Diet composition and plastic ingestion in Poecilia reticulata from urban streams.

Fish are excellent bioindicators and can reveal the presence of plastic in the environment. Diagnosing the composition and abundance of polymers in the fish diet makes it possible to evaluate their point sources and possible trophic transfers. We aimed to use the gastrointestinal contents of Poecilia reticulata in subtropical urban streams to detect the occurrence, shape, color, size, and chemical composition of polymers. For this, the diet of 240 individuals was analyzed using the volumetric method, and the microplastics (MPs; < 5 mm) recorded were characterized using Raman spectroscopy. Individuals predominantly consumed organic detritus and aquatic macroinvertebrates, with higher proportions of Diptera. A total of 111 plastic particles (< 0.5 to 12 mm) were recorded, and a subset of 14.4% was subjected to a micro-Raman spectrometer (830 nm excitation). The occurrence of polyethylene terephthalate (PET) and polypropylene (PP) with phthalocyanine dye was recorded. Some fragments could not be identified by Raman, but they contained indigo blue dye. Poecilia reticulata had a predominantly detritivorous diet with a record of plastic consumption, reflecting environmental pollution. Our results demonstrate that individuals of P. reticulata have ingested MPs in urban streams. This reinforces the need for future studies on the relationship between the presence of MPs in fish and the level of pollution in streams, comparisons with species of different feeding habits, and the potentially harmful effects on the entire biota.

Diagnosis, isolation and description of a novel amnoonvirus recovered from diseased fancy guppies, Poecilia reticulata.

The guppy, Poecilia reticulata, is one of the most common cultured ornamental fish species, and a popular pet fish highly desired by hobbyists worldwide due to its availability of many brilliantly coloured fish of many varieties. The susceptibility of guppies to diseases presents a remarkable concern for both breeders and hobbyists. In this study, we report the emergence of disease in fancy guppies caused by a previously uncharacterized virus in the USA. This virus was isolated from moribund guppies in two separate outbreaks in California and Alabama, from December 2021 to June 2023. The infected guppies presented with acute morbidity and mortality shortly after shipping, displaying nonspecific clinical signs and gross changes including lethargy, anorexia, swimming at the water surface, gill pallor, mild to moderate coelomic distension and occasional skin lesions including protruding scales, skin ulcers and hyperaemia. Histological changes in affected fish were mild and nonspecific; however, liver and testes from moribund fish were positive for Tilapia lake virus (TiLV), the single described member in the family Amnoonviridae, using immunohistochemistry and in situ hybridization, although the latter was weak. A virus was successfully recovered following tissue inoculation on epithelioma papulosum cyprini and snakehead fish cell lines. Whole genome sequencing and phylogenetic analyses revealed nucleotide and amino acid homologies from 78.3%-91.2%, and 78.2%-97.7%, respectively, when comparing the guppy virus genomes to TiLV isolates. Based on the criteria outlined herein, we propose the classification of this new virus, fancy tailed guppy virus (FTGV), as a member of the family Amnoonviridae, with the name Tilapinevirus poikilos (from the Greek 'poikilos', meaning of many colours; various sorts, akin to 'poecilia').

Identification of Philaster apodigitiformis in aquaculture and functional characterization of its ß-PKA gene and expression analysis of infected Poecilia reticulata.

Cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) is a distinctive member of the serine­threonine protein AGC kinase family and an effective kinase for cAMP signal transduction. In recent years, scuticociliate has caused a lot of losses in domestic fishery farming, therefore, we have carried out morphological and molecular biological studies. In this study, diseased guppies (Poecilia reticulata) were collected from an ornamental fish market, and scuticociliate Philaster apodigitiformis Miao et al., 2009 was isolated. In our prior transcriptome sequencing research, we discovered significant expression of the ß-PKA gene in P. apodigitiformis during its infection process, leading us to speculate its involvement in pathogenesis. A complete sequence of the ß-PKA gene was cloned, and quantified by quantitative reverse transcription-polymerase chain reaction to analyse or to evaluate the functional characteristics of the ß-PKA gene. Morphological identification and phylogenetic analysis based on small subunit rRNA sequence, infection experiments and haematoxylin­eosin staining method were also carried out, in order to study the pathological characteristics and infection mechanism of scuticociliate. The present results showed that: (1) our results revealed that ß-PKA is a crucial gene involved in P. apodigitiformis infection in guppies, and the findings provide valuable insights for future studies on scuticociliatosis; (2) we characterized a complete gene, ß-PKA, that is generally expressed in parasitic organisms during infection stage and (3) the present study indicates that PKA plays a critical role in scuticociliate when infection occurs by controlling essential steps such as cell growth, development and regulating the activity of the sensory body structures and the irritability system.

Investigation of extragonadal teratomas in two Poecilia wingei.

Teratoma is a rare tumour in fish consisting of tissues from more than one germ layer, that may be located in either the gonads or extragonadal sites. Teratomas in many fish species remain poorly understood. In this work, we performed the first histological examinations of extragonadal teratomas in Poecilia wingei and also examined the influence of a large teratoma on male sexual activity. The studied teratomas shared general organizational features, but they also had variations in both external and internal features. In teratomas, the most common and highly differentiated tissues were striated muscle and nervous tissue. Despite the tumour, the male P. wingei exhibited normal mating behaviour and retained the ability for successful copulation. The structural features of extragonadal teratomas in guppy fish indicate a possible connection between these tumours and a failure of conserved processes operating in the embryonic germline.

Cylindrospermopsin exposure promotes redox unbalance and tissue damage in the liver of Poecilia reticulata, a neotropical fish species.

There is a growing concern regarding the adverse risks exposure to cylindrospermopsin (CYN) might exert on animals and humans. However, data regarding the toxicity of this cyanotoxin to neotropical fish species are scarce. Using the fish species Poecilia reticulata, the influence of CYN concentrations equal to and above the tolerable for drinking water may produce on liver was determined by assessing biomarkers of antioxidant defense mechanisms and correlated to qualitative and semiquantitative histopathological observations. Adult females were exposed to 0.0 (Control); 0.5, 1 and 1.5 µg/L pure CYN for 24 or 96 hr, in triplicate. Subsequently the livers were extracted for biochemical assays and histopathological evaluation. Catalase (CAT) activity was significantly increased only by 1.5 µg/L CYN-treatment, at both exposure times. Glutathione -S-transferase (GST) activity presented a biphasic response for both exposure times. It was markedly decreased after exposure by 0.5 µg/L CYN treatment but significantly elevated by 1.5 µg/L CYN treatment. All CYN treatments produced histopathological alterations, as evidenced by hepatocyte cords degeneration, steatosis, inflammatory infiltration, melanomacrophage centers, vessel congestion, and areas with necrosis. Further, an IORG >35 was achieved for all treatments, indicative of the presence of severe histological alterations in P. reticulata hepatic parenchyma and stroma. Taken together, data demonstrated evidence that CYN-induced hepatotoxicity in P. reticulata appears to be associated with an imbalance of antioxidant defense mechanisms accompanied by histopathological liver alterations. It is worthy to note that exposure to low environmentally-relevant CYN concentrations might constitute a significant risk to health of aquatic organisms.

Structural and immunohistochemical characterization of pancreas of Molly fish (Poecilia sphenops), with a special reference to its immune role.

Recently, teleost species have been considered important model systems for investigating different research areas including immunologic one. The available literature provides poor data about the localization and the structure of pancreas in Molly fish. Moreover, little attention has been paid to the immunologic role of pancreatic tissue of teleost, particularly Molly fish; therefore, this study aimed to highlights the description of pancreatic tissue in Molly fish using light- and electron- microscopy, focusing on the role of pancreatic immune cells and pancreatic acinar cells in immune responses. Microscopic analysis revealed that the pancreas of Molly fish was composed of intrahepatic, disseminated and compact parts. Exocrine pancreatic tissue was diffusely extended within the hepatic tissue forming hepatopancreas. The disseminated pancreas appeared as several irregular nodules of pancreatic tissue localized within the mesenteric adipose tissue. The compact pancreas appeared as an oval shaped body embedded within the mesenteric adipose tissue between the spleen and the intestinal loops. Several telocytes and melanomacrophages were detected within the disseminated pancreatic nodules. Moreover, dendritic cells were found in a close association to the exocrine pancreatic acini. The pancreatic acinar cells showed strong immunoreactivity to APG5, TGF-ß, IL-1ß, NF-κB, Nrf2, and SOX9 in both hepatopancreas and disseminated pancreas of Molly fish. S100 protein revealed a strong expression in the exocrine pancreatic acinar cells of disseminated pancreas and also in the endocrine cells of the compact pancreas. In conclusion, findings of this study suggest the potential role of the pancreas of the Molly fish in cell proliferation and differentiation, proinflammatory cytokines stimulation, and regulation of both innate and adaptive immunity. RESEARCH HIGHLIGHTS: Telocytes and melanomacrophages were detected in the disseminated pancreatic nodules of the Molly fish. In Molly fish, dendritic cells were found in a close association to the exocrine pancreatic acini. Strong immunoreactivity of the pancreatic acinar cells of the Molly fish to APG5, TGF-ß, IL-1ß, NF-κB, Nrf2, SOX9, and S100.

Co-exposure of iron oxide nanoparticles with glyphosate herbicides in Poecilia reticulata: Fish liver damages is reversible during iron accumulation and elimination period.

Iron oxide nanoparticles (IONPs) are advanced materials for water remediation technologies. It is therefore relevant to evaluate the cellular and tissue behavior of fishes in response to IONPs and their associations with agrochemicals such as glyphosate (GLY) and glyphosate-based herbicides (GBHs). Iron accumulation, tissue integrity and lipid distribution in the hepatocytes of Poecilia reticulata (guppy) were investigated in a control group and in groups exposed to soluble iron ions, namely IFe (0.3 mgFe/L), IONPs (0.3 mgFe/L), and IONPs, associated with GLY (0.65 mg/L), GBHs 0.65 mgGLY/L (IONPs + GBH1), and 1.30 mgGLY/L (IONPs + GBH2), for 7, 14, and 21 days, followed by an equal period of postexposure in clean reconstituted water. The results showed that the accumulation of iron was greater in the subjects in the IONP treatment group when compared to that in the Ife group. In addition, the subjects in the mixtures with GBHs had a greater accumulation of iron than those in the IONP + GLY treatment group. Tissue integrity assessments demonstrated an intense accumulation of lipids, formation of necrotic zones and leukocyte infiltrates in all the treated groups, with a greater quantity of lipids in the animals treated with IONP + GLY and IFe. During postexposure, the results indicated an elimination of iron in all treated groups, reaching the same level as the control group, throughout the 21 days postexposure. Thus, the damage caused to animal livers by IONP mixtures is reversible, providing promising results for the development of safe environmental remediation practices using nanoparticles.

Microplastic exposure and consumption increases susceptibility to gyrodactylosis and host mortality for a freshwater fish.

Microplastics have been found in all surveyed ecosystems and in the diet of multiple species. Detrimental health impacts of microplastic consumption include reduced growth and fecundity, metabolic stress and immune alterations for both invertebrates and vertebrates. Limited information exists, however, on how disease resistance may be affected by microplastic exposure and consumption. Here, the impact of microplastic (0.01 and 0.05 mg l-1 of polypropylene) on fish host susceptibility to disease and mortality was assessed using the guppy Poecilia reticulata-gyrodactylid Gyrodactylus turnbulli system. Fish exposed to and/or consuming microplastic at both concentrations demonstrated significantly higher pathogen burdens over time compared with fish fed a plastic-free diet. Furthermore, microplastic (at both tested concentrations) was associated with increased mortality events for fish within all treatments, regardless of host infection status. This study adds to the growing body of evidence showing that microplastic pollution can be detrimental to fish welfare by reducing disease resistance.

Multispecies collective waving behaviour in fish.

Collective behaviour is widely accepted to provide a variety of antipredator benefits. Acting collectively requires not only strong coordination among group members, but also the integration of among-individual phenotypic variation. Therefore, groups composed of more than one species offer a unique opportunity to look into the evolution of both mechanistic and functional aspects of collective behaviour. Here, we present data on mixed-species fish shoals that perform collective dives. These repeated dives produce water waves capable of delaying and/or reducing the success of piscivorous bird attacks. The large majority of the fish in these shoals consist of the sulphur molly, Poecilia sulphuraria, but we regularly also found a second species, the widemouth gambusia, Gambusia eurystoma, making these shoals mixed-species aggregations. In a set of laboratory experiments, we found that gambusia were much less inclined to dive after an attack as compared with mollies, which almost always dive, though mollies dived less deep when paired with gambusia that did not dive. By contrast, the behaviour of gambusia was not influenced by the presence of diving mollies. The dampening effect of less responsive gambusia on molly diving behaviour can have strong evolutionary consequences on the overall collective waving behaviour as we expect shoals with a high proportion of unresponsive gambusia to be less effective at producing repeated waves. This article is part of a discussion meeting issue 'Collective behaviour through time'.

Effects of ammonia exposure on anxiety behavior, oxidative stress and inflammation in guppy (Poecilia reticulate).

Ammonia is one of the most important aquatic environmental factors, which is of great concern. In order to evaluate the effect of ammonia on guppy (Poecilia reticulate), fish were exposed to increased concentrations (0, 12.50, 25.00, 41.67, 62.50 mg/L) of ammonia for 48 h. After exposure, we measured the anxiety behavior, antioxidant enzymes and pro-inflammation genes (TNF-α, IL-1ß and IL-6) of guppy. The results showed that ammonia stress induced fish anxiety, which was manifested by the increased latency to enter the upper half and decreased time spent in upper half compared with control fish. The guppy showed oxidative stress after 48 h of ammonia stress as evidenced by decreases in the activities of antioxidant enzymes and an increase in lipid hydroperoxide content. With prolonged ammonia stress, the expressions of HSP70, HSP90, TNF-α, IL-1ß and IL-6 mRNA at first had an increasing trend, and then decreased, all of which were significantly higher than the control levels at 12 h and 24 h after ammonia stress (P < 0.05). Ammonia significantly upregulated these genes mRNA levels after 48 h exposure, suggesting that heat shock proteins and innate immune system may try to protect cells from oxidative stress induced by ammonia stress. Our study showed that higher ammonia exposure induced oxidative stress in exposed fish, since inhibition of antioxidant enzymes activity and increases in lipid peroxidation, and inflammation occurred. Furthermore, the results will be helpful to understand the mechanism of ammonia toxicity in guppys.

Effects of 4-n-nonylphenol in liver of male and female viviparous fish (Poecilia vivipara).

4-n-Nonylphenol (NP) is one of the most toxic alkylphenols found in the environment. To evaluate the transcriptional effects of NP in the viviparous fish Poecilia vivipara, a hepatic transcriptome and qPCR analysis of genes were carried out. Guppies separated by sex were injected with two doses of NP (15 µg/g and 150 µg/g) or peanut oil (control). After 24 h, analysis of transcriptional level of Aryl Hydrocarbon Receptor (AhR), Estrogen Nuclear Receptor Alpha (ESR1), Pregnane X Receptor (PXR), Cytochromes P450 (CYP1A, CYP2K1 and CYP3A30), Glutathione S-transferase A3 and Mu 3 (GSTa3 and GSTMu3), SRY-Box Transcription Factor 9 (SOX9), Vitellogenin-1 (VIT), ATP Binding Cassette Subfamily C Member 1 (ABCC1), Multidrug Resistance-Associated Protein 2 (MRP2) and UDP Glucuronosyltransferase Family 1 Member A1 (UGT1A1) was evaluated. 205,046 transcripts were assembled and protein prediction resulted in 203,147 predicted peptides. In females, no significant changes were detected in the transcription of some phase I biotransformation and ABC transporter genes. AhR, PXR, GSTa3 and SOX9 genes where higher in the lower dose group (15 µg/g) compared to control. In male fish, no changes were observed in the transcript levels of the nuclear receptors, in endocrine disruption and phase I biotransformation genes. GSTa3 showed lower transcription in fish treated with both doses. ABCC1 was higher in guppies treated with the lower dose while MRP2 showed less transcripts. This short-term and low-dose exposure to NP caused changes that could serve as early indicators of deleterious processes. These results indicate P. vivipara as a good sentinel in biomonitoring programs.

Co-exposure of iron oxide nanoparticles and glyphosate-based herbicide promote liver toxicity in guppy (Poecilia reticulata): A histochemical and ultrastructural approach.

Citrate functionalized iron oxide nanoparticles (IONPs) are employed for various purposes-including environmental remediation but the interaction of IONPs with aquatic contaminants is poorly understood. Among those, glyphosate-based herbicides are toxic and affect target organs such as the liver. Evaluations of livers of female Poecilia reticulata by exposures to IONPs at a concentration of 0.3 mg/L were performed with association to: (1) 0.65 mg of glyphosate per litter and (2) 1.3 mg of glyphosate per litter of Roundup Original, and (3) glyphosate P.A at 0.65 mg/L. These associations were carried out progressively, after 7, 14, and 21 days. We detected circulatory disturbances, inflammatory responses, activation of the immune system, regressive changes, and progressive responses with changes in the connective tissue and decreased glycogen reserve from days 14 to 21. Ultrastructural changes in the Disse space and microvilli of hepatocytes indicated decreased contact surface area. In general, the damage was time and concentration dependent, increasing from 7 to 14 days and tending to stabilize from 14 to 21 days. Therefore, herbicide-associated IONPs functioned as xenobiotics inducing intense cellular detoxification processes and activation of hepatic immune responses.

Evolution of the Degenerated Y-Chromosome of the Swamp Guppy, Micropoecilia picta.

The conspicuous colour sexual dimorphism of guppies has made them paradigmatic study objects for sex-linked traits and sex chromosome evolution. Both the X- and Y-chromosomes of the common guppy (Poecilia reticulata) are genetically active and homomorphic, with a large homologous part and a small sex specific region. This feature is considered to emulate the initial stage of sex chromosome evolution. A similar situation has been documented in the related Endler's and Oropuche guppies (P. wingei, P. obscura) indicating a common origin of the Y in this group. A recent molecular study in the swamp guppy (Micropoecilia. picta) reported a low SNP density on the Y, indicating Y-chromosome deterioration. We performed a series of cytological studies on M. picta to show that the Y-chromosome is quite small compared to the X and has accumulated a high content of heterochromatin. Furthermore, the Y-chromosome stands out in displaying CpG clusters around the centromeric region. These cytological findings evidently illustrate that the Y-chromosome in M. picta is indeed highly degenerated. Immunostaining for SYCP3 and MLH1 in pachytene meiocytes revealed that a substantial part of the Y remains associated with the X. A specific MLH1 hotspot site was persistently marked at the distal end of the associated XY structure. These results unveil a landmark of a recombining pseudoautosomal region on the otherwise strongly degenerated Y chromosome of M. picta. Hormone treatments of females revealed that, unexpectedly, no sexually antagonistic color gene is Y-linked in M. picta. All these differences to the Poecilia group of guppies indicate that the trajectories associated with the evolution of sex chromosomes are not in parallel.

Can spike fragments of SARS-CoV-2 induce genomic instability and DNA damage in the guppy, Poecilia reticulate? An unexpected effect of the COVID-19 pandemic.

The identification of SARS-CoV-2 particles in wastewater and freshwater ecosystems has raised concerns about its possible impacts on non-target aquatic organisms. In this particular, our knowledge of such impacts is still limited, and little attention has been given to this issue. Hence, in our study, we aimed to evaluate the possible induction of mutagenic (via micronucleus test) and genotoxic (via single cell gel electrophoresis assay, comet assay) effects in Poecilia reticulata adults exposed to fragments of the Spike protein of the new coronavirus at the level of 40 µg/L, denominated PSPD-2002. As a result, after 10 days of exposure, we have found that animals exposed to the peptides demonstrated an increase in the frequency of erythrocytic nuclear alteration (ENA) and all parameters assessed in the comet assay (length tail, %DNA in tail and Olive tail moment), suggesting that PSPD-2002 peptides were able to cause genomic instability and erythrocyte DNA damage. Besides, these effects were significantly correlated with the increase in lipid peroxidation processes [inferred by the high levels of malondialdehyde (MDA)] reported in the brain and liver of P. reticulata and with the reduction of the superoxide dismutase (SOD) and catalase (CAT) activity. Thus, our study constitutes a new insight and promising investigation into the toxicity associated with the dispersal of SARS-CoV-2 peptide fragments in freshwater environments.

Altered phagocytic capacity due to acute exposure and long-term post-exposure to pesticides used for vector-borne disease as dengue.

Spinosad and temefos are widely used pesticides for chemical control of dengue vector-borne disease (Aedes aegypti). The aim of this study was to compare the effect of acute exposure (7 days) to spinosad (0.5 mg A.I. L-1) and temefos (10 mg A.I. L-1), concentrations used by the Mexican Ministry of Health, on phagocytic capacity (PC) of mononuclear cells of guppies fish (Poecilia reticulata), as well as to assess PC in fish, at 96 days after exposure to those pesticides. Obtained results indicated that spinosad did not alter PC, while an acute exposure to temefos significantly affected phagocytosis and this parameter was maintained downed even 96 days after the acute exposure, suggesting that the immunotoxic effects of temefos may be chronic.

Phenotypic responses to oil pollution in a poeciliid fish.

Pollution damages ecosystems around the globe and some forms of pollution, like oil pollution, can be either man-made or derived from natural sources. Despite the pervasiveness of oil pollution, certain organisms are able to colonise polluted or toxic environments, yet we only have a limited understanding of how they are affected by it. Here, we analysed phenotypic responses to oil pollution in guppies (Poecilia reticulata) living in oil-polluted habitats across southern Trinidad. We analysed body-shape and life-history traits for 352 individuals from 11 independent populations, six living in oil-polluted environments (including the naturally oil-polluted Pitch Lake), and five stemming from non-polluted habitats. Based on theory of, and previous studies on, responses to environmental stressors, we predicted guppies from oil-polluted waters to have larger heads and shallower bodies, to be smaller, to invest more into reproduction, and to produce more but smaller offspring compared to guppies from non-polluted habitats. Contrary to most of our predictions, we uncovered strong population-specific variation regardless of the presence of oil pollution. Moreover, guppies from oil-polluted habitats were characterised by increased body size; rounder, deeper bodies with increased head size; and increased offspring size, when compared to their counterparts from non-polluted sites. This suggests that guppies in oil-polluted environments are not only subject to the direct negative effects of oil pollution, but might gain some (indirect) benefits from other concomitant environmental factors, such as reduced predation and reduced parasite load. Our results extend our knowledge of organismal responses to oil pollution and highlight the importance of anthropogenic pollution as a source of environmental variation. They also emphasise the understudied ecological heterogeneity of extreme environments.

Recovery trend to co-exposure of iron oxide nanoparticles (γ-Fe2O3) and glyphosate in liver tissue of the fish Poecilia reticulata.

Citrate-coated iron oxide nanoparticles (IONPs) have potential use in environmental remediation, with possibilities in decontaminating aquatic environments exposed to toxic substances. This study analyzed IONPs associated to Roundup Original, a glyphosate-based herbicide (GBH), and pure glyphosate (GLY), through ultrastructural and histopathological biomarkers in liver tissue, from females of Poecilia reticulata exposed to: iron ions (0.3 mg/L) (IFe) and IONPs (0.3 mgFe/L) associated with GLY (0.65 mg/L) and GBH (0.65 mgGLY/L (IONP + GBH1) and 1.30 mgGLY/L (IONP + GBH2)) for a period of 7, 14 and 21 days, followed by an equal post-exposure period only in reconstituted water. For the assays, the synthetized IONPs had crystalline and rounded shape with an average diameter of 2,90 nm, hydrodynamic diameter 66,6 mV, zeta potential -55,4 and diffraction profile of maghemite (γ-Fe2O3). The data obtained by biomarkers indicated a high inflammatory response in all treatments. These same parameters, considered during the post-exposure period indicated recovery in reaction patterns of circulatory disturbances and regressive changes, resulting in average reductions of 37,53 points in IFe, 21 points in IONP + GBH1, 15 points in IONP + GBH2 and 11 points in IONP + GLY in total histopathological index of liver after 21 days post-exposure. However, although the cellular and tissue responses were significant, there was no change in the condition factor and hepatosomatic index, denoting resilience of the experimental model.

The extraordinary partnership of Geoff Burnstock and Mollie Holman.

Here, we recognise some of the extraordinary accomplishments of the partnership between Geoff Burnstock and Mollie Holman, and the everlasting impact they both made in autonomic neuroscience in Australia. Much of strength today in autonomic neuroscience can be traced back to a time when Geoff and Mollie commenced their seminal studies on autonomic neuroscience, initially at Oxford, then at The University of Melbourne in the mid 1960's. Mollie and Geoff published their first paper together, at Oxford, with their then mentor, and doyenne of smooth muscle, Professor Edith Bülbring. They did not always agree on the interpretation of their own scientific findings. Geoff was convinced early on that Adenosine triphosphate (ATP), or a related purine, was an excitatory neurotransmitter at peripheral sympathetic neuroeffector junctions. Mollie was reticent for decades. However, she began to take the notion seriously that ATP maybe a neurotransmitter, when receptors for purines were identified in the 1990's. What the partnership between Mollie and Geoff taught us in Australia was to not fear respectful criticism, but rather to be receptive to and embrace objective, collegial and constructive scientific peer-review. One of the many great legacies of Geoff and Mollie was the large number of researchers, who were fortunate disciples of their supervision, and who have now themselves gone on to make significant discoveries in autonomic and visceral neuroscience. This review summarizes some of their major legacies and represents a very personal historical perspective of the two authors, pupils respectively of Mollie and Geoff.

Co-exposure of iron oxide nanoparticles and glyphosate-based herbicide induces DNA damage and mutagenic effects in the guppy (Poecilia reticulata).

Iron oxide nanoparticles (IONPs) have been tested to remediate aquatic environments polluted by chemicals, such as pesticides. However, their interactive effects on aquatic organisms remain unknown. This study aimed to investigate the genotoxicity and mutagenicity of co-exposure of IONPs (γ-Fe2O3 NPs) and glyphosate-based herbicide (GBH) in the fish Poecilia reticulata. Thus, fish were exposed to citrate-functionalized γ-Fe2O3 NPs (0.3 mg L-1; 5.44 nm) alone or co-exposed to γ-Fe2O3 NPs (0.3 mg L-1) and GBH (65 and 130 µg of glyphosate L-1) during 14 and 21 days. The genotoxicity (DNA damage) was analyzed by comet assay, while the mutagenicity evaluated by micronucleus test (MN test) and erythrocyte nuclear abnormalities (ENA) frequency. The co-exposure induced clastogenic (DNA damage) and aneugenic (nuclear alterations) effects on guppies in a time-dependent pattern. Fish co-exposed to NPs and GBH (130 µg glyphosate L-1) showed high DNA damage when compared to NPs alone and control group, indicating synergic effects after 21 days of exposure. However, mutagenic effects (ENA) were observed in the exposure groups after 14 and 21 days. Results showed the potential genotoxic and mutagenic effects of maghemite NPs and GBH co-exposure to freshwater fish. The transformation and interaction of iron oxide nanoparticles with other pollutants, as herbicides, in the aquatic systems are critical factors in the environmental risk assessment of metal-based NPs.

Bio-robotic cues show how the Trinidadian guppy male recognises the morphological features of receptive females.

Sensory fusion is used by the males of several animal species to discriminate the mating status of females by evaluating their phenotypic traits. The predominant trait used is olfactory cues, and the role of visual cues is not yet fully understood. The ability of Poecilia reticulata males to evaluate females' receptivity based on visual cues was investigated. Guppy males adopt two different mating strategies, courtship displays and forced copulation, towards receptive and pregnant females, respectively. Robotic counterparts mimicking receptive and pregnant females were developed to test whether males relied only on visual information to determine a females' mating status. Exposure to the robotic receptive females evoked courtship behaviours, while forced copulation attempts were more frequent towards the robotic pregnant females. When the robotic fish were simultaneously exposed, regardless of the presence or absence of receptive-female olfactory cues, males expressed their preference for the receptive female. Visual processing showed that fish social interactions played a strategic role in their collection of information, especially when other stimuli were not available. The proposed ethorobotic approach allowed for assessing the role of visual cues in the mating choice of P. reticulata males and highlighting the information processing methods and cognition in aquatic animals.