Giardia intestinalis: Expression of ubiquitin, glucosamine-6-phosphate and cyst wall protein genes during the encystment process

We determined the relative expression of ubiquitin (ub), glucosamine-6-phosphate-isomerase (gn6pi) and cyst wall protein (cwp) genes during encystment of the Portland-1 and Portland-1R strains of Giardia intestinalis. Encystment was induced with bile for different time periods. The presence of encystment-specific vesicles (ESVs) and the relative expression of genes (log₁₀ΔRn) were determined by transmission electron microscopy and real-time PCR, respectively. Our results demonstrated the gene expression and the presence of ESVs after 6 h of encystment. Values of cwp2 gene expression increased by 591-fold in strain Portland-1 and 78.2-fold in strain Portland-1R at this time point compared to values at 0 h, after which values gradually decreased until reaching basal values between 8 and 18 h after the encystment started. Expression of gn6pi was 43.5- and 46.3-fold higher than basal values, in Portland-1 and Portland-1R, respectively. Ub gene expression was 82.25-fold higher than its basal levels at 4 h, after which expression decreased gradually until reaching basal values after 16 h. Conclusions: This work showed the relationship between the presence of ESVs and encystment gene expression at 6 h, and resistance to albendazole does not inhibit the encystment process. The results revealed important knowledge with implications in the control of parasite dissemination for preventing parasite transmission.     

Formation of the Giardia Cyst Wall: Studies on Extracellular Assembly Using Immunogold Labeling and High Resolution Field Emission SEM

Encystment of the intestinal protozoan, Giardia, is a key step in the life cycle that enables this parasite to be transmitted from host to host via either fecal oral, waterborne, or foodborne transmission. The process of encystment was studied by localizing cyst wall specific antigens with immunofluorescence for light microscopy and immunogold staining for field emission scanning electron microscopy. Chronological sampling of Giardia cultures stimulated with endogenous bile permitted identification of an intracellular and extracellular phase in cyst wall formation, a process which required a total of 14-16 h. The intracellular phase lasted for 8-10 h, while the extracellular phase, involved the appearance of cyst wall antigen on the trophozoite membrane, and the assembly of the filamentous layer, a process requiring an additional 4-6 h for completion of mature cysts. The extracellular phase was initiated with the appearance of cyst wall antigen on small protrusions of the trophozoite membrane (-15 nm), which became enlarged with time to caplike structures ranging up to 100 nm in diameter. Caplike structures involved with filament growth were detected over the entire surface of the trophozoite including the adhesive disc and flagella. Encysting cells rounded up, lost attachment to the substratum, and became enclosed in a layer of filaments. Late stages in encystment included a “tailed” cyst in which flagella were not fully retracted into the cyst. Clusters of cysts were seen in which filaments at the surface of one cyst were connected with the surface of adjacent cysts or the “tailed” processes of adjacent cysts, suggesting that the growth of cyst wall filaments may be at the terminal end. In conclusion, the process of encystment has been shown to consist of two morphologically different stages (intracellular and extracellular) which requires 16 h for completion. Further investigation of the extracellular stage with regard to assembly of the filamentous layer of the cyst wall may lead to innovative methods for interfering with production of an intact functional cyst wall, and thereby, regulation of viable Giardia cyst release from the host.     

The effects of temperature on the distribution and establishment of Echinoparyphium recurvatum metacercariae in Lymnaea peregra

The establishment and distribution of Echinoparyphium recurvatum metacercariae in the second intermediate host, Lymnaea peregra, were investigated at a temperature range of 5-29 degrees C. Preliminary studies on the survival and infectivity of E. recurvatum cercariae showed that both parameters were temperature dependent. No cercarial transmission occurred at 5 or 10 degrees C. Nevertheless, the transmission efficiency (1/H0) indicated that transmission was temperature independent in the temperature range 17-25 degrees C and was much lower than in previous studies using this host-parasite system. These differences were attributed to low cercarial densities used in this study. The effect of temperature on encystment site choice (mantle cavity, kidney, pericardium) by metacercariae showed that the mantle cavity was the prime site of encystment, followed by the pericardium and the kidney. Temperatures at the lower and upper ranges (14 and 29 degrees C), however, caused a significant reduction in encystment in the mantle cavity but not in the pericardium or kidney. The importance of cercarial densities, the physiological mechanisms influencing metacercarial distribution and their implications for parasite transmission to the definitive host are discussed.     

Life cycle truncation in a trematode: does higher temperature indicate shorter host longevity?

The typical three-host life cycle of most trematodes creates transmission challenges for which a variety of adaptations have evolved to increase the probability of transmission. Some species can abbreviate their life cycle via progenesis, the precocious maturation of the parasite in the second intermediate host resulting in the production of eggs through self-fertilisation without requiring a definitive host. Adoption of the progenetic life cycle may be a conditional strategy in response to different environmental cues related to low probability of transmission to the definitive host. Using high water temperature and/or limited diet as experimental stressors, we tested the effect of body condition and life span of the fish second intermediate host on facultative truncation of the typical three-host life cycle by progenesis in Stegodexamene anguillae. The results suggest that environmental cues, such as temperature and encystment site, may signal transmission opportunities to the parasite so that it may adjust its developmental strategy accordingly. Indeed, a greater proportion of worms became progenetic at higher temperatures, and progenesis was more common among worms encysted in the gonads or body cavity of their fish hosts than among those in other host tissues. These findings highlight the often unrecognised plasticity in parasite developmental and transmission strategies.     

Regulation of attachment, germination, and appressorium formation by zoospores ofLagenidium giganteum and related oomycetes by chitin, chitosan, and catecholamines

Summary Lagenidium giganteum (Oomycetes: Lagenidiales), a facultative parasite of mosquito larvae, infects the larval stage of most species of mosquitoes and a very limited number of alternate hosts. Host infection by this and other members of Oomycetes is initiated by motile, laterally biflagellate zoospores. Chemical bases for the various degrees of host specificity exhibited by these parasites is not known, but presumably involves receptors on the zoospore surface recognizing compounds either secreted by or on the surface of their hosts. Surface topography had no detectable effect onL. giganteum encystment or appressorium formation. Scanning electron microscopy documented the detachment of flagella during zoospore encystment. Bulbous knobs at the basal end of the detached flagellum were interpreted as encysting zoospores dropping the axoneme and/or the basal body and associated structures to which flagella are attached. Multiple signals appear to be involved in the initial steps ofL. giganteum host invasion. Zoospores of this parasite did not encyst on powdered preparations of chitin or chitosan (deacetylated chitin). Upon dissolution of chitosan in dilute acid followed by drying these solutions to form thin, transparent films, zoospores readily encysted. The degree of reacetylation of these films and the spacing of acetylated and deacetylated residues had no significant effect on zoospore encystment. Zoospores of a strain ofLagenidium myophilum isolated from marine shrimp, that also infects mosquito larvae, encysted on chitosan films. No encystment of spores of the plant parasitePhytophthora capsici was observed on chitin or chitosan films. Simulation of cuticle sclerotization by incubating chitosan films with different catecholamines and tyrosinase significantly reduced zoospore encystment. Zoospores that encysted on chitosan films did not germinate in distilled water. Germination could be induced by adding microgram quantities of bovine serum albumin or proteins secreted by motile zoospores into the water, and to a lesser degree by some amino acids, but not by various cations. Zoospores encysted and germinated on the pupal stage of some mosquito species. Appressoria were occasionally formed, but most subsequently sent out another mycelial branch, apparently without attempting to pierce the pupal cuticle. Methylation of pupal exuviae with ethereal diazomethane or methanol/HCl significantly increased zoospore encystment. Modification of chitin by catecholamines, lipids and protein on the epicuticular larval surface all affected host invasion.Abbreviations BSA bovine serum albumin - CID collision-induced dissociation - DOPA 3,4-dihydroxyphenylalanine - ESI-MS electrospray mass spectrometry - ESI-MS/MS tandem electrospray mass spectrometry - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - WGA wheat germ agglutinin - ZAP zoospore aggregation pheromone     

Influence of cadmium exposure on the incidence of first intermediate host encystment by Echinoparyphium recurvatum cercariae in Lymnaea peregra

The effect of cadmium exposure of the snail first intermediate host Lymnaea peregra on the incidence of encystment of Echinoparyphium recurvatum (Digenea: Echinostomatidae) cercariae without emergence from the snail was investigated. Exposure to 100 microg l(-1) Cd for 72 h caused a significant increase in the incidence of first host encystment when compared to controls. In addition, autometallographic staining of E. recurvatum daughter rediae and developing cercariae showed that there was metal accumulation within their body tissues. The significance of these findings to parasite transmission in metal-polluted environments is discussed.     

De novo synthesis of sphingolipids plays an important role during in vitro encystment of Entamoeba invadens

Entamoeba invadens is a protozoan, which causes multiple damages in reptiles and is considered a prototype for the study of the Entamoeba encystment in vitro. Here we report for the first time the role of the de novo synthesis pathway of sphingolipids during the encystment of E. invadens. In silico analysis showed that this parasite has six putative genes coding for ceramide synthases (CerS), all of them coding for proteins containing the Lag1p motif, a region conserved in the ceramide synthases of multiple organisms, suggesting that they might be bona fide CerS. The six genes of E. invadens are differentially expressed at different time intervals in both stages trophozoite and cyst, based on the results obtained through qRT-PCR assays, the genes involved in the synthesis of sphingolipids with long-chain fatty acids CerS 2,3,4 (EIN_046610, EIN_097030, EIN_130350) have maximum points of relative expression in both stages of the E. invadens life cycle, which strongly suggest that the signaling exerted from the synthesis pathway of sphingolipids is essential for the encystment of E. invadens, since the generation of the more abundant sphingomyelin (SM) subspecies with long-chain fatty acids are fundamental for the parasite to reach its conversion from trophozoite to cyst. When myriocin was used as an inhibitor of serine palmitoyl CoA transferase (SPT), first enzyme in the de novo biosynthesis of sphingolipids, the trophozoites of E. invadens were unable to reach the encystment. Since the effect of myriocin was reversed with exogenous d-erythrosphingosine (DHS), it was demonstrated that the inhibition was specific and it was confirmed that the synthesis of sphingolipids play an essential role during the encystment process of E. invadens.     

The differentiation of cellular structure during encystment in the soil hypotrichous ciliate Australocirrus cf. australis (Protista,Ciliophora)

Ciliates are able to form resting cysts as a survival strategy in response to stressful environmental factors. Studies on the characteristics of cellular structure during encystment may provide useful information for further understanding of the regulatory mechanism of cellular patterns and supply new clues regarding the phylogeny of ciliates. Scanning and transmission electron microscopies were used to observe the ultrastructure of cells during encystment of the soil ciliate Australocirrus cf. australis. The dedifferentiation of ciliature was revealed for the first time. Ciliary shafts first shortened, and the remaining ciliature, including basal bodies and the fibrillar cirral basket, retracted into the cytoplasm and was surrounded by the autophagic vacuoles and then gradually digested. A large number of autophagic vacuoles were observed in mature resting cysts. Autophagy might not only be necessary for the differentiation of cellular structures during encystment but might also be important to sustain the basic life activities in the resting stage. Australocirrus cf. australis formed a kinetosome-resorbing cyst and contained four layers in the cyst wall: the ectocyst, mesocyst, endocyst and granular layer. The ciliature resorbing state and the number of layers in the cyst wall were consistent with those found in other oxytrichous ciliates. However, the phenomenon wherein the two macronuclear nodules are not fused during encystment is not commonly observed among oxytrichids. Additionally, the octahedral granules in the mesocyst of this species exhibit different morphology from the congeners.     

Structural and developmental studies of Chlamydomyzium oviparasiticum from Rhabditis nematodes and in culture

The oomycete (Peronosporomycete) Chlamydomyzium oviparasiticum, previously recorded as a parasite of rotifer eggs, was found infecting Rhabditis nematodes in a sample of rotting garden compost. For the first time C. oviparasiticum was cultured in liquid media, which enabled more detailed studies of zoospore behaviour and facilitated the use of confocal microscopy. Rhabditis nematodes were successfully re-infected from liquid-cultured inoculum. Light (including video) microscopy and transmission electron microscopy were used to document details of thallus development, zoospore release and resting spore morphology to enable comparison with other oomycete species. This species showed several significant saprolegnialian characters such as the ‘achlyoid’ pattern of spore formation, centrifugal cleavage and structured encystment vesicles. In contrast, spore release into a transient vesicle was a peronosporalean characteristic. The thick-walled resting spores showed relatively poor cytoplasmic preservation and had a thick multi-layered wall. It was still not possible to unequivocably decide whether these were chlamydospores or parthenogenically formed oospores. The phylogenetic significance of these observations is discussed.     

Different transmission strategies of a parasite in male and female hosts

We investigated whether a parasite with two routes of transmission responds to the different transmission opportunities offered by male and female hosts by using different transmission strategies in the two sexes. The parasite Ascogregarina culicis, which infects the mosquito Aedes aegypti, can be transmitted as its host’s pupa transforms into an adult or when a female lays its eggs. As the latter transmission route is missing in males, we expected, and found, that the parasite releases a greater proportion of its infectious forms during emergence when it is within a male than when it infects a female. The transmission route, which influences the parasite’s dispersal and the evolution of its virulence, was also affected by the dose of infection and the parasite’s previous transmission route. Our results emphasize the complexity underlying the development of parasites and show their ability to tune their strategy to their environment.     

The Characterization of an Adrenergic Signalling System Involved in the Encystment of the Ocular Pathogen Acanthamoeba spp.

The aim of this study was to identify and characterize the receptor system involved in controlling encystment in Acanthamoeba using specific agonists and antagonists and to examine whether endogenous stores of catecholamines are produced by the organism. Acanthamoeba trophozoites suspended in axenic growth medium were exposed to adrenoceptor agonists and antagonists to determine which compounds promoted or prevented encystment. Second, trophozoites were cultured in medium containing a catecholamine synthesis inhibitor to investigate the effect this had on natural encystment. Nonspecific adrenoceptor agonists including epinephrine, isoprotenerol, and the selective β1 adrenoceptor agonist dobutamine were found to cause > 90% encystment of Acanthamoeba trophozoites compared to < 30% with the controls. The selective β1 antagonist metoprolol was able to inhibit epinephrine mediated encystment by > 55%. Cultures of Acanthamoeba with the catecholamine synthesis inhibitor α‐methyl‐p‐tyrosine significantly reduced the level of amoebic encystment compared to controls. In conclusion, Acanthamoeba appear to contain a functional adrenergic receptor system of unknown structure which is involved in initiating the encystment process that can be activated and blocked by β1 agonists and antagonists respectively. Furthermore, the presence of this receptor system in Acanthamoeba indicates that topical β adrenoceptor blockers may be effective adjunct therapy by reducing the transformation of trophozoites into the highly resistant cyst stage.     

Starvation and Encystment of a Soil Amoeba Hartmannella castellanii*

SYNOPSIS. The behavior of the amoeba H. castellanii was investigated in various carbon and nitrogen deficient media with a view to developing a satisfactory replacement medium for the study of encystment and excystment. Media which had been devised for other soil amoebae did not cause H. castellanii to encyst. In these media there was an efflux of material from the cells which was independent of osmolarity but which was minimized by the addition of magnesium. Maximal encystment occurred in a medium containing magnesium chloride alone. The cysts produced in the magnesium chloride replacement medium are viable and readily excyst when resuspended in the growth medium. The cysts contain cellulose, which is not present in the vegetative amoebae, and differ from the amoebae in their greater resistance to induced lysis and mechanical injury.     

Electron microscopical observations onPseudaphelidium drebesii Schweikert and Schnepf, a parasite of the centric diatomThalassiosira punctigera

Summary Ultrastructural observations revealed details of the infection process and the fine structure ofPseudaphelidium drebesii Schweikert and Schnepf, a parasite of the marine centric diatomThalassiosira punctigera (Castracane) Hasle. After attachment and encystment of the zoospore an intracellular infection tube is generated. This structure is everted between the overlap of the girdle bands or the gap between the valve and the girdle band of the host diatom. The bulk of thePseudaphelidium protoplast enters the silica shell of the diatom via the infection tube but does not pierce the host plasma membrane. Parts of the host cytoplasm are phagocytized with microfilament involvement.Pseudaphelidium drebesii is characterized by unusual microbodies containing tubular inclusions and by closed mitosis with a perinuclear spindle. The taxonomic position ofP. drebesii is discussed.     

Balamuthia mandrillaris: Role of galactose in encystment and identification of potential inhibitory targets

Balamuthia mandrillaris is a causative agent of granulomatous encephalitis that almost always proves fatal. A major concern during the course of therapy is that B. mandrillaris can transform into cysts. Cysts are highly resistant to physical and chemical conditions and present a problem in successful antimicrobial chemotherapy. However, the underlying mechanisms of B. mandrillaris transformation into cysts are not known. In this study, we examined the effects of exogenous sugars on B. mandrillaris encystment. The findings revealed that free exogenous galactose, but not other sugars, enhanced parasite differentiation into cysts, and apparently a galactose-binding protein is involved in B. mandrillaris encystment. Cytoskeletal re-arrangements and phosphatidylinositol 3-kinase (PI3K)-mediated pathways are involved in B. mandrillaris encystment based on inhibitor studies. Dual functionality of galactose-binding protein in B. mandrillaris pathogenesis and encystment is discussed further.     

Geographic variation in life cycle strategies of a progenetic trematode

Numerous parasite species have evolved complex life cycles with multiple, subsequent hosts. In trematodes, each transmission event in multi-host life cycles selects for various adaptations, one of which is facultative life cycle abbreviation. This typically occurs through progenesis, i.e., precocious maturity and reproduction via self-fertilization within the second intermediate host. Progenesis eliminates the need for the definitive host and facilitates life cycle completion. Adopting a progenetic cycle may be a conditional strategy in response to environmental cues related to low probability of transmission to the definitive host. Here, the effects of environmental factors on the reproductive strategy of the progenetic trematode Stegodexamene anguillae were investigated using comparisons among populations. In the 3-host life cycle, S. anguillae sexually reproduces within eel definitive hosts, whereas in the progenetic life cycle, S. anguillae reproduces by selfing within the metacercaria cyst in tissues of small fish intermediate hosts. Geographic variation was found in the frequency of progenesis, independent of eel abundance. Progenesis was affected by abundance and length of the second intermediate fish host as well as encystment site within the host. The present study is the first to compare life cycle strategies among parasite populations, providing insight into the often unrecognized plasticity in parasite developmental strategies and transmission.     

Characterization of zoospore and cyst surface structure in saprophytic and fish pathogenicSaprolegnia species (oomycete fungal protists)

Summary The importance of the surface structure and chemistry in zoospores and cysts of oomycetes is briefly reviewed and the organelle systems associated with encystment described. The surface structure and chemistry of primary and secondary zoospores and cysts ofSaprolegnia diclina (a representative saprophytic species) andS. parasitica (a representative salmonid fish pathogen) were explored using the lectins concanavilin A (Con A) and wheat germ agglutinin (WGA) and monoclonal antibodies (MAbs) raised against a mixed zoospore and cyst suspension ofS. parasitica. The binding of lectins and antibodies to spores was determined using immunofluorescence microscopy with fluorescein isothiocyanate-labelled probes and with electron microscopy with gold-conjugated probes applied to spore suspensions post-fixation. In both species Con A, which is specific for glucose and mannose sugars, bound to both the surface of primary and secondary zoospores (the surface glycocalyx) and their cyst coats and readily induced zoospore encystment. The binding to the cysts appeared to be mainly associated with the matrix material released from the primary and secondary encystment vesicles and which appeared to diminish with time. No binding to germ tube walls was observed with this lectin. The MAb labelling showed a generally similar binding pattern to the primary and secondary cysts to that observed with Con A, although the binding to zoospores was more variable. Primary zoospores bound the antibodies but secondary zoospores appeared less reactive. It is suggested that the MAbs share a common epitope with one or more of the Con A-binding components. In both species WGA, which is specific for amongst other things the sugar N-acetyl glucosamine, bound to localised apical patches on the primary zoospores. This lectin also binds to the ventral groove region of secondary zoospores ofS. diclina, which were induced to encyst by this lectin. In contrast secondary zoospores ofS. parasitica were not induced to encyst by the addition of WGA and showed a patchy dorsal binding with this lectin. WGA also binds to both the inner wall of discharged primary cysts and the young germ tube walls of both species. These observations are discussed both in relation to other oomycete spores and to their possible functional and ecological significance.Abbreviations BSA bovine serum albumin - Con A Concanavalin A - DBA Dolichos biflorus agglutinin - ELISA enzyme-linked immunosorbent assay - EM electron microscope - EV encystment vesicles - FCS foetal calf serum - FITC Fluorescein isothiocyanate - FV peripheral fibrillar vesicles - G+F 0.2% glutaraldehyde and 2.0% formaldehyde primary fixative solution - 2G 2% glutaraldehyde primary fixative - LM light microscopy - MAbs monoclonal antibodies - LPV large peripheral vesicles - PBS phosphate buffered saline - PCV flattened peripheral cisternae - PEV primary encystment vesicle - PIPES piperazine-N,N1-bis(2-ethane sulfonic acid) - PNA Ricinus communis agglutinin - RAM-FITC/Au_10–20 Fluorescein isothiocyanate/gold (10 or 20 nm) labelled rabbit anti-mouse immunoglobulin - RCA Ricinus communis agglutinin - SEM scanning electron micrograph - SBA soybean agglutinin - SEV secondary encystment vesicles - TEM transmission electron micrograph - UEA I Ulex europaeus agglutinin - WGA wheat germ agglutinin     

Morphological Study of the Encystment and Excystment of Vermamoeba vermiformis Revealed Original Traits

Free‐living amoebae are ubiquitous protozoa commonly found in water. Among them, Acanthamoeba and Vermamoeba (formerly Hartmannella) are the most represented genera. In case of stress, such as nutrient deprivation or osmotic stress, these amoebae initiate a differentiation process, named encystment. It leads to the cyst form, which is a resistant form enabling amoebae to survive in harsh conditions and resist disinfection treatments. Encystment has been thoroughly described in Acanthamoeba but poorly in Vermamoeba. Our study was aimed to follow the encystment/excystment processes by microscopic observations. We show that encystment is quite rapid, as mature cysts were obtained in 9 h, and that cyst wall is composed of two layers. A video shows that a locomotive form is likely involved in clustering cysts together during encystment. As for Acanthamoeba, autophagy is likely active during this process. Specific vesicles, possibly involved in ribophagy, were observed within the cytoplasm. Remarkably, mitochondria rearranged around the nucleus within the cyst, suggesting high needs in energy. Unlike Acanthamoeba and Naegleria, no ostioles were observed in the cyst wall suggesting that excystment is original. During excystment, large vesicles, likely filled with hydrolases, were found in close proximity to cyst wall and digest it. Trophozoite moves inside its cyst wall before exiting during excystment. In conclusion, Vermamoeba encystment/excystment displays original trends as compare to Acanthamoeba.     

Life history and virulence are linked in the ectoparasitic salmon louse Lepeophtheirus salmonis

Models of virulence evolution for horizontally transmitted parasites often assume that transmission rate (the probability that an infected host infects a susceptible host) and virulence (the increase in host mortality due to infection) are positively correlated, because higher rates of production of propagules may cause more damages to the host. However, empirical support for this assumption is scant and limited to microparasites. To fill this gap, we explored the relationships between parasite life history and virulence in the salmon louse, Lepeophtheirus salmonis, a horizontally transmitted copepod ectoparasite on Atlantic salmon Salmo salar. In the laboratory, we infected juvenile salmon hosts with equal doses of infective L. salmonis larvae and monitored parasite age at first reproduction, parasite fecundity, area of damage caused on the skin of the host, and host weight and length gain. We found that earlier onset of parasite reproduction was associated with higher parasite fecundity. Moreover, higher parasite fecundity (a proxy for transmission rate, as infection probability increases with higher numbers of parasite larvae released to the water) was associated with lower host weight gain (correlated with lower survival in juvenile salmon), supporting the presence of a virulence–transmission trade‐off. Our results are relevant in the context of increasing intensive farming, where frequent anti‐parasite drug use and increased host density may have selected for faster production of parasite transmission stages, via earlier reproduction and increased early fecundity. Our study highlights that salmon lice, therefore, are a good model for studying how human activity may affect the evolution of parasite virulence.     

A correlated response of a parasite’s virulence and life cycle to selection on its host’s life history

We demonstrate a correlated response of the virulence and the mode of transmission of the microsporidian parasite Edhazardia aedis to selection on the age at pupation of its host, the mosquito Aedes aegypti. We selected three lines of mosquitoes each for early or late pupation and exposed the larvae after zero, two and four generations of selection to a low and a high concentration of the parasite’s spores. Before selection the parasites induced a similar level of mortality in the six lines; after four generations of selection mortality was higher in the mosquitoes selected for late pupation than in those selected for early pupation. Overall, parasite-induced mortality was positively correlated with the mean age at pupation of the matching uninfected line. When they died, mosquitoes selected for early pupation harboured mostly binucleate spores, which are responsible for vertical transmission. Mosquitoes selected for late pupation were more likely to harbour uninucleate spores, which are responsible for horizontal transmission. The parasite enhanced this tendency for horizontal transmission by prolonging the larval period in the lines selected for late pupation, but not in the ones selected for early pupation. These results suggest that the genetic basis of the mosquito’s age at pupation helps to determine the parasite’s mode of transmission: parasites in rapidly developing mosquitoes are benign and transmit vertically, while parasites in slowly developing mosquitoes are virulent and transmit horizontally. Thus, as the host’s life history evolves, the parasite’s performance changes, because the host’s evolution changes the environment in which the parasite develops.     

The Physiology of Encystment of Hartmannella castellanii*

SYNOPSIS. Some aspects of the physiology of encystment of the soil amoeba Hartmannella castellanii in a replacement encystment medium consisting of 5 × 10^-2 M MgCl₂ have been investigated. It is suggested that measurement of the cellulose produced during encystment in the synthesis of the cyst wall is a more reliable measure of the process than other methods tried. The degree of encystment was dependent on the physiologic state of the amoebae and the composition of the growth medium, but the initial pH of the encystment medium (C. 4.0-8.5) had little effect on the process. The requirement for Mg during encystment was probably not due to its deficiency during growth. Encystment was inhibited to varying extents by inhibitors of protein synthesis, tetracycline and chloramphenicol and also by arsenate, arsenite and iodoacetate; sodium fluoride, malonate and 2, 4-dinitrophenol were without marked effect. Addition of glucose and α-ketoglutarate to the replacement medium led to improvement in the encystment response. The presence of glutamate and histidine during encystment led to cell death. Other carbon and nitrogen sources had no effect. During encystment there was an increase in the metabolic activity of the amoebae, as measured by their oxygen consumption. This was accompanied by a decrease of about 40% in cellular dry weight and protein content. Of the other chemical components, there were marked initial increases in the levels of total carbohydrates and pentose which were followed by their depletion during cellulose synthesis. Encystment was completed after about 64 hr when the synthesis of cellulose was complete and the oxygen uptake of the amoebae fell to an immeasurable level.