The 1996 NSTA Share the Wealth Session




Hunting Bears with a Microscope


Steve Case
Olathe East High School
Olathe, Kansas
E-mail: click here




In this study, students will use lichens and tardigrades (water bears) to investigate their use as bioindicators of key air pollutants. When lichens are exposed to some kinds of air pollutants, especially to sulfur dioxide (SO2), the lichens are injured and die. The lichen coverage in a specified area should be a good indicator of the level of air quality. Tardigrades are macroinvertebrates living in and on lichens. The diversity of the tardigrade species on the lichens will be used to develop another level for bioindication of air quality.


Introduction

Lichens are small, nondescript, often overlooked organisms. Once we notice them growing on the surfaces of trees, rocks, up the sides of buildings, and in mossy carpets in our forests, we cannot fail to admire the way in which they color our world. Lichens are actually two organisms existing in a symbiotic relationship know as mutalism. Lichens are typically composed of a green algae and a fungus. Lichens can also be composed blue-green algae, yellow-green algae, or cynobacteria living in association with different kinds of fungi. Lichens are classified based on the fungal component. The fungi are either Ascomycetes (sac fungi), Basidiomycetes (bracket fungi), or Deuteromycetes ( imperfect fungi). Lichens appear in a wide variety of habitats because they can tolerate, and in fact thrive, under difficult environmental conditions. The alga manufactures and provides itself and the fungus with carbohydrates and some vitamins. Some blue-green algae can even fix nitrogen from the air. In turn, the fungus provides the alga with certain physical protection, it obtains water vapor from the air, providing moisture for the algae. In addition the fungus converts the carbohydrate produced by the algae into a sugar alcohol for food storage. In addition to this relationship between the two organisms, lichens have special adaptations permitting them to withstand moisture and temperature extremes. When moisture is available, it is taken up by the fungus leading to a mechanical change which allows more light to get through, triggering algal photosynthesis. When the atmosphere is dry the lichen is dormant and does not grow.

Tardigrades live in lichens and are unusual organisms that most people would rarely see. In fact, lodged within the lichens is a surprising diversity of microscopic invertebrates and protozoan that awaits reviving. These animals can survive periodic drying that occurs naturally in lichens by entering a special metabolic state called cryptobiosis. During cryptobiosis microinvertebrates weather dry conditions by suspending all but the most vital life functions. These microscopic animals range from 0.1 to 1.0 millimeters and live wherever trees and lichens grow.

Samples of lichens with as many as five species of tardigrades as well as rotifers, nematodes mites, small insect larvae and various protozoa have been found. Tardigrades are distributed worldwide and thrive in diverse habitats including marine, terrestrial and freshwater environments. Terrestrial tardigrades become active only when surrounded and rehydrated by water. Reanimated tardigrades cling to substrate and search slowly for food. These "water bears" as they are commonly called, use four pairs of stumpy, clawed legs to lumber through the water. When surrounding water evaporates, tardigrades can eliminate as much as 90% of their body water. This loss of body water is called anhydrobiosis and leads to a cryptobiotic state in which tardigrades assume a desiccated form called a tun. Tardigrades can survive for months or even years as tuns, remaining inactive until reanimated with water. When moistened, a 120 year-old museum specimen of dried moss yielded tardigrades showing signs of life.

The hardy lichens, and the community living on them, may provide a useful bioindicator for air pollution since they derive their water and essential nutrients mainly from the atmosphere rather then from the soil. It also helps that they are evergreen and able to react to air pollutants year round. In addition, compared with most physical/chemical monitors, they are inexpensive. They can also be used to measure toxic elemental pollutants and radioactive metals because they bind these substances in their fungal threads where they concentrate over time.


Sulfur Dioxide and Lichens

Lichens are injured by sulfur dioxide (SO2 ). Rose (1975) has calculated that more than one-third of England and Wales has lost nearly all its epiphytic lichens, the most delicate shrubby lichens, largely due to the sulfur-dioxide emissions of coal-burning power plants. In Northern Siberia, an area of the Soviet Union which is very polluted, the number of lichen species has fallen from 50 to about 3, and the lichen production in general stands at about 1 or 2% of normal levels, threatening the reindeer diet; in Alaska there are similar concerns about lichen reduction and the caribou diet.

Losses in other parts of the world reflect the increasingly poor quality of the earth's air and the need for early warning bioindicators such as lichens. This pollutant has natural sources, such as volcanic eruptions and sea spray. By far the largest source for it, however, is the combustion of fossil fuels, automobile emissions, and some industrial processes. The pollutant is carried in the atmosphere until rained out or deposited as dry particles or as gas. Sulfur dioxide combines with moisture in the atmosphere to form sulfurous acid (H2SO3) or sulfuric acid (H2SO4). When this happens with rainwater, the result is acid rain. All these forms of sulfur are harmful to lichens and plants. Lichens have also shown sensitivity to some other pollutants, such as heavy metals and ozone, but for the most part lichen damage can be attributed to SO2 .

The effect of pollution upon lichens can depend on the pH of the substrate, the surface on which the lichen grows. In general, an alkaline substrate such as basic bark or limestone counteracts the acidity of SO2 pollution. As acid rain falls on a substrate, one kind of lichen growth form will often be replaced by another more tolerant form. In areas of high pollution lichens may be found only on sites such as wounds on trees and on sandstone walls, which have high (basic) pH. Scientists have found that, with considerable SO2 pollution in an area;

  • The first loss of the same pH-sensitive lichens occurs on birches and conifers
    (acid bark and low buffering capacity);
  • The next loss on oaks and sycamore (intermediate acidity and buffering capacity);
  • The last on trees like elm (alkaline bark and high buffering capacity).

Lichen communities are either weakened or killed by pollution with a consequent loss of species diversity. Lacking the protective cuticle of higher plants, lichens accumulate sulfur dioxide in their thalli bodies in sufficient concentrations to quickly injure or kill them. They also accumulate metals, some of which are toxic, and as they store these toxic metals safely in their hyphal cell walls, they can be professionally evaluated for toxin levels.

The lichen's symptoms of sulfur dioxide death are distinctive. The lichen turns from its usual gray or green hues to the more unusual brown, yellow, pink or white as its chlorophyll is lost. It starts to peel away from its substrate. The sizes of lichen thalli often decrease, especially in fruticose lichens. Fewer spores are produced. The centers of circular lichen colonies usually die first, leaving rings and crescent patterns on rocks or trees.


Sampling Procedure for Lichen Coverage

The following procedures are intended to standardize collection so results can be more easily compared with those of other sites.

  • Use trees that are within one kilometer of your school site.

  • Record the latitude and longitude of your school site on the data collection table.

  • Try to choose trees with alkaline bark, preferably ash, but if not ash, then elm or sycamore. If need be, use trees with acid bark, preferably oak, but if not oak, then beech or birch.

  • If you do not know the pH of your trees, scrape some bark into water and measure with a pH probe, if you have access to one, or with a pH strip test.

  • Select 10 mature trees within the one kilometer radius around your school. These ten trees should be of the same species, if possible. Mark the tree for later identification. At each selected mature tree of your selected species, tie a string around the trunk at a height of one meter.



For each tree, fill in the information for your site on the following lines.

School Name___________________________________________________

Latitude _____________________________________________________

Longitude _____________________________________________________

Date _________________________________________________________

Tree Species___________________________________________________

pH of the Bark _________________________________________________


To estimate the degree of cover we will use a belt transect with accurate determination of cover. Record your tree's identification number on the chart below. Make sure your string around the tree is one meter above the ground at all points. Determine North, South, East, and West using a compass and mark these points on the tree. Copy the 100-dot grid at the end of this lab onto acetate, making a transparent copy. Place the transparent grid so that it's lower edge touches the string on the tree and is in the center of each quadrant, i.e., north, south, east, west. To observe and record percentage cover by each type of lichen, simply count what is showing through each of the 100 small circles and record on the chart below. Each column should add up to 100%. Repeat this procedure for all 10 trees.



Tree _______ North South East West
Crustose



Foliose



Fructicose



Moss



Bare Bark



Other





Tardigarde Sampling

Students collect samples of lichens from the bark of area trees. The samples are taken to the classroom and submerged in water to reanimate the tardigrades living in the lichens. The exercise provides students the opportunity to make basic ecological calculations and introduces the concept of diversity.

The following procedure will be helpful in collecting lichens for use in tardigrade sampling. Lichens are sensitive slow growing organisms. Collection should be limited to necessary samples.


Sampling Procedure for Tardigardes

  1. The lichens should be collected from the same 10 trees used for the lichen coverage study, but not from the transect area.

  2. Use an area of bark that is 100% covered by lichens. Use a large cork borer 1 3/32 inch, or as large as you can find, to collect the lichens. Some samples will be easily collected while others will require that the bark of the tree be collected also. The student should record the tree identification number.

  3. When students return to the classroom they should invert the lichen samples in Petri dishes (lichen down), half full with filtered spring water, deionized, or with distilled water. Each lichen sample should have it's own Petri dish. The water rehydrates desiccated tardigrades and other invertebrates that the students will observe. The soaking will reanimate these animals in 24 hours but 48 or 72 hours will yield better results.

  4. Have the students remove the lichens and search the Petri dishes for tardigrades (water bears). The search should be systematic and uniform, following a simple pattern throughout the dish. Ask the students to construct a data table to record numbers of tardigrades in each sample.

  5. Help them develop a method of categorizing the tardigrades into different species or types. These categories may be based on the tardigrade's size, color, smooth or ornamented cuticle, alive or dead, presence of eggs, or any meaningful separation of observable characteristics the students wish to use. If the students wish to use a more advanced method of classification a good reference is : Morgan, C.I., & P.E. King (1976) British Tardigrades: Keys and notes for the identification of the species. New York: Academic Press. Counts should be made three times and recorded on the chart below.

  6. Calculate the average of each type of tardigrade and the total of each count.



Tree_____ Species Count 1 Count 2 Count 3 Average
Tardigrade A




Tardigrade B




Tardigrade C




Tardigrade D




Tardigrade E




Total






To calculate the density of tardigrades students should determine the lichen surface area for the sample they are observing. If a cork borer was used to collect the lichens, the area of the lichen sample is:

Lichen Area = (3.14)(radius of the sample) 2

Density of the tardigrades is calculated by dividing the number of tardigrades observed by the area of the lichen sample. Density is calculated for each type of tardigrade by dividing the number of each type of tardigrade observed by the total surface area of the lichen.

Tardigrade Density Type A = Number of tardigrades Type A/Lichen Area
Tardigrade Density Type B = Number of tardigrades Type B/Lichen Area

Calculate the mean density by summing the densities of all the types of tardigrades and dividing by the number of types found.

Mean Density = Density of type A + Density of type B

2



Calculating diversity using the Simpson Diversity Index

Calculate the proportion of the total number of tardigrades of each type (Pi).

Proportion type A (Pa) = number of Type A/Total number of tardigrades
Proportion type B(Pb) = number of Type B/Total number of tardigrades

To calculate the Simpson Diversity Index = 1-Pi2

For example, if you looked at 50 tardigrades classified as type A and 20 tardigrades of type B you would calculate:

Diversity Index = 1- ((50/70)2 + (20/70)2)
Diversity Index = .41

This index ranges from zero to one and is literally a measure of the probability that two tardigrades taken at random from the sample are different species. A number close to zero means low diversity and it is likely you will get the same species of tardigrade and a number close to one means high diversity.


Interpretation of results

Once data have been obtained, a follow-up discussion in the classroom can correlate the results with environmental influences such as light, wind direction, exposure to pollution, and other factors.

With this data in hand, you may want to;

  • Collaborate with other schools in your district to create a local lichen coverage map.
  • Collaborate with other ecological regions to create a larger scale lichen coverage map.

Laying out a grid of the data permits a more systematic sampling of an area. Use the data from many sites to produce a map of lichen coverage over a large area and to observe patterns.

Find someplace where SO2 is being monitored and correlate these measurement with your lichen data.



References

This research activity has been developed by relying on the Lichen Investigations of the TERC Global Lab project and on an original article, Diversity in a Hidden Community: Tardigrades in Lichens by Marcia Shofner and Darrell Vodopich, appearing in The American Biology Teacher, October 1993.