Gregory A. Sword

Evaluation of potential reference genes for reverse transcription-qPCR studies of physiological responses in Drosophila melanogaster

Drosophila melanogaster is one of the most important genetic models and techniques such as reverse transcription quantitative real-time PCR (RT-qPCR) are being employed extensively for deciphering the genetics basis of physiological functions. In RT-qPCR, the expression levels of target genes are estimated on the basis of endogenous controls. The purpose of these reference genes is to control for variations in RNA quantity and quality. Although determination of suitable reference genes is essential to RT-qPCR studies, reports on the evaluation of reference genes in D. melanogaster studies are lacking. We analyzed the expression levels of seven candidate reference genes (Actin, EF1, Mnf, Rps20, Rpl32, Tubulin and 18S) in flies that were injured, heat-stressed, or fed different diets. Statistical analyses of variation were determined using three established software programs for reference gene selection, geNorm, NormFinder and BestKeeper. Best-ranked references genes differed across the treatments. Normalization candidacy of the selected candidate reference genes was supported by an analysis of gene expression values obtained from microarray datasets available online. The differences between the experimental treatments suggest that assessing the stability of reference gene expression patterns, determining candidates and testing their suitability is required for each experimental investigation.

Do outbreaks affect genetic population structure? A worldwide survey in Locusta migratoria, a pest plagued by microsatellite null alleles

An understanding of the role of factors intrinsic to a species’ life history in structuring contemporary genetic variation is a fundamental, but understudied, aspect of evolutionary biology. Here, we assessed the influence of the propensity to outbreak in shaping worldwide genetic variation in Locusta migratoria, a cosmopolitan pest well known for its expression of density‐dependent phase polyphenism. We scored 14 microsatellites in nine subspecies from 25 populations distributed over most of the species’ range in regions that vary in the historical frequency and extent of their outbreaks. We rejected the hypothesis that L. migratoria consists of two genetically distinct clusters adapted to habitats either rarely (nonoutbreaking) or cyclically (outbreaking) favourable to increases in population density. We also invalidated the current subspecific taxonomic classification based on morphometrics. Bayesian inferences indicated evidence of a homogenizing effect of outbreaks on L. migratoria population structure. Geographical and ecological barriers to gene flow in conjunction with historical events can also explain the observed patterns. By systematically assessing the effects of null alleles using computer simulations, we also provide a template for the analysis of microsatellite data sets characterized by a high prevalence of null alleles.

Density-dependent warning coloration

Predators learn to avoid unpalatable prey more quickly when it is conspicuous than when it is cryptic. When they are rare, however, conspicuous individuals suffer a greater risk of discovery by predators than the cryptic majority, and their frequency is still too low for predators to learn to avoid them. We might therefore expect selection to favour unpalatable prey that is cryptic at low local densities and conspicuous at high local densities. I have found that such density-dependent warning coloration occurs in Schistocerca emarginata (=lineata) (Orthoptera: Acrididae) grasshoppers, providing insight into the biology of the desert locust, Schistocerca gregaria, and a new perspective on the evolution of warning coloration (aposematism).

Density–dependent aposematism in the desert locust

The ecological processes underlying locust swarm formation are poorly understood. Locust species exhibit phenotypic plasticity in numerous morphological, physiological and behavioural traits as their population density increases. These density‐dependent changes are commonly assumed to be adaptations for migration under heterogeneous environmental conditions. Here we demonstrate that density‐dependent nymphal colour change in the desert locust Schistocerca gregaria (Orthoptera: Acrididae) results in warning coloration (aposematism) when the population density increases and locusts consume native, toxic host plants. Fringe‐toed lizards (Acanthodactylus dumerili (Lacertidae)) developed aversions to high‐density‐reared (gregarious‐phase) locusts fed Hyoscyamus muticus (Solanaceae). Lizards associated both olfactory and visual cues with locust unpalatability, but only gregarious‐phase coloration was an effective visual warning signal. The lizards did not associate low rearing density coloration (solitarious phase) with locust toxicity. Predator learning of density‐dependent warning coloration results in a marked decrease in predation on locusts and may directly contribute to outbreaks of this notorious pest.

Mitochondrial genomes reveal the global phylogeography and dispersal routes of the migratory locust

The migratory locust, Locusta migratoria, is the most widely distributed grasshopper species in the world. However, its global genetic structure and phylogeographic relationships have not been investigated. In this study, we explored the worldwide genetic structure and phylogeography of the locust populations based on the sequence information of 65 complete mitochondrial genomes and three mitochondrial genes of 263 individuals from 53 sampling sites. Although this locust can migrate over long distances, our results revealed high genetic differentiation among the geographic populations. The populations can be divided into two different lineages: the Northern lineage, which includes individuals from the temperate regions of the Eurasian continent, and the Southern lineage, which includes individuals from Africa, southern Europe, the Arabian region, India, southern China, South‐east Asia and Australia. An analysis of population genetic diversity indicated that the locust species originated from Africa. Ancestral populations likely separated into Northern and Southern lineages 895 000 years ago by vicariance events associated with Pleistocene glaciations. These two lineages evolved in allopatry and occupied their current distributions in the world via distinct southern and northern dispersal routes. Genetic differences, caused by the long‐term independent diversification of the two lineages, along with other factors, such as geographic barriers and temperature limitations, may play important roles in maintaining the present phylogeographic patterns. Our phylogeographic evidence challenged the long‐held view of multiple subspecies in the locust species and tentatively divided it into two subspecies, L. m. migratoria and L. m. migratorioides.

A role for phenotypic plasticity in the evolution of aposematism

The evolution of warning coloration (aposematism) has been difficult to explain because rare conspicuous mutants should suffer a higher cost of discovery by predators relative to the cryptic majority, while at frequencies too low to facilitate predator aversion learning. Traditional models for the evolution of aposematism have assumed conspicuous prey phenotypes to be genetically determined and constitutive. By contrast, we have recently come to understand that warning coloration can be environmentally determined and mediated by local prey density, thereby reducing the initial costs of conspicuousness. The expression of density–dependent colour polyphenism is widespread among the insects and may provide an alternative pathway for the evolution of constitutive aposematic phenotypes in unpalatable prey by providing a protected intermediate stage. If density–dependent aposematism can function as an adaptive intermediate stage for the evolution of constitutive aposematic phenotypes, differential reaction norm evolution is predicted among related palatable and unpalatable prey populations. Here, I present empirical evidence that indicates that (i) the expression of density–dependent colour polyphenism has differentially evolved between palatable and unpalatable populations of the grasshopper Schistocerca emarginata (= lineata) (Orthoptera: Acrididae), and (ii) variation in plasticity between these populations is commensurate with the expected costs of conspicuousness.

The Entomopathogenic Fungal Endophytes Purpureocillium lilacinum (Formerly Paecilomyces lilacinus ) and Beauveria bassiana Negatively Affect Cotton Aphid Reproduction under Both Greenhouse and Field Conditions

The effects of two entomopathogenic fungal endophytes, Beauveria bassiana and Purpureocillium lilacinum (formerly Paecilomyces lilacinus), were assessed on the reproduction of cotton aphid, Aphis gossypii Glover (Homoptera:Aphididae), through in planta feeding trials. In replicate greenhouse and field trials, cotton plants (Gossypium hirsutum) were inoculated as seed treatments with two concentrations of B. bassiana or P. lilacinum conidia. Positive colonization of cotton by the endophytes was confirmed through potato dextrose agar (PDA) media plating and PCR analysis. Inoculation and colonization of cotton by either B. bassiana or P. lilacinum negatively affected aphid reproduction over periods of seven and 14 days in a series of greenhouse trials. Field trials were conducted in the summers of 2012 and 2013 in which cotton plants inoculated as seed treatments with B. bassiana and P. lilacinum were exposed to cotton aphids for 14 days. There was a significant overall effect of endophyte treatment on the number of cotton aphids per plant. Plants inoculated with B. bassiana had significantly lower numbers of aphids across both years. The number of aphids on plants inoculated with P. lilacinum exhibited a similar, but non-significant, reduction in numbers relative to control plants. We also tested the pathogenicity of both P. lilacinum and B. bassiana strains used in the experiments against cotton aphids in a survival experiment where 60% and 57% of treated aphids, respectively, died from infection over seven days versus 10% mortality among control insects. Our results demonstrate (i) the successful establishment of P. lilacinum and B. bassiana as endophytes in cotton via seed inoculation, (ii) subsequent negative effects of the presence of both target endophytes on cotton aphid reproduction using whole plant assays, and (iii) that the P. lilacinum strain used is both endophytic and pathogenic to cotton aphids. Our results illustrate the potential of using these endophytes for the biological control of aphids and other herbivores under greenhouse and field conditions.

Host plant-associated genetic differentiation in the snakeweed grasshopper, Hesperotettix viridis (Orthoptera: Acrididae)

Studies of herbivorous insects have played a major role in understanding how ecological divergence can facilitate genetic differentiation. In contrast to the majority of herbivorous insects, grasshoppers as a group are largely polyphagous. Due to this relative lack of intimate grasshopper–plant associations, grasshopper–plant systems have not played a large part in the study of host‐associated genetic differentiation. The oligophagous grasshopper, Hesperotettix viridis (Thomas), is endemic to North America and feeds on composites (Asteraceae) within the tribe Astereae. Previous work has shown both preference and performance differences between H. viridis individuals feeding on either Solidago mollis or Gutierrezia sarothrae. Using 222 AFLP markers, we examined the genetic relationships among 38 H. viridis individuals feeding on these plants both in sympatry and allopatry. Neighbour‐joining analysis resulted in two distinct host‐associated clades with 71% bootstrap support for host‐associated monophyly. Analyses of molecular variation (amova) revealed significant genetic structuring with host plant accounting for 20% of the total genetic variance while locality accounted for 0%. Significant genetic differentiation was detected between S. mollis‐feeders and G. sarothrae‐feeders even when the two were present at the same locality. These results are consistent with observed differences in preference and performance between H. viridis grasshoppers feeding on either G. sarothrae or S. mollis and indicate that H. viridis is comprised of at least two genetically distinct host plant‐associated lineages.

Radiotelemetry reveals differences in individual movement patterns between outbreak and non‐outbreak Mormon cricket populations

Abstract.  1. Outbreaks of insect pest populations are common and can have devastating effects on natural communities and on agriculture. Little is known about the causes of these outbreaks or the causes of en masse migrations during outbreaks.

Tasty on the outside, but toxic in the middle: grasshopper regurgitation and host plant-mediated toxicity to a vertebrate predator

Regurgitation by arthropods is often considered to be a rudimentary form of defense against predators. In phytophagous insects, regurgitate composition will vary with diet, and plant secondary compounds from host plants can contribute to the effectiveness of regurgitate deterrence. Regurgitation in response to predator attack is particularly common in grasshoppers. However, there is little empirical evidence in favor of grasshopper regurgitation as an effective antipredator mechanism in natural predator-prey systems. In particular, studies of the effect of grasshopper diet on regurgitate deterrence to vertebrate predators are lacking. This study investigated the relationship between diet and predator defense in the grasshopper, Schistocerca emarginata (=lineata) (Orthoptera: Acrididae). Using the insectivorous lizard, Anolis carolinensis (Iguanidae), as a predator, I demonstrate that consumption of Ptelea trifoliata (Rutaceae) by S. emarginata can confer distastefulness as well as toxicity. Regurgitate deterrence is mediated strictly by host plant material in the gut and does not require an enteric contribution from the grasshopper. Regurgitation by Ptelea-fed S. emarginata can result in rejection prior to ingestion by A. carolinensis and can enable grasshoppers to survive predator attacks.