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Eggshell Composition, Formation and Function

Egg Shell Composition and Structure
Reptiles (Lepidophores and Testudines)
A mineralized egg shell membrane evolved as a synapamorphy for reptilia (1). The mineral content and structure of eggshells vary among reptilian species. Egg shells of many reptiles consist of a fibrous shell membrane connected to the albumen, as well as a calcareous layer attached to the outer surface of the membrane. The inorganic content of the reptilian shell involves mainly the outer portion and is comprised of calcium carbonate in the form of calcite (5). Reptilian eggs can be separated into three separate groups based on the structure of their shells: flexible shells with no calcareous layer, flexible shells with a thick calcareous layer, and rigid shells with a well-developed calcareous layer. Egg shell structures can be examined and studied with the use of a Scanning Electron Microscope (SEM). The SEM analysis of eggshells is used to differentiate important taxonomic characters to determine relationships among taxa.
Squamate reptiles mainly lay eggs with flexible shells. In fact, all squamate eggs are flexible except for two subfamilies of keggonid lizards (1). Some of these flexible shells, such as those of Diposaurus doralis, Anolis auratus, Anolus limiforns and Natrix natrix contain no calcareous layer, only a shell membrane. This fibrous membreane is organized into an irregular series of ridges and a finely woven mat of fibers on the outer surface.
In other squamates, the outer surface of the flexible shell contains a relatively thin calcareous layer. This can be shown in the network of spheres found in the SEM pictures of Figure 3A and B (3). This type of shell is found in Anolis carolinensis and Callisaurus draconoides (3). Pogona barbata is a squamate with a unique egg shell structure; it is the only flexible shell squamate egg in which the calcite crystals form columns that penetrate into the fibrous layer. This type of egg shell is also typical for the lepidosaurian sphenodon punctuates, or Tuatara (5). A study done by Osborne and Thompson on the chemical composition and structure of the eggshell of three oviparous lizards analyzes the typical flexible eggshells of squamates including the scincid lizards Lamprophois guichenoti and Laprophois delicate, as well as anagamid Physignathus lesuerurri. This study used SEM to determine that the structure of the shells of L. guichenoti, L. delecata and P. lesueurii were very similar. Their shell structure consisted of an outer calcareous layer, a fibrous shell membrane and an inner boundary layer. The calcareous layer appeared uneven at low magnification, but showed a surface consisting of protruding calcareous segments which touch to form a smooth appearance at higher magnifications. Also visible were pore like structures and fibers that were laid down in layers with alternating direction. Osborne and Thompson’s study also did an x-ray analysis using an Energy Dispersive Analysis by X-ray (EDAX) to determine the distribution of ions within the eggshell. This study showed that most of the inorganic content of the shell, including magnesium, sodium and potassium occurred in the calcareous layer.
Some turtles also have flexible shelled eggs. These eggs are made of a defined calcareous layer that is as thick as the shell membrane. This calcareous layer is variable however. For example, the eggs of sea turtles have a poorly ordered, open matrix calcareous layer with undefined shell components and pores. In the flexible shelled eggs of Emydids and chelydrids, however, the calcareous layer is highly structured with well defined shell componants and pores (3). Rigid-shelled eggs (laid by crocodilian, some chelonians, dibamids and gekkonids) have a well developed calcareous layer that makes up most of the eggshell and a thin shell membrane. These eggshells are organized into shell units that fit together tightly and interlock. Turtles do not have only flexible eggs however; shell structure can be described as pliable, hard-expansible or brittle (1).
Birds (Aves)
Birds generate heavily mineralized, rigid egg shells (2). The shell of avian eggs consists of shell membranes along with an external layer of calcite crystals (A). Avian egg shells are composed of mostly inorganic materials with an organic matrix. The shell is made of two separate layers, the palisade and the cone layer, which make up the bulk of the shell. The palisade and the cone layer are comprised of calcite. The outer part of the palisade layer is formed from dense crystalline material. Magnesium and phosphate are minor constituents to the avian eggshell. Magnesium levels are thought to increase outwards from the inner shell toward the palisade layer and a relationship between the magnesium content and strength and hardness of the eggshell of the domestic hen has been found by a study completed by Board and Scott (c). Surrounding the true shell are shell accessory materials comprised of a distinct cover and cuticle made of organic and inorganic material. Pore canals are present connecting the cones and extend across the palisade layer to the external surface of the shell. The type of pore canals is a major variation in avian eggs. There are five types of pore systems found in avian eggshells: open, occluded, plugged, capped and reticulate. Most pores are non-branched; exceptions to this are mainly in thick shells such as those of swans, ratites, and ostriches. The simplest open pore systems can be found in the in the eggshells of pigeons and doves. Occluded pore systems have accessory materials that form a skin on the outer surface of the shell but are considered to be a variant of the simple shell form. Plugged pore systems are found in Rhea Americana, Micropara capensi, and Cuculus canorus ©. The plugs themselves are made of variable materials. Capped pore systems have pores covered with accessory materials in the form of sphere like layers. Capped pore systems include the eggshells of Guira guira, Crotophagia ani, Podiceps cristatus and Numidia meleagris ©. The reticulate pore system includes a pore in which the outer part of the palisade layer is extensively modified. This type of pore system can be found in Casuarius casuarius, and Dromaius nouvaehollandiea. ( c).
The eggs of currently living oviparous mammals are composed of soft, leathery, flexible shells. The monotreme egg shell is not mineralized and lacks a calcareous covering. The parchment- like egg shell is white in color, soft and compressible (9). Little studies have been done on the egg shell composition and structure and therefore will not be mentioned.
Although it is known that reptiles ovulate all eggs at the same time, the process of shell formation in reptiles is not well studied. For example, a study of the prolonged egg retention in the turtle Deirochelys reticularia in South Carolina by Buhlmann, Lynch, Gibbons and Greene were not able to determine if eggshell formation begins sequentially on each egg or if formations begins only when all of the eggs are in the oviduct. (11) It is known that formation of the shell membrane and the calcareous layer happens in the uterus. In order to form the egg shell a supersaturated solution of ions needs to be present along with a site of nucleation which acts to overcome the free energy barrier of crystal growth. These nucleators differ depending on the egg type.

Genetic Investigation of Corn

According to Mendel’s Law of Segregation, phenotypic ratios may be influenced by dominance of one allele compared to another. The alleles separate when an organism produces gametes via meiosis. This experiment investigated
INTRODUCTION In order to conduct this experimental, Mendel’s laws of inheritance were to be studied in order to understand genetics. Mendel’s first law (the principle of segregation), is where two alleles of a homologous pair segregate during the formation of gametes, via meiosis, and each gamete only receives one allele and the phenotype ratios are influenced by the dominance of one allele compared to another. Mendel’s second law is the principle of independent assortment where alleles of a pair of genes arrange themselves independently of the other gene pairs on heterozygous chromosomes.
Corn cobs were provided for the experiment and each cob had more than one phenotype. Corn plants pollinate via wind, therefore, each kernel may be the product of a different cross. All kernels within a cob share the same female parent but could have many different male parents. By looking at Figure 1 below you can see the aleurone layer. This layer can be all sorts of different colours due to the anthocyanin pigments that are contained within it.
Genes are the fundamental biophysical unit of hereditary information. It occupies the locus of a chromosome, and when it is copied it affects the phenotype. Genes are able mutate and various allelic forms can be produced. Genes are contained within the DNA (deoxyribonucleic acid) of an organism, for bacteria and viruses it is kept within RNA. Alleles are an alternative version of a gene that produces distinguishable phenotypic effects. If the alleles that are produced are identical to each other, the individual has the homozygous trait. However, if it is made of two different alleles the individual is heterozygous.
In order to determine the type of cross and genes responsible for what a corn can look like, the colouration and texture of the kernels were looked at. The four phenotypes identified were red and smooth (RS), red and wrinkled (Rs), yellow and smooth (rS), and yellow and wrinkled (rs).
The aim of this experimental was to examine the behaviour of two different genes for colouration and texture within corn kernels. The experiment investigated the F2 generation results from two monohybrid crosses, RS Rs rS rs x RS Rs rS rs. A null hypothesis was proposed, H0, there is no difference between the phenotype of the observed class results and the expected class results. There will be a phenotypic ratio of 3:1, red to yellow phenotypes with the crosses RS Rs x rS rs. As an alternative hypothesis, H1, the phenotypical ratio between the observed and expected class results are different to that predicted ratio of 3:1.
P generation
F1 generation
F2 generationThe trait investigated in the first section is the kernel colour. A monohybrid cross is the product of a single pair of alleles. The red colour (R) is the dominant gene, whereas the recessive is the yellow colour (r). The P generation represents the parental, F1 and F2 generations represent the first filial and second filial generations.
RR (homozygous) x rr (homozygous)
Rr (heterozygous) x Rr (heterozygous)
RR Rr rr
In a test cross, the individual with the unknown genotype is crossed with a homozygous individual that expresses the recessive trait, and punnett squares are used to predict the possible outcomes (refer to results and discussion) (Campbell et al., 2008). This monohybrid test cross involved several plants from a pure line of plants that produced all yellow kernels, and one individual plant that only produced red seeds. The red genotype could be RR but since the R (red) allele is dominant to the r (yellow) allele, it could produce the phenotype Rr.
The dihybrid cross had for grain phenotypes in the ear of genetic corn and they were red and smooth (RS), red and wrinkled (Rs), yellow and smooth (rS), and yellow and wrinkled (rs). In addition to our previous dominant and recessive genes of red (R) and (r), S represents a smooth texture dominant to s which is a wrinkled texture.
The difference texture characteristics is because of the gene controlling storage within the endosperm (protective layer that surrounds the embryo in seed plants) (Figure 1). The endosperm can contain either sugar or starch. If it encases starch it will appear full, smooth and rounded (S), however, if it is sugar it will look wrinkly (s).
Determine the expected frequencies of the genotypes and phenotypes in the F2 generation of the monohybrid cross, by filling in the genotypes, transferring them, and calculating genotype and phenotype frequencies.
Count the kernels on one ear of corn, classifying them as either red or yellow. Keep track of your results, and when you are done, add them to the class results. Use the table to keep track of your results. To prevent counting kernels twice, use pins to mark your position: one for the row you started on and one for the row you are currently counting.
Compare the numbers of each phenotype on your kernels and on the class kernels with the numbers you would expect based on the outcome of a monohybrid cross. Expected numbers may be calculated for each phenotypic class by multiplying the total number of kernels counted (by you and by the class) times the expected fraction for that phenotypic class.
Carry out a test on the class results.
Count the kernels on one ear of corn from the monohybrid test cross set, using the same techniques as previously. Keep track of your results.
Construct punnet squares in order to determine whether the parent that grew from a red seed had the genotype RR or the genotype Rr.
Determine which expected frequency best fits the data you observed. This does not require a statistical test.
First use a punnet square to examine the theoretical outcome of the heterozygous x heterozygous dihybrid cross. Remember that each box represents a genotype possibility for an offspring. Determine the outcome as phenotypic ratios.
Obtain an ear of corn that is the result of a cross that was heterozygous x heterozygous for both traits. Count the kernels using the same techniques as previously.
Now calculate the ratio for the cross. The phenotype with the least number of individuals you will call 1. Place the 1 in the space below the appropriate phenotype. Now divide the other count numbers by the number of individuals from the phenotype you called 1, and round your answers to the nearest whole number. Compare your results with the theoretical answers you obtained for the cross.
Data from the monohybrid test cross did support the predicted ratio of 3:1. The monohybrid phenotypic ratio of 3 red seeds versus 1 yellow seed is derived from a punnett square (see tables 1 and 2). The observed values were 263 red kernels and 133 yellow kernels, while the class observations were 363 red and 143 yellow. The chi-squared value was used to interpret the data and came to 3.09. Also, the chi-squared value for p0.05 was calculated and came to 3.03. Therefore, the null hypothesis was accepted and there was no difference between the phenotypes of the observed and expected class results.
Punnett squares were constructed in order to determine whether the parent that grew from a red seed had the genotype RR or the genotype Rr. The expected frequency had to be determined by using punnett squares (tables 5 and 6). My results of counting the kernels on this corn cob were 325 red seeds and 146 yellow seeds.
For the dihybrid cross examination, a punnett square was used, first to calculate the theoretical outcome of heterozygous x heterozygous dihybrid cross (table 8). Then, a phenotypic ratio was produced which was 9:3:3:1. A corn cob was then counted using the same techniques that were used for the other corn cobs. There were 111 RS, 52 Rs, 341 rS, and 87 rs kernels. The ratio for the cross was calculated and supported the original phenotypic ratio of 9:3:3:1. Therefore, it is easy to say that the dihybrid cross followed Mendel’s law of independent assortment.