¿ìè¶ÌÊÓƵ

OST OF the animals in the world are protozoa. This is not immediately
obvious, but anyone with access to a microscope will soon realise that every
drop of water in the ocean, every bog, moist tree hole and thin film of
water in soil or leaf litter is alive with them. Protozoa are the smallest
of all animals: each individual consists of a single cell, and the cell
is often less than one-tenth of a millimetre long.

Being so small protozoa do not always have much choice about what they
eat. If they are very hungry they might eat their cousins or any other organic
morsel they can catch and swallow. But protozoa usually feed on organisms
that are even smaller than themselves, especially bacteria and small algae.
They spend a lot of time processing the water that surrounds them, trapping
or sieving out particles of food.

This is probably all that we might expect from primitive hunter-gatherers:
they catch and eat the microbes they find, digest them and convert them
into new protozoa. But some protozoa have gone a stage further by domesticating
certain types of microbes. The protozoa may harvest these microbes for food,
or the microbes may photosynthesise and release sugars into the cytoplasm
of the host. Still other microbes, nurtured in the host’s cytoplasm, recycle
the by-products of the protozoon’s metabolism. These ‘waste disposal organisms’
grow on the waste and are then digested by the protozoon.

Protozoa seem to obtain microbes and their products in at least two
entirely different ways: they feed rather unspecifically on the free-living
forms and they exploit specific types living in or on their own cells. These
specific consortia are extremely varied. In some, the protozoon takes the
lion’s share of the benefits; in others, the benefits are more evenly distributed.

Sandy marine sediments are a rich source of protozoa, especially the
thin, ribbon-like forms that can squeeze between sand particles. One such
organism is a ciliate called Kentrophoros. Most ciliates have a mouth, but
Kentrophoros does not and it never shows signs of having ingested any microbes
from the sediment. Another curious feature is that Kentrophoros carries
on its back a dense coat of large bacteria with refractive inclusions.

The bacteria are rod-shaped, and lined up perpendicularly to the surface
of the protozoon. Bacteria in the process of dividing into two are obvious,
but rather than dividing in the traditional way across the shorter axis,
they divide lengthwise, and the bacterial coat grows over the surface of
the protozoon. We never imagined that this bacterial coat was incidental
to the lifestyle of Kentrophoros, but only recently did we discover its
function: it is, in fact, a microbial kitchen garden. The ciliate regularly
invaginates the surface of its cell, bringing with it the attached bacteria.
Inside the protozoon, the bacteria are wrapped up in food vacuoles and digested.
This is how the mouthless ciliate obtains a meal: it simply harvests some
of the bacteria growing on its back.

But how do the bacteria manage to make a living? To find out, we incubated
the living consortium in the presence of two radioactive isotopes (C₁4
bicarbonate and S35 sulphide). Later, we tracked down where the isotopes
had accumulated by covering them with a thin layer of photographic emulsion
and developing it. The bacteria can oxidise hydrogen sulphide to elemental
sulphur (the refractive blobs inside the cells). They use the energy of
the reaction to convert carbon dioxide into cell material. So the bacteria
are autotrophs, that is, they manufacture complex organic materials from
simple inorganic ones. To do this, they need three things: oxygen, hydrogen
sulphide and carbon dioxide. Oxygen and hydrogen sulphide are rarely found
together because the sulphide is easily oxidised. In marine sediments, however,
they do co-exist in one narrow zone. The sulphide diffuses up from the anaerobic
depths of the sediment and oxygen diffuses down from the overlying water.
In the zone where they meet, low concentrations of both persist.

Fertiliser for the garden

If the bacteria living on the back of Kentrophoros were transported
to this zone, they would have everything they need. Kentrophoros obliges.
It does not seek out hydrogen sulphide but it does move towards places where
there is still a small amount of oxygen. In these zones there is also just
enough hydrogen sulphide for the bacteria, so Kentrophoros’s behaviour takes
it to the chemical environment where its kitchen garden flourishes best.

Kentrophoros is the only protozoon known to have established a symbiotic
relationship with autotrophic bacteria. This is somewhat surprising because
a range of protozoa live in the zone between the oxidised and reduced layers
of sediments where natural selection might favour the evolution of such
symbiotic relationships.

At least one other marine protozoon, Parablepharisma, has a dense coat
of bacteria. Like Kentrophoros, this ciliate ingests the bacteria through
the surface of its cell although it does have a mouth and it feeds on other
sorts of microbes.

Parablepharisma is anaerobic, that is, it lives without oxygen, and
it is possible that the bacteria (whose identity is unknown) depend on products
of fermentation, such as hydrogen and acetate, that might diffuse out of
the ciliate, but the rest is pure guesswork.

The bacteria attached to Kentrophoros are bathed in hydrogen sulphide,
which they oxidise to obtain energy. The many types of bacteria that live
inside protozoa must also find sources of energy but, in most cases, biologists
know very little about how they do this. There is one notable exception,
which takes the story of protozoan kitchen gardens a stage further.

Some anaerobic bacteria produce methane gas (CH₄) as an end
product of their metabolism. Methane is usually produced either by coupling
the oxidation of hydrogen to the reduction of carbon dioxide or by splitting
acetate (CH₃COOH), a common product of anaerobic metabolism.
Bacteria that produce methane, the methanogens, also have unique coenzymes
which make them fluoresce blue-green when they are excited with violet light.
A few years ago, Johan van Bruggen, of Nijmegen University in the Netherlands,
used fluorescence microscopy to examine anaerobic protozoa. He found that
each organism contained hundreds of fluorescing particles. Tests showed
that the particles were methanogens. Van Bruggen, among others, immediately
suggested a role for these bacteria.

A few years earlier, Miklos Muller and Donald Lindmark at Rockefeller
University in New York had begun to unravel the biochemistry of hydrogenosomes,
small bodies that produce energy in many anaerobic protozoa. Hydrogenosomes
are similar in size to mitochondria (the organelles that produce most of
the energy in aerobic organisms). They have a similar internal structure,
with a folded inner membrane, but their biochemistry is unique. Hydrogenosomes
produce carbon dioxide and acetate, but their most striking characteristic
is that they release hydrogen gas. (In mitochondria, hydrogen is transferred
to oxygen to produce water.) This organelle within the anaerobic protozoon
produces the basic requirements of methanogens. In this case, life inside
the protozoon has obvious benefits for the methanogen: hydrogen is a particularly
scarce commodity in the natural environment. But what does the protozoon
get out of the relationship? The protozoa probably enjoy two benefits: waste
disposal and a vigorous internal kitchen garden. The hydrogenosomes work
efficiently only if the concentration of hydrogen in the protozoon is kept
very low. This task falls to the methanogens, which scavenge hydrogen; their
presence increases the efficiency with which the protozoon produces energy.
In ciliates such as Metopus, the methanogens are actually connected to the
hydrogenosomes which makes the transfer of hydrogen more efficient. The
bacteria grow rapidly and reproduce faster than the ciliate, producing an
excess of methanogens. These become a source of food, channelled into food
vacuoles and digested. Metopus recycles and eventually eats its own waste.

The bacteria associated with Kentrophoros or Metopus probably do not
provide their hosts with anything other than digestible microbial matter.
But green microbes (usually called algae) have something else to offer.
These microbes are green because they contain chlorophyll, a pigment that
traps the energy in sunlight. Like green plants, the microbes photosynthesise,
using the trapped energy to convert carbon dioxide into cell matter. Algae
have a curious tendency to release into the surrounding water some of the
organic compounds they produce by photosynthesis. The main beneficiaries
are bacteria, which readily assimilate these compounds, and protozoa, which
consume the bacteria. But many protozoa benefit in a more subtle and direct
way.

Some species of the freshwater ciliate Euplotes maintain stable associations
with specific types of algae (usually Chlorella). The ciliates enclose the
algae within vacuoles, each enveloped in a protective membrane, and they
encourage the algae to photosynthesise. The object of the exercise is to
‘milk’ the algae of the sugars that they would normally release into the
water. The ciliates create an internal environment that maximises the ‘milk’
yield; they control both the number of symbiotic algae and the rate at which
the algae release the sugar maltose.

The algae grow only if they are supplied with nitrogen, which the protozoa
release to the algae in the form of ammonium (NH₄⁺). But the protozoa keep
a tight control on the supply of ammonium: if it is too great, the algae
grow too fast, but if it is restricted, the algae cannot manufacture new
proteins and instead release the excess sugar they produce.

Ammonium has another role in this relationship: it increases the pH
of the environment around the algae. The algae release most maltose, perhaps
80 per cent of all they produce, at a pH of about 4. By restricting the
supply of ammonium to the algae, the protozoon both restricts the growth
of the algae and maximises the amount of maltose they release. To accomplish
this, however, the protozoon must also provide the symbionts with their
other main requirement, carbon dioxide. Some carbon dioxide reaches the
algae by diffusion from the surrounding water, while the protozoon itself
supplies some. The algae would have a guaranteed supply if the protozoa
carried them down to the sediment, where the decomposition of organic material
generates most of the carbon dioxide in the lake. But sediments are usually
dark – and green algae need light. The algae need resources that come from
opposite directions, carbon dioxide from below and light from above.

The protozoa offer a compromise. During the summer the water at the
surface of lakes and ponds contains plenty of oxygen, but deeper down the
water often becomes stagnant and anaerobic. The oxygenated water is illuminated
but the anaerobic zone contains most of the carbon dioxide. The ciliates
accumulate where the two zones meet. In some respects this behaviour is
similar to the performance of Kentrophoros, which seeks out the best place
to receive resources (oxygen and sulphide) diffusing from opposite directions.
Kentrophoros takes its external kitchen garden to where it grows best; Euplotes
and Frontonia take their internal dairy herds to a zone where they might
yield the most.

All these ciliates reach their preferred environment by responding to
the same chemical cue; they perceive the amount of dissolved oxygen in the
water around them. Where the concentration is low (between 1 and 5 per cent
of the saturation value), they slow down significantly, until they hardly
move at all. As a result, they remain in the place where conditions are
best.

The symbiotic algae that live inside protozoa are complete organisms
capable of an independent existence. The cost of maintaining these organisms
is the price the protozoa must pay for exploiting what the symbionts can
produce. The only part of the symbiont that a protozoon really needs, however,
is the photosynthetic machinery, the sugarproducing apparatus. The best
of both worlds would be to be able to consume other organisms and also have
chloroplasts. The surface waters of the world’s oceans contain many protozoa
that have made valiant efforts in that direction.

Oligotrichs are planktonic ciliates that live in the surface layers
of oceans and lakes, where the water is well mixed and contains plenty of
oxygen. Chemical gradients in the water are never steep enough for something
as small as a protozoon to detect. The water always contains some small
algae that can provide a meal for oligotrichs but at times these algae are
in short supply. The oligotrichs display two obvious adaptations. First,
they swim quickly, which increases their chances of bumping into food particles.
And, second, they steal the chloroplasts of captured algae. Inside the protozoon,
the algae are cracked open, and the chloroplasts retrieved. The chloroplasts
are implanted just inside the oligotrich’s cell membrane, where they can
capture light, fix carbon dioxide and produce sugars for the host. The chloroplasts
eventually wear out and are then digested. There is no doubt about the benefits
to the protozoon; the amount of carbon the chloroplasts fix may be a significant
proportion of the protozoan carbon budget, especially when food is scarce.
The phenomenon is quite common. Diane Stoecker, at the Woods Hole Oceanographic
Institution, in Massachusetts, found that more than 40 per cent of planktonic
ciliates have chloroplasts during spring and summer.

At first sight it appears that the protozoon enslaves the chloroplasts
and invests very little in their maintenance. This is not quite true. Isolated
chloroplasts continue to function for only a few hours but when the oligotrich
sequesters a chloroplast it may carry on working for several weeks. The
chloroplasts seem to be integrated in some way into the metabolism of the
host.

Biologists are continually discovering new consortia of protozoa. Indeed,
it is becoming increasingly difficult to find protozoa, at least of the
large species, that do not have other organisms or bits of organisms living
inside them or on their surfaces. The role of these consortia in the natural
environment varies greatly. The Kentrophoros consortium is an amusing reminder
of the amazing diversity of nature but its discovery is unlikely to cause
a radical rethink of the marine sulphur cycle. Other relationships may be
more important. Methane is an important greenhouse gas which is building
up in the atmosphere. It would be rash to suggest that most of this methane
emanates from protozoan consortia, but they could make a significant contribution
in some habitats, especially in the anaerobic layers of marine sands and
in lake sediments. And if the planktonic foraminifera of the world’s oceans
did not milk their symbiotic algae, they would not be so common or so abundant,
and their shells would not sink to the seabed to form the oozes so familiar
to petroleum geologists.

In the era of biotechnology we can probably expect the protozoa to teach
us something about exploiting microbes. A useful first step would be to
find out more about the range of types of consortia that exist. If the number
of recent discoveries is anything to go by, we will soon be impressed by
the number of guests at the ‘everlasting picnic’.

Bland Finlay is a principal scientific officer at the Institute of Freshwater
Ecology, Ambleside. Tom Fenchel is professor of marine biology at Copenhagen
University and director of the Marine Biological Laboratory, Helsingr, Denmark.

* * *

PROTOZOA, THE FIRST ANIMALS

THE protozoa are an extraordinarily varied group of tiny aquatic animals.
Biologists have described about 20 000 species, although many of these are
extinct forms. Protozoa range in size from 0.002 to 2 millimetres and they
usually eat other microbes.

The flagellates are the smallest and the most abundant. They have one
or more whiplike extensions that they use in feeding and moving about. The
sarcodines include the amoebas and the foraminiferans so loved by oil geologists.
They have no permanent mouth and engulf their prey by wrapping extensions
of their body, pseudopodia, around them. Ciliates have a covering of hundreds
of short, flexible hairs (cilia) that both propel them through the water
and generate currents that bring supplies of food.

Protozoa flourish wherever their microbial food is plentiful. As many
as 100 000 protozoa may live in a millilitre of sediment from the bottom
of a pond. In a sewage treatment plant, the number is often closer to a
million.