UNIVERSITY OF NOVA GORICA SCHOOL OF ENVIRONMENTAL SCIENCES

EFFECTS OF POTENTIAL CLIMATE CHANGES ON THE BEHAVIOUR, FEEDING RATE AND REPRODUCTION OF SELECTED SOIL INVERTEBRATES

MASTER'S THESIS

Nijat Rahimli

Mentor: Assist. prof. dr. Suzana Žižek

Nova Gorica, 2018 II ACKNOWLEDGEMENTS

I would like to express my gratitude to my mentor assist. prof. dr. Suzana Žižek for her support. Her great ideas and broad knowledge helped me to complete my master’s thesis. I learnt a lot from her. I am very thankful also to Infinity project in the framework of the EU Erasmus Mundus Action 2 for financial support

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IV EFFECTS OF POTENTIAL CLIMATE CHANGES ON THE BEHAVIOUR, FEEDING RATE AND REPRODUCTION OF SELECTED SOIL INVERTEBRATES

ABSTRACT

As a consequence of global climate change, the biodiversity of soil invertebrates is impacted. Elevated temperatures and moisture alterations in soil have deleterious effects on soil invertebrates. These organisms are important bioindicators of changes in soil ecosystems. Therefore, we investigated the effect of soil moisture, as potential impacts of climate change, on the behaviour, feeding rate and reproduction rates of two soil invertebrate species: woodlice (Porcellio scaber) and earthworms (Eisenia andrei) in laboratory experiments. Our results indicate that soil invertebrates are highly sensitive to desiccation. The feeding activity of woodlice and the reproduction rate of earthworms are likely dependent on soil moisture.

KEY WORDS

Climate change, Soil invertebrates, Soil ecology, Isopods, Earthworms

V VPLIV POTENCIALNIH KLIMATSKIH SPREMEMB NA OBNAŠANJE, PREHRANJEVANJE IN REPRODUKCIJO TALNIH NEVRETENČARJEV

IZVLEČEK

Klimatske spremembe zelo vplivajo na biotsko raznovrstnost nevretenčarjev v tleh. Povišane temperature, ter spremembe količine vlage v tleh, negativno vplivajo na nevretenčarje. Talni nevretenčarji, so bioindikatorji, ki se odzivajo na spremembe v ekosistemu. Raziskovali smo, morebitne vplive klimatskih sprememb na obnašanje, prehranjevanje in reprodukcijo dveh talnih nevretenčarjev, in sicer: mokric (red enakonožci) in deževnikov. Doseženi rezultati so pokazali, da so nevretenčarji zelo občutljivi na izsušenost tal. Vlažnost tal pa zelo vpliva na prehranjevanje mokric in reprodukcijo deževnikov.

KLJUČNE BESEDE

Klimatske spremembe, Talni nevretenčarji, Ekologija tal, Enakonožci, Deževniki

VI VII TABLE OF CONTENTS

1. INTRODUCTION ...... - 1 - 2. THEORETICAL BACKROUND ...... - 2 - 2.1 Climate change ...... - 2 - 2.2 Soil Organisms ...... - 6 - 2.2.1 Bacteria...... - 7 - 2.2.2 Microfauna ...... - 8 - 2.2.3 Mesofauna ...... - 9 - 2.2.4 Macrofauna...... - 11 - 2.3 Ecological functions of soil invertebrates ...... - 14 - 2.3.1 Nutrient cycling ...... - 15 - 2.4 Climate change and soil invertebrates ...... - 19 - 3. MATERIALS AND METHODS ...... - 21 - 3.1 Methods ...... - 21 - 3.1.1 Experiment - Isopod avoidance behaviour ...... - 22 - 3.1.2 Feeding activity experiment with woodlice ...... - 23 - 3.1.3 Experiment - Earthworm avoidance behaviour ...... - 26 - 3.1.4 Earthworm reproduction test ...... - 28 - 3.1.2 Statistical analyses...... - 31 - 4. RESULTS AND DISCUSSION ...... - 31 - 4.1 Results of the isopod avoidance behaviour experiment ...... - 31 - 4.2 Results of the feeding activity experiment of woodlice ...... - 34 - 4.3 Results of the earthworm avoidance behaviour ...... - 36 - 4.4 Results of the earthworm reproduction test ...... - 38 - 5. CONCLUSION ...... - 39 - REFERENCES ...... - 41 -

VIII LIST OF FIGURES

Figure 1: Global land-ocean temperature index (climate.nasa.gov)...... - 4 - Figure 2: Global sea level rise (climate.nasa.gov)...... - 5 - Figure 3: Rough woodlouse (Porcellio scaber) ...... - 22 - Figure 4: Experimental setup for the isopod avoidance experiment...... - 23 - Figure 5: Counting the animals at the end of experiment...... - 23 - Figure 6: Woodlice in the sample...... - 24 - Figure 7: Prepared samples for the feeding activity experiment with woodlice...... - 25 - Figure 8: Partially consumed piece of leaf by woodlice in the sample...... - 25 - Figure 9: Earthworms (Eisenia andrei) ...... - 26 - Figure 10: Prepared samples for the earthworm avoidance experiment...... - 27 - Figure 11: Container with dividers in place at the end of the earthworm avoidance experiment...... - 27 -

IX LIST OF TABLES

Table 1: Results of the avoidance behaviour experiment...... - 32 - Table 2: Weight of woodlice (Porcellio scaber) before and after experiment (g). .... - 35 - Table 3: Food consumption of woodlice during four weeks (g)...... - 35 - Table 4: Results of the earthworm avoidance experiment...... - 37 - Table 5: Results of the experiment ...... - 38 -

X 1. INTRODUCTION

Soil is one of the key life support systems of the planet and an important component of terrestrial ecosystems that plays an important role also in human life. Soil doesn't only play a buffer role for the ecosystem services but also underpins social, economic and environmental well-being of humans. Jonas et al. (2009) have reported some crucial functions of soil on our planet:  supply of water and nutrients for plant growth and food production (natural ecosystems, agriculture and forestry);  regulation of the water cycle;  nutrient cycles, storage of carbon and regulation of greenhouse gases;  trapping of contaminants (buffering capacity);  source of raw material (e.g. clay minerals);  preservation of cultural heritage;  habitats for animal and plant species, maintaining their biological and genetic diversity;  supports human settlements, providing a basis for buildings and infrastructures, disposal of waste material, slope stability.

Soil is therefore an indispensable treasure for our planet, and it is very important to know the processes taking place in soil. Soil is a multiphase system that contains minerals, organic materials, liquids, different gases and soil organisms. All these components are related to one another and together perform the soil processes. The soil processes are divided into 3 categories:  Physical;  Chemical;  Biological (Hüseynov A. and Hüseynov N., 2012).

The physical soil processes are based on water movement though soil, root penetration, water-logging and so on. Soil is the source of chemical elements for living organisms. Different chemical substances undergo chemical reactions in soil. And finally, biological processes, which are the topic of this thesis. Biological processes of soil depend on soil fauna and microflora (Mirsal, 2008).

Soil invertebrates are an essential part of soil fauna that contribute to soil productivity and enrichment. They regulate decomposition and nutrient mineralization, stimulate the activity of microbial organisms, altering physical properties such as soil structure and porosity and increase water holding capacity of soil (Mirsal, 2008).Their effects are generally greater in tropical ecosystems than in temperate regions because of the more moderate climate and also their influences are greater in deciduous forests than coniferous because of higher contribution from leaves (König and Varma, 2006).

Climate change is a very complex issue, which has at least seven theories about its cause. The most widespread consensus among scientists attributes global warming to increased concentration of greenhouse gases in the atmosphere (Bast, 2010). However, Bast (2010) listed another six theories:  Bio-thermostat - feedbacks from biological and chemical processes contribute to global temperature rise;  Cloud formation and albedo - changes in the formation and albedo of clouds create negative feedback;

- 1 -  Land use - large-scale changes in land use (forestry, irrigation and building cities) affects climate;  Ocean currents - the ocean’s Thermohaline Circulation (THC) affects climate;  Planetary motion - natural gravitational and magnetic oscillations of the solar system induced by the planet’s movement through space drive climate change.  Solar variability - changes in the coronal ejections and magnetic fields of the sun cause changes in cloud formation, ocean currents, and wind that cause climate to change.

Climate is one of the main drivers of the soil formation process (Məmmədov, 2007); therefore, every climate change affects the biological, physical and chemical characteristics of soil during its development (Mirsal, 2008). Abiotic factors such as temperature and moisture can affect the activity and the performance of soil organisms and these negative influences can alter the direction and the development of the soil ecosystem. The impacts of climate change on soil invertebrates can be direct or indirect and present as alterations in the phenology, abundance and diversity of species, habitat preference and ecosystem function, and increases in invasive species (Hopkins et al., 2007).

Compared to above-ground communities, soil invertebrates are less well known or covered in scientific literature, because these organisms have less scientific attention than the high-profile above-ground organisms (Cock et al., 2011).

In this thesis I focused on soil invertebrates, tried to explain their role in soil and generally in the ecosystem, with regard to the predicted and observed climate change and how rapidly it is occurring, and I attempt to determine directly or indirectly the impacts of climate change on these soil invertebrates.

We expect that soil invertebrates will react while content of ambient conditions namely temperature and moisture changes. In case of moisture content decreases we expect that feeding activity and reproduction rate will likely increase till one point. Generally, we expect soil invertebrates will have lower abundance in minimum and maximum rate of ambient conditions, however in optimum conditions their abundance will likely be higher.

2. THEORETICAL BACKROUND

2.1 Climate change

In different periods of Earth history our planet was subjected to many episodes of dramatic climate changes. There have been periods during which the whole planet was covered with ice and at other times it was extremely hot and dry (Berger, 2012). Climate change is a serious reason for concern, because it affects every person living on the planet. Usually climate is defined as the “average weather” in a place and it includes patterns of temperature, precipitation, humidity, wind and seasons. We expect the weather to change a lot from day to day or during the week and month, but we expect the climate regularly to remain constant. If it does not stay constant, we call it climate change.

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What are global climate changes? How do we know that they exist?

Warming of the Earth’s climate is unequivocal, and since 1950, most of the observed changes of the Earth’s climate are unprecedented over decades. These changes were observed as warming of atmosphere and ocean, diminishing of snow and ice sheets, and rising of global sea level. There are some aspects that cause Earth’s climate to change. These are:  Changes in atmosphere;  Natural processes (volcanoes, changes in the Sun system, tectonic plate movement etc.);  Human activities (all activities that cause greenhouse gas emissions) (Bast, 2010).

Most scientists agree that climate changes are occurring because of the human expansion of the greenhouse effect. Gases that contribute to the greenhouse effect include:  Water vapour;  Carbon dioxide;  Methane;  Nitrous oxide;  Chlorofluorocarbons.

There are some indications that people are directly responsible for the global warming. Worldwide, the output of greenhouse gases is a source of grave concern: from the time the Industrial Revolution began to the present. Methane levels have increased 148 % during this time period (NASA, 2016). About half of total CO2 emissions between 1750 and 2011 have occurred in the last 40 years. The main origins of these emissions are fossil fuel combustion, flaring, cement production, and forestry and other land use (FOLU) activities. Electricity and heat production, agriculture, forestry and other land use, buildings, transport, industry and other energy are the main economic sectors behind the greenhouse effect that is currently influencing the climate. Total CO2 emissions from burning of fossil fuel, flaring, and cement production have tripled, and total CO2 emissions from FOLU have increased about 40 %, since 1970. Cumulative anthropogenic greenhouse gas (GHG) emissions from 2000 to 2010 were the highest (49 GtCO2–eq/yr) in human history. The most significant drivers of increases CO2 emissions from fossil fuel combustion are explained as economic and population growth (IPCC, 2014).

Global temperature rise – Since 1880 the Earth started warming, and the period between 1983 and 2012 was the warmest 30-year time period of last 800 years. From 1880 to 2012 land and ocean surface temperature of the Earth increased by 0.85 °C on average. Total temperature increase in the periods 1850-1900 and 2003-2012 is 0.78 °C on average (IPCC, 2014). Most scientists agree that the main cause of global temperature rise is the greenhouse effect that is accelerated by human activity. Because of global warming, 2014 was the warmest year on record and 10 of the hottest years have all come after 1998 (NASA, 2016) (Figure 1).

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Figure 1: Global land-ocean temperature index (climate.nasa.gov).

Warming oceans – The oceans are able to absorb excess heat from atmosphere. The top few meters of ocean stores as much heat as Earth’s whole atmosphere, so the reason for the warming of the planet is that the ocean receives extra energy. In the upper 75 m of ocean surface temperature has increased by 0.11 °C on average per decade between 1971 and 2010. The upper ocean (0-700 m) has likely warmed during 1971-2010, 700-2000 m between 1957 and 2009, from 3000 m till bottom between 1992 and 2005 (IPCC, 2014).

Sea level rise – Sea level can rise due to two factors related to global warming. These are: the added water coming from melting land ice and the expansion of sea water. Global sea level rises 3.4 mm per year (NASA, 2016) (Figure 2). Compared to the last two millennia, the mid-19 century has larger rate of sea level rise. Global sea level rose by about 0.19 m between 1901 and 2010. On average global sea level rise was 1.7 mm/yr during this time period. Over the time period 1993- 2010 global sea level rise was 3.2 mm/yr. Since 1970, about 75 % of observed global sea level rise is explained as loss of glacier mass and thermal expansion of ocean from warming. Rates of sea level rise over the regions of the Earth can differ from global mean sea level rise. For instance, since 1993, the regional rates of sea level rise for Western Pacific are about three times larger than global mean sea level rise, while for Eastern Pacific these rates are zero or negative (IPCC, 2014).

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Figure 2: Global sea level rise (climate.nasa.gov).

Shrinking ice sheets – The Greenland and Antarctic ice sheets have decreased during the last century. According to the data from NASA (2016), Greenland lost 150 to 250 cubic kilometres of ice per year between 2002 and 2006, while Antarctica lost about 152 cubic kilometres of ice between 2002 and 2005. It is more likely that the rate of ice mass loss from Greenland ice sheets increased between 1992 and 2011, while the rate of mass loss is larger over 2002 to 2011 compared to the time period 1992-2011.

Declining Arctic sea ice – The ice thickness of Arctic sea has decreased rapidly during the last decades. Every September it reaches its minimum level. Compared to the 1981-2010 average, it decreases 13.3 % per decade (NASA, 2016). This declining was observed from the northern Antarctic Peninsula and the Amundsen Sea sector of West Antarctica over 2002 to 2011 (IPCC, 2014).

Glacial retreat – All around the world there are glaciers that are currently retreating. They are found in the Alps, Himalayas, Andes, Rockies, Alaska and Africa. The cause of this process is also global warming (NASA, 2016).

Ocean acidification – The advent of industrial revolution accelerated the acidity of surface ocean waters because of access CO2 dissolving in water and forming carbonic acid. Acidity increased by about 30 % during this period. As a result of high emitted carbon dioxide by humans, the amount of carbon dioxide absorbed by the upper layer of the oceans is increasing by about 2 billion tons per year (NASA, 2016).

Decreased snow cover – According the satellite observations the snow cover of the Northern Hemisphere has decreased over the last five decades because of melting (NASA, 2016). Observations show that snow cover of Northern Hemisphere is decreased by 1.6 % per decade for March and April, and 11.7 % per decade for June

- 5 - between 1967 and 2012. According to increased surface temperature, permafrost temperatures have increased in the regions of Northern Hemisphere that has resulted in the reduction in thickness and areal extent in some regions.

These processes are all evidences of global climate change. Of course these big changes have several consequences on the delicate equilibrium of our planet.

What are the consequences of climate change?

The Intergovernmental Panel on Climate Change (IPCC) predicts that global climate change will produce harmful impacts in some regions (NASA, 2016). Future effects of climate change include:  Change will continue through this century and beyond;  Temperatures will continue to rise;  Frost-free season (and growing season) will lengthen;  Changes in precipitation patterns;  More droughts and heat waves;  Hurricanes will become stronger and more intense;  Sea level will rise 30-122 cm by 2100;  Arctic likely to become ice-free.

These climate change impacts will also occur at the level of ecosystems. Temperature and precipitation changes will generate big impacts on soil invertebrates. In our study, we tried to determine some of the potential influences of these changes on soil invertebrates.

2.2 Soil Organisms

Soil organisms are divided into three groups, namely microorganisms, soil invertebrates and vertebrates. Regarding to importance of their ecosystem role mostly microorganisms and soil invertebrates undertake major and the most significant processes of soil. Soil invertebrates are an important component of soil biodiversity. This group of animals live in soil and contribute a large variety of species. In soil ecosystems microorganisms and soil invertebrates interact and their interaction completes the biological and biogeochemical processes of soil. They determine the structure and the essential functions of natural ecosystems (Cock et al., 2011). The exact number of species of soil invertebrates is not known, because there is no soil where all the resident invertebrate species can be determined and quantified (Wall et al., 2005).

There are two main classifications of soil invertebrates (Cock et al., 2011). The first classification is the most generally used, where soil invertebrates are classified by their body size (Swift et al., 1979) into three main groups:

 Micro-invertebrates or micro-fauna – their body length is less than 0.2 mm. Nematodes and protozoa are representatives of this group. Soil micro-fauna is an irreplaceable part of soil fauna. This group is the most numerous of the three groups;  Meso-invertebrates or meso-fauna – their body length is between 0.2 - 2 mm. Mites, springtails, featherwing beetle, Pseudoscorpions, Diplura, and Protura

- 6 - are representatives of this group. The meso-fauna is the major consumer group of soil fauna. Mostly they are microscopic non-segmented roundworms with abundance of about 10-20 million/m2 in soil. There are two groups of soil meso- fauna: free-living and parasitic organisms. Mostly they live in the leaf litter layer on the soil surface;  Macro-invertebrates or macro-fauna – their body length is larger than 2 mm. Earthworms, ants, millipedes, isopods, termites, ground beetles, and centipedes are representatives of this group.

The second classification of soil invertebrates was designed by Lavelle et al. (2004). In this classification soil invertebrates are classified by their functions and processes that they mediate:  Micro-predators - This group of soil organisms are the smallest invertebrates. Their basic role is to contribute to the mineralization of soil organic matter by preying upon microorganisms (Couteaux et al., 1991). In turn, they feed the further groups of animals in the soil food web. Micro-predators do not produce biogenic structures.  Litter-transformers - This group of invertebrates are members of meso-fauna and macro-fauna and live in the leaf litter layer. They undertake some essential processes such as decomposition of plant litter.  Ecosystem engineers - This group of invertebrates produce solid organo- mineral physical structures that directly impact the physical and chemical properties of soil. They have the ability to change direction and speed of basic soil processes such as hydric function, organic-matter dynamics, soil chemical fertility and plant growth. Their activities in soil can also change the abundance and structure of other soil organisms.

Soil fauna is one of the soil formation factors (Mirsal, 2008). Organic matter, fallen tree leaves and other plant parts are broken down by bacteria, fungi, insects, earthworms and other representatives of soil fauna (Bardgett, 2005). In this way they increase soil nutrients. They also change some substances such as sulphur and nitrogen compounds into different forms, which are usable for plants (Mandal and Neenu, 2012). Soil fertility is therefore strongly dependent on soil fauna (Singh, 2011). They accelerate the humification and mineralization of plant residues, change the salt regime of soil, enhance porosity and permeability of soil to water and air and help the mixing of soil layers (Bardgett, 2005).

2.2.1 Bacteria

Bacteria are the most abundant and dominant organisms in soil. One teaspoon of soil contains 10,000 different species of bacteria – on average there are from 1,000,000 to 10,000,000 bacteria per gram of soil. The concentration of bacteria in soil depends on the physical, chemical and biological conditions of soil. The size of soil bacteria varies from 0.5 to 1.0 micron in diameter and 1.0 to 10.0 microns in length. Soil bacteria belong to three groups according to their morphological structure (Bardgett, 2005):  Cocci (round/spherical);  Bacilli (rod-shaped);  Spirilla (cells with long wavy chains).

- 7 - Bacteria are also classified on the basis of their physiological activity and mode of nutrition (Bardgett, 2005):  Autotrophic bacteria - this type of bacteria are able to synthesize their food from inorganic matter. They utilize CO2 from atmosphere as carbon source. The genera Nitrobacter, Nitrosomonas and Thiobacillus are the main autotrophic bacteria in soil;  Heterotrophic bacteria - this type of bacteria are the main group in soil and they get their energy and carbon from organic matter. They obtain their nitrogen from nitrates in soil and other nutrients from soil or decomposing organic matter.

Bacteria play an important role in the formation of soil and in soil reactions and processes. They are the most important biological group of soil formation factors. Most processes and reactions in soil depend on bacteria. For instance, they have an irreplaceable role in water dynamic processes, nutrient cycling, and disease suppression. One of the most important processes in soil is biodegradation and bacteria and fungi are the two groups of organisms that perform this process. For example, Actinomycetes are very effective at breaking down complex substances like cellulose and chitin.

2.2.2 Microfauna

Soil microfauna is the smallest size group of invertebrates. The most important representatives of this group are protozoans and nematodes.

2.2.2.1 Protozoa

In one gram of soil there are approximately 10,000 protozoans. Their body diameter is 5 to 500 μm. More than 15,000 species of protozoa are known. Main groups of protozoa are rhizopods, flagellates, ciliates and parasitic sporozoans (König and Varma, 2006). Regarding to their body shape they are classified into three groups:  Ciliates – hair-like cilia. They are the largest protozoans;  Amoebae – changeable shape. They are classified into two groups, namely testate amoebae and naked amoebae. They are also quite large;  Flagellates – whip-like flagella. The smallest group of protozoans (Ingham, 2016.).

Protozoa feed mainly on bacteria (König and Varma, 2006), but they can eat other protozoa, soluble organic matter and sometimes fungi (Ingham, 2016). Due to their feeding on bacteria, they accumulate in the rhizosphere (König and Varma, 2006). Foster and Dormaar (1991) reported that amoebae ingest approximately 10,000 bacteria per day.

Meyer et al. (1989) reported that approximately one-third of the biomass of the soil fauna is made up by protozoa. If soil respiration is considered this number is doubled. Protozoans are main contributors to soil respiration among soil organisms. Approximately two-thirds of respiration by soil organisms is accomplished by protozoans.

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2.2.2.2 Nematodes

Nematodes are non-segmented worms and are the most widely distributed invertebrate group on earth. They have simple body structure with 50 μm body diameter and 2 mm body length. Richness of nematodes is high. Approximately 20,000 species of nematodes have been described, but it is likely to be half a million according to estimations (König and Varma, 2006). Their abundance in soil is quite high. In one teaspoon of forest soil there are several hundred nematodes, in agricultural soils it is less than 100 and in grasslands one teaspoon of soil holds 50 to 500 nematodes (Ingham, 2016).

Nematodes play an important role in the soil food web. They have diverse feeding needs, as some feed on plants and algae, some on bacteria and fungi (grazers), and some on other nematodes (Ingham, 2016.). König and Varma (2006) classified them regarding their feeding types:  Bacterial feeders;  Fungal feeders;  Plant feeders;  Predatory nematodes;  Omnivorous nematodes;  Plant associated nematodes;  Entomopathogenic nematodes.

2.2.3 Mesofauna

The mesofauna are mostly micro-arthropods. Their body diameter is below 2 mm and their body length is below 4 mm. The most significant representatives of soil mesofauna are Enchytraeidae, Pseudoscorpionida, Acari, Symphyla, Pauropoda, Collembola, Protura, and Diplura. Some groups of pterygote insects such as Psocoptera and Hymenoptera are also included in the mesofauna. The abundance of mesofauna in soil is quite high, they exceed macrofauna abundance by few times. Especially in extreme soil environments the abundance of mesofauna increases and they become the dominant group of organisms in soil (König and Varma, 2006).

2.2.3.1 Pseudoscorpionida – False Scorpions

False scorpions are tiny organisms with reddish or brown oval or teardrop shaped body (Hahn and Kells, 2016a). Their body size is 1 to 5 mm. There are approximately 2,400 species. False scorpions get their name from the similarity with scorpions, but they do not have the tail stinger. They mostly have two or four eyes, but some representatives are eyeless. They have eight legs and two long chelate pedipalps equipped with sensory hairs that they use to detect the location of prey. False scorpions are very quick organisms and they can move forwards, backwards or sideways and change directions very quickly (König and Varma, 2006).

- 9 - False scorpions like high humidity, therefore they are mostly found in leaf litter, moss, under the tree bark or stones. They have quite diverse prey, since they can feed on other micro-arthropods such as springtailes, psocids, thrips, beetle larvae, mites, ants, flies (Hahn and Kells, 2016a) and nematodes (König and Varma, 2006). Most of them pursue their prey very aggressively while some prefer to ambush their prey. Most representatives of false scorpions have poison glands that help to paralyze their prey (Hahn and Kells, 2016a).

Pseudoscorpions have a two or three year lifespan and they produce one or two generations per year (Hahn and Kells, 2016a).

2.2.3.2 Acari – Mites

Mites are a significant and diverse mesofaunal group of soil invertebrates. Their body size is 200 nm to 2 mm. More than 40,000 species have been described. They have two main lines namely the Parasitiformers and the Acariformers. Mites are a very important group of organisms in soil ecosystems (König and Varma, 2006).

Predatory mites are widely distributed in soil and they prefer dark habitats. They do not have eyes and they avoid light. They have special mechanism like a catapult for hunting. They use the pincers on their head very skilfully to catch their prey. Predatory mites feed mainly on nematodes and collembolans (König and Varma, 2006).

Oribatids are eyeless mites that live in the upper layer of soil under the leaf and debris litter. Instead of eyes they are equipped with sensilla called trichobothria to detect and receive signals from their surroundings. In soils of Central Europe 20 to 80 species are described, but worldwide there are more than 6,500 species. The Oribatids feed mainly on plant debris, bacteria, fungi, lichens and living plants (König and Varma, 2006).

2.2.3.3 Collembola – Springtailes

The body size of this microarthropod group varies between 200 μm and 10 mm. More than 6,500 species are distributed in terrestrial habitats. Collembolans are found at high densities in all climatic zones. They are especially common in humid tropical and temperate zones. Collembolans have high densities in forest soils (König and Varma, 2006). In one hectare of soil the abundance of springtails is few millions of organisms (Hahn and Kells, 2016). They can move vertically between the soil and vegetation layer. Three orders of Colembolans are described, namely Arthropleona, Neelipleona and Symphyleona (König and Varma, 2006).

Collembolans feed on fungi, pollen, algae or decomposed organic matter (Hahn and Kells, 2016b).

2.2.3.4 Protura

Proturans are primitive hexapods with three body parts: the head, thorax and abdomen. Their body is slender and elongated with size 0.5 to 2 mm. The body of Proturans is unpigmente. Proturans do not have eyes or antennae. They have forelegs

- 10 - that function as antennae. Instead of eyes they are equipped with a sensory organ pseudoculus which is located on the head (König and Varma, 2006).

Proturans are the smallest class of the Arthropoda phylum with more than 500 species distributed worldwide. They are classified into two groups, Ametabola and Apterygota. Proturans are inhabitants of humid soils and they are mainly found under the leaf litter (Meyer, 2016).

2.2.3.5 Diplura

Diplura are unpigmented soil inhabitants found under stones, bark and in the deeper layers of soil. They are distributed as groups of small colonies in humid soil. Diplura like moist and dark habitats. They are very sensitive to desiccation. More than 500 Diplura species are distributed worldwide and 50 species in Central Europe. They have narrow and colourless body with size 2 to 5 mm. They do not have eyes. Most species of Diplura are herbivores, but the Diplura species that have pincer-like cerci are carnivorous. They feed on small arthropods (König and Varma, 2006).

2.2.4 Macrofauna

The body size of macrofauna varies from 4 to 80 mm. These organisms are distinguished by their four body types:  A short and compact body with a solid, semi-solid or soft trunk (e.g. isopods, glomerids, termites);  An elongated, rod-like body with a solid trunk (e.g. Julids, Poydesmidis, larvae of carabids);  A soft wormlike body with a flexible trunk (e.g. Earthworms, some larvae and snails);  A semi-solid wormlike body with a flexible trunk (e.g. Chilopods).

The most important representatives of macrofauna are Enchytraeidae, Isopoda, Diplopoda, Arnaeida, Lumbricidae, Chilopoda, Diptera, Coleoptera, Isoptera and insect larvae (König and Varma, 2006).

2.2.4.1 Earthworms (Lumbricina)

Earthworms are one of the most important groups of soil organisms. They are the most significant macro invertebrates of soil in humid temperate zones. Earthworms live in different layers of soil but mostly top layer because food is most abundant there. Generally they prefer moist non-acid soils. Earthworms also exist in extreme zones. Normally earthworm community consists of 8-11 species in extreme habitats (Lavelle et al., 1995). These hermaphrodite worms have different size and length. There are about 3000 species but this number can still increase because new species are constantly found (Bardgett, 2005).

- 11 - According to the opinion of Aristotle, earthworms are “the intestines of the earth”. They have an irreplaceable role in soil. They have been called “soil ecosystem engineers” because they are able to change the structure of the soil ecosystem. They can ingest 50 to 1000 tons of soil per hectare in one year. Different types of earthworms live in different layers of soil. The earthworm burrows create pores in soil which are very important for the entrance of oxygen and water into the soil, as well as for the exit of carbon dioxide from soil (Bardgett, 2005).

Earthworm excreta contain essential plant nutrients. Earthworms produce 10-15 tons of excreta each year, containing 50 % organic matter, traces of some minerals and more important the plant macronutrients nitrogen, phosphorus and potassium. The polysaccharide mucus that the earthworms excrete helps to determine and maintain soil structure by forming soil aggregates (Scheu et al., 1987).

Earthworms also play an important role in the decomposition processes, as they break down dead organic matter. This process increases the amount of nutrients in soil; in this way earthworms make soil fertile and provide other soil animals with food. By breaking down larger particles they provide food for fungi and bacteria. For this reason, the abundance of fungi and bacteria is directly proportional to the abundance of earthworms. Furthermore, earthworms mix organic matter with soil particles. In this way they play a “ploughing” role in soil and mix soil layers (Bardgett, 2005).

Bouché (1977) has reported three ecotypes of earthworms regarding their life style:  Epigeic earthworms – Bright red and red-brown in colour these worms live in the top layer of soil where there is much leaf litter. Dendrobaena octaedra, Dendrobaena attemsi, Dendrodrilus rubidus, Eisenia andrei, Eiseniella tetraedra, Heliodrilus oculatus, Lumbricus rubellus, Lumbricus castaneus, Lumbricus festivus, Lumbricus friendi, and Satchellius mammalis are the species of this ecotype.  Endogeic earthworms – Grey, light pink, green and blue, these worms play an important role in the soil structure. They make burrows in soil and when they move they make pores; in this way they improve porosity of soils. Allolobophora chlorotica, Apporectodea caliginosa, Apporectodea icterica, Apporectodea rosea, Murchieona muldali, Octolasion cyaneum and Octolasion lacteum are species of endogeic earthworms.  Anecic earthworms – Dark coloured and paler-tailed these worms affect the fertility of soil. Their excreta are found at the entrance of their burrows and sometimes, it is possible to see them in grasslands. Lumbricus terrestris and Apporectodea longa are the species of this ecotype.

Eisenia andrei is one of the most wide distributed groups among the earthworm species. These reddish earthworms are easily handled. Therefore, they are commonly used in ecotoxicology studies. Short life cycle, high reproduction rate, high rate of consumption and assimilation of organic matter are main characteristics of E. andrei (Dominguez, 2004).

According to Elvira et al. (1996) deposition of further generation of cocoons range from 45 to 51 days and they get mature in 31-30 days. The rate of cocoon production of E. andrei ranges from 0.35 to 1.3 per day. Incubation period varies at the range of 18-26 days. Hatching rate and life cycle of E. andrei is dependent on ambient temperature. On average their survival time period is 594 days at 28°C and 589 days at 18°C. It

- 12 - takes 21-30 days to reach maturity for young earthworms that are recently emerged from cocoons (Edwards and Bohlen, 1996).

2.2.4.2 Terrestrial Isopoda – Woodlice

These soil macroinvertebrates also are found throughout the world. Approximately 3,500 species of woodlice are distributed worldwide. These grey (or dark blue, sometimes orange) soil invertebrates pass their entire life cycle in terrestrial habitats. The body size of woodlice varies from 2 to 20 mm. They do not have a waxy cuticle. Therefore, their physiological activity depends on external ambient conditions. The body of woodlice is equipped with antennae, mouthparts, thoracopods, pleopods and uropods (König and Varma, 2006).

Terrestrial isopods typically live in dark environments and avoid light. These organisms like humid environment, which is why they are found on the upper part of moist soil layers. Woodlice are hydrophilic organisms because their cuticle does not have a lipid layer. When the ratio of humidity is unfavourable for them, they quickly move to more favourable environments. In summer, when soil moisture decreases in forests they move to the cover of trees where there is more moisture. They are very susceptible to drought. Especially Ligidium hypnorum and Trichoniscus pusillus can lose 70 % of their water per hour during drought. Oniscus asellus, Porcellio scaber and Armadillidium vulgare are the most tolerant to water loss (König and Varma, 2006).

Terrestrial isopods play an important role in soil. They are detritivores and they degrade dead material such as decaying leaves and old wood on soil. They are significant organisms in the degradation process of organic matter. Their food choice depends on the quality of food, the moisture level and the decomposition stage (König and Varma, 2006). Woodlice are also coprophagous (Bauer and Christian, 1995). Coprophagy has benefits for isopods, especially if the quality of dead organic matter, plant litter or leaves is very low (Keutz et al., 2002).

The abundance of woodlice has been connected to higher nutrient content in soil. They rapidly break down organic particles and speed up the process of microbial degradation by colonising particles that pass their intestine with their gut micro-flora. The normal abundance of woodlice is approximately 30 animals m-2 (Dunger, 1983).

Porcellio scaber is mostly known as common rough woodlouse is one of the most abundant groups among terrestrial isopod species. Common rough woodlice prefer temperate environments, though they are widely distributed through all continents of the world and considered as native species of natural ecosystem of Europe. They are found mostly in dark and humid places under leaves in forests, beneath rocks, sometimes in meadows, gardens and greenhouses (Harding and Sutton, 1985; Hutchins et al., 2003).

They have compact, flat, elliptical-shaped and rough-surfaced body with mostly brown and brick-orange body colour. Their colours are deep slate-blue in males and mottled in females and juveniles. Their body length can reach to 17 mm. Body of P. scaber is contained seven segments. Each segment contains a pair of legs. Body weight of P. scaber is dependent on water content. P. scaber has two compound eyes and a pair of antennae on its head (Hutchins et al., 2003).

- 13 - Common rough woodlouse has 12-36 offspring per year. Number of broods is commonly one to three. Reproduction occurs mostly in spring and summer when the days are lengthened. On average after 35 days of gravid, hatchlings occur and they reach maturity within 14-22 months (Hutchins et al. 2003; Warburg et al., 1984).

These invertebrates are mainly feed on decaying organic matter, because of the higher amount of microbes. Bacteria are the essential part of their diet, because they break down nutrient that are not easily absorbed. Common woodlouse also feeds on their faeces. Generally common rough woodlice are included into groups of detritivores, saprophagous, mycophagous and comprophagous (Hutchins et al., 2003; Kostanjšek et al., 2004).

Common rough woodlice are active mostly at night. Their activity depends on speed of the wind and evaporation rates. They prefer humid ambient conditions and avoid light and dry environments. Common rough woodlice move to trees in summer and spring, and to soil in autumn and winter (Hutchins et al., 2003).

2.2.4.3 Diplopoda – Millipedes

Millipedes are cylindrical and long-bodied macro-invertebrates. Worldwide 10,000 species of Diplopoda are distributed. Millipedes have a dark brown body with size that varies from 20 to 40 mm. König and Varma (2006) has reported 5 different body types of millipedes:  Rammer bulldozer type (e.g. Iulidae);  Globular type (e.g. Glomeridae);  Borer type (e.g. Poyzonidae)  Wedge type (e.g. Polydesmidae);  Soft bark-dweller (e.g. Polyxenidae).

Millipedes have two pairs of legs per body segment, totally up to 400. Therefore they move very slowly. But their box-like construction of body helps them to move through the soil skilfully. Their movement in soil has benefits such as mixing of soil; therefore they are also considered “soil ecosystem engineers”. Millipedes are primarily decomposers. They feed on decaying organic matter. As other macro-invertebrates millipedes like moist and dark environments (König and Varma, 2006).

2.3 Ecological functions of soil invertebrates

Soil invertebrates accomplish important processes in ecosystems and provide the life- cycling of not only soil ecosystem but the whole biosphere. For instance, they take over nutrient cycling as the primary driving agents and as a result they regulate the dynamics of soil organic matter. They decompose organic matter into different forms. As the result they release soil carbon as a form of CO2. Therefore they regulate carbon sequestration and accomplish important role not only in soil ecosystem but also in biosphere. They also contribute the modification of soil physical properties and water regimes. Soil organisms enhance the amount and efficiency of nutrient acquisition by the vegetation and plant health (De Groot et al., 2002; Mandal and Neenu, 2012).

- 14 -

Therefore, these ecosystem services provide the maintenance of ecosystem integrity (Brussaard, 1998). Soil organisms interact in soil food web that is based on the degradation of roots and dead organic matter. Each trophic level of soil organisms serves as food for the further one and this function is very necessary in soil ecosystem. The ecological stability of soil ecosystems depends on the soil food web (Mandal and Neenu, 2012).

Each of the three size groups of soil invertebrates has its own functions in soil ecosystems. For instance, soil meso-fauna contributes to the breakdown of organic matter, stimulation of microorganisms and deposition of faeces, which increases soil fertility. Some of them feed on bacteria, fungi and algae and some scavenge on degraded organic matter. Soil meso-fauna plays an important role in the carbon cycle, which is why it is directly related to climate change (Cock et al., 2011).

The micro-fauna plays an important role in the cycling of plant nutrients, mineralization and remobilisation of N, P and S, accumulation and stabilisation of organic carbon, enhancing of nitrification rates, suppressing of bacterial and fungal pathogens etc. Ingham et al. (2017) reported that soil invertebrates take over some functions of the micro-fauna, such as - they:

 Decompose organic matter;  Release nutrients into plant available forms;  Degrade pesticides;  Form symbiotic associations with plant roots;  Bio-control plant pathogens;  Affect the weathering and solubilisation of minerals;  Contribute to soil formation, structure and aggregation.

Macro-invertebrates also have a vital role in soil. They enhance physical properties of soil, neutralise soil pH, increase the availability of many nutrients, stimulate microbial populations, reduce levels of harmful nematodes and play an important role in forming soil structure (Cock et al., 2011). Earthworms supply the soil with organo-mineral complexes. They improve the hydraulic conductivity of soil (Joschko et al., 1989). Their activities can alter the physico-chemical and biological status of soil. The soil zone affected by earthworms is called drilosphere (König and Varma, 2006). Earthworms also stimulate microbial activity in soil. Many microorganisms are present in their faeces and casts (Edwards, 2016).

Therefore the role of soil invertebrates is very significant and their existence provides the stabile performance for the ecosystem.

2.3.1 Nutrient cycling

2.3.1.1 Carbon cycle

Carbon cycle is one of the most significant nutrient cycles due its relation to the theory of climate change (Mandal and Neenu, 2012). Carbon cycle is driven by the process of

- 15 - photosynthesis when carbon dioxide is fixed into organic form in the green cells of plants (Huntingford et al., 2000).

Although plants are the most well-known organisms that perform this process, some microbial organisms such as algae, cyanobacteria and other forms of bacteria are also able of photosynthesis (Mandal and Neenu, 2012). In the carbon cycle, fixed carbon moves through trophic levels in soil as photosynthetic organisms are grazed upon by primary consumers or herbivores and these can be eaten by secondary consumers and etc. The world’s soils have two times more carbon than atmosphere and that is why small changes in the amount of soil carbon impact the concentration of carbon dioxide in the atmosphere, as a result this creates a positive feedback on climate (Smith and Fang, 2010). On a global basis, organic carbon in soil is estimated to be approximately 1,395 × 1015 g (Post et al., 1982).

The release of carbon from soil to atmosphere is accomplished by micro-, meso-, and macro-invertebrates and heterotrophic microorganisms. There are two essential pathways of the loss of carbon from soil, namely through microbial respiration and through methanogenesis. In the aerobic process of microbial respiration, CO2 is generated and released into atmosphere. The rate of released CO2 depends on the age and quality of organic matter, as particulate organic matter is the easiest to decompose, while humus decomposes more slowly (Hernandez and Hobbie, 2010; Skjemstad et al., 2004). The generation or methane (CH4) is accomplished by methanogens in the anaerobic process of microbial respiration. CH4 is about 25 times more effective than CO2 as a greenhouse gas (IPCC, 2007). Methane is also consumed by methanotrophs that are interdependent microbial communities living in soil and ocean sediment.

2.3.1.2. Nitrogen cycle

Nitrogen (N) is one of the most important elements of the ecosystem and a necessary nutrient for the plants and animals. Although 79 % of atmosphere consists of nitrogen (N2), plants are not able to use atmospheric nitrogen directly. Plants require nitrogen to be converted to nitrogenous compounds and this process is accomplished by some soil microorganisms (such as cyanobacteria and various genera of bacteria and actinomycetes, or by symbiotic microbes such as Rhizobium which form root nodules in legumes) and called nitrogen fixation (Berntson and Bazzaz, 1997). Therefore, the nitrogen cycle depends on the soil biota (Mandal and Neenu, 2012). The process of nitrogen fixation converts atmospheric nitrogen into ammonia (NH3).

There are nine different forms of nitrogen in soil depending on its oxidation degree (Paul 2007). Nitrous oxide (N2O) and nitric oxide (NO) are greenhouse gases and are generated during two microbial processes, nitrification and denitrification.

+ Nitrification is an oxidation process of transforming ammonia (NH3 ) into other ionic forms of nitrogen, such as nitrosonium (NO−), nitrite (NO2−) and then nitrate (NO3−). This process is accomplished by autotrophic and heterotrophic nitrifiers. The autotrophic nitrifiers are divided into two taxonomic groups called ammonia oxidisers and nitrite oxidisers. The main ammonia oxidisers are bacteria from the genera Nitrosmonas, Nitrosolobus, Nitrospira, and Nitrosovibrio (Paul, 2007). The nitrite oxidisers are Nitrobacter, Nitrospina, Nitrospira, and Nitrosococcus. Heterotrophic

- 16 - nitrifiers are fungi that include the common soil fungi Fusarium, Aspergillus and Penicillium (Lin et al., 2008). Nitrification process is a complex process which consists of two steps (Hüseynov A. and Hüseynov N., 2012):

 The first step is the oxidation of NH3 to nitric acid. This step is performed by bacteria from the genus Nitrosomonas: 2NH3+ 3O2→2HNO2 + 2H2O + 273 kJ  The second step is the oxidation of nitric acid to nitrate acid. This step is performed by Nitrobacter: 2HNO2 + O2 → 2HNO3 + 70 kJ

These forms of nitrogen can be converted into atmospheric nitrogen in a process of denitrification. Denitrification occurs in anaerobic conditions where bacteria use nitrogen, due to the absence of oxygen, for anaerobic respiration. This process is carried out by denitrifiers from the genera Pseundomonas, Algaligenes, Bacillus, Agribacterium and Flavibacterium (Paul, 2007). The denitrifiers are major greenhouse gas produces. The reaction of this process is (Hüseynov A. and Hüseynov N., 2012):

5C6H12O6 +24KNO3 → 24KCO3 + 6CO2 + 12N2 + 18H2O + 1722 kJ

The nitrogen cycle has a very important role in the environment and agriculture. In terrestrial ecosystem nitrogen (Mandal and Neenu, 2012) and phosphorus (Ruttenberg, 2003) are the most common limiting nutrients for crop growth, which is why fertility of soils depends mainly on the nitrogen and phosphorus cycles.

2.3.1.3 Phosphorus cycle

Phosphorus is one of the most essential elements in ecosystem. It is very important nutrient for all life forms. Phosphorus is the main component of DNA (Deoxyribonucleic acid), RNA (Ribonucleic acid) and ATP (Adenosine triphosphate). Photosynthetic organisms intake energry from the Sun and use dissolved phosphorus to make different essential organic compounds which are mainly characterized by weak P-O-C or stabile P-C ester bonds (Richey, 1983). Therefore the availability of phosphorus can limit the productivity of producers. Carbon dioxide fixation is directly dependent on availability of phosphorus. As a result phosphorus cycle has a large scale influence on global climate (Paytan, 2009).

In terrestrial systems phosphorus is significant element for primary production and found in bedrocks, soils and living organisms. The amount of phosphorus is accounted approximately 0.1-0.12 % of weight of Earth’s crust. Majority of phosphorus is mainly in a form of inorganic phosphate minerals. Phosphorus-containing organic compounds also exist in terrestrial systems (Paytan, 2009). Bedrock minerals such as apatite which is the most abundant P-mineral are considered primary sources of phosphorus (Ruttenberg, 2003). The main phosphorus minerals include:

 Apatite Ca5(PO4)3(F,Cl,OH) ;  Frankolite Ca10-a-b ; NaaMgb(PO4)6-x(CO3)x-y-z(CO3F)y(SO4)zF2;  Lazulite (Mg,Fe)Al2(PO4)2(OH)2;  Monazite (Ce,La,Y,Th)PO4;  Pyromorphite Pb5(PO4)3Cl;  Strengite FePO4·2H2O;  Triphylite Li(Fe,Mn)PO4;  Turquoise CuAl6(PO4)4(OH)8·5H2O;  Variscite AlPO4·2H2O;

- 17 -  Vauxite FeAl2(PO4)2(OH)2·6H2O;  Vivianite Fe3(PO4)2·8H2O;  Wavellite Al3(PO4)2(OH)3·5H2O (Paytan, 2009)..

According to Lerman et al. (1975) the amount of phosphorus in soil is 200.000 Tg, while Pierrou (1976) noticed 160.000 Tg. Weathering P-minerals are subjected to some physical, chemical and biological processes in the further step of P cycling. Inorganic forms of phosphorus are assimilated by living organisms and converted into organic forms of phosphorus. Plant material is considered as major source of organic phosphorus in terrestrial systems. Microbes convert unavailable form of phosphorus into bioavailable form of phosphorus which process is called ‘mineralization’ of phosphorus (Paytan, 2009). Soil invertebrates have special and very significant role in this step of P cycling. Chapius-Lardy et al. (2009) noticed that soils which are suspected to earthworm activities have higher potential of phosphorus availability than uningested soils. Soils that are ingested by earthworms contain earthworm castings. Earthworm castings does not only contain higher amount of organic phosphorus (Chapius-Lardy et al., 1998) but also stimulate microbial activity (Le Bayon and Binet, 2006). López-Hernández (2001) reported the importance of termites on the transformation of phosphorus within organic compounds. Therefore soil invertebrates play an important role in phosphorus cycle.

2.3.1.4 Waste recycling, biodegradation

Saprotrophic organisms decompose dead organic matter such as leaves to obtain their energy and nutrients. This heterotrophic process is called decomposition. It is a biological “cascade” process and plays a key function role in soil ecosystem (Cock et al., 2011). Swift et al. (1979) explained decomposition as interactions of three components: soil organisms, physical environment and the quality of the decomposing resources. The importance of these three components and their action of space and time are not equal. Lavelle et al. (1993) proposed a hierarchical model to ascertain microbial decomposition processes in soil ecosystems at decreasing space and time scales: climate > clay mineralogy + nutrient status of soil > quality of decomposing resources > effect of macro-organisms. Regarding this hierarchy, factors which are acting higher time and space scales tend to be dominant over those acting at smaller scales of time and space (Allen and Hoekstra, 1992).

The primary decomposers such as bacteria, fungi and some invertebrates (earthworms etc.) play a very significant role in waste recycling. Other soil invertebrates such as isopods, millipedes and collembolans are called detritivores. The difference between the two groups is that detritivores are not able to digest the wide range of compounds that primary decomposers are capable of digesting. Bacteria are decomposers of dead animals and fungi are primary decomposers of plant litter (Swift et al., 1979).

2.3.1.5 Soil formation process

As a complex feedback cycle soil forming processes occur between the mineral fraction of soil, environment and soil biota (Mandal and Neenu, 2012). Soil formation process is dependent on five important factors, namely climate, organisms, relief, parent material and time (Bockheim et al., 2014). According to the founder of soil science V.V. Dokuchaev and his followers, climate and organisms are two most

- 18 - important factors among others. Soil organisms include vegetation, bacteria, meso-, micro-, and macro-fauna (White, 1979) and have extensive and various functions in the soil formation process. Soil organisms accomplish soil weathering process which is the primary source of essential elements (except carbon and nitrogen) for soil organisms. As a biological effect factor, soil organisms have never been absent in any soil forming process during weathering and they always accompany the evaluation of soil. For instance, the mechanical forces exerted by roots or the enormous work of worms and rodents in mixing and disintegrating rock bodies in the upper surface environment show the importance of biological agents in soil formation (Mirsal, 2008).

Representatives of meso- and macro-fauna such as earthworms, termites, ants, beetles and others have a significant role in soil formation process. However, earthworms are the most important soil forming organisms in temperate regions, while termites, ant, and beetles have greater importance in sub-humid and semiarid regions. Earthworms mix mineral particles with organic litter by digestion and establish stone- free layer on the soil surface (White, 1979) and accelerate the weathering process (Carpenter et al., 2007). For instance, the transformation of smectite to illite (two forms of clay minerals) by earthworms has already been proven (Mandal and Neenu, 2012).

Saprotrophic and mutualistic fungi increase the rates of mineral weathering at ecological and evolutionary time scales of soil system (Hoffland et al., 2004). Soil microorganisms derive their energy from the decomposition of organic matter and they convert complex organic molecules to simple inorganic forms in the process of mineralisation (White, 1979).

2.4 Climate change and soil invertebrates

Soil organisms are dependent on ambient conditions. Impacts of climate changes on soil invertebrates may include temperature and moisture responses.

As mentioned in paragraph 2.1 temperature is likely rise in the future that will alter global climate. This process will create negative impacts on terrestrial ecosystems. IPCC (2014) have reported that global climate change will derive risks on composition, structure and function of terrestrial ecosystems. Global climate change will likely cause some alterations on terrestrial species, such as:  Geographical ranges;  Seasonal activities;  Migration patterns;  Abundance;  Species interactions.

Jones et al. (2009) have reported that global temperature rise and moisture reduction will tend decomposition of carbon in soil. As a result amount of elevated CO2 into atmosphere will increase and contribute to global warming. Cole et al. (2002) reported the role of enchytraeids in the dynamics of carbon in soil. Reproduction of enchytraeids increases at higher temperature (Briones et al., 1997) that contributes to the exploitation of carbon sources in soil (Briones and Ineson, 2002). McCulley et al. (2005) stated that moisture is a limiting factor for net primary productivity (NPP) and decomposition of soil organic matter included soil organic carbon. As a consequence of

- 19 - global warming precipitation is likely increased and soil moisture is likely decreased. This process will derive some implications that are harmful for soil invertebrates.

The rise of atmospheric temperature causes increased activity of soil invertebrates. The activity of soil invertebrates decreases in winter when atmospheric temperature is low, but in summer when temperature is high, the activity of soil invertebrates increases. For that reason we can say that seasonal dynamics can change soil ecosystems. But this does not mean that soil organisms require high temperature. Extremely high temperature is very deleterious for many soil invertebrates (Schadt et al., 2003). As all organisms, soil invertebrates have a temperature minimum, maximum and optimum. Maximum temperature is the highest temperature that the animals can survive (Bardgett, 2005).

Elevated temperature is likely to affect essential ecological services such as decomposition of organic matter that is accomplished by soil organisms. Römbke et al. (2011) have reported that elevated temperature affects the feeding activity and mortality of two isopod species – Porcellio scaber and Porcellionides pruinosus. When the temperature increases, moisture content of soil starts to decrease due to evaporation. Therefore, organisms are affected by moisture stress. Consequently, at higher temperature the mortality rate of Porcellio scaber increases significantly. Elevated temperature also increases the food consumption of organisms due to their higher energy needs.

The food preferences of soil invertebrates are very important to investigate, because some soil invertebrates such as Porcellio scaber can distinguish between leaves from different tree species (Nair et al., 1994). The effects of increased temperature on tree species that are the food source for soil invertebrates have not been well investigated.

In addition to temperature, soil moisture also plays a very important role in soil ecosystem and affects soil invertebrates directly or indirectly. Soil moisture can indirectly affect the quality and quantity of plant litter production, and can be observed on plant microbes and “engineers”- microbes’ interactions (Swift et al., 1979).

The seasonal patterns of micro-fauna depend on two most significant abiotic factors: temperature and moisture. The optimal temperature for survival of micro-invertebrates is above 20°C and higher temperature limit is about 50°C. Temperature tolerance of invertebrates can differ depending on their habitats. For instance, species which live on the litter surface have more tolerance to drought than species living in lower layers of soil or generally species that live in warm areas have higher tolerance for high temperature than species living in temperate and cold areas. Some microarthropods such as springtails and mites have quite high temperature tolerance. Temperature factor affects development and reproduction rates of springtails and this is observed on population growth. The developmental stages (Chown and Nicolson, 2004) and species specificity (Christiansen, 1964) are other factors which must be considered when the temperature and moisture needs of microarthropods are studied.

Respiration of soft-bodied soil organisms such as earthworms, collembolans and enchytraeids take place through their thin, moist and vascular skin. Therefore, these organisms are very sensitive to ambient conditions, especially to desiccation (Didden, 1993). According to Jucevica and Melecis (2005) different ecomorphological life forms of Collembola have different reactions to climate changes. Thus Euedaphic (soil dwelling organism) species are more sensitive to temperature responses, but hemiedaphic species are mostly affected by moisture conditions. Frampton et al.

- 20 - (2000) have reported that drought is a limiting factor for Collembola. Richness of species in soil depends on moisture conditions, thus drought can decrease richness of Collembola species in soil (Jucevica and Melecis, 2005).

The effects of microclimate on nematodes and microarthropods have been observed and showed that sensitivity of nematodes to temperature and soil moisture depends on their metabolic state (Ruess, et al., 1999; Hoschitz and Kaufmann, 2004). When there are extremal temperature and moisture climate conditions they start to become cysts and enter dormant stages which can survive in this way (Wall and Virginia, 1999; McSorley, 2003). Blankinship et al. (2011) have shown that increasing precipitation favoured the fungal component of the soil food web and CO2 enrichment favoured the bacterial component. The frequency of wet-dry, freeze-thaw cycles can modify different components of soil such as the aggregation of soil, organic part of soil and also activity of soil invertebrates (Mandal and Neenu, 2012).

The respiration of soil invertebrates is a very important process in soil ecosystem and it can be affected by climate change due to increasing temperature. As a result, various changes in soil ecosystem can occur, because the intensity of decomposition of organic matter and the carbon storage process, which are essential for soil ecosystem, depend on respiration of soil microorganisms (Mandal and Neenu, 2012). More respiration means faster decomposition of organic matter and a parallel increase in the release of CO2.

Increasing temperature also affects nematodes via its influence on soil moisture (Hoschitz and Kaufmann, 2004). In this case, bio-geographical zone and original hydrological conditions are main factors that determine the sensitivity of soil moisture (Papatheodorou et al., 2004; Strong et al., 2004).

Under unfavourable dry conditions soil animals start to avoid drought. Consequently, some move into deeper layers of soil (Verhoef and Van Selm, 1983) and some can enter inactive stages, or survive as dormant eggs (Hopkin, 1997). According to Norton (1994) moisture content of the litter affects the ability of juveniles to penetrate substrates successfully. Moisture alteration in soil may cause indirect effects on the oviposition of oribatid mites (Hågvar 1998).

3. MATERIALS AND METHODS

3.1 Methods

We performed four experiments – two with earthworms (Eisenia andrei) and two with woodlice (Porcellio scaber). The fist experiment with woodlice was done to ascertain what soil moisture they prefer and which environments they would avoid in case of different ambient moisture conditions. The second experiment with woodlice was conducted to ascertain their feeding activity at two different moisture conditions. The first experiment with earthworms focused on their moisture preference. The second experiment with earthworms was conducted to ascertain their reproduction rate at two different moisture conditions.

- 21 - 3.1.1 Experiment - Isopod avoidance behaviour

Materials: 480g soil; 12 petri dishes; 60 rough woodlice (Porcellio scaber); water; pipette; balance.

Experimental setup:

Before starting the experiment we prepared petri dishes for the experiment by dividing them into 2 equal parts with plastic material in order not to mix different humidity soil samples. We made 12 petri dishes for this. Then we prepared soil samples. We used the standardised Lufa 2.2 soil (Speyer, Germany), which is commonly used in ecotoxicity and environmental studies. It is a loamy sand soil. Two days before the experiment we air dried 480 g of soil. The water holding capacity (WHC) of the soil is 43.3 g/100g. For the experiment we gave the animals a choice of drier and wetter soil. For control samples, we prepared petri dishes with soil containing water at 50% WHC on both sides of the dish. This was done in order to verify if the animals chose the sides randomly and not because of some extraneous environmental condition such as light. The experimental petri dishes were prepared with 50% WHC – 40% WHC and 50% WHC – 60% WHC. Each treatment was prepared in 4 replicates. For experiment we chose rough woodlouse which is typical for Slovenian forests and gardens (Figure 3). After preparing soil samples we added 5 animals into each petri dish. The invertebrates could choose the moisture they preferred. The animals were left in the laboratory (Figure 4) for 48 hours with natural day/light cycle and at 20°C. At the end of the experiment we placed a plastic divider (Figure 5) in the middle of each petri dish and counted the organisms on each side of the dish.

Figure 3: Rough woodlouse (Porcellio scaber)

- 22 -

Figure 4: Experimental setup for the isopod avoidance experiment.

Figure 5: Counting the animals at the end of experiment.

3.1.2 Feeding activity experiment with woodlice

Materials:

8 glass jars; 250 g Lufa 2.2 soil;

- 23 - Distilled water; Pipette; Balance; Analytical balance; Dry hazel (Corylus avellana) leaves; 40 rough woodlice (Porcellio scaber).

Experimental setup:

We prepared soil samples with 50 % and 60 % Water Holding Capacity (WHC). We used the Lufa 2.2 standardised soil. We placed soil samples with 50 % and 60 % WHC in glass jars in four replicates. Then 5 rough woodlice per sample were weighed and placed inside the jars (Figure 6).

We provided them with dry hazel leaves as a food source. Leaf pieces of 5 mg were placed into each jar. We closed the jars with perforated lids and left the samples at 20 °C for 4 weeks (Figure 7).

Figure 6: Woodlice in the sample.

- 24 -

Figure 7: Prepared samples for the feeding activity experiment with woodlice.

We regulated the moisture of samples two times a week, weighing the jars and replacing lost water. Every week leaves (Figure 8) were taken, air dried and weighed, and new leaves were added

Figure 8: Partially consumed piece of leaf by woodlice in the sample.

- 25 - 3.1.3 Experiment - Earthworm avoidance behaviour

Materials:

4 plastic containers - 77 x 20 x 17 cm; 36 L potting soil; 6 Cardboard dividers; Distilled water; Pipette; Analytical balance; 16 sample holders; 80 earthworms.

Experimental setup:

Before starting the experiment we prepared plastic containers and cardboard dividers for the experiment. For this we marked containers in order to divide them into seven equal parts (the length of each part was 11cm). Six cardboard dividers were prepared to separate these parts from one another at the end of the experiment.

Then we placed approximately 9 L of soil in each container. We used potting soil containing wood fibres, compost from crop residues, peat, composted bark, and clay minerals. In the next step of the experiment we added distilled water into the soil samples. For this, we took 500 ml distilled water per container and added this water in an amount of 200 ml, 150 ml, 100 ml, 50 ml in order to the 1st, 2nd, 3rd and 4th part of the containers. This was done to ensure variable moisture along the containers.

We selected the animals for the experiment (Figure 9) and added 20 earthworms per container (Figure 10), then the containers were covered with polyethylene to minimise evaporation and placed in the laboratory at 20 °C for 72 hours.

Figure 9: Earthworms (Eisenia andrei)

- 26 -

Figure 10: Prepared samples for the earthworm avoidance experiment.

After three days, the cardboard dividers were placed into the borders of the parts of the containers and we counted the earthworms in each part (Figure 11).

Soil samples were taken from each part of containers to measure the moisture con- tent.

Figure 11: Container with dividers in place at the end of the earthworm avoidance experiment.

Soil Moisture Measurements

Soil Moisture was measured by gravimetric technique. Aluminium vessels were numbered and weighed. Then soil samples were placed inside and the vessels were weighed again and placed in an oven at 105°C for 72 hours. After drying the samples were taken out and placed into the desiccator.

- 27 - After 20 minutes samples were re-weighed and the amount of dry soil was recorded. The moisture content of samples was determined by the difference in weight between wet and dry soil samples.

3.1.4 Earthworm reproduction test

Materials:

9 plastic cuboid containers with lids; 2,700 g soil; Distilled water; Desiccated horse manure; 45 earthworms; Analytic scale; Scale; Water bath.

Experimental setup:

The earthworm reproduction experiment followed the guidelines set in the OECD test No. 222 (OECD, 2004). Transparent square plastic food containers were taken and we made holes in their lids to permit gas exchange. We prepared soil samples with 40 %, 50 % and 80 % water holding capacity. We used the standardised Lufa 2.2 soil in this experiment. In the next step we placed soil samples into the plastic containers. We prepared testing samples in three replicates. One day before the experiment 45 adult earthworms (Eisenia andrei) were counted and acclimatised in the same soil that was used for the experiment. Then we weighed groups of 5 worms and randomly placed them into the containers. Before weighing the earthworms were washed with distilled water (Figure 12) and placed on the filter paper to remove the excess water. At the end we added 5 g of commercially available organic desiccated horse manure moistened with de-ionised water (Figure 13) per sample. Prepared samples were left in the laboratory at 20 °C during four weeks (Figure 14).

- 28 -

Figure 12: Washing of earthworms with de-ionised water.

Figure 13: Moistened horse manure.

- 29 -

Figure 14: Prepared samples for the earthworm reproduction experiment.

During the experiment we assured constant soil moisture by replenishing evaporated water three times a week. We provided food once a week during four weeks. At the end of 28 days we removed the adult earthworms form the containers and left the cocoons (Figure 15.). Earthworms were washed with de-ionised water and weighed. The soil samples (with cocoons) were replaced into the containers. The experiment was continued for another four weeks. During these four weeks soil moisture was kept constant and food was provided only once in the beginning of the second part of the experiment. At the end of the experiment (Day 56) we placed the samples into a water bath (Figure 16.) at 60 °C. After a period of 20 minutes juvenile earthworms started to appear on the soil surface. They were removed from the top of the soil and counted.

Figure 15: Earthworm cocoons at the end of 28th day of the earthworm reproduction experiment.

- 30 -

Figure 16: Samples of earthworm reproduction experiment in the water bath.

3.1.2 Statistical analyses

We calculated standard deviation for the average food consumption of woodlice and the distribution of earthworms at different moisture contents. We used Microsoft Excel 2010 for the calculation of standard deviations. Standard deviation shows us how far the values are spread above and below the mean.

4. RESULTS AND DISCUSSION

4.1 Results of the isopod avoidance behaviour experiment

During the experiment mortality of animals was not observed. The distribution of animals in the 50% - 50%, 50%-40% and 50%-60% WHC treatments in the replicates is presented in Table 1.

- 31 - Table 1: Results of the avoidance behaviour experiment. Moisture of Number of woodlice in samples samples Sample №1 Sample №2 Sample №3 Sample №4 50% - 50% 2-3 0-5 4-1 1-4

50% - 40% 5-0 0-5 4-1 2-3

50% - 60% 0-5 1-4 0-5 0-5

The results of the experiment showed that the animals had no clear preference for either side of the dish in the 50%-50% WHC treatment (Figure 17.). So we can conclude that light conditions did not affect the experimental results. In the 50%-40% WHC treatment, we can see that there was also no clear preference for either soil moisture level (Figure 18). In the 50%-60% WHC treatment 19 rough woodlice chose 60% of soil moisture and only one chose 50% soil moisture (Figure 19). We can say that if rough woodlice have a choice of 40%, 50% and 60% WHC, they will immediately choose 60% soil moisture condition.

During the experiment at 40% WHC side of 50%-40% treatment the aggregation of woodlice was observed on the surface of the soil. They were tightly aggregated at the wall of petri dish. This behaviour of woodlice is explained as their protection against desiccation. We can say that in case of desiccation, aggregation behaviour of woodlice will likely be increased. Similar findings have been studied before. Hassall et al. (2010) reported that the aggregation behaviour of woodlice increases in a case of desiccation.

The absence of mortality can be explained by the duration of experiment. Thus, it is most likely related to short duration of the experiment. However, Rombke et al. (2011) have found mortality cases of woodlice in similar tests. Therefore, long-duration experiments are needed to understand more clearly the relations between the mortality of woodlice and soil moisture.

The results of the experiment showed that woodlice activity decreases at lower soil moisture contents. Soil moisture content is the result of temperature, rainfall, water- holding capacity of soil and soil texture. As it is mentioned in Chapter 2.1 the mean temperature of the climate of the Earth is increasing, therefore the moisture content of soils will likely be decreased. This will likely lead the avoidance of some moisture dependent soil invertebrates as well as woodlice.

- 32 -

Figure 17: Distribution of woodlice by replicates in the 50%-50% WHC treatment.

Figure 18: Distribution of woodlice on the 50% - 40% WHC petri dishes.

Figure 19: Distribution of woodlice on the 50% - 60% WHC petri dishes.

- 33 - 4.2 Results of the feeding activity experiment of woodlice

During the experiment mortality of animals was observed. Generally, at 50 % WHC the mortality rate of woodlice was 30 %, while at 60 % WHC the mortality rate of woodlice was 25 %. There is no big difference between the two results. Woodlice are sensitive animals and their mortality could be due to moulding, cannibalism, or stress during handling. It is likely that WHC did not affect the mortality rate of woodlice, as already concluded in previous experiment.

The weights of animals were recorded before and after experiment. The weights of animals before and after the experiments are given in Table 2. The results indicate that average weight of animals at 50 % WHC and 60 % WHC did not change during the experiment.

The amount of total food consumption of woodlouse is presented in Table 3. The total food consumption of woodlice was 0.07 g at 50% WHC, while at 60% WHC it was 0.107 g (Figure 20). At 50% WHC average food consumption of woodlice was 0.017±0.002 g, while at 60% WHC it was 0.027±0.002 g during four weeks (Figure 21). Results of our experiment indicate that food consumption of woodlice at 60% WHC is higher than food consumption at 50% WHC. Morgado et al. (2015) have reported that the food consumption of woodlice at high moisture conditions is higher than at lower moisture conditions. So the results of these two experiments let us conclude that woodlice consume more food at higher soil moisture conditions.

In the case of global climate warming desiccation will likely increase in terrestrial ecosystems that will also lead to decreased moisture contents in soils. As the consequence of lower moisture conditions the feeding activity of woodlice will likely be decreased that will also derive adverse implications in the terrestrial ecosystems. For instance, lower consumption of woodlice means lower available food for other soil organisms such as bacteria. This process will impact negatively on the global cycles of carbon and nitrogen. Therefore, whole ecosystem will be affected by adverse impacts directly or indirectly.

During the experiment, few juveniles were observed in some test jars and it is highly likely that body weights and rate of total food consumption were influenced by this factor.

- 34 - Table 2: Weight of woodlice (Porcellio scaber) before and after experiment (g).

Sample № WHC Weight of animals Weight of animals after (%) before experiment (g) experiment (g)

1 50 0.108 0.109 2 50 0.122 0.122 3 50 0.109 0.108 4 50 0.12 0.121 5 60 0.109 0.109 6 60 0.118 0.118 7 60 0.100 0.101 8 60 0.101 0.101

Table 3: Food consumption of woodlice during four weeks (g). Weeks Consumption rate of woodlice (g)

50 % WHC 60 % WHC

1/4 2/4 3/4 4/4 1/4 2/4 ¾ 4/4 1 0.004 0.006 0.003 0.006 0.010 0.008 0.008 0.008

2 0.004 0.006 0.004 0.003 0.006 0.007 0.007 0.008

3 0.004 0.002 0.005 0.006 0.006 0.005 0.007 0.008

4 0.004 0.003 0.005 0.005 0.007 0.006 0.008 0.007

Total 0.016 0.017 0.017 0.02 0.02 0.026 0.03 0.031

Average 0.017 0.027

- 35 -

Figure 20: Total food consumption of woodlice at 50 and 60% WHC during four weeks.

Figure 21: Average food consumption of woodlice at 50 and 60% WHC during four weeks.

4.3 Results of the earthworm avoidance behaviour

During the experiment no mortality of earthworms was observed. At the moisture below 50% no animals were found. 52.5 % of all earthworms (42 of 80) chose moisture content between 55 and 60 %, while 43.75 % (35 of 80) chose moisture content between 60 and 65 % Only 3.75 % (3 of 80) of all earthworms chose moisture content 50-55 % (Table 4).

On average 0.8±0.5 earthworms were found at moisture between 50 and 55 %, while 10±8 earthworms at moisture between 55 and 60 %, and 9±8 earthworms at moisture between 60 and 65 % were found (Figure 22).

- 36 -

Our results showed that earthworms prefer moist environments and if earthworms have a choice of soil moisture contents, they will prefer moisture levels higher than 55% WHC. It is more likely that water levels below this value in soil are suboptimal conditions for earthworms. Several studies (Grant, 1955; Loehr et al., 1985; Berry and Jordan, 2000; Eggleton et al., 2009) have been done on the moisture preference of earthworms. In these studies it was highlighted that soil moisture content is a limiting ambient factor for earthworms and in case of desiccation effects on earthworms, such as horizontal migration from desiccated areas are observed.

The absence of mortality in our experiment is most likely related to the soil moisture content, which was not too low. Wever et al. (2000) have reported that the survival rate of Aporrectodea tuberculate decreases when the soil moisture decreases. In case of global warming it is more likely that earthworms will move from upper layers of soil (dry conditions) towards deeper layers (moist conditions) or they will move horizontally from lower moisture environments towards moist environments as a consequence of desiccation in the upper parts of the soil. We can therefore expect that soil organic matter degradation will be severely affected at lower soil moisture.

Table 4: Results of the earthworm avoidance experiment. Moistur Number of earthworms e (%)

1st replicate 2nd replicate 3rd replicate 4th replicate Total 30-50 0 0 0 0 0

50-55 1 1 1 0 3

55-60 16 0 9 17 42

60-65 3 19 10 3 35

Figure 22: Distribution of earthworms at different moisture contents.

- 37 - 4.4 Results of the earthworm reproduction test

During the experiment, no mortality of earthworms was observed. Total number of juveniles at 40 % WHC was 55, at 50% WHC 212 and at 80% WHC 558 (Table 5). The result of the experiment indicates that at 40 % WHC reproduction rate was quite low and on the contrary, at 80 % WHC reproduction rate of earthworms was much high (Figure 23). So, we can say that low WHC had a negative effect on the reproduction rate of earthworms. It is likely that high reproduction rate of earthworms demands WHC more than 50 %. Below 50 % WHC earthworms are likely stressed due to lack of water.

Table 5: Results of the experiment Number of juveniles WHC (%) 1st replicate 2nd replicate 3rd replicate Total 40 6 26 23 55

50 81 76 55 212

80 187 177 194 558

Figure 23: Reproduction of earthworms at 40 %, 50 % and 80 % WHC.

The results of both earthworm avoidance behaviour and earthworm reproduction experiments showed us that earthworms are highly dependent on soil moisture. Changed moisture content likely derives some implications on earthworms. Previously, Berry and Jordan (2000) have stated that soil moisture content affects the growth of earthworms. Growth rate of earthworms is positively related to moisture content. Similarly, Dominguez and Edwards (1996) have compared growth rate of earthworms

- 38 - at different moisture contents. They found out that growth rate of earthworms was the highest at 85% soil moisture content and the lowest at 65% moisture content (minimum moisture content was 65% in the experiment). Eggleton et al. (2009) have reported that abundance of earthworms is higher during very wet period of the year and lower during the dry period of the year.

Though moisture is considered as a limiting factor for earthworms, adaptation of earthworm species to changing climate conditions is not so clear yet. It is likely that some species can adapt to changing climate and tolerate suboptimal ambient conditions. Grant (1955) has reported that Aporrectodea caliginosa can resist desiccation in slowly drying soil. Additional investigations must however be done to find answers to the questions: do some species adapt changing climate? If the answer is yes, are they able to take over the ecosystem role of other species?

5. CONCLUSION

Soil invertebrates such as earthworms and isopods are highly dependent on ambient conditions. It is likely that climate change has some implications on the survival, abundance, richness, locomotion, feeding parameters, avoidance behaviour and other performances of soil invertebrates.

Based on the results of the experiments presented in this thesis, we can conclude that isopods show high preference to 60% WHC soil. At the 60% WHC food consumption of isopods was by 1.59-times higher than at 50% WHC. Similarly, earthworms showed higher preference for soil with higher moisture content (i.e. 55-60% soil WHC). Their reproduction rate was also higher (by 10-times) at 80% soil WHC as compared to 40% WHC.

In case of global warming moisture content of soils will decrease. As the result of this process activity of woodlice will likely be decreased and the avoidance behaviour of some moisture dependent soil invertebrates as well as woodlice will likely be observed. Desiccation will also very likely decrease the feeding activity of woodlice which will also derive adverse implications in the terrestrial ecosystems.

Due to desiccation, it is more likely that the migration of earthworms from upper layers of soil (dry conditions) towards deeper layers (moist conditions) or horizontally from lower moisture environments towards moist environments will be observed. It is also expected that soil organic matter degradation will be severely affected at lower soil moisture.

It is accepted that organisms require optimum temperature and moisture for their normal living. However, the adaptation of soil invertebrates is not understood clearly yet. As the result of climate change a drastic variation in ecosystems richness, with long-term consequences on nature and economy will very likely be observed.

The potential impacts of climate change on soil invertebrates were investigated less and limited studies have been published.

- 39 - In our future work, we will focus on the effects of climate change on soil biodiversity by sampling organisms in the field and comparing the soil community structure with past results published in literature. Part of our work will also be on determining the effects of climate change on soil fertility.

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