CUTTING EDGE INNOVATION: DISSECTING THE GENETIC BASIS OF A PLANT-PIERCING OVIPOSITOR IN AN HERBIVOROUS

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Authors RAY, JULIANNE FLORENCE

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Link to Item http://hdl.handle.net/10150/613574 CUTTING EDGE INNOVATION: DISSECTING THE GENETIC BASIS OF A

PLANT-PIERCING OVIPOSITOR IN AN HERBIVOROUS FLY

By

JULIANNE FLORENCE RAY

______

A Thesis Submitted to The Honors College

In Partial Fulfillment of the Bachelors Degree

With Honors In

Molecular and Cellular Biology

THE UNIVERSITY OF ARIZONA

M A Y 2 0 1 6

Approved By:

______

Dr. Noah K. Whiteman

Department of Ecology and Evolutionary Biology

Department of Integrative Biology

University of California at Berkeley OVIPOSITOR GENETICS IN FLAVA | 1

Abstract

The evolution of herbivory within an lineage is often enabled by novel morphological innovations. The ancestor of Scaptomyza flava developed a serrated ovipositor nearly six million years ago, associated with an evolutionary transition to herbivory, that allows these to cut into mustard plants deposit eggs into the wound.

We aim to identify candidate genes associated with ovipositor peg development in S. flava using a genome-wide association study (GWAS). GWAS methods are only appropriate for heritable, variable traits. Dissection and photographic profiling of ovipositors from over 700 female flies revealed variation in the number of serrated pegs within natural populations. Mother-daughter profiling showed this variation was heritable

(h2 = 46%). Peg number variation among individuals followed a normal distribution, suggesting multiple genes likely influence this trait. Sequencing genomes of pools of individuals with the most and fewest ovipositor pegs from two populations identified four candidate loci affecting ovipositor peg number in S. flava. Many of these loci contribute to neural development in Drosophila melanogaster, consistent with the hypothesis that ovipositor pegs are hardened, innervated bristles. Overall, this project sets the stage for understanding the genetic and developmental basis of a key evolutionary innovation – a leaf-cutting ovipositor – in herbivorous .

OVIPOSITOR GENETICS IN SCAPTOMYZA FLAVA | 2

Introduction

Half of all insects, the most diverse class in the kingdom, are herbivorous.

Insects with herbivorous feeding patterns face the challenge of successfully attaching eggs to or near food sources suitable for the emerging offspring: living plants. Solutions between species vary, but one common solution is the evolution of an ovipositor capable of cutting into living plant material and placing an egg within the plant (Aluja and Norrbom 2000).

The fruit fly Scaptomyza flava is a promising model organism for identifying genes involved in the evolution of cutting ovipositor. S. flava diverged from common ancestors within the genus Drosophila () to become one of the few herbivorous fruit flies between six and sixteen million years before present (Whiteman et al. 2012). A key innovation linked to this drastic change in feeding behavior, the chitinous ovipositor structure used to lay eggs, is critical to parental and offspring survival and is a defining morphological feature of this species (Seraj 1994). Female S. flava utilize sharp pegs along their egg-laying ovipositor to cut into leaves and eat the contents of the damaged area before laying an egg within the cut (Figure 1).

The sharp, hardened, cutting ovipositor of S. flava is an example of an evolutionary innovation enabling the evolution of herbivory. Over two years, this project addressed several questions: (1) Is the number of pegs on an ovipositor variable within and between populations? (2) Is the number of pegs on an ovipositor heritable? (3)

What genes are associated with morphological variation in the peg-covered ovipositor of

S. flava? OVIPOSITOR GENETICS IN SCAPTOMYZA FLAVA | 3

As pegs are thought to be modified sensory bristles (Aluja and Norrbom 2000,

Atallah et al. 2014, McKay and Lyman 2005), we predicted that genes involved in neural cell development would be associated with peg number variation along the ovipositor.

The results of this project could be relevant to future genetic study of a related species, Drosophila suzukii. This destructive fly devastates grape and other fruit crops worldwide by cutting into tough-skinned fruit with a sharp ovipositor and laying eggs beneath the skin inside the bored wound (Walsh et al. 2011). The cuts induce rot in the fruit before larvae emerge, rearing bacterial colonies for larvae to consume and ruining crops with a similar tool to the more-bristled ovipositor of S. flava. A deeper understanding of genes that contribute to the cutting ovipositor of S. flava could contribute to future genomic studies of this widespread fruit pest.

Methods

Phenotypic variation in morphology

S. flava populations were founded by Andrew Gloss in July 2014 from approximately 75 (NH1 colony) and 58 (NH2 colony) wild-collected larvae near Dover,

New Hampshire. Flies used in phenotypic profiling were second generation, lab-reared offspring of the wild-collected flies.

Excisions of ovipositors from more than 1000 female flies were performed using a Zeiss Stemi 2000C scope and dissection lighting. Ovipositors were mounted by placing the ovipositor with ventral pegs facing away from the slide towards the coverslip OVIPOSITOR GENETICS IN SCAPTOMYZA FLAVA | 4

on 900 uL of Permount spread over two square centimeters of the slide. The coverslip was slowly brought down on the glued area and the ovipositor's position was monitored through the Zeiss scope at 50x. Each slip was allowed to dry for at least one day before measurements were taken. Individuals used for dissection were carefully individually preserved in 96-well plates in 100% ethanol at -20o C.

Measurements of excised and carefully mounted ovipositor photographs taken on a Canon EOS Rebel T3i mounted on the Zeiss Stemi 2000 were completed using the program ImageJ for ovipositor serration cord length and a 1000 uM scale bar for scale calibration. Wing cord length was measured with ImageJ from the base of the musculature to the wing apex following the third longitudinal vein. Pegs were counted along the ventral edge of the ovipositor from the smallest peg at the anterior to the longest peg at the posterior apex in their linear position along the ovipositor. Along the dorsal edge of the ovipositor, peg number varied by only one peg in all specimens studied, so these pegs were not included in analysis. Peg counts and length measurements were performed manually twice for each specimen and averaged to reduce measurement error.

Heritability

More than 50 single-pair matings of one male and one virgin female fly from the combined NH1 and NH2 colonies were conducted on single Turritis glabara plants in

Magenta boxes (Sigma-Aldrich). Each box was provisioned with cotton balls soaked in

10% honey solution to improve survivorship. For 30 matings that yielded daughters, ovipositor length and peg count was profiled (as described earlier) for every mother and OVIPOSITOR GENETICS IN SCAPTOMYZA FLAVA | 5

at least one of her daughters. Narrow-sense heritability of ovipositor peg number was calculated by regressing the phenotype of each mother against the average phenotype of her daughters. Narrow-sense heritability estimates for each trait were calculated by doubling the slope of each regression, since ovipositor traits could be measured only in mothers and not fathers. Heritability was estimated for ovipositor length, which we aimed to eliminate as a confounding variable in our search for genes underlying peg number variation.

DNA extraction and genome sequencing

Pools of individuals for sequencing were formed by regressing peg number against ovipositor length and selecting individuals with extreme residual peg number values for sequencing . The top and bottom 20% extreme residual flies were pooled separately. This created density-corrected pools: ovipositor peg number differed between pools, but ovipositor size did not.

DNA extractions were performed with the user-developed protocol DY11 for use with the Qiagen DNeasy® Blood & Tissue Kit and TissueLyser using the thoraxes of extreme individuals separated into smaller pools that were later combined into the

"high" and "low" sets for each population.

Two separate populations were sequenced to approximately 40x coverage using these pools on an Illumina HiSeq 2500.

Genome mapping OVIPOSITOR GENETICS IN SCAPTOMYZA FLAVA | 6

Best practices were performed for pooled sequencing analysis recommended by Schlotterer et al. (2014). Low quality reads were removed, and low quality regions were trimmed, using Trimmomatic (Bolger et al. 2014). Reads were mapped to the S. flava genome using bwa (Li and Durbin 2009). Duplicates were removed using Picard

(http://picard.sourceforge.net/). Allele frequency differences among the pools with high and low peg number were tested for using the Cochran-Mantel-Haenszel test in

Popoolation2 (Kofler et al. 2011). P values were Bonferroni-corrected to control the type

I error rate.

Results

Variation was present within both populations of S. flava (Fig.2). Narrow-sense heritability (h2) of ovipositor peg number, estimated using mother-daughter regression, was 46% (Fig. 3).

Four SNPs were at significantly different frequencies among the pools of flies with high and low peg number (Bonferroni corrected P< 0.5) (Fig. 4). These SNPs closely neighbored genes whose orthologs are involved in cell fate specification or migration in Drosophila melanogaster (FlyBase gene annotations, Table 1). One such neighboring gene is sloppy paired (slp2), a gene involved in neuron and axon cell fate and differentiation in D. melanogaster (Fig. 5). Minor allele frequencies (maf) values of slp2 are 4% in the whole population, 27% in the low pool, and 0% in the high pool

(Table 1). OVIPOSITOR GENETICS IN SCAPTOMYZA FLAVA | 7

Discussion

This study sought to gain new insight into the genetic basis of hardened ovipositor pegs, a key innovation shared by many herbivorous insects (Aluja and

Norrbom 2000), in S. flava. Ovipositor pegs are thought to be modified bristles in flies

(Aluja and Norrbom 2000, Atallah et al. 2014, McKay and Lyman 2005), so we expected that patterns of variation in ovipositor peg number, and the genetic basis of this variation, would be similar to those for well-studied bristle phenotypes in Drosophila.

Previous genetic studies of bristle variation in Drosophila have revealed that genes involved in nervous system development predominately underlie variation in bristle number (MacKay and Lyman 2005).

Variation in peg number within populations of S. flava roughly followed a normal distribution. Bristle number in Drosophila is polygenic (MacKay and Lyman 2005), and polygenic traits are typically normally distributed (Lande 2005), so peg number is likely polygenic as well. Narrow-sense heritability (h2) estimated from mother-daughter regressions for peg count was 46%, close to the heritability estimate of thorax bristle number of 50% in other Drosophila species (MacKay and Lyman 2005). Both S. flava ovipositor peg number variation and Drosophila bristle number variation are associated with genes involved in cell development, and more specifically nervous system development of neural cells (Norga et al. 2003).

Among the four SNPs associated with variation in ovipositor peg number, one of the most interesting neighboring genes is sloppy paired (slp2), a gene involved in axon OVIPOSITOR GENETICS IN SCAPTOMYZA FLAVA | 8

and neuron differentiation and cell fate determination. The minor allele associated with low peg number near slp2 is completely absent in high peg pools, suggesting this allele strongly reduces peg number. Studies of medulla formation in D. melanogaster (Li et al.

2013) indicate slp2 as a critical gene in early determination of cell fate. As bristles in D. melanogaster are known to be innervated (McKay 2005), ovipositor pegs could be co- opted bristles or may have become innervated through other pathways.

OVIPOSITOR GENETICS IN SCAPTOMYZA FLAVA | 9

Figure 1. Female Scaptomyza flava has a green-colored abdomen, indicating that plant exudates are consumed as a food source. This consumption is dependent on the serrated ovipositor located at the posterior of the abdomen.

serrated ovipositor

digesting plant exudates

Figure 2. Ovipositor peg number variation within two S. flava populations, New Hampshire 1 and New Hampshire 2, is roughly normally distributed.

OVIPOSITOR GENETICS IN SCAPTOMYZA FLAVA | 10

Figure 3. Mother-Daughter regressions for ovipositor peg count and ovipositor length. If a trait is heritable, a positive correlation is expected. Peg count heritability was roughly 50%, (h2 = 0.46), while ovipositor length was not heritable. P = 0.046 P = 0.31 4 h2 = 0.46 250 2 0 230 ï daughters, mean daughters, mean ovipositor length (um) ï size-corrected peg count 210 ï ï 0 2 4 230 240 250 260 270 mother, size-corrected mother, ovipositor peg count length (um)

Figure 4. Manhattan plot showing Cochran-Mantel-Haenszel test P values comparing allele frequencies between two replicate pools of S. flava females with high and low peg number. Only the four genomic scaffolds with significant SNPs are shown. The dashed line indicates the Bonferroni-corrected significance cutoff.

10.0

7.5 ( P )

10 5.0 ïORJ 2.5

0.0

VFDIIROG VFDIIROG VFDIIROG VFDIIROG

scale: 0.5 Mbp OVIPOSITOR GENETICS IN SCAPTOMYZA FLAVA | 11

Table 1. Genes neighboring the four SNPs associated with ovipositor peg variation in S. flava. The function of the D. melanogaster ortholog is indicated for each gene. Minor allele frequency (maf) indicates the frequency of the rarest allele in each population.

Figure 5. A SNP near slp2 was significantly associated with variation in ovipositor peg number in S. flava. slp2 is an essential transcription factor involved in specifying neuron and axon fate in D. melanogaster (Li et al. 2013).

RhoGAP93B 10.0 slp2 (weak orthology)

7.5

( P )

10 5.0

ïORJ 2.5

0.0 0.125 0.130 0.135 0.140 0.145 position (Mbp) OVIPOSITOR GENETICS IN SCAPTOMYZA FLAVA | 12

Acknowledgments

This work was funded by a Templeton Foundation grant to Noah Whiteman, an

NSF dissertation improvement grant to Andrew Gloss, and a University of Arizona

Honors College research grant and UBRP fellowship to Julianne Ray.

I thank Andrew Gloss for aid in conducting the statistical analyses. I thank Bruce

Walsh for advice regarding the heritability study design. I also thank Rick LaPointe for contributing the photograph in Figure 1 of S. flava. I thank the Whiteman Laboratory for their advice regarding ovipositor extractions and statistical analysis, and Timothy

O'Connor for dissection training.

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