Anthropogenic Xenobiotics Research Articles

Анализ природных и антропогенных факторов, влияющих на способность Lepidium sativum L. адаптироваться к токсичности триазольных ксенобиотиков

The effects of natural factors (temperatures +34°C and +20°C) and anthropogenic xenobiotics (1,2,4-TriH; 1-(CH3SO2)-1H-1,2,4-Tri; 4-(PhSO2)-4H-1,2,4-Tri; 4-(TolSO2)-4H-1,2,4-Tri) on watercress Lepidium sativum L. was studied. For this, 4 series of experiments were carried out. In the first two series, the effect of concentrations was studied: 0.001, 0.01, 0.1 mg/ml during germination at different temperatures for three days. It was found that the ability of the triazole compounds to inhibit the germination of watercress seeds increased with increasing concentration of the solutions. The magnitude of lipophilicity was the determining factor in their toxicity. Differences between the influence of the studied temperatures on the energy of seed germination weren’t revealed. The ability of the synthesized triazoles to inhibit the growth of watercress roots and stems increased with increasing their concentration. At the same time, their toxicity was significantly higher at the higher of the studied temperatures. The next two series examined the ability of Lepidium sativum to adapt to the toxicity of triazoles. To do this, the seeds were germinated during the day at contrasting temperatures in solutions with a concentration of 0.001 mg/ml, then they were germinated in solutions of 0.1 mg/ml for two days. It turned out that triazoles had significantly different effects on the development of the adaptive response in plants. Only 4-(PhSO2)-4H-1,2,4-Tri; 4-(TolSO2)-4H-1,2,4-Tri caused a significant development of an adaptive response, the magnitude of which also depended on the cultivation temperature. We found that the value of lipophilicity of the studied triazoles was of significant importance during their direct action, and the values of their molecular weight and molecular volume were important for the development of preadaptation.

Xenobiotic responses in insects.

Insects have evolved a powerful detoxification system to protect themselves against environmental and anthropogenic xenobiotics including pesticides and nanoparticles. The resulting tolerance to insecticides is an immense problem in agriculture. In this study, we summarize advances in our understanding of insect xenobiotic responses: the detoxification strategies and the regulation mechanisms against xenobiotics including nanoparticles, the problem of response specificity and the potential usefulness of this study field for an elaborate pest management. In particular, we highlight that versatility of the detoxification system relies on the relatively unspecific recognition of a broad range of potential toxic substances that trigger either of various canonical xenobiotic responses signaling pathways, including CncC/Keap1, HR96, AHR/ARNT, GPCR, and MAPK/CREB. However, it has emerged that the actual response to an inducer may nevertheless be specific. There are two nonexclusive possibilities that may explain response specificity: (1) differential cross-talk between the known pathways and (2) additional, yet unidentified regulators and pathways of detoxification. Hence, a deeper and broader understanding of the regulation mechanisms of xenobiotic response in insects in the future might facilitate the development and application of highly efficient and environmentally friendly pest control methods, allowing us to face the challenge of the world population growth.

The influence of anthropogenic xenobiotics on freshwater fish organisms

The influence of anthropogenic xenobiotics on freshwater fish organisms

Volatile scent chemicals in the urine of the red fox, Vulpes vulpes

The red fox is a highly adaptable mammal that has established itself world-wide in many different environments. Contributing to its success is a social structure based on chemical signalling between individuals. Urine scent marking behaviour has long been known in foxes, but there has not been a recent study of the chemical composition of fox urine. We have used solid-phase microextraction and gas chromatography-mass spectrometry to analyze the urinary volatiles in 15 free-ranging wild foxes (2 female) living in farmlands and bush in Victoria, Australia. Foxes here are routinely culled as feral pests, and the urine was collected by bladder puncture soon after death. Compounds were identified from their mass spectra and Kovats retention indices. There were 53 possible endogenous scent compounds, 10 plant-derived compounds and 5 anthropogenic xenobiotics. Among the plant chemicals were several aromatic apocarotenoids previously found in greater abundance in the fox tail gland. They reflect the dietary consumption of carotenoids, essential for optimal health. One third of all the endogenous volatiles were sulfur compounds, a highly odiferous group which included thiols, methylsulfides and polysulfides. Five of the sulfur compounds (3-isopentenyl thiol, 1- and 2-phenylethyl methyl sulfide, octanethiol and benzyl methyl sulfide) have only been found in foxes, and four others (isopentyl methyl sulfide, 3-isopentenyl methyl sulfide, and 1- and 2-phenylethane thiol) only in some canid, mink and skunk species. This indicates that they are not normal mammalian metabolites and have evolved to serve a specific role. This role is for defence in musteloids and most likely for chemical communication in canids. The total production of sulfur compounds varied greatly between foxes (median 1.2, range 0.4-32.3 μg 'acetophenone equivalents'/mg creatinine) as did the relative abundance of different chemical types. The urinary scent chemistry may represent a highly evolved system of semiochemicals for communication between foxes.

The assessment of the environmental hazard of isomers of 1,2,4-triazole phenolic derivatives for natural ecosystems

Its necessary to synthesize homologues of compounds frequently used in practice and to analyze their biological activity in laboratory experiments using screening tests that provide an integral assessment of biological responses to assess the effect of anthropogenic xenobiotics with different structures on ecosystems adjacent to agricultural complexes. We analyzed alcohol solutions of 2-(1H-1,2,4-triazolyl-methyl)phenol (ortho-isomer) and 4-(1H-1,2,4-triazolyl-methyl)phenol (para-isomer) in three concentrations: 0,0001; 0,001; 0.01 mg/ml using the Allium-test. The solvent was 0,1% isopropyl alcohol; the test object was Allium fistulosum L. The duration of the experiment was 5 days. Triazolide solutions significantly inhibited seed germination at all investigated concentrations. However, no significant differences were found between the isomers and the studied concentrations. Both isomers inhibited root growth at all concentrations tested. The toxicity of a triazolide containing an OH group in the para-position didnt change over the selected concentration range. For its ortho-isomer, toxicity increased with increasing concentration, reaching the toxicity of its homologue at a dose of 0,01 mg/ml. Both tested compounds significantly inhibited the proliferation of meristem cells as compared to the control. At the same time, no differences were observed in the effect of homologues with the OH-group in the para- and ortho-position on the value of the mitotic index. However, we found a paradoxical reaction: both homologues showed maximum cytotoxicity at a minimum concentration of 0,0001 mg/ml, and cytotoxicity decreased with increasing concentration compared to control. A triazolid containing an OH group in the para-position caused a block at the metaphase and anaphase stages at the lowest concentration. The specificity of its action disappeared with an increase in concentration, which was expressed in a general prophase and metaphase block. Its ortho-isomer inhibited cell division at all concentrations at the prophase stage. Both compounds are mutagenic. The number of chromosomal aberrations depended on both the structure of the compounds and their concentration. The para-homologue is less mutagenic than ortho. In the ortho-homologue, mutagenicity decreased slightly with increasing concentration. The highest mutagenicity was found for the ortho-homologue at its lowest concentration. The paper discusses possible mechanisms of action of isomers and their negative impact on plant organisms in ecosystems.

Comparative study of esterase activities in different tissues of marine fish species Trachurus trachurus, Merluccius merluccius and Trisopterus luscus

Pesticides are one of the most frequently anthropogenic xenobiotics detected in water. Among these, the organophosphorus pesticides (OPs) are very widely used in agriculture due to their broad spectrum of activity and their low price, but they also have high potent effects as neurotoxic compounds in non-target organisms. The aim of this study was to evaluate biomarkers acetylcholinesterase (AChE), butyrylcholinesterase (BChE), propionylcholinesterase (PChE) and carboxylesterase (CbE) in the representative Atlantic fish species Trachurus trachurus, Merluccius merluccius and Trisopterus luscus from “Rías Gallegas”, a traditional Spanish fishing area. These esterase activities were evaluated in the brain, muscle and liver to determine the most adequate tissue to measure such enzymatic activities. The sensitivity of AChE and CbE activities from different tissues the widely used organophosphorus insecticide chlorpyrifos (CP), and its toxic metabolite (CP-oxon) was also tested. AChE activity was predominant in all tissues of the analysed species (particularly in brain constituting from 78.33%, 89.83% and 88.43% of total ChEs in Trachurus trachurus, Merluccius merluccius and Trisopterus luscus, respectively). Under in vitro exposure, esterases were shown to be highly sensitive to CP and especially to CP-oxon. Moreover, a similar effect observed on AChE and CbE activities could suggest that CbE activity might contribute efficiently against the toxic effects of CP, especially in muscle and the liver. The presence of BChE, PChE and upper CbE activities in muscle and the liver and their OP-sensibilities can be used to study their function in the pesticide biochemical detoxification pathways with a prominent role as a safeguarding mechanism against pesticide toxicity.

Assessment of the integrated reaction of onions of different types for the action of xenobiotics for use in ecological-genetic monitoring

The influence of many anthropogenic xenobiotics constantly falling into natural ecosystems is poorly understood, so laboratory experiments are needed to understand the consequences of such actions. It is especially important to study this effect on plants that are unable to avoid uncomfortable conditions. It is necessary to use a variety of test objects and screening tests that give an integral assessment of biological responses. Such tests include the Allium-test, which allows to evaluate toxicity at both the organismic and cellular levels, as well as mutagenicity of xenobiotics. We analyzed the response of three species of the genus Allium (Allium cepa, Allium ramosum, Allium fistulosum) to exposure to alcohol solutions of benzotriazole in extremely low concentrations. This heterocyclic compound is widely used in industry as a light stabilizer for polymers and as an anti-corrosion agent protecting various metals and alloys. The biological activity of two concentrations (0,0001; 0,001 mg/ml) was studied. The solvent was 0,5% isopropyl alcohol. Allium test was used to analyze a biological response of the selected onions. The duration of the experiment was 5 days. The effect of benzotriazole stimulated growth processes in A. cepa and A. ramosum in the lowest concentration and inhibited growth processes in a higher dose compared to the control. Benzotriazole had a stimulating effect on A. fistulosum in both of the concentrations studied, however, the stimulating effect in a high concentration was less pronounced. The solution in low concentration stimulates the proliferative activity in the cells of the root meristem of all species. However, cells of A. cepa and A. ramosum meristem are not sensitive to the action of a higher concentration of benzotriazole solution, unlike A. fistulosum, in which cell division is inhibited in comparison with the control. It was found that A. fistulosum, in which benzotriazole causes a block at the anaphase stage in both concentrations studied, is the most sensitive to the mitosis-modifying action of benzotriazole. In the other two species, only exposure to benzotriazole in high concentration led to inhibition of mitosis at the prophase stage. A.

A Survey of Pesticide Accumulation in a Specialist Feeder, the Koala (Phascolarctos cinereus)

To maintain profitability in Australia's agricultural and urban landscapes pesticides are used throughout the range of koala habitats. The koala is a specialist feeder, reliant on metabolic enzyme capacities to utilise a toxic diet of eucalypt leaves and is potentially prone to adverse effects when xenobiotic interactions between dietary and anthropogenic xenobiotics occur. The aim of this study was to investigate accumulation of frequently used pesticides in wild koalas in 4 areas of New South Wales and Queensland. Liver samples of 57 deceased koalas were collected from care facilities and analysed using a modified QuEChERS extraction method followed by GCMSMS, HRLCMS and LCMSMS. No accumulation of any of the 166 investigated pesticides was found. Data indicate hepatic accumulation of pesticides in this species is uncommon even with close interactions with intensive land use. Despite the lack of hepatic bioaccumulation, this study cannot exclude a direct effect on hepatocellular metabolic pathways.

Multi-residue analysis of 36 priority and emerging pollutants in marine echinoderms (Holothuria tubulosa) and marine sediments by solid-liquid extraction followed by dispersive solid phase extraction and liquid chromatography–tandem mass spectrometry analysis

Marine echinoderms are filter-feeding invertebrates widely distributed along the coasts, and which are therefore extensively exposed to anthropogenic xenobiotics. They can serve as good sentinels for monitoring a large variety of contaminants in marine ecosystems. In this context, a multi-residue analytical method has been validated and applied to Holothuria tubulosa specimens and marine sediments for the determination of 36 organic compounds, which belong to some of the most problematic groups of emerging and priority pollutants (perfluoroalkyl compounds, estrogens, parabens, benzophenones, plasticizers, surfactants, brominated flame retardants and alkylphenols). Lyophilization of samples prior to solvent extraction and clean-up of extracts with C18, followed by liquid chromatography–tandem mass spectrometry analysis, is proposed. A Box–Behnken design was used for optimization of the most influential variables affecting the extraction and clean-up steps. For validation, matrix-matched calibration and recovery assay were applied. Linearity (% r2) higher than 99%, recoveries between 80% and 114% (except in LAS and NP1EO), RSD (precision) lower than 15% and limits of quantification between 0.03 and 12.5ngg−1 dry weight (d.w.) were achieved. The method was applied to nine samples of Holothuria collected along the coast of Granada (Spain), and to marine sediments around the animals. The results demonstrated high bioaccumulation of certain pollutants. A total of 25 out of the 36 studied compounds were quantified, being surfactants, alkylphenols, perfluoroalkyl compounds, triclocarban and parabens the most frequently detected. Nonylphenol was found in the highest concentration (340 and 323ngg−1 d.w. in sediment and Holothuria samples, respectively).

Using aquatic fungi for pharmaceutical bioremediation: Uptake of acetaminophen by Mucor hiemalis does not result in an enzymatic oxidative stress response

The increasing anthropogenic pollution of aquatic environments and fresh water scarcity worldwide have prompted the development of low-cost and effective water treatment alternatives. One example of a highly released anthropogenic xenobiotics is acetaminophen (APAP), which has been detected in surface waters at concentrations as high as 5 μg L−1. To date, traditional water treatment plants were unable to remove all pharmaceutical xenobiotics and as in the case with APAP, the breakdown products are toxic. Phytoremediation has proved to remove xenobiotics efficiently producing no toxic breakdown products, however, they are often restrained in their application range. Therefore, it was necessary to find alternate remediation tools to extend and complement the application ranges of existing bioremediation techniques. With the success of mycoremediation as well as the adaptability of fungi, Mucor hiemalis was investigated in terms of its APAP uptake capabilities. The investigation included the examination of concentration- and time-dependent uptake studies to examine the effects of each of these parameters independently. Additionally, the extracellular peroxidase activity of M. hiemalis was measured with exposure to APAP to evaluate possible breakdown and the antioxidative stress enzymes, catalase, glutathione peroxidase, and glutathione reductase, were assayed to investigate whether APAP caused oxidative stress. The results showed that M. hiemalis was able to internalize between 1 and 2 μg APAP per g dried fungal biomass when exposed to 5, 10, 50 and 100 ng mL−1 APAP for 24–48 h, but not beyond this time frame. Further, exposure to APAP did not result in elevated extracellular peroxidase activity or oxidative stress. The findings led to the conclusion that M. hiemalis could be integrated in bioremediation systems, for short-term degradation at low concentrations of APAP with effective management.

Physiological responses of Cladophora glomerata to cyanotoxins: a potential new phytoremediation species for the Green Liver Systems

Due to increased pollution of potable water sources as a consequence of eutrophication and anthropogenic xenobiotics, sustainable water purification is an essential concern. Therefore, the Green Liver System, a natural, economic and sustainable water purification system employing the biotransformation capabilities of aquatic plants, was developed. To expand the capacities and applications of this system, new aquatic plants are continually evaluated for their potential to remediate various aquatic pollutants. In this study, the potential of Cladophora glomerata to internalize cyanotoxins, microcystins (MCs) and anatoxin-a, and consequently its ability to cope with the subsequent oxidative stress associated with toxin-uptake were investigated. C. glomerata was able to take up all three of the tested MC congeners as well as anatoxin-a, similarly to previous toxin internalizations reported for aquatic plants such as Ceratophyllum demersum, Myriophyllum spicatum and Hydrilla versiculata. The antioxidative stress defense of C. glomerata proved to efficiently endure the toxin-uptake with no adverse effects. Subsequently, the uptake potential of C. glomerata was investigated at lab-scale by exposure to the three MC congeners and anatoxin-a collectively. After a period of seven days, 95–97% of the MCs and 100% of anatoxin-a were removed from the exposure media. C. glomerata therefore, is a suitable candidate to be incorporated in future Green Liver Systems.

Alterations in gills of Lepomis gibbosus, after acute exposure to several xenobiotics (pesticide, detergent and pharmaceuticals): morphometric and biochemical evaluation

In recent decades, scientific research about the effects of anthropogenic xenobiotics on non-target organisms has increased. Among the likely effects, some studies reported the evaluation of biochemical and morphological changes in specific tissues or organs of fishes, such as gills, which are key organs for the direct action of pollutants in the aquatic environment. This work intended to assess biochemical [oxidative stress/phase II conjugation isoenzymes glutathione S-transferase (GSTs)] and morphological [secondary lamellar length (SLL), secondary lamellar width (SLW), interlamellar distance (ID), basal epithelial thickness (BET) and proportion of the secondary lamellae available for gas exchange (PAGE)] changes in gills, after acute exposure to the pesticide chlorfenvinphos, the detergent sodium dodecylsulphate (SDS) and to the anticholinesterasic pharmaceuticals (neostigmine and pyridostigmine). Our results point to a significant, eventually hormetic, effect in the activity of GSTs following exposure to chlorfenvinphos that significantly increased the activity of GSTs at concentration of 0.2 mg/L. The activity of GSTs increased significantly after exposure to 100 mg/L of neostigmine. Considering the morphometric analysis of the gills, the data obtained showed that chlorfenvinphos exerted mainly minor architectural alterations in gills, with the exception of the highest tested concentration of chlorfenvinphos that produced also a slight decrease of the PAGE. The overall conclusions point to a null or negligible toxicity of the selected toxicants towards L. gibbosus, which may be reverted if exposure is withdrawn.

<i>In vivo</i> effects of genistein, herbimycin a and geldanamycin on rat hepatic cytochrome P4501A

Cytochrome P4501A (the CYP1A1 and CYP1A2 enzymes) are regulated through the aryl hydrocarbon receptor (AhR)-dependent signal transduction pathway and are generally known as enzymes which metabolize anthropogenic xenobiotics such as dioxin to carcinogenic and mutagenic compounds. However, recently the facts of CYP1A activation under physiological conditions or under action of non-dioxin-like compounds appear. In the present study we show that genistein, herbimycin A and geldanamycin (the protein-tyrosine kinase inhibitors) affect in vivo to CYP1A1 activity, the CYP1A1 mRNA level and the CYP1A1 protein level. These data provide insight into the role of protein kinases in CYP1A regulation may facilitate the understanding of CYP1A regulation.

Molecular mechanisms of cold-induced CYP1A activation in rat liver microsomes

Cytochrome P4501A (the CYP1A1 and CYP1A2 enzymes) is known to metabolize anthropogenic xenobiotics to carcinogenic and mutagenic compounds. CYP1A1 transcriptional activation is regulated via the aryl hydrocarbon receptor (AhR)-dependent signal transduction pathway. CYP1A2 activation may occur through the AhR-dependent or AhR-independent signal transduction pathways. We used male Wistar rats to explore possible mechanisms of CYP1A activation induced by exposure to cold and the effects of the protein-tyrosine kinase inhibitors genistein, herbimycin A, and geldanamycin on the properties of hepatic CYP1A1 and CYP1A2 proteins following exposure to cold and to classic CYP1A inducers. The molecular mechanisms of cold-induced CYP1A1 and CYP1A2 activation are different. The CYP1A2 activation apparently occurs at the post-transcriptional level. The CYP1A1 activation, whether caused by exposure to cold or by classic CYP1A inducers, is AhR-dependent and occurs at the transcriptional level. Protein tyrosine kinase inhibitors have no effect on benzo(a)pyrene-induced CYP1A expression but alter cold-induced CYP1A1 activity and the CYP1A1 mRNA level. Thus, treatment with herbimycin A or geldanamycin leads to an increase in CYP1A1 activity, while treatment with genistein increases CYP1A1 mRNA expression and decreases CYP1A2 activity. These data elucidate the molecular mechanisms of cold-induced CYP1A activation and the role of protein kinases in the regulation of CYP1A during exposure to cold. Our results can also help identify the differences between the molecular mechanisms underlying the effects of the classic CYP1A inducers and the effects of cooling.

Quantitation of major protein constituents of murine intestinal fluid

The gastrointestinal tract is a hostile biological environment, yet not all ingested materials are destroyed. The minute differences that determine whether a substance persists or is digested, liberated, adsorbed, excreted, or taken up are still poorly understood. Most attempts to investigate the events occurring during an orogastrointestinal passage rely on simplified in vitro systems where an analyte is exposed to artificial intestinal fluids. To closely mimic the events in the gastrointestinal tract, the exact intestinal fluid composition and the in vivo concentration of its constituents must be known. The widely used lavage procedures, however, dilute the intestinal fluids to an extent that precludes recalculation to the original concentrations. Thus, we developed procedures with which undiluted murine intestinal fluid can be harvested; determined the in vivo concentrations of the digestive enzymes trypsin, chymotrypsin, and elastase and the adsorbents mucin and immunoglobulin A in small intestinal fluid of fasted and unfasted female Balb/c mice; and identified chymotrypsin and immunoglobulin A as valid endogenous dilution markers for the recalculation of aqueous lavages. With these technologies and information at hand, more reliable investigations on the fate of allergens, pathogens, food, and anthropogenic xenobiotics in the gastrointestinal tract will be possible.

Production and sedimentation of peptide toxins nodularin-R and microcystin-LR in the northern Baltic Sea

This seven-year survey was primarily targeted to quantification of production of nodularin-R (NOD-R), a cyclic pentapeptide hepatotoxin, in Baltic Sea cyanobacteria waterblooms. Additionally, NOD-R and microcystin-LR (MC-LR; a cyclic heptapeptide toxin) sedimentation rates and NOD-R sediment storage were estimated. NOD-R production (70-2450 microg m(-3); approximately 1 kg km(-2) per season) and sedimentation rates (particles; 0.03-5.7 microg m(-2)d(-1); approximately 0.3kg km(-2) per season) were highly variable over space and time. Cell numbers of Nodularia spumigena did not correlate with NOD-R quantities. Dissolved NOD-R comprised 57-100% of total NOD-R in the predominantly senescent, low-intensity phytoplankton blooms and seston. Unprecedentedly intensive MC-LR sedimentation (0.56 microg m(-2)d(-1)) occurred in 2004. Hepatotoxin sedimentation rates highly exceeded those of anthropogenic xenobiotics. NOD-R storage in surficial sediments was 0.4-20 microg kg(-1) ( approximately 0.1 kg km(-2)). Loss of NOD-R within the chain consisting of phytoplankton, seston and soft sediments seemed very effective.

Role of Chelating Agents for Prevention, Intervention, and Treatment of Exposures to Toxic Metals

Role of Chelating Agents for Prevention, Intervention, and Treatment of Exposures to Toxic Metals

Toxicological and ecological implications of biotransformation enzymes in the tropical teleost Chaetodon capistratus

Hepatic cytochromes P450 (phase I monooxygenases) and glutathione transferases (phase II conjugating enzymes) were investigated in Chaetodon capistratus (Linnaeus) collected in Florida and Belize in June and December 1991, respectively. These biotransformation enzymes play major roles in the detoxification of xenobiotics by converting lipophilic chemicals to more hydrophilic, readily excretable metabolites. Content of total microsomal P450 (0.501 to 0.821 nmol mg-1 microsomal protein) and rates of NADPH-cytochrome c (P450) reductase (270.7 to 330.2 nmol min-1 mg-1 microsomal protein) and glutathione transferase (2.81 to 3.12 μg min-1 mg-1 cytosolic protein) in these fish were greater than in most untreated fish species, i.e., fish that have not been exposed to PAHs (polycyclic aromatic hydrocarbons) or PCBs (polycyclic biphenyls). Ethoxyresorufin O-deethylase (EROD) rates (0.029 to 0.171 nmol min-1 mg-1) were also comparable to those in most untreated marine fish. Immunoblot analysis with monoclonal antibody (MAb) 1-12-3 to scup P450E (the EROD catalyst and a teleost representative of the PAH-inducible CYP1A gene subfamily) showed slight amounts of cross-reacting protein in C. capistratus liver microsomes. Hepatic CYP1A content and EROD activity did not differ significantly between fish collected in Florida and Belize, suggesting that the two sites differed little in contamination by CYP1A inducers. Immunochemical analyses with polyclonal antibodies to scup P450B (a teleost representative of the CYP2B subfamily) and human CYP3A4 cross-reacted strongly with C. capistratus hepatic proteins. The CYP2B and CYP3A subfamilies in mammals are believed to have partially evolved in response to toxic dietary allelochemicals. C. capistratus regularly feeds on terpenoid-rich gorgonian corals, suggesting that biotransformation enzymes may be involved in the metabolism of dietary allelochemicals as well as anthropogenic xenobiotics in this species.