SUMMARY
Molecular information on biological organisms is currently being produced at an astonishing pace. Therefore, it is important that future biologists learn the craft of using such information. However, many undergraduates lack interest towards molecular issues, and still carry that molecularphobia after graduation. This turns student's motivation into a priority goal of biomolecular teachers.
In the University of Azores, many of the incoming Biology students demonstrate an evident interest in marine biology and biodiversity issues. We considered using a marine animal model motivate students to learn the fundamentals of two molecular sciences. This work describes the biochemical part of an interdisciplinary approach in which Biochemistry and Genetics were taught recurring to the most abundant Azorean periwinkles — Littorina striata and Melarhaphe neritoides. The approach includes field and laboratory work that provides results which can be used to teach the fundamentals of protein structure and behaviour, keeping in mind species diversity. The benefits of using local models to motivate students for the science of biology are discussed.
KEYWORDS: Undergraduate motivation, molecular subjects, local models, Littorinidae
INTRODUCTION
Why aim at motivation?
Second year Biology undergraduates at the University of Azores attend two molecular subjects in the same semester: Structural Biochemistry and Genetics. Many confess their reluctance to study a molecular world which they cannot see and is difficult to imagine, and for which they fail to see any future professional interest. Every year several students evidence severe difficulties in concluding any of the disciplines. These recurrent themes have given birth to a tradition: new students are already worried and unmotivated, even before actually starting the syllabus. In the subject of Biochemistry, this was evident not only in tutorials but also in practicals. Such a molecularphobia has important negative consequences for students scientific development (Wood 1996) making more difficult their comprehension of basic information required to interpret molecular literature or devise molecular approaches to biological problems.
Biochemistry practicals consisted on laboratory exercises in which students worked mainly with chemicals and would generally focus on the use a particular laboratory method to answer a biochemical question. Even though the conclusions could be transposed to a biological context, students rarely were successful in doing so, performing the exercises exclusively for their technical interest or curricular obligation.
It occurred to us that one way of overcoming the lack of motivation would be to let students experience Biochemistry and Genetics in a different context, of higher relevance to them. We decided to create and explore a molecular approach that would start from field observation of model organisms and end with laboratory analysis of some of their biomolecules. We chose marine organisms, taking into consideration students. We ended up in littorinids - Mollusca, Gastropoda - since they are abundant all over the azorean rocky shores (Morton et al. 1998), easy to collect and to work with. A diversity of studies have been carried out on these organisms which might be found motivating by the students (for example, taxonomy (e.g. Medeiros et al. 1998), population genetics (e.g. De Wolfe et al. 1998) and biogeography (e.g. De Wolfe et al. 1997)). Using these organisms, we could also profit from the knowhow on these organisms gathered in our University. One last important aspect was that we felt that the work would allow students to work with beings which are part of their regular life, and would thus contribute to the development of links between important concepts an methods, and student's biological context (Grifiths and Moon 2000, Ramakrishnam 2000).
Interestingly, littorinids myoglobin-like proteins are very easy to obtain from the organism's isolated radular muscle (Reid 1996). Furthermore, myoglobin and hemoglobin are classical textbook examples (Zubay 1998, Stryer 1995) explored in tutorials on protein structure and myoglobin sequences of many organisms have been deposited in publicly available data banks and its role as phylogenetic markers has been investigated (Medeiros et al. 1998, Jones 1972, Wium-Andersen 1970). Taken together its theoretical importance, availability of specific data, and student's motivation for marine issues, littorinids myoglobin-like proteins looked like adequate instruments to underly an integrative approach to biochemistry and genetics. The whole approach is oriented towards two teaching goals: (1) molecules can be used to get data on biological diversity, many times in simple ways (Nordell 1999); (2) the potential of using biochemical knowledge in a biological field.
This paper describes an approach in which differences between the main azorean littorinid species were investigated through the analysis of myoglobin-like proteins of the organisms' radular muscles, in practicals of Biochemistry. The approach allows discussion of the basic fundamentals of simple and advanced laboratory techniques (from obtaining and handling of molecular samples to electrophoresis) and observations on protein structure, and how it influences protein laboratory behavior. Results can be used to stimulate students to conclude on how molecular approaches can be applied to biodiversity issues.
METHOD
The biological questions
Three main questions were advanced: (1) do different periwinlkles inhabit the azorean rocky intertidal?; (2) if so, are species differences also evident at the molecular level?; (3) can differences be found on the laboratory behavior of myoglobins of Azorean species of periwinkles?
Field work
Students are organized in groups that developed the field work under teacher's supervision. Field work is easy to perform in low tide, since these inhabit the upper intertidal (Morton et al. 1998) and individuals of different morphologies are quickly identified: Melarhaphe neritoides (smooth, oblong and dark shell - height: 5.0 - 10.0 mm ) and Littorina striata (striated, more spherical and greenish shell - height 5.0-19.4 mm) (Reid, 1996). The specimens are brought to the laboratory and frozen for future work.
Sample extracts
Organisms are thawed, removed from their shell and dissected under a magnifying lens. Students follow the anatomical sketch provided by Reid (1996) to expose radular muscles, bilobular, heart shaped bright red organs, found under animals' heads. Students weigh the organs on 1,5 ml eppendorfs, and add the correct volume of homogeneization buffer (Medeiros et al. 1998). Afterwards students disrupt the organs, crashing them against the tube's walls with 0,5 ml eppendorf fitted in glass rods, until a crude homogenate is obtained. By this time, student's obtain pale red supensions which are centrifuged, the supernatant being retained for further analysis. Experimental mistkes in weighing or measuring buffer volume may result in colorless extracts.
Spectrophotomerty
The extracts are analysed spectrophotometrically revealing that both species evidence the same visible spectra (Costa and Brito, unpublished) which are compatible with a myoglobin-like identity (Ernesto Iorio 1981). These results allow the introduction and discussion of why molecules absorb light, and about the structural meaning of having the same absortion spectra, and its consequences for the use of spectrophotometry to obtain data on biological molecules and organisms.
Electrophoresis
The work consists of 3 different types of electrophoresis - SDS PAGE (Costa and Brito, unpublished), native PAGE (Medeiros et al. 1998) and IEF (Medeiros et al. 1998) — the 2 latter stained by a myoglobin-like specific peroxidase protocol (Medeiros et al. 1998). SDS PAGE reveals the molecular mass for the major band contained in the extracts, again a value close to what would be expected for myoglobin-like proteins (Zubay 1998). Notably, SDS PAGE and PAGE fail to reveal any differences between myoglobins form the 2 species in contrast with IEF that reveals a 3 banded pattern for L. striata and a single band for M. neritoides. Students are initially puzzled on why they can find no differences between SDS PAGE migrations of both extracts. This problem will assist them in learning some fundamentals of working with proteins.
CONTRIBUTION OF THE MODEL FOR SKILL DEVELOPMENT
Students first use their skills on the preparation of sample records. Different data on each specimen are continuously gathered, requiring the use of synthetic and systematized of records, two skills difficult to practice in "one-stand" practicals. Hand dexterity, organization in the laboratory and being able to work in groups within a limited time period, are all extremely important skills (Wood 1996), that are practised in the steps of organism dissection, measuring weight and volumes, and electrophoresis (Reed et al. 1998).
Knowing how to use theoretical information on protein structure, is vital to interpret the different results obtained in the various electrophoretic techniques. Students must understand what it means to be native or denatured, to explain the different migrations between native and denaturing PAGE. Furthermore, they must understand the relatively minor amount of biological information stored in a protein’s molecular mass, when compared to the information that is given by amino acid composition (a concept many times reinforced in textbooks, but seldom evidenced in practicals).
Taken together, the laboratory exercises allow students to progressively construct their comprehension on theoretical fundamentals, as they are experiencing the making of observations in a progressive, student-centered integrative way. If the former is compared to classical practical classes in which a single and different goal is introduced per class, one realizes that students have increased opportunities of developing integrative.
STUDENT'S REACTIONS
This model was introduced this year for the first time, resulting in more interested attitudes from students towards the classes - instead of dully assisting some of the practicals, most were dynamically engaged in their endeavors. Student's opinions were also gathered in written questionaires handed in the last practical, from which the following considerations can be made, bearing in mind that only 29,6% of the questionaires have been returned yet. 89% felt the approach contributed positively for their understanding of the laboratory procedures, 61% discoverwed unexpected links between biochemistry and genetics and 67% confessed to have thought of biology in ways yet unexplored. Finally, 83% of the students felt motivated by the approach.
COMMENTS
The fact that this molecular approach deals with marine organisms, may have contributed to the arousal of new motivation in some of the students. Since Littorinids exist worldwide (Reid 1996), the model can be reproduced in other places, as long as there is access the shore. The obtention of the organisms, muscles and extracts is very cheap and easy to acomplish. As to the other techniques, they are quite straightforward and reproducible, once basic molecular analytical equipment is available.
FINAL REMARKS
Our final messsage does not have to do directly with this model, but with the incredible diversity of biological models that await to be explored outside the biology classroom. Our experience is that they can be important in getting students actively involved in learning, as they experience their own bilogical context..
REFERENCES
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R. Medeiros, L. Serpa, C. Brito, H. De Wolf, K. Jordaens, B. Winnepenninckx and T. Backeljau, "Radular myoglobin and protein variation within and among some littorinid species (Mollusca: Gastropoda)", Hydrobiologia, 378, 1998, 43-51.
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