RESUMEN

Entomopathogenic fungi may interact with insects' symbiotic bacteria during infection. We hypothesized that topical infection with Beauveria bassiana may alter the microbiota of the Colorado potato beetle (CPB) and that these modifications may alter the course of mycoses. We used a model with two concentrations of conidia: (1) high concentration that causes rapid (acute) pathogenesis with fast mortality followed by bacterial decomposition of insects; (2) lower concentration that leads to prolonged pathogenesis ending in conidiation on cadavers. The fungal infections increased loads of enterobacteria and bacilli on the cuticle surface and in hemolymph and midgut, and the greatest increase was detected during the acute mycosis. By contrast, stronger activation of IMD and JAK-STAT signaling pathways in integuments and fat body was observed during the prolonged mycosis. Relatively stable (nonpathogenic) conditions remained in the midgut during both scenarios of mycosis with slight changes in bacterial communities, the absence of mesh and stat expression, a decrease in reactive oxygen species production, and slight induction of Toll and IMD pathways. Oral administration of antibiotic and predominant CPB bacteria (Enterobacteriaceae, Lactococcus, Pseudomonas) led to minor and mainly antagonistic effects in survival of larvae infected with B. bassiana. We believe that prolonged mycosis is necessary for successful development of the fungus because such pathogenesis allows the host to activate antibacterial reactions. Conversely, after infection with high concentrations of the fungus, the host's resources are insufficient to fully activate antibacterial defenses, and this situation makes successful development of the fungus impossible.

RESUMEN

The Siberian frog Rana amurensis has a uniquely high tolerance to hypoxia among amphibians, as it is able to withstand several months underwater with almost no oxygen (0.2 mg/liter) vs. several days for other studied species. Since it was hypothesized that hypoxia actives the antioxidant defense system in hypoxia-tolerant animals, one would expect similar response in R. amurensis. Here, we studied the effect of hypoxia in the Siberian frog based on the transcriptomic data, activities of antioxidant enzyme, and content of low-molecular-weight antioxidants. Exposure to hypoxia upregulated expression of three relevant transcripts (catalase in the brain and two aldo-keto reductases in the liver). The activities of peroxidase in the blood and catalase in the liver were significantly increased, while the activity of glutathione S-transferase in the liver was reduced. The content of low-molecular-weight antioxidants (thiols and ascorbate) in the heart and liver was unaffected. In general, only a few components of the antioxidant defense system were affected by hypoxia, while most remained unchanged. Comparison to other hypoxia-tolerant species suggests species-specific adaptations to hypoxia-related ROS stress.

Asunto(s)

RESUMEN

Schiff bases and similar molecules forming metal complexes may cause redox effects, which may also be influenced by light. Anthraquinones such as doxorubicin and idarubicin are widely used antitumor agents, which can generate reactive oxygen species (ROS), stimulated by both the presence of iron and copper ions and also by light. The generated ROS can cause DNA scission, cell membrane oxidation, and many other toxic effects. The redox activity of the quinone-quinoline chelator 2-phenyl-4-(butylamino)naphtho [2,3-h]quinoline-7,12-dione (Q1) was investigated in the presence of iron, copper, and zinc. The influence of light in these interactions was also examined. The chemically induced dynamic nuclear polarization (CIDNP), nuclear magnetic resonance (NMR), and electron paramagnetic resonance (EPR) methods were used to elucidate the molecular changes and ROS generation effects of the Q1 metal interactions. A model electron transfer reaction system between 1,4-dihydropyridine and Q1 was utilized to demonstrate that the chelate complexes of Q1 with both Fe(III) and Cu(II) ions were more redox active than Q1 itself. Similarly, CIDNP and NMR data showed that the concentration dependence of the free radicals yield is much higher in the presence of Fe(III) and Cu(II) ions, in comparison to Zn(II), and also that it increased in the presence of light. These findings underline the role of transition metal ions and Q1 in cyclic redox chain reactions and increase the prospect of the development of copper- and iron-based chelating agents, including Q1 and its derivatives, for anticancer therapy. Furthermore, these findings also signify the effect of light on enhancing ROS formation by Q1 and the prospect of utilizing such information for designing target specific anticancer drugs for photodynamic therapy.

RESUMEN

Fungal infections and toxicoses caused by insecticides may alter microbial communities and immune responses in the insect gut. We investigated the effects of Metarhizium robertsii fungus and avermectins on the midgut physiology of Colorado potato beetle larvae. We analyzed changes in the bacterial community, immunity- and stress-related gene expression, reactive oxygen species (ROS) production, and detoxification enzyme activity in response to topical infection with the M. robertsii fungus, oral administration of avermectins, and a combination of the two treatments. Avermectin treatment led to a reduction in microbiota diversity and an enhancement in the abundance of enterobacteria, and these changes were followed by the downregulation of Stat and Hsp90, upregulation of transcription factors for the Toll and IMD pathways and activation of detoxification enzymes. Fungal infection also led to a decrease in microbiota diversity, although the changes in community structure were not significant, except for the enhancement of Serratia. Fungal infection decreased the production of ROS but did not affect the gene expression of the immune pathways. In the combined treatment, fungal infection inhibited the activation of detoxification enzymes and prevented the downregulation of the JAK-STAT pathway caused by avermectins. The results of this study suggest that fungal infection modulates physiological responses to avermectins and that fungal infection may increase avermectin toxicosis by blocking detoxification enzymes in the gut.

Asunto(s)

RESUMEN

Entomopathogenic fungi form different strategies of interaction with their insect hosts. The influence of fungal infection on insect physiology has mainly been studied for generalists (Metarhizium, Beauveria), but studies of specialized teleomorphic species, such as Cordyceps militaris, are rare. We conducted a comparative analysis of the immune reactions of the wax moth Galleria mellonella after injection with blastospores of C. militaris (Cm) and Metarhizium robertsii (Mr) in two doses (400 and 4000 per larva). Cm-injected insects died more slowly and were more predisposed to bacterial infections than Mr-injected insects. It was shown that Cm infection led to a predominance of necrotic death of hemocytes, whereas Mr infection led to apoptotic death of cells. Cm-infected insects produced more dopamine and reactive oxygen species compared to Mr-infected insects. Moreover, Cm injection led to weak inhibition of phenoloxidase activity and slight enhancement of detoxification enzymes compared to Mr-injected insects. Blastospores of Cm that were cultivated in artificial medium (in vitro) and proliferated in wax moth hemolymph (in vivo) were characterized by equal intensity of fluorescence after staining with Calcofluor White. In contrast, Mr blastospores that proliferated in the wax moth had decreased fluorescence intensity compared to Mr blastospores grown in medium. The results showed that insects combat Cm infection more actively than Mr infection. We suggest that Cm uses fewer universal tools of killing than Mr, and these tools are available because of specific interactions of Cm with hosts and adaptation to certain host developmental stages.

Asunto(s)

RESUMEN

Most invertebrate species exhibit immunological responses that can inactivate and eliminate penetrating parasites. Such immune responses in particular involve the formation of potentially toxic reactive oxygen species (ROS). We explored the immune capabilities of the first-generation (F1) offspring of naturally infected freshwater snails, Lymnaea stagnalis, in response to infection by trematode cercariae under laboratory conditions. The rates of ROS formation and peroxidase activity in the hemolymph of the F1 offspring of L. stagnalis parents infected by an asexual stage of trematodes were significantly higher than in F1 offspring of uninfected parents. Compared to offspring from uninfected parents, the growth rate of F1 snails from infected parents was higher, but survival was lower. After infection of F1 snails by trematode cercariae of Echinoparyphium aconiatum under laboratory conditions, the rate of ROS formation and peroxidase activity in the hemolymph of F1 offspring of uninfected parents increased compared to control snails. This pattern persisted throughout the entire 3-week observation period. In contrast, the rate of ROS formation in the hemolymph of F1 snails from infected parents after experimental infection by E. aconiatum cercariae did not differ from controls, and peroxidase activity even decreased. Thus, trematode parthenitae infection of parents could alter the immune response of their offspring.

Asunto(s)

RESUMEN

The lipid peroxidation process in hemocytes, activities of phenoloxidase and key enzymatic antioxidants (superoxide dismutase, glutathione-S-transferase, catalase) and nonenzymatic antioxidants (thiols, ascorbate) in hemolymph of the greater wax moth Galleria mellonella L. (Lepidoptera: Pyralidae) were studied during the encapsulation process of nylon implants. It has been established that as soon as 15 min after piercing a cuticle with the implant, a capsule is formed on its surface. Active melanization of the capsule has been shown to last for 4 h. During the first hours after incorporating the implant, an increase in phenoloxidase activity and lipid peroxidation in the insect hemocytes has been revealed. Adhesion and degranulation on the surface of foreign object lead to the depletion of total hemocytes count (THC). Our results indicated that thiols and ascorbate molecules take part in the immediate antioxidant response, during later stages of encapsulation process hemolymph glutathione-S-transferase detoxifies and protects insect organism thereby restoring the internal redox balance. We suggest that nonenzymatic and enzymatic antioxidants of hemolymph plasma play a key role in the maintenance of redox balance during encapsulation of foreign targets.

Asunto(s)

RESUMEN

SIGNIFICANCE: Chemotherapy is currently the principal method for treating many malignancies. Thus, the development of improved antitumor drugs with enhanced efficacy and selectivity remains a high priority. Recent Advances: Anthracycline antibiotics (AAs), for example, doxorubicin, daunomycin, and mitomycin C, belong to an important family of antitumor agents widely used in chemotherapy. These compounds are all quinones. They are, thus, capable of being reduced by appropriate chemicals or reductases. One of their important properties is that under aerobic conditions their reduced forms undergo oxidation, with concomitant generation of reactive oxygen species (ROS), namely, superoxide anion radicals, hydrogen peroxide, and hydroxyl radicals. The presence of metal ions is essential for the generation of ROS by AAs in biological systems. CRITICAL ISSUES: A fundamental shortcoming of the AAs is their high cardiotoxicity. We have proposed, and experimentally realized, a new type of quinones that is capable of coordinating metal ions. We have demonstrated in vitro that they can be reduced by electron transfer chains and glutathione with concomitant generation of ROS. They can also produce ROS under photo-excitation. The mechanisms of these reactions have been characterized by using nuclear magnetic resonance and electron paramagnetic resonance. FUTURE DIRECTIONS: To enhance their therapeutic effectiveness, and decrease cardiotoxicity and other side effects, we intend to conjugate the quinone chelators with monoclonal antibodies and peptide hormones that are specifically targeted to receptors on the cancer cell surface. Some such candidates have already been synthesized. An alternative approach for delivery of our compounds involves the use of specific peptide-based nanoparticles. In addition, our novel approach for treating malignancies is also suitable for photodynamic therapy. Antioxid. Redox Signal. 28, 1394-1403.

Asunto(s)

RESUMEN

The study has demonstrated a dual effect of nitric oxide on phenoloxidase (PO)-mediated DOPA oxidation and melanization process. NO generated at low rates proportionally increased in PO-mediated DOPA oxidation. Competitive PO inhibitor, phenylthiourea, resulted in significant inhibition of NO-mediated DOPA oxidation. Further analysis using fluorescent and EPR methods demonstrated that the effect of NO on DOPA oxidation is explained by oxidation of NO to NO2 at the active site of PO followed by oxidation of DOPA by NO2. On the contrary, the bolus addition of NO gas solution resulted in a significant decrease in observed PO activity. Similar dose-dependent effect of NO was observed for the insect's haemocytes quantified as percentage of melanized cells after treatment with nitric oxide. In conclusion, the results of the study suggest that NO may have a significant regulatory role on melanization process in invertebrates as well as in human and result in protective or damaging effects.

Asunto(s)

RESUMEN

Ectoparasitoids inject venom into hemolymph during oviposition. We determined the influence of envenomation by the parasitoid, Habrobracon hebetor, on the hemocytes of its larval host, Galleria mellonella. An increase in both intracellular Са(2+) content and phospholipase C activity of the host hemocytes was recorded during 2 days following envenomation by the parasitoid. The decreased hemocyte viability was detected 1, 2, and 24 h after the envenomation. Injecting of the crude venom (final protein concentration 3 µg/ml) into the G. mellonella larvae led to the reduced hemocyte adhesion. The larval envenomation caused a decrease in transmembrane potential of the hemocytes. These findings document the suppression of hemocytic immune effectors in the parasitized host larvae.

Asunto(s)

RESUMEN

The generation of nitric oxide by Galleria mellonella larvae haemocytes has been investigated. For this purpose, a fluorescent method, specific for detection of NO, has been developed. The method is based on the application of fluorescence probe DAF-FM diacetate and nitronyl nitroxyl radical, NNR, which accelerates the NO-dependent formation of fluorescence product, DAF-FM triazole. The key feature of the method is the registration and analysis of differential kinetics, namely, the difference of kinetics obtained in samples with NNR and without NNR. This approach allows us to exclude any other kinetic processes not related to triazole formation. The differential kinetics were calibrated versus NO generation rate and the resulting low limit of method sensitivity was obtained as about 0.4-0.5 nM/min. The generation of nitric oxide by the haemocytes of insects treated with LPS in vivo has been detected at a rate of 0.5-0.7 nM/min. However, the production of NO in haemocyte suspensions treated with both the substrate, l-arginine, and the inhibitor, l-NAME, of NOS, has not been detected within method sensitivity. These data provide only the upper level of NO generation by haemocytes but cannot be used to draw definite conclusions about NOS as a source of NO. Meanwhile, it is known, that NO can be formed by NOS independent mechanism. Indeed, we have observed a significant increase in NO generation in the samples of haemocytes intracellularly loaded with nitrite. Moreover, adding nitrite to lysed haemocytes results in the higher NO generation rate. After addition of 500 µM nitrite, the rates of NO generation in the samples are determined to be 2 and 20-30 nM/min, respectively. The nitrite/nitrate content of haemocytes and lymph were found to be 5 and 50 µM, respectively. The detected nitrite reduction activity of haemocytes allowed us to estimate the generation rate of nitric oxide as 0.05-0.5 nM/min from endogenous nitrite. It is thus assumed that the observed nitrite reduction activity in haemocytes is dominant in the increased NO production during immune response of the G. mellonella larvae.

Asunto(s)

RESUMEN

The method for measurement of trans-membrane potential of cell membrane was evaluated for the case of low potential value using fluorescence probe 4-(4-dimethylaminostyryl)-1-methylpyridinium, DSM. The method is based on comparative titration of cells with probe in buffers containing Na(+) or K(+). The apparent trans-membrane potential obtained with this way is a result of K(+)-Na(+) pump activity. The presented approach allowed measuring the low value of potential with 1-2 mV of accuracy without additional calibration procedures. The method was applied for investigation of potential of cell membrane of haemocytes of Galleria mellonella larvae. The value of potential of intact insect's haemocytes was found in the range from -10 to -20 mV. The change of potential value of haemocytes was investigated under model immune response and natural envenomation and parasitizing. The obtained deviations of cell membrane potential were in good correlation with changes of activity of main immune reactions, described in literature and obtained by us earlier.

Asunto(s)

RESUMEN

Phenoloxidase is a key enzyme of melanization catalyzing the oxidation of phenols. Phenylthiourea (PTU) is the well-known and widely used inhibitor of phenoloxidase. However, the mechanism of its action is not quite clear. In the present work, the effect of PTU on the enzymatic oxidation of 3-(3,4-dihydroxyphenyl)-l-alanine (DOPA) by phenoloxidase was studied by spectrophotometric methods. The inhibition constant of PTU was estimated as 0.21 ± 0.09 µM and the competitive type of inhibition was determined for this reaction.

Asunto(s)

RESUMEN

Generation of superoxide anion and hydrogen peroxide during enzymatic oxidation of 3-(3,4-dihydroxyphenyl)-DL-alanine (DOPA) has been studied. The ability of DOPA to react with O2*- has been revealed. EPR spectrum of DOPA-semiquinone formed upon oxidation of DOPA by O2*- was observed using spin stabilization technique of ortho-semiquinones by Zn2+ ions. Simultaneously, the oxidation of DOPA by O2*- was found to produce hydrogen peroxide (H2O2). The analysis of H2O2 formation upon oxidation of DOPA by O2*- using 1-hydroxy-3-carboxy-pyrrolidine (CP-H), and SOD as competitive reagents for superoxide provides consistent values of the rate constant for the reaction between DOPA and O2*- being equal to (3.4+/-0.6)x10(5) M(-1) s(-1). The formation of H2O2 during enzymatic oxidation of DOPA by phenoloxidase (PO) has been shown. The H2O2 production was found to be SOD-sensitive. The inhibition of H2O2 production by SOD was about 25% indicating that H2O2 is produced both from superoxide anion and via two-electron reduction of oxygen at the enzyme. The attempts to detect superoxide production during enzymatic oxidation of DOPA using a number of spin traps failed apparently due to high value of the rate constant for DOPA interaction with O2*-.

Asunto(s)

RESUMEN

The formation of reactive oxygen metabolites in haemolymph of Galleria mellonella larvae was studied by ESR spectroscopy. The inhibition of the production of the reactive oxygen metabolites of DOPA in haemolymph under the action of fungal infection was shown using spin trap 1-hydroxy-3-carboxy-pyrrolidine. This inhibition correlated with decrease of phenoloxidase activity in haemolymph of infected insects. Simultaneously, the decrease of production of DOPA-semiquinone was detected using method of spin stabilization by diamagnetic metal ions. Moreover, it was shown that the formation of DOPA-quinone was slowed down in haemolymph of infected insects. Our results suggest that the DOPA-derived quinones/semiquinones may be involved in immune response of insects as part of its defense mechanism.

Asunto(s)