Interactions among juveniles of two freshwater crayfish species and a predatory fish

Two freshwater crayfish species, Astacus astacus L. and Pacifastacus leniusculus Dana, co-occur in some Swedish lakes. Observational studies indicate that the introduced, North American species P. leniusculus may gradually replace the native A. astacus, but the mechanism behind the replacement is not known. This study examined the direct effects of interspecific competition between the crayfish, and indirect effects of competitive interactions and fish (European perch, Perca fluviatilis L.) predation. Three different experiments with young-of-the-year (YOY) crayfish were performed. P. leniusculus was strongly dominant over similar-sized A. astacus in interference competition for shelter in a laboratory experiment. However, in a 35-day experiment in outdoor pools, A. astacus growth and survival were about equally affected by interactions with conspecifics and P. leniusculus. In contrast, P. leniusculus was significantly more affected by intraspecific competition than by competition with A. astacus, suggesting asymmetric competition between the two species. The presence of perch in outdoor ponds with mixed-species groups of the two crayfish species resulted in considerably higher predation rates on A. astacus than on P. leniusculus. Both species showed strong antipredator responses to perch by increasing refuge use. I suggest that higher perch predation rates on A. astacus originate from P. leniusculus being the superior species in interspecific competition for shelter. Because of displacement from refuges, A. astacus individuals become more exposed to the predator. This indirect effect of interactions among the two cray-fish species and the predator may be important in the observed in situ replacement of A. astacus by P. leniusculus.     

Ultrastructure of the penetration of the crayfish integument by the fungal parasite, Aphanomyces astaci, Oomycetes

In the crayfish, Astacus astacus, susceptible to the crayfish plague fungus, penetration of the cuticle by the parasite occurred in the soft cuticle. The zoospore lysed the surface lipid layer, tore it away, and formed an infection peg (germ tube) that penetrated through the epicuticle. A septum was formed in the infection peg, and a hypha was formed below the inner epicuticular surface. In the endocuticle, hyphae grew preferrentially parallel to the surface, occassionally perpendicular to it. Growth direction in relation to cuticle architecture is discussed. Subsequently, some hyphae started to penetrate out through the epicuticle. This process was preceded by the swelling of the hyphal tip touching the inner side of the epicuticle. The hypha penetrating out through the epicuticle was much thicker than the infection peg. Histolytic activity, combined with mechanical penetration, seems to be evident in all stages and levels except in the outward penetration of the epicuticular lipid surface layer, where only mechanical rupture could be seen. Differences in the protoplasmic ultrastructure were found between the spore and the penetrant hyphae. Penetration of the cuticle of a resistant crayfish was essentially identical to that in susceptible ones. However, inward penetration of intact epicuticle was too scarce to allow for ultrastructural studies.     

Replacement of the native crayfish Astacus astacus by the introduced species Pacifastacus leniusculus in a small, enclosed Finnish lake: a 30-year study

The native noble crayfish Astacus astacus L., and the introduced North American signal crayfish Pacifastacus leniusculus Dana, co‐occur in Slickolampi, a small lake in southern Finland. That both species have lived side‐by‐side for 30 yr without any signs of crayfish plague Aphanomyces astaci, indicates that the P. leniusculus population must be plague‐free. According to annual trap catches and population size estimates, A. astacus was clearly dominant in the 1970s and most of the 1980s. At the end of the 1980s, however, there was a shift in the relative abundances of the two species, and in the 1990s, P.leniusculus became dominant. As the 1990s drew to a close, it accounted for >98% of total catches. Originating from a minor stocking (only 900 2nd stage juveniles) P. leniusculus has not augmented the existing fauna in this lake but has almost completely replaced A. astacus. Both species seem to a great extent to prefer the same types of biotope but P. leniusculus is distinctly more demanding and was encountered less often than A. astacus on gently‐sloping soft shores. The proportion of A. astacus with chelae injuries (16 yr, mean 17.3%) was nearly twice that of P. leniusculus (9.3%), suggesting that agonistic interspecific encounters do occur and that P. leniusculus is much more competitive. However, the consistent weakening of A. astacus, even at sites with only a low density of P. leniusculus, indicates that the elimination of A. astacus is not adequately explained by competitive exclusion. We suggest that its disappearance is governed by a combination of several interacting mechanisms, of which harvest (≥100 mm specimens of both species) and interspecific competition with P.leniusculus were initially the main reasons for the decline in the population. The ultimate reason for the collapse of A. astacus seems to have been the almost complete cessation of successful reproduction, presumably due to reproductive interference between the two species. Interspecific mating results in females laying sterile eggs. Although both species suffer from the ensuing loss of recruitment, the consequences are less serious for P. leniusculus, which has a higher capacity for population increase than A. astacus: the smaller the proportion of A. astacus, the greater the role played by reproductive interference as a replacement mechanism.     

Diurnal variations in physiology and behaviour of the noble crayfish Astacus astacus and the signal crayfish Pacifastacus leniusculus

Heart rate, locomotor activity, and oxygen consumption were recorded simultaneously and continuously in seven individuals of the noble crayfish Astacus astacus (Linneus 1758) and seven individuals of the signal crayfish Pacifastacus leniusculus (Dana 1852). The recordings were made in the laboratory over 7 days at 15°C under a 12?:?12?h dark?:?light regime. Circadian rhythms in heart rate, locomotor activity and oxygen consumption were found both in A. astacus and P. leniusculus. Increased heart rate, locomotor activity, and oxygen consumption levels during night time in both A. astacus and P. leniusculus illustrated expression of nocturnal behaviour. No differences in oxygen consumption levels were observed between A. astacus and P. leniusculus. Also, no significant difference between heart rate levels or heart rate variances was found in A. astacus and P. leniusculus at night. During day, however, heart rate levels, heart rate variances and locomotor activity were higher in P. leniusculus than in A. astacus. The higher activity level in P. leniusculus than in A. astacus during daytime indicates that P. leniusculus is less strictly nocturnal than is A. astacus.     

Associations between stream habitat characteristics and native and alien crayfish occurrence

Human mediated introductions of non-indigenous crayfish species (NICS) are responsible for their rapid colonisation of European freshwaters. The introduction of North American crayfish is furthermore linked to the spread of crayfish plague and the decline of indigenous crayfish species (ICS). As the management of ICS and NICS have become necessary, a detailed knowledge on their distribution and ecological requirements is needed. We studied the current range of native noble crayfish Astacus astacus and stone crayfish Austropotamobius torrentium, as well as alien signal crayfish Pacifastacus leniusculus in Carinthia (Austria) and evaluated environmental and physical habitat features in streams with and without crayfish. Meanwhile, the loss of many ICS populations was recorded and alien P. leniusculus was found to be widespread in this region. Most of the habitat features of streams having crayfish differed significantly from sites lacking crayfish for at least one investigated native or alien species. Furthermore, multivariate and regression analyses showed specific differences in the habitat use of the investigated crayfish. Our results showed that the presence of alien P. leniusculus was associated with larger and smoother sloped lowland rivers, while the occurrence of the two native species was confined to smaller streams either at higher altitudes and with distinct physical habitat conditions (A. torrentium) or with moderate water temperatures (A. astacus). This study helps to identify potential refuge areas for the endangered native species and to predict the further spread of the most common non-native crayfish species in European streams.     

Effects of crayfish on leaf litter breakdown and shredder prey: are native and introduced species functionally redundant?

Recent increases in biological invasions frequency may have important consequences on native communities. However, functional redundancy between invasive and native species could reduce non-native species effects on native ecosystems. Despite this, even small differences in functional traits between these species may still have unpredictable effects on colonized ecosystems. Invasive crayfish, as ecosystem engineers, potentially have wide and complex effects on recipient ecosystems, even when replacing a native counterpart. We used laboratory microcosms to test whether native (Astacus astacus) and invasive crayfish species (Orconectes limosus, Pacifastacus leniusculus and Procambarus clarkii) are actually functionally redundant in their effects on prey/shredder density and leaf litter breakdown. Results show that crayfish strongly influenced macroinvertebrate numbers and leaf litter breakdown and indicate that differences in direct (prey and leaf litter consumption) and indirect (prey habitat use and leaf litter breakdown) effects between crayfish species do exist. While the replacement of A. astacus by O. limosus may have induced only minor changes in freshwater ecosystems, invasions by the larger and more aggressive P. clarkii and P. leniusculus will likely have strong effects on invaded ecosystem. Overall, there seems to be no functional redundancy between these four species and outcomes of crayfish invasion will likely be species specific.     

Comparative ultrastructure of the spermatozoa of three crayfish species: Austropotamobius torrentium,Pacifastacus leniusculus,and Astacus astacus (Decapoda: Astacidae)

This study reports about the spermatozoal ultrastructure of three species of astacid crayfish, i.e., the stone crayfish Austropotamobius torrentium, signal crayfish Pacifastacus leniusculus, and noble crayfish Astacus astacus. The acrosome is a cup shaped and electron‐dense structure at the anterior of the spermatozoon and comprises three layers of differing electron densities filled with parallel filaments that extend from the base to the apical zone. The acrosome was significantly longer in A. astacus than in P. leniusculus and the shortest acrosome belongs to A. torrentium. The width of the acrosome was significantly narrower in A. torrentium than in P. leniusculus and the widest acrosome belongs to A. astacus. The L:W ratio was significantly greater in A. torrentium than in P. leniusculus and the lowest ratio belongs to A. astacus. Radial arms are visible on each side of the acrosome or nucleus in sagittal view and wrap around the spermatozoon. Each radial arm comprises a parallel bundle of microtubules arranged along the long axis within a sheath. The nucleus, with decondensed material, is located in the posterior of the cell. All parts of the spermatozoon are tightly enclosed within an extracellular capsule. Despite a well‐conserved general structure and similarity of pattern among these spermatozoa, differences in the dimensions of the acrosome within the studied species may be useful to help distinguish the different crayfish species. J. Morphol., 2013. © 2013 Wiley Periodicals, Inc.     

Phenol oxidation in soft cuticle and blood of crayfish compared with that in other arthropods and activation of the phenol oxidases by fungal and other cell walls

Phenol oxidase from blood as well as from soft cuticle of different crayfish was highly activated by cell walls of a number of fungi and other plants, but the enzymes from other crustaceans tested and from some insects were not. Attachment of the phenol oxidase on blood and soft cuticle to the cell wall surface of certain plants and subsequent heavy melanization of the walls with natural or added substrate may be a unique property of crayfish since it did not occur in any other arthropods tested. Interestingly, blood and cuticle of crayfish showed the same specificity in these processes and the mechanisms might therefore be similar, if not the same, in both. The activity of the nonadsorbed enzyme dispersed in the blood was higher in a crayfish species resistant to fungal attack than in susceptible ones, but the relative degree of activation by adding cell walls was greater in the latter.The natural substrate for the phenol-oxidase activity in crayfish blood was released or mobilized by the blood cells. The substrate for the activity in cuticle was not available in the intact cuticle itself. It appeared only after a “signal” had been transmitted through the cuticle to the cells inside. The possibility that these mechanisms are involved in the cuticular as well as the blood defense system against fungal and other microbial parasites is discussed.     

Non-indigenous signal crayfish <Emphasis Type="Italic">Pacifastacus leniusculus</Emphasis> are now common in Danish streams: preliminary status for national distribution and protective actions

North American signal crayfish (Pacifastacus leniusculus) are invasive in Europe and pose a serious threat to indigenous European crayfish such as the noble crayfish (Astacus astacus). This is mainly because signal crayfish is the carrier of crayfish plague agent, Aphanomyces astaci, which freshwater crayfish from all other continents are highly susceptible to. Until recently, the distribution of signal crayfish in Danish streams has been considered local and restricted to a small geographical area. Here we present data demonstrating that signal crayfish are now widespread in Denmark, including the largest Danish river, River Gudenå. For one of the rivers where co-existing signal crayfish and indigenous noble crayfish were documented, sensitive molecular tests could not detect the crayfish plague agent Aphanomyces astaci in either species. Hence, it seems that not all signal crayfish are chronic carriers of the disease. For the remaining freshwater systems with the introduced signal crayfish, the infection status is presently unknown. Large areas of the freshwater systems in Denmark also remain unexplored with respect to presence/absence of signal crayfish and noble crayfish. Nevertheless, our preliminary data that covers about 14% of the Danish rivers, strongly suggests that signal crayfish should be considered as a common invader that poses an increased threat to the biota in Danish streams, in particular for the indigenous noble crayfish.     

First records of the non-indigenous signal crayfish (<Emphasis Type="Italic">Pacifastacus leniusculus</Emphasis>) and its threat to noble crayfish (<Emphasis Type="Italic">Astacus astacus</Emphasis>) populations in Estonia

This study gives an overview of status and distribution of signal crayfish (Pacifastacus leniusculus), the first NICS in Estonia and its influence on native noble crayfish (Astacus astacus) populations. The first specimen of signal crayfish was caught during the monitoring of noble crayfish in North Estonia in 2008. The signal crayfish has since been found in three additional sites. Test fishing has indicated that the abundance of signal crayfish has been fluctuating between years and among localities. It has had strong negative impact on abundance of one noble crayfish population. The disconnected distribution of signal crayfish strongly suggests that these populations are the result of human-assisted introductions. Real-time PCR analyses proved that signal crayfish carry the causative agent of the crayfish plague, an oomycete Aphanomyces astaci, thus contributing to its spread. Mortalities in noble crayfish populations had been caused by A. astaci strains from A, B and E genotype group.     

Does the introduced signal crayfish occupy an equivalent trophic niche to the lost native noble crayfish in boreal lakes?

The introduced North-American signal crayfish (Pacifastacus leniusculus) has become widespread throughout Europe where it has often replaced the native noble crayfish (Astacus astacus). The impact of this replacement on ecosystem processes in boreal lakes is still largely unknown. We compared the trophic niches of these two crayfish species in 16 small to medium sized boreal lakes in southern Finland; eight lakes with noble crayfish and eight lakes where the native crayfish populations had been lost and replaced by signal crayfish. We analysed carbon and nitrogen stable isotopes from samples of the crayfish and their putative food sources, and used stable isotope models to compare trophic niche widths of the two species of crayfish and to quantify the food sources used by them. At species level the signal crayfish exhibited a substantially larger trophic niche than that of the noble crayfish, but within-lake populations of the species did not differ in their niche widths. The isotopic niches of the two species strongly overlapped, and while the estimated proportions of food resources (profundal and littoral macroinvertebrates, terrestrial leaf detritus and macrophytes) used by crayfish varied considerably among individual populations, they did not differ consistently between the species. Our results suggest that, contrary to often expressed concerns, replacement of lost noble crayfish populations by the signal crayfish may not greatly alter the littoral food web structure in boreal lakes.     

The first record of the non-indigenous signal crayfish <Emphasis Type="Italic">Pasifastacus leniusculus</Emphasis> in Norway

The non-indigenous signal crayfish Pasifastacus leniusculus was registered for the first time in Norway in October 2006. The location represents an isolated pond about 100 km in air line from the nearest known signal crayfish population in the neighbouring country Sweden without any connecting watercourses. The occurrence is therefore undoubtedly caused by human introduction. Molecular analyses confirmed that tested individuals from the signal crayfish population were carriers of the crayfish plague agent Aphanomyces astaci. Disease carrying signal crayfish represents a severe threat to the indigenous and endangered noble crayfish Astacus astacus. Norwegian authorities are currently considering actions for the eradication of the signal crayfish population.     

A comparative study of the physiological condition of native and invasive crayfish in Croatian rivers

The physiological condition of two invasive crayfish species identified in Croatian rivers (Orconectes limosus and Pacifastacus leniusculus) was compared with that of native crayfish populations (Astacus astacus and A. leptodactylus). Hepatosomatic, gonadosomatic and abdominal muscle indices were examined for all four species and within a specific invadernative pair. The results demonstrate that invasive crayfish had better overall physiological condition when compared to native species, with a higher energy content of the hepatopancreas, abdominal muscle and gonads in invasive than in native species, with the exception of A. leptodactylus females which demonstrated an increased ability to allocate energy to reproduction in comparison with other species. These results also indicate that A. astacus is physiologically more unstable than A. leptodactylus and thus more likely to be outcompeted and replaced by non-indigenous crayfish species.     

Growth conditions influence melanization of Brazilian clinical Sporothrix schenckii isolates

Sporothrix schenckii is known to produce DHN melanin on both conidial and yeast cells, however little information is available regarding the factors inducing fungal melanization. We evaluated whether culture conditions influenced melanization of 25 Brazilian S. schenckii strains and one control strain (ATCC 10212). Tested conditions included different media, pH, temperature, incubation time, glucose concentrations, and presence or absence of tricyclazole or L-DOPA. Melanization was reduced on Sabouraud compared to defined chemical medium. The majority of strains produced small amounts of melanin at 37 °C and none melanized at basic pH. Increased glucose concentrations did not inhibit melanization, rather increasing glucose enhanced pigment production in 27% of strains. Melanin synthesis was also enhanced by the addition of L-DOPA and its addition to medium with tricyclazole, an inhibitor of melanin synthesis, resulted in fungal melanization, including hyphal melanin production. Our results suggest that different S. schenckii strains have distinct control of melanization and that this fungus can use phenolic compounds to enhance melanization in vitro.     

Solid-state NMR Reveals the Carbon-based Molecular Architecture of Cryptococcus neoformans Fungal Eumelanins in the Cell Wall

Melanin pigments protect against both ionizing radiation and free radicals and have potential soil remediation capabilities. Eumelanins produced by pathogenic Cryptococcus neoformans fungi are virulence factors that render the fungal cells resistant to host defenses and certain antifungal drugs. Because of their insoluble and amorphous characteristics, neither the pigment bonding framework nor the cellular interactions underlying melanization of C. neoformans have yielded to comprehensive molecular-scale investigation. This study used the C. neoformans requirement of exogenous obligatory catecholamine precursors for melanization to produce isotopically enriched pigment “ghosts” and applied 2D ¹³C-¹³C correlation solid-state NMR to reveal the carbon-based architecture of intact natural eumelanin assemblies in fungal cells. We demonstrated that the aliphatic moieties of solid C. neoformans melanin ghosts include cell-wall components derived from polysaccharides and/or chitin that are associated proximally with lipid membrane constituents. Prior to development of the mature aromatic fungal pigment, these aliphatic moieties form a chemically resistant framework that could serve as the scaffold for melanin synthesis. The indole-based core aromatic moieties show interconnections that are consistent with proposed melanin structures consisting of stacked planar assemblies, which are associated spatially with the aliphatic scaffold. The pyrrole aromatic carbons of the pigments bind covalently to the aliphatic framework via glycoside or glyceride functional groups. These findings establish that the structure of the pigment assembly changes with time and provide the first biophysical information on the mechanism by which melanin is assembled in the fungal cell wall, offering vital insights that can advance the design of bioinspired conductive nanomaterials and novel therapeutics.     

Post-mating spermatophore storage strategies in two species of crayfish: implications for broodstock management

Female crayfish stores male gametes after mating until the beginning of egg laying and fertilization. The aim of the present study was to investigate the duration of post-mating spermatophore storage as well as the timing and temperature of spawning in two crayfish species of economic importance, namely the signal crayfish Pacifastacus leniusculus and the noble crayfish Astacus astacus. Results showed that the average duration of the post-mating spermatophore storage is significantly (P<0.05) longer in the noble crayfish (34.6±1.7 days, range: 19 to 60 days) than the signal crayfish (3.9±0.5 days, range: 1 to 18 days). The highest percentages of the post-mating spermatophore storage duration in the signal crayfish (46.5%) and the noble crayfish (44.5%) were 1 and 31 to 40 days, respectively. While there is an overlap in the timings of mating and egg laying in the signal crayfish, these two reproductive processes were not observed at the same days in the noble crayfish and there was at least 2 weeks interval between last mating and first egg laying individuals. Average mating and egg laying temperatures were significantly (P<0.05) higher in the signal crayfish than the noble crayfish. The average temperatures for mating in both species were significantly (P<0.05) higher than the temperatures that they utilized for egg laying. In conclusion, female noble crayfish stores post-mating spermatophores a longer duration compared with the signal crayfish. Also, the signal crayfish mates and lays egg in temperatures that are higher than the noble crayfish. Spawning season is shorter in the signal crayfish compared with the noble crayfish. The results of present study provide information contributing to the crayfish broodstock management in aquaculture.     

Cryptococcus neoformans Can Utilize the Bacterial Melanin Precursor Homogentisic Acid for Fungal Melanogenesis

Cryptococcus neoformans melanizes in the environment and in mammalian tissues, but the process of melanization in either venue is mysterious given that this microbe produces melanin only from exogenous substrates. Understanding the process of melanization is important because melanization is believed to protect against various stresses in the environment, including UV radiation, and pigment production is associated with virulence. Melanization in C. neoformans requires the availability of diphenolic precursors. In contrast, many bacteria synthesize melanin from homogentisic acid (HGA). We report that C. neoformans strains representing all four serotypes can produce a brown pigment from HGA. The brown pigment was acid resistant and had the electron paramagnetic resonance spectrum of a stable free radical, qualities that identified it as a melanin. Melanin “ghost”-like particles obtained from pigmented C. neoformans cells were hydrophobic, fluorescent under a variety of irradiation wavelengths, negatively charged, insoluble in organic solvents and alcohols, resistant to degradation by strong acids, and vulnerable to bleaching. HGA melanization was laccase dependent and repressed by high concentrations of glucose. The ability of C. neoformans to utilize a bacterial melanin precursor compound suggests a new substrate source for melanization in the environment.     

The Mosquito Melanization Response Is Implicated in Defense against the Entomopathogenic Fungus Beauveria bassiana

Mosquito immunity studies have focused mainly on characterizing immune effector mechanisms elicited against parasites, bacteria and more recently, viruses. However, those elicited against entomopathogenic fungi remain poorly understood, despite the ubiquitous nature of these microorganisms and their unique invasion route that bypasses the midgut epithelium, an important immune tissue and physical barrier. Here, we used the malaria vector Anopheles gambiae as a model to investigate the role of melanization, a potent immune effector mechanism of arthropods, in mosquito defense against the entomopathogenic fungus Beauveria bassiana, using in vivo functional genetic analysis and confocal microscopy. The temporal monitoring of fungal growth in mosquitoes injected with B. bassiana conidia showed that melanin eventually formed on all stages, including conidia, germ tubes and hyphae, except the single cell hyphal bodies. Nevertheless, melanin rarely aborted the growth of any of these stages and the mycelium continued growing despite being melanized. Silencing TEP1 and CLIPA8, key positive regulators of Plasmodium and bacterial melanization in A. gambiae, abolished completely melanin formation on hyphae but not on germinating conidia or germ tubes. The detection of a layer of hemocytes surrounding germinating conidia but not hyphae suggested that melanization of early fungal stages is cell-mediated while that of late stages is a humoral response dependent on TEP1 and CLIPA8. Microscopic analysis revealed specific association of TEP1 with surfaces of hyphae and the requirement of both, TEP1 and CLIPA8, for recruiting phenoloxidase to these surfaces. Finally, fungal proliferation was more rapid in TEP1 and CLIPA8 knockdown mosquitoes which exhibited increased sensitivity to natural B. bassiana infections than controls. In sum, the mosquito melanization response retards significantly B. bassiana growth and dissemination, a finding that may be exploited to design transgenic fungi with more potent bio-control activities against mosquitoes.     

The imd gene is required for local Cecropin expression in Drosophila barrier epithelia

Surfaces of higher eukaryotes are normally covered with microorganisms but are usually not infected by them. Innate immunity and the expression of gene-encoded antimicrobial peptides play important roles in the first line of defence in higher animals. The immune response in Drosophila promotes systemic expression of antimicrobial peptides in response to microbial infection. We now demonstrate that the epidermal cells underlying the cuticle of larvae respond to infected wounds by local expression of the genes for the antimicrobial peptide cecropin A. Thus, the Drosophila epidermis plays an active role in the innate defence against microorganisms. The immune deficiency (imd) gene was found to be a crucial component of the signal-induced epidermal expression in both embryos and larvae. In contrast, melanization, which is part of the wound healing process, is not dependent on the imd gene, indicating that the signalling pathways promoting melanization and antimicrobial peptide gene expression can be uncoupled.     

The Role of L-DOPA on Melanization and Mycelial Production in Malassezia Furfur

Melanins are synthesized by organisms of all biological kingdoms and comprise a heterogeneous class of natural pigments. Certain of these polymers have been implicated in the pathogenesis of several important human fungal pathogens. This study investigated whether the fungal skin pathogen Malassezia furfur produces melanin or melanin-like compounds. A melanin-binding monoclonal antibody (MAb) labelled in vitro cultivated yeast cells of M. furfur. In addition, melanization of Malassezia yeasts and hyphae was detected by anti-melanin MAb in scrapings from patients with pityriasis versicolor. Treatment of Malassezia yeasts with proteolytic enzymes, denaturant and concentrated hot acid yielded dark particles and electron spin resonance spectroscopy revealed that these particles contained a stable free radical compound, consistent with their identification as melanins. Malassezia yeasts required phenolic compounds, such as L-DOPA, in order to synthesize melanin. L-DOPA also triggered hyphal formation in vitro when combined with kojic acid, a tyrosinase inhibitor, in a dose-dependent manner. In this respect, L-DOPA is thought to be an essential substance that is linked to both melanization and yeast-mycelial transformation in M. furfur. In summary, M. furfur can produce melanin or melanin-like compounds in vitro and in vivo, and the DOPA melanin pathway is involved in cell wall melanization.