Evolutionary biology and genetic techniques for insect control

The requirement to develop new techniques for insect control that minimize negative environmental impacts has never been more pressing. Here we discuss population suppression and population replacement technologies. These include sterile insect technique, genetic elimination methods such as the release of insects carrying a dominant lethal (RIDL), and gene driving mechanisms offered by intracellular bacteria and homing endonucleases. We also review the potential of newer or underutilized methods such as reproductive interference, CRISPR technology, RNA interference (RNAi), and genetic underdominance. We focus on understanding principles and potential effectiveness from the perspective of evolutionary biology. This offers useful insights into mechanisms through which potential problems may be minimized, in much the same way that an understanding of how resistance evolves is key to slowing the spread of antibiotic and insecticide resistance. We conclude that there is much to gain from applying principles from the study of resistance in these other scenarios – specifically, the adoption of combinatorial approaches to minimize the spread of resistance evolution. We conclude by discussing the focused use of GM for insect pest control in the context of modern conservation planning under land-sparing scenarios

Male reproductive success and population control in the Mediterranean fruit fly, ceratitis capitata

The traits that determine male reproductive success in Ceratitis capitata (medfly) are largely unknown. This comes despite decades of research into the reproductive behaviour of this agricultural pest. An understanding of what makes a successful male is of great importance in this lekking species, as one male has the ability to dominate access to females. In addition species-specific pest control techniques such as the Sterile Insect Technique (SIT) and Release of Insects carrying a Dominant Lethal (RIDL) rely on the mating competitiveness of mass-reared ‘sterile’ males to disrupt natural mating systems and reduce population numbers. However, it is well known that the mass-rearing process required to generate these individuals produces less competitive males, despite this an understanding of the sexual selection processes that are involved is severely lacking. In this thesis I used the medfly as a model organism to investigate the context-dependent nature of male reproductive success. In Chapters 2 and 3 I investigate the effect of manipulating the adult sex ratio on pre- and post-mating reproductive traits. Chapter 2 uses proximate manipulation of the adult sex ratio to show how the relationship between pre- and post-mating success is affected by the levels of male competition. Chapter 3 shows that selection lines are unable to select for the traits that predict male reproductive success under altered levels of male competition. Chapter 4 describes the attempts to produce RIDL genetic constructs with embryonic lethality. Chapter 5 demonstrates that RIDL lines of medfly display characteristics that make them suitable for wild population control and may exceed the performance of existing SIT lines. Chapter 6 shows that manipulation of the larval diet can alter the gut microbiota of adults; however these manipulations have no effect on reproductive behaviour. Instead there was a significant effect of high-sugar larval rearing on mating success and body mass. This effect persisted for a single generation in offspring of these flies, even when they were reared on an inferior starch-based larval diet. Finally Chapter 7 summarises the thesis and discusses the implications of this work and future directions for both research in the reproductive behaviour of the medfly, and the future of RIDL technology in medfly population control

Testing for assortative mating by diet in Drosophila melanogaster

Experimental studies of the evolution of reproductive isolation in real time are a powerful way to reveal the way that fundamental processes, such as mate choice, initiate divergence. Mate choice, while frequently described in females, can occur in either sex, and can be affected by the genetics or environment of an individual. Here we describe simple protocols for assessing mating outcomes in fruit flies, which in this context can be used to assess reproductive isolation derived from rearing on different diets over multiple generations

Diet, Gut Microbes and Host Mate Choice:Understanding the significance of microbiome effects on host mate choice requires a case by case evaluation

All organisms live in close association with microbes. However, not all such associations are meaningful in an evolutionary context. Current debate concerns whether hosts and microbes are best described as communities of individuals or as holobionts (selective units of hosts plus their microbes). Recent reports that assortative mating of hosts by diet can be mediated by commensal gut microbes have attracted interest as a potential route to host reproductive isolation (RI). Here we discuss logical problems with this line of argument. We briefly review how microbes can affect host mating preferences and evaluate recent findings from fruitflies. Endosymbionts can potentially influence host RI given stable and recurrent co-association of hosts and microbes over evolutionary time. However, observations of co-occurrence of microbes and hosts are ripe for misinterpretation and such associations will rarely represent a meaningful holobiont. A framework in which hosts and their microbes are independent evolutionary units provides the only satisfactory explanation for the observed range of effects and associations

Expanding the CRISPR toolbox in Culicine mosquitoes: in vitro validation of Pol III promoters

CRISPR–Cas9-based “gene drive” technologies have been proposed as a novel and effective means of controlling human diseases vectored by mosquitoes. However, more complex designs than those demonstrated to date—and an expanded molecular toolbox with which to build them—will be required to overcome the issues of resistance formation/evolution and drive spatial/temporal limitation. Foreseeing this need, we assessed the sgRNA transcriptional activities of 33 phylogenetically diverse insect Polymerase III promoters using three disease-relevant Culicine mosquito cell lines (Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus). We show that U6 promoters work across species with a range of transcriptional activity levels and find 7SK promoters to be especially promising because of their broad phylogenetic activity. We further show that U6 promoters can be substantially truncated without affecting transcriptional levels. These results will be of great utility to researchers involved in developing the next generation of gene drives

Contribution of maternal effects to dietary selection in Mediterranean fruit flies

Individual responses to dietary variation represent a fundamental component of fitness, and nutritional adaptation can occur over just a few generations. Maternal effects can show marked proximate responses to nutrition, but whether they contribute to longer term dietary adaptation is unclear. Here, we tested the hypotheses that maternal effects: (i) contribute to dietary adaptation, (ii) diminish when dietary conditions are constant between generations, (iii) are trait-specific and (iv) interact with high- and low-quality food. We used experimental evolution regimes in the medfly (Ceratitis capitata) to test these predictions by subjecting an outbred laboratory-adapted population to replicated experimental evolution on either constant high calorie sugar (‘A’) or low-calorie starch (‘S’) larval diets, with a standard adult diet across both regimes. We measured the contribution of maternal effects by comparing developmental and adult phenotypes of individuals reared on their own diet with those swapped onto the opposite diet for either one or two generations (high and low maternal effect conditions, respectively), both at the start and after 30 generations of selection. Initially, there were strong maternal effects on female body mass and male mating success but not larval survival. Interestingly, the initial maternal effects observed in female body mass and male mating success showed sex-specific interactions when individuals from high calorie regimes were tested on low calorie diets. However, as populations responded to selection, the effects of maternal provisioning on all traits diminished. The results broadly supported the predictions. They show how the contribution of maternal effects to dietary responses evolves in a context-dependent manner, with significant variation across different fitness-related traits. We conclude that maternal effects can evolve during nutritional adaptation and hence may be an important life history trait to measure, rather than to routinely minimize

Genetic elimination of field-cage populations of Mediterranean fruit flies

The Mediterranean fruit fly (medfly, Ceratitis capitata Wiedemann) is a pest of over 300 fruits, vegetables and nuts. The sterile insect technique (SIT) is a control measure used to reduce the reproductive potential of populations through the mass release of sterilized male insects that mate with wild females. However, SIT flies can display poor field performance, due to the effects of mass-rearing and of the irradiation process used for sterilization. The development of female-lethal RIDL (release of insects carrying a dominant lethal) strains for medfly can overcome many of the problems of SIT associated with irradiation. Here, we present life-history characterizations for two medfly RIDL strains, OX3864A and OX3647Q. Our results show (i) full functionality of RIDL, (ii) equivalency of RIDL and wild-type strains for life-history characteristics, and (iii) a high level of sexual competitiveness against both wild-type and wild-derived males. We also present the first proof-of-principle experiment on the use of RIDL to eliminate medfly populations. Weekly releases of OX3864A males into stable populations of wild-type medfly caused a successive decline in numbers, leading to eradication. The results show that genetic control can provide an effective alternative to SIT for the control of pest insects

Transmission efficiency drives host–microbe associations

Sequencing technologies have fuelled a rapid rise in descriptions of microbial communities associated with hosts, but what is often harder to ascertain is the evolutionary significance of these symbioses. Here, we review the role of vertical (VT), horizontal (HT), environmental acquisition and mixed modes of transmission (MMT), in the establishment of animal host–microbe associations. We then model four properties of gut microbiota proposed as key to promoting animal host–microbe relationships: modes of transmission, host reproductive mode, host mate choice and host fitness. We found that: (i) MMT led to the highest frequencies of host–microbe associations, and that some environmental acquisition or HT of microbes was required for persistent associations to form unless VT was perfect; (ii) host reproductive mode (sexual versus asexual) and host mate choice (for microbe carriers versus non-carriers) had little impact on the establishment of host–microbe associations; (iii) host mate choice did not itself lead to reproductive isolation, but could reinforce it; and (iv) changes in host fitness due to host–microbe associations had a minimal impact upon the formation of co-associations. When we introduced a second population, into which host–microbe carriers could disperse but in which environmental acquisition did not occur, highly efficient VT was required for host–microbe co-associations to persist. Our study reveals that transmission mode is of key importance in establishing host–microbe associations

Gut microbiomes and reproductive isolation in Drosophila

Experimental studies of the evolution of reproductive isolation (RI) in real time are a powerful way in which to reveal fundamental, early processes that initiate divergence. In a classic speciation experiment, populations of Drosophila pseudoobscura were subjected to divergent dietary selection and evolved significant positive assortative mating by diet. More recently, a direct role for the gut microbiome in determining this type of RI in Drosophila melanogaster has been proposed. Manipulation of the diet, and hence the gut microbiome, was reported to result in immediate assortative mating by diet, which could be eliminated by reducing gut microbes using antibiotics and recreated by adding back Lactobacillus plantarum. We suggest that the evolutionary significance of this result is unclear. For example, in D. melanogaster, the microbiome is reported as flexible and largely environmentally determined. Therefore, microbiome-mediated RI would be transient and would break down under dietary variation. In the absence of evolutionary coassociation or recurrent exposure between host and microbiome, there are no advantages for the gut bacteria or host in effecting RI. To explore these puzzling effects and their mechanisms further, we repeated the tests for RI associated with diet-specific gut microbiomes in D. melanogaster. Despite observing replicable differences in the gut microbiomes of flies maintained on different diets, we found no evidence for diet-associated RI, for any role of gut bacteria, or for L. plantarum specifically. The results suggest that there is no general role for gut bacteria in driving the evolution of RI in this species and resolve an evolutionary riddle