Dichlorodiphenyltricholorethane (DDT) resistance mechanisms in Drosophila melanogaster and applications for the cowpea pest Clavigralla tomentosicollis

Steele, Laura Diane

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Description

Title

Dichlorodiphenyltricholorethane (DDT) resistance mechanisms in Drosophila melanogaster and applications for the cowpea pest Clavigralla tomentosicollis

Author(s)

Steele, Laura Diane

Issue Date

2017-04-20

Director of Research (if dissertation) or Advisor (if thesis)

Pittendrigh, Barry R.

Doctoral Committee Chair(s)

Pittendrigh, Barry R.

Committee Member(s)

• Berenbaum, May R.
• Francis, Bettina M.
• Paige, Ken N.

Department of Study

Entomology

Discipline

Entomology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Insecticide resistance
• Adaptive genomics
• Signatures of selection
• Genome re-sequencing
• Dichlorodiphenyltricholorethane (DDT)
• Resistance
• 91-C
• 91-R
• Drosophila melanogaster
• Mitochondria
• Mitogenome
• Reactive oxygen species (ROS)
• Clavigralla tomentosicollis
• Pod-sucking bug
• Cowpea
• Vigna unguiculata
• Coreoidea

Abstract

Control of insects by insecticides has led to resistance in both medical and agricultural pests as well as health and environmental concerns, and improved insect pest management (IPM) programs are necessary. The arrival of next-generation sequencing (NGS) and lowered sequencing costs provides the tools for elucidating how insects evolve resistance to pesticides and the opportunity for developing molecular markers to characterize pest populations and their dynamics. One relatively effective control method for anopheline mosquitoes that are vectors of malaria continues to be dichlorodiphenyltricholorethane (DDT) in indoor residual spraying in some developing countries, even after it has been discontinued in many countries due to concerns about health impacts, bioaccumulation and biomagnification in the environment, and resistance Studies demonstrated DDT to directly affect the mitochondria, including the disruption of oxidative phosphorylation and the electron transport chain. The fruit fly, Drosophila melanogaster (Drosophila), functions as a key model organism to study the molecular effects of xenobiotics, including DDT, and, in particular, DDT resistance mechanisms. Beyond model organisms, there are many examples of the need to develop a better understanding of pest insect dynamics toward making better decisions within the context of integrated pest management programs. For example, the staple legume crop cowpea, Vigna unguiculata (L.) Walp, constitutes a key source of dietary protein and nutrition across sub-Saharan Africa, a region where approximately 220 million people are undernourished. Cowpea, in addition to a food source for both humans and livestock, has significant economic value. Insect damage has severe impacts on the overall crop production, with a range of insects feeding on the plant. A primary insect pest of cowpea is Clavigralla tomentosicollis (Stäl) (Hemiptera: Coreidae), with feeding by nymph and adult stages on cowpea pods inflicting up to 100% yield loss. Most studies of C. tomentosicollis to date have focused on control methods, including chemical and natural insecticidal options. Additionally, the majority of prior molecular data of pests of cowpea focused on the lepidopteran Maruca vitrata, with the exception of one study that isolated potential molecular genetic markers for four additional pests of cowpea, including C. tomentosicollis, highlighting the gap in knowledge for this critical cowpea pest. Insect mitochondrial genomes (mitogenomes) provide useful information for defining evolutionary relationships between species, and in some cases species-complexes within populations, as well as contain the cytochrome c oxidase subunit 1 (cox1 or COI) gene, the standard gene for DNA barcoding. Additionally, in some pest species the cox1 gene has been used to study pests at a population level, providing insights that have the potential for use in IPM strategies. This dissertation contains four chapters that highlight how genomic techniques and approaches can be used to improve the understanding of insect responses to xenobiotics and to utilize genomic data of insect pests of key food crops to improve control methods. Chapter 1 is an introduction that provides background information about the use of DDT as an insecticide, DDT resistance in Drosophila, and Drosophila as a model system for other pest species. Additionally, an overview of the mitochondria and its corresponding mitochondrial genome (mitogenome) are outlined specifically for Chapters 3 and 4. This introduction also summarizes the current knowledge of control methods and the minimal available molecular data for the brown pod-sucking bug, C. tomentosicollis, a pest of cowpea. Both Chapter 2 and Chapter 3 examine molecular differences between two Drosophila fly lines, the highly DDT-resistant 91-R and the DDT-susceptible 91-C. Chapter 2 provides a whole-genome selective sweep analysis to identify loci with additive genetic effects in the highly DDT resistant 91-R. This study revealed only two previously identified genes related to DDT-resistance (NinaC and Cyp4g1), suggesting a new set of candidate genes for future study. One gene of particular interest was MDR49, an ATP-binding cassett transporter that contains a structural mutation. Identifying these genes suggest the potential for discovery of unknown DDT-mechanisms and provide additional support that DDT-resistance in Drosophila is polygenic. DDT has been previously shown to disrupt the function of the mitochondria, particularly affecting oxidative phosphorylation, components of the electron transport chain, and causing cell apoptosis. Chapter 3 explores the effects of high selective pressure for DDT resistance on the mitochondrial genome (mitogenome) as well as nuclear genes associated encoding mitochondrial proteins. Additionally, a set of genes of interest, identified through a literature search of studies examining mitochondrial responses to DDT exposure, was analyzed. This analysis identified eight genes that are differentially expressed in 91-R, six of which were over-expressed (Cyp12d1-d, Cyp12a4, cyt-c-d, COX5BL, COX7AL, CG17140) and two of which were under-expressed (dif, Rel). Four of the identified genes are considered the result of gene duplications. Predicted functions of these genes (or in some cases, their parent genes) suggest 91-R may be up-regulating existing pathways that regulate reactive oxygen species (ROS), which DDT is known to increase. This study sets the groundwork for further experiments examining the specific processes influenced by these differentially expressed genes, as well as provides an additional 212 candidate genes, to gain insight into the effects of DDT on insect mitochondria. Chapter 4 characterizes the complete mitogenome sequence of C. tomentosicollis. C. tomentosicollis is of importance as it is a key pest of the staple food crop cowpea of sub-Saharan Africa and damage by this insect can reduce yield by 80-100%. Additionally, I completed a comparative analysis of the C. tomentosicollis mitogenome to the published mitogenomes of six other species from the superfamily Coreoidea: Riptortus pedestris (Alydidae), Aeschyntelus notatus (Rhopalidae), Stictopleurus subviridis (Rhopalidae), Corizus tetraspilus (Rhopalidae), Dicranocephalus femoralis (Stenocephalidae) and Hydaropsis longirostris (Coreidae), the only other species from the family Coreidae. This chapter improves on the limited C. tomentosicollis molecular data available and provides the basis for future molecular work for this pest species. In conclusion, the results from Chapters 2 and 3 of this dissertation provide the necessary foundation for whole genome studies examining insects exposed to intense selective pressure by insecticides. Additionally, I identified novel candidate genes within Drosophila that require further analysis to discern their potential roles in conferring DDT-resistance. The completed C. tomentosicollis mitogenome presented in Chapter 4 provides a basis for future molecular studies, such as investigating polymorphisms across populations, to continue to fill in the gap in knowledge for this insect pest, such as population structure and movement patterns of this species in the West African agro-ecosystem. The ultimate aim will be the ability to improve pest management choices through an increased understanding of population dynamics and migration patterns for a pest of a crop that is an important food source across sub-Saharan Africa.

Graduation Semester

2017-05

Type of Resource

text

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Copyright 2017 Laura Steele

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