Hemolytic activity and siderophore production in different Aeromonas species isolated from fish

The hemolytic activity and siderophore production of several strains of motile aeromonads were determined. The hemolytic activity of Aeromonas caviae and Aeromonas eucrenophila was enhanced after trypsinization of the samples. The enhancement of hemolysis was observed in strains that carried an aerolysin-like gene, detected by a PCR procedure. Siderophore production was demonstrated in all but one strain of Aeromonas jandaei. No apparent relationship was observed between the presence of plasmid DNA and hemolysis or siderophore production.     

Hemolytic Factor Production by Clinical Isolates of Candida Species

Most cases of fungal bloodstream infections (BIs) are attributed to Candida albicans; however, non-Candida albicans Candida species have recently been identified as common pathogens. Although hemolytic factor is known to be putative virulence factor contributing to pathogenicity in Candida species, its production is poorly evaluated. The present study was undertaken to analyze the production of hemolytic factor by C. albicans (10), C. tropicalis (13), and C. parapsilosis (8) isolates associated with BIs. Data of hemolysis zones on plate assay revealed that the majority of C. albicans isolates produced mild hemolytic activity whereas the majority of C. tropicalis produced strong activity. None of the tested C. parapsilosis isolates exhibited hemolysis on plate assay. We also evaluated the hemolytic activity in the cell-free broth. There were no significant differences (P > 0.05) in the secreted hemolytic activity among intra-species isolates. Different levels of secreted hemolytic factor were observed for Candida species, where C. tropicalis exhibited the highest production of hemolytic factor (P < 0.05) followed by C. albicans and C. parapsilosis. Inhibition of hemolysis (up to 89.12 %) from culture supernatant, following incubation with the lectin Concanavalin A (Con A), was observed for all three Candida species. This finding suggests that the secreted hemolytic factor of C. tropicalis and C. parapsilosis may be a mannoprotein, similar to that described for C. albicans.     

Quantification of Siderophore and Hemolysin from Stachybotrys chartarum Strains, Including a Strain Isolated from the Lung of a Child with Pulmonary Hemorrhage and Hemosiderosis

A strain of Stachybotrys chartarum was recently isolated from the lung of a pulmonary hemorrhage and hemosiderosis (PH) patient in Texas (designated the Houston strain). This is the first time that S. chartarum has been isolated from the lung of a PH patient. In this study, the Houston strain and 10 strains of S. chartarum isolated from case (n = 5) or control (n = 5) homes in Cleveland were analyzed for hemolytic activity, siderophore production, and relatedness as measured by random amplified polymorphic DNA analysis.     

Mutational analysis of TlyA from Brachyspira hampsonii reveals two key residues conserved in pathogenic bacteria responsible for oligomerization and hemolytic activity

BackgroundTlyA proteins are expressed in a variety of pathogenic bacteria and possess dual hemolytic and ribosomal RNA methyltransferase functions. While the mechanism of TlyA mediated rRNA methylation is well understood, relatively little is known about the mechanism of TlyA induced hemolysis.MethodsTlyA protein from the pig pathogen Brachyspira hampsonii was heterologously expressed and purified from an E. coli host. Hemolytic activity and rRNA methylation were assessed in vitro. Site-directed mutagenesis was used to mutate amino acids believed to be involved in TlyA mediated hemolysis.ResultsPurified TlyA-His protein exhibited both hemolytic and rRNA methyltransferase activities in vitro, with partial inhibition of hemolysis observed under reducing conditions. Mutation of cysteine 80 to alanine impaired hemolytic activity. A C27A/C93A mutant was capable of dimerizing under non-reducing conditions, indicating that a C80-C80 disulfide bond is involved in TlyA oligomerization. A mutation conserved in several avirulent Brachyspira species (S9K) completely abolished hemolytic activity of TlyA. This loss of activity was attributed to impaired oligomerization in the S9K mutant, as assessed by ITC and size-exclusion chromatography experiments.ConclusionsOligomeric assembly and hemolytic activity of TlyA from Brachyspira hampsonii is dependent on the formation of an intermolecular C80-C80 disulfide bond and noncovalent interactions involving serine 9. The conservation of these amino acids in TlyA proteins from pathogenic bacteria suggests a correlation between tlyA gene mutations and bacterial virulence.General significanceOur results further elucidate the mechanisms underlying TlyA mediated hemolysis and provide evidence of a conserved mechanism of oligomerization for TlyA family proteins.     

Quantitative Metabolomics Reveals an Epigenetic Blueprint for Iron Acquisition in Uropathogenic Escherichia coli

Bacterial pathogens are frequently distinguished by the presence of acquired genes associated with iron acquisition. The presence of specific siderophore receptor genes, however, does not reliably predict activity of the complex protein assemblies involved in synthesis and transport of these secondary metabolites. Here, we have developed a novel quantitative metabolomic approach based on stable isotope dilution to compare the complement of siderophores produced by Escherichia coli strains associated with intestinal colonization or urinary tract disease. Because uropathogenic E. coli are believed to reside in the gut microbiome prior to infection, we compared siderophore production between urinary and rectal isolates within individual patients with recurrent UTI. While all strains produced enterobactin, strong preferential expression of the siderophores yersiniabactin and salmochelin was observed among urinary strains. Conventional PCR genotyping of siderophore receptors was often insensitive to these differences. A linearized enterobactin siderophore was also identified as a product of strains with an active salmochelin gene cluster. These findings argue that qualitative and quantitative epi-genetic optimization occurs in the E. coli secondary metabolome among human uropathogens. Because the virulence-associated biosynthetic pathways are distinct from those associated with rectal colonization, these results suggest strategies for virulence-targeted therapies.     

Bifidobacteria strains isolated from stools of iron deficient infants can efficiently sequester iron

Background

Bifidobacteria is one of the major gut commensal groups found in infants. Their colonization is commonly associated with beneficial effects to the host through mechanisms like niche occupation and nutrient competition against pathogenic bacteria. Iron is an essential element necessary for most microorganisms, including bifidobacteria and efficient competition for this micronutrient is linked to proliferation and persistence. For this research we hypothesized that bifidobacteria in the gut of iron deficient infants can efficiently sequester iron. The aim of the present study was to isolate bifidobacteria in fecal samples of iron deficient Kenyan infants and to characterize siderophore production and iron internalization capacity.

Results

Fifty-six bifidobacterial strains were isolated by streaking twenty-eight stool samples from Kenyan infants, in enrichment media. To target strains with high iron sequestration mechanisms, a strong iron chelator 2,2-dipyridyl was supplemented to the agar media. Bifidobacterial isolates were first identified to species level by 16S rRNA sequencing, yielding B. bifidum (19 isolates), B. longum (15), B. breve (11), B. kashiwanohense (7), B. pseudolongum (3) and B. pseudocatenulatum (1). While most isolated bifidobacterial species are commonly encountered in the infantile gut, B. kashiwanohense was not frequently reported in infant feces. Thirty strains from culture collections and 56 isolates were characterized for their siderophore production, tested by the CAS assay. Siderophore activity ranged from 3 to 89% siderophore units, with 35 strains (41%) exhibiting high siderophore activity, and 31 (36%) and 20 (23%) showing intermediate or low activity. The amount of internalized iron of 60 bifidobacteria strains selected for their siderophore activity, was in a broad range from 8 to118 μM Fe. Four strains, B. pseudolongum PV8-2, B. kashiwanohense PV20-2, B. bifidum PV28-2a and B. longum PV5-1 isolated from infant stool samples were selected for both high siderophore activity and iron internalization.

Conclusions

A broad diversity of bifidobacteria were isolated in infant stools using iron limited conditions, with some strains exhibiting high iron sequestration properties. The ability of bifidobacteria to efficiently utilize iron sequestration mechanism such as siderophore production and iron internalization may confer an ecological advantage and be the basis for enhanced competition against enteropathogens.

Electronic supplementary material

The online version of this article (doi:10.1186/s12866-014-0334-z) contains supplementary material, which is available to authorized users.     

Hemolytic activity of Serratia marcescens

A cell-bound hemolytic activity was found in several strains of Serratia marcescens. One Serratia cell per ten erythrocytes was sufficient to cause complete lysis of human erythrocytes within 2 h in the liquid assay. The hemolytic activity resided in the membrane fraction and could be inactivated by incubating cells with proteases. The hemolytic activity was greatly enhanced in actively metabolizing Serratia cells and was partially controlled by the iron supply. Hemolysis was accompanied by degradation of erythrocyte membrane proteins (band 3 and 6, glycophorin) and was independent of the blood group. The exoprotease secreted by S. marcescens in large amounts was not involved in hemolysis. Comparison with various hemolytic strains of Escherichia coli showed that hemolysis of erythrocytes was more pronounced with S. marcescens than with E. coli. In contrast to hemolysis by E. coli, lysis of erythrocytes by S. marcescens was not enhanced by Ca²⁺ ions.Dedicated to Professor Dr. Gerhart Drews on the occasion of his 60th birthday     

How much do we know about hemolytic capability of pathogenic <Emphasis Type="Italic">Candida</Emphasis> species?

Hemolytic factor production by pathogenic Candida species is considered an important attribute in promoting survival within the mammal host through the ability to assimilate iron from the hemoglobin-heme group. Hemolytic capability has been evaluated for Candida species based on hemolysis zones on plate assay, analysis of hemolytic activity in liquid culture medium, and hemolysis from cell-free culture broth. The production of hemolytic factor is variable among Candida species, where C. parapsilosis is the less hemolytic species. In general, no intraspecies differences in beta-hemolytic activities are found among isolates belonging to C. albicans, C. glabrata, C. krusei, C. tropicalis, and C. parapsilosis. The production of hemolytic factor by Candida species is affected by several factors such as glucose supplementation in the culture medium, blood source, presence of erythrocytes and hemoglobin, and presence of electrolytes. On the basis of existing achievements, more researches are still needed in order to extend our knowledge about the biochemical nature of hemolytic molecules produced by distinct Candida species, the mechanism of hemolysis, and the molecular basis of the hemolytic factor expression.     

Potential of Siderophore Production by Bacteria Isolated from Heavy Metal: Polluted and Rhizosphere Soils

Recently, heavy metals have been shown to have a stimulating effect on siderophore biosynthesis in various bacteria. In addition, several studies have found that siderophore production is greater in bacteria isolated from soil near plant roots. The aim of this study was to compare the production of siderophores by bacterial strains isolated from heavy metal-contaminated and uncontaminated soils. Chrome azurol sulphonate was used to detect siderophore secretion by several bacterial strains isolated from heavy metal-contaminated and rhizosphere-uncontaminated soils with both a qualitative disc diffusion method and a quantitative ultraviolet spectrophotometric method. Siderophore production by rhizosphere bacteria was significantly greater than by bacteria isolated from contaminated soil. The Pearson’s correlation test indicated a positive correlation between the amount of siderophore produced by bacteria isolated from the rhizosphere using the quantitative and qualitative detection methods and the amount of heavy metal in the soil. However, a significant negative correlation was observed between the amount of siderophore produced by bacteria isolated from heavy metal-contaminated soil and the amount of heavy metal (r value of ?0.775, P < 0.001).     

水稻种子内生泛菌(Pantoea spp.)系统发育多样性及其促生功能

【目的】Pantoea菌株是广泛分布在自然界中的一类功能多样的细菌。本研究对分离自水稻种子内生的Pantoea菌株进行系统发育分析及功能评价,从而确定分类地位、种类多样性、分布特征及功能特性。【方法】采用乙醇-次氯酸钠联合灭菌方法进行水稻种子的表面灭菌,进行内生细菌的分离与纯化;其次将纯化后的菌株进行16Sr RNA基因PCR扩增及序列分析,通过MEGA7软件构建系统发育树;将分离得到的菌株进行功能实验检测,如溶磷、产IAA、产铁载体、拮抗病原真菌等特性,最后检测菌株的溶血性;水稻分型采用SSR方法,并对水稻农学性状如分蘖数、株高、植株重及产量进行调查。【结果】本研究对分离自8个不同基因型水稻种子中的146株内生Pantoea菌株进行系统发育分析及功能评价,结果发现所分离到的泛菌菌株主要属于Pantoea dispersa、Pantoea agglomerans、Pantoea cypripedii以及Pantoea brenneri四个种,其中P. dispersa的菌株数量最多,分布最广,并且存在于所有的8个水稻种子样品中。对其中66株菌进行功能检测,发现86.3%和69.7%的菌株具有溶磷和产IAA能力,有7株菌具有产铁载体能力,未发现对真菌病害Fusarium moniliforme有拮抗作用的菌株,并发现3株菌具有溶血性;本实验未发现泛菌组成与水稻系统发育及农学性状存在明显的相关性。【结论】本研究首次对水稻种子中泛菌的多样性及其功能进行报道,发现不同基因型的水稻种子所含Pantoea种类及组成存在差异,种子选择性地积累了Pantoea类群,大部分菌株具有一定的促生特性。该研究结果有助于进一步探究微生物与植物的共进化、种子微生物的传播途径及作用方式。     

Propionibacterium jensenii Produces the Polyene Pigment Granadaene and Has Hemolytic Properties Similar to Those of Streptococcus agalactiae

The red polyene pigment granadaene was purified and identified from Propionibacterium jensenii. Granadaene has previously been identified only in Streptococcus agalactiae, where the pigment correlates with the hemolytic activity of the bacterium. A connection between hemolytic activity and the production of the red pigment has also been observed in P. jensenii, as nonpigmented strains are nonhemolytic. The pigment and hemolytic activity from S. agalactiae can be extracted from the bacterium with a starch extraction solution, and this solution also extracts the pigment and hemolytic activity from P. jensenii. A partial purification of the hemolytic activity was achieved, but the requirement for starch to preserve its activity made the purification unsuccessful. Partially purified hemolytic fractions were pigmented, and the color intensity of the fractions coincided with the hemolytic titer. The pigment was produced in a soluble form when associated with starch, and the UV-visual spectrum of the extract gave absorption peaks of 463 nm, 492 nm, and 524 nm. The pigment could also be extracted from the cells by a low-salt buffer, but it was then aggregated. The purification of the pigment from P. jensenii was performed, and mass spectrometry and nuclear magnetic resonance analysis revealed that P. jensenii indeed produces granadaene as seen in S. agalactiae.     

Siderophore-Mediated Iron Sequestering by Shewanella putrefaciens

The iron-sequestering abilities of 51 strains of Shewanella putrefaciens isolated from different sources (fish, water, and warm-blooded animals) were assessed. Thirty strains (60%) produced siderophores in heat-sterilized fish juice as determined by the chrome-azurol-S assay. All cultures were negative for the catechol-type siderophore, whereas 24 of the 30 siderophore-producing strains tested positive in the Csáky test, indicating the production of siderophores of the hydroxamate type. Siderophore-producing S. putrefaciens could to some degree cross-feed on the siderophores of other S. putrefaciens strains and on compounds produced by an Aeromonas salmonicida strain under iron-limited conditions. The siderophores of S. putrefaciens were not sufficiently strong to inhibit growth of other bacteria under iron-restricted conditions. However, siderophore-producing Pseudomonas bacteria were always inhibitory to S. putrefaciens under iron-limited conditions. Growth of siderophore-producing strains under iron-limited conditions induced the formation of one major new outer membrane protein of approximately 72 kDa. Two outer membrane proteins of approximately 53 and 23 kDa were not seen when iron was restricted.     

Social evolution of toxic metal bioremediation in Pseudomonas aeruginosa

Bacteria are often iron-limited, and hence produce extracellular iron-scavenging siderophores. A crucial feature of siderophore production is that it can be an altruistic behaviour (individually costly but benefitting neighbouring cells), thus siderophore producers can be invaded by non-producing social ‘cheats’. Recent studies have shown that siderophores can also bind other heavy metals (such as Cu and Zn), but in this case siderophore chelation actually reduces metal uptake by bacteria. These complexes reduce heavy metal toxicity, hence siderophore production may contribute to toxic metal bioremediation. Here, we show that siderophore production in the context of bioremediation is also an altruistic trait and can be exploited by cheating phenotypes in the opportunistic pathogen Pseudomonas aeruginosa. Specifically, we show that in toxic copper concentrations (i) siderophore non-producers evolve de novo and reach high frequencies, and (ii) producing strains are fitter than isogenic non-producing strains in monoculture, and vice versa in co-culture. Moreover, we show that the evolutionary effect copper has on reducing siderophore production is greater than the reduction observed under iron-limited conditions. We discuss the relevance of these results to the evolution of siderophore production in natural communities and heavy metal bioremediation.     

Catecholate Siderophores Protect Bacteria from Pyochelin Toxicity

Background

Bacteria produce small molecule iron chelators, known as siderophores, to facilitate the acquisition of iron from the environment. The synthesis of more than one siderophore and the production of multiple siderophore uptake systems by a single bacterial species are common place. The selective advantages conferred by the multiplicity of siderophore synthesis remains poorly understood. However, there is growing evidence suggesting that siderophores may have other physiological roles besides their involvement in iron acquisition.

Methods and Principal Findings

Here we provide the first report that pyochelin displays antibiotic activity against some bacterial strains. Observation of differential sensitivity to pyochelin against a panel of bacteria provided the first indications that catecholate siderophores, produced by some bacteria, may have roles other than iron acquisition. A pattern emerged where only those strains able to make catecholate-type siderophores were resistant to pyochelin. We were able to associate pyochelin resistance to catecholate production by showing that pyochelin-resistant Escherichia coli became sensitive when biosynthesis of its catecholate siderophore enterobactin was impaired. As expected, supplementation with enterobactin conferred pyochelin resistance to the entE mutant. We observed that pyochelin-induced growth inhibition was independent of iron availability and was prevented by addition of the reducing agent ascorbic acid or by anaerobic incubation. Addition of pyochelin to E. coli increased the levels of reactive oxygen species (ROS) while addition of ascorbic acid or enterobactin reduced them. In contrast, addition of the carboxylate-type siderophore, citrate, did not prevent pyochelin-induced ROS increases and their associated toxicity.

Conclusions

We have shown that the catecholate siderophore enterobactin protects E. coli against the toxic effects of pyochelin by reducing ROS. Thus, it appears that catecholate siderophores can behave as protectors of oxidative stress. These results support the idea that siderophores can have physiological roles aside from those in iron acquisition.     

Increased Bacterial Hemolytic Activity is Conferred by Expression of TlyA Methyltransferase but not by its 2′-O-methylation of the Ribosome

Bacterial 2′-O-methyltransferase TlyA methylates either both nucleotide C1409 of 16S rRNA and C1920 of 23S rRNA or only the C1920. Both ribosomal methylations increase bacterial susceptibility to ribosome-targeting antibiotics capreomycin and viomycin. However, TlyA has been suggested to also function as a hemolysin. Here, heterologous expression of TlyA from six diverse bacteria (including Mycobacterium tuberculosis and M. smegmatis) was found to increase hemolytic ability in the Escherichia coli host. Characterizing E. coli strains expressing mycobacterial TlyA with mutated rRNA recognition domain and impaired rRNA methylations showed that the abolished C1409 methylation altogether with significantly reduced C1920 methylation did not affect E. coli hemolytic activity. Thus, the increased bacterial hemolytic function is not likely a consequence of TlyA-mediated methylations of the ribosome. Purified water-soluble TlyA showed a weak concentration-dependent hemolysis in vitro. Therefore, the TlyA isoform alone is not a potent hemolysin. The results suggested that the bacterial hemolytic function might relate to the over-expression of TlyA and its interaction to other non-ribosomal target that is associated with the hemolytic ability.     

Effects of freshwater bacterial siderophore on Microcystis and Anabaena

Six siderophore-producing bacterial strains were isolated from the freshwater, in which five strains belonged to Pseudomona genus, and the other belonged to Stenotrophomonas genus. The strain, Stenotrophomonas maltophilia 15, which produced hydroxamate-type siderophore, was selected for siderophore preparation. Its siderophore production was inhibited by FeCl₃, especially when FeCl₃ concentration was higher than 20 μM. Effects of siderophore on cyanobacteria Microcystis aeruginosa FACHB-905 and Anabaena flos-aquae FACHB-245 were studied. Compared to the control, almost all the treated groups showed decrease in growth rate and chlorophyll a, carotenoids, phycocyanin, soluble protein, microcystin content, which was attributed the low iron bioavailability in the culture medium. In the study, S. maltophilia 15 showed algicidal activities by secreting siderophore and could inhibit cyanobacterial growth, especially when iron bioavailabity is very low. The two cyanobacterial strains showed distinct demand for iron. It was deduced that in the freshwater the competition between bacteria and cyanobacteria existed for the low-bioavailable iron, which may relate to the replacement of dominant cyanobacteria.     

Acquisition of iron from host sources by mesophilic Aeromonas species.

The mesophilic Aeromonas species are opportunistic pathogens that produce either of the siderophores amonabactin or enterobactin. Acquisition of iron for growth from Fe-transferrin in serum was dependent on the siderophore amonabactin; 50 of 54 amonabactin-producing isolates grew in heat-inactivated serum, whereas none of 30 enterobactin-producing strains were able to grow. Most isolates (regardless of siderophore produced) used haem as a sole source of iron for growth; all of 33 isolates grew with either haematin or haemoglobin and 30 of these used haemoglobin when complexed to human haptoglobin. Mutants unable to synthesize a siderophore used iron from haem, suggesting that this capacity was unrelated to siderophore production. Some members of the mesophilic Aeromonas species have evolved both siderophore-dependent and -independent mechanisms for acquisition of iron from a host.     

Characterization of Fluorescent and Nonfluorescent Peptide Siderophores Produced by Pseudomonas syringae Strains and Their Potential Use in Strain Identification

Nonfluorescent highly virulent strains of Pseudomonas syringae pv. aptata isolated in different European countries and in Uruguay produce a nonfluorescent peptide siderophore, the production of which is iron repressed and specific to these strains. The amino acid composition of this siderophore is identical to that of the dominant fluorescent peptide siderophore produced by fluorescent P. syringae strains, and the molecular masses of the respective Fe(III) chelates are 1,177 and 1,175 atomic mass units. The unchelated nonfluorescent siderophore is converted into the fluorescent siderophore at pH 10, and colors and spectral characteristics of the unchelated siderophores and of the Fe(III)-chelates in acidic conditions are similar to those of dihydropyoverdins and pyoverdins, respectively. The nonfluorescent siderophore is used by fluorescent and nonfluorescent P. syringae strains. These results and additional mass spectrometry data strongly suggest the presence of a pyoverdin chromophore in the fluorescent siderophore and a dihydropyoverdin chromophore in the nonfluorescent siderophore, which are both ligated to a succinamide residue. When chelated, the siderophores behave differently from typical pyoverdins and dihydropyoverdins in neutral and alkaline conditions, apparently because of the ionization occurring around pH 4.5 of carboxylic acids present in β-hydroxyaspartic acid residues of the peptide chains. These differences can be detected visually by pH-dependent changes of the chelate colors and spectrophotochemically. These characteristics and the electrophoretic behavior of the unchelated and chelated siderophores offer new tools to discriminate between saprophytic fluorescent Pseudomonas species and fluorescent P. syringae and P. viridiflava strains and to distinguish between the two siderovars in P. syringae pv. aptata.     

Involvement of SpoVG in hemolysis caused by Bacillus subtilis

Bacillus subtilis is a facultative anaerobic Gram-positive non-pathogenic bacterium that includes members displaying hemolytic activity. To identify the genes responsible for hemolysis, a random mariner-based transposon insertion mutant library of B. subtilis 168 was constructed. More than 20,000 colonies were screened for the hypohemolytic phenotype on blood agar plates. One mutant showed significantly less pronounced hemolytic phenotype than the wild type. DNA sequencing and Southern blot analysis showed this mutant has a single transposable element inserted into the open reading frame (ORF) of the spoVG gene; complementation of the spoVG-disrupted mutant with a wild-type copy restored its hemolytic phenotype. It was therefore concluded that the spoVG gene, which plays a role in regulating asymmetric septation during sporulation in B. subtilis, is involved in hemolysis by B. subtilis.     

Comparison of the hemolysis machinery in two evolutionarily distant blood-feeding arthropod vectors of human diseases

Host blood protein digestion plays a pivotal role in the ontogeny and reproduction of hematophagous vectors. The gut of hematophagous arthropods stores and slowly digests host blood and represents the primary gateway for transmitted pathogens. The initial step in blood degradation is induced lysis of host red blood cells (hemolysis), which releases hemoglobin for subsequent processing by digestive proteolytic enzymes. The activity cycles and characteristics of hemolysis in vectors are poorly understood. Hence, we investigated hemolysis in two evolutionarily distant blood-feeding arthropods: The mosquito Culex pipiens and the soft tick Argas persicus, both of which are important human and veterinary disease vectors. Hemolysis in both species was cyclical after blood meal ingestion. Maximum digestion occurs under slightly alkaline conditions in females. Hemolytic activity appears to be of lipoid origin in C. pipiens and enzymatic activity (proteolytic) in A. persicus. We have assessed the effect of pH, incubation time, and temperature on hemolytic activity and the hemolysin. The susceptibility of red blood cells from different hosts to the hemolysin and the effect of metabolic inhibition of hemolytic activity were assessed. We conclude that in C. pipiens and A. persicus midgut hemolysins control the amplitude of blood lysis step to guarantee an efficient blood digestion.