Wolbachia infections of the butterfly Eurema mandarina interfere with embryonic development of the sawfly Athalia rosae.

Although maternally transmitted microorganisms such as Wolbachia are well known to have a variety of effects on the reproduction of diverse insect species, little is known about the underlying mechanisms of actions. Artificial transfer of Wolbachia between taxonomically distant host species may provide insights into Wolbachia-induced manipulations of hosts. Here we performed a cross-order transfer of feminizing Wolbachia derived from a butterfly, Eurema mandarina. The Wolbachia were propagated in the Eurema hecabe cell line, called NTU-YB, and then used to inject prepupal/pupal females of a Wolbachia-free hymenopteran sawfly, Athalia rosae. The 14 females that emerged as adults looked morphologically and behaviorally healthy, and ovarian development appeared normal on dissection. However, in contrast to the control, none of the 333 eggs harbored by the seven Wolbachia-injected females developed successfully. Similarly, none of the 140 eggs laid on host plant by the four Wolbachia-injected females, which were mated with males, showed any signs of development. Wolbachia infection was detected from whole-body samples of the inoculated individuals, but not from the eggs they produced. Disruption of embryonic development despite the absence of Wolbachia in the egg cytoplasm may represent a new phenotype involving maternal effects that result in female sterility.

A new spiroplasma isolate from the field cricket (Gryllus bimaculatus) in Taiwan.

We briefly described the morphology and transmission pathway of a Spiroplasma sp. isolated from the field cricket, Gryllus bimaculatus in Taiwan, followed by the phylogenetic analysis based on the 16S rRNA gene sequence. The cricket spiroplasma infected the hemolymph, gut, muscle tissues and tracheal cells; therefore we suggest that the pathogen invaded tissues and organs from the hemolymph through the tracheal system and the endoplasmic reticular system. Based on 16S rRNA gene sequences and the phylogeny, this spiroplasma was most closely related to Spiroplasma platyhelix (Identity=95%) isolated from the dragonfly Pachydiplax longipennis and belongs to the Ixodetis clade.

A new microsporidium, Triwangia caridinae gen. nov., sp. nov. parasitizing fresh water shrimp, Caridina formosae (Decapoda: Atyidae) in Taiwan.

A new microsporidium was isolated from the endemic, Taiwanese shrimp, Caridina formosae (Decapoda, Atyidae) from northern Taiwan. A conspicuous symptom of infection was presence of opaque white xenomas located in the proximity of the alimentary tract, the surface of the hepatopancreas, and the gills. A fully developed xenoma consisted of a hard, thick capsule filled with sporophorous vesicles containing multiple spores. Microsporidia developed synchronously within the same sporophorous vesicle, although the stage of parasite development differed among the vesicles. Fresh spores were pyriform, mononucleated and measured 6.53 × 4.38 µm. The polar filament was anisofilar with 9-11 coils. Phylogenetic analysis based on the small subunit ribosomal DNA sequence showed that the isolate is most similar to the fish microsporidian clade containing the genera Kabatana, Microgemma, Potaspora, Spraguea, and Teramicra. The highest sequence identity, 80%, was with Spraguea spp. Based on pathogenesis, life cycle and phylogenetic analysis, we erect a new genus and species, Triwangia caridinae for the novel microsporidium.

Nosema ceranae infection intensity highly correlates with temperature.

Nosema ceranae, a microsporidian entomopathogen, was first reported from honey bees, Apis mellifera, in 2005 in Taiwan (Huang et al., 2007) and has become a major concern in apiculture worldwide. In Taiwan, we found one infection peak for N. ceranae during the winter months, compared to two peaks in spring and fall reported in 1980 for Nosema apis. N. ceranae infection intensity in apiaries reached a high level earlier than N. apis, a possible factor in replacement. We found a significant negative correlation of N. ceranae pathogen load with temperature; the highest spore counts were recorded at an average temperature of approximately 15 °C and infection intensity equaled the annual average at 23.8 °C. This model corresponds with published results but is most reliable for subtropical to tropical climates.

A new cell line (NTU-SE) from pupal tissues of the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae), is highly susceptible to S. exigua multiple nucleopolyhedrovirus (SeMNPV) and Autographa californica MNPV (AcMNPV).

A new continuous cell line, NTU-SE, was established from the pupal tissues of an economically important pest, the beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae). This cell line contains four major morphologic types: round, polymorphic, spindle-shaped, and comma-shaped cells. The population doubling time of this new line in TNM-FH medium supplemented with 8% fetal bovine serum (FBS) at 28°C is 35.5h. The chromosomal spread from NTU-SE cells is typical to the chromosomal morphology of lepidopteran cell lines. Confidently, NTU-SE cell line is a new cell line that exhibits distinct isozyme patterns of esterase, lactate dehydrogenase (LDH), and malate dehydrogenase (MDH) from those of the other insect cell lines. In addition, the DNA sequence of the nuclear ribosomal internal transcribed spacer (ITS) region of NTU-SE cells is above 96% identical to that sequence of S. exigua larvae, as compared to only 66% identical to that of S. litura larvae. The NTU-SE cell line is highly susceptible to S. exigua multiple nucleopolyhedrovirus (SeMNPV) and Autographa californica MNPV (AcMNPV). Therefore, a highly virulent SeMNPV strain, SeMNPV-1, had been successfully isolated and propagated in NTU-SE cells. We conclude that the NTU-SE cell line will be a useful tool for the selection and mass production of highly virulent SeMNPV strains for the S. exigua biocontrol and the baculovirus based recombinant protein expression systems.

Genomic sequencing and analyses of Lymantria xylina multiple nucleopolyhedrovirus.

BACKGROUND: Outbreaks of the casuarina moth, Lymantria xylina Swinehoe (Lepidoptera: Lymantriidae), which is a very important forest pest in Taiwan, have occurred every five to 10 years. This moth has expanded its range of host plants to include more than 65 species of broadleaf trees. LyxyMNPV (L. xylina multiple nucleopolyhedrovirus) is highly virulent to the casuarina moth and has been investigated as a possible biopesticide for controlling this moth. LdMNPV-like virus has also been isolated from Lymantria xylina larvae but LyxyMNPV was more virulent than LdMNPV-like virus both in NTU-LY and IPLB-LD-652Y cell lines. To better understand LyxyMNPV, the nucleotide sequence of the LyxyMNPV DNA genome was determined and analysed. RESULTS: The genome of LyxyMNPV consists of 156,344 bases, has a G+C content of 53.4% and contains 157 putative open reading frames (ORFs). The gene content and gene order of LyxyMNPV were similar to those of LdMNPV, with 151 ORFs identified as homologous to those reported in the LdMNPV genome. Two genes (Lyxy49 and Lyxy123) were homologous to other baculoviruses, and four unique LyxyMNPV ORFs (Lyxy11, Lyxy19, Lyxy130 and Lyxy131) were identified in the LyxyMNPV genome, including a gag-like gene that was not reported in baculoviruses. LdMNPV contains 23 ORFs that are absent in LyxyMNPV. Readily identifiable homologues of the gene host range factor-1 (hrf-1), which appears to be involved in the susceptibility of L. dispar to NPV infection, were not present in LyxyMNPV. Additionally, two putative odv-e27 homologues were identified in LyxyMNPV. The LyxyMNPV genome encoded 14 bro genes compared with 16 in LdMNPV, which occupied more than 8% of the LyxyMNPV genome. Thirteen homologous regions (hrs) were identified containing 48 repeated sequences composed of 30-bp imperfect palindromes. However, they differed in the relative positions, number of repeats and orientation in the genome compared to LdMNPV. CONCLUSION: The gene parity plot analysis, percent identity of the gene homologues and a phylogenetic analysis suggested that LyxyMNPV is a Group II NPV that is most closely related to LdMNPV but with a highly distinct genomic organisation.

A new microsporidian species, Vairimorpha ocinarae n. sp., isolated from Ocinara lida Moore (Lepidoptera: Bombycidae) in Taiwan.

A new microsporidium was isolated from Ocinara lida Moore (Lepidoptera: Bombycidae), a pest of Ficus microcarpa L. f. in Taiwan. The microsporidium produces systemic infections in O. lida larvae; the midgut epithelium, Malpighian tubules, and midgut muscle tissues were the target tissues for this isolate, and atrophied fat body tissues were found in heavily infected larvae. Two types of spores were observed, diplokaroytic spores with 11-13 coils of polar tube, and monokaryotic spores with 12 coils of the polar tube that developed within a sporophorous vesicle to form octospores. Electron-dense granules were abundant in the episporontal space of the sporophorous vesicles, and were similar to those of Vairimorpha invictae isolated from Solenopsis invicta, but different from granules or inclusions of other Vairimorpha species. Based on the phylogenetic analysis of the small subunit ribosomal DNA sequence, this isolate is unique within the Vairimorpha complex. Morphological and genetic characters showed this isolate to be a new species. It is placed in the genus Vairimorpha and is described as Vairimorpha ocinarae n. sp.

Morphological and molecular studies of a microsporidium (Nosema sp.) isolated from the thee spot grass yellow butterfly, Eurema blanda arsakia (Lepidoptera: Pieridae).

A microsporidium possessing molecular and morphological characteristics of the genus Nosema was isolated from larvae of the thee-spot grass yellow butterfly, Eurema blanda arsakia. The complete rRNA gene sequences of the E. blanda isolate contained 4,428 base pairs (GenBank Accession No. EU338534). The organization of the rRNA genes is LSU rRNA-ITS-SSU rRNA-IGS-5S, which corresponds with that of Nosema species closely related to Nosema bombycis. Phylogenetic analysis based on rRNA gene sequences show that this isolate is closely related to Nosema bombycis, Nosema plutellae, Nosema spodopterae, and Nosema antheraeae. The ultrastructure of all developmental stages of this microsporidium confirmed its placement in the genus Nosema. The isolate was successfully propagated in cell lines IPLB-LD652Y (Lymantria dispar) and NTU-LY (Lymantria xylina) and, in the in vitro system, it was frequently found to develop in the nuclei of the host cells, a circumstance that seldom occurs in other Nosema species. An extra-cellular vegetative stage of this microsporidium was also observed in the culture medium after 14 days of infection. The ECMDFs might be released from disrupted host cells.

A new nucleopolyhedrovirus strain (LdMNPV-like virus) with a defective fp25 gene from Lymantria xylina (Lepidoptera: Lymantriidae) in Taiwan.

A new multiple nucleopolyhedrovirus strain was isolated from casuarina moth, Lymantria xylina Swinhoe, (Lepidoptera: Lymantriidae) in Taiwan. This Lymantria-derived virus can be propagated in IPLB-LD-652Y and NTU-LY cell lines and showed a few polyhedra (occlusion bodies) CPE in the infected cells. The restriction fragment length polymorphism (RFLP) profiles of whole genome indicated that this virus is distinct from LyxyMNPV and the virus genome size was approximately 139 kbps, which was smaller than that of LyxyMNPV. The molecular phylogenetic analyses of three important genes (polyhedrin, lef-8 and lef-9) were performed. Polyhedrin, LEF-8 and LEF-9 putative amino acid analyses of this virus revealed that this virus belongs to Group II NPV and closely related to LdMNPV than to LyxyMNPV. The phylogenetic distance analysis was further clarified the relationship to LdMNPV and this virus provisionally named LdMNPV-like virus. A significant deletion of a 44bp sequence found in LdMNPV-like virus was noted in the fp25k sequences of LdMNPV and LyxyMNPV and may play an important role in the few polyhedra CPE. In ultrastructural observations, the nuclei of the infected LD host cells contained large occlusion bodies (OBs), and few OBs, which presented as one or two OBs in a nucleus that was otherwise filled with free nuclocapsids and virions. We concluded that this LdMNPV-like virus is a new LdMNPV strain from L. xylina.

Characterization of a new insect cell line (NTU-YB) derived from the common grass yellow butterfly, Eurema hecabe (Linnaeus) (Pieridae: Lepidoptera) and its susceptibility to microsporidia.

A new lepidopteran cell line, NTU-YB, was derived from pupal tissue of Eurema hecabe (Linnaeus) (Pieridae: Lepidoptera). The doubling time of YB cells in TNM-FH medium supplemented with 8% FBS at 28 degrees C was 26.87h. The chromosome numbers of YB cells varied widely from 21 to 196 with a mean of 86. Compared to other insect cell lines, the YB cells produced distinct esterase, malate dehydrogenase, and lactate dehydrogenase isozyme patterns. Identity of the internal transcribed spacer region-I (ITS-I) of YB cells to E. hecabe larvae was 96% and to Eurema blanda larvae (tissue isolated from head) was 81%. The YB cells were permissive to Nosema sp. isolated from E. blanda and the infected YB cells showed obvious cytopathic effects after 3weeks post inoculation. The highest level of spore production was at 4weeks post inoculation when cells were infected with the Nosema isolate, and spore production was 1.34+/-0.9x10(6)spore/ml. Ultrastructrual studies showed that YB cells can host in vitro propagation of the E. blanda Nosema isolate, and developing stages were observed in the host cell nuclei as observed in the natural host, E. blanda. The NTU-YB cell line is also susceptible to Nosema bombycis.

Imaging polyhedral inclusion bodies of nuclear polyhedrosis viruses with second harmonic generation microscopy.

We studied the polarization anisotropy of second harmonic generation (SHG) in polyhedral inclusion bodies (PIBs) of nuclear polyhedrosis viruses (NPV). Due to a body-centered-cubic arrangement of polyhedrin trimers, a characteristic SHG polarization property with a mixture of I23 and I3 symmetry was measured from PIBs. With this characteristic SHG anisotropy, it provides an intrinsic nonlinear signal for virus infection studies in living cells. With multimodal harmonic generation microscopy, we also demonstrated 3D imaging on PIBs of NPV in living cells. The distribution and the number of PIBs in intact infected cells can be revealed without the help of fluorescent labeling.

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay.

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell culture system. This system was developed to express foreign genes under the control of the baculoviral promoter in transient expression plasmids. Furthermore, this system can be applied to a functional assay of either the baculovirus itself or foreign proteins. The most widely and commercially available transient gene expression system is developed based on the immediate-early gene (IE) promoter of Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV). However, a low expression level of foreign genes in insect cells was observed. Therefore, a transient gene expression system was constructed for improving protein expression. In this system, recombinant plasmids were constructed to contain the target sequence under the control of the Drosophila heat shock 70 (Dhsp70) promoter. This protocol presents the application of this heat shock-based pDHsp/V5-His (V5 epitope with 6 histidine)/Spodoptera frugiperda cell (sf9 cell) system; this system is available not only for gene expression but also for evaluating the anti-apoptotic activity of candidate proteins in insect cells. Furthermore, this system can be either transfected with one recombinant plasmid or co-transfected two potentially functionally antagonistic recombinant plasmids in insect cells. The protocol demonstrates the efficiency of this system and provides a practical case of this technique.

The 5' untranslated region of Perina nuda virus (PnV) possesses a strong internal translation activity in baculovirus-infected insect cells.

A bicistronic baculovirus expression vector and fluorescent protein-based assays were used to identify the sequences that possess internal translation activity in baculovirus-infected insect cells. We demonstrated that the 5' untranslated region (5'UTR; 473 nucleotides) of Perina nuda virus (PnV) and the 5'UTR (579 nucleotides) of Rhopalosiphum padi virus (RhPV), but not the IRES sequence of Cricket paralysis virus, have internal translation activity in baculovirus-infected Sf21 cells. In addition, we found that including the first 22 codons of the predicted PnV open reading frame (ORF; a total of 539 nucleotides) enhanced internal translation activity by approximately 18 times. This is the first report of internal translation activity for a baculovirus expression system (BEVS) in the iflavirus 5' sequence and may facilitate the development of polycistronic baculovirus transfer vectors that can be used in BEVS for the production of multiple protein complexes.

Evidence from molecular markers and population genetic analyses suggests recent invasions of the Western North Pacific region by biotypes B and Q of Bemisia tabaci (Gennadius).

Invasive events by Bemisia tabaci (Gennadius) biotypes in various parts of the world are of continuing interest. The most famous is biotype B that has caused great economic losses globally. In addition, biotype Q has also recently been reported to be a new invasive pest. These two biotypes have been monitored for some time in the Western North Pacific region, but the invasive events and population genetic structures of these two biotypes are still not clear in this region. In this study, the mitochondrial cytochrome oxidase I (COI) gene was used to reconstruct a phylogenetic tree for identifying biotypes B and Q and to study the relationships between invasive events and ornamental plants. Population genetic analyses of mtCOI sequences were also used to study the genetic relationships within and between populations. A combination of a phylogenetic tree and haplotype analysis suggested the recent invasion of biotype Q in this region is related to the international ornamental trade from the Mediterranean region. Low levels of haplotype diversity and nucleotide diversity indicate that the presence of biotypes B and Q in the Western North Pacific region are caused by multiple invasions. Hierarchical analysis of molecular variance supports the hypothesis of multiple invasions. In addition, high sequence identities and low genetic distances within and between populations of the two biotypes revealed that these invasive events occurred recently. The low levels of genetic differentiation revealed by pairwise F (ST) values between populations also suggests the invasions were recent. Therefore, results of this study suggested that biotypes B and Q entered this region through multiple recent invasions. A quarantine of agricultural crops may be necessary to prevent further invasions.

Complete sequence and structure of ribosomal RNA gene of Heterosporis anguillarum.

The ribosomal RNA (rRNA) gene region of the microsporidium Heterosporis anguillarum has been examined. Complete DNA sequence data (4060 bp, GenBank Accession No. AF402839) of the rRNA gene of H. anguillarum are presented for the small subunit gene (SSU rRNA: 1359 bp), the internal transcribed spacer (ITS: 37 bp), and the large subunit gene (LSU rRNA: 2664 bp). The secondary structures of the H. anguillarum SSU and LSU rRNA genes are constructed and described. This is the first complete sequence of an rRNA gene published for a fish-infecting microsporidian species. In the phylogenetic analysis, the sequences, including partial SSU rRNA, ITS, and partial LSU rRNA sequences of the fish-infecting microsporidia, were aligned and analysed. The taxonomic position of H. anguillarum as suggested by Lom et al. (2000; Dis Aquat Org 43:225-231) is confirmed in this paper.

White spot syndrome virus (WSSV) PCR-positive Artemia cysts yield PCR-negative nauplii that fail to transmit WSSV when fed to shrimp postlarvae.

Positive results were obtained with nested white spot syndrome virus (WSSV) diagnostic PCR performed on 5 commercial brands of dry-packed Artemia cysts using several WSSV genomic sequence-specific primers. In 2 brands, PCR and nucleotide sequence analysis found C-->T and C-->G point mutations in the pms 146 WSSV amplicon, but in all 5 brands, the nucleotide sequences that were successfully amplified by the rrl, rr2 and tk-tmk gene-specific primer sets were identical to those of Penaeus monodon WSSV. However, despite the inarguable presence of WSSV or WSSV-like template DNA, we were unable to detect WSSV by PCR in hatched nauplii derived from PCR-positive cysts or in P. monodon postlarvae fed Artemia nauplii hatched from such cysts. Most simply, these results suggested that the cysts were externally contaminated with WSSV or WSSV-like template material that was removed during hatching and washing of the nauplii. Given the small sequence variations found, it may also have been a variety of WSSV non-infectious for P. monodon or Artemia and derived from other crustaceans or arthropods in the Artemia environment. However, we could not establish this conclusively and a small possibility remained that the PCR template in these tests was derived from WSSV template present internally in the cysts and derived from infected Artemia adults. However small, this possibility must be vigorously tested, given the impact that a positive outcome could have on the shrimp industry.

Development of a prokaryotic-like polycistronic baculovirus expression vector by the linkage of two internal ribosome entry sites.

Recombinant baculoviruses are suitable for the high-level production of large multi-protein complexes. A tri-cistronic expression vector was constructed by the inclusion of two internal ribosome entry sites (IRESs). In this novel polycistronic vector, one single polyhedrin promoter controlled the transcription of a tri-cistronic transcript. Also, the first cistron was translated through a cap-dependent mechanism, while the second and third cistrons were translated by the IRESs derived from the 5' UTR of Rhopalosiphum padi virus (RhPV) and Perina nuda virus (PnV), respectively. The ratio of tri-cistronic gene expression levels produced by the three translational initiation modules is about 2:1:1 (cap:PnV IRES:RhPV IRES). This study indicates that polycistronic genes can be co-expressed at the translational level as in prokaryotic expression system by baculovirus biotechnology.

Genomic and host range studies of Maruca vitrata nucleopolyhedrovirus.

The complete genome of the Maruca vitrata nucleopolyhedrovirus (MaviNPV) isolated from the legume pod borer, Maruca vitrata (Lepidoptera: Pyralidae), was sequenced. It was found to be 111 953 bp in length, with an overall 39 % G+C content, and contained 126 open reading frames (ORFs) encoding predicted proteins of over 50 aa. The gene content and gene order of MaviNPV have the highest similarity to those of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and their shared homologous genes are 100 % collinear. In fact, MaviNPV seems to be a mini-AcMNPV that is native to Taiwan and possesses a smaller genome with fewer auxiliary genes than the AcMNPV type species. Except for one ORF (Mv74), all of the MaviNPV ORFs have homologues in the AcMNPV genome. MaviNPV is the first lepidopteran-specific baculovirus to lack homologues of vfgf and odv-e66. In addition, MaviNPV lacks the baculovirus repeat ORF (bro) gene that corresponds to AcMNPV ORF2. Five homologous regions (hrs) were located within the MaviNPV genome, and these contained a total of 44 imperfect palindromes. Phylogenetic analysis of the whole genome revealed that MaviNPV was separated from the common ancestor of AcMNPV and Bombyx mori nucleopolyhedrovirus before these two viral species diverged from each other. Moreover, replication of MaviNPV in several cell lines and an egfp-MaviNPV infection assay revealed that IPLB-LD-652Y cells are only partially permissive to MaviNPV, which supports our conclusion that MaviNPV is a distinct species of the group I lepidopteran NPVs.

The comparison of rDNA spacer regions of Nosema ceranae isolates from different hosts and locations.

Nosema ceranae is a common microsporidian pathogen, one of two Nosema species that cause "nosema disease" in honeybees, Apis cerana and Apis mellifera. Samples of N. ceranae rDNA from isolates collected in different locations were sequenced and one 5S rRNA was found to be upstream of SSUrRNA. The rDNA arrangement, 5'-5S rRNA-IGS-SSUrRNA-ITS-LSUrRNA-3', was found in all isolates. In order to better understand the distribution relationship between N. ceranae isolates from A. cerana and A. mellifera, their rRNA spacer regions were also sequenced for analysis. Results showed that there are no significant differences between the IGS sequences of the isolates and no difference in the ITS sequence with the exception of one transition found in an isolate from Martinique. These isolates showed consistency in the IGS phylogenic analysis suggesting that no transmission barrier exists between A. mellifera and A. cerana and there is no difference between isolates from geography separated areas.

Phylogenetic analysis of two putative Nosema isolates from Cruciferous Lepidopteran pests in Taiwan.

In this study, a new microsporidian, PX2, was isolated from the diamondback moth, Plutella xylostella, and then compared with another isolate (PX1), and with Nosema spodopterae and N. bombycis. Sequence data showed that the rRNA gene organizations of PX1 and PX2 exhibited a typical Nosema-specific organization: 5'-LSUrRNA (large subunit ribosomal RNA)-ITS (internal transcribed spacer)-SSUrRNA-IGS (intergenic spacer)-5S-3'. Phylogenetic analysis (maximum likelihood, neighbor joining, maximum parsimony, and Bayesian analysis) of the LSUrRNA and SSUrRNA gene sequences, and the sequences of the alpha-tubulin, beta-tubulin, and RPB1 (DNA dependent RNA polymerase II largest subunit) genes found that PX1 was closer to N. bombycis and N. spodopterae than to PX2. Comparison of the identities of the rRNA domains and of the other three genes showed a high divergence in the sequences of the rRNA spacer regions (ITS and IGS). This is consistent with the hypothesis that PX2, if not PX1, might represent a new Nosema species.