biochemical technique in taxonomic studies

Biochemical techniques

1. Biochemical Technique in Taxonomy

2. Taxonomy The term is derived from the Greek word - taxis (“arrangement”) and nomos (“law”) . Taxonomy is the methodology and principles of systematic botany and zoology and sets up arrangements of the kinds of plants and animals in hierarchies of superior and subordinate groups . (Tapan and Dutta, 2017)

3. Biochemical Taxonomy This method extensively studied in plants than in animals . de Candolle (1813) first initiated the use of such characters in taxonomy in 1813 to differentiate close1y related species of plants. Even Lankester (1871) speculated that The chemical differences of different species and genera of animals and plants are certainly as significant for the history of their origin as the differences of form .

4. The animal contains a large number of complex compounds like hormones. enzymes and other proteins with peptides, nucleic acids, amino acids and sugars . The biochemical taxonomic techniques are probably less subject to direct environmental influences and thus are more likely to reflect genetic divergence than many of the classic morphological analyses.

5. The principal work of a biochemical taxonomist concerns the comparison and contrasting of compounds of the same class and performing the same function in different animal species, with regard to their properties as well as to their distribution in different organs of the body. The species can be differentiated based on the amino acid sequences in the proteins of an organism and on differences between these as found in different species. Crick (1958) called it 'Protein Taxonomy' .

6. Turner (1966) preferred to divide it into two taxonomy Microinolecular Taxonomy : This is stress upon the distribution and biosynthetic interrelationships of small molecular weight compounds such as free amino acids, alkacids, terpenes, flavonoids, etc., commonly referred to as secondary compounds . This approach is especially useful in resolving systematic problems where hybridisation has been a factor.

7. Macromolecular Taxonomy: This is concerned with the polymeric molecules like DNA, RNA, polysaccharides and proteins . This approach is useful in resolving some of the more intractable systematic problems especially those involving relationships among higher categories.

8. Kinds of Approaches of Chemical Taxonomy These studies are taken up in five ways a) Immunological b) Chromatographic c) Electrophoresis d) Infrared spectrophotometry e) Histochemical

9. Immunological Approach Immunological: This approach is based on the precipitin reaction preferred for the study of soluble antigens, such as those contained in animal sera or tissue extracts from plants or animals. It was first discovered by Rudolph Krauss (1897) in respect or microorganisms. Nuttal (1901) was the first its use in animal systematics. Its application is based on the fact that "the proteins of one organism will show a stronger antibody reaction to the proteins of a closely related organism than they will to those of a more distantly related organism".

10. Identifying predators, antibodies against S. frugiperda eggs were generated by inoculating rabbits with macerated S. frugiperda eggs, and the production of antibodies against S. frugiperda egg proteins was verified by double immunodiffusion (DID). These antibodies were then utilized in another serological technique, counterimmunoeletrophoresis (CIE), to identify insects that could have ingested S. frugiperda eggs. This study shows that the CIE technique is efficient in the identifi cation of insects that fed on eggs of S. frugiperda. Dorus luteipes Lagria villosa Use of Serological Techniques for Determination of Spodoptera frugiperda (J E Smith) Predators (Lepidoptera: Noctuidae)-Joaquim et al. (2010).

11. Counterimmunoelectrophoresis in 0.7% agar gel Double immunodiffusion in 0.9% agar gel with pure antigen

12. Chromatographic Approach Chromatography: It is a technique by which the constituents of a complex mixture can be separated and subsequently identified. It depends on the "different rates at which the compounds in a double mixture move along a porous medium, i.e., a piece of paper (paper chromatography) or a column of powdered chalk (column chromatography).”

13. Paper chromatography has been widely used for comparing the chemical composition of closely related species, especially with regard to amino acids and peptides through ninhydrin treatment (spray), and purines, pyrimidines, or other compounds which either fluoresce or absorb ultraviolet light. Florkin and Jeuniaux (1964) discovered that the primitive hemimetabolous insects have low concentration of free amino acids in their haemolymph as compared to high concentration in holometabolous insects.

14. Paper chromatography One- dimensional chromatograms of Hemiptera (T. gerstaeckeri, A; T. infestans, B), Orthoptera (Periplaneta americana, C; Blattella germanica, D; Supella supellectilium, E) and Diptera (C. fatigans, F and G). The chromatogram on the left was run in propanol- NH3 and the one on the right in propanol-diethylamine. (Micks, 1956)

15. Gas chromatography Cuticular hydrocarbons as a tool for the identification of insectspecies: Puparial cases from Sarcophagidae- (Braga et al ., 2013) Hierarchical tree made by clustering of Bray– Curtis similarity index among hydrocarbon profiles analyzed by GC–MS. Representation of the general phylogenetic relationships Peckia and among Peckia chrysostoma, intermutans, Sarcodexia lambens Sarcophaga ruficornis .

16. Electrophoresis Approach Electrophoresis: involving a similar movement of dissolved substances through a fixed medium, but here the movement is brought about by electrical potential differences . It is based on the fact that the "components of mixtures carry electric charges of varying amounts and so will move at different rates in salt solution through which a current is passed".

17. Such techniques were first used by Tiselius (1937) to distinguish multiple fractions of serum proteins migrating through solution under the influence of an electric current . Since then these techniques have been greatly refined to permit even large numbers of different proteins in the same cyanine.

18. Electrophoretic Studies of Insect Esterases – (Arurkar and Knowles, 1967) Zone electrophoresis in acrylamide gel was used to separate the soluble esterases from homogenates of 6 insect species. Esterases were classified according to their response to certain organophosphate and carbamate compounds. Zymograms resulting from separation of esterases present in homogenates of house cricket nymphs in acrylamide gel.

19. DNA barcoding, species- specific PCR and real- time PCR techniques for the identification of six Tribolium pests of stored products (Zhang et al ., 2016)

20. Infrared spectrophotometry Approach It is based on the principle of absorption of infrared light by biological materials. The patterns thus formed depend upon their chemical composition and bring to light many features of taxonomic importance. So far, this approach is mainly applied to microorganisms (Norris, 1959). Micks and Benedict (1953) for the first time applied this technique in the identification of mosquitoes .

21. A near- infrared spectroscopy routine for unambiguous identification of cryptic ant species – ( Kinzner et al ., 2015) Mean spectra of four cryptic Tetramorium species. All spectra showed a similar curve progression, and differences were not detectable by visual evaluation; chemometric analysis was required. R, reflectance.

22. Histochemical Approach Histochemical studies: When the same kind of tissues from different animal species may exhibit apparently the same functions, histochemical differences between them may be observed which could be of taxonomic value . This can also help in the recognition of infraspecific groupings. These techniques have been employed in the qualitative and semi- quantitative analysis of proteins, free amino acids, enzymes, carbohydrates, liquids and nucleic acids including metal ions.

23. A method for paraffin sectioning and identification of indoleamines in the brain of insects with a sclerotized cuticle- ( Freelance et al ., 2017 ) Brightfield images of stained longitudinal sections through cricket head

24. Advantages of Biochemical Taxonomy Basu and Chatterjee (1969) demonstrated phylogenetic relationship among various orders of insects and birds on the basis of the quantitative analysis of ascorbic acid . In some birds it is produced in the kidney; in some, in the liver; in some, in both liver and kidney; and in others, in neither. Accordingly they clarified that the ancestral enzyme systems involved occurred first in kidney, were later somehow transferred to the liver, and finally, in some of the more evolved passerine birds, completely lost.

25. Brand et al . (1972) established the phylogeny of a group of fireants using biochemical characters of the highly unique fireant venoms . Walbank and Waterhouse (1970) corrected the phylogenetic affinities (based earlier on morphological data) of certain genera of Australian cockroaches after analysing their defence secretions.

26. Disadvantages of Biochemical Taxonomy Such studies are possible only in the existing organisms and therefore it is difficult to trace the course of evolutionary history . It cannot lead to definite judgements with regard to the phylogeny of any organism whose fossil records are inadequate or lacking. Most of the biochemical taxonomic works are based on qualitative and quantitative differences in single chemical constituent of whole organisms or one of their tissues. Like morphological characters, chemical characters are also variable.

27. Brand, J. M., M. S. Blum, H. M. Fales, and J. G. MacConnell. (1972). Fire ant venoms: comparative analyses of alkaloidal components. Toxicon 10: 259 Ð271. Chaudhuri CR, Chatterjee IB. (1969). L- ascorbic acid synthesis in birds: phylogenetic trend. Science.164:435–6. [PubMed: 5777214]. Wallbank, B. E.; Waterhouse, D. F. (1970- 11- 01). The defensive secretions of Polyzosteria and related cockroaches. Journal of Insect Physiology. 16 (11): 2081–2096. doi:10.1016/0022-1910(70)90081- 8. ISSN 0022- 1910. Nuttall, G. H. F. (1901b). A further note on the biological test for blood and its impoitance in zoological classification. British Medical Journal. 2: 669. Tiselius, A (1937). Electrophoresis of serum globulin II. Electrophoretic analysis of normal and immune sera. Biochemical Journal 31, 1464–1477. Norris, K. P. (1959). Infra- red spectroscopy and its application to microbiology. The Journal of hygiene, 57(3), 326–345. doi:10.1017/s0022172400020192.