Perhaps the best known species of the genus has beenSmittium culisetae. It is widespread and found in several host species, especially mosquitoes. It has been used often in laboratory research.(Wikipedia 2015)
While S. culisetae is commonly isolated from Culicidae (mosquito larvae), it has a relatively large host range and can be found in Simuliidae (black flies), Chironomidae (non-biting midges), Ceratopogonidae (sandflies) and Tipulidae (crane flies) (Alencar et al 2003). Williams and Lichtwardt (1972) found S. culisetae to be relatively non-host specific, as specificity of the fungus is absent at the species and genus level for the mosquitos tested. The hosts used in this study were also obtained from diverse regions of the globe.
Williams and Lichtwardt (1972) found that S. culisetae can grow in mosquito larvae without the presence of other microorganisms, showing that other organisms such as bacteria are not needed for S. culisetae to associate with the mosquito larvae. S. culisetae has been shown however to need to be ingested by mosquito larvae in order to germinate (Williams and Lichtwardt 1972).
The holdfast of S. culisetae is attached to the surface of the arthropod gut and does not penetrate it (Horn and Lichtwardt 1981), which supports many researchers’ claims that S. culisetae does not act as a parasite of its arthropod host.
It has been shown that when Culicidae change instar status, they lose infestation of S. culisetae (the hindgut, containing the fungus, is shed). S. culisetae spores have been observed weakly attached to the hindgut at a half hour after larval ingestion of trichospores, and S. culisetae germination has been observed after just one and a half hours after larval ingestion of trichospores (Williams and Lichtwardt 1972). After germination and maturation of trichospores, these spores are released into the gut lumen and combined in the column with the host feces (Vojvodic and McCreadie 2009). This rapid process strongly matches the lifecycle of mosquitos, as mosquitos reach their second instar in just 23-30 hours. When mosquitos reach the second instar they shed their hindgut (containing the fungus). For this reason, it is important that the fungus associate with the hindgut quickly and sporulate before that shedding step. Horn and Lichtwardt (1981) also have hypothesized that infestations in later instars are due to presence of initial infecting trichospores in the environment or from spores that arose from earlier, infected instars that successfully reached sporulation. Also, any thalli that are shed with the hindgut lining may still grow and produce trichospores outside of the host (Vojvodic and McCreadie 2009).
Experimentation has been conducted in vitro to assess the nutritional exchange between S. culisetae and its arthropod host. Results of these trials can be found within the “Smittium culisetae Cultivation and Behavior In Vitro” section.
Smittium culisetae is a fungus within the class Trichomycetes (Alencar et al. 2003), therefore it is largely characterized as an obligate (Horn and Lichtwardt 1981) inhabitant of the digestive tracts of arthropods. The genus Smittium was one of the first genera of obligate gut fungi to be discovered and the organisms in the genus are exceptional because they are readily cultured. Additionally, Smittium is the most species-rich genus within the Harpellales (Wang et al 2014).
The first cultures of Smittium were established by Clark, Kellen and Lindegren in 1963 from mosquito larvae. S. culisetae specifically was first described by Lichtwardt in 1964 from Culisetae impatiens, and since then it has been reported across the globe in many hosts.
S. culisetae has been reported in Japan, Australia, New Zealand, Hawaii, continental United States, France, Argentina, Costa Rica, and Brazil (Alencar et al. 2003).
The common region used for sequence comparison in fungi, the ITS region, is not able to differentiate species within Smittium.
The genes 18S rDNA, 28S rDNA, RPB1, RPB2, and MCM7 have been used (Wang et al 2014) to analyze species within Smittium, as well as organisms closely related to Smittium.
S. culisetae can be easily identified by its relatively small trichospores that have a slight bulge less than half way down the length of the trichospore (Alencar et al. 2003). Trichospores have a short collar and the “plasma membrane of the trichospore is loosely appressed to the spore wall” (Williams and Lichtwardt 1972). These spores are located at the ends of branched thalli (Farr and Lichtwardt 1967). When the trichospore germinates, the protoplast moves out of the apical end of the trichospore and is normally observed to be longer than the spore from which it was exuded. A dark region of this protoplast migrated downward and settles at the base of protoplasm, forming a supposed holdfast (Williams and Lichtwardt 1972). The thalli of S. culisetae is branched (Wang et al 2014), verticillate, and whorled.
A structure termed “holdfast” is the point of attachment of the fungus to the chitinous arthropod gut wall (Horn and Lichtwardt 1981).
Farr and Lichtwardt (1967) describe the ultrastructure of S. culisetae as similar to that of other groups of fungi. There is no Golgi body in S. culisetae which is again common to many groups of fungi. The mycelium however, is unique in that it contains swollen septal pores. Interestingly, the spores contain a swollen septal pore identical to those observed in the vegetative hyphae (Farr and Lichtwardt 1967). This septal pore feature of S. culisetae is comparable to those present in basidiomycetes, but without a septal pore cap.
Valle and Santamaria (2004) describe the trichospores of S. culisetae as elongate-ovoid, and with a collar of 1–2 mm. The dimensions of trichospores depend largely on the host on which the fungus is growing and the environment (such as temperature).
Phase-contrast microscopy is a common way to visualize the morphology of S. culisetae.
A study by Wang et al. (2014) used 99 taxa (in which Smittium, Smittium allies, Non-Smittium Harpellales, and Kickxellales were included), and five genes (18S rDNA, 28S rDNA, RPB1, RPB2, and MCM7) to create a supported and updated phylogeny. They found that their results supported previous trees of these organisms. There is the presence of a Non-Smittium clade that includes the genera Harpella, Bojamyces, Capniomyces, Genistelloides, Lancisporomyces, Legeriomyces, Pteromaktron, and Zancudomyces. There is also the presence of a Smittium clade that has two branches: true Smittium and Parasmittium. The organisms within the true Smittium group are characterized by having a tapering holdfast and non-verticillate thallus, while the organisms within the Parasmittium group are characterized by their pathogenic tendency.
The results of this study (Wang et al. 2014) also show the non-verticillate thallus to be the ancestral state of the overall Smittium clade.
Cultures of S. culisetae can be started by rinsing the fungus with a penicillin-streptomycin antibiotic solution and plating the fungus on 1/10th strength brain heart infusion agar, with a thin layer of distilled water atop the agar (Alencar et al 2003). To obtain cultures rich in trichospores, S. culisetae may be grown on tryptone glucose agar in Erlenmeyer flasks with constant shaking for 4 days at 24 C (Horn and Lichtwardt 1981). Furthermore, trichospores can be isolated by filtering with #1 Whitman filter papers (Horn and Lichtwardt 1981) or with a glass-wool 4d later (Vojvodic and McCreadie 2009).Farr and Lichtwardt (1967) found that the maximal growth of S. culisetae occurred at 10 C in 16 days, and at 22-28 C at seven days although S. culisetae can be grown successfully in vitro over a 27 degree range. This large temperature range and associated growth is thought to be due to the large host range of S. culisetae, as each host has a different temperature optimum (Vojvodic and McCreadie 2009). Additionally, Farr and Lichtwardt (1967) found the optimal pH for S. culisetae was 8.3 (at nine days).
To induce the germination of S. culisetae trichospores, potassium and increased pH should be implemented in the medium followed by reduced pH and potassium (Beard and Adler 2003).
Horn and Lichtwardt (1981) studied the nutritional relationship between Culicidae larvae and S. culisetae in a variety of completely sterile, isolated environments. When A. aegypti larvae (mosquito larvae) was grown on undefined media with and without S. culisetae, it was shown that the addition of S. culisetae led to an increased pupation of mosquitos. However, when Horn and Lichtwardt (1981) grew A. aegypti larvae out on a semi-defined media with and without S. culisetae, it was shown that the addition of S. culisetae led to an increased mortality of mosquitos. Horn and Lichtwardt (1981) explain this result by stating that number of spores, competing microorganisms, and nutrient availability can influence largely if S. culisetae is a beneficial or detrimental symbiont of mosquitos and other hosts. It was also thought that when mortality was increased by S. culisetae, it was due to S. culisetae thalli blocking the larvae hindgut. In both of these nutritional experiments there was no mortality in the first mosquito instar with the presence of S. culisetae, which shows that the larvae readily supports the fungus, supporting the hypothesis that S. culisetae is an established symbiont in some capacity. Additionally, it should be noted that the growth rate of larvae grown in the presence of S. culisetae was not affected by the fungus.
Horn and Lichtwardt (1981) found when Culicidae was grown without select compounds (riboflavin, pyridoxine, or nicotinamide), the addition of S. culisetae inoculum helped the insect reach an additional instar, which suggests S. culisetae may positively affect its insect host. When the same procedure was carried out for studying the insect-fungus relationship with the removal of compounds thiamine or Ca pantothenate, no additional instar was reached, showing that the positive interaction between S. culisetae and Culicidae may be dependent on environment. When the same procedure was yet again repeated but with the removal of sterols, one of 45 larvae pupated with S. culisetae (as opposed to zero pupating without S. culisetae).Horn and Lichtwardt (1981) hypothesize this relative success of larvae with S. culisetae is due to sterols present in the mycelium of S. culisetae. It was also experimentally shown that S. culisetae satisfies the sterol requirement of the mosquito and that desmosterol is the predominate sterol produced by S. culisetae.
Additionally, S. culisetae can attach to the external cuticle of Culicidae, which is noted as unusual by Horn and Lichtwardt (1981) since Smittium is a group of fungi characterized as obligate, internal symbionts of arthropods.
S. culisetae does not currently hold any economic purpose, nor is it used in human medicine.
