External morphology of Lagenidium giganteum zoospores (Oomycetes: Lagenidiales)

The external morphology of Lagenidium giganteum was studied with the aid of Nomarski differential interference microscopy, scanning electron microscopy and unidirectional shadow-casting techniques. Zoospores were ovoid, bluntly pointed and flattered on one side at the anterior end. On one side of the zoospore, a groove ran from end to end parallel to the long axis. Protruding from the groove were two flagella, a posterior whiplike flagellum and an anterior tinsel flagellum with lateral hair. Features of the zoospore surface included an irregular reticulate pattern of ridges and mounds marked by deep irregular pits scattered randomly over the zoospore surface.This investigation received financial support from UNDP/World bank/WHO Special Programme for research and training in Tropical Diseases under the Scientific Working Group on Biological Control of Vectors.Rhode Island Agricultural Experiment Station Contribution N 2287.     

In vitro production of zoospores by the mosquito pathogen Lagenidium giganteum (Oomycetes: Lagenidiales) on solid media

Reliable, large-scale production of Lagenidium giganteum zoospores was obtained on solid media. The fungus was grown for 7 days in a liquid medium of wheat germ, hemp seed, yeast extract, and glucose, then placed onto hemp-seed agar. Zoosporogenesis was induced on agar by immersing the fungal cultures into water. Zoospore production began 10 hr postimmersion, peaked at 18 hr, and ceased by 36 hr. A single, 10-cm Petri dish of fungus on hemp-seed agar produced 1.7?3.8 × 10⁷ zoospores during the 26 hr of zoosporogenesis. Optimal zoospore production occurred with 4- to 7-day-old cultures; cultures older than 10 days produced few zoospores. The temperature range for zoosporogenesis was 15–35°C. The extent of zoosporogenesis was directly related to the volume of water used to induce zoospore formation and inversely proportional to agar thickness. Bioassay of zoospores against second instar Culex quinquefasciatus larvae yielded an LD₅₀ of 400 zoospores/ml.     

Effects of salinity, pH, organic solutes, anaerobic conditions, and the presence of other microbes on production and survival of Lagenidium giganteum (Oomycetes: Lagenidiales) zoospores

The effects of several environmental factors on the production of viable zoospores by Lagenidium giganteum were determined by counts of germlings produced after induction of zoosporogenesis by suspension of mycelium in various substances. NaCl at a concentration of 0.6 g/liter virtually eliminated zoosporogenesis. At 0.2 g/liter NaCl there was a significant reduction in four of the five fungal isolates tested. Transmission of fungus between mosquito larvae at 0.8 g/liter NaCl suggests that estimates of solute effects from in vitro studies are exaggerated. Zoosporogenesis took place from pH 4.5 to 8.4 by three isolates and from 4.5 to 8 by two other isolates (±0.2). Anaerobic conditions halted zoosporogenesis. Reintroduction of oxygen within 7 days allowed the process to resume, but with reduced production for each day under anaerobic conditions. Five species of bacteria inhibited zoosporogenesis at concentrations of 10⁷–10⁸ cells/ml. The yeast, Saccharomyces cerevisiae, was inhibitory at 10⁶ cells/ml. Three sugars, a sugar alcohol, three amino acids, and peptone all reduced zoosporogenesis at levels that generally correlated positively with their nutritional values for the fungus. Peptone, which can support lush vegetative growth of L. giganteum in the absence of other nutrients, was far more effective in inhibiting zoosporogenesis than the other solutes.     

The extracellular proteolytic enzymes of the mosquito-parasitizing fungus Lagenidium giganteum

Lagenidium giganteum produces extracellular proteases when grown in peptone-yeast extract-glucose medium. The production of the enzymes required a suitable inducer such as protein, and was repressed by glucose. The total filtrate proteases had a pH optimum of 8.4, and were heat denatured t 60°C. The use of specific substrates and inhibitors demonstrated the presence of collagenase, trypsin-like protease, and a weak elastase. Trypsin-like activity was present in the medium after 38 h of growth, whereas collagenase appeared in 43 h. The roles of these enzymes in pathogenesis were discussed.     

Sequence analysis of duplicated actin genes in Lagenidium giganteum and Pythium irregulare (Oomycota)

Southern analysis of genomic DNA identified multiple-copy actin gene families in Lagenidium giganteum and Pythium irregulare (Oomycota). Polymerase chain reaction (PCR) protocols were used to amplify members of these actin gene families. Sequence analysis of genomic coding regions demonstrated five unique actin sequences in L. giganteum (Lg-Ac 1, 2, 3, 4, 5) and four unique actin sequences in P. irregulare (Pi-Acl, 2, 3, 4); none were interrupted by introns. Maximum parsimony analysis of the coding regions demonstrated a close phylogenetic relationship between oomycetes and the chromophyte alga Costaria costata. Three types of actin coding regions were identified in the chromophyte/oomycete lineage. The type 1 actin is the single-copy coding region found in C. costata. The type 2 and type 3 actins are found in the oomycetes and are the result of a gene duplication which occurred soon after the divergence of the oomycetes from the chromophyte algae. The type 2 coding regions are the single-copy sequence of Phytophthora megasperma, the Phytophthora infestans actB gene, Lg-Ac5 and Pi-Ac2. The type 3 coding regions are the single-copy sequence of Achlya bisexualis, the P. infestans actA gene, Lg-Ac1, 2, 3, 4 and Pi-Acl, 3, 4. Correspondence to: D. Bhattacharya     

The extracellular acid phosphatase of the mosquito-parasitizing fungus Lagenidium giganteum

The mosquito-parasitizing fungus Lagenidium giganteum secreted a soluble acid phosphatase and beta-D-glucosidase into the growth medium. The acid phosphatase was isolated and purified to single component, and some of its physicochemical properties were determined. The enzyme exhibited a pH optimum of 5.6 in phthalate buffer with p-nitrophenyl phosphate and was temperature-inactivated at 55 degrees C. Enzyme activity seems to be limited to phenyl-phosphate substrates. A molecular weight of 42,800 was found and the amino acid content was also determined. A Km for p-nitrophenyl phosphate of 1.6 x 10(-7) M was found. The possible involvement of the enzyme in the infective process was discussed.     

Solute effects on Lagenidium giganteum: zoospore motility and bioassay reproducibility

The zoospores of Lagenidium giganteum rapidly lose motility when dispersed in deionized water. Several organic solutes were tested for the ability to prolong zoospore activity. Peptone at 0.2 and 0.05 g/liter was more effective than methionine and glucose, individually or in combination. The use of 0.2 g/liter of peptone as a medium for bioassays of L. giganteum against 3-day-old Aedes aegypti reduced the mean LD₅₀ to 12.9 zoospores/ml as compared to 133 with field water and 124 with deionized water. The use of peptone also dramatically improved the reproducibility of the assays and the goodness of fit of the resultant probit regression lines. The mean χ² values were 7.4 for 0.2 g/liter of peptone, 26.8 for field water, and 47.8 for deionized water. It is suggested that the erratic results obtained from use of deionized water are due to variation in the osmotic stress to which the zoospores were exposed, depending on the amount of debris that is introduced into the assays along with the mosquito larvae.     

Description of three novel Lagenidium (Oomycota) species causing infection in mammals

BackgroundRecent molecular phylogenetic analysis of Lagenidium strains recovered from subcutaneous lesions in cats, dogs, and a human with lagenidiosis resolved into four clades; one of them was Lagenidium giganteum, but three others were novel.AimsDue to the recent increase in L. giganteum infections from mammals, we studied 21 Lagenidium strains isolated from dogs and a human available in our collection.MethodsMolecular phylogenetic studies and phenotypic characteristics were used to characterize the strains.ResultsWe report the finding of three novel species, herein designated as Lagenidium ajelloi, sp. nov., Lagenidium albertoi sp. nov, and Lagenidium vilelae sp. nov. Their morphological and growth features are also presented.ConclusionsOur study revealed the presence of three novel Lagenidium species infecting mammals.     

Comparative utilization of sterols by the OomycetesLagenidium giganteum and Lagenidium callinectes

The mosquito-parasitizing OomyceteLagenidium giganteum requires exogenous sterols before it can produce the infective zoospores. In contrast,L. callinectes will synthesize small quantities of cholesterol and produce zoospores. Since the former is a promising biological control agent it was important to delineate the metabolic role(s) and fate(s) of these sterols in zoosporogenesis. Twenty-three different sterols were administered to growing cultures of both organisms. These compounds were reisolated after 7 days, along with any metabolites produced from them. This approach was designed so as to lead to the outline of some of the metabolic sequences which were present. The data show that both organisms possess very similar patterns of sterol transformation. The apparent metabolic route leading to cholesterol is cycloartenol →→ fucosterol →→ cholesterol, which is similar to the overall metabolic sequence observed in many algae. Some transformations that were indicative of obvious intermediate biosynthetic steps were apparent. BothLagenidium spp. transformed 24-alkylidene but not 24-alkyl sterols to cholesterol. The organisms reduced cholesta-5,7-dienol to cholesterol, metabolized cholestanol to cholesterol, and transformed 3-ketosteroids to 3β-hydroxysterols. Coprostanol was changed to cholestanol.     

Preliminary Characterization of Phospholipase A2 in Lagenidium giganteum

MacKichan, J. K., Tuininga, A. R., and Kerwin, J. L. 1994. Preliminary characterization of phospholipase A₂ in Lagenidium giganteum. Experimental Mycology 18, 180-192. Phospholipase A₂ (PLA₂) hydrolyses the fatty acyl ester bond at the sn-2 position in glycerophospholipids. To better understand its regulatory roles, factors affecting PLA₂ activity in Lagenidium giganteum were investigated: divalent ions; chelators: inhibitors; pH; and substrate concentration. PLA₂ activity of L. giganteum whole cell homogenates was determined using 1-stearoyl-2-[1-¹⁴C]arachidonoyl phosphatidylcholine as substrate. The divalent cations Ca²⁺, Mg²⁺, and Mn²⁺ all enhanced PLA² activity, while Co²⁺, Fe²⁺, and Zn²⁺ were either slightly inhibitory or without effect. High concentrations of EGTA enhanced activity, low concentrations of the chelators were slightly inhibitory, while high concentrations of EDTA had little effect. EGTA, which has a higher affinity for Ca²⁺ and Mn²⁺ than Mg²⁺, reduced hydrolysis less than a comparable concentration of EDTA. Two pH optima were found, at both acid (ca. 5.5) and alkaline (ca. 11.5) levels. Four classical inhibitors, nordihydroguaiaretic acid, ellagic acid, gossypol, and 4-bromophenacylbromide, reduced PLA₂ activity by about 80% at 5 mM concentration, 50% with 1 mM inhibitor, and had no effect at 100 μM. The relatively high levels of these compounds needed to inhibit PLA₂ hydrolysis may have been due to the presence of a cocktail of enzymes, some of which were not susceptible to inhibition. All inhibitors at 1 mM concentration in live cell cultures effectively shut down oosporogenesis, without adverse effects to the mycelia. PLA₂ activity was highest in the late oospore stage of the life cycle, although the enzymes were probably not metabolically active in these stationary cultures. Cultures grown on cholesterol-supplemented defined media had significantly higher levels of PLA₂ activity relative to cultures grown on sterol-free media. The enzyme was found to be associated primarily with microsomal membranes, but there was significant activity in cytosolic fractions. Separation of cell homogenates by column chromatography revealed that there were at least nine enzymes capable of cleaving fatty acids in the sn -2 position of phospholipids.     

Defense reactions by larvae of Aedes aegypti during infection by the aquatic fungus Lagenidium giganteum (Oomycete)

Summary The adherence of zoospores of Lagenidium giganteum to the cuticle of mosquito larvae is the initial step in the infection process. Subsequently, a germ tube penetrates the integument, inducing a rapid melanization of the injured cuticle and epidermis. After entering the hemocoel the developing hyphae are occasionally encapsulated locally. This process is slow (6 to 12 h postincubation) and most frequently cell-free, although it can be mediated by circulating hemocytes. Sporadic hemocyte mediation of the humoral encapsulation process in larval stages of Culicidae adds a previously unreported dimension to this unusual type of defense reaction. The defense reactions of larvae of Aedes aegypti were ineffective against observed infection by Lagenidium giganteum.     

The kinetics of Lagenidium giganteum growth in liquid and solid cultures

AIMS: Production of the mosquito biolarvacide Lagenidium giganteum in solid culture has been proposed as an economic alternative to production in liquid culture because of observations of improved shelf life and efficacy upon storage. Understanding the differences between these production systems and estimating growth rate in solid culture are important for commercialization. In order to address these needs a logistic model was developed to describe the growth kinetics of L. giganteum produced in solid and liquid cultures. METHODS AND RESULTS: Kinetic parameters in the logistic model were estimated by nonlinear regression of CO2 evolution rate (CER) and biomass data from solid and liquid cultivation experiments. Lagenidium giganteum biomass was measured using DNA extracted directly from samples. The logistic model was fit to experimental biomass and CER data with low standard errors for parameter estimates. The model was validated in two independent experiments by examining prediction of biomass using on-line CER measurements. CONCLUSIONS: There were significant differences between maximum biomass density, maintenance coefficients, and specific growth rates for liquid and solid cultures. The maximum biomass density (mg dw ml-1) was 11 times greater for solid cultivation compared with liquid cultivation of L. giganteum; however, the maintenance coefficient (mg CO2 h-1 (mg dw)-1) was six times greater for liquid cultivation than in solid cultivation. The specific growth rate at 30 degrees C was approximately 30% greater in liquid cultivation compared with solid cultivation. Slower depletion of substrate and lower endogenous metabolism may explain the longer shelf life of L. giganteum produced in solid culture. SIGNIFICANCE AND IMPACT OF THE STUDY: A simple logistic model was developed which allows real-time estimation of L. giganteum biomass from on-line CER measurements. Parameter estimates for liquid and solid cultivation models also elucidated observations of longer shelf life for production in solid culture.     

Observations of in vitro gametangial copulation and oosporogenesis in Lagenidium giganteum

An in vitro liquid culture oospore production method yielding 5 × 10³ oospores/ml was used to follow the sequential events of gametangial copulation and oospore formation in Lagenidium giganteum. Observations were made with Nomarski differential interference microscopy and scanning electron microscopy. After septation and division of fungal thalli into a chain-like series of links, certain individual subthalli differentiated into gametaniga, oogonia, and antheridia. Antheridia issued a fertilization tube which made contact with, and fused to a single oogonium. Copulative behavior was relatively synchronous and necessitated physical contact between thalli. Sexual reproduction was manifested by the migration and condensation of gametes. Plasmogamy was achieved following the introduction of the male gamete into the oogonium. The fused gametes gave rise to a zygote. Small amounts of periplasm remained in the oogonium. Zygote maturation into a fully developed oospore was characterized by the deposition of a multilaminated oospore wall, the coalescence of lipids into a highly refractive central reserve globule surrounded by a layer of fine-grained cytoplasm.     

A predictor variable for efficacy of Lagenidium giganteum produced in solid-state cultivation

Lagenidium giganteum was cultivated on solid media in the absence of free water and evaluated for efficacy against second-instar Aedes aegypti mosquito larvae in 100-ml bioassays. Bioassay variables included level of media addition, CO₂ evolution rate (CER) and cell density. Logistic regression was performed on bioassay infection observations to determine if the tested variables were correlated to the probability of attaining at least 80% infection. Both CER (p=0.003) and number of cells (p=0.017) were significantly correlated, while level of media addition was not (p=0.42). Although media addition did not correlate with efficacy, media levels greater than 2 g/l reduced water clarity and infection. Media from cultures younger than 3 days performed poorly under all conditions tested. Journal of Industrial Microbiology & Biotechnology (2001) 27, 203–207. Received 16 November 2000/ Accepted in revised form 28 June 2001     

Oosporogenesis by Lagenidium giganteum: induction and maturation are regulated by calcium and calmodulin

Induction and maturation of the sexual stage (oospores) of the facultative mosquito parasite Lagenidium giganteum (Oomycetes: Lagenidiales) are complex developmental processes regulated by calcium-dependent events. Use of developmentally synchronized cultures of L. giganteum allowed stage-specific disruption of calcium metabolism. A calcium chelator (EGTA), an ionophore (chlortetracycline), and inhibitors of the calcium-binding protein calmodulin (dibucaine, trifluoperazine, chlorpromazine) disrupted several discrete developmental steps associated with oosporogenesis: induction of antheridia, gametangial fusion, meiosis, oospore wall formation, and subsequent spore maturation. Extracellular calcium is necessary for oosporogenesis to proceed normally and under some conditions magnesium has a synergistic effect with calcium on oospore induction. Results are discussed in relation to calcium mediation of fusion events in a number of model membrane and biological systems.