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

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

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

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

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

Accentuating the positive and eliminating the negative: Efficacy of TiO2 as digestibility index marker for poultry nutrition studies

Inert digestibility index markers such as titanium dioxide are universally accepted to provide simple measurement of digestive tract retention and relative digestibility in poultry feeding trials. Their use underpins industry practice: specifically dosing regimens for adjunct enzymes added to animal feed. Among these, phytases, enzymes that degrade dietary phytate, inositol hexakisphosphate, represent a billion-dollar sector in an industry that raises ca. 70 billion chickens/annum. Unbeknown to the feed enzyme sector, is the growth in cell biology of use of titanium dioxide for enrichment of inositol phosphates from extracts of cells and tissues. The adoption of titanium dioxide in cell biology arises from its affinity under acid conditions for phosphates, suggesting that in feeding trial contexts that target phytate degradation this marker may not be as inert as assumed. We show that feed grade titanium dioxide enriches a mixed population of higher and lower inositol phosphates from acid solutions. Additionally, we compared the extractable inositol phosphates in gizzard and ileal digesta of 21day old male Ross 308 broilers fed three phytase doses (0, 500 and 6000 FTU/kg feed) and one inositol dose (2g/kg feed). This experiment was performed with or without titanium dioxide added as a digestibility index marker at a level of 0.5%, with all diets fed for 21 days. Analysis yielded no significant difference in effect of phytase inclusion in the presence or absence of titanium dioxide. Thus, despite the utility of titanium dioxide for recovery of inositol phosphates from biological samples, it seems that its use as an inert marker in digestibility trials is justified—as its inclusion in mash diets does not interfere with the recovery of inositol phosphates from digesta samples

CRISPR-based gene drives generate super-Mendelian inheritance in the disease vector Culex quinquefasciatus

Culex mosquitoes pose a significant public health threat as vectors for a variety of diseases including West Nile virus and lymphatic filariasis, and transmit pathogens threatening livestock, companion animals, and endangered birds. Rampant insecticide resistance makes controlling these mosquitoes challenging and necessitates the development of new control strategies. Gene drive technologies have made significant progress in other mosquito species, although similar advances have been lagging in Culex. Here we test a CRISPR-based homing gene drive for Culex quinquefasciatus, and show that the inheritance of two split-gene-drive transgenes, targeting different loci, are biased in the presence of a Cas9-expressing transgene although with modest efficiencies. Our findings extend the list of disease vectors where engineered homing gene drives have been demonstrated to include Culex alongside Anopheles and Aedes, and pave the way for future development of these technologies to control Culex mosquitoes

Genetic pest management and the background genetics of release strains

Genetic pest management (GPM) methods involve releasing modified versions of a pest species to mate with wild pests in the target area. Proposed for a wide range of applications in public health, agriculture and conservation, most progress has been made with pest insects. Offspring of the released modified insects and wild pests carry the modification—which might be transgenes, artificially introduced Wolbachia or genetic damage from radiation, for example—but they also carry a complete haploid genome from their laboratory-reared parent, as well as one from their wild parent. Unless these F1 hybrids are completely unable to reproduce, further mating will lead to introgression of DNA sequences from the release strain into the wild population. We discuss issues around strain selection and the potential consequences of such introgression. We conclude that such introgression is probably harmless in almost all circumstances, and could, in theory, provide specific additional benefits to the release programme. We outline population monitoring approaches that could be used, going forward, to determine how background genetics may affect GPM

Toward a CRISPR-Cas9-based gene drive in the diamondback moth Plutella xylostella

Promising to provide powerful genetic control tools, gene drives have been constructed in multiple dipteran insects, yeast, and mice for the purposes of population elimination or modification. However, it remains unclear whether these techniques can be applied to lepidopterans. Here, we used endogenous regulatory elements to drive Cas9 and single guide RNA (sgRNA) expression in the diamondback moth (DBM), Plutella xylostella, and test the first split gene drive system in a lepidopteran. The DBM is an economically important global agriculture pest of cruciferous crops and has developed severe resistance to various insecticides, making it a prime candidate for such novel control strategy development. A very high level of somatic editing was observed in Cas9/sgRNA transheterozygotes, although no significant homing was revealed in the subsequent generation. Although heritable Cas9-medated germline cleavage as well as maternal and paternal Cas9 deposition were observed, rates were far lower than for somatic cleavage events, indicating robust somatic but limited germline activity of Cas9/sgRNA under the control of selected regulatory elements. Our results provide valuable experience, paving the way for future construction of gene drives or other Cas9-based genetic control strategies in DBM and other lepidopterans