Specimen 327a: Suzanne Scott and Derek Randolph

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Contents

TEST

Molecular Introduction

paragraph 1 paragraph 2

Location of collection=

GGC site 2

Date of collection

Fall 2012

Shannon-Wiener Index of Biodiversity

H' 3.5788

Photo

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Distinguishing morphological features of Order

When identifying plecoptera, the important features to look for are (1):

  • Elongated body
  • Cerci(face shield)are well developed often long and many segmented
  • Wings are forward usually with a series of cross veins appearing ladder like
  • Hind wing usually with large anal lobe
  • Tarsi is three segmented
  • Threadlike antennae
  • Chewing mouthparts

Sub-order ? Family ? After sequencing, genus? and species?

  • Family: Coleophoridae
  • Genus: Coleophora
  • Species: Duplicisgroup

Geographical Distribution

Coleophora is a very large genus of moths of the family Coleophoridae. It contains some 1,350 described species. The genus is represented on all continents, but the majority are found in the Nearctic and Palaearctic regions.

Life cycle

Sexual dimorphism

What it eats:Larval/nymphal stage Adult stage

As with most members of the family, the larvae initially feed on the seeds, flowers or leaves of the host plant, but when larger, they feed externally and construct distinctive protective silken cases, often incorporating plant material. Many species have specific host plants; discarded larval cases are often scattered thickly on affected plants.

Habitat: Larval/nymphal stage Adult stage

Ecological Importance: Larval/nymphal stage Adult stage

Economic/agricultural/human health importance: Larval/nymphal stage Adult stage

DNA isolation

The purpose of this project was to extract DNA from an insect specimen collected by Ecology student Suzanne Scott and have its DNA analyzed to discover the insect’s genus and species. The first part of the project was to isolate the DNA from the rest of the insect and amplify the mitochondrial gene CO1 of the DNA. The DNA was then separated and purified of other material. Next, the DNA was sent off to be sequenced at the University of Georgia. When the results returned they were compared to databases to find the species it most closely related to if any. The start of the project began with extracting and isolating the DNA from insect # 327. Extraction broke apart the insect to get to cellular material. The DNA was then isolated to separate it from the other cellular material. With no test to verify, it was assumed that the final product was DNA from the insect. With the final product of DNA, the next step was to amplify the gene needed.

Polymerase Chain Reaction/Gel electrophoresis

The section of DNA wanted was the CO1 gene from the mitochondria of the cell. This gene is involved in cell respiration and is very distinguishable and unique to different species. The Polymerase Chain Reaction (PCR) targets the CO1 gene and replicates it multiple times. To assure that the procedure worked accurately controls were used. One control contained DNA designed to show the same results as the previously isolated DNA and another control contained no DNA that was designed to not show anything. The two controls would be compared to the isolated DNA to see if there was contamination of any kind during any of the previous processes. In the next step gel electrophoresis would show if the PCR had worked. The final product of PCR was DNA that had been replicated multiple times to give lots of the CO1 gene to be put through the next process gel electrophoresis. The gel electrophoresis separated the DNA into different nucleotide lengths by size with electrical currents. A stain was added in this process so the DNA could then be viewed under a UV light. The result, as seen in (Figure I), was that the band wanted was not very bright and a second unwanted band appeared. This was a result of possibly not having enough DNA from PCR or contamination during previous processes. Therefore the PCR and gel electrophoresis would be done again to get better results. The second PCR used three different concentrations of DNA to insure different results from the first PCR. The results of the second PCR came back with brighter bands that were wanted as seen in (Figure II). With lane 4 having the brightest band of the three lanes, it was chosen to be purified of all other materials.

327pcr.JPG

Sequencing

Before the DNA could be sent to the University of Georgia to be sequenced, the DNA needed to be cleaned up of all unnecessary material. The Exosapit removed excess DNA and free nucleotides making the DNA much easier to sequence. The DNA was then ready to send to the University of Georgia to be sequenced and the results would return two weeks later. The results that came back were a forward and a reverse sequence (Figures III (a) & (b)). The sequences were run through a computer program to put them into chromatograms or picture representations of the sequences (Figures IV (a) & (b)). The sequences were now ready to compare to online databases.

327sequence.JPG
327chromatogram.JPG

Bioinformatics

The sequences needed to be compared to databases to find out the genus and species of the insect from which the DNA was taken. Both of the sequences were put into the BOLD database and Blast database and the results came back with the most related genus and species. The BOLD database results came back for the forward sequence as genus: Coleophora and species: duplicisgroup (Table I) and the reverse sequence was genus: Aegla and species: neuquensis (Table II). The BLAST database results had no similarities for the forward sequence (Figure V) and the reverse sequence was genus: Coleophora and species: Intermediella (Table III).

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