Alex T. Ford

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.

The biological effects of antidepressants on the molluscs and crustaceans: a review

Antidepressants are among the most commonly detected human pharmaceuticals in the aquatic environment. Since their mode of action is by modulating the neurotransmitters serotonin, dopamine, and norepinephrine, aquatic invertebrates who possess transporters and receptors sensitive to activation by these pharmaceuticals are potentially affected by them. We review the various types of antidepressants, their occurrence and concentrations in aquatic environments, and the actions of neurohormones modulated by antidepressants in molluscs and crustaceans. Recent studies on the effects of antidepressants on these two important groups show that molluscan reproductive and locomotory systems are affected by antidepressants at environmentally relevant concentrations. In particular, antidepressants affect spawning and larval release in bivalves and disrupt locomotion and reduce fecundity in snails. In crustaceans, antidepressants affect freshwater amphipod activity patterns, marine amphipod photo- and geotactic behavior, crayfish aggression, and daphnid reproduction and development. We note with interest the occurrence of non-monotonic dose responses curves in many studies on effects of antidepressants on aquatic animals, often with effects at low concentrations, but not at higher concentrations, and we suggest future experiments consider testing a broader range of concentrations. Furthermore, we consider invertebrate immune responses, genomic and transcriptomic sequencing of invertebrate genes, and the ever-present and overwhelming question of how contaminant mixtures could affect the action of neurohormones as topics for future study. In addressing the question, if antidepressants affect aquatic invertebrates at concentrations currently found in the environment, there is strong evidence to suggest the answer is yes. Furthermore, the examples highlighted in this review provide compelling evidence that the effects could be quite multifaceted across a variety of biological systems.

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.

Epibenthic diversity in the North Sea

In 1999 the epibenthic fauna of the North Sea was investigated using the 3rd quarter ‘International Bottom Trawl Survey’ of five European countries. Altogether 241 stations were sampled covering 143 ICES rectangles.The objectives of the project were (i) to analyse epibenthic diversity patterns in the North Sea, (ii) to identify the spatial distribution of faunal communities and (iii) to relate environmental factors as well as fishing effort to species diversity.Epibenthic fauna was clearly divided between the southern North Sea and the central-northern North Sea, roughly along the 50 m depth line. The separation was based on an overall higher number of species in the central and northern North Sea and a change in the species composition from north to south.Sessile fauna including erect, branching species like bryozoans and hydrozoans were particularly diverse along a corridor in the central-northern North Sea between 56° and 58°N, coinciding with the area between the 50 m and 100 m depth line. Cluster analysis, based on the structure of the community, confirmed the north-south gradient found for species diversity. Separation of clusters was driven to a great extent by species occurring predominantly or exclusively north of the 50 m contour line. Few species were exclusive to the south, but a number of scavenging species were found here more frequently and in higher numbers.Depth was positively correlated with the diversity of free-living fauna, whereas the type of sediment showed no significant relationship with variations in numbers of species. Beam-trawling effort was negatively correlated with the diversity of sessile fauna.

Infertility in male aquatic invertebrates: A review

As a result of endocrine disruptor studies, there are numerous examples of male related reproductive abnormalities observed in vertebrates. Contrastingly, within the invertebrates there have been considerably less examples both from laboratory and field investigations. This has in part been due to a focus of female related endpoints, inadequate biomarkers and the low number of studies. Whether contaminant induced male infertility is an issue within aquatic invertebrates and their wider communities therefore remains largely unknown and represents a key knowledge gap in our understanding of pollutant impacts in aquatic wildlife. This paper reviews the current knowledge regarding pollutants impacting male infertility across several aquatic invertebrate phyla; which biomarkers are currently being used and where the science needs to be expanded. The limited studies conducted so far have revealed reductions in sperm numbers, examples of poor fertilisation success, DNA damage to spermatozoa and inhibition of sperm motility that can be induced by a range of environmental contaminants. This limited data is mainly comprised from laboratory studies with only a few studies of sperm toxicity in natural populations. Clearly, there is a need for further studies in this area, to include both laboratory and field studies from clean and reference sites, with a focus on broadcast spawners and those with direct fertilisation. Biomarkers developed for measuring sperm quantity and quality in vertebrates are easily transferable to invertebrates but require optimisation for particular species. We discuss how sperm tracking and techniques for measuring DNA strand breaks and sperm viability have been successfully transferred from human infertility clinics to aquatic invertebrate ecotoxicology. Linking sperm toxicity and male infertility effects to higher level impacts on the reproductive biology and dynamics of populations requires a much greater understanding of fertilisation dynamics and sperm competition/limitation for invertebrate species and represents the next challenge in our understanding of male toxicity effects in natural populations.

Infertility in a marine crustacean: have we been ignoring pollution impacts on male invertebrates?

Invertebrate infertility has been under-explored as a potential ecological issue or biomarker of stress within ecotoxicology. To date, the majority of studies focussing on contaminant induced infertility have centred on vertebrate groups. This study aimed to address the question whether industrial pollution has the ability to influence the sperm counts and testicular morphology of male amphipod, Echinogammarus marinus (Leach). In addition, the sperm counts of normal and intersex specimens were compared to assess the potential impact of a crustacean with a disrupted endocrine system. Specimens of E. marinus were collected at one industrially impacted (Inverkeithing) and two reference (Thurso and Loch Fleet) sites along the north and eastern coasts of Scotland. Significantly higher sperm counts ( approximately 20%) were observed from normal males collected from reference sites compared to the industrially impacted site. Higher proportions (30%) of intersex specimens were observed at the industrially impacted site compared to 17 and 6% male intersexuality observed at Thurso and Loch Fleet, respectively. Intersex male specimens from Thurso had lower mean sperm counts ( approximately 15%) than normal male specimens, however, this result was not significant (P=0.089). No significant differences in sperm counts were observed between normal and intersex males at Inverkeithing. Our results indicate that industrial pollution does have the potential to affect the sperm counts of male crustaceans. Whether the quality of sperm in Crustacea from contaminated sites is also compromised or whether this is an endocrine mediated effect is yet to be confirmed. To date, many of the studies of endocrine disruption in crustaceans have, surprisingly, focussed on female fecundity parameters, growth and moulting despite many of the vertebrate studies initially focussing on the male gender. Whether this could be an ecological issue needs to be addressed through further field and laboratory based studies. It is recommended that, strategically, considerable information regarding developing methodologies could be gained from scientists working in the crustacean aquaculture industries and crustacean taxonomists.

Intersexuality in Crustacea: an environmental issue?

This paper aims to give a historical overview of current understanding about intersexuality in crustaceans, assesses gaps in our knowledge and asks whether it should be an environmental concern. The oldest known cases of intersexuality come from 70 million year old fossil crabs whilst the oldest published case of intersex crustacean stems from a 1730 Royal Society report of a gynandromorph lobster. Many crustacean species are sequential hermaphroditic or simultaneous hermaphrodites. Consequently, there has been confusion as to whether accounts of intersex in the literature are correct. Intersexuality is fairly common throughout the Crustacea and it has been suggested that intersex may arise through different mechanisms. For example, sexual gynandromorphism may arise through disruption in early embryonic development whereas intersexuality may also arise through perturbations of androgenic gland hormone and sexual differentiation in later development. The causes of intersex are multifaceted and can occur through a number of mechanisms including parasitism, environmental sex determination, genetic abnormalities and increasingly pollution is being implicated. Despite many studies on the effects of endocrine disrupters on crustaceans, very few have focussed on wild populations or male related endpoints; rather many laboratory studies have been attempting to assess biomarkers of feminisation. This is surprising as many of the seminal papers on endocrine disruption focussed on effects found in the wild and male specimens. This paper argues that we might have been addressing the right questions (i.e. pollution induced intersex), but in the wrong way (feminisation); and therefore gives recommendations for future directions for research. Biomarker development has been hampered by paucity of genomic and endocrine knowledge of many crustacean model species; however this is rapidly changing with the advent of cheaper affordable genomic techniques and high throughput sequencing.

The effects of antidepressants appear to be rapid and at environmentally relevant concentrations

The effects of antidepressants on wildlife are currently raising some concern because of an increased number of publications indicating biological effects at environmentally relevant concentrations (<100 ng/L). These results have been met with some scepticism because of the higher concentrations required to detect effects in some species and the perceived slowness to therapeutic effects recorded in humans and other vertebrates. Because their mode of action is thought to be by modulation of the neurotransmitters serotonin, dopamine, and norepinephrine, aquatic invertebrates that possess transporters and receptors sensitive to activation by these pharmaceuticals are potentially affected by them. The authors highlight studies on the effects of antidepressants, particularly on crustacean and molluskan groups, showing that they are susceptible to a wide variety of neuroendocrine disruptions at environmentally relevant concentrations. Interestingly, some effects observed in these species can be observed within minutes to hours of exposure. For example, exposure of amphipod crustaceans to several selective serotonin reuptake inhibitors can invoke changes in swimming behavior within hours. In mollusks, exposure to selective serotonin reuptake inhibitors can induce spawning in male and female mussels and foot detachment in snails within minutes of exposure. In the light of new studies indicating effects on the human brain from selective serotonin reuptake inhibitors using magnetic resonance imaging scans, the authors discuss possible reasons for the discrepancy in former results in relation to the read-across hypothesis, variation in biomarkers used, modes of uptake, phylogenetic distance, and the affinity to different targets and differential sensitivity to receptors.

Notes on the occurrence of intersex in Amphipods

It has long been assumed that intersex is common amongst the Amphipoda. An investigation of museum specimens, and a review of relevant literature were conducted with the aim of assessing how widespread intersexuality was amongst this group of Crustacea. We demonstrate that intersex is, indeed, widespread throughout the Amphipoda, occurring in at least 20 different species covering several different families, from marine to freshwater habitats, and from Arctic, Temperate and Mediterranean environments.

Can you feminise a crustacean

The ability of anthropogenic chemicals to cause reproductive disorders has been the focus of toxicologists for many years. Whilst the focus of endocrine disrupting chemicals has mainly been associated with vertebrate groups, there have been continued calls for more research on the invertebrates. Surprisingly, within the Crustacea, many studies have focussed on female or growth/moulting related endpoints despite many of the vertebrate studies highlighting male related effects such as abnormal male reproductive development. Furthermore, a large number of the invertebrate studies have focussed on vertebrate estrogens or their mimics. Considering the biology of the crustacean endocrine systems, this paper shall argue that unlike the vertebrates, it is a lot more difficult to feminise a crustacean than it is to de-masculinise one. Consequently, crustacean toxicologists, by following the tact of vertebrate biologists, may have been trying to address the right questions, but in the wrong way. Studies have shown that intersexuality in crustaceans may arise through the masculinisation of heterogametic (WZ) females or the de-masculinisation of males through aberrations in male androgenic gland activity. It is recommended that the focus be put on understanding the mechanisms of sex determination in Crustacea, and the expression of male secondary sexual characteristics at the molecular, biochemical and physiological level are fully explored so that appropriate assessments can be made as to whether sexual endocrine disruption is occurring in this ecologically important group.