BIO320

Lecture by: Mdm. Nurul Iman Mohamad

 CONTENT

• Protozoa – Amoebas, Forams, Actinopods,

Zooflagellates, Ciliates, Apicomplexans
• Algae – Euglenoids, Dinoflagellates, Diatoms,
Golden Algae, Brown Algae, Green Algae, Red
Algae
• Molds – Plasmodials Slime Molds, Celullar
Slime Molds and Water Molds
Evolution of The Eukaryotes
• First eukaryotic cell evolved from ancestral prokaryote
which appeared 1.5 to 1.6 billion years ago.
• Endosymbiont Theory: certain eukaryotic organelles
(eg. mitochondria and chloroplasts) arose from symbiotic
relationship between larger cells and smaller prokaryotes
that were incorporated and lived within them.
• Mitochondria was believed to be originated from aerobic
eubacteria.
• Chloroplast was believed to be formed from the
incorporation of an ancient cyanobacterium within a host
cell.
 INTR ODUCTION
• Consists of vast organisms,
extremely diverse in body forms
and sizes, types of reproduction,
modes of nutrition and lifestyle.

• Difficult to characterize. They are

neither plants, animals nor fungi.

• Unicellular, colonial, coenocytes

(multinucleate cell/polynuclear
cell), or simple multicellular
organisms.
• coenocytic –multinucleate mass of cytoplasm

ADDITIONAL INFO
 INTR ODUCTION

• Divide to 3 main groups:

– animal-like (protozoa)
– plant-like (algae)
– fungus-like (Slime molds
& water molds )
PROTISTA VVIP
• Protists have evolved diversity in their:
a. means of locomotion
b. ways of obtaining nutrients
c. interactions with other organisms
d. habitats
e. modes of reproduction
a. Means of locomotion
• Most are motile at some point in their life cycle
& have various means of locomotion.

• They may move by pushing out cytoplasmic

extension (pseudopodia) as an amoeba does; by
flexing individual cells; by gliding over surfaces;
by waving cilia (short, hairlike organelles); or by
lashing flagella (long, whiplike organelles).

• Some use a combination of two or more means

of locomotion, eg, flagella & pseudopodia.
b. Ways of obtaining nutrients
• Most algae – autotrophic & photosynthesize as
plants do.

• Some heterotrophic protists obtain their

nutrients by absorption, as fungi do.

• Some heterotrophic protists ingest food, as

animals do.
c. Interactions with other organisms
• Many protists are free-living.

• Some form stable symbiotic associations with

unrelated organisms: mutualism (both partners
benefit); commensalism (one partner benefit
and the other unaffected); parasitism (one
partner-parasite lives on or in another - the
host).
 d. Habitat
• Most protists are aquatic and live in the ocean or in
freshwater ponds, lakes, and streams. They make up
most of the plankton.

• Other aquatic protists attach to rocks or other surfaces

in the water.

• Even parasitic protists are aquatic because they live in

the watery environments of other organisms’ body
fluids.

• Terrestrial protists are restricted to damp places such

as soil, cracks in bark, and leaf litter.
e. Modes of reproduction
• Almost all protists reproduce asexually, and
many also reproduce sexually, often by syngamy
(the union of gametes).

• Most protists do not develop multicellular

reproductive organs and do not form embryos as
the way complex organisms do.
Others:
• Protists are important source of food for other
organisms.
• Photosynthetic protists supply oxygen to aquatic
and terrestrial ecosystem.
• Some are economically important; some cause
diseases.
 ANIMAL-LIKE (PROTOZOA)
• In Latin means “the first animal”

• The “first eukaryotes” to evolve.

• Originally given to animal-like

organism that are unicellular.

• Currently, the term is used for

protists that are heterotrophic and
ingest food (as animals do).
 ANIMAL-LIKE (PROTOZOA)

• In this chapter, six phyla of

protozoa are discussed:

– Amoebas
– Foraminiferans
– Actinopods
– Zooflagellates
– Ciliates
– Apicomplexans
Amoebas (Phylum Rhizopoda)

• Unicellular organisms found in soil,

fresh water, ocean and parasitic in
other organisms.

• Asymmetric body form and

continually change shape as they
move (because of the plasma
membrane extreme flexibility).
Amoebas (Phylum Rhizopoda)

• Moves by pushing out temporary

cytoplasmic projections called
pseudopodia from the cell
surface.

• Pseudopodia are also used

to capture and engulf food
by phagocytosis.

• Reproduce asexually by
binary fission.
Amoebas (Phylum Rhizopoda)

• Example of amoeba is Entamoeba

hystolitica, which caused human
dysentery (severe diarrhea with
blood and ulcers in intestinal wall)

• It is transmitted as cyst (resistant,

resting stage in life cycle of protists)
in contaminated drinking water.

• Acanthamoeba can cause eye

infections in contact lens users.
Infection of Acanthamoeba
To avoid any eye
infection, the CDC
advises:
•Never reuse or top
off old solution.
•Never use saline
solution or rewetting
drops to disinfect
lenses.
•Be sure to clean, rub,
and rinse your lenses
each time you remove
your lenses.
•Store reusable lenses
in the proper storage
case.
Forams (Phylum Foraminifera)

• Marine organisms that secretes

chalky, many-chambered tests
(shells) with pores through
which cytoplasmic projections
can be extended.

• The cytoplasmic projections

(protruding pseudopods) form
a sticky, interconnected net
– entangles prey.
Forams (Phylum Foraminifera)

• Many are endosymbiotic with

unicellular algae
– provide food by photosynthesis.

• Most foram species live on the ocean

floor, others are part of the plankton.

• e.g. Globigerina, a common organism

in plankton.
Globigerina, a
foram with a
snail–like test.
Threadlike
pseudopodia
extend through
pores in the test
(LM). The inset
SEMshows a
calcareous foram
test.

*calcareous =
containing
calcium
carbonate; chalky.
• Dead forams settle on the
bottom of the ocean. Their tests
form a gray mud that is gradually
transformed into chalk.

• With geological uplifting, the

chalk formation can become part
of the land, like the White Cliffs of
Dover in England.

• Forams can be used as index

fossils (guide fossils or indicator
fossils), markers to help identify
sedimentary rock layers.
Actinopods (Phylum Actinopoda)
• Mostly are marine plankton
organisms with long, filamentous
cytoplasmic projections called
axopods that protrude through
pores in their shells.

• A cluster of microtubules
strengthens each axopod.

• Prey become entangled in

these axopods and engulfed
outside the main body of
actinopods.
Actinopods (Phylum Actinopoda)
• Axopods increase the surface area
that contact with surrounding water
• Help organism float and function
in feeding
• Cytoplasmic streaming brings
prey back within the shell

• Most of actinopods e.g. Actinophrys

have algal endosymbiont

• During binary fission, test splits in

half, each daughter cell regenerates
• Some actinopods known as
radiolarians secrete
elaborate and beautiful
glassy shells made of silica.

• Radiolarians are important

constituent of marine
plankton.

• When radiolarians and other

actinopods die, their shells
settle and become an ooze
(sediment) several meters
thick on the ocean floor.
A radiolarian.
Numerous
threadlike
axopodia radiate
from the central
body of this
radiolarian,
which is found in
the Red Sea (LM).
A radiolarian.
Beautiful glassy
shells made of
silica
 Zooflagellates (Phylum Zoomastigina)

• Mostly unicellular (a few

are colonial) with spherical
or elongated body, a single
central nucleus, and
one/many long whiplike
flagella.

• Move by lashing flexible

flagella (located at anterior
end).
 Zooflagellates (Phylum Zoomastigina)

• Some engulf food by

forming pseudopodia like
amoeba; some by means
of a definite “mouth”, or
oral groove, and “throat”,
or cytopharinx.

• Heterotrophic. May be free-

living or endosymbionts. e.g
trichonymphs live in the guts
of termites.
 Zooflagellates (Phylum Zoomastigina)

Trichonymphs, complex
specialized zooflagellates
with many flagella, which live
in the gut of termites and
digest cellulose in the wood
the termites eat. Both the
termite and flagellates obtain
their nutrients from this
source. Termites would starve
without this endosymbionts
• Some parasitic zooflagellates cause
disease e.g. Trypanosoma; causes
African sleeping sickness and
transmitted by tsetse flies.

• Other parasit are Giardia or

diplomonad e.g. Giardia intestinalis
causes backpackers’ diarrhea.

• Meanwhile, Choanoflagellates are

permanently attached by thin stalk
to bacteria-rich debris. Their single
flagellum is surrounded by collar of
microvillus
Symptoms of African Sleeping Sickness
• General symptoms include:
• Anxiety
• Drowsiness during the day
• Fever
• Headache
• Insomnia at night
• Mood changes
• Sleepiness (may be uncontrollable)
• Sweating
• Swollen lymph nodes all over the body
• Swollen, red, painful nodule at site of fly bite
• Weakness
Zooflagellates (Phylum Zoomastigina)

Commonly among the campers and

hikers. Giardia is eliminated as a
resistant cyst in the feces of many
vertebrate animals.

The cysts are common

contaminant in mountain streams.
Infection starts when the campers
drink or wash their dishes with the
“clean” mountain rivers.

The symptoms are interference in

Giardia – inhabit
the absorption of nutrient, usually
digestive tract of
in intestinal tracts causing weight
wide variety of
loss, abdominal cramps and
domestic and wild
diarrhea.
animal species,
including humans.
Zooflagellates (Phylum Zoomastigina)

Trypanosoma, the
kinetoplastid that
causes sleeping
sickness.
The squiggles
among these red
blood cells are the
trypanosomes
(colorized SEM).
Life cycle of Trypanosoma
 Zooflagellates (Phylum Zoomastigina)

• A protozoan called
as Trichomonas
vaginalis cause
trichomoniasis
which is a most
common sexually
transmitted
infection (STI)
worldwide
Ciliates (Phylum Ciliophora)

• Unicellular organisms, possess

flexible outer coverage called
pellicle to gives a definite but
changeable shape.

• Cell surface covered with

thousand fine, short, hairlike
cilia that extend through pores
in pellicle and permit
movement.
Ciliates (Phylum Ciliophora)
• Many have
trichocysts,
organelles that
discharge filaments
to aid trapping and
holding prey.

• Some are sessile, and

although motile they
prefer to remain
attach to a rock or
other surface.
 Ciliates (Phylum Ciliophora)
• Ciliates have a wide range of
habits and diets; most ingest
bacteria or other tiny protists.

• Their cilia draw the food into a

simple opening (in some
species) or funnel-like oral
groove (some other species).

• A vacuole forms around the

food at the end of the opening,
and the food is digested.
 Ciliates (Phylum Ciliophora)

• Water regulation is controlled

by contractile vacuoles.

• Being hypertonic to their

environment, freshwater
ciliates continually take water
by osmosis; the contractile
vacuole continually expels
water back to the environment.
• Ciliates have two kinds of nuclei; one/many
small, diploid micronuclei that function in
reproduction and larger, polyploid
macronucleus that control cell metabolism and
growth

• Most ciliates are capable of a sexual process

called conjugation: two individuals come together
and exchange genetic material.
Apicomplexans (Phylum Apicomplexa)

• Large group of parasitic,

spore- forming protozoa,
some of which cause
diseases in human.

• Lack of specific structures

e.g. cilia, flagella or
pseudopodia for locomotion,
but move by flexing.

• Possess an apical complex of

microtubules (only visible using
SEM) that attaches the parasite
to its host cell.
Apicomplexans (Phylum Apicomplexa)

• At some stage in their life

cycle, they produce
sporozoits (small infective
agents transmitted to the next
host).

• Many apicomplexans spend

part of their life cycle in
different host species.
Apicomplexans (Phylum Apicomplexa)

• Their life cycle has both

sexual and asexual stages.
They require two or more
different host species for
completion. Eg: Plasmodium
sp. (cause malaria)
• Mosquito (Anopheles sp.)
• Human
• e.g. Plasmodium which cause
malaria in human:
VVIP
• Plasmodium sporozoits enter human
blood through the bite of an infected
female Anopheles mosquito.
• Plasmodium first enters liver cell,
where its multiplies and then RBC,
where its proliferates (multiply
rapidly)
• When each infected RBC bursts,
many new parasites are release.
• The simultaneous bursting of millions
RBC causes the symptoms of
malaria.
 VVIP

The two–host life cycle of

Plasmodium, the
apicomplexan that causes
malaria.
 Plasmodium Life Cycle

sporozoites

Gametes form sporozoites

in mosquito gut,
combine to form
zygotes

merozoite

Offspring
enter blood,
cause malarial
Male and female symptoms
gametocytes
in blood
  Infected female Anopheles mosquito
bites uninfected human and transmits
Plasmodium sporozoites to human blood VVIP

 Zygote embeds in   Sporozoites enter liver

mosquito’s stomach lining cells and divide to
and produces sporozoites  produce merozoites, then
(spores) which are release from liver cells
released and migrate to  infect red blood cells.
salivary glands.

 In the mosquito’s    In blood cells, more

digestive tract, merozoites produced and
gametocytes develop into infect more RBCs. Some
gametes, and fertilization form gametocytes.
occurs. 

 Uninfected female Anopheles

mosquito bites infected person and
obtains Plasmodium gametocytes
along with human blood.
 PLANT-LIKE PROTIST(ALGAE)

• Group of mostly photosynthetic

protists; range in size,
uni/multicellular.

• Not considered as plant because

lack of plant structures (root, stem,
leave, cuticle, etc.).
 PLANT-LIKE PROTIST(ALGAE)
‘Alga’ – Latin, seaweed.
• Lack of cuticle (it doesn’t need to reduce water
lose); habitat restricted to damp or wet
environment
• All alga posses chlorophyll a, yellow and orange
carotenoids (same as plants), and other pigments in
different algal phyla.
• Mode of reproduction may involved both sexual
and asexual types. – produce single-cell
gametangia
Gametangia
 PLANT-LIKE PROTIST(ALGAE)
• Seven phyla of algae will be discussed:
– Euglenoids
– Dinoflagellates
– Diatom
– Golden algae
– Brown algae
– Green algae
– Red algae
PLANT-LIKE PROTIST(ALGAE)

• Algae are classified based on:

1) pigment composition
2) type of storage product/ energy
reserved
3) cell wall composition
4) the number and placement of
flagella
5) chloroplast morphology
 Euglenoids (Phylum Euglenophyta)
• Most are unicellular flagellates,
about one-third are photosynthetic

• Generally possess 2 flagella:

– one long and whiplike
– one so short that it does not
extend outside the cell.

• Reproduce asexually by
longitudinal cell division (binary
fission)
Euglenoids (Phylum Euglenophyta)

• Closely related to zooflagellates but consider

as algal protists because many of them have
chlorophyll a and b and carotenoids.

• Energy reserved stored as paramylon

(kind of polysaccharide).

• When grown in dark, some loses chlorophyll

and become heterotrophic by ingesting
organic matter or engulf bacteria by
phagocytosis.
 Euglenoids (Phylum Euglenophyta)
• Euglenoids inhabit freshwater
ponds and puddles,
particularly those with large
concentrations of organic
materials.
• For that they are used as
indicator species of
organic pollution.

• Some can be found in marine

and mud flats.
Pellicle
 Dinoflagellates (Phylum Dinoflagella)
• Most are unicellular, a few are
colonial.
• Their cell often have intracellular
shells of interlocking cellulose
impregnate with silicates.
• Typical dinoflagellates have 2
flagella - one wrap around a transverse
groove; one lies in longitudinal groove &
projects behind the cell.

• The undulation of these flagella

propels them like a spinning top.
Dinoflagellates (Phylum Dinoflagella)

• Most are photosynthetic (have

chlorophyll a and c, and
carotenoids including
fucoxanthin)
• Energy reserves store
as oils &
polysaccharides.
• Many are endosymbiont
(zooxanthellae) that reside in
mollusks, jellyfish and corals.
Pfiesteria shumwayae, a dinoflagellate. Beating of the spiral flagellum,
which lies in a groove that encircles the cell, makes this alveolate spin
(colorized SEM).
 Dinoflagellates (Phylum Dinoflagella)

• Reproduction is primarily asexual by

longitudinal cell division, some are
sexually reproduce.

• A few dinoflagellates have

occasional populations (blooms)
which are frequently color coastal
waters orange, red or brown (red
tides).
 Ecological importance of
dinoflagellates

• Important producers in marine

ecosystem – population explosion or
bloom which colors the water as
orange, red or brown known as red
tides
• Red tides produces toxin to marine
organisms and to human who
consumes the contaminated
organisms such as fish and filter
feeding mollusks (oyster, mussels
and clams)
• The toxin is a neurotoxin that Red Tides
impairs breathing in human –
paralytic shellfish poisoning
 Diatoms (Phylum Bacillariophyta)
• Unicellular, a few exist in
colonies.

• Cell wall of each diatoms consists

of two overlap shell (like petri
dish) which deposited silica.

• Two basic groups of diatoms:

– radial symmetry (wheel-
shaped) and bilateral
symmetry (boat or needle-
shaped).
 Diatoms (Phylum Bacillariophyta)
• Some are part of floating
plankton, others live on rocks
and sediments.

• Move by gliding, facilitated by

secretion of a slimy material
from a small groove along the
shell.

• Common in freshwater, abundant

in cool ocean water.
• Contain chlorophyll a and c and carotenoids including
fucoxanthin a.k.a xantophyll

• Energy reserves stored as oil or chrysolaminarin.

• Reproduce asexually by cell division, each shell

become larger half of new cell. Because the glass
cannot grow, diatom get smaller with succeeding
generation.

• Sexual reproduction trigger to restore the original size

by producing shell-less gametes (n). The resulting
zygote (2n) from gamete fusion grows before
producing new shells.
 Golden Algae (Phylum Chrysophyta)
• Complex group found in freshwater
and marine

• Most species are biflagellate and

unicellular, although some are colonial.

• A few lack of flagella and are similar of

amoeba except they contain chloroplasts.

• Produce the same photosynthetic

pigments as diatoms - chlorophyll a and c and
carotenoids including fucoxanthin
 Golden Algae (Phylum Chrysophyta)

• Reproduction is asexual and involves

the production of flagellated, motile
spore called zoospores.

• Energy reserves stored as oils &

carbohydrates (cellulose but some
alginates and fucoidan).
 Golden Algae (Phylum Chrysophyta)
• A few species ingest bacteria and particles of food.
• Important group of producers in marine ecosystems.
They comprise a significant portion of the ocean’s
nanoplankton.
Dinobryon, a
colonial golden
alga found in
fresh water
(LM).
 Brown Algae (Phylum Phaeophyta)

• The largest and most complex of

all algae (commonly called
seaweed), multicellular, size
range from few cm to 75 m.

• Kelps, the largest brown algae

posses leaflike blades,
stemlike stipes and rootlike
holdfasts. Often have gas-filled
floats to provide buoyancy.
 Brown Algae (Phylum Phaeophyta)

• Have chlorophyll a and c,

carotenoids including
fucoxanthin.
• Energy reserves stored as
laminarin (carbohydrate).
• Not free floating marine algae
species-holdfast
• Colour range from brown to dark
brown
 Brown Algae (Phylum Phaeophyta)
• Occur primarily at temperate and cool water
• Smallest - Ralfsia, largest-Pelagophycus (bull
kelp)- may grow in water deeper 100 feets
(30meters) and extend to surface
• Reproduction is varied
and complex; the
reproductive cells, both
asexual zoospores and
sexual gametes are Flagellated zoospores

usually flagellated.

• Essential as primary
food producers and
provide habitat for
invertebrates, fish and
mammals.
A kelp forest.
The great kelp beds of
temperate coastal
waters provide
habitat and food for a
variety of organisms,
including many fish
species caught by
humans. Macrocystis,
a kelp common along
the Pacific coast of
the United States, can
grow more than
60m in a single
season, the fastest
linear growth
recorded in any
organism.
 Green Algae (Phylum Chlorophyta)
• Have pigments, energy reserve
products and cell wall that are
chemically identical to plants.

• Photosynthetic with chlorophyll a

and b and carotenoids.

• Main energy reserves stored as

starch.

• Possess cell walls with

cellulose, although some lack
walls.
 Green Algae (Phylum Chlorophyta)
• Most are flagellated or produce
flagellated cells during life
history.
• Believed to be the ancestors to
land pants.
• Body form:unicellular, colonial,
coenocytic, filaments and sheets
(simple multicellular).
• Found in both aquatic and
terrestrial environments
• Important as the base of the
food web
• Reproduction is varied – sexual as well as
asexual – and their life cycle exhibit alternation
of generation.

• Asexual may be binary fission, fragmentation

and formation of spore. Sexual reproduction
involve formation of gamete in unicellular
gametangia.

• 3 types of sexual reproductions:

a) Isogamous
b) Anisogamous
c) Oogamous
Green Algae (Phylum Chlorophyta)
• Three types of syngamy:
1. Isogamous: fusion of two flagellated
gametes identical in size and appearance.
2. Anisogamous: fusion of two flagellated
gametes of different sizes.
3. Oogamous: fusion of a non-motile egg and
a flagellated male gamete.
• Some perform conjugation.
Green Algae (Phylum Chlorophyta)
Life cycle of Chlamydomonas
The life cycle of Chlamydomonas, a unicellular chlorophyte.
 Red Algae (Phylum Rhodophyta)

• Multicellular, composed of
interwoven filament

• Attach to rocks or other

substrate by basal holdfast

• Complex reproduction, with

alternation of sexual and
asexual stage – no flagellated cell
in life history.
 Red Algae (Phylum Rhodophyta)
• Chloroplast contain
phycoerythrin, phycocyanin,
chlorophyll a and b,
carotenoids.

• Energy stored as floridean

starch (like glycogen).

• Found in warm tropical ocean,

fresh water and in soil
 Red Algae (Phylum Rhodophyta)

• The cell walls contain thick,

sticky polysaccharide that have
commercial value – agar - as a
culture medium, food thickener
• Carrageenan - to stabilize
chocolate milk, provide thick,
creamy texture to ice cream
Red Algae (Phylum Rhodophyta)
WHY FUNGUS-LIKE
Resembles fungi because :
they are not photosynthetic
some have fungus-like bodies consisting of
threadlike structures called hyphae

NOT fungi because:

they have centrioles (fungi lack centrioles)
produce cellulose for cell wall (fungi have
chitin as cell wall)
WHY FUNGUS-LIKE

Three phyla are discussed:

Plasmodial Slime Molds (Myxomycota)
Cellular Slime Molds (Acrasiomycota)
Water Molds (Oomycota)
 Slime molds Fungi

Cell wall = cellulose

Cell wall = chitin

No septate
Has septate

Vegetative state = diploid (2n)

Vegetative state = haploid (n)
nuclei
nuclei

Has centrioles + flagella No centrioles + no flagella

1. Plasmodial Slime Molds (Phylum Myxomycota)

Feeding stage is
plasmodium
[DON’T CONFUSE WITH
MALARIA Plasmodium]
multinucleate mass & still
unicellular:
slimy
streams over damp, leaf litter,
etc.
Plasmodia form a network and as it
creeps along wet surface:
ingests bacteria, yeasts, spores
and decaying organic matter.
1. Plasmodial Slime Molds (Phylum Myxomycota)

Under insufficient food and moisture,

plasmodium produce haploid spore
within sporangia that are resistant
to unfavourable conditions.

When the condition is favourable,

the haploid spore germinates and
acts as gamete and fuse to form a
zygote.
Physarum polycephalum,
a plasmodial slime mold.
The life cycle of a plasmodial slime mold.
2. Cellular Slime Molds (Phylum Acrasiomycota)

During feeding stage, each

cellular slime mold is an
individual amoeboid cell that
behaves as solitary organisms.

Each amoeboid have haploid

cell and reproduces by binary
fission like amoeba
2. Cellular Slime Molds (Phylum Acrasiomycota)

When moisture & food become

inadequate, cells aggregate to
hundreds-thousands
(pseudoplasmodium) but
each still retains individual
identity.

The pseudoplasmodium constructs

a fruiting body that bears spore.
2. Cellular Slime Molds (Phylum Acrasiomycota)

After being released, each

spore opens and single
haploid amoeboid cell
emerges (asexual
reproduction). The haploid
amoeboid cell can survive as
the feeding stage

The life cycle lack of

flagellated stage (sexual
stage).
The life cycle of Dictyostelium, a
cellular slime mold.
CELLULAR SLIME MOLDS – LIFE CYCLE
 3. Water Molds (Phylum Oomycota)
• Have coenocytic (aseptate)
mycelium and the cell wall may be
composed of cellulose, chitin or
both.

• Reproduce asexually by forming

zoospores when food is plentiful. Saprolegnia sp.

• Zoospores developed into mycelium

When environmental condition

worsen, they reproduce sexually
by forming oospores. Phytophthora infestans.
WATER MOLDS LIFE CYCLE
The life cycle of a water mold. Water molds help decompose dead insects, fishes, and other
animals in fresh water. (Note the hyphal mass on the goldfish in the inset.)
End of chapter