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Table of Contents
Year : 2021  |  Volume : 58  |  Issue : 4  |  Page : 346-351

Production of conidia using different culture media modifies the virulence of the entomopathogenic fungus Metarhiziumys against Aedes aegypti larvae

1 Universidade Estadual do Norte Fluminense Darcy Ribeiro, CCTA-LEF Av. Alberto Lamego 2000 Campos dos Goytacazes RJ 28013-602, Brazil
2 UFSC Faculdade de Biociências, Florianópolis, SC, Brazil

Date of Submission29-Jun-2020
Date of Acceptance21-Jan-2021
Date of Web Publication25-Mar-2022

Correspondence Address:
Richard Ian Samuels
UENF-CCTA-LEF, Av. Alberto Lamego 2000, Campos dos Goytacazes, RJ 28013-602
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0972-9062.318315

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Background & objectives: Entomopathogenic fungi are being investigated for the biological control of a range of mosquitoes. Metarhizium conidiospores (conidia) effectively kill Aedes aegypti larvae and could be deployed as an alternative to chemical insecticides. Conidial yield and virulence of fungi when cultured on three different types of solid media, was investigated.
Methods: Three culture media were tested: a) Sabouraud dextrose agar (SDA); b) rice flour yeast agar (RYA) and c) rice grains. Conidia produced using these substrates were tested for virulence against Ae. aegypti larvae obtained from field collected eggs. Larvae (2nd – 3rd instar) were exposed to aqueous conidial suspensions and survival monitored over 7 days. Survival analysis was performed using Log-Rank and Kaplan Meier tests, while fungal growth and conidial yields were analyzed using a two-way ANOVA.
Results: There were only small differences between growth rates on RYA and SDA; however, ESALQ 818 showed the highest conidial yield on rice. Conidia produced on rice grains were more virulent, rapidly reducing survival rates of mosquito larvae. ESALQ 818 conidia produced on rice grains, RYA and SDA killed 100% of the larvae on the 2nd, 3rd and 4th day of exposure, respectively. IP 46 virulence of was consistently lower than ESALQ 818 for all the media tested.
Interpretation & conclusion: The choice of culture media can influence the virulence of fungal conidia to Ae. aegypti larvae, demonstrating the importance of not only selecting the most virulent isolate but also standardizing growth conditions when screening for virulence.

Keywords: Culture media; Entomopathogenic fungus; Virulence; Infection; Biological control

How to cite this article:
Carolino AT, Teodoro TB, Gomes SA, Silva CP, Samuels RI. Production of conidia using different culture media modifies the virulence of the entomopathogenic fungus Metarhiziumys against Aedes aegypti larvae. J Vector Borne Dis 2021;58:346-51

How to cite this URL:
Carolino AT, Teodoro TB, Gomes SA, Silva CP, Samuels RI. Production of conidia using different culture media modifies the virulence of the entomopathogenic fungus Metarhiziumys against Aedes aegypti larvae. J Vector Borne Dis [serial online] 2021 [cited 2022 May 21];58:346-51. Available from: https://www.jvbd.org/text.asp?2021/58/4/346/318315

  Introduction Top

Aedes aegypti Linnaeus, 1762 (Diptera: Culicidae) is a vector of four highly important etiological agents: dengue, chikungunya, Zika and urban yellow fever[1]. Application of synthetic insecticides is currently the most important method used to control these insects and reduce the incidence of diseases[2]. However, the cumulative effects of using chemical insecticides, which select resistant insects, as well as damage the environment and cause human health concerns, have encouraged the search for alternative methods of mosquito control[3]. Alternative control methods could help reduce the negative impact on the environment caused by the action of chemical agents[4],[5]. The biological control of vector insects is promising, especially with new developments in the deployment and formulation of entomopathogenic fungi[6]. Fungal encapsulation techniques[7], combining fungi with attractive baits[8] and the use of blastospores rather than conidia[9], are some of the new approaches being investigated to improve the efficiency of fungal biological control agents. Furthermore, the bioactive metabolites produced by entomopathogenic fungi could also be a source of novel insecticidal compounds. A recent study showed that Metarhizium anisopliae metabolites had larvicidal activity against three species of vector mosquitoes, Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus[10].

Conidiospores (conidia) are the most highly investigated and utilized agents for the microbial control of insects, although some studies have shown that blastospores can be more virulent than conidia[9],[11]. The virulence of conidia against larvae and adult Ae. aegypti has been demonstrated under laboratory and field conditions[11],[12]. Metarhizium anisopliae (Metschn.) Sorokin (Hypocreales: Clavicipitaceae) conidia differ from blastospores of the same fungus in that they possess a thick cell wall, which makes this structure more tolerant to the stress of natural environmental conditions[13].

The use of different culture media has been investigated with the aim of improving the efficiency of conidia and blastospores[14],[16]. The influence of different substrates on virulence against Ae. aegypti larvae and adults was investigated for two species of entomopathogenic fungi, M. anisopliae and Beauveria bassiana[11]. Maldonado-Blanco and co-workers[11] demonstrated that the highest levels of virulence were recorded when M. anisopliae was cultured in casamino acids medium. Culture media can exert an effect on the production of key enzymes involved in the infection process, such as Pr1 and Pr2[18], and these enzymes are considered as virulence factors[19]. For mass production of conidia, the use of rice grains as a substrate is economical and highly efficient[20], although alternative substrates have also been investigated such as sorghum, wheat bran[21], barley[22], sugar-cane bagasse[23] and millet[24]. When Metarhizium anisopliae was cultured on millet, the conidia were shown to have potential for the control of the Asian tiger mosquito Aedes albopictus larvae[24]. However, rice continues to be an excellent substrate, producing high concentrations of infectious propagules[25].

The current study investigated the effect of three different culture media on conidial production and virulence. The virulence of two species of Metarhizium (Metarhizium anisopliae and Metarhizium humberi) was evaluated against Ae. aegypti larvae. The results highlight the importance of the correct choice of conidial production media when considering specific insect targets, not only to optimize yield, but also to maximize virulence.

  Material & Methods Top

Mosquito collection and rearing

All of the experiments used larvae which were reared from eggs collected in the field (University Campus). One of the aims of this study was to test the virulence of fungal isolates against mosquito larvae from natural populations, rather than laboratory lines. It is probable that laboratory mosquito lines are not as fit as natural populations and therefore any new control strategies should be tested against genotypes that are present in the field.

Aedes aegypti eggs were collected in the field using “ovitraps”. Ovitraps were black plastic plant pots (12 cm in diameter x 15 cm in height) with 4 wooden strips (3 x 12 cm) placed vertically within the pots, which act as attractive surfaces for mosquito oviposition. About 300 mL of tap water was added to each ovitrap. The ovitraps were placed near the University Insectary (Latitude: -21°45’8.11”S; Longitude: -41°19’49.58”W) in positions that were protected from rain and direct sunlight. After 5 days, the wooden strips were dried at room temperature for 24 h. The strips were examined for the presence of eggs. One strip could normally collect on average up to 100 eggs. This technique specifically collects Aedes eggs and adult emergence studies by our group have shown that > 90% of the eggs were Aedes aegypti and <10% were Aedes albopictus (unpublished results).

Strips with eggs were then submerged in water to stimulate larval emergence. Larvae were maintained in plastic trays (approximately 100 larvae per 100 mL) and feed on freshly ground and autoclaved commercial fish food (Alcon®, Camboriú, SC, Brazil; 0.05 g per L). Second and third instar larvae were selected for bioassays as there was sufficient time before pupation occurred to evaluate differences in survival. Fourth and fifth instar larvae are not appropriate for this type of bioassay.

Fungal isolates

Metarhizium anisopliae (ESALQ 818) was obtained from the Universidade de São Paulo - Escola Superior de Agricultura “Luiz de Queiroz” (ESALQ) collection in Piracicaba, São Paulo, Brazil. Metarhizium humberi (IP 46) was obtained from the Institute of Tropical Pathology and Public Health, at the Federal University of Goiás, Goiânia, Brazil. ESALQ 818 was chosen for this study as virulence of this isolate against Ae. aegypti larvae has been demonstrated by our research group[26]. IP 46 was chosen for this comparative study as it is a promising candidate for mosquito biological control programs.

Fungal culture

For the virulence assays, three different media were used: (1) Sabouraud Dextrose Agar (SDA); (2) rice flour + yeast Agar media (RYA) and (3) rice grains. SDA (dextrose 10 g; peptone 2.5 g; yeast extract 2.5 g; agar 20 g in 1 L H20) was prepared following manufacture instructions (LABSYNTH Ltd. São Paulo, Brazil) and autoclaved for 20 min at 121°C. For RYA, 20 g of rice flour (SADIA Ltd. São Paulo, Brazil) was mixed with 2.5 g of yeast extract (LABSYNTH Ltd.) and 20 g of agar (KASVI Ltd. São Paulo, Brazil) in 1L H20 and autoclaved as described for SDA. Five hundred μL of conidial suspensions (1x107 conidia mL-1) of both Metarhizium isolates were inoculated onto SDA and RYA plates.

For rice grain media, 50g of parboiled white grain rice (Tio João, São Paulo, Brazil) were presoaked in 30 mL of water for 30 min, placed in 250 mL conical flasks and then autoclaved as above. A volume of 500 μL of conidial suspensions of either ESALQ 818 or IP 46 adjusted to 1x107 conidia mL-1, following conidial concentration evaluation using a Neubauer hemocytometer, was inoculated into the previously sterilized conical flasks containing rice. All fungal cultures were incubated at 27°C and 12L:12D photoperiod for 15 days before harvesting the conidia by rinsing the Petri dishes with aqueous Tween 80 (0.05%). These suspensions were used in the subsequent experiments.

Growth and conidi alyield of fungal isolates on different culture media

In order to determine the effect of different media on fungi growth and conidial yield, 10 μL of conidial suspensions of ESALQ 818 or IP 46 (1x106 conidia mL-1) were inoculated in the center of plastic Petri dishes (9 cm diameter) containing SDA or RYA media. All plates were incubated at 27 °C and 12L: 12D photoperiod. Conidial production on rice grains was determined following inoculation of 10 μL of fungal suspensions (1x106 conidia mL-1) onto 5 g of sterilized parboiled rice in glass vials (25 mL). The total radial growth (cm) of the colonies on SDA and RYA media were measured on the 15th day after fungal inoculation using a steel ruler. All experiments were performed three times.

The conidial production of both isolates on the three different media was estimated on the 15th day after inoculation. Conidia were harvested from SDA and RYA using a spatula and added to 10 mL of Tween (0.05%). The conidia grown on rice were rinsed off the grains by adding 10 mL water + Tween 80 to each tube and the tubes were vigorously agitated using a vortex mixer for 1 min. The conidial concentration in each suspension was then estimated using a Neubauer hemocytometer.

Survival rates of larvae exposed to conidia produced on different substrates

The bioassays were conducted to investigate the virulence of conidia produced on three different substrates. The experiments were carried out using Ae. aegypti larvae (L2-3) obtained from eggs collected in the field using a standard procedure[11]. Conidia of both isolates cultivated on three substrates were harvested using a spatula and suspended in sterile distilled water + Tween 80 (0.01%). Conidial concentration was determined using a Neubauer hemocytometer and adjusted to 1x106 conidia mL-1 by serial dilution.

The assays were conducted by placing ten larvae in each plastic cup (8.4 cm height x 6.3 cm diameter) with 50 mL of conidial suspensions. Therefore, larvae were constantly exposed to the conidial suspension. Larval mortality was recorded daily for 7 days. The assays were conducted at room temperature (25–27 °C). Control treatments were carried out using Tween 80 (0.01%) only. For each treatment group 40 larvae were used. Therefore, a total of 160 larvae were used per isolate per repetition (40 larvae for each of the three substrates + 40 control larvae). All experiments were carried out four times.

Statistical analysis

GraphPad Prism 8 software was used for survival curve comparisons (Log-rank Mantel-Cox) and median survival time (S50) estimated from Kaplan-Meier analysis. Insects which survived the 7-day experimental period were regarded as “censored”. The data for fungal growth and spore production were submitted to normality (Shapiro-Wilk) and equality of variance tests. Normally distributed data was then analyzed using a two-way ANOVA and Tukey’s post-hoc test using SigmaPlot 12.

  Results Top

Radial growth and conidial production

The study was conducted evaluate the radial growth rates of both species of fungi when cultured on either SDA or RYA. The Shapiro-Wilk test confirmed normality of this data (P= 0.899) and equality of variance (P = 0.412). The two-way ANOVA showed that there was a significant effect of fungal species on the colony growth (diameter) (F (1) = 7.511, P= 0.025), but no significantly effect of the different media on growth (F (1) = 2.844, P = 0.130). The average radial growth of ESALQ 818 on day 15 was 5.76 cm (± 0.21 SEM) on SDA and 6.03 cm (± 0.16) on RYA. The average mycelial growth of IP 46 was higher than that of ESALQ 818 with a mean of 6.20 cm (± 0.15) on SDA and 6.46 cm (± 0.03) 15 days after inoculation on RYA.

The data for conidial production on all three substrates (SDA, RYA and rice grains) was normally distributed (Shapiro-Wilk; P = 0.225). The result showed that there was a significant difference between conidial yields when comparing the two species of Metarhizium tested here cultured on the three different substrates (F (1) =1318.33; P < 0.001). ESALQ cultured on rice produced significantly more conidia (approximately x5) than IP 46 cultured on rice [Table 1].
Table 1: Conidial yield of Metarhizium anisopliae (ESALQ 818) and Metarhizium humberi (IP 46) when cultured on different substrates

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There was also a significant difference between the conidial yields when considering each fungus grown on the three substrates (F (2) = 1457.78; P < 0.001). ESALQ 818 produced the highest conidial yields when grown on rice grains when compared to SDA or RYA [Table 1]. IP 46 also produced the highest yields when grown on rice [Table 1].

Effect of culture media on virulence

ESALQ 818 conidia produced all three types of culture media caused significant reductions in Ae. aegypti larval survival when tested at a concentration of 1x106 conidia mL-1 [Figure 1]. The survival curves were significantly different when comparing all treatments, including control survival, 48h after exposing the larvae to conidia (χ2 = 552.9, df = 3, P < 0.001).
Figure 1: Survival curves of Aedes aegypti larvae exposed to ESALQ 818 conidia which had been produced on SDA, RYA and Rice grains. Larvae were exposed to a concentration of 1x106 conidia mL-1. The bars represent the standard error of the mean.

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Conidia produced on rice grains were more virulent than conidia produced on the two other substrates, causing 100% of mortality within 2 days (S50 = 1 day). When larvae were exposed to conidia produced on RYA and SDA, 100% mortality was only observed on day 3 and day 4, respectively (S50 = 2 days for both treatments). Control survival was high throughout the experiment (96.2 % on day seven).

The virulence of IP 46 conidia produced on rice grains was significantly different to that of conidia produced using the other two substrates (χ2 = 570.4, df = 3, P < 0.0001). These comparisons were made using Log-rank survival curve analysis [Figure 2]. When larvae were exposed to conidia produced on rice, 100% died within 72 hours. Conidia produced on SDA and RYA were less virulent, with 80% and 82% of the larvae still alive after 24 hours, respectively (S50 = 3 days for both treatments). At the end of the bioassay (day 7), conidia produced on RYA had killed higher percentages of the larvae (3% survival) than conidia produced on SDA (11% survival). Control larval survival was 96.8 % on day 7 [Figure 2].
Figure 2: Survival curves of Aedes aegypti larvae exposed to IP 46 conidia which had been produced on SDA, RYA and Rice grains. Larvae were exposed to a concentration of 1x106 conidia mL-1. The bars represent the standard error of the mean.

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  Discussion Top

Different entomopathogenic fungi, even when closely related, can display different levels of virulence towards the same target insect[24]. In the current study, when comparing the virulence of Metarhizium anisopliae (isolate ESALQ 818) to that of Metarhizium humberi (isolate IP 46),M. anisopliae was significantly more virulent than M. humberi against Ae. aegypti larvae. Differences in virulence of entomopathogenic fungi have also been observed when modifying culture conditions. However, few studies have investigated the effect of different culture media on the virulence of fungi against disease vectors such as Ae. aegypti. When testing the virulence of Metarhizium and Beauveria spores produced using a range of liquid substrates, no significant differences were seen in spore yield but only spores produced in one of the substrates (casamino acids) were virulent to Ae. aegypti larvae[17]. The virulence levels seen in the current study were much higher than those observed by Maldonado-Blanco and co-workers[17].

Virulence of the two species of fungi investigated here was influenced by cultivation on different substrates. Cultivation of both species of fungi on rice grains resulted in the highest virulence (lowest S50 values) against natural populations of Ae. aegypti larvae, and in the case of ESALQ 818, the use of rice media also resulted in significantly greater conidial yields [Table 1].

One of the most commonly used media for fungal culture is SDA[27]. However, as shown here, the virulence of conidia produced on this media was lower than that of conidia produced on either RYA or rice grains. It is therefore important to evaluate virulence of conidia produced on rice grains otherwise possible candidates for biological control programs could be underestimated. An interesting alternative for small scale conidial production in the laboratory is the use of RYA. In all of the experiments carried out here, the virulence of conidia produced on this substrate was higher than that of conidia produced on SDA and larger amounts of conidia were also obtained using RYA when compared to SDA for both species of fungi [Table 1].

It is known that nutritional factors can directly affect virulence. The amount of carbon and nitrogen stored in conidia can alter the ability of the fungus to germinate and infect organisms[28]. In addition, the level of stress under which entomopathogens are cultured may also influence germination and production of cuticle degrading enzymes with consequences for virulence levels[29]. When tested against Tenebrio molitor (Coleoptera), M. anisopliae cultivated under nutritive stress on minimal medium, supplemented with lactose, produced highly virulent conidia with enhanced rates of adhesion to the host cuticle when compared to conidia produced using nutrient rich media[30].

Metarhizium humberi (IP 46) is considered to be a highly promising candidate for vector control as it has been shown to be pathogenic to adult malaria mosquitoes Anopheles gambiae s.s. and Anopheles arabiensis[3]. It is also pathogenic to Ae. aegypti eggs[32], larvae[33] and adults[34]. However, those studies were carried out using conidia produced using SDA and PDA (potato dextrose agar) media, which could have negatively influenced the results. Here, we have shown that IP 46 conidia produced on SDA media were significantly less virulent than conidia produced on RYA or rice grains when tested against Ae. aegypti larvae. These results highlight the need to standardize the substrates used when comparing fungal virulence.

It is not known which factors provided by rice flour increased virulence when compared to nutrient rich SDA, although studies have shown that limiting nutrients can have a positive effect on virulence[35]. Conidia produced on rice grains tolerate stress and produce higher levels of destruxins and proteases such as Pr1 when compared to conidia cultivated on other substrates[29]. Rice is the most commonly used substrate for commercial conidial production. It is economical and has positive effects on fungal germination, providing a surface area that allows rapid fungal colonization and has the capacity to absorb moisture, contributing to faster germination and high conidial viability[20]. Another factor to be considered is post-harvest storage. A study of the effect of different substrates on conidial production showed that storage conditions were more critical to virulence against Culex pipiens pipiens larvae than the substrate used to produce the conidia[35].

  Conclusion Top

The present study showed that the choice of substrate used for conidial production is an important consideration for optimizing virulence against Ae. aegypti larvae. Further studies should be carried out to determine whether other substrates could further increase the virulence of the fungi, with concomitant improvement in their potential for use in mosquito biological control strategies.

Conflict of interest: None

  Acknowledgements Top

We wish to thank UENF for the post-doctoral research grant awarded to ATC. We also thank FAPERJ (E26/201.336/2016 & E26/202.923/2019) and CNPq (431766/2016-9) for financial support. RIS is a CNPq Research Fellow.

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  [Figure 1], [Figure 2]

  [Table 1]


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