T h e more common hyphomycetous pathogens produce spores abun
dantly and release them in situations conducive to efficient dispersal.
Speare (1912) calculated that one adult of Rhabdocnemis obscura (Bois-duval) killed by M. anisopliae gives rise to 66.4 million spores. Frequently diseased insects die in elevated or exposed positions. Further, particu
larly in insects killed on or just in the soil, the spores are sometimes formed on coremia, of which one insect may give rise to several (Siemaszko, 1937). T h e fertile tips of these coremia are raised into ex
posed positions as a result of their growth toward the light (Boczkowska, 1934). Coremia are sometimes encountered in species not known charac
teristically to form them, such as B. densa and B. bassiana (Petch, 1930, 1932). Schaerffenberg (1957a, 1959) has recently claimed that ascospores of B. bassiana, B. densa, and M. anisopliae, discharged from perithecia 2 to 3 weeks after the death of the insect, germinated on the corpse to form conidiophores which aggregated as coremia. These interesting ob
servations, however, remain to be confirmed.
A high humidity is essential for spore production on the mummified insect. It affects both the rate and density of development of the sporu-lating mycelium (Toumanoff, 1933). Burnside (1930) found that if bees killed by aspergilli were kept in too dry an atmosphere, the fungus sporulated within the exoskeleton instead of on its surface.
T h e reproductive structures of the pathogen itself may become as
sociated with or attacked by secondary fungi (e.g., see Morrill and Back, 1912; Blunck, 1939; Aoki, 1957). Petch (1931) warns that it cannot be concluded that all the fungi found on an insect at the same time are stages of the same fungus, nor that the most obvious fungus on an insect is the one that killed it.
IX. NATURAL L I F E CYCLE OF THE PATHOGEN
Since similar conditions favor infection and sporulation, simple in
sect-to-insect infection cycles can flourish when these conditions prevail.
However, at the end of an outbreak of disease the surviving insects may be few and widely dispersed. H o w does the pathogenic fungus then survive? Several theoretically possible ways exist. T h e fungus may persist
in the insect p o p u l a t i o n at a very low incidence; it may successively in
fect different susceptible species; it may possess durable resting stages;
it may persist as d o r m a n t infections; or it may enter a saprophytic phase, e.g., in the soil. Since comparatively little systematic study has been made of natural life cycles, those characteristics which underlie survival by the above methods will be reviewed. T h e y are the degree of host specificity, the possession of resting stages, d u r a t i o n of viability of different stages, ability to form d o r m a n t infections, and capacity to live saprophytically.
Species of Beauveria, Metarrhizium, Spicaria, and Aspergillus are generally able to attack many different host species. It is possible though that specialized races exist within the different fungal species. Only a particular strain of M. anisopliae attacks larvae of Oryctes rhinoceros (Linnaeus) ( R a d h a et al., 1956), while this species isolated from the cara-bid Amara obesa (Say) h a d little virulence for wireworms, unlike a strain isolated from an elaterid (Rockwood, 1951). Similarly B. bassiana iso
lated from Loxostege sticticalis was more virulent toward this species than a strain from Agrotis segetum Schiffermüller (Volkoff, 1938). If host specialization did not exist, one would expect epizootics of mycoses to involve more than the one insect species in the same environment, b u t there are virtually no records of this happening.
Although conidia are not characteristically long-lived structures, they sometimes survive for fairly long periods, depending on conditions.
Unfortunately reports of d u r a t i o n of viability have not always specified these conditions. I n fungi generally, low temperatures and low humidi
ties are most conducive to long survival of spores. At 4°C, dry spores of B. bassiana survived u p to nearly two and a half years, whereas at 23°C they survived no more than 12 weeks (Steinhaus, 1960a). M. anisopliae conidia have been reported as surviving from more than one year to even three years (Glaser, 1926; Vouk and Klas, 1931; Boczkowska, 1935) although Masera (1957) found that they usually lost their virulence in the laboratory after 4 months. Spores of S. farinosa verticillotdes and Aspergillus ochraceus Wilhelm can survive about a year (Voukassovitch,
1925; Burnside, 1930). It would be rare, however, for a spore to en
counter conditions ideal for long survival in nature. Dampness, solar radiation, and climatic extremes take their toll. As little as 3 hours' di
rect exposure to the sun destroys the infectivity of spores of B. bassiana and B. globulifera (Toumanoff, 1933). Besides conidia, certain Hypho
mycetes such as Verticillium cinnamomeum Petch, a pathogen of citrus whitefly (Dialeurodes citri), and Sorosporella uvella form special spores suited to surviving adverse conditions (Morrill and Back, 1912; Speare,
1920).
Probably the most important resting stage in the natural life cycle
258 Μ. F. MADELIN
of most pathogenic Hyphomycetes is the S c l e r o t i u m within the m u m m i fied insect. T h e r e appear to be n o records of their m a x i m u m periods of survival. Reports of survival for several months are surely m u c h below the m a x i m u m (Harrar and McKelvey, 1942; Jaynes a n d Marucci, 1947).
Spicaria farinosa verticilloides in mummified chrysalids of P. botrana can survive for more t h a n a year in dry places a n d can in moist con
ditions produce several successive lots of conidiophores before it exhausts its nutritive reserves (Voukassovitch, 1925).
Reports of d o r m a n t infections have been noted above (Section IV, A), b u t how widespread these are and what conditions lead to their estab
lishment are not known.
Most insect-pathogenic Hyphomycetes readily grow saprophytically on artificial media, b u t they may do so less readily in nature. T h e vege
tative mycelium of some species, notably of Beauveria, spreads exten
sively from buried insect cadavers into the surrounding soil (Giard, 1892a; Rockwood, 1916; T i m o n i n , 1939; H u r p i n and Vago, 1958).
Blunck (1939) observed the mycelium of B. densa to spread 5 to 6 cm in radius and cites records u p to 10 cm. H o w m u c h this is growth at the expense of the dead insect and how m u c h is utilization of nutrients in the soil is not known. H u b e r (1958) concluded from his experiments that the spores of B. bassiana could not germinate in fresh unsterilized soil. Fungistasis in the soil is apparently a widespread p h e n o m e n o n (Dobbs et al., 1960). Nevertheless B. bassiana has been isolated from the soil (e.g., Sewell, 1959) as also has M. anisopliae (Miller et al., 1957;
Meyer, 1959), a n d Billings a n d Glenn (1911) demonstrated widespread uniform natural occurrence of B. globulifera in soil in Kansas. I n France, Dieuzeide (1925) discovered B. effusa in soil in regions where it was known to be active against Leptinotarsa decemlineata. O n e does not, however, know the state in which these fungi existed in these soils.
L i n d e m a n (1926) found that the proportion of Cleonus punctiventris killed by M. anisopliae in the soil was independent of the spore dose added, whereas with Sorosporella uvella it was dependent. H e inter
preted this as indicating that S. uvella could not multiply without its host, whereas M. anisopliae could grow saprophytically so that its abun
dance became independent of the original dose of spores. Ready sapro
phytic growth in n a t u r e appears characteristic of at least some patho
genic aspergilli (Lepesme, 1938; Ogloblin and Jauch, 1943).
T h e evidence in general thus suggests that some pathogenic Hypho
mycetes grow saprophytically in nature. H o w readily they resume a para
sitic existence is not known. Lepesme (1938) noted that a strain of A. flavus isolated from moldy grain was only a quarter as pathogenic to Schistocerca gregaria, by an unspecified assessment, as one isolated from locusts at the height of an epizootic caused by the fungus.
X . OPPOSITION TO FUNGAL INVASION
T h e resistance that the live insect presents to fungi operates at many levels. A n element of opposition has been attributed to cleaning move
ments and molting, both of which may remove adherent spores (Oglob-lin and Jauch, 1943). T h e first major barrier to infection lies in the in
tegument. If this is by-passed by direct injection of spores, even normally innocuous species can prove lethal (Burnside, 1930; Boczkowska, 1935;
Jolly, 1959). M u c h of this in tegumental resistance appears to be located in the epicuticle (Sussman, 1951b; Koidsumi, 1957). Koidsumi found that lipids in the exuviae of silkworms inhibited A. flavus, which was pathogenic toward these insects, and considers it highly probable that free medium-chain length unsaturated fatty acids in the cuticle, presum
ably caprylic or capric, contribute to its effectiveness as a barrier against fungi. T h e r e might also be resistive factors in the chitin itself. Lihnell (1944) found that M. anisopliae could digest chitin prepared from Cossus cossus larvae, b u t not that from elytra of Melolontha hippocastani Fa
bricius. Kawase (1958) isolated protocatechuic acid (3,4-dihydroxybenzoic acid) from the exuviae of silkworms at their p u p a t i o n period. I n onion bulbs the natural presence of.-this substance has been found to confer resistance to fungal attack. T h e possibility of a similar role in insects clearly exists.
Although it has been demonstrated that volatile materials able to kill fungal spores are produced by adults of Tribolium confusum Duval u n d e r stress conditions (van Wyk et al., 1959) it remains to be shown whether in any instance similar materials protect insects from fungal pathogens.
F u r t h e r defensive reactions occur at the hypodermal level in the form of aggregation of blood cells about the invader (Glaser, 1926; Paillot, 1930; Boczkowska, 1935). Phagocytosis of fungal cells in the hemolymph appears to be a general p h e n o m e n o n in mycoses, but, confronted with virulent pathogens, it generally proves ineffectual, for the ingested cell sometimes parasitizes the surrounding phagocyte (Speare, 1920; Paillot, 1930). Different insect species differ in their capacities to phagocytose the same fungus. Speare found that in order to kill silkworms and Lachnosterna species with S. uvella, to which they were normally resist
ant, it was necessary to inject into the blood enough spores apparently to exceed the ingestive capacity of their phagocytes.
It appears that if a fungus is able successfully to penetrate the integu
m e n t from the outside and gain access to the body cavity, generally it will sooner or later overcome the defensive factors in the blood and kill the insect. However, when a fungus has passively entered the body cavity by artificial injection or by way of a wound, it may sometimes
260 Μ. F. MADELIN
thereby have entered an environment in which its own lack of virulence renders it very vulnerable to the defensive factors in the blood, in which case it will soon be eradicated and the insect will survive. Recovery of an insect that has been invaded through the intact integument appears to be rare.
A r n a u d (1927) presents graphs showing the rate at which four species of fungi killed silkworms. T h e asymmetrical long-tailed mortality curves, particularly for B. globulifera and a Spicaria species, suggest the possi
bility that the longer the infected insect survives, the more it can retard the progress of the pathogen. T r u e acquired immunity to fungal diseases does not appear to exist (Paillot, 1930; Boczkowska, 1935).
X I . HOST SPECIFICITY OF THE PATHOGEN
T h e bases of host specificity are probably the defense systems located at the cuticular and hypodermal levels. T h e demonstration of antifungal lipids in insect cuticles by Japanese workers (Koidsumi and Wada, 1955; Koidsumi, 1957; W a d a , 1957) suggests that differences between the cuticles of different insect species in respect of these lipids and in the sensitivity of fungi to such substances might govern particular host-para
site combinations. T h e Japanese workers found that ether extractives from the integuments of live larvae and p u p a e of muscardine-resistant races of the silkworm were more strongly antifungal in vitro than those from susceptible races. W a d a (1957) studied the antifungal activity of lipids from the entire bodies of field insects in relation to their suscepti
bility in nature to two different species of Spicaria. Four insect species were resistant to S. prasina (Maublanc) Sawada b u t susceptible to S.
farinosa, while three were the reverse. T h e antifungal action of body lipids from S. pragma-resistant insects was stronger against 5. prasina than against S. farinosa, whereas with one exception the reverse was true of lipids from S. / a r m osa-resistant species. Lihnell (1944) suggested also that a further factor may be inability of hyphae to penetrate the chitin of certain species. A possible nutritional basis of specificity emerges from the work of MacLeod (1954a), who found that while fungi like B. bas-siana, M. anisopliae, a n d S. farinosa have simple nutritional require
ments, certain species of Spicaria and Hirsutella, each apparently limited to particular host species, have more exacting requirements.
Because the pathogen must contact the insect before it can infect, certain instances of apparent immunity in the field are probably of the nature of disease escape. Males of Stephanoderes hampei (Ferrari) are rarely attacked by Botrytis stephanoderis Bally (—Beauveria bassiana) be
cause unlike the females they do not leave the coffee berry in which they have developed (Pascalet, 1939). T h e rarity of disease in cerambycids is
believed to be related to the isolation of the i m m a t u r e stages throughout development (Gardiner and MacLeod, 1959).
Although it has been reported that certain predatory and parasitic insects are i m m u n e to pathogenic fungi (Dresner, 1949; Box a n d Pontis Videla, 1952) there are equally reports of their susceptibility (Voukas-sovitch, 1925; Nishikawa, 1930; Lefevre, 1948). T h e r e is no evidence that these insects are in general more resistant to mycoses than others. Indeed there has been work in J a p a n on the use of Spicaria fumoso-rosea for the microbial control of the tachinid silkworm parasite, Sturmia sericariae R o n d a n i (Maki, 1940; review in Aoki, 1957).
As it develops, an insect changes m u c h in form, physiology a n d habits, so that a particular fungal pathogen tends to be restricted in n a t u r e to a certain span in the host's life cycle. T h e later stages of de
velopment are commonly the more susceptible (Snow, 1896; Billings and Glenn, 1911; Paillot, 1930; Misra, 1952), b u t there are exceptions (Get-zin, 1961). T h e apparently greater susceptibility of some stages to spon
taneous infection may be related to their greater duration ( H u r p i n and Vago, 1958).
T h e eggs of insects are generally resistant to fungi which can attack other stages, although sometimes, particularly u n d e r d a m p conditions in the laboratory, they prove susceptible. T h e r e are, nevertheless, a num
ber of Hyphomycetes which are not parasites of other stages yet attack eggs. These include Oospora ovorum T r a b u t (Delassus, 1931) and species of Aspergillus (Ingram and Douglas, 1932), Fusarium (Arndt and Dozier, 1931), Macrosporium (Geyer, 1947), a n d Penicillium (Pickles, 1930).
X I I . PHYSIOLOGICAL CHARACTERISTICS OF THE PATHOGEN
Only those aspects of the physiology of parasitic Hyphomycetes which appear to be directly related to the parasitic habit will be considered.
Conditions in an insect are probably rarely optimal for the Hypho
mycetes able to attack it; the generally faster and more profuse develop
m e n t of these fungi in artificial culture attests to this. Such fungi are probably pathogenic chiefly because they are able to tolerate those chem
ical and physical conditions which are presented by live insect bodies, and thereby are able to escape from their competitors. It is significant that they generally attain the climax of their development—sporulation
—only after the death of their hosts. It is therefore not to be expected that conditions optimal for their growth in vitro will closely resemble conditions which exist within the usual host insects.
Much of the difference between fungi in respect of their nutrition lies in the range of extracellular enzymes with which they are equipped to convert insoluble food materials into diffusible nutrients which they
262 Μ. F. Μ ADELIN
can absorb. It is in relation to the indiffusible nutrients in insects that adaptation of pathogenic fungi might be found. T h e following data are drawn from the work of Burnside (1930), Vouk and Klas (1931), Lihnell (1944), Masera (1957), and H u b e r (1958). B. bassiana, M. anisopliae, and A. flavus can digest chitin, glycogen, proteins, and fats. T h o u g h they hydrolyze chitin to iV-acetylglucosamine, they apparently do not convert this to glucosamine, which is a poorly used nutrient. Other usable nu
trients include glycerol and fatty acids. Besides organic nitrogen sources, all three can use sources as simple as nitrate salts. T h e y require no sup
ply of growth factors, unlike the more host-specific parasite, Hirsutella gigantea (MacLeod, 1960).
It is unlikely that the p H of the body fluids of insects ever exceeds the limits tolerated by at least the more common pathogens. These grow from below p H 3.3 to above p H 8.5 (Lihnell, 1944; H u b e r , 1958). It is possible that certain physiological processes important in pathogenesis may be more sensitive to p H than growth as a whole. Although the role, if any, which it plays in pathogenesis is unknown, it is interesting that oxalic acid is produced in vitro by M. anisopliae only above p H 6 and most abundantly at p H 8 (Lihnell, 1944).
Reports differ in the way the virulence of pathogenic Hyphomycetes is affected by long periods in artificial culture. D i m i n u t i o n of virulence is reported for B. densa by Giard (1891a), for M. anisopliae by Fox and Jaques (1958), for S. farinosa by Kerner (1959), and for Cephalosporium lecanii by G a n h ä o (1956). However, no change was observed in B. bas
siana by A r n a u d (1927) and Lefebvre (1931); in B. bassiana, S. farinosa, and A. flavus by Toumanoff (1933); and in Metarrhizium brunneum by Rockwood (1951). A r n a u d suggested that h a d her test of virulence not been conducted under conditions ideal for infection, a difference might perhaps have been observed. Indeed, Voukassovitch (1925) de
tected a small change in virulence of S. farinosa verticilloides only when infection tests were conducted without high humidity. It is possible that in n a t u r e the capacity to infect u n d e r suboptimal conditions is an important factor.
Just as cultivation on artificial media in at least some instances re
duces virulence, so too repeated passage through insect hosts and re-isolation enhances the virulence of at least S. farinosa (Kerner, 1959) and A. flavus (Lepesme, 1938).
It is generally believed that the way a fungus has been artificially cultured influences its virulence, b u t though the belief is probably true there are few supporting data. I n a very small experiment, Voukassovitch (1925) found that spores of S. farinosa verticilloides from a peptone me
d i u m killed silkworms whereas those from a potato m e d i u m did not.
Schaerffenberg (1957b) considered that to conserve the virulence of pathogenic fungi, particularly B. bassiana, it was essential to cultivate them on proteinaceous media, a procedure adopted also by Wallengren and Johansson (1929) with M. anisopliae.
X I I I . R O L E OF STRESS IN THE INCIDENCE OF MYCOSES
Steinhaus (1958, 1960b) defined stress as a state, manifested by a syndrome or bodily changes, which is caused by some force, condition, or circumstance in or on an insect or on one of its physiological or anatomical systems. Stress tends to disrupt the homeostasis of an insect and make it more p r o n e to disease. T h e fact that fungi can sometimes infect a species of insect in the laboratory which they can
not, or can only with difficulty, infect in the field may often be because the artificial conditions favor the fungus, b u t may sometimes be because they directly stress the insect. Bryce (1923) found evidence that M. anisopliae attacks larvae of Oryctes rhinoceros only after they have been in captivity for a long time and have suffered a loss of vitality;
and Janisch (1938) found that resistance of Lymantria monacha (Lin
naeus) to Aspergillus versicolor (Vuillemin) Tiraboschi decreased as en
vironmental conditions deviated from the o p t i m u m . Telenga (1959) reported that B. bassiana and Μ . anisopliae combined with insecticidal sprays were more effective against certain pests than either used alone.
It is possible that pathogens may m a k e insects more susceptible to chemi
cal poisoning, rather than vice versa (Steinhaus, 1956). Nevertheless, Tielecke (1952) found that watering beet plants with a diluted parathion concentrate, which killed some larvae of Cleonus punctiventris, was fol
lowed by a high incidence of an u n k n o w n fungus disease of this pest.
An unidentified stress factor may have been responsible for the fact that Watson (1916) could produce epizootics of Spicaria rileyi (Farlow) Charles in captive populations of Anticarsia gemmatilis H ü b n e r only at that time of the year when the disease regularly became epizootic in the field.
XIV. CONCLUSION
Because there are so many hyphomycetous pathogens and potential host species, there is a multiplicity of mycoses. Of these, only a very small proportion has been closely studied. It is therefore at present diffi
cult to gauge how general are the pathogenic p h e n o m e n a observed.
Much work remains to be done. Although studies of mycoses u n d e r con
ditions ideal for their development are necessary, m u c h of relevance to conditions in n a t u r e should emerge if attention is also directed to disease u n d e r suboptimal conditions.
2 6 4 Μ. F. MADELIN
REFERENCES
Akbar, Κ., Hague, Η., and Abbas, Η. Μ. 1958. Fusarium acridiorum, a parasite
Akbar, Κ., Hague, Η., and Abbas, Η. Μ. 1958. Fusarium acridiorum, a parasite