Microsporidian intrasporal sugars and their role in germination

The hypothesis that spores of terrestrial and aquatic microsporidia differ in their utilization of sugars was tested by evaluating the sugars in germinated and ungerminated spores of several species in each category. The aquatic species tested were Vavraia culicis, Edhazardia aedis, and Nosema algerae and the terrestrial species were Vairimorpha necatrix, Nosema disstriae, Nosema apis, Vairimorpha lymantriae, and Nosema spp. from Spodoptera exigua and Plutella xylostella. The percentage germination varied between species, ranging between 40 and 92%. Total sugars (anthrone reactive) and reducing sugars (Nelson's test) remained unchanged through germination in the three terrestrial species tested; however, reducing sugars increased significantly in the aquatic species. High-performance liquid chromatography and gas chromatography revealed a preponderance of trehalose in all species and large quantities of sorbitol in all species except N. algerae and E. aedis. Other sugars were present in some species in much lower concentrations. After germination no changes in sugar content were observed in terrestrial species; however, all aquatic species lost trehalose with a concomitant increase in fructose and/or glucose concentrations. Increased osmotic potential from breakdown of trehalose has been postulated to induce germination of the aquatic species, but another explanation must be found for the terrestrial species.     

Trehalose catabolism in microsporidia Nosema grylli spores

Some differences in trehalose catabolism were found for terrestrial and aquatic microsporidian species (Undeen, Van der Meer, 1999). In microsporidia species from aquatic hosts, the spore extrusion causes the intrasporal trehalose hydrolysis by trehalase that is followed by the drastic rise of reducing sugars (glucose) concentration. On the contrary, in tested terrestrial microsporidian species, total and reducing sugars remain unchanged through the germination. In this study we demonstrate by means of the enzymatic and paper chromatography methods, that in spores of microsporidia Nosema grylli, infecting fat bodies of crickets Gryllus bimaculatus, neither an increase of glucose concentration nor a reduction in intrasporal trehalose content takes place during the spore discharge. In this respect N. grylli is close to other terrestrial species. However, we have revealed in N. grylli spores activity of alpha,alpha-trehalase (EC 3.2.1.28) with acid pH-optimum like it was found by other authors in spores of aquatic microsporidia N. algerae. This result differs from the neutral pH-optimum (7.0) of trehalse of other terrestrial microsporidia N. apis. Concentration of trehalose in N. grylli spores reduces during long-term storage. All attempts to detect an activity of trehalose phosphorylase (synthase) (K phi 2.4.1.64), other potential key enzyme for trehalose catabolism in N. grylli spores have failed. The absence of changes of the sugar content in terrestrial microsporidian spores during the extrusion indicates, that the main physiological role of trehalose hydrolysis by trehalase in these species is catabolism of energy reserves for providing the long-term survival in the environment.     

Fixation of microsporidian spores for electron microscopy

Fresh and frozen spores of the microsporidia Nosema apis and Nosema bombi were fixed using various fixatives at different times and temperatures. Paraformaldehyde and technical formaldehyde gave results comparable to or better than glutaraldehyde. Increased fixation temperature improved the fixation of spores from terrestrial hosts. Freezing did not destroy the cytology of the spore.     

Physiological host specificity: a model using the European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae) and microsporidia of row crop and other stalk-boring hosts

We investigated vertical and horizontal transmission as means by which entomopathogenic microsporidia may be isolated in their hosts. Ostrinia nubilalis larvae were challenged with microsporidia isolated from other stalk-boring and row crop Lepidoptera and were susceptible to seven species. Two species were horizontally transmitted. A Nosema sp. from Eoreuma loftini was transmitted among O. nubilalis larvae but not among larvae of the E. loftini host. This species was also vertically transmitted to the offspring of infected O. nubilalis females. An rDNA sequence showed the E. loftini isolate to be Nosema pyrausta, a naturally occurring species in O. nubilalis. Our results suggest that both horizontal and vertical transmission provide physiological barriers to host switching in the microsporidia, thus restricting the natural host range.     

Diversity of Nosema associated with bumblebees (Bombus spp.) from China

Bumblebees (Bombus spp.) are important pollinators of many economically important crops and microsporidia are among the most important infections of these hosts. Using molecular markers, we screened a large sample (n=1,009 bees) of workers of 27 different Bombus spp. from China (Sichuan, Qinghai, Inner Mongolia, and Gansu provinces). The results showed that 62 individuals representing 12 Bombus spp. were infected by microsporidia with an overall prevalence of 6.1%. Based on the haplotypes (ssrRNA sequences), we confirmed the presence of Nosema bombi, Nosema ceranae and (likely) Nosema thomsoni. In addition, four new putatively novel taxa were identified by phylogenetic reconstruction: Nosema A, Nosema B-complex, Nosema C-complex and Nosema D-complex. In many cases, hosts were infected by more than one Nosema taxon. Possible caveats of sequence analyses are discussed.     

Small Subunit Ribosomal DNA Phylogeny of Various Microsporidia with Emphasis on AIDS Related Forms

ABSTRACT. Phylogenetic analysis of the small subunit ribosomal DNA of a broad range of representative microsporidia including five species from humans ( Enterocytozoon bieneusi, Nosema corneum, Septata intestinalis, Encephalitozoon hellem and Encephalitozoon cuniculi ), reveals that human microsporidia are polyphyletic in origin. Septata intestinalis and E. hellem are very similar to the mammalian parasite E. cuniculi . Based on the results of our phylogenetic analysis, we suggest that S. intestinalis be designated Encephalitozoon intestinalis . Furthermore, analysis of our data indicates that N. corneum is much more closely related to the insect parasite Endoreticulatus schubergi than it is to other Nosema species. This finding is supported by recent studies which have shown a similarity between E. schubergi and N. corneum based on the origin and development of the parasitophorous vacuole. Thus these opportunistic microsporidian parasites can originate from hosts closely or distantly related to humans. Finally, the phylogeny based on small subunit ribosomal DNA sequences is highly inconsistent with traditional classifications based on morphological characters. Many of the important morphological characters (diplokaryon, sporophorous vesicle, and meiosis) appear to have multiple origins.     

Comparative Analysis of the Proteins with Tandem Repeats from 8 Microsporidia and Characterization of a Novel Endospore Wall Protein Colocalizing with Polar Tube from Nosema bombycis

As a common feature of eukaryotic proteins, tandem amino acid repeat has been studied extensively in both animal and plant proteins. Here, a comparative analysis focusing on the proteins having tandem repeats was conducted in eight microsporidia, including four mammal‐infecting microsporidia (Encephalitozoon cuniculi, Encephalitozoon intestinalis, Encephalitozoon hellem and Encephalitozoon bieneusi) and four insect‐infecting microsporidia (Nosema apis, Nosema ceranae, Vavraia culicis and Nosema bombycis). We found that the proteins with tandem repeats were abundant in these species. The quantity of these proteins in insect‐infecting microsporidia was larger than that of mammal‐infecting microsporidia. Additionally, the hydrophilic residues were overrepresented in the tandem repeats of these eight microsporidian proteins and the amino acids residues in these tandem repeat sequences tend to be encoded by GC‐rich codons. The tandem repeat position within proteins of insect‐infecting microsporidia was randomly distributed, whereas the tandem repeats within proteins of mammal‐infecting microsporidia rarely tend to be present in the N terminal regions, when compared with those present in the C terminal and middle regions. Finally, a hypothetical protein EOB14572 possessing four tandem repeats was successfully characterized as a novel endospore wall protein, which colocalized with polar tube of N. bombycis. Our study provided useful insight for the study of the proteins with tandem repeats in N. bombycis, but also further enriched the spore wall components of this obligate unicellular eukaryotic parasite.     

鳞翅目昆虫病原微孢子虫研究进展

微孢子虫广泛存在于鳞翅目昆虫中,是一类重要的病原微生物。微孢子虫病一方面影响野外昆虫种群的自然平衡,另一方面对家蚕、柞蚕等经济和资源昆虫造成了严重的危害。微孢子虫分子生物学研究基础相对薄弱,再加上微孢子虫表面坚厚的孢壁,无疑增加了研究难度。随着核酸、蛋白质等生物大分子分离制备方法和高通量测序技术的不断更新发展,基于各种组学(Omics)研究微孢子虫的工作方兴未艾,并且有了一些重要的发现。本文综述了微孢子虫与鳞翅目昆虫寄主的相互作用及寄生于鳞翅目昆虫的病原微孢子虫基因组、转录组和蛋白质组进展情况,以期为微孢子虫的深入研究提供参考。这些昆虫微生物研究将为鳞翅目害虫生物防治提供新的思路,并对家蚕等经济昆虫微粒子病的诊断、防控及治疗产生积极影响。     

Nosema chrysorrhoeae n. sp. (Microsporidia), isolated from browntail moth (Euproctis chrysorrhoea L.) (Lepidoptera, Lymantriidae) in Bulgaria: characterization and phylogenetic relationships

A new microsporidian parasite Nosema chrysorrhoeae n. sp., isolated in Bulgaria from the browntail moth (Euproctis chrysorrhoea L.), is described. Its life cycle includes two sequential developmental cycles that are similar to the general developmental cycles of the Nosema-like microsporidia and are indistinguishable from those of two Nosema spp. from Lymantria dispar. The primary cycle takes place in the midgut tissues and produces binucleate primary spores. The secondary developmental cycle takes place exclusively in the silk glands and produces binucleate environmental spores. N. chrysorrhoeae is specific to the browntail moth. Phylogenetic analysis based on the ssu rRNA gene sequence places N. chrysorrhoeae in the Nosema/Vairimorpha clade, with the microsporidia from lymantriid and hymenopteran hosts. Partial sequences of the lsu rRNA gene and ITS of related species Nosema kovacevici (Purrini K., Weiser J., 1975. Natürliche Feinde des Goldafters, Euproctis chrysorrhoea L., im Gebiet von Kosovo, FSR Jugoslawien. Anzeiger fuer Sch?dlingskunde, Pflanzen-Umweltschutz, 48, 11-12), Nosema serbica Weiser, 1963 and Nosema sp. from Lymantria monacha was obtained and compared with N. chrysorrhoeae. The molecular data indicate the necessity of future taxonomic reevaluation of the genera Nosema and Vairimorpha.     

Incidence of Wolbachia in aquatic insects

Wolbachia is a genus of intracellular bacteria typically found within the reproductive systems of insects that manipulates those systems of their hosts. While current estimates of Wolbachia incidence suggest that it infects approximately half of all arthropod species, these estimates are based almost entirely on terrestrial insects. No systematic survey of Wolbachia in aquatic insects has been performed. To estimate Wolbachia incidence among aquatic insect species, we combined field‐collected samples from the Missouri River (251 samples from 58 species) with a global database from previously published surveys. The final database contained 5,598 samples of 2,687 total species (228 aquatic and 2,459 terrestrial). We estimate that 52% (95% CrIs: 44%–60%) of aquatic insect species carry Wolbachia, compared to 60% (58%–63%) of terrestrial insects. Among aquatic insects, infected orders included Odonata, Coleoptera, Trichoptera, Ephemeroptera, Diptera, Hemiptera, and Plecoptera. Incidence was highest within aquatic Diptera and Hemiptera (69%), Odonata (50%), and Coleoptera (53%), and was lowest within Ephemeroptera (13%). These results indicate that Wolbachia is common among aquatic insects, but incidence varies widely across orders and is especially uncertain in those orders with low sample sizes such as Ephemeroptera, Plecoptera, and Trichoptera.     

The preservation of infective spores of Octosporea muscaedomesticae in Phormia regina,of Nosema algerae in Anopheles stephensi,and of Nosema whitei in Tribolium castaneum by lyophilization

Infective spores of three species of microsporidia were subjected to the lyophilization process by employing varying media as cryoprotectants. The infectivity of the lyophilized spores was then tested against a standard fresh spore preparation in the appropriate host insect. Spores of Octosporea muscaedomesticae served as an experimental model and were rendered noninfective in host Phormia regina (Calliphoridae: Diptera) after lyophilization with the following cryoprotective agents: skim milk (12%), ascorbic acid (5%) combined with thiourea (5%), glycerol (10%), mesoinositol (5%), and equine serum. Spores of O. muscaedomesticae lyophilized or vacuum-dried in 50% sucrose as well as in the hosts' tissues remained highly infective for as long as 2 years at a dose of 10⁶ spores/fly and a trial length of 12 days. At a dose of 5 × 10⁴ spores/fly there was a slight decrease in infectivity of the spores which had been lyophilized in the host's abdomen after a 2-year storage period compared with that of fresh, nonlyophilized spores. Naked spores of Nosema algerae suspended in 50% sucrose and lyophilized produced infection in 50% of the host population of Anopheles stephensi (Culicidae: Diptera) compared with 70% infection produced by fresh non-lyophilized spores. Spores of Nosema whitei lyophilized within its host larva Tribolium castaneum (Tenebrionidae: Coleoptera) remained 100% infective at a dose of 5 × 10⁵ spores/gram diet. It is concluded that an aqueous solution of 50% sucrose and/or the host's tissues are excellent protectants for the cryogenic or vacuum-drying process of the above-named spores, and their protective function may apply also to other microsporidian species.     

Heterorhabditis spp., Neoaplectana spp., and Steinernema kraussei: Interspecific and intraspecific differences in infectivity for insects

The effectiveness of various dosages of different species/strains of nematodes was compared for Galleria mellonella and various pest insects that live in or pupate in soil. Neoaplectana feltiae (= carpocapsae), the only nematode species tested by most other workers, was never the most infective for any of the insect species tested and was least infective for two. All species/strains of nematode were able to kill insects of each species. The degree of infectivity of each of the nematode species/strains for different hosts varied considerably, and no one species/strain of nematode was the most infective for all insect species. This indicates the importance of testing a number of nematode species against any particular insect before commencing field evaluations for biological control.     

Microsporidian infections in Lymantria dispar larvae: interactions and effects of multiple species infections on pathogen horizontal transmission

The interactions in multiple species infections and effects on the horizontal transmission of three microsporidian species, Vairimorpha disparis, Nosema lymantriae and Endoreticulatus schubergi, infecting Lymantria dispar were evaluated in the laboratory. Simultaneous and sequential inoculations of host larvae were performed and the resulting infections were evaluated. Test larvae were exposed to the inoculated larvae to measure horizontal transmission. Dual species infections demonstrated interspecific competition between Nosema and Vairimorpha in the host larvae, but no observable competition occurred between Endoreticulatus and either of the other microsporidian species. Timing of inoculation was an important factor determining the outcome of competition between Nosema and Vairimorpha. The species inoculated first showed a higher rate of successful establishment; a time lag of 7 days between inoculations allowed the first species to essentially exclude the second. The microsporidia differed in efficiency of horizontal transmission. Nosema and Endoreticulatus were transmitted at very high rates, close to 100%. Horizontal transmission of Vairimorpha was less efficient, ranging from 25% to a maximum of 75%. The patterns of infection observed in inoculated larvae were reflected in the test larvae that acquired infections in the horizontal transmission experiments. Competition with Vairimorpha suppressed horizontal transmission of Nosema after simultaneous and sequential inoculation. In simultaneous inoculation experiments Endoreticulatus had no effect on transmission of Nosema and Vairimorpha.     

Accumulation of heat-shock proteins 70 in insect fat bodies as evidence of stress in Microsporidia-infected insects

At present a concept prevails that pathological alterations in insect hosts infected with microsporidia, and those associated with hormone imbalance may be explained by the production of juvenile hormone-like (JH) substances by microsporidia. According to another view point, this pathology is a consequence of the host response. We suggested that the microsporidian infection can provoke a stress reaction in insects, which may cause JH secretion by these insects. To confirm this hypothesis, we have analysed major stress protein Hsp70 levels in the infected insects. Using affinity chromatography on ATP-agarose and immunoblotting, we have shown that Hsp70 was accumulated in infected crickets, and that it was the host protein. The consequence of events accompanying the infection in the insects is discussed in relation to the response of hormonal system of the host organism.     

Plant and animal rhabdovirus host range: a bug's view

Rhabdoviruses affect human health, terrestrial and aquatic livestock and crops. Most rhabdoviruses are transmitted by insects to their vertebrate or plant hosts. For insect transmission to occur, rhabdoviruses must negotiate barriers to acquisition, replication, movement, escape and inoculation. A better understanding of the molecular interactions of rhabdoviruses with insects will clarify the complexities of rhabdovirus infection processes and epidemiology. A unique opportunity for studying how insects become hosts and vectors of rhabdoviruses is provided by five maize-infecting rhabdoviruses that are differentially transmitted by one or more related species of two divergent homopteran families.     

Transfer of Nosema locustae (Microsporidia) to Antonospora locustae n. comb. based on molecular and ultrastructural data

Nosema locustae is a microsporidian parasite of grasshopper pests that is used as a biological control agent, and is one of the emerging model systems for microsporidia. Due largely to its diplokaryotic nuclei, N. locustae has been classified in the genus Nosema, a large genus with members that infect a wide variety of insects. However, some molecular studies have cast doubt on the validity of certain Nosema species, and on the taxonomic position of N. locustae. To clarify the affinities of this important insect parasite we sequenced part of the rRNA operon of N. locustae and conducted a phylogenetic analysis using the complete small subunit rRNA gene. Nosema locustae is only distantly related to the nominotypic N. bombycis, and is instead closely related to Antonospora scoticae, a recently described parasite of bees. We examined the ultrastructure of mature N. locustae spores, and found the spore wall to differ from true Nosema species in having a multi-layered exospore resembling that of Antonospora (one of the distinguishing features of that genus). Based on both molecular and morphological evidence, therefore, we propose transferring N. locustae to the genus Antonospora, as Antonospora locustae n. comb.     

Diversity and abundance of insect herbivores foraging on seedlings in a rainforest in Guyana

1. Free-living insect herbivores foraging on 10 000 tagged seedlings representing five species of common rainforest trees were surveyed monthly for more than 1 year in an unlogged forest plot of 1 km² in Guyana. 2. Overall, 9056 insect specimens were collected. Most were sap-sucking insects, which represented at least 244 species belonging to 25 families. Leaf-chewing insects included at least 101 species belonging to 16 families. Herbivore densities were among the lowest densities reported in tropical rainforests to date: 2.4 individuals per square metre of foliage. 3. Insect host specificity was assessed by calculating Lloyd’s index of patchiness from distributional records and considering feeding records in captivity and in situ. Generalists represented 84 and 78% of sap-sucking species and individuals, and 75 and 42% of leaf-chewing species and individuals. In particular, several species of polyphagous xylem-feeding Cicadellinae were strikingly abundant on all hosts. 4. The high incidence of generalist insects suggests that the Janzen–Connell model, explaining rates of attack on seedlings as a density-dependent process resulting from contagion of specialist insects from parent trees, is unlikely to be valid in this study system. 5. Given the rarity of flushing events for the seedlings during the study period, the low insect densities, and the high proportion of generalists, the data also suggest that seedlings may represent a poor resource for free-living insect herbivores in rainforests.     

Two species of feminizing microsporidian parasite coexist in populations of Gammarus duebeni

The amphipod crustacean Gammarus duebeni hosts two species of vertically transmitted microsporidian parasites, Nosema granulosis and Microsporidium sp. A. Here it is demonstrated that these co-occurring parasite species both cause infected females to produce female-biased broods. A survey of European G. duebeni populations demonstrates that these two parasites co-occur in six of 10 populations. These findings contrast with the theoretical prediction that two vertically transmitted feminizing parasites should not coexist in a panmictic population of susceptible hosts at equilibrium. Possible explanations for the co-occurrence of the two feminizing microsporidia in G. duebeni include the recent invasion of a new parasite, horizontal transmission of one or both parasites and the spread of alleles for resistance to the dominant parasite in host populations.     

Detection of relationships among microsporidan isolates by electrophoretic analysis: hydrophobic extracts

Hydrophobic spore proteins were extracted from 11 microsporidan isolates obtained from 9 species of insects for which these microorganisms are pathogenic. Hydrophobic protein spectra were found to be stable when (1) two different genera of hosts were used for spore propagation, (2) hosts were reared at a variety of temperatures, or (3) protein was extracted from spores harvested at different stages of sporogenesis. Five consistent and distinct electrophoretic spectra were observed. Spectrum I was represented by 6 isolates including Nosema necatrix, Thelohania diazoma, Nosema plodiae, and Nosema sphingidis; spectrum II by Pleistophora sp; Spectrum III by Nosema whitei; spectrum IV by Thelohania legeri; and spectrum V by Nosema trichoplusia. The highly reproducible nature of these analyses indicated polyacrylamide gel disc electrophoresis of hydrophobic extracts can be used for the identification of Microsporida. Moreover, these analyses do not support the present classification, based mainly on the number of spores in a pansporoblast, inasfar as (1) some species of Nosema have the same pattern (I) as a species of Thelohania and (2) two species of Nosema have different patterns (III and V) in contrast to the Nosema species showing pattern I.     

Species richness of insect herbivore communities on Ficus in Papua New Guinea

Insect herbivores were sampled from the foliage of 15 species of Ficus (Moraceae) in rainforest and coastal habitats in the Madang area, Papua New Guinea. The collection included 13 193 individuals representing 349 species of leaf-chewing insects and 44 900 individuals representing 430 species of sap-sucking insects. Despite a high sampling intensity, the species accumulation curve did not reach an asymptote. This pattern was attributed to the highly aggregated distribution of insects on individual host trees. The number of insect species collected on a particular Ficus species ranged from 34 to 129 for leaf-chewing and from 51 to 219 for sap-sucking insects. Two Ficus species growing on the seashore sustained less speciose insect communities than their counterparts growing in forest. For the forest figs, significant predictors of insect species richness included leaf palatability and leaf production for leaf-chewing insects (40% of the variance explained), and tree density and leaf expansion for sap-sucking insects (75%). The high faunal overlap among Ficus communities and the importance of local resources for insect herbivores suggest that highly specialized interactions between insect herbivores and Ficus in Papua New Guinea have not been conserved in evolutionary time. This is at variance with the dogma of old, extremely specialized and conservative interactions between insect herbivores and their hosts, providing numerous ecological niches in the floristically rich tropics.