water
Article
Artemia spp. (Crustacea, Anostraca) in Crimea: New Molecular
Genetic Results and New Questions without Answers
Anastasia Lantushenko 1 , Yakov Meger 1 , Alexandr Gadzhi 1 , Elena Anufriieva 1,2, * and Nickolai Shadrin 1,2
1 Sevastopol State University, 33 Universitetskaya Street, 299053 Sevastopol, Russia
2 A.O. Kovalevsky Institute of Biology of the Southern Seas of RAS, 2 Nakhimov Ave.,
299011 Sevastopol, Russia
* Correspondence: ; Tel.: +7-8692-54-5550
Abstract: Many works have been devoted to the study of the molecular genetic diversity of Artemia
in different regions; however, there are regions such as Crimea, the largest peninsula in the Black
Sea, which has seen few studies. Artemia specimens from several Crimean hypersaline lakes were
analyzed using the mitochondrial marker cytochrome oxidase C (COI). The analyzed individuals
from bisexual populations formed clades with the species A. salina, A. urmiana, A. sinica, and A. monica
(=A. franciscana). A. sinica and A. monica had not been recorded in Crimea previously. In Lake
Adzhigol, the three species A. urmiana, A. sinica, and A. monica were found at the same time, which
has not been noted anywhere before. In the Crimean lakes, a total of 10 haplotypes were found,
six of them for the first time: Once for A. monica, once for A. sinica, and four for A. salina. Those
haplotypes may be regarded as endemic to Crimea. In the 1990s, experiments were carried out in
Lake Yanyshskoe using mainly purchased cysts of Artemia, so A. monica and A. sinica were introduced
into Crimea and could then have easily been spread by birds to other Crimean lakes.
Keywords: Artemia; cytochrome oxidase C (COI); haplotypes; hypersaline lakes; invasive species
Citation: Lantushenko, A.; Meger, Y.;
Gadzhi, A.; Anufriieva, E.; Shadrin,
N. Artemia spp. (Crustacea,
Anostraca) in Crimea: New 1. Introduction
Molecular Genetic Results and New
Other than in Antarctica, Artemia spp. are the most common and abundant animals
Questions without Answers. Water
in hypersaline waters worldwide [1,2]. They belong to Anostraca, the most primitive and
2022, 14, 2617. https://doi.org/
ancient group among living crustaceans and have one of the most advanced osmoregulation
10.3390/w14172617
systems among all animals, which allows them to exist in an extremely wide range of
Academic Editor: Jun Yang salinity [3,4]. Due to this, they play a key multidimensional role in most ecosystems of
Received: 10 July 2022
hypersaline waters of the planet [1,5–8]. The existence of several water bird species depends
Accepted: 23 August 2022
on the development of Artemia populations [9–11]. Artemia biomass and its cysts are of great
Published: 25 August 2022
commercial value [1,12]. These crustaceans are also considered convenient test objects in
ecotoxicology [13,14], as well as model species to study various issues in different branches
Publisher’s Note: MDPI stays neutral
of biology [3,4,15,16].
with regard to jurisdictional claims in
It is therefore not difficult to understand the existing theoretical and practical interest
published maps and institutional affil-
in the study of Artemia, including the study of its diversity and the factors that determine
iations.
it [5,6,8,17]. Recent studies show that, along with parthenogenetic populations, there
are five species of bisexual Artemia in the world: A. salina (Linnaeus, 1758), A. urmiana
Günther, 1890, A. monica Verrill, 1869 (=A. franciscana Kellogg, 1906), A. sinica Cai, 1989,
Copyright: © 2022 by the authors.
and A. persimilis Piccinelli and Prosdocimi, 1968. Regarding the species A. monica and
Licensee MDPI, Basel, Switzerland. A. franciscana, there is currently no consensus, as some researchers believe that both species
This article is an open access article are valid while others believe that this is one species.
distributed under the terms and Many works have been devoted to the study of the molecular genetic diversity of
conditions of the Creative Commons Artemia in different regions [5,18,19]; however, there are still some practically unexplored
Attribution (CC BY) license (https:// regions. One of these is Crimea, the largest peninsula in the Black Sea (27,000 km2 ). The
creativecommons.org/licenses/by/ existence of Artemia in Crimea, thanks to P. Pallas, was already determined in the 18th
4.0/). century [20]. In the 19th century, there were four different species described, including the
Water 2022, 14, 2617. https://doi.org/10.3390/w14172617 https://www.mdpi.com/journal/water
, Water 2022, 14, 2617 2 of 11
species A. salina, A. arietina Fischer, 1851, A. milhausenii Fischer de Waldheim, 1834, and
A. koeppeniana Fischer, 1851 [21,22]. A. arietina is now recognized as a variety of A. salina,
and A. milhausenii and A. koeppeniana are recognized as synonyms of A. urmiana [8].
In the second half of the 19th century, it was experimentally shown that salinity
causes a high level of Artemia morphological variability [23–25], and, proceeding from this,
all Artemia species in Crimea were reduced to one species, A. salina [26]. A revision of
the diversity of Artemia in Crimea using electron microscopy showed that bisexual brine
shrimp on the peninsula mainly belong to the species A. salina, but several males in Lake
Sasyk-Sivash belonged to another species [27]. Later, another species was found in Lake
Koyashskoe, identified by morphological characteristics as A. urmiana [28], which was
confirmed using molecular genetic methods [29]. Previously, based on morphological
similarity, it was suggested that A. mulhausinii corresponds to A. urmiana described from
Lake Urmia [30]. Crimea was regarded as unique due to having a relatively small territory,
and its hypersaline lakes host at least two bisexual Artemia species and their parthenogenetic
populations [30]. So, the conclusion was made that Crimea may be considered a remnant
of the center of the Artemia biodiversity origin near the ancient Tethys Ocean [30,31].
To date, it is known that Artemia populations exist in Crimea in more than fifty water
bodies, including Bay Sivash, the world’s largest Artemia habitat (2560 km2 ), which are
represented by two bisexual native species and parthenogenetic populations of different
ploidy [7,32–34]. Nevertheless, the existence of only one bisexual species, A. urmiana, was
confirmed using the molecular genetic approach [29].
The main objectives of this study are (1) to analyze Artemia specimens from different
lakes of Crimea using the mitochondrial marker cytochrome oxidase C (COI), and (2) to test
the hypotheses about the existence of at least two bisexual Artemia species on the peninsula
and the possibility of coexistence of two bisexual Artemia species in one water body.
2. Materials and Methods
On the Crimean Peninsula, there are many hypersaline water bodies (Figure 1), which
differ in size, ranges of salinity fluctuations, and biological diversity [35,36].
Figure 1. Distribution of the hypersaline lakes in Crimea, including the location of the studied lake.
((A) — the European scale, (B) — the Crimean scale).
All lakes are shallow (up to 1.5 m deep), polymyxic, and characterized by high seasonal
and long-term variability. In this study, 14 specimens of bisexual Artemia from three lakes
were analyzed, the general characteristics of which are given in Table 1. Sampling was
carried out by standard methods, by filtering water through a plankton net [34,35]. Live
crustaceans were delivered to the laboratory. Simultaneously with sampling in lakes, water
Article
Artemia spp. (Crustacea, Anostraca) in Crimea: New Molecular
Genetic Results and New Questions without Answers
Anastasia Lantushenko 1 , Yakov Meger 1 , Alexandr Gadzhi 1 , Elena Anufriieva 1,2, * and Nickolai Shadrin 1,2
1 Sevastopol State University, 33 Universitetskaya Street, 299053 Sevastopol, Russia
2 A.O. Kovalevsky Institute of Biology of the Southern Seas of RAS, 2 Nakhimov Ave.,
299011 Sevastopol, Russia
* Correspondence: ; Tel.: +7-8692-54-5550
Abstract: Many works have been devoted to the study of the molecular genetic diversity of Artemia
in different regions; however, there are regions such as Crimea, the largest peninsula in the Black
Sea, which has seen few studies. Artemia specimens from several Crimean hypersaline lakes were
analyzed using the mitochondrial marker cytochrome oxidase C (COI). The analyzed individuals
from bisexual populations formed clades with the species A. salina, A. urmiana, A. sinica, and A. monica
(=A. franciscana). A. sinica and A. monica had not been recorded in Crimea previously. In Lake
Adzhigol, the three species A. urmiana, A. sinica, and A. monica were found at the same time, which
has not been noted anywhere before. In the Crimean lakes, a total of 10 haplotypes were found,
six of them for the first time: Once for A. monica, once for A. sinica, and four for A. salina. Those
haplotypes may be regarded as endemic to Crimea. In the 1990s, experiments were carried out in
Lake Yanyshskoe using mainly purchased cysts of Artemia, so A. monica and A. sinica were introduced
into Crimea and could then have easily been spread by birds to other Crimean lakes.
Keywords: Artemia; cytochrome oxidase C (COI); haplotypes; hypersaline lakes; invasive species
Citation: Lantushenko, A.; Meger, Y.;
Gadzhi, A.; Anufriieva, E.; Shadrin,
N. Artemia spp. (Crustacea,
Anostraca) in Crimea: New 1. Introduction
Molecular Genetic Results and New
Other than in Antarctica, Artemia spp. are the most common and abundant animals
Questions without Answers. Water
in hypersaline waters worldwide [1,2]. They belong to Anostraca, the most primitive and
2022, 14, 2617. https://doi.org/
ancient group among living crustaceans and have one of the most advanced osmoregulation
10.3390/w14172617
systems among all animals, which allows them to exist in an extremely wide range of
Academic Editor: Jun Yang salinity [3,4]. Due to this, they play a key multidimensional role in most ecosystems of
Received: 10 July 2022
hypersaline waters of the planet [1,5–8]. The existence of several water bird species depends
Accepted: 23 August 2022
on the development of Artemia populations [9–11]. Artemia biomass and its cysts are of great
Published: 25 August 2022
commercial value [1,12]. These crustaceans are also considered convenient test objects in
ecotoxicology [13,14], as well as model species to study various issues in different branches
Publisher’s Note: MDPI stays neutral
of biology [3,4,15,16].
with regard to jurisdictional claims in
It is therefore not difficult to understand the existing theoretical and practical interest
published maps and institutional affil-
in the study of Artemia, including the study of its diversity and the factors that determine
iations.
it [5,6,8,17]. Recent studies show that, along with parthenogenetic populations, there
are five species of bisexual Artemia in the world: A. salina (Linnaeus, 1758), A. urmiana
Günther, 1890, A. monica Verrill, 1869 (=A. franciscana Kellogg, 1906), A. sinica Cai, 1989,
Copyright: © 2022 by the authors.
and A. persimilis Piccinelli and Prosdocimi, 1968. Regarding the species A. monica and
Licensee MDPI, Basel, Switzerland. A. franciscana, there is currently no consensus, as some researchers believe that both species
This article is an open access article are valid while others believe that this is one species.
distributed under the terms and Many works have been devoted to the study of the molecular genetic diversity of
conditions of the Creative Commons Artemia in different regions [5,18,19]; however, there are still some practically unexplored
Attribution (CC BY) license (https:// regions. One of these is Crimea, the largest peninsula in the Black Sea (27,000 km2 ). The
creativecommons.org/licenses/by/ existence of Artemia in Crimea, thanks to P. Pallas, was already determined in the 18th
4.0/). century [20]. In the 19th century, there were four different species described, including the
Water 2022, 14, 2617. https://doi.org/10.3390/w14172617 https://www.mdpi.com/journal/water
, Water 2022, 14, 2617 2 of 11
species A. salina, A. arietina Fischer, 1851, A. milhausenii Fischer de Waldheim, 1834, and
A. koeppeniana Fischer, 1851 [21,22]. A. arietina is now recognized as a variety of A. salina,
and A. milhausenii and A. koeppeniana are recognized as synonyms of A. urmiana [8].
In the second half of the 19th century, it was experimentally shown that salinity
causes a high level of Artemia morphological variability [23–25], and, proceeding from this,
all Artemia species in Crimea were reduced to one species, A. salina [26]. A revision of
the diversity of Artemia in Crimea using electron microscopy showed that bisexual brine
shrimp on the peninsula mainly belong to the species A. salina, but several males in Lake
Sasyk-Sivash belonged to another species [27]. Later, another species was found in Lake
Koyashskoe, identified by morphological characteristics as A. urmiana [28], which was
confirmed using molecular genetic methods [29]. Previously, based on morphological
similarity, it was suggested that A. mulhausinii corresponds to A. urmiana described from
Lake Urmia [30]. Crimea was regarded as unique due to having a relatively small territory,
and its hypersaline lakes host at least two bisexual Artemia species and their parthenogenetic
populations [30]. So, the conclusion was made that Crimea may be considered a remnant
of the center of the Artemia biodiversity origin near the ancient Tethys Ocean [30,31].
To date, it is known that Artemia populations exist in Crimea in more than fifty water
bodies, including Bay Sivash, the world’s largest Artemia habitat (2560 km2 ), which are
represented by two bisexual native species and parthenogenetic populations of different
ploidy [7,32–34]. Nevertheless, the existence of only one bisexual species, A. urmiana, was
confirmed using the molecular genetic approach [29].
The main objectives of this study are (1) to analyze Artemia specimens from different
lakes of Crimea using the mitochondrial marker cytochrome oxidase C (COI), and (2) to test
the hypotheses about the existence of at least two bisexual Artemia species on the peninsula
and the possibility of coexistence of two bisexual Artemia species in one water body.
2. Materials and Methods
On the Crimean Peninsula, there are many hypersaline water bodies (Figure 1), which
differ in size, ranges of salinity fluctuations, and biological diversity [35,36].
Figure 1. Distribution of the hypersaline lakes in Crimea, including the location of the studied lake.
((A) — the European scale, (B) — the Crimean scale).
All lakes are shallow (up to 1.5 m deep), polymyxic, and characterized by high seasonal
and long-term variability. In this study, 14 specimens of bisexual Artemia from three lakes
were analyzed, the general characteristics of which are given in Table 1. Sampling was
carried out by standard methods, by filtering water through a plankton net [34,35]. Live
crustaceans were delivered to the laboratory. Simultaneously with sampling in lakes, water
