ACTA PROTOZOOLOGICA Testate amoebae: a review on their multiple uses as bioindicators

Abstract

Testate amoebae (TA) are unicellular protozoans enclosed in a test capable of indicating a wide variety of environmental conditions. Among others, characteristics such as short life cycle, great sensitivity and worldwide distribution makes them adequate bioindicators. As a complement to physical and chemical measurements, biomonitoring can be a cheaper and fastest way of environmental monitoring. This research sought to evaluate the extent of TA use in biomonitoring and the responses given by them to environmental features. The research was conducted in Scielo, Science Direct, Online Library, Google Scholar and Capes Journal Portal and yielded 211 papers. TA bioin-dication is able to provide information on metal, trace element and atmospheric pollution, and to point out different trophic states, pH, and evidence on characteristics of hydrology. Further, TA can be used in paleoenvironmental reconstruction as they reflect climate, volcanic and even sea level change phenomena. Sometimes, together with other organisms in environmental analysis, they have shown to be an important complement to biomonitoring. Additionally, a functional traits approach has been recently included as a promising tool. Methodological adjustments that have been conducted throughout the years are allowing TA use to be more reliable and precise. This review provides insight on the many possible functions of TA in bioindication studies, highlighting their wide use as bioindicators.

Full text

Acta Protozool. (2022) 61: 1–9
www.ejournals.eu/Acta-Protozoologica
doi:10.4467/16890027AP.22.001.15671
ACTA
PROTOZOOLOGICA
Testate amoebae: a review on their multiple uses as bioindicators
Yasmin de Góes Cohn Freitasa, Beatriz Rodrigues D’Oliveira Ramosa*, Yemna Gomes da Silvaa,
Gabriela Silva Sampaioa, Letícia da Silva Nascimentoa,b, Christina Wyss Castelo Brancoa, Viviane
Bernardes dos Santos Mirandaa
a Neotropical Limnology Group, Department of Zoology, Federal University of the State of Rio de Janeiro, 22290-240 Rio de Janeiro,
RJ, Brazil (yasmingcfreitas@edu.unirio.br; beatriz.ramos@edu.unirio.br; yemnasilva@edu.unirio.br; gaaabisam@edu.unirio.br;
cbranco@unirio.br; v.bernardesbio@gmail.com)
b Intitute of Biological Sciences, Instituto Federal do Rio de Janeiro, 20270-021 Rio de Janeiro, RJ, Brazil (leticia.nascimento2232@
gmail.com)
* Corresponding author
Abstract: Testate amoebae (TA) are unicellular protozoans enclosed in a test capable of indicating a wide variety of environmental condi-
tions. Among others, characteristics such as short life cycle, great sensitivity and worldwide distribution makes them adequate bioindicators.
As a complement to physical and chemical measurements, biomonitoring can be a cheaper and fastest way of environmental monitoring.
This research sought to evaluate the extent of TA use in biomonitoring and the responses given by them to environmental features. The re-
search was conducted in Scielo, Science Direct, Online Library, Google Scholar and Capes Journal Portal and yielded 211 papers. TA bioin-
dication is able to provide information on metal, trace element and atmospheric pollution, and to point out dierent trophic states, pH, and
evidence on characteristics of hydrology. Further, TA can be used in paleoenvironmental reconstruction as they reect climate, volcanic and
even sea level change phenomena. Sometimes, together with other organisms in environmental analysis, they have shown to be an important
complement to biomonitoring. Additionally, a functional traits approach has been recently included as a promising tool. Methodological
adjustments that have been conducted throughout the years are allowing TA use to be more reliable and precise. This review provides insight
on the many possible functions of TA in bioindication studies, highlighting their wide use as bioindicators.
Keywords: biomonitor, thecamoebian, bioindication, protozoan, hydrology, restoration
oering qualitative information about the quality of the
ecosystem (Markert et al. 2003, Sumudumali and Jaya-
wardana 2021). Bioindicator species may show chang-
es in their population dynamics and stability when re-
sponding to environmental modications, which can
be measured through variations in abundance, distribu-
tion, age structure, reproductive eort and success, and
growth (Burger and Gochfeld 2001). The biomonitor-
ing approach is an important complement to physical
and chemical measurements as it’s a fast, ecient and
a Neotropical Limnology Group, Department of Zoology, Federal
University of the State of Rio de Janeiro, 22290-240 Rio de Janeiro,
RJ, Brazil. E-mail address: beatriz.ramos@edu.unirio.br (B.R.D.
Ramos)
1. INTRODUCTION
In a biomonitoring study, an organism, a part of it, or
a community is used as bioindicators pointing out en-
vironmental changes in a specic moment and habitat,

Y. de Góes Cohn Freitasa et al.
2
cost-eective method (Dale and Beyeler 2001), provid-
ing a more continuous data, sometimes including ex-
treme events that have not been detected by standard
methods (Nguyen-Viet et al. 2007).
The testate amoebae (TA) are a polyphyletic group
that comprises ameboid protozoans enclosed in a test
test, common in freshwater (sediments, plankton), and
terrestrial habitats (Kosakyan et al. 2020). In order to
feed and locomote, thecamoebians protrude lose or lo-
bose pseudopodia through the test’s aperture, the pseu-
dostome (Cavalier-Smith 2004, Adl et al. 2019). TA
present key characteristics to act as reliable bioindica-
tors, such as: 1) short life cycle that allows fast response
to ecological change (Foissner 1999); 2) persistent test,
that can resist fossilizing processes (Charman 2001,
Patterson and Kumar 2002); 3) sensitivity to a wide
range of environmental variables (Nasser et al. 2020);
4) great abundance and diversity in dierent environ-
ments (Foissner 1999, Lansac-Tôha et al. 2007, Alves
et al. 2010, Miranda et al. 2020); 5) worldwide distri-
bution and established as bioindicators throughout dif-
ferent biotopes (Nasser et al., 2020). These organisms
have been studied and used as bioindicators, answering
to past and present conditions (Payne 2013, Roe and
Patterson 2014, Amesbury et al. 2016) in the many bio-
topes in which those species inhabit.
In freshwater ecosystems, TA inhabit rivers (Cos-
ta et al. 2015), lakes (Nasser et al. 2020), reservoirs
(Misailidis et al. 2018) and phytotelms (Kratina et al.
2017); but they can be also found in brackish envi-
ronments, such as estuaries (Eichler et al. 2006), salt-
marshes (Barnett et al. 2017) and marine sand supral-
ittoral (Golemansky 2007). In terrestrial habitats, TA
inhabit soils (Wanner et al. 2020), Sphagnum and mosses
(Basińska et al. 2020), phytotelms (Kratina et al. 2017),
and are also present in wastewater treatment systems
(Hu et al. 2013). Thecamoebians occupy key roles in
the trophic webs, varying in feeding types and trophic
positions (Gilbert et al. 1998a, b; Lamentowicz et al.
2013a). They feed on bacteria, cyanobacteria, microal-
gae, fungi, humus, or even small thecamoebians, cili-
ates, rotifers and nematodes (Schroeter 2001, Gilbert et
al. 2003, Han et al. 2008, Jassey et al. 2012). TA can
be directly aected by environmental changes such as
moisture uctuation, and indirectly by water chemistry,
via the impact on the organism’s TA prey on (Mitchell
et al. 2000a). Thecamoebians also participate in chemi-
cal and nutrient cycling such as carbon, nitrogen and
silicon cycles (Puppe 2020). All above highlight the-
camoebians role in both ecological health functioning
and structure (Burger and Gochfeld 2001).
Anthropogenic impacts throughout the globe aect
Earth system’s resilience by processes like land-system
changes, freshwater uses, changes in biosphere integrity
and in biogeochemical ows, such as carbon, nitrogen
and phosphorus (Steen et al. 2015). Considering this,
bioindicators can oer an early warning of environmen-
tal changes, in a way to spotlight specic damages and
trends in the habitat’s conditions (Paoletti 1999, Burger
and Gochfeld 2001). Thus, allowing restoration and
conservation management tools to be intelligently as-
sociated with the environment condition, and by pro-
viding an assessment on ecological health, bioindica-
tion will keep track of goods and services provided by
it contributing to human health (Paoletti 1999, Burger
and Gochfeld 2001).
The present study aimed to review the current
available scientic literature on testate amoeba’s use
as bioindicators throughout dierent habitats and pur-
poses, focusing on the knowledge gathered about the
responses given by these protozoans to environmental
changes.
2. MATERIALS AND METHODS
The search has been conducted in the following scientic data-
bases: Scielo, Science Direct, Online Library, Google Scholar and
Capes Journal Portal. The keywords used were (“testate amoebae”
OR “testate amoeba” OR “thecamoebian”) AND (“bioindicator”
OR “biomonitoring” OR “bioindication” OR “paleoecology” OR
“transfer function”), paired with Boolean Operators. Papers pub-
lished until 2020 were gathered, with no inferior limit, in order to
access a greatest number of papers. During the article’s survey, an
evaluation of the title and abstract were carried out, in order to select
the papers that utilized thecamoebians as a tool in biomonitoring
research, those being the focus or used as support with other or-
ganisms. Were also selected articles taken from previous gathered
paper’s references. Afterwards, a full text assessment was made, in
order to detail what responses the thecamoebians gave in each envi-
ronment and to dierent variables.
3. RESULTS AND DISCUSSION
The database search encountered a total of 3.481
results. There were selected 211 papers that present
testate amoebae as eligible or potentially indicators
in environmental monitoring and paleoenvironmen-
tal reconstructions at peatlands, bogs, lakes, soil, wet-

Testate amoebae 3
lands, rivers, streams, saltmarsh and paleocryosols. A
total of 70 under genera taxa, with specic bioindica-
tion properties including several environmental and
paleoenvironmental features (Figure 1), were cited in
this paper. The specic tolerance and/or indicator prop-
erty to some environmental variables are presented in
Table 1. These protozoans are further used to develop
transfer functions in order to reconstruct palaeohydrol-
ogy, paleoclimate and sea-level changes. Another use
is in sewage treatment, where, amongst other microor-
ganisms, thecamoebians indicate the health of activated
sludge plants and water quality of wastewater treatment
systems.
3.1. Organic matter, pH and nutrients
Environmental impacts, especially those caused by
human activities, have been causing a series of losses in
water quality (Cardoso and Novaes 2013). In lakes, riv-
ers, reservoirs and streams, TA have their communities
aected by the pollution caused by high concentration
of nutrients (McCarthy et al. 1995, Qin et al. 2009, Nev-
ille et al. 2010, Qin et al. 2016, Schwind et al. 2019),
minerals (Casper and Schonborn 1985), thaw salts (Qin
et al. 2013), acidication of streams (Costan and Planas
1986) and industrial and domestic untreated euents
(Costa et al. 2015).
Acidication in streams can decrease thecamoebians
density, which can be explained by environment modi-
cation (e.g., potassium liberation from the sediments)
and by the disturbance caused on the mechanical os-
motic regulators of TA (Costan and Planas 1986). The
pollution of lakes by thaw salts, especially sodium chlo-
ride (NaCl), is another impact that water resources may
suer, causing lower diversity of TA (Roe and Patterson
2014). In these places, the increase of brackish tolerant
species as Arcella vulgaris, Centropyxis constricta spinosa
and Centropyxis aculeata aculeata is reported, being
those recommended for tracking salt increase in healthy
lakes (Roe and Patterson 2014). On the other hand, a
higher diversity of TA was registered in alkaline lakes,
pointing out strong correlations of the community with
water pH. Pontigulasia elisa, Pontigulasia compressa and
Lesquereusia modesta were the most abundant taxa, mak-
ing them indicators of higher pH (Qin et al. 2013).
Suspended matter can be considered an inuence
to TA communities in aquatic environments, since it
is mostly composed of minerals used in the test con-
struction (Pereira et al. 2006, Du Châtelet et al. 2010,
Schwind et al. 2019). In a lake with high levels of cal-
cite, the species Diugia limnetica replaced the sand
grains on its carapace for calcite grains (Casper and
Schonborn 1985), being considered a good indicator
of calcite precipitation in lakes (Casper and Schonborn
1985). In particular, the exogenous species respond sig-
nicantly to the suspended matter in the water column,
which can reect in the abundance and occurrence of
individuals (Schwind et al. 2019). Positive responses
to higher concentrations of suspended inorganic mat-
ter were reported for Diugia pseudogramen, Diugia
lobostoma and Centropyxis ecornis. Conversely, Protocu-
curbitella coroniformes, Cucurbitella crateriformis and Cu-
curbitella dentata f. quinquilobata responded negatively to
increases of suspended inorganic matter (Schwind et al.
2019).
Anthropic impacts increase and speed up processes
that lead to high concentrations of suspended matter in
water resources (Souza and Knoppers 2003), being eu-
trophication one of the problems that strikes the water
environments the most, being able to limit heterotroph-
ic microorganism’s metabolic activities (Smith and
Schindler 2009). In rivers contaminated by industrial
and domestic untreated euents, the low abundance of
Diugia distenda was considered an indicative of im-
pact at the water quality (Costa et al. 2015). Heleopera
sphagni and Nebela collaris were conspicuous in lakes
with high concentrations of organic matter (McCarthy
et al. 1995). In comparison, a more diverse community
was observed in hypereutrophic and mesotrophic grass-
land regions lakes, with Diugia oblonga and Cucubitella
tricuspis being dominant (Neville et al. 2010). However,
the greatest diversity was reported for the boreal for-
est, where most of the lakes are eutrophic (Neville et
al. 2010). In lakes with high concentrations of nutrients
and heavy metals, the community of TA had its rich-
ness reduced, being the genera Centropyxis and Diugia
the most tolerant to high nutrient’s concentrations, and
dominant in all the pollution gradients (Qin et al. 2016).
Cucurbitella tricuspis was also pointed out as commonly
associated with high levels of solid waste (Escobar et al.
2005). However, there is still a lack of studies about the
ecology of the group in polluted lakes, which makes it
hard to explain the dominance of certain taxa in a spe-
cic condition (Qin et al. 2016).
In lakes and rivers, there have been recorded correla-
tions between TA and ammonia, nitrate, phosphate and
chlorophyll-a content (Arrieira et al. 2015, Arrieira et
al. 2016, Schwind et al. 2017); interfering in their size
and morphometry (Arrieira et al. 2016) and frequency
and abundance (Schwind et al. 2017). Those nutrients
are linked to the environment’s productivity, impact-

Y. de Góes Cohn Freitasa et al.
4
Fig. 1. Diverse bioindication properties shown by testate amoebae in lakes, peatlands, soil and sea marshes. (a) Variables liable to be iden-
tied by testate amoebae. (b) Paleoenvironmental variables recorded by testate amoebae. N: nitrogen, P: phosphorus, K: potassium, Mn:
magnesium, Hg: mercury, Sc: scandium, Pb: lead, Fe: iron, Ba: barium, Cr: chromium, Zn: zinc, Co: cobalt, As: arsenic, Cu: copper, Ca:
calcium.

Testate amoebae 5
Table 1. Testate amoebae specic tolerance and/or indicator property to some environmental variables.
Testate Amoebae
Brackish tolerant
Acidic pH
Alkaline pH
Temperature
Calcite precipitation in
lakes
Concentration of heavy
metals and/or semimetals
Concentration of organic
matter
High concentrations of sus-
pended inorganic matter
Chlorophyll-a
Good water quality
Very high water content in
peatlands
High-water content
Low-water content
Burning indicator
Non-shaded habitats
Atmospheric pollution and
NO2
1Alabasta militaris * * *
2Amphitrema wrightianum * * * *
3Arcella * *
4Arcella catinus *
5Arcella discoides * * * *
6Arcella hemisphaerica *
7Arcella megastoma *
8Arcella vulgaris * * * *
9Archerella avum * * * * * *
10 Assulina *
11 Assulina muscorum * * *
12 Bullinularia indica * * *
13 Centropyxis * *
14 Centropyxis aculeata * * * * * *
15 Centropyxis aculeata ‘aculeata’ *
16 Centropyxis cassis * *
17 Centropyxis constricta * *
18 Centropyxis constricta ‘spinosa’ *
19 Centropyxis discoides *
20 Centropyxis ecornis * * * *
21 Corythion * *
22 Corythion dubium * * * *
23 Cucurbitella crateriformis *
24 Cucurbitella dentata f. quinquilobata *
25 Cucurbitella tricuspis * *
26 Cyclopyxis arcelloides * * *
27 Diugia * * *
28 Diugia acuminata * * * *
29 Diugia amphoralis * *
30 Diugia bacillifera *
31 Diugia bicornis * *
32 Diugia biwae *
33 Diugia distenda *
34 Diugia elegans * * * *
35 Diugia gramen *
36 Diugia helvetica * *
37 Diugia kempnyi *

Y. de Góes Cohn Freitasa et al.
6
38 Diugia limnetica *
39 Diugia lobostoma ***
40 Diugia oblonga * * *
41 Diugia parva * *
42 Diugia proteiformis *
43 Diugia pseudogramen *
44 Diugia schuurmani * *
45 Diugia urceolata * *
46 Diugia ventricosa *
47 Euglypha diliociformis *
48 Euglypha laevis * *
49 Euglypha rotunda * *
50 Euglypha strigosa * *
51 Euglypha tuberculata * *
52 Heleopera petricola * * *
53 Heleopera sphagni * * * * * *
54 Hyalosphenia elegans * * * *
55 Hyalosphenia papilio * * * * * *
56 Hyalosphenia subava * * *
57 Lesquereusia globulosa *
58 Lesquereusia modesta * *
59 Lesquereusia ovalis *
60 Mediolus corona * * *
61 Nebela carinata * * * * *
62 Nebela collaris * *
63 Nebela tincta * * *
64 Netzelia tuberculata *
65 Paraquadrula irregularis *
66 Phryganella acropodia * * *
67 Placocista spinosa ***
68 Pontigulasia compressa * *
69 Pontigulasia elisa *
70 Protocucurbitella coroniformes *
71 Quadrulella symmetrica * *
72 Trigonopyxis arcula * * * * *
73 Trinema * *
74 Trinema enchelys *
75 Trinema lineare *
References: 1 – Tolonen et al. 1994; Mitchell et al. 1999; Booth 2002; Lamentowicz and Mitchell 2005; Nguyen-Viet et al. 2008; Heine-
meyer and Swindles 2018; 2 – Mitchell et al. 1999; Charman et al. 2000; Mieczan 2009; Turner et al. 2013; Lamentowicz and Mitchell
2005; Niedzwiecki et al. 2016; Mieczan and Tarkowska-Kukuryk 2017; Heinemeyer and Swindles 2018; Creevy et al. 2018; 3 – Song et
al. 2014; Lamentowicz and Mitchell 2005; 4 – Nguyen-Viet et al. 2008; 5 – Charman and Warner 1997; Warner et al. 2007; Nguyen-Viet
et al. 2008; Mieczan 2009; Qin et al. 2016; Heinemeyer and Swindles 2018; 6 – Madoni 1994; Hu et al. 2013; 7 – Misailidis et al. 2018;
8 – Patterson et al. 1996; Kumar and Patterson 2000; Escobar et al. 2005; Patterson et al. 2013; Turner et al. 2013; Roe and Patterson 2014;
Swindles et al. 2015; Amesbury et al. 2016; Mieczan and Tarkowska-Kukuryk 2017; 9 – Charman and Warner 1992; Tolonen et al. 1994;
Warner and Charman 1994; Mitchell et al. 1999; Mieczan 2009; Turner et al. 2013; Marcisz et al. 2014a; Lamentowicz and Mitchell 2005;
Swindles et al. 2015; Swindles et al. 2016; Marcisz et al. 2016; Niedzwiecki et al. 2016; Ratclie et al. 2017; Mieczan and Tarkowska-

Testate amoebae 7
Kukuryk 2017; Lamentowicz et al. 2020; 10 – Song et al. 2014; Payne et al. 2016; 11 – Warner and Chmielewski 1992; Mitchell et al.
1999; Laggoun-Défarge et al. 2008; Mieczan and Tarkowska-Kukuryk 2013; Lamentowicz and Mitchell 2005; Niedzwiecki et al. 2016;
Mieczan and Tarkowska-Kukuryk 2017; Lamentowicz et al. 2020; 12 – Tolonen et al. 1992; Mitchell and Gilbert 2004; Mieczan 2009;
Lamentowicz and Mitchell 2005; 13 – Qin et al. 2016; Nasser et al. 2020; Asada and Warner 2009; 14 – McCarthy et al. 1995; Patterson et
al. 1996; Mitchell et al. 1999; Nguyen-Viet et al. 2008; Yang et al. 2011; Qin et al. 2013; Song et al. 2014; Qin et al. 2016; Lamentowicz
and Mitchell 2005; Mieczan and Tarkowska-Kukuryk 2017; 15 – Roe and Patterson 2014; 16 – Asada and Warner 2009; Qin et al. 2013;
Qin et al. 2016; 17 – Patterson et al. 1996; McCarthy et al. 1995; 18 – Roe and Patterson 2014; 19 – Yang et al. 2011; 20 – Lamentowicz
and Mitchell 2005; Qin et al. 2013; Qin et al. 2016; Amesbury et al. 2016; Schwind et al. 2019; 21 – Mieczan 2009; Song et al. 2014; 22 –
Mitchell et al. 1999; Laggoun-Défarge et al. 2008; Nguyen-Viet et al. 2008; Lamentowicz and Mitchell 2005; Payne et al. 2012; Mieczan
and Tarkowska-Kukuryk 2017; 23 – Schwind et al. 2019; 24 – Schwind et al. 2019; 25 – Escobar et al. 2005; Qin et al. 2016; Nasser et
al. 2020; 26 – Mitchell et al. 1999; Booth 2002; Lamentowicz and Mitchell 2005; Asada and Warner 2009; Yang et al. 2011; Turner et al.
2013; Heinemeyer and Swindles 2018; 27 – Qin et al. 2016; Song et al. 2014; Nasser et al. 2020; 28 – Qin et al. 2013; Schwind et al. 2017;
Qin et al. 2016; 29 – Qin et al. 2016; Schwind et al. 2017; 30 – Charman and Warner 1997; 31 – Qin et al. 2016; 32 – Qin et al. 2016;
33 – Costa et al. 2015; 34 – Lamentowicz and Mitchell 2005; Qin et al. 2013; Niedzwiecki et al. 2016; Mieczan and Tarkowska-Kukuryk
2017; Nasser et al. 2020; 35 – Misailidis et al. 2018; 36 – Schwind et al. 2017; 37 – Schwind et al. 2017; 38 – Casper and Schonborn 1985;
39 – Schwind et al. 2019; Schwind et al. 2017; 40 – Qin et al. 2016; Misailidis et al. 2018; McCarthy et al. 1995; 41 – Schwind et al. 2017;
42 – Patterson et al. 1996; 43 – Schwind et al. 2019; 44 – Schwind et al. 2017; 45 – McCarthy et al. 1995; Qin et al. 2016; 46 – Schwind et
al. 2017; 47 – Nguyen-Viet et al. 2007; 48 – Mitchell et al. 1999; Yang et al. 2011; 49 – Mitchell et al. 1999; Mieczan 2009; Mieczan and
Tarkowska-Kukuryk 2013; Wanner et al. 2020; 50 – Mitchell et al. 1999; Lamentowicz and Mitchell 2005; Meyer et al. 2012; Nguyen-Viet
et al. 2008; 51 – Madoni 1994; Lamentowicz and Mitchell 2005; Mieczan 2009; Hu et al. 2013; 52 – Mitchell et al. 1999; Lamentowicz
and Mitchell 2005; Niedzwiecki et al. 2016; Heinemeyer and Swindles 2018; 53 – McCarthy et al. 1995; Lamentowicz and Mitchell 2005;
Nguyen-Viet et al. 2008; Marcisz et al. 2014a; Marcisz et al. 2016; Niedzwiecki et al. 2016; Lamentowicz et al. 2020; 54 – Charman and
Warner 1992; Tolonen et al. 1994; Mitchell et al. 1999; Lamentowicz and Mitchell 2005; Charman et al. 2007; Warner et al. 2007; Mieczan
2009; Niedzwiecki et al. 2016; Ratclie et al. 2017; Creevy et al. 2018; 55 – Charman and Warner 1992; Tolonen et al. 1994; Mitchell et al.
1999; Lamentowicz and Mitchell 2005; Charman et al. 2007; Warner et al. 2007; Marcisz et al. 2014a; Niedzwiecki et al. 2016; Payne et al.
2016; Marcisz et al. 2016; Niedzwiecki et al. 2016; Ratclie et al. 2017; Creevy et al. 2018; Lamentowicz et al. 2020; Basińska et al. 2020;
56 – Charman and Warner 1992; Booth 2002; Turner and Swindles 2012; Turner et al. 2013; Niedzwiecki et al. 2016; 57 – Misailidis et al.
2018; 58 – Qin et al. 2013; Qin et al. 2016; 59 – Schwind et al. 2017; 60 – McCarthy et al. 1995; Misailidis et al. 2018; Qin et al. 2013; Qin
et al. 2016; 61 – Charman and Warner 1997; Mitchell et al. 1999; Lamentowicz and Mitchell 2005; Nguyen-Viet et al. 2008; Mieczan 2009;
Mieczan and Tarkowska-Kukuryk 2017; 62 – McCarthy et al. 1995; Mitchell et al. 1999; Lamentowicz and Mitchell 2005; 63 – Mitchell et
al. 1999; Booth 2002; Lamentowicz and Mitchell 2005; Laggoun-Défarge et al. 2008; Mieczan 2009; Meyer et al. 2012; 64 – Niedzwiecki et
al. 2016; Misailidis et al. 2018; 65 – Nguyen-Viet et al. 2004; 66 – Mitchell et al. 1999; Mitchell 2004; Heinemeyer and Swindles 2018; 67
– Mitchell et al. 1999; Warner et al. 2007; 68 – Qin et al. 2013; Qin et al. 2016; 69 – Qin et al. 2013; 70 – Schwind et al. 2019; 71 – Mitchell
et al. 1999; Lamentowicz and Mitchell 2005; Nguyen-Viet et al. 2007; 72 – Mitchell et al. 1999; Charman et al. 2000; Lamentowicz and
Mitchell 2005; Turner and Swindles 2012; Meyer et al. 2012; Turner et al. 2013; Swindles et al. 2015; Mieczan and Tarkowska-Kukuryk
2017; Heinemeyer and Swindles 2018; 73 – Song et al. 2014; 74 – Wanner et al. 2020; 75 – Wanner et al. 2020; Nguyen-Viet et al. 2008.
ing TA by the food web (Arrieira et al. 2015, Arrieira
et al. 2016). Diugia parva, D. acuminata, Lesquereusia
ovalis, Diugia schuurmani and Diugia Helvetica were
positively correlated with chlorophyll-a (Schwind et al.
2017). D. parva, D. acuminata and D. ventricosa are indica-
tor organisms that are positively associated with phos-
phorus concentrations, unlike D. lobostoma (Schwind
et al. 2017). Regarding nitrogen, D. helvetica and D.
schuurmani are positively associated with high concen-
trations, while D. lobostoma, negatively (Schwind et al.
2017). The bioindicator species that expressed posi-
tive associations with chlorophyll-a, phosphorus and
nitrogen, responded preferentially to higher concentra-
tions of them commonly found in eutrophic conditions
(Schwind et al. 2017).
3.2. The inuence of hydrology
Moisture/humidity is the main factor inuencing
thecamoebians composition at bogs. In addition, micro-
topography, water chemistry and seasonality have some
impact on testate amoeba’s distribution (Warner et al.
2007).
Capable of causing more than one third of variance
in testate amoeba communities (Song et al. 2018), wa-
ter-table depth has been pointed out as the main factor
inuencing these communities at peatlands (Lamento-
wicz and Mitchell 2005, Mitchell et al. 2008, Li et al.
2015, Payne et al. 2016, Zhang et al. 2017, Song et al.
2018). Seasonality plays an important role in those en-
vironments, as it changes water availability to TA (Heal
1964, Gilbert et al. 2003, Warner et al. 2007). Thus, TA

Y. de Góes Cohn Freitasa et al.
8
communities can suggest water table uctuation, or it’s
stability over prolonged time scales (van Bellen et al.
2018, Lamentowicz et al. 2019, Marcisz et al. 2020).
Also, seasonality can have an impact on TA communi-
ties through changes in temperature (Lamentowicz et
al. 2013a), light abundance (Marcisz et al. 2014a) and
even in combination with nutritional conditions (Mitch-
ell 2004, Payne and Mitchell 2007, Mieczan 2007,
Mieczan 2010, Elliott et al. 2012, Jassey et al. 2013,
Song et al. 2018). The last combination could be in-
ferred also when correlating TA with Sphagnum species,
due to chemical compounds released by the plants (Jas-
sey et al. 2013, Jassey et al. 2014, Marcisz et al. 2014b).
The above cited variables can be related to testate
amoebae size structure, as smaller species have been
more abundant at drier periods of the year (Warner et al.
2007, Jassey et al. 2014, Fournier et al. 2015, Marcisz et
al. 2016), while the biggest ones where more common
at the wettest seasons (Lamentowicz et al. 2013a).
In a wet open bog, Argynnia vitraea, D. oblonga, Neb-
ela carinata were characteristic species, while Spheno-
deria lenta, Cryptodiugia oviformis, and Nebela lageni-
formis inhabited a drier forested swamp (Warner et al.
2007). During the season with wetter conditions, Hya-
losphenia papilio, Hyalosphenia elegans, Arcella discoides,
and Placocista spinosa were found, reinforcing their re-
lation to high soil water content (Warner et al. 2007).
In relation to spatial patterns, microtopography of the
Sphagnum have aected testate amoebae’s assemblages,
reecting ecological gradients that, otherwise, wouldn’t
be visible macroscopically. Albeit, ecological vari-
ables cannot be inferred precisely by it (Mitchell et al.
2000b). The taxa Archerella avum, H. papilio, and H.
elegans were pointed out as good high paleo-moisture
indicators, while Hyalosphenia subava, was related to
the drier sampling points (Charman and Warner 1992,
Tolonen et al. 1994, Warner and Charman 1994). Spe-
cies of narrow ecological amplitude are A. discoides,
Diugia bacillifera, N. carinata, Nebela griseola, Nebela
marginata, Cryptodiugia sacculus, Quadrulella symmet-
rica, Amphitrema stenostoma, and S. lenta (Charman and
Warner 1997). In a Chile peatland, Centropyxis was the
only dominant genera during all the four seasons of the
year, being pointed out as generalist and opportunist
(Fernández and Zapata 2011).
The community of testate amoebae is aected by a
hydroseral gradient, changing according to the stage,
from open water to fen and pioneer raised mire to om-
brotrophic bog (Elliott et al. 2012). It was also suggest-
ed that thecamoebians could be used alongside plant
macrofossils with the potential for delimiting salin-
ity changes, as they responded more quickly and were
more sensitive to nutrient gradients than the plant com-
munities (Elliott et al. 2012). In Moorlands, subject to
a lot of changes and degradations over the past centu-
ries, hydrology was the strongest environmental factor
controlling the communities of TA, allowing their use
as monitors of this ecosystem. (Turner and Swindles
2012).
Characteristic taxa of wet Sphagnum habitats are Am-
phitrema wrightianum, Nebela carinata, Archerella avum
and Hyalosphenia papilio, Assulina species (Charman et
al. 2000, Booth 2002, Lamentowicz and Mitchell 2005,
Mazei and Tsyganov 2006). Characteristic taxa of dry
Sphagnum habitats are Trigonopyxis arcula, Bullinularia
indica, Nebela tincta, Alabasta militaris (Charman et al.
2000, Booth 2002, Lamentowicz and Mitchell 2005,
Mazei and Tsyganov 2006). Other abundant and nota-
ble taxa from the wetter end of the water table were
Pseudodiugia fulva type, and Diugia pulex type, H.
papilio, Assulina seminulum type, Arcella catinus type and
H. elegans for the dryer end (Charman et al. 2007). Stud-
ies in mosses indicate that the species A. wrightianum, A.
vulgaris and Archerella avum were indicative of a more
humid environment (Turner et al. 2013, Swindles et al.
2015, Amesbury et al. 2016, Heinemeyer and Swindles
2018). On the other hand, C. arcelloides and Heleopera
petricola indicate intermediate humidity (Amesbury et
al. 2016). Conversely, B. indica, A. militaris, C. ecornis, C.
arcelloides, H. petricola, Phryganella acropodia and Trigo-
nopyxis arcula were indicative of drier locations (Turner
et al. 2013, Swindles et al. 2015, Amesbury et al. 2016,
Heinemeyer and Swindles 2018). Species with spines,
such as Centropyxis aculeata, species from the Arcella ge-
nus and belonging to the genus Diugia, are also use-
ful for indicating higher humidity (Song et al. 2014).
In contrast to the genus Trinema, Assulina and Corythion,
linked to the drier places (Song et al. 2014).
3.3. Ecosystem restoration
Testate amoebae indicate changes in regenerating
peatlands and oodplains. Aorestation, drainage and
harvest at peatlands are concerns in these ecosystems,
as they cause ecological damage and carbon loss to the
atmosphere (Lachance et al. 2005). Eorts have been
made to restore such environments, allowing the peat to
re-growth (Parry et al. 2014). Simultaneously, in ood-
plains, anthropic impacts have created the necessity to
apply restoration projects (Sudduth et al. 2007), and
strategies for tracking recovery are welcome.

Testate amoebae 9
At dierent levels of succession, peatlands can be
inhabited by dierent assemblages of TA that respond
to physical and chemical properties of the peat (But-
tler et al. 1996). As the peatland becomes drier and
more acidic, species richness and abundance increased,
while density, biomass and average thecamoebian size
decreased (Laggoun-Défarge et al. 2008). N. tincta, As-
sulina muscorum and Corythion dubium were character-
istic species of the advanced stage environment, being
indicators of dry and acidic conditions (Mitchell et al.
1999, Laggoun-Défarge et al. 2008). A. muscorum and
C. dubium have already increased in abundance after
one and a half to two years from drainage in a forested
mire, showing their capability to monitor soil microen-
vironmental eects (Warner and Chmielewski 1992).
Thecamoebian communities were able to show impacts
from harvest, even when secondary succession surface
vegetation appeared to be similar to the natural site’s
vegetation. These protists are an alternative to continu-
ous measurement in the eld, in order to indicate wa-
ter table depth and pH (Laggoun-Défarge et al. 2008).
Also, in a restoring peatland, management have par-
tially caused the TA community changes, but besides it,
the weather has also contributed (Swindles et al. 2016).
With the management intervention, wet indicator spe-
cies appeared, such as Amphitrema stenostoma, Archerella
avum, Arcella discoides type, Diugia bacillifera and D.
bacillarium (Swindles et al. 2016).
TA can further be used as bioindicators in peatlands
undergoing forest-to-bog restoration (Creevy et al.
2018). By comparing the community inhabiting open,
forested and forest-to-bog sites, signicant dierences
in relative abundance between them were observed. Al-
though diversity was lower in the open sites (a factor
that was inuenced by peatland microtopography), the
richness was higher than at forested and forest-to-bog
ones (Creevy et al. 2018). Commercial forestry prac-
tices have caused the loss of mixotrophic taxa in the
forested sites. This result was related especially with
the light condition, as mixotrophic taxa persisted in un-
disturbed open bog because of their need for light to
survive. A. discoides was considered a potential indica-
tor in forested sites and it was recommended that future
sampling designs encompass microtopography (Creevy
et al. 2018).
Thecamoebians functional traits (FT) related to their
carapace were indicated as candidates for bioindica-
tion of woody debris impact on shaping the community
at a restored oodplain in Switzerland (Fournier et al.
2012). Taking into consideration the multiple environ-
mental pressures selecting testate amoeba’s FT, and the
potential of FT to complement classical indices, further
research regarding TA FT has been encouraged (Fourni-
er et al. 2015).
3.4. Agricultural practices
Soil quality is essential in the sustainable perfor-
mance of agricultural practices (Carter 2002); however,
the increased use of pesticides, herbicides and fertiliz-
ers have side eects on the environment (Nesbitt and
Adl 2014). Many protists are sensitive to pesticides and
other components commonly used in agricultural prac-
tices, and insecticides are more harmful to these organ-
isms than herbicides (Foissner 1994, 1997). A. vulgaris,
in presence of the fungicide Fundasol had the growth
stimulated, but some hours later all species in the study
were dead (Todorov and Golemansky 1992).
Testate amoebae in an Arctic Tundra had abundance
and biomass reduced by 77 and 84% with long-term ni-
trogen (N) and phosphorus (P) fertilization. Some spe-
cies vanished from the fertilized plots, while P. acropodia
increased (Mitchell 2004). These results were probably
related to the deterioration of the mosses caused by fer-
tilization and the availability of preys (Mitchell 2004).
Furthermore, when inhabiting moss fertilized with N,
Bullinaria indica showed greater abundance (Mitchell
and Gilbert 2004), but this response needs further inves-
tigation, as the species has already been related to the
lowest preferences of N at peat (Tolonen et al. 1992). In
a Sphagnum peatland N and P fertilization experiment,
testate amoeba’s richness and density decreased (Miec-
zan et al. 2015), which could be related to a decrease
in oxygen concentration. The testacean C. dubium pre-
sented lower abundance in a heathland when the sites
were treated with huge concentrations of nitrogen for
20 years (Payne et al. 2012). Although it may highlight
the species as a N bioindicator, it is discussed that the
response may have been indirect, more related to plant
community changes (Payne et al. 2012).
3.5. Fire events and volcanic activities
Fire events have already shown to impact testate
amoebae communities in moorlands, where, after char-
coal deposition, water inltration may be reduced and
drought and erosion increased (Turner and Swindles
2012). Hyalosphenia subava and T. arcula have already
been pointed out as potential burning indicators, being
related to the driest sites (Turner and Swindles 2012,
Turner et al. 2013). Although Qin et al (2017) regis-
tered a growth at thecamoebians with xenosomes af-

ter a wildre episode, in a multi-proxy study, TA with
idiosomes with organic coating became more abundant
after re (Marcisz et al. 2019). Also, re events caused
a selection of smaller TA species (Marcisz et al. 2016)
and a substantial decrease of mixotrophs (Marcisz et
al. 2016, Marcisz et al. 2019). The last, may be caused
by the water table depth lowering after the re, along-
side changes at the site’s vegetation and ash fallout
shade, that diminished the photosynthetic capacity of
the mixotrophs (Fournier et al. 2015, Lamentowicz et
al. 2015a, Marcisz et al. 2016). On the other hand, when
re occurs on the peatland’s surrounding vegetation, it
may increase water table depth, as the plants cease to
absorb water from the soil. These events can contribute
to a change in TA assemblages to wet indicator species
(Lamentowicz et al. 2007, Marcisz et al. 2015).
Deposition of sulphuric acid by anthropogenic or
volcanogenic activities have presented an impact to TA
communities (Payne 2010), such as deposition of sodi-
um sulphate (Payne et al. 2010). Although it is not clear
by which mechanisms these protozoans were aected,
they can be related to changes in the ecosystem’s troph-
ic web, with a sulphate-reducing bacteria (SRB) and
methanogenic archaea (MA) shift (Payne 2010, Payne
et al. 2010).
Thecamoebians are indicators of past and current
volcanic activity, in a way that could be used as prox-
ies of both frequency and extent of eruptions, dating
and correlating paleoenvironmental data. This is pos-
sible by the ability of TA to be one of the rst protists
in the succession to inhabit volcanic ash fallout (Smith
1985), incorporating particles with less than 125μm,
called cryptotephra, into they’re tests. This character-
istic facilitates the extraction and counting of these
minerals (Delaine et al. 2016). Moreover, the construc-
tion of thecamoebian’s carapace using foreign material
can turn them into indicators of microplastic pollution.
Thecamoebians from order Arcellinida were capable of
incorporating 4μm polystyrene spheres into their tests
under laboratory conditions (Bian et al. 2019). With mi-
croplastics being accumulated year after year in fresh-
water ecosystems to hundreds or thousands of particles
per litre (Li et al. 2020), it’s bioavailability may impact
the test construction.
3.6. Metal and semimetal contamination
In areas previously impacted by mining, protozoa
were the rst eukaryotic organisms to colonize the en-
vironment (Wanner and Dunger 2001). This way, TA
are aected and responsive to metals and semimetals,
pointing out contamination. When reclaiming an open-
cast coal mining in Germany, dierent assemblages of
testate amoebae demonstrated sensitivity to dierent
stages of soil recovery, relating to characteristics that
alter the quality of the substrate (Wanner and Dunger
2001). In lake’s sediments and water columns aected
by gold mining, the techniques implemented for meas-
uring arsenic are not ecient to point out ecological
impacts (Nasser et al. 2020). Notwithstanding, strong
correlations between thecamoebians and arsenic (As)
have already been pointed out (Nasser et al. 2016), with
this group being considered a good indicator of con-
tamination by As. When related to that, the least toler-
ant group was mainly represented by the genus Diugia
and by the species Cucurbitella tricuspis; and the greater
tolerance, represented by some species of Centropyxis
and Diugia elegans (Nasser et al. 2020). In addition,
these protists were also correlated with phosphorus (P),
barium (Ba), carbon (C) and calcium (Ca) (Nasser et
al. 2016).
The concentration of heavy metals in lakes were
positively correlated with the taxa Mediolus corona, Dif-
ugia bicornis, C. tricuspis and P. compressa, and nega-
tively with Diugia acuminata and L. modesta. However,
the research only points to the inuence of heavy metals
under the structure of the community (Qin et al. 2016).
The specie A. vulgaris was registered in great abundance
in lakes with high heavy metals concentrations and low
pH, which shows that this specie was tolerant to stress-
ing conditions, being able to present dierent altera-
tions in tropical environments (Patterson et al. 2013,
Kumar and Patterson 2000, Escobar et al. 2005). The
species Centropyxis aculeata, C. constricta and A. vulgaris
were shown to be tolerant in a lake heavily polluted by
mercury and arsenic (Patterson et al. 1996). Although
the genus Centropyxis and A. vulgaris showed anity
with metals, only A. vulgaris was considered a good
indicator, as it was not being favoured by the trophic
state and salinity of the lake. Moreover, a relationship
between mercury and arsenic levels with the distribu-
tion of Diugia proteiformis is notorious, being present
at highly contaminated sites (Patterson et al. 1996).
The presence of potentially toxic trace elements in a
reservoir substrate (As, Cr, Cu, Fe, Mn, Ni, Pb, Sc and
Zn) aected the abundance and diversity of the com-
munity of thecamoebians (Misailidis et al. 2018). Plac-
es with low rates of contamination presented a higher
number of species, and regions with aggregations of
roots and low concentrations of pollutants are dominat-
ed by Diugia corona, D. oblonga and D. gramen. As the

Testate amoebae 11
concentration increases, species that were more toler-
ant remain and dominate. They were: Arcella megastoma
and D. corona, in contrast with the most sensitive ones:
D. gramen, Lesquereusia globulosa and Netzelia tuberculata
(Misailidis et al. 2018). The identication to the lowest
taxonomic resolution leads to a conclusion less uncer-
tain and more clear of the biomonitoring properties of
testaceans, especially in dynamic habitats as reservoirs,
besides contributing with ecological information re-
garding genera and species (Vieira et al. 2017).
In a oodplain contaminated by heavy metals (Cu,
Pb, Zn, Co) in Japan, the diversity of TA was lower in
the most polluted places, where the concentrations of
Cu, Pb and Zn were higher (Wanner et al. 2020). Their
results showed in areas contaminated by heavy metals,
species such as Euglypha rotunda, Trinema enchelys and
Trinema lineare as indicators of the impact on the bio-
geochemical cycle in proterozoic silicon wells due to
the self-secrete composition of their test (Wanner et al.
2020).
C. arcelloides and Centropyxis cassis were abundant in
peatlands areas where copper (Cu) concentration was
high, suggesting that the genera could be tolerant to
high concentrations of this metal. However, the diver-
sity of TA in general was lower, pointing out Centropyxis
species’s tolerance as an exception. It is believed that
the high concentrations of metals, such as Cu, might
reduce the abundance of prey (e.g., algae and bacteria)
and only species tolerant to food shortage would sur-
vive (Asada and Warner 2009). In stream mosses, C.
aculeata, C. discoides, C. arcelloides, and Euglypha lae-
vis can be potentially used to measure Cu pollution in
water, while mercury (Hg) causes community’s abun-
dance to decline (Yang et al. 2011). Euglypha strigosa
was also pointed out as sensitive to Cu (Meyer et al.
2012). When the moss retains such pollutants, a dier-
ent picture was found at 63 Canadian lakes, where low
concentration of Hg didn’t have an impact on TA (Yang
et al. 2011, Neville et al. 2013).
Increasing concentration of lead (Pb) accumulated
in Barbula indica moss diminished thecamoebians spe-
cies richness, abundance and Shannon index. Pb signi-
cantly decreased abundance of some Euglypha, Trinema,
Centropyxis and Tracheleuglypha species, while Quadrule-
lla symmetrica and Euglypha diliociformis have suggested
resistance to lead (Nguyen-Viet et al. 2007). TA had
their richness, total density and total biomass decreased
as Pb concentration and time of exposition increased in
a controlled environment. N. carinata, E. strigosa, and H.
sphagni were the most sensitive taxa, while A. catinus,
A. discoides, A. militaris, C. dubium, T. lineare, and C. acu-
leata are the most resistant (Nguyen-Viet et al. 2008).
3.7. Atmospheric inuence and its pollution
Moss with inhabiting testate amoebas are good
bioindicators for atmospheric pollution since they are
in subaerial environments, directly exposed to atmos-
pheric pollutants (Nguyen-Viet et al. 2007). Parameters
as appearance/extinction of species or decrease in bio-
mass/abundance can be used to identify the atmospher-
ic condition (Meyer et al. 2012). The contamination
with nitrogen dioxide (NO2) can impact the richness,
abundance and biomass of the testate amoebae commu-
nity (Meyer et al. 2009, Meyer et al. 2012), with the
species richness being signicantly correlated with NO2
concentration (Nguyen-Viet et al. 2004). Changes in
interactions at microbial level, especially predation, en-
hanced this eect (Meyer et al. 2009). Paraquadrula ir-
regularis was pointed out as an indicator of atmospheric
pollution caused by this gas, what could be related to
acidication of the environment, and calcium leaching
of its calcareous test (Nguyen-Viet et al. 2004). N. tincta
and Trygonopyxis arculla were also sensitive to NO2, and
the community that lives in Pseudoscleropodium purum
moss can be used to dierentiate sources of pollution
(industrial or urban ones) (Meyer et al. 2012). In an
experiment with atmospheric carbon dioxide (CO2) in-
crease, Sphagnum TA decreased in biomass (Mitchell
et al. 2003). Moreover, road trac pollution was able
to cause a decrease in abundance and diversity of soil
TA (Balik 1991). The locations with the greatest im-
pact, also had a dierent composition. From a total of
42 recorded TA species, 16 were restricted to less pol-
luted locations (Balik 1991), showing their relationship
with atmospheric pollution (Nguyen-Viet et al. 2004).
Testate amoeba assemblages from a brown coal com-
bustion polluted peatland were impacted by it, with Al
and Cu being indicated as the most toxic elements to
them (Fiałkiewicz-Kozieł et al. 2015). The pollution
decreased mixotrophic and larger TA species, but an in-
crease in Centropyxis aerophila and Phryganella acropodia
species (Fiałkiewicz-Kozieł et al. 2015).
Environmental variations caused by climate change
such as increase in temperature and precipitation altera-
tions have an impact on peatland microbiota, and TA
are pointed out as candidates to monitor them (Jassey
et al. 2011b). At the Sphagnum upper segments, the-
camoebians biomass and density increased together
with temperature. Although more studies in this sub-
ject are necessary, in this condition they fed on an ex-

pressive range of prey and react to their changes (e.g.,
bacteria) (Jassey et al. 2011b). Temperature increase
altered the trophic web, as it has the Sphagnum poly-
phenols selecting dierently the microbial community
and shortening the food web. This led to a faster carbon
and nutrient recycling by decreasing predators and om-
nivores (including larger TA, with bigger pseudostome)
while increasing autotrophs and decomposers (Jassey
et al. 2013). H. papilio was an indicator of temperature
increase, and could be an important tool in monitoring
during the reconstruction of Sphagnum peatlands, since
it’s a mixotrophic thecamoebian inhabiting the top lay-
ers of Sphagnum (Basińska et al. 2020).
The species C. constricta, C. aculeata, Diugia urceo-
lata, D. oblonga and D. corona, presented high density
in core studies from three lakes in Atlantic Canada as
a result of an increase of the temperature (McCarthy
et al. 1995). In lakes of rocky mountain regions with
low temperatures, a low diversity of TA was registered,
with dominance of the taxa C. aculeata and C. constricta
(Neville et al. 2010).
3.8. Paleoenvironmental reconstruction
Fossil thecamoebian shells are very often well pre-
served and abundant in peatlands, making them special
as bioindicators when compared to conventional fos-
sil indicators (Warner and Charman 1994). Modern
and fossil assemblages of testate amoeba paired with
depth to water table analysis and radiocarbon-dating
were used to construct transfer functions, which pro-
vides palaeohydrological data when applied to the cor-
responding site (Warner and Charman 1994, Charman
et al. 2007). Those have been applied to infer centen-
nial scale climate patterns (Charman and Hendon 2000,
Hendon et al. 2001, Booth 2002), anthropic impacts
(Patterson et al. 2002, Ndayishimiye et al. 2020) and
sea level change (Barnett et al. 2017). Regional trans-
fer functions have already been developed throughout
Europe (Tolonen et al. 1992, 1994; Schnitchen et al.
2006; Swindles et al. 2015; Lamentowicz et al. 2020),
to Russia (Willis et al. 2015, Lamentowicz et al. 2015b,
Kurina and Li 2019), China (Qin et al. 2013, Li et al.
2015), New Zealand (Charman 1997), United States
and Canada (Booth 2001, 2008; Amesbury et al. 2013;
Lamarre et al. 2013), Peru (Swindles et al. 2014) and
Panama (Swindles et al. 2018).
Functional trait-based reconstructions have been
increasing recently, being conducted over several
landscapes (Marcisz et al. 2020), and provide reliable
analysis when there is no transfer function and inde-
pendently from taxonomic identication (Lamentow-
icz et al. 2015a, van Bellen et al. 2017, Krashevska et
al. 2020). By constructing statistical models based on
functional traits, identication errors and taxonomic
change inaccuracies are diminished, while there is a
bigger chance of comprehending community dynamics
and functional processes over long time-scales (Mar-
cisz et al. 2020). Also, a multi-proxy approach involv-
ing functional-traits could benet paleoenvironmental
reconstructions by correlating ecological processes to
functional roles (Lamentowicz et al. 2015a, van Bellen
et al. 2017, Galka et al. 2017, Marcisz et al. 2020, Kra-
shevska et al. 2020).
Water table depth (WTD) was the main variable
to which thecamoebians responded, along with point-
ing out microtopography dierentiation in peatlands
(Warner and Charman 1994, Krashevska et al. 2020).
TA species could indicate increase, instability, and low-
ering of the water table (even when it happens by sharp
declines of the WTD) (Galka et al. 2014, Galka et al.
2015). In these environments, moisture content has been
primarily taken into consideration (Booth 2002), but, as
it changes more often, even during a day, WTD is the
main variable observed (Charman and Warner 1997,
Warner et al. 2007). More reliable reconstructions can
be carried out during wetter conditions phases, as taxa
with dry optima can be wider distributed than those
with wet optima. During dry conditions phases, the ab-
sence of a wet indicator becomes more reliable than the
encounter of a dry one (Charman et al. 2007).
On the other hand, WTD inuence becomes second-
ary when most of the sample is collected from min-
erotrophic mires, and not ombrotrophic ones. In those
cases, the trophic state of a mire, that can be reected
by pH (Payne et al. 2006, Markel et al. 2010), conduc-
tivity and calcium content, will have a bigger impact
on the thecamoebians assemblages (Lamentowicz et al.
2013b). This way, when applied to WTD reconstruc-
tion through transfer functions, assemblages from om-
brotrophic and minerotrophic mires should be regarded
to the construction of dierent transfer functions to
dierent peatland development stages (Kurina et al.
2020). When constructing a transfer function to an om-
brotrophic peatland, the bog stage of its development
should be reconstructed with an ombrotrophic model,
and a minerotrophic model should t the earlier fen-
bog and fen transition stages (Kurina et al. 2020).
Thecamoebian’s assemblages are pointed out as
paleolimnological and paleoecological indicators at
lakes, being able to reect climate changes (McCa-

Testate amoebae 13
rthy et al. 1995). During the late glacial and Holocene,
ve assemblages identied the beginning of the epoch,
alongside with climate phenomena that palynology
alone could not indicate (McCarthy et al. 1995). These
protists could also indicate lake depth (Tsyganov et al.
2019), sedimentation rates and pH changes throughout
the Holocene (Ellison 1995). In a multi-proxy approach
study, a thecamoebians based WTD reconstruction
paired peaks in the bog surface water with periods with
lower sunspot activity (Turner et al. 2013). In an East
Siberian Arctic permafrost study, thecamoebians spe-
cies composition from the Late Pleistocene-Holocene
were used to identify dierences between both periods
and between their temperatures (Bobrov et al. 2004).
Regarding the diculty of separating climate
change from human impact; when testate amoebae are
integrated with micro and macrofossils, such as pol-
len, microcharcoal, local plant community, spores and
dendroecological analysis, in a multi-proxy approach,
it can be possible to separate these two factors in peat-
lands and lakes (Patterson et al. 2002, Lamentowicz et
al. 2008, Lamentowicz et al. 2015a, Galka et al. 2017,
Lamentowicz et al. 2019, Lamentowicz et al. 2020).
Moreover, TA showed to be very responsive to hydro-
logical uctuations, while macrofossils may be delayed
(Lamentowicz et al. 2008). At peatlands, TA assem-
blages structure dierentiated after land use change,
caused by deforestation and implementation of agricul-
ture (Marcisz et al. 2020). Because of changes in water
absorption, a bigger availability of water may increase
WTD. In this case, testate amoeba size and biovolume
(Marcisz et al. 2020), pseudostome size and position
are aected by WTD (Lamentowicz et al. 2015a), and
may there will be an increase in mixotrophs, if light
becomes more available (Galka et al. 2017, Marcisz et
al. 2020). Deforestation may also lead to terrestrializa-
tion, as it increases dust deposition and nutrient ow to
the peatland (Ireland and Booth 2012, Lamentowicz et
al. 2020); this way, a shift in the plant community can
also impact TA assemblages (Ireland and Booth 2012).
Furthermore, in a scenario of WTD lowering, after peat
extraction, TA size and mixothrops decreased (Marcisz
et al. 2016). Further, TA with smaller (Krashevska et
al. 2020) and more hidden pseudostome increased with
dryness (Marcisz et al. 2016). Thecamoebians core as-
semblages from Swan Lake (Canada), have been able
to show the dierent uses of the land around it (Pat-
terson et al. 2002). The dierent communities were
compared to palynological data, and reected the great
impact of deforestation for agriculture and human set-
tlement, even in underwater life. The Deforestation
Assemblage, presented dominance of stress indicator
taxa, such as C. aculeata and A. vulgaris (Patterson et
al. 2002). C. tricuspis, in contrast, appeared in the Eu-
trophication Assemblages, a period after World War II
with high use of chemical fertilizers (Patterson et al.
2002). Although erosion was also present in the latter
study, it’s combination with nutrient input at Lake Erie
(Canada) inferred a bigger impact in the community,
being indicated by Diugia biddens presence (Scott and
Medioli 1983). Soil erosion also played a key role mod-
elling thecamoebians assemblages in lake Lugu (China)
where three characteristic assemblages were identied
through 2500 years (Ndayishimiye et al. 2020). As-
semblages also shifted by change in nutrient input, by
forest growth and eutrophication inuenced by human
activities (Ndayishimiye et al. 2020). All those papers
point out that these protozoans are proxies for land use
changes.
A salt-marsh is a wet biotope located next to the
coast regions, ooded by salt waters by the high tide
(Viswanathan et al. 2020). Despite the known impor-
tance of these organisms, there is a scarcity of studies
that use this group as an indicator in the salt marsh bio-
tope (Barnett et al. 2017). A few species inhabit these
regions with strong zonation, from the high salt-marsh
environments transitioning into the supratidal zone
(Barnett et al. 2013, Barnett et al. 2017). In salt-marsh-
es, the TA were paleoenvironmental indicators related
to the reconstruction of the sea level and being used
to construct tendencies of it in the current geological
period (Barnett et al. 2015), being more precise in its
indication when compared to foraminifera (Gehrels
2000, Gehrels et al. 2006). Throughout the North At-
lantic, a thecamoebian’s assemblages-based transfer-
function could indicate, with great precision, sea level
uctuations, revealing information when other proxies
are missing. Moreover, when paired with foraminifera-
based data, the reconstruction capability extends back
in time (Barnett et al. 2017).
3.9. Sewage Treatment
The biological sewage treatment process is based
on the formation of suspended bacterial akes, which
have a diverse microfauna associated, being segregated
from the euent in the sedimentation tanks (Zhou et
al. 2006). The microbiota is sensitive to environmental
variations, so changes in the community are responsi-
ble for disassembling the trophic web, aecting the bio-
logical performance of the treatment station (Madoni

2003). The microfauna protists perform a vital func-
tion, mainly controlling microbial population density
through predation, assuring by that the quality of the
euent (Curds and Cockburn 1970).
Testate Amoebae of the genera Arcella, Diugia and
Euglypha were bioindicators (Madoni 1994, Nicolau et
al. 2005, Pérez-Uz et al. 2010, Hu et al. 2013) found
in greater abundance in older sludge plants, as their
growth was favoured (Madoni et al. 2000). In addition,
specic system characteristics, such as low loading,
long retention time and high aeration rate of the tank
inuence their abundances. These variables indicate
the euent quality and the high biological activity of
sludge plants (Chierici and Madoni 1991). Therefore,
TA can be indicators of the functioning quality of a
sewage treatment system. In treatment places, TA with
less than 20μm that were associated with ocs help-
ing to improve productivity of nitrifying bacteria that
lives inside those, since those little thecamoebians are
bacteria predators (Pérez-Uz et al. 2010). Furthermore,
this group of organisms responds to nitrication in the
treatment system, once its occurrence decreases with
the decrease of the removal of nitrogen, mostly in spe-
cies of the genera Arcella (Pérez-Uz et al. 2010). Species
that stood out in this biotope were Arcella hemisphaerica
and Euglypha tuberculata, that respond to the system ni-
trication (Madoni 1994, Hu et al. 2013). However, in
an activated sludge plant, it was shown that the rst
one was related to good settlement of sludge plants and
should be used as an indicator of the eectiveness of
the plant, because its abundance decreased when the
euent’s quality dropped (Zhou et al. 2006). Nowa-
days, the ecological importance that TA play in these
systems is known, and according to the advance of the
decades, studies have emphasized these organisms and
their bioindicator potential. However, studies on this is-
sue are still scarce.
3.10. Taxonomic and methodology issues
Biomonitoring using zooplankton has indicated
that it’s identication and analysis, including TA, can
be done to genera level (Machado et al. 2015, Souza
et al. 2019). Identication to the genera level has been
an economic strategy to overcome the lack of specic
taxonomists to certain groups. However, TA studies did
not obtain a high agreement between the data provided
by the level of genera and of species, which excludes
them from that strategy (Alarcão et al. 2014, Gomes et
al. 2015). The lack of specialists can be explained as a
result of a certain negligence with testate amoebae in
comparison to other members of zooplankton, mainly
regarding its lack of a specic methodology for data
collection and analysis (Leipnitz et al. 2006), as well
as a recognized relevance to biodiversity in biosystems
(Corliss 2002, Han et al. 2011).
Some concerns regarding methodology include
sampling. For instance, signicant dierences between
upper and lower Sphagnum thecamoebians, raise con-
cern about how deep the samplings should occur. As
it would change community composition, diversity
and dominance, providing an incomplete answer of
the site’s thecamoebians if not taken into consideration
(Booth 2002, Jassey et al. 2011a, Ireland and Booth
2012). Upper and lower portions shelter restricted taxa,
and lower assemblages have shown to have bigger
richness than shallower ones, as it accumulates tests
of thecamoebians from upper portions and from previ-
ous years (Booth 2002). Mixotrophs are dominant in
surface layers, while heterotrophic species are more
abundant in deeper layers, but the use of xenosomes to
build the carapace also plays a part in the vertical dis-
tribution, as Amphitrema wrightianum where more abun-
dant in intermediate segments of the Sphagnum rather
than in the top layers (Jassey et al. 2011a). Upper layers
of Sphagnum are more exposed to water table uctua-
tions, and may indicate it dierently than deeper layers
(Basińska et al. 2020). Although upper taxa would be
more representative of environmental variables at the
sample time, collecting all the vertical extension of tes-
tate amoeba distribution could be the best way to char-
acterize its relations with environment variables (Booth
2002, Ireland and Booth 2012).
4. CONCLUSION
Testate amoebae are reliable indicators when applied
to dierent uses in a diversity of biotopes. They can
be an important tool as biological indicators in various
conditions ranging from water, soil and atmospheric
pollution, to sea level uctuations, land use change and
paleoclimate reconstruction. A multi-proxy approach
and inclusion of functional trait-based analysis more
recently developed for the testaceans should contribute
to a crescent importance and extension of their use. It is
clear that, although testate amoebae’s use as indicators
has gained space through dierent elds, in all the areas
where applied, there is still room for growth.

Testate amoebae 15
Acknowledgments. The authors would like to thank the Under-
graduate Program in Biological Sciences of the Federal University
of the State of Rio de Janeiro – UNIRIO, (Brazil) for laboratory
facilities. The authors Beatriz Ramos, Gabriela Sampaio and Yemna
Silva were supported by UNIRIO scholarships.
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Received on 23rd March, 2021; revised on 21st September, 2021; ac-
cepted on 5th March, 2022