Search Results (362)

Gene delivery to target cells is required for bioengineering or medical/agricultural applications. However, the hydrophobicity of the cell membrane always makes it resistant to polar nucleic acids. This physicochemical barrier is usually overcome by a nano-formulation to hide the polarity. This study evaluated a specific formulation called star polycation (SPc) regarding its efficacy in DNA/RNA delivery to insect cells. The delivery efficiency of the SPc formulation was assessed by transient expression of green fluorescence protein (GFP) in Sf9 cells, in which SPc formulation significantly enhanced the gene expression compared with an unformulated vector. In vivo transient expression (IVTE) was performed by injection of the expression construct with the SPc formulation into larvae of S. exigua. Fluorescence was detected in all four tissues, namely, epidermis, midgut, hemocyte, and fat body, where the SPc formulation enhanced the expression in most tissues except epidermis. Under this IVTE condition, an additional injection of SPc-formulated dsRNA specific to GFP suppressed the gene expression significantly more than the unformulated vector. The enhanced RNA interference (RNAi) efficiency caused by the SPc formulation was confirmed against four endogenous genes of S. exigua, namely, Snf7, PSMB5, vATPase, and α-tubulin, by either injection or feeding of dsRNA. These RNAi treatments were lethal to S. exigua, in which dsRNA specific to vATPase formulated with SPc resulted in almost 80% mortality through oral delivery. A similar oral toxicity by SPc-formulated dsRNA was demonstrated in another lepidopteran Plutella xylostella. The oral delivery of dsRNA was applied to control sucking insects such as aphids and thrips by spraying SPc-formulated dsRNA onto plant surfaces. The sprayed dsRNA labeled with Cy3 fluorescence was detected in the internal tissues of plant leaves, in which the penetration of dsRNA into the plant tissues was further accelerated by SPc formulation. The SPc formulation of dsRNA specific to vATPase was effective at killing the sucking insects by spraying on plant surfaces. These results suggest the application of an SPc formulation to deliver DNA/RNA to insect cells. Full article

20-Hydroxyecdysone (20E) is the active form of the steroid hormone ecdysone and is responsible for regulating insect development, molting, and metamorphosis. Infection with the entomopathogenic Beauveria bassiana enhances innate immunity and reduces 20E titers in Lymantria dispar. To further explore the role of the 20E signaling pathway in antifungal immunity, we combined exogenous hormone assays, proteomic profiling, and RNAi-mediated knockdown. In this study, exogenous hormone assays revealed that 20E accelerated the death of B. bassiana-infected larvae, whereas juvenile hormone (JH) markedly delayed it. 20E promoted larval weight gain and food utilization efficiency in L. dispar during fungal infection compared to the B. bassiana group. Moreover, the 20E + B. bassiana group significantly decreased the expression of the Toll pathway-related genes, Attacin2, Gloverin4, and Lysozyme4, while reducing hemocyte counts and Pvf2 expression. Furthermore, the 20E + B. bassiana group increased the number of hyphae in the hemolymph, but had no obvious effect on hemocyte encapsulation capacity compared to the B. bassiana group. Proteomic analysis showed that Cyp306a1 and Cyp314a1 were significantly down-regulated in L. dispar at 48 h post-infection with B. bassiana. Knockdown of Cyp306a1 delayed molting and increased larval mortality in L. dispar, but did not alter the mortality rate upon B. bassiana infection compared to the dsGFP group. Intriguingly, dsCyp306a1 + B. bassiana group had significantly increased the expression of Toll3, Toll5, Toll7, Gloverin4 and Cecropin5 compared to the B. bassiana group. In conclusion, these findings suggest that reduced 20E signaling shifts L. dispar from growth toward immune activation during B. bassiana infection. This study provides valuable insights into the 20E-mediated trade-off between growth and immunity. Full article

Tropical coastal ecosystems are highly productive but increasingly threatened by anthropogenic pressures, making environmental monitoring tools essential to determine the health status of the environment. In this study, the health status of three sites along the Pacific coast of Panama (Río Santa María, Cuchilluyo, and Montijo) was assessed using the bivalve mollusc Anadara tuberculosa as a sentinel species. A multilevel biomarker approach was employed, combining chemical analyses with biochemical and histological biomarkers. Metal concentrations in whole soft tissues were generally low-to-moderate, except for arsenic, and polycyclic aromatic hydrocarbon (PAH) concentrations were low in all sites, being dominated by low-molecular-weight compounds. Biomarker responses were consistent with the chemical data, with cockles from Montijo exhibiting lower glutathione S-transferase (GST) activity and reduced oxidative DNA damage. Histological analyses revealed only mild alterations, including hemocytic infiltration in the connective tissue of digestive glands and atrophy, with no major reproductive impairments. Overall, results indicate low to moderate pollution levels in the studied areas, with Montijo representing the least impacted site. The study supports the suitability of A. tuberculosa as a sentinel species and the relevance of a multilevel biomarker approach for assessing environmental health in tropical coastal ecosystems. Full article

In vertebrates, circulating and tissue-resident immune cells contribute to host defense and tissue homeostasis. Their balance is maintained through hematopoiesis and anatomical reservoirs. In mollusks, this balancing is poorly investigated. Thus, the dynamics of circulating and tissue-resident hemocytes after a hemolymph withdrawal were analyzed in Pomacea canaliculata, a snail that allows repeated hemolymph withdrawals and possesses posterior kidney (PK) cell aggregates known as hemocyte islets. In the absence of specific hemocyte markers, flow cytometry data revealed dynamic changes in circulating hemocyte number and populations within 48 h following hemolymph withdrawal. In addition, computer-assisted image analysis of histological sections showed a significant transient reduction in PK hemocyte-islet area. Analyses of publicly available P. canaliculata transcriptomes revealed predominant and highly variable expression of two hematopoiesis-related genes, namely Transglutaminase (PcTGase) and Hematopoietically expressed homeobox protein (PcHhex), in circulating hemocytes and PK. Fluorescence in situ hybridization highlighted constitutive PcTGase and PcHhex expression within PK hemocyte islets. RT-qPCR experiments confirmed transcriptomic data on PcTGase and PcHhex constitutive expression, without significant modulation following hemolymph withdrawal. Collectively, these findings provide the first evidence of parallel recovery dynamics involving circulating hemocytes and PK hemocyte islets in P. canaliculata. Full article

Microplastics pose a growing threat to marine ecosystems as they can accumulate in aquatic organisms, interfering with essential physiological processes including reproduction. This study analyzed the effects of short-term exposure (48 h) to two concentrations (0.5 and 1 µg/mL) of 5 µm diameter polystyrene microplastic bead particles in female Mytilus galloprovincialis mussels, a bioindicator species of the Mediterranean Sea. Histological analyses revealed progressive oocyte degeneration and the development of hypertrophic PAS-positive mucous cells, indicative of a stress response, in a dose-dependent manner. Changes in hemocyte classes, which are major effectors of bivalve immunity, were evidenced by the May–Grünwald Giemsa reaction. Biochemical data showed that microplastics increased levels of stress proteins, such as HSP70 and p53, and altered the composition of ovarian glycoproteins. Metabolomic analysis based on ¹H NMR spectrometry revealed significant alterations in metabolites involved in energy (glucose, glycogen, and malonate) and amino acid (branched-chain amino acids arginine, glycine, glutamate, histidine, betaine, and choline) metabolism, suggesting impairment to bioenergetic and antioxidant pathways. Overall, these results suggest that even short-term exposure to polystyrene microplastic beads can alter the ovarian function and metabolism of female M. galloprovincialis, highlighting their vulnerability. Full article

In the context of global climate change, intensified salinity fluctuations driven by altered precipitation, extreme rainfall events, and typhoons have emerged as a major threat to coastal mollusk aquaculture. In this study, integrated physiological and transcriptomic analyses were performed to investigate the responses of Pacific abalone (DD, Haliotis discus hannai) and its hybrid (DF, H. discus hannai ♀ × H. fulgens ♂) to hypo- and hypersalinity stress. Two salinity breakpoints (BPS1 for hyposalinity, BPS2 for hypersalinity) were identified using heart rate monitoring to indicate the osmotic tolerance thresholds of the abalone. The BPS1 and BPS2 values did not differ significantly between the DD and DF groups. However, a subsequent 30-day culture trial confirmed that exposure to the salinity level corresponding to BPS1 significantly reduced growth and survival of both DD and DF groups. To explore the molecular mechanisms underlying these two salinity breakpoints in abalone, the transcriptomes of hemocytes and gill tissues were profiled under both stress conditions. Both hypo- and hypersalinity stress induced pronounced transcriptomic responses in abalone, accompanied by upregulated differentially expressed genes (DEGs) significantly enriched in pathways like TNF and NF-κB signaling, including genes like piap, diap2, birc7-a, birc2, and birc3. However, abalone exhibited more intense responses to hypersalinity stress, as reflected by a greater number of annotated differentially expressed genes (DEGs) and more complex transcriptional regulation. Overall, this study integrates physiological assessment based on heart rate monitoring, aquaculture trials, and transcriptomic analysis to advance our mechanistic understanding of osmotic stress adaptation in abalone, while laying a scientific foundation for the sustainable development of abalone aquaculture under global climate change. Full article

Invertebrates rely exclusively on innate immunity but exhibit memory-like responses termed immune priming or trained immunity. In the commercially vital whiteleg shrimp (Litopenaeus vannamei), infection by Vibrio parahaemolyticus causes severe economic losses, yet the molecular networks driving secondary immune recall remain poorly understood. In this study, we established a two-step immune challenge model in L. vannamei using formaldehyde-inactivated V. parahaemolyticus and performed transcriptomic analysis on hemocytes to compare primary and secondary immune responses. Differentially expressed gene (DEG) screening and enrichment analyses (GO, KEGG, and GSEA) suggest that shrimp hemocytes undergo a broad and coordinated transcriptional reprogramming rather than uniform upregulation of immune genes. Transcriptomic data show potential associations between secondary immune priming and the modulation of cell fate processes: genes related to cell cycle progression (e.g., CDK1, CCNB3) and spindle assembly (e.g., MPS1) were significantly upregulated alongside apoptosis inhibition (CASP6 downregulation). Concurrently, metabolic remodeling was observed through the upregulation of lipid synthesis (SREBF1, FASN) and carbohydrate uptake pathways, potentially providing anabolic support for hemocyte growth and immune activation. Furthermore, the humoral effector responses appear to be strengthened, characterized by upregulated antimicrobial peptides (PEN, ALF) and the proPO melanization cascade (PPAF3, PPO3), whereas the expression of intracellular NLR was relatively suppressed, which might help mitigate excessive immune inflammation and immunopathological damage. Collectively, these transcriptomic findings identify a putative coordinated transcriptional signature of hemocyte recall responses in L. vannamei. This study expands our understanding of innate immune memory in invertebrates and provides candidate molecular markers for further study in disease-resistant breeding research in shrimp aquaculture. Full article

Invasive candidiasis caused by drug-resistant Candida species represents a critical global health challenge, with few novel therapeutic scaffolds under development. Here, silver nanoparticles were synthesized using a 21-day fermented Chun Mee kombucha tea extract (K-AgNPs) and characterized by UV-Vis spectroscopy, transmission electron microscopy, nanoparticle tracking analysis, and Fourier-transform infrared spectroscopy. LC-MS/MS profiling of the kombucha substrate documented a phytochemical landscape dominated by epigallocatechin (up to 122,631 µg/mL) and epigallocatechin gallate (up to 415 µg/mL), with a progressive ~80% decline in epicatechin and concomitant increases in gallic acid and chlorogenic acid across the 21-day fermentation. K-AgNPs obtained were spherical, 19.4 nm (±7.9 nm SD) in diameter, with a surface plasmon resonance peak at 415 nm. FTIR confirmed phenolic, carboxylate, and glycosidic surface capping. Antifungal susceptibility testing against eight Candida species, including the WHO critical–priority pathogen Candidozyma auris, showed concordant minimum inhibitory and minimum fungicidal concentrations of 0.80–1.60 µg/mL, confirming fungicidal activity. In vivo evaluation in Galleria mellonella larvae across six infection models demonstrated that K-AgNP treatment at the species-specific MIC significantly improved larval survival versus untreated infected controls (p < 0.01–0.001), while nanoparticle-only groups maintained ≥98% survival, indicating negligible toxicity. Co-treatment amplified total hemocyte mobilization, and K-AgNP-only larvae maintained hemocyte viability above 96% at all time points, indistinguishable from negative controls. Together, these findings demonstrate antifungal activity of K-AgNPs across the genus Candida in standardized in vitro and in vivo settings and provide justification for further investigation, including head-to-head comparison against licensed antifungals and physicochemical validation of nanoparticle stability under assay conditions. Full article

To identify novel field control strategies against Pyrrhalta aenescens (Coleoptera: Chrysomelidae) and provide scientific support for its biocontrol in urban tree management, this study investigated the virulence of Beauveria bassiana against this pest under laboratory conditions, as well as its physiological and biochemical effects. Bioassays using the dipping method showed that B. bassiana was pathogenic to all developmental stages of P. aenescens, with the highest virulence observed against early-instar larvae (1st and 2nd instars). For these stages, corrected mortality and mycosis rate were positively correlated with conidial concentration, and the median lethal time (LT₅₀) was the shortest. In contrast, pupae and eggs exhibited the strongest resistance to fungal infection. In leaf-disk choice tests, larvae significantly preferred untreated leaves or those treated with low concentrations of B. bassiana, displaying a concentration-dependent repellent response to the fungus. Physiological measurements revealed that larval body length and weight gain were significantly inhibited following fungal exposure. Further analysis indicated that B. bassiana infection markedly reduced total hemocyte counts and triggered intense melanization and nodulation responses, particularly in younger larvae. Overall, these results suggest that B. bassiana has strong potential for the biological control of P. aenescens. Control measures targeting early-instar larvae are recommended for cost-effective management, providing a scientific basis for developing eco-friendly control technologies based on this entomopathogenic fungus. Full article

In many parasitoid insects, especially hymenopteran parasitoids, venom is delivered around oviposition to override host physiology. Once viewed as a narrowly immunosuppressive secretion, parasitoid venom is now recognized as a multifunctional effector system targeting host immunity, development, metabolism, and behavior. This review synthesizes the venom apparatus, identification strategies, mechanisms of host manipulation, evolutionary processes, and biocontrol prospects, drawing primarily on Hymenoptera. A consistent pattern emerges: comparable host phenotypes arise from divergent molecular scaffolds, indicating functional rather than molecular convergence. Mechanistic evidence is strongest along six interlocking axes: inhibition of the prophenoloxidase cascade and melanization, disruption of cellular and humoral immunity, developmental arrest, metabolic reprogramming, and neuromuscular or behavioral manipulation. Outside Hymenoptera, the coleopteran ectoparasitoid Dastarcus helophoroides offers one of the clearest comparative cases: parasitization inhibits host melanization, alters phenoloxidase activity, and reduces antibacterial activity and circulating hemocyte abundance. As of today, fifty putative venom-like candidate proteins have been identified from larval whole-body proteomics, yet the secretory tissue remains anatomically unresolved, and no individual effector has been functionally validated. The central challenge is therefore the transition from candidate catalogs to mechanistically resolved repertoires, particularly outside Hymenoptera. Closing this gap will be important for evolutionary inference and the rational development of venom-based biocontrol tools. Full article

Background/Objectives: Hypericum perforatum L. (St. John’s Wort) is extensively utilized in ethnopharmacology due to its anti-inflammatory and immunomodulatory properties. However, its effects on the interaction between innate immunity and oxidative homeostasis remain incompletely characterized, particularly in alternative invertebrate models. This study aimed to evaluate the effects of H. perforatum extract on oxidative homeostasis and protein metabolism using the Galleria mellonella model, a 3R-compliant and ethically sustainable platform for preliminary immunological and redox-related screening. Methods: Last instar G. mellonella larvae were administered increasing concentrations of H. perforatum extract (0.001–20 mg mL⁻¹) by intrahemocoelic injection. After 24 h, hemolymph samples were analyzed for total protein (TP), total hemocyte count (THC), encapsulation and melanization responses, superoxide dismutase (SOD) activity, catalase (CAT) activity, and malondialdehyde (MDA) levels. The phytochemical profile of the extract was additionally evaluated using GC–MS analysis. Results: Significant group-dependent alterations were observed in TP levels and THC values, with the HP-2 group demonstrating the highest hemocyte counts and enhanced strong encapsulation responses. Higher extract concentrations, particularly HP-4, were associated with increased weak encapsulation profiles, suggesting altered cellular immune organization. Melanization responses became significantly elevated at 24 h following treatment. In contrast, SOD activity, CAT activity, and MDA levels did not differ significantly among groups, indicating preservation of oxidative homeostasis under the tested conditions. Conclusions: H. perforatum extract induced dose-dependent modulation of cellular and humoral immune responses in G. mellonella without evidence of detectable oxidative disruption during acute exposure. These findings support the utility of the G. mellonella model for preliminary evaluation of botanical immunomodulators and suggest that H. perforatum may influence immunophysiological pathways independently of overt oxidative toxicity. Full article

EpOMEs suppress excessive and unnecessary immune responses at late infection stage in insects. This immune resolvin activity of EpOMEs includes the cell lysis of hemocytes by causing apoptosis. This cytolytic toxicity of EpOMEs suggests a potential insecticidal activity of their stable analogs. In fact, a propoxy derivative of 12,13-EpOME called AS56 mimicked EpOME activity by suppressing immune responses and exhibited insecticidal activity against a lepidopteran insect, Spodoptera exigua. This study evaluated the potency of the EpOME analogs by modifying their functional groups. PD28 is a racemic mixture of propoxy analog at the twelfth carbon. FD25 is a racemic butoxy mixture at the twelfth carbon of AS56. AS61 is saturated at the ninth carbon of AS56. The effects of the EpOME analogs were assessed on two cellular immune responses: hemocyte-spreading and nodule formation. All four analogs inhibited the cellular immune responses; however, AS56 was the most potent in inhibiting the immune responses. FD25 and AS61 were significantly less potent compared to AS56, suggesting crucial roles of the double bond at the ninth carbon and the propoxy chain at the twelfth carbon. The stronger inhibitory activity of AS56 compared to PD28 also suggests the functional role of the stereoisomeric form in physiological functions. The cytotoxicity of AS56 was also the most potent among the analogs, suggesting its insecticidal activity. Subsequent insecticidal bioassays on AS56 toxicity were performed against different insect species. These EpOME analogs were potent against lepidopteran insects (S. exigua and Plutella xylostella) but not against coleopteran (Tenebrio molitor) or thysanopteran (Frankliniella occidentalis) species. Among the EpOME analogs, AS56 was the most toxic against the lepidopteran insects. Spraying AS56 in cabbage fields infested by lepidopteran insects led to acute and high control efficacy against two lepidopteran pests, similar to that of a chemical insecticide, fluxametamide. The AS56-intoxicated larvae exhibited over-excitation in their behavior around 12 h post-treatment. This study indicates that AS56 exhibits an oral toxicity against lepidopteran insects with its cytotoxicity and behavioral over-excitation. Full article

Environmental exposures can influence phenotypes across generations, yet the cellular routes by which somatic stress signals reach the germline remain poorly defined. piRNAs are attractive candidates for transgenerational epigenetic inheritance because they silence transposable elements, guide chromatin regulation, carry a stabilizing 3′ 2′-O-methyl modification, and participate in self-reinforcing amplification pathways, including ping-pong amplification in animals and RNA-dependent RNA polymerase (RdRP)-mediated secondary small-RNA amplification in systems such as C. elegans. This review examines evidence linking piRNAs, macrophage biology, and environmentally induced inheritance. We first summarize small-RNA inheritance in animals, plants, and ciliates, emphasizing C. elegans piRNA-triggered epigenetic memory and plant RNA-directed DNA methylation as parallel small-RNA-based inheritance systems. We then discuss emerging evidence that macrophage polarization states contain distinct piRNA signatures and release extracellular vesicles carrying non-coding RNAs. Finally, we revisit the Drosophila ectopic large bristle outgrowth (ELBO) phenotype as a possible example of macrophage-like hemocytes linking stress, tissue remodeling, and heritable morphological variation. We propose the macrophage-mediated morphological evolution (M3) model as a testable framework connecting environmental stress to transgenerational phenotypes. Full article

This study investigated the individual and combined effects of mercury (as HgCl₂) and Bisphenol S (BPS) on the blue crab Callinectes sapidus, focusing on biomarkers measured in hemolymph, gills, and hepatopancreas and ultrastructure (transmission electron microscopy, TEM) observations. Adult males were exposed for seven days to environmentally relevant concentrations of each contaminant, alone and in mixture. Results revealed clear tissue-specific responses. In hemolymph, both contaminants reduced total hemocyte count and affected immune-related parameters, including hemocyte proliferation and enzymatic activities. In gills, mercury significantly decreased total antioxidant capacity, while both contaminants increased lipid peroxidation, indicating oxidative stress. BPS and the mixture stimulated catalase activity, whereas electron transport system activity increased only under combined exposure. In contrast, the hepatopancreas showed limited biochemical alterations, suggesting a higher resilience to medium-term exposure. TEM observations revealed a general decrease in granularity of hemocytes from crabs exposed to BPS and mixture, suggesting cell degranulation. In addition, infiltration of hemocytes into gills was observed in crabs exposed to experimental conditions, suggesting migration of hemocytes from hemolymph to peripheral tissues, while alterations of the microvilli arrangement were detected in digestive cells of BPS-treated crabs. Overall, the findings suggested that hemolymph and gills were more sensitive to contaminant exposure, while the hepatopancreas appears more resistant, at least under the experimental conditions tested. The study also suggests potential non-additive interactions between mercury and BPS. Full article

Background/Objectives: The increasing prevalence of antifungal resistance among Candida albicans (C. albicans) strains necessitates the development of alternative therapeutic strategies. Curcumin (CUR), a natural polyphenolic compound, has attracted attention due to its antimicrobial and immunomodulatory properties. This study aimed to evaluate the antifungal activity of curcumin (CUR) and its effects on cellular and nuclear morphometric parameters (area, width, height, and perimeter), cytoplasmic area, and the nuclear-to-cytoplasmic ratio in an in vivo Galleria mellonella (G. mellonella) infection model. Methods: The experimental design consisted of four groups: (i) healthy control receiving phosphate-buffered saline (PBS), (ii) C. albicans-infected group (1.5 × 10⁸ CFU/mL), (iii) infected group treated with CUR (10 mg/kg), and (iv) healthy group treated with CUR. Survival was monitored for 96 h. Hemolymph samples were collected from larvae, smeared onto slides, and stained using May–Grünwald–Giemsa and Giemsa methods. Morphological evaluation and cytomorphometric analyses, including cellular area, nuclear area, cytoplasmic area, perimeter, width, height, and nucleus-to-cytoplasm ratio, were performed. Results: A higher dose (100 mg/kg) resulted in 100% mortality within 24 h and was therefore defined as lethal, whereas 10 mg/kg showed no toxicity in healthy larvae. Hemolymph was collected from surface-sterilized larvae, pooled per group, and a 10 µL aliquot was smeared onto slides. Conclusions: CUR exhibited significant antifungal activity against C. albicans and modulated host immune cell morphometry in the G. mellonella model. Its effects were dose-dependent, with potential cytotoxicity at higher concentrations. Further studies involving quantitative fungal burden analyses and mammalian models are required to clarify its therapeutic potential. Full article