Hipomagnesemia grave secundaria a inhibidores de la bomba de protones, dos casos clínicos / Severe hypomagnesaemia secondary to proton pump inhibitors: a presentation of two cases

Se describen dos casos clínicos de pacientes con hipomagnesemia grave secundaria a inhibidores de la bomba de protones. Los datos de laboratorio nos pueden ayudar a diferenciar entre la hipomagnesemia producida por pérdida intestinal de magnesio y la producida por pérdidas renales de magnesio. La hipomagnesemia asociada al uso prolongado de inhibidores de la bomba de protones es un efecto adverso que suele pasar inadvertido y que puede ocasionar manifestaciones clínicas graves, siendo conveniente realizar controles periódicos de magnesemia en pacientes que precisen de tratamiento prolongado con inhibidores de la bomba de protones, fundamentalmente en el uso concomitante de antiarrítmicos o diuréticos y en aquellos pacientes con hipocalcemia y/o hipopotasemia. La posibilidad de este y de otros efectos adversos es un motivo más para valorar periódicamente el tratamiento con inhibidores de la bomba de protones a largo plazo (AU)

Two clinical cases are presented on patients with severe hypomagnesaemia induced by proton pump inhibitors. Laboratory data can help us to differentiate between hypomagnesaemia caused by intestinal loss of magnesium and the one produced by renal magnesium losses. Hypomagnesaemia associated with a prolonged use of proton pump inhibitors is an inadvertent adverse effect that can lead to severe clinical manifestations, therefore it is advisable to perform periodic monitoring of magnesium in the blood of patients who require prolonged treatment with proton pump inhibitors, especially in the concomitant use of antiarrhythmic or diuretic drugs, and in those patients with hypocalcaemia and/or hypokalaemia. The possibility of this and other adverse effects is one more reason to periodically evaluate long-term proton pump inhibitors treatment (AU)

Biochemical Analysis of Microbial Rhodopsins.

Ion-pumping rhodopsins transfer ions across the microbial cell membrane in a light-dependent fashion. As the rate of biochemical characterization of microbial rhodopsins begins to catch up to the rate of microbial rhodopsin identification in environmental and genomic sequence data sets, in vitro analysis of their light-absorbing properties and in vivo analysis of ion pumping will remain critical to characterizing these proteins. As we learn more about the variety of physiological roles performed by microbial rhodopsins in different cell types and environments, observing the localization patterns of the rhodopsins and/or quantifying the number of rhodopsin-bearing cells in natural environments will become more important. Here, we provide protocols for purification of rhodopsin-containing membranes, detection of ion pumping, and observation of functional rhodopsins in laboratory and environmental samples using total internal reflection fluorescence microscopy. © 2016 by John Wiley & Sons, Inc.

Western blot confirmation of the H+/K+-ATPase proton pump in the human larynx and submandibular gland.

OBJECTIVE: The authors have previously demonstrated the H(+)/K(+)-ATPase (proton pump) in human larynx and lung glands via immunohistochemistry (IHC). The present hypothesis is that the proton pump is expressed in other seromucinous glands of the digestive tract that can be confirmed by IHC and Western blot analysis. STUDY DESIGN: Prospective controlled tissue analysis study. SETTING: Academic medical institution. METHODS: Ten anonymous fresh-frozen donor specimens were obtained, comprising 3 submandibular glands, 4 larynges, and 3 normal stomach specimens for control. Submandibular gland sections were immunostained with 2 monoclonal antibodies selectively reactive with α or ß subunits of the H(+)/K(+)-ATPase. Western blot analysis was performed on all specimens. RESULTS: Consistent IHC staining was observed in the submandibular gland specimens for both α and ß subunits. Western blot analysis revealed very strong expression for the stomach at 100 kDa, corresponding to the α protein, and weak but notable banding for all larynx and submandibular gland specimens. Similar findings were noted for the 60- to 80-kDa glycosylated ß subunit protein, as well as the 52-kDa ß subunit precursor for all specimens. CONCLUSION: The H(+)/K(+)-ATPase (proton) pump is present in the human larynx and submandibular gland although in much lower concentrations than in the stomach. Proton pump involvement in human aerodigestive seromucinous glands may have a role in protecting mucosa from acid environments (local or systemic), explain heightened laryngeal sensitivity in those patients with laryngopharyngeal reflux, and be a site of action for proton pump inhibitor pharmacotherapy.

Inhibidores de la bomba de protones y riesgo de infección / Proton pump inhibitors and infection risk

Los fármacos antisecretores gástricos, y en especial los inhibidores de la bomba de protones, se encuentran entre los fármacos más usados tanto en el medio ambulatorio como en el hospital, y su prescripción no siempre se ajusta a las indicaciones establecidas. Existen datos experimentales que sugieren que la inhibición de la secreción ácida gástrica y los efectos de estos fármacos sobre el sistema inmune pueden favorecer la aparición de infecciones. En los últimos años se han publicado un número de estudios observacionales que encuentran una asociación independiente entre el uso de inhibidores de la bomba de protones y un riesgo aumentado de infecciones gastrointestinales, incluyendo las causadas por Clostridium difficile, y de neumonía comunitaria y nosocomial. En esta revisión se discute la evidencia existente, se plantean los posibles mecanismos patogénicos implicados y se presentan recomendaciones para la investigación futura y la práctica clínica actual(AU)

Gastric antisecretory drugs, especially proton pump inhibitors, are among the most used drugs both in ambulatory and hospital settings, and prescription does not always follows approved indications. Experimental data suggest that gastric acid inhibition and the effects of proton pump inhibitors on the immune system can promote the development of infections. In recent years a number of observational studies have found an independent association between the use of proton pump inhibitors and an increased risk of gastrointestinal infections, including those caused by Clostridium difficile, and community and nosocomial pneumonia. This review discusses the current evidence, raises the potential pathogenic mechanisms involved and makes recommendations for current clinical practice and future research(AU)

Gastric acid is the key modulator in the pathogenesis of non-steroidal anti-inflammatory drug-induced ulceration in rats.

1. In the present study, we investigated the role of gastric acid (GA) secretion on non-steroidal anti-inflammatory drug (NSAID)-induced ulcerogenesis in vivo. Rats were administered single oral doses of selective cyclo-oxygenase (COX)-1 (SC-560; 2.5 mg/kg), COX-2 (DFU; 25 mg/kg) or non-selective COX (indomethacin; 25 mg/kg) inhibitors. Three groups (basal, histamine-stimulated and histamine with lansoprazole) were pylorus ligated 2 h after inhibitor administration and killed 2 h later. Another group without pylorus ligation received only inhibitors and was killed after 18 h. 2. At 4 h, indomethacin increased the ulcer index (UI) and myeloperoxidase (MPO) activity in basal and histamine-stimulated states, whereas SC-560 only increased MPO activity. Histamine-stimulated, but not basal, GA was further enhanced by indomethacin and SC-560 via increased proton pump expression. Lansoprazole (10 mg/kg) reduced the UI, MPO activity and GA to basal levels with SC-560 and DFU and to near basal with indomethacin. Indomethacin and SC-560 significantly inhibited prostaglandin (PG) E(2), without significantly affecting COX-1 and COX-2 expression. Although DFU inhibited PGE(2) by one-third, it did not affect COX expression. 3. At 18 h, indomethacin significantly increased the UI and MPO activity, whereas PGE(2) synthesis was less inhibited, indicating a return to control levels. In contrast, PGE(2) synthesis was higher than control with SC-560. Furthermore, COX-2 expression was significantly elevated with indomethacin and SC-560, explaining the source of augmented PGE(2) synthesis. Proton pump expression remained elevated, comparable with 4 h levels, with indomethacin and SC-560. However, DFU had no significant effect on the aforementioned parameters. 4. The data suggest that NSAID-induced ulcerogenesis is dependent on the amount of GA secretion derived from increased proton pump expression and requires inhibition of both COX-1 and COX-2.

Negative inotropy of the gastric proton pump inhibitor pantoprazole in myocardium from humans and rabbits: evaluation of mechanisms.

BACKGROUND: Proton pump inhibitors are used extensively for acid-related gastrointestinal diseases. Their effect on cardiac contractility has not been assessed directly. METHODS AND RESULTS: Under physiological conditions (37 degrees C, pH 7.35, 1.25 mmol/L Ca2+), there was a dose-dependent decrease in contractile force in ventricular trabeculae isolated from end-stage failing human hearts superfused with pantoprazole. The concentration leading to 50% maximal response was 17.3+/-1.3 microg/mL. Similar observations were made in trabeculae from human atria, normal rabbit ventricles, and isolated rabbit ventricular myocytes. Real-time polymerase chain reaction demonstrated the expression of gastric H+/K+-adenosine triphosphatase in human and rabbit myocardium. However, measurements with BCECF-loaded rabbit trabeculae did not reveal any significant pantoprazole-dependent changes of pH(i). Ca2+ transients recorded from field-stimulated fluo 3-loaded myocytes (F/F0) were significantly depressed by 10.4+/-2.1% at 40 microg/mL. Intracellular Ca2+ fluxes were assessed in fura 2-loaded, voltage-clamped rabbit ventricular myocytes. Pantoprazole (40 microg/mL) caused an increase in diastolic [Ca2+]i by 33+/-12%, but peak systolic [Ca2+]i was unchanged, resulting in a decreased Ca2+ transient amplitude by 25+/-8%. The amplitude of the L-type Ca2+ current (I(Ca,L)) was reduced by 35+/-5%, and sarcoplasmic reticulum Ca2+ content was reduced by 18+/-6%. Measurements of oxalate-supported sarcoplasmic reticulum Ca2+ uptake in permeabilized cardiomyocytes indicated that pantoprazole decreased Ca2+ sensitivity (Kd) of sarcoplasmic reticulum Ca2+ adenosine triphosphatase: control, Kd=358+/-15 nmol/L; 40 microg/mL pantoprazole, Kd=395+/-12 nmol/L (P<0.05). Pantoprazole also acted on cardiac myofilaments to reduced Ca2+-activated force. CONCLUSIONS: Pantoprazole depresses cardiac contractility in vitro by depression of Ca2+ signaling and myofilament activity. In view of the extensive use of this agent, the effects should be evaluated in vivo.

Impacto de las actividades de uso racional del medicamento en médicos que nunca han recibido esta información / Impact of activities designed to improve the rational use of drugs on doctors who have never received the information before

Los médicos del Instituto Social de la Marina en España, a partir de enero de 2005, se incorporaron a trabajar para los sistemas de salud de la comunidad autónoma donde trabajaban, en el caso de Andalucía al Sistema Andaluz de Salud. Desde ese momento integraron en su trabajo los objetivos del nuevo sistema sanitario al que pertenecen, entre ellos el Uso Racional del Medicamento. El presente artículo explica la metodología seguida para difundir e inculcarles la importancia de una prescripción de calidad, siguiendo los indicadores establecidos por grupos de expertos. Tras catorce meses trabajando en su nueva plaza, se observa una disminución general del importe de un 9,77% y una mejora muy considerable en los indicadores cualitativos de prescripción, fundamentalmente en lo referente a la prescripción por principio activo, al empleo de omeprazol como antiulceroso y de simvastatina como hipolipemiante de elección

From January 2005 all doctors employed by the “Instituto Social de la Marina” in Spain were incorporated into the health services of the corresponding Autonomous Communities in which they worked: in the case of Andalousia, the “Sistema Andaluz de Salud”. Henceforward, they were required to assimilate all the objectives of the health system to which they now belonged, the Rational Use of Drugs included. This paper sets out the methodology employed to transmit the information and to highlight the importance of quality prescription in accordance with indicators established by groups of experts. After fourteen months in their new positions, an overall decrease in cost of 9.77% has been noted, along with a very appreciable improvement in quality prescription indicators, primarily with regard to active ingredient prescription, the use of Omeprazol in the treatment of ulcers and of Simvastatin as the drug of choice in the treatment of hyperlipidaemias

Utilizing adsorbed proteoliposomes trapped in a non-ruptured state on SiO2 for amplified detection of membrane proteins.

The quartz crystal microbalance with dissipation (QCM-D) technique was used to monitor the formation of supported phospholipid bilayers (SPBs) on SiO2 using proteoliposomes with reconstituted proton translocating nicotinamide nucleotide transhydrogenase (TH). Exposure of the surface to such proteoliposomes creates a lipid film composed of a mixture of proteolipid bilayers and adsorbed non-ruptured proteoliposomes, where the fraction of the latter is reduced if the TH-liposomes are pretreated with trypsin to remove the water soluble domains of TH [Langmuir 19 (2003) 842]. In the present work, the latter study is complemented by investigating the influence of trypsin treatment of the mixed adlayer (proteolipid bilayer + non-ruptured proteoliposomes) after adsorption on the surface. This demonstrates how trypsin-cleavage induced rupture of adsorbed TH-liposomes can be utilized to detect the presence of less than 0.04 pmol/cm2 of immobilized TH.

Determination of pantoprazole by adsorptive stripping voltammetry at carbon paste electrode.

A voltammetric method was described for the determination of pantoprazole by differential-pulse adsorptive stripping voltammetry at a carbon paste electrode. Accumulation of pantoprazole was found to be optimized in Britton-Robinson buffer (0.04 M, pH 4.0) solution following 5 min accumulation time at open circuit condition. Under optimized conditions, the current showed a linear dependence with concentration in the range 1.0 x 10(-7)-1.0 x 10(-5) M. The detection limit was 2.0 x 10(-8) M. The method was applied successfully for the analysis of pantoprazole in tablet dosage form. The results of accuracy and precision were comparable to those obtained by spectrophotometry.

Sodium and chloride transport in soft water and hard water acclimated zebrafish (Danio rerio).

While the zebrafish is commonly used for studies of developmental biology and toxicology, very little is known about their osmoregulatory physiology. The present investigation of Na(+) and Cl(-) transport revealed that the zebrafish is able to tolerate extremely low ambient ion concentrations and that this is achieved at least in part by a greatly enhanced apparent uptake capacity and affinity for both ions. Zebrafish maintain plasma and whole body electrolyte concentrations similar to most other freshwater teleosts even in deionized water containing only 35 microM NaCl, i.e soft water. We recorded an extremely low transport affinity constant (K(m)) of 8+/-1 microM for the active uptake of Cl(-) in soft water acclimated fish, while other transport kinetic parameters were in agreement with reports for other freshwater organisms. While both Na(+) and Cl(-) uptake in soft water clearly depends on apical proton pump activity, changes in abundance and possibly localization of this protein did not appear to contribute to soft water acclimation. Active Cl(-) uptake was strongly dependent on branchial carbonic anhydrase (CA) activity regardless of water type, while the response of Na(+) transport to a CA inhibitor was more variable. Differential response of Na(+) uptake to amiloride depending on acclimation medium suggests that different Na(+) transport mechanisms are employed by zebrafish acclimated to soft and hard water.

Molecular cloning and characterization of Atp6n1b: a novel fourth murine vacuolar H+-ATPase a-subunit gene.

The 116-kDa a-subunit of the vacuolar proton pump (H(+)-ATPase) exists as several isoforms encoded by different genes and with different patterns of tissue expression. Its function within the multisubunit pump complex has yet to be elucidated. To date, three isoforms have been identified in mouse (designated a1-a3). We now report the cloning and characterization of Atp6n1b, encoding a novel fourth murine isoform (a4). Murine a4 has 833 residues and shows 85% amino acid identity to the human kidney-specific ATP6N1B protein in which loss-of-function alterations cause autosomal recessive distal renal tubular acidosis. The human and murine genes have similar genomic organization; furthermore, Atp6n1b maps to a region of mouse chromosome 6 that is syntenic with the segment of human 7q33-34 containing ATP6N1B. Together these findings establish the two genes as orthologs. The mouse a4 protein is 61, 52, and 47% identical to a1, a2, and a3, respectively. Phylogenetic analysis confirms that among vertebrates there are four a-subunit families, with a4 most resembling a1. Northern blot analysis of Atp6n1b reveals a 3.7-kilobase a4 transcript in kidney but not other major organs, and a reverse transcription polymerase chain reaction in 12 mouse tissues detects expression in kidney alone. Immunofluorescence studies in murine kidney demonstrate high intensity a4 staining at the surface of intercalated cells, with additional expression in the proximal tubule (not previously reported in human kidney). Similar apical a4 immunostaining is also present in male genital tissue. Identification of this novel murine kidney-enriched 116-kDa a-subunit provides a molecular tool for investigation of the currently unknown role of this protein, which is essential for proper function of the apical renal vacuolar H(+)-ATPase in man.

Learning from hydrogenases: location of a proton pump and of a second FMN in bovine NADH--ubiquinone oxidoreductase (Complex I).

Hydrogenases have clear evolutionary links to the much more complex NADH-ubiquinone oxidoreductases (Complex I). Certain membrane-bound [NiFe]-hydrogenases presumably pump protons. From a detailed comparison of hydrogenases and Complex I, it is concluded here that the TYKY subunit in these enzymes is a special 2[4Fe-4S] ferredoxin, which functions as the electrical driving unit for a proton pump. The comparison further revealed that the flavodoxin fold from [NiFe]-hydrogenases is presumably conserved in the PSST subunit of Complex I. It is proposed that bovine Complex I and the soluble NAD(+)-reducing hydrogenase from Ralstonia eutropha each contain a second FMN group.

Flow cytometry of apoptotic cell death.

The term apoptosis or programmed cell death defines a genetically encoded cell death program, which is morphologically and biochemically distinct from necrosis or accidental cell death. The characteristic morphological signs of apoptosis (cellular shrinkage, membrane blebbing, nuclear condensation and fragmentation) are the final results of a complex biochemical cascade of events which is an integral part of physiological homeostasis. Techniques designed to identify, quantitate and characterize apoptosis are numerous, but flow cytometry (FCM) remains the methodology of choice to study the apoptotic cascade in relation to cell type, trigger and time. This review outlines the main stages of the apoptotic cascade together with current FCM methods. All FCM apoptosis assays described have a solid experimental basis and have been used successfully in basic research on molecular and biochemical mechanisms of apoptosis. In various clinical settings the ability to follow the apoptotic process in patient samples may offer the rationale for optimal treatment schedules.

NBC3 expression in rabbit collecting duct: colocalization with vacuolar H+-ATPase.

We have recently cloned and characterized a unique sodium bicarbonate cotransporter, NBC3, which unlike other members of the NBC family, is ethylisopropylamiloride (EIPA) inhibitable, DIDS insensitive, and electroneutral (A. Pushkin, N. Abuladze, I. Lee, D. Newman, J. Hwang, and I. Kurtz. J. Biol. Chem. 274: 16569-16575, 1999). In the present study, a specific polyclonal antipeptide COOH-terminal antibody, NBC3-C1, was generated and used to determine the pattern of NBC3 protein expression in rabbit kidney. A major band of approximately 200 kDa was detected on immunoblots of rabbit kidney. Immunocytochemistry of rabbit kidney frozen sections revealed specific staining of the apical membrane of intercalated cells in both the cortical and outer medullary collecting ducts. The pattern of NBC3 protein expression in the collecting duct was nearly identical to the same sections stained with an antibody against the vacuolar H+-ATPase 31-kDa subunit. In addition, the NBC3-C1 antibody coimmunoprecipitated the vacuolar H+-ATPase 31-kDa subunit. Functional studies in outer medullary collecting ducts (inner stripe) showed that type A intercalated cells have an apical Na+-dependent base transporter that is EIPA inhibitable and DIDS insensitive. The data suggest that NBC3 participates in H+/base transport in the collecting duct. The close association of NBC3 and the vacuolar H+-ATPase in type A intercalated cells suggests a potential structural/functional interaction between the two transporters.

Distribution of V-ATPase in rat salivary glands.

Using immunohistochemistry we have investigated the presence and cellular distribution of the 31-kDa subunit of vacuolar-type H+-ATPase (V-ATPase) in secretory endpieces and the duct system of rat major salivary glands. In all three salivary glands studied the 31-kDa subunit of V-ATPase was not expressed in secretory endpieces. In rat parotid gland V-ATPase was luminally located in main excretory and striated duct cells. In contrast, both rat submandibular and sublingual glands showed a diffuse intracellular V-ATPase distribution. The differences in V-ATPase immunolocalization in rat salivary glands probably reflect the structural heterogeneity of the different glands. The data also suggest that the duct systems of major salivary glands may modify the H+ and HCO3- concentration of the final saliva in different ways.

Medical treatment of metastasizing carcinoid tumors.

Long-acting somatostatin analogs, such as octreotide, comprise the therapeutic modality of choice for the symptomatic relief of flush and diarrhea in patients with carcinoid syndrome. The sequelae of gastric acid hypersecretion in patients with gastrin-producing duodenal carcinoids (gastrinoma) are perfectly controlled by proton pump inhibitors. Antiproliferative medical strategies to control the growth of metastatic carcinoid tumors include long-acting somatostatin analogs, interferon alpha, and the combination of the two. However, the success rate is less than 50%, and it is questionable whether true tumor regression can be expected. Controlled prospective studies are mandatory to address the question whether interferon or somatostatin analogs or the combination of the two should be used as first-line medical strategies and if hepatic artery embolization in patients with liver metastases should be performed before beginning medical therapy. Chemotherapy, including etoposide and cisplatin, has been shown to be effective only for purely differentiated neuroendocrine carcinomas and not for slowly growing carcinoids.

The pegs on the decorated tubules of the contractile vacuole complex of Paramecium are proton pumps.

Our previous study has shown that the decorated tubules (collectively known as the decorated spongiome) of the contractile vacuole complex (CVC) in Paramecium are the site of fluid segregation, as the binding of microinjected monoclonal antibody (mAb) DS-1 to the tubules reduced the CVC's fluid output. In this study, we showed by immunogold labeling on cryosections that the antigenic sites for mAb DS-1 were located on the 15 nm 'pegs' protruding from the cytosolic surface of the decorated tubules. In immunofluorescence studies, both polyclonal antibodies against the subunits of the V-ATPase of Dictyostelium discoideum and against the 57 kDa B-subunit of the V-ATPase of chromaffin granules gave identical labeling patterns to that produced by mAb DS-1. On cryosections, all three antigens were located most consistently near or on the pegs of the decorated tubules. These data support the notion that the pegs on the membrane of the decorated tubules represent the V1 complex of a proton pump. Concanamycin B, a potent inhibitor of V-ATPase activity and of acidification of lysosomes and endosomes, strongly and reversibly inhibited fluid output from the CVC but had minimal effect on the integrity of the decorated spongiome as observed by immunofluorescence. Such inhibition suggests that a V-ATPase is intimately involved in fluid segregation. Exposing Paramecium to 12 degrees C or 1 degrees C for 30 minutes resulted in the dissociation of the decorated tubules from the smooth spongiome that borders the collecting canals; thus the DS-1-reactive A4 antigen, the 75 kDa and 66 kDa antigens were all found dispersed in the cytosol.(ABSTRACT TRUNCATED AT 250 WORDS)

Interference with the endosomal acidification by a monoclonal antibody directed toward the 116 (100)-kD subunit of the vacuolar type proton pump.

A monoclonal antibody (OSW2) was prepared by using human osteosarcoma cells. OSW2 was found to be directed toward the 116 (also called 100)- kD protein that uniquely associates to the vacuolar-type proton pump. The antibody specifically localized acidic membrane compartments that could be visualized with acridine orange in many types of human cells. It also reacted with the surface and was internalized along the endosomal pathway. Monitoring the endosome pH by using FITC-dextran and acridine orange suggested that the antibody interfered with low pH. Cell-free experiments indicated that the ATP-dependent acidification was inhibited in endosomes associated with OSW2. In contrast, the antibody gave little effect on the ATPase activity of the solubilized H+ pump. The internalization of OSW2 reduced infectivity of certain enveloped viruses (influenza, SFV, VSV) by 50 to 80%. Inhibition of viral fusion was directly demonstrated by monitoring the fate of octadecylrhodamine-labeled influenza virus fluorescence. These results indicate that the 116 (100)-kD protein is necessary for the control of pH. The antibody represents a novel probe for understanding the role of the endosomal compartments in cellular physiology.

Lipid composition and proton transport in Penicillium cyclopium and Ustilago maydis plasma membrane vesicles isolated by two-phase partitioning.

Plasma membranes have been isolated and purified from two species of fungi, Penicillium cyclopium and Ustilago maydis, using a two-phase aqueous polymer technique. The membranes were characterised using marker enzyme assays (e.g., vanadate-sensitive (Mg(2+)-K+)-ATPase and glucan synthetase II) and lipid composition (sterol enrichment, increased phosphatidylethanolamine/phosphatidylcholine ratio, and the absence of diphosphatidylglycerol). The proton-pumping activities of the plasma membrane-bound H(+)-ATPases from these species were compared. H(+)-ATPase activity was found to be greater in U. maydis than in P. cyclopium, which was attributed to differences in orientation of the plasma membrane vesicles. There was evidence to suggest the presence of redox chain activity in the plasma membranes of both species.