PROBLEMS SOLUTION OF SPECIAL IDENTIFICATION OF EISENIA CULTURAL POPULATIONS
Introduction. Intensification of various aspects of modern agricultural production, based on the use of a large number of mineral fertilizers and chemical means of plant and animal protection, actualizes the development and mastering of natural-like methods for restoring the quality of soils and bottom deposits of inland water bodies. The goal is their successful use in cultivating cultivated plants and conducting remediation measures.
One such approach is vermiculturing – breeding of earthworms of the family Lumbricidae, for biotransformation of depleted soils and organic wastes in order to obtain biohumus.
Relevance. Earthworms differ significantly in biological characteristics from animals, traditionally bred in agriculture. When working with them, a selection problem arises to assess their phenotypes – it is difficult to identify individuals and assess their performance. In this regard, the main task of effective selection becomes more complicated.
Today in scientific sources on the study of the genetic structure of earthworms natural populations, their karyotypes and morphological features are not few data . But the development of vermiculture, based on breeding and industrial use for biotransformation of earthworms, requires a clear species identification of cultural lines of invertebrate animals of the family Lumbricidae.
Research objective. The aim of the study was to investigate the morphological and cytogenetic features of the Eisenia worms population that is being forms.
Materials and methods. While forming of the new population, the worms that were purchased by the Department of Biotechnology of the UGHTU in the association "Bioconversion" and previously described as E. foetida were used.
Of the array of these animals, 6 worms were selected that became the founders of the new population. After increasing the number of up to 300 animals, we formed groups of 20 individuals, each group irradiated with a laser of the LGN-208b type With a power of 1 mW, a wavelength of 633 nm, a beam diameter of 14 mm, various exposures in time from 5 to 30 min. The control was not irradiated. Irradiated animals were bred in separate groups and studied morphological, biochemical and cytogenetic features.
Morphological study of the species affiliation was carried out that was determined and compared with the descriptions of these species given in the works of foreign taxonomy . The intensity of the pigmentation of the integument of the body was determined in animals. All further morpho-metric studies were carried out on worms fixed in 75% ethanol. By the method of microscopy, parameters such as length and diameter of the body, total number of segments, location of the segments of the girdle, pubertal ridges and the first dorsal pore, type of setae were analyzed.
Karyological analysis was performed with worms selected at the time of highest sexual activity. Preparations were prepared from the tissue of the seminal sacks by the method previously successfully used to study karyotypes of lumbricids . The worms were injected with 0.1% solution of colchicine into the pre-lobe zone for 19 hours. 20 min to the autopsy. The animals were immobilized in 75% ethanol solution and digested along the median spinal line. Removed spermatic bags hypotone 50 min. in the distillate and fixed in three steps in a mixture of acetic acid and ethanol in a ratio of 1:3. Chromosome preparations were made by imprinting.
Genetic markings were performed by electrophoresis in a 7.5% polyacrylamide gel Tris-EDTA • Na2-borate system with pH = 8.5  1 hour 20 minutes at a voltage of 200 V and a current strength of 140–mA. Extract of enzymes and proteins was obtained by grinding the final segments of the body with a size of 5–10 mm in the distillate in the ratio 1:1. After switching of the electric current, the gel was treated with a solution containing a special substrate that specifically reacts with the enzyme under study, forming spots corresponding to the spectra of the enzymes on the gels. The genotype of the individual according to the locus encoding the enzyme being studied is determined by the nature of the distribution of the spots on the gel .
Results of the research. The carried out researches have shown that the groups of animals under study belong to the species E. foetida, and by other attributes to the species E. venetta, which caused the problem of the species affiliation of the earthworm array when working to form a new population.
Thus, the morphological indices studied have revealed that animals for all morphological features refer to the species Eisenia foetida. During the study of cytogenetic, it was found that the karyotype of the animals was 36 chromosomes but it should be 22. Just kind of Eisenia foetida is composed of 22 chromosomes and karyotypes of other species of the genus Eisenia have 36 chromosomes.
Therefore, biochemical gene marking was carried out on enzyme systems, in particular nonspecific esterases. Nonspecific esterases of different species of the genus Eisenia differ in molecular weight. Esterаs of E. foetida have a lower mass than esterases of E. veneta.
Our studies showed that in the place of the locus of spectra of nonspecific esterases, the individuals under study belong to the species E. veneta.
Nonspecific esterases of different species of the genus Eisenia differ in molecular weight. Esterases E. foetida have a lower mass than E. veneta esterase. Our studies have shown that individuals at the locus of spectra of non-specific esterases belong to the species E. veneta.
Conclusions The research may be a theoretical hypothesis for certain types of animal identification in vermiculture and creating biological diversity in its population. Despite the fact that it originates from 6 individuals obtained from a single array of animals, polymorphism by esterases showed a fairly high level of genetic variability in the forming line that indicates the reserve of its genetic variability and allows it to hope for its successful development in the future.
2. Verner, М. Ustoychyvoe zemledelye y ekolohiya dozhdevoho chervya – Sustainable agriculture and ecology of earthworm. Per. s anhl. N. Stepanova – Translated from english by N. Stepanova. www.vermik.narod.ru
3. Mezhzheryn, S., Y. Kotsyuba, and O. Harbar. 2010. Henetycheskaya struktura populyatsii partohenetycheskoho dozhdevoho chervya Eiseniela Tetraedra (Savigny, 1826) v estestvennoy y urbanyzyrovannoy srede obytanyya – Тhe genetic structure of populations of the partogenetic earthworm Eiseniela Tetraedra (Savigny, 1826) in a natural and urbanized habitat. Naukovyy visnyk Uzhhorodskoho universytety – Scientific Bulletin of Uzhgorod University. – 28:135–138 (in Russian).
4. Vsevolodova-Perel, T. 1997. Dozhdevyye chervi fauny Rossii. Kadastr i opredilitel – Rainworms of the fauna of Russia. Cadastre and determinant. M.: Nauka, 104 (in Russian).
5. Bouchе, M. 1972. Lombriciens de France. Ecologie et systеmatique. 1972. Ann. Zool. – ecol. Anim. 72(2):1–671.
6. Garbar, A., and R. Vlasenko. 2006. Kariotipy treh vidov roda Aporrectodea (Oligochaeta : Lumbricidae) fauny Ukrainy – Karyotypes of three species of the genus Aporrectodea (Oligochaeta : Lumbricidae) of Ukrainian fauna. Kariosistematikа bespozvonochnykh zhivotnyh – Karyosystematics of invertebrate animals. – Sankt-Peterburg, 16 (in Russian).
7. Peacock, F., S. Bunting, and K. Queen. 1965. Serum protein electrophoresis in acrilamyde gel patterns from normal human subjects. Science. – 147:1451–1455.
8. Garbar, O. V. 2001. Kompleksne kariologichne ta morfologichne doslidzhennya stavkovikiv fauni Ukraini – Comprehensive karyological and morphological study of pond fauna of Ukraine: Author. Dis. forobtaining Sciences. degree candidate. Biological sciences. Kiev, 20 (in Ukraine).
9. Perel, T., and A. Grafodatskiy. 1984. Novie vidy roda Eisenia (Lumbricidae, Oligochaeta) i ih hromosomnye nabory – New species of the genus Eisenia (Lumbricidae, Oligochaeta) and their chromosome sets. Zool. Zhurnal – Journal of Zoology. 63(4):610–612 (in Russian).
10. Ivantsiv, V. 1989. Shchetinkoviy apparat dozhdevyh chervey (Lumbricidae, Oligochaeta) i ego ispolzovanie v diagnostike taksonov vidovoy gruppy – The bristle apparatus of earthworms (Lumbricidae, Oligochaeta) and its use in the diagnosis of species group taxa. Vestnik zoologii – Vestnik of Zoologу. 3:76–77 (in Russian).
11. Logvinenko, B., O. Kodolova, and N. Bolotetskiy 1984. Sravnenye nekotoryh vidov dozhdevyh chervey semeystva Lumbricidae (Oligochaeta) po elektroforeticheskim spektram obshchikh belkov – Comparison of some species of earthworms of the family Lumbricidae (Oligochaeta) by electrophoretic spectra of common proteins. Dokl. AN SSSR – Dokl. Academy of Sciences of the USSR. 275 (5):1253–1255 (in Russian).
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