Albert H. Undeen

A proposed mechanism for the germination of microsporidian (Protozoa: Microspora) spores

A hypothesis for the mechanism of microsporidian spore germination is presented which is consistent with the experimental evidence available. Based upon concentrations of the disaccharide, trehalose, in germinated and ungerminated spores, stimulation by anions and cations and the high osmotic pressures involved in germinating spores, the following explanation is proposed: monovalent ions diffuse passively into the spores, stimulating changes which cause trehalase to come into contact with trehalose, possibly through an intermediary calcium exchange or release. As trehalose is degraded into higher concentrations of smaller molecules, the osmotic pressure increases until the filament begins its emergence. Once started, expulsion is completed by the osmotic movement of water into the spore, maintaining the pressure as the filament and sporoplasm are forced out. This hypothesis is discussed in light of existing data and hypotheses on microsporidian spore germination and the role of trehalose in the germination of fungal spores. Methods for testing some predictions arising from this hypothesis are suggested.

Germination of Nosema algerae (Microspora) Spores: Conditional Inhibition by D2O, Ethanol and Hg2+ Suggests Dependence of Water Influx upon Membrane Hydration and Specific Transmembrane Pathways

ABSTRACT. 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° C to 40° C range) or in ionic strength (2‐ 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 μM 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.

Epizootiology and transmission of a newly discovered baculovirus from the mosquitoes Culex nigripalpus and C. quinquefasciatus

Reports of mosquito baculoviruses are extremely uncommon and epizootics in field populations are rarely observed. We describe a baculovirus that was responsible for repeated and extended epizootics in field populations of Culex nigripalpus and C. quinquefasciatus over a 2 year period. These mosquito species are important vectors of St Louis and Eastern equine encephalitis in the United States. Our initial attempts to transmit this baculovirus to mosquitoes in the laboratory were unsuccessful. A salt mixture similar to that found in water supporting infection in the field was used in laboratory bioassays and indicated that certain salts were crucial to transmission of the virus. Further investigations revealed conclusively that transmission is mediated by divalent cations: magnesium is essential, whereas calcium inhibits virus transmission. These findings represent a major advancement in our understanding of the transmission of baculoviruses in mosquitoes and will allow characterization of the virus in the laboratory. In addition, they can explain, in great part, conditions that support epizootics in natural populations of mosquitoes that vector life-threatening diseases of man and animals.

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.

Conversion of Intrasporal Trehalose into Reducing Sugars During Germination of Nosema algerae (Protista: Microspora) Spores: A Quantitative Study

ABSTRACT. Carbohydrates were extracted from dormant, stimulated and germinated spores of Nosema algerae. Concentrations of total sugars were measured by the Anthrone test. Non‐reducing sugars were quantified by NaOH hydrolysis followed by the Anthrone reaction, and reducing sugars by the Nelsons test. Glucose was measured by the o‐toluidine test and a glucose oxidase assay. The concentrations of trehalose in the cytoplasm of the dormant, ungerminated spore was estimated to be in excess of 1.0 M. Trehalose decreased by 70% during the five‐minute course of germination. All of the lost trehalose was converted to reducing sugar of which 70–78% was glucose. The osmotic potential increase caused by catabolism of trehalose appears to be sufficient for germination.

Effect of anions on the germination of Nosema algerae (Microspora: Nosematidae) spores

Abstract Solutions of many monovalent salts induce optimal Nosema algerae spore germination at p H 9.5. The order of anion efficacy is Br − > Cl − > I − > F − , suggesting that the anion, as well as the cation, is important to germination. Percentage germination is correlated with the concentration of sodium fluoride under acidic conditions. This small, neutral molecule enters the spore instead of F − , but within the spore, equilibrium favors the fluoride ion which, along with the cation, stimulates germination.

Trehalose levels and trehalase activity in germinated and ungerminated spores of Nosema algerae (Microspora: Nosematidae)

Abstract Gas chromatographic analysis of carbohydrates in ungerminated Nosema algerae spores revealed trehalose at concentrations above 0.4 m and traces of glucose. After germination in KCl at pH 9.5, only one-third of the trehalose remained, and a large increase in glucose was found. A significant amount of glucose was also detected when germination was inhibited by NH4Cl, but the trehalose concentration was unchanged. Trehalase activity in the soluble and insoluble fractions of crude N. algerae spore homogenate, its response to temperature, pH, and trehalose concentration, and its relationship to spore germination were investigated. The temperature optima are 43.7°C for the supernatant and 43.9°C for the residue fraction. The Kms are 26 m m for the supernatant and 25 m m for the residue. Thirty-minute temperature tolerance tests showed 50% activity reductions at 35°C for the supernatant and 38°C for the residue. The pH optima were 5.5 for the supernatant and 5.25 for the residue fractions. A significantly greater proportion of the total enzyme activity was associated with the supernatant fraction in germinated than in ungerminated spores. Neither NH4Cl nor γ radiation appeared to exert inhibitory effects directly through trehalase.

In vitro and in vivo germination of Nosema locustae (Microspora: Nosematidae) spores.

Abstract Spores of Nosema locustae (E. U. Canning, Parasitology, 43, 287–290, 1953) were induced to germinate in vitro by a two-step procedure. Spores were first activated by drying in air, dehydrated in hyperosmotic solutions of sucrose or polyethylene glycol 400 (PEG), or by extended treatments in low concentrations of PEG. Germination occurred after transfer of the activated spores to an alkaline salt solution, with a pH optimum of 9.0–10.0. Percentage germination in vitro varied from 2 to 50% among different production batches of spores. Germination in the midgut of Melanoplus sanguinipes was consistently near 50% in all batches and dehydration prior to ingestion was not necessary. The in vitro germination stimulus described here is probably not the physiologically normal one.

Monovalent Cations Induce Microsporidian Spore Germination In Vitro

ABSTRACT The relative capacity of Na+, K+ and Cl‐ to stimulate germination of spores of the microsporidian Nosema algerae, a pathogen of mosquitoes, was examined by ion substitution experiments. Sodium at 0.1 M was ineffective to produce the high percentage of germination that typically occurs with 0.1 M NaCl (the normal stimulation solution) if Cl‐ was substituted with the usually impermeant anions SO42‐, HPO42‐, or the organic acids oxalate, cacodylate, EGTA, MES and HEPES. However, substantial concentration‐ and pH‐dependent germination was seen with Na2SO4 in the 0.2‐0.8 M Na+ range. Similar results were obtained with solutions of K+ accompanied by impermeant anions. In contrast, the chloride salts of usually impermeant cations, like choline and triethanolamine, failed to germinate spores even at 0.8 M unless Na+ or K+ was independently added. The presence of 0.5 M choline chloride in the medium reduced the levels of Na2SO4 required to produce germination down to equivalence with those of Na+ in the normal stimulation solution. Monensin, a Na+ ionophore, facilitated the germination induced by a medium‐level stimulus (0.04 M NaCl) in sonicated samples. These findings indicate that N. algerae spores germinate in response to the alkali metal cations, while CI‐ plays a passive role by diffusing to maintain internal electroneutrality during cation influx. A possible mechanism of cation action in spore germination is suggested on the basis of these results and observations on other systems of intracellular motility.

Spectrophotometric measurement of Nosema algerae (Microspora: Nosematidae) spore germination rate

Abstract The visible process of polar tube emergence and sporoplasm expulsion, during which the Nosema algerae spore also becomes phase-contrast dark, takes only 1–2 sec. This rapid germination event occurs, at 30°C in 100 m m NaCl at pH 9.5, after a lag time of 1–4 min. Presence of the germinant (NaCl) was required only during the first 35% of the lag time. Maximum percentage germination (ca. 85%) causes 55% reduction in the optical density (OD) of a suspension of spores. Percentage germination is highly correlated with OD reduction (r = 0.946). Continuous spectrophotometric recording of a suspension of germinating spores produces a sigmoid curve, regardless of the final percentage germination. Germination rates (slopes) and lag times (LT50) were obtained through linear regression of probit-transformed percentage changes in OD. The relative effects of CaCl2 inhibition and reduced temperatures on maximum OD change, slope, and LT50 were different. These results demonstrate that kinetic studies might provide some insight on the germination mechanism.