Milen Marinov

Diverse circular ssDNA viruses discovered in dragonflies (Odonata: Epiprocta)

Viruses with circular ssDNA genomes that encode a replication initiator protein (Rep) are among the smallest viruses known to infect both eukaryotic and prokaryotic organisms. In the past few years an overwhelming diversity of novel circular Rep-encoding ssDNA (CRESS-DNA) viruses has been unearthed from various hosts and environmental sources. Since there is limited information regarding CRESS-DNA viruses in invertebrates, this study explored the diversity of CRESS-DNA viruses circulating among insect populations by targeting dragonflies (Epiprocta), top insect predators that accumulate viruses from their insect prey over space and time. Using degenerate PCR and rolling circle amplification coupled with restriction digestion, 17 CRESS-DNA viral genomes were recovered from eight different dragonfly species collected in tropical and temperate regions. Nine of the genomes are similar to cycloviruses and represent five species within this genus, suggesting that cycloviruses are commonly associated with insects. Three of the CRESS-DNA viruses share conserved genomic features with recently described viruses similar to the mycovirus Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1, leading to the proposal of the genus Gemycircularvirus. The remaining viruses are divergent species representing four novel CRESS-DNA viral genera, including a gokushovirus-like prokaryotic virus (microphage) and three eukaryotic viruses with Reps similar to circoviruses. The novelty of CRESS-DNA viruses identified in dragonflies using simple molecular techniques indicates that there is an unprecedented diversity of ssDNA viruses among insect populations.

Dragonfly cyclovirus, a novel single-stranded DNA virus discovered in dragonflies (Odonata: Anisoptera)

Dragonfly cyclovirus (DfCyV), a new species of ssDNA virus discovered using viral metagenomics in dragonflies (family Libellulidae) from the Kingdom of Tonga. Metagenomic sequences of DfCyV were similar to viruses of the recently proposed genus Cyclovirus within the family Circoviridae. Specific PCRs resulted in the recovery of 21 DfCyV genomes from three dragonfly species (Pantala flavescens, Tholymis tillarga and Diplacodes bipunctata). The 1741 nt DfCyV genomes share >95 % nucleotide identity and are classified into 11 subtypes representing a single strain. The DfCyV genomes share 48-63 % genome-wide nucleotide identity with cycloviruses identified in human faecal samples. Recombination analysis revealed three recombinant DfCyV genomes, suggesting that recombination plays an important role in cyclovirus evolution. To our knowledge, this is the first report of a circular ssDNA virus identified in insects, and the data may help elucidate evolutionary links among novel Circoviridae recently identified in animals and environmental samples.

High global diversity of cycloviruses amongst dragonflies

Members of the family Circoviridae, specifically the genus Circovirus, were thought to infect only vertebrates; however, members of a sister group under the same family, the proposed genus Cyclovirus, have been detected recently in insects. In an effort to explore the diversity of cycloviruses and better understand the evolution of these novel ssDNA viruses, here we present five cycloviruses isolated from three dragonfly species (Orthetrum sabina, Xanthocnemis zealandica and Rhionaeschna multicolor) collected in Australia, New Zealand and the USA, respectively. The genomes of these five viruses share similar genome structure to other cycloviruses, with a circular ~1.7 kb genome and two major bidirectionally transcribed ORFs. The genomic sequence data gathered during this study were combined with all cyclovirus genomes available in public databases to identify conserved motifs and regulatory elements in the intergenic regions, as well as determine diversity and recombinant regions within their genomes. The genomes reported here represent four different cyclovirus species, three of which are novel. Our results confirm that cycloviruses circulate widely in winged-insect populations; in eight different cyclovirus species identified in dragonflies to date, some of these exhibit a broad geographical distribution. Recombination analysis revealed both intra- and inter-species recombination events amongst cycloviruses, including genomes recovered from disparate sources (e.g. goat meat and human faeces). Similar to other well-characterized circular ssDNA viruses, recombination may play an important role in cyclovirus evolution.

Identification of diverse circular single-stranded DNA viruses in adult dragonflies and damselflies (Insecta: Odonata) of Arizona and Oklahoma, USA

Next generation sequencing and metagenomic approaches are commonly used for the identification of circular replication associated protein (Rep)-encoding single stranded (CRESS) DNA viruses circulating in various environments. These approaches have enabled the discovery of some CRESS DNA viruses associated with insects. In this study we identified and recovered 31 viral genomes which represent 24 distinct CRESS DNA viruses from seven dragonfly species (Rhionaeschna multicolor, Erythemis simplicicollis, Erythrodiplax fusca, Libellula quadrimaculata, Libellula saturata, Pachydiplax longipennis, and Pantala hymenaea) and two damselfly species (Ischnura posita, Ischnura ramburii) sampled in various locations in the states of Arizona and Oklahoma of the United States of America (USA). We also identified Sclerotinia sclerotiorum hypovirulence-associated DNA virus-1 (SsHADV-1) in P. hymenaea, E. simplicicollis and I. ramburii sampled in Oklahoma, which is the first report of SsHADV-1 in the New World. The genome architectures of the CRESS DNA viruses recovered vary, but they all have at least two major open reading frames (ORFs) that have either a bidirectional or unidirectional arrangement. Four of the viral genomes recovered, in addition to the three isolates of SsHADV-1, show similarities to viruses of the proposed gemycircularvirus group. Analysis of the Rep encoded by the remaining 24 viral genomes reveals that these are highly diverse and allude to the fact that they represent novel CRESS DNA viruses.

Description of the larva of Trineuragrion percostale Ris (Odonata: Megapodagrionidae) with a key to the larvae of New Caledonian genera of Megapodagrionidae

The larva of Trineuragrion percostale is described and illustrated based on an exuvia collected with the teneral female. Four more exuviae sampled without imagines are added for comparison and discussion of morphological variations. This is the first time the larva has been associated with the correct species, although the larval description was given earlier. Lieftinck included it in his analysis of New Caledonian Odonata larval fauna under the name Caledargiolestes uniseries (by supposition). Variations in the morphological features between Lieftincks specimens and those reported here are discussed. The key morphological features that differentiate larva of T. percostale from other Megapodagrionidae in the country are indicated.

Geometric morphometrics and molecular systematics of Xanthocnemis sobrina (McLachlan, 1873) (Odonata: Coenagrionidae) and comparison to its congeners

The taxonomy of the damselfly genus Xanthocnemis is revised, with particular focus on populations inhabiting the North Island of New Zealand. Earlier studies revealed two species: X. sobrina, restricted to cool, shaded streams in kauri forests and other forested areas, and X. zealandica, a common species throughout New Zealand except the Chatham and subantarctic islands. A field study encompassing aquatic habitats throughout the whole North Island was carried out to establish the relationship between morphological variation (body size and various morphological traits over the entire body) observed by previous researchers with ecological conditions and/or geographical location. The main aim was to propose reliable diagnostic features that could be used in future studies. Morphological and molecular variation was assessed. Morphological examination included assigning landmarks for all body parts corresponding to the external morphological features that are usually used in Odonata taxonomy. Molecular analysis targeted fragments of the 28S and 16S rRNA genes. Congruence was sought between both types of data, statistical support for two morphological types previously described as different species and a maximum likelihood phylogenetic tree in conjunction with a pairwise genetic distance matrix constructed from the DNA sequences obtained from the sampled specimens. Geometric morphometrics revealed statistically significant differentiation between specimens identified as X. zealandica and X. sobrina for four traits: (1) dorsal view of the head for both sexes as well as male appendages from (2) dorsal, (3) ventral and (4) lateral views. Wings appeared different when analysed for males only. Molecular analysis, however, grouped all specimens into a single undifferentiated cluster with very low mean pairwise distance (<0.01) between them showing almost no variation at the molecular level among the sampled populations on the North Island. Therefore, an additional analysis of the mitochondrial cytochrome c-oxidase I gene was carried out comparing randomly selected North Island specimens to Xanthocnemis specimens targeted in other molecular studies (Nolan et al. 2007, Amaya-Perilla et al. 2014). The analysis of the COI gene confirmed that all North and South Island isolates of Xanthocnemis cluster together in a well-supported clade with pairwise identity >96% and ~93% pairwise identity with X. tuanuii sequences obtained from the Chatham Island specimens. A careful investigation of the thin plate spline deformations generated for the geometric morphometric landmarks showed that the significant variations in the appendages of the Xanthocnemis specimens appeared to be the result of size, rather than shape, differences. Therefore, X. sobrina is proposed as a synonym of X. zealandica. Recently Amaya-Perilla et al. (2014) synonymised X. sinclairi with X. zealandica and confirmed the status of the Chatham Island X. tuanuii as a distinct species. It is therefore proposed that the genus Xanthocnemis consists of two species only: zealandica occurring all over the North, South and Stewart Islands, and tuanuii, endemic to Chatham and Pitt islands. Considering several statistical tests involving body measurements and ecological variables recorded during the field study, as well as various discussion points from similar studies of other species of Odonata, two alternative hypotheses are proposed for future testing. The first hypothesis synonymises X. sobrina with X. zealandica and suggests a possible explanation for the evolution of the two morphological traits that have previously been considered diagnostic for these species. The second hypothesis suggests that as typical X. sobrina were not sampled during this study this could represent a species that is now extinct, unless future studies prove it otherwise.

Anormogomphus kiritshenkoi Bartenev, 1913 (Odonata: Gomphidae): a literature review of the variable spelling of the species epithet, choice of the correct spelling and notes on the type locality of the species

The spelling of the specific name of an Anormogomphus species in its original description by Bartenev (1913) was variable, kiritshenkoi (5 times) vs kiritschenkoi (1 time). Bartenev himself did not mention this species in his further publications. Later authors proceeded to use different spelling versions of this name, including those not in the original paper. According to the International Code of Zoological Nomenclature (ICZN), the valid spelling should be chosen by the First Reviser. Acting as such, we choose as valid the name spelling Anormogomphus kiritshenkoi Bartenev, 1913, since it predominates in the original description, appears in the species subtitle, and is a proper transliteration from Cyrillic to Latin. It also corresponds to the spelling of his name used by A.N. Kiritshenko himself. Information of the type locality of the species is provided.