STUDY OF THE DISTRIBUTION AND COMPOSITION OF MOLECULES IN GALL-FORMING INSECTS AT DIFFERENT STAGES OF DEVELOPMENT BY MEANS OF RAMAN SPECTROSCOPY
M. I. Nikelshparg1, E. I. Nikelshparg2, V. V. Anikin3, D. N. Bratashov3
1 Gymnasium No. 3
2 M.V. Lomonosov Moscow State University
3 N.G. Chernyshevsky Saratov State National Research University
Abstract
The use of Raman spectroscopy (RS) for the study of biological objects has many advantages. One of them is the non-invasiveness of the method. This is especially important for studying the development of living insects, which go through several completely different stages in their development. One of the most difficult molecules to study is the carotenoid pigment. Phytophagous insects are believed to obtain carotenoids from plants during feeding. The aim of our work was to determine the composition of carotenoids at various stages of the development of insects of the family Hymenoptera using the RS method as well as to assess the possibility of development and survival of insects after repeated irradiation.
For the experiment, we took small (1-3 mm) gall-forming insects, the larvae of which form a gall on the plant - a tumor, within which they develop and hibernate. Some gall-formers are pests of cultivated plants, while others enter into symbiotic relationships. The authors collected galls on the hawk plant (Hieracium x robustum), formed by the gall-former Aulacidea hieracii l., 1758 (Hymenoptera: Cynipidae), in July 2019, January, February 2020. in the Volzhsky district of Saratov. We used an inVia Renishaw (UK) Raman microspectrometer with a 532 nm laser, x5 objective, numerical aperture 0.75, signal accumulation time of 60 seconds at a power not exceeding 0.3 mW, laser spot size 5.4 μm. Experimental living larvae or pupae of gall-formers were removed from the galls onto a glass slide and the Raman spectra were immediately recorded. At the same time, two control groups of living gall larvae were formed, which remained intact. After the first study, the slide with the experimental larvae was put into a Petri dish and left for growing (continuation of development). After a week, both experimental and control larvae successfully pupated. We repeated the RS experiment with experimental pupae, but this time pupae of the first control group were also studied, the second control group was left without influence. All three Petri dishes were kept under observation for insect development for four months.
The Raman spectra of larvae and pupae of gall wasps contain characteristic peaks of carotenoids, lipids, and proteins. For the first time, we discovered a statistically significant heterogeneity in the arrangement of carotenoids within one individual, both larvae and pupae. Moreover, the distribution of carotenoids in summer and wintering larvae and in pupae was different. As a result of the study, healthy adults emerged from pupae in all experimental Petri dishes, which lived a standard period of time for this species. Thus, we were convinced of the safety and non-invasiveness of the Raman method for these animals.
Speaker
Nikelshparg Matvey
MAOU Gymnasium 3
Russia
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