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Article

Effect and Mechanism of Lepista nuda Mycelia Obtained via In Vitro Culture on the Lifespan of Drosophila melanogaster

School of Pharmaceutical Engineering, Xinyang Agriculture and Forestry University, Xinyang 464000, China
*
Author to whom correspondence should be addressed.
Fermentation 2023, 9(3), 212; https://doi.org/10.3390/fermentation9030212
Submission received: 1 February 2023 / Revised: 17 February 2023 / Accepted: 19 February 2023 / Published: 23 February 2023
(This article belongs to the Special Issue New Research on Fungal Secondary Metabolites)

Abstract

:
To provide a theoretical basis for biogenic fly-killing pesticides, in this study, we sought to examine the lethal effects of Lepista nuda mycelium-supplemented diets in Drosophila melanogaster. In doing so, we also studied the effects of Lepista nuda mycelium-supplemented diets on lifespan, antioxidant enzyme activity, peroxide content, relative transcript amounts of antioxidant enzyme genes, signaling pathways, and lifespan. Lower Lepista nuda mycelium-supplemented diets activated the antioxidant system and prolonged lifespan, while higher mycelium-supplemented diets had a significant toxic effect. After the administration of mycelium-supplemented diets for 24 h, the highest corrected mortality (41.96%) and lifespan inhibition rates (96.50%) were observed. In addition, the antioxidant enzyme activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px); the relative transcript amounts of the key antioxidant enzyme genes SOD, CAT, and TrxR-1; and the lifespan genes Hep and Nrf2 were found to be the lowest, while the contents of the oxidation products malondialdehyde (MDA), protein carbonylation product (PCO), and triglyceride (TG); the relative transcript amounts of the signaling pathway-related genes S6K, TOR, and Keap-1; and the lifespan gene MTH were observed to be the highest after 48 h. Higher Lepista nuda mycelium-supplemented diets significantly inhibited lifespan, acting via the initiation of oxidative stress systems.

Graphical Abstract

1. Introduction

Drosophila melanogaster has an extensive host range, reproduces rapidly, and has the capacity to live in a wide range of climatic conditions, all of which have contributed to the global spread of this economically injurious insect [1]. Recent control measures largely rely on chemical insecticide applications, which are toxic to people, livestock, wild animals, soil, water, food, and the human living environment [2]. These are not sustainable strategies due to administrative constraints and the potential for insecticide resistance [3,4]. For these reasons, it is vital to explore novel sources of environmentally friendly biopesticides. Many secondary metabolites of plants and fungal possess remarkable biological activities [5].
Today, edible-mushroom-derived biocides are often used in place of conventional synthetic pesticides. The hydroalcoholic extract of Pleurotus ostreatus exhibits contact toxicity and has been shown to impact the propagation and spawning of Sitophilus zeamais [6]. Ganoderma lucidum contains compounds that are toxic against Tribolium castaneum and D. melanogaster [7]. Cordycepin from the fruiting body of Cordyceps militaris is toxic against the larvae of the Colorado potato beetle Leptinotarsa decemlineata [8] and has been shown to kill Plutella xylostella [9]. Amanita muscaria (Agaricales, Amanitaceae) can be used against the mosquito Culex quinquefasciatus (Diptera, Culicidae) [10]. Amanita muscaria (L.) Lam. can be used for catching flies when soaked in milk or water [11]. Trametes odorata (Wulfen) Fr. powder keeps insects away from clothing, and among the 175 different species of fungi tested, 79 were found to inhibit insect development [12]. The observed results demonstrate that certain fungi contain repellent, antifeedant, and even toxic compounds that act against insect pests.
Lepista nuda (Bull. ex Fr.) Cooke (Clitocybe genus, Tricholomataceae, Agaricomycetes) is an edible mushroom with a rich and subtle flavor [13] and violet coloration. Recent studies indicate that L. nuda has a wide range of pharmacological functions, including antitumor [14] and antimicrobial [15] properties. L. nuda extracts have been shown to inhibit HIV-1 reverse transcriptase [16] and biofilm production [17] to a certain extent. In fact, extracts of Clitocybe genus fruiting bodies have been shown to exhibit potent insecticidal activity against D. melanogaster [18]. In addition, L. nuda has exhibited insecticidal activity against D. melanogaster larvae [19]. To the best of our knowledge, no previous study has investigated the insecticidal mechanism of L. nuda.
Studies from around the world show that macrofungi and their secondary metabolites have the advantages of strong activity in natural environments, good compatibility with the environment, mixing easily with other drugs, and safety for humans and animals when used correctly [20]. In China in recent years, wild macrofungi specimens have been extensively collected as their habitats are being continually destroyed [21]. Using in vitro cultures minimizes the overexploitation of endangered, rare, or valuable species, and thus represents a methodology that prioritizes sustainable conservation and the rational utilization of biodiversity [22].
Previous studies have shown that exposure of D. melanogaster to 3 Gy electron beam irradiation, 15 mM paraquat (Pq), and the rare-earth element cerium (Ce) can induce oxidative stress, which specifically manifests as a significant increase in malanoldialdehyde (MDA) content. In addition, radiation/Pq/Ce-induced free radicals have been shown to impair antioxidant defense mechanisms, leading to a reduction in superoxide dismutase (SOD) and catalase (CAT) activity and glutathione (GSH) levels. Furthermore, Pq-treated flies exhibited severe locomotor impairments, with 84% of flies unable to fly [23]. There was a significant decrease in mean lifespan, maximum lifespan, and reproductive output with increasing doses of cerium [24]. Recent studies have illustrated that metabolic signaling pathways can mediate age and longevity [25]. For example, target of rapamycin (TOR) is a pivotal regulator of cell proliferation and affects senescence, S6K is a downstream effector of TOR kinase [26], and the Jun kinase (JNK) signaling pathway circuitously conveys the cellular oxidative stress response and extends longevity. Moreover, Hep is a homolog of Drosophila Jun kinase kinase (JNKK), and Drosophila carrying the Hep mutation were shown to be more susceptible to oxidative stress and to have shorter lifespans [27].
At present, the research on D. melanogaster biocontrol with macrofungi is limited. In this study, the toxicity of L. nuda mycelia obtained via in vitro culture was examined against D. melanogaster. Its effects on lifespan, antioxidant enzyme activity, peroxide content, and gene expression related to antioxidant enzymes, signaling pathways, and lifespan were compared, providing a reference for the development and utilization of macrofungi as biological pesticides.

2. Materials and Methods

2.1. Materials

2.1.1. Insects

Adult wild-type D. melanogaster, captured in a local cherry orchard, were reared in classic corn yeast medium and propagated in a light incubator at 25 °C.

2.1.2. Macrofungi Strain

L. nuda were collected from among fallen leaves in the summer of 2021 from the forests of the Ta-pieh Mountain region of China.

2.2. Methods

2.2.1. Isolation and Purification of Mycelia

L. nuda were sterilized, cultivated in a PDA culture using tissue isolation, and incubated at 26 °C. The well-grown slant strains were inserted into a PD in vitro medium without agar, placed in a rotating shaker, and incubated for 6 d at 28 °C at 130 r/min. After in vitro culturing, the mycelia were collected via centrifugation at 6000 r/min for 20 min and rinsed 3 times with distilled water, dried to constant weight at 50 °C, and weighed.

2.2.2. Experimental Design and Grouping

The mycelia obtained from the in vitro culture were mixed at a mass ratio of 1:4 with pure water, homogenized via ultrasonic (240 W) crushing for 1 h, bottled at 4 °C, and sealed. The treatment medium was configured on the basis of the corn yeast medium, and the corn yeast medium was used as the control (CK). The corn flour and distilled water in the experimental treatment groups were subtracted from original masses of 2.5, 5.0, 10, 20, and 40%, and the total mass of the two was replaced with the same mass of L. nuda mycelia homogenate so that the added mass concentrations of L. nuda mycelia were 0.23, 0.46, 0.91, 1.82, and 3.64 g/mL, for a total of five experimental treatment groups (A, B, C, D, and E). Thereafter, the medium was divided into 100 mL triangular flasks and set aside.

2.2.3. Determination of Lethal Effect against D. melanogaster

Two female and two male virgin flies that had been starved within the previous 8 h were inserted into bottles. A total of 80 flies were selected for each experimental group and were incubated at 25 ℃. They were observed 6, 12, 18, and 24 h after being placed in the bottles. D. melanogaster with wings at 45° to the body were considered dead, and the number of dead flies was recorded. Mortality rate (MR, %) and corrected mortality rate (CMR, %) were calculated according to the following formulae:
MR = number of dead flies/number of test flies × 100%,
CMR = (treatment mortality − control mortality)/(1 − control mortality) × 100%.

2.2.4. Measurement of D. melanogaster Lifespan

From the time the virgin flies were introduced into the treatment medium, they were regularly observed and were counted every 6 h. After 24 h of rearing flies using the macrofungal treatment medium, the remaining surviving flies were transferred to the corn yeast medium for further rearing. When pupae were formed, the flies were again transferred to corn yeast medium until all were dead. Median lethal time (LT50, h), mean lifespan (MLS, h), maximum mean lifespan (MMLS, h), and lifespan inhibition rate (LIR, %) were calculated according to the following formulae:
LT50 = the time at which half of each group of flies were dead,
MLS = the sum of the survival time of each group of flies/number of flies in the group,
MMLS = the mean of the lifespan of the last 10 dead flies in each group,
LIR = (mean lifespan of control group − mean lifespan of test group)/mean lifespan of control group × 100%.

2.2.5. Determination of Antioxidant Enzyme Activity and Peroxidation Product Content in D. melanogaster

In addition, 100 mg of female and male flies reared with the treatment medium for 24 and 48 h were weighed. Each group of flies was combined with 0.9 mL of saline solution and homogenized at 2000 r/min for 10 s in an ice bath with an interval of 10 s. This was repeated three times to produce a homogenate. The activity of SOD, CAT, and glutathione peroxidase (GSH-Px), and the MDA content, protein carbonylation product (PCO), and triglyceride (TG) were calculated with an enzyme marker according to the kit’s instructions (Nanjing Jiancheng Institute of Biological Engineering, Nanjing, China.).

2.2.6. Determination the Relative amounts of Transcripts in D. melanogaster

Total mRNA was extracted from D. melanogaster using the Trizol method, as described in the Trizol kit’s instructions (Invitrogen, Waltham, MA, USA). cDNA synthesis was performed according to the instructions of the PrimeScriptTM RT-PCR Kit. The primer sequences used for the quantitative analysis were synthesized by Shanghai Meiji Biomedical Technology Co. (Shanghai, China). Fluorescence real-time quantitative PCR was performed using a QuantStudio 3 real-time quantitative PCR system, the reagents were used with a DyNAmoTM SYBRR Green qPCR kit, and the experimental operations were performed according to the reagent instructions. Data were collected and processed using CFX-Manager, and the relative expressions of the target genes and internal reference genes were calculated using the Ct (2−△△Ct) method. Using ribosomal protein (RP49) as an internal reference gene, high-throughput fluorescent quantitative PCR was used to determine the relative amounts of transcripts of antioxidant enzyme genes, including superoxide dismutase (SOD), catalase (CAT), thioredoxin reductase (TrxR-1), and lifespan-related genes, including methuselah (MTH), nuclear factor erythroid-2-related factor 2 (Nrf2), hemipterus (Hep), and signaling-pathway-related genes, including RPS6-p70-protein kinase (S6K), target of rapamycin (TOR), and Kelch-like ECH-associated protein 1 (Keap-1).

2.2.7. Statistical Analysis

All operations in this experiment were repeated three times (n = 3), and data are expressed as the mean ± standard deviation (SD) of each sample. The significance of difference between means was assessed using one-way ANOVA, followed by post hoc Tukey’s testing using the software package SPSS v26 (SPSS Inc., Chicago, IL, USA). Different lowercase letters in the same column indicate least significant differences (LSDs) at the 5% level (p < 0.05) for each treatment.

3. Results

3.1. Lethal Effects of L. nuda Mycelium-Supplemented Diets on D. melanogaster

The mortality and corrected mortality of flies after receiving L. nuda mycelium-supplemented diets are shown in Table 1. It can be seen from the table that after 6, 12, and 18 h, the mortality of treatment groups A, B, and C was not significantly different from that of the control group. As the time spent receiving the mycelium-supplemented diets increased, the mortality of the five treatment groups exhibited a gradually increasing trend. The corrected mortality of the five treatment groups reached its maximum after 24 h, and the corrected mortality of group E was the highest at 41.96%.

3.2. Effects of L. nuda Mycelium-Supplemented Diets on the Lifespan of D. melanogaster

The lifespans and inhibition rates of flies after receiving L. nuda mycelium-supplemented diets for 24 h are shown in Table 2. It can be seen from Table 2 that, as compared with the control, the LT50, MLS, and MMLS of the A, B, and C groups increased, and the increase in groups B and C was significant as compared with the control. The MLS in group C was the highest (1716 h), i.e., a 21.18% increase as compared with the control. The LT50, MLS, and MMLS in groups D and E were significantly lower than those of the control group, while the MLS in group E was the lowest, i.e., a 96.5% reduction as compared with the control group.

3.3. Effects of L. nuda Mycelium-Supplemented Diets on the Antioxidant Activity of D. melanogaster

The activities of the antioxidant enzymes SOD, CAT, and GSH-Px in male and female flies after receiving L. nuda mycelium-supplemented diets are shown in Figure 1. It can be seen that the activity of the three antioxidant enzymes in both male and female flies exhibited an increasing and then decreasing trend with the increase in the mycelium-supplemented diets after 48 h. As compared with the control group, the enzyme activities in group C increased significantly, and the antioxidant enzyme activities in the males were higher than in the females, i.e., the SOD, CAT, and GSH-Px activities were higher by 9, 142, and 13 U/mg, respectively. As compared with the control group, the enzyme activity in group E decreased significantly, and the enzyme activity at 48 h was lower than at 24 h.

3.4. Effects of L. nuda Mycelium-Supplemented Diets on Peroxidation Product Content of D. melanogaster

As shown in Figure 2, with the gradient increase in the L. nuda mycelium-supplemented diets, the content of the three peroxidation products exhibited a decreasing and then increasing trend in both the male and female flies. The MDA content in treatment group C and the PCO content in groups B and C decreased significantly as compared with the control after 48 h, while the TG contents in groups A, B, and C were not significant. All three peroxisomes in groups D and E significantly increased, and group E exhibited the highest peroxisomal content, with males having higher levels than females.

3.5. Effects of L. nuda Mycelium-Supplemented Diets on the Levels of Antioxidant-Related Gene Transcripts in D. melanogaster

It can be seen from Figure 3 that with the gradient increase in L. nuda mycelium-supplemented diets, the levels of the antioxidant enzymes SOD, CAT, and TrxR-1 key gene transcripts in female and male flies exhibited an increasing and then decreasing trend. The gene transcription in group C was the highest, while that of group E was the lowest. As compared with the control, after 24 h and 48 h, the levels of SOD and TrxR-1 key gene transcripts in female and male flies in groups A, B, and C significantly increased, and gene transcription at 48 h was higher than at 24 h. The levels of SOD, CAT, and TrxR-1 key gene transcripts in groups D and E were lower at 48 h than at 24 h.

3.6. Effects of L. nuda Mycelium-Supplemented Diets on Levels of Signaling Pathway Gene Transcripts in D. melanogaster

It can be seen from Figure 4 that with the gradient increase in L. nuda mycelium-supplemented diets, the levels of S6K, TOR, and Keap-1 signaling pathway gene transcripts all exhibited a decreasing and then increasing trend. The levels of S6K, TOR, and Keap-1 transcripts in groups A, B, and C were significantly reduced, and those in group E were significantly increased. Those in group D were not compared with the control after 24 h.
The levels of S6K, TOR, and Keap-1 transcripts in the five treatment groups at 48 h were higher than those at 24 h. As compared with the control, the levels in group D and E significantly increased. Furthermore, the levels of signaling-pathway-related gene transcripts in group E at 48 h were the highest. The level of S6K transcripts in female flies was 3.07 times higher than in male flies, and the levels of TOR and Keap-1 transcripts in male flies were 2.87 and 2.34 times higher than in female flies, respectively.

3.7. Effects of L. nuda Mycelium-Supplemented Diets on the Levels of Lifespan-Related Gene Transcripts in D. melanogaster

It can be seen from Figure 5 that with the gradient increase in L. nuda mycelium-supplemented diets, the levels of the Hep and Nrf2 lifespan-related gene transcripts exhibited an increasing and then decreasing trend. In addition, MTH expression exhibited a decreasing and then increasing trend. The levels of Hep and Nrf2 transcripts in groups A, B, and C increased significantly as compared with the control after 48 h, while those in group E decreased significantly. The levels of MTH transcripts in groups A, B, and C decreased significantly as compared with the control after 24 h. Moreover, MTH transcription in group C decreased significantly as compared with the control after 48 h, while that in groups D and E increased significantly. The levels of MTH transcripts in group E were the highest, i.e., they were 1.77 times for female flies and 2.37 times for male flies.

4. Discussion

The urgent global need for new pesticides can only be met if we strive to find biologically active secondary metabolites [28]. Various ceramide components [29], sterols, and triterpenoids [30] have been isolated from L. nuda. However, existing studies suggest that lucidenic acid O, lactone [31], lectins [32], fungal cyclic peptides [33], and hemolysins are potential fungal insecticides. Clitocine, a novel nucleoside produced by Clitocybe inversa, has exhibited strong insecticidal activity against the pink bollworm Pectinophora gossypiella [34]. Clitocypin, a fungal cysteine protease inhibitor, exerts an insecticidal effect against Colorado potato beetle larvae by inhibiting their digestive cysteine proteases [35]. Cnispin, a protease inhibitor that inhibits a serine protease called trypsin, is toxic to D. melanogaster as they mainly use serine proteases for digestion [36]. It has been shown that the insecticidal biological function of lectins and protease inhibitors is regulated by the b-Trefoil structure, which enables interactions between lectins and protease inhibitors [37].
In this study, D. melanogaster was fed with varying gradients of L. nuda mycelium-supplemented diets. The results show that L. nuda mycelium-supplemented diets had a lethal effect on D. melanogaster, and the corrected mortality rate was positively correlated with time and concentration. The corrected mortality rate indicated a dose-dependent decrease as the mycelium-supplemented diets increased (Table 1). This dose-dependence is consistent with the virulence of other insecticides against D. melanogaster. Within a certain dose range, the mortality and knockdown times of pyrethroid combined with piperonyl butoxide were higher than those observed for pyrethroid alone, in a dose-dependent manner [38].
Genetic alterations have been documented to interfere with lifespan [39]. To date, a large number of longevity-related genes have been identified in D. melanogaster [40]. There are six possible key pathways associated with lifespan, including the longevity regulatory pathway, the peroxisome pathway, the mTOR signaling pathway, the FOXO signaling pathway, the diabetic complication AGE-RAGE signaling pathway, and the TGF-b signaling pathway. The expression of six representative key pathway genes, including Cat, Ry, S6k, Sod, Tor, Tsc1, and the predicted genes Jra, Kay, and Rheb were significantly altered in aging D. melanogaster as compared to young flies [41]. In this study, after receiving L. nuda mycelium-supplemented diets for 48 h, group E, which received the highest dose, exhibited the lowest levels of SOD, CAT, and GSH-Px; the lowest relative transcript amounts of the key antioxidant enzyme genes SOD, CAT, and TrxR-1; and the lowest levels of the lifespan genes Hep and Nrf2 (Figure 1, Figure 3 and Figure 5). In addition, in this group, the contents of the oxidation products MDA, PCO, and TG; the relative transcript amounts of the signaling pathway-related genes S6K, TOR, Keap-1; and the lifespan gene MTH were the highest (Figure 2, Figure 4 and Figure 5). At 48 h, this group exhibited the highest corrected mortality rate of 41.96% (Table 1) and the highest lifespan inhibition rate of 96.50% (Table 2). The Methuselah (MTH) gene has long been considered to be a determinant of longevity. In addition, various studies have shown that cranberry anthocyanin extract can extend lifespan by downregulating MTH in D. melanogaster [42]. It has been suggested that the activation of TOR [43] and S6K [44] expression can reduce lifespan via rapamycin in D. melanogaster, which is consistent with the results of the present study.
The overconsumption of L. nuda mycelium-supplemented diets by starving D. melanogaster led to a large accumulation of chemical components, such as cnispin and clitocine, in the body. This inhibited the transcription of the key antioxidant enzyme genes SOD, CAT, and TrxR-1, resulting in a large accumulation of the oxidation products MDA, PCO, and TG, which activated the transcription of the signaling-pathway-related genes S6K, TOR, and Keap-1. The enrichment of signaling pathway factors downregulated the transcripts of the longevity genes Hep and Nrf2 and upregulated the transcripts of the lifespan gene MTH, leading to cellular senescence and apoptosis. This eventually led to the lethal effect of the L. nuda mycelium-supplemented diets on D. melanogaster, and the shortened lifespan.
Both female and male D. melanogaster from group C exhibited significant differences in terms of the observed decreases in MDA and PCO contents as compared to the control after L. nuda mycelium-supplemented diets for 24 h (Figure 2). The SOD, CAT, and GSH-Px activities in group C exhibited significant differences in terms of the observed increases as compared to the control after 48 h (Figure 1). Transcripts of the key antioxidant enzyme genes SOD, CAT, and TrxR-1 and the longevity genes Hep and Nrf2 were upregulated at both 24 h and 48 h (Figure 3 and Figure 5).
It has been previously demonstrated that the increased antioxidant enzyme activity of L. nuda mycelia [45] can protect against ROS-induced cell damage, reduce lipid peroxidation, stabilize cell membrane integrity, and upregulate antioxidant and lifespan genes. The transcription and regulation of this activity are described above. Recent research has demonstrated that a Lentinus edodes mycelia polysaccharide (LEMP) exerts potential antiaging effects in vivo and antioxidant activities in vitro [46]. The addition of LEMP to the diet significantly extended the average lifespan of D. melanogaster, reduced MDA levels, and downregulated the expression of the S6K, TOR, and MTH genes [47], which is consistent with the conclusions of this investigation. Thus, L. nuda mycelia can be applied as a fly-killing biopesticide and small amounts of pesticide residues on the surface of fruits are harmless to the environment and humans.

5. Conclusions

In summary, in this study, D. melanogaster received L. nuda mycelium-supplemented diets. Herein, we demonstrated that lower mycelium-supplemented diets activated the antioxidant system and prolonged lifespan, but higher mycelium-supplemented diets had a significant toxic effect on D. melanogaster. Excessive consumption of L. nuda mycelium inhibited the expression of key antioxidant enzyme genes, resulting in a large accumulation of oxidative products, which activated the expression of genes related to the signaling pathway. The enrichment of signaling pathway factors downregulated the longevity of the Hep and Nrf2 gene transcripts, resulting in a reduced lifespan. These results provide a reference for the development of biogenic fly-killing pesticides using L. nuda mycelium. Hence, further studies should consider transcriptome analysis to achieve greater insight into the toxicological mechanisms.

Author Contributions

Resources, data curation, writing—original draft preparation, J.L.; conceptualization, methodology, software, validation, formal analysis, investigation, Y.H.; writing—review and editing, visualization, supervision, D.W.; project administration, N.Z.; funding acquisition, X.Q. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Henan Provincial Science and Technology Research Project, grant number 202102110176, Henan Province Young Backbone Teacher Training Program, grant number 2019GGJS263, Doctoral Start-up Fund of Xinyang Agriculture and Forestry University, and Innovative Research Team of Research on Traditional Chinese Medicine Resources and Series Products Development of Ta-pieh Mountains in Xinyang Agriculture and Forestry University, grant number XNKJTD-009.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

The authors would like to thank Henan Innovation Strategic Alliance of Edible-Medicinal Fungi Industrial Technology and Xinyang Aojite Edible Fungus Development Co., Ltd. for technical assistance and guidance.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Mean (±SD) of the activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) of D. melanogaster after L. nuda mycelium-supplemented diets for 24, 48 h. Means with different letters differ significantly at p < 0.05 (Tukey’s test). CK: control—received basal diet without L. nuda mycelium. A, B, C, D, and E—received L. nuda mycelium-supplemented diet at 0.23, 0.46, 0.91, 1.82, and 3.64 g/mL, respectively.
Figure 1. Mean (±SD) of the activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) of D. melanogaster after L. nuda mycelium-supplemented diets for 24, 48 h. Means with different letters differ significantly at p < 0.05 (Tukey’s test). CK: control—received basal diet without L. nuda mycelium. A, B, C, D, and E—received L. nuda mycelium-supplemented diet at 0.23, 0.46, 0.91, 1.82, and 3.64 g/mL, respectively.
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Figure 2. Mean (±SD) of the content of malondialdehyde (MDA), protein carbonylation product (PCO), triglyceride (TG) of D. melanogaster after L. nuda mycelium-supplemented diets for 24, 48 h. Means with different letters differ significantly at p < 0.05 (Tukey’s test). CK: control—received basal diet without L. nuda mycelium. A, B, C, D, and E—received L. nuda mycelium-supplemented diet at 0.23, 0.46, 0.91, 1.82, and 3.64 g/mL, respectively.
Figure 2. Mean (±SD) of the content of malondialdehyde (MDA), protein carbonylation product (PCO), triglyceride (TG) of D. melanogaster after L. nuda mycelium-supplemented diets for 24, 48 h. Means with different letters differ significantly at p < 0.05 (Tukey’s test). CK: control—received basal diet without L. nuda mycelium. A, B, C, D, and E—received L. nuda mycelium-supplemented diet at 0.23, 0.46, 0.91, 1.82, and 3.64 g/mL, respectively.
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Figure 3. Mean (±SD) of the relative amounts of transcripts of antioxidant enzyme genes, including superoxide dismutase (SOD), catalase (CAT), thioredoxin reductase (TrxR-1) of D. melanogaster after L. nuda mycelium-supplemented diets for 24, 48 h. Means with different letters differ significantly at p < 0.05 (Tukey’s test). CK: control—received basal diet without L. nuda mycelium. A, B, C, D, and E—received L. nuda mycelium-supplemented diet at 0.23, 0.46, 0.91, 1.82, and 3.64 g/mL, respectively.
Figure 3. Mean (±SD) of the relative amounts of transcripts of antioxidant enzyme genes, including superoxide dismutase (SOD), catalase (CAT), thioredoxin reductase (TrxR-1) of D. melanogaster after L. nuda mycelium-supplemented diets for 24, 48 h. Means with different letters differ significantly at p < 0.05 (Tukey’s test). CK: control—received basal diet without L. nuda mycelium. A, B, C, D, and E—received L. nuda mycelium-supplemented diet at 0.23, 0.46, 0.91, 1.82, and 3.64 g/mL, respectively.
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Figure 4. Mean (±SD) of the relative amounts of transcripts of signaling pathway-related genes, including RPS6-p70-protein kinase (S6K), target of rapamycin (TOR), Kelch-like ECH-associated protein 1 (Keap-1) of D. melanogaster after L. nuda mycelium-supplemented diets for 24, 48 h. Means with different letters differ significantly at p < 0.05 (Tukey’s test). CK: control—received basal diet without L. nuda mycelium. A, B, C, D, and E—received L. nuda mycelium-supplemented diet at 0.23, 0.46, 0.91, 1.82, and 3.64 g/mL, respectively.
Figure 4. Mean (±SD) of the relative amounts of transcripts of signaling pathway-related genes, including RPS6-p70-protein kinase (S6K), target of rapamycin (TOR), Kelch-like ECH-associated protein 1 (Keap-1) of D. melanogaster after L. nuda mycelium-supplemented diets for 24, 48 h. Means with different letters differ significantly at p < 0.05 (Tukey’s test). CK: control—received basal diet without L. nuda mycelium. A, B, C, D, and E—received L. nuda mycelium-supplemented diet at 0.23, 0.46, 0.91, 1.82, and 3.64 g/mL, respectively.
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Figure 5. Mean (±SD) of the relative amounts of transcripts of lifespan-related genes, including methuselah (MTH), nuclear factor erythroid-2 related factor 2 (Nrf2), hemipterus (Hep) of D. melanogaster after L. nuda mycelium-supplemented diets for 24, 48 h. Means with different letters differ significantly at p < 0.05 (Tukey’s test). CK: control—received basal diet without L. nuda mycelium. A, B, C, D, and E—received L. nuda mycelium-supplemented diet at 0.23, 0.46, 0.91, 1.82, and 3.64 g/mL, respectively.
Figure 5. Mean (±SD) of the relative amounts of transcripts of lifespan-related genes, including methuselah (MTH), nuclear factor erythroid-2 related factor 2 (Nrf2), hemipterus (Hep) of D. melanogaster after L. nuda mycelium-supplemented diets for 24, 48 h. Means with different letters differ significantly at p < 0.05 (Tukey’s test). CK: control—received basal diet without L. nuda mycelium. A, B, C, D, and E—received L. nuda mycelium-supplemented diet at 0.23, 0.46, 0.91, 1.82, and 3.64 g/mL, respectively.
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Table 1. Mean (±SD) of mortality rate (MR) and corrected mortality rate (CMR) of Drosophila melanogaster after Lepista nuda mycelium-supplemented diets for 6, 12, 18, and 24 h. Flies with wings at 45° to the body were considered dead. Means with different letters differ significantly at p < 0.05 (Tukey’s test). CK: control—received basal diet without L. nuda mycelium. A, B, C, D, and E—received L. nuda mycelium-supplemented diet at 0.23, 0.46, 0.91, 1.82, and 3.64 g/mL, respectively.
Table 1. Mean (±SD) of mortality rate (MR) and corrected mortality rate (CMR) of Drosophila melanogaster after Lepista nuda mycelium-supplemented diets for 6, 12, 18, and 24 h. Flies with wings at 45° to the body were considered dead. Means with different letters differ significantly at p < 0.05 (Tukey’s test). CK: control—received basal diet without L. nuda mycelium. A, B, C, D, and E—received L. nuda mycelium-supplemented diet at 0.23, 0.46, 0.91, 1.82, and 3.64 g/mL, respectively.
Groups6 h12 h18 h24 h
MR/%CMR/%MR/%CMR/%MR/%CMR/%MR/%CMR/%
CK0.00 ± 0.00 c0.00 ± 0.00 c0.00 ± 0.00 c1.67 ± 0.72 d
A0.00 ± 0.00 c0.00 ± 0.00 c0.00 ± 0.00 c0.00 ± 0.00 c0.83 ± 0.72 c0.83 ± 0.72 c1.67 ± 0.72 d0.00 ± 0.00 d
B0.00 ± 0.00 c0.00 ± 0.00 c0.00 ± 0.00 c0.00 ± 0.00 c0.42 ± 0.72 c0.42 ± 0.72 c2.08 ± 1.44 d0.43 ± 0.74 d
C0.00 ± 0.00 c0.00 ± 0.00 c0.00 ± 0.00 c0.00 ± 0.00 c2.08 ± 0.72 c2.08 ± 0.72 c5.83 ± 0.72 c4.24 ± 0.72 c
D5.00 ± 1.25 b5.00 ± 1.25 b8.75 ± 2.50 b8.75 ± 2.50 b11.25 ± 2.50 b11.25 ± 2.50 b17.08 ± 2.60 b15.67 ± 3.11 b
E8.75 ± 1.25 a8.75 ± 1.25 a20.83 ± 1.91 a20.83 ± 1.91 a31.25 ± 2.17 a31.25 ± 2.17 a42.92 ± 3.15 a41.96 ± 2.82 a
Table 2. Mean (±SD) of median lethal time (LT50, h), mean lifespan (MLS, h), maximum mean lifespan (MMLS, h), lifespan inhibition rate (LIR, %) of D. melanogaster after L. nuda mycelium-supplemented diets for 24 h. Means with different letters differ significantly at p < 0.05 (Tukey’s test). CK: control—received basal diet without L. nuda mycelium. A, B, C, D, and E—received L. nuda mycelium-supplemented diet at 0.23, 0.46, 0.91, 1.82, and 3.64 g/mL, respectively.
Table 2. Mean (±SD) of median lethal time (LT50, h), mean lifespan (MLS, h), maximum mean lifespan (MMLS, h), lifespan inhibition rate (LIR, %) of D. melanogaster after L. nuda mycelium-supplemented diets for 24 h. Means with different letters differ significantly at p < 0.05 (Tukey’s test). CK: control—received basal diet without L. nuda mycelium. A, B, C, D, and E—received L. nuda mycelium-supplemented diet at 0.23, 0.46, 0.91, 1.82, and 3.64 g/mL, respectively.
GroupsLT50/hMLS/hMMLS/hLIR/%
CK1360.00 ± 45.83 c1418.00 ± 54.11 b1956.00 ± 70.74 b
A1340.00 ± 45.83 c1426.00 ± 51.73 b1970.00 ± 60.00 b−0.61 ± 3.46 d
B1492.00 ± 58.28 b1660.00 ± 39.04 a2096.00 ± 54.11 a−17.15 ± 4.29 c
C1582.00 ± 40.84 a1716.00 ± 26.15 a2152.00 ± 58.28 a−21.18 ± 6.45 c
D518.00 ± 42.14 d558.00 ± 30.00 c852.00 ± 45.30 c60.59 ± 3.03 b
E42.00 ± 12.00 e50.00 ± 15.10 d96.00 ± 18.00 d96.50 ± 0.93 a
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Li, J.; Huang, Y.; Wang, D.; Zhu, N.; Qiao, X. Effect and Mechanism of Lepista nuda Mycelia Obtained via In Vitro Culture on the Lifespan of Drosophila melanogaster. Fermentation 2023, 9, 212. https://doi.org/10.3390/fermentation9030212

AMA Style

Li J, Huang Y, Wang D, Zhu N, Qiao X. Effect and Mechanism of Lepista nuda Mycelia Obtained via In Vitro Culture on the Lifespan of Drosophila melanogaster. Fermentation. 2023; 9(3):212. https://doi.org/10.3390/fermentation9030212

Chicago/Turabian Style

Li, Jinzhe, Yaqin Huang, Dezhi Wang, Nailiang Zhu, and Xinrong Qiao. 2023. "Effect and Mechanism of Lepista nuda Mycelia Obtained via In Vitro Culture on the Lifespan of Drosophila melanogaster" Fermentation 9, no. 3: 212. https://doi.org/10.3390/fermentation9030212

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