Laurence Blondin

Assessment and validation of a suite of reverse transcription-quantitative PCR reference genes for analyses of density-dependent behavioural plasticity in the Australian plague locust

BackgroundThe Australian plague locust, Chortoicetes terminifera, is among the most promising species to unravel the suites of genes underling the density-dependent shift from shy and cryptic solitarious behaviour to the highly active and aggregating gregarious behaviour that is characteristic of locusts. This is because it lacks many of the major phenotypic changes in colour and morphology that accompany phase change in other locust species. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the most sensitive method available for determining changes in gene expression. However, to accurately monitor the expression of target genes, it is essential to select an appropriate normalization strategy to control for non-specific variation between samples. Here we identify eight potential reference genes and examine their expression stability at different rearing density treatments in neural tissue of the Australian plague locust.ResultsTaking advantage of the new orthologous DNA sequences available in locusts, we developed primers for genes encoding 18SrRNA, ribosomal protein L32 (RpL32), armadillo (Arm), actin 5C (Actin), succinate dehydrogenase (SDHa), glyceraldehyde-3P-dehydrogenase (GAPDH), elongation factor 1 alpha (EF1a) and annexin IX (AnnIX). The relative transcription levels of these eight genes were then analyzed in three treatment groups differing in rearing density (isolated, short- and long-term crowded), each made up of five pools of four neural tissue samples from 5th instar nymphs. SDHa and GAPDH, which are both involved in metabolic pathways, were identified as the least stable in expression levels, challenging their usefulness in normalization. Based on calculations performed with the geNorm and NormFinder programs, the best combination of two genes for normalization of gene expression data following crowding in the Australian plague locust was EF1a and Arm. We applied their use to studying a target gene that encodes a Ca2+ binding glycoprotein, SPARC, which was previously found to be up-regulated in brains of gregarious desert locusts, Schistocerca gregaria. Interestingly, expression of this gene did not vary with rearing density in the same way in brains of the two locust species. Unlike S. gregaria, there was no effect of any crowding treatment in the Australian plague locust.ConclusionArm and EF1a is the most stably expressed combination of two reference genes of the eight examined for reliable normalization of RT-qPCR assays studying density-dependent behavioural change in the Australian plague locust. Such normalization allowed us to show that C. terminifera crowding did not change the neuronal expression of the SPARC gene, a gregarious phase-specific gene identified in brains of the desert locust, S. gregaria. Such comparative results on density-dependent gene regulation provide insights into the evolution of gregarious behaviour and mass migration of locusts. The eight identified genes we evaluated are also candidates as normalization genes for use in experiments involving other Oedipodinae species, but the rank order of gene stability must necessarily be determined on a case-by-case basis.

Demographic processes shaping genetic variation of the solitarious phase of the desert locust

Between plagues, the solitarious desert locust (Schistocerca gregaria) is generally thought to exist as small populations, which are particularly prone to extinction events in arid regions of Africa and Asia. Given the high genetic structuring observed in one geographical area (the Eritrean coast) by former authors, a metapopulation dynamics model involving repeated extinction and colonization events was favoured. In this study, we assessed the validity of a demographic scenario involving temporary populations of the solitarious phase of the desert locust by analysing large‐scale population genetic data. We scored 24 microsatellites in 23 solitarious population samples collected over most of the species range during remission. We found very little genetic structuring and little evidence of declining genetic diversity. A Bayesian clustering method distinguished four genetically differentiated units. Three groups were largely consistent with three population samples which had undergone recent bottleneck events. Nevertheless, the last genetically homogeneous unit included all individuals from the remaining 18 population samples and did not show evidence of demographic disequilibrium. An approximate Bayesian computation treatment indicated a large population size for this main genetic group, moderately reduced between plague and remission but still containing tens of thousands of individuals. Our results diverge from the hypothesis of a classical metapopulation dynamics model. They instead support the scenario in which large populations persist in the solitarious phase of the desert locust.

Taxa-specific heat shock proteins are over-expressed with crowding in the Australian plague locust

Most heat shock proteins (Hsps) function as molecular chaperones that help organisms to cope with stress. Although the best empirical evidence is related to heat shock, there is evidence that Hsps and their encoding genes are involved in resistance to other ecologically relevant types of stresses such as those imposed by high population density. We quantified density-dependent gene expression of large (i.e. Hsp40, Hsc70 and Hsp90) and small (Hsp20.5, Hsp20.6 and Hsp20.7) heat shock genes in neural tissue of fifth-instar nymphs of the Australian plague locust, Chortoicetes terminifera, using reverse transcription-quantitative PCR. Locusts are of particular interest when studying the influence of stress induced by high population density since they show an extreme form of phenotypic plasticity changing from a cryptic solitarious phase to a swarming gregarious phase. Crowding led to a synchronous and sustained 2-3 fold increase in the expression of only two Hsp genes, Hsp20.5 and Hsp20.7, which do not BLAST with any known animal sequences and therefore are likely to be unique to members of the Orthoptera. This study opens a range of experiments to investigate the possibility of specific roles for these two small Hsps in the resistance to stressful conditions imposed by crowded environments and/or the expression of gregarious behavior as well as their evolutionary significance to locusts whose populations are regularly exposed to high density conditions in the field.

Isolation of microsatellite markers in the Calliptamus genus (Orthoptera, Acrididae).

Abstract The Calliptamus genus (Orthoptera: Acrididae) includes locust and grasshopper species, some of which have a high economic impact. Using an enriched methodology, 10 microsatellite markers have been developed from two species, Calliptamus italicus and Calliptamus barbarus. These polymorphic markers were tested on different populations of three Calliptamus species: C. italicus, C. barbarus, C. wattenwylianus. Two markers were amplified on the three species, as well as four on C. barbarus and two on C. italicus. In each species, 9 to 23 alleles per locus were observed. These molecular markers might prove to be a new and interesting tool for Calliptamus population genetics and dispersion studies.

Genetic and Morphological Variation in Non-Polyphenic Southern African Populations of the Desert Locust

This study addresses geographic variation of body size and shape and of allele frequencies at 21 microsatellite loci in southern African populations of the desert locust, Schistocerca gregaria. These populations, which belong to the subspecies S. g. flaviventris, lack the capacity to change phase and to swarm relative to the northern populations of the nominate subspecies, S. g. gregaria. We reported overall genetic and morphological similarities among localities that cover most of the subspecies range. Of particular interest, the level of genetic diversity was moderately lower than in the swarming subspecies of the northern range. In addition, S. g. flaviventris populations were genetically homogeneous, such as observed in the northern range of the nominate subspecies. This result can be explained by north—southwest seasonal migration to follow rainfall.

Microsatellite evolutionary rate and pattern in Schistocerca gregaria inferred from direct observation of germline mutations.

Unravelling variation among taxonomic orders regarding the rate of evolution in microsatellites is crucial for evolutionary biology and population genetics research. The mean mutation rate of microsatellites tends to be lower in arthropods than in vertebrates, but data are scarce and mostly concern accumulation of mutations in model species. Based on parent–offspring segregations and a hierarchical Bayesian model, the mean rate of mutation in the orthopteran insect Schistocerca gregaria was estimated at 2.1e−4 per generation per untranscribed dinucleotide locus. This is close to vertebrate estimates and one order of magnitude higher than estimates from species of other arthropod orders, such as Drosophila melanogaster and Daphnia pulex. We also found evidence of a directional bias towards expansions even for long alleles and exceptionally large ranges of allele sizes. Finally, at transcribed microsatellites, the mean rate of mutation was half the rate found at untranscribed loci and the mutational model deviated from that usually considered, with most mutations involving multistep changes that avoid disrupting the reading frame. Our direct estimates of mutation rate were discussed in the light of peculiar biological and genomic features of S. gregaria, including specificities in mismatch repair and the dependence of its activity to allele length. Shedding new light on the mutational dynamics of grasshopper microsatellites is of critical importance for a number of research fields. As an illustration, we showed how our findings improve microsatellite application in population genetics, by obtaining a more precise estimation of S. gregaria effective population size from a published data set based on the same microsatellites.

Subspecific taxonomy of the desert locust, #Schistocerca gregaria# (Orthoptera: Acrididae), based on molecular and morphological characters

We evaluated the validity of the subspecific designation for Schistocerca gregaria gregaria (Forskål) and Schistocerca gregaria flaviventris (Burmeister), isolated in distinct regions along the north–south axis of Africa. Towards this goal, we assessed the variation of multiple morphological and molecular traits within species. We first used elliptic Fourier and landmark‐based relative warps analyses to compare the size and shape of two internal and two external structures of male genitalia. We provide a discriminant function which classified the specimens with 100% accuracy and selected shape elements of the external structures only (cercus and epiproct). We also tested eight molecular markers, and because of either absence of variation or contamination by mitochondrial DNA (mtDNA)‐like sequences, we used a clone‐and‐sequence analysis of the standard cytochrome c oxidase subunit I mitochondrial DNA barcode only. We differentiated 185 true mitochondrial sequences from 66 mitochondrial DNA‐like sequences, most of which were from S. g. gregaria specimens. On the dataset of mitochondrial origin, we identified three characteristic point mutations that diagnosed the two allopatric subspecies with 94% accuracy. Minimum spanning network and parsimony tree analyses identified S. g. flaviventris as a monophyletic lineage distinct from the nominate subspecies. Accordingly, microsatellite data indicate rarely occurring admixture events only, showing that independent evolutionary history is the norm.

Extra Molting and Selection on Nymphal Growth in the Desert Locust

In insects, extra-molting has been viewed as a compensatory mechanism for nymphal growth that contributes to optimize body weight for successful reproduction. However, little is known on the capacity of extra-molting to evolve in natural populations, which limits our understanding of how selection acts on nymphal growth. We used a multi-generational pedigree, individual monitoring and quantitative genetics models to investigate the evolution of extra-molting and its impact on nymphal growth in a solitarious population of the desert locust, Schistocerca gregaria. Growth compensation via extra-molting was observed for 46% of the females, whose adult weight exceeded by 4% that of other females, at a cost of a 22% longer development time. We found a null heritability for body weight threshold only, and the highest and a strongly female-biased heritability for extra molting. Our genetic estimates show that (1) directional selection can act on growth rate, development time and extra-molting to optimize body weight threshold, the target of stabilizing selection, (2) extra-molting can evolve in natural populations, and (3) a genetic conflict, due to sexually antagonistic selection on extra-molting, might prevent its fixation. Finally, we discuss how antagonistic selection between solitarious and gregarious environments and/or genetic correlations between growth and phase traits might also impact the evolution of extra-molting in locusts.

Isolation and Characterization of Twelve Polymorphic Microsatellite Loci for the Cocoa Mirid Bug Sahlbergella Singularis

Mirids are the primary pests affecting cocoa production in Africa, but no genetic studies have been conducted on these insects. Here we report the isolation and characterization of 12 polymorphic microsatellite loci for Sahlbergella singularis. A microsatellite-enriched genomic DNA library was developed and screened to identify marker loci. Twelve polymorphic loci were identified by screening 28 individuals collected from one presumed population in cocoa plantations in Southern Cameroon. The number of alleles ranged from 5 to 25, whereas the observed and the expected heterozygosities ranged from 0.179 to 0.786 and from 0.671 to 0.946, respectively. Tests showed significant deviations from HW equilibrium for four loci, but no linkage disequilibrium was detected at any of the loci. No cross-species amplification was observed in two other mirid pests in Africa.

Microsatellite markers for the Chameleon grasshopper (Kosciuscola tristis) (Orthoptera: Acrididae), an Australian Alpine Specialist.

A set of polymorphic loci was characterised using an enrichment library for the Australian alpine specialist, the chameleon grasshopper (Kosciuscola tristis), an atypical grasshopper known for its remarkable temperature-controlled colour change. The number of alleles per locus ranged from three to 20 and observed heterozygosity from 0.16 to 0.76. These are the first microsatellite markers for a non-endangered Australian alpine animal and will inform questions of gene flow across the sky islands of this unique and threatened region.