Regina G. Kleespies

The Genome of Gryllus bimaculatus Nudivirus Indicates an Ancient Diversification of Baculovirus-Related Nonoccluded Nudiviruses of Insects

ABSTRACT The Gryllus bimaculatus nudivirus (GbNV) infects nymphs and adults of the cricket Gryllus bimaculatus (Orthoptera: Gryllidae). GbNV and other nudiviruses such as Heliothis zea nudivirus 1 (HzNV-1) and Oryctes rhinoceros nudivirus (OrNV) were previously called “nonoccluded baculoviruses” as they share some similar structural, genomic, and replication aspects with members of the family Baculoviridae. Their relationships to each other and to baculoviruses are elucidated by the sequence of the complete genome of GbNV, which is 96,944 bp, has an AT content of 72%, and potentially contains 98 predicted protein-coding open reading frames (ORFs). Forty-one ORFs of GbNV share sequence similarities with ORFs found in OrNV, HzNV-1, baculoviruses, and bacteria. Most notably, 15 GbNV ORFs are homologous to the baculovirus core genes, which are associated with transcription (lef-8, lef-9, lef-4, vlf-1, and lef-5), replication (dnapol), structural proteins (p74, pif-1, pif-2, pif-3, vp91, and odv-e56), and proteins of unknown function (38K, ac81, and 19kda). Homologues to these baculovirus core genes have been predicted in HzNV-1 as well. Six GbNV ORFs are homologous to nonconserved baculovirus genes dnaligase, helicase 2, rr1, rr2, iap-3, and desmoplakin. However, the remaining 57 ORFs revealed no homology or poor similarities to the current gene databases. No homologous repeat (hr) sequences but fourteen short direct repeat (dr) regions were detected in the GbNV genome. Gene content and sequence similarity suggest that the nudiviruses GbNV, HzNV-1, and OrNV form a monophyletic group of nonoccluded double-stranded DNA viruses, which separated from the baculovirus lineage before this radiated into dipteran-, hymenopteran-, and lepidopteran-specific clades of occluded nucleopolyhedroviruses and granuloviruses. The accumulated information on the GbNV genome suggests that nudiviruses form a highly diverse and phylogenetically ancient sister group of the baculoviruses, which have evolved in a variety of highly divergent host orders.

Susceptibility of North-American and European crickets to Acheta domesticus densovirus (AdDNV) and associated epizootics

The European house cricket, Acheta domesticus L., is highly susceptible to A. domesticus densovirus (AdDNV). Commercial rearings of crickets in Europe are frequently decimated by this pathogen. Mortality was predominant in the last larval stage and young adults. Infected A. domesticus were smaller, less active, did not jump as high, and the adult females seldom lived more than 10-14 days. The most obvious pathological change was the completely empty digestive caecae. Infected tissues included adipose tissue, midgut, epidermis, and Malpighian tubules. Sudden AdDNV epizootics have decimated commercial mass rearings in widely separated parts of North America since the autumn of 2009. Facilities that are producing disease-free crickets have avoided the importation of crickets and other non-cricket species (or nonliving material). Five isolates from different areas in North America contained identical sequences as did AdDNV present in non-cricket species collected from these facilities. The North American AdDNVs differed slightly from sequences of European AdDNV isolates obtained in 1977, 2004, 2006, 2007 and 2009 and an American isolate from 1988. The substitution rate of the 1977 AdDNV 5kb genome was about two nucleotides per year, about half of the substitutions being synonymous. The American and European AdDNV strains are estimated to have diverged in 2006. The lepidopterans Spodoptera littoralis and Galleria mellonella could not be infected with AdDNV. The Jamaican cricket, Gryllus assimilis, and the European field cricket, Gryllus bimaculatus, were also found to be resistant to AdDNV.

Type IV secretion system components as phylogenetic markers of entomopathogenic bacteria of the genus Rickettsiella

The genus Rickettsiella (class Gammaproteobacteria; order Legionellales; family Coxiellaceae) comprises intracellular bacterial pathogens of a wide range of arthropods that are currently classified in the three recognized species, Rickettsiella popilliae, Rickettsiella grylli, and Rickettsiella chironomi. Rickettsiella bacteria contain a type IVB secretion system (T4SS) known to be a key virulence factor of the related genus Legionella. Providing the first respective sequence information for the nomenclatural type species, R. popilliae, the three T4SS components DotA, IcmB, and IcmQ were used as phylogenetic markers to test hypotheses implicit in the currently accepted taxonomic organization of Rickettsiella at the species, genus, and family level. These results, firstly, firmly corroborate the previous 16S rRNA gene-based coassignment of the species R. grylli and R. popilliae to the gamma-proteobacterial order Legionellales and, secondly, support the current classification of the investigated R. grylli and R. popilliae strains in different species of the same genus. In contrast, the analysis of intergeneric sequence distances does not lend support to the current taxonomic classification of the genus Rickettsiella in the family Coxiellaceae, but is consistent with a hierarchically neutral family-level assignment within the order Legionellales.

A Rickettsiella Bacterium from the Hard Tick, Ixodes woodi: Molecular Taxonomy Combining Multilocus Sequence Typing (MLST) with Significance Testing

Hard ticks (Acari: Ixodidae) are known to harbour intracellular bacteria from several phylogenetic groups that can develop both mutualistic and pathogenic relationships to the host. This is of particular importance for public health as tick derived bacteria can potentially be transmitted to mammals, including humans, where e.g. Rickettsia or Coxiella act as severe pathogens. Exact molecular taxonomic identification of tick associated prokaryotes is a necessary prerequisite of the investigation of their relationship to both the tick and possible vertebrate hosts. Previously, an intracellular bacterium had been isolated from a monosexual, parthenogenetically reproducing laboratory colony of females of the hard tick, Ixodes woodi Bishopp, and had preliminarily been characterized as a “Rickettsiella-related bacterium”. In the present molecular taxonomic study that is based on phylogenetic reconstruction from both 16 S ribosomal RNA and protein-encoding marker sequences complemented with likelihood-based significance testing, the bacterium from I. woodi has been identified as a strain of the taxonomic species Rickettsiella grylli. It is the first time that a multilocus sequence typing (MLST) approach based on a four genes comprising MLST scheme has been implemented in order to classify a Rickettsiella-like bacterium to this species. The study demonstrated that MLST holds potential for a better resolution of phylogenetic relationships within the genus Rickettsiella, but requires sequence determination from further Rickettsiella-like bacteria in order to complete the current still fragmentary picture of Rickettsiella systematics.

Basic techniques in insect virology

Viruses are a highly diverse group of pathogens, which are widespread among insects. Many viruses are of importance in the natural regulation of insect populations, some of them, such as baculoviruses, are used as biological control agents. During the last two decades, the scope of methods for analyzing insect viruses has expanded from disease diagnosis and virus characterization to the functional dissection of the virus replication cycle and the analysis of virus interaction on the population level. The initial characterization of a new virus requires a broad range of methods for describing the morphology and cytopathology, for determining the biological activity and infection parameters and for analyzing the genome and genetic functions. The following chapter aims to provide essential methods important in virus identification by microscopy and molecular means, the propagation and quantification of insect viruses, the quantification of their in vivo activity, essential cell culture techniques, as well as recent developments in reverse genetics to study virus gene functions.

Sequencing of the large dsDNA genome of Oryctes rhinoceros nudivirus using multiple displacement amplification of nanogram amounts of virus DNA

The genomic sequence analysis of many large dsDNA viruses is hampered by the lack of enough sample materials. Here, we report a whole genome amplification of the Oryctes rhinoceros nudivirus (OrNV) isolate Ma07 starting from as few as about 10 ng of purified viral DNA by application of phi29 DNA polymerase- and exonuclease-resistant random hexamer-based multiple displacement amplification (MDA) method. About 60 microg of high molecular weight DNA with fragment sizes of up to 25 kbp was amplified. A genomic DNA clone library was generated using the product DNA. After 8-fold sequencing coverage, the 127,615 bp of OrNV whole genome was sequenced successfully. The results demonstrate that the MDA-based whole genome amplification enables rapid access to genomic information from exiguous virus samples.

Genetic and electron-microscopic characterization of Rickettsiella tipulae, an intracellular bacterial pathogen of the crane fly, Tipula paludosa☆

Rickettsiella tipulae is an intracellular bacterial pathogen of larvae of the crane fly, Tipula paludosa (Diptera: Tipulidae) and has previously been claimed to represent an independent species within the genus Rickettsiella. Recently, this taxon has been reorganized and transferred as a whole from the alpha-proteobacterial order Rickettsiales to the gamma-proteobacterial order Legionellales. Here we present the electron-microscopic identification of this rickettsial pathogen together with the first DNA sequence information for R. tipulae. The results of our 16S rDNA-based phylogenetic analysis demonstrate that the transfer to the order Legionellales is justified for R. tipulae. However, there is no phylogenetic basis to consider R. tipulae an independent species, but instead conclusive evidence substantiating its species level co-assignment with Rickettsiella melolonthae. Furthermore, implications of our results for a possible reorganization of the internal structure of the genus Rickettsiella are discussed.

Diseases of insects and other arthropods: results of diagnostic research over 55 years

Abstract Arthropods and especially insects are attacked by many arthropod-pathogenic organisms belonging to viruses, bacteria including rickettsiae, fungi, microsporidia, protists, and nematodes. They may cause lethal or chronic diseases. For 55 years, research on all kinds of arthropod diseases was conducted at the Institute for Biological Control, Darmstadt, formerly belonging to the Federal Biological Research Centre for Agriculture and Forestry (BBA) that became part of the newly founded Julius Kühn-Institute. Special attention is still being paid to diagnostics. During this period, about 1900 accessions comprising many thousands of dead or diseased specimens from 21 different arthropod orders were examined and the causative agents diagnosed. In the present paper, about 450 arthropod species, mainly insects, are listed together with the group and scientific name of the diagnosed arthropod pathogens and the origin (country) of the accession.

Genetic and Electron-Microscopic Characterization of ‘Rickettsiella agriotidis’, a new Rickettsiella Pathotype Associated with Wireworm, Agriotes sp. (Coleoptera: Elateridae)

Wireworms, the polyphagous larvae of click beetles belonging to the genus Agriotes (Coleoptera: Elateridae), are severe and widespread agricultural pests affecting numerous crops. A previously unknown intracellular bacterium has been identified in a diseased Agriotes larva. Microscopic studies revealed the subcellular structures characteristic of Rickettsiella infections. Molecular phylogenetic analysis based on 16S ribosomal RNA and signal recognition particle receptor (FtsY) encoding sequences demonstrates that the wireworm pathogen belongs to the taxonomic genus Rickettsiella. Therefore, the new pathotype designation ‘R. agriotidis’ is proposed to refer to this organism. Moreover, genetic analysis makes it likely that—on the basis of the currently accepted organization of the genus Rickettsiella—this new pathotype should be considered a synonym of the nomenclatural type species, Rickettsiella popilliae.

Morphological and molecular investigations of a microsporidium infecting the European grape vine moth, Lobesia botrana Den. et Schiff., and its taxonomic determination as Cystosporogenes legeri nov. comb.

We have isolated a microsporidium from a laboratory stock of the European grape vine moth, Lobesia botrana Den. et Schiff. (Lepidoptera, Tortricidae). Screening of this stock showed an infection rate of more than 90%, whereas field collected larvae from three different locations in Rhineland-Palatinate (Germany) did not demonstrate any signs of infection. Light and electron microscopic investigations of infected insects showed that gross pathology, morphology, and ultrastructure of the microsporidium are similar to those described earlier for Pleistophora legeri. Comparative phylogenetic analysis of the small subunit rDNA using maximum likelihood, maximum parsimony, and neighbour joining distance methods showed that our isolate was closely related to Cystosporogenes operophterae. Based on our morphological and molecular investigations we propose to rename this species Cystosporogenes legeri nov. comb.