Maintenance of redox balance by antioxidants in hemolymph of the greater wax moth Galleria mellonella larvae during encapsulation response.

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.

VENOM FROM THE ECTOPARASITIC WASP Habrobracon hebetor ACTIVATES CALCIUM-DEPENDENT DEGRADATION OF Galleria mellonella LARVAL HEMOCYTES.

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.

Quantitative determination of nitric oxide production in haemocytes: nitrite reduction activity as a potential pathway of NO formation in haemolymph of Galleria mellonella larvae.

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.

Express method for determination of low value of trans-membrane potential of living cells with fluorescence probe: application on haemocytes at immune responses.

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.