Non-concordant epigenetic and transcriptional responses to acute thermal stress in western mosquitofish (Gambusia affinis).

Climate change is intensifying the frequency and severity of extreme temperatures. Understanding the molecular mechanisms underlying the ability to cope with acute thermal stress is key for predicting species' responses to extreme temperature events. While many studies have focused on the individual roles of gene expression, post-transcriptional processes and epigenetic modifications in response to acute thermal stress, the relative contribution of these molecular mechanisms remains unclear. The wide range of thermal limits of western mosquitofish (Gambusia affinis) provides an opportunity to explore this interplay. Here, we quantified changes in gene expression, alternative splicing, DNA methylation and microRNA (miRNA) expression in muscle tissue dissected from mosquitofish immediately after reaching high (CTmax) or low thermal limit (CTmin). Although the numbers of genes showing expression and splicing changes in response to acute temperature stress were small, we found a possibly larger and non-redundant role of splicing compared to gene expression, with more genes being differentially spliced (DSGs) than differentially expressed (DEGs), and little overlap between DSGs and DEGs. We also identified a small proportion of CpGs showing significant methylation change (i.e. differentially methylated cytosines, DMCs) in fish at thermal limits; however, there was no overlap between DEGs and genes annotated with DMCs in both CTmax and CTmin experiments. The weak interplay between epigenetic modifications and gene expression was further supported by our discoveries of no differentially expressed miRNAs. These findings provide novel insights into the relative role of different molecular mechanisms underlying immediate responses to extreme temperatures and demonstrate non-concordant responses of epigenetic and transcriptional mechanisms to acute temperature stress.

Neurotoxic effects of carbamazepine on the mosquitofish Gambusia affinis.

In recent years, the presence of pharmaceuticals in the aquatic environment has gained a significant attention. Carbamazepine, a commonly prescribed antiepileptic drug, has been consistently found in aquatic environments at concentrations ranging from nanograms to micrograms, raising concerns about its potential negative impacts on aquatic organisms. The study examined the acute and chronic neurotoxic effects of environmentally relevant and sublethal concentrations of carbamazepine in the mosquitofish Gambusia affinis. After a 96-hour exposure period, the median lethal concentration (LC50) of carbamazepine for G. affinis was determined as 24 mg L - 1. For the current study, sublethal concentrations i.e., one-tenth (2.4 mg L - 1) and one-fifth (4.8 mg L - 1) of the LC50 value were chosen for assessing the neurotoxic effects along with the environmentally relevant concentration (13 ng L - 1). The research findings indicated that carbamazepine had a disruptive impact on the typical growth and behavior of the fish. During the acute exposure phase, physical deformities were observed in the fish, resulting in neonatal and postneonatal fatalities. Furthermore, the neurotoxic effects of carbamazepine were clearly demonstrated through alterations in various neurological parameters, including acetylcholinesterase, dopamine, gamma-aminobutyric acid, serotonin, monoamine oxidase, 5-hydroxyindole acetic acid, adrenaline, and nor-adrenaline. These findings raise concerns about the survival of fish populations in their natural environment.

Effects of TLR2/4 signalling pathway in western mosquitofish (Gambusia affinis) after Edwardsiella tarda infection.

Gambusia affinis is regarded as an important animal model. Edwardsiella tarda is one of the most serious pathogens affecting aquaculture. The study explores the effects of TLR2/4 partial signalling pathway in G. affinis of E. tarda infection. The study collected the brain, liver, and intestine after E. tarda LD50 and 0.85% NaCl solution challenge at different times (0 h, 3 h, 9 h, 18 h, 24 h, and 48 h). In these three tissues, the mRNA levels of PI3K, AKT3, IRAK4, TAK1, IKKß, and IL-1ß were substantially enhanced (p < .05) then returned to normal levels. Additionally, Rac1 and MyD88 in liver had different trend with other genes in brain and intestine, which displayed significantly indifference. The overexpression of IKKß, and IL-1ß indicated that E. tarda also caused immune reaction in intestine and liver, which would be consistent with delayed edwardsiellosis, which causes intestinal lesions and liver and kidney necrosis. Additionally, MyD88 plays a smaller role than IRAK4 and TAK1 in this signalling pathways. This study could enrich the understanding of the immune mechanism of the TLR2/4 signalling pathway in fish and might help to prescribe preventive measures against E. tarda to prevent infectious diseases in fish.

Effects of norethindrone on the growth, behavior, and thyroid endocrine system of adult female western mosquitofish (Gambusia affinis).

Progestins are mainly used in pharmacotherapy and animal husbandry and have received increasing attention as they are widely detected in various aquatic ecosystems. In this study, adult female western mosquitofish (Gambusia affinis) were exposed to different concentrations of norethindrone (NET) (solvent control, 5.0 (L), 50.0 (M), and 500.0 (H) ng/L) for 42 days. Behaviors, morphological parameters, histology of the thyroid, thyroid hormone levels (TSH, T3, and T4), and transcriptional levels of nine genes in the hypothalamic-pituitary-thyroid (HPT) axis were examined. The results showed that NET decreased sociality but increased the anxiety of G. affinis. Sociality makes fish tend to cluster, and anxiety may cause G. affinis to reduce exploration of new environments. Female fish showed hyperplasia, hypertrophy, and glial depletion in their thyroid follicular epithelial cells after NET treatment. The plasma levels of TSH and T4 were significantly reduced, but T3 concentrations were significantly increased in the fish from the H group. In addition, the transcripts of genes (tshb, tshr, tg, dio1, dio2, thrb) in the brains of fish in the M and H treatments were significantly stimulated, while those of trh and pax2a were suppressed. Our results suggest that NET may impact key social behaviors in G. affinis and interfere with the entire thyroid endocrine system, probably via affecting the transcriptional expression of upstream regulators in the HPT axis.

Drivers and Cascading Ecological Consequences of Gambusia affinis Trait Variation.

AbstractPhenotypic trait differences among populations can shape ecological outcomes for communities and ecosystems. However, few studies have mechanistically linked heritable and plastic components of trait variation to generalizable processes of ecology, such as trophic cascades. Here, we assess morphological and behavioral trait variation in nine populations of common garden-reared western mosquitofish (Gambusia affinis) from three distinct ancestral predator environments (three populations per environment), each reared in the presence and absence of predator cues. We then use a pond mesocosm experiment to examine the ecological consequences of mosquitofish trait variation and density variation. Our results show significant among-population trait variation, but this variation was generally unrelated to ancestral predator environment. When traits did vary congruently with respect to ancestral predator environment, this trait variation was driven by gene-by-environment interactions. Variation in several mosquitofish traits altered the cascading effects of mosquitofish on zooplankton and primary producers, but the effect of any given trait was typically weaker than that of density. We note that the relatively stronger ecological effects of density may mask the effects of traits in some systems. Our example here shows that trait variation can be highly noncongruent with respect to a perceived selective agent, phenotypic change is a product of complex interactions between genes and the environment, and numerous interacting phenotypes generate significant but potentially cryptic cascading ecological change.

The legacy of predator threat shapes prey foraging behaviour.

Predators exert strong selection on prey foraging behaviour such that prey responses may reflect a combination of ancestral effects of predators (genetic and nongenetic transgenerational effects), past individual experience with predators (phenotypic plasticity), and current exposure to predators (behavioural response). However, the importance of these factors in shaping prey foraging behaviour is not well understood. To test the relative effects of ancestry, prior experience, and current exposure, we measured foraging rates and food size preference of different ancestry and exposure groups of Western mosquitofish in the presence and absence of immediate threat from predatory largemouth bass. Our results confirm that mosquitofish had lower foraging rate in the immediate presence of predator threat. Mosquitofish also foraged at a lower rate if they had ancestry with predators, regardless of immediate threat. In contrast, individual prior experience with predators only caused reduced foraging rates in the immediate presence of a predator. This suggests that phenotypic plasticity could carry a lower risk of maladaptive antipredator responses-i.e., reduced food intake-in the complete absence of a predator. Finally, in the presence of a predator, mosquitofish with both ancestry and experience with predators consumed larger, presumably more energetically valuable, food items. Overall, our results show that non-consumptive effects of predators on prey behaviour can persist within and across generations, such that the legacy of past predator exposure-or "the ghost of predation past"-may continue to shape prey behaviour even when predators are no longer around.

Color Discrimination Provides Insight into the Relationship between Personality Cognition and Brain Morphology in the Western Mosquitofish (Gambusia affinis).

Recent studies on the differences in cognitive ability between individuals focused on two aspects: one is whether the individual differences in cognitive ability are related to brain size, the other is whether they pertain to certain personality traits. To explore these two hypotheses, we tested the personality traits, cognitive abilities, and brain volumes of western mosquitofish (Gambusia affinis). First, a color preference test was conducted to select two unbiased colors for G. affinis for subsequent cognitive tests. The results showed that G. affinis had a great preference for red and green to yellow and blue; therefore, the red-green combination was selected for the study of cognitive abilities. Then, we explored the relationship among cognition, personality, and brain morphology through cognitive abilities tests, personality traits, and brain volume measurements. We found that there was a trade-off among cognition, personality, and brain morphology. For example, more active individuals found food faster, but had also poor memory; Those individuals with larger corpus cerebelli were bolder while they were less likely to find food; The individuals that found food faster were more active and had a smaller inferior lobe. The color preference test provides a reliable way for selecting unbiased colors for behavioral studies in G. affinis. Meanwhile, our study indicates that there exists a balance mechanism among cognition, personality, and brain morphology.

Consumer trait responses track change in resource supply along replicated thermal gradients.

Rising temperatures may alter consumer diets through increased metabolic demand and altered resource availability. However, current theories assessing dietary shifts with warming do not account for a change in resource availability. It is unknown whether consumers will increase consumption rates or consume different resources to meet increased energy requirements and whether the dietary change will lead to associated variation in morphology and nutrient utilization. Here, we used populations of Gambusia affinis across parallel thermal gradients in New Zealand (NZ) and California (CA) to understand the influence of temperature on diets, morphology and stoichiometric phenotypes. Our results show that with increasing temperature in NZ, mosquitofish consumed more plant material, whereas in CA mosquitofish shifted towards increased consumption of invertebrate prey. In both regions, populations with plant-based diets had fuller guts, longer relative gut lengths, better-orientated mouths and reduced body elemental %C and N/P. Together, our results show multiple pathways by which consumers may alter their feeding patterns with rising temperatures, and they suggest that warming-induced changes to resource availability may be the principal determinant of which pathway is taken.

Male size and reproductive performance in three species of livebearing fishes (Gambusia spp.): A systematic review and meta-analysis.

The genus Gambusia represents approximately 45 species of polyandrous livebearing fishes with reversed sexual size dimorphism (i.e. males smaller than females) and with copulation predominantly via male coercion. Male body size has been suggested as an important sexually selected trait, but despite abundant research, evidence for sexual selection on male body size in this genus is mixed. Studies have found that large males have an advantage in both male-male competition and female choice, but that small males perform sneaky copulations better and at higher frequency and thus may sire more offspring in this coercive mating system. Here, we synthesized this inconsistent body of evidence using pre-registered methods and hypotheses. We performed a systematic review and meta-analysis of summary and primary (raw) data combining both published (n = 19 studies, k = 106 effect sizes) and unpublished effect sizes (n = 17, k = 242) to test whether there is overall selection on male body size across studies in Gambusia. We also tested several specific hypotheses to understand the sources of heterogeneity across effects. Meta-analysis revealed an overall positive correlation between male size and reproductive performance (r = 0.23, 95% confidence interval: 0.10-0.35, n = 36, k = 348, 4,514 males, three Gambusia species). Despite high heterogeneity, the large-male advantage appeared robust across all measures studied (i.e. female choice, mating success, paternity, sperm quantity and quality), and was considerably larger for female choice (r = 0.43, 95% confidence interval: 0.28-0.59, n = 14, k = 43). Meta-regressions found several important factors explaining heterogeneity across effects, including type of sperm characteristic, male-to-female ratio, female reproductive status and environmental conditions. We found evidence of publication bias; however, its influence on our estimates was attenuated by including a substantial amount of unpublished effects, highlighting the importance of open primary data for more accurate meta-analytic estimates. In addition to positive selection on male size, our study suggests that we need to rethink the role and form of sexual selection in Gambusia and, more broadly, to consider the ecological factors that affect reproductive behaviour in livebearing fishes.

Mosquito oviposition and larvae development in response to kairomones originated by different fish.

Predator-released kairomones indicating the presence of predatory fish are known to alter the behaviour and life-history traits of several aquatic herbivores. Culex mosquitoes (Diptera: Culicidae) respond to such cues by altering oviposition habitat selection and larval development time. These responses differ among fish species indicating composition differences among fish-released cues, but the recognition pattern is not clear. This study tested the dependence of fish recognition to co-evolution and the level of threat to larvae. We compared Culex responses to three fish, the invasive larvivorous Gambusia affinis (Cyprinodontiformes: Poeciliidae), the native larvivorous Aphanius mento (Cyprinodontiformes: Cyprinodontidae) and the native algivorous Garra rufa (Cypriniformes: Cyprinidae). The effects of cues released by the three fish on Culex oviposition and life-history shifts were compared in field mesocosms and lab tests. Our results showed that ovipositing females were significantly repelled only by cues originated from G. affinis, while developing larvae response was a more general one. Our results support the idea that fish-released kairomones differ in composition or quantities among different species.

Synonymous single nucleotide polymorphism in arsenic (+3) methyltransferase of the Western mosquitofish (Gambusia affinis) and its gene expression among field populations.

Naturally occurring arsenic is toxic at extremely low concentrations, yet some species persist even in high arsenic environments. We wanted to test if these species show evidence of evolution associated with arsenic exposure. To do this, we compared allelic variation across 872 coding nucleotides of arsenic (+3) methyltransferase (as3mt) and whole fish as3mt gene expression from three field populations of Gambusia affinis, from water sources containing low (1.9 ppb), medium-low (3.3 ppb), and high (15.7 ppb) levels of arsenic. The high arsenic site exceeds the US EPA's Maximum Contamination Level for drinking water. Medium-low and high populations exhibited homozygosity, and no sequence variation across all animals sampled. Eleven of 24 fish examined (45.8%) in the low arsenic population harbored synonymous single nucleotide polymorphisms (SNPs) in exons 4 and/or 10. SNP presence in the low arsenic population was not associated with differences in as3mt transcript levels compared to fish from the medium-low site, where SNPs were noted; however, as3mt expression in fish from the high arsenic concentration site was significantly lower than the other two sites. Low sequence variation in fish populations from sites with medium-low and high arsenic concentrations suggests greater selective pressure on this allele, while higher variation in the low population suggests a relaxed selection. Our results suggest gene regulation associated with arsenic detoxification may play a more crucial role in influencing responses to arsenic than polymorphic gene sequence. Understanding microevolutionary processes to various contaminants require the evaluation of multiple populations across a wide range of pollution exposures.

The pivotal protein profile between the conjoined twins and normal mosquitofish Gambusia affinis based on iTRAQ proteomic analysis.

The fish abnormal embryonic development has attracted public attention in the recent few years. In this study, an iTRAQ proteomic analysis of mosquitofish between conjoined twins and normal fishes is applied for the first time by using the genome database of mosquitofish. Three thousand four hundred ninety proteins were identified with 304 differentially expressed proteins (DEPs). One hundred six differentially upregulated proteins (DUPs) and 198 differentially downregulated proteins (DDPs) were identified between the conjoined twins and normal mosquitofish groups. Notably, the proteins related to lipid and proteolysis were the important GO terms for the DUPs while response to light stimulus and response to radiation were the most enriched GO terms for the DDPs. The proteins related to lysosome, apoptosis, autophagy, and phagosome were the functional KEGG pathway for the DUPs while most of the pathways were related to cardiovascular for the DDPs. This study expatiated a pivotal protein profile between the conjoined twins and normal mosquitofish which can provide a conference for fish embryonic development.

Sex differences in cognitive performance and style across domains in mosquitofish (Gambusia affinis).

Given that the sexes often differ in their ecological and sexual selection pressures, sex differences in cognitive properties are likely. While research on sexually dimorphic cognition often focuses on performance, it commonly overlooks how sexes diverge across cognitive domains and in behaviors exhibited during a cognitive task (cognitive style). We tested male and female western mosquitofish (Gambusia affinis) in three cognitive tasks: associative learning (numerical discrimination), cognitive flexibility (detour task), and spatio-temporal learning (shuttlebox). We characterized statistical relationships between cognitive performances and cognitive style during the associative learning task with measures of anxiety, boldness, exploration, reaction time, and activity. We found sex differences in performance, cognitive style, and the relationships between cognitive domains. Females outperformed males in the spatio-temporal learning task, while the sexes performed equally in associate learning and cognitive flexibility assays. Females (but not males) exhibited a 'fast-exploratory' cognitive style during associative learning trials. Meanwhile, only males showed a significant positive relationship between domains (associative learning and cognitive flexibility). We propose that these sexually dimorphic cognitive traits result from strong sexual conflict in this taxon; and emphasize the need to explore suites of sex-specific cognitive traits and broader comparative work examining sexual selection and cognition.

Immune-related genes expression analysis of Western mosquitofish (Gambusia affinis) challenged with Aeromonas hydrophila.

The great Gambusia affinis (G. affinis) is considered as an important animal model to study the endocrine disruption, ecological behavior, and environmental pollutant. The present study aims to build a new promising infection model with Aeromonas hydrophila (A. hydrophila) in aquaculture. The mRNA expression of Rac1, MyD88, IRAK4, TAK1, IKKß, and IL-1ß in G. affinis were significance higher (P < 0.05) in the liver of G. affinis than that of brain and intestine. And the PI3K mRNA expression level was significant lower (P < 0.05) in the intestine than that of brain and liver. The mRNA levels of AKT3 were significant higher (P < 0.05) in the brain than that of liver and intestine. And then the brain, liver, and intestine were collected at different time points (0 h, 3 h, 9 h, 18 h, 24 h, 48 h) after post injection of LD50 of A. hydrophila. The 0.85% NaCl was used as a negative control for the LD50 of A. hydrophila. The RT-PCR results showed that mRNA expressions of TLR2/4 pathway downstream genes MyD88, IRAK4, TAK1, Rac1, IKKß, and IL-1ß were firstly significantly up-regulated (P < 0.05) and were then backed to the 0 h group levels in three tissues. In contrast, mRNA expressions of TLR2/4 pathway downstream genes PI3K and AKT3 were firstly significantly decreased (P < 0.05) and were then increased to the 0 h group levels in brain and intestine. In summary, the results indicated that A. hydrophila could cause inflammatory reaction in intestinal and brain. In addition, the liver showed a provocative reaction when infected with A. hydrophila.

Prey adaptation along a competition-defense tradeoff cryptically shifts trophic cascades from density- to trait-mediated.

Trophic cascades have become a dominant paradigm in ecology, yet considerable debate remains about the relative strength of density- (consumptive) and trait-mediated (non-consumptive) effects in trophic cascades. This debate may, in part, be resolved by considering prey experience, which shapes prey traits (through genetic and plastic change) and influences prey survival (and therefore density). Here, we investigate the cascading role of prey experience through the addition of mosquitofish (Gambusia affinis) from predator-experienced or predator-naïve sources to mesocosms containing piscivorous largemouth bass (Micropterus salmoides), zooplankton, and phytoplankton. These two sources were positioned along a competition-defense tradeoff. Results show that predator-naïve mosquitofish suffered higher depredation rates, which drove a density-mediated cascade, whereas predator-experienced mosquitofish exhibited higher survival but fed less, which drove a trait-mediated cascade. Both cascades were similar in strength, leading to indistinguishable top-down effects on lower trophic levels. Therefore, the accumulation of prey experience with predators can cryptically shift cascade mechanisms from density- to trait-mediated.

Susceptibility of betanodavirus in a newly established vertebra-derived cell line from Mosquitofish (Gambusia affinis).

In the present study, a new cell line from the vertebra of mosquitofish Gambusia affinis was successfully established and characterized. The cell line is named as bone Gambusia affinis (BGA) and subcultured for more than 55 passages in Leibovitz's/L15 medium supplemented with 15% FBS at 28°C. The cell line has a modal chromosome number of 48. Molecular characterization of the partial sequence of the coi gene confirmed the origin of the BGA cell line from mosquitofish. These cells exhibited epithelial morphology confirmed by the cytokeratin marker. The BGA cells showed mineralization of their extracellular matrix when stained with alizarin red and von Kossa stain. BGA cells were found to be susceptible to RGNNV and SJNNV strains of betanodavirus (NNV) showing cytopathic effect with multiple vacuolations in the cells. The RT-PCR confirmed the betanodavirus infections in BGA cells. The SEM micrograph showed the morphological changes observed in the cell during virus infection. The in vivo challenge experiment also showed the viral replicating efficiency in the Gambusia affinis with increasing viral titre. Thus, our present results show that the BGA cell line is a useful tool for isolating betanodavirus and could be used to investigate bone cell differentiation and extracellular matrix mineralization.

Cytochrome P450 1A mRNA in the Gambusia affinis and Response to Several PAHs.

Cytochrome P4501A (CYP1A) has been used as a specific biomarker for monitoring water contamination such as PAHs, PCBs and dioxins. In the present study, the cyp1a gene of Gambusia affinis was cloned and sequenced and their expressions under PAHs exposure were characterized. The newly identified cyp1a encodes a protein with 521 amino acids that shared 96-80% identity with other Cyprinodontiformes fishes. RT-PCR analysis revealed that the basal mRNA level of cyp1a was highly expressed in liver and intestine. The expression level of cyp1a was significantly induced by exposure to 100 µg/L 3, 4-Benzopyrene (BaP) for 5 days in the muscle, testis, brain, liver and intestine of adult male fish. Except in the testis, the induced mRNA level of cyp1a ultimately decreased after prolonging the exposure time to 25 days. As for testis, the induced mRNA level of cyp1a was maintained at a high level during the entire exposure time under 100 µg/L BaP exposure. Furthermore, the expression of cyp1a increased with exposure time under a relatively low exposure concentrations 1 µg/L. Regarding the effects of other PAHs, D(a,h)A, BbF, and BaA showed a statistically significant effect of induction on mRNA level of cyp1a in the muscle, testis, brain, liver and intestine.

Rational choice of social group size in mosquitofish.

Choice of social group can affect the likelihood of survivorship and reproduction for social species. By joining larger social groups-shoals-small freshwater fish like the mosquitofish Gambusia affinis can reduce predation risk and forage more efficiently. We tested shoal choice in mosquitofish to determine whether such choices are economically rational, i.e. consistent and optimal. Although many studies of decision-making assume rational choice, irrational decision-making is common and occurs across contexts. We tested rationality of shoaling decisions by testing the constant ratio rule, which states that the relative preference for two options should not change in the presence of a third option. Female mosquitofish upheld this rule when tested for shoal preference based on group size. Our results contrast with other studies showing violations of the constant ratio rule in foraging and mate choice decision-making contexts. These results suggest that decisions that immediately influence survivorship or decision-making along a single dimension may favour rational decision-making.

Transcriptomic and physiological changes in western mosquitofish (Gambusia affinis) after exposure to norgestrel.

Norgestrel (NGT) is a synthetic progestin used in human and veterinary medicine. Adult female mosquitofish were exposed to NGT for 42 d at 377 ng L-1. The fin morphology and the liver transcriptome were assessed. NGT exposure increased ray 4:6 length ratio. As compared to the control, NGT treatment affected the expression of 11,772 annotated transcripts in female mosquitofish. Specifically, we found 5780 were repressed while 5992 were significantly induced. Gene ontology (GO) analysis showed that 53 KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways and 158 GO terms were significantly over expressed. Genes showing the largest magnitude of expression changes were related to fin development, androgen biosynthesis, and lipid and fatty acid metabolisms, suggesting the involvement of these biological processes in response to NGT exposure in G. affinis. This first comprehensive study on the transcriptomic alterations by NGT in G. affinis not only provides valuable information on the development of molecular markers but also opens new avenues for studies on the molecular mechanisms of effects of NGT in particular and possibly other progestins in G. affinis.

Effect of the exposure to Mn-doped ZnS nanoparticles on biomarkers in the freshwater western mosquitofish Gambusia affinis.

Synthesized Mn-doped ZnS nanoparticles, with 10% of Mn dopant, were used to investigate their environmental toxicity. Mn-doped ZnS quantum dots (QDs) stabilized by 3-mercaptopropionic acid (MPA) were synthesized in a basic aqueous solution using the nucleation doping. The optical properties and structure of the obtained Mn (10%):ZnS QDs have been characterized by X-ray diffraction, UV-vis, photoluminescence spectroscopies and transmission electron microscopy. The brain, gills and liver stained sections from Gambusia affinis were dissected. Antioxidant enzyme activities (acetylcholinesterase and catalase), as well as malondialdehyde and H2O2 levels, were determined after exposure (94 h) to 14 and 28 mg/L of nanoparticles. The obtained nearly monodisperse Mn(10%):ZnS@MPA QDs have an average diameter of ca. 2.8 nm and a zinc-blende crystal structure. Mn-doped ZnS acts differently on the activities of the biomarkers in a dose-dependent manner. The recorded alterations varied between organs. Such findings provide information on the biological target of nanoparticles and their behaviour within the environment.