Beyond the Brain: Perinatal Exposure of Rats to Serotonin Enhancers Induces Long-Term Changes in the Jejunum and Liver.

Serotonin (5-hydroxytryptamine, 5HT) homeostasis is essential for many physiological processes in the central nervous system and peripheral tissues. Hyperserotonemia, a measurable sign of 5HT homeostasis disruption, can be caused by 5HT-directed treatment of psychiatric and gastrointestinal diseases. Its impact on the long-term balance and function of 5HT in the peripheral compartment remains unresolved and requires further research due to possible effects on human health. We explored the effects of perinatal 5HT imbalance on the peripheral organs responsible for serotonin metabolism-the jejunum, a synthesis site, and the liver, a catabolism site-in adult rats. Hyperserotonemia was induced by subchronic treatment with serotonin precursor 5-hydroxytryptophan (5HTP) or serotonin degradation inhibitor tranylcypromine (TCP). The jejunum and liver were collected on postnatal day 70 and analyzed histomorphometrically. Relative mRNA levels of 5HT-regulating proteins were determined using qRT-PCR. Compared to controls, 5HTP- and TCP-treated rats had a reduced number of 5HT-producing cells and expression of the 5HT-synthesising enzyme in the jejunum, and an increased expression of 5HT-transporter accompanied by karyomegaly in hepatocytes, with these differences being more pronounced in the TCP-treated animals. Here, we report that perinatal 5HT disbalance induced long-term cellular and molecular changes in organs regulating 5HT-metabolism, which may have a negative impact on 5HT availability and function in the periphery. Our rat model demonstrates a link between the developmental abnormalities of serotonin homeostasis and 5HT-related changes in adult life and may be suitable for exploring the neurobiological substrates of vulnerability to behavioral and metabolic disorders, as well as for modeling the adverse effects of the prenatal exposure to 5HT enhancers in the human population.

Combined effects of valproate and naringin on kidney antioxidative markers and serum parameters of kidney function in C57BL6 mice.

Valproate is known to disturb the kidney function, and high doses or prolonged intake may cause serum ion imbalance, kidney tubular acidosis, proteinuria, hyperuricosuria, polyuria, polydipsia, and dehydration. The aim of this in vivo study was to see whether naringin would counter the adverse effects of high-dose valproate in C57Bl/6 mice and to which extent. As expected, valproate (150 mg/kg bw a day for 10 days) caused serum hyperkalaemia, more in male than female mice. Naringin reversed (25 mg/kg bw a day for 10 days) the hyperkalaemia and activated antioxidative defence mechanisms (mainly catalase and glutathione), again more efficiently in females. In males naringin combined with valproate was not as effective and even showed some prooxidative effects.

The first cavefish in the Dinaric Karst? Cave colonization made possible by phenotypic plasticity in Telestes karsticus.

Cave animals are an excellent model system for studying adaptive evolution. At present, however, little is known about the mechanisms that enable surface colonizers to survive in the challenging environment of caves. One possibility is that these species have the necessary genetic background to respond with plastic changes to the pressures of underground habitats. To gain insight into this process, we conducted a comparative study with the fish species Telestes karsticus, which occurs in a hydrological system consisting of an interconnected stream and a cave. Results showed that T. karsticus resided year-round and spawned in Susik cave, making it the first known cavefish in the Dinaric Karst. Cave and surface populations differed in morphological and physiological characteristics, as well as in patterns of gene expression without any evidence of genetic divergence. To test whether observed trait differences were plastic or genetic, we placed adult fish from both populations under light/dark or constant dark conditions. Common laboratory conditions erased all morphometric differences between the two morphs, suggesting phenotypic plasticity is driving the divergence of shape and size in wild fish. Lighter pigmentation and increased fat deposition exhibited by cave individuals were also observed in surface fish kept in the dark in the laboratory. Our study also revealed that specialized cave traits were not solely attributed to developmental plasticity, but also arose from adult responses, including acclimatization. Thus, we conclude that T. karsticus can adapt to cave conditions, with phenotypic plasticity playing an important role in the process of cave colonization.

The Effects of Nettle Extract Consumption on Liver PPARs, SIRT1, ACOX1 and Blood Lipid Levels in Male and Female C57Bl6 Mice.

The aim of this study was to evaluate how nettle (Urtica dioica L.) water extract consumption would interact with regulators of peroxysomal lipid oxidation, histone deacetylase, and markers of oxidative stress in the liver and blood lipid levels in male and female C57Bl6 mice. Metabolically unchallenged (healthy) mice (n = 5 per sex) were treated with a nettle extract in a dose of 40 mg of total polyphenols in the extract per kg mice body weight. The nettle extract was applied daily along with normal diet for 15 days. The serum triglycerides, cholesterol, HDL, LDL, and liver PPAR-α, PPAR-γ, PGC-1-α, ACOX1, SIRT1, MDA, SOD, CAT, and GSH were compared between exposed and unexposed (control) animals. In males, the PPAR-α, PGC1-α, and ACOX1 levels together with systemic HDL cholesterol were significantly (p ≤ 0.05) increased while the LDL cholesterol decreased (p ≤ 0.05). In females, no changes in PPAR-α and PGC1-α or serum lipids were noted, but the ACOX1 content in the liver was significantly (p ≤ 0.05) increased. The SIRT1 activity increased (p ≤ 0.05) only in females. In both sexes, the PPAR-γ levels were not significantly (p ≤ 0.05) affected in either sex. The results indicate that nettle plant extract has the potential to modulate selected transcriptional factors and histone deacetylase in vivo, with certain sex differences, which should be studied further in similar models.

Effects of naringin and valproate interaction on liver steatosis and dyslipidaemia parameters in male C57BL6 mice.

Valproate is a common antiepileptic drug whose adverse effects include liver steatosis and dyslipidaemia. The aim of our study was to see how natural flavonoid antioxidant naringin would interact with valproate and attenuate these adverse effects. For this reason we treated male C57BL6 mice with a combination of 150 mg/kg of valproate and 25 mg/kg naringin every day for 10 days and compared their serum triglycerides, cholesterol, LDL, HDL, VLDL, and liver PPAR-alpha, PGC-1 alpha, ACOX1, Nrf2, SOD, CAT, GSH, and histological signs of steatosis. Valproate increased lipid peroxidation parameters and caused pronounced microvesicular steatosis throughout the hepatic lobule in all acinar zones, but naringin co-administration limited steatosis to the lobule periphery. In addition, it nearly restored total serum cholesterol, LDL, and triglycerides and liver ACOX1 and MDA to control levels. and upregulated PPAR-alpha and PGC-1 alpha, otherwise severely downregulated by valproate. It also increased SOD activity. All these findings suggest that naringin modulates key lipid metabolism regulators and should further be investigated in this model, either alone or combined with other lipid regulating drugs or molecules.

Disturbance in invasion? Idiopathic necrotizing hepatopancreatitis in the signal crayfish Pacifastacus leniusculus (Dana, 1852) in Croatia.

As the most successful crayfish invader and possible vector for infectious agents, signal crayfish Pacifastacus leniusculus is among the major drivers of the native crayfish species decline in Europe. We describe histopathological manifestation and frequency of newly detected idiopathic necrotizing hepatopancreatitis along the invasion range of the signal crayfish in the Korana River in Croatia. Our results show extremely high prevalence of necrotizing hepatopancreatitis (97.3%), with 58.9% of individuals displaying mild and 31.5% moderate histopathological changes in the hepatopancreas, also reflected in the lower hepatosomatic index of analysed animals. Recorded histopathological changes were more frequent in the invasion core where population density is higher. Our preliminary screening of co-occurring native narrow-clawed crayfish Pontastacus leptodactylus showed lower incidence (33.3%) and only mild hepatopancreatic lesions, but potentially highlighted the susceptibility of native crayfish populations to this disease. Pilot analyses of dissolved trace and macro elements in water, sediment fractions and crayfish hepatopancreas do not highlight alarming or unusually high concentrations of analysed elements. Hepatopancreas microbiome analysis, using 16S rRNA gene amplicon sequencing, identified taxonomic groups that should be further investigated, along with impacts of the disease on health and viability of both invasive and native crayfish populations.

Characterization of oral and cloacal microbial communities of wild and rehabilitated loggerhead sea turtles (Caretta caretta).

BACKGROUND: Microbial communities of wild animals are being increasingly investigated to provide information about the hosts' biology and promote conservation. Loggerhead sea turtles (Caretta caretta) are a keystone species in marine ecosystems and are considered vulnerable in the IUCN Red List, which led to growing efforts in sea turtle conservation by rescue centers around the world. Understanding the microbial communities of sea turtles in the wild and how affected they are by captivity, is one of the stepping stones in improving the conservation efforts. Describing oral and cloacal microbiota of wild animals could shed light on the previously unknown aspects of sea turtle holobiont biology, ecology, and contribute to best practices for husbandry conditions. RESULTS: We describe the oral and cloacal microbiota of Mediterranean loggerhead sea turtles by 16S rRNA gene sequencing to compare the microbial communities of wild versus turtles in, or after, rehabilitation at the Adriatic Sea rescue centers and clinics. Our results show that the oral microbiota is more sensitive to environmental shifts than the cloacal microbiota, and that it does retain a portion of microbial taxa regardless of the shift from the wild and into rehabilitation. Additionally, Proteobacteria and Bacteroidetes dominated oral and cloacal microbiota, while Kiritimatiellaeota were abundant in cloacal samples. Unclassified reads were abundant in the aforementioned groups, which indicates high incidence of yet undiscovered bacteria of the marine reptile microbial communities. CONCLUSIONS: We provide the first insights into the oral microbial communities of wild and rehabilitated loggerhead sea turtles, and establish a framework for quick and non-invasive sampling of oral and cloacal microbial communities, useful for the expansion of the sample collection in wild loggerhead sea turtles. Finally, our investigation of effects of captivity on the gut-associated microbial community provides a baseline for studying the impact of husbandry conditions on turtles' health and survival upon their return to the wild.

An emergency situation for pen shells in the Mediterranean: The Adriatic Sea, one of the last Pinna nobilis shelters, is now affected by a mass mortality event.

We identified areas with high individual densities of the pen shell, Pinna nobilis, in two areas along the Croatian Adriatic coast. The surveys carried out in 2018 and 2019 showed population densities of approximately 9 to 13 individuals/100 m2. However, in 2019 a mass mortality event (MME) causing 36% to 100% mortality of this bivalve species was observed in the surveyed Croatian bays. The parasite Haplosporidium pinnae was identified by histological and molecular methods in all affected sampled individuals, while Mycobacterium sp. and Gram negative bacilli were detected in some affected and live bivalves. This finding constitutes the first record of these pathogens affecting P. nobilis in the middle Adriatic, confirming the continuous spread of the disease. Previously, the Adriatic water body was considered to be a natural shelter against the MME caused by pathogens in pen shell populations because of its distinct ecological features. The Adriatic Sea is a semi-closed water body with the largest continental shelf in the Mediterranean Sea, and due to its geomorphology and bathymetry, it is a sea with distinct characteristics. Monitoring plans and further studies in the Adriatic bays are now a priority for mitigating the high risk of extinction and working toward the conservation of this protected species.

Phenotypic plasticity as a mechanism of cave colonization and adaptation.

A widely accepted model for the evolution of cave animals posits colonization by surface ancestors followed by the acquisition of adaptations over many generations. However, the speed of cave adaptation in some species suggests mechanisms operating over shorter timescales. To address these mechanisms, we used Astyanax mexicanus, a teleost with ancestral surface morphs (surface fish, SF) and derived cave morphs (cavefish, CF). We exposed SF to completely dark conditions and identified numerous altered traits at both the gene expression and phenotypic levels. Remarkably, most of these alterations mimicked CF phenotypes. Our results indicate that many cave-related traits can appear within a single generation by phenotypic plasticity. In the next generation, plasticity can be further refined. The initial plastic responses are random in adaptive outcome but may determine the subsequent course of evolution. Our study suggests that phenotypic plasticity contributes to the rapid evolution of cave-related traits in A. mexicanus.

The Mexican tetra is a fish that has two forms: a surface-dwelling form, which has eyes and silvery grey appearance, and a cave-dwelling form, which is blind and has lost its pigmentation. Recent studies have shown that the cave-dwelling form evolved rapidly within the last 200,000 years from an ancestor that lived at the surface. The recent evolution of the cave-dwelling form of the tetra poses an interesting evolutionary question: how did the surface-dwelling ancestor of the tetra quickly adapt to the new and challenging environment found in the caves? 'Phenotypic plasticity' is a phenomenon through which a single set of genes can produce different observable traits depending on the environment. An example of phenotypic plasticity occurs in response to diet: in animals, poor diets can lead to an increase in the size of the digestive organs and to the animals eating more. To see if surface-dwelling tetras can quickly adapt to cave environments through phenotypic plasticity, Bilandzija et al. have exposed these fish to complete darkness (the major feature of the cave environment) for two years. After spending up to two years in the dark, these fish were compared to normal surface-dwelling and cave-dwelling tetras. Results revealed that surface-dwelling tetras raised in the dark exhibited traits associated with cave-dwelling tetras. These traits included changes in the activity of many genes involved in diverse processes, resistance to starvation, metabolism, and levels of hormones and molecules involved in neural signaling, which could lead to changes in behavior. However, the fish also exhibited traits, including an increase in the cells responsible for pigmentation, that would have no obvious benefit in the darkness. Even though the changes observed require no genetic mutations, they can help or hinder the fish's survival once they occur, possibly determining subsequent evolution. Thus, a trait beneficial for surviving in the dark that appears simply through phenotypic plasticity may eventually be selected for and genetic mutations that encode it more reliably may appear too. These results shed light on how species may quickly adapt to new environments without accumulating genetic mutations, which can take hundreds of thousands of years. They also may help to explain how colonizer species succeed in challenging environments. The principles described by Bilandzija et al. can be applied to different organisms adapting to new environments, and may help understand the role of phenotypic plasticity in evolution.

Oxidative stress, polarization of macrophages and tumour angiogenesis: Efficacy of caffeic acid.

Macrophage polarization is a process when macrophage expresses different functional programs in response to microenvironmental signals and two extreme forms exist; M1 and M2 macrophages. M1 macrophages are highly microbicidal and anticancer with enhanced ability to kill and phagocytose pathogens, upregulate pro-inflammatory cytokines and reactive molecular species, and present antigens; M2 macrophages and the related tumour associated macrophages (TAMs) regulate tissue remodelling and promote tissue repair and angiogenesis and can amplification of metabolic pathways that can suppress adaptive immune responses. It is demonstrated that ROS production, critical for the activation and functions of M1 macrophages, is necessary for the differentiation of M2 macrophages and TAMs, and that antioxidant therapy blocks TAMs differentiation and tumorigenesis in mouse models of cancer. In order to study how caffeic acid (CA), a natural antioxidant, affects macrophage function, polarization, angiogenesis and tumour growth we injected mice with Ehrlich ascites tumour (EAT) cells and treated them for 10 days with CA in a dose of 40 and/or 80 mg kg(-1.) Macrophage polarization was further characterized by quantifying secreted pro- and anti-inflammatory cytokines, nitric oxide and arginase 1 activity. CA may increase the cytotoxic actions of M1 macrophages and inhibit tumour growth; inhibitory activity on TAMs may be mediated through its antioxidative activity. Taken together, we conclude that the antitumour activity of CA was the result of the synergistic activities of different mechanisms by which CA acts on proliferation, angiogenesis, immunomodulation and survival. The continuous administration of CA efficiently blocked the occurrence of TAMs and markedly suppressed tumorigenesis in mouse cancer models. Targeting TAMs by antioxidants can be a potentially effective method for cancer treatment.

Croatian aquatic dance flies (Diptera: Empididae: Clinocerinae and Hemerodromiinae): species diversity, distribution and relationship to surrounding countries.

A checklist of aquatic Empididae (dance flies) from Croatia (36 species in subfamily Clinocerinae and 14 species in subfamily Hemerodromiinae) is presented, including information related to the Ecoregions in which species were found and specific species traits. Clinocerinae are represented by five genera, with Wiedemannia Zetterstedt being most species rich (20 species) and Clinocerella Engel least numerous with only one species. In Hemerodromiinae there are 8 species of Chelifera Macquart and 6 species and Hemerodromia Meigen. In addition, a discussion related to the species included and excluded from the list is provided. Most species are univoltine with adults emerging in Spring and Summer, although Kowarzia barbatula Mik and Wiedemannia (Eucelidia) zetterstedti (Fallén) are present throughout the year and Wiedemannia (Chamaedipsia) aequilobata Mandaron occurrs in Winter. The Croatian species assemblage is similar to the well-studied fauna of neighboring Slovenia (63 spp.). It is recommended that some rare species and the streams they inhabit should be considered for greater protection.

Gastrointestinal helminth community of loggerhead sea turtle Caretta caretta in the Adriatic Sea.

We analysed the intestinal helminth community of 70 loggerhead sea turtles Caretta caretta with a curved carapace length ranging from 25 to 85.4 cm, recovered dead in neritic foraging habitats in the Adriatic Sea in 1995 to 2004. The overall prevalence of infection was high (70.0%), with a mean abundance of 36.8 helminth parasites per turtle. Helminth fauna comprised 5 trematodes (Calycodes anthos, Enodiotrema megachondrus, Orchidasma amphiorchis, Pachypsolus irroratus, Rhytidodes gelatinosus) and 3 nematodes (Sulcascaris sulcata, Anisakis spp., Hysterothylacium sp.), with 6 taxa specific for marine turtles. In terms of infection intensity and parasite abundance, O. amphiorchis was the dominant species (mean intensity: 49.8; mean abundance: 12.8), followed by R. gelatinosus (30.5 and 8.3, respectively) and P. irroratus (23.5 and 7.0, respectively), while larval Anisakis spp. exhibited the highest prevalence (34.3%). The intensity of helminth infection ranged from 1 to 302 (mean: 52.6 ± 69.1) and was not correlated with the size of turtles; this relationship held for all species, except R. gelatinosus (rS = 0.556, p < 0.05). In comparison to other marine habitats, the helminth community of Adriatic loggerheads is characterised by higher species diversity (Shannon-Wiener H' = 1.58) and evenness (E = 0.76), and lower dominance values (Berger-Parker d = 0.35), which can be attributed to the life history and feeding ecology of sea turtles in recruited neritic grounds and the diversity of their benthic prey.

Accumulation of organochlorine contaminants in loggerhead sea turtles, Caretta caretta, from the eastern Adriatic Sea.

We analyzed 15 polychlorinated biphenyls (PCBs) and five organochlorine pesticides (OCPs) in fat tissue of 27 loggerhead sea turtles (Caretta caretta) from eastern Adriatic Sea with the curved carapace length ranging from 25.0 to 84.5 cm. The PCB profile was dominated by hexa- and hepta-chlorinated congeners, with PCB-153 (median: 114.9 ng g⁻¹ wet mass), PCB-138 (80.0 ng g⁻¹ w.m.), and PCB-180 (26.7 ng g⁻¹ w.m.) detected with the highest levels. Toxic mono-ortho congeners PCB-118, PCB-105 and PCB-180 with dioxin-like toxicity were found in > 70% samples. 4,4'-DDE (81.0 ng g⁻¹ w.m.) prevailed the OCP signature, accounting for 80% of the total DDTs. A significant increasing trend in accumulation with size was found for PCB-52 (r(s) = 0.512, p < 0.01) and PCB-114 (r(s) = 0.421, p < 0.05). Comparison of our results with organochlorine contaminant (OC) pattern in some prey taxa suggest that contamination occurs primarily through the food web, with biomagnification factors of 0.14-6.99 which were positively correlated with the octanol-water partition coefficient (logK(ow); p < 0.05). This study, based upon a fairly large sample size collected mostly from incidentally captured animals over a short time period (June 2001-November 2002), present a temporal snapshot of OC contamination in wild, free-ranging loggerheads from Adriatic feeding grounds and provide a baseline for monitoring the regional OC trends in this endangered species.

Ingestion of marine debris by loggerhead sea turtles, Caretta caretta, in the Adriatic Sea.

We examined the occurrence of marine debris in the gastrointestinal tract of 54 loggerhead sea turtles (Caretta caretta) found stranded or incidentally captured dead by fisheries in the Adriatic Sea, with a curved carapace length of 25.0-79.2 cm. Marine debris was present in 35.2% of turtles and included soft plastic, ropes, Styrofoam and monofilament lines found in 68.4%, 42.1%, 15.8% and 5.3% of loggerheads that have ingested debris, respectively. The dry mass of debris per turtle was low, ranging from <0.01 to 0.71 g, and the ingestion was not significantly affected by sex or body size (all p>0.05). Marine debris averaged 2.2 ± 8.0% of dry mass of gut content, with a maximum of 35% found in a juvenile turtle that most likely died due to debris ingestion. Considering the relatively high occurrence of debris intake and possible sub-lethal effects of even small quantities of marine debris, this can be an additional factor of concern for loggerheads in the Adriatic Sea.