Galleria mellonella is used as a model organism to study the innate immune response of insects. In this study, the humoral immune response was assessed by examining phenoloxidase activity, fungal burden, and the expression of phenoloxidase and antimicrobial peptide genes at different time point following separate and combined injections of Hypericum perforatum extract and a nonlethal dose of Candida albicans. The administration of a plant extract at low doses increased phenoloxidase activity, while higher doses had no effect. Similarly, co‐injection of a low dose of the extract with the pathogen allowed half of the yeast cells to survive after 24 h. Co‐injection of plant extract with the pathogen decreased the phenoloxidase activity at the end of 4 h compared to C. albicans mono‐injection. The phenoloxidase gene expressions was reduced in all experimental conditions with respect to the control. When plant extracts and the pathogen were administered together, gallerimycin and hemolin gene expressions were considerably higher compared to mono‐injections of plant extracts and the pathogen. The results of this study reveal that gene activation and regulatory mechanisms may change for each immune gene, and that recognition and signaling pathways may differ depending on the involved immunoregulator.

Insects have an effective innate immune system that includes both cellular and humoral responses for defense against pathogens. Antimicrobial peptides like gallerimycin and galiomycin, as well as immune proteins like hemolin, are the important effectors of the humoral immune response in Galleria mellonella L. (Lepidoptera:Pyralidae). Encapsulation, on the contrary, is one of the important cellular immune responses. This study investigated the tissue‐specific expression of an immune effector in G. mellonella larvae after injection with Candida albicans (C.P. Robin) (Ascomycota: Debaryomycetaceae) and silica beads. The gene expression of gallerimycin, galiomycin, and hemolin was examined in total larvae, hemocytes, and fat bodies at 4 and 24 h following injection. Our findings indicate that hemocytes serve as the main site for AMP synthesis, especially after bead injection, implying a more effective immune recognition mechanism relative to pathogen injection. Furthermore, we detected higher hemolin expression in hemocytes than fat tissue, indicating its role in hemocyte‐mediated immune responses. Encapsulation rates were also evaluated in bead‐injected larvae. At 4 h post‐injection, most beads were weakly encapsulated, whereas by 24 h, the majority were strongly encapsulated, reflecting a time‐dependent maturation of the immune response. These results show that G. mellonella has a unique immune system, with hemocytes playing a key role in regulating AMP production and immune responses during infection. This study provides deeper insights into the molecular and cellular mechanisms of insect immunity, positioning G. mellonella as a valuable model for studying host–pathogen interactions.

Gunay M, Genc TT. 2023. Molecular identification of yeasts from Turkish traditional cheeses: Extracellular enzyme activities and physiological properties important for dairy industry. Nusantara Bioscience 15: 1-11. The determination of yeast microbiota in cheeses and the physiological properties of yeasts are very important for the dairy industry. In addition, the physiological features, proteolytic and lipolytic activities, and stress tolerance of yeasts have a significant role in the selection of starter yeast species for cheese ripening. This study aimed to determine industrially important yeasts isolated from cheese samples. Molecular techniques identified the isolated yeast strains. The yeast strains’ extracellular enzyme activities, fermentation capacities, and thermotolerance and osmotolerance properties were also evaluated. A total of 81 yeast strains were isolated and characterized from three types of cheese samples. PCR-RFLP determined the isolated yeast strains and sequence analysis of ITS1-5.8S-ITS2 and 26S rDNA regions. A maximum parsimony tree was constructed by MEGA X software to evaluate the phylogenetic relationship of identified yeast strains. Candida intermedia, Candida parapsilosis, Clavispora lusitaniae, Debaryomyces hansenii, Kluyveromyces marxianus, Pichia kudriavzevii, and Wickerhamomyces anomalus yeast species were identified on cheese samples. The distribution of identified yeast species on cheese samples was determined as 48.1% for W. anomalus, 17.3% for K. marxianus, 14.8% for C. parapsilosis, 8.6% for D. hansenii, 4.9% for Cl. lusitaniae, 3.7% for C. intermedia and 2.5% for P. kudriavzevii. The W. anomalus yeast species was common in three cheese types. All strains of W. anomalus and P. kudriavzevii yeast species, three C. parapsilosis, and two Cl. lusitaniae yeast strains have important physiological properties for industrial applications. These yeast strains have the potential to be used in combination as starter cultures to improve cheese maturation in the future. This comprehensive study identifies yeast species by ITS1-5.8S-ITS2 and 26S rDNA regions and determines industrially important yeast species using multiple criteria (extracellular enzyme activity, stress tolerance, and fermentation capacity).

Saccharomyces cerevisiae accumulates trehalose as a stress metabolite in adverse environmental conditions. The trehalose synthesis and breakdown are important for the regulation of trehalose levels within the yeast cell. Therefore, TPS1 and NTH1 gene expressions are tightly regulated during transcription and also translation. Since both genes contain Stress Response Elements (STRE) in the promoter regions, they are co-activated under stress conditions. However, the presence of similar regulatory elements in the promoter of both genes shows that these genes undergo a different regulation at the transcriptional level. In our study, the role of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex in the transcriptional regulation of TPS1 and NTH1 genes was determined in nutrient-poor environment. For that purpose, the wild type and Δada1 mutant yeast cells, where Ada1p is a member of the SAGA complex, were grown in normal and nitrogen starvation conditions. In addition, trehalose level was detected enzymatically in both wild type and mutant yeast cells. In silico promoter analysis of TPS1 and NTH1 promoters revealed that the STRE sequences required for binding of Msn2/4 transcription factors are closed by nucleosomes at the NTH1 promoter, but open at the TPS1 promoter. In the absence of Ada1p, stress-induced promoter activation in the TPS1 gene was observed, while NTH1 gene expression was not activated. According to these results, the nucleosomes spanning the STRE sequences could not be mobilized in the absence of Ada1 protein, and therefore the Msn2/4 transcription factors cannot bind to the promoter and activate the NTH1 gene expression under stress conditions. It was also observed that in the absence of Ada1p, trehalose accumulation was reduced regardless of stress conditions.

Genc TT, Günay M. 2020. Internal transcribed spacer (ITS) sequence-based identification of yeast biota on pomegranate surface and determination of extracellular enzyme profile. Nusantara Bioscience 12: 59-67. Yeasts are the most significant organisms to produce fermented products from different types of fruits such as grape, strawberry and pomegranate. The native yeasts on these fruits contribute to beverages’ quality and aroma during fermentation. Pomegranate is used in fruit juice and wine production because of high antioxidant characteristic. In order to determine yeast microbiota on the pomegranate fruits collected from Gallipoli (Gelibolu), Çanakkale-Turkey, ITS-5.8S rDNA gene region have been utilized. Also, phylogenetic relationships among identified yeast species were assigned by using sequences of ITS-5.8S rDNA gene region. In addition, extracellular enzyme activity of identified yeast strains was detected by using API-ZYM. Kluyveromyces lactis, Aureobasidium pullulans, Hanseniaspora uvarum, Candida zeylanoides, Kwoniella sp., and Metschnikowia pulcherrima and Metschnikowia ziziphicola yeast species were identified on pomegranate surface. Phylogenetic analysis, carried out in ITS-5.8S rDNA gene region of identified yeast strains, revealed the presence of five clades. Kwoniella sp., H. uvarum, M. pulcherrima, and Kl. lactis yeast strains revealed high leucine arylamidase activity. Also valine arylamidase activity was determined in M. pulcherrima and Kl. lactis yeast species. Acid phosphatase activity was determined in H. uvarum and K. lactis yeast species. Uncultured Kwoniella sp. and H. uvarum yeast species displayed high β-galactosidase and β-glucosidase activities, respectively.

The immunomodulatory effects of Ziziphus jujuba Mill. fruit extracts were investigated using Galleria mellonella as an insect immune model. The expression of antimicrobial peptides, hemolin, phenoloxidase genes (PO-I and PO-II), and enzyme activity was measured in response to Candida albicans , silica beads, and Z. jujuba fruit extract. Responses were found to be stimulus- and time-dependent. Gallerimycin and galiomycin, key antimicrobial peptides, exhibited distinct expression patterns, with gallerimycin showing a more pronounced response to pathogens and beads. The Z. jujuba extract stimulated an early but balanced immune activation, likely due to its bioactive compounds. Hemolin expression varied between larvae and haemocytes depending on the type and duration of the challenge, supporting its role in immune recognition and opsonisation. Phenoloxidase activity and gene expression were also enhanced, supporting their role in promoting melanisation processes. Docking analyses suggested that hemolin contributes to phenoloxidase activation by stabilising PO-I and interacting with the phenoloxidase-activating factor-1-like protein (PAP1). The findings suggest that Z. jujuba extract effectively modulates immune responses, promoting enhanced protection while maintaining immune balance. GC-MS analysis revealed multiple bioactive compounds potentially contributing to immune modulation. This study highlights the significant immunomodulatory effects of Z. jujuba fruit extract on the immune system of G. mellonella . The findings suggest its potential as a natural immunostimulant and warrant further investigation of the underlying molecular mechanisms and bioactive components.