Selective type II TRK inhibitors overcome xDFG mutation mediated acquired resistance to the second-generation inhibitors selitrectinib and repotrectinib.

Neurotrophic receptor kinase (NTRK) fusions are actionable oncogenic drivers of multiple pediatric and adult solid tumors, and tropomyosin receptor kinase (TRK) has been considered as an attractive therapeutic target for "pan-cancer" harboring these fusions. Currently, two generations TRK inhibitors have been developed. The representative second-generation inhibitors selitrectinib and repotrectinib were designed to overcome clinic acquired resistance of the first-generation inhibitors larotrectinib or entrectinib resulted from solvent-front and gatekeeper on-target mutations. However, xDFG (TRKAG667C/A/S, homologous TRKCG696C/A/S) and some double mutations still confer resistance to selitrectinib and repotrectinib, and overcoming these resistances represents a major unmet clinical need. In this review, we summarize the acquired resistance mechanism of the first- and second-generation TRK inhibitors, and firstly put forward the emerging selective type II TRK inhibitors to overcome xDFG mutations mediated resistance. Additionally, we concluded our perspectives on new challenges and future directions in this field.

Transmitted drug resistance to antiretroviral drugs in Spain during the period 2019-2021.

To evaluate the prevalence of transmitted drug resistance (TDR) to nucleoside and nonnucleoside reverse transcriptase inhibitors (NRTI, NNRTI), protease inhibitors (PI), and integrase strand transfer inhibitors (INSTI) in Spain during the period 2019-2021, as well as to evaluate transmitted clinically relevant resistance (TCRR) to antiretroviral drugs. Reverse transcriptase (RT), protease (Pro), and Integrase (IN) sequences from 1824 PLWH (people living with HIV) were studied. To evaluate TDR we investigated the prevalence of surveillance drug resistance mutations (SDRM). To evaluate TCRR (any resistance level ≥ 3), and for HIV subtyping we used the Stanford v.9.4.1 HIVDB Algorithm and an in-depth phylogenetic analysis. The prevalence of NRTI SDRMs was 3.8% (95% CI, 2.8%-4.6%), 6.1% (95% CI, 5.0%-7.3%) for NNRTI, 0.9% (95% CI, 0.5%-1.4%) for PI, and 0.2% (95% CI, 0.0%-0.9%) for INSTI. The prevalence of TCRR to NRTI was 2.1% (95% CI, 1.5%-2.9%), 11.8% for NNRTI, (95% CI, 10.3%-13.5%), 0.2% (95% CI, 0.1%-0.6%) for PI, and 2.5% (95% CI, 1.5%-4.1%) for INSTI. Most of the patients were infected by subtype B (79.8%), while the majority of non-Bs were CRF02_AG (n = 109, 6%). The prevalence of INSTI and PI resistance in Spain during the period 2019-2021 is low, while NRTI resistance is moderate, and NNRTI resistance is the highest. Our results support the use of integrase inhibitors as first-line treatment in Spain. Our findings highlight the importance of ongoing surveillance of TDR to antiretroviral drugs in PLWH particularly with regard to first-line antiretroviral therapy.

The potential for development of clinically relevant microbial resistance to rifaximin-α: a narrative review.

Rifaximin-α is a gut-targeted antibiotic indicated for numerous gastrointestinal and liver diseases. Its multifaceted mechanism of action goes beyond direct antimicrobial effects, including alterations in bacterial virulence, cytoprotective effects on host epithelial cells, improvement of impaired intestinal permeability, and reduction of proinflammatory cytokine expression via activation of the pregnane X receptor. Rifaximin-α is virtually non-absorbed, with low systemic drug levels contributing to its excellent safety profile. While there are high concentrations of drug in the colon, low water solubility leads to low colonic drug bioavailability, protecting the gut microbiome. Rifaximin-α appears to be more active in the bile-rich small bowel. Its important biologic effects are largely at sub-inhibitory concentration. Although in vitro testing of clinical isolates from rifaximin recipients has revealed rifaximin-resistant strains in some studies, the risk of emergent rifaximin-α resistance appears to be lower than for many other antibiotics. Rifaximin-α has been used for many years for traveler's diarrhea with no apparent increase in resistance levels in causative pathogens. Further, rifaximin-α retains its efficacy after long-term and recurrent usage in chronic gastrointestinal disorders. There are numerous reasons why the risk of microbial resistance to rifaximin-α may be lower than that for other agents, including low intestinal bioavailability in the aqueous colon, the mechanisms of action of rifaximin-α not requiring inhibitory concentrations of drug, and the low risk of cross transmission of rifaximin-α resistance between bacterial species. Reported emergence of vancomycin-resistant Enterococcus in liver-disease patients maintained on rifaximin needs to be actively studied. Further studies are required to assess the possible correlation between in vitro resistance and rifaximin-α efficacy.

Clinical Impact of New Treatment Strategies for HER2-Positive Metastatic Breast Cancer Patients with Resistance to Classical Anti-HER Therapies.

HER2-positive breast cancer accounts for 15-20% of all breast cancer cases. This subtype is characterized by an aggressive behavior and poor prognosis. Anti-HER2 therapies have considerably improved the natural course of the disease. Despite this, relapse still occurs in around 20% of patients due to primary or acquired treatment resistance, and metastasis remains an incurable disease. This article reviews the main mechanisms underlying resistance to anti-HER2 treatments, focusing on newer HER2-targeted therapies. The progress in anti-HER2 drugs includes the development of novel antibody-drug conjugates with improvements in the conjugation process and novel linkers and payloads. Moreover, trastuzumab deruxtecan has enhanced the efficacy of trastuzumab emtansine, and the new drug trastuzumab duocarmazine is currently undergoing clinical trials to assess its effect. The combination of anti-HER2 agents with other drugs is also being evaluated. The addition of immunotherapy checkpoint inhibitors shows some benefit in a subset of patients, indicating the need for useful biomarkers to properly stratify patients. Besides, CDK4/6 and tyrosine kinase inhibitors are also included in the design of new treatment strategies. Lapitinib, neratinib and tucatinib have been approved for HER2-positive metastasis patients, however clinical trials are currently ongoing to optimize combined strategies, to reduce toxicity, and to better define the useful setting. Clinical research should be strengthened along with the discovery and validation of new biomarkers, as well as a deeper understanding of drug resistance and action mechanisms.

Switch type I to type II TRK inhibitors for combating clinical resistance induced by xDFG mutation for cancer therapy.

TRK xDFG mutation-induced acquired resistance of 1st generation inhibitors larotrectinib and entrectinib remains an unmet clinical need. Here we report a series of 6-(pyrrolidin-1-yl)imidazo[1,2-b]pyridazine-based derivatives as selective type II TRK inhibitors by hybridization. A representative compound 12d potently inhibited TRKA/B/C and TRKAG667C with IC50 values of 3.3, 6.4, 4.3 and 9.4 nM, respectively. 12d potently suppressed proliferation of a panel of Ba/F3 cells stably transformed with wild type, xDFG as well as solvent-front (SF) mutant TRK fusion proteins. Compared with larotrectinib and selitrectinib, 12d displayed superior inhibitory activity towards Ba/F3 cells harboring CD74-TRKAG667C and ETV6-TRKCG696C with IC50 values of 2.6 and 6.1 nM, respectively. Moreover, 12d also exhibited potent antiproliferation activity against Ba/F3-ETV6-TRKCG623R and Ba/F3-ETV6-TRKCG623E mutants with IC50 values of 31.0 and 28.2 nM, respectively. This work provided a new potential type II TRK inhibitor-based lead compound for the treatment of TRK driven cancers.

Factors promoting and limiting antimicrobial resistance in the environment - Existing knowledge gaps.

The dissemination of multidrug-resistant bacteria strains and genes carrying antibiotic resistance is currently considered to be one of the most important global problem. The WHO calls for the need to contain the spread of Antimicrobial Resistance (AMR) from all possible sources. There have been many international actions grouping scientists studying this phenomenon, and quite a lot of scientific projects devoted to this problem have already been carried out. As well, so far several strategies have been developed that can inhibit the AMR spread. In this mini-review, we highlight overlooked aspects that seem to be crucial for creating a comprehensive picture of AMR, especially in the context of One Health approach.

Discovery of 3-pyrazolyl-substituted pyrazolo[1,5-a]pyrimidine derivatives as potent TRK inhibitors to overcome clinically acquired resistance.

Several secondary tropomyosin receptor kinase (TRK) mutations located in the solvent front, xDFG, and gatekeeper regions, are a common cause of clinical resistance. Mutations in the xDFG motif in particular limit sensitivity to second-generation TRK inhibitors, which represent an unmet clinical need. We designed a series of 3-pyrazolyl-substituted pyrazolo[1,5-a]pyrimidine derivatives toward these secondary mutations using ring-opening and scaffold-hopping strategies. Compound 5n was the most potent, with IC50 values of 2.3 nM, 0.4 nM, and 0.5 nM against TRKAG667C, TRKAF589L, and TRKAG595R, compared to selitrectinib with IC50 values of 12.6 nM, 5.8 nM, and 7.6 nM, respectively (approximately 5.4, 14.5, and 15.2-fold increases). Furthermore, 5n displayed favorable pharmacokinetic properties and satisfactory antitumor efficacy (tumor growth inhibition of 97% at 30 mg/kg and 73% at 100 mg/kg) in TRKAWT and TRKAG667C xenograft mouse models. Collectively, 5n is a promising TRK inhibitor lead compound for overcoming clinically acquired resistance to second-generation inhibitors, particularly for resistant tumors harboring the TRKAG667C mutation in the xDFG motif.

Cellular Growth Arrest and Efflux Pumps Are Associated With Antibiotic Persisters in Streptococcus pyogenes Induced in Biofilm-Like Environments.

Streptococcus pyogenes (group A Streptococcus-GAS) is an important pathogen for humans. GAS has been associated with severe and invasive diseases. Despite the fact that these bacteria remain universally susceptible to penicillin, therapeutic failures have been reported in some GAS infections. Many hypotheses have been proposed to explain these antibiotic-unresponsive infections; however, none of them have fully elucidated this phenomenon. In this study, we show that GAS strains have the ability to form antimicrobial persisters when inoculated on abiotic surfaces to form a film of bacterial agglomerates (biofilm-like environment). Our data suggest that efflux pumps were possibly involved in this phenomenon. In fact, gene expression assays by real-time qRT-PCR showed upregulation of some genes associated with efflux pumps in persisters arising in the presence of penicillin. Phenotypic reversion assay and whole-genome sequencing indicated that this event was due to non-inherited resistance mechanisms. The persister cells showed downregulation of genes associated with protein biosynthesis and cell growth, as demonstrated by gene expression assays. Moreover, the proteomic analysis revealed that susceptible cells express higher levels of ribosome proteins. It is remarkable that previous studies have reported the recovery of S. pyogenes viable cells from tissue biopsies of patients presented with GAS invasive infections and submitted to therapy with antibiotics. The persistence phenomenon described herein brings new insights into the origin of therapeutic failures in S. pyogenes infections. Multifactorial mechanisms involving protein synthesis inhibition, cell growth impairment and efflux pumps seem to play roles in the formation of antimicrobial persisters in S. pyogenes.

Design, synthesis, and biological evaluation of Bcr-Abl PROTACs to overcome T315I mutation.

Bcr-Abl threonine 315 to isoleucine 315 (T315I) gatekeeper mutation induced drug resistance remains an unmet clinical challenge for the treatment of chronic myeloid leukemia (CML). Chemical degradation of Bcr-AblT315I protein has become a potential strategy to overcome drug resistance. Herein, we first described the design, synthesis, and evaluation of a new class of selective Bcr-AblT315I proteolysis-targeting chimeric (PROTAC) degraders based on GZD824 (reported as Bcr-AblT315I inhibitor by our group). One of the degrader 7o with 6-member carbon chain linkage with pomalidomide exhibits the most potent degradation efficacy with DR of 69.89% and 94.23% at 100 and 300 nmol/L, respectively, and has an IC50 value of 26.8 ± 9.7 nmol/L against Ba/F3T315I cells. Further, 7o also displays substantial tumor regression against Ba/F3-Bcr-AblT315I xenograft model in vivo.

The Role of Efflux Pumps in the Transition from Low-Level to Clinical Antibiotic Resistance.

Antibiotic resistance is on the rise and has become one of the biggest public health challenges of our time. Bacteria are able to adapt to the selective pressure exerted by antibiotics in numerous ways, including the (over)expression of efflux pumps, which represents an ancient bacterial defense mechanism. Several studies show that overexpression of efflux pumps rarely provides clinical resistance but contributes to a low-level resistance, which allows the bacteria to persist at the infection site. Furthermore, recent studies show that efflux pumps, apart from pumping out toxic substances, are also linked to persister formation and increased spontaneous mutation rates, both of which could aid persistence at the infection site. Surviving at the infection site provides the low-level-resistant population an opportunity to evolve by acquiring secondary mutations in antibiotic target genes, resulting in clinical resistance to the treating antibiotic. Thus, this emphasizes the importance and challenge for clinicians to be able to monitor overexpression of efflux pumps before low-level resistance develops to clinical resistance. One possible treatment option could be an efflux pump-targeted approach using efflux pump inhibitors.

The best type of inoculum for testing the antifungal drug susceptibility of Microsporum canis: In vivo and in vitro results.

BACKGROUND: Data correlating in vitro drug susceptibility of Microsporum canis with clinical outcomes of its infections are lacking as well as the most suitable inoculum and incubation time in broth microdilution assays. OBJECTIVES AND METHODS: Microsporum canis strains were collected from animal hosts that tested positive (Group I; n = 13) and negative (Group II; n = 14) to this pathogen following itraconazole (ITC) therapy. In vitro ITC susceptibility was assessed according to the Clinical Laboratory Standards Institute (CLSI M38-A2) methodology using conidia, hypha-conidia and arthroconidia at 3 and 7 days of incubation in order to assess the most suitable inoculum and incubation time. Successively, ketoconazole (KTC), voriconazole (VRC), terbinafine (TRB), posaconazole (PSZ), fluconazole (FLC) and griseofulvin (GRI) susceptibilities were assessed using the chosen inoculum. RESULTS: The MIC values of ITC after three-day incubation were equal than those recorded after 7-day incubation. Itraconazole MICs were ≤1 µg/mL for strains from Group II and >1 µg/mL for those of Group II only when conidia were used. All strains showed high susceptibility to VRC, POS, TEB and low susceptibility to ITC, KTC, GRI and FLC regardless of the source and incubation time. CONCLUSIONS AND CLINICAL IMPORTANCE: Results suggest that correlation between the in vitro results and clinical outcome was observed only by incubating conidia for 3 days at 30 ± 2°C. These conditions might be most suitable to assess in vitro susceptibility of M. canis and assist in determining the occurrence of drug resistance and cross-resistance phenomena.

Trypanothione Reductase Gene Mutations in Meglumine Antimoniate Resistant Isolates from Cutaneous Leishmaniasis Patients Using Molecular Dynamics Method.

BACKGROUND: In this study, mutations in the tripanothion reductase of Leishmania tropica isolated from Iran was investigated using sequencing and simulation of the enzyme by the molecular dynamic method. METHODS: Fifteen susceptible and 15 clinical resistant L. tropica specimens were collected from skin lesions from different regions of Iran in 2017. After DNA extraction, trypanothione reductase (TRYR or TPR), gene fragment was amplified using PCR and sequencing methods. In the case of structural mutations, the components were simulated by molecular dynamics using the GROMACS software. RESULTS: Some structural mutations were observed in 9 amino acids surrounding the active site of the TRYR gene of L. tropica with three-dimensional trypanothione reductase alteration. CONCLUSION: Change in the active site of TRYR of L. tropica, could probably contribute to the development of resistant L. tropica to glucantime. Because of the likely occurrence of mutations in glucantime as well as the ease of development of L. tropica resistant populations, more samples are needed to demonstrate the relationship between mutations in this enzyme and clinical resistance to glucantime. On the other hand, it is recommended that enzymatic studies be performed to confirm the role of mutation in the function and expression of trypanothione reductase in glucantime resistant and susceptible populations.

Assessment of trimethoprim-sulfamethoxazole susceptibility testing methods for fastidious Haemophilus spp.

OBJECTIVES: To compare the determinants of trimethoprim-sulfamethoxazole resistance with established susceptibility values for fastidious Haemophilus spp., to provide recommendations for optimal trimethoprim-sulfamethoxazole measurement. METHODS: We collected 50 strains each of Haemophilus influenzae and Haemophilus parainfluenzae at Bellvitge University Hospital. Trimethoprim-sulfamethoxazole susceptibility was tested by microdilution, E-test and disc diffusion using both Mueller-Hinton fastidious (MH-F) medium and Haemophilus test medium (HTM) following EUCAST and CLSI criteria, respectively. Mutations in folA, folP and additional determinants of resistance were identified in whole-genome-sequenced isolates. RESULTS: Strains presented generally higher rates of trimethoprim-sulfamethoxazole resistance when grown on HTM than on MH-F, independent of the methodology used (average MIC 2.6-fold higher in H. influenzae and 1.2-fold higher in H. parainfluenzae). The main resistance-related determinants were as follows: I95L and F154S/V in folA; 3- and 15-bp insertions and substitutions in folP; acquisition of sul genes; and FolA overproduction potentially linked to mutations in -35 and -10 promoter motifs. Of note, 2 of 19 H. influenzae strains (10.5%) and 9 of 33 H. parainfluenzae strains (27.3%) with mutations and assigned as resistant by microdilution were inaccurately considered susceptible by disc diffusion. This misinterpretation was resolved by raising the clinical resistance breakpoint of the EUCAST guidelines to ≤30 mm. CONCLUSIONS: Given the routine use of disc diffusion, a significant number of strains could potentially be miscategorized as susceptible to trimethoprim-sulfamethoxazole despite having resistance-related mutations. A simple modification to the current clinical resistance breakpoint given by the EUCAST guideline for MH-F ensures correct interpretation and correlation with the reference standard method of microdilution.

Immunogenicity Associated with Botulinum Toxin Treatment.

Botulinum toxin (BoNT) has been used for the treatment of a variety of neurologic, medical and cosmetic conditions. Two serotypes, type A (BoNT-A) and type B (BoNT-B), are currently in clinical use. While considered safe and effective, their use has been rarely complicated by the development of antibodies that reduce or negate their therapeutic effect. The presence of antibodies has been attributed to shorter dosing intervals (and booster injections), higher doses per injection cycle, and higher amounts of antigenic protein. Other factors contributing to the immunogenicity of BoNT include properties of each serotype, such as formulation, manufacturing, and storage of the toxin. Some newer formulations with purified core neurotoxin devoid of accessory proteins may have lower overall immunogenicity. Several assays are available for the detection of antibodies, including both structural assays such as ELISA and mouse-based bioassays, but there is no consistent correlation between these antibodies and clinical response. Prevention and treatment of antibody-associated non-responsiveness is challenging and primarily involves the use of less immunogenic formulations of BoNT, waiting for the spontaneous disappearance of the neutralizing antibody, and switching to an immunologically alternate type of BoNT.

The first multilocus genotype analysis of Giardia intestinalis in humans in the Czech Republic.

To date, genotyping data on giardiasis have not been available in the Czech Republic. In this study, we characterized 47 human isolates of Giardia intestinalis from symptomatic as well as asymptomatic giardiasis cases. Genomic DNA from trophozoites was tested by PCR-sequence analysis at three loci (ß-giardin, glutamate dehydrogenase and triose phosphate isomerase). Sequence analysis showed assemblages A and B in 41 (87.2%) and six (12.8%) isolates, respectively. Two of the 41 assemblage A samples were genotyped as sub-assemblage AI, and 39 were genotyped as sub-assemblage AII. Four previously identified multilocus genotypes (MLGs: AI-1, AII-1, AII-4 and AII-9) and six likely novel variations of MLGs were found. In agreement with previous studies, sequences from assemblage B isolates were characterized by a large genetic variability and by the presence of heterogeneous positions, which prevent the definition of MLGs. This study also investigated whether there was a relationship between the assemblage and clinical data (including drug resistance). However, due to the large number of genotypes and the relatively small number of samples, no significant associations with the clinical data were found.

Novel Molecular Challenges in Targeting Anaplastic Lymphoma Kinase in ALK-Expressing Human Cancers.

Targeting anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase receptor initially identified as a potent oncogenic driver in anaplastic large-cell lymphoma (ALCL) in the form of nucleophosmin (NPM)-ALK fusion protein, using tyrosine kinase inhibitors has shown to be a promising therapeutic approach for ALK-expressing tumors. However, clinical resistance to ALK inhibitors invariably occurs, and the molecular mechanisms are incompletely understood. Recent studies have clearly shown that clinical resistance to ALK inhibitors is a multifactorial and complex mechanism. While few of the mechanisms of clinical resistance to ALK inhibitors such as gene mutation are well known, there are others that are not well covered. In this review, the molecular mechanisms of cancer stem cells in mediating resistance to ALK inhibitors as well as the current understanding of the molecular challenges in targeting ALK in ALK-expressing human cancers will be discussed.

Synthesis and identification of GZD856 as an orally bioavailable Bcr-AblT315I inhibitor overcoming acquired imatinib resistance.

Bcr-AblT315I induced drug resistance remains a major challenge to chronic myelogenous leukemia (CML) treatment. Herein, we reported GZD856 as a novel orally bioavailable Bcr-AblT315I inhibitor, which strongly suppressed the kinase activities of both native Bcr-Abl and the T315I mutant with IC50 values of 19.9 and 15.4 nM, and potently inhibited proliferation of corresponding K562, Ba/F3WT and Ba/F3T315I cells with IC50 values of 2.2, 0.64 and 10.8 nM. Furthermore, GZD856 potently suppressed tumor growth in mouse bearing xenograft K562 and Ba/F3 cells expressing Bcr-AblT315I. Thus, GZD856 may serve as a promising lead for the development of Bcr-Abl inhibitors overcoming acquired imatinib resistance.

The fungal resistome: a risk and an opportunity for the development of novel antifungal therapies.

The risks for toxicity of novel antifungal compounds, together with the emergence of resistance, makes the use of inhibitors of resistance, in combination with antifungal compounds, a suitable strategy for developing novel antifungal formulations. Among them, inhibitors of efflux pumps are suitable candidates. Increasing drug influx or interfering with the stress response may also improve the efficacy of antifungals. Therapies as induction of fungal apoptosis or immunostimulation are also good strategies for reducing the risks for resistance and to improve antifungals' efficacy. Understanding the effect of the acquisition of resistance on the fungal physiology and determining the collateral sensitivity networks are useful for the development of novel strategies based on combination of antifungals for improving the efficacy of the therapy.

Hybrid pyrimidine alkynyls inhibit the clinically resistance related Bcr-Abl(T315I) mutant.

A series of pyrimidine alkynyl derivatives were designed and synthesized as new Bcr-Abl inhibitors by hybriding the structural moieties from GNF-7, ponatinib and nilotinib. One of the most potent compounds 4e strongly suppresses Bcr-Abl(WT) and Bcr-Abl(T315I) kinase with IC50 values of 5.0 and 9.0 nM, and inhibits the proliferation of K562 and murine Ba/F3 cells ectopically expressing Bcr-Abl(T315I) cells with IC50 values of 2 and 50 nM, respectively. It also displays good pharmacokinetics properties with an oral bioavailability of 35.3% and T(1/2) value of 48.7 h, and demonstrates significantly suppression on tumor growth in xenografted mice of K562 and Ba/F3 cells expressing Bcr-Abl(T315I). These inhibitors may serve as lead compounds for further developing new anticancer drugs overcoming the clinically acquired resistance against current Bcr-Abl inhibitors.

Environmental azole fungicide, prochloraz, can induce cross-resistance to medical triazoles in Candida glabrata.

Acquisition of azole resistance by clinically relevant yeasts in nature may result in a significant, yet undetermined, impact in human health. The main goal of this study was to assess the development of cross-resistance between agricultural and clinical azoles by Candida spp. An in vitro induction assay was performed, for a period of 90 days, with prochloraz (PCZ) - an agricultural antifungal. Afterward, the induced molecular resistance mechanisms were unveiled. MIC value of PCZ increased significantly in all Candida spp. isolates. However, only C. glabrata developed cross-resistance to fluconazole and posaconazole. The increased MIC values were stable. Candida glabrata azole resistance acquisition triggered by PCZ exposure involved the upregulation of the ATP binding cassette multidrug transporter genes and the transcription factor, PDR1. Single mutation previously implicated in azole resistance was found in PDR1 while ERG11 showed several synonymous single nucleotide polymorphisms. These results might explain why C. glabrata is so commonly less susceptible to clinical azoles, suggesting that its exposure to agricultural azole antifungals may be associated to the emergence of cross-resistance. Such studies forward potential explanations for the worldwide increasing clinical prevalence of C. glabrata and the associated worse prognosis of an infection by this species.