Transposon-mediated insertional mutagenesis unmasks recessive insecticide resistance in the aphid Myzus persicae.

The evolution of resistance to insecticides threatens the sustainable control of many of the world's most damaging insect crop pests and disease vectors. To effectively combat resistance, it is important to understand its underlying genetic architecture, including the type and number of genetic variants affecting resistance and their interactions with each other and the environment. While significant progress has been made in characterizing the individual genes or mutations leading to resistance, our understanding of how genetic variants interact to influence its phenotypic expression remains poor. Here, we uncover a mechanism of insecticide resistance resulting from transposon-mediated insertional mutagenesis of a genetically dominant but insecticide-susceptible allele that enables the adaptive potential of a previously unavailable recessive resistance allele to be unlocked. Specifically, we identify clones of the aphid pest Myzus persicae that carry a resistant allele of the essential voltage-gated sodium channel (VGSC) gene with the recessive M918T and L1014F resistance mutations, in combination with an allele lacking these mutations but carrying a Mutator-like element transposon insertion that disrupts the coding sequence of the VGSC. This results in the down-regulation of the dominant susceptible allele and monoallelic expression of the recessive resistant allele, rendering the clones resistant to the insecticide bifenthrin. These findings are a powerful example of how transposable elements can provide a source of evolutionary potential that can be revealed by environmental and genetic perturbation, with applied implications for the control of highly damaging insect pests.

Holocentric chromosomes.

Holocentric chromosomes possess multiple kinetochores along their length rather than the single centromere typical of other chromosomes [1]. They have been described for the first time in cytogenetic experiments dating from 1935 and, since this first observation, the term holocentric chromosome has referred to chromosomes that: i. lack the primary constriction corresponding to centromere observed in monocentric chromosomes [2]; ii. possess multiple kinetochores dispersed along the chromosomal axis so that microtubules bind to chromosomes along their entire length and move broadside to the pole from the metaphase plate [3]. These chromosomes are also termed holokinetic, because, during cell division, chromatids move apart in parallel and do not form the classical V-shaped figures typical of monocentric chromosomes [4-6]. Holocentric chromosomes evolved several times during both animal and plant evolution and are currently reported in about eight hundred diverse species, including plants, insects, arachnids and nematodes [7,8]. As a consequence of their diffuse kinetochores, holocentric chromosomes may stabilize chromosomal fragments favouring karyotype rearrangements [9,10]. However, holocentric chromosome may also present limitations to crossing over causing a restriction of the number of chiasma in bivalents [11] and may cause a restructuring of meiotic divisions resulting in an inverted meiosis [12].

Comparative Gene Mapping as a Tool to Understand the Evolution of Pest Crop Insect Chromosomes.

The extent of the conservation of synteny and gene order in aphids has been previously investigated only by comparing a small subset of linkage groups between the pea aphid Acyrthosiphon pisum and a few other aphid species. Here we compared the localization of eight A. pisum scaffolds (covering more than 5 Mb and 83 genes) in respect to the Drosophila melanogaster Muller elements identifying orthologous loci spanning all the four A. pisum chromosomes. Comparison of the genetic maps revealed a conserved synteny across different loci suggesting that the study of the fruit fly Muller elements could favour the identification of chromosomal markers useful for the study of chromosomal rearrangements in aphids. A. pisum is the first aphid species to have its genome sequenced and the finding that there are several chromosomal regions in synteny between Diptera and Hemiptera indicates that the genomic tools developed in A. pisum will be broadly useful not only for the study of other aphids but also for other insect species.

Genomic and Cytogenetic Localization of the Carotenoid Genes in the Aphid Genome.

Data published in the scientific literature suggests a possible link between chromosomal rearrangements involving autosomes 1 and 3 and the presence of red morphs in the peach-potato aphid Myzus persicae (Sulzer). In order to begin a study of this relationship, we analysed the genomic and chromosomal location of genes involved in carotenoid biosynthesis in M. persicae and the pea aphid, Acyrthosiphon pisum (Harris), since carotenoids are the basis of the colour in many aphid species. Genomic analysis identified a DNA sequence containing carotenoid genes in synteny between the 2 species. According to the results obtained using in situ PCR, carotenoid genes were located in a subterminal portion of autosome 1 in both species. The same localization has also been observed in the onion aphid Neotoxoptera formosana Takahashi that, as M. persicae and A. pisum, belongs to the tribe Macrosiphini, thereby suggesting a synteny of this chromosomal region in aphids. In situ PCR experiments performed on 2 M. persicae asexual lineages bearing heterozygous translocations involving autosomes 1 and 3 revealed that carotenoid genes were located within chromosomal portions involved in recurrent rearrangements. We also verified by bioinformatics analyses the presence of fragile sites that could explain these recurrent rearrangements in M. persicae.

Occurrence of Rabl-like telomere clustering in the holocentric chromosomes of the peach potato aphid Myzus persicae (Hemiptera; Aphididae).

Several studies demonstrated that chromosome anchoring to nuclear structures is involved in the organization of the interphase nucleus. The Rabl configuration, a well-studied chromosome organization in the interphase nucleus, has been deeply studied in organisms with monocentric chromosomes but just slightly touched in species with holocentric chromosomes. In the present paper, by means of the isolation and chromosomal mapping of the C0t DNA fraction and chromatin immunoprecipitation with anti-LEM-2 antibodies, we evidenced the presence of few foci where telomeres and subtelomeric regions cluster in the aphid interphase nuclei, suggesting the occurrence of a Rabl-like chromosome configuration. The same experimental approaches also evidenced that most of the repetitive DNA of the 2 X chromosomes is located at the periphery of the nucleus, whereas the ribosomal genes, located at 1 telomere of each X chromosome, are present towards the inner portion of the nucleus, favoring their transcriptional activity.

Characterization of non-LTR retrotransposable TRAS elements in the aphids Acyrthosiphon pisum and Myzus persicae (Aphididae, Hemiptera).

A non-LTR TRAS retrotransposon (identified as TRASAp1) has been amplified in the pea aphid Acyrthosiphon pisum and its presence has been assessed also in the peach potato aphid Myzus persicae. This TRAS element possesses 2 overlapping ORFs (a gag-ORF1 and a pol-ORF2 containing the reverse transcriptase and the endonuclease domains) that show a similarity ranging from 40% to 48% to proteins coded by other TRAS elements identified in insects (including the beetle Tribolium castaneum and the moth Bombyx mori). The study of the TRAS chromosomal insertion sites, performed by standard fluorescent in situ hybridization (FISH) and fiber FISH, showed that TRAS elements were located in a subtelomeric position, just before the telomeric (TTAGG) n repeats. In both the aphid species, TRAS elements were present at all termini of autosomes, but the 2 X chromosome telomeres show a clear-cut structural difference. Indeed, cromomycin A3 staining, together with FISH using a TRAS probe, revealed that TRAS signals only occur at the telomere opposite to the NOR-bearing one. Lastly, the analysis of the distribution of TRAS retrotransposons in a M. persicae strain possessing spontaneous fragmentations of the X chromosomes assessed that TRAS elements were not involved in the healing of de novo telomeres.

A1-3 chromosomal translocations in Italian populations of the peach potato aphid Myzus persicae (Sulzer) not linked to esterase-based insecticide resistance.

Esterase-based resistance in the peach-potato aphid, Myzus persicae (Sulzer), is generally due to one of two alternative amplified carboxylesterase genes, E4 or FE4 (fast E4). The E4 amplified form is distributed worldwide and it is correlated with a particular translocation between autosomes 1 and 3, whereas the FE4 form, which has hitherto not been found to be associated with chromosomal rearrangements, is typical of the Mediterranean regions. In this study, we present for the first time cytogenetic and molecular data on some M. persicae parthenogenetic lineages, which clearly show a chromosomal A1-3 translocation associated with esterase FE4 genes and unrelated to high levels of esterase-based resistance.

Continuous occurrence of intra-individual chromosome rearrangements in the peach potato aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae).

Analysis of the holocentric mitotic chromosomes of the peach-potato aphid, Myzus persicae (Sulzer), from clones labelled 50, 51 and 70 revealed different chromosome numbers, ranging from 12 to 14, even within each embryo, in contrast to the standard karyotype of this species (2n = 12). Chromosome length measurements, combined with fluorescent in situ hybridization experiments, showed that the observed chromosomal mosaicisms are due to recurrent fragmentations of chromosomes X, 1 and 3. Contrary to what has generally been reported in the literature, X chromosomes were frequently involved in recurrent fragmentations, in particular at their telomeric ends opposite to the nucleolar organizer region. Supernumerary B chromosomes have been also observed in clones 50 and 51. The three aphid clones showed recurrent fissions of the same chromosomes in the same regions, thereby suggesting that the M. persicae genome has fragile sites that are at the basis of the observed changes in chromosome number. Experiments to induce males also revealed that M. persicae clones 50, 51 and 70 are obligately parthenogenetic, arguing that the reproduction by apomictic parthenogenesis favoured the stabilization and inheritance of the observed chromosomal fragments.

Presence of a functional (TTAGG)(n) telomere-telomerase system in aphids.

The structure of the telomeres of four aphid species (Acyrthosiphon pisum, Megoura viciae, Myzus persicae and Rhopalosiphum padi) was evaluated by Southern blotting and fluorescent in situ hybridization, revealing that each chromosomal end consists of a (TTAGG)(n) repeat. The presence of a telomerase coding gene has been verified successively in the A. pisum genome, revealing that aphid telomerase shares sequence identity ranging from 12% to 18% with invertebrate and vertebrate homologues, and possesses the two main domains involved in telomerase activity. Interestingly, telomerase expression has been verified in different somatic tissues suggesting that, in aphids, telomerase activity is not as restricted as in human cells. The study of telomeres in a M. persicae strain with a variable chromosome number showed that aphid telomerase can initiate the de novo synthesis of telomere sequences at internal breakpoints, resulting in the stabilization of chromosomal fragments.

Unlocking holocentric chromosomes: new perspectives from comparative and functional genomics?

The presence of chromosomes with diffuse centromeres (holocentric chromosomes) has been reported in several taxa since more than fifty years, but a full understanding of their origin is still lacking. Comparative and functional genomics are nowadays furnishing new data to better understand holocentric chromosome evolution thus opening new perspectives to analyse karyotype rearrangements in species with holocentric chromosomes in particular evidencing unusual common features, such as the uniform GC content and gene distribution along chromosomes.

The foraging Gene Is Involved in the Presence of Wings and Explorative Behaviours in Parthenogenetic Females of the Aphid Myzus persicae.

The foraging gene (for) encodes for a cyclic guanosine monophosphate (cGMP)-dependent protein kinase involved in behavioural plasticity in aphids and in other insects. In this paper, we analysed the complete for sequence in eight clones of the peach potato aphid Myzus persicae, reporting the presence of nonsense and frameshift mutations in three studied clones characterized by a reduced number of winged females and by the absence of exploratory behaviours. Quantitative PCR experiments evidenced similar results in clones possessing for genes with a conserved coding sequence, but low expression levels. The comparison of the for transcriptional level in Myzus persicae persicae and Myzus persicae nicotianae showed very different expression in the two studied M. p. nicotianae clones so that our data did not support a previous hypothesis suggesting that a differential for expression was related to ecological specialization of M. p. nicotianae. In view of its role in both the dispersal of winged females and exploratory behaviours, the screening of the for sequences could be useful for predicting invasions of cultivated areas by peach potato aphids.

Distribution and molecular composition of heterochromatin in the holocentric chromosomes of the aphid Rhopalosiphum padi (Hemiptera: Aphididae).

In order to study the structure of holocentric chromosomes in aphids, the localization and the composition of Rhopalosiphum padi heterochromatin and rDNA genes have been evaluated at cytogenetic and molecular level. In particular, heterochromatin resulted located on all the chromosomes both in intercalary and telomeric positions. Moreover, enzymatic digestion of R. padi genome put in evidence a DraI satellite DNA which has been isolated, cloned and sequenced. FISH experiments showed that this satellite DNA clusters in an intercalary C-positive band on the two X chromosomes.

Evolutionary insights into the aphid genome: Aphid genomics between quality problems and intriguing perspectives.

In the last decade the genomes of several aphid species have been sequenced allowing a better understanding of their biology and evolution. Unfortunately, as frequently occurs with the next generation sequencing technologies, several aphid genomes consist in fragmented assemblies that contain thousands of genomic scaffolds of reduced length. In order to improve the quality of the published genomic data, several research groups are currently resequencing aphid DNA making possible to take the full advantage of genomics to face complex biological problems, such as aphid diversification. This review is aimed to discuss the current state of art in aphid genomics focusing in particular on the aspects that could improve our knowledge of their evolution.

The Evolutionary History and Functional Divergence of Trehalase (treh) Genes in Insects.

Trehalases (treh) have been found in different organisms, such as bacteria, fungi, yeast, nematodes, insects, vertebrates, and plants. Their biochemical properties are extremely variable and not yet fully understood. Gene expression patterns have shown differences among insect species suggesting a potential functional diversification of trehalase enzymes during their evolution. A second gene family encoding for enzymes with hypothetical trehalase activity has been repeatedly annotated in insect genome as acid trehalases/acid trehalase-like (ath), but its functional role is still not clear. The currently available large amount of genomic data from many insect species may enable a better understanding of the evolutionary history, phylogenetic relationships and possible roles of trehalase encoding genes in this taxon. The aim of the present study is to infer the evolutionary history of trehalases and acid trehalase genes in insects and analyze the trehalase functional divergence during their evolution, combining phylogenetic and genomic synteny/colinearity analyses.

Comparative Analysis of Intra- and Inter-Specific Genomic Variability in the Peach Potato Aphid, Myzus persicae.

The availability of genomic data in the last decade relating to different aphid species has allowed the analysis of the genomic variability occurring among such species, whereas intra-specific variability has hitherto very largely been neglected. In order to analyse the intra-genomic variability in the peach potato aphid, Myzus persicae, comparative analyses were performed revealing several clone-specific gene duplications, together with numerous deletions/rearrangements. Our comparative approach also allowed us to evaluate the synteny existing between the two M. persicae clones tested and between the peach potato aphid and the pea aphid, Acyrthosiphon pisum. Even if part of the observed rearrangements are related to a low quality of some assembled contigs and/or to the high number of contigs present in these aphid genomes, our evidence reveals that aphid clones are genetically more different than expected. These results suggest that the choice of performing genomes sequencing combining different biotypes/populations, as revealed in the case of the soybean aphid, Aphis glycines, is unlikely to be very informative in aphids. Interestingly, it is possible that the holocentric nature of aphid chromosomes favours genome rearrangements that can be successively inherited transgenerationally via the aphid's apomictic (parthenogenetic) mode of reproduction. Lastly, we evaluated the structure of the cluster of genes coding for the five histones (H1, H2A, H2B, H3 and H4) in order to better understand the quality of the two M. persicae genomes and thereby to improve our knowledge of this functionally important gene family.

Analysis of the extent of synteny and conservation in the gene order in aphids: A first glimpse from the Aphis glycines genome.

In the last decade several insect genomes have been sequenced, but for most the chromosomal mapping of the identified scaffolds/annotated genes is not available. The lack of this information makes it difficult to analyse various genetic aspects, including the presence of genome rearrangements and the extent of synteny within and across species. We mapped five multigenic DNA families (major and minor rDNAs, histone gene cluster, esterases and carotenoid desaturases) and seven scaffolds corresponding to 9 Mb of the soybean aphid, Aphis glycines, genome and identified loci spanning the four soybean aphid chromosomes. A comparative analysis of the localization of the annotated A. glycines genes with respect to the peach potato aphid, Myzus persicae, and the fly, Drosophila melanogaster, evidenced a lower degree of synteny between the two aphid species than in the aphid-fly comparison. Only 1.4 genes per syntenic block were observed in aphids in contrast to 2.3 genes per block in flies. This higher chromosomal rearrangement rate in aphids could be explained considering that they possess holocentric chromosomes that can favour the stabilization and inheritance of chromosomal rearrangements. Lastly, our experiments did not detect the presence of chimeric assemblies in the newly available A. glycines biotype 1 genome, differently from what reported in assembled genome of other aphid species, suggesting that chromosomal mapping can be used to ascertain the quality of assembled genomes.

Fertility and markers of male reproductive function in Inuit and European populations spanning large contrasts in blood levels of persistent organochlorines.

OBJECTIVE: We synthesized the main findings from an international epidemiologic study on the impact of biopersistent organic pollutants (POPs) on human reproductive function. DATA SOURCES AND EXTRACTION: We used a database with interview and biological data from 2,269 women and their spouses, and 18 published core papers. DATA SYNTHESIS: The study did not provide direct evidence of hormone-like activity of the polychlorinated biphenyl (PCB) congener CB-153 and the main dichlorodiphenyltrichloroethane (DDT) metabolite, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE), as serum concentrations of these compounds were not consistently related to either endogenous or exogenous hormone activity in serum. Nevertheless several links bewteen POP exposure and biomarkers of male reproductive function were identified. First, an association between high CB-153 serum levels and low sperm counts was detected within a subgroup of men with short androgen receptor CAG repeat length. Second, a relationship between increased CB-153 serum concentrations and decreased sperm motility was seen in all four studied regions, and indications of reduced neutral alpha-glucosidase activity in seminal plasma point to a post-testicular effect. Third, damage of sperm chromatin integrity was considerably less frequent in Greenlandic Inuits compared with that in European groups, and only in the latter was impairment of sperm chromatin integrity related to POPs. Despite these effects, fertility in terms of time taken to conceive was not related to POPs except in Inuits. A likely explanation of the latter was not identified. CONCLUSIONS: POPs may interfere with male reproductive function without major impact on fertility. The data do not provide direct evidence for endocrine disruption, hence other mechanisms should also be considered.

Xenobiotic activity in serum and sperm chromatin integrity in European and inuit populations.

Lipophilic persistent organic pollutants (POPs) are ubiquitous in the environment and suspected to interfere with hormone activities and reproduction. In previous studies we demonstrated that POP exposure can affect sperm DNA integrity and differences between Inuits and Europeans in sperm DNA integrity and xenobiotic activity were observed. The aim of this study was to investigate possible relations between human sperm chromatin integrity and the xenobiotic serum activity of lipophilic POPs assessed as effects on the estrogen (ER), androgen (AR), and/or aryl hydrocarbon (AhR) receptors. Human sperm chromatin integrity was assessed as DNA fragmentation index (%DFI) and high DNA stainability (%HDS) using the flow cytometric sperm chromatin structure assay (SCSA). Xenobiotic receptor activities were determined using chemically activated luciferase gene expression (CALUX) assay. The study included 53 Greenlandic Inuits and 247 Europeans (Sweden, Warsaw (Poland) and Kharkiv (Ukraine)). A heterogeneous pattern of correlations was found. For Inuits, ER and AhR activities and %DFI were inversely correlated, whereas a positive correlation between AR activity and %DFI was found for Europeans. In contrast, no correlation between receptor activities and %HDS was observed for Inuits but for Europeans positive and negative correlations were observed between ER and AR activities and %HDS, respectively. We suggest that the different patterns of xenobiotic serum activities, in combination with diet associated factors and/or genetics, might be connected to the observed differences in sperm chromatin integrity between the Inuits and Europeans.

Xenoandrogenic activity in serum differs across European and Inuit populations.

BACKGROUND: Animal and in vitro studies have indicated that human male reproductive disorders can arise as a result of disrupted androgen receptor (AR) signalling by persistent organic pollutants (POPs). Our aim in the present study was to compare serum xenoandrogenic activity between study groups with different POP exposures and to evaluate correlations to the POP proxy markers 2,2',4,4',5,5'-hexachlorobiphenyl (CB-153) and 1,1-dichloro-2,2-bis(p-chlorophenyl)-ethylene (p,p'-DDE). METHODS: We determined xenoandrogenic activity in the serum fraction containing the lipophilic POPs but free of endogenous hormones. Adult male serum (n = 261) from Greenland, Sweden, Warsaw (Poland), and Kharkiv (Ukraine) was analyzed. Xenoandrogenic activity was determined as the effect of serum extract alone (XAR) and in the presence of the synthetic AR agonist R1881 (XARcomp) on AR transactivated luciferase activity. RESULTS: The study groups differed significantly with respect to XARcomp activity, which was increased in the Inuits and decreased in the European study groups; we observed no difference for XAR activity. We found the highest level of the AR antagonist p,p'-DDE in Kharkiv, and accordingly, this study group showed the highest percent of serum samples with decreased XARcomp activities. Furthermore, the percentage of serum samples with decreased XARcomp activities followed the p,p'-DDE serum level for the European study groups. No correlations between serum XAR or XARcomp activities and the two POP markers were revealed. CONCLUSIONS: The differences in XARcomp serum activity between the study groups suggest differences in chemical exposure profiles, genetics, and/or lifestyle factors.

Reproductive hormone levels in men exposed to persistent organohalogen pollutants: a study of inuit and three European cohorts.

OBJECTIVE: Persistent organohalogen pollutant (POP) exposure may have a negative impact on reproductive function. The objective of this study was to assess the impact of POP exposure on the male hypothalamo-pituitary-gonadal axis. PARTICIPANTS: Participants included 184 Swedish fishermen and spouses of pregnant women from Greenland (n = 258), Warsaw, Poland (n = 113) , and Kharkiv, Ukraine (n = 194). EVALUATIONS/MEASUREMENTS: Serum levels of 2,2,4,4,5,5-hexachlorobiphenyl (CB-153) and dichlorodiphenyl dichloroethene (p,p -DDE) were determined in the four populations, showing different exposure patterns: Swedish fishermen, high CB-153/low p,p -DDE; Greenland, high CB-153/high p,p -DDE; Warsaw, low CB-153/moderate p,p -DDE; Kharkiv, low CB-153/high p,p -DDE. Serum was also analyzed for testosterone, estradiol, sex hormone-binding globulin (SHBG) , inhibin B, luteinizing hormone (LH) , and follicle-stimulating hormone (FSH) . Free testosterone levels were calculated based on testosterone and SHBG. RESULTS: We found significant center-to-center variations in the associations between exposure and the outcomes. The most pronounced effects were observed in Kharkiv, where statistically significant positive associations were found between the levels of both CB-153 and p,p -DDE and SHBG, as well as LH. In Greenland, there was a positive association between CB-153 exposure and LH. In the pooled data set from all four centers, there was positive association between p,p -DDE and FSH levels [beta = 1.1 IU/L; 95% confidence interval (CI), 1.0-1.1 IU/L]. The association between CB-153 levels and SHBG was of borderline statistical significance (beta = 0.90 nmol/L; 95% CI, -0.04 to 1.9 nmol/L). CONCLUSIONS: Gonadotropin levels and SHBG seem to be affected by POP exposure, but the pattern of endocrine response is the subject of considerable geographic variation.