Many species of trichomycetes are available in axenic culture, and this has permitted the design and analysis of various experiments, some strictly in vitro, others in vivo. Mosquito larvae have been the host of choice for studies with Smittium spp. because of the relative ease with which the larvae can be manipulated and raised axenically in the laboratory, their short developmental cycles, and the background of information on their growth and physiology available to investigators. The fungi used in experimental studies have been Amoebidium parasiticum, Austrosmittium biforme, Furculomyces boomerangus, Smittium culisetae, S. culicis, S. simulii, S. mucronatum, and several isolates of unidentified Smittium species. Molecular systematic studies have involved representatives of all genera and species available in culture at the time of the investigations (Grigg and Lichtwardt, 1996; Gottlieb and Lichtwardt, 2001). Currently available trichomycete cultures are listed on the web page www.nhm.ku.edu/~fungi, and methods for their isolation and maintenance in culture are given in Chapter 3.
The first of these fungi to be isolated axenically, by Whisler (1960), was the ectocommensal Amoebidium parasiticum. This feat was followed by the isolation of two species of Smittium by Clark et al. (1963) from the guts of mosquito larvae, and Lichtwardt's (1964) isolation of species of the same genus from larval mosquitoes and blackflies. Since that time a large number of isolates of Smittium spp., and some of A. parasiticum and new endocommensal genera, have been obtained from a variety of hosts and geographic localities around the world.
It has been stated previously, but bears stressing here, that the nutrients
and culture conditions that satisfy the currently available isolates have
proved to be unsatisfactory for the in vitro isolation or stable
growth of a large number of other trichomycetes. All species of Smittium
are not amenable to the isolation techniques that have been employed with
the culturable species. Further, the authors have been unable to make isolations
from some natural populations of those same species currently in culture,
leading to the conclusion that in some cases there may be cultivation idiosyncrasies
among strains.
Whisler (1962) found that A. parasiticum grew well in a shaken
liquid medium consisting of 1% Bacto-tryptone, 0.3% glucose, and inorganic
salts. The addition of thiamine (200 µg/liter) to the semidefined
medium increased growth more than sixfold, and it was later found to be
the only vitamin required. A defined medium was devised by substituting
methionine (0.01%) for tryptone and making some adjustments in the levels
of ammonium and phosphate. This defined medium provided thallial dry weights
of only 21-55 mg/50 ml medium, as compared with maximum dry weight levels
of about 150 mg/50 ml medium with the tryptone-containing formula, but
it permitted a more precise determination of the organism's nutritional
requirements. Replacement of methionine with cystine or sulfate was not
successful, nor did nitrate serve as a nitrogen source in place of ammonium.
Mannose supported good growth as did also glucose, and the organism was
able to utilize fructose fairly well as a carbon source. From Whisler's
study it is clear that A. parasiticum has nutritional requirements
that do not differ from those of many aquatic phycomycetes and other organisms,
and therefore its natural growth on living aquatic arthropods does not
appear to be determined by nutritional factors alone.
The type isolate of Smittium culisetae (COL-18-3) has been used almost exclusively as a model for studies on nutrition of endocommensal trichomycetes, as well as for other biological parameters to be covered in subsequent sections of this chapter. This fungus grows well on a tryptone-glucose medium (TGv) containing 2% tryptone, 0.5% glucose, inorganic salts, and vitamins (see Chapter 3 for the formula), a modification of Whisler's (1962) medium. Dry weight yields of around 170 mg/50 ml medium were obtainable in shaken cultures (Williams and Lichtwardt, 1972b). A satisfactory defined medium capable of yielding good growth has not been devised yet, for there are unidentified components in tryptone that greatly stimulate growth of S. culisetae (Williams, 1971). Significantly, Bacto-tryptone (Difco) in the TGv medium provided almost two and one-half times the amount of growth as did Fisher tryptone, and more than eight times the growth obtained with BBL trypticase. According to a quantitative analysis provided by Difco, Bacto-tryptone (a pancreatic digest of casein) contains 18 amino acids and five vitamins, but growth was not satisfactory when tryptone was reconstituted from the given ingredients. The addition of thiamine in concentrations as low as 10 µg/liter to the basal TG medium with 2% tryptone stimulated growth considerably. The growth stimulant in tryptone can be eluted with warm ethanol and apparently is not a vitamin; ethanol-washed tryptone yielded inferior growth even when supplemented with thiamine, and growth levels were restored by the addition of air-dried ethanol extract of tryptone to the washed medium.
Williams (1971) obtained no positive effects on growth of S. culisetae when he added an auxin (naphthylacetic acid), a cytokinin (N-6-benzyladenine) and gibberellic acid (GA) in various concentrations at which these plant hormones are used with higher plants. At relatively high concentrations, the hormones suppressed both dry weight yields and spore production.
Glucose produced better growth in S. culisetae than 18 other carbohydrate sources (Williams and Lichtwardt, 1972b). In these tests tryptone was reduced to 1% in the medium, and dry weights were compared against the baseline growth level without carbohydrate supplementation. The fungus did not ferment glucose. Glycerine, mannose, and fructose were assimilated but were less satisfactory carbon sources. Trehalose, an important blood sugar in insects, supported almost no growth, as was true of soluble starch even though hydrolysis of starch by S. culisetae was demonstrated on starch plates. Synergistic effects on growth were not found when the other carbohydrate sources were combined individually with glucose.
Smittium culisetae was found to utilize ammonium compounds and urea when grown on TGv medium containing 0.1% tryptone (to provide at least minimal growth). Cultures with nitrate and methionine as the major nitrogen supplements produced dry weights only slightly greater than the controls. Asparagine was not utilized, and nitrite was inhibitory to growth. Ammonium and urea are commonly excreted into insect guts (Chapman, 1966), and therefore may be major sources of nitrogen for the gut fungi in vivo as well.
The studies cited show that the nutrition of A. parasiticum and
S.
culisetae is very similar with respect to carbon, nitrogen, and vitamin
requirements. The growth of both species is greatly stimulated by tryptone.
Unknown components of tryptone are necessary for growth of
S. culisetae
in an otherwise defined medium, whereas methionine can replace tryptone
with A. parasiticum to provide satisfactory, but less, growth. These
species will grow well on a number of other undefined media commonly used
with fungi (Lichtwardt, unpublished). Dilute brain-heart infusion (BHI/10)
is one of the best, but has been used mostly as a primary isolation and
culture maintenance medium.
Growth rates of cultured trichomycetes compare favorably with many
other fungi. Under good conditions, growth rates as high as 220 mg dry
weight/day (150 ml medium in 500-ml flasks) have been measured for S.
culisetae (Williams and Lichtwardt, 1972b), and 120 mg/day (50 ml medium
in 125-ml flasks) for A. parasiticum (Whisler, 1962). Maximum dry
weights were attained in about 4 days for both species. As is to be expected,
growth rates and dry weight yields can vary among species of Smittium
(Fig. 9.1), just as there are measurable
species and strain differences in other physiological properties in this
genus. Whisler reported satisfactory growth of A. parasiticum over
a temperature range of 15 to 30oC. Farr and Lichtwardt (1967)
grew S. culisetae (COL-18-3) in stationary cultures and obtained
best growth rates between 22 and 28oC; however, maximum dry
weight yields were produced at 10oC (Fig.
9.2), with satisfactory growth occurring at the more extreme temperatures
of 7 and 32oC. Chapman (1966), also using unshaken cultures,
found a generally similar pattern of growth as a function of temperature
with another strain of S. culisetae (HAW-5-7) and with S. simulii
(JAP-51-5), but S. culicis (WYO-51-11) produced considerably slower
although steady growth at 12, 18, and 26oC with the growth curves
still rising after 27 days and with the best growth at the lowest temperature.
(This unorthodox isolate of S. culicis came from a larva of Aedes
sticticus living in water at 8oC in a small pool at 2750
m altitude.) Studies of several strains of Smittium in shaken culture
corroborate the general pattern of slower growth rates but with higher
dry weight yields at lower temperatures (Williams and Lichtwardt, 1972b;
El-Buni and Lichtwardt, 1976a).
The shaking of cultures in flasks increases both the rate and the amount of growth substantially. Whisler (1962) reported increased growth of A. parasiticum about 162% over stationary cultures when he used a rotary shaker. With S. culisetae the results have been even more dramatic (Williams and Lichtwardt, 1972b): in 250-ml flasks with 50 ml of TGv medium, growth was almost 360% greater in shaken flasks (Fig. 9.1), and in 500 ml flasks with 150 ml of the same medium, shaking resulted in more than 450% increase in dry weight. Maximum yields were obtained in 4 days in both flask sizes that were shaken, and in 8 days in stationary flasks. It would therefore appear that Smittium species growing in the guts of larvae, where oxygen potentials can be expected to be low, do not fulfill their growth potentials. However, prolific vegetative growth of thalli in the guts would not be necessarily advantageous to the fungi and could even be lethal to the larvae.
The hindgut contents of mosquito larvae tend to be slightly acidic (pH 6.4) to alkaline (pH 8.0) (Clements, 1963), depending on the species and probably many other factors. In vitro stationary cultures of S. culisetae (COL-18-3) grown by Farr and Lichtwardt (1967) using 10 different initial pH values ranging from 4.0 to 9.6 that were unadjusted during growth in TGv medium, produced better growth in those media with initial values on the alkaline side, with the best growth occurring at pH 8.3. No growth was produced at either extreme of that pH range. A subsequent study of the same isolate by Williams and Lichtwardt (1972b) indicated that stationary culture conditions lead to a considerable drop in pH of the TGv medium over time (Fig. 9.3), which might account for better growth at the initially higher pH values in stationary cultures. Shaken cultures, on the other hand, became more alkaline after a slight drop in pH. They found that significantly more growth was obtained in shaken TGv medium (initial pH 6.7) when pH values were allowed to change autonomously over time than when the values were maintained constant at pH 6.5, 7.5, and 8.5; least growth was measured at the highest pH value. With A. parasiticum grown in shaken flasks at constant pH values in a similar medium, optimum growth was obtained at pH 7.0, and the growth range was approximately pH 6.0-9.0 (Whisler, 1962).
It would appear that pH is not a limiting factor to vegetative growth of either A. parasiticum or S. culisetae in nature, because of their tolerances. How these fungi respond to their immediate natural environment with respect to pH influences is not clear, however, since the data obtained experimentally are quite artificial. But the data do suggest that those fungi within the confines of the gut might exert some influence on the acidity of the lumen if they are growing luxuriantly.
The only study on growth and the effects of pH in culture using genera
other than Smittium was conducted by Misra (2000) who used two cultures,
Furculomyces
boomerangus and Austrosmittium biforme, isolated from Australian
Chironomidae. These species also produced more biomass and trichospores
under aerated conditions, but trichospore production was considerably lower
than had been found in Smittium culisetae, and the percentage of
viable trichospores upon release was also considerably less. He concluded
that the two species from Australia were not as tolerant to culture conditions
as S. culisetae.
Starr et al. (1979) did a study of 14 isolates of Smittium spp.,
two strains of A. parasiticum, and two Kickxellales (Dipsacomyces
acuminosporus and Linderina pennispora) to determine
and compare their ability to synthesize sterols. They were grown for 7
days in shaken TGv medium, which was determined to be sterol free, and
mycelial extracts were analyzed by gas chromatography and mass spectroscopy.
The major free sterol component of the Smittium species was found
to be desmosterol, a rare sterol in fungi. A few of the isolates also produced
ergosterol, and some contained one of two unidentified sterols that had
trimethysilyl ethers with parent ions at either m/e 470 or 498.
Strain COL-18-3 of S. culisetae was analyzed after harvesting at
days 3, 7, and 14, and it was shown that the sterol content varied both
quantitatively and qualitatively during growth (Table
9. 1). The increase in total sterols in the thalli from day 3 to day
7 coincides with increases in spore production after maximum dry weight
has been achieved (Figs. 9.1 and 9.4),
and the decrease in sterols on day 14 occurs at the time culture dry weights
are decreasing and lytic activities are presumably in progress. Cholesterol
was found only in this one strain of Smittium, but it was present,
along with ergosterol, in the two strains of A. parasiticum.
Cholesterol was the sole sterol produced by the two Kickxellales. Only
Smittium spp. produced desmosterol. The Kickxellales (Zygomycetes)
were included in the study because they have been shown to have a slight
serological relationship with Smittium spp. (see
later section on Serology). The sterol content of Smittium spp.,
based on the dry weight of fungal tissue, was as high as 0.34%. Sterols
are an essential nutrient of insects, and the possible role of these gut
fungi in furnishing sterols and vitamins to nutritionally deprived mosquito
larvae is presented in the next section of this chapter.
The lipid components of S. culisetae were studied by Patrick
et al. (1973), who found that 12.9% of the extractable lipid (about 1.28%
of the total mycelial dry weight) consisted of unspecified steroids. They
grew their fungus on dilute brain-heart infusion (Difco) which, according
to a later analysis by Starr et al. (1979), contains 0.715 mg of cholesterol
per gram of dry medium. As a consequence, it cannot be determined whether
the steroids in the thalli were totally synthesized or partly incorporated
from the medium. The lipids extracted by Patrick et al. from 10-day-old
cultures of the fungus accounted for 9.9% of the total weight, and consisted
of 76.3% neutral lipids and 23.7% polar lipids, with triglycerides comprising
26.6% of the lipid classes (Table 9.2).
Of the fatty acids, palmitoleic was present in unusually high concentrations
(38.7% of total fatty acids), followed by palmitic (34.3%) and oleic acids
(16.3%).
The common occurrence of trichomycetes in such a wide range of arthropods
tempts one to postulate that these commensals are not always completely
neutral with respect to the welfare of their hosts, even though their effects
may not be readily observed. Death of infested mosquito larvae has been
recorded in several instances, as described in Chapter 8. In this section
the possible beneficial aspects will be considered. The only experimental
evidence along these lines was obtained by Horn (1980) and published by
Horn and Lichtwardt (1981) using larval Aedes aegypti to study the
nutritional relationship with cultured S. culisetae (COL-18-3).
The nutritional requirements of these mosquito larvae have been studied
(e.g., Trager, 1935; Lea et al., 1956; Singh and Brown, 1957; Akov, 1962;
Lang et al., 1972). The larvae can be raised axenically simply by surface
sterilizing the eggs.
Experiments by Horn were conducted using a semidefined phosphate-buffered medium consisting of purified egg albumin, vitamins, cholesterol, RNA, and minerals. Additional controls were run concurrently with each experiment on an undefined (yeast-pork liver) medium, which provided assurance that the larvae were capable of development at an optimal rate. Vitamins and cholesterol were deleted individually from the semidefined medium to compare the rate of development and survivability of larvae infested with S. culisetae versus uninfested ones raised on the deficient media. Infestation was accomplished by adding a large measured number of washed fungal spores to test tubes containing 1st instar larvae. Larvae were kept singly in tubes in order to record the time of molting and mortality, if it occurred.
In the absence of riboflavin in the medium only a few uninfested larvae survived to the 4th instar, and none pupated, whereas almost 50% of the infested larvae were alive at the 4th instar and almost half of these pupated (Fig. 9.5). In pyridoxine-deficient medium, no uninfested larvae survived beyond the 2nd instar, while a few of the infested ones reached the 4th instar but did not pupate. Without nicotinamide the development was similar, except that only a small percentage of infested larvae reached the 3rd instar. In these vitamin-deficient media, the infested larvae thus were able to attain one or two developmental stages beyond what the uninfested ones attained, presumably due to the presence of the fungus. No such improvement in infested larval development was found when thiamine or calcium pantothenate was omitted from the medium.
When cholesterol was omitted, none of the uninfested larvae pupated, whereas one out of 45 larvae with Smittium did. It was further found that when lipid extracts of 3-day-old S. culisetae cultures equivalent to 3.1 and 6.2 mg dry weight of mycelium were provided for each larva, the rate of development of those larvae was as good as that on the complete medium with cholesterol. Extracts equivalent to 37.2 mg mycelium per larva inhibited pupation, however, but this is not unexpected since it is known that excessive amounts of lipids or cholesterol can be toxic to larvae (Golberg and De Meillon, 1948; Akov, 1962). Purified desmosterol, the principal sterol synthesized by Smittium spp., when substituted for cholesterol in the medium provided rates of development comparable to cholesterol through the four larval stages; however, only 63% of the larvae pupated with desmosterol as compared to 97% with cholesterol. When heat-killed thalli of S. culisetae were added to sterol-free medium (4.4 mg/mosquito), the rate of development was greater than the controls and more than 50% of the larvae achieved pupation; those not fed dead thallial material did not develop beyond the 3rd instar. Some of those larvae whose development had stalled in the 3rd instar eventually pupated after dead Smittium thalli were added to the medium.
Comparison of infested and uninfested larvae raised on complete semidefined medium showed that about 26% of the infested ones died before reaching the 2nd instar. The same general level of mortality also occurred after the 1st- or 2nd-instar stages more or less consistently in all of the experiments with vitamin- or sterol-deficient media (see Fig. 9.5). This was attributable in most cases to excess growth of the fungus in the larval gut (Fig. 9.6), presumably caused by the ingestion of too many spores and resulting in unsuccessful ecdysis. On the undefined (yeast-liver) medium, this mortality did not occur in infested larvae fed similar amounts of inoculum. Later instars rarely became overinfested, and in fact infestations were not always maintained through the 4th instar. Williams and Lichtwardt (1972a) also found mortality (5-10%) in artificially infested 1st-instar larvae raised on Akov's (1962) defined medium.
Trichospores of the S. culisetae isolate used (COL-18-3), unlike most isolates of this genus, are capable of extruding their sporangiospores in vitro (see a later section on Sporangiospore Extrusion and Trichospore Survival).
Sparse thallial growth was observed by Horn in tubes with larvae whose development became stalled and which were held for up to 21 days. Larvae apparently ate some of this fungal material, and it is not known to what extent this may have influenced their nutrition in the later stages of those experiments. An unusual observation was made: Thalli of S. culisetae could attach to the external cuticle of some larvae that survived several weeks when reared on the semidefined medium lacking riboflavin, pyridoxine, nicotinamide, or sterols (Fig. 9.6). This is the first instance of external attachment reported in any Harpellales.
These experiments with S. culisetae, although not unequivocal,
offer some support to the hypothesis that mosquito larvae growing in vitamin-
and
sterol-deficient media may develop more favorably if infested with
Smittium.
In natural environments, it is more likely that the deficiencies would
be only partial, and it is probable that the amounts of inocula would not
be so high as to produce the kind of mortality observed in the experiments.
If the results are corroborated by other similar investigations, and if
one extrapolates this to other gut fungi, one could envisage some trichomycetes
living commensalistically within their hosts under conditions of good nutrition,
but assuming a more mutualistic relationship when the arthropods become
nutritionally stressed. From an evolutionary point of view, this would
suggest that those populations of arthropods that are infested with trichomycetes
would, over time, have some selective advantage.
Adequate rates of spore production in gut fungi that are frequently
expelled from their hosts due to ecdysis are probably more critical to
their survival than is the amount of vegetative growth they produce. The
rate of spore production in the holocarpic thalli of A. parasiticum
in culture is equal to the generation time, but in Smittium spp.
there normally is appreciable growth before sporulation commences, and
spores will be formed at the tips of some branches more or less continuously
as the thalli grow. The authors have seen in some isolates of Smittium
the most abbreviated structural form of sporulation possible: A spore will
extrude some time after release in culture and the extruded inner cell
directly becomes a generative cell that produces a trichospore (Fig.
9.7). Apparently this kind of development does not occur regularly
but only under cultural conditions that are inimical to vegetative growth.
A study by EI-Buni and Lichtwardt (1976a) of ten isolates of Smittium grown in shaken TGv medium at 22oC showed that three isolates of S. culisetae used were more prolific (5.0-7.5 x 106 spores/ml) than any of the isolates of S. culicis, S. simulii, or S. mucronatum (0.5-2.5 x 106 spores/ml). Several cultural parameters affecting sporulation were studied, including medium composition. Glucose was inhibitory to sporulation at all levels above 0.2% in TGv medium, even at concentrations that produced maximum dry weight (Fig. 9.8). Glucose proved to be the best carbohydrate source of 12 tested (at 0.5% w/v) for spore production in S. culisetae, S. culicis, and S. mucronatum. Considerably different effects were detected depending on the temperature used. For instance, with S. mucronatum fructose provided almost as many spores at 14oC as glucose did, but relatively fewer spores developed with fructose at 22oC, at which temperature other carbohydrates induced more spores. When tryptone concentration was varied in TGv medium, a substantial increase in spore production in S. culisetae occurred in 2% tryptone, the increase coinciding with a sharp rise in final pH of the medium (Fig. 9.9). It was found that brain-heart infusion prepared as recommended by the manufacturer (3.7%) almost totally inhibited spore production in three species of Smittium; all three sporulated best when the medium was diluted to 0.74% or one-fifth of the recommended concentration. The addition of 20 mg/liter of sitosterol acetate and P-sitosterol to an undefined medium increased sporulation in S. mucronatum twofold or more, but ergosterol and cholesterol were both inhibitory to spore production.
El-Buni and Lichtwardt also showed that optimum temperatures for sporulation can be quite different among species. In TGv medium, Smittium culisetae had maximum sporulation at 22oC, and S. culicis and S. mucronatum produced most spores at 14oC (Fig. 9.10). Thus, while vegetative growth is good at room temperature in all three species (Fig. 9.1), cultures must be grown at considerably lower temperatures in the laboratory if maximum spore production is desired in the latter two species. These same three isolates were grown at 22oC at different initial pH values spanning the range that has been reported in mosquito hindguts (Clements, 1963), and all three had maximum sporulation at pH 6.9 or slightly lower (Fig. 9.11), indicating that unadjusted TGv medium (initial pH 6.7) is satisfactory for spore production. Just as aeration in shaken cultures increases vegetative growth in vitro (Fig. 9.1), so does it increase spore production (Fig. 9.4). When S. culisetae was shaken in cotton-plugged flasks (the usual procedure) and compared against similar shaken replicates with rubber stoppers, the spore numbers were almost fivefold greater in the cotton-plugged flasks. It was not determined what role carbon dioxide accumulation may play in reducing spore production.
As mentioned in a previous section of this Chapter, Misra (2000) found
viable trichospore production in Furculomyces boomerangus and Austrosmittium
biforme to be quite low under culture conditions. The greater number
of spores produced by S. culisetae isolates does not necessarily
mean that this species puts more of its resources in terms of biomass into
reproduction, because the spores of S. culisetae are smaller than
those of any of the other Smittium species used in these sporulation
experiments. The results obtained in the experiments are useful to the
investigator in selecting appropriate media and culture conditions to obtain
adequate numbers of spores. How the data apply to these same species growing
in the gut, where the rate and quantity of spore production are undoubtedly
of importance to their transmission and survival, is less clear. Since
the hosts are poikilothermic, the temperatures at which the fungi grow
are close to those of the environment in the hosts' aquatic habitats. Temperature
tolerances for spore production may well be a major factor in the degree
to which infestation is sustained in any given population of suitable arthropod,
and could account for shifts in the percentage of hosts infested as temperatures
rise or fall in aquatic habitats during different seasons of the year.
Extrusion of sporangiospores from the trichospore (which is a unispored
sporangium) is an especially sensitive adaptive process in the life cycle
of Harpellales, for it must occur within the short time span that the trichospore
moves through the gut after it is ingested. Trichospores in natural situations
do not normally extrude in the outer aquatic environment, for such an occurrence
would be counterproductive to survival. When ingested, they are stimulated
in the digestive tract to initiate development. Williams and Lichtwardt
(1972a) determined that trichospores of S. culisetae can
extrude and attach to the gut cuticle in Aedes aegypti larvae in
as little as 30 min. The question is, why do trichospores that are ingested
by a compatible host extrude their inner spore, which then attaches and
develops in the gut, whereas alien trichospores pass through the gut unaffected?
As pointed out by Lichtwardt (1996), streams with abundant and varied insect
faunas almost always contain an assortment of gut fungi. For instance,
a 20-m stretch of one high elevation second-order stream in the Colorado
Rocky Mountains was found to have 9 genera and 18 species of Harpellales
plus several species of Paramoebidium (Lichtwardt and Williams 1988).
It can be assumed that the insect larvae in that stream, several species
of which were often found clinging to the same benthic substrate, were
exposed to a smorgasbord of different trichospores.
Horn (1989a, 1989b, 1990) addressed the problem of what host stimuli induce sporangiospore extrusion through in vitro and in vivo studies using Aedes aegypti larvae and two species of Smittium, S. culisetae and S. culicis. The sporangiospore of S. culisetae extrudes from the sporangium (the trichospore) by bursting through the apex of the sporangium under pressure, and the extruding sporangiospore approximately doubles its original length (Fig. 9.12, Fig. 9.17). This process usually takes but 2-10 sec (Horn 1989a; Williams 1983). A preliminary holdfast is secreted through a field of canals in the tip of the extruded sporangiospore wall. The origin of this adhesive holdfast substance is a series of membrane-bound and usually spherical "apical spore bodies" within the tip of the spore (Fig. 9.12, Fig. 9.16) (Horn 1989a, 1989b; Moss and Lichtwardt 1976; Williams 1983).
Horn (1989a) obtained up to 98% spore extrusion in vitro with S. culisetae by subjecting cultured trichospores to two sequential treatments. In Phase I, trichospores were exposed to a minimum of 20 mM potassium chloride at pH 10 for about 15 min, followed by Phase II which consisted of shifting the buffer to pH 7 (6-8). Phase II led to S. culisetae spore extrusion within several minutes. The extrusion process, once started, took 10 sec or less. These stimuli mimic the physiological conditions an ingested trichospore would encounter in the mosquito gut. Some potassium, excreted from the Malpighian tubules in primary urine, flows forward and is present in the midgut lumen where the alkalinity is maintained at approximately pH 10. Upon reaching the pyloric chamber and hindgut region, the pH drops abruptly to seven, which stimulates spore extrusion and holdfast formation.
Extrusion in S. culicis was slightly different, for it occurred during Phase I (or in the midgut of larvae), but holdfast formation followed Phase II treatment (hindgut conditions). Furthermore, although spore extrusion was sometimes apical, as in S. culisetae, extrusion in S. culicis was often subbasal or basal (Fig. 9.17). There were no visible ultrastructural changes in S. culisetae trichospores during Phase I treatment. However, an interwall layer may play a part in aiding sporangiospore extrusion, for it swells in Phase II (Horn 1990), and in S. culisetae (only) it forms a sleeve-like structure (Fig. 9.12, Fig. 9.17) connecting the trichospore wall and the expanded and extruded sporangiospore (Horn 1989b).
Pressure within the trichospore of S. culisetae increased by 1.0 MPa during Phase I, though this increase was not sufficient to extrude the sporangiospore and could not be attributed to potassium uptake (Horn 1990). It was possible to inhibit extrusion in Phase II by lowering the osmotic potential of the solution to £-2.7 MPa. Horn hypothesized that potassium might stimulate the breakdown of certain organic solutes in trichospores during Phase I, leading to a pressure increase and subsequent sporangiospore extrusion.
Only a few of the more than 200 axenic isolates of Smittium available are known to extrude spontaneously in culture, perhaps a result of their having been transferred repeatedly and the ability to extrude having been a selective factor under such artificial conditions. Horn (1989a) used Phase I and Phase II treatments on 29 harpellids belonging to four genera and ten species, comparing extrusion in treated and untreated trichospores. Among these harpellids were three species of Smittium that are notorious for their wide dipteran host ranges: S. culisetae and S. culicis (usually in Culicidae), and S. simulii (usually in Chironomidae or Simuliidae, but occasionally in Culicidae). The extrusion response to treatment of 22 isolates, with a few exceptions, was generally fair to excellent. The other seven species (belonging to four genera) showed no response to this specific trichospore treatment. These studies indicate that trichospores are attuned to chemical stimuli and may serve as a first level of host recognition by species of harpellids. It is not known what elicits sporangiospore extrusion in other families of aquatic insects.
Holdfasts do not form under ordinary culture conditions, but in some situations the first end of the spore to emerge may produce a swelling (Fig. 9.17) surrounded at times by a secreted substance barely visible with phase microscopy, or a narrow rhizoidal extension may be seen, which branches or coils. In some cultured strains capable of extruding there is no further in vitro development into thalli, whereas a few produce highly branched fertile thalli with the old trichospore wall sometimes remaining at the point of origin.
EI-Buni and Lichtwardt (1976b) found five strains of three species in the culture collection of Smittium that had better than 20% spore extrusion. The best extrusion occurred in three strains of S. culisetae, with COL-18-3 topping the list by producing up to 82% extrusion. TGv medium was superior to BHI as an extrusion medium, and incubating the spores in still cultures resulted in better extrusion than shaken cultures in almost all tests. They found that 24-72 hr were required for spores to extrude, depending on various culture parameters; thus, extrusion in vitro takes considerably longer than in vivo. Temperatures between 24 and 30oC produced maximum extrude in S. culisetae (HAW-5-7), as well as the shortest times to achieve those maxima (30-40 hr) under the experimental conditions used (Fig. 9. 13). At least 10% extrusion in strain COL-18-3 occurred over a range of pH values from 5.5 to 8.5, with an optimum (77%) near pH 7.2 (Fig. 9.14). This is slightly more alkaline than the optima for growth and sporulation cited previously. EI-Buni and Lichtwardt also found that spore extrusion was dependent on spore concentration to some extent, the extrusion percentages decreasing more or less linearly in TGv medium from a maximum of about 79% extrusion with 0.2 x 106 spores/ml to 31% with 4 x 106 spores/ml.
The duration of spore viability is of interest both because of the need for maintaining cultures in the laboratory and the implication such data may have for understanding survival of spores in nature as a source of inoculum. Freeze-drying has not succeeded as a method for long-term laboratory storage, but storage in liquid nitrogen appears to be satisfactory with all isolates of Smittium and Amoebidium, provided the cultures are initially cooled down slowly (1oC/min) to below freezing before plunging the sealed ampules into liquid nitrogen (El-Buni, 1975).
It has been noted from the time axenic isolates were first obtained that the longevity of refrigerated cultures varies considerably among the isolates. Vegetative cells of thalli seem to die much sooner than trichospores, as expected, and it is conceivable that the apparent lack of viability in stored cultures sometimes may actually be the result of spores not extruding, although viable, when cultures are transferred to new medium. This possibility has not been thoroughly tested, although El-Buni (1975) fed mosquito larvae with 2-year-old cultures of four species of Smittium, and obtained no infestation. In further studies on storability, El-Buni and Lichtwardt (1976b) harvested new trichospores from six isolates of four Smittium species and fed them to different instars of Aedes aegypti before and after the trichospores were refrigerated in distilled water at 5oC for 8 and 12 months. Spores from S. culisetae strains COL-18-3 and HAW-5-7 infested mosquito larvae well to moderately well after 12 months of storage, but spores of strains CAN-X-I and WYO-51-11 (S. culicis) and JAP-33-2 (S. simulii) did not survive either storage period at 5oC. FRA12-3 (S. mucronatum) infested no larvae, even before refrigeration.
In the case of S. simulii and S. mucronatum, the lack of extrusion in the guts may have been due to the fact that A. aegypti is not a suitable host for those particular strains (see Table 9.3). The indications are, however, that strains of S. culicis have much shorter viability time spans than S. culisetae. El-Buni and Lichtwardt conducted further tests on S. culisetae (COL-18-3) using in vitro extrusion, and found that when spores were stored in water at 5oC, extrusion decreased linearly from 80% to 14% over an 18-month period. At -5oC, extrusion dropped abruptly to 20% in 2 weeks, and at 4 and 8 weeks it decreased to 10% and 8%, respectively. Williams (1983a) demonstrated that trichospores of S. culisetae placed in moist soil and stored in a freezer (-10oC) for 5 months were capable of infesting 71% of A. aegypti larvae, whereas lower infestation resulted from subsets stored in moist soil that had been refrigerated (-4oC) or kept at room temperature. These data suggest there might be considerable loss of viability in overwintering spores, although natural substrates obviously were not duplicated in these experiments.
It should be noted that in vivo extrusion of sporangiospores
after trichospore ingestion sometimes occurs prematurely in branched harpellids
that normally extrude in the hindgut where their thalli develop. Precocious
extrusion occurs instead in the midgut, with attachment and minimal development
of the extruded sporangiospore that becomes attached to the peritrophic
membrane. This phenomenon has been observed not infrequently in Smittium
longisporum and S. perforatum (Lichtwardt et al., 1997) and
in a few other harpellids (Lichtwardt, unpubl.) Confusion occurs when these
structures are erroneously interpreted to be young thalli of Stachylina
(Moss, 1979). In all instances involving such precocious extrusion, the
extruded sporangiospores produce an elongated basal growth that penetrates
the peritrophic membrane, but develop no further and no septa are formed.
A few Stachylina spp. do penetrate the peritrophic membrane (S.
minima, S. pedifer, S. penetralis, S. queenslandia), but the growth
from the Stachylina thallus that penetrates is shorter than in the
aberrant extruded sporangiospores of Smittium, and soon after attachment
septa begin to form as the Stachylina thalli grow and eventually
sporulate.
The availability of many axenic Smittium isolates obtained
from different dipteran hosts and geographic localities has provided the
opportunity to study selected representatives for their ability to infest
a "foreign" host. This was first done by Chapman (1966), who fed sporulating
thalli (and spores alone of one isolate) to larvae of Aedes triseriatus.
She found that all of the tests resulted in infestation of that larval
species. Her studies were expanded and refined by Williams and Lichtwardt
(1972a), who used spores of 12 isolates and four species of Smittium
and determined their infestibility in two instar stages of A. aegypti
(Table 9.3). The results showed that
the isolates that originated from mosquito larvae infested A. aegypti
well, but so did two isolates from blackflies, CAN-X-I and CLW2-44. Smittium
culisetae and S. culicis almost always have been found infesting
species of mosquito larvae, but exceptions are known (see Axenic
Isolates List). Smittium mucronatum has been found only in chironomid
larvae, and while the cultured isolate did not infest A. aegypti
well in these experiments, Coste-Mathiez (1970) reported some growth of
S. mucronatum in Culex pipiens after infested chironomid
larvae were placed in the same containers with the mosquitoes (see Chapter
6). In nature S. simulii has been found mostly in chironomid
and blackfly larvae, but one of the isolates, CLW-2-44, readily infested
A. aegypti. These cross-infestation experiments, together with data
on field isolation of species from different families of hosts, show quite
clearly that some species of Smittium are not highly host specific,
and therefore their taxonomic determinations cannot be based on the host
in which they are found.
Amoebagenesis is the term used by Whisler for the process of inducing
thalli of Amoebidium parasiticum to release amoeboid cells instead
of sporangiospores. Several earlier investigators had noted that amoebae
were normally produced only when the host molted or was injured. Whisler's
earlier study (1966) was concerned with exploring the nature of the chemical
inducer and environmental conditions that influence amoebagenesis in
vitro using axenic isolates. He developed a quantitative assay by growing
cultures of A. parasiticum in shaken tryptone-glucose broth, harvesting
the thalli during the logarithmic phase of growth by centrifugation, washing
them in a dilute salts solution, then keeping the thalli in shaken dilute
salts for 16 hr. This provided a homogeneous culture of starved young thalli
against which various substances could be tested to record the percentage
of thalli that released amoebae. Out of a long list of defined and undefined
substances tried, all but one showed no more than a trace of amoebagenic
activity. Only an extract of dried Daphnia (commercially available
as a fish food) produced appreciable (17%) release. He was able to obtain
very high release of amoebae (80-100%) within 16 hr when he used a dialysate
of the daphnid extract, and showed that the release was proportional to
the concentration used. The inducing substance was heat stable, had a relatively
small molecular size, and was not soluble in ether, acetone, or chloroform.
He further showed that amoebagenesis in his system was optimal near pH
8.0 and 30oC.
In 1968 Whisler was able to identify more precisely the amoebagenic
factors by fractionating the daphnid concentrate through a Sephadex G-10
column. This, together with other tests, implicated calcium, glucose, and
various amino acids as essential for amoebagenesis. Histidine and methionine
promoted amoeba formation more than other individual amino acids, but highest
release was obtained with a combination of 10 amino acids. Calcium was
optimal around 0.01 M, and he speculated that this relatively high concentration
might somehow interfere with wall synthesis around the cleaved protoplasts
within the thallus such that amoebae rather than sporangiospores result.
Studies on chemical components of trichomycete cell walls have been
few, and consist mostly of tests performed on uncultured species (Table
7.1). In this section two analyses of wall material from cultured species
will be described. Sangar and Dugan (1973) used a strain of Smittium
culisetae (HAW-13-2), and isolated wall fragments mechanically.
They found glucosamine to be the predominant component (35%) in hydrolyzed
walls, with glucose (13%), mannose (5.5%), and galactose (4%) comprising
the other monosaccharides. The overall analysis indicated 65% carbohydrate,
15% protein, 13% lipid, and 3% ash. The presence of chitin was substantiated
by digesting the acid- and alkali-resistant portion of wall material with
chitinase, which released N-acetylglucosamine, and by infrared spectroscopy.
The presence of cellulose was not detected using the standard IKI-H2SO4
test.
A study by Trotter and Whisler (1965) on Amoebidium parasiticum
revealed neither chitin nor cellulose in the walls. Their analysis gave
30% galactosamine, 10% galactose, 3% xylose, 30% protein, and 4% ash. Acid
hydrolysates of the wall material gave no evidence of glucose or glucosamine.
The presence of hemicellulose was indicated. Thus, it appears that A.
parasiticum not only differs from S. culisetae in its wall composition,
but the apparent lack of chitin and cellulose raised the possibility of
a lack of relationship of A. parasiticum with other groups of fungi
as well. Recent DNA evidence confirms that A. parasiticum is a protozoan
(Benny and O'Donnell; Ustinova et al., 2000).
In 1972 Sangar et al. published the results of an immunological
study on trichomycetes and Zygomycetes designed to test their serological
relatedness. Antigens were extracted from 21 isolates of Smittium representing
what were later determined to be four species, from one isolate of Amoebidium
parasiticum, and from six Zygomycetes: Linderina pennispora Raper
& Fennell, Dipsacomyces acuminosporus Benjamin, Syzygites
megalocarpus Ehrenb. ex Fr., Entomophthora apiculata (Thaxter)
Gustafsson, E. virulenta Hall & Dunn, and Basidiobolus ranarum
Eidam.
Antisera were raised in eight rabbits against seven of the Smittium
isolates
(one duplicate), and the eight antisera were tested against all 28 antigens
by immunoelectrophoresis and immunodiffusion. The data were processed with
a computerized, numerical taxonomic program that included cluster and principal
component analyses.
The immunodiffusion tests by Sangar et al. indicated that the antigens of L. pennispora reacted strongly with the antibodies of one isolate of S. simulii (93%, based on 100% reference reaction), and D. acuminosporus reacted fairly strongly with five of the eight antisera (22-52%). These same two species of Kickxellales produced lesser reactions against Smittium antibodies in the immunoelectrophoretic tests (15-18% against three antisera and 15-22% against five antisera, respectively, for L. pennispora and D. acuminosporus). The other Zygomycetes gave no, or very low, reactions with the Smittium antisera in both kinds of tests. Amoebidium parasiticum, significantly, reacted likewise, producing no precipitates.
In general, immunoelectrophoresis resulted in more distinct groupings in the analyses than did immunodiffusion. Figure 9.15 shows a projection of the first three principal components of variation derived from the correlation matrix for all 28 cultures using immunoelectrophoresis. Smittium culisetae clustered farthest from the other Smittium species, and is also morphologically most distinctive. Smittium simulii clustered into what Sangar et al. interpreted as two closely related groups, but they can be considered one species inasmuch as the morphological differences among those isolates also are very slight. The immunoelectrophoretic tests were not able to distinguish between the six S. culicis isolates and the single isolate of S. mucronatum (for which no antiserum was developed); the latter species is close to S. culicis on a morphological basis as well. In Fig. 9.15 the nontrichomycete species are clustered together, with the two Kickxellales paired and slightly apart from the others; this clustering was considered to be due to an artifact resulting from the failure of those antigens to react consistently and strongly with any of the antisera, and perhaps due to the limited number of antisera available for comparison with a large number of antigens. Ideally, antisera would have been obtained against each of the 28 antigens in order to do reciprocal serological tests throughout, but this was not practicable at the time.
The study by Sangar et al. showed a greater serological affinity among isolates within each Smittium species than between species (except for S. mucronatum), as expected. But unexpected was the serological relatedness, though at a low average level, of the two Kickxellales with some Smittium isolates, and the virtual lack of common antigens between Amoebidium and Smittium. The possible phylogenetic significance of these and other data are discussed in Chapter 12.
Peterson (1984; Peterson and Lichtwardt, 1986) used crossed and rocket
immunoelectrophoresis to test precipitin reactions of 12 antisera with
antigens extracted from 47 isolates of Harpellales, a few other fungi,
and Amoebidium parasiticum. The Harpellales included 29 cultures
of S. culisetae derived from a wide variety of hosts from different
geographical areas, plus isolates of five other species of Smittium,
and Capniomyces stellatus and Genistelloides hibernus. A
cluster diagram of the relationships of the isolates based on the number
of precipitins formed in crossed-immunoelectrophoresis showed good concordance
with the taxonomy of genera and species of Harpellales. Smittium culisetae
formed a distinct cluster but with no distinct groupings within the species
that could be attributed to geography or host origins. Antibodies of Linderina
pennispora (Kickxellales) had cross reactivity with some S. culisetae
isolates.
Porter and Smiley (1979) undertook a study of ribosomal RNA molecular
weights using a relatively large number of isolates in an attempt to clarify
phylogenetic relatedness of cultured trichomycetes and a wide representation
of Zygomycetes. They used nine isolates of Smittium (4 species),
one isolate of A. parasiticum, 15 species of Mucorales representing
six families, three Kickxellales, and one Entomophthorales. The molecular
weights for the heavy (25S) and light (18S) rRNA constituents were determined
by their relative electrophoretic mobility in polyacrylamide gels. The
average rRNA molecular weights for all Smittium isolates (calculated
from their tables) were 1.47 (1.41-1.51) x 106 and 0.75 (0.72-0.78)
x 106 for the 25S and 18S molecules, respectively; for A.
parasiticum, they were 1.36 and 0.69 x 106; and for all
the Zygomycetes, they averaged 1.29 (1.26-1.33) x 106 and 0.70
(0.67-0.73) x 106 Thus, each group of species appeared
to be quite distinct on this basis. As the rRNA molecules are considered
to be conservative in terms of evolution, Porter and Smiley concluded that
the genus Smittium is not phylogenetically related to either A.
parasiticum or the Zygomycetes. An overall evaluation of phylogenetic
relationships is given in Chapter 12.
Walker (1984) investigated 5S rRNA sequences of 11 Zygomycota that included the Harpellales species Smittium culisetae, Genistelloides hibernus,Capniomyces stellatus, and three Kickxellales (Dipsacomyces acuminosporus, Coemansia mojavensis, and Linderina macrospora), as well as Amoebidium parasiticum and two Chytridiales. In one of his several analyses of the data set, the dendrogram resulted in close grouping of the three Harpellales, but did not support a closely shared relationship with the Kickxellales. There were several incongruities in the dendrogram, suggesting that the 5S rRNA gene, as analyzed, was not satisfactory for resolving phylogenies of the fungi he studied.
The first detailed analyses of the phylogenetic relationship between Harpellales and Kickxellales were done by O'Donnell et al. (1998) using species representing four genera of Harpellales (Capniomyces, Furculomyces, Genistelloides, Smittium) and genera of Kickxellales, with the outgroup represented by Chytridiales. Morphological characters were also utilized in some of the analyses. 18S rDNA gene trees supported monophyletic lineages in the Harpellales and Kickxellales (but with Spiromyces forming a separate sister clade). O'Donnell et al. pointed out the need to obtain molecular data from the unculturable Asellariales and Eccrinales to test the monophyly of the Trichomycetes as an order. Nonetheless, Harpellales and Kickxellales had a close phylogenetic relationship.
Benny (2001), in a cladogram based in 18S rDNA sequences, incorporated the four same genera and species of Harpellales utilized by O'Donnell et al., and Amoebidium parasiticum, along with Kickxellales and other representatives of fungal classes and protozoans. These represented all five of Cavalier-Smith's (1998) eukaryotic kingdoms. Benny's cladogram showed that Harpellales and Kickxellales are sister groups, and provided clear evidence that A. parasiticum is not a fungus but part of a protozoan assemblage known as DRIPs (also see Benny et al., 2000, Ustinova et al., 2000). He also stressed the need for sequencing data of the other two trichomycete orders, Asellariales and Eccrinales.
Gottlieb (Gottlieb and Lichtwardt, 2001) used 82 axenic cultures of
Harpellales that included Capniomyces stellatus, Furculomyces boomerangus,
Genistelloides hibernus, and 77 isolates of Smittium representing
most of the species in the University of Kansas Culture Collection including
a number of unnamed species. She found that the internal transcribed spacers
ITS 1 and ITS 2 using RFLP analysis of PCR-amplified fragments were not
suitable for comparing isolates within Smittium. A more selective
representation of Harpellales and other Zygomycota, using the 18S rDNA
gene, produced five lineages within Smittium that suggested the
genus is not monophyletic. Smittium spp. are identified as branched
harpellids that produce trichospores with a collar and a single appendage.
In those species where zygospores have been found, they are Type II. Consequently,
species within the genus, as it now stands, warrants reexamination. Her
cladograms showed an affinity of Harpellales and Kickxellales, but did
not strongly support a kickxellid origin of Harpellales.
Like most fungi maintained in culture, trichomycetes may produce
aberrant structures not normally seen when they are in their natural environment.
In Smittium isolates this may include abortive trichospores, chlamydospore-like
vegetative cells, and other thallial deformities. Such abnormalities are
more frequent when the fungi are grown on agar media with liquid overlayers,
and predominate in older cultures.
One interesting variant was found by Chapman (1966). She noted in a petri dish of BHI/10 agar with a water overlayer and seeded with S. culisetae isolate HAW-14-5 that numerous small colonies of the Smittium were developing around a fungus contaminant growing under the agar layer. Examination of the Smittium revealed that the numerous colonies came from extruding trichospores (spores of this isolate had not been found to extrude in vitro), and that the trichospores were virtually all asymmetrical, often bent to one side. Colonies that developed from the abnormal trichospores produced trichospores with similar bizarre shapes. The contaminant was isolated and identified as Aspergillus amstelodami (Mangin) Thom & Church. Normal subcultures of the S. culisetae isolate remained normal when grown adjacent to the contaminant species in test plates of BHI/10 medium and when grown on medium that had previously supported growth of the contaminant. A subculture of the abnormal strain of S. culisetae was fed to larval Aedes triseriatus, and the resulting thalli in the gut produced spores predominantly, but not entirely, of the normal type. The abnormal strain was kept in culture with repeated transfers, and after many years it eventually reverted to producing mostly normal spores. The possibility of some type of viral infection transmitted from A. amstelodami was not investigated.
The authors have found that some strains of Amoebidium parasiticum also may produce aberrant structures. These are more pronounced in still cultures than shaken ones, but the precise conditions that lead to such growth have not been investigated. One form of abnormality involves the formation of giant, thick-walled cells resembling cysts. These apparently do not arise from the amoeba-cyst phase; rather, they seem to be vegetative cells (thalli) that enlarge abnormally and become thick walled. Although these are usually spherical, in some instances they may have irregular shapes. Another form of giant thallus seen in isolates contains two or more generations of cells within a thin outer wall. In this instance it appears that sporangiospores are not released from a mother thallus, and these in turn enlarge and produce unreleased sporangiospores, and so on. Meanwhile, the older thalli keep enlarging to accommodate the internal generations.
The authors have had several strains of A. parasiticum that originally grew well in vitro but began to decline over time and eventually died out. During this process some abnormally shaped cells were observed as well as cells that appeared to be lysed. That such cultures might have had viral infections is a possibility, in view of the presence of virus-like particles in Paramoebidium spp. (see Chapter 7). Current cultures of A. parasiticum have been examined by Lichtwardt (unpubl.), but no virus-like particles were detected with the electron microscope.