| Abstract |
The olive fruit fly Bactrocera oleae (B. oleae) is considered the major insect pest in olive orchards,
causing great damage in the quality and the quantity of the olive production worldwide. However,
its pest management approaches have proven difficult and inefficient, for a variety of reasons, a
fact that has brought about a need for alternative tools and approaches. The objective of this
thesis is to provide new insights and characterize mechanisms, potentially implicated in
insecticide resistance, one of the limiting factors for an efficient pest management, as well as in
the interactions between the olive fly and its symbiotic bacteria, especially of the species
Candidatus Erwinia dacicola (Ca. E. dacicola), towards the establishment of innovative olive fly
control strategies, which will target the symbiotic relationship with the bacterial partners
(dysbiosis).
The present study is divided in 3 sections. In the first, the aim was to develop and apply a highly
precise genome editing tool for Bactrocera oleae, and particularly, a CRISPR/Cas9 technology-
based approach. We chose to target the scarlet gene in B. oleae, which provides an easy to
screen eye color phenotype, in order to demonstrate that this technology is applicable to this non-
model organism. We report the development of a CRISPR/Cas9 gene editing tool, using the well-
known eye color marker gene scarlet. Two synthetic guide RNAs targeting the coding region of
the scarlet gene were synthesized and shown to work efficiently in vitro. These sgRNAs along
with purified Cas9 protein were then micro-injected into early-stage embryos. Successful
CRISPR- induced mutations of both copies of the scarlet gene showed a striking yellow eye
phenotype, indicative of gene disruption. Multiple successful CRISPR events were confirmed by
PCR and sequencing. The establishment of an efficient CRISPR/Cas9-based gene editing tool in
B. oleae will enable the study of critical molecular pathways in olive fly biology and physiology.
The availability of such a genetic tool will enable a better understanding concerning the potent
roles of various genes and mechanisms (i.e. critical symbiosis-based interactions with bacterial
partners, insecticide resistance), towards the future development and application of novel pest
control strategies.
In the second section of this thesis, we investigated potential mechanisms which are implicated
in insecticide resistance of B. oleae. Olive fly pest management in Greece has relied mostly on
the use of chemical (small-molecule) insecticides. The long overuse of organophosphorus-based
(OP) insecticides has resulted in the development of resistance to this compound. OPs target the
acetylcholinesterase enzyme (AChE) and suppress its function, which is the hydrolysis of the
neurotransmitter acetylcholine (ACh), in order to prevent neuro-toxicity and subsequent death of
the insect. In the first part of this second section, we attempted the functional validation of a target
site mutation in the AChE of B. oleae, namely Δ3Q, which has been associated with OP resistance
in B. oleae, after field screens and in vitro experiments, since 2008. The deletion of these three
amino acids in its last exon, has been proposed to provide a better anchoring of the enzyme on
the cell membrane, and as a result, the available molecules that hydrolyze ACh and interact with
the insecticide are increased, resulting in survival of the olive fly at higher doses of insecticide,
conferring resistance to OP compounds. However, this hypothesis has not been supported with
in vivo evidence yet. The aim of this study was to investigate this hypothesis in vivo, and more
specifically by introducing the Δ3Q mutation, with the CRISPR/Cas9 gene editing tool developed
in the previous section, in a susceptible genetic background of a laboratory-reared B. oleae strain
and study the mutant phenotype upon insecticide application.
A strategy was implemented in which purified and commercially available Cas9 protein along with
multiple sgRNAs and a synthetic donor ssODN DNA template (IDT) were introduced into early
embryos with microinjections, following the protocol that was established in the previous section,
in order to knock-in the Δ3Q modification, by homologous recombination (HDR mechanism). The
results showed that although in vitro, five out of the total seven sgRNAs direct the Cas9 to the
desired sites, in order to cleave the DNA and potently integrate the donor template (including the
Δ3Q instead of the WT 5Q in the end of AChE), the corresponding result was not accomplished
in vivo;; approximately 2,174 olive fly embryos were injected and sequenced in groups.
Sequencing results did not show any DNA cleavage, suggesting an insufficient Cas9 RNP uptake
by the oocytes, resulting in a micro-injection protocol with very low efficiency. We conclude that
the knock-in method requires further improvements, in order to successfully introduce mutations
and study them functionally with the CRISPR/Cas9 genome modification tool.
In the second part of the second section of this thesis, we searched for potent gene candidates,
implicated in pyrethroid resistance of olive flies, using transcriptome sequencing on olive fly
malpighian tubules (MTs). The aim of this study was to identify genes not immediately apparent
in the already existing whole organism RNA sequencing data, through a gene expression
comparison in MTs, one of the proposed detoxification tissues in insects, dissected out of
pyrethroid resistant and susceptible olive flies. Sequencing of the extracted RNA was performed
using the Illumina platform, in three biological replicates for each one of the two populations.
Sequencing reads were then aligned back to the olive fly genome reference sequence, gene
expression levels were calculated and the up- and down-regulated genes, both in resistant and
susceptible samples were identified. As expected, many genes that are well known to be
implicated in insecticide resistance were identified, such as cytochrome P450s (CYPs),
glutathione S-transferases (GSTs) and UDP-glucuronosyltransferases (UGTs). Moreover,
several other genes were also identified, but their role in insecticide resistance remains to be
elucidated via further investigation.
The over-expression of two P450 genes (CYP4P6 and CYP6G28) that were highlighted in the
RNAseq data, was quantitatively validated with qPCR analysis and the functional validation of
one of them, through gene silencing upon dsRNA injections (using the RNA interference
technology, RNAi), revealed a promising phenotype, upon insecticide treatment;; specifically, a
30% down-regulation of the CYP4P6 gene (compared to the control levels of the gene) conferred
a 21% mortality to the injected olive flies, upon α-cypermethrin application, compared to the
control group (4%). However, despite the promising phenotype conferred upon silencing of the
CYP4P6 gene, foreshadowing a possible implication of this gene in pyrethroid resistance, the
season-dependent limitation factor of this pest species did not allow the export of a complete and
statistically significant conclusion, within the framework of this PhD thesis.
A general enrichment in the transcription of several genes from the five major detoxification gene
families was observed in the MT-specific RNA dataset of resistant olive flies, providing new
insights for the detoxification of insecticides in this species. However, further functional
characterization studies are required. Following the encouraging preliminary results reported in
the current study (concerning the role of the CYP4P6 gene), which require further validation steps,
the interesting gene cases can be further investigated with in vitro insecticide metabolism assays,
in order to confirm a potent implication in the detoxification procedures of insecticides. Therefore,
such knowledge may contribute in the development of effective strategies for controlling this
destructive pest and protecting the olive trees, the cultivation of which is of great regional
importance, based on improved molecular diagnostics tools.
Moreover, in the third section of this thesis, we discuss the unique ability of olive flies to utilize
unripe olives for their development, which has been associated with interactions with symbiotic
bacteria. In this chapter, the aim was to investigate and define critical aspects of the unique
symbiotic relationship, between the olive fly host and the Ca. E. dacicola bacterial symbiont. The
objective of the current approach was to acquire further information for this unique symbiosis,
through microscopy and transcriptomics analysis. At first, the determination of the relative
abundance of Ca. E. dacicola during the life cycle of the olive fly, from the larval to the adult stage,
comparing flies developing in unripe and ripe olives, was performed using real time quantitative
PCR (RT-qPCR) and the data revealed that the bacterial titer is fluctuating between different
developmental stages. Furthermore, we report, through confocal scanning imaging techniques,
the localization of the bacterial symbionts in a specific part of the olive fly midgut, namely the
gastric caeca, during the larval stages of the host. Noticeably, gastric caeca transform
morphologically, depending on the developmental stage of the larva. Afterwards, a pairwise
comparison was set, in order to define critical aspects, concerning the transformation of the
gastric caeca during development, at a gene level. Gastric caeca which were dissected out of
second and third instar larvae, revealed many genes potentially involved in the olive fly
development. Moreover, comparative analysis between gastric caeca from second instar larvae
developing in olives as well as in artificial diet, identified genes of the host, which are potentially
involved in the establishment and the regulation of this symbiosis, since wild-type animals contain
huge numbers of the symbiont partner, while the laboratory-reared do not. Subsequently,
significant changes in transcript expression levels were reported and a detailed analysis of the
data was undertaken, focusing on certain groups of genes that potentially participate in the
symbiosis, as well as in the developmental transformation of the gastric caeca.
The new insights that are reported in this study, concerning the olive fly development and its
interaction with this vertically transmitted and obligate symbiont partner, Ca. E. dacicola, can be
exploited for the future development of symbiosis-based pest management strategies, concerning
the olive fly control. Particularly, a better understanding of this symbiotic relationship will serve as
the basis for the future development of novel olive fly control approaches, targeting this or other
bacterial partners, by using molecular and classical tools in smart applications. Taken together,
the data that are provided through this work, namely the establishment of a precise genome
editing tool for B. oleae (CRISPR/Cas9) and the resistance-specific and symbiosis-specific
released gene datasets, generate the opportunity to address the molecular basis of insecticide
resistance and symbiosis with bacterial partners mechanisms of the olive flies, in a systemic
manner and, moreover, utilize the acquired knowledge towards the development of innovative
pest control strategies, which will go beyond the traditional approaches and that will efficiently
control this destructive pest species.
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Characterization of insecticide resistance mechanisms of the olive fly (Bactrocera oleae) and its interactions with symbiotic bacteria of the species Candidatus Erwinia dacicola
