Characterization of a broad range antimicrobial substance from Bacillus cereus

AIMS: The aim of this research was to isolate and characterize an antimicrobial substance from the Bacillus cereus type strain ATCC 14579. METHODS AND RESULTS: A substance with antimicrobial activity was isolated from B. cereus ATCC 14579. The substance was produced during late exponential growth and well into the stationary phase with a maximum 9 h after inoculation. The inhibitory substance was purified by reverse-phase HPLC and shown to be highly active against closely related Bacillus spp. Clinically relevant species such as Staphylococcus aureus and Micrococcus luteus were also inhibited. The substance was characterized as a bacteriocin-like inhibitory substance (BLIS) with a molecular mass of ca 3.4 kDa. The BLIS was very heat stable, and sensitive only to pronase E and proteinase K. Antimicrobial activity was stable and high in the pH range of 2.0-9.0, and relatively unaffected by organic chemicals. CONCLUSIONS: An antimicrobial substance produced by the B. cereus type strain ATCC 14579 was characterized, with a wide spectrum of activity and the potential to be applied as a control agent against pathogenic bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study is the first report of a substance with antimicrobial activity from the B. cereus type strain.     

A novel multiplex PCR for the identification of Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus

AIM: To establish a simple multiplex polymerase chain reaction (PCR) that will identify Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus. METHODS AND RESULTS: A total of 429 Vibrio spp. from various origins were tested with the novel primers targeting toxR. The reverse primers were all designed to be species specific, while the forward primer was universal. The primers correctly identified all the V. parahaemolyticus, V. cholerae and V. vulnificus isolates tested. CONCLUSIONS: The toxR multiplex PCR works well when the initial colony morphology is known. If not, Vibrio alginolyticus might represent a diagnostic obstacle. SIGNIFICANCE AND IMPACT OF THE STUDY: The method provides a fast and reliable way of identifying the main Vibrio spp. involved in food-borne disease. The method could prove very useful for laboratories working with identification of these Vibrio spp.     

Isolation and preliminary characterization of bacteriocins produced by Vibrio vulnificus

AIMS: The aim of this work was to isolate bacteriocins from the environment that would be effective in neutralizing Vibrio vulnificus in seafood. METHODS AND RESULTS: Water samples from Wilmington (NC, USA) were plated to determine total viable counts and to isolate presumptive Vibrio spp. Isolates containing plasmids were checked for antimicrobial activity which was not due to lytic bacteriophage or small, non-specific molecules. Three bacteriocin producers were detected and their inhibitory spectra determined: IW1 inhibited few strains of V. vulnificus; BC1 inhibited several strains of V. vulnificus, V. cholerae and V. parahaemolyticus and BC2 inhibited all tested Vibrio spp., Plesiomonas shigelloides and Escherichia coli. Loss of inhibitory activity coincided with loss of the bacteriocinogenic plasmid. The bacteriocins were found to be between 1.3 and 9.0 kDa. IW1 was heat labile, while BC1 was moderately stable except at extreme temperatures. BC2 was very stable and maintained its activity when frozen, autoclaved or exposed to extreme pH values. CONCLUSIONS: Bacteriocins have been isolated from environmental isolates of V. vulnificus and V. cholerae. BC2, with its broad spectrum and stability, may be useful in neutralizing V. vulnificus. SIGNIFICANCE AND IMPACT OF THE STUDY: The results have significance in relation to reducing the occurrence of food poisoning caused by V. vulnificus.     

Environmental Vibrio parahaemolyticus DNA signatures validation

Insignia is a novel DNA computational system which uses highly efficient algorithms to compare bacterial genomes and to identify specific DNA signatures to distinguish a target bacterium, or group of bacteria, from all other known bacterial species. It is currently being validated using different bacterial groups, including Vibrio spp. In this study, the genomic analysis by Insignia was conducted on Vibrio parahaemolyticus, a halophilic gram-negative bacteria which constitutes a leading cause of seafood-borne disease. Insignia was used to identify 37 V. parahaemolyticus-specific signatures and to design PCR assays to validate the representative signature sequences by TaqMan essays. The 37 assays targeted loci distributed around the genome and detected genes coding for hypothetical proteins and for proteins involved in adhesion, starvation and virulence. A panel of V. parahaemolyticus environmental strains isolated from the North Adriatic Sea (Italy) and from the Black Sea (Georgia) was used to validate the selected signatures. The signature assays revealed both sensitive and specific and the method allowed a more accurate identification of the tested bacterial strains at the species level when compared to biochemical and PCR standard methods. Using Insignia, it was possible to distinguish two different groups among the strains previously identified as V. parahaemolyticus: most of the strains were included in a "V. parahaemolyticus-like group" showing nearly all of the signatures assayed while a small group of 10 strains contained only a few of the signatures tested. By sequencing the 16S rDNA of this latter group, it was confirmed that they were not V. parahaemolyticus but in fact belonged to other Vibrio species. No significant genome-wide differences were detected between the strains isolated in Italy and in Georgia though the very different geographical origin.     

Catecholamine-induced stimulation of growth in Vibrio species

AIMS: To evaluate the effect of norepinephrine (NE) and related compounds on the growth of bacteria, we have examined the effect of the neuroendocrine hormone NE and related compounds on the growth of Vibrio parahaemolyticus and other human-pathogenic Vibrio species (Vibrio cholerae, Vibrio vulnificus, and Vibrio mimicus). METHODS AND RESULTS: The effects on bacterial growth were examined using the serum-based medium and viable cells were counted using agar plates. We have shown that NE and its related compounds stimulate growth of V. parahaemolyticus in serum-based medium. This NE-induced growth stimulation was dependent upon the presence of transferrin. NE also stimulated growth of V. mimicus, but not V. cholerae and V. vulnificus. CONCLUSIONS: These results suggest that the Vibrio species differ in their ability to respond to NE. SIGNIFICANCE AND IMPACT OF THE STUDY: It is possible that NE and related compounds modulate the pathogenicity of V. parahaemolyticus and V. mimicus.     

Investigation of seven Vibrio virulence genes among Vibrio alginolyticus and Vibrio parahaemolyticus strains from the coastal mariculture systems in Guangdong, China

AIMS: To investigate the distribution of the virulence of two Vibrio species among different strains obtained from the mariculture systems on the coast of Guangdong in China and the correlation between the virulence strains and the virulence genes among Vibrio alginolyticus. METHODS: Besides three strains, 72 V. alginolyticus strains and seven Vibrio parahaemolyticus strains were examined by PCR or semi-nested PCR for the virulence genes (tlh, trh, tdh, toxR, toxRS, ctxA, VPI). Additionally, the virulence of 18 V. alginolyticus strains was tested. SIGNIFICANCE AND IMPACT OF THE STUDY: Virulence genes homologous to those in the V. parahaemolyticus and Vibrio cholerae are widely distributed among V. alginolyticus and V. parahaemolyticus in the coastal mariculture systems in Guangdong, China. Some of the V. alginolyticus strains are pathogenic to aquatic animals, and might have derived their virulence genes from V. parahaemolyticus or V. cholerae, representing a possible reservoir of these genes. However, there is no correlation between presence and absence of the virulence genes used to investigate V. alginolyticus and its virulent strains. In this report, we also show that tlh is distributed among V. alginolyticus.     

Effect of Hydrostatic Pressure on Growth and Viability of Vibrio parahaemolyticus

Six strains of marine bacteria, including three strains of Vibrio parahaemolyticus, two Vibrio spp isolated from coastal regions, and the deep ocean isolate Pseudomonas bathycetes, were examined for ability to survive and grow at deep ocean hydrostatic pressures. V. parahaemolyticus and the coastal Vibrio spp. were unable to survive or grow at 200, 400, 600, 800, or 1,000 atm of pressure. In contrast, the deep ocean isolate P. bathycetes was capable of survival and growth at these pressures. The evidence strongly supports the neritic or estuarine origin and habitat for V. parahaemolyticus.     

Characteristics of bacteriocin-like inhibitory substances from dochi-isolated Enterococcus faecium D081821 and D081833

AIMS: To characterize bacteriocin-like inhibitory substances (BLIS) from two dochi-isolated Enterococcus faecium. METHODS AND RESULTS: Enterococcus faecium D081821 and D081833 were isolated from dochi (a traditional fermented food in Taiwan) and found to produce BLIS with inhibitory activities against Listeria monocytogenes, Clostridium perfringens, and Bacillus cereus. Strains D081821 and D081833 showed different growth temperatures and their BLIS showed different sensitivities to heat, proteolytic enzymes, and antibacterial spectra. Both BLIS were collected, partially purified, and analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE showed that both partially purified BLIS were approximately 3.0 kDa in size. CONCLUSIONS: These results indicate that E. faecium D081821 and D081833 produce different BLIS with strong antibacterial actions against the tested pathogenic bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study suggest that two different BLIS from dochi-isolated lactic acid bacteria have potential for use as food preservatives.     

用基于TaqMan探针的Real-time PCR技术定量检测副溶血弧菌

副溶血弧菌是一种引起食源性疾病的重要病原菌,传统的鉴定方法费时费力且容易出现假阴性,建立一种定量检测副溶血弧菌基因的方法尤为重要。根据GenBank公布的副溶血弧菌的gyrB基因序列设计一对引物和TaqMan探针,建立了基于TaqMan探针的RealtimePCR方法。通过对9种细菌(12株菌株)的DNA进行扩增,结果所有4株副溶血弧菌均可产生扩增曲线,其他8株非副溶血弧菌均不产生扩增曲线,证明了引物和探针具有很高的特异性。细菌纯培养物品和人工布菌的检测敏感度分别为1CFUPCR反应体系和10CFUPCR反应体系,相关系数均为0.99(r2=0.99),整个试验可在1h内完成。建立的方法可用于海产品中副溶血弧菌的快速定量检测。     

Comparison of different primers for rapid detection of Vibrio parahaemolyticus using the polymerase chain reaction

AIMS: The aim of this study was to compare different primers for rapid and effective detection of Vibrio parahaemolyticus by polymerase chain reaction (PCR). METHODS AND RESULTS: A total of four pairs of primers, three previously published and one based on a newly developed V. parahaemolyticus metalloprotease (vpm) gene, have been assayed for PCR detection of V. parahaemolyticus. They have been tested for specificity and sensitivity on a total of 101 strains including reference and environment isolates belonging to V. parahaemolyticus and other species in Vibrio. Of the four sets of primers tested, the one designed on the basis of the metalloprotease gene (675 bp) gave optimal results with bacterial strains examined as they only amplified the specific fragment in strains that had been genetically and biochemically assessed as V. parahaemolyticus and the limit of detection was 4 pg of purified target DNA. CONCLUSIONS: The primers designed on the metalloprotease gene gave optimal results for specific, sensitive and rapid detection of V. parahaemolyticus by PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR amplification with the optimal primer set VPM1/VPM2 could facilitate the rapid diagnosis and surveillance of potentially pathogenic strains of V. parahaemolyticus and reduce food-borne illness.     

霍乱弧菌和副溶血弧菌分离株的gyrB基因系统发育分析

依据gyrB基因部分编码序列构建系统发育树以分类和鉴别霍乱弧菌和副溶血弧菌,并探讨其种系发生关系。扩增并测序13株霍乱弧菌、8株副溶血弧菌、2株嗜水气单胞菌及1株类志贺邻单胞菌的gyrB基因(编码DNA促旋酶B亚单位)序列,并采用距离法与最大似然法构建系统发育树。两种方法所构建的树结构完全一致,霍乱弧菌、副溶血弧菌、嗜水气单胞菌及类志贺邻单胞菌各自形成一个独立的簇。其中,霍乱肠毒素基因(ctxA)阳性的霍乱弧菌(8株O139群与2株O1群ElTor型)聚类成一分枝;3株副溶血弧菌临床株(1株2002年流行株,2株2004年分离株)与1日本菌株及2001年1株自环境分离的毒力株聚类。系统发育分析靶分子gyrB基因可以良好区分上述4种常见病原菌。产毒O139群霍乱弧菌与产毒O1群ElTor型霍乱弧菌关系密切。副溶血弧菌环境毒力株与本地区临床主要流行株在系统发育关系上较为接近,可能是潜在的致病菌。     

The ability of two different Vibrio spp. bacteriophages to infect Vibrio harveyi, Vibrio cholerae and Vibrio mimicus

AIMS: To determine the host range of the Vibrio harveyi myovirus-like bacteriophage (VHML) and the cholera toxin conversion bacteriophage (CTX Phi) within a range of Vibrio cholerae and V. mimicus and V. harveyi, V. cholerae and V. mimicus isolates respectively. METHODS AND RESULTS: Three V. harveyi, eight V. cholerae and five V. mimicus isolates were incubated with VHML and CTX Phi. Polymerase chain reaction (PCR) was used to determine the presence of VHML and CTX Phi in infected isolates. We demonstrated that it was possible to infect one isolate of V. cholerae (isolate ACM #2773/ATCC #14035) with VHML. This isolate successfully incorporated VHML into its genome as evident by positive PCR amplification of the sequence coding part of the tail sheath of VHML. Attempts to infect all other V. cholerae and V. mimicus isolates with VHML were unsuccessful. Attempts to infect V. cholerae non-01, V. harveyi and V. mimicus isolates with CTX Phi were unsuccessful. CONCLUSIONS: Bacteriophage infection is limited by bacteriophage-exclusion systems operating within bacterial strains and these systems appear to be highly selective. One system may allow the co-existence of one bacteriophage while excluding another. VHML appears to have a narrow host range which may be related to a common receptor protein in such strains. The lack of the vibrio pathogenicity island bacteriophage (VPI Phi) in the isolates used in this study may explain why infections with CTX Phi were unsuccessful. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study has demonstrated that Vibrio spp. bacteriophages may infect other Vibrio spp.     

Application of polymerase chain reaction for detection of Vibrio parahaemolyticus associated with tropical seafoods and coastal environment

AIMS: To study the incidence of Vibrio parahaemolyticus in seafoods, water and sediment by molecular techniques vs conventional microbiological methods. METHODS AND RESULTS: Of 86 samples analysed, 28 recorded positive for V. parahaemolyticus by conventional microbiological method, while 53 were positive by the toxR-targeted PCR, performed directly on enrichment broth lysates. While one sample of molluscan shellfish was positive for tdh gene, trh gene was detected in three enrichment broths of molluscan shellfish. CONCLUSIONS: Direct application of PCR to enrichment broths will be useful for the rapid and sensitive detection of potentially pathogenic strains of V. parahemolyticus in seafoods. SIGNIFICANCE AND IMPACT OF THE STUDY: Vibrio parahaemolyticus is an important human pathogen responsible for food-borne gastroenteritis world-wide. As, both pathogenic and non-pathogenic strains of V. parahaemolyticus exist in the seafood, application of PCR specific for the virulence genes (tdh & trh) will help in detection of pathogenic strains of V. parahaemolyticus and consequently reduce the risk of food-borne illness.     

Detection and identification of Vibrio parahaemolyticus by multiplex PCR and DNA-DNA hybridization on a microarray

In this paper,we developed a rapid and accurate method for the detection of Vibrio parahaemolyticus strains,using multiplex PCR and DNA-DNA hybridization.Multiplex PCR was used to simultaneously amplify three diagnostic genes(tlh,tdh and fia)that serve as molecular markers of V.parahaemolyticus.Biotinylated PCR products were hybridized to primers immobilized on a microarray,and detected by chemiluminesce with avidin-conjugated alkaline phosphatase.With this method,forty-five samples were tested.Eight known virulent strains (tlh+/tdh+/fia+)and four known avirulent strains(tlh+/tdh-/fla+)of the V.parahaemolyttcus were successtuny aetectea,ana no non-spectnc hybridization and cross-hybridization reaction were found from fifteen closely-related strains(tin-/tdh-/fta+)or the Vibrio spp.In addition,all the other eighteen strains of non-Vibrio bacteria(tlh-/tdh-/fla-)gave negative results.The DNA microarray successfully distinguished V.parahaemolyticus from other Vibrio spp.The results demonstrated that this was an efficient and robust method for identifying virulent strains of V.parahaemolyticus.     

Sensitivity of Vibrio and Aeromonas to antibiotics]

The antibiotic sensitivity of 696 cultures belonging to the family Vibrionaceae (V. cholerae O1, V. cholerae non-O1, V. albensis, V. parahaemolyticus, V. alginolyticus and Aeromonas spp.) was studied and general regularities of the antibiotic sensitivity were shown: a high sensitivity to broad-spectrum antibiotics (tetracycline and chloramphenicol) and a low sensitivity to ++beta lactams (carbenicillin and ampicillin). The comparative examinations revealed similarity in the antibioticograms of V. cholerae O1 (el Tor++), V. cholerae non-O1 and V. albensis, especially the latter two groups, as well as the tested halophilic Vibrio cultures by the range of the MICs, Mo, Me and the nature of the antibiotic resistance. Cultures of V. cholerae and luminescent Vibrio tended to preserve a high sensitivity. High resistance levels were noted in the halophilic Vibrio and Aeromonas cultures. No significant differences in the sensitivity of the strains of various origin (from man and environmental objects) were detected. However, several more resistant strains were isolated from the environmental objects.     

Effect of Mg(2+) ion in protein secretion by magnesium-resistant strains of Pseudomonas aeruginosa and Vibrio parahaemolyticus isolated from the coastal water of Haldia port

A rapidly growing industrial complex including oil refineries and chemical industries has developed around the coastal area of Haldia port in the district of Midnapore, West Bengal, India. The coastal water is highly polluted with industrial wastes along with petroleum hydrocarbons. The bacteria isolated from the different sites of the coastal waters were Escherichia coli, Alcaligenes, Acinetobacter, Klebsiella spp., Micrococcus spp., Vibrio spp., Pseudomonas aeruginosa and Vibrio parahaemolyticus. The salinity of the water during the time of collection of samples around the port area was 8. 2 ppt. Among the isolated organisms, only two isolates, P. aeruginosa and V. parahaemolyticus, showed growth at 300 mM Mg(2+) ion concentration. However, a 3 mM Mg(2+) concentration was detected in the coastal water whereas other metal ion concentrations were less than 3x10(-5) mM. Resistance to Mg(2+) (300 mM) was determined by a 5.5-kb plasmid. A large amount of a 40-kDa outer membrane protein, which was highly soluble in 1 M MgCl(2), was isolated from both V. parahaemolyticus and P. aeruginosa. The secretion of proteins in the culture supernatant of V. parahaemolyticus was highly increased when the cells were grown in the presence of 300 mM Mg(2+), whereas very low secretion was observed in the same concentration of Mg(2+) in the case of P. aeruginosa. Mg(2+) may act as a specific release factor in protein secretion by V. parahaemolyticus strains.     

Vibrios isolated from the cultured manila clam (Ruditapes philippinarum): numerical taxonomy and antibacterial activities

AIMS: A numerical taxonomic study of halophilic Vibrio isolated from healthy and brown ring disease (BRD) affected manila clams (Ruditapes philippinarum), harvested from the Atlantic coast of south-western Spain, was performed. METHODS AND RESULTS: Characterization of 123 presumptive Vibrio spp. was carried out using 94 phenotypic tests. Simple matching and Jaccard similarity coefficients were used for numerical analysis. Cluster analysis by the unweighted pair group method with arithmetic averages yielded 15 phena defined at 0.81 similarity. Large phena corresponded to Vibrio tubiashii, V. splendidus biotype I and V. harveyi (phena 1, 5 and 9, respectively). The species V.splendidus biotype II, V. natriegens, V. mediterranei and V. alginolyticus were also represented. The inhibitory effect of diffusible extracellular products of the isolates against 27 strains of V.tapetis, the aetiological agent of BRD, was also investigated. Only five V. tubiashii isolates inhibited the growth of V. tapetis strains. The antimicrobial effect was inhibited by heating and depended on the culture medium. CONCLUSIONS: The main Vibrio species associated with manila clams were V. tubiashii, V.spendidus and V. harveyi. The antagonistic relationship established between V. tapetis and the Vibrio spp. clam microbiota may explain the failure of isolation in plating medium of V.tapetis from BRD-affected clams on the south Atlantic coast of Spain. SIGNIFICANCE AND IMPACT OF THE STUDY: Some of the strains isolated from manila clams correspond to agarolytic strains that constitute phenon 7 and they do not fit into any of the currently described Vibrio species.     

Molecular typing of Vibrio parahaemolyticus strains isolated from the Philippines by PCR-based methods

AIM: The main aim of the present study was to use three PCR-based techniques for the analysis of genetic variability among Vibrio parahaemolyticus strains isolated from the Philippines. METHODS AND RESULTS: Seventeen strains of V. parahaemolyticus isolated from shrimps (Penaeus monodon) and from the environments where these shrimps are being cultivated were analysed by random amplified polymorphic DNA PCR (RAPD-PCR), enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) and repetitive extragenic palindromic PCR (REP-PCR). The results of this work have demonstrated genetic variability within the V. parahaemolyticus strains that were isolated from the Philippines. In addition, RAPD, ERIC and REP-PCR are suitable rapid typing methods for V. parahaemolyticus. All three methods have good discriminative ability and can be used as a rapid means of comparing V. parahaemolyticus strains for epidemiological investigation. Based on the results of this study, we could say that REP-PCR is inferior to RAPD and ERIC-PCR owing to the fact that it is less reproducible. Moreover, the REP-PCR analysis yielded a relatively small number of products. This may suggests that the REP sequences may not be widely distributed in the V. parahaemolyticus genome. CONCLUSIONS: Genetic variability within V. parahaemolyticus strains isolated in the Philippines has been demonstrated. The presence of ERIC and REP sequences in the genome of this bacterial species was confirmed. SIGNIFICANCE AND IMPACT OF THE STUDY: The RAPD, ERIC and REP-PCR techniques are useful methods for molecular typing of V. parahaemolyticus strains. To our knowledge this is the first study of this kind carried out on V. parahaemolyticus strains isolated from the Philippines.     

Inhibition of Vancomycin and High-Level Aminoglycoside-Resistant Enterococci Strains and Listeria monocytogenes by Bacteriocin-Like Substance Produced by Enterococcus faecium E86

Three hundred and thirty nine lactic bacteria strains isolated from food samples were screened for antimicrobial activity. Only one strain isolated from meat pie and identified as Enterococcus faecium produced a bacteriocin-like inhibitory substance (BLIS) showing activity against Enterococcus, Leuconostoc, Lactobacillus, Listeria, Corynebacterium and Staphylococcus aureus. The BLIS produced was resistant to acid and alkali treatment and 121oC for 15 min. The addition of BLIS in BHI contaminated with Listeria monocytogenes decreased the contamination in 4.8 log cycles in 24 h. The inhibition of listeria was also obtained in milk. Forty multiresistant enterococci strains were inhibited in the well-diffusion test. Two vancomycin resistant strains tested in liquid with BLIS were also inhibited. The BLIS producer showed no pathogenicity marker.     

Bactericidal effect of lactoferrin and lactoferrin chimera against halophilic Vibrio parahaemolyticus

Infections caused by Vibrio parahaemolyticus, an halophilic member of the genus Vibrio, have increased globally in the last 5 years. Diarrhea caused by V. parahaemolyticus results from eating raw or undercooked seafood. The aim of this work was to investigate whether lactoferrin and some lactoferrin-peptides have bactericidal activity against Vibrio parahaemolyticus ATCC 17802, the pandemic strain O3:K6, and the multidrug resistant isolate 727, as well as against Vibrio cholerae strains O1 and non-O1. Whereas both peptides lactoferricin (17-30) and lactoferrampin (265-284) did not have bactericidal activity, 40 microM of lactoferrin chimera (a fusion of the two peptides) inhibited the growth of all Vibrio tested to the same extent as the antibiotic gentamicin. The cidal effect of LFchimera showed a clear concentration response in contrast to bovine lactoferrin which showed higher inhibition at 10 microM than at 40 microM. FITC-labeled LFchimera bound to the bacterial membranes. Moreover LFchimera permeabilized bacterial cells and membranes were seriously damaged. Finally, in experiments with the multidrug resistant isolate 727, sub-lethal doses of LFchimera strongly reduced the concentrations of ampicillin, gentamicin or kanamicin needed to reach more than 95% growth inhibition, suggesting synergistic effects. These data indicate that LFchimera is a potential candidate to combat the multidrug resistant pathogenic Vibrio species.