Benomyl: Effectiveness against the microsporidian Nosema heliothidis in the corn earworm,Heliothis zea

The fungicide benomyl was studied as a possible antimicrobial agent for obtaining Nosema heliothidis-free laboratory colonies of Heliothis zea. Newly hatched, transovarially infected larvae were placed on artificial diets containing 250, 500, or 1000 ppm benomyl. While late-stage larvae were found to be free of Nosema spores, low-level infections were found in pupae and newly emerged adults. The reduced intensity of infection in adults reared as larvae on treated diets was not correlated with a significant reduction in the efficiency of transovarian transmission. The chemical effect of benomyl was manifested by aberrant spores and vegetative stages and a rapid reduction in the number of microsporidian stages. However, small, isolated centers of infection in various host tissues resulted in a rapid resurgence of the microsporidiosis in pupae and adults. Thus, at the concentrations tested, benomyl was not effective in eliminating infection by N. heliothidis in H. zea. A discussion of the necessity for careful evaluation of the apparent suppression of microsporidioses by antimicrobial agents is also presented.     

Effect of the microsporidian Pleistophora schubergi on Anaitis efformata (Lepidoptera: Geometridae) and its elimination from a laboratory colony

Infection by the microsporidian Pleistophora schubergi was lethal to most larvae of Anaitis efformata, while sublethal infection retarded development and led to reduced longevity, weight, fecundity, and mating success in adults. Fumagillin had no effect on the level of P. schubergi infection. Benomyl suppressed infection for as long as treatment was given, but the microsporidian resurged during the nonfeeding pupal stage. P. schubergi was not transmitted transovarially and this facilitated its elimination from the colony of A. efformata in one generation through selective individual mating and rearing of the moth.     

Influence of Dimilin on a microsporidian infection in the gypsy moth Lymantria dispar (L.) (Lepidoptera: Lymantriidae)

Bioassay studies were conducted to investigate the influence of Dimilin (diflubenzuron), a chitinsynthetase inhibitor used for insecticidal control of the gypsy moth, Lymantria dispar, on the development and viability of a microsporidian pathogen of L. dispar. Before or after an infection with a Nosema species, L. dispar larvae were fed Dimilin in sublethal dosages. Dimilin fed to L. dispar larvae at 0.65 ng/cm² diet surface resulted in a total larval mortality of 53%. Although the microsporidian infection alone did not cause high mortality rates (9%), mortality increased to 96% when L. dispar larvae were inoculated with both Dimilin and Nosema spores. When Dimilin was fed to the larvae 24 h before or 6 days after inoculation with the microsporidium, the number of mature spores produced was significantly reduced. When Dimilin was fed to the larvae 24 h after microsporidian inoculation, the number of spores produced was not significantly reduced. Spores that were produced in larvae after Dimilin had been ingested with the diet were less infectious than spores produced in control larvae; the experimental infection rate decreased from 94% when spores obtained from control larvae were used, to 48 or 10% when spores obtained from larvae fed Dimilin 24 h or 6 days after Nosema inoculation, respectively, were used. Mature microsporidian spores washed in Dimilin solution prior to oral inoculation, however, were as infectious as spores stored in liquid nitrogen. We have shown that Dimilin interferes with the establishment of the parasite in its host. In addition, when Nosema sp. succeeds in infecting the L. dispar host despite treatment with Dimilin, the microsporidium does not develop optimally and spore production is reduced.     

Infection of Drosophila melanogaster by Tubulinosema kingi: stage-specific susceptibility and within-host proliferation

Despite its importance as a model organism very little is known about the interaction between Drosophila and its microsporidian pathogens. Here we report on the relative susceptibility of Drosophila melanogaster life history stages to infection by Tubulinosema kingi, and on patterns of pathogen proliferation. We find that only larvae can be infected, and that this susceptibility decreases with larval age. Following infection, the pathogen shows little subsequent proliferation in larvae, a limited amount in pupae while it replicates greatly in adults. We present evidence that the host launches a cellular immune response after infection with the pathogen, although its effectiveness remains to be demonstrated.     

Microsporidia Are Natural Intracellular Parasites of the Nematode Caenorhabditis elegans

For decades the soil nematode Caenorhabditis elegans has been an important model system for biology, but little is known about its natural ecology. Recently, C. elegans has become the focus of studies of innate immunity and several pathogens have been shown to cause lethal intestinal infections in C. elegans. However none of these pathogens has been shown to invade nematode intestinal cells, and no pathogen has been isolated from wild-caught C. elegans. Here we describe an intracellular pathogen isolated from wild-caught C. elegans that we show is a new species of microsporidia. Microsporidia comprise a large class of eukaryotic intracellular parasites that are medically and agriculturally important, but poorly understood. We show that microsporidian infection of the C. elegans intestine proceeds through distinct stages and is transmitted horizontally. Disruption of a conserved cytoskeletal structure in the intestine called the terminal web correlates with the release of microsporidian spores from infected cells, and appears to be part of a novel mechanism by which intracellular pathogens exit from infected cells. Unlike in bacterial intestinal infections, the p38 MAPK and insulin/insulin-like growth factor (IGF) signaling pathways do not appear to play substantial roles in resistance to microsporidian infection in C. elegans. We found microsporidia in multiple wild-caught isolates of Caenorhabditis nematodes from diverse geographic locations. These results indicate that microsporidia are common parasites of C. elegans in the wild. In addition, the interaction between C. elegans and its natural microsporidian parasites provides a system in which to dissect intracellular intestinal infection in vivo and insight into the diversity of pathogenic mechanisms used by intracellular microbes.     

Quantifying horizontal transmission of Nosema lymantriae, a microsporidian pathogen of the gypsy moth, Lymantria dispar (Lep., Lymantriidae) in field cage studies

Nosema lymantriae is a microsporidian pathogen of the gypsy moth, Lymantria dispar that has been documented to be at least partially responsible for the collapse of L. dispar outbreak populations in Europe. To quantify horizontal transmission of this pathogen under field conditions we performed caged-tree experiments that varied (1) the density of the pathogen through the introduction of laboratory-infected larvae, and (2) the total time that susceptible (test) larvae were exposed to these infected larvae. The time frame of the experiments extended from the early phase of colonization of the target tissues by the microsporidium to the onset of pathogen-induced mortality or pupation of test larvae. Upon termination of each experiment, the prevalence of infection in test larvae was evaluated. In the experiments performed over a range of pathogen densities, infection of test larvae increased with increasing density of inoculated larvae, from 14.2 ± 3.5% at density of 10 inoculated per 100 larvae to 36.7 ± 5.7% at 30 inoculated per 100 larvae. At higher densities, percent infection in test larvae appeared to level off (35.7 ± 5.5% at 50 inoculated per 100 larvae). When larval exposure to the pathogen was varied, transmission of N. lymantriae did not occur within the first 15 d post-inoculation (dpi) (11 d post-exposure of test larvae to inoculated larvae). We found the first infected test larvae in samples taken 20 dpi (16 d post-exposure). Transmission increased over time; in the cages sampled 25 dpi (21 d post-exposure), Nosema prevalence in test larvae ranged from 20.6% to 39.2%.     

Pathogenicity of a Microsporidium Isolate from the Diamondback Moth against Noctuid Moths:Characterization and Implications for Microbiological Pest Management

Background

Due to problems with chemical control, there is increasing interest in the use of microsporidia for control of lepidopteran pests. However, there have been few studies to evaluate the susceptibility of exotic species to microsporidia from indigenous Lepidoptera.

Methodology/Principal Findings

We investigated some biological characteristics of the microsporidian parasite isolated from wild Plutella xylostella (PX) and evaluated its pathogenicity on the laboratory responses of sympatric invasive and resident noctuid moths. There were significant differences in spore size and morphology between PX and Spodoptera litura (SL) isolates. Spores of PX isolate were ovocylindrical, while those of SL were oval. PX spores were 1.05 times longer than those of SL, which in turn were 1.49 times wider than those of the PX. The timing of infection peaks was much shorter in SL and resulted in earlier larval death. There were no noticeable differences in amplicon size (two DNA fragments were each about 1200 base pairs in length). Phylogenetic analysis revealed that the small subunit (SSU) rRNA gene sequences of the two isolates shared a clade with Nosema/Vairimorpha sequences. The absence of octospores in infected spodopteran tissues suggested that PX and SL spores are closely related to Nosema plutellae and N. bombycis, respectively. Both SL and S. exigua (SE) exhibited susceptibility to the PX isolate infection, but showed different infection patterns. Tissular infection was more diverse in the former and resulted in much greater spore production and larval mortality. Microsporidium-infected larvae pupated among both infected and control larvae, but adult emergence occurred only in the second group.

Conclusion/Significance

The PX isolate infection prevented completion of development of most leafworm and beet armyworm larvae. The ability of the microsporidian isolate to severely infect and kill larvae of both native and introduced spodopterans makes it a valuable candidate for biocontrol against lepidopteran pests.     

Nippostrongylus brasiliensis: effects of immunity on the pre-intestinal and intestinal larval stages of the parasite

Nippostrongylus brasiliensis: effects of immunity on the pre-intestinal and intestinal larval stages of the parasite. International journal for Parasitology4: 183–191. Migration of the pre-intestinal larval stages of N. brasiliensis was studied in rats undergoing either primary or challenge infections. In rats undergoing a primary infection, more than 67 percent of larvae successfully migrated from the skin to the oesophagus by 70 h after infection, and subsequently over 90 per cent of these larvae became established in the small intestine as sexually mature adults. In immune rats undergoing a second infection, 46 per cent of larvae completed migration to the oesophagus by 70 h and of these, only 1·6 per cent became established in the intestine to produce eggs. These inhibitory effects on the pre-intestinal and intestinal larval stages were even more pronounced in immune rats undergoing a third or fourth infection and in addition, there was a prolonged sojourn and substantial retention of larvae in their lungs. There was no evidence that the immune response had an adverse effect on oesophageal fourth stags larvae as these organisms (obtained from immune donors) were able to establish and develop to maturity when transferred per os to normal animals.Syngeneic transfer of immune mesenteric lymph node cells to normal recipients, caused expulsion of parasites from the intestine but failed to effect migration of pre-intestinal larval stages. The implications of these findings are discussed in the context of current knowledge of the mechanisms of immunity to helminths.     

Hyperparasitism has wide-ranging implications for studies on the invertebrate phase of myxosporean (Myxozoa) life cycles

All of the actinospore releasing oligochaetes collected in an environmental sample were found to be infected with the microsporidian Neoflabelliforma aurantiae n. gen. n. sp. Ultrastructural and phylogenetic studies on this microsporidian indicated similarities with Flabelliforma magnivora but not with the type species Flabelliforma montana, necessitating the formation of a new genus Neoflabelliforma and reassignment of F. magnivora as Neoflabelliforma magnivora n. comb. The development of N. aurantiae is described both parasitising the oligochaete worm and hyperparasitising the concurrent myxosporean infection. The effect of N. aurantiae on the myxosporeans was deleterious and progressive, eventually stopping all actinospore formation. Its discovery has the potential to impact on areas examining the phase of myxosporean life cycles in the invertebrate host, from transmission studies and epidemiology to re-evaluating the basic steps of intra-oligochaete development. Recent evidence has suggested that studies using invertebrate systems should consider possible adverse effects that co-infections can have on experimental outcomes. The discovery of N. aurantiae highlights the need for careful screening of experimental animals to help circumvent erroneous results.     

Modeling horizontal transmission of microsporidia infecting gypsy moth,Lymantria dispar (L.), larvae

We developed a simulation model that describes the horizontal transmission of three different microsporidia, Endoreticulatus schubergi, Nosema lymantriae and Vairimorpha disparis and their insect host, the gypsy moth, Lymantria dispar. The model describes the stage specific development and mortality of uninfected, latently infected or infectious hosts, the food consumption, the infection by spore-laden feces of E. schubergi and N. lymantriae and by spore-laden cadaver of N. lymantriae and V. disparis. Model results were compared to percent infection of L. dispar test larvae published in earlier studies using caged oak trees and potted oak-plants. When feces were selected as the source of spores for transmission of E. schubergi or N. lymantriae, the model estimated a percent infection in susceptible larvae that was in the range of the experimental studies. When spore-laden cadavers were the source of spores of N. lymantriae or V. disparis, the model did not correctly predict the experimentally measured percent infection in susceptible larvae. The most critical points of the simulation model are exact calculation of spore release, mortality and exact determination of the transmission coefficients when cadavers were included as a source for microsporidian infection.     

A microsporidium,Nosema pilicornis sp. n., of the purslane sawfly,Schizocerella pilicornis

A new microsporidian species, Nosema pilicornis, which infects the purslane sawfly, Schizocerella pilicornis, is described. This microsporidium infects most body tissues of the host. N. pilicornis was compared to other microsporidian species infecting Hymenoptera and to a group of similar microsporidia infecting Lepidoptera. N. pilicornis could be distinguished from all other microsporidian species on the basis of host range and ultrastructural characteristics of the spore. Spores were oval, containing 11 to 12 polar filament coils, and the polar filament had an angle of tilt of about 80°. N. pilicornis infected lepidopteran larvae, but only when heavy spore dosages were fed to early larval instars. S. pilicornis is a good but sporadic biological control agent of common purslane, Portulaca oleracea, a pernicious weed of vegetable, ornamental, and orchard crops. N. pilicornis, which is transovarially transmitted and causes high mortality in infected larvae, affects the performance of S. pilicornis as a biological control agent.     

Immunologic studies on Heligmosomoides polygyrus infection in the mouse: the dynamics of single and multiple infections and the effect of DDT upon acquired resistance

Neilson J.T. McL., Forrester D.J. and Thompson N.P. 1973. Immunologic studies on Heligmosomoides polygyrus infection in the mouse: The dynamics of single and multiple infections and the effect of DDT upon acquired resistance. International Journal for Parasitology3: 371–378. Swiss Webster mice were given infections of 100,200, 300 and 400 Heligmosomoides polygyrus (= Nematospiroides dubius) larvae respectively at intervals of 4 weeks. Where appropriate, the preceding infection was terminated with anthelmintic 7 days prior to the subsequent infection. Animals were killed at regular inteivals following each infection and the worm burdens compared with those found in control mice given a primary infection of similar size. The expulsion of worms in mice given three previous infections occurred after day 3 and before day 7 postinfection indicating that those larvae moulting from the fourth to fifth stages may be most susceptible to the host's resistance mechanisms. The administration of p,p'-DDT to hyperinfected mice did not interfere with the immunologic expulsion of worms.     

Biological Control of the Leafminer Liriomyza trifolii (Burgess): Implications for Intraguild Predation between Diglyphus begini Ashmead and Steinernema carpocapsae (Weiser)

Studies were conducted on the combined use of the eulophid parasitoid wasp Diglyphus begini Ashmead and the entomopathogenic nematode Steinernema carpocapsae (Weiser) for control of the leafminer Liriomyza trifolii (Burgess) on chrysanthemums. Several factors indicated that these two agents were suitable for combined use: adult D. begini were not susceptible to nematode infection, leafminer larvae parasitized by the wasp were less susceptible to nematode infection, adult wasps detected and tended to avoid ovipositing on nematode-infected leafminer larvae, nematode-infected larvae served as host-feeding sources for the adult wasps, and nematodes showed equal orientation toward paralyzed/parasitized leafminer larvae and healthy leafminer larvae. However, interspecific interference and intraguild predation (IGP) between the agents were found. Infection of D. begini larval stages by nematodes was seen in petri dishes and in intact leaf mines. The presence of nematodes in mines with wasp eggs decreased the chance of wasp survival to adulthood. IGP may be minimized through proper timing of natural enemy releases.     

Effects of temperature and dosage on Vairimorpha sp. 696 spore morphometrics,spore yield,and tissue specificity in Heliothis virescens

The effects of temperature and dosage on a new microsporidian species, Vairimorpha sp. 696, were examined in H. virescens. The pathogen was evaluated for tissue specificity, spore size, cumulative percentage mortality, and spore production. All tissues examined bore infection at 32°C. Spore length was significantly longer at 19°C (5.9 μm) than at 32°C (4.7 μm). Spore widths at these two temperatures did not differ significantly. Octospores were not found at either temperature at 8 or 12 days postinoculation. One hundred percent mortality was attained in all dosages administered, but the initial rate of mortaily was more rapid in the higher dosages. Finally, spore yield was greater in larvae administered lower dosages. Maximum spore yield at 27°C was 4.87 × 10⁹ spores/larva.     

Stages of Infection during the Tripartite Interaction between Xenorhabdus nematophila, Its Nematode Vector, and Insect Hosts

Bacteria of the genus Xenorhabdus are mutually associated with entomopathogenic nematodes of the genus Steinernema and are pathogenic to a broad spectrum of insects. The nematodes act as vectors, transmitting the bacteria to insect larvae, which die within a few days of infection. We characterized the early stages of bacterial infection in the insects by constructing a constitutive green fluorescent protein (GFP)-labeled Xenorhabdus nematophila strain. We injected the GFP-labeled bacteria into insects and monitored infection. We found that the bacteria had an extracellular life cycle in the hemolymph and rapidly colonized the anterior midgut region in Spodoptera littoralis larvae. Electron microscopy showed that the bacteria occupied the extracellular matrix of connective tissues within the muscle layers of the Spodoptera midgut. We confirmed the existence of such a specific infection site in the natural route of infection by infesting Spodoptera littoralis larvae with nematodes harboring GFP-labeled Xenorhabdus. When the infective juvenile (IJ) nematodes reached the insect gut, the bacterial cells were rapidly released from the intestinal vesicle into the nematode intestine. Xenorhabdus began to escape from the anus of the nematodes when IJs were wedged in the insect intestinal wall toward the insect hemolymph. Following their release into the insect hemocoel, GFP-labeled bacteria were found only in the anterior midgut region and hemolymph of Spodoptera larvae. Comparative infection assays conducted with another insect, Locusta migratoria, also showed early bacterial colonization of connective tissues. This work shows that the extracellular matrix acts as a particular colonization site for X. nematophila within insects.     

Parasitism of Western Corn Rootworm Larvae and Pupae by Steinernema carpocapsae

Virulence and development of the insect-parasitic nematode, Steinernema carpocapsae (Weiser) (Mexican strain), were evaluated for the immature stages of the western corn rootworm, Diabrotica virgifera virgifera LeConte. Third instar rootworm larvae were five times more susceptible to nematode infection than second instar larvae and 75 times more susceptible than first instar larvae and pupae, based on laboratory bioassays. Rootworm eggs were not susceptible. Nematode development was observed in all susceptible rootworm stages, but a complete life cycle was observed only in second and third instar larvae and pupae. Nematode size was affected by rootworm stage; the smallest infective-stage nematodes were recovered from second instar rootworm larvae. Results of this study suggest that S. carpocapsae should be applied when second and third instar rootworm larvae are predominant in the field.     

Proteome of Aedes aegypti larvae in response to infection by the intracellular parasite Vavraia culicis

We report on the modification of the Aedes aegypti larval proteome following infection by the microsporidian parasite Vavraia culicis. Mosquito larvae were sampled at 5 and 15 days of age to compare the effects of infection when the parasite was in two different developmental stages. Modifications of the host proteome due to the stress of infection were distinguished from those of a more general nature by treatments involving hypoxia. We found that the major reaction to stress was the suppression of particular protein spots. Older (15 days) larvae reacted more strongly to infection by V. culicis (46% of the total number of spots affected; 17% for 5 days larvae), while the strongest reaction of younger (5 days) larvae was to hypoxia for pH range 5-8 and to combined effects of infection and hypoxia for pH range 3-6. MALDI-TOF results indicate that proteins induced or suppressed by infection are involved directly or indirectly in defense against microorganisms. Finally, our MALDI-TOF results suggest that A. aegypti larvae try to control or clear V. culicis infection and also that V. culicis probably impairs the immune defense of this host via arginases-NOS competition.     

Cryptic microsporidian parasites differentially affect invasive and native Artemia spp.

We investigated the host specificity of two cryptic microsporidian species (Anostracospora rigaudi and Enterocytospora artemiae) infecting invasive (Artemia franciscana) and native (Artemia parthenogenetica) hosts in sympatry. Anostracospora rigaudi was on average four times more prevalent in the native host, whereas E. artemiae was three times more prevalent in the invasive host. Infection with An. rigaudi strongly reduced female reproduction in both host species, whereas infection with E. artemiae had weaker effects on female reproduction. We contrasted microsporidian prevalence in native A. franciscana populations (New World) and in both invaded and non-invaded Artemia populations (Old World). At a community level, microsporidian prevalence was twice as high in native compared with invasive hosts, due to the contrasting host-specificity of An. rigaudi and E. artemiae. At a higher biogeographical level, microsporidian prevalence in A. franciscana did not differ between the invaded populations and the native populations used for the introduction. Although E. artemiae was the only species found both in New and Old World populations, no evidence of its co-introduction with the invasive host was found in our experimental and phylogeographic tests. These results suggest that the success of A. franciscana invasion is probably due to a lower susceptibility to virulent microsporidian parasites rather than to decreased microsporidian prevalence compared with A. parthenogenetica or to lower microsporidian virulence in introduced areas.     

The levels of natural Nosema spp. infection in Apis mellifera iberiensis brood stages

Nosema ceranae is the most prevalent endoparasite of Apis mellifera iberiensis and it is a major health problem for bees worldwide. The infective capacity of N. ceranae has been demonstrated experimentally in honey bee brood, however no data are available about its prevalence in brood under natural conditions. Thus, brood combs from 10 different hives were analyzed over two consecutive years, taking samples before and after winter. A total of 1433 larvae/pupae were analyzed individually and N. ceranae (3.53%) was the microsporidian most frequently detected, as opposed to Nosema apis (0.42%) which was more frequently detected in conjunction with N. ceranae (0.71%). The active multiplication of both microsporidians was confirmed by the expression (real-time-PCR) of the N. ceranae polar tube protein 3 gene and/or the N. apis RNA polymerase II gene in 24% of the brood samples positive for Nosema spp. Both genes are related to microsporidian multiplication. As such, N. ceranae multiplication was confirmed in 1.06% of the samples, while N. apis multiplication was only observed in co-infections with N. ceranae (0.07%). Brood cells were analyzed for the presence of Nosema spp., as those are the immediate environment where the brood stages develop. The brood samples infected by Nosema spp. were in brood cells in which that microsporidians were not detected, while brood cells positive for N. ceranae hosted brood stages that were not apparently infected, indicating that this is unlikely to be the main pathway of infection. Finally, the colonies with brood infected by N. ceranae showed higher levels (numbers) of infected adult bees, although the differences were not significant before (P = 0.260), during (P = 0.055) or after (P = 0.056) brood sampling. These results show that N. ceranae is a bee parasite ubiquitous to all members of the colony, irrespective of the age of the bee. It is also of veterinary interest and should be considered when studying the epidemiology of the disease.