Exposure to the antidepressant fluoxetine reduces mating behaviour in the freshwater isopod Asellus aquaticus

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Linköping University | Department of Physics, Chemistry and Biology Bachelor thesis, 16 hp | Biology programme: Physics, Chemistry and Biology Spring term 2019 | LITH-IFM-G-EX—19/3703--SE

Exposure to the antidepressant

fluoxetine reduces mating behaviour in

the freshwater isopod Asellus aquaticus

Hanna Norén

Examinator, Jordi Altimiras, IFM Biologi, Linköpings universitet Tutor, Hanne Løvlie, IFM Biologi, Linköpings universitet

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Department of Physics, Chemistry and Biology Linköping University Datum Date 2019-06-18 Språk Language Svenska/Swedish Engelska/English ________________ Rapporttyp Report category Licentiatavhandling Examensarbete C-uppsats D-uppsats Övrig rapport _____________ ISBN ISRN: LITH-IFM-G-EX--19/3703--SE _________________________________________________________________

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Titel

Title

Exposure to the antidepressant fluoxetine reduces mating behaviour in the freshwater isopod Asellus aquaticus Författare

Author

Hanna Norén

Nyckelord

Keyword

Fluoxetine, SSRI, long-term exposure, Asellus aquaticus, mating behaviour, wastewater Sammanfattning

Abstract

Worldwide, pharmaceutical compounds continue to increase in our aquatic environment. The predominant route into nature is through wastewater treatment plants since the elimination of residual pharmaceuticals is still not mainstream in WWTPs. Fluoxetine is an antidepressant which is commonly prescribed to treat human depression. Wastewater residual fluoxetine is typically found in waters around the world, and can thus affect exposed organisms, such as fish and invertebrates. However, how fluoxetine may affect mating behaviour in exposed organisms remains poorly understood, and particularly so in invertebrates. This is hampering our understanding of the consequences of our medicine leaking into nature because mating behaviour often affect fitness, and invertebrates are key organisms in food chains. Therefore, I here experimentally investigated long-term effects of environmental relevant concentration of fluoxetine (20 ng L-1_{) on mating behaviours of male and female freshwater isopod Asellus aquaticus. I demonstrate that} fluoxetine reduced male mating attempts with receptive females. Further, there was a tendency for fluoxetine exposure to increase latency to form pre-copula. There was no effect of fluoxetine exposure on male latency to encounter females or female responses toward males. These results indicate that fluoxetine also can affect isopods by reducing mating behaviour. In the long-term, if reproduction is delayed or reduced, it may cause a reduction in populations and thus, alter the whole ecosystem.

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1. Abstract

Worldwide, pharmaceutical compounds continue to increase in our aquatic environment. The predominant route into nature is through wastewater treatment plants (WWTPs) since the elimination of residual pharmaceuticals is still not mainstream in WWTPs. Fluoxetine is an antidepressant which is commonly prescribed to treat human depression. Wastewater residual fluoxetine is typically found in waters around the world, and can thus affect exposed organisms, such as fish and invertebrates. However, how fluoxetine may affect mating behaviour in exposed organisms remains poorly understood, and particularly so in invertebrates. This is hampering our understanding of the consequences of our medicine leaking into nature because mating behaviour often affect fitness, and invertebrates are key organisms in food chains. Therefore, I here experimentally investigated long-term effects of environmental relevant concentration of fluoxetine (20 ng L-1_{) on mating behaviours of male and female freshwater}

isopod Asellus aquaticus. I demonstrate that fluoxetine reduced male mating attempts with receptive females. Further, there was a tendency for fluoxetine exposure to increase latency to form pre-copula. There was no effect of fluoxetine exposure on male latency to encounter females or female responses toward males. These results indicate that fluoxetine also can affect isopods by reducing mating behaviour. In the long-term, if reproduction is delayed or reduced, it may cause a reduction in populations and thus, alter the whole ecosystem.

2. Introduction

A major challenge in ecology is the increasing amount of pharmaceuticals that enter the environment, and their effects on wild organisms (Mazzitelli et al. 2018). Pharmaceuticals are human medical products that are commonly used in both hospitals and at home. As a side effect of its use, pharmaceuticals are released into the environment. They are released especially through wastewater treatment plants (WWTPs) by excretion from urine and faeces, spreading to different aquatic communities (The Swedish Environmental Protection Agency 2017). WWTPs are normally not constructed to eliminate pharmaceuticals (The Swedish Environmental Protection Agency 2017; Daughton & Ternes 1999). In addition to human use of pharmaceuticals, veterinary medicines for agriculture and other domestic animals are a remarkable source of residual pharmaceuticals leaking into the environment (reviewed in Heberer 2002). Therefore, pharmaceuticals remain a considerable problem in the environment and the effluents of pharmaceuticals are only increasing.

A common pharmaceutical is fluoxetine, which is the most commonly prescribed antidepressant (Daughton & Ternes 1999; Weinberger & Klaper 2014; reviewed in Fong & Ford 2014) and dominated in hospital wastewater (Frédéric & Yves 2014). Fluoxetine is the active ingredient in the human pharmaceutical Prozac (Weinberger & Klaper 2014) and a selective serotonin reuptake inhibitor (SSRI), that causes an increase in the neurotransmitter serotonin in the brain (Foran et al. 2003; Winder et al. 2009; reviewed in Fong & Ford 2014). The underlying mechanism is that fluoxetine inhibits the reuptake of serotonin by binding to

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serotonin reuptake receptors (Foran et al. 2003; Winder et al. 2009). Fluoxetine has been reported in wastewater treatment plants influents and effluents and also in surface water (reviewed in Silva et al. 2012). Fluoxetine concentration in WWTPs effluents range from 2-99 ng L-1_{in North America and 0.12-929 ng L}-1_{in Europe, while the fluoxetine concentration in}

stream waters range from 4-141 ng L-1_{in North America and 8-44 ng L}-1_{in Europe (reviewed}

in Silva et al. 2012).

Environmentally relevant concentrations can impact various species in aquatic systems (e.g. De Lange et al. 2006; Lazzara et al. 2012; Martin et al. 2017; Weinberger & Klaper 2014). For example, fluoxetine affected anti-predator behaviour in eastern mosquitofish (Gambusia

holbrooki) through reduced freezing behaviour (Martin et al. 2017). On the other hand, exposed

wild guppies (Poecilia reticulata) remained longer in their freezing behaviour after a simulated predator attack (Saaristo et al. 2017). In fathead minnows (Pimephales promelas), fluoxetine impacted mating behaviour by decreasing male mating attempts (Weinberger & Klaper 2014) and concentration of fluoxetine as low as 1 ng/L affected the efficiency of learning in cuttlefish (Sepia officinali, Di Poi et al. 2013). In invertebrates, exposure to fluoxetine caused a greater attraction to light in Dapnia magna (Rivetti et al. 2016), induced spawning in zebra mussel (Dreissena polymorpha, Lazzara et al. 2012) and increased swimming velocity in Gammarus

pulex (De Castro-Català et al. 2017). On the other hand, a decrease in activity when exposed to

fluoxetine was reported in Gammarus pulex (De Lange et al. 2006). Therefore, it is important to further investigate the effects of fluoxetine as these can have mixed effects on behaviour. In both invertebrates and vertebrates, neurotransmitters affect several biological processes including behaviour, reproduction, maturation, immunity, growth, metabolism and colour physiology (Daughton & Ternes 1999; reviewed in Fong & Ford 2014). Due to the importance of mating behaviour for fitness, it is important to investigate the influence of SSRIs in organisms being unintentionally exposed to them.

Many insects and crustaceans have a pre-copulatory passive phase, where males guard females before mating (Hargeby et al. 2004; Parker, 1974; Ridley & Thompson 1979). This mate guarding is considered as a strategy where males remain attached to the female until she is ready to mate (Parker 1974). In isopods, males hold females between their legs with the fourth pair of paraepods (legs) and the pre-copula remains for six to eight days (Ridley & Thompson 1979). During this pre-copula, males are capable to crawl unlimited and both individuals can feed normally (Manning 1975). The number of females and which females that are encountered by males are important, because the time spent with a given female will decrease males’ opportunities to further encountered other females (Parker 1974). Females can respond to these mating attempts by being either receptive or unreceptive (i.e. accept or reject males). Female and male interactions thus can play a major role affecting fitness.

In this study, the macroinvertebrate Asellus aquaticus was used. Macroinvertebrates are consumers at the intermediate trophic level and thus play a vital role in freshwater ecosystems due to top-down and bottom-up actions such as decomposition (Wallace & Webster 1996) and prey for predator invertebrates or fishes (Hargeby 2004). Moreover, A. aquaticus is important for bioturbation of sediments that influence nutrient cycling and recruitment of zooplankton

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(Gyllström et al. 2008; Ståhl-Delbanco & Hansson 2002). There is a risk that fluoxetine or other pharmaceuticals can alter the behaviour of A. aquaticus and thus have indirect effects on aquatic ecosystems. Moreover, there are few studies that examine long-term exposure to fluoxetine in

A. aquaticus, especially when it comes to mating behaviour. Therefore, it is important to

examine effects of fluoxetine on mating behaviour in A. aquaticus.

In the present study, the aim was to investigate how the antidepressant fluoxetine affects the mating behaviour in an isopod. Four different behaviours were studied: (1) male latency to encounter a female, (2) male mating attempts, (3) female response to male mating attempts, and (4) latency to form pre-copula. The applied concentration of fluoxetine (20 ng L-1_{) was}

environmentally relevant for lakes and wastewater effluents in Sweden (Helmfrid & Eriksson 2010). According to previous findings (Weinberger & Klaper 2014; De Lange et al. 2006), fluoxetine is expected to change the mating behaviour of the isopods negatively. Males are predicted to decrease their mate search and attempting to mate with females while females are predicted not to change their behaviour towards males.

3. Materials and methods

3.1. Study organism

The experiment includes the isopod Asellus aquaticus. It is a wide-spread organism and lives in different freshwater systems, such as ponds, lakes and slow-flowing streams (Hargeby et al. 2004) throughout North America, Europe and Russia (Maltby 1991). A. aquaticus is a detritivore and feeds on leaf litter (Zimmer & Bartholme 2003), but it can also feed on periphyton (Arakelova 2001). Since A. aquaticus is a non-swimming isopod, it is a slow colonizer of new habitats (Hargeby 1990).

Before mating, males carry females, which is called precopulatory stage (amplexus, Hargeby et al. 2004). Amplexus remains a few days until the female has moulted to maturity, which means the process where the A. aquaticus is changing the old exoskeleton to form a new and hard exoskeleton (Fingerman 1987). Then, when an oviduct pouch is formed, she is ready to mate (Hargeby et al. 2004).

On 21st_{of March 2019, around 200 isopods were retrieved from the freshwater lake Tåkern (N}

6466063; E 489255) in Östergötland, Sweden. It was made by raking up vegetation, mainly stoneworts (Chara tormentosa), from the bottom at ca 1.2 metres depth. Isopods were then shaken off in a sieve (0.5 mm). After they were collected, they were taken to Linköping University and kept in a cold (5 ºC) and dark room in five plastic boxes (40 cm x 30 cm x 17 cm; length x width x height) for five days before the experiment started. These plastic boxes were filled with filtrated water from Lake Tåkern. The water was filtrated to remove other organisms. Freshwater was stored for at least 96 hours before use, to get rid of odour from predators (Peacor 2006).

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3.2. Experimental design

On 27th_{of March 2019, a fluoxetine stock solution was made by dissolving solid fluoxetine}

hydrochloride (F132, Sigma-Aldrich) in deionized water to a concentration of 1 mg L-1_{. In the}

experiment, a concentration of 20 ng L-1_{of fluoxetine was used to reflect observed}

concentrations in Swedish WWTPs effluents and lakes (Helmfrid & Eriksson 2010; Swedish Environmental Protection Agency 2008). In lake Tåkern, there are no measurements of its fluoxetine concentration. However, in a nearby lake, Roxen, surface water samples showed 20 ng L-1_{of fluoxetine in the year 2008 (Helmfrid & Eriksson 2010). I therefore chose to use this}

concentration. During my experiment, the stock solution was stored in darkness at 5 ℃. The half-life of fluoxetine ranges from 102-385 days when fluoxetine is solved in a buffer solution depending on the pH-value (Kwon & Armbrust 2006), meaning that fluoxetine should not break down during the course of my experiment.

One day prior to the experiment, on 26th_{of March 2019, isopods were separated into two groups}

depending on sex, males (larger, and longer legs) and females (smaller, and with eggs). Individuals who were captured in mating pairs were carefully separated by hand. Isopods were placed in tanks (16 cm x 14 cm x 14 cm; length x width x height) with filtrated freshwater, and were fed with decaying elm and alder leaves, which had been colonized with microorganisms for about three weeks. Isopods were acclimatized in room temperature for 24 hours, before exposure to the chemical treatment. The following day, each of the two groups were divided into two treatments (control, and exposed to fluoxetine). For each group and treatment, isopods were divided into at least two replicates, depending on the number of individuals available for each group. Around 5-15 isopods, depending on the total individuals in each group, were placed into each group with ca 2 litres of filtrated freshwater. Lastly, fluoxetine (20 ng L-1_{) was added.}

In my control treatment, the same amount of freshwater was added to the control groups. Each tank was oxygenated with an air pump, and decaying elm and alder leaves were added as needed. Once a week, the water was changed, and 20 ng L-1_{of fluoxetine was added. Light and}

dark regimes mimicked natural conditions through windows and the water temperature was 17-18 ℃ during the experiment, which is a common temperature when testing the behaviour of A.

aquaticus (Augusiak & Van den Brink 2016; Eroukhmanoff & Svenssson 2009; Hargeby &

Erlandsson 2006; Harris et al. 2013; Jormalainen & Merilaita 1995; Karlsson et al. 2010).

3.3. Behavioural observations

Behavioural observations were performed in daylight (14:00-19:30), 18 days after the exposure to fluoxetine had started. For these observations, 54 females and 54 males from both control and exposure animals were used, and formed twenty-seven pairs (fluoxetine males with fluoxetine females, and control males with control females). Behavioural observations were carried out in novel tanks (ø 6,5 cm x 4 cm) and animals were exposed to their original treatments (i.e. either freshwater, or water with fluoxetine, 20 ng L-1_{). Observations were}

alternated between pairs of each treatment to reduce any effect of time of day when testing occurred. Individuals were randomly selected to the experiment. Females were in the novel tank for 30 seconds before males were added, and the observations started. Four behaviours were

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recorded with the camera Sony HDR-as50: (1) Male latency to encounter a female (measured in seconds from the time the male was placed in the tank until he encountered the female with his antennas), (2) male mating attempts (whether the males tried to mate), (3) female response to male mating attempts (whether the female was receptive toward the male’s mating attempt), and (4) latency to form pre-copula where a male carries a female (measured in seconds from when the male encountered the female to that he held her between his legs).

3.3.1. Male latency to encounter a female

To test if fluoxetine affected male latency to encounter a female, I recorded it as a measure of male mate searching ability. In another crustacean, antennas play a major role in detecting a female during mate search (Dahl et al. 1970; Dunn 1998). When a male encounter any part of a female with his antennae, he was regarded to have sexually detected the female (Vandel 1926, in Manning 1975).

3.3.2. Male mating attempts

To test if fluoxetine affected male mating attempts, I recorded if males tried to mate and the number of mating attempts. Observations started after the male had encountered the female for the first time with his antennas. Thereafter, I noticed if the male attempted to pre-copulate by struggling to place the female correctly under him. Males’ responses to attempt to pre-copulate was reported as 0 (not trying) or 1 (trying), and for males which were trying to form pre-copula, the number of attempts were counted.

3.3.3. Female response to male mating attempts

To test if fluoxetine affected female receptivity, the female´s behaviour towards a male was recorded. If the female was unreceptive, she either ran away or turned over on her back after the male encountered her with his antennas. In another isopod, Idotea balthica, females resist copulation by kicking and bending their bodies (Jormalainen & Merilaita 1993). Females´ responses toward males were reported as zeros or ones (0 = unreceptive, 1 = receptive).

3.3.4. Latency to form pre-copula

In A. aquaticus, the male places the female between the legs and holds the female by the fourth pair of paraepods (legs) (De Geer 1778, in Ridley & Thompson 1979). To test if fluoxetine affected male latency to form pre-copula (i.e. amplexus), the time it took for the male to form pre-copula from the time he detected the female, was recorded. Maximum time was set to 240 seconds if there was no formation of amplexus.

3.4. Statistical analyses

Due to data not meeting assumption for parametric statistics, I used non-parametric statistics. To investigate eventual correlations between male latency to encounter a female and latency to form pre-copula, a Spearman rank correlation test was used. Spearman rank correlation test was performed for each treatment group (fluoxetine or control). There was no correlation between

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male latency to encounter a female and latency to form pre-copula in fluoxetine (rs = 0.27, p =

0.18) or in control (rs = -0.09, p = 0.65). Therefore, both behaviours were individually analysed

further.

To investigate effects of fluoxetine on mating behaviour in A. aquaticus, different Mann-Whitney U tests were used to examine whether male latency to encounter a female and latency to form pre-copula were affected of fluoxetine. Mann-Whitney U test was also performed to examine if fluoxetine impacted the number of male mating attempts. In each test, fluoxetine and control were independent groups while the behaviour that was measured was the dependent variable. Fisher´s exact test were used to examine whether males were trying to form pre-copula or not. I analysed the difference of the number of males who were trying to form a pre-copula between fluoxetine exposure and control. Fisher´s exact test was also applied to examine female response to male mating attempts, if they were receptive or unreceptive to form pre-copula. I analysed the difference of the number of females who were receptive to form a pre-copula between fluoxetine exposure and control.

All statistical analyses were performed in IBM SPSS statistics (version 25).

4. Results

4.1. Male latency to encounter a female

There was no effect of fluoxetine on male latency to encounter a female, compared to control males (fluoxetine males, mean ± SE: 34.00 ± 7.22 s; control males, mean ± SE: 20.09 ± 5.09 s; U = 296.50, p = 0.24, Figure 1).

Figure 1. Male latency to encounter a female after exposure to fluoxetine (black), or not (white) in A. aquaticus. Columns show mean ± SE, n = 27.

4.2. Male mating attempts

Fluoxetine exposure reduced male mating attempts to form pre-copula (fluoxetine males, frequency: 0.74; control males, frequency: 0.96: p = 0.05, Figure 2). However, fluoxetine exposure did not affect the number of mating attempts males made (fluoxetine males, mean ± SE: 1.18 ± 0.19; control males, mean ± SE: 1.41 ± 0.18; U = 306.50, p = 0.37).

0 10 20 30 40 50 Fluoxetine Control Tim e (s )

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Figure 2. Frequency of males attempting to form pre-copula after exposure to fluoxetine (black), or not (white) in A. aquaticus, n = 27.

4.3. Female response to male mating attempts

Fluoxetine exposure did not affect female receptivity to mate, compared to control females (fluoxetine females, frequency: 0.52; control females: frequency: 0.59; p = 0.78, Figure 3).

Figure 3. Frequency of females which were receptive after exposure to fluoxetine (black), or not (white) in A. aquaticus, n = 27.

4.4. Latency to form pre-copula

There was a tendency that fluoxetine increased the time it took for males to form pre-copula with females (U = 260.00, p = 0.053). Pre-copula formation in exposed males was either more delayed or failed, compared to the control group (fluoxetine pairs, mean ± SE: 190.21 ± 15.32 s; control pairs, mean ± SE: 127.87 ± 20.06 s, Figure 4).

0 0,2 0,4 0,6 0,8 1 1,2 Fluoxetine Control Fre q u en cy 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 Fluoxetine Control Fre q u en cy

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Figure 4. Latency to form pre-copula after exposure to fluoxetine (black), or not (white) in A. aquaticus. Columns show mean ± SE, n = 27.

5. Discussion

I have here shown that exposure of fluoxetine did affect mating behaviour in Asellus aquaticus. Male mating attempts with receptive females were reduced when males had been exposed to fluoxetine for 18 days, compared to control males that had not been exposed to the antidepressant. Furthermore, there was a tendency that fluoxetine exposure increased male latency to form a pre-copula, compared to control males. However, fluoxetine had no impact on male latency to encounter a female or female response to male mating attempts.

My results showed that there was no effect of fluoxetine on male latency to encounter a female. Males use their antennas to detect females (Dahl et al. 1970; Dunn 1998). However, the small arena (ø 6.5 cm x 4 cm) used may have contributed to males randomly encountered females more than searched for her, masking any difference fluoxetine exposure may have caused. Studies have shown that male latency to encounter a female the first time decreased with the size of the experimental tank (Bertin & Cézilly 2005). Males run faster to encounter a female in small tanks (5 × 5 × 3.5 cm) compared to medium (10 × 10 × 3.5 cm) and large tanks (28 × 42 × 8 cm) (Bertin & Cézilly 2005). This suggest that the size of experimental arenas is important when studying male latency to encounter a female for the first time, and future studies should take this into consideration.

A previous study has shown that A. aquaticus has an average pre-copulation time at 512 seconds (Eroukhmanoff et al. 2009). In some cases, it may take hours before a male pre-copulate with a female (Eroukhmanoff et al. 2009; Bertin & Cézilly 2003). In the present study, a maximum time was set to 240 seconds for all the four behavioural observations. Therefore, this may have resulted in that not all males had the chance to form pre-copula, and thus affected the results. In total for both treatments, half of the pairs (27 of 54) formed pre-copula. If males had been given longer time, more of them may have formed a pre-copula and then, it may have been a greater difference between exposed and unexposed males. This should also be considered by future experiments.

Fluoxetine exposure affected male mating attempts to carry a female in pre-copula, where fewer males attempted to mate when they had been exposed to fluoxetine. However, there was no

0 50 100 150 200 250 Fluoxetine Control Tim e (s )

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effect of fluoxetine exposure on the number of male mating attempts for the males that tried to mate. This indicates that fluoxetine influenced the act of attempting to mate, but not the number of mating attempts. In the fish, fathead minnow (Pimephales promelas), fluoxetine exposure affected males by a decrease in frequency of attempts to mate with females (Weinberger & Klaper 2014). This finding is in line with my result, where fluoxetine exposure reduced male mating attempts to form pre-copula. It is also similar to my result where there was a tendency for fluoxetine exposure to increase latency to form pre-copula. The effect of fluoxetine exposure may also be linked to a decrease in activity. Fluoxetine is known to invoke a decrease in activity in another crustacean, Gammarus pulex (De Lange et al. 2006) and the Mediterranean field cricket (Gryllus bimaculatus, Abbey-Lee et al. 2018). On the other hand, in eastern mosquitofish (Gambusia holbrooki) exposure to fluoxetine led to more copulation attempts by males, compared to unexposed males (Bertram et al. 2018). This difference suggests that fluoxetine exposure differs for different organisms, but also for different concentrations of fluoxetine. Fluoxetine appears to be dose-dependent, showing effects for both high and low concentrations (Weinberger & Klaper 2014). If fluoxetine can affect the mating behaviour of isopods, it may also mean that fluoxetine can affect the mating behaviour of animals due to similarities in monoaminergic systems across animals. Fluoxetine is only one of the pharmaceutical residues and together with other chemicals it may cause cocktail effects, and the effects may be increased. Therefore, it gives increased knowledge that pharmaceutical residues in nature have adverse effects.

Previous findings and my results in this study shows that fluoxetine somehow alter behaviour in several organisms, and this is probably due to that fluoxetine is a selective serotonin reuptake inhibitor (SSRI). SSRIs act by imitate and modulate effects of serotonin (5-HT, reviewed in Silva et al. 2012) by inhibiting the reuptake of serotonin by binding to serotonin reuptake receptors (Foran et al. 2003; Winder et al. 2009). Fluoxetine is made to treat human disorders and not to, for example, affect aquatic organism. The question is if the serotonergic system is similar for both vertebrates and invertebrates. In vertebrates, the 5-HT receptors belong to G protein-coupled receptors (GPCRs) and ligand-gated ion-channel families (reviewed in Vleugels et al. 2015). There are seven classes of 5-HT receptors in vertebrates and these receptors are similar to the receptors found in invertebrates, but all the receptors in vertebrates cannot be compared to the invertebrates’ receptors (reviewed in Blenau & Baumann 2001, Tierney 2001 and Vleugels et al. 2015). Even the pharmacological characteristics of receptors differ between vertebrates and invertebrates (reviewed in Blenau & Baumann 2001, Tierney 2001 and Vleugels et al. 2015). The serotonin transport protein (SERT) in humans is homologous (around 90 %) to other vertebrates, but only homologous with 44-53 % with invertebrates (Murphy 2004). This may suggest that fluoxetine acts on other receptors or affects SERT in a different way, and therefore causes effects in invertebrates. However, fluoxetine has a side effect in humans which result in a decreased sexual desire (FASS 2015). This can be compared to the result in this study where male mating attempts were reduced when exposed to fluoxetine. This, however, shows that the serotonergic system is similar for both vertebrates and invertebrates.

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Females did not behave differently with fluoxetine exposure compared to control females. It was only the males’ behaviours that were affected by fluoxetine. This outcome is in line with results on fathead minnows (Pimephales promelas, Weinberger & Klaper 2014). In many organisms, there is a sexual dimorphism, which also occurs in Asellus aquaticus (Bertin et al. 2002). Further, males and females have differences in physiology. This may translate into that fluoxetine affects the sexes through different behavioural changes in females and males. Different behavioural changes in this case, when males go slow and females do not change, may translate into an imbalance between their interactions. This may lead to males failing to detect when females are receptive and ready to mate, and thus, reduce reproduction.

Benthic invertebrates are important for bioturbation, especially the isopods Asellus aquaticus (Gyllström et al. 2008; Ståhl-Delbanco & Hansson 2002). If there is a decrease in number of isopods, it may reduce the recruitment of zooplankton due to a decrease in bioturbation (Gyllström et al. 2008). This can lead to an imbalance in trophic levels and thus affect ecosystems. An influence on mating behaviour in isopods can thus affect also other aquatic organisms, and the effect of fluoxetine may have consequences on a larger scale.

In conclusion, I demonstrate that exposure to an experimentally relevant concentration of the antidepressant fluoxetine for 18 days altered mating behaviour in the freshwater isopod Asellus

aquaticus. Fluoxetine exposure reduced male mating attempts which is the first study, to my

knowledge, to examine the impact of fluoxetine on mating behaviour in Asellus aquaticus. This suggests that human medicine leaking out in the environment can alter mating behaviour which, in turn, may have negative ecological consequences. Reduced male mating attempts may result in delaying or reducing reproduction in this key organism. A potential reduction in populations of isopods can, in turn, cause other trophic levels to be impacted because each trophic level is interdependent. In the long-term, the whole ecosystem may be altered.

5.1. Social and ethical aspects

It is known that antidepressants are found in our wastewater and disperse further in the environment. This is a problem for aquatic ecosystems considering that antidepressants can affect many kinds of organisms. If antidepressants affect one organism, it may even cause ecosystem effects because organisms are interdependent. Knowledge about how the antidepressant fluoxetine affects the freshwater isopod Asellus aquaticus can identify potential effects of fluoxetine for example other insects or fish. Therefore, it is important to investigate how antidepressants can affect aquatic organisms, like Asellus aquaticus.

Asellus aquaticus is an animal which is important for our ecosystems. However, according to 7

chapter 6 § in Swedish regulation of animal welfare (SFS 2019:66), no ethical permission is required for animal experimentation with invertebrates. Even though the law approves you, one should always handle organisms with care because of the stress that can occur within this type of experiment.

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6. Acknowledgement

First, I want to send a big thank you to my tutor Hanne Løvlie for your guidance, advise and help to make this possible, and of course, thank you to Anders Hargeby for all support and help in the laboratory with Asellus aquaticus. I also want to thank my fellow students, Erik Isakson and Lisa Andrésen, for the cooperation with Asellus and for all discussions throughout the project. I must not forget Kristoffer Lundgren, who helped us to prepare fluoxetine-solution, and Løvlie group for discussion. A big thank you for that.

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