Optimization of protease production by the fungusMonacrosporium thaumasium and its action againstAngiostrongylus vasorum larvae

Otimização da produção de protease pelo fungoMonacrosporium thaumasium e sua ação contra larvas deAngiostrongylus vasorum

Filippe Elias de Freitas Soares Fabio Ribeiro Braga Jackson Victor de Araújo Walter dos Santos Lima Lanuze Rose Mozzer José Humberto de Queiroz About the authors

Abstracts

The objectives of this study were to optimize protease production from the nematophagous fungus Monacrosporium thaumasium (NF34a) and evaluate its larvicidal activity and biological stability. An isolate of the nematophagous fungus Monacrosporium thaumasium (NF34a) was used to produce the enzyme. The Plackett-Burman design was used in order to scan which components of the culture medium could have a significant influence on protease production by the fungus NF34a. An in vitro assay was also performed to evaluate the larvicidal activity of NF34a. It was observed that only one component of the culture medium (yeast extract), at the levels studied, had any significant effect (p < 0.05) on protease production. There was a reduction (p < 0.01) in the mean number of larvae recovered from the treated groups, compared with the control groups. The results confirm previous reports on the efficiency of nematophagous fungi for controlling nematode larvae that are potentially zoonotic. Thus, given the importance of biological control, we suggest that further studies should be conducted on the protease produced by the fungus Monacrosporium thaumasium.

Nematophagous fungi; Monacrosporium thaumasium; protease; Angiostrongylus vasorum


O objetivo deste trabalho foi otimizar a produção de proteases do fungo nematófago Monacrosporium thaumasium (NF34a), avaliar sua atividade larvicida e sua estabilidade biológica. Foi utilizado um isolado do fungo nematófago Monacrosporium thaumasium (NF34a) para a produção de enzima. O delineamento Plackett-Burman foi utilizado com o objetivo de se escanear quais componentes do meio de cultura, poderiam ter influência significava na produção de protease pelo fungo NF34a. Foi realizado um ensaioin vitro para avaliar a atividade larvicida de NF34a. Observou-se que apenas um dos componentes do meio de cultura (extrato de levedura), nos níveis avaliados, apresentou um efeito significativo (p < 0,05) sobre a produção de protease. Houve reducão (p < 0,01) entre as médias de larvas recuperadas dos grupos tratados em relação ás dos grupos controle. Os resultados confirmam trabalhos anteriores da eficiência de fungos nematófagos no controle de larvas de nematóides potencialmente zoonóticos. Assim, devido á importãncia do controle biológico, os autores sugerem estudos mais aprofundados sobre a protease produzida pelo fungo Monacrosporium thaumasium.

Fungos nematófagos; Monacrosporium thaumasium; protease; Angiostrongylus vasorum


Nematophagous fungi have been shown to be potential sources of enzyme production (MEYER; WIEBE, 2003Meyer WJ, Wiebe MG. Enzyme production by the nematode-trapping fungus, Duddingtonia flagrans. Biotechnol Lett 2003; 25(10): 791-795. http://dx.doi.org/10.1023/A:1023580621840
http://dx.doi.org/10.1023/A:102358062184...
; ESTEVES et al., 2009Esteves I, Peteira B, Atkins SD, Magan N, Kerry B. Production of extracellular enzymes by different isolates of Pochonia chlamydosporia. Mycol Res 2009; 113(8): 867-876. http://dx.doi.org/10.1016/j.mycres.2009.04.005
http://dx.doi.org/10.1016/j.mycres.2009....
). In laboratory practice, optimal culture media have been successfully used for producing proteases (BRAGA et al., 2011aBraga FR, Araújo JV, Soares FEF, Araujo JM, Geniêr HLA, Silva AR, et al. Optimizing protease production from an isolate of the nematophagous fungus Duddingtonia flagrans using response surface methodology and its larvicidal activity on horse cyathostomins. J Helminthol 2011a; 85(2): 164-170. http://dx.doi.org/10.1017/S0022149X10000416
http://dx.doi.org/10.1017/S0022149X10000...
). To achieve high levels of proteolytic activity, the culturing conditions need to be optimized. One strategy for optimization is to use the Plackett-Burman statistical experimental design (PLACKETT; BURMAN, 1946Plackett RL, Burman JP. The design of optimum multifactorial experiments. Biometrika 1946; 33(4): 305-325. http://dx.doi.org/10.1093/biomet/33.4.305
http://dx.doi.org/10.1093/biomet/33.4.30...
), in which the components of the culture medium are scanned in order to select the most significant ones for protease production.

There is increasing evidence showing that proteolytic enzymes may be involved in penetration of the cuticle and cellular digestion of the host nematode (LIANG et al., 2011Liang L, Liu S, Yang J, Meng Z, Lei L, Zhang K. Comparison of homology models and crystal structures of cuticle-degrading proteases from nematophagous fungi: structural basis of nematicidal activity. FASEB J 2011; 25(6): 1894-1902. http://dx.doi.org/10.1096/fj.10-175653
http://dx.doi.org/10.1096/fj.10-175653...
). On the other hand, there are few reports about the molecular mechanism for the pathogenic action of fungi against nematodes, which means that this has not been fully elucidated (SOARES et al., 2012Soares FE, Braga FR, Araújo JV, Lima WS, Mozer LR, Queiróz JH. In vitro activity of a serine protease from Monacrosporium thaumasium fungus against first-stage larvae of Angiostrongylus vasorum. Parasitol Research 2012; 110(6): 2423-2427. http://dx.doi.org/10.1007/s00436-011-2781-x
http://dx.doi.org/10.1007/s00436-011-278...
). The protease Mt1 of the nematophagous fungusMonacrosporium thaumasium has been used successfully to combat the first-stage larvae of Angiostrongylus vasorum, a potentially zoonotic nematode (SOARES et al., 2012Soares FE, Braga FR, Araújo JV, Lima WS, Mozer LR, Queiróz JH. In vitro activity of a serine protease from Monacrosporium thaumasium fungus against first-stage larvae of Angiostrongylus vasorum. Parasitol Research 2012; 110(6): 2423-2427. http://dx.doi.org/10.1007/s00436-011-2781-x
http://dx.doi.org/10.1007/s00436-011-278...
). However, there are no reports on optimization of the production of this enzyme, or on its biological stability. Thus, there is a need for knowledge regarding some significant factors that could be used under natural conditions. Among these are (1) culture medium composition and (2) biological stability of these enzymes.

The objectives of this work were to optimize protease production from the nematophagous fungus Monacrosporium thaumasium (NF34a) and to evaluate its larvicidal activity and biological stability.

We used an isolate of the nematophagous fungus Monacrosporium thaumasium (NF34) for producing this enzyme. This isolate originated from Brazilian soil and has been maintained under laboratory conditions, through continual transfer to solid culture media in the dark for 10 days.

For the present study, fungal mycelia from this isolate were transferred to previously autoclaved flasks containing 50 mL of liquid medium. The mixtures were kept under continual stirring at 120 rpm for six days using the method described by Soares et al. (2012)Soares FE, Braga FR, Araújo JV, Lima WS, Mozer LR, Queiróz JH. In vitro activity of a serine protease from Monacrosporium thaumasium fungus against first-stage larvae of Angiostrongylus vasorum. Parasitol Research 2012; 110(6): 2423-2427. http://dx.doi.org/10.1007/s00436-011-2781-x
http://dx.doi.org/10.1007/s00436-011-278...
, with modifications.

Next, first-stage larvae (L1) of A. vasorum were obtained from positive feces from dogs that had been kept in the Parasitology Department, Federal University of Minas Gerais, using the technique described by Lima et al. (1985)Lima WS, Costa HMA, Guimarães MP, Leite ACR. Angiostrongylus vasorum (Baillet, 1866) Nematoda: Protostrongylidae, em cães de Minas Gerais, Brasil. Mem Inst Oswaldo Cruz 1985; 80(2): 233-235. http://dx.doi.org/10.1590/S0074-02761985000200015
http://dx.doi.org/10.1590/S0074-02761985...
.

The protease Mt1, produced by the nematophagous fungus M. thaumasium (NF34a), was purified in accordance with the protocol described by Soares et al. (2012)Soares FE, Braga FR, Araújo JV, Lima WS, Mozer LR, Queiróz JH. In vitro activity of a serine protease from Monacrosporium thaumasium fungus against first-stage larvae of Angiostrongylus vasorum. Parasitol Research 2012; 110(6): 2423-2427. http://dx.doi.org/10.1007/s00436-011-2781-x
http://dx.doi.org/10.1007/s00436-011-278...
. The proteolytic activity was measured by means of the caseinolytic method, as described by Braga et al. (2011a)Braga FR, Araújo JV, Soares FEF, Araujo JM, Geniêr HLA, Silva AR, et al. Optimizing protease production from an isolate of the nematophagous fungus Duddingtonia flagrans using response surface methodology and its larvicidal activity on horse cyathostomins. J Helminthol 2011a; 85(2): 164-170. http://dx.doi.org/10.1017/S0022149X10000416
http://dx.doi.org/10.1017/S0022149X10000...
. A tyrosine standard curve was constructed to quantify enzyme activity. One unit of enzyme was defined as the amount of enzyme required to release 1.0 µg of tyrosine per minute under the assay conditions (60 °C and pH 8.0).

The Plackett-Burman design (PLACKETT; BURMAN, 1946Plackett RL, Burman JP. The design of optimum multifactorial experiments. Biometrika 1946; 33(4): 305-325. http://dx.doi.org/10.1093/biomet/33.4.305
http://dx.doi.org/10.1093/biomet/33.4.30...
) was used in order to scan the components of the culture medium and determine which of them might have significant influence on protease production by the fungus NF34a. The Minitab Release 15 software was used for this analysis. The variables studied were: glucose (g/L), yeast extract (g/L), K2HPO4 (g/L), MgSO4 (g/L), ZnSO4 (g/L), FeSO4 (g/L), CuSO4 (g/L) and pH. Based on the Plackett-Burman factorial design, each factor was analyzed on two levels: −1 to lower level and +1 to higher level. The analysed factors and respective levels were: glucose −1: 5 g/L, +1: 15 g/L; yeast extract −1 (5g/L), +1 (15 g/L); K2HPO4−1 (2.5 g/L), +1 (7.5 g/L); MgSO4 −1 (0.1 g/L), +1 (0.3 g/L); ZnSO4 −1 (0.0025 g/L), +1 (0.0075 g/L); FeSO4−1 (0.0005 g/L), +1 (0.002 g/L); CuSO4 −1 (0.000125 g/L), +1 (0.0005 g/L). The choice of levels to study was based on the work of Braga et al. (2011a)Braga FR, Araújo JV, Soares FEF, Araujo JM, Geniêr HLA, Silva AR, et al. Optimizing protease production from an isolate of the nematophagous fungus Duddingtonia flagrans using response surface methodology and its larvicidal activity on horse cyathostomins. J Helminthol 2011a; 85(2): 164-170. http://dx.doi.org/10.1017/S0022149X10000416
http://dx.doi.org/10.1017/S0022149X10000...
. The study variables and their respective levels are shown in Table 1. The model is given by the following equation:

)1)

Where Y is the response (proteolytic activity in units), βo is the intercept of the model, βi is the linear coefficient and xi is the level of the independent variable. The proteolytic activity was measured in triplicate and the mean of these values was used as the response Y (MUKHERJEE; RAI, 2011Mukherjee AK, Rai SK. A statistical approach for the enhanced production of alkaline protease showing fibrinolytic activity from a newly isolated Gram-negative Bacillus sp. strain AS-S20-I. N Biotechnol 2011; 28(2): 182-189.). The model selection was performed with the help of the Minitab Release 15 software.

Table 1.
Matrix of Plackett-Burman experimental design.

The in vitro assay was based on previous work by Braga et al. (2011b)Braga FR, Araujo JM, Tavela AO, Araújo JV, Soares FEF, Geniêr HLA, et al. Atividade larvicida do extrato bruto enzimático do fungo Duddingtonia flagras sobre larvas de primeiro estádio de Angiostrongylus vasorum. Rev Soc Bras Med Trop 2011b; 44(3): 383-385.. The data obtained in this assay were interpreted by means of analysis of variance, with a significance levels of 1 and 5% probability. The efficiency of predatory action on L1 in relation to the control was measured by means of the Tukey test at 1% probability (AYRES et al., 2003Ayres M, Ayres JRM, Ayres DL, Santos AS. Aplicações estatísticas nas áreas de ciências biológicas. Belém: Sociedade civil mamirauá; Brasília CNPq; 2003. p. 290.). Subsequently, the mean percentage reduction of larvae was calculated in accordance with the following equation:

)2)

(2)

In order to determine the degree of stability of biological activity of the crude extract and the protease Mt1 (SOARES et al., 2012Soares FE, Braga FR, Araújo JV, Lima WS, Mozer LR, Queiróz JH. In vitro activity of a serine protease from Monacrosporium thaumasium fungus against first-stage larvae of Angiostrongylus vasorum. Parasitol Research 2012; 110(6): 2423-2427. http://dx.doi.org/10.1007/s00436-011-2781-x
http://dx.doi.org/10.1007/s00436-011-278...
), these were incubated at 28 °C for 7 days. Every 24 hours, aliquots were removed and the enzyme activity was determined.

The Plackett-Burman design was used to investigate the significance of the components of the culture medium used for protease production from the fungus M. thaumasium (NF34a). In the present work, it was observed that one component of the culture medium (yeast extract), at the levels studied, had a significant effect (p < 0.05) on protease production (Table 2). The R2 of the model was 93.01%, thus demonstrating that it is a reliable model. It was also observed that the variable of pH was significant (p < 0.1) with regard to protease production.

Table 2.
Factors studied in the Plackett-Burman design.

Regarding the enzymatic activity on A. vasorum L1(in vitro assay), over a 24-hour interval, the crude extract produced by NF34a showed a reduction of 77.4% in larval recovery rate. A difference was noted (p < 0.01) regarding the action of this extract from the isolate, in relation to the larvae present in the control group. On the other hand, it was demonstrated that both the crude extract and the protease Mt1 maintained intact biological activity after incubation at 28 °C for 7 days.

Soil contaminated with infective stages (eggs and/or larvae) has been shown to be one of the major sources of contamination by geohelminths (GORTARI et al., 2007Gortari C, Cazau C, Hours R. Hongos nematófagos de huevos de Toxocara canis en un paseo público de La Plata, Argentina. Rev Iberoam Micol 2007; 24(1): 24-28. http://dx.doi.org/10.1016/S1130-1406(07)70005-0
http://dx.doi.org/10.1016/S1130-1406(07)...
). Fungal structures such as conidia have been shown to be effective in controlled trials against A. vasorumL1 (BRAGA et al., 2009Braga FR, Carvalho RO, Araujo JM, Silva AR, Araújo JV, Lima WS, et al. Predatory activity of the fungi Duddingtonia flagrans, Monacrosporium thaumasium, Monacrosporium sinense and Arthrobotrys robusta on Angiostrongylus vasorum first stage larvae. J Helminthol 2009; 83(4): 303-308. http://dx.doi.org/10.1017/S0022149X09232342
http://dx.doi.org/10.1017/S0022149X09232...
, 2011bBraga FR, Araujo JM, Tavela AO, Araújo JV, Soares FEF, Geniêr HLA, et al. Atividade larvicida do extrato bruto enzimático do fungo Duddingtonia flagras sobre larvas de primeiro estádio de Angiostrongylus vasorum. Rev Soc Bras Med Trop 2011b; 44(3): 383-385.). Moreover, use of crude extract is also an artifice, albeit recent, that can be used to combat the eggs and/or larvae of geohelminths (BRAGA et al., 2011aBraga FR, Araújo JV, Soares FEF, Araujo JM, Geniêr HLA, Silva AR, et al. Optimizing protease production from an isolate of the nematophagous fungus Duddingtonia flagrans using response surface methodology and its larvicidal activity on horse cyathostomins. J Helminthol 2011a; 85(2): 164-170. http://dx.doi.org/10.1017/S0022149X10000416
http://dx.doi.org/10.1017/S0022149X10000...
, bBraga FR, Araujo JM, Tavela AO, Araújo JV, Soares FEF, Geniêr HLA, et al. Atividade larvicida do extrato bruto enzimático do fungo Duddingtonia flagras sobre larvas de primeiro estádio de Angiostrongylus vasorum. Rev Soc Bras Med Trop 2011b; 44(3): 383-385.).

Proteases produced by nematophagous fungi have been studied because they are important in relation to degradation of the cuticle of nematodes (LIANG et al., 2011Liang L, Liu S, Yang J, Meng Z, Lei L, Zhang K. Comparison of homology models and crystal structures of cuticle-degrading proteases from nematophagous fungi: structural basis of nematicidal activity. FASEB J 2011; 25(6): 1894-1902. http://dx.doi.org/10.1096/fj.10-175653
http://dx.doi.org/10.1096/fj.10-175653...
). Esteves et al. (2009)Esteves I, Peteira B, Atkins SD, Magan N, Kerry B. Production of extracellular enzymes by different isolates of Pochonia chlamydosporia. Mycol Res 2009; 113(8): 867-876. http://dx.doi.org/10.1016/j.mycres.2009.04.005
http://dx.doi.org/10.1016/j.mycres.2009....
stated that there might be differences in the results from enzyme production between different isolates from nematophagous fungi. Furthermore, they stated that culturing conditions seemed to play an important role and should always be standardized so that significant comparisons can be made. Cayrol et al. (1989)Cayrol JC, Djian C, Pijarowski L. Study of the nematocidal properties of the culture filtrate of the nematophagous fungus Paecilomyces lilacinus. Revue Nématolol 1989; 12(4): 331-336. argued that the culturing conditions might affect the toxic properties of a fungus. In a recent study, it was demonstrated that the nematophagous fungus M. thaumasium (NF34a) produces a serine protease (Mt1) with importance in relation to the infection process of first-stage larvae ofA. vasorum (SOARES et al., 2012Soares FE, Braga FR, Araújo JV, Lima WS, Mozer LR, Queiróz JH. In vitro activity of a serine protease from Monacrosporium thaumasium fungus against first-stage larvae of Angiostrongylus vasorum. Parasitol Research 2012; 110(6): 2423-2427. http://dx.doi.org/10.1007/s00436-011-2781-x
http://dx.doi.org/10.1007/s00436-011-278...
). However, the present study is the first to report on optimization of protease production from M. thaumasium.

The crude extract of M. thaumasium (NF34) was efficient in destroying A. vasorum L1. Braga et al. (2009)Braga FR, Carvalho RO, Araujo JM, Silva AR, Araújo JV, Lima WS, et al. Predatory activity of the fungi Duddingtonia flagrans, Monacrosporium thaumasium, Monacrosporium sinense and Arthrobotrys robusta on Angiostrongylus vasorum first stage larvae. J Helminthol 2009; 83(4): 303-308. http://dx.doi.org/10.1017/S0022149X09232342
http://dx.doi.org/10.1017/S0022149X09232...
showed that the isolate (NF34) was effective (p > 0.05) in capturing and destroying A. vasorum L1 under laboratory conditions. Those authors reported that, at the end of the experiment (seven days), the percentage reduction was 74.5%. In that study, the efficiency of predatory action was measured in a solid culture medium, with the fungus already grown. In the present study, the observed percentage reduction was similar (77.4%). However, it needs to be noted that in the present study, this percentage reduction was achieved over a short interval of time (24 hours). These results may eventually contribute towards discovery of new methodologies that might assist in decontamination of geohelminth-infested environments.

In relation to time, the results showed that the enzymatic action of the nematophagous fungus NF34a was effective by the end of 24 hours, thus demonstrating that use of this protease is promising. Furthermore, even after seven days of exposure to conditions resembling natural conditions, the biological activity of this enzyme remained intact. This is an interesting point, because it justifies undertaking capture and destruction of infective larvae of nematodes under laboratory conditions and possibly in the field. This may in the future make a contribution as a tool for further research to combat the infectious forms of nematodes that are potentially zoonotic.

Regarding predatory nematophagous fungi, few reports on production of enzymes and their larvicidal activity have been published (BRAGA et al., 2011aBraga FR, Araújo JV, Soares FEF, Araujo JM, Geniêr HLA, Silva AR, et al. Optimizing protease production from an isolate of the nematophagous fungus Duddingtonia flagrans using response surface methodology and its larvicidal activity on horse cyathostomins. J Helminthol 2011a; 85(2): 164-170. http://dx.doi.org/10.1017/S0022149X10000416
http://dx.doi.org/10.1017/S0022149X10000...
, 2013). The results obtained confirmed that nematophagous fungi were efficient for controlling nematode larvae that are potentially zoonotic. Thus, given the importance of biological control, we suggest that further studies should be conducted on the protease produced by the fungus Monacrosporium thaumasium and its larvicidal activity.

The authors would like to thank CAPES, FAPEMIG, and CNPq for their financial support.

References

  • Ayres M, Ayres JRM, Ayres DL, Santos AS. Aplicações estatísticas nas áreas de ciências biológicas. Belém: Sociedade civil mamirauá; Brasília CNPq; 2003. p. 290.
  • Braga FR, Araújo JV, Soares FEF, Araujo JM, Ferreira SR, Tavela AO, et al. Proteolytic action of the crude extract of Duddingtonia flagrans on Cyathostomins (Nematoda: Cyathostominae) in coprocultures. Rev Bras Parasitol Vet 2013; 22. In press.
  • Braga FR, Araújo JV, Soares FEF, Araujo JM, Geniêr HLA, Silva AR, et al. Optimizing protease production from an isolate of the nematophagous fungus Duddingtonia flagrans using response surface methodology and its larvicidal activity on horse cyathostomins. J Helminthol 2011a; 85(2): 164-170. http://dx.doi.org/10.1017/S0022149X10000416
    » http://dx.doi.org/10.1017/S0022149X10000416
  • Braga FR, Araujo JM, Tavela AO, Araújo JV, Soares FEF, Geniêr HLA, et al. Atividade larvicida do extrato bruto enzimático do fungo Duddingtonia flagras sobre larvas de primeiro estádio de Angiostrongylus vasorum. Rev Soc Bras Med Trop 2011b; 44(3): 383-385.
  • Braga FR, Carvalho RO, Araujo JM, Silva AR, Araújo JV, Lima WS, et al. Predatory activity of the fungi Duddingtonia flagrans, Monacrosporium thaumasium, Monacrosporium sinense and Arthrobotrys robusta on Angiostrongylus vasorum first stage larvae. J Helminthol 2009; 83(4): 303-308. http://dx.doi.org/10.1017/S0022149X09232342
    » http://dx.doi.org/10.1017/S0022149X09232342
  • Cayrol JC, Djian C, Pijarowski L. Study of the nematocidal properties of the culture filtrate of the nematophagous fungus Paecilomyces lilacinus. Revue Nématolol 1989; 12(4): 331-336.
  • Esteves I, Peteira B, Atkins SD, Magan N, Kerry B. Production of extracellular enzymes by different isolates of Pochonia chlamydosporia. Mycol Res 2009; 113(8): 867-876. http://dx.doi.org/10.1016/j.mycres.2009.04.005
    » http://dx.doi.org/10.1016/j.mycres.2009.04.005
  • Gortari C, Cazau C, Hours R. Hongos nematófagos de huevos de Toxocara canis en un paseo público de La Plata, Argentina. Rev Iberoam Micol 2007; 24(1): 24-28. http://dx.doi.org/10.1016/S1130-1406(07)70005-0
    » http://dx.doi.org/10.1016/S1130-1406(07)70005-0
  • Lima WS, Costa HMA, Guimarães MP, Leite ACR. Angiostrongylus vasorum (Baillet, 1866) Nematoda: Protostrongylidae, em cães de Minas Gerais, Brasil. Mem Inst Oswaldo Cruz 1985; 80(2): 233-235. http://dx.doi.org/10.1590/S0074-02761985000200015
    » http://dx.doi.org/10.1590/S0074-02761985000200015
  • Liang L, Liu S, Yang J, Meng Z, Lei L, Zhang K. Comparison of homology models and crystal structures of cuticle-degrading proteases from nematophagous fungi: structural basis of nematicidal activity. FASEB J 2011; 25(6): 1894-1902. http://dx.doi.org/10.1096/fj.10-175653
    » http://dx.doi.org/10.1096/fj.10-175653
  • Meyer WJ, Wiebe MG. Enzyme production by the nematode-trapping fungus, Duddingtonia flagrans. Biotechnol Lett 2003; 25(10): 791-795. http://dx.doi.org/10.1023/A:1023580621840
    » http://dx.doi.org/10.1023/A:1023580621840
  • Mukherjee AK, Rai SK. A statistical approach for the enhanced production of alkaline protease showing fibrinolytic activity from a newly isolated Gram-negative Bacillus sp. strain AS-S20-I. N Biotechnol 2011; 28(2): 182-189.
  • Plackett RL, Burman JP. The design of optimum multifactorial experiments. Biometrika 1946; 33(4): 305-325. http://dx.doi.org/10.1093/biomet/33.4.305
    » http://dx.doi.org/10.1093/biomet/33.4.305
  • Soares FE, Braga FR, Araújo JV, Lima WS, Mozer LR, Queiróz JH. In vitro activity of a serine protease from Monacrosporium thaumasium fungus against first-stage larvae of Angiostrongylus vasorum. Parasitol Research 2012; 110(6): 2423-2427. http://dx.doi.org/10.1007/s00436-011-2781-x
    » http://dx.doi.org/10.1007/s00436-011-2781-x

Publication Dates

  • Publication in this collection
    Apr-June 2013

History

  • Received
    1 Apr 2012
  • Accepted
    20 Sept 2012
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