Neuroendocrine pathways at risk? Simvastatin induces inter and transgenerational disruption in the keystone amphipod Gammarus locusta

1 Neuroendocrine pathways at risk? Simvastatin induces inter and transgenerational disruption in the 1 keystone amphipod Gammarus locusta 2 3 T. Neupartha,£,⁎, N. Alvesa,b,£, A.M. Machadoa,b, M. Pinheiroa,b, R. Montesd, R. Rodild, S. Barrosa,c, R. 4 Ruivoa, L. Filipe C. Castroa,b, J.B. Quintanad, M.M. Santosa,b,⁎ 5 6 a CIIMAR—Interdisciplinary Centre of Marine and Environmental Research, University of Porto, 7 Avenida General Norton de Matos, S/N, 4450-208 Matosinhos, Portugal 8 b FCUP – Department of Biology, Faculty of Sciences, University of Porto, Porto, Portugal 9 c CITAB – Centre for the Research and Technology of Agro-Environmental and Biological Sciences, 10 Quinta de Prados – Ed. Blocos Laboratoriais C1.10, 5000-801, Vila Real, Portugal 11 d Department of Analytical Chemistry, Nutrition and Food Sciences, IAQBUS – Institute of Research on 12 Chemical and Biological Analysis, Universidade de Santiago de Compostela, R. Constantino Candeira 13 S/N, 15782 Santiago de Compostela, Spain 14 * Corresponding authors: CIIMAR - Group of Endocrine Disruptors and Emerging Contaminants, 15 University of Porto, Avenida General Norton de Matos S/N, 4450-208, Matosinhos, Portugal (T. 16 Neuparth) and FCUP - Department of Biology, Faculty of Sciences, University of Porto, Porto, Portugal 17 (M.M. Santos). 18 E-mail addresses: tneupar[email protected]t (T. Neuparth), miguel.san[email protected]t (M.M. Santos) 19 £ T. Neuparth and N. Alves contributed equally to this study. 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 This is the postprint (accepted manuscript) version of the article published in Aquatic Toxicology http://dx.doi.org/10.1016/j.aquatox.2022.106095, © 2022, Elsevier. This manuscript version is made available under the CC-BY-NC-ND 4.0 license: http://creativecommons.org/licenses/by-nc-nd/4.0/ Highlights -Simvastatin Inter and transgenerational effects are not well understood in aquatic organisms -It´s unexplored if simvastatin effects are associated with neuroendocrine system disruption -Simvastatin inter/transgenerational exposure severely impacts G.locusta neuroendocrine regulation -Ecdysteroid, Dopamine, NO/cGMP/PKG, GABA and Cholinergic signaling pathways are affected - These findings improve risk assessment of biological active compounds, such as Simvastatin 2 ABSTRACT 37 The primary focus of environmental toxicological studies is to address the direct effects of chemicals 38 on exposed organisms (parental generation – F0), mostly overlooking effects on subsequent non39 exposed generations (F1 and F2 – intergenerational and F3 transgenerational, respectively). Here, we 40 addressed the effects of Simvastatin (SIM), one of the most widely prescribed human pharmaceuticals 41 for the primary treatment of hypercholesterolemia, using the keystone crustacean Gammarus locusta. 42 We demonstrate that SIM, at environmentally relevant concentrations, has significant inter and 43 transgenerational (F1 and F3) effects in key signalling pathways involved in crustaceans´ 44 neuroendocrine regulation (Ecdysteroids, Catecholamines, NO/cGMP/PKG, GABAergic and Cholinergic 45 signalling pathways), concomitantly with changes in apical endpoints, such as depressed reproduction 46 and growth. These findings are an essential step for improving hazard and risk assessment of biological 47 active compounds, such as SIM and highlight the importance of studying the transgenerational effects 48 of environmental chemicals in animals’ neuroendocrine regulation. 49 Keywords: Simvastatin; Gammarus locusta; Neuroendocrine signalling pathways; Transcriptomic; Inter 50 and Transgenerational effects; Regulatory agencies. 51 52 1.INTRODUCTION 53 Aquatic environments are the ultimate reservoirs for most anthropogenic chemicals. Accordingly, 54 aquatic organisms are chronically exposed during critical periods in early life stages or even for an 55 entire life cycle (Capela et al., 2016; Groh et al., 2015; Major et al., 2020). Knowledge of the hazards 56 and risks associated with chronic exposure to anthropogenic chemicals has grown considerably over 57 the last decades. However, contrasting with the extensive literature dealing with the direct effects of 58 chemicals on parental exposed organisms (F0), little is known about the effects that parental exposure 59 (F0) exerts on indirectly exposed (F1 and F2) or truly non-exposed (F3) generations – intergenerational 60 and transgenerational effects (Bhandari et al., 2015; Kalichak et al., 2019; Neuparth et al., 2020a). 61 Yet, given the potentially broad ecological impacts of such effects in aquatic ecosystems, a growing 62 scientific interest has emerged (Shaw et al., 2017; Van Cauwenbergh et al., 2020). Still, only a limited 63 number of inter and transgenerational studies are available in the literature, focusing on a few 64 endocrine disrupting chemicals (EDCs; e.g. Bisphenol A, some pesticides and phthalates), known for 65 their ubiquity in the environment and associated with reproductive endocrine disorders (Brevik et al., 66 2018; Li et al., 2020; Santangeli et al., 2019). However, many other natural and synthetic substances, 67 including pharmaceuticals, are suspected to hijack endocrine functions and disrupt reproduction, 68 development, neuronal processes and/or other important physiological responses; not only in exposed 69 individuals, but also in subsequent non-exposed generations (Gillette et al., 2018; Tuscher and Day, 70 3 2019; Walker and Gore, 2011). One of such pharmaceuticals is the hypocholesterolaemic 71 pharmaceutical simvastatin (SIM). SIM is among the most prescribed human pharmaceuticals. 72 Hovever, only a few studies have examined its concentrations in surface waters. According with the 73 available information, SIM can reach the aquatic environments, in the vicinity of urban areas, with 74 concentrations above 100 ng/L (Norway 108 ng/L - Langford and Thomas, 2011; India 414.9 ng/L - Khan 75 et al., 2021 and South Africa 1585 ng/L - Tete et al., 2020). SIM mode of action operates through 76 inhibition of the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), a rate-limiting 77 step of the mevalonate pathway (MP), responsible for the de novo synthesis of cholesterol in 78 vertebrates and the synthesis of the methyl farnesoate hormone (MF) in crustaceans, with a key role 79 in the regulation of reproduction and molting (Bellés X. et al., 2005; Fent et al., 2006; Li et al., 2010; 80 Santos et al., 2016). Although the final steps of the mevalonate pathway differ between vertebrates 81 and crustaceans, most of the enzymatic cascade is highly conserved among metazoans (Santos et al., 82 2016). In our previous research (Neuparth et al., 2020a), G. locusta was exposed for 4 consecutive 83 generations (F0 to F3) to environmentally relevant concentrations of SIM (64 and 320 ng/); 84 simultaneously, the offspring of the F0-exposed generation were raised in SIM-free water during three 85 subsequent generations (non-exposed F1 to F3). Our findings showed that reproduction and growth 86 were particularly impacted by environmentally relevant concentrations of SIM, not only in directly 87 exposed animals (exposed F0 to F3), but also in the generations raised in SIM-free water (non-exposed 88 F1 and F2, intergenerational, and non-exposed F3, transgenerational effects). In addition, a significant 89 decrease in MF levels was recorded in exposed and non-exposed females during four generations (F0 90 to F3). Although the molecular mechanism(s) underlying the observed transgenerational effects of SIM 91 are not fully defined, epigenetic remodeling stands as the most plausible explanation for these 92 findings, since most of environmental chemicals do not have the ability to alter DNA sequences or 93 produce direct genetic mutations in such a short timeframe. In agreement, Alves et al. (2021) reported 94 a significant downregulation of DNA methyltransferase 1 (DNMT1) gene expression upon exposure of 95 G. locusta to 320 and 8000 ng/L of SIM, concomitantly with global DNA hypomethylation. 96 Although severe effects on reproduction, growth, and development are known to occur in arthropods, 97 including amphipods, following exposure to SIM or other statins (Dahl et al., 2006; Liu et al., 2019; 98 Neuparth et al., 2014; Ortiz de García et al., 2014; Zapata et al., 2003), the absence of mechanistic 99 association between exposure and observed effects limits our understanding, notably regarding the 100 contribution of endocrine disruption processes. 101 Crustaceans utilize a variety of neuroendocrine signaling cascades to regulate molting, growth, and 102 reproduction, with most being unique to crustaceans and arthropods in general (Rodríguez et al., 2007; 103 Waye and Trudeau, 2011). The crustacean X-organ–sinus gland complex is the major neuroendocrine 104 structure, responsible for the release of an array of neuropeptides hormones: including MIH (molt 105 inhibiting hormone), GIH (gonad inhibiting hormone), MOIH (mandibular organ inhibiting hormone) 106 4 and CHH (crustacean hyperglycemic hormone), hormones that have been extensively studied over the 107 last decades and demonstrated to have a central role in homeostasis (Gismondi and Joaquim-Justo, 108 2019; Hyne, 2011). With the recent advances in next-generation sequencing, as well as the growing 109 availability of arthropods transcriptomes and genomes, new neuropeptides genes have been 110 cataloged in different crustacean species, some of which with a foreseen role in reproduction and 111 development, such as Allatostatins, SIFamide, Prohormones, among others (Nguyen et al., 2016). 112 Besides X-organ–sinus gland neuropeptides hormones, the sesquiterpenoid hormone MF is 113 synthetized in the mandibular organ, whereas various types of steroidal hormones implicated in 114 molting, such as ecdysteroids, are produced by the Y-organ. These hormones, involved in the 115 endocrine system of crustaceans, are physiologically linked to the X-organ–sinus gland complex 116 (Subramoniam, 2000). Apart from these arthropod-specific neuropeptides, sesquiterpenoid and 117 steroidal hormones, other neurotransmitters, like biogenic amines (e.g. serotonin, dopamine), are 118 associated with the endocrine regulation of physiological functions that control upstream 119 neurosecretory processes shared by both arthropods and vertebrates (Campbell et al., 2004; Vaudry 120 and Kah, 2017). In crustaceans, the biogenic amines are linked to the X-organ–sinus gland complex by 121 controlling the release of the neuro-hormones MIH and CHH. 122 In addition to disrupting the homeostasis of endocrine hormones, different EDCs were also reported 123 to produce multiple actions in the neural system, being neurotoxic particularly in early life stages (Gu 124 et al., 2019; Sun et al., 2016; Xu and Yin, 2019). Several studies in humans and rodents have provided 125 evidence for positive associations between pre- /post-natal exposure to certain EDCs (e.g., bisphenol 126 A, phthalates and pesticides) and impaired neurodevelopmental outcomes in offspring (e.g. emotional, 127 cognitive, motor and muscular disorders) leading to lifelong or even transgenerational dysfunctions 128 (Lupu et al., 2020; Repouskou et al., 2020). In fact, recent epidemiological and toxicological studies 129 established a robust link between human neurological diseases and exposure to certain EDCs 130 (Trasande et al., 2015). Given the importance of neuroendocrine control for the maintenance of 131 homeostasis, further efforts should be made to address the effects of EDCs at this level. Yet, it is not 132 clear whether the observed effects occur as direct neurotoxicity of EDCs or result from an indirect 133 effect through disruption of endocrine functions (Knigge et al., 2021; Waye and Trudeau, 2011). 134 The emergence of new genomic tools, which can provide a high throughput screening of gene 135 expression changes, are revolutionizing the way ecotoxicologists address the impact of environmental 136 stressors on model organisms (Chen and Li, 2016; Merrick, 2019; Wang and Chang, 2018). These 137 “omics” technologies provide robust approaches to further advance the mechanistic linkage between 138 exposure and effects of potential endocrine disruptors like SIM, in crustaceans. Therefore, considering 139 the findings of Neuparth et al. (2020a), reporting a marked reproductive and growth effect in G. locusta 140 after inter and transgenerational exposure to SIM, the present study performed a comprehensive 141 investigation that simultaneously assessed the inter and transgenerational (F1 and F3) molecular 142 5 changes of genes/pathways related with the regulation of the endocrine system, with a special focus 143 on the neuroendocrine system and brain functions. In order to improve our knowledge on the 144 molecular mechanisms related with the potential endocrine/neurotoxic efects of SIM throughout G. 145 locusta generations, we addressed the effects particularly in five key signalling pathways involved in 146 crustaceans´ neuroendocrine regulation, i.e., Ecdysteroids, Catecholamines, NO/cGMP/PKG, 147 GABAergic and Cholinergic signalling pathways. 148 149 2. Material and Methods 150 2.1. Experimental design 151 To thoroughly analyze the effects of environmentally relevant concentrations of SIM on endocrine and 152 neural systems of G. locusta in an inter and transgenerational context (non-exposed F1 and F3), we 153 took advantage of the experiment conducted by Neuparth et al. (2020a). Briefly, the experiment 154 started with one-week-old G. locusta offspring (parental generation - F0) continuously exposed to an 155 environmentally relevant concentration of SIM (320 ng/L prepared in 0.0005% acetone), plus control 156 (0.0005% acetone in filtered natural seawater). Fifty offspring, from our laboratory culture, were 157 randomly allocated in 7L aquaria (four per treatment) and exposed to SIM up to adulthood (55-65 158 days). In each aquarium, with a 1cm layer of natural clean sediment, the amphipods were kept in 159 filtered natural saltwater (33-35‰ salinity) with a temperature and photoperiod set to 20°C and 16:8 160 hours (light:dark) and feed ad libitum with Ulva sp. Water renewal in each aquarium occurred every 161 three days and final SIM concentration was re-established after the SIM solution was spiked directly in 162 the aquaria and properly stirred. The actual SIM concentration was monitored in the course of the 163 assay (Neuparth et al., 2020b). In order to evaluate the putative inter and transgenerational effects of 164 SIM, the F0 offspring of each treatment were allocated in SIM-free water (natural filtered seawater) 165 for three consecutive generations (F1, F2 and F3). Each generation was initiated with fifty offspring of 166 the previous one and kept for 55 to 65 days. At the end of each generation, Females were sampled 167 immediately after the third reproduction after maturity to determine growth, reproduction and 168 methyl-farnesoate (MF) levels; the last measured according to the methods developed by Montes et 169 al. (2017). Furthermore, three randomly selected females from control groups (F1 and F3) and from 170 320 ng/L SIM inter/transgenerational groups (F1 and F3), were preserved in RNA later at −80°C until 171 individual use in RNA-sequencing analyses. Further methodological details are available in Neuparth 172 et al., (2020a,b). 173 174 2.2. Analyses of the disrupted endocrine and neural genes in F1 and F3 G. locusta females’ 175 transcriptome 176 6 The comprehensive transcriptome assembly produced in Neuparth et al. (2020a,b) was used in the 177 current study to thoroughly evaluate the functional annotated genes of G. locusta females from inter 178 and transgenerational exposure to SIM (non-exposed F1 and F3 generations, respectively). Briefly, to 179 produce the G. locusta females transcriptome reported in Neuparth et al. (2020a,b), RNA was 180 extracted from 3 randomly selected G. locusta females per condition [control and SIM 181 inter/transgenerational groups (F1 and F3)] and individually sequenced using the Illumina 182 HiSeq2500paired-end (2x150) system. Notably, the whole amphipod body was used rather than 183 specific organs, because G. locusta females are too small to fully separate the organs of interest 184 without contamination from other tissues. The RNA-Seq datasets were de novo assembled using the 185 Trinity assembler and functionally annotated with the Trinotate suite. Lastly, the differential gene 186 expression (DEGs) analyses (False Discovery Rate – corrected (FDR) p-value < 0.05, log2|fold change| 187 ≥ 2 and blast e-value less than 1×10-29) were performed using the Degust platform and KEGG pathways 188 scrutinized with the KAAS webserver (detailed information available in Neuparth et al., 2020b, sections 189 2.3 to 2.7). 190 In the present study, all the F1 and F3 functional annotated genes from the Neuparth et al. (2020b) 191 transcriptome were first manually analized and a high number of genes involved in neuroendocrine 192 control were identified. Based on these findings, we performed a data-driven approach searching for 193 the main molecular components of the neuroendocrine network from all functional annotaned genes. 194 We focused particulary on the metabolic pathways related with molting, reproduction and neural 195 regulation (Ecdysteroid, Catecholamines, NO/cGMP/PKG, GABAergic and Cholinergic signalling 196 pathways). Considering that crustacean neuroendocrine data is limited in KEGG databases, to properly 197 study these specialized physiological processes in G. locusta, the functional annotated genes related 198 with the neuroendocrine system were systematically reviewed using a manual approach, thus avoiding 199 misrepresentation of G. locusta transcriptomic data due to annotation omission. 200 Thus, to perform a gene functional analysis, we first searched known protein names within several 201 platforms such as Uniprot (https://www.uniprot.org/ accessed in March 2020), Genecard 202 (https://www.genecards.org/ accessed in March 2020), NCBI (https.//www.ncbi.nlm.nih.gov/ 203 accessed in March 2020) and Nextprot (https://www.nextprot.org/ accessed in March 2020). When 204 available, the information was obtained from species with the closest taxonomy to G. locusta. We then 205 used different databases (NCBI-https://www.ncbi.nlm.nih.gov/gene accessed in April 2020; Reactome206 https://reactome.org/ accessed in April 2020; Biogrid v4.4-https://thebiogrid.org/ accessed in April 207 2020; STRING v11.5https://string-db.org/ accessed in April 2020; IntAct208 https://www.ebi.ac.uk/intact/ accessed in April 2020 and Rheahttps://www.rhea-db.org/ accessed 209 in April 2020) to retrieve potential direct and/or indirect biological interactions between proteins 210 encoded by selected DEGs related to the neuroendocrine system. All putative interactions recorded 211 by the databases above were confirmed through a detailed review of functional studies in the 212 7 literature with a special focus on crustaceans and when the information for crustaceans was scarce, 213 on insects (e.g. Hyne, 2011; Sterkel and Oliveira, 2017; Covi et al., 2009; Mykles et al., 2010; Zhang et 214 al., 2014b; Nako et al., 2018; Takesian and Hensch, 2013). Additionally, we used arthropods´ dedicated 215 functional studies that were available in the literature to find proteins encoded by the selected DEGs 216 of interest related with ecdysteroids and molting processes that were not found in the databases used 217 (e.g. Qian et al., 2014; Street et al., 2019; Webster, 2015a,b; Zhou et al., 2019; Guittard et al., 2011). 218 All this data was used to manually design the neuroendocrine pathways presented in the present study 219 (Figure 1). 220 8 221 Figure 1 – Study workflow 222 3. Results and discussion 223 Due to the extensive use of industrial, agricultural and pharmaceutical products, tens of thousands of 224 chemicals are released annually into aquatic environments (Evans et al., 2019; Rzymski, 2017; Wang 225 and Yang, 2016). However, to date, only an infinitesimal fraction of environmental chemicals has been 226 assessed for their endocrine-disrupting potential in wildlife (Brack et al., 2018; Santos et al., 2018). 227 15 (Avramov et al., 2013; Chang et al., 2015; Gallo et al., 2016). Catecholamines, particularly DA, mediate 399 the release of several critical neuro-hormones, such as Crustacean Hyperglycaemic Hormone (CHH) 400 and alterations on CHH levels can lead to disruption of important biological processes such as sugar 401 and lipid metabolism (Bulau et al., 2003; Chang, 2005) and osmoregulation (Chung and Webster, 2006). 402 In our transcriptome, besides the downregulation of TH and VSP mentioned above, we also detected, 403 in F1, a downregulation of Dopamine Bhydroxylase (DBH) that catalyzes the conversion of DA to 404 NEP/EP, and a downregulation of CHH. 405 406 Figure 4 – Catecholamines pathway in Crustaceans. Genes modulated by SIM and non-responsive genes in F1 and F3 407 generations are presented in boxes. Red text inside the boxes indicates gene expression downregulation in F1 and/or F3 408 generations and black text inside the boxes indicates unchanged gene expression in F1 and F3 generations. No gene was 409 found to be upregulated in this pathway. . TH –Tyrosine hydroxylase; DHPR – dihydropteridine reductase; BH4 – 410 tetrahydrobiopterin; L-DOPA – L-3,4-dihydroxyphenylalanine; CPs – cuticle proteins; ProPO – Pro phenol oxidase; PO – Phenol 411 oxidase hyperglycemic hormone; VSP – venom serine protease Bi-vsp; PPAE – prophenoloxidase activating enzyme; CHH – 412 crustacean hyperglycemic hormone; DBH – dopamine beta-hydroxylase. 413 414 3.3. NO/cGMP/PKG signalling pathway 415 As aforementioned, MIH can negatively control the synthesis of ecdysteroids. MIH regulates the 416 NO/cGMP/PKG signalling, critical for ecdysis (Lee and Mykles, 2006). There is consensus that cyclic 417 guanosine monophosphate (cGMP) acts as an intracellular signalling messenger of MIH-mediated 418 action (Chen et al., 2018; Covi, 2012; Francis et al., 2010; Nagai et al., 2009; Xu et al., 2019), leading to 419 an activation of cGMP-dependent protein kinase G (PKG) and subsequent inhibition of 420 ecdysteroidogenesis (Covi et al., 2009; Mykles et al., 2010). Covi et al. (2009) found that MIH induces 421 a sustained increase in cGMP, by the activation of membrane-bound guanylyl cyclases (GYC32E and 422 16 GUCY1B1) that convert guanosine triphosphate (GTP) to cGMP. In the present study, we observed a 423 downregulation of GYC32E and GUCY1B1 in F1 and F3, indicative of a decrease in cGMP, althougth the 424 receptor-type guanylate cyclase Gyc76C (GYC76C) and the soluble guanylate cyclase 89Db (GYC89DB) 425 remained unchanged (Figure 5). Interestingly, a downregulation of cGMP-specific 3',5'-cyclic 426 phosphodiesterase (PDE5A), that prevents the conversion of cGMP to its inactive form guanosine 427 monophosphate (GMP), was also downregulated in F1 and F3, suggesting a cGMP triggered action 428 (Figure 5), even though 3',5'-cyclic phosphodiesterase pde-4 (PDE4) was non-responsive. Also, the 429 guanylate kinase-like isoform X1 (GUK1), involved in cGMP and GMP recycling, was unchanged. 430 However, a downregulation of neprilysin-1 (NEP1) that promotes the degradation of atrial natriuretic 431 peptide (ANP) was observed in F1 and F3 which indicates an activation of cGMP, since more ANP is 432 available for cGMP synthesis. However, the atrial natriuretic peptide receptor B (ANR) was found to 433 be downregulated in F1 and F3, suggesting a decrease of cGMP, since the binding of ANP to its receptor 434 activates the synthesis of cGMP (Chen et al., 2008; Piggott et al., 2006) (Figure 5). Thus, with these 435 findings taken together, we hypothesize that SIM triggers a feedback mechanism to control cGMP 436 levels and subsequent inhibition of ecdysteroidogenesis through the balances between the action of 437 GUCYB1/GYC32E and PDE5A and between NEP1 and ANR in F1 and F3. 438 439 Figure 5 – NO/cGMP/PKG signalling pathway in Crustaceans. Genes modulated by SIM and non-responsive genes in F1 and 440 F3 generations are presented in boxes. Red text inside the boxes indicates gene expression downregulation in F1 and/or F3 441 generations and black text inside the boxes indicates unchanged gene expression in F1 and F3 generations. No gene was 442 found to be upregulated in this pathway.. MIH – molt inhibiting hormone; GTP – guanosine triphosphate; cGMP – cyclic 443 guanosine monophosphate; PKG – cGMP-dependent protein kinase; GYC32E – guanylate cyclase 32E; GUCY1B1 – guanylate 444 cyclase soluble subunit beta-1; GYC76C – receptor-type guanylate cyclase Gyc76C; GYC89DB – soluble guanylate cyclase 445 89Db; PDE5A – cGMP-specific 3',5'-cyclic phosphodiesterase; PDE4 – 3',5'-cyclic phosphodiesterase pde-4; GMP – guanosine 446 monophosphate; GUK1 – guanylate kinase isoform X1; GDP – guanosine diphosphate; NEP1 – neprilysin1; ANP – atrial 447 natriuretic peptide; ANPR – atrial natriuretic peptide receptor B. 448 449 17 3.4. Cholinergic synapse pathway 450 Given the importance of neural regulation for the homeostatic control (Legradi et al., 2018), we also 451 assessed whether additional pathways/genes related with neural/behavioral regulation were 452 modulated by SIM. Interestingly, the transcriptomic analysis showed that several genes related with 453 the synthesis, transport, and release of the neurotransmitter acetylcholine (Ach), which plays a key 454 role in the regulation of behavior, cognition, and control of muscle contraction, is downregulated in F1 455 and/or F3 (Figure 6). The cholinergic synapse pathway is a highly conserved evolutionary pathway in 456 eukaryotes, vital for the normal function of the sensorial and neuromuscular systems (Bossy et al., 457 1988; Zhang et al., 2014b). Even though the choline transporter (CHT2) and some acetylcholine 458 receptors subunits (ACRb1 and CHRNAa4) were found to be non-responsive to SIM, we observed that 459 SIM severely disrupts the cholinergic synapse pathway, as choline O-acetyltransferase (ChAT), choline 460 (Ch) and acetylcholine transporters (SLC5A7 and VAChT, respectively), acetylcholine receptors - 461 nicotinic α7-like subunits (CHRNA7 and ACR-16) and Acetylcholinesterase (AChE) are all 462 downregulated in F1 and ChAT, SLC5A7, VAChT, CHRNA7 and AChE are also downregulated in F3 463 (Figure 6). Since this pathway is critical for the normal function of cholinergic transmission—i.e., the 464 communication of neurons in the nervous system, including motor neurons, that will activate muscle 465 contraction—we hypothesize that inter and transgenerational exposure to SIM could modulate the 466 muscle system of G. locusta. Interestingly, previous studies reported the occurrence of adverse 467 neuromuscular reaction upon administration of SIM, possibly mediated by the nicotinic acetylcholine 468 receptor (nAChRs) affecting nerve transmission across the synapse and resulting in muscle weakness 469 and pain by neuromuscular degeneration (Grajales-Reyes et al., 2013). Our findings also suggest that 470 the observed SIM modulation of the cholinergic synapse pathway could potentially disturb the molting 471 cycle of G. locusta in F1 and F3 generations. In fact, simultaneous contractions of specialized muscle in 472 arthropods are required to split the old cuticle during molting and generate pressure pulses for body 473 expansion (Nako et al., 2018). Molting, as described above, is a key process for crustaceans´ growth 474 and reproduction. Therefore, the inter and transgenerational disruption of the cholinergic 475 synapse pathway could be potentially linked with the observed effects of SIM on G. locusta growth 476 and reproduction previously reported (Figure 2) (Neuparth et al., 2020a; Neuparth et al., 2014) and be 477 potentially related with the ecdysteroid cascade disturbance described above, also involved in the 478 regulation of molting, reproduction and growth. 479 18 480 Figure 6 – Cholinergic synapse pathway in Crustaceans. Genes modulated by SIM and non-responsive genes in F1 and F3 481 generations are presented in boxes. Red text inside the boxes indicates gene expression downregulation in F1 and/or F3 482 generations and black text inside the boxes indicates unchanged gene expression in F1 and F3 generations. No gene was 483 found to be upregulated in this pathway. . Ch – Choline; Ach – Acetylcholine; ChAT – choline O-acetltransferase; ACR16 – 484 acetylcholine receptor subunit alpha-type ACR-16-like; ACR b1 – acetylcholine receptor subunit beta; CHRNA7 – acetylcholine 485 receptor subunit alpha-7-like; CHRNA α4 – acetylcholine receptor subunit alpha-4; SLC5A7 – high affinity choline transporter 486 1; CHT2 – choline transporter protein 2; VAChT – vesicular acetylcholine transporter 1; AChE – acetylcholinesterase. 487 488 489 3.5. GABAergic signalling pathway 490 A downregulation of several proteins involved in the neurotransmitter γ-aminobutyric acid (GABA) 491 cycle was also observed. GABA is primary involved in hyperpolarising inhibitory synaptic transmission 492 by counterbalancing the depolarization of neuronal membranes (Crowley et al., 2016) and is 493 synthesized from L-glutamate, the principal excitatory neurotransmitter involved in processes such as 494 synaptic memory and plasticity (Benarroch, 2010; Fagg and Foster, 1983; Fonnum, 1984). Its action 495 reduces the excessive glutamatergic signalling and subsequent excitotoxicity, acting as a feedback 496 control system (Gonzalez-Burgos and Lewis, 2008; Lau and Tymianski, 2010), regulating neural activity 497 and energy metabolism and thus, modulating brain homeostasis (Takesian and Hensch, 2013; Xu et al., 498 2011). Figure 7 displays the transcriptomic results of GABA signalling pathway where downregulation 499 of genes coding for key proteins in F1 was observed: i.e., glutaminase liver, mitochondrial-like (GLS), 500 4-aminobutyrate aminotransferase (ABAT), GABA receptor delta (GABA-R) and glutaminase, sodium 501 and chloride dependent GABA transporter 1-like (GAT1) that are involved in the action, transport and 502 degradation of GABA. On the other hand, it was also observed that some genes related with the 503 interaction, anchoring and organization of GABA receptors with the cytoskeleton, such as gephyrin 504 19 (GPHN) and gamma-aminobutyric acid receptor-associated protein (GABARAP), and the formation of 505 ketoglutarate from glutamate, i.e., mitochondrial glutamate dehydrogenase 1 (GAD), were non506 responsive to SIM. 507 508 Figure 7 – GABAergic signalling pathway in Crustaceans. Genes modulated by SIM and non-responsive genes in F1 and F3 509 generations are presented in boxes. Red text inside the boxes indicates gene expression downregulation in F1 and/or F3 510 generations and black text inside the boxes indicates unchanged gene expression in F1 and F3 generations. No gene was 511 found to be upregulated in this pathway.. GLS – glutaminase liver mitochondrial-like; GAD – mitochondrial glutamate 512 dehydrogenase 1; ABAT – 4-aminobutyrate aminotransferase; GPHN – gephyrin; GABA-R – GABA receptor delta; GABARAP – 513 gamma-aminobutyric acid receptor-associated protein; GAT1 – sodium and chloride dependent GABA transporter-1-like. 514 515 Besides the neuroendocrine pathways discussed above, table 2 displays additional genes involved in 516 the regulation of nervous and neuromuscular functions that showed a shift in gene expression profile 517 after inter and transgenerational SIM exposure. 518 519 Table 2 –Genes related with neurotoxic effects and their respective function 520 Gene F1 status F3 status Evalue/ Bit score Function Species prohormone-4-like isoform X1 - 1.3e-86 329.3 Cleaved into the following six chains: Brain peptides IDLSRFYGHFNT, IDLSRFYGHFN, IDLSRFYGHF, DLSRFYGHFNT, DLSRFYGHFN or Brain peptide DLSRFYGHF Ame putative lachesin-like 2.9e-94 356.7 Functions as a homophilic cell-adhesion molecule. May play a role in early neuronal differentiation and axon outgrowth Dme prohormone-3-like - 1.6e-34 156.0 Cleaved into Brain peptide ITGQGNRIF Ame prohormone-1-like 3.1e-31 144.8 Cleaved into three chains: Brain peptide LRNQLDIGDLQ, Brain peptide LRNQLDIGDL and Brain peptide SYWKQCAFNAVSCF-amide Ame synaptobrevin-1-like isoform X4 - 1.1e-48 202.6 Acts in neuronal exocytosis of synaptic transmission. Potential role in cholinergic transmission Cbr protein unc-79 homolog 8.5e-299 1036.9 Component of the NALCN channel is responsible for Na(+) leak currents and activated by neuropeptides Hsa neuropilin-1-like isoform X1 2.1e-99 372.5 Receptor involved in the formation of certain neuronal circuits and in organogenesis outside the nervous system. Hsa neprilysin-1-like 5.9e-109 405.2 Metalloendoprotease which functions in fertility and memory formation. Dme 20 zwei Ig domain protein zig8-like 3.2e-98 368.6 Required postembryonically to maintain the position of several neuron cell bodies and ventral nerve cord axons of specific neurons Cel neurotrophin 1-like 1.3e-90 342.4 Ligand for the Toll-related receptors, promoting motor axon targeting and neuronal survival Dme putative neural-cadherin 2 2.9e-261 912.5 Cell adhesion proteins involved in the transmission of developmental information Dme contactin-6-like 0.0e+00 1128.6 Mediate cell surface interactions. Participates in oligodendrocytes generation by acting as a ligand of NOTCH1. Hsa potassium voltage-gated channel subfamily B member 1-like isoform X1 1.3e-54 223.4 Mediates transmembrane transport in excitable membranes Regulation of action potential in neurons, muscle and endocrine cells Hsa irregular chiasm Croughest protein-like 2.5e-308 1067.8 Required for correct axonal pathway formation in the optic lobe and for programmed cell death in the developing retina. Dme putative neurotrimin-like 9.2e-69 271.2 Neural cell adhesion molecule Hsa protein RUFY3-like isoform X1 - 1.0e-176 630.2 Plays a role in the generation of neuronal polarity formation and axon growth Hsa Down syndrome cell adhesion molecule-like protein Dscam2 0.0e+00 1729.9 Cell adhesion molecule. Involved in axon guidance. Dme ninjurin-2-like isoform X2 - 6.0e-39 170.6 Homophilic cell adhesion molecule that promotes axonal growth. May play a role in nerve regeneration and in the formation and function of other tissues Hsa protein turtle homolog Blike isoform X7 - 1.3e-53 220.7 Abundantly expressed in interneurons, where it may regulate inhibitory synapse development Hsa Down syndrome cell adhesion molecule-like protein 1 homolog, partial 1.4e-196 696.8 Cell adhesion molecule that plays a role in neuronal self-avoidance. Hsa sodium-coupled monocarboxylate transporter 1-like - 2.8e-210 741.5 May play a critical role in an electrochemical Na+ gradient on neurons. Maintenance of the energy status and function of neurons. Hsa putative neurotrypsin-like - 3.0e-274 954.5 Plays a role in neuronal plasticity and is associated with learning and memory operations Hsa neuroligin-4, Y-linked-like - 0.0e+00 1387.1 Neuronal cell surface protein involved in cell-cell-interactions Hsa neuroligin-4, X-linked like 1.8e-295 1025.4 Neuronal cell surface protein involved in cell-cell-interaction Hsa neuroligin-2-like, partial 1.4e-219 772.3 Involved in cell-cell interactions via its interactions with neurexin family members. Mediates cell-cell interactions both in neurons and in other types of cells Hsa synaptotagmin-11-like isoform X2 5.7e-179 637.1 Plays an important role in dopamine transmission by regulating endocytosis and the vesicle-recycling process Hsa arylsulfatase B-like 2.8e-78 302.0 Regulator of neurite outgrowth and neuronal plasticity Hsa RNA-binding protein Musashi homolog Rbp6like 4.3e-66 261.2 May play a role in the proliferation and maintenance of stem cells in the central nervous system Dme ionotropic receptor 25alike - 0.0e+00 1389.4 Integral part of various neural sensory systems in the antenna that provide the neural basis for the response to environmental changes Dme serine/threonine-protein kinase BRSK2-like isoform X2 - 3.4e-296 1027.7 Serine/threonine-protein kinase that plays a key role in polarization of neurons and axonogenesis, Hsa innexin inx1-like isoform X2 - 1.2e-31 145.6 Structural component of the gap junctions Dme myelin P2 protein-like - 4.5e-49 203.8 May play a role in lipid transport protein in Schwann cells Hsa calcium-activated potassium channel slowpoke-like 1.9e-252 881.3 Potassium channel activated by both membrane depolarization or increase in cytosolic Ca2+ that mediates export of K+. Its activation dampens the excitatory events that elevate the cytosolic Ca2+ concentration and/or depolarize the cell membrane. It therefore contributes to repolarization of the membrane potential. Dme peptidyl-glycine alphaamidating monooxygenase B-like - 4.9e-32 147.5 Bifunctional enzyme that catalyzes the post-translational modification of inactive peptidylglycine precursors to the corresponding bioactive alpha-amidated peptides, a terminal modification in biosynthesis of many neural and endocrine peptides Hsa peptidyl-alphahydroxyglycine-alphaamidating lyase I - 1.8e-76 296.2 Probable lyase that catalyzes an essential reaction in C-terminal alpha-amidation of neuropeptides Dme peptidyl-alphahydroxyglycine alphaamidating lyase 2-like - 2.8e-88 335.5 Probable lyase that catalyzes an essential reaction in C-terminal alpha-amidation of neuropeptides Dme neuropeptide SIFamide receptor-like - 2.8e-159 573.5 Controls different reproductive behaviours, e.g., sexual receptivity in females, dominance and aggression behaviours Dme Red indicates downregulation, green indicates upregulation; (-) indicates no changes in gene expression; Hsa – Homo sapiens; 521 Ame – Apis mellifera, Dme – Drosophila melanogaster, Cbr - Caenorhabditis briggsae, Cel - Caenorhabditis elegans 522 523 21 4. Conclusions 524 Overall, we demonstrate here that inter and transgenerational exposure to SIM, at environmentally 525 relevant levels, has significant effects in the regulation of key signaling pathways involved in crustacean 526 neuroendocrine regulation, concomitantly to changes in apical endpoints, such as depressed 527 reproduction and growth. The neuroendocrine system plays a major role in metazoan homeostatic 528 control, particularly in taxa displaying a central neuroendocrine system: regulating behavior, 529 reproduction, molting, among other major biological processes. The neuroendocrine metabolic 530 pathways here designed provide additional genomic information in this poorly annotated species and 531 will represent a reference source for further and more focused neuroendocrine molecular analysis on 532 this and other amphipod species. 533 Yet, despite the biological importance of neuroendocrine regulation, the impact of environmental 534 chemicals at this level has been poorly studied. Therefore, the findings reported here support the 535 importance of addressing the effects of environmental chemicals in neuroendocrine regulation, as a 536 step towards improving hazard and risk assessment of biological active compounds. Importantly, the 537 disruption in signaling pathways reported here was not only observed in F1 but also in the F3 538 generation, which implies transgenerational effects. Neuparth et al. (2020a,b) reported the 539 modulation of several genes involved in the regulation of the epigenome in F3, which may explain the 540 observed transgenerational effects. A detailed evaluation of the epigenetic machinery involved in this 541 disruption should be disclosed in the future, given that environmental chemicals inducing 542 transgenerational effects over several non-exposed generations are of major concern. 543 544 5. Authorship contribution statement 545 T. Neuparth: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, 546 Data curation, Writing – original draft, Writing - review & editing, Supervision, Project administration, 547 Funding acquisition. N. Alves: Conceptualization, Methodology Formal analysis, Investigation, Data 548 curation, Data curation, Writing – original draft, Writing - review & editing. A.M. Machado: Formal 549 analysis, Investigation, Data curation. M. Pinheiro: Formal analysis, Investigation, Data curation, 550 Writing - review & editing. R. Montes: Investigation, Writing - review & editing. R. Rodil: Investigation, 551 Writing - review & editing. S. Barros: Formal analysis, Investigation, Data curation, Writing - review & 552 editing. R. Ruivo: Formal analysis, Investigation, Data curation, Writing - review & editing. L. Filipe C. 553 Castro: Validation, Writing - review & editing. J.B. Quintana: Investigation, Writing - review & editing, 554 Funding acquisition. M.M. Santos: Conceptualization, Methodology, Validation, Formal analysis, 555 Investigation, Resources, Data curation, Writing - original draft, Writing - review & editing, Supervision, 556 Project administration, Funding acquisition. 557 558 6. Declaration of Competing Interest 559 The authors declare that they have no known competing financial interests or personal relationships 560 that could have appeared to influence the work reported in this paper. 561 562 563 22 7. Acknowledgments 564 This work was financially supported by: i) Transobesogen Project – “Trans-phyletic obesogenic 565 responses: from epigenetic modules to transgenerational environmental impacts” reference: 566 PTDC/CTA-AMB/31544/2017 – NORTE-01-0145-FEDER-031544, co-financed by the Portuguese 567 Foundation for Science and Technology (FCT), North Regional Operational Program (NORTE 2020) and 568 the European Regional Development Fund (FEDER). ii) Nor-Water Project – “Poluentes emergentes nas 569 águas da Galiza-Norte de Portugal: novas ferramentas para gestão de risco” reference: 570 0725_NOR_WATER_1_P financed by INTERREG VA Spain-Portugal cooperation programme, Cross571 Border North Portugal/Galizia Spain Cooperation Program (POCTEP) 2014–2020. iii) the National Funds 572 through FCT under the projects (UIDB/04423/2020; 573 UIDP/04423/2020). Nélson Alves acknowledges FCT for his Ph.D. grant DFA/BD/6218/2020. Andre M 574 Machado acknowledges FCT for his Ph.D. grant DFA/BD/8069/2020. Marlene Pinheiro acknowledges 575 FCT for her Ph.D. grant SFRH/BD/147834/2019. Susana Barros acknowledges FCT for her Ph.D. grant 576 PD/BD/143090/2018. Financial support by Xunta de Galicia (ED431C2021/06) and the Spanish Agencia 577 Estatal de Investigación - AEI (PID2020-117686RB-C32) is also gratefully acknowledged. 578 579 580 8. References 581 Alves, N., Neuparth, T., Barros, S., and Santos, M.M. (2021). The anti-lipidemic drug simvastatin 582 modifies epigenetic biomarkers in the amphipod Gammarus locusta. Ecotoxicology Environmental 583 Safety 209, 111849. 10.1016/j.ecoenv.2020.111849. 584 Arakane, Y., Noh, M.Y., Asano, T., and Kramer, K.J. (2016). Tyrosine Metabolism for Insect Cuticle 585 Pigmentation and Sclerotization. In Extracellular Composite Matrices in Arthropods, E. Cohen, and B. 586 Moussian, eds. 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Machado, Rosa Montes and Miguel M. Santos; Validation, Teresa Neuparth, André M. Machado, L. Filipe C. Castro and Miguel M. Santos; Formal analysis, Teresa Neuparth, André M. Machado, Rosa Montes, Rosario Rodil, Susana Barros, Nelson Alves., Raquel Ruivo, L. Filipe C. Castro, José B.Quintana and Miguel M. Santos; Investigation, Teresa Neuparth, André M. Machado, Rosa Montes, Rosario Rodil, Susana Barros, Nelson Alves., Raquel Ruivo, L. Filipe C. Castro, José B.Quintana and Miguel M. Santos; Software, Teresa Neuparth, André M. Machado; Resources, Teresa Neuparth, L. Filipe C. Castro and Miguel M. Santos; Data curation, Teresa Neuparth, André M. Machado, Rosa Montes, Rosario Rodil, Susana Barros, Nelson Alves., Raquel Ruivo, L. Filipe C. Castro, José B.Quintana and Miguel M. Santos; Writing-original draft preparation, Teresa Neuparth and Miguel M. Santos; Writing-review and editing Teresa Neuparth, André M. Machado, Raquel Ruivo, L.Filipe C. Castro, José B.Quintana and Miguel M. Santos; Supervision, Teresa Neuparth and Miguel M. Santos; Project administration, Teresa Neuparth and Miguel M. Santos Funding acquisition, José B.Quintana and Miguel M. Santos