Vairimorpha Species Research Articles

Occurrence and distribution of the microsporidium Vairimorpha (Nosema) spp. in apiaries in Brazil - Systematic review

Nosemosis is one of the main diseases of bees, caused by microsporidia of the genus Vairimorpha (Nosema). The main etiologic agents are Vairimorpha apis and Vairimorpha ceranae. These pathogens are widely distributed around the world, and in Brazil, they have already been reported in some states, but these data have never been gathered in a review. The objective of this study was to perform a systematic review of the occurrence and distribution of the microsporidium Vairimorpha spp. in apiaries in Brazil. The search was done in the SciELO, PubMed, DOAJ, and Capes Journals (Scopus) databases. 14 articles were selected and published in English and Portuguese. In the included publications, only eight Brazilian states were referred (Bahia, Espírito Santo, Goiás, Minas Gerais, Piauí, Rio Grande do Sul, Santa Catarina, and São Paulo). No work was conducted in the northern of the country, and most of the publications are concentrated in the southern and southeastern. Regarding the Vairimorpha species, only V. apis and V. ceranae were investigated, and V. ceranae was predominantly detected. Most of the studies were done only with the Africanized bee Apis mellifera. This review showed which Brazilian regions have never been explored in terms of incidence and distribution of these pathogens, revealing a gap to be filled by the country's beekeeping surveillance and health systems. In addition, because of the great Brazilian biodiversity, it is evident that the occurrence and distribution of these pathogens also need to be investigated with other bee species.

Fall Treatment with Fumagillin Contributes to an Overwinter Shift in Vairimorpha Species Prevalence in Honey Bee Colonies in Western Canada.

(1) Background: Microsporidiosis (nosemosis) is an intestinal disorder of adult honey bees caused by the microsporidian pathogens Vairimorpha apis and Vairimorpha ceranae. In Canada, fumagillin is an approved antibiotic used to treat this disease. However, the recommended dosage is based on efficacy studies for V. apis, the native pathogen in European honey bees. Since the detection of V. ceranae in Apis mellifera, V. ceranae became more prevalent in managed European honey bees and seems to have replaced V. apis due to yet unknown reasons. (2) Methods: This colony study investigated the efficacy of fumagillin administered in the fall to colonies infected with both V. apis and V. ceranae and its effects on the Vairimorpha species' prevalence overwinter. Spore loads in control and fumagillin-treated colonies were analysed by microscopy; Vairimorpha species prevalence was determined molecularly and infection and treatment effects on colony productivity were assessed. (3) Results: Fall fumagillin treatment was associated with a temporary reduction in spore load, but there was no difference in spore loads between treated and control colonies the following spring. Interestingly, fumagillin-treated colonies had a significantly greater prevalence of V. ceranae relative to V. apis the following spring, suggesting fumagillin is less effective in controlling V. ceranae.

A new microsporidium, Vairimorpha subcoccinellae n. sp. (Microsporidia: Burenellidae), isolated from Subcoccinella vigintiquatuorpunctata L. (Coleoptera: Coccinellidae).

A new microsporidium was isolated from Subcoccinella vigintiquatuorpunctata L. (Coleoptera: Coccinellidae), a pest of Galega officinalis L. in Turkey. Infection in larval and adult stages was systemic with mature spores produced in the midgut, gonads, Malpighian tubules and, most extensively, fat body tissues. The microsporidium was polymorphic with two sporulation sequences producing two types of spores, binucleate spores with 13-15 coils of the polar tube, and uninucleate spores with 7 coils of the polar tube that developed within a sporophorous vesicle (SPV) to form meiospores. The 16S small subunit rRNA (SSU rRNA) gene of the microsporidium was sequenced and compared with twenty-seven microsporidian sequences from GenBank. Based on the phylogenetic analysis of the SSU rRNA sequence, this microsporidium is unique within the Vairimorpha group. Morphological and genetic characters indicate that the described microsporidium is dissimilar to all known Vairimorpha species, and so is named here as Vairimorpha subcoccinellae n. sp.

Morphological and molecular studies of Vairimorpha necatrix BM, a new strain of the microsporidium V. necatrix (microsporidia, burenellidae) recorded in the silkworm, Bombyx mori

Vairimorpha sp. BM (2012) is a recent isolate of the microsporidia from the silkworm in Shandong, China. The ultrastructure, tissue pathology and molecular characterization of this isolate is described in this study. This pathogenic fungus causes pebrine disease in silkworms which manifests as a systemic infection. Meanwhile, the silkworm eggs produced by the infected moths were examined using a microscope and PCR amplification. Neither spores nor the expected PCR band were observed, suggesting that no vertical transmission occurred in Bombyx mori. In addition, the ultrastructure of the isolate was studied by light microscopy and transmission electron microscopy. Two types of spores were observed: diplokaroytic spores with 13–17 coils of polar tubes and monokaryotic spores with less coils of polar tubes which could form octospores; however, no sporophorous vesicles were observed. Finally, phylogenetic analysis of the small subunit rRNA genes of Vairimorpha species showed that this isolate has a closer relationship to Vairimorpha necatrix than the other species studied. This result also is supported by phylogenetic analysis based on their actin genes, heat shock protein 70 (HSP70) and RNA polymerase II (RPB1). Based on the information gained during this study, we propose that this microsporidian species infecting B. mori should be given the name V. necatrix BM.

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.

Vairimorpha disparis n. comb. (Microsporidia: Burenellidae): A Redescription and Taxonomic Revision of Thelohania disparis Timofejeva 1956, a Microsporidian Parasite of the Gypsy Moth Lymantria dispar (L.) (Lepidoptera: Lymantriidae)

Investigation of pathogens of populations of the gypsy moth, Lymantria dispar (L.) in Central and Eastern Europe revealed the existence of a microsporidium (Fungi: Microsporidia) of the genus Vairimorpha. The parasite produced three spore morphotypes. Internally infective spores are formed in the gut and adjacent muscle and connective tissue; single diplokaryotic spores and monokaryotic spores grouped by eight in sporophorous vesicles develop in the fat body tissues. The small subunit rDNA gene sequences of various isolates of the Vairimorpha microsporidia, obtained from L. dispar in various habitats in the investigated region, revealed their mutual identity. In phylogenetic analyses, the organism clustered with other L. dispar microsporidia that form only diplokaryotic spores in the sporogony cycle. The octospores of certain microsporidia infecting Lepidoptera that were previously described as Thelohania spp., have recently been shown to be one of the several spore morphotypes produced by species in the genus Vairimorpha. Because the description and drawings of a parasite described as Thelohania disparis by Timofejeva fit the characteristics of Vairimorpha, and all octospore-producing microsporidia collected from L. dispar since 1985 are genetically identical Vairimorpha species, it is believed that the parasite characterized here is identical to T. disparis Timofejeva 1956, and is herein redescribed, characterized, and transferred to the genus Vairimorpha as the new combination Vairimorpha disparis n. comb.

Phylogenetic relationships of three new microsporidian isolates from the silkworm, Bombyx mori

The pathogenicity, mode of transmission, tissue specificity of infection and the small subunit rRNA (SSU-rRNA) gene sequences of the three new microsporidian isolates from the silkworm Bombyx mori were studied. Out of the three, NIK-2r revealed life cycle features and SSU-rRNA gene sequence similar to Nosema bombycis, suggesting that it is N. bombycis. The other two, NIK-4m and NIK-3h, differed from each other as well as from N. bombycis. NIK-4m was highly pathogenic and did not show any vertical transmission, in accordance with the apparent lack of gonadal infection, whereas NIK-3h was less pathogenic and vertical transmission was not detected but could not be excluded. Phylogenetic analysis based on SSU-rRNA gene sequence placed NIK-3h and NIK-4m in a distinct clade that included almost all the Vairimorpha species and Nosema species that infect lepidopteran and non-lepidopteran hosts, while NIK-2r was included in a clade containing almost all the Nosema isolates that infect only lepidopteran hosts. Thus, we have presented molecular evidence that one of the three isolates is in fact the type species N. bombycis, while the other two isolates are Vairimorpha spp. There was distinct separation of microsporidian isolates infecting only lepidopteran hosts and those infecting lepidopteran and non-lepidopteran hosts, reflecting possible co-evolution of hosts and microsporidian isolates.

Ultrastructural characteristics and small subunit ribosomal DNA sequence of Vairimorpha cheracis sp. nov., (Microspora: Burenellidae), a parasite of the Australian yabby, Cherax destructor (Decapoda: Parastacidae)

This is the first record of a species of Vairimorpha infecting a crustacean host. Vairimorpha cheracis sp. nov. was found in a highland population of the Australian freshwater crayfish, Cherax destructor. The majority of spores and earlier developmental stages of V. cheracis sp. nov. were found within striated muscle cells of the thorax, abdomen, and appendages of the crayfish. Only octosporoblastic sporogony within sporophorous vesicles (SPVs) was observed. Diplokaryotic sporonts separated into two uninucleate daughter cells, each of which gave rise to four sporoblasts in a rosette-shaped plasmodium, so that eight uninucleate spores were produced within the persistent ovoid SPV. Ultrastructural features of stages in the octosporoblastic sequence were similar to those described for Vairimorpha necatrix, the type species. Mature spores were pyriform in shape and averaged 3.4 × 1.9 μm in dimensions. The anterior polaroplast was lamellar in structure, and the posterior polaroplast vesicular. The polar filament was coiled 10–12 times, lateral to the posterior vacuole. The small subunit ribosomal DNA (SSU rDNA) of V. cheracis sp. nov. was sequenced and compared with other microsporidia. V. cheracis sp. nov. showed over 97% sequence identity with Vairimorpha imperfecta and five species of Nosema, and only 86% sequence identity with V. necatrix. The need for a taxonomic revision of the Nosema/Vairimorpha group of species is discussed.

Vairimorpha imperfecta n.sp., a microsporidian exhibiting an abortive octosporous sporogony in Plutella xylostella L. (Lepidoptera: Yponomeutidae)

The microsporidian genus Nosema is characterized by development in direct control with host cell cytoplasm, diplokaryotic nuclei throughout development and disporous sporogony. The genus Vairimorpha exhibits the same features plus an octoporous sporogony producing uninucleate spores in a sporophorous vesicle. A microsporidium from diamondback moth, Plutella xylostella, falls between Nosema and Vairimorpha in that it initiates but fails to complete the octosporous sequence in this host. The name Vairimorpha imperfecta n.sp. is proposed. Merogony is mainly by formation of buds from multinucleate meronts, the buds remaining attached in chains. Diplokaryotic spores measure 4.3 x 2.0 microns (fresh) and have 15.5 coils of the polar tube in 1 rank. The octosporous sporogony is aborted owing to irregular formation of nuclear spindles, incomplete cytoplasmic fission and bizarre deposition of electron-dense episporontal secretions. Phylogenetic analyses of the sequences of the small subunit rRNA genes of V. imperfecta and of several Nosema and Vairimorpha spp. place V. imperfecta in a clade with Nosema spp. from Lepidoptera rather than in the clade containing the more typical species of Vairimorpha. It is suggested that the ancestors of the Vairimorpha/Nosema complex of species exhibited both disporous and octosporous sporogonies, as does the type species of Vairimorpha, Vairimorpha necatrix. It would follow that true Nosema spp. have lost the ability to express an octosporous sequence and that V. imperfecta is in the process of losing it. It is proposed that the genera Nosema and Vairimorpha be placed in the same family Nosematidae Labbé 1899, rather than in separate families and orders as at present.

Phylogenetic Relationships among Vairimorpha and Nosema Species (Microspora) Based on Ribosomal RNA Sequence Data

A portion (approximately 350 nucleotides) of the large subunit ribosomal RNA (rRNA) 5′ to the 580 region (Escherichia coli numbering) was sequenced using the reverse transcriptase dideoxy method and compared for several species of Nosema and Vairimorpha. Comparison among Nosema species suggests that this genus is composed of several unrelated groups. The group which includes the type species, Nosema bombycis, consists of closely related species found primarily in Lepidoptera. Other Nosema species sequenced (Nosema kingi, Nosema algerae, and Nosema locustae ) do not appear to be closely related to each other or to the lepidopteran Nosema group. Comparison among the Vairimorpha species indicates that two distinct but very closely related groups exist. The Lymantria group consists of species isolated from the gypsy moth, Lymantria dispar , while the Vairimorpha necatrix group consists of species isolated from other Lepidoptera. Intergeneric comparison of the sequence data suggests that the lepidopteran Nosema species are closely related to the Vairimorpha species.

An Ultrastructural Study of the Episporontal Inclusions Produced during Octosporogony by Five Species/Isolates of Vairimorpha (Microspora: Microsporida)

Two described species of Vairimorpha, Vairimorpha plodiae and Vairimorpha heterosporum , and three undescribed isolates from the lepidopteran species Heliothis armigera, Utethesia bella, and Alabama argillacea, respectively Ha, Ub, and 78-5, were studied by electron microscopy with emphasis on episporontal inclusions. Early secretory products appeared as small electron-dense, amorphous masses which usually became vesiculated or vacuolated in appearance. More significant differences were noted in the appearance of the late-stage lamellae formed by each isolate. In V. plodiae the lamellae exhibited a whorled or fingerprint configuration, while in V. heterosporum the lamellae were thickened and hinged in appearance being somewhat similar to those produced by isolate 78-5. The lamellae of isolate Ha were thin and organized in a regularly layered manner, while those of isolate Ub occurred as randomly oriented, curved structures coated on one or both surfaces with a less dense layer of material. Episporontal tubules were also produced by each isolate, generally adjacent to the sporont plasmalemma. Although the tubules of each isolate were similar in appearance and were coated with exospore material during sporoblast formation, the coated tubules for isolates Ha and Ub were found to be polygonal in cross section. As spores matured, these episporontal inclusions gradually disappeared. A discussion is presented of the potential value of such episporontal inclusions as taxonomic criteria at the species level.

Pulsed-Field Gel Electrophoresis of DNA from Four Microsporidian Isolates

Pulsed-field gel electrophoresis is used to visualize chromosome-sized DNA molecules of four microsporidian isolates from pest insects. Vavraia oncoperae isolated from Wiseana spp. caterpillars had 16 resolvable bands and the same species isolated from grass grubs had 14 resolvable bands. Chromosomal bands were more difficult to resolve for Nosema costelytrae and a Vairimorpha species from cabbage white butterflies, but electrophoresis revealed that these species had a minimum of 8 bands. The microsporidian DNA molecules resolved by this method are estimated to be between 130 and 1930 kilobases in size.

A species-specific enzyme-linked immunosorbent assay for Nosema furnacalis (Microspora: Nosematidae)

A serological assay has been developed that specifically reacts with Nosema furnacalis proteins. N. furnacalis spores were harvested from laboratory-reared Ostrinia nubilalis and purified by centrifugation through a Percoll gradient. Spores were germinated and soluble proteins were used to immunize rabbits and mice. Antibodies from hyperimmune animals were purified and used to develop an indirect enzyme-linked immunosorbent assay (ELISA). The assay detected N. furnacalis proteins in infected O. nubilalis larvae and pupae, in Helicoverpa zea larvae and pupae, and in H. zea cell culture. Latex beads sensitized with antibody did not agglutinate spores of N. furnacalis or N. pyrausta, indicating that the antigens are probably internal sporoplasm proteins rather than soluble surface proteins. No cross reaction was observed when the assay was tested against five species of Nosema: N. pyrausta in O. nubilalis; N. apis in Apis mellifera; N. locustae in Melanoplus femurrubrum, M. bivitattis, and M. lakinus; N. bombycis in Bombyx mori; and an undescribed species from Malacosoma americanum. Three species of Vairimorpha were also tested, all showing no cross reaction: V. necatrix, another pathogen of O. nubilalis, and two undescribed Vairimorpha spp. in Hyphantria cunea and Alabama argillacea. Western blot analysis confirmed specificity of the ELISA, distinguishing among N. furnacalis infected, N. pyrausta infected, and unifected O. nubilalis.

Impact of Vairimorpha necatrix and Vairimorpha sp. (Microspora: Microsporida) on Bonnetia comta (Diptera: Tachinidae) within Agrotis ipsilon (Lepidoptera: Noctuidae) hosts

The effect of Vairimorpha necatrix and Vairimorpha sp. on Bonnetia comta developing within pathogen-treated black cutworm, Agrotis ipsilon, hosts was studied. Parasitism by B. comta did not interfere with the near 100% mortality of A. ipsilon caused by either Vairimorpha species. Both microsporidia decreased the number of B. comta able to pupate from a. ipsilon hosts, the days required for adult parasitoid eclosion, and the weights of the puparia. Only in the V. necatrix treatments, however, were these effects significant ( P < 0.05) as determined by Duncan's (1955) multiple range test. The impact of each microsporidium on the parasitoid increased with an increase in the concentration of spores to which A. ipsilon was exposed. Both Vairimorpha species had a more detrimental effect on female B. comta than they did on male B. comta. Histological examination of B. comta maggots within V. necatrix- and Vairimorpha sp.-infected A. ipsilon showed the spores primarily restricted to the gut lumen. In older B. comta, dissected from dead Vairimorpha-infected hosts, the guts containing spores were much distended, some to the point that most other parasitoid tissues were absent. This suggests that the detrimental impact the host microsporidian infections had on the parasitoid were related to a nutritional deficiency caused by the accumulation of nondigestable spores in the parasitoid's gut lumen.

Interactions between Vairimorpha necatrix, Vairimorpha sp., and a nuclear polyhedrosis virus from Rachiplusia ou in Agrotis ipsilon larvae

Vairimorpha sp. and V. necatrix were assayed in combination with one another, and independently, with a nuclear polyhedrosis virus (Ro MNPV) from a mint looper, Rachiplusia ou, against neonate and third-stage black cutworm larvae, Agrotis ipsilon. Initially the effect of Vairimorpha sp. was subadditive, additive, or slightly inhibiting to the V. necatrix in the dual microsporidian assays; later, V. necatrix antagonized the effect of the Vairimorpha sp. In combination with Vairimorpha sp. gradients, V. necatrix significantly ( P < 0.05) reduced, in most instances, the LT 50 values in both neonate and third-stage larval assays. Ro MNPV assayed against neonate and third instars, in combination with either Vairimorpha sp. or V. necatrix gradients, usually significantly reduced the LT 50 values ( P < 0.05). Ro MNPV, when combined with either Vairimorpha species, had varying effects on its pathology. In all assays, the particular relationship that was expressed seemed to be a function of the concentration of each pathogen, which may indicate that the two microorganisms compete for entry and/or infective sites within the larval host. Larval size, which was significantly reduced ( P < 0.05) by both microsporidia, would be involved in such competition because it would limit the tissue mass available for infection. Histological examinations of larvae with dual infections revealed pathogens in the tissues that they normally infect. Vairimorpha sp. primarily infected epithelial, fat body, and Malpighian tubule tissue and, occasionally, muscle tissue. Viral polyhedral inclusion bodies were found in the same fat body tissues as the Vairimorpha spores.

Variation in Spore Polypeptides from Four Species of Vairimorpha

Spore polypeptides from four species of Vairimorpha were separated with sodium dodecyl sulfate polyacrylamide gel electrophoresis for identification and classification. The spore profile for each Vairimorpha species is unique and reproducible. The unweighed pair-group method with arithmethic means is used to construct a phenogram to summarize the relationship of the Vairimorphe species.