CUTTING EDGE INNOVATION: DISSECTING THE GENETIC BASIS … · 2020. 4. 2. · fruit before larvae emerge, rearing bacterial colonies for larvae to consume and ruining ... longest peg

CUTTING EDGE INNOVATION: DISSECTINGTHE GENETIC BASIS OF A PLANT-PIERCING

OVIPOSITOR IN AN HERBIVOROUS FLY

Item Type text; Electronic Thesis

Authors RAY, JULIANNE FLORENCE

Publisher The University of Arizona.

Rights Copyright © is held by the author. Digital access to this materialis made possible by the University Libraries, University of Arizona.Further transmission, reproduction or presentation (such aspublic display or performance) of protected items is prohibitedexcept with permission of the author.

Download date 06/07/2021 19:17:25

Link to Item http://hdl.handle.net/10150/613574

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 SCAPTOMYZA FLAVA | 1

Abstract

The evolution of herbivory within an insect 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 flies 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 insects.


Introduction

Half of all insects, the most diverse class in the animal 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 (Drosophilidae) 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?


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


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


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


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).


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


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.


serrated ovipositor

digesting plant exudates

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.

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


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.

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.

0.0

2.5

5.0

7.5

10.0

scale: 0.5 Mbp

10(P

)

230 240 250 260 270

210

230

250

mother, ovipositor length (um)

daug

hter

s, m

ean

ovip

osito

r len

gth

(um

) P = 0.31

0 2 4

02

4

mother, size-corrected peg count

daug

hter

s, m

ean

size

-cor

rect

ed p

eg c

ount P = 0.046

h2 = 0.46


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).

0.0

2.5

5.0

7.5

10.0

0.125 0.130 0.135 0.140 0.145position (Mbp)

10(P

)

slp2RhoGAP93B(weak orthology)


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.

References

Aluja M, Norrbom AL (2000) Fruit flies (Tephritidae): phylogeny and evolution of behavior. CRC Press, Boca Raton. Atallah J, Teixeira L, Salazar R, Zaragoza G, Kopp A (2014) The making of a pest: the evolution of a fruit-penetrating ovipositor in Drosophila suzukii and related species. Proceedings of the Royal Society B: Biological Sciences, 281, 20132840–20132840. Bernays EA, Jarzembowski EA, Malcolm SB (1991) Evolution of Insect Morphology in Relation to Plants [and Discussion]. Philosophical Transactions of the Royal Society B: Biological Sciences, 333, 257–264. Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics, 30, 2114–2120. DNEasy® Blood & Tissue Kit, User-Developed Protocol DY11 (2006) DNEasy® Blood & Tissue Kit, User-Developed Protocol DY11.


Kofler R, Pandey RV, Schlotterer C (2011) PoPoolation2: identifying differentiation between populations using sequencing of pooled DNA samples (Pool-Seq). Bioinformatics, 27, 3435–3436. Lande R (2007) The maintenance of genetic variability by mutation in a polygenic character with linked loci. Genetical Research Genet. Res., 89, 373. Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics, 25, 1754–1760. Li X, Erclik T, Bertet C et al. (2013) Temporal patterning of Drosophila medulla neuroblasts controls neural fates. Nature, 498, 456–462. Mackay TFC, Lyman RF (2005) Drosophila bristles and the nature of quantitative genetic variation. Philosophical Transactions of the Royal Society B: Biological Sciences, 360, 1513–1527. Norga KK, Gurganus MC, Dilda CL et al. (2003) Quantitative Analysis of Bristle Number in Drosophila Mutants Identifies Genes Involved in Neural Development. Current Biology, 13, 1388–1396. Seraj AA (1994) Biology and host plant relationships of Scaptomyza flava leaf miner: a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philiosophy in entomology, Plant Science Department, Massey University, Palmerston North, New Zealand. Schlötterer C, Tobler R, Kofler R, Nolte V (2014) Sequencing pools of individuals — mining genome-wide polymorphism data without big funding. Nat Rev Genet Nature Reviews Genetics, 15, 749–763. Walsh DB, Bolda MP, Goodhue RE et al. (2011) Drosophila suzukii (Diptera: Drosophilidae): Invasive Pest of Ripening Soft Fruit Expanding its Geographic Range and Damage Potential. Journal of Integrated Pest Management j integ pest manage, 2, 1–7. Whiteman NK, Gloss AD, Sackton TB et al. (2012) Genes Involved in the Evolution of Herbivory by a Leaf-Mining, Drosophilid Fly. Genome Biology and Evolution, 4, 788–804.

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CUTTING EDGE INNOVATION: DISSECTING THE GENETIC BASIS … · 2020. 4. 2. · fruit before larvae emerge, rearing bacterial colonies for larvae to consume and ruining ... longest peg