Sex-induction and zygote germination protocols for volvocine algae

Last updated 12/20/2006

Matt Herron, University of Arizona

· Astrephomene gubernaculifera (Brooks 1966)

o Heterothallic

o Induction: mixing of mating types

o Storage:

§ Zygotes plated on growth medium + 1% agar

§ 20-25° in darkness for 6-10 days

o Germination:

§ Zygotes isolated and replated

§ Returned to normal growth conditions

· Astrephomene gubernaculifera (Stein 1958)

o Heterothallic

o Induction: 4-7 day old cultures of both mating types mixed

o Storage/Germination: not described

· Eudorina elegans (Szostak et al. 1973)

o Heterothallic

o Induction: sexual inducer isolated from male strain

o Storage/germination: not described

· Eudorina elegans (Goldstein 1964)

o Induction:

§ Actively growing ♂ and ♀ cultures (2-3 mL each) + equal volume soilwater mixed in a watch glass

§ Watch glass placed in a petri dish with 5% NaHCO₃ in the bottom

o Method 1:

§ Storage:

· Watch glass kept in bright light for 5-7 days

· Zygotes plated on soilwater + 1% agar

· Plates inverted over a petri dish of chloroform for 30s to kill vegetative material

· 37° oven for 2-3 days

§ Germination: plates returned to 20° and 250-350 fc

o Method 2:

§ Storage:

· Watch glass kept in 250-350 fc for 7-10 days

· Watch glass transferred to darkness for 1 week (room temp.)

· Zygotes allowed to dry on watch glass

§ Germination

· After 1-2 days of drying, zygotes rewetted with soilwater

· Transferred to light (room temp.)

· Supernatant replaced with fresh soilwater every 2-3 days until germination (usually only required 1 replacement)

· Zygotes on dried watch glasses remained viable for at least a year.

· Eudorina elegans var. elegans (Nozaki 1983)

o Heterothallic

o Induction:

§ Actively growing (4day old) mixture of ♂ and ♀ concentrated from 12 mL to 0.5 mL.

§ Concentrated culture mixed with 3 mL Eudorina mating medium

o Storage:

§ 3-10 day-old zygotes plated on SVM + 0.6% agar

§ Darkness for “more than three months”

o Germination:

§ Transfer to liquid medium with N₂ substituted for air

§ Light filtered through red cellophane

· Eudorina elegans var. synoica (Nozaki 1986b)

o Homothallic, dioecious

o Induction:

§ Sexual colonies developed spontaneously under normal culture conditions “several days” after transfer to fresh medium.

§ Zygotes turned reddish-brown after about a week.

o Storage:

§ Plated on growth medium + 0.6% agar

§ Darkness for one month

o Germination:

§ Transfer to liquid medium, normal growth conditions

· Eudorina minodii (Nozaki & Krienitz 2001)

o Homothallic, dioecious

o Induction:

§ Actively growing (4-6 day old) culture concentrated from 12 mL to 0.2 mL.

§ Concentrated culture mixed with 0.5-0.8 mL AFM medium (Nakazawa et al. 2001) in a watch glass in a petri dish.

§ Sexual reproduction took place within 24 h of transfer to AFM

§ Reddish-brown zygotes 22-28 μm in diameter

o Storage/germination: not described

· Gonium quadratum (Nozaki 1993)

o Heterothallic, isogamous

o Induction:

§ Actively growing 4-8 day-old culture condensed to 1.5-2.0 mL

§ Condensed cultures of both mating types mixed in a watch glass

o Storage: darkness for 50-90 days

o Germination: as in Nozaki & Kuroiwa (1991a)

· Gonium multicoccum (Nozaki & Kuroiwa 1991a)

o Homothallic, isogamous

o Induction:

§ Actively growing 4-day-old culture condensed to 0.1-0.2 mL

§ Condensed culture, 1.5 mL Pleodorina mating medium (Nozaki et al. 1989) added to watch glass in a petri dish with water in the bottom

§ Normal growth conditions

o Storage:

§ 4-7 day old zygotes plated on growth medium + 0.6% agar minus sodium acetate

§ 25° in darkness for 30-90 days

o Germination:

§ Zygotes returned to fresh growth medium

§ Normal growth conditions

· Gonium viridistellatum (Nozaki 1989)

o Heterothallic, isogamous

o Induction: mixing of two mating types

o Storage/germination: as in Nozaki & Kazaki (1979)

· Pandorina colemaniae (Nozaki & Kuroiwa 1991b)

o Heterothallic

o Induction:

§ Actively growing 4-6 day-old culture condensed to 0.5 mL

§ Condensed cultures of both mating types and Pleodorina mating medium (Nozaki et al. 1989) added to watch glass in a petri dish with water in the bottom

o Storage:

§ Week-old zygotes plated on growth medium + 0.6% agar minus sodium acetate

§ 25° in darkness for 60-90 days

o Germination:

§ Zygotes returned to liquid medium and normal growth conditions

· Pandorina morum (Coleman 1977)

o Heterothallic

o Induction:

§ Small inocula of both mating types mixed in a tube of fresh medium

o Storage/germination: as in Coleman (1959, 1975)

· Pandorina morum (Nozaki & Kazaki 1979)

o Heterothallic

o Induction: 1 mL each of both mating types mixed in a watch glass in a petri dish with water in the bottom

o Storage:

§ Zygotes plated on growth medium + 0.6% agar

§ Darkness for one month

o Germination: zygotes transferred to fresh liquid medium, normal growth conditions

· Pandorina unicocca (Nozaki 1981)

o Heterothallic

o Induction: mixing of mating types

o Storage:

§ Zygotes plated on growth medium + agar

§ Darkness (time not specified)

o Germination:

§ Zygotes returned to liquid growth medium and normal growth conditions

· Platydorina caudata (Harris & Starr 1969)

o Heterothallic, oogamous

o Induction:

§ Actively growing (10-12 day old) ♂ and ♀ cultures mixed in spot plate in a petri dish with 5% NaHCO₃ in the bottom

§ NaHCO₃ “…to prevent evaporation and to increase the degree of the sexual response.”

§ Normal lighting, 20° or 31°

§ After fertilization, a “light maturation period” of 24-36 h at 250-350 fc

o Storage:

§ Zygotes transferred to covered centrifuge tube

§ Darkness for >5 days (viable for at least 3 months)

o Germination:

§ Zygotes spread thinly on a watch glass

§ Watch glass inverted over chloroform for 30 s to destroy vegetative cells

§ Fresh growth medium added to watch glass, normal growth conditions

· Pleodorina starii (Nozaki et al. 2006)

o Heterothallic

o Induction:

§ Actively growing ♂ and ♀ cultures concentrated from 12 mL to 0.2-0.4 mL

§ Concentrated cultures mixed in a watch glass with 0.5-0.8 mL Pleodorina mating medium (Nozaki et al. 1989)

§ Mating reaction occurred 1-2 days after mixing

§ Zygotes turned reddish-brown in about a week

· Tetrabaena socialis (Nozaki 1986a)

o Homothallic, dioecious

o Induction:

§ 12 mL medium inoculated with 1 mL actively growing culture

§ 4-day-old culture concentrated to 0.2-0.3 mL

§ Condensed culture added to watch glass with 1.5 mL Eudorina mating medium (Nozaki 1983) minus sodium acetate

§ Watch glass in a petri dish with water in the bottom

§ Normal growth conditions for 1-2 weeks

o Storage:

§ 1-2 week old zygotes plated on growth medium + 0.6% agar

§ 4° for 3 months, 20° for 3 months, all 6 months in darkness

o Germination

§ Zygotes returned to growth medium and normal conditions

§ Germination in 2-3 days

· Volvox carteri f. kawasakiensis (Nozaki 1988)

o Heterothallic, oogamous

o Induction:

§ 1 mL ♂ or ♀ culture and 0.5 mL sexual inducer added to a tube of fresh medium

§ Sexual colonies developed 2-3 days after induction

§ Mature ♂ and ♀ cultures mixed

o Storage: not described (apparently not necessary)

o Germination:

§ Week-old zygotes transferred into fresh growth medium in a watch glass in a petri dish with water in the bottom

· Volvulina compacta (Nozaki & Kuroiwa 1990)

o Heterothallic

o Induction:

§ Actively growing (3-6 day old) cultures condensed to 1 mL

§ Colonies of both mating types mixed in a watch glass in a petri dish with water in the bottom

o Storage:

§ Zygotes transferred to growth medium + 0.6% agar

§ 20° in darkness for 1 month

o Germination:

§ Zygotes transferred to fresh liquid medium, normal growth conditions

· Volvulina steinii (Nozaki 1982)

o Heterothallic

o Induction:

§ Colonies of both mating types mixed in a watch glass in a petri dish with water in the bottom

o Storage:

§ 2-10 day old zygotes plated on growth medium + agar

§ Darkness for “more than three months”

o Germination:

§ Zygotes transferred to new liquid medium

§ N₂ substituted for air

§ Tube wrapped in red cellophane, normal lighting

· Volvulina steinii (Stein 1958)

o Heterothallic

o Induction: 5-7 day old cultures of both mating types mixed

o Storage/Germination: not described

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Species^1	Condensed^2	Medium^3	Induction^4	Plated^5	Dark Treatment^6	Germination^7	Reference
Astrephomene gubernaculifera (H)	No	Growth	Mixing	Agar	6-10 d	Normal	Brooks 1966
Astrephomene gubernaculifera (H)	No	Growth	Mixing	-	-	-	Stein 1958
Eudorina elegans (H)	No	Growth	SI	-	-	-	Szostak et al 1973
Eudorina elegans (H)	No	Growth	Mixing, NaHCO3	Agar	Chloroform, 37°, 2-3 d	20°, 250-350 fc	Goldstein 1964
Eudorina elegans (H)	No	Growth	Mixing, NaHCO3	Watch glass	1 week	Normal	Goldstein 1964
Eudorina elegans var. elegans (H)	Yes	EMM	Mixing	Agar	>3 mo	N2, red	Nozaki 1983
Eudorina elegans var. synoica (D)	No	Growth	Spontaneous	Agar	1 mo	Normal	Nozaki 1986b
Eudorina minodii (D)	Yes	AFM	Medium	-	-	-	Nozaki & Krienitz 2001
Gonium quadratum (H)	Yes	Growth	Mixing	-	50-90 d	-	Nozaki 1993
Gonium multicoccum (D)	Yes	PMM	Medium	Agar no NaOAc	25°, 30-90d	Normal	Nozaki & Kuroiwa 1991a
Gonium viridistellatum (H)	No	Growth	Mixing	-	-	-	Nozaki 1989
Pandorina colemaniae (H)	No	PMM	Medium	Agar no NaOAc	25°, 30-90d	Normal	Nozaki & Kuroiwa 1991b
Pandorina morum (H)	No	Growth	Mixing	-	-	-	Coleman 1977
Pandorina morum (H)	No	Growth	Mixing	Agar	1 mo	Normal	Nozaki & Kazaki 1979
Pandorina unicocca (H)	No	Growth	Mixing	Agar	Not specified	Normal	Nozaki 1981
Platydorina caudata (H)	No	Growth	Mixing, NaHCO3	Tube	>5 d	Normal	Harris & Starr 1969
Pleodorina starii (H)	Yes	PMM	Medium	-	-	-	Nozaki et al 2006
Tetrabaena socialis (D)	Yes	EMM no NaOAc	Medium	Agar	4° for 3 mo, 20° for 3 mo	Normal	Nozaki 1986a
Volvox carteri f. kawasakiensis (H)	No	Growth	SI	-	-	Normal	Nozaki 1988
Volvulina compacta (H)	Yes	Growth	Mixing	Agar	20° for 1 mo	Normal	Nozaki & Kuroiwa 1990
Volvulina steinii (H)	No	Growth	Mixing	Agar	>3 mo	N2, red	Nozaki 1982
Volvulina steinii (H)	No	Growth	Mixing	-	-	-	Stein 1958

1. H = heterothallic; D = homothallic dioecious; M = monoecious.
2. Actively growing cultures condensed to <1 mL by centrifugation.
3. Growth = normal growth medium; EMM = Eudorina mating medium (Nozaki 1983); AFM = modified AF-6 (Nakazawa et al. 2001); PMM = Pleodorina mating medium (Nozaki et al. 1989).
4. Mixing = mixing of ♂ and ♀ strains; SI = sexual inducer (filtered supernatant from sexual culture); NaHCO₃ = 5% NaHCO₃ added to bottom of petri dish; Spontaneous = sexual colonies appear in normal growth conditions; Medium = sex induced by low-nutrient medium.
5. Agar = growth medium with 0.6 or 1.0% agar; watch glass = zygotes allowed to dry on watch glass; Agar no NaOAc = growth medium minus sodium acetate with 0.6 or 1.0% agar.
6. Chloroform = plates inverted for 30 s over chloroform to kill vegetative material.
7. Normal = return to normal lighting and fresh growth medium; N₂ = air replaced with nitrogen gas; red = light filtered through red cellophane.

Reference List

Brooks, A. E. 1966. The sexual cycle and intercrossing in the genus Astrephomene. Journal of Protozoology, 13: 367-375.

Coleman, A. W. 1977. Sexual and genetic isolation in the cosmopolitan algal species Pandorina morum. American Journal of Botany, 64: 361-368.

Goldstein, M. 1964. Speciation and mating behavior in Eudorina. Journal of Protozoology, 11: 317-344.

Harris, D. O. and R. C. Starr. 1969. Life history and physiology of reproduction in Platydorina caudata Kofoid. Archiv für Protistenkunde, 111: S138-S155.

Nakazawa, A., L. Krienitz and H. Nozaki. 2001. Taxonomy of the unicellular green algal genus Vitreochlamys (Volvocales), based on comparative morphology of cultured material. European Journal of Phycology, 36: 113-128.

Nozaki, H. 1981. The life history of Japanese Pandorina unicocca (Chlorophyta, Volvocales). Journal of Japanese Botany, 56: 65-72.

Nozaki, H. 1982. Morphology and reproduction of Japanese Volvulina steinii (Chlorophyta, Volvocales). Journal of Japanese Botany, 57: 105-113.

Nozaki, H. 1983. Sexual reproduction in Eudorina elegans (Chlorophyta, Volvocales). The Botanical Magazine, Tokyo, 96: 103-110.

Nozaki, H. 1986a. Sexual reproduction in Gonium sociale (Chlorophyta, Volvocales). Phycologia, 25: 29-35.

Nozaki, H. 1986b. Zygote germination in Eudorina elegans var. synoica (Chlorophyta, Volvocales). Journal of Japanese Botany, 61: 316-320.

Nozaki, H. 1988. Morphology, sexual reproduction and taxonomy of Volvox carteri f. kawasakiensis f. nov. (Chlorophyta) from Japan. Phycologia, 27: 209-220.

Nozaki, H. 1989. Morphological variation and reproduction in Gonium viridistellatum (Volvocales, Chlorophyta). Phycologia, 28: 77-88.

Nozaki, H. 1993. Asexual and sexual reproduction in Gonium quadratum (Chlorophyta) with a discussion of phylogenetic relationships within the Goniaceae. J. Phycol., 29: 369-376.

Nozaki, H. and H. Kazaki. 1979. The sexual process of Japanese Pandorina morum Bory (Chlorophyta). Journal of Japanese Botany, 54: 363-370.

Nozaki, H. and L. Krienitz. 2001. Morphology and phylogeny of Eudorina minodii (Chodat) Nozaki et Krienitz, comb. nov. (Volvocales, Chlorophyta) from Germany. European Journal of Phycology, 36: 23-28.

Nozaki, H., H. Kuroiwa, T. Mita and T. Kuroiwa. 1989. Pleodorina japonica sp. nov. (Volvocales, Chlorophyta) with bacteria-like endosymbionts. Phycologia, 28: 252-267.

Nozaki, H. and T. Kuroiwa. 1990. Volvulina compacta sp. nov. (Volvocaceae, Chlorophyta) from Nepal. Phycologia, 29: 410-417.

Nozaki, H. and T. Kuroiwa. 1991a. Morphology and sexual reproduction of Gonium multicoccum (Volvocales, Chlorophyta) from Nepal. Phycologia, 30: 381-393.

Nozaki, H. and T. Kuroiwa. 1991b. Pandorina colemaniae sp. nov. (Volvocaceae, Chlorophyta) from Japan. Phycologia, 30: 449-457.

Nozaki, H., F. D. Ott and A. W. Coleman. 2006. Morphology, molecular phylogeny and taxonomy of two new species of Pleodorina (Volvocaceae, Chlorophyceae). J. Phycol., 42: 1072-1080.

Stein, J. R. 1958. A morphologic and genetic study of Gonium pectorale. American Journal of Botany, 45: 664-672.

Szostak, J. W., J. Sparkuhl and M. E. Goldstein. 1973. Sexual induction in Eudorina: effects of light, nutrients, and conditioned medium. J. Phycol., 9: 215-218.