JOURNAL OF BACTERIOLOGY, Apr. 1975, p. 288-294Copyright © 1975 American Society for Microbiology

Vol. 122, No. 1Printed in U.S.A.

Sexual Reproductive Cycle of Monascus in SubmergedShaken Culture

MARIETTE CARELS AND DAVID SHEPHERD*Research and Development Department, Nestle Products Technical Assistance Co. Ltd., CH-1350 Orbe,

Switzerland

Received for publication 12 January 1975

A strain of Monascus was grown in submerged, shaken culture using aglucose-salts medium. The formation of ascospores was studied by microscopicexamination of samples taken at regular intervals for 6 days. All the stages of'sexual reproduction previously described for Monascus were observed. Theunusual feature of the strain studied was that it produced sexual structures andcleistothecia of greatly varying sizes.

Sexual reproduction in the genus Monascus,like many other Ascomycetes, has been ob-served and studied for many years. After theisolation of the first species by van Tieghem (7)in 1884, a long debate followed concerning thenomenclature and sequence of the differentstages of the sexual cycle. Went (8), Barker (1),Dangeard (3), Ikeno (4), Kuyper (5), andSchikorra (6) were the principle investigators tosuggest different interpretations for the se-quence of the sexual cycle. It was not until 1931that Young (9) proposed an interpretation thathas remained virtually unchallenged. An an-theridium arises at a hyphal tip by septation. Aprotuberance grows out from the cell beneaththe septum and becomes the female organ. Thetwo organs grow towards each other, the femaleorgan pushing the antheridium to one side,becoming the main growing point. A septumseparates the female organ into ascogonium andtrichogyne. Male nuclei migrate from the an-theridium via the trichogyne through a smallopening into the ascogonium, but the nuclei donot fuse. The ascogonium swells and sterilehyphae grow out from the stalk below theascogonium, rapidly forming a protective rind.Ascogenous hyphae arise near the base of' theascogonium; they grow round and lie close tothe surface of the ascogonium inside the protec-tive rind. The ascogenous hyphae become di-vided into cells, each containing a male andfemale nucleus. Asci are formed, then the twonuclei fuse, and a meiotic, followed by a mi-totic, division results in the formation of eightdaughter nuclei. Each nucleus becomes thecenter of a spore by the extension of a wall,which delimits a volume of cytoplasm. Theascus wall disintegrates, releasing the sporesinto the ascocarpal cavity. The ascocarp, a

stalked cleistothecium, then breaks open, re-leasing the ascospores, which germinate to givea new hypha.During our studies on different Monascus

species in shaken culture, several structureswere observed resembling those described byYoung. This paper describes the results of anattempt to f'ollow the reproductive cycle of aMonascus species in shaken cultures.

MATERIALS AND METHODS

The strain Monascus sp. ATCC 16436 was obtainedfrom the American Type Culture Collection, Rock-ville, Md. It was transferred every 3 weeks on MP Iagar slants containing 4.0% glucose, 0.3% KH2PO4,1.0% yeast extract (Difco), and 1.5% agar (Difco)made up in distilled water. These were incubated at25 C for 6 to 7 days and then stored at 15 C. (Allchemicals were obtained from Merck and were analyt-ical grade).

Liquid cultures were prepared by homogenizing themycelium from an MP I agar slant with about 30 ml ofMP I liquid medium in a 50-ml Sorvall Omnimixerhomogenizer chamber for 45 s. This homogenate wastransferred into a 500-ml baffled shake flask contain-ing 150 ml of MP I liquid medium. The cultures wereincubated at 25 C for 48 h at 150 rpm on a NewBrunswick G53 rotary shaker with a 3-inch (about7.5-cm) throw. After this time, 10 ml of culture wastransferred into another flask containing MP I liquidmedium and grown for a further 24 h.

Test cultures were prepared by inoculating 10 ml ofthis 24-h culture into 150 ml of MP II mediumcontaining 2%7c glucose, 0.3% NaNO3, 0.1c7c KH2PO4,0.05. MgSO4 7H20, 0.05% KCl, and 0.01WFeSO4 7H20 made up in distilled water. The pH aftersterilization was 6.7. These cultures were grown for168 h, using the conditions described for the prepara-tion of the MPI liquid medium cultures.

Microscopic observation. Samples were takenafter 8, 24, 48, 72, 96, 120, and 168 h. For observationwith the light microscope (Leitz Orthoplan), a small

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SEXUAL REPRODUCTIVE CYCLE OF MONASCUS

amount of mycelium was placed on a glass slide and acover glass was placed over it. The hyphae thusremained alive and unharmed and were observed atlow, medium, and high magnification with phasecontrast and normal illumination. Throughout thisstudy all photographs were taken with a Leitz Or-thomat camera and Kodak Plus-X Pan film (22 DIN).

RESULTSThe photographs in Fig. 1 through 28 illus-

trate the sequence of events in the production ofascospores by Monascus. Figures 1, 2, and 3show the early stages of development of theantheridium and ascogonium. It was neverpossible to observe a stage earlier than thoseshown in these figures. Many hyphae had theirterminal cell cut off by a septum, but it wasimpossible to distinguish the hyphal tips thatwould develop into a sexual structure.

Figure 4 shows the next stage, where theascogonium has divided into two cells and thetrichogyne has grown towards the antheridium.It is interesting to note that this strain producessexual structures of different sizes (Fig. 4 and6). It appears that if the basal cell swells, theascogonium is larger whereas the antheridium isshorter. The size of the hyphae from which thesestructures are formed seems to remain aboutthe same. The trichogyne wall dissolved, form-ing a passage (U1) that allows the migration ofthe male nuclei from the antheridium into theascogonium (Fig. 5 and 7).

Figures 8 through 16 show how the sterileinvesting hyphae develop, while the ascogoniumswells and the antheridium diminishes in size.From these photographs it is difficult to seeexactly where the investing hyphae arise, but itseems that they come from the septum dividingthe basal cell from the ascogonium. The invest-ing hyphae form a dense layer around theascogonium, eventually forming the protectiverind of the young cleistothecium. During thisstage the wall is very dense and it is impossibleto distinguish any structures inside the develop-ing cleistothecium.

Figures 14 through 16 are photographs of thesame young cleistothecium taken at three dif-ferent focal planes. It can be seen that once thewall is formed it becomes more transparent andstructures can be distinguished inside the cleis-tothecium. Figure 15 shows asci that are start-ing to develop, and in Fig. 16 ascogenoushyphae can be seen. These could not be distin-guished in earlier stages because of the forma-tion of a dense layer around the ascogonium bythe investing hyphae.

In Fig. 17 the outlines of the developing ascican just be seen and the development is fol-

lowed through Fig. 18, until in Fig. 19 and 20distinct spores can be seen inside the asci. Acleistothecium containing a single ascus can beseen in Fig. 21. Figure 22 shows a cleistotheciumfull of spores that have been released from theirasci. Figures 23 through 27 show the release ofspores from the mature cleistothecium. It is ofinterest to see that the cleistothecium alwaysseems to break at a point opposite the stalk andthat the diameter of the cleistothecia variesgreatly. Figures 24 to 26 show the emptying ofthe cleistothecium, and in Fig. 27 the cleisto-thecium is empty. Even when the cleisto-thecium is empty it keeps its rigid sphericalshape, underlining the solidity of the cleistothe-cial wall. Several filamentous structures can beseen inside the empty cleistothecium, whichcould be ascogenous hyphae that did not de-velop into asci or the remains of the ascal stalks.Finally, in Fig. 28 the ascospores are found freein the medium. They are all the same size andnearly all the same shape. A conidium seen nextto the ascospores illustrates the different formand dimensions of the two types of sporesproduced by this strain of Monascus.

DISCUSSIONBefore drawing any conclusions from these

observations, it must be pointed out that sincewe wished to study the development of Monas-cus in shaken culture, we could not follow thesame sexual structure from its formation toascospore production, rendering estimation ofthe time sequence rather difficult.

Unlike an earlier investigator (8), we wereable to observe the complete sexual cycle of astrain of Monascus in submerged culture. Theconditions were of course different in that theculture was shaken, which suggests that aera-tion could play a role in the initiation of thesexual cycle. The use of a different medium mayalso have been significant, as has been shownfor other fungi (2). One unusual feature of thisstrain is that it can continually produce sexualstructures. It seems to possess some constitutivemechanism for their formation, because al-though the first sexual structures shown in Fig.1 through 7, which develop into cleistothecia,are seen after 8 h of cultivation, new sexualstructures still appear after 120 h.The different stages in the formation of the

young cleistothecium (Fig. 8-14) appeared be-tween 24 and 48 h, whereas the development ofcleistothecia where spores were visible took 96h. It therefore seems that the first stages, untilthe young cleistothecia are formed, are quiterapid, whereas the maturation and release of

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FIG. 1. Hyphal tip is cut off from the rest of the hypha by a septum. The penultimate cell forms aprotuberance that becomes the growing point. The antheridium (an) is pushed to one side.

FIG. 2. As above, but the protuberance is more developed.FIG. 3. Ascogonium (as) is cut off from the original hypha and divides into two, forming the trichogyne (tr) at

the tip of the protuberance.FIG. 4. Septation in the ascogonium becomes more visible and the trichogyne grows down to touch the

antheridium.FIG. 5. As above, but the passage (11) between the trichogyne and the antheridium is visible.FIG. 6. Sexual structure where the basal cell (b) is much bigger than in other structures and the antheridium

much shorter.FIG. 7. Large sexual structure showing the passage (1I) between the antheridium and trichogyne, indicating

that these structures are capable of normal development.

290 J. BACTERIOL.

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FIG. 8. Ascogonium (as) swells and changes shape. The antheridium (an) and trichogyne (tr) are still visibleand, although smaller, do not change shape.

FIG. 9. Ascogonium continues to become rounded and the surface irregular, but the investing sterile hyphaehave not yet developed.

FIG. 10. Investing hyphae (ih) start to surround the ascogonium (as) to form a protective layer.FIG. 11-13. Investing hyphae continue to grow around the ascogonium, forming a dense layer of hyphae.FIG. 14-16. Young cleistothecium taken at three different focal planes. (Fig. 14) The amorphous surface of the

protective rind is visible. (Fig. 15) The interior with asci starting to develop. (Fig. 16) Ascogenous hyphae (ah)which develop from the ascogonium and lie against it inside the protective rind.

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FIG. 17. Cleistothecium with developing asci (ac). The spores are not yet visible.FIG. 18. Asci (ac) develop, eventually filling the interior of the cleistothecium. The spores are not yet visible.FIG. 19. Well-developed asci with clearly visible spores (s).FIG. 20. Two well-developed asci containing spores. It would appear that only two asci have developed in this

cleistothecium.FIG. 21. Another cleistothecium that is smaller and appears to have a single ascus in it.FIG. 22. Mature cleistothecium. The asci have broken down, releasing the spores that now fill the whole

structure.

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FIG. 23. Cleistothecial wall starts to disintegrate and the spores are liberated. It seems that the cleistotheciumbreaks open at a point opposite the stalk.

FIG. 24. Cleistothecium partly empty.FIG. 25. Small cleistothecium showing that the small structures are capable of producing spores.FIG. 26 and 27. Empty cleistothecium. There is not a total disintegration of the cleistothecial wall during

spore liberation.FIG. 28. Ascospores. A conidium (c) is shown for comparison.

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CARELS AND SHEPHERD

ascospores from the cleistothecium takes muchlonger.No germination of the free ascospores found

in the medium was observed, probably due tothe absence of essential nutrients.

It has already been mentioned that the di-mensions of the developing sexual structuresand the cleistothecia vary greatly. On studyingthe ascospores released from cleistothecia ofdifferent diameters, however, all are seen to bethe same size. This being so, the number ofspores in each cleistothecium must vary widely.The small ascogonia probably produce severalascogenous hyphae and hence several asci, butat this stage only a few of the asci develop to fillthe cleistothecial cavity while the rest degener-ate. It could also be that the smaller ascogoniacontain fewer nuclei, so that even though sev-eral ascogenous hyphae may be formed, someare devoid of nuclei. This would limit thenumber of asci that develop spores and couldexplain the filamentous structures seen in theempty cleistothecium. Even if one of theseexplanations is correct, it is still not knownwhat determines the size of the sexual struc-tures, since they are all formed on hyphae ofsimilar dimensions.Although it is not possible to follow the same

structure during its development, the ease withwhich culture conditions can be controlled andthe evolution of the medium followed make this

method very suitable for studies of the sexualcycle of Monascus.To what extent the cultural conditions have

an influence on the variation in size of thestructures we have described or whether this isunder genetic control is also not known but willbe the subject of further study.

ACKNOWLEDGMENTSWe would like to thank H. Bauer and E. Dentan for their

many useful suggestions during the course of this work.

LITERATURE CITED1. Barker, B. T. P. 1903. Morphology and development of the

ascocarp in Monascus. Ann. Bot. 17:167-236.2. Barnett, H. C., and V. G. Lilly. 1955. The effects of

humidity, temperature and carbon dioxide on the sporu-lation of Choanephora cucurbitarum. Mycologia47:26-29.

3. Dangeard, P. A. 1907. Recherches sur le developpement duperithbce chez les Ascomycetes. Botaniste 10:177-216.

4. Ikeno, S. 1903. Uber die Sporenbildung and systematischeStellung von Monascus purpureus Went. Ber. Dtsch.Bot. Ges. 21:259.

5. Kuyper, H. P. 1905. Die Perithecien Entwicklung vonMonascus purpureus Went und Monascus barkeri Dang.und die systematische Stellung dieser Pilze. Ann. My-col. 3:32-81.

6. Schikorra, W. 1909. Uber die Entwicklungsgeschichte vonMonascus. Z. Bot. 1:379-410.

7. van Tieghem, M. 1884. Monascus genre nouveau de l'ordredes Ascomycetes. Bull Soc. Bot. France 31:226-231.

8. Went, F. A. F. C. 1895. Monascus purpureus, le champi-gnon de l'Ang-Quac, une nouvele ThMl1bolbe. Ann.Sci. Nat. Bot. Ser. VIII 1:1-18.

9. Young, E. M. 1931. The morphology and cytology ofMonascus ruber. Am. J. Bot. 7:499-517.

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