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Endophytic fungus Phomopsis sp. as a source of 3-nitropropionic acid with larvicidal activity against Aedes aegypti (Linnaeus 1762, Diptera: Culicidae)

ABSTRACT

Background:

Aedes aegypti is the primary vector of viruses, such as Zika, chikungunya, yellow fever, and dengue. In this context, a biomonitored chemical study was conducted to evaluate the activity of the crude extract of the endophytic fungus Phomopsis sp. against the larvae of Aedes aegypti.

Methods:

Crude extract, fractions, and isolated substances were evaluated in in-vitro assays against third-stage larvae of Aedes aegypti.

Results:

We isolated 3-nitropropionic acid with an LC50 of 15.172 ppm and LC90 of 18.178 ppm after 24 hours of larval exposure.

Conclusions:

The results indicated that 3-nitropropionic acid exerted larvicidal activity.

Keywords:
Biomonitored chemical; Secondary metabolite; Dengue

The mosquito Aedes aegypti (Linnaeus 1762; Diptera: Culicidae) is responsible for the transmission of the Zika, chikungunya, yellow fever, and dengue viruses, all of which are a cause for concern in health sectors around the world11. Pilz-Junior HL, Lemos AB, Almeida KN, Corção G, Schrekker HS, Silva CE, et al. Microbiota potentialized larvicidal action of imidazolium salts against Aedes aegypti (Diptera: Culicidae). Sci Rep. 2019;9(1):1-8.. In Brazil, the dengue virus is considered hyperendemic with the circulation of all four serotypes (DENV-1 to DENV-4)22. Garcia GA, David MR, Martins AJ, Freitas RM, Linss JGB, Araújo SC, et al. The impact of insecticide applications on the dynamics of resistance: The case of four Aedes aegypti populations from different Brazilian regions. PLoS Negl Trop Dis. 2018;12(2):1-20.. According to the epidemiological bulletin of the Brazil Ministry of Health33. Ministério da Saúde (MS). Secretaria de Vigilância em Saúde - Monitoramento dos casos de arboviroses até a semana epidemiológica 18 de 2022. 2022. 40 p. Available from: https://www.gov.br/saude/pt-br/centrais-de-conteudo/publicacoes/boletins/boletins-epidemiologicos/edicoes/2022/boletim-epidemiologico-vol-53-no18/view.
https://www.gov.br/saude/pt-br/centrais-...
, in the first 4 months of 2022 alone there was a 151.4% increase in registered dengue cases compared with the same period of the previous year.

Synthetic insecticides are the main means of controlling the vectors of these diseases; however, the continuous use of these products has triggered resistance in mosquito populations, as well as affecting non-target organisms and contaminating the environment11. Pilz-Junior HL, Lemos AB, Almeida KN, Corção G, Schrekker HS, Silva CE, et al. Microbiota potentialized larvicidal action of imidazolium salts against Aedes aegypti (Diptera: Culicidae). Sci Rep. 2019;9(1):1-8.. Therefore, it is critical to find new ways to combat transmitting agents, such as natural fungal products, which have shown great potential.

Fungi of the genus Phomopsis are widely distributed and have been identified as a rich source of new compounds with several biological activities44. Flores AC, Pamphile JA, Sarragiotto MH, Clemente E. Production of 3-nitropropionic acid by endophytic fungus Phomopsis longicolla isolated from Trichilia elegans A. JUSS ssp. elegans and evaluation of biological activity. World J Microbiol Biotechnol. 2013;29(5):923-32.. Accordingly, 3-nitropropionic acid (3-NPA) has been isolated from fungal strains of the genus Phomopsis44. Flores AC, Pamphile JA, Sarragiotto MH, Clemente E. Production of 3-nitropropionic acid by endophytic fungus Phomopsis longicolla isolated from Trichilia elegans A. JUSS ssp. elegans and evaluation of biological activity. World J Microbiol Biotechnol. 2013;29(5):923-32.. This acid is known as a neurotoxic substance in mammals and is frequently used as an inducer of Huntington's disease symptoms for evaluation in in-vivo animal models55. Polonio JC, Ribeiro AS, Rhoden SA, Sarragiotto MH, Azevedo JL, Pamphile JA. 3-Nitropropionic acid production by the endophytic Diaporthe citri: Molecular taxonomy, chemical characterization, and quantification under pH variation. Fungal Biol. 2016;120(12):1600-8..

The current literature provides few studies involving endophytic fungi with activity against the larvae of Aedes aegypti. Therefore, the aim of this study was to conduct a biomonitored chemical analysis and obtain a pure substance that could be used as an alternative control for the transmission agent of Zika, chikungunya, yellow fever, and dengue viruses.

The endophytic fungus Phomopsis sp. was isolated from the plant species Passovia stelis (L.) Kuijt (Lorantahaceae) following the method of Oliveira et al.66. Oliveira CM, Silva GH, Regasini LO, Flausino O, López SN, Abissi BM, et al. Xylarenones C-E from an Endophytic Fungus Isolated from Alibertia macrophylla. J Nat Prod. 2011;74(6)1353-7. The plant was collected in February 2012 on the campus of the Federal University of Amazonas (UFAM; 3°08'57"S, 58°26'38"W) in the city of Itacoatiara, Amazonas, Brazil. The species was identified by Dr. Welma Sousa Silva Carneiro of the UFAM Institute of Exact Sciences and Technology. A voucher specimen (No. 244061) was deposited in the herbarium of the National Research Institute of the Amazon (INPA; Manaus, Brazil).

The fungus was identified according to the morphological characteristics of the culture and by comparing the genetic sequencing of rDNA extracted from the mycelia. The extraction was performed according to the protocol described by Ribeiro et al.77. Ribeiro S, Garcia AC, Santos HED, Montoya QV, Rodrigues A, et al. Antimicrobial activity of crude extracts of endophytic fungi from Oryctanthus alveolatus ( Kunth ) Kuijt ( Mistletoe ). African J Microbiol. 2018;12(11):263-8. The internal transcribed spacer region was amplified and bidirectionally sequenced using Sanger sequencing. The generated sequences were compared with reference data available in the GenBank database, indicating 98% similarity with the genus Phomopsis sp.77. Ribeiro S, Garcia AC, Santos HED, Montoya QV, Rodrigues A, et al. Antimicrobial activity of crude extracts of endophytic fungi from Oryctanthus alveolatus ( Kunth ) Kuijt ( Mistletoe ). African J Microbiol. 2018;12(11):263-8.

To obtain the crude extract, Phomopsis sp. was grown in 72 500 mL Erlenmeyer flasks, each containing 250 mL of the aqueous culture medium, dextrose potato broth, previously autoclaved at 121°C for 15 minutes. The endophyte was grown in Petri dishes with a potato dextrose agar medium for 5 days in a biochemical oxygen demand incubator. After sterilization, five pieces (1 cm2) of the endophyte were inoculated. The 72 inoculated vials were incubated at 25°C for 20 days. After this period, the broth was separated from the mycelium by filtering and subjected to liquid-liquid partition with ethyl acetate (EtOAc) (3 × 125 mL). The organic phase was concentrated under reduced pressure, resulting in 2.94 g of crude extract. After confirmation of larvicidal activity, the crude extract was fractionated in a silica normal phase cartridge using an increasing polarity gradient (dichloromethane, EtOAc, and methane [MeOH]), resulting in 19 fractions. Fractions with similar patterns in the comparative thin layer chromatography were collected from F1 to F8 and subjected to evaluation of selective larvicidal activity, with F4 being the most active. This was fractionated by column chromatography packaged with octyldecylsilane and eluted with MeOH:H2O 9:1 (v/v), resulting in 10 subfractions (F4-1 to F4-10). The F4-1 subfraction showed better purity and, through spectroscopic analysis and comparison with data available in the literature, it was possible to identify 3-NPA, which was subjected to a bioassay against the larvae of Aedes aegypti.

The larvicidal bioassays were conducted according to the protocol recommended by the World Health Organization88. World Health Organization (WHO). Guidelines for laboratory and field testing of mosquito larvicides. World Health Organization. 2005. Available from: http://whqlibdoc.who.int/hq/2005/WHO_CDS_WHOPES_GCDPP_2005.13.pdf?ua=1.
http://whqlibdoc.who.int/hq/2005/WHO_CDS...
. The bioassays were conducted at a temperature of 26 ± 2°C and relative humidity of 85%. The selective bioassay was conducted with extracts and fractions at concentrations of 100 and 500 ppm, previously diluted in dimethyl sulfoxide (DMSO), also used as the negative control, at 1% (v/v). The assay was conducted in triplicate in 50 mL cups, each of which contained 10 third-stage larvae of Aedes aegypti and 40 µL of larval food. Mortality was determined at 24, 48, and 72 hours after the start of the bioassay.

Evaluations of 3-NPA were done at concentrations of 20, 18, 16, 14, and 12 ppm, in triplicate. The bioassay was performed in 50 mL cups containing 9.8 mL of well water, 100 µL of larval food, 10 third-stage larvae of Aedes aegypti, and 100 µL of solutions of 3-NPA previously diluted in 1% DMSO (v/v). The negative control was assembled of 9.8 mL of well water, 10 third-stage larvae of Aedes aegypti, 100 µL of larval food, and 100 µL DMSO, corresponding to a concentration of 1% (v/v). The positive control was assembled using Bacillus thuringiensis israelensis (Bti) at 1 ppm. Larval mortality readings were performed 24 and 48 hours after contact with 3-NPA. Larvae that did not respond to the artificial stimuli were considered dead.

The Chi-square, slope ± SE, and R2 tests were used to indicate the non-significance of the lethal concentration values, growth up the curve of mortality, and the linear correlation between concentrations and mortality, calculated using Poloplus software version 1.0. (LeOra Software). The data were analyzed by one-way analysis of variance (ANOVA) and Tukey’s test (p<0.05) using GraphPad Prism software (version 6.0; San Diego, CA, USA) and were expressed as mean (%) ± standard deviation.

The extract caused the death of 18 of 30 larvae at a concentration of 100 ppm, representing 60% mortality after 72 hours of exposure. At 500 ppm, 100% mortality was observed within 24 hours. Fraction 4 showed 100% larval mortality at the two tested concentrations (100 and 500 ppm) in the first 24 hours. No larval death was observed in the control group.

The biomonitored investigation of the crude extract of Phomopsis sp. allowed the isolation of 3-NPA (Figure 1). Its structure was elucidated based on 1H NMR spectroscopic data and by comparison with data obtained by Flores et al.44. Flores AC, Pamphile JA, Sarragiotto MH, Clemente E. Production of 3-nitropropionic acid by endophytic fungus Phomopsis longicolla isolated from Trichilia elegans A. JUSS ssp. elegans and evaluation of biological activity. World J Microbiol Biotechnol. 2013;29(5):923-32. (Table 1).

FIGURE 1:
Chemical structure of 3-nitropropionic acid.

TABLE 1:
1H NMR data of 3-nitropropionic acid obtained at 300 MHz.

The 1H NMR spectrum showed two triplet signals at δ H 2.87 (2H, t, J = 5.9 Hz, H-2) and 4.69 (2H, t, J = 5.9 Hz, H-3), attributed to the coupling 3 J of the vicinal methylene hydrogens H-2 and H-3. The chemical displacement at δ H2 2.87 is characteristic of carboxylic α-hydrogens (-COOH) under greater deprotection caused by the nitro group (-NO2) in position b at H-2. Similarly, the δ H3 signal 4.69 suffers the greatest influence of deprotection due to the position a of the nitro group (-NO2).

The results of the larvicidal bioassays with 3-NPA (Table 2) indicated an LC50 of 15.172 ppm and an LC90 of 18.178 ppm against third-stage larvae of Aedes aegypti. No larval mortality was observed in the negative control, indicating that the observed larvicidal activity was related to the action of 3-NPA.

TABLE 2:
Percentages of mortality of A. aegypti larvae and lethal doses of 3-nitropropionic acid after 24 h of larval exposure.

The secondary metabolism of both plants and endophytic fungi produce 3-NPA and this may result from the interaction of both; therefore, it can be found in different parts of the host species, especially in the leaves, due to the higher occurrence of endophytic species in this organ44. Flores AC, Pamphile JA, Sarragiotto MH, Clemente E. Production of 3-nitropropionic acid by endophytic fungus Phomopsis longicolla isolated from Trichilia elegans A. JUSS ssp. elegans and evaluation of biological activity. World J Microbiol Biotechnol. 2013;29(5):923-32.. Furthermore, 3-NPA of fungal origin is a precursor of other bioactive molecules of interest to the industrial sector and may present antifungal, antibacterial, antiviral, and insecticidal properties, among others55. Polonio JC, Ribeiro AS, Rhoden SA, Sarragiotto MH, Azevedo JL, Pamphile JA. 3-Nitropropionic acid production by the endophytic Diaporthe citri: Molecular taxonomy, chemical characterization, and quantification under pH variation. Fungal Biol. 2016;120(12):1600-8..

At 20 ppm, 3-NPA caused 100% mortality in Aedes aegypti larvae after 24 hours of exposure. These results were compared with the activity of the bacterium Bti in the positive control, which also caused the death of all exposed larvae at 1 ppm. This bacterium exhibits high toxicity to the larvae of the genera Aedes, Culex, and Anopheles, and is one of the most suitable larvicides for the biological control of Aedes aegypti99. Masi M, Cimmino A, Tabanca N, Becnel JJ, Bloomquist JR, Evidente A. A survey of bacterial, fungal and plant metabolites against Aedes aegypti (Diptera: Culicidae), the vector of yellow and dengue fevers and Zika virus. Open Chem. 2017;15(1):156-66.. Rotation of this bacterium with temephos is indicated to prevent resistance1010. Ministério da Saúde (MS). Secretaria de Vigilância em Saúde. Departamento de Vigilância Epidemiológica - Diretrizes Nacionais para a Prevenção e Controle de Epidemias de Dengue. Brasília: MS; 2009. 160 p.. The World Health Organization1111. World Health Organization (WHO). Guidelines for drinking-water quality. 2017. 631 p. recommends the use of Bti at concentrations ranging from 1 to 5 ppm, and the concentration used in this study was within the recommended range.

Temephos is an organophosphorus pesticide that is widely applied in domestic water reserves to control Aedes aegypti at a concentration of 1 ppm1111. World Health Organization (WHO). Guidelines for drinking-water quality. 2017. 631 p.. Its effects on human health are still uncertain; however, studies by Verdín-Betancourt et al.1212. Verdín-Betancourt FA, Figueroa M, López-González ML, Gómez E, Bernal-Hernández YY, Rojas-García AE, et al. In vitro inhibition of human red blood cell acetylcholinesterase (AChE) by temephos-oxidized products. Sci Rep. 2019;9 (14758):1-11. have shown that in chlorinated water, a stable product of temephos oxidation is a potent inhibitor of human acetylcholinesterase, suggesting high toxicological potential. Furthermore, organophosphates are not selective and affect non-target organisms, such as aquatic invertebrates, to which they can cause biological feeding and reproductive dysfunctions1313. Sandoval-Herrera N, Mena F, Espinoza M, Romero A. Neurotoxicity of organophosphate pesticides could reduce the ability of fish to escape predation under low doses of exposure. Sci Rep. 2019;9(10530):1-11.. Therefore, it is important to search for other control products, and 3-NPA is considered a possible alternative.

Few studies have investigated secondary metabolites of endophytic fungi that can be applied to the larvae of Aedes aegypti. To date, the study by Masi et al.99. Masi M, Cimmino A, Tabanca N, Becnel JJ, Bloomquist JR, Evidente A. A survey of bacterial, fungal and plant metabolites against Aedes aegypti (Diptera: Culicidae), the vector of yellow and dengue fevers and Zika virus. Open Chem. 2017;15(1):156-66. is the only study that evaluates the larvicidal activity of 3-NPA. Their results indicated that 3-NPA isolated from the endophytic fungus Diaporthe gulyae causes 100% mortality in the first-stage larvae of Aedes aegypti at concentrations of 1000 ppm and 500 ppm, and 33.3% mortality at 250 ppm. These values are higher than those obtained in the present study where 3-NPA at 20 ppm caused 100% mortality of the third-stage larvae of Aedes aegypti. This disparity may be related to the different susceptibilities of the larval stages to 3-NPA, as observed in larvicidal assays with the compound fosinopril1414. Hasan ZIA, Williams H, Ismail NM, Othman H, Cozier GE, Acharya KR, et al. ISSAC, E. The toxicity of angiotensin converting enzyme inhibitors to larvae of the disease vectors Aedes aegypti and Anopheles gambiae. Sci Rep. 2017;7(45409):1-10.. The results of this study showed that first-stage larvae were more resistant than second- and third-stage larvae, even after 72 hours of treatment with fosinopril.

Masi et al.99. Masi M, Cimmino A, Tabanca N, Becnel JJ, Bloomquist JR, Evidente A. A survey of bacterial, fungal and plant metabolites against Aedes aegypti (Diptera: Culicidae), the vector of yellow and dengue fevers and Zika virus. Open Chem. 2017;15(1):156-66. did not evaluate lethal concentrations of 3-NPA; however, cytochalasin A and gliotoxin isolated from the fungus Neosartorya pseudofischeri (anamorph Aspergillus thermomutatus) were among the most effective substances evaluated against the first-stage larvae of Aedes aegypti, with LC50 of 85.4 ppm and 25.7 ppm, respectively in 24 hours. The results of the present study indicated that 3-NPA presented a lower lethal dose after 24 hours than the substances mentioned, as, at 15,172 ppm, it was able to kill 50% of the third-stage larvae of the species.

Other substances from natural sources, such as plants, have also been evaluated against the larvae of Aedes aegypti. In a study by Nobsathian et al.1515. Nobsathian S, Bullangpoti V, Kumrungsee N, Wongsa N, Ruttanakum D. Larvicidal effect of compounds isolated from Maerua siamensis (Capparidaceae) against Aedes aegypti (Diptera: Culicidae) larvae. Chem Biol Technol Agric. 2018;5(8):4-10., lethal doses of cinnamic and 3,4-dimethoxybenzoic acids, isolated from Maerua siamensis (Capparidaceae) and applied in larvicidal assays on third-stage larvae of Aedes aegypti, were determined 48 hours after larval exposure. The acids presented LC50 of 22.79 and 72.54 ppm, and LC90 of 1581.11 and 2732.78 ppm, respectively1515. Nobsathian S, Bullangpoti V, Kumrungsee N, Wongsa N, Ruttanakum D. Larvicidal effect of compounds isolated from Maerua siamensis (Capparidaceae) against Aedes aegypti (Diptera: Culicidae) larvae. Chem Biol Technol Agric. 2018;5(8):4-10.. In general, when considering the lethal doses of 3-NPA (LC50 = 15.172 ppm and LC90 = 18.178 ppm), this acid was more effective than the substances used in the aforementioned study.

The results of this study suggest that 3-NPA can be used as a larval control agent for Aedes aegypti. Further studies should be conducted to investigate the differences in susceptibility of the larval stages of the species and establish the appropriate concentrations for these different stages. The results of the present study may be useful in the search for new larvicidal compounds as alternatives to temephos, and subsequently minimize larval resistance and environmental impacts, especially on aquatic organisms.

ACKNOWLEDGMENTS

The authors thank INCT Bionat (#465637/2014-0) for the financial support. FAPEAM and CNPq for the grants awarded. We also thank Dr. Welma Sousa Silva (ICET-UFAM) for her support in identifying the plant material.

REFERENCES

  • 1
    Pilz-Junior HL, Lemos AB, Almeida KN, Corção G, Schrekker HS, Silva CE, et al. Microbiota potentialized larvicidal action of imidazolium salts against Aedes aegypti (Diptera: Culicidae). Sci Rep. 2019;9(1):1-8.
  • 2
    Garcia GA, David MR, Martins AJ, Freitas RM, Linss JGB, Araújo SC, et al. The impact of insecticide applications on the dynamics of resistance: The case of four Aedes aegypti populations from different Brazilian regions. PLoS Negl Trop Dis. 2018;12(2):1-20.
  • 3
    Ministério da Saúde (MS). Secretaria de Vigilância em Saúde - Monitoramento dos casos de arboviroses até a semana epidemiológica 18 de 2022. 2022. 40 p. Available from: https://www.gov.br/saude/pt-br/centrais-de-conteudo/publicacoes/boletins/boletins-epidemiologicos/edicoes/2022/boletim-epidemiologico-vol-53-no18/view
    » https://www.gov.br/saude/pt-br/centrais-de-conteudo/publicacoes/boletins/boletins-epidemiologicos/edicoes/2022/boletim-epidemiologico-vol-53-no18/view
  • 4
    Flores AC, Pamphile JA, Sarragiotto MH, Clemente E. Production of 3-nitropropionic acid by endophytic fungus Phomopsis longicolla isolated from Trichilia elegans A. JUSS ssp. elegans and evaluation of biological activity. World J Microbiol Biotechnol. 2013;29(5):923-32.
  • 5
    Polonio JC, Ribeiro AS, Rhoden SA, Sarragiotto MH, Azevedo JL, Pamphile JA. 3-Nitropropionic acid production by the endophytic Diaporthe citri: Molecular taxonomy, chemical characterization, and quantification under pH variation. Fungal Biol. 2016;120(12):1600-8.
  • 6
    Oliveira CM, Silva GH, Regasini LO, Flausino O, López SN, Abissi BM, et al. Xylarenones C-E from an Endophytic Fungus Isolated from Alibertia macrophylla. J Nat Prod. 2011;74(6)1353-7.
  • 7
    Ribeiro S, Garcia AC, Santos HED, Montoya QV, Rodrigues A, et al. Antimicrobial activity of crude extracts of endophytic fungi from Oryctanthus alveolatus ( Kunth ) Kuijt ( Mistletoe ). African J Microbiol. 2018;12(11):263-8.
  • 8
    World Health Organization (WHO). Guidelines for laboratory and field testing of mosquito larvicides. World Health Organization. 2005. Available from: http://whqlibdoc.who.int/hq/2005/WHO_CDS_WHOPES_GCDPP_2005.13.pdf?ua=1
    » http://whqlibdoc.who.int/hq/2005/WHO_CDS_WHOPES_GCDPP_2005.13.pdf?ua=1
  • 9
    Masi M, Cimmino A, Tabanca N, Becnel JJ, Bloomquist JR, Evidente A. A survey of bacterial, fungal and plant metabolites against Aedes aegypti (Diptera: Culicidae), the vector of yellow and dengue fevers and Zika virus. Open Chem. 2017;15(1):156-66.
  • 10
    Ministério da Saúde (MS). Secretaria de Vigilância em Saúde. Departamento de Vigilância Epidemiológica - Diretrizes Nacionais para a Prevenção e Controle de Epidemias de Dengue. Brasília: MS; 2009. 160 p.
  • 11
    World Health Organization (WHO). Guidelines for drinking-water quality. 2017. 631 p.
  • 12
    Verdín-Betancourt FA, Figueroa M, López-González ML, Gómez E, Bernal-Hernández YY, Rojas-García AE, et al. In vitro inhibition of human red blood cell acetylcholinesterase (AChE) by temephos-oxidized products. Sci Rep. 2019;9 (14758):1-11.
  • 13
    Sandoval-Herrera N, Mena F, Espinoza M, Romero A. Neurotoxicity of organophosphate pesticides could reduce the ability of fish to escape predation under low doses of exposure. Sci Rep. 2019;9(10530):1-11.
  • 14
    Hasan ZIA, Williams H, Ismail NM, Othman H, Cozier GE, Acharya KR, et al. ISSAC, E. The toxicity of angiotensin converting enzyme inhibitors to larvae of the disease vectors Aedes aegypti and Anopheles gambiae Sci Rep. 2017;7(45409):1-10.
  • 15
    Nobsathian S, Bullangpoti V, Kumrungsee N, Wongsa N, Ruttanakum D. Larvicidal effect of compounds isolated from Maerua siamensis (Capparidaceae) against Aedes aegypti (Diptera: Culicidae) larvae. Chem Biol Technol Agric. 2018;5(8):4-10.
  • Financial Support: The authors thank INCT Bionat (#465637/2014-0) for the financial support. FAPEAM and CNPq for the grants awarded.

Publication Dates

  • Publication in this collection
    24 Oct 2022
  • Date of issue
    2022

History

  • Received
    12 Jan 2022
  • Accepted
    14 Sept 2022
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