21 Jul 2010

Sample Essay: The Intraspecific Variation by Study of Electrophorectic Banding Patterns Derived From Striated Muscle Samples in Herring (Clupea)

Genus Clupea

Fishes of genus Clupea, which are classified as C. pallasi (Pacific herring) or C. harengus (Atlantic herring), are basal marine teleosts known for their less stringent requirements to reproduce. Members of C. pallasi are commonly found along the eastern coast of Bering Sea and northeast Pacific ocean (Williams and Quinn, 2000). This has lead to a variety of populations living at different conditions. Variations in living conditions among their habitats eventually lead to differences in migration cycles, age structure, growth rate, and age of maturation among populations (Semenova, et al., 2009). Because of its market value, it has been a subject of many researches originally focused on describing their adaptation mechanisms and finding means to manage their population. These initial efforts resulted to a well-defined wide range of spawning season and specific spawning locations. Gradually, studies using members of Clupea as animal model have lead to researches about epigenetics and development, physiology, and evolution (Geffen, 2009).

Role of molecular biology in ecology

Study of molecular biology of Clupea, or other animal groups, is important in population maintenance. Since its conceptualization, the use of molecular markers has gradually overtaken the role of morphology in assessing differences among populations, because morphological traits overlap between populations. For example, Atlantic and Pacific herring were differentiated based on the number of vertebrates. The multivertebrate Atlantic herring has, on an average, 57 vertebrae, while the oligovertebrate Pacific herring has 53. However, this character proves to be unreliable in population studies because the ranges of the number of vertebrates in Atlantic and Pacific herrings considerably overlap: 51-60 vertebrae for Atlantic herrings and 47-57 vertebrae for Pacific herrings (Semenova, et al., 2009).

Landscape genetics

Inferring specific environmental factors that may have caused the persisting genetic structures and their differences among populations, or predicting future genetic structures of populations based on current environmental pressures can be done through landscape genetics (Jorgensen, et al., 2005). Landscape genetics works on the presumption that genetic drift, is a reflection of habitat reduction and fragmentation, because such conditions hamper the continuity of flow of adaptive genes (Jorgensen, et al., 2005). Genetic drift is a natural evolutionary process in which a population’s allele frequencies change. The effects of genetic drift are much more magnified when the population size becomes drastically reduced. The resulting population may not represent the original population’s gene pool, may overrepresent alleles, and may subsequently not contain in them other alleles (Campbell and Reece, 2002). So how can a sudden reduction in population size and subsequent reduction in genetic monotony be prevented? By allowing migration to seek refuge and/or subsequent interpopulation interactions to allow adaptive genes to be passed on. Indeed, the genetic make-up among populations reflects the interplay of genetic drift and gene flow (Jorgensen, et al., 2005). The set of genes that allows evolutionary success changes from time to time, depending on the present environmental conditions. The variety in one’s genetic structure increases the possibility that it contains the genes encoding for proteins or mRNA that allow survival in the persisting conditions. In fishes, genetic differentiation among populations of the same species can be expected if they have (1) large spawning population sizes without considerable genetic drift, (2) freedom to migrate, (3) highly dispersive migratory life stages to increase the gene flow (Jorgensen, et al., 2005). On the contrary, isolated populations tend to reduce their genetic variability and their ability to adapt to environmental changes because they are not exposed to other populations that may have a genetic make-up different from theirs. Thus, adaptive genes, and an increased possibility of having them, are important factors ensuring survival because it allows resilience against disturbances (Piorski et al., 2008).

Genetics are also important in establishing intraspecific variations within a species, which are usually designated by morphological similarities or interbreeding ability. For such purposes, use of molecular markers is inevitable because members of a species usually look the same in terms of their morphological character (Chinain and Germain, 1997).

Molecular markers for landscape genetics

In determining genetic variations among populations of Clupea, lactate dehydrogenase (LDH) -1* and LDH-2* are two of the four polymorphous loci determined to be most informative. For example, C. harengus and C. pallasi can be differentiated just by LDH loci. Using LDH-1* locus, LDH-1* 100 allele can only be found in C. harengus, while LDH-1* 200 is only found in C. pallasi (Semenova, et al., 2009).

Just like any other scientific techniques, use of genetic markers has its drawbacks. First, compared to proteins, DNA are much harder to extract. The genetic material, especially of eukaryotes, is packed tightly with histones to fit into the nucleus. On the other hand, enzymes, such as LDH, for example, are just in the cytoplasm. In addition, the ratio of gene: protein in any cell is definitely much lower than its reciprocal. You can thus get lower amounts of gene than protein from a certain volume of tissue homogenate. Second, the DNA sequence should have already been more or less established. This is necessary in producing primers, which attach to the DNA at its complementary site. Primers will then allow selective DNA replication through PCR, or to present substrates for nucleases to cleave desired potions of the DNA. Because of these, other molecular markers have already been explored for use in population genetics.

Isoenzymatic markers

Aside from genetic markers, one of the molecular makers through which fish population is studied is through isoenzymatic markers because members of the same species can be different biochemically (Chinain and Germain, 1997). In addition, differences in proteins are reflection of differences in genes encoding them. Isoenzymes are molecular modifications of an enzyme resulting from the presence of multiple genes coding for the enzyme. This multiple genes are a result of substitution and insertion/deletion mutations in the DNA (Piorski et al., 2008).

Lactate dehydrogenase (LDH): function and structure

Table 1. Comparison of metabolic processes between normal oxygen and hypoxic conditions
Normal oxygen levels Hypoxic condtions
Glycolysis (2 ATP)

Glucose + NAD+          2 pyruvate + NADH

Entry of pyruvate into mitochondria

pyruvate           2 acetyl coA

Lactic acid fermentation by LDH

2 pyruvate + 2 NADH          2 NAD+ + 2 lactate

Krebs cycle (2 ATP)

= energy from 2 acetyl coA transferred to NADH and FADH2

Electron transport chain and oxidative phosphorylation (34 ATP)

= Energy from NADH and FADH2 converted into ATP

Total number of ATP produced:  38 ATP 2 ATP

LDH is a NAD-dependent tetramer protein responsible for lactic acid fermentation, regulating pyruvate and lactate interconversions through redox reactions during hyper- and hypoxic conditions (Gronczewska, et al., 2003; Meany, 2007). After glycolysis, pyruvate is processed depending on the availability of oxygen. Table 1 summarizes the metabolic pathways at different oxygen concentrations. At physiological oxygen levels, pyruvate enter the mitochondria continues into Krebs cycle, after which energy is processed through an electron transport chain that utilizes oxygen in one of its steps. At hypoxic conditions, pyruvate cannot enter the Krebs cycle. Energy production is thus depended solely on glycolysis, when glucose is converted into pyruvate. What happens next is that LDH, with reducing agent as NADH, reduces pyruvate to lactate. It is not an energy-producing process, but this is necessary to continue reproducing NAD+ that is involved in glycolysis. Accumulation of lactate is harmful, as can be attested by the muscle pain humans feel after prolonged exercise that utilized lactic acid fermentation, because it can be converted to lactic acid, which affects physiological pH and causes lactic acidosis. In addition, aerobic respiration is much more efficient than glycolysis paired with lactic acid fermentation. Thus, when normal oxygen levels are restored, LDH catalyzes the oxidation of lactate by NAD+ to make pyruvate available again for metabolism (Campbell and Reece, 2002; Meany; 2007 Zietara, et al., 2009).

LDH in Clupea

LDH exists in different anatomical structures of Clupea. In spermatozoa, its main role is to reoxidize NADH and to subsequently speed up glycolysis. Lactate metabolism and NADH reoxidation will then power sperm motility even at low levels of oxygen (Gronczewska, et al., 2003). In Clupea harengus, LDH is present in skeletal muscle, cardiac muscle, and semen. The enzyme’s three isoforms have been identified as B4, A2B2, and A4. Cardiomyocytes contain all three isoforms (Gronczewska, et al., 2003), while in their spermatozoa, only B4 and A2B2 LDH isoforms can be isolated, although not as much as found in the heart. (Zietara, et al., 2009; Gronczewska, et al., 2003). The skeletal muscle has the least varied LDH, with A4 as the only type of LDH found in this tissue. It may be inferred that A4 is specific for myocytes, although it is not the main LDH isoform in cardiac muscles (Figure 1).

Figure 1. Electrophoretic profile of lactate dehydrogenase (LDH) from different homogenates of Clupea harengus. The three isoforms, A4, A2B2, and B4, were all seen from the starch gel. Wells are designated as (1) for skeletal muscle, (2) for heart, and (3) for semen. Both the amount and isoenzyme composition of LDH are different among (1), (2), and (3). Cardiomyocytes has the most varied LDH, as it contains all three isoforms, while skeletal muscle has the least as it is only made up of A4 isoform. Semen, on the other hand, has the least amount of LDH. Figure from Gronczewska et al., 2003, p. 402.

Isoenzyme electrophoresis

Isozyme electrophoresis is a useful technique in establishing population variations. This was first used in diatoms, and soon after applied on macroalgae, dinoflagellates (Chinain and Germain, 1997). Two electrophoresis techniques are used for the study of LDH. To screen for the presence of LDH in tissue homogenates, horizontal starch gel electrophoresis is performed. Zietara and Skorkowsi (1993) has identified 14% starch gel ran at 27 mA for 4 hours as the optimal conditions at which LDH electrophoresis should be performed. An LDH-specific stain composed of 30 mM Na-lactate, 0.25 mM NAD+, 0.1 mg/ml p-nitro blue tetrazolium chloride and 0.1 mg/ml phenasine methosulfate in 0.1 M Tris-HCl buffer, pH 8.0 is being used to incubate the gel. Once purified, the LDH-containing sample is run through polyacrylamide gel electrophoresis, and the gel is stained with Coomassie Brilliant Blue R-250 to determine its amount and to confirm differences among samples, if present, that were initially observed during starch gel electrophoresis (Gronczewska, et al., 2003).

It is the aim of this study to determine and elucidate differences among herring populations in terms of (1) LDH isoforms and (2) how their LDH respond to changes in temperature. Most studies and findings have been elucidating the effects of temperature on metabolism. Because the maximum amount of dissolved oxygen depends on how hot or how cold the water is, water temperature is a telling factor whether or not metabolism undergoes an aerobic route. When water temperature is high, water molecules have higher kinetic energy. This subsequently lessens the amount of intermolecular spaces which oxygen molecules can fill. When conditions are changed from normoxic to hypoxic conditions, it was found that metabolism gears toward the production of lactate at a much faster rate when the temperature is at 20°C, than when it is at 5°C. In addition, fishes at 20°C seem to be much more responsive to lowering oxygen levels than at the lower temperature (Li, 2008). Although these changes in metabolism may be traced to changes in LDH, it is possible that other factors contributed to increased lactate production at high temperatures and hypoxic conditions. For example, the fish, as one of its mechanisms, has undergone decreased activity to conserve energy and prevent unnecessary ATP production through lactic acid fermentation. It is thus interesting to find out whether or not changes in LDH really lead to changes in metabolic activity among populations of herring.


Campbell, NA and Reece, JB 2002, Biology, Fourth edition, Benjamin Cummings, San Francisco.

Chinain, M and Germain, M 1997, ‘Intraspecific variation in the dinoflagellate Gambierdiscus toxicus (Dinophyceae). I. Isozyme analysis’  Journal of Phycology, 33, 36-43.

Geffen, AJ 2009, ‘Advances in herring biology: from simple to complex, coping with plasticity and adaptability’ ICES Journal of Marine Science, 66, 1-8.

Gronczewska, J, Zietara, MS, Biegniewska, A, and Skorkowski, EF 2003, ‘Enzyme activities in fish spermatozoa with focus on lactate dehydrogenase isoenzymes from herring Clupea harengusComparative Biochemistry and Physiology, 134B, 399-406.

Gronczewska, J, Biegniewska, A, Zietara, MS, and Skorkowski, EF 2004, ‘Inhibition by tributyltin of herring skeletal muscle lactate dehydrogenase activity’ Comparative Biochemistry and Physiology, 137C, 307-311.

Jorgensen, HBH, Hansen, MM, Bekkevold, D, Ruzzante, DE, and Loeschcke, V 2005, ‘Marine landscapes and population genetic structure of herring (Clupea harengus L.) in the Baltic Sea’ unpublished

Li, VB 2008, ‘Physiological and behavioural responses of Largemouth Bass yearlings (Micropterus salmoides) to hypoxia at summer and winter temperatures,’ MSc. Thesis, Queen’s University

Meany, JE 2007, ‘Lactate dehydrogenase catalysis: Roles of keto, hydrated, and enol pyruvate’ Journal of Chemical Education, 84(9), 1520-1523.

Piorski, NM, Sanches, A, Carvalho-Costa, LF, Hatanaka, T, Carrillo-Avila, M, Freitas, PD, and Galetti Jr., PM 2008, ‘Contribution of conservation genetics in assessing neotropical freshwater fish diversity’ Brazilian Journal of Biology, 68(4, suppl.), 1039-1050.

Semenova, AV, Andreeva, AP, Karpov, AK, and Novikov, GG 2009, ‘An analysis of allozyme variation in Herring Clupea pallasi from the White and Barents Seas’ Journal of Ichthyology, 49(4), 313-330.

Williams, EH and Quinn II, TJ 2000, ‘Pacific herring, Clupea pallasi, recruitment in the Bering Sea and northeast Pacific ocean, II: relationships to environmental variables and implications to forecasting’ Fisheries Oceanpgraphy, 9(4), 300-315.

Zietara, MS, Biegniewska, A, Rurangwa, E, Scwierczynski, J, Ollevier, F, and Skorkowski, EF 2009, ‘Bioenergetics of fish spermatozoa during seme storage’ Fish Physiol Biochem, 35, 607-614.

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