Genomic exaptation enables <Emphasis Type="Italic">Lasius niger</Emphasis> adaptation to urban environments

Background

The world is rapidly urbanizing, and only a subset of species are able to succeed in stressful city environments. Efficient genome-enabled stress response appears to be a likely prerequisite for urban adaptation. Despite the important role ants play in the ecosytem, only the genomes of ~13 have been sequenced so far. Here, we present the draft genome assembly of the black garden ant Lasius niger – the most successful urban inhabitant of all ants – and we compare it with the genomes of other ant species, including the closely related Camponotus floridanus.

Results

Sequences from 272 M Illumina reads were assembled into 41,406 contigs with total length of 245 MB, and N50 of 16,382 bp, similar to other ant genome assemblies enabling comparative genomic analysis. Remarkably, the predicted proteome of L. niger is significantly enriched relative to other ant genomes in terms of abundance of domains involved in nucleic acid binding, DNA repair, and nucleotidyl transferase activity, reflecting transposable element proliferation and a likely genomic response. With respect to environmental stress, we note a proliferation of various detoxification genes, including glutatione-S-transferases and those in the cytochrome P450 families. Notably, the CYP9 family is highly expanded with 19 complete and 21 nearly complete members - over twice as many compared to other ants. This family exhibits the signatures of strong directional selection, with eleven positively selected positions in ligand-binding pockets of enzymes. Gene family contraction was detected for several components of the olfactory system, accompanied by instances of both directional selection and relaxation.

Conclusions

Our results suggest that the success of L. niger in urbanized areas may be the result of fortuitous coincidence of several factors, including the expansion of the CYP9 cytochrome family due to coevolution with parasitic fungi, the diversification of DNA repair systems as an answer to proliferation of retroelements, and the reduction of olfactory system and behavioral preadaptations from non-territorial subdominant life strategies found in natural environments. Diversification of cytochromes and DNA repair systems along with reduced odorant communication are the basis of L. niger pollutant resistance and polyphagy, while non-territorial and mobilization strategies allows more efficient exploitation of large but patchy food sources.

     

On the biology of anthocorid bugs (Heteroptera,Anthocoridae), predators of grass aphids in the forest-steppe zone of West Siberia

Four species of the heteropteran family Anthocoridae, Orius niger Wolff., O. majusculus Reut., O. minutus L., and Anthocoris confusus Reut., feeding on grass aphids on grains and bird cherry occur in the foreststeppe zone of Western Siberia. Orius niger and O. majusculus possess the greatest population numbers, with 98% of all the bugs collected in 1987 and 1988 belonging to these species. In 1998–2000, O. niger was predominant (97–100%). Data on the bionomics, seasonal abundance, feeding intensity, and role of anthocorids in the control of aphids are reported. Density of Orius bugs on wheat has been shown to be independent of the preceding culture. Permanent wheat is a preferable habitat of the predatory anthocorids. Mass reproduction of Orius bugs on pea-oat and alfalfa has been recorded.     

Regulating the regulators: responses of four plant growth regulators during clonal propagation of <Emphasis Type="Italic">Lachenalia montana</Emphasis>

Geraniol, the precursor of terpenoid indole alkaloids can be converted to the 10-hydroxy geraniol by the function of geraniol 10-hydroxylase. In our study, for the first time, a full-length cytochrome P450 monooxygenase (P450) geraniol 10-hydroxylase (PgCYP76C9) cDNA was isolated and characterized from Panax ginseng Meyer. The gene has an open reading frame (ORF) of 1503 base pairs and encodes a precursor protein of 501 amino acids residues. The calculated molecular mass of the protein is approximately 56.3 kDa with a predicated isoelectric point of 8.45. Amino acid identities between PgG10H and other P450s of the CYP76 family in the database had revealed that the deduced amino acid of PgG10H sharing a higher sequence homology with geraniol 10-hydroxylase-like proteins encoded by Cinchona calisaya and Lonicera japonica. We implemented a molecular modeling method to evaluate the possible interaction of geraniol with PgG10H active site. Our finding showed that the geraniol was the potential ligand for PgG10H in P. ginseng. Expression of PgG10H gene was tissue-regulated and showed high expression in 3-year-old ginseng flowers and roots. Expression of PgG10H was differentially induced in ginseng, not only during Pseudomonas syringae infection and wounding but also after exposure to methyl jasmonate and salt stress. Furthermore, overexpression of the newly identified ginseng geraniol 10-hydroxylase P450 gene in Arabidopsis caused terpenoid indole alkaloid dihydrositsirikine production and also conferred enhanced resistance to P. syringae.     

Genomic Signatures of Selection between Urban and Rural Populations of Black Garden Ant <Emphasis Type="Italic">Lasius niger</Emphasis>

Black garden ant L. niger is known as a dominant species of urban ant fauna in Europe. Successful propagation of L. niger in transformed ecosystems was associated with behavioral traits in many surveys; however, molecular and genetic basis of such adaptation was never studied. In the present study, genomes of populations from the city of Moscow and natural habitats of Moscow oblast are compared. Pooled samples from rural and urban habitats are collected and sequenced with Illumina HiSeq. SNP frequency, Tajima’s D, and fixation index are estimated with PoPoolation and Popoolation2 software. SNP frequencies are significantly different in 64 genes according to Fisher’s exact test. Some of these genes are detected as affected by recent selection. Out of 64 genes, 26 encode retrotransposon proteins. The genes of immune response to viral and fungal infections, fatty acid synthases, and elements of the Hippo/Fat pathway have different SNP frequencies between populations. Certain retrotransposon genes also can be under selection. It is important to note that repressors and mediators of RNA polymerase II are significantly different between urban and rural populations of L. niger. These genes can influence retrotransposon mobility.     

Typification of urban habitats,with ant assemblages of Moscow city taken as an example

A typification of Moscow city habitats is undertaken, based on their consideration as mosaic of patches and using such fundamental parameters as habitat origin (soil type), floristic composition, vegetation structure, and area of the biotopes. Altogether, 11 habitat types are distinguished: lawns, agrocenoses, xerophytic and mesophytic meadows, tall weeds, boulevards, small degenerative parks, small oppressed artificial parks, landscape parks, forest parks, and technocenoses. Such a classification is primarily useful for studying ants. The present paper describes the basic structure of ant assemblages in most types of urban biocenoses. The main pool of Moscow’s ant species ranked by their occurrence is as follows: Lasius niger (87%), Myrmica rugulosa (44%), Myrmica rubra (33%), Formica cunicularia (11%), Myrmica ruginodis (10%), etc. Leaf litter removal with a rake was shown to negatively affect the numbers, biomass, and species diversity of ant communities in urban areas with trees. The most stable two-species ant community revealed in Moscow City, termed an “elementary urban community,” consists of L. niger and M. rugulosa, with the former always outnumbering the latter.     

A key enzyme of animal steroidogenesis can function in plants enhancing their immunity and accelerating the processes of growth and development

Background

The initial stage of the biosynthesis of steroid hormones in animals occurs in the mitochondria of steroidogenic tissues, where cytochrome P450_SCC (CYP11A1) encoded by the CYP11A1 gene catalyzes the conversion of cholesterol into pregnenolone – the general precursor of all the steroid hormones, starting with progesterone. This stage is missing in plants where mitochondrial cytochromes P450 (the mito CYP clan) have not been found. Generating transgenic plants with a mitochondrial type P450 from animals would offer an interesting option to verify whether plant mitochondria could serve as another site of P450 monooxygenase reaction for the steroid hormones biosynthesis.

Results

For a more detailed comparison of steroidogenic systems of Plantae and Animalia, we have created and studied transgenic tobacco and tomato plants efficiently expressing mammalian CYP11A1 cDNA. The detailed phenotypic characterization of plants obtained has shown that through four generations studied, the transgenic tobacco plants have reduced a period of vegetative development (early flowering and maturation of bolls), enlarged biomass and increased productivity (quantity and quality of seeds) as compared to the only empty-vector containing or wild type plants. Moreover, the CYP11A1 transgenic plants show resistance to such fungal pathogen as Botrytis cinerea. Similar valuable phenotypes (the accelerated course of ontogenesis and/or stress resistance) are also visible in two clearly distinct transgenic tomato lines expressing CYP11A1 cDNA: one line (No. 4) has an accelerated rate of vegetative development, while the other (No. 7) has enhanced immunity to abiotic and biotic stresses. The progesterone level in transgenic tobacco and tomato leaves is 3–5 times higher than in the control plants of the wild type.

Conclusions

For the first time, we could show the compatibility in vivo of even the most specific components of the systems of biosynthesis of steroid hormones in Plantae and Animalia. The hypothesis is proposed and substantiated that the formation of the above-noted special phenotypes of transgenic plants expressing mammalian CYP11A1 cDNA is due to the increased biosynthesis of progesterone that can be considered as a very ancient bioregulator of plant cells and the first real hormone common to plants and animals.

     

Mass Occurrence and Dominant Behavior of the European Ant Species <Emphasis Type="Italic">Formica fuscocinerea</Emphasis> (Forel)

Recently, masses of the ant Formica (Serviformica) fuscocinerea (Forel) have been occurring at numerous sites in Southern Germany. Although F. fuscocinerea is native to Southern Germany, these mass occurrences resemble ant invasions in density and dominance. This study aimed to investigate the underlying mechanisms that promote sudden mass occurrence of a previously inconspicuous ant species within its native range. To estimate the competitive dominance of F. fuscocinerea, species occurrence and abundance considering biotic and abiotic parameters were studied in a natural habitat where F. fuscocinerea co-occurred with two other common ant species, Myrmica ruginodis (Nylander) and Lasius niger (Linnaeus). To understand the species’ distribution in the field, laboratory experiments on interspecific competition were conducted. Finally, the colony structure of F. fuscocinerea was investigated with intraspecific aggression tests. Formica fuscocinerea dominated an area that, as indicated by strongly frequented foraging trails on the trees, provided important food sources, e.g. trophobionts, to the ants. Other ant species coexisted only at the periphery of the F. fuscocinerea range. Laboratory experiments revealed F. fuscocinerea as highly dominant species. Additionally, F. fuscocinerea showed a complete lack of intraspecific aggression between ants originating from distances up to 58 km, indicating weak or nonexistent behavioral boundaries among ants of physically separated nests. Since extraordinarily high worker densities, strong interspecific dominance and a lack of colony boundaries within supercolonies are considered to be important traits of several invasive ant species we conclude that the same traits also promote the dominance of F. fuscocinerea.     

Isolation a P450 gene in <Emphasis Type="Italic">Pinus armandi</Emphasis> and its expression after inoculation of <Emphasis Type="Italic">Leptographium qinlingensis</Emphasis> and treatment with methyl jasmonate

In conifers, the cytochrome P450 monooxygenase of the CYP720B family plays an important role in the synthesis of diterpene resin acids, which are specialised metabolites of the oleoresin defence. We isolated CYP720B19 from Pinus armandi and found that it belongs to the CYP720B family. The CYP720B19 open reading frame of 1467 bp encodes a protein of 488 amino acid residues with high similarity to abietadienol/abietadienal oxidase. Differential expression of the CYP720B19 gene significantly changed when plants were inoculated with the fungus Leptographium qinlingensis, mechanically wounded, or treated with methyl jasmonate (MeJa) and Tween at 4 days and 8 days. Increased expression of the CYP720B19 gene after these treatments suggested that the gene was involved in pine defence against inoculation by L. qinlingensis, MeJa treatment and mechanical injury.     

Effect of colchicine-induced polyploidy on morphological characteristics and essential oil composition of ajowan (<Emphasis Type="Italic">Trachyspermum ammi</Emphasis> L.)

Cyclophilins (CYPs) belong to the immunophilin superfamily, having the peptidyl prolyl cis/trans isomerase activity that can catalyze the cis/trans isomerisation process of proline residues. Previous studies have shown their importance in plants, but no comprehensive analysis of maize CYP family has been reported. In the present study, a whole-genome-wide analysis of maize CYP family was performed and 39 ZmCYP genes (ZmCYP1 to ZmCYP39) were identified from maize genome, which were unequally distributed on maize ten chromosomes. Phylogenetic analysis revealed a weak relationship among these ZmCYP genes. Furthermore, their gene structure and motif patterns also displayed variant within the gene family. Four segmental and one tandem duplicated gene pairs were found from 39 ZmCYP genes, respectively, indicating their roles in the expansion of maize CYP family. Expression analysis of 39 ZmCYP genes in maize tissues showed their differential tissue specific expression patterns. Quantitative real-time PCR analysis of 19 selected ZmCYP genes under salinity stress indicated their stress-inducible expression profile. Heterologous expression of ZmCYP15 in E. coli enhanced tolerance against abiotic stress. Subcellular localization analysis indicated ZmCYP15 was located in nucleus and cytoplasm. Our study describes the importance of the maize CYP gene family in stress response, and provides a reference for future study and application for maize genetic improvement.     

Phenotypic and phylogenetic characterization of actinomycetes isolated from <Emphasis Type="Italic">Lasius niger</Emphasis> and <Emphasis Type="Italic">Formica cunicularia</Emphasis> ants

Multiple actinomycete strains were isolated from two ant species, Lasius niger and Formica cunicularia, and their phenotypic properties and phylogenetic position were studied. Partial sequencing of 16S rRNA assigned the greater part of them to the genus Streptomyces, but only one belonged to Nocardia. However, some isolates had significant color and morphological differences from their closest phylogenetic relatives. The abundance and biodiversity of actinomycete communities isolated from L. niger ants greatly exceeded those found for F. cunicularia. All of the actinomycetes associated with F. cunicularia ants demonstrated cellulolytic activity, but only one had such ability among the strains associated with black ants.     

Antifungal activity of 3-acetylbenzamide produced by actinomycete WA23-4-4 from the intestinal tract of <Emphasis Type="Italic">Periplaneta americana</Emphasis>

Actinomycetes are well-known for producing numerous bioactive secondary metabolites. In this study, primary screening by antifungal activity assay found one actinomycete strain WA23-4-4 isolated from the intestinal tract of Periplaneta americana that exhibited broad spectrum antifungal activity. 16S rDNA gene analysis of strain WA23-4-4 revealed close similarity to Streptomyces nogalater (AB045886) with 86.6% sequence similarity. Strain WA23-4-4 was considered as a novel Streptomyces and the 16s rDNA sequence has been submitted to GenBank (accession no. KX291006). The maximum antifungal activity of WA23-4-4 was achieved when culture conditions were optimized to pH 8.0, with 12% inoculum concentration and 210 ml ISP2 medium, which remained stable between the 5^th and the 9^th day. 3-Acetyl benzoyl amide was isolated by ethyl acetate extraction of WA23-4-4 fermentation broth, and its molecular formula was determined as C₉H₉NO₂ based on MS, IR, ¹H, and ¹³C NMR analyses. The compound showed significant antifungal activity against Candida albicans ATCC 10231 (MIC: 31.25 μg/ml) and Aspergillus niger ATCC 16404 (MIC: 31.25 μg/ml). However, the compound had higher MIC values against Trichophyton rubrum ATCC 60836 (MIC: 500 μg/ml) and Aspergillus fumigatus ATCC 96918 (MIC: 1,000 μg/ml). SEM analysis showed damage to the cell membrane of Candida albicans ATCC 10231 and to the mycelium of Aspergillus niger ATCC 16404 after being treatment with 3-acetyl benzoyl amide. In conclusion, this is the first time that 3-acetyl benzoyl amide has been identified from an actinomycete and this compound exhibited antifungal activity against Candida albicans ATCC 10231 and Aspergillus niger ATCC 16404.     

Fen meadows on the move for the conservation of <Emphasis Type="Italic">Maculinea (Phengaris) teleius</Emphasis> butterflies

In the Netherlands, a single population of the obligate myrmecophilic butterfly Maculinea (Phengaris) teleius has survived on only 3 ha of habitat for more than 25 years, whereas at least 40 ha of habitat are thought to be required for a sustainable metapopulation. Therefore, 170 ha of farmland is being restored to wet meadows within a LIFE?+?project by large-scale soil excavation and hay inoculation. For successful restoration, the habitat requirements of the butterfly, with Sanguisorba officinalis as host plant and its particular life cycle as parasite of the ant species Myrmica scabrinodis, have to be taken into account. We tested whether colonization of nests of this ant species in the restoration areas is facilitated by translocation of sods collected from fen meadows. We divided 54 sods, each sized 1 m², randomly over six patches and measured vegetation development and ant presence in the sods and surrounding control plots for 2 years. In the first summer, significantly more Myrmica ants were found in the transplanted sods in comparison to the surrounding area. Herb cover had a significant positive effect on Myrmica ant presence while it did not affect the presence of the pioneer ant species Lasius niger. In the second year, Myrmica ants were found in the surrounding control plots as well. This study contributes to the knowledge-base required for the design of restoration projects aimed at expanding the habitat of the critically endangered butterfly Maculinea (Phengaris) teleius.     

Purification,characterization, gene cloning and sequencing of a new β-glucosidase from <Emphasis Type="Italic">Aspergillus niger</Emphasis> BE-2

The cDNA gene (BgL1), encoding GH3 family β-glucosidase (EC 3.2.1.21) from Aspergillus niger BE-2 (abbreviated to BgL1), was amplified and inserted into the yeast expression pPIC9K vector at the site of Bln I (Avr II) and NotI. The recombinant expression vector, designated as pPIC9K-BgL1, was transformed into Pichia pastoris GS115. The transformants were screened on minimal dextrose plates, which inoculated on geneticin G418-containing yeast extract-peptone-dextrose plates. The transformants expressed the high β-glucosidase activity of 22.6 U/mL. SDS-PAGE demonstrated that the BgL1 was extracellularly expressed with an apparent molecular weight of 90.0 kDa. The purified BgL1 displayed the maximum activity at pH 6.0 and 60°C. It was highly stable at a broad pH range of 4.0–7.5 and temperature of 60°C. The BgL1 displayed high similarity to the β-glucosidases of A. niger FN430671 and A. niger DQ655704, the members of the GH3 family. Its three-dimensional structure was predicted using http://swiss-model.expasy.org/ on-line programs based on the crystal structure of Aspergillus aculeatus β-glucosidase.     

Structural and inhibition analysis of novel sulfur-rich 2-mercaptobenzothiazole and 1,2,3-triazole ligands against <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> DprE1 enzyme

Mycobacterium tuberculosis decaprenylphosphoryl-β-d-ribose oxidase (MtbDprE1) acts in concert with decaprenylphosphoryl-β-d-ribose 2-epimerase (MtbDprE2) and catalyzes the epimerization of DPR into DPA. DPA is the sole precursor for synthesis of arabinogalactan and lipoarabinomannan in the mycobacterial cell wall. MtbDprE1 is a unique antimalarial drug target and many covalent and non-covalent inhibitors against MtbDprE1 have been studied for their antituberculosis activities. In the current study, we have purified MtbDprE1 enzyme and synthesized six sulfur-rich 2-mercaptobenzothiazole and 1, 2, 3-triazole conjugated ligands and performed binding analysis with MtbDprE1. All ligands have shown competitive binding, as observed for other covalently and noncovalently bound MtbDprE1 inhibitors. Molecular docking analysis of six ligands with MtbDprE1 shows that they occupy the substrate binding pocket of MtbDprE1 and are stabilized by hydrogen bonds and van der Waals interactions. Our study shows that sulfur-rich 2-mercaptobenzothiazole ligands act as specific inhibitors against MtbDprE1 and could be used as antituberculosis agents.     

Adaptive evolution of osmoregulatory-related genes provides insight into salinity adaptation in Chinese mitten crab, <Emphasis Type="Italic">Eriocheir sinensis</Emphasis>

Osmoregulation is an important mechanism by which euryhaline crustaceans regulate osmotic and ionic concentrations. The Chinese mitten crab (Eriocheir sinensis) is a strong osmoregulating animal model among crustacean species, as it can maintain its hemolymph composition and survives well in either seawater or freshwater. Osmoregulation by E. sinensis during physiological adaptation has been studied extensively. However, the genetic basis of osmoregulation in E. sinensis for acclimating to changing salinities remains unclear. The current study investigated five genes involved in E. sinensis osmoregulation and compared them with a representative marine crab Portunus trituberculatus to test whether adaptive evolution has occurred changing salinity conditions. The results showed that carbonic anhydrase (CA), cytochrome P450 4C (CYP4C), glutamate dehydrogenase (GDH), and the Na⁺/H⁺ exchanger (NHE) have undergone positive selection (i.e., directional selection) in E. sinensis. Thus, the positive selection in CA and NHE suggests that E. sinensis has enhanced capacity for maintaining systemic acid-base balance and ion regulation. GDH and CYP4C also demonstrated positive selection in E. sinensis, suggesting that E. sinensis might have acquired an enhanced capacity to metabolize glutamate and synthesize ecdysteroids in response to a change in osmotic concentration. The present study provides new insight into the molecular genetic basis of salinity adaption in E. sinensis.     

Ant–aphid relations in the south of Western Siberia (Hymenoptera: Formicidae; Hemiptera: Aphididae)

Aphids, the main suppliers of energy-rich honeydew, play an important role in the life of ants. However, the data on the trophobiotic ant–aphid associations in the majority of regions are still limited. We present the first data on the ant–aphid relations in the south of Western Siberia. Investigations were carried out in the most typical biotopes of forest-steppe and steppe zones in the territory of Novosibirsk and Kurgan regions (Russia) during 1993–2014. There were revealed 35 species of ants and 198 species of aphids. Detected 456 ant–aphid associations involved 28 ant species and 134 myrmecophilous aphids. Seven ant species were found to consume honeydew of 9 non-myrmecophilous aphids, scraping it from the plant. This behaviour is typical of subdominant and subordinate ants which do not protect their foraging areas. Ants associate with various numbers of aphid species. About 36% of ants attended aphid colonies of less than 5 species. The largest number of myrmecophilous aphids is associated with L. niger (Linnaeus, 1758) (103 species), Formica pratensis Retzius, 1783 (50), Formica rufa group (25–33), F. (Serviformica) fusca Linnaeus, 1758 (26) and F. (S.) cunicularia Latreille, 1798 (27). Different ants play unequal roles in the formation of trophobiotic interactions with aphids. Due to complex territorial and foraging behaviour, including high functional specialization among honeydew collectors, dominant ants of Formica s. str. are one of the leaders in this process. The role of L. niger and Formica ants of the subgenus Serviformica requires further detailed investigation.