Identification of antimicrobial peptide genes from transcriptomes in Mandarin fish (Siniperca chuatsi) and their response to infection with Aeromonas hydrophila.

Mandarin fish (Siniperca chuatsi) is a valuable freshwater fish species widely cultured in China. Its aquaculture production is challenged by bacterial septicaemia, which is one of the most common bacterial diseases. Antimicrobial peptides (AMPs) play a critical role in the innate immune system of fish, exhibiting defensive and inhibitory effects against a wide range of pathogens. This study aimed to identify the antimicrobial peptide genes in mandarin fish using transcriptomes data obtained from 17 tissue in our laboratory. Through nucleotide sequence alignment and protein structural domain analysis, 15 antimicrobial peptide genes (moronecidin, pleurocidin, lysozyme g, thymosin ß12, hepcidin, leap 2, ß-defensin, galectin 8, galectin 9, apoB, apoD, apoE, apoF, apoM, and nk-lysin) were identified, of which 9 antimicrobial peptide genes were identified for the first time. In addition, 15 AMPs were subjected to sequence characterization and protein structure analysis. After injection with Aeromonas hydrophila, the number of red blood cells, hemoglobin concentration, and platelet counts in mandarin fish showed a decreasing trend, indicating partial hemolysis. The expression change patterns of 15 AMP genes in the intestine after A. hydrophila infection were examined by using qRT-PCR. The results revealed, marked up-regulation (approximately 116.04) of the hepcidin gene, down-regulation of the piscidin family genes expression. Moreover, most AMP genes were responded in the early stages after A. hydrophila challenge. This study provides fundamental information for investigating the role of the different antimicrobial peptide genes in mandarin fish in defense against A. hydrophila infection.

Gastrointestinal perforation in extremely low birth weight infants: A single center retrospective study in China.

BACKGROUND: Gastrointestinal perforation in extremely low birth weight infants, characterized by its rapid onset, multiple complications, and critical condition, poses a significant risk of infant mortality. The aim of this study was to investigate the clinical characteristics of pneumoperitoneum in extremely low birth weight infants (ELBWI) and explore the risk factors associated with gastrointestinal perforation in very low birth weight preterm infants. Additionally, we shared our surgical experiences in managing gastrointestinal perforation among extremely low birth weight infants. METHODS: The Department of Neonatology at Chengdu Women and Children's Central Hospital conducted a retrospective study on gastrointestinal perforation in extremely low birth weight infants (birth weight <1000 g) who were admitted between 2014 and 2021. After baseline analysis and comparing it with the control group, we identified the risk factors associated with gastrointestinal perforation in ELBWI by multiple logistic regression analysis. The Kaplan-Meier analysis was performed to assess the adverse effect of gastrointestinal perforation for survival in ELBW infants. Cox multivariate regression analysis was used to evaluate hazard level of different variables for ELBW infants survival. RESULTS: Hemodynamically significant patent ductus arteriosus (hsPDA)(p = 0.043, OR = 2.779) and sepsis (p = 0.014, OR = 2.265) were significant risk factors for gastrointestinal perforation in extremely low birth weight infants. The Cox proportional hazard model revealed that intraventricular hemorrhage (HR = 2.854, p＜0.001) Sepsis (HR = 1.645, p = 0.015) and gastrointestinal perforation (HR = 1.876, p = 0.008) had detrimental effects on the survival of extremely low birth weight infants; conversely, ibuprofen (HR = 0.304, p＜0.001) and blood transfusion (HR = 0.372, p＜0.001) are beneficial factors for their survival. The preoperative indicators of infection in infants with spontaneous intestinal perforation (SIP) were significantly better than those in the necrotizing enterocolitis (NEC) group (p < 0.05). CONCLUSIONS: Gastrointestinal perforation poses a significant threat the survival of extremely low birth weight (ELBW) infants, with hsPDA and sepsis serving as predisposing factors for gastrointestinal perforation. The gastrointestinal perforation caused by various diseases exhibits distinct clinical characteristics, necessitating tailored surgical approaches based on operative conditions.

Corn silk (Zea mays L.), a source of natural antioxidants with α-amylase, α-glucosidase, advanced glycation and diabetic nephropathy inhibitory activities.

The oxidative stress plays a critical role in the progression of diabetes mellitus (DM) and its complications. Corn silk is a traditional medicine used to treat DM. The aim of this study is to investigate the antioxidant capacity of corn silk, as well as its inhibitory potential on DM and diabetic nephropathy (DN). The ethanol extract of corn silk (CS) was liquid-liquid fractionated to get petroleum ether fraction (PCS), ethyl acetate fraction (ECS), n-butanol fraction (BCS) and water fraction (WCS). The Folin-Ciocalteu and AlCl3 assay showed that silk corn contained considerable amount of phenolics and flavonoids, ECS and BCS were the two phenolic-enriched fractions with highest TPC and TFC values. The ECS and BCS showed the highest total antioxidant activity and reducing power, as well as the strongest scavenging activity against DPPH and hydroxyl radicals, compared to CS and other fractions. The ECS and BCS displayed appreciable anti-hyperglycaemic effect indicated by the significant inhibition on α-amylase and α-glucosidase in enzymatic assays. In BSA-glucose model, ECS and BCS effectively inhibited the formation of advanced glycation end products (AGEs). In addition, the anti-diabetic nephropathy activity assay displayed that CS, ECS and BCS significantly inhibited the production of Col IV, FN and IL-6 in high-glucose stimulated mesangial cells at 200 µg/mL. These findings suggested the antioxidant activities of corn silk could contribute, at least in part, to its traditionally claimed therapeutic benefits on DM and DN. The phenolic-enriched CS fractions could be considered as a source of natural antioxidants and further developed for the prevention and treatment of DM and its complications including DN.

Mitochondrial genome annotation and phylogenetic placement of Oreochromis andersonii and O. macrochir among the cichlids of southern Africa.

Genetic characterization of southern African cichlids has not received much attention. Here, we describe the mitogenome sequences and phylogenetic positioning of Oreochromis andersonii and O. macrochir among the African cichlids. The complete mitochondrial DNA sequences were determined for O. andersonii and O. macrochir, two important aquaculture and fisheries species endemic to southern Africa. The complete mitogenome sequence lengths were 16642 bp and 16644 bp for O. andersonii and O. macrochir respectively. The general structural organization follows that of other teleost species with 13 protein-coding genes, 2 rRNAs, 22 tRNAs and a non-coding control region. Phylogenetic placement of the two species among other African cichlids was performed using Maximum Likelihood (ML) and Bayesian Markov-Chain-Monte-Carlo (MCMC). The consensus trees confirmed the relative positions of the two cichlid species with O. andersonii being very closely related to O. mossambicus and O. macrochir showing a close relation to both species. Among the 13 mitochondrial DNA protein coding genes ND6 may have evolved more rapidly and COIII was the most conserved. There are signs that ND6 may have been subjected to positive selection in order for these cichlid lineages to diversify and adapt to new environments. More work is needed to characterize the southern Africa cichlids as they are important species for capture fisheries, aquaculture development and understanding biogeographic history of African cichlids. Bio-conservation of some endangered cichlids is also essential due to the threat by invasive species.

The complete mitochondrial genome of Choristoneura longicellana (Lepidoptera: Tortricidae) and phylogenetic analysis of Lepidoptera.

To better understand the diversity and phylogeny of Lepidoptera, the complete mitochondrial genome of Choristoneura longicellana (=Hoshinoa longicellana) was determined. It is a typical circular duplex molecule with 15,759bp in length, containing the standard metazoan set of 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and an A+T-rich region. All of the inferred tRNA secondary structures show the common cloverleaf pattern, with the exception of trnS1(AGN), which lacks the DHU arm. The rrnL of C. Longicellana is the longest in sequenced lepidopterans. C. Longicellana has the same gene order as all lepidopteran species currently available in GenBank. There are 5 overlapping regions ranging from 1bp to 8bp and 14 intergenic spacers ranging from 1bp to 48bp. In addition, there are four similar tandem macro-satellite regions with the lengths of 101bp, 98bp, 92bp, and 92bp respectively in the A+T-rich regions of C. longicellana. We sampled 89 species representing 13 superfamilies, and reconstructed their relationship among Lepidoptera by Bayesian Inference and Maximum Likelihood analysis. The topology of the two phylogenetic analysis trees is identical roughly, except for Cossoidea in different locations, the positions of Cossoidea, Copromorphoidea, Gelechioidea, Zygaenoidea were not determined based the limited sampling. (Geometroidea+(Noctuoidea+Bombycoidea)) form the Macrolepidoptera "core". Pyraloidea group with the "core" Macrolepidoptera. Papilionoidea are not Macrolepidoptera. The Hesperiidae (represent Hesperioidea) is nested in the Papilionoidea, and closely related to Pieridae and Papilionidae. The well-known relationship of (Nymphalidae+(Riodinidae+Lycaenidae)) is recovered in this paper.

Role of miR-21 in alkalinity stress tolerance in tilapia.

MicroRNAs (miRNAs) are a class of short, evolutionary conserved non-coding RNA molecules, which are shown as the key regulators of many biological functions. External stress can alter miRNA expression levels, thereby changing the expression of mRNA target genes. Here, we show that miR-21 is involved in the regulation of alkalinity tolerance in Nile tilapia. Alkalinity stress results in a marked reduction in miR-21 levels. miR-21 loss of function could affect ion balance regulation, ROS production, and antioxidant enzyme activity in vivo. Moreover, miR-21 knockdown protects cell against alkalinity stress-induced injury in vitro. miR-21 directly regulates VEGFB and VEGFC expression by targeting the 3'-untranslated regions (UTRs) of their mRNAs, and inhibition of miR-21 significantly increases the levels of VEGFB and VEGFC expression in vivo. Taken together, our study reveals that miR-21 knockdown plays a protective role in alkalinity tolerance in tilapia.

Molecular characterization and functional analysis of IRF3 in tilapia (Oreochromis niloticus).

Interferon regulatory factor 3 (IRF3) plays a key role in interferon (IFN) response and binding to the IFN stimulatory response elements (ISREs) within the promoter of IFN and IFN-stimulated genes followed by virus infection. In the current study, we discovered one IRF3 homologue in tilapia genome and analyzed the characterizations and functions of tilapia IRF3. Tilapia IRF3 contains 1368 bp with an ORF of 455 aa. Structurally, tilapia IRF3 protein typically shares the conserved characterizations with other species' IRF3 homologues, displaying conserved DNA-binding domain, IRF association domain, serine-rich C terminal domain, and tryptophan residue cluster. Phylogenetic analysis illustrated that tilapia IRF3 belongs to the IRF3 subfamily. Real-time PCR revealed a broad expression pattern of tilapia IRF3 in various tissues. Subcellular localization analysis showed that tilapia IRF3 mainly resides in the cytoplasm, Western blot demonstrated that IRF3 was distributed in the cytoplasmic fraction. Functionally, IRF3 was found to be transcriptionally up-regulated by the poly I:C stimulation. Moreover, reporter assay elucidated that tilapia IRF3 serves as a regulator in mediating IFN response by increasing the activity of IFN-ß and ISRE-containing promoter. These data supported the view that tilapia IRF3 is a potential molecule in IFN immune defense system against viral infection.

The complete mitochondrial genome of Acleris fimbriana (Lepidoptera: Tortricidae).

The yellow tortrix, Acleris fimbriana belongs to Tortricidae in Lepidoptera. We described the complete mitogenome of A. fimbriana, which is typical circular duplex molecules and 15,933 bp in length containing the standard metazoan set of 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and an A + T-rich region with macro-repeat sequences. All the inferred tRNA secondary structures show the common cloverleaf pattern, with the exception of trnS1(AGN) which lacks the DHU arm. The A. fimbriana mitochondrial genome has the same gene order with other lepidopterans.

The complete mitochondrial genome of Carposina sasakii (Lepidoptera: Carposinidae).

The peach fruit moth, Carposina sasakii belongs to Carposinidae in Lepidoptera. In this paper, we described the complete mitogenome of C. sasakii. It is 15,611 bp in length, including 13 PCGs, 2 rRNAs, 22 tRNAs and a major noncoding A + T-rich region, which revealed the typical gene content found in other metazoan mitogenomes. The overall base composition is 42.0% A, 39.5% T, 7.75% G and 10.75% C. The A + T-rich region is located between rrnS and trnM. There is a motif ATAGA in downstream of rrnS followed by a 19 bp Poly-T stretch. The Poly-A is not found in upstream of trnM, and the position of Poly-A is replaced by a stem-loop structure. There are eight mononucleotide repeat sequences (Tn/An) with the length of 7 bp-19bp, three dinucleotide repeat sequences (TA)n/(AT)n, and a longer repeat sequence (AATATATA)5 in A + T-rich region. The mononucleotide repeat sequences occur repeatedly in A + T-rich reigion of C. sasakii, which is special in insects sequenced of Lepidoptera.

Copper amine oxidase-catalysed hydrogen peroxide involves production of nitric oxide in darkness-induced stomatal closure in broad bean.

Hydrogen peroxide is an important intermediate in darkness-induced stomatal closure. In the present work, we provide evidence that copper amine oxidase (CuAO) was involved in H2O2 production in darkness-induced stomatal closure in Vicia faba L. Darkness activated CuAO in intercellular washing fluid from leaves. Aminoguanidine (AG) and 2-bromoethylamine (BEA), which were both irreversible inhibitors of CuAO, significantly suppressed darkness-induced stomatal closure and H2O2 generation. The effects of AG and BEA were reversed only by H2O2 but not by other products of CuAO. These results indicate that CuAO participates in darkness-induced stomatal closure through its reaction product, H2O2. Furthermore, darkness-induced nitric oxide (NO) production and cytosolic alkalinisation were obviously inhibited by AG and BEA, and only H2O2, among the products of CuAO, could reverse the effects, implying that the CuAO-catalysed product H2O2 is required for NO production and cytosolic alkalinisation to a large extent in darkness-induced stomatal closure. In addition, butyric acid blocked but methylamine enhanced the ability of H2O2 to reverse the effect of BEA on NO production, suggesting that cytosolic alkalinisation is involved in CuAO-mediated NO generation in darkness-induced stomatal closure.

miRNA-directed regulation of VEGF in tilapia under hypoxia condition.

The Nile tilapia represents an excellent model for hypoxia tolerance. Vascular endothelial growth factor (VEGF) plays a key role in physiological blood vessel formation and pathological angiogenesis under hypoxia conditions. Tight regulation of VEGF level is necessary for hypoxia adaptation in tilapia. MicroRNAs (miRNAs) function as important regulators of gene expression at the post-transcriptional level, which are usually involved in stress responses. We reasoned that VEGF level could be regulated by miRNAs. Through bioinformatics analysis, we identified a putative miR-204 binding site in the VEGF mRNA. We found that hypoxia leads to a marked up-regulation in VEGF level, but a decrease in miR-204 level. miR-204 directly regulates VEGF expression by targeting its 3'-UTR, and inhibition of miR-204 substantially increases VEGF level in vivo. Moreover, we found that miR-204 loss of function could affect blood O2-carrying capacity, anaerobic metabolism, and antioxidant enzyme activity. Taken together, miR-204 is an endogenous regulator of VEGF expression, which participates in a regulatory circuit that allows rapid gene program transitions upon hypoxia stress.

MicroRNA repertoire for functional genome research in tilapia identified by deep sequencing.

The Nile tilapia (Oreochromis niloticus; Cichlidae) is an economically important species in aquaculture and occupies a prominent position in the aquaculture industry. MicroRNAs (miRNAs) are a class of noncoding RNAs that post-transcriptionally regulate gene expression involved in diverse biological and metabolic processes. To increase the repertoire of miRNAs characterized in tilapia, we used the Illumina/Solexa sequencing technology to sequence a small RNA library using pooled RNA sample isolated from the different developmental stages of tilapia. Bioinformatic analyses suggest that 197 conserved and 27 novel miRNAs are expressed in tilapia. Sequence alignments indicate that all tested miRNAs and miRNAs* are highly conserved across many species. In addition, we characterized the tissue expression patterns of five miRNAs using real-time quantitative PCR. We found that miR-1/206, miR-7/9, and miR-122 is abundantly expressed in muscle, brain, and liver, respectively, implying a potential role in the regulation of tissue differentiation or the maintenance of tissue identity. Overall, our results expand the number of tilapia miRNAs, and the discovery of miRNAs in tilapia genome contributes to a better understanding the role of miRNAs in regulating diverse biological processes.

Inhibition of ABA-induced stomatal closure by fusicoccin is associated with cytosolic acidification-mediated hydrogen peroxide removal.

BACKGROUND: Fusicoccin (FC), a fungal phytotoxin produced by Fusicoccum amygdale, causes the inhibition of ABA-induced stomatal closure. The mechanism of inhibition is remaining unclear. We analyzed the role of hydrogen peroxide (H2O2) and relationship between H2O2 removal and cytosolic pH changes during inhibition of ABA-induced stomatal closure by FC. RESULTS: According to the results, ABA treatment induced H2O2 production and stomatal closure, but FC inhibited the effects of ABA on these two parameters. Treatment with catalase (CAT) and NADPH oxidase inhibitor diphenylene iodonium (DPI) mimicked the effect of FC. These data suggest that inhibition of ABA effect by FC is related to the decrease of H2O2 levels in guard cells. Furthermore, similar to CAT, FC not only suppressed stomatal closure and H2O2 levels in guard cells treated with exogenous H2O2, but also reopened the stomata which had been closed by ABA and reduced the level of H2O2 that had been produced by ABA, indicating that FC causes H2O2 removal in guard cells. The butyric acid treatment simulated the effects of FC on the stomatal aperture and H2O2 levels in guard cells treated with exogenous H2O2 and had been closed by ABA, and both FC and butyric acid reduced cytosolic pH in guard cells of stomata treated with H2O2 and had been closed by ABA, which demonstrate that cytosolic acidification mediates FC-induced H2O2 removal. CONCLUSION: These results suggest that FC causes cytosolic acidification in guard cells, then induces H2O2 removal and reduces H2O2 levels in guard cells, finally inhibits stomatal closure induced by ABA.

microRNA regulation of skin pigmentation in fish.

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play crucial roles in numerous biological processes. However, the role of miRNAs in skin color determination in fish has not been completely determined. Here, we identified that 13 miRNAs are differentially expressed between red and white skin. The analysis of miRNA spatial and temporal expression patterns suggests that miR-429 is a potential regulator of skin pigmentation. miR-429 silencing results in an obvious change in skin pigmentation. Bioinformatics analysis and a luciferase reporter assay show that miR-429 directly regulates expression of Foxd3 by targeting its 3'-untranslated (3'-UTR) region. miR-429 silencing leads to a substantial increase in the expression of Foxd3 in vivo, thereby repressing the transcription of MITF and its downstream genes, such as TYR, TYRP1 or TYRP2. These findings would provide a novel insight into the determination of skin color in fish.

Mechanism of osmoregulatory adaptation in tilapia.

The shortage of freshwater resource in many countries leads to a shift to develop aquaculture in brackish water and sea water. Tilapias are euryhaline that can thrive from freshwater to full sea water. They and their hybrids are the best candidate species for cultivation in brackish habitats. Thus, understanding their osmoregulatory mechanisms will help to breed or genetically engineer salt tolerant species. In this paper, we review recent progress in understanding the mechanisms of osmoregulatory adaptations in tilapia.

miR-206 regulates the growth of the teleost tilapia (Oreochromis niloticus) through the modulation of IGF-1 gene expression.

MicroRNAs (miRNAs) are ~22-nucleotide noncoding RNAs that play a crucial role in regulating muscle development. Our previous study shows that miR-206 is specifically expressed in tilapia skeletal muscle, and exhibits a dynamic expression pattern at different developmental stages. Here, we reveal that miR-206 emerges as a crucial regulator of tilapia growth. miR-206 loss of function leads to the acceleration of tilapia growth. IGF-1 is identified as the target gene of miR-206. miR-206 directly changes IGF-1 expression by targeting its 3' UTR, and inhibition of miR-206 substantially increases the IGF-1 mRNA level in vivo. Thus, miR-206 could be developed as a molecular marker to assist fish breeding.

miR-203b: a novel regulator of MyoD expression in tilapia skeletal muscle.

MyoD is one of the helix-loop-helix proteins regulating muscle-specific gene expression in tilapia. Tight regulation of the MyoD protein level is necessary for the precise regulation of skeletal muscle development. MicroRNAs (miRNAs) are a class of regulatory RNAs that post-transcriptionally regulate gene expression. An increasing amount of evidence has suggested that miRNAs play an important role in regulating skeletal muscle development. We reasoned that MyoD expression may be regulated by miRNAs. Predictions from bioinformatics have identified a putative miR-203b target site in the 3'-UTR of the MyoD gene. Interestingly, miR-203b expression is negatively correlated with MyoD expression, whereas miR-203b suppression leads to a significant increase in MyoD expression, thereby activating MyoD downstream genes. A 3'-UTR luciferase reporter assay further verifies the direct interaction between miR-203b and MyoD. Taken together, our results reveal a novel molecular mechanism in which miRNA participates in transcriptional circuits that regulate gene expression in tilapia skeletal muscle.

miR-429 regulation of osmotic stress transcription factor 1 (OSTF1) in tilapia during osmotic stress.

The Nile tilapia represents an excellent model for osmoregulation study. Osmotic stress transcription factor 1 (OSTF1) identified in tilapia gill epithelium is a critical element of osmosensory signal transduction by means of transcriptional regulation. Thus, tight regulation of OSTF1 level is necessary for tilapia osmotic adaptation. microRNAs (miRNAs), have emerged as a crucial regulator of gene expression at post-transcriptional level. We reasoned that OSTF1 expression could be regulated by miRNAs. By bioinformatics analysis, we identified a putative miR-429 binding site in the OSTF1 mRNA. Interestingly, miR-429 is down-regulated in tilapia upon osmotic stress, consistent with OSTF1 protein up-regulation. miR-429 directly regulates OSTF1 expression by targeting its 3'-UTR, and inhibition of miR-429 substantially increases OSTF1 level in vivo. Moreover, miR-429 loss of function could influence the regulation of plasma osmolality and ion concentration responding to osmotic stress. Taken together, miR-429 is an endogenous regulator of OSTF1 expression, which participates in a regulatory circuit that allows rapid gene program transitions in response to osmotic stress.

The complete mitochondrial genome of Leucoptera malifoliella Costa (Lepidoptera: Lyonetiidae).

The mitochondrial genome (mitogenome) of Leucoptera malifoliella (=L. scitella) (Lepidoptera: Lyonetiidae) was sequenced. The size was 15,646 bp with gene content and order the same as those of other lepidopterans. The nucleotide composition of L. malifoliella mitogenome is highly A+T biased (82.57%), ranked just below Coreana raphaelis (82.66%) (Lepidoptera: Lycaenidae). All protein-coding genes (PCGs) start with the typical ATN codon except for the cox1 gene, which uses CGA as the initiation codon. Nine PCGs have the common stop codon TAA, four PCGs have the common stop codon T as incomplete stop codons, and nad4l and nad6 have TAG as the stop codon. Cloverleaf secondary structures were inferred for 22 tRNA genes, but trnS1(AGN) was found to lack the DHU stem. The secondary structure of rrnL and rrnS is generally similar to other lepidopterans but with some minor differences. The A+T-rich region includes the motif ATAGA, but the poly (T) stretch is replaced by a stem-loop structure, which may have a similar function to the poly (T) stretch. Finally, there are three long repeat (154 bp) sequences followed by one short repeat (56 bp) with four (TA)(n) intervals, and a 10-bp poly-A is present upstream of trnM. Phylogenetic analysis shows that the position of Yponomeutoidea, as represented by L. malifoliella, is the same as traditional classifications. Yponomeutoidea is the sister to the other lepidopteran superfamilies covered in the present study.

MiR-30c: a novel regulator of salt tolerance in tilapia.

miRNAs comprise a class of ~22 nt noncoding RNAs that modulate the stability and/or translational potential of their mRNA targets. Emerging data suggest that stress conditions can alter the biogenesis of miRNAs, thereby changing the expression of mRNA targets. Here, we reveal that miR-30c, a kidney-enriched miRNA, emerges as a crucial osmoregulator in Nile tilapia. miR-30c loss of function leads to an inability to respond to osmotic stress. We identify HSP70 as one of the direct regulatory targets of miR-30c. miR-30c directly regulates HSP70 by targeting its 3'-UTR, and inhibition of miR-30c substantially increases HSP70 mRNA level in vivo. Taken together, our experiments suggest that miRNAs participate in a regulatory circuit that allows rapid gene program transitions in response to osmotic stress. miR-30c may be developed as a molecular marker to assist to breed or genetically engineer salt tolerant species.