D. V. Mukha

The family Parvoviridae

A set of proposals to rationalize and extend the taxonomy of the family Parvoviridae is currently under review by the International Committee on Taxonomy of Viruses (ICTV). Viruses in this family infect a wide range of hosts, as reflected by the longstanding division into two subfamilies: the Parvovirinae, which contains viruses that infect vertebrate hosts, and the Densovirinae, encompassing viruses that infect arthropod hosts. Using a modified definition for classification into the family that no longer demands isolation as long as the biological context is strong, but does require a near-complete DNA sequence, 134 new viruses and virus variants were identified. The proposals introduce new species and genera into both subfamilies, resolve one misclassified species, and improve taxonomic clarity by employing a series of systematic changes. These include identifying a precise level of sequence similarity required for viruses to belong to the same genus and decreasing the level of sequence similarity required for viruses to belong to the same species. These steps will facilitate recognition of the major phylogenetic branches within genera and eliminate the confusion caused by the near-identity of species and viruses. Changes to taxon nomenclature will establish numbered, non-Latinized binomial names for species, indicating genus affiliation and host range rather than recapitulating virus names. Also, affixes will be included in the names of genera to clarify subfamily affiliation and reduce the ambiguity that results from the vernacular use of “parvovirus” and “densovirus” to denote multiple taxon levels.

Population Genetic Structure in German Cockroaches (Blattella Germanica): Differentiated Islands in an Agricultural Landscape

Although a number of species live syanthropically with humans, few rely entirely on humans for their survival and distribution. Unlike other cosmopolitan human commensals, the German cockroach (Blattella germanica), an insect of both public and livestock health concern, is considered incapable of dispersal outside human dwellings. Patterns of genetic association are therefore constrained and may not be associated with distance. Analogies with other human-commensal species are therefore impossible to draw with any degree of accuracy. In the past 2 decades, B. germanica has become a prominent pest within the US swine production system. Swine production is mainly carried out through contracted producers, each associated with a management company. It has been hypothesized that cockroach populations will be genetically structured based on association to a specific management company. Here, we tested this hypothesis using microsatellite genotypes (8 polymorphic loci) from 626 individual cockroaches collected from 22 farms in southeastern North Carolina representing 3 management companies. Significant genetic differentiation was detected (F(ST) = 0.171), most of which was partitioned among the 22 farms rather than the 3 management groups. All pair-wise population comparisons yielded F(ST) values significantly greater than zero. Our results reveal that structure does not correspond to management company of origin, but instead it may be regional and influenced strongly by the unintentional movement of cockroaches by farm workers.

Population Genetic Structure of the German Cockroach (Blattodea: Blattellidae) in Apartment Buildings

ABSTRACT The German cockroach, Blattella germanica (L.) (Blattodea: Blattellidae), is a major residential pest with the potential to vector various pathogens and produce and disseminate household allergens. Understanding population genetic structure and differentiation of this important pest is critical to efforts to eradicate infestations, yet little is known in this regard. Using highly polymorphic microsatellite markers, we investigated patterns of genetic diversity and differentiation within and among 18 apartments from six apartment complexes located in Raleigh, NC. No departure from panmixia was found between rooms within apartments, indicating that active dispersal resulting in gene flow may occur among rooms within apartment units. Alternatively, aggregations within apartments may exist in relative isolation under a metapopulation framework, derived from a recent, common source. Thus, in the event of population control practices leading to incomplete cockroach eradication within an apartment, recolonization of shelters and rooms is likely to occur from a genetically similar aggregation. A pattern of isolation-by-distance across the six apartment complexes indicated that dispersal was more common within complexes than among them, and F statistics suggested greater genetic similarity between apartments in a single building than between separate buildings of an apartment complex. Similarly, neighbor-joining tree and Bayesian clustering analyses were able to cluster only those apartments that were within a single building, indicating higher dispersal with associated gene flow within buildings than between them. The lack of any broader connectivity, as indicated by significant FST and G-tests suggests that human-mediated dispersal of B. germanica between buildings of an apartment complex or between complexes occurs infrequently enough to have negligible effects on gene flow.

Analysis of intraspecies polymorphism in the ribosomal DNA cluster of the cockroach Blattella germanica

HindIII restriction digests of the rDNA repeat unit of the German cockroach, Blattella germanica, reveal significant intraspecies sequence polymorphism. This variability is probably caused by structural differences within the nontranscribed spacer regions (NTS) of the ribosomal repeat unit. HindIII rDNA fragment polymorphisms in three cockroach strains show that individuals from different populations may have different HindIII rDNA patterns, whereas individuals within populations exhibit relatively similar rDNA patterns. We suggest that HindIII restriction fragment polymorphisms within cockroach ribosomal DNA will be a valuable tool for measuring population‐level parameters within and between natural cockroach populations.

Expression Strategy of Densonucleosis Virus from the German Cockroach, Blattella germanica

ABSTRACT Blattella germanica densovirus (BgDNV) is an autonomous parvovirus that infects the German cockroach. BgDNV possesses three mRNAs for NS proteins, two of which are splice variants of the unspliced transcript. The unspliced variant encodes open reading frame 5 (ORF5) (NS3), while NSspl1 encodes ORF3 (NS1) and ORF4 (NS2) and NSspl2 encodes the C-proximal half of NS1. BgDNV possesses three VP transcripts, one of which (VP) is unspliced, while the other two (VPspl1 and VPspl2) are generated by alternative splicing. The unspliced VP transcript contains both ORF1 and ORF2, while in VPspl1, ORF1 and ORF2 are joined in frame. The transcription of NS genes begins at an earlier stage of the virus life cycle than the transcription of VP genes. NS and VP transcripts overlap by 48 nucleotides (nt). BgDNV is characterized by two additional NS transcripts overlapping by more than 1,650 nt with VP-coding transcripts. Four different bands (97, 85, 80, and 57 kDa) corresponding to three BgDNV capsid proteins were detected on SDS-PAGE. Mass spectrometry analysis showed that the amino acid composition of the 85-kDa and 80-kDa proteins is the same. Moreover, both of these proteins are ubiquitinated. The BgDNV PLA2 domain, which is critical for cellular uptake of the virus, is located in ORF2 and is present only in VP1. In contrast to all of the parvoviruses studied in this respect, VP2 has a unique N terminus that is not contained within VP1 and VP3. In situ recognition with NS1- and VP-specific antibodies revealed an uneven pattern of NS1 expression resembling a halo within the nuclear membrane.

Intraspecific and Interspecific Variation of the Mitochondrial Gene of Cytochrome Oxidase I in Ladybirds (Coleoptera: Coccinellidae)

Intergeneric, interspecific, and intraspecific genetic variation of the 310-bp 3′-end region of the mitochondrial gene of cytochrome oxidase I (COI) has been assessed in ladybirds (Coleoptera: Coccinnellidae). The phylogenetic distances between eight species of ladybirds have been determined. Mitochondrial DNA (mtDNA) nucleotide sequences have been compared in Adalia bipunctata L. differing in the elytron and pronotum colors that have been sampled from several geographically remote populations. The taxonomic statuses of two morphs from the genus Adalia, A. bipunctata bipunctata and A. bipunctata fasciatopunctata, have been identified.

Hierarchical genetic analysis of German cockroach (Blattella germanica) populations from within buildings to across continents.

Understanding the population structure of species that disperse primarily by human transport is essential to predicting and controlling human-mediated spread of invasive species. The German cockroach (Blattella germanica) is a widespread urban invader that can actively disperse within buildings but is spread solely by human-mediated dispersal over longer distances; however, its population structure is poorly understood. Using microsatellite markers we investigated population structure at several spatial scales, from populations within single apartment buildings to populations from several cities across the U.S. and Eurasia. Both traditional measures of genetic differentiation and Bayesian clustering methods revealed increasing levels of genetic differentiation at greater geographic scales. Our results are consistent with active dispersal of cockroaches largely limited to movement within a building. Their low levels of genetic differentiation, yet limited active spread between buildings, suggests a greater likelihood of human-mediated dispersal at more local scales (within a city) than at larger spatial scales (within and between continents). About half the populations from across the U.S. clustered together with other U.S. populations, and isolation by distance was evident across the U.S. Levels of genetic differentiation among Eurasian cities were greater than those in the U.S. and greater than those between the U.S. and Eurasia, but no clear pattern of structure at the continent level was detected. MtDNA sequence variation was low and failed to reveal any geographical structure. The weak genetic structure detected here is likely due to a combination of historical admixture among populations and periodic population bottlenecks and founder events, but more extensive studies are needed to determine whether signatures of global movement may be present in this species.

Phylogenetic Information Content of Copepoda Ribosomal DNA Repeat Units: ITS1 and ITS2 Impact

The utility of various regions of the ribosomal repeat unit for phylogenetic analysis was examined in 16 species representing four families, nine genera, and two orders of the subclass Copepoda (Crustacea). Fragments approximately 2000 bp in length containing the ribosomal DNA (rDNA) 18S and 28S gene fragments, the 5.8S gene, and the internal transcribed spacer regions I and II (ITS1 and ITS2) were amplified and analyzed. The DAMBE (Data Analysis in Molecular Biology and Evolution) software was used to analyze the saturation of nucleotide substitutions; this test revealed the suitability of both the 28S gene fragment and the ITS1/ITS2 rDNA regions for the reconstruction of phylogenetic trees. Distance (minimum evolution) and probabilistic (maximum likelihood, Bayesian) analyses of the data revealed that the 28S rDNA and the ITS1 and ITS2 regions are informative markers for inferring phylogenetic relationships among families of copepods and within the Cyclopidae family and associated genera. Split-graph analysis of concatenated ITS1/ITS2 rDNA regions of cyclopoid copepods suggested that the Mesocyclops, Thermocyclops, and Macrocyclops genera share complex evolutionary relationships. This study revealed that the ITS1 and ITS2 regions potentially represent different phylogenetic signals.

Characterization of Cyclops kolensis inter-simple sequence repeats in germline and postdiminution somatic cells

337 Chromatin diminution is programmed elimination of a part of the genome from presumptive somatic cells during early embryogenesis of some animal species. Chromatin diminution was described for a fairly small number of species of different groups of animals, such as ascarides, myxines, dipterans, Cyclops , and protozoans [2, 3, 6, 8, 10, 13, 14]. Although a great variety of chromatin diminution forms is known, it seems most promising to study this process in Cyclops kolensis Lill., a member of the family Cyclopidae. This freshwater crustacean contains a record-breaking (~94%) amount of eliminated DNA (eDNA) compared to other multicellular animals. In this species, DNA is eliminated from chromosomes of somatic cells during the fourth cleavage division, with the diploid chromosome number remaining unchanged [2, 3]. In this study, we demonstrated that chromatin diminution in C. kolensis is not accompanied by total elimination of nonfunctional genomic regions. This finding broadens our notion on chromatin diminution in C. kolensis . Currently, the molecular mechanisms ensuring diminution and determine the eDNA type remain largely obscure. However, it was shown that the C. kolensis eDNA contains noncoding sequences, some of which have an intricate mosaic structure of repeats [7]. It is known that Mesocyclops edax , another member of the family Cyclopidae, loses a considerable part of satellite DNA sequences during chromatin diminution [9]. The so-called microsatellite sequences are also classified with the satellite DNA. Microsatellite, or short tandem repeats (simple sequence repeats, SSRs), are a characteristic component of the eukaryotic genome. Today, an ample body of evidence regarding the structure of this component and the mechanisms that ensure their size polymorphism and increase in the number of their copies has been accumulated [15]. The consistent patterns of microsatellite distribution over the genome has been studied and their predominant concentration in the noncoding region of the genome has been shown [11]. An average distance between microsatellites in heterochromatin regions varies from several hundreds to several thousands of nucleotide pairs [12], which makes it possible to amplify the DNA fragments located between microsatellites (the so-called inter-simple sequence repeats, or ISSRs sequences) by polymerase chain reaction (PCR). It was shown earlier that, in some species, the majority of heterochromatin regions in chromosomes is eliminated from the genome of somatic cells in the course of chromatin diminution [10, 13]. It can be assumed that a considerable part of heterochromatin, including the microsatellite sequences, is eliminated from the C. kolensis genome during chromatin diminution. In this study, we amplified, cloned, and sequenced five C. kolensis DNA sequences located between the (GA) n microsatellite sequences. A comparative analysis of the nucleotides comprising the cloned sequences was performed using special software programs. Unique internal primer pairs selected for four out of five cloned sequences made it possible to amplify by PCR the corresponding DNA fragments initially located between the (GA) n microsatellites. We performed a comparative analysis of the amplification products of these sequences in C. kolensis before and after chromatin diminution. It was found that three out of four intermicrosatellite loci studied remain in the genome of somatic cells after chromatin diminution and only one of them is eliminated during diminution. Female C. kolensis caught in the pond situated on Vorob’evy Gory (Moscow) were determined according to Monchenko [4]. The prediminution stage of

R1 and R2 Retrotransposons of German Cockroach Blatella germanica : A Comparative Study of 5'-Truncated Copies Integrated into the Genome

For the first time, extended fragments (5′-truncated copies) of R1 and R2 retrotransposons integrated into the Blattella germanica genome were identified, cloned, and sequenced. Structural comparison of the clones revealed two distinct R1 subfamilies. However, all R1 clones had two common features: poly(T) tails and similar target site duplications. R1 retrotransposons are the first known mobile elements with poly(T) tails on the 3′-ends. The structure and nucleotide sequences of five sequenced R2 fragments were similar to each other. Nucleotide sequence analysis of R2 retrotransposons revealed typical deletions at the 3′ ends of the target sites and the lack of homopolynucleotide tails.