitats, little is known about the molecular basis of this abnormal sexual differentiation and any associated sexual dysfunction. To increase the relevance of crustaceans to environmental toxicologists, we comprehensively analyzed gene expression in amphipods presenting parasite- and nonparasite-associated intersexuality. Our findings reveal existing vertebrate biomarkers of feminization should not be applied to crustaceans, as orthologous genes are not induced in feminized amphipods. Furthermore, in contrast to vertebrates, where feminization and intersexuality is often associated with deleterious demasculinization, we find males maintain masculinity even when unambiguously feminized. This reveals a considerable regulatory separation of the gene pathways responsible for male and female characteristics and demonstrates that evidence of feminization (even if detected with appropriate biomarkers) is not a proxy for demasculinization in crustaceans. This study has also produced a comprehensive spectrum of potential molecular biomarkers that, when combined with our new molecular understanding, will greatly facilitate the use of crustaceans to monitor aquatic habitats.

Population screening and transmission experiments indicate paramyxid-microsporidian co-infection in Echinogammarus marinus represents a non-hyperparasitic relationship between specific parasite strains.

Phylogenetically distant parasites often infect the same host. Indeed, co-infections can occur at levels greater than expected by chance and are sometimes hyperparasitic. The amphipod Echinogammarus marinus presents high levels of co-infection by two intracellular and vertically transmitted parasites, a paramyxid (Paramarteilia sp. Em) and a microsporidian strain (Dictyocoela duebenum Em). This co-infection may be hyperparasitic and result from an exploitative 'hitchhiking' or a symbiotic relationship between the parasites. However, the best-studied amphipod species are often collected from contaminated environments and may be immune-compromised. Immune-challenged animals frequently present co-infections and contaminant-exposed amphipods present significantly higher levels of microsporidian infection. This suggests the co-infections in E. marinus may result from contaminant-associated compromised immunity. Inconsistent with hyperparasitism, we find that artificial infections transmit Paramarteilia without microsporidian. Our population surveys reveal the co-infection relationship is geographically widespread but find only chance co-infection between the Paramarteilia and another species of microsporidian, Dictyocoela berillonum. Furthermore, we identify a haplotype of the Paramarteilia that presents no co-infection, even in populations with otherwise high co-infection levels. Overall, our results do not support the compromised-immunity hypothesis but rather that the co-infection of E. marinus, although non-hyperparasitic, results from a relationship between specific Paramarteilia and Dictyocoela duebenum strains.

Behavioural and transcriptional changes in the amphipod Echinogammarus marinus exposed to two antidepressants, fluoxetine and sertraline.

In the past decade, there have been increasing concerns over the effects of pharmaceutical compounds in the aquatic environment, however very little is known about the effects of antidepressants such as the selective serotonin re-uptake inhibitors (SSRIs). Many biological functions within invertebrates are under the control of serotonin, such as reproduction, metabolism, moulting and behaviour. The effects of serotonin and fluoxetine have recently been shown to alter the behaviour of the marine amphipod, Echinogammarus marinus (Leach, 1815). The purpose of this study was to observe behavioural and transcriptional modifications in this crustacean exposed to the two most prescribed SSRIs (fluoxetine and sertraline) and to develop biomarkers of neurological endocrine disruption. The animals were exposed to both drugs at environmentally relevant concentrations from 0.001 to 1µg/L during short-term (1h and 1day) and medium-term (8 days) experiments. The movement of the amphipods was tracked using the behavioural analysis software during 12min alternating dark/light conditions. The behavioural analysis revealed a significant effect on velocity which was observed after 1h exposure to sertraline at 0.01µg/L and after 1 day exposure to fluoxetine as low as 0.001µg/L. The most predominant effect of drugs on velocity was recorded after 1 day exposure for the 0.1 and 0.01µg/L concentrations of fluoxetine and sertraline, respectively. Subsequently, the expression (in this article gene expression is taken to represent only transcription, although it is acknowledged that gene expression can also be regulated at translation, mRNA and protein stability levels) of several E. marinus neurological genes, potentially involved in the serotonin metabolic pathway or behaviour regulation, were analysed in animals exposed to various SSRIs concentrations using RT-qPCR. The expression of a tryptophan hydroxylase (Ph), a neurocan core protein (Neuc), a Rhodopsin (Rhod1) and an Arrestin (Arr) were measured following exposure to fluoxetine or sertraline for 8 days. The levels of Neuc, Rhod1 and Arr were significantly down-regulated to approximately 0.5-, 0.29- and 0.46-fold, respectively, for the lower concentrations of fluoxetine suggesting potential changes in the phototransduction pathway. The expression of Rhod1 tended to be up-regulated for the lower concentration of sertraline but not significantly. In summary, fluoxetine and sertraline have a significant impact on the behaviour and neurophysiology of this amphipod at environmentally relevant concentrations with effects observed after relatively short periods of time.

Paramyxean-microsporidian co-infection in amphipods: is the consensus that Microsporidia can feminise their hosts presumptive?

The current consensus is that Microsporidia belong to a select group of parasites capable of causing both intersexuality and complete sex reversal in their hosts. Indeed, species such as Nosema granulosis and Dictyocoela duebenum, which infect amphipod crustaceans, are regularly referred to as 'feminising microsporidians'. This categorisation is based on a combination of findings: that these species are vertically transmitted and occur at a high prevalence of infection in intersex and female amphipods, that infected amphipod populations are female-biased, and that infected females have significantly female-biased broods with no concurrent increase in mortality. In this study, we report on a population of the amphipod Echinogammarus marinus presenting both female bias and high levels of intersexuality, which are infected with D. deubenum. In keeping with its feminising classification, infection is prevalent in animals presenting female and intersex phenotypes. However, a further screen revealed the presence of a previously unknown paramyxean parasite related to organisms of the genus Marteilia, a group known to cause catastrophic sexual dysfunction in bivalves. We found that the paramyxean parasite was also vertically transmitted, with infections being more prevalent in females and intersex animals. Critically, every animal infected with D. deubenum was also co-infected with the paramyxean, with few animals presenting an independent paramyxean infection. In contrast, co-infection of E. marinus with a paramyxean and the non-feminising microsporidian Dictyocoela berillonum rarely occurred. These observations raise a new hypothesis, namely, that D. duebenum and other feminising microsporidians are not actually capable of host feminisation but instead 'hitch-hike' together with a feminising paramyxean parasite.

Anti-depressants make amphipods see the light.

The effects of serotonin altering parasites, serotonin, the anti-depressant fluoxetine, plus two other highly prescribed pharmaceuticals (carbamazepine and diclofenac) on the behaviour of the marine amphipod, Echinogammarus marinus were investigated. Acanthocephalan parasites are known to alter the swimming behaviour in their amphipod hosts through changes in serotonergic activity resulting in increased predation. Behavioural assays were adapted to record changes in phototaxis and geotaxis behaviour in male E. marinus following 7, 14 and 21 days exposure to serotonin and each pharmaceutical compound at 4 concentrations compared to a control (between 10 ng/L and 10 microg/L). E. marinus infected with acanthocephalans parasites had both significantly higher phototaxis and geotaxis scores than those of uninfected specimens. Phototaxis and geotaxis behaviour increased significantly in a concentration-dependent manner with exposure to serotonin. Fluoxetine significantly altered phototaxis and geotaxis activity in what appeared to be a non-monotonic concentration response curve with the greatest behavioural changes observed at 100 ng/L. The main patterns of these behavioural responses were consistent between two trials and the 3 weeks exposure with specimens spending more time within the light and occurring higher in the water column. No obvious trends could be concluded in the phototaxis and geotaxis scores from individuals exposed to carbamazepine or diclofenac as might be expected from their known mode of action. From this study phototaxis and geotaxis behaviour have been observed to be affected by exposure to serotonin modulators. Parasite studies have shown strong links between changes in behaviour and increased predation risk correlating with changes in serotonergic activity. This study has highlighted the potential for highly prescribed anti-depressant drugs to change the behaviour of an ecologically relevant marine species in ways which could conceivably lead to population level effects.