Insect Pigment Patterning: A Genome-Wide Study of Bombus melanopygus Using tan Gene

Andrew Walrond

Abstract
Insect pigmentation is a phenomenon that has garnered the attention of the general public and scientists alike. From the intricate patterning of butterflies to the amazing color pattern variation in bumblebees, it is a very interesting topic. The mode by which various patterns and pigmentation occurs differs among organisms, but is similar amongst species. Drosophila melanogaster is a specie which has been studied extensively to determine the mechanism of melanization, tan is a gene that has been indicated to play a role in the pathway. In our study tan was selected to determine if it is the gene that affects patterning in Bombus melanopygus. Using genetic sequencing and genome association to map genomic regions contributing to the variation in pigment patterning, we attempt to determine if this locus is indeed involved in the selected specie. Only two locations show single nucleotide polymorphisms (SNPs) that sort according to phenotype, all other locations indicate sorting by location. This is a great method to determine if a gene is associated with pigmentation patterning and will prove to be very useful if applied across the genome of many individuals, and different species.

Introduction
When studying evolutionary and developmental biology, insect pigmentation is a diverse, yet in many respects, common trait that can lead to clues in answering various questions. One of the most basic ways to study the development and subsequent evolution of species is by looking at genetic changes. Genes interact at the molecular level in order to produce great variation at the phenotypic level. Studying the mechanism and which genes are involved in these phenotypes will provide insight into how different characteristics are not only inherited but how they have changed over time. Pigmentation research spans all disciplines of biology: from ecology, to physiology, even to genetics. All of these have contributed to a greater understanding of why these phenotypic traits are inherited, when they were inherited, and how they are inherited. More recently, however, studies regarding pigmentation have started to center around the evolutionary and developmental fields of biology. In insects, pigments are produced by epidermal cells through a developmental process that includes pigment patterning and synthesis1. Extensive studies have gone into drosophila pigmentation and butterfly wing patterns. In a study completed by True, J. et al. the Drosophila melanogaster locus, tan, was found to encode a novel enzyme required for two cellular functions2. One of the cellular functions involves cuticular melanization. Tan is a gene that has been hypothesized to have an effect on the melanization pathway, including the selective loss of expression of certain pigmentation. In a study composed by Jeong, S. it was demonstrated that tan gene expression was eliminated through the mutation inactivation of a specific cis-regulatory element and subsequent loss of pigmentation3. Studying the developmental process by which pigmentation occurs will address evolutionary questions as well as provide a basis for future examination of various genes hypothesized to affect coloration.

In our study we (1) explored the mechanisms of pigmentation in insects, (2) decided upon a gene thought to be involved in the melanization pathway, (3) performed various genetic analyses on the gene selected, (4) discuss what obtained data conveys about the selected gene. The gene that was selected was tan, as previously discussed tan has been determined to have a part in the melanin synthesis of D. melanogaster by reversing the synthesis of Dopamine to NBAD1 the production of brown melanin is achieved. With this information we hypothesize that tan is the gene responsible for the coloration pattern in the four samples covered in this study.

Materials and Methods

Most mutations that are believed to cause morphological variation are found in the cisregulatory region of developmental genes4. Exploring the DNA sequence of B. melanopygus, specifically, the gene tan should provide evidence as to how the patterns of pigmentation arise. Twelve individuals from the boarder of Oregon and California were selected and their DNA was extracted. To begin our study of the tan gene the sequence had to be retrieved. To retrieve the sequence we used www.flybase.org, upon reaching the website a BLAST was performed in which we searched for tan. Upon finding the gene that we were looking for we downloaded the cds in FASTA format. That data was then entered into a www.beebase.org BLAST. From there we retrieved the scaffold in which the gene was located which was scf_0019. The scaffold was a strong hit with the expected value of 2e-46, the next hit was .37 which allowed us to draw the conclusion that this was our gene. The data was then downloaded and each of the exons from the hit was labeled in the following fashion: tan_exon1..2..3.._Bimp. The genomic region was then downloaded by clicking on the gene track in gbrowse. The genomic region was also retrieved for Bombus terrestris scaffold. In order to double check that we had the proper gene we took one of the exons and placed it into a tbastx on flybase, this was repeated once more with a different exon just to be completely certain. Certain we had the right gene the exons that we previously obtained were then BLAST in Genbank under tblastn which generated an annotated bumblebee gene. The FASTA sequence was saved for both impatiens and terrestris. To make sure we had the correct CDS we did another BLAST on flybase. The FASTA sequences that were obtained were then added to text wrangler to ensure they were saved in simple text format. The files were then imported into geneious a sequencing program and saved separately.

Exons

This figure depicts the alignment of the exons to the genome. The terrestris genome, exons, and impatiens genome were all aligned using the map to reference command. This alignment was then used to generate primers for the qPCR performed later in the experiment. The next step was to design primers, general guidelines were used in which: the length is 18-22 bp (20 was used for the primers we designed), the melting temperature is in the range of 52-58 C, and the annealing temperature is in the range of 52-60 C. All other conditions were determined using the website www.primer3plus.com which provides a score of different categories such as self-binding where 0 is the best and 5 or higher is the worst, GC which gives a percentage of the G and C nucleotide content, and various other options which will aid in one choosing the proper primer.

Primer 1 F

Primer 1 R

Primer 2 F

Primer 2 F

Once all of the information was gathered we proceeded with the PCR reaction. Qiagen products were used in this PCR reaction. A stock solution was prepared which contained 30l of Taq Master Mix, 1.2 l each of the two primers that were designed, and 21.6 l of water (Qiagen supplied). To that stock solution 1.5l of bombus melanopygus DNA was added. The DNA that was supplied was previously extracted by members of the lab. All contents when not being used were kept in a freezer to ensure minimal degrading and reactions, also while working with any

reagent, DNA, etc. they were all kept on ice. When everything was added to the solution the contents were finger vortexed to ensure proper mixing, then centrifuged to allow the contents on the walls to enter the mix. A 3-step PCR was used for the reaction with the parameters: 1) 95 for 2min, 2) 95 for 30sec, 3) 52 for 30sec, 4) 72 for 30sec, 5) 72 for 5min, 6) 4 as the holding temperature. To visualize the DNA gel electrophoresis was used a 1% gel which contained 1g agarose and 50ml TAE buffer, this was heated for ~1:00 in the microwave or until a clear product was obtained to which 2.5 l of DNA visualization liquid was added (gel-star). The liquid was then transported into a gel mold until it solidified. From our newly obtained PCR sample we took 2 l and added it to 2 l of loading dye. These samples as well as a DNA ladder were added to the wells of the agarose gel. The DNA migration was analyzed under UV light by taking a photograph using the UV pro program. The next step was to purify the PCR sample; to do this an exosap reagent was used in a 60 l exosap: 30 l H2O dilution. From that dilution 4.3 l was added to 13 l of the PCR sample. Once that was completed it was cycled at 37 C for 30min, 80 C for 15min, and 10 C as the holding temperature. Once that product was finished cycling it was sent to be sequenced.

Results
UV pro was used to visualize the gel. The first set of data collected was from an initial trial with DNA samples from BmelR_002, BmelR_005, BmelB_001, and BmelB_004. The following gel was obtained:

Depicted above is the gel visualization for the first set of primers run for tan.

This sample was run under the conditions outlined in the materials and methods section. From this visualization we can see that the DNA has migrated, but there is a faint set of bands at the bottom which indicates primer dimer. A second trial was then run on the same sample, but some of the conditions were changed. The annealing temperature was changed to 51 C and the DNA concentration was increased from 1.5l to 2 l. This produced a visualization which was clearer and had less primer dimer in the product. The next samples that were run were the EF1- gene samples which would serve as our baseline, originally the sample was run at an annealing temperature of 58 C, but that proved to be too high and there were no bands in the gel when visualized. To rectify that the annealing temperature was lowered to 52 C, which then produced visible bands. This method of visualizing the PCR before sequencing was repeated for every batch of DNA that was going to be sequenced. Once the samples were sent out the results were then imported into geneious and edited. Consensus sequences were obtained for all samples after editing was completed. Once all consensus sequences had been obtained they were then aligned to the bombus impatiens genome for individuals R_002, R_005, B_001, and B_004. An analysis for where the SNPs were located was then performed:

T T T T C A A A A G C C C C T T T T C C C C C C C C G G G G T T T T This table depicts the SNPs found in the exons of the tan gene.

Locati on 29228 30421 31057 31156 31231 31518 31754 32049

B1 Genomic

R2 Genomic

B4 Genomic

R5 Genomic

Exon 1

Exon 2

Exon 3

Exon 4

Exon 5

Exon 6

Exon 7

T C T -

T -

A -

Locat ion 723 724 742 1030 11598 11906 14440 18292 22856 24070 28950 30472 30479 30474 30475 30476 30477 30478 30489 30480 30481 30485 30486 30488 30489

B1 Genomi c G T C T G T G A T T A A T G T T A A G G A T C A G

R2 Genomi c G T C T A T A G G A A A T G T T A A G G A T C A G

R5 Genomi c C G A G G C G A T T C A T G T T A A G G A T C A G

B4 Genomi c C G A G A C A G G A C A T G T T A A G G A T C A G

Tan 1 B1 A T G T T A A G G A T C A G

Tan 1 R2 A T G T T A A G G A T C A G

Tan 1 R5 C G A A T A A G G A T C A G

Tan 2 B1 C G A A A T G C A T A T T A

Tan 2 R2 -

Tan 2 R5 -

G G G G G G G A 30490 A A A A A A A G 30491 A A A A A A A C 30492 C C C C C C C G 30493 G G G G G G G T 30494 G G G G G G G A 30495 G G G G G G G T 30498 A A A A A A A T 30497 C C C C C C C G 30500 G G G G G G G C C 30501 G G G G G G G T T 30502 G G G G G G G A G 30504 A A A A A A A T A 30508 G G G G G G G C G 30511 T T T T T T T C T 30512 A A A A A A A T A 30445 T T T T T T T T T C 30540 T T C C T T C T T C 30970 G G G G G G G C G C 30985 C C C C C C C C C C 31011 T T T T T T T A T T 31045 G G G G G G G G G T 31056 G G G G G G A A G A 31075 A A G G 33169 G G A A 34708 C T T C 44135 A C C A 46451 A T A T 48387 A C A C 48738 C T T C 49736 C C T T 52272 T T C C 54150 This table depicts the SNPs across the region of the genome that we selected; this table was modified just to view the haplotypes that occur across the genome.

The exons are fairly conserved, we only observe eight SNPs across the seven exons obtained. We see a stretch of haplotypes that sort B1, R2 and B4, R5 from bp 723-742, but then this sequence is broken up to B1, R5 and R2, B4. There are only two instances in which we get soring by phenotype (B1, B4 and R2, R5) and that occurs at bp 44135 and 49736. In the primer sequences that were obtained there seems to be a lot of variation in B1 of the second set of tan primers, as well as R2 of the second set of tan primers. Overall the association of SNPs seems to

be by location, with only two instances of phenotypic sorting. At bp 30970 the area surrounding that location is fairly conserved, but at that bp we found a SNP which sorted R5, B4.

Discussion
The main goal of this research was to determine if the gene tan which has been implied to have an effect on the melanization pathway is involved with bumblebee patterns. Through genetic sequencing we observed whether there is a pattern with the base sorting between the four individuals R2, R5, B1, and B4. SNPs should occur mostly in non coding regions or regions where selection is happening, if a SNP is observed repeatedly over a portion of the genome then there is hypothesized to be an association. In our study there was a recurring pattern in the sorting of B1, R5 and R2, B4. Unfortunately this is not a sorting by phenotype which is what we would like to see; instead this is a sorting by location. Having a sorting by location is not very informative because the variation in habitats is most likely the overlying reason as to why they sort in that manner. Unfortunately for the designed primers both sequences for B4 did not produce any data and could not be used in the analysis. Degraded DNA, poor sequencing, or issues with amplification could have produced this issue. The primer sequence variation that was observed could be due to many factors including: improper design of primers, bad sequencing, or actual variation between the samples. The primer sequences that were obtained did match the genomic sequence, so there wasnt an issue with the primer design. Since there was no issue with the primer the main source of error was most likely in the sequencing, when editing the sequences clear peaks were not observed and the percentage was very low. The culmination of all this data implies that tan is not the gene that we are looking for to be involved in color patterning. Of course multiple trials and expansion of the gene to more samples could prove otherwise, but for the purposes of this experiment there does not seem to be an association between tan and color pattern. There are many genes which have been identified in the melanization pathway such as bric a brac, ddc, pale, and countless others. Future researchers could explore these options as the target gene in the melanin pathway.

References
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