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.

Systemic microsporidiosis in inland bearded dragons (Pogona vitticeps).

One laboratory-hatched and -reared inland bearded dragon (Pogona vitticeps) (No. 1) and two privately owned inland bearded dragons (Nos. 2 and 3) died, showing nonspecific signs of illness. Light microscopic examination of hematoxylin and eosin-stained tissue sections from lizard No. 1 revealed severe hepatic necrosis with clusters of light basophilic intracytoplasmic microorganisms packing and distending hepatocytes and free in areas of necrosis. Similar microorganisms were within cytoplasmic vacuoles in distended renal epithelial cells, pulmonary epithelial cells, gastric mucosal epithelial cells, enterocytes, and capillary endothelial cells and ventricular ependymal cells in the brain. In lizard Nos. 2 and 3, microorganisms of similar appearance were in macrophages in granulomatous inflammation in the colon, adrenal glands, and ovaries. The microorganism was gram positive and acid fast and had a small polar granule that stained using the periodic acid-Schiff reaction. Electron microscopic examination of deparaffinized liver of lizard No. 1 revealed merogonic and sporogonic stages of a protozoan compatible with members of the phylum Microspora. This report provides the first description of microsporidiosis in bearded dragons and is only the second report of this infection in a lizard.

Germination of Nosema algerae (Microspora) spores: conditional inhibition by D2O, ethanol and Hg2+ suggests dependence of water influxupon membrane hydration and specific transmembrane pathways.

The germination of microsporidian spores under conditions expected to affect water flow across the plasma membrane-wall complex was studied by assessing their responses to in vitro stimulation with Na+ or K+. Partial or full substitution of common water with D2O, which more effectively coats ions and electrostatically-charged cell surfaces with relatively stable hydration layers, delayed and inhibited spore germination in a concentration-dependent manner; yet, preincubation in 100% D2O did not change the normal response to standard stimulation. Water structure-breaking conditions, such as an increase in temperature (within the 15 degrees C to 40 degrees C range) or in ionic strength (1- to 10-fold normal), opposed the inhibition by D2O and allowed significant stimulation by Li+, the monovalent cation with the largest hydration diameter and a usually weak stimulant action on the spores. Ethanol, known to reduce water permeation across cell membranes and phospholipid bilayers, also caused a powerful and dose-dependent (1% to 4% v/v) inhibition of spore germination, but pretreatment with ethanol did not affect the normal response. HgCl2, an inhibitor of specific water channels, blocked spore germination at just 250 microM in the normal stimulation solution irrespective of the temperature, and permitted only a delayed response in high salt stimulation solutions. However,the inhibition by Hg2+ was abolished by the simultaneous presence of 2-mercaptoethanol in the medium. These results suggest (1) that spore germination is keenly dependent upon the hydration states of both the plasma membrane-wall complex and the stimulant ions, and (2) that osmotic water flows into the spores through specific transmembrane pathways with critical sulfhydryl groups, i.e., analogous to the water channels that facilitate water movements across the plasma membranes of highly permeable cells.

Egg production by Strelkovimermis spiculatus (Nematoda:Mermithidae).

Egg production by the mermithid nematode Strelkovimermis spiculatus, a parasite of mosquitoes, was examined over a period of 34 days. Oviposition did not occur in the absence of males. Egg production was best when males were continuously present (6.4 x 10(3) +/- 0.9 x 10(3) eggs/female). Fewer eggs were produced when males were removed after 7 days (2.8 x 10(3) +/- 0.2 x 10(3) eggs/female) and oviposition partially recovered after males were returned 11 days later (4.4 x 10(3) +/- 0.5 x 10(3) eggs/female). The nematodes deposited substantially more eggs in sand (6.4 x 10(3) +/- 0.9 x 10(3)/female) than in water (1.9 x 10(3) +/- 0.3 x 10(3)/(female).

Use of stream width for determining the dosage rates of Bacillus thuringiensis var. israelensis for larval black fly (Diptera: Simuliidae) control.

Data from several operational black fly abatement programs using Bacillus thuringiensis var. israelensis (B.t.i.) in New York State's Adirondack Mountains were analyzed to determine what the concentration of formulation in the stream might have been if application rates were determined by a stream's width rather than its discharge. There was a high correlation (magnitude of R = 0.87) between discharge and width measured at 315 treatment sites. Had the applications been based upon the stream width, 96% of the actual concentrations in the streams would have been within plus or minus a factor of 5 and none exceeding a factor of 10, a range in which B.t.i. remains environmentally safe and effective.

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.

A device for monitoring populations of larval mosquitoes in container habitats.

A device was developed for repetitive sampling of mosquito larvae without undue disruption of the larval habitat. The sampler is a 3-oz. (ca. 100-ml capacity) transparent plastic cup with a hole in the center of its convex bottom. The device is buoyed by corks so that the water level is 15 mm above the bottom rim of the cup and 5 mm above the hole. There was significant correlation between 24-h samples of Aedes albopictus and Culex quinquefasciatus 4th-instar larvae in the larval sampling device and populations in tires. Greater numbers of immature mosquitoes were found per unit surface area of the sampling device than the tire as a whole, demonstrating that immature mosquitoes were trapped by the sampler.

Effects of host resistance and injury on the susceptibility of Aedes taeniorhynchus to mosquito iridescent virus.

An iridescent virus is found at low prevalence in population of Aedes taenirhynchus. Attempts at experimental transmission produced low levels of infection, regardless of the dosage applied. In a test for genetic resistance in colonized Ae. taeniorhynchus, the mean infection rates +/- SD for groups of randomly selected and sibling larvae were compared. The standard deviation of the sibling groups was not higher than the random groups (4.0 +/- 3.1% and 3.0 +/- 2.1%), rendering genetic resistance unlikely. Injury to the larvae by feeding silicon carbide fibers consistently caused higher infection rates (4.8 +/- 2.0% by virus alone and 17.5 +/- 5.3% by virus and fibers concurrently). Similar results were obtained for vertical transmission. These results support the hypothesis that this virus has no active means of penetration, invading only through random breaks in the cuticle or peritrophic membrane.

Influence of temperature on developmental parameters of the parasite/host system Edhazardia aedis (Microsporida: Amblyosporidae) and Aedes aegypti (Diptera: Culicidae).

Larvae of Aedes aegypti, transovarially infected with Edhazardia aedis, were reared between 20 and 36 degrees C to determine the influence of temperature on the development of the parasite and the infected host. Development of the parasite was evaluated based on spore yield and size. The predicted optimum temperature for maximum spore production of E. aedis in A. aegypti was 30.8 degrees C. The results demonstrate that the E. aedis-A. aegypti system has a wide temperature tolerance; whereas spore yield will be lower at unfavorable temperatures, the host will remain infected. Additionally, spores were significantly smaller from individual reared at 34 degrees C than those reared at either 20 or 27 degrees C. Development of the infected host was evaluated based on pupal weight and time of pupation. Infected pupae were significantly larger than uninfected pupae. There was also a significant difference in the pupation rate between controls and infected A. aegypti larvae. Controls had a 50% cumulative pupation time (CPT50) of 65.7 degree days and infected individuals a CPT50 of 76.6 degree days.

Effect of Nosema algerae on the house fly Musca domestica (Diptera: Muscidae).

Larvae of Musca domestica were exposed to spores of Nosema algerae on the surface of their diet. Infective concentrations (IC50 and IC90) for the larvae were 3.6 x 10(4) and 1.6 x 10(6) spores/cm2, respectively. The disease appeared to cause no larval mortality, but the longevity of adult females was reduced. At 30 days post-infection, there were at least 1 x 10(7) spores per fly in all dosage groups. At lower dosages, the development of spores was delayed and fewer spores were produced.

Structural alteration of the plasma membrane in spores of the microsporidium Nosema algerae on germination.

The fine structure of the plasma membrane in spores of the microsporidium Nosema algerae, a pathogen of mosquitoes, was examined in the resting condition and after the spores were stimulated to germinate in vitro. Slow penetration of resin caused collapse of the germinated spores. Thin sections of germinated spores showed peculiar membrane infoldings that were never found in ungerminated samples. Analogous germination-dependent configurations of the plasma membrane were observed in freeze-fractured preparations of spores either fixed and impregnated with glycerol prior to freezing, or rapidly frozen with liquid propane while in the process of germination. In every case, the replicas presented germinated spores with indentations in the protoplasmic face of the plasma membrane, and apparently complementary blunt spines on the external face, that were absent in ungerminated spores. It suggests that these alterations of the plasma membrane result from a structural adjustment to a spontaneous contraction of the spore case after germination. We discuss this interpretation with regard to conflicting views on the nature of such morphological features.

The role of osmotic pressure in the germination of Nosema algerae spores.

Both the lag period and the time required for the filament and sporoplasm to emerge from Nosema algerae spores were prolonged when germination occurred under hyperosmotic conditions. Polyethylene glycol (PEG) and sucrose inhibited germination, first by preventing eversion of the filament, and then at higher concentrations by preventing stimulation. The size of the spore cases decreased by about 21% following germination, indicating an elastic spore wall and turgor pressure in the dormant spores. Increased pressure during germination was indicated by less osmotically-induced shrinkage in stimulated than in dormant spores and by higher concentration of solutes in the homogenates of germinated than ungerminated spores. These results are consistent with the hypothesis of a pressure increase during germination that is caused by an endogenous increase in solute concentration.

Horizontal transmission of Parathelohania anophelis to the copepod, Microcyclops varicans, and the mosquito, Anopheles quadrimaculatus.

The copepod Microcyclops varicans was infected with Parathelohania anophelis by unincleate meiospores from a field-collected fourth instar Anopheles quadrimaculatus larva. Large numbers of unincleate, pyriform spores developed in the copepod. These spores were fed to early instar A. quadrimaculatus larvae, infecting both males and females, resulting in the production of cylindrical, binucleate spores in the adult. These spores were responsible for vertical transmission, through the eggs, to the larvae. The original spore type collected from the field was found in the male progeny from the infected females. Another P. anophelis-infected mosquito colony was established by feeding spores from a single, field-collected, infected copepod to A. quadrimaculatus larvae. The microsporidium was continuously maintained by vertical transmission in newly established infected colonies.

The effect of ultraviolet radiation on the germination of Nosema algerae Vávra and Undeen (Microsporida: Nosematidae) spores.

Spores of Nosema algerae Vávra and Undeen were subjected to various dosages of 254 nm ultraviolet radiation (UV). Very high dosages of UV were required to block germination. Germination was normal immediately after UV dosages of 0.2 to 1.0 J/cm2, followed by a delayed effect in which both percentage germination and the intrasporal concentration of trehalose decreased with time after UV exposure. Although a few spores were germinated, most of them were inactivated (rendered temporarily unable to germinate) by exposure to UV of 1.1 J/cm2. Ultraviolet radiation between 1.1 and 3.4 J/cm2 stimulated spores to germinate. However, spores were completely unable to germinate immediately after exposure to dosages above 3.8 J/cm2. Ammonia had little effect on stimulation by UV but was inhibitory to germination after stimulation had occurred. These results demonstrate that UV behaves like a germination stimulus and are discussed in terms of the hypothesis that germination is initiated by the breakdown of barriers between trehalose and trehalase.

The effect of Bacillus sphaericus upon the susceptibility of Anopheles quadrimaculatus to Plasmodium berghei.

Larvae of Anopheles quadrimaculatus were exposed to Bacillus sphaericus. The surviving adults took blood meals on hamsters infected with Plasmodium berghei. Fewer mosquitoes were infected than were the paired controls. The inhibitory action appeared to occur during the early stages of the infection in the mosquito gut.

The isolation of microsporidia and other pathogens from concentrated ditch water.

Water from a mosquito larval habitat in Florida was collected periodically for one year. After removing debris and macroscopic organisms, the small particles were concentrated by continuous flow centrifugation and examined microscopically. Anopheles quadrimaculatus, Culex salinarius and Heliothis zea larvae were exposed to the concentrates. The microsporidia isolated were Nosema, Pleistophora, Telomyxa, Vavraia and Vairimorpha. In addition to these microsporidia, a Helicosporidium (Protozoa), a Metarrhyzium (fungi) and two cytoplasmic polyhedrosis viruses were also isolated.