Exudate from sporulating cultures of Hirsutella thompsonii inhibit oviposition by the two-spotted spider mite Tetranychus urticae

The acaricidal mycopathogen Hirsutella thompsonii has been found to secrete metabolites that are active against femaleTetranychus urticae. Specifically, the rose-colored exudate produced on sporulating cultures of Mexican HtM120I strain sterilized female spider mites in a dose-dependent fashion. Topical application of the exudate resulted in a 100% reduction in mite fecundity over the initial six days of experimentation. Depending upon the exudate dosage, mites partially recovered within 3 and 6 d post-treatment and produced a limited number of eggs. The spider mite active HtM120I exudate contained less detectable HtA toxin than the HtM120I broth filtrate, and it was innocuous when injected into the greater wax moth Galleria mellonella L. larvae. Broth filtrates of HtM120I cultures, although toxic to assayed G. mellonella larvae, did not inhibit mite oviposition to the degree or duration of the exudate preparations. These findings suggest that the factor responsible for suppressing oviposition in female spider mites is linked to the sporulation process and is distinct from the well-characterized HtA produced by vegetative cells. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of fungal culture filtrates of Verticillium lecanii (Lecanicillium spp.) hybrid strains on Heterodera glycines eggs and juveniles

Many nematode-antagonistic fungi produce secondary metabolites and enzymes that demonstrate toxicity against plant-parasitic nematodes. The objective of this study was to evaluate the effects of fungal culture filtrates of Verticillium lecanii hybrid strains on mature eggs, embryonated eggs (eggs fertilized but without development of juveniles), and second-stage juveniles (J2) of Heterodera glycines and to compare these effects with those of their parental strains. The fungal culture filtrates of certain hybrid strains inhibited egg hatch of mature eggs. Furthermore, the fungal culture filtrates of two hybrid strains, AaF23 and AaF42, exhibited high toxicity against embryonated eggs of H. glycines. However, most of the fungal culture filtrates of V. lecanii did not inactivate J2. These results suggested that enzymes or other active compounds produced by the fungal culture filtrates of V. lecanii exhibit activity against specific stages in the H. glycines life cycle. In addition, based on a visual assessment of the morphological changes in eggs caused by filtrates of each strain, there were differences between the hybrid strains and their respective parental strains with regard to the active substances produced by V. lecanii against the embryonated eggs. As a result of promoting recombination of whole genomes via protoplast fusion, several hybrid strains may have enhanced production of active substances that are different from those produced by their parental strains. It was concluded that natural substances produced by V. lecanii are one of the important factors involved in the suppression of H. glycines damage.     

Extraction and Characterization of the Insecticidal Toxin Hirsutellin A Produced by Hirsutella thompsonii var. thompsonii

Liu, W.-Z., Boucias, D. G., and McCoy, C. W. 1995. Extraction and characterization of the insecticidal toxin hirsutellin A produced by Hirsutella thompsonii var. thompsonii. Experimental Mycology 19, 254-262. Hirsutellin A (HtA) produced by Hirsutella thompsonii var. thompsonii (strain JAB-04) was extracted and purified using a combination of ion-exchange, gel-permeation, and immunoaffinity chromatography. The identity of the purified HtA was confirmed by amino acid analysis and N-terminal sequencing. Monoclonal antibodies prepared against HtA were capable of detecting 25-50 ng of HtA by direct sandwich ELISA. In addition, utilizing Western blot methods, the antibodies were shown to be specific to HtA. The production of HtA was monitored during submerged fermentation. The peak level of exocellular HtA (13-14 μg/ml) was during the late exponential growth phase (39-45 h), determined by utilizing a combination of densitometric analysis of the 16.3-kDa bands on SDS-PAGE gels and ELISA. HtA production was directly correlated with mycelial growth. Twenty-one-hour culture filtrates were highly toxic to larvae of the greater wax moth. Pure HtA at a final concentration of 40 pmol was highly toxic to Galleria mellonella larvae.     

Hirsutella thompsonii and Metarhizium anisopliae as potential microbial control agents of Varroa destructor,a honey bee parasite

The potential of Hirsutella thompsonii Fisher and Metarhizium anisopliae (Metschinkoff) as biological control agents of the parasitic mite, Varroa destructor Anderson and Trueman was evaluated in the laboratory and in observation hives. In the laboratory, time required for 90% cumulative mortality of mites (LT(90)) was 4.16 (3.98-4.42) days for H. thompsonii and 5.85 (5.48-7.43) days for M. anisopliae at 1.1 x 10(3) conidia mm(-2). At a temperature (34+/-1 degrees C) similar to that of the broodnest in a honey bee colony, Apis mellifera L., H. thompsonii [LC(90)=9.90 x 10(1) (5.86-19.35) conidia mm(-2) at Day 7] and M. anisopliae [LC(90)=7.13 x 10(3) (2.80-23.45) conidia mm(-2) at Day 7] both showed significant virulence against V. destructor. The applications of H. thompsonii to observation hives resulted in significant mortality of mites, and reduction of the number of mites per bee 21 and 42 days post-treatments. The treatments did not significantly affect the mite population in sealed brood. However, the fungus must have persisted because infected mites were still observed [82.97+/-(0.6)%] 42 days post-treatment. In addition, the fungus was found to sporulate on the host. A small percentage [2.86+/-(0.2)%] of dead mites found in the control hives also showed fungal infection, suggesting that adult bees drifted between hives and disseminated the fungus. H. thompsonii was harmless to the honey bees at the concentrations applied and did not have any deleterious effects on the fecundity of the queens. Microbial control with fungal pathogens provides promising new avenues for control of V. destructor and could be a useful component of an integrated pest management program for the honey bee industry.     

Virulence and site of infection of the fungus,Hirsutella thompsonii,to the honey bee ectoparasitic mite,Varroa destructor

The Varroa mite, Varroa destructor, is recognized as the most serious pest of both managed and feral Western honey bee (Apis mellifera) in the world. The mite has developed resistance to fluvalinate, an acaricide used to control it in beehives, and fluvalinate residues have been found in the beeswax, necessitating an urgent need to find alternative control measures to suppress this pest. Accordingly, we investigated the possibility of using the fungus, Hirsutella thompsonii, as a biocontrol agent of the Varroa mite. Among the 9 isolates of H. thompsonii obtained from the University of Florida and the USDA, only the 3 USDA isolates (ARSEF 257, 1947 and 3323) were infectious to the Varroa mite in laboratory tests. The mite became infected when it was allowed to walk on a sporulating H. thompsonii culture for 5 min. Scanning electron micrographs revealed that the membranous arolium of the mite leg sucker is the focus of infection where the fungal conidia adhered and germinated. The infected mites died from mycosis, with the lethal times to kill 50% (LT(50)s) dependent on the fungal isolates. Thus, the LT(50)s were 52.7, 77.2, and 96.7h for isolates 3323, 257, and 1947, respectively. Passage of H. thompsonii through Varroa mite three times significantly reduced the LT(50)s of isolates 257 and 1947 (P<0.05) but not the LT(50) of isolate 3323.The fungus did not infect the honey bee in larval, prepupal, pupal, and adult stages under our laboratory rearing conditions. Our encouraging results suggest that some isolates of H. thompsonii have the potential to be developed as a biocontrol agent for V. destructor. However, fungal infectivity against the mites under beehive conditions needs to be studied before any conclusion can be made.     

Efficacy of culture filtrates of <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> against larvae of <Emphasis Type="Italic">Anopheles stephensi</Emphasis> and <Emphasis Type="Italic">Culex quinquefasciatus</Emphasis>

Efficacy of culture filtrates of five strains of Metarhizium anisopliae isolated from insects were evaluated against Anopheles stephensi and Culex quinquefasciatus. The culture filtrates released from the strains of M. anisopliae in the YpSs and chitin broths were filtered and used for the bioassays after a growth of 7 days. Among the culture filtrates of five strains, M. anisopliae 892 was found to be more effective against both the mosquitoes. The LC(50) values of culture filtrates of M. anisopliae 892 in chitin broth was lower than the LC(50) of culture filtrates in YpSs broth against first and fourth instars of both the mosquitoes. The LC(50) values of culture filtrates were significantly different between first and fourth instars of A. stephensi (t test; P = 0.0001) and C. quinquefasciatus (t test; P = 0.02). The larvae of A. stephensi were more susceptible than C. quinquefasciatus except in two cases. This is the first report of efficacy of culture filtrates produced by M. anisopliae in chitin broth against mosquitoes and have potential as a biological control agent of mosquitoes.     

The insecticidal protein hirsutellin A from the mite fungal pathogen Hirsutella thompsonii is a ribotoxin

The mite fungal pathogen Hirsutella thompsonii produces a single polypeptide chain, insecticidal protein named hirsutellin A (HtA) that is composed of 130 amino acid residues. This protein has been purified from its natural source and produced as a recombinant protein in Escherichia coli. Spectroscopic analysis has determined that the two protein forms are indistinguishable. HtA specifically inactivates ribosomes and produces the alpha-fragment characteristic of ribotoxin activity on rRNA. Behaving as a cyclizing ribonuclease, HtA specifically cleaves oligonucleotides that mimick the sarcin/ricin loop of the ribosome, as well as selected polynucleotides and dinucleosides. HtA interacts with phospholipid membranes as do other ribotoxins. As a consequence of its ribonuclease activity and its ability to interact with cell membranes, HtA exhibits cytotoxic activity on human tumor cells. On the basis of these results, HtA is considered to be a member of the ribotoxin group of proteins, although it is significantly smaller (130 aa) than all known ribotoxins that are composed of 149/150 amino acids. Ribotoxins are members of a larger family of fungal ribonucleases whose members of smaller size (100/110 aa) are not cytotoxic. Thus, the characterization of the fungal ribotoxin HtA represents an important milestone in the study of the diversity and the function of fungal ribonucleases.     

Distribution of the entomopathogenic nematodes from La Rioja (Northern Spain)

Entomopathogenic nematodes (EPNs) distribution in natural areas and crop field edges in La Rioja (Northern Spain) has been studied taking into account environmental and physical-chemical soil factors. Five hundred soil samples from 100 sites of the most representative habitats were assayed for the presence of EPNs. The occurrence of EPNs statistically fitted to a negative binomial distribution, which pointed out that the natural distribution of these nematodes in La Rioja was in aggregates. There were no statistical differences (p < or = 0.05) in the abundance of EPNs to environmental and physical-chemical variables, although, there were statistical differences in the altitude, annual mean air temperature and rainfall, potential vegetation series and moisture percentage recovery frequency. Twenty-seven samples from 14 sites were positive for EPNs. From these samples, twenty isolates were identified to a species level and fifteen strains were selected: 11 Steinernema feltiae, two S. carpocapsae and two S. kraussei strains. S. kraussei was isolated from humid soils of cool and high altitude habitats and S. carpocapsae was found to occur in heavy soils of dry and temperate habitats. S. feltiae was the most common species with a wide range of altitude, temperature, rainfall, pH and soil moisture, although this species preferred sandy soils. The virulence of nematode strains were assessed using G. mellonella as insect host, recording the larval mortality percentage and the time to insect die, as well as the number of infective juveniles produced to evaluate the reproductive potential and the time tooks to leave the insect cadaver to determinate the infection cycle length. The ecological trends and biological results are discussed in relationship with their future use as biological control.     

Temporal association of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) and bacteria

Galleria mellonella L. larvae were infected with three species (seven strains) of Steinernema spp. or three species (three strains) of Heterorhabditis spp. Infected larvae were incubated at 22, 27, and 32 degrees C. Larvae were dorsally dissected every 6h over a 48-h period. Hemolymph was collected and streaked on tryptic soy agar plates. Several non-symbiotic bacterial species were identified from infected insect cadavers: Enterobacter gergoviae, Vibrio spp., Pseudomonas fluorescens type C, Serratia marcescens, Citrobacter freundii, and Serratia proteomaculans. At 18-24 h incubation, the nematode-associated symbiont occurred almost exclusively. Bacterial associates generally appeared outside the 18-24 h window. Infective juveniles of Steinernema feltiae (Filipjev) (27), Steinernema riobrave Cabanillas, Poinar, and Raulston (Oscar), or Steinernema carpocapsae (Weiser) (Kapow) were left untreated, or surface sterilized using thimerosal, then pipetted under sterile conditions onto tryptic soy agar plates. Several additional species of associated bacteria were identified using this method compared with the less extensive range of species isolated from infected G. mellonella. There was no difference in bacterial species identified from non-sterile or surface sterilized nematodes, suggesting that the bacteria identified originated from either inside the nematode or between second and third stage juvenile cuticles. Infective juveniles of S. feltiae (Cowles), S. carpocapsae (Cowles), and H. bacteriophora Poinar (Cowles) were isolated from field samples. Nematodes were surface-sterilized using sodium hypochlorite, mixed with G. mellonella hemolymph, and pipetted onto Biolog BUG (with blood) agar. Only the relevant symbionts were isolated from the limited number of samples available. The nematodes were then cultured in the laboratory for 14 months (sub-cultured in G. mellonella 7-times). Other Enterobacteriaceae could then be isolated from the steinernematid nematodes including S. marcescens, Salmonella sp., and E. gergoviae, indicating the ability of the nematodes to associate with other bacteria in laboratory culture.     

Interaction of microbial populations in Steinernema (Steinernematidae,Nematoda) infected Galleria mellonella larvae

Infection of Galleria mellonella larvae with the entomopathogenic nematodes Steinernema feltiae (A21 and R strains) and Steinernema glaseri (Dongrae) resulted in several species of bacteria, including the respective bacterial symbiont, Xenorhabdus spp., growing in the infected insect cadavers. These other bacteria were Enterococcus in all three nematode infections studied and Acinetobacter in the S. feltiae infections. The respective populations of these bacteria changed with time. Following infection of G. mellonella larvae with any one of the Steinernema sp., only Enterococcus bacteria were detected initially in the dead larvae. Between 30 and 50h post-infection Xenorhabdus bacteria were detected and concurrent with this Enterococcus population declined to zero. This was probably due to secondary metabolites with antibacterial properties that were produced by Xenorhabdus. In the S. feltiae (both R and A21 strains) infections a third bacterium, Acinetobacter, appeared at about 130h (in S. feltiae A21 infections) or 100h (in S. feltiae R infections) and increased in population size to approximately that of Xenorhabdus. It was demonstrated that Enterococcus, orginating from the G. mellonella digestive tract, was sensitive to the organically soluble antimicrobials produced by Xenorhabdus but Acinetobacter, which was carried by the nematode, was not.     

Plasmid transfer between strains of Bacillus thuringiensis infecting Galleria mellonella and Spodoptera littoralis

To determine the possibility of plasmid transfer occurring between strains of Bacillus thuringiensis in infected lepidopterous larvae, Galleria mellonella and Spodoptera littoralis were infected with two or more strains of B. thuringiensis and the resulting bacteria from the dead insects were examined for plasmid transfer. Transfer rates of plasmids coding for crystal production and tetracycline resistance were high, reaching levels similar to those obtained in laboratory broth cultures. Transfer was higher in G. mellonella than S. littoralis, probably due to the greater ability of B. thuringiensis to colonize the larvae. In broth cultures, B. thuringiensis was also able to transfer plasmids into sporeforming bacteria present in soil samples. The results suggest that plasmid transfer between strains of B. thuringiensis occurs in nature, resulting in the production of new combinations of delta-endotoxins within populations of the bacteria.     

Distribution and diversity of cry genes in native strains of Bacillus thuringiensis obtained from different ecosystems from Colombia

Colombia is a tropical country located at the north of South America. It is considered to be one of the most important countries in terms of its biodiversity worldwide. One hundred and eight soil samples obtained from agricultural crops and wild ecosystems were evaluated in terms of the presence of Bacillus thuringiensis (Bt) native strains. One hundred and eight different Bt strains were isolated and characterized by the presence of crystal proteins by SDS-PAGE and a multiplex PCR with general and specific primers for cry1 and cry3, cry7, and cry8 gene detection. Most of the Bt strains (73%) reacted with the cry1 general primers; 27.8% of the Bt strains reacted with cry3, cry7, and cry8 general primers and 17.8% of strains did not react with any of these two sets of primers. Thirty different PCR profiles were found in the strains with cry1 genes when they were analyzed with specific primers (cry1A to cry1F). A high frequency of joint occurrence was observed for cry1Aa/cry1Ab, cry1Aa/cry1Ac, cry1Ab/cry1Ac, and cry1C/cry1D genes with a Pearson coefficient of 0.88, 0.74, 0.76, and 0.87, respectively. Other distinctive characteristics were found in the Colombian collection as the presence of 22.2% of native strains which presented, at the same time, lepidopteran and coleopteran active genes. Interesting relations were found as well between the cry gene distribution and the geographical areas sampled. Finally, some strains with moderate to high biopesticide activity against Spodoptera frugiperda (Lepidoptera) and Premnotrypes vorax (Coleoptera) insects were identified, this being important to explore future microbial strategies for the control of these crop pests in the region.     

Plasmid transfer between strains of Bacillus thuringiensis infecting Galleria mellonella and Spodoptera littoralis.

To determine the possibility of plasmid transfer occurring between strains of Bacillus thuringiensis in infected lepidopterous larvae, Galleria mellonella and Spodoptera littoralis were infected with two or more strains of B. thuringiensis and the resulting bacteria from the dead insects were examined for plasmid transfer. Transfer rates of plasmids coding for crystal production and tetracycline resistance were high, reaching levels similar to those obtained in laboratory broth cultures. Transfer was higher in G. mellonella than S. littoralis, probably due to the greater ability of B. thuringiensis to colonize the larvae. In broth cultures, B. thuringiensis was also able to transfer plasmids into sporeforming bacteria present in soil samples. The results suggest that plasmid transfer between strains of B. thuringiensis occurs in nature, resulting in the production of new combinations of delta-endotoxins within populations of the bacteria.     

Viability of Heterodera glycines Exposed to Fungal Filtrates

Filtrates from nematode-parasitic fungi have been reported to be toxic to plant-parasitic nematodes. Our objective was to determine the effects of fungal filtrates on second-stage juveniles and eggs of Heterodera glycines. Eleven fungal species that were isolated from cysts extracted from a soybean field in Florida were tested on J2, and five species were tested on eggs in vitro. Each fungal species was grown in Czapek-Dox broth and malt extract broth. No toxic activity was observed for fungi grown in Czapek-Dox broth. Filtrates from Paecilomyces lilacinus, Stagonospora heteroderae, Neocosmospora vasinfecta, and Fusarium solani grown in malt extract broth were toxic to J2, whereas filtrates from Exophiala pisciphila, Fusarium oxysporum, Gliocladium catenulatum, Pyrenochaeta terrestris, Verticillium chlamydosporium, and sterile fungi 1 and 2 were not toxic to J2. Filtrates of P. lilacinus, S. heteroderae, and N. vasinfecta grown in malt extract broth reduced egg viability, whereas F. oxysporum and P. terrestris filtrates had no effect on egg viability.     

Txp40, a ubiquitous insecticidal toxin protein from Xenorhabdus and Photorhabdus bacteria

Xenorhabdus and Photorhabdus are gram-negative bacteria that produce a range of proteins that are toxic to insects. We recently identified a novel 42-kDa protein from Xenorhabdus nematophila that was lethal to the larvae of insects such as Galleria mellonella and Helicoverpa armigera when it was injected at doses of 30 to 40 ng/g larvae. In the present work, the toxin gene txp40 was identified in another 59 strains of Xenorhabdus and Photorhabdus, indicating that it is both highly conserved and widespread among these bacteria. Recombinant toxin protein was shown to be active against a variety of insect species by direct injection into the larvae of the lepidopteran species G. mellonella, H. armigera, and Plodia interpunctella and the dipteran species Lucilia cuprina. The protein exhibited significant cytotoxicity against two dipteran cell lines and two lepidopteran cell lines but not against a mammalian cell line. Histological data from H. armigera larvae into which the toxin was injected suggested that the primary site of action of the toxin is the midgut, although some damage to the fat body was also observed.     

Mycobacterium tuberculosis Rv2536 protein implicated in specific binding to human cell lines

The gene encoding the Mycobacterium tuberculosis Rv2536 protein is present in the Mycobacterium tuberculosis complex (as assayed by PCR) and transcribed (as determined by RT-PCR) in M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. bovis BCG, and M. africanum strains. Rabbits immunized with synthetic polymer peptides from this protein produced antibodies specifically recognizing a 25-kDa band in mycobacterial sonicate. U937 and A549 cells were used in binding assays involving 20-amino-acid-long synthetic peptides covering the whole Rv2536 protein sequence. Peptide 11207 (161DVFSAVRADDSPTGEMQVAQY180) presented high specific binding to both types of cells; the binding was saturable and presented nanomolar affinity constants. Cross-linking assays revealed that this peptide specifically binds to 50 kDa U937 cell membrane and 45 kDa A549 cell membrane proteins.     

Studies on the Host-finding Mechanisms of Neotylenchus linfordi

The plant-parasitic nematode, Neotylenchus linlordi, congregated around colonies or filtrates from mycelia of Gliocladium roseum, Rhizoctonia solani, Pyrenochaeta terrestris and Chaetomium indicum. The average time required for the nematodes to reach the fungal colonies ranged from less than 4 hr for G. roseum to 20 hr for R. solani. Nematodes first circled near the point of introduction, then moved toward the fungus or filtrate. Several methods of measuring the response of N. linfordi to G. roseum culture filtrate were evaluated. The response was strongest when the test materials were assayed on an agar disk submerged in water agar and the introduced nematodes suspended in agar in a center well midway between the test materials. Filtrates obtained from cultures of G. roseum incubated between 12 and 21 days in potato dextrose broth, were most active. The attractants were small thermostable molecules, soluble in methyl alcohol and unaffected by pH. A yellow pigment with properties similar to a mixture of aurantiogliocladin, rubrogliocladin, and gliorosein was shown to be one of the active materials. The response of N. linfordi to the G. roseum filtrate was not associated with any nutritive factors which would result in reproduction.     

Comparative Biological Efficacy of Hirsutella thompsonii and Neoseiulus teke for Cassava Mite (Mononychellus tanajoa) Suppression

The biological control potential of Hirsutella thompsonii Fisher (Deuteromycetes: Monilianes) was compared with that of Neoseiulus teke Pritchard and Baker (Acari: Phytoseiidae) for cassava mite, Mononychellus tanajoa (Acari:Tetranychidae), suppression. Mite infestation levels (egg and mite motile counts and damage rating) were lower on H. thompsonii- and N. teke- treated plants than on plants sprayed with water. N. teke reduced the pest attack to a comparable level to that of H. thompsonii . In the field, applications of aqueous suspensions of H. thompsonii at concentrations of 1.2 1011 conidia/ha (CPH) and 6.0 1010 CPH significantly lowered mite infestation on a number of recording dates. As rainfall levels increased, mite infestation and the number of cadavers on the leaves declined. The study showed that H. thompsonii has promise for controlling M. tanajoa when the fungus is applied in harmony with other natural mortality factors.     

Molecular studies of co-formulated strains of the entomopathogenic fungus,Beauveria bassiana

A 28S rDNA intron was used as a molecular marker to distinguish between two single spore strains of Beauveria bassiana, Bb123 and Bb151. When co-formulated and assayed against larvae of Galleria mellonella these strains exhibited no synergistic increase in virulence, rather Bb123 usually dominated. This study shows that the success of any strain to infect Galleria is dependent on the dose and method of inoculation (injection versus immersion). The result of co-formulated strains grown on solid culture also showed that usually one strain dominated, i.e., strain displacement could happen both in vivo and in vitro. The speed by which one strain was displaced following successive sub-culturing on PDA partly depended on the ratio of Bb151 and Bb123. The co-formulated inoculum could widen the window over which parent strains would be active on different water activity media. Co-infection did result in heterokaryosis within the Galleria host. Molecular studies also showed that the heterokaryon was not stable and could revert back to the parent strain.     

Implication of an Asp residue in the ribonucleolytic activity of hirsutellin A reveals new electrostatic interactions at the active site of ribotoxins

Ribotoxins are fungal extracellular ribonucleases that specifically cleave ribosomes leading to cell-death via apoptosis. α-Sarcin is the ribotoxin studied in deepest detail, and therefore constitutes the referential protein for the whole family. It has been demonstrated that ribotoxin activity depends on a very precise structural microenvironment in which electrostatic interactions among residues in the active site are of the highest importance. Hirsutellin A (HtA) has been recently described as the smallest ribotoxin known to date, encompassing all the abilities of previously characterized members of this family into a shorter sequence. Comparison of HtA and α-sarcin three-dimensional structures suggested that residues presumably forming the catalytic triad of HtA would be His 42, Glu 66, and His 113. Within this same idea, the presence of an Asp residue (Asp 40) in a position equivalent to α-sarcin Tyr 48 is highlighted as a novelty in this field. In this work, substitution mutants H42Q, E66Q and H113Q, as well as double and triple mutants in all possible combinations, are studied regarding their ribonucleolytic activity and cytotoxicity. Implication of these three residues in the ribotoxin activity of HtA is confirmed, though none of them is strictly essential for ribosomal cleavage. Studies with mutants D40N and D40N/E66Q demonstrate an important role for Asp 40 in the activity of HtA and establish a new set of electrostatic interactions different from the one described for already known ribotoxins.