Genome assembly and transcriptomic analysis to elucidate the ability of Nasonovia ribisnigri to break host plant resistance.

Aphid genomic resources enable the study of complex life history traits and provide information on vector biology, host adaption and speciation. The currant-lettuce aphid (Nasonovia ribisnigri (Hemiptera: Aphididae) (Mosley)) is a cosmopolitan pest of outdoor lettuce (Lactuca sativa (Asterales: Asteraceae) (Linnaeus)). Until recently, the use of resistant cultivars was an effective method for managing N. ribisnigri. A resistant cultivar containing a single gene (Nr-locus), introduced in the 1980s, conferred complete resistance to feeding. Overreliance of this Nr-locus in lettuce resulted in N. ribisnigri's ability to break resistance mechanism, with first reports during 2003. Our work attempts to understand which candidate gene(s) are associated with this resistance-breaking mechanism. We present two de novo draft assembles for N. ribisnigri genomes, corresponding to both avirulent (Nr-locus susceptible) and virulent (Nr-locus resistant) biotypes. Changes in gene expression of the two N. ribisnigri biotypes were investigated using transcriptomic analyses of RNA-sequencing (RNA-seq) data to understand the potential mechanisms of resistance to the Nr-locus in lettuce. The draft genome assemblies were 94.2% and 91.4% complete for the avirulent and virulent biotypes, respectively. Out of the 18,872 differentially expressed genes, a single gene/locus was identified in N. ribisnigri that was shared between two resistant-breaking biotypes. This locus was further explored and validated in Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) experiments and has predicted localisations in both the cytoplasm and nucleus. This is the first study to provide evidence that a single gene/locus is likely responsible for the ability of N. ribisnigri to overcome the Nr-locus resistance in the lettuce host.

Contrasting population structure and demographic history of cereal aphids in different environmental and agricultural landscapes.

Genetic diversity of populations has important ecological and evolutionary consequences, whose understanding is fundamental to improve the sustainability of agricultural production. Studies of how differences in agricultural management and environment influence the population structure of insect pests are central to predict outbreaks and optimize control programs. Here, we have studied the population genetic diversity and evolution of Sitobion avenae and Sitobion miscanthi, which are among the most relevant aphid pests of cereals across Europe and Asia, respectively. We have used genotyping by sequencing (GBS) to identify genome-wide single nucleotide polymorphisms (SNPs) to infer the geographic structure and migration patterns. In the present study, we show that the population structure in present-day populations is different from that described in previous studies, which suggest that they have evolved recently possibly as a response to human-induced changes in agriculture. This study shows that S. avenae in England is predominantly parthenogenetic and there has been a demographic and spatial expansion of a single genetic cluster, which could correspond with the insecticide resistance superclone identified in previous studies. Conversely, in China, S. miscanthi populations are mostly cyclical parthenogenetic, with one sexual stage in autumn to produce overwintering eggs, and there are six genetically differentiated subpopulations and high genetic differentiation between geographic locations, which suggests that further taxonomical research is needed. Unlike S. avenae in England, there is no evidence for insecticide resistance and there is no predominance of a single lineage in S. miscanthi in China.

Population genetic structure and predominance of cyclical parthenogenesis in the bird cherry-oat aphid Rhopalosiphum padi in England.

Genetic diversity is the determinant for pest species' success and vector competence. Understanding the ecological and evolutionary processes that determine the genetic diversity is fundamental to help identify the spatial scale at which pest populations are best managed. In the present study, we present the first comprehensive analysis of the genetic diversity and evolution of Rhopalosiphum padi, a major pest of cereals and a main vector of the barley yellow dwarf virus (BYDV), in England. We have used a genotyping-by-sequencing approach to study whether (a) there is any underlying population genetic structure at a national and regional scale in this pest that can disperse long distances; (b) the populations evolve as a response to environmental change and selective pressures; and (c) the populations comprise anholocyclic lineages. Individual R. padi were collected using the Rothamsted Insect Survey's suction-trap network at several sites across England between 2004 and 2016 as part of the RIS long-term nationwide surveillance. Results identified two genetic clusters in England that mostly corresponded to a North-South division, although gene flow is ongoing between the two subpopulations. These genetic clusters do not correspond to different life cycle types, and cyclical parthenogenesis is predominant in England. Results also show that there is dispersal with gene flow across England, although there is a reduction between the northern and southern sites with the south-western population being the most genetically differentiated. There is no evidence for isolation by distance and other factors such as primary host distribution, uncommon in the south and absent in the south-west, could influence the dispersal patterns. Finally, results also show no evidence for the evolution of the R. padi population, and it is demographically stable despite the ongoing environmental change. These results are discussed in view of their relevance to pest management and the transmission of BYDV.

The genome of the biting midge Culicoides sonorensis and gene expression analyses of vector competence for bluetongue virus.

BACKGROUND: The new genomic technologies have provided novel insights into the genetics of interactions between vectors, viruses and hosts, which are leading to advances in the control of arboviruses of medical importance. However, the development of tools and resources available for vectors of non-zoonotic arboviruses remains neglected. Biting midges of the genus Culicoides transmit some of the most important arboviruses of wildlife and livestock worldwide, with a global impact on economic productivity, health and welfare. The absence of a suitable reference genome has hindered genomic analyses to date in this important genus of vectors. In the present study, the genome of Culicoides sonorensis, a vector of bluetongue virus (BTV) in the USA, has been sequenced to provide the first reference genome for these vectors. In this study, we also report the use of the reference genome to perform initial transcriptomic analyses of vector competence for BTV. RESULTS: Our analyses reveal that the genome is 189 Mb, assembled in 7974 scaffolds. Its annotation using the transcriptomic data generated in this study and in a previous study has identified 15,612 genes. Gene expression analyses of C. sonorensis females infected with BTV performed in this study revealed 165 genes that were differentially expressed between vector competent and refractory females. Two candidate genes, glutathione S-transferase (gst) and the antiviral helicase ski2, previously recognized as involved in vector competence for BTV in C. sonorensis (gst) and repressing dsRNA virus propagation (ski2), were confirmed in this study. CONCLUSIONS: The reference genome of C. sonorensis has enabled preliminary analyses of the gene expression profiles of vector competent and refractory individuals. The genome and transcriptomes generated in this study provide suitable tools for future research on arbovirus transmission. These provide a valuable resource for these vector lineage, which diverged from other major Dipteran vector families over 200 million years ago. The genome will be a valuable source of comparative data for other important Dipteran vector families including mosquitoes (Culicidae) and sandflies (Psychodidae), and together with the transcriptomic data can yield potential targets for transgenic modification in vector control and functional studies.

Distinctive patterns of evolution of the δ-globin gene (HBD) in primates.

In most vertebrates, hemoglobin (Hb) is a heterotetramer composed of two dissimilar globin chains, which change during development according to the patterns of expression of α- and ß-globin family members. In placental mammals, the ß-globin cluster includes three early-expressed genes, ε(HBE)-γ(HBG)-ψß(HBBP1), and the late expressed genes, δ (HBD) and ß (HBB). While HBB encodes the major adult ß-globin chain, HBD is weakly expressed or totally silent. Paradoxically, in human populations HBD shows high levels of conservation typical of genes under strong evolutionary constraints, possibly due to a regulatory role in the fetal-to-adult switch unique of Anthropoid primates. In this study, we have performed a comprehensive phylogenetic and comparative analysis of the two adult ß-like globin genes in a set of diverse mammalian taxa, focusing on the evolution and functional divergence of HBD in primates. Our analysis revealed that anthropoids are an exception to a general pattern of concerted evolution in placental mammals, showing a high level of sequence conservation at HBD, less frequent and shorter gene conversion events. Moreover, this lineage is unique in the retention of a functional GATA-1 motif, known to be involved in the control of the developmental expression of the ß-like globin genes. We further show that not only the mode but also the rate of evolution of the δ-globin gene in higher primates are strictly associated with the fetal/adult ß-cluster developmental switch. To gain further insight into the possible functional constraints that have been shaping the evolutionary history of HBD in primates, we calculated dN/dS (ω) ratios under alternative models of gene evolution. Although our results indicate that HBD might have experienced different selective pressures throughout primate evolution, as shown by different ω values between apes and Old World Monkeys + New World Monkeys (0.06 versus 0.43, respectively), these estimates corroborated a constrained evolution for HBD in Anthropoid lineages, which is unlikely to be related to protein function. Collectively, these findings suggest that sequence change at the δ-globin gene has been under strong selective constraints over 65 Myr of primate evolution, likely due to a regulatory role in ontogenic switches of gene expression.

SERPINA2 is a novel gene with a divergent function from SERPINA1.

Serine protease inhibitors (SERPINs) are a superfamily of highly conserved proteins that play a key role in controlling the activity of proteases in diverse biological processes. The SERPIN cluster located at the 14q32.1 region includes the gene coding for SERPINA1, and a highly homologous sequence, SERPINA2, which was originally thought to be a pseudogene. We have previously shown that SERPINA2 is expressed in different tissues, namely leukocytes and testes, suggesting that it is a functional SERPIN. To investigate the function of SERPINA2, we used HeLa cells stably transduced with the different variants of SERPINA2 and SERPINA1 (M1, S and Z) and leukocytes as the in vivo model. We identified SERPINA2 as a 52 kDa intracellular glycoprotein, which is localized at the endoplasmic reticulum (ER), independently of the variant analyzed. SERPINA2 is not significantly regulated by proteasome, proposing that ER localization is not due to misfolding. Specific features of SERPINA2 include the absence of insoluble aggregates and the insignificant response to cell stress, suggesting that it is a non-polymerogenic protein with divergent activity of SERPINA1. Using phylogenetic analysis, we propose an origin of SERPINA2 in the crown of primates, and we unveiled the overall conservation of SERPINA2 and A1. Nonetheless, few SERPINA2 residues seem to have evolved faster, contributing to the emergence of a new advantageous function, possibly as a chymotrypsin-like SERPIN. Herein, we present evidences that SERPINA2 is an active gene, coding for an ER-resident protein, which may act as substrate or adjuvant of ER-chaperones.

The Drosophila melanogaster methuselah gene: a novel gene with ancient functions.

The Drosophila melanogaster G protein-coupled receptor gene, methuselah (mth), has been described as a novel gene that is less than 10 million years old. Nevertheless, it shows a highly specific expression pattern in embryos, larvae, and adults, and has been implicated in larval development, stress resistance, and in the setting of adult lifespan, among others. Although mth belongs to a gene subfamily with 16 members in D. melanogaster, there is no evidence for functional redundancy in this subfamily. Therefore, it is surprising that a novel gene influences so many traits. Here, we explore the alternative hypothesis that mth is an old gene. Under this hypothesis, in species distantly related to D. melanogaster, there should be a gene with features similar to those of mth. By performing detailed phylogenetic, synteny, protein structure, and gene expression analyses we show that the D. virilis GJ12490 gene is the orthologous of mth in species distantly related to D. melanogaster. We also show that, in D. americana (a species of the virilis group of Drosophila), a common amino acid polymorphism at the GJ12490 orthologous gene is significantly associated with developmental time, size, and lifespan differences. Our results imply that GJ12490 orthologous genes are candidates for developmental time and lifespan differences in Drosophila in general.

Drosophila americana as a model species for comparative studies on the molecular basis of phenotypic variation.

Understanding the molecular basis of within and between species phenotypic variation is one of the main goals of Biology. In Drosophila, most of the work regarding this issue has been performed in D. melanogaster, but other distantly related species must also be studied to verify the generality of the findings obtained for this species. Here, we make the case for D. americana, a species of the virilis group of Drosophila that has been diverging from the model species, D. melanogaster, for approximately 40 Myr. To determine the suitability of this species for such studies, polymorphism and recombination estimates are presented for D. americana based on the largest nucleotide sequence polymorphism data set so far analyzed (more than 100 data sets) for this species. The polymorphism estimates are also compared with those obtained from the comparison of the genome assembly of two D. americana strains (H5 and W11) here reported. As an example of the general utility of these resources, we perform a preliminary study on the molecular basis of lifespan differences in D. americana. First, we show that there are lifespan differences between D. americana populations from different regions of the distribution range. Then, we perform five F2 association experiments using markers for 21 candidate genes previously identified in D. melanogaster. Significant associations are found between polymorphism at two genes (hep and Lim3) and lifespan. For the F2 association study involving the two sequenced strains (H5 and W11), we identify amino acid differences at Lim3 and Hep that could be responsible for the observed changes in lifespan. For both genes, no large gene expression differences were observed between the two strains.

Phylogenetic patterns of geographical and ecological diversification in the subgenus Drosophila.

Colonisation of new geographic regions and/or of new ecological resources can result in rapid species diversification into the new ecological niches available. Members of the subgenus Drosophila are distributed across the globe and show a large diversity of ecological niches. Furthermore, taxonomic classification of Drosophila includes the rank radiation, which refers to closely related species groups. Nevertheless, it has never been tested if these taxonomic radiations correspond to evolutionary radiations. Here we present a study of the patterns of diversification of Drosophila to test for increased diversification rates in relation to the geographic and ecological diversification processes. For this, we have estimated and dated a phylogeny of 218 species belonging to the major species groups of the subgenus. The obtained phylogenies are largely consistent with previous studies and indicate that the major groups appeared during the Oligocene/Miocene transition or early Miocene, characterized by a trend of climate warming with brief periods of glaciation. Ancestral reconstruction of geographic ranges and ecological resource use suggest at least two dispersals to the Neotropics from the ancestral Asiatic tropical disribution, and several transitions to specialized ecological resource use (mycophagous and cactophilic). Colonisation of new geographic regions and/or of new ecological resources can result in rapid species diversification into the new ecological niches available. However, diversification analyses show no significant support for adaptive radiations as a result of geographic dispersal or ecological resource shift. Also, cactophily has not resulted in an increase in the diversification rate of the repleta and related groups. It is thus concluded that the taxonomic radiations do not correspond to adaptive radiations.

A comparative study of the short term cold resistance response in distantly related Drosophila species: the role of regucalcin and frost.

The molecular basis of short term cold resistance (indexed as chill-coma recovery time) has been mostly addressed in D. melanogaster, where candidate genes (Dca (also known as smp-30) and Frost (Fst)) have been identified. Nevertheless, in Drosophila, the ability to tolerate short term exposure to low temperatures evolved several times independently. Therefore, it is unclear whether variation in the same candidate genes is also responsible for short term cold resistance in distantly related Drosophila species. It should be noted that Dca is a candidate gene for cold resistance in the Sophophora subgenus only, since there is no orthologous gene copy in the Drosophila subgenus. Here we show that, in D. americana (Drosophila subgenus), there is a north-south gradient for a variant at the 5' non-coding region of regucalcin (a Dca-like gene; in D. melanogaster the proteins encoded by the two genes share 71.9% amino acid identities) but in our D. americana F2 association experiment there is no association between this polymorphism and chill-coma recovery times. Moreover, we found no convincing evidence that this gene is up-regulated after cold shock in both D. americana and D. melanogaster. Size variation in the Fst PEST domain (putatively involved in rapid protein degradation) is observed when comparing distantly related Drosophila species, and is associated with short term cold resistance differences in D. americana. Nevertheless, this effect is likely through body size variation. Moreover, we show that, even at two hours after cold shock, when up-regulation of this gene is maximal in D. melanogaster (about 48 fold expression change), in D. americana this gene is only moderately up-regulated (about 3 fold expression change). Our work thus shows that there are important differences regarding the molecular basis of cold resistance in distantly related Drosophila species.

Resolving the phylogenetic relationships and evolutionary history of the Drosophila virilis group using multilocus data.

The Drosophila virilis group is one of the major lineages of Drosophila previously recognised and it has been used as a model for different types of studies. It comprises 13 species whose phylogenetic relationships are not well resolved. In the present study, six nuclear genes (Adh, fused, Gpdh, NonA, CG9631 and CG7219) and the mitochondrial ribosomal RNA genes (12S-16S) have been used to estimate the evolutionary tree of the group using different methods of phylogenetic reconstruction. Different competing evolutionary hypotheses have also been compared using the Approximately Unbiased test to further evaluate the robustness of the inferred trees. Results are, in general, consistent with previous studies in recovering the four major lineages of the group (D. virilis phylad, Drosophila montana subphylad, Drosophila kanekoi subphylad and Drosophila littoralis subphylad), although D. kanekoi, D. littoralis and Drosophila ezoana are here inferred to be more closely related to the D. virilis phylad than to the D. montana subphylad. The age of the crown group, estimated with a Bayesian method that assumes a relaxed molecular clock, is placed in the late Miocene (∼ 10 Mya). The oldest lineages also appeared during this period (∼ 7.5 to ∼ 8.9 Mya), while the ages of the basal nodes of the montana subphylad and the virilis phylad are located in the early Pliocene (∼ 4.9 and ∼ 4.1 Mya). Major cladogenesis events correlate to geological and palaeoclimatic occurrences that most likely affected the freshwater and deciduous forests where these species are found. The inferred biogeographical history of the group, based on the statistical dispersal-vicariance analysis, indicates that the last common ancestor of the group had a Holarctic distribution from which the North American and the Eurasian lineages evolved as a result of a vicariant event.

Molecular systematics of filarial parasites, with an emphasis on groups of medical and veterinary importance, and its relevance for epidemiology.

Filarial parasites are members of the Phylum Nemata that comprise several species of medical and veterinary importance. Among the human diseases caused by members of this group of nematodes are river blindness and lymphatic filariasis, which afflict millions of people in the tropics. These diseases not only have an impact on the health of the people affected but also bear a great socioeconomic burden. Despite their relevance, the systematics of the filarial parasites is not well understood yet, and additional molecular phylogenetic studies are required to comprehend the evolution of these parasites. Identifying the patterns of evolution of these parasites will be of relevance in preventing emerging zoonoses. The present review examines the information about the molecular systematics of filarial parasites available in the literature and evaluates the relevance of the different directions of future research. Furthermore, it is also intended to highlight the relevance of molecular systematic studies in the molecular epidemiology research area.

An old bilbo-like non-LTR retroelement insertion provides insight into the relationship of species of the virilis group.

In Drosophila, at the population and species level, fixation of a TE insertion is an unlikely fate. Of the few reported fixations at the species level most involve non-LTR retroelements. In this work we report the fixation of a non-LTR retroelement in five species (Drosophila littoralis, Drosophila virilis, Drosophila lummei, Drosophila americana and Drosophila novamexicana) of the virilis group of Drosophila. In most species, this TE insertion is being lost through the accumulation of small deletions, but there is also evidence for the accumulation of large deletions. In the americana lineage an insertion of about 900 bp of the non-LTR retroelement is a marker for the Xc inversion. This insertion is, at most, 80 kb away from the basal Xc inversion breakpoint. The presence of a bilbo-like element in D. littoralis but not in D. kanekoi, suggests that D. littoralis is more closely related to species of the virilis phylad than to species of the montana phylad, which is in contrast with the traditional view. Nevertheless, the phylogenetic analyses here performed using a 7 gene dataset suggest that D. littoralis is indeed more closely related to species of the virilis phylad than to species of the montana phylad. The re-evaluation of the phylogenetic relationship of the species of the virilis group, under the assumption of a relaxed molecular clock, results in an estimated age of the bilbo-like element insertion of at least 7.5 Mya.

Inferring the evolutionary history of Drosophila americana and Drosophila novamexicana using a multilocus approach and the influence of chromosomal rearrangements in single gene analyses.

The evolutionary history of closely related organisms can prove sometimes difficult to infer. Hybridization and incomplete lineage sorting are the main concerns; however, genome rearrangements can also influence the outcome of analyses based on nuclear sequences. In the present study, DNA sequences from 12 nuclear genes, for which the approximate chromosomal locations are known, have been used to estimate the evolutionary history of two forms of Drosophila americana (Drosophila americana americana and Drosophila americana texana) and Drosophila novamexicana (virilis group of species). The phylogenetic analysis of the combined data set resulted in a phylogeny showing reciprocal monophyly for D. novamexicana and D. americana. Single gene analyses, however, resulted in incongruent phylogenies influenced by chromosomal rearrangements. Genetic differentiation estimates indicated a significant differentiation between the two species for all genes. Within D. americana, however, there is no evidence for differentiation between the chromosomal forms except at genes located near the X/4 fusion and Xc inversion breakpoint. Thus, the specific status of D. americana and D. novamexicana is confirmed, but there is no overall evidence for genetic differentiation between D. a. americana and D. a. texana, not supporting a subspecific status. Based on levels of allele and nucleotide diversity found in the strains used, it is proposed that D. americana has had a stable, large population during the recent past while D. novamexicana has speciated from a peripheral southwestern population having had an ancestral small effective population size. The influence of chromosomal rearrangements in single gene analyses is also examined.

A preliminary analysis of the population genetics and molecular phylogenetics of Onchocerca volvulus (Nematoda: Filarioidea) using nuclear ribosomal second internal transcribed spacer sequences.

Nuclear internal transcribed spacer 2 (ITS2) rDNA sequences were used for a molecular phylogenetics analysis of five Onchocerca species. The sister species of the human parasite O. volvulus was found to be the cattle parasite O. ochengi and not O. gibsoni, contrary to chromosomal evidence. The genetic differentiation of two African populations (representing the two African strains) and a Brazilian population of O. volvulus was also studied. Phylogenetic and network reconstruction did not show any clustering of ITS2 alleles on geographic or strain grounds. Furthermore, population genetics tests showed no indication of population differentiation but suggested gene flow among the three populations.

A preliminary analysis of the population genetics and molecular phylogenetics of Onchocerca volvulus (Nematoda: Filarioidea) using nuclear ribosomal second internal transcribed spacer sequences

Nuclear internal transcribed spacer 2 (ITS2) rDNA sequences were used for a molecular phylogenetics analysis of five Onchocerca species. The sister species of the human parasite O. volvulus was found to be the cattle parasite O. ochengi and not O. gibsoni, contrary to chromosomal evidence. The genetic differentiation of two African populations (representing the two African strains) and a Brazilian population of O. volvulus was also studied. Phylogenetic and network reconstruction did not show any clustering of ITS2 alleles on geographic or strain grounds. Furthermore, population genetics tests showed no indication of population differentiation but suggested gene flow among the three populations.

Different positively selected sites at the gametophytic self-incompatibility pistil S-RNase gene in the Solanaceae and Rosaceae (Prunus, Pyrus, and Malus).

In this work we perform a comparative study on the location of positively selected sites (those likely responsible for defining specificity differences) at the S-RNase gene, the pistil component of the gametophytic self-incompatibility system. For Plantaginaceae and Rosaceae (Prunus and Pyrus/Malus) this is the first study of this kind. A clear sign of positive selection was observed for 13, 17, and 27 amino acid sites in Solanaceae, Prunus, and Pyrus/Malus, respectively, using two different methodologies. In Plantaginaceae no clear positively selected sites were identified. Possible reasons for this result are discussed. Indirect experimental evidence suggests that the identified positively selected amino acid sites play a role in specificity determination. The percentage of positively selected sites is similar in Solanaceae and Rosaceae but the location of those sites is different.

Comparative polytene chromosome maps of D. montana and D. virilis.

Chromosomal inversion polymorphism was characterized in Finnish Drosophila montana populations. A total of 14 polymorphic inversions were observed in Finnish D. montana of which nine had not been described before. The number of polymorphic inversions in each chromosome was not significantly different from that expected, assuming equal chance of occurrence in the euchromatic genome. There was, however, no correlation between the number of polymorphic inversions and that of fixed inversions in each chromosome. Therefore, a simple neutral model does not explain the evolutionary dynamics of inversions. Furthermore, in contrast to results obtained by others, no significant correlation was found between the two transposable elements (TEs) Penelope and Ulysses and inversion breakpoints in D. montana. This result suggests that these TEs were not involved in the creation of the polymorphic inversions seen in D. montana. A comparative analysis of D. montana and Drosophila virilis polytene chromosomes 4 and 5 was performed with D. virilis bacteriophage P1 clones, thus completing the comparative studies of the two species.

The evolutionary history of the transposable element Penelope in the Drosophila virilis group of species.

We have used phylogenetic techniques to study the evolutionary history of the Penelope transposable element in the Drosophila virilis species group. Two divergent types of Penelope have been detected, one previously described, clade I, and a new one which we have termed clade III. The phylogeny of some copies of the Penelope clade I element was partially consistent with the species phylogeny of the D. montana subphylad, suggesting cospeciation and allowing the estimation of the evolutionary rate of Penelope. Divergence times of elements found in different species are younger than the age of the species, suggesting horizontal transfer events.