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Citation: Klimov, P.B.; Kolesnikov,
V.B.; Khaustov, V.A.; Khaustov, A.A.
Thyreophagus tauricus sp. n., a New
Subcortical Mite Species (Acari:
Acaridae), with a COX1 DNA
Sequence Analysis of Several
Economically Important Species of
Thyreophagus.Animals 2023,13, 3546.
https://doi.org/10.3390/
ani13223546
Academic Editor: Michael E. Davis
Received: 16 October 2023
Revised: 2 November 2023
Accepted: 10 November 2023
Published: 16 November 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
animals
Article
Thyreophagus tauricus sp. n., a New Subcortical Mite Species
(Acari: Acaridae), with a COX1 DNA Sequence Analysis of
Several Economically Important Species of Thyreophagus †
Pavel B. Klimov 1, 2, *, Vasiliy B. Kolesnikov 1,3 , Vladimir A. Khaustov 1and Alexander A. Khaustov 1
1X-Bio Institute, Tyumen State University, 10 Semakova Str., 625003 Tyumen, Russia;
jukoman@yandex.ru (V.B.K.); kh4ustov93@yandex.ru (V.A.K.); alkhaustov@mail.ru (A.A.K.)
2Lilly Hall of Life Sciences, Purdue University, G-225, 915 W State St., West Lafayette, IN 47907, USA
3Federal Public Budgetary Scientific Institution, All-Russian Research Institute of Plant Protection,
396030 Voronezh, Russia
*Correspondence: pklimov@purdue.edu
†urn:lsid:zoobank.org:pub:37D3DEB8-69F6-4EC6-9BDB-338F4A987BB9.
Simple Summary:
In recent years, there has been a growing interest in finding sustainable and
environmentally friendly solutions to combat agricultural pests while minimizing the adverse impacts
of chemical pesticides. Species of the genus Thyreophagus have emerged as a valuable asset in this
pursuit. These mites are utilized as factitious prey for the mass rearing of predatory mites. Predatory
mites, in turn, play a crucial role in biological pest control—they feed on a variety of agricultural pests
such as spider mites, thrips and other small arthropods that harm crops. Despite their significance,
we do not know much about Thyreophagus mites because many species live in hidden habitats and
are difficult to study. As part of our survey, we discovered a new species, Thyreophagus tauricus, and
provide detailed descriptions of its different life stages. Genetic sequencing was also performed to
identify this new species and compare it with others: Thyreophagus corticalis (broadly distributed
Palearcic species), Th. calusorum,Th. entomophagus (economically important factitious prey mites). We
also correct some mistakes in mite identification, particularly the economically important species Th.
entomophagus, which can be crucial for future studies and biocontrol applications.
Abstract:
As part of a survey of the subcortical astigmatic mites of Crimea, we discovered a new
sexual acarid species, Thyreophagus tauricus sp. n. This species was cultured in the laboratory to
correlate the adult and deutonymphal stages. Using specimens obtained by these rearing experiments,
we provide a detailed description of Th. tauricus (light microscopy, SEM) based on females, males and
heteromorphic deutonymphs. Furthermore, to facilitate molecular identification, the entire COX1
gene was also sequenced for this and three other Palearctic species reared in the lab as pure cultures.
Adults of Th. tauricus sp. n. are distinct among all other species of the genus by the presence of
flattened, button-shaped or minute spiniform setae sIII and IV, which are well-developed spiniform
in all other known species of Thyreophagus. Heteromorphic deutonymphs of Th. tauricus are distinct
from all other species of Thyreophagus by the presence of well-developed setae cm on the dorsal part
of the subcapitular remnant (absent all other species). Th. tauricus is morphologically very similar to
Th. corticalis; however, COX1 K2P distances between these two species were large, 19.8%. COX1 K2P
distances between Th. tauricus and other species (Th. entomophagus,Th. “entomophagus” NC 066986.1,
Th. calusorum,Th. corticalis) ranged between 20.1 and 24.3%. We show that the GenBank sequence of
Th. “entomophagus” from China (NC 066986.1) was probably misidentified.
Keywords:
astigmatid mites; sexual species; morphology; molecular identification; Crimea; laboratory
culture
Animals 2023,13, 3546. https://doi.org/10.3390/ani13223546 https://www.mdpi.com/journal/animals
Animals 2023,13, 3546 2 of 26
1. Introduction
The genus Thyreophagus Rondani, 1874 (Acari: Acaridae) is distributed worldwide,
except Antarctica [
1
–
4
]. Various species of Thyreophagus occur in subcortical habitats, stored
food, in association with scale insects, and nests of wasps and bees [
1
–
8
]. Some Thyreopha-
gus species are beneficial or economically and medically important [
9
–
14
]. For example,
Thyreophagus entomophagus (Laboulbène and Robin, 1862) is widely used as factitious prey
for mass rearing of phytoseiid predatory mites for biocontrol applications [
9
]; additional
beneficial species have been recently identified, tested and used in industrial settings, such
as Thyreophagus calusorum Klimov, Demard, Stinson, Duarte, Wäckers et Vangansbeke,
2022 [
15
] and Thyreophagus cracentiseta Barbosa, OConnor and Moraes, 2016 [
7
]. In the
genus Thyreophagus, there are 34 nominal species and one subspecies; however, the actual
diversity of the genus Thyreophagus may be underappreciated, as most species prefer hidden
habitats, and can be easily overlooked [
1
]. Living in habitats that have a limited number
of natural predators is also a key biological feature that makes these mites well suited as
factitious prey in industrial settings. Mites of the genus Thyreophagus, known for their slow
movement and a lack of certain natural defenses (like long setae), are especially useful as
prey for mass produced phytoseiid mites.
Most commonly, mite taxonomists collect and describe females, while other taxonomi-
cally important stages, males (absent in asexual species) and heteromorphic deutonymphs
are omitted. These ontogenetic stages can be obtained through rearing in the lab, but this
is rarely performed. As a result, only four species are currently recognizable from both
adult and deutonymphal stages: Th. australis Clark, 2009, Th. corticalis (Michael, 1885), Th.
entomophagus and Th. calusorum [1,16–18].
Because of its economic importance and the presence of potentially interesting bio-
logical features related to asexuality, a comprehensive study of the Thyreophagus species
morphological and molecular diversity is needed. Such a study should be based on different
stages (females, males, heteromorphic deutonymphs) obtained through rearing experi-
ments of pure cultures and/or through the correlation of ontogenetic stages in the wild
populations. Rearing in the lab is also important to confirm if a species is indeed asexual.
As part of a survey of the subcortical astigmatic mites, we discovered a new sexual
species, Thyreophagus tauricus sp. n., from Crimea. This new species was cultured in the
laboratory to correlate the adult and deutonymphal stages. Based on specimens obtained by
these rearing experiments, we provide a detailed description (light microscopy, SEM) based
on females, males and heteromorphic deutonymphs. To facilitate molecular identification
and species delimitation, we sequenced the entire COX1 gene (a DNA barcoding gene) of
four Palearctic species, including Th. tauricus its closely related species, Th. corticalis. We
used these and GenBank sequence data to compare the genetic distances among different
species of Thyreophagus.
2. Materials and Methods
Fallen twigs of different species of deciduous trees were collected, transferred into a
laboratory and examined for the mites under a dissecting microscope. Mites were collected
using a camel brush and preserved in 96% ethanol, cleared in lactic acid 80% for 1–2 days
and mounted in Hoyer’s medium, followed by 7-day drying at 60 ◦C.
For rearing in the lab, live mite specimens were transferred into rearing units and
maintained on a mixture of yeast and bran as a food source. The purity of a culture was
confirmed via morphological identification of a large of number of mites (n = 50) harvested
from the same culture.
Cultures were established for following species: Thyreophagus calusorum—USA: Florida,
Fort Pierce, branch on ground, stick2, 12 October 2020, Emilie Demard, 27
◦
25
0
34.5
00
N
80
◦
24
0
22.7
00
W, PBK 20-0101-007); Thyreophagus entomophagus—Russia: Tyumenskaya oblast’,
Tyumen, culture from a Russian biocontrol company, 20 November 2021, Vladimir Khaustov,
PBK 20-0101-199; Thyreophagus corticalis—Russia: Voronezhskaya oblast’, Voronezh, mixed
Animals 2023,13, 3546 3 of 26
forest, under bark of Acer platanoides, 14 November 2021, Vasiliy Kolesnikov, PBK 20-0101-
061; Thyreophagus tauricus sp. n. (see below).
Images were taken from multiple focal planes and assembled in Helicon Focus 7.6.4
Pro (algorithm B, rarely A) with subsequent manual editing (retouching) of misassembled
regions. Individual, partially overlapping images were merged into a full panorama in
Adobe Photoshop 22.2.0. Line drawings were made in Photoshop 22.2.0 using micropho-
tographs as the background. Background images were taken using a Euromex Color
HD-Ultra camera and a Bioptic C-400 (Bioptic, Moscow, Russian Federation) microscope
equipped with bright field and differential interference contrast optics (DIC). Publication-
quality microphotographs were taken using an Axio Imager A2 (Carl Zeiss, Oberkochen,
Germany) compound microscope equipped with DIC and phase contrast optics and an Ax-
iocam 506 color (Carl Zeiss, Oberkochen, Germany) digital camera. For scanning electron
microscope imaging, alcohol-preserved mites were dried in a JFD 320 freeze dryer (JEOL,
Tokyo, Japan), dusted with gold, and scanned using a JEOL-JSM-6510LV SEM microscope.
Specimens used for SEM were not preserved.
In descriptions, idiosomal chaetotaxy follows [
19
]; the terminology of coxisternal
setae follows [
20
]; for appendages, the chaetotaxy and solenidiotaxy follow Grandjean for
palps [
21
] and legs [
22
]. Designations of tarsal dorsoapical setae of legs III–IV follow [
8
].
All measurements are given in micrometers (µm).
For each cultured species, genomic DNA was extracted from 200 females obtained
from a pure culture (see above), using a QIAamp DNA Micro kit (Qiagen, Venlo, The
Netherlands) with modifications as described here [
23
]. Illumina sequencing libraries were
generated and sequenced commercially on an Illumina NovaSeq 6000 sequencing system.
Short Illumina reads were assembled in SPAdes v.3.15.5 [
24
] as follows: metaspades.py
-t 24 -m 240 -1 ${name}_R1_001.fastq -2 ${name}_R2_001.fastq -o ${name}. Full-length
COX1 sequences were found using a local NCBI BLAST search and then deposited into the
GenBank database, accession IDs: OR640973-OR640976. Genetic distances were calculated
in PAUP v.4a168 [
25
] as follows: begin paup; dset distance = p; savedist format = tabtext
undefined = asterisk file = 1_p_distances.tab; dset distance = k2p; savedist format = tabtext
undefined = asterisk file = 2_k2p_distances.tab; end.
3. Results
3.1. Molecular Identification
For the COX1 gene of Th. tauricus, the top blastx hit (translated nucleotide to protein
analysis, genetic code = invertebrate mitochondrial) was Thyreophagus entomophagus from
China (NC_066986.1), with a 90.31% amino acid sequence similarity. Our sequence of
Th. tauricus was therefore classified in the genus Thyreophagus correctly. However, the Th.
entomophagus GenBank entry did not match our sequence of Th. entomophagus, with K2P
COX1 nucleotide distance = 0.212 (21.2%) (Table 1). Since our sequence was obtained from
specimens from a pure culture and given the careful identification of our morphological
co-vouchers (Fain, 1982), we believe that the GenBank sequence of Th. entomophagus
(NC_066986.1) may be misidentified.
Table 1.
COX1 nucleotide distances of four species of the genus Thyreophagus. Uncorrected p-distances
are in the upper diagonal, K2P distances are in the lower diagonal.
Th. “entomophagus”Th. calusorum Th. entomophagus Th. corticalis Th. tauricus
Th. “entomophagus” China
NC 066986.1 - 0.1686 0.1834 0.1699 0.2059
Th. calusorum OR640973 0.1929 - 0.1705 0.1705 0.1905
Th. entomophagus OR640974 0.2116 0.1942 - 0.1538 0.1750
Th. corticalis OR640975 0.1934 0.1984 0.1732 - 0.1725
Th. tauricus OR640976 0.2428 0.2216 0.2008 0.1980 -
Animals 2023,13, 3546 4 of 26
Given our dataset, Th. tauricus has the closets match to Th. corticalis, COX1 K2P
nucleotide distance = 0.198 (19.8%) vs. 0.201–0.243 for other species (Table 1). This re-
sult makes sense because these two species have only minor morphological differences
(see below).
3.2. Morphological Description
3.2.1. Genus Thyreophagus Rondani, 1874
Thyreophagus Rondani, 1874: 67 (=Moneziella Berlese, 1897; Monetiella Berlese, 1897;
Monieziella Berlese, 1897; Fumouzea Zachvatkin, 1953; Michaelopus Fain and Johnston, 1974).
Type species Thyreophagus entomophagus (Laboulbène and Robin, 1862) (=Acarus ento-
mophaus Laboulbène, 1852 (nomen nudum)), by monotypy.
3.2.2. Thyreophagus tauricus sp. n.
Description
Female (Figures 1–6,7A–C, 8and 9). Idiosoma elongate, 500
×
280 (holotype),
400–530 ×190–270
(paratypes, n = 9), 1.8 (2.0–2.1) times longer than wide. Idiosomal
cuticle smooth. Subcapitular setae (h) long, widened basally; palp tibial setae (a), lateral
dorsal palp tibial setae (sup), dorsal palp tarsal seta (cm) filiform; supracoxal seta elcp
present; terminal palp tarsal solenidion
ω
short; external part of terminal eupathidium ul”
dome-shaped; terminal eupathidium ul’ small, rounded. Prodorsal sclerite 100 (84–102)
long, 97 (75–90) wide, 1.0 (1.1) times longer than wide, with setae vi (situated at anterior
part of shield, alveoli separated), rounded anterolateral incisions, and elongate midlateral
incisions (insertion points of setae ve). Prodorsal sclerite smoothly punctate except large
lineate central region; posterior end of sclerite with lineate pattern. Grandjean’s organ
(GO) with seven membranous finger-shaped processes. Supracoxal setae (scx) smooth,
sword-shaped, widened and flattened, tapering at tip. Idiosomal setae (vi,se,c
p
,d
2
,e
2
,
h
1
,h
2
,h
3
,ps
3
) smooth, filiform and short; opisthosomal gland openings slightly anteriad
setal bases e
2
. Three pairs of fundamental cupules (ia,im and ih) present, ip not observed.
Ventral idiosoma with four pairs of coxal setae (1a,3a,4a and 4b) and 1 pair of genital setae
(g). Shape of coxal sclerites as in Figures 1B and 4E,F. Genital region situated between
coxal fields III and IV; genital valves form an inverted Y; epigynal and medial apodemes
well-developed. Diameter of genital papillae approximately 0.3–0.4 the length of coxal and
genital setae. Anal opening terminal. Copulatory tube present, situated anterodorsally
to anus, with developed opening. Canal of spermatheca long, slender tube-like, leading
from copulatory opening to spermatheca, uniformly wide, wider at entrance to spermath-
eca. Sclerotized vase-shaped atrium of spermatheca with length greater than width, base
3–4 times
wider than end of atrium at junction with sclerites of oviducts. Paired Y-shaped
sclerites of oviducts, small, elongated.
Legs short, all segments free. Trochanters I–III each with long, filiform seta, pR I–II,
sR III; trochanter IV without setae. Femoral setation 1-1-0-1; setae vF I–II and wF IV long,
filiform. Genual setation 2-2-0-0; setae mG and cG I–II long, filiform; seta nG III absent.
Tibial setation 2-2-1-1; setae hT I-II represented by alveoli or minute setae; setae gT I–II and
kT III–IV elongate, somewhat spiniform. Tarsal setation 10-10-10-10; pretarsi consists of
hooked empodial claws attached to short paired condylophores. Tarsus I and II with setae
ra,la,fand dfiliform, e,u,vspiniform, pand qrepresented by small triangular rudiments,
sflattened, button-shaped or minute spiniform; setae wa absent. Tarsus III with setae f,d,r
filiform, e,u,v,p,qspiniform, sflattened, button-shaped or minute spiniform, wflattened,
button-shaped. Tarsus IV similar to tarsus III, except wfiliform. Solenidion
ω1
on tarsus I
cylindrical, with clavate apex, curved; solenidion
ω1
on tarsus II simple, cylindrical, with
clavate apex, not bent, shorter and wider than
ω1
on tarsus I. Solenidion
ω2
on tarsus I
shorter than
ω1
, cylindrical, with rounded apex, slightly widened distally, situated slightly
distal to
ω1
. Solenidion
ω3
on tarsus I cylindrical, with rounded tip, subequal to
ω1
, longer
than
ω2
. Famulus (
ε
) of tarsus I wide, spiniform, with broadly rounded apex, widest
at middle. Solenidia
ϕ
of tibiae I–III elongate, tapering, well extending beyond apices
Animals 2023,13, 3546 5 of 26
of respective tarsi with ambulacra; solenidion
ϕ
IV shorter, shorter than tarsus IV (with
ambulacra). Solenidia
σ0
and
σ00
on genu I elongate, tapering, subequal in length, slightly
not reaching bases of
ϕ
I. Solenidion
σ
on genu II more than 6–7 times longer than its
width) with rounded tip. Solenidion σof genu III absent.
Male (n = 4, paratypes). (Figures 7D–F and 10–15). Idiosoma elongate,
300–400 ×160–210
,
1.9–2.1 times longer than wide. Idiosomal cuticle smooth. Gnathosoma as in female.
Prodorsal sclerite 63–78 long, 58–73 wide, 1.1–1.2 times longer than wide, with setae vi,
incisions and ornamented as in female. Grandjean’s organ (GO) with
5–7 membranous
processes. Supracoxal seta (scx) as in female. Idiosomal setae (vi,se,c
p
,d
2
,e
2
,h
1
,h
2
,h
3
)
smooth, filiform and short; opisthosomal gland openings slightly anteriad setal bases e
2
.
Three pairs of fundamental cupules (ia,im and ih) present, ip not observed. Opisthonotal
shield smoothly punctate; ventral part extends to anal suckers. Ventral idiosoma with
four pairs of coxal setae (1a,3a,4a and 4b) and one pair of genital setae (g). Shape of coxal
sclerites on Figures 10B and 13E,F. Genital region between coxisternal fields IV; arms of
genital capsule rounded; aedeagus short, not protruding beyond anterior edge of genital
capsule. Diameter of genital papillae approximately 0.3–0.4 the length of coxal and genital
setae. Anal suckers rounded in outline. Setae ps1–3 very short.
Animals 2023, 13, x FOR PEER REVIEW 5 of 31
Figure 1. Thyreophagus tauricus sp. n., female, holotype: (A)—dorsal view; (B)—ventral view. Scale
bar 100 µm.
Figure 1.
Thyreophagus tauricus sp. n., female, holotype: (
A
)—dorsal view; (
B
)—ventral view. Scale
bar 100 µm.
Animals 2023,13, 3546 6 of 26
Animals 2023, 13, x FOR PEER REVIEW 6 of 31
Figure 2. Thyreophagus tauricus sp. n., female, paratype: (A)—right leg I, posterior view; (B)—tarsus
I, anterior view; (C)—right leg II, posterior view; (D)—tarsus II, anterior view; (E)—right leg III,
anterior view; (F)—tarsus III, posterior view; (G)—right leg IV, anterior view; (H)—tarsus IV, pos-
terior view; (I)—supracoxal sclerite and Grandjeans organ; (J)—spermatheca. Scale bar 100 µm.
Figure 2.
Thyreophagus tauricus sp. n., female, paratype: (
A
)—right leg I, posterior view; (
B
)—tarsus I,
anterior view; (
C
)—right leg II, posterior view; (
D
)—tarsus II, anterior view; (
E
)—right leg III, anterior
view; (
F
)—tarsus III, posterior view; (
G
)—right leg IV, anterior view; (
H
)—tarsus IV, posterior view;
(I)—supracoxal sclerite and Grandjean’s organ; (J)—spermatheca. Scale bar 100 µm.
Animals 2023,13, 3546 7 of 26
Animals 2023, 13, x FOR PEER REVIEW 7 of 31
Figure 3. Thyreophagus tauricus sp. n., female, paratypes, DIC images: (A)—dorsal views; (B)—ven-
tral views. Scale bar 100 µm.
Figure 3.
Thyreophagus tauricus sp. n., female, paratypes, DIC images: (
A
)—dorsal views; (
B
)—ventral
views. Scale bar 100 µm.
Legs I-III as female, except solenidion
ω3
on tarsus I very short, truncated; and
solenidion
σ00
about two-times longer than
σ0
. Trochanter and genu IV without setae, femur
IV with wF IV long, filiform, tibia IV with kT IV elongate, somewhat spiniform. Tarsus IV
with 10 setae, of them, f,r,wfiliform, dand erepresented by suckers, u,v,p,qspiniform, s
flattened, button-shaped or minute spiniform. Solenidion ϕon tibia IV short and wide.
Phoretic deutonymph (n = 4, paratypes) (Figures 16–21). Body elongate, 1.33–1.44
times longer than wide, widest in sejugal region; idiosomal length 220–240 width 153–180.
Gnathosoma short, subcapitulum and palps fused, bearing palpal solenidia (
ω
) apically
and filiform apicodorsal setae (sup); setae hpresent, minute (Figure 20C) or absent (their
positions marked by somewhat refractile spots), setae cm present.
Dorsum. Idiosoma smoothly punctate; distinct linear pattern present on anterior and
lateral sides of prodorsal sclerite and hysterosomal shield. Apex of propodosoma anterior to
anterior border of prodorsal sclerite, with apical internal vertical setae (vi) (bases separated)
and a pair of band-like sclerites coalescing anteriorly. A pair of lateral, widely separated
ocelli (distance 40–49) present on prodorsum; lenses and pigmented spots present; maximal
diameter of lenses 18–20. External vertical setae (ve) absent; external scapular setae se
situated below lenses; internal scapular setae (si) distinctly posterior and medial to external
scapulars (se). Supracoxal setae of legs I (scx) filiform, with extended base, positioned below
si and anterolaterad to ocelli. Sejugal furrow well developed. Prodorsal sclerite 67–73,
hysterosomal shield 150–160, ratio hysterosoma shield/prodorsal sclerite
length = 2.2–2.3
.
Hysterosoma with 11 pairs of simple, filiform setae on hysterosomal shield (c
1
,c
2
,c
p
,d
1
,d
2
,
e
1
,e
2
,f
2
,h
1
,h
2
,h
3
), setae h
3
distinctly longer than others. Opisthonotal gland openings (gla)
situated ventrally on hysterosomal shield, slightly posterior to setae c
3
. Of four fundamental
Animals 2023,13, 3546 8 of 26
pairs of cupules, three pairs were observed: ia posteriomediad setae c
2
,im ventral, laterad
of trochanters IV and ih ventral, laterad posterior sides of attachment organ.
Animals 2023, 13, x FOR PEER REVIEW 8 of 31
Figure 4. Thyreophagus tauricus sp. n., female, paratypes, DIC images: (A,B)—prodorsal shields;
(C,D)—gnathosoma, ventral views; (E)—coxisternal fields I–II; (F)—coxisternal fields III–IV. Scale
bar 20 µm.
Figure 4.
Thyreophagus tauricus sp. n., female, paratypes, DIC images: (
A
,
B
)—prodorsal shields;
(
C
,
D
)—gnathosoma, ventral views; (
E
)—coxisternal fields I–II; (
F
)—coxisternal fields III–IV. Scale bar
20 µm.
Animals 2023,13, 3546 9 of 26
Animals 2023, 13, x FOR PEER REVIEW 9 of 31
Figure 5. Thyreophagus tauricus sp. n., female, paratypes, DIC images: (A)—left leg I, posterior view;
(B)—left leg I, anterior view; (C)—left leg II, posterior view; (D)—left leg II, anterior view; (E)—left
leg III, anterior view; (F)—left leg III, posterior view; (G)—left leg IV, posterior view; (H)—left leg
IV, anterior view. Scale bar 20 µm.
Figure 5.
Thyreophagus tauricus sp. n., female, paratypes, DIC images: (
A
)—left leg I, posterior view;
(
B
)—left leg I, anterior view; (
C
)—left leg II, posterior view; (
D
)—left leg II, anterior view; (
E
)—left
leg III, anterior view; (
F
)—left leg III, posterior view; (
G
)—left leg IV, posterior view; (
H
)—left leg IV,
anterior view. Scale bar 20 µm.
Animals 2023,13, 3546 10 of 26
Animals 2023, 13, x FOR PEER REVIEW 10 of 31
Figure 6. Thyreophagus tauricus sp. n., female, paratypes, DIC images: (A–D)—spermatheca. Scale
bar 20 µm.
Figure 6.
Thyreophagus tauricus sp. n., female, paratypes, DIC images: (
A
–
D
)—spermatheca. Scale
bar 20 µm.
Venter. Coxal fields sclerotized, smoothly punctate. Anterior apodemes of coxal fields I
fused forming sternum; sternum not reaching posterior border of sternal shield by distance
exceeding its length. Posterior border of sternal shield not sclerotized. Anterior apodemes
of coxal fields II curved medially. Posterior apodemes of coxal fields II weakly developed,
thin, curved medially. Sternal and ventral shield contiguous. Anterior apodemes of
coxisternal fields III free, connected by thin transverse sclerotization. Posterior medial
apodeme in area of coxisternal fields IV weakly developed. Posterior apodemes IV absent.
Subhumeral setae (c
3
) filiform, situated on ventral surface between legs II–III, adjacent to
region separating sternal and ventral shields. Coxal setae 1a,3a reduced, represented by
minute structures or filiform, situated in alveoli (Figure 20G). Setae 4b,gfiliform; 4a in
form of small, rounded conoids. Genital region in posterior portion of coxisternal fields IV;
opening elongate, with two pairs of genital papillae within genital atrium; papillae two-
segmented, with rounded apices. Coxal setae (4b) situated at tips anterior coxal apodemes
IV; genital setae (g) laterad of genital opening. Attachment organ posterior to coxisternal
fields IV. Anterior suckers (ad
3
) round, median suckers (ad
1+2
) distinctly larger, with paired
vestigial alveoli (not situated on common sclerite); pair of small refractile spots anterolateral
to median suckers (ps
3
); lateral conoidal setae of attachment organ (ps
2
) situated distinctly
posterior to line joining centers of median suckers, slightly anterior to conoidal setae (ps
1
);
anterior and posterior lateral and posterior median cuticular conoids well developed; anus
situated between anterior suckers (ad3).
Animals 2023,13, 3546 11 of 26
Animals 2023, 13, x FOR PEER REVIEW 11 of 31
Figure 7. Thyreophagus tauricus sp. n., SEM images: (A)—female, lateral view; (B)—female, dorsal
view; (C)—female, ventral view; (D)—male, dorsal view; (E)—male, ventral view; (F)—male, lateral
view.
Figure 7.
Thyreophagus tauricus sp. n., SEM images: (
A
)—female, lateral view; (
B
)—female, dor-
sal view; (
C
)—female, ventral view; (
D
)—male, dorsal view; (
E
)—male, ventral view; (
F
)—male,
lateral view.
Animals 2023,13, 3546 12 of 26
Animals 2023, 13, x FOR PEER REVIEW 12 of 31
Figure 8. Thyreophagus tauricus sp. n., female, SEM images: (A)—prodorsal shield, dorsal view; (B)—
genital area and legs III–IV, ventral view; (C,E)—gnathosoma and legs I, lateral views; (D)—anus,
ventral view; (F)—gnathosoma and legs I, ventral view.
Figure 8.
Thyreophagus tauricus sp. n., female, SEM images: (
A
)—prodorsal shield, dorsal
view;
(B)—genital
area and legs III–IV, ventral view; (
C
,
E
)—gnathosoma and legs I, lateral views;
(D)—anus, ventral view; (F)—gnathosoma and legs I, ventral view.
Animals 2023,13, 3546 13 of 26
Animals 2023, 13, x FOR PEER REVIEW 13 of 31
Figure 9. Thyreophagus tauricus sp. n., female, SEM images: (A)—Grandjeans organ; (B)—supracoxal
seta; (C)—copulatory tube; (D)—tarsus III, posterior view; (E)—tarsus II, posterior view; (F)—tarsus
II, anterior view; (G)—solenidion σ I; (H)—tarsus I, posterior view.
Figure 9.
Thyreophagus tauricus sp. n., female, SEM images: (
A
)—Grandjean’s organ; (
B
)—supracoxal
seta; (
C
)—copulatory tube; (
D
)—tarsus III, posterior view; (
E
)—tarsus II, posterior view; (
F
)—tarsus
II, anterior view; (G)—solenidion σI; (H)—tarsus I, posterior view.
Animals 2023,13, 3546 14 of 26
Animals 2023, 13, x FOR PEER REVIEW 15 of 31
Figure 10. Thyreophagus tauricus sp. n., male, paratype: (A)—dorsal view; (B)—ventral view. Scale
bar 100 µm.
Figure 10.
Thyreophagus tauricus sp. n., male, paratype: (
A
)—dorsal view; (
B
)—ventral view. Scale
bar 100 µm.
Legs. Legs elongate, all segments free. Trochanters I–III each with long, filiform
seta, pR I–II, sR III. Femoral setation 1-1-0-1; setae vF I–II and wF IV long, filiform. Genual
setation 2-2-0-0; setae mG and cG I–II filiform, seta nG III absent. Tibial setation 2-2-1-1; setae
hT I somewhat spiniform; setae gT I filiform; setae gT and hT II spiniform, setae gT II longer
than hT II; setae kT III spiniform and with distinct prong; setae kT IV somewhat spiniform,
shorter than kT III, with short prong. Tarsal setation 8-9-8-8. All pretarsi consisting of
hooked empodial claws attached to short paired condylophores. Tarsus I with setae ra,
la,p,q,d,fnarrowly lineolate; eslightly spoon-shaped; seta delongate, its base at level
of ra and la; seta srepresented by alveolus; setae wa,aa and ba I absent; tarsus II similar
to tarsus I except seta ba present, filiform, situated close to
ω1
. Tarsus III with setae w,r,
s,p,q,e,fand dsmooth; all setae, except for dIII, more or less foliate; seta dlonger than
leg. Tarsus IV similar to tarsus III, except seta rlonger, filiform; seta wfiliform and with
distinct prong. Solenidia
ω1
on tarsi I–II cylindrical, with slightly clavate apices;
ω3
on
tarsus I slightly shorter than
ω1
, with rounded apex, situated slightly distal to
ω1
;
ω1
and
ω3
separated by bulbous famulus (
ε
); solenidion
ω2
of tarsus I expanding slightly apically,
situated somewhat more basal and posterior to
ω1
+
ε
+
ω3
group; solenidia
ϕ
of tibiae
I–III elongate, tapering;
ϕ
I longer than tarsus I;
ϕ
II shorter than tarsus II;
ϕ
III reaching
tip of tarsus III without ambulacrum;
ϕ
IV short;
σ
of genu I elongate, slightly tapering,
nearly reaching tip of tibia I;
σ
of genu II much shorter, cylindrical, not reaching midlength
of tibia II; σof genu III absent.
Animals 2023,13, 3546 15 of 26
Animals 2023, 13, x FOR PEER REVIEW 16 of 31
Figure 11. Thyreophagus tauricus sp. n., male, paratype: (A)—right leg I, dorsal view; (B)—tarsus I,
ventral view; (C)—left leg IV, anterior view; (D)—tarsus IV, posterior view; (E)—genitalia. Scale bar
100 µm.
Figure 11.
Thyreophagus tauricus sp. n., male, paratype: (
A
)—right leg I, dorsal view; (
B
)—tarsus I, ventral
view; (C)—left leg IV, anterior view; (D)—tarsus IV, posterior view; (E)—genitalia. Scale bar 100 µm.
Animals 2023, 13, x FOR PEER REVIEW 17 of 31
Figure 12. Thyreophagus tauricus sp. n., male, paratypes, DIC images: (A)—dorsal views, (B)—ventral
views. Scale bar 100 µm.
Figure 12.
Thyreophagus tauricus sp. n., male, paratypes, DIC images: (
A
)—dorsal views, (
B
)—ventral
views. Scale bar 100 µm.
Animals 2023,13, 3546 16 of 26
Animals 2023, 13, x FOR PEER REVIEW 18 of 31
Figure 13. Thyreophagus tauricus sp. n., male, paratypes, DIC images: (A)—prodorsal shield; (B)—
anal region, ventral view; (C)—opisthonotal shield, dorsal view; (D)—genital region, ventral view;
(E)—coxisternal fields I–II; (F)—coxisternal fields III–IV. Scale bar 20 µm.
Figure 13.
Thyreophagus tauricus sp. n., male, paratypes, DIC images: (
A
)—prodorsal shield;
(B)—anal
region, ventral view; (
C
)—opisthonotal shield, dorsal view; (
D
)—genital region, ventral view;
(E)—coxisternal fields I–II; (F)—coxisternal fields III–IV. Scale bar 20 µm.
Animals 2023,13, 3546 17 of 26
Animals 2023, 13, x. hps://doi.org/10.3390/xxxxx www.mdpi.com/journal/animals
Figure 14. Thyreophagus tauricus sp. n., male, paratypes, DIC images: (A)—left leg I, anterior view;
(B)—left leg I, posterior view; (C)—left leg II, anterior view; (D)—left leg II, posterior view; (E)—
left legs III–IV, dorsal view; (F)—left legs III–IV, ventral view. Scale bar 20 µm.
Figure 14.
Thyreophagus tauricus sp. n., male, paratypes, DIC images: (
A
)—left leg I, anterior view;
(
B
)—left leg I, posterior view; (
C
)—left leg II, anterior view; (
D
)—left leg II, posterior view; (
E
)—left
legs III–IV, dorsal view; (F)—left legs III–IV, ventral view. Scale bar 20 µm.
Animals 2023,13, 3546 18 of 26
Animals 2023, 13, x FOR PEER REVIEW 20 of 31
Figure 15. Thyreophagus tauricus sp. n., male, SEM images: (A)—gnathosoma, legs I and coxisternal
fields I, ventral view; (B)—anogenital area, ventral view; (C)—tibia and tarsus I, dorsal view; (D)—
tarsus I, anterior view; (E)—leg IV, dorsal view; (F)—tarsus IV, anterior view.
Legs I-III as female, except solenidion ω3 on tarsus I very short, truncated; and
solenidion σ″ about two-times longer than σ′. Trochanter and genu IV without setae, fe-
mur IV with wF IV long, filiform, tibia IV with kT IV elongate, somewhat spiniform. Tarsus
IV with 10 se tae, of them, f, r, w filiform, d and e represented by suckers, u, v, p, q spiniform,
s flaened, buon-shaped or minute spiniform. Solenidion φ on tibia IV short and wide.
Phoretic deutonymph (n = 4, paratypes) (Figures 16–21). Body elongate, 1.33–1.44
times longer than wide, widest in sejugal region; idiosomal length 220–240 width 153–180.
Figure 15.
Thyreophagus tauricus sp. n., male, SEM images: (
A
)—gnathosoma, legs I and coxister-
nal fields I, ventral view; (
B
)—anogenital area, ventral view; (
C
)—tibia and tarsus I, dorsal view;
(D)—tarsus I, anterior view; (E)—leg IV, dorsal view; (F)—tarsus IV, anterior view.
Animals 2023,13, 3546 19 of 26
Animals 2023, 13, x FOR PEER REVIEW 21 of 31
Gnathosoma short, subcapitulum and palps fused, bearing palpal solenidia (ω) apically
and filiform apicodorsal setae (sup); setae h present, minute (Figure 20C) or absent (their
positions marked by somewhat refractile spots), setae cm present.
Figure 16. Thyreophagus tauricus sp. n., phoretic deutonymph, paratype, dorsal view. Scale bar 100
µm.
Figure 16.
Thyreophagus tauricus sp. n., phoretic deutonymph, paratype, dorsal view. Scale bar 100
µ
m.
Type material. Holotype (female) and paratypes (14 females, 9 males and 4 heteromor-
phic deutonymphs) from lab culture; culture started from specimens collected in Crimea,
vicinity of Yalta, under the bark of fallen twigs of Tilia sp., 6 April 2022, 44.483333 N,
34.083333 E, coll. Khaustov V.A, PBK 20-0101-065.
Depository. The holotype and paratypes (11 females, 6 males and 1 heteromorphic deu-
tonymphs) are deposited in the Museum of Zoology, Tyumen State University, Russia. The
remaining paratypes (4 females, 3 males and 3 heteromorphic deutonymphs) are deposited
in the Zoological Institute, Russian Academy of Sciences, Saint Petersburg, Russia.
Animals 2023,13, 3546 20 of 26
Animals 2023, 13, x FOR PEER REVIEW 22 of 31
Figure 17. Thyreophagus tauricus sp. n., phoretic deutonymph, paratype, ventral view. Scale bar 100
µm.
Figure 17.
Thyreophagus tauricus sp. n., phoretic deutonymph, paratype, ventral view. Scale bar 100
µ
m.
Etymology. Tauricus (of Taurica, Lat. adjective). Taurica is a historical name of the
Crimean Peninsula used by the Greeks and Romans. This is a nomen in supposition.
Diagnosis. Adults of Thyreophagus tauricus are distinct among all other species of the
genus by the presence of flattened, button-shaped or minute spiniform (much less than v
and p) setae sIII-IV (Figure 9F) (vs. well-developed spiniform (not much less than vand p)
in all other species, the adults of which are known). The new species is close to Th. corticalis
(patterns of prodorsal sclerite, length of dorsal setae and shaped of legs setae, except s
III–IV), but differs from it in having the vase-shaped atrium of the spermatheca, which is
Animals 2023,13, 3546 21 of 26
3–4 times wider at the basal part than at junction with sclerites of oviducts (vs. 1.5–2 times
wider in Th. corticalis).
Heteromorphic deutonymphs of Th. tauricus are distinct from all other species of
Thyreophagus by the presence of the well-developed setae cm on the dorsal part of the
subcapitular remnant (vs. absent all other species). Thyreophagus tauricus has a prong on
kT III (as in Th. australis Clark, 2009 and Th. sminthurus (Fain and Johnston, 1974)), but it
differs from Th. australis in having the larger ocelli 20 (vs. 10 in Th. australis). Thyreophagus
tauricus differs from Th. sminthurus in having the wider idiosoma, which is 1.33–1.44 times
longer than wide (vs. 2.1 in T. sminthurus), and the presence of tibial setae hT I, II (vs. absent
in Th. sminthurus).
Animals 2023, 13, x FOR PEER REVIEW 23 of 31
Figure 18. Thyreophagus tauricus sp. n., phoretic deutonymph, paratype: (A)—right leg I, posterior
view; (B)—tarsus I, anterior view; (C)—right leg II, dorsal view; (D)—tarsus II, ventral view; (E)—
right leg III, anterior view; (F)—right leg IV, ventral view; (G)—gnathosoma, ventral view. Scale bar
100 µm.
Figure 18.
Thyreophagus tauricus sp. n., phoretic deutonymph, paratype: (
A
)—right leg I, poste-
rior view; (
B
)—tarsus I, anterior view; (
C
)—right leg II, dorsal view; (
D
)—tarsus II, ventral view;
(E)—right
leg III, anterior view; (
F
)—right leg IV, ventral view; (
G
)—gnathosoma, ventral view. Scale
bar 100 µm.
Animals 2023,13, 3546 22 of 26
Animals 2023, 13, x FOR PEER REVIEW 24 of 31
Figure 19. Thyreophagus tauricus sp. n., phoretic deutonymph, paratype, DIC images: (A)—dorsal
view; (B)—ventral view. Scale bar 100 µm.
Figure 19.
Thyreophagus tauricus sp. n., phoretic deutonymph, paratype, DIC images: (
A
)—dorsal
view; (B)—ventral view. Scale bar 100 µm.
Animals 2023, 13, x FOR PEER REVIEW 25 of 31
Figure 20. Thyreophagus tauricus sp. n., phoretic deutonymph, paratype, DIC images: (A)—prodor-
sum; (B)—propodosoma, ventral view; (C)—gnathosoma, ventral view; (D,E)—gnathosoma, dorsal
views; (F,G)—hysterosoma, part, ventral view. Scale bar 20 µm.
Figure 20.
Thyreophagus tauricus sp. n., phoretic deutonymph, paratype, DIC images:
(A)—prodorsum
;
(
B
)—propodosoma, ventral view; (
C
)—gnathosoma, ventral view;
(D,E)—gnathosoma
, dorsal views;
(F,G)—hysterosoma, part, ventral view. Scale bar 20 µm.
Animals 2023,13, 3546 23 of 26
Animals 2023, 13, x FOR PEER REVIEW 26 of 31
Figure 21. Thyreophagus tauricus sp. n., phoretic deutonymph, paratype, DIC images: (A)—legs I,
dorsal view; (B)—legs I, ventral view; (C)—left leg II, dorsal view; (D)—left leg II, ventral view;
(E)—right legs III–IV, dorsal view; (F)—right legs III–IV, ventral view. Scale bar 20 µm.
Dorsum. Idiosoma smoothly punctate; distinct linear paern present on anterior and
lateral sides of prodorsal sclerite and hysterosomal shield. Apex of propodosoma anterior
to anterior border of prodorsal sclerite, with apical internal vertical setae (vi) (bases sepa-
rated) and a pair of band-like sclerites coalescing anteriorly. A pair of lateral, widely sep-
arated ocelli (distance 40–49) present on prodorsum; lenses and pigmented spots present;
maximal diameter of lenses 18–20. External vertical setae (ve) absent; external scapular
Figure 21.
Thyreophagus tauricus sp. n., phoretic deutonymph, paratype, DIC images: (
A
)—legs I,
dorsal view; (
B
)—legs I, ventral view; (
C
)—left leg II, dorsal view; (
D
)—left leg II, ventral view;
(E)—right legs III–IV, dorsal view; (F)—right legs III–IV, ventral view. Scale bar 20 µm.
4. Discussion
In recent years, there has been a growing interest in finding sustainable and environ-
mentally friendly solutions to combat agricultural pests while minimizing the adverse
impacts of chemical pesticides. Mite species of the genus Thyreophagus represent an exciting
alternative to synthetic pesticides in the realm of pest control in agriculture. For example,
Thyreophagus entomophagus and Thyreophagus calusorum are widely used as factitious prey
Animals 2023,13, 3546 24 of 26
for mass rearing of phytoseiid predatory mites for biocontrol applications [
9
,
15
], while
Thyreophagus cracentiseta, has been proposed as such a species based on its performance in
laboratory experiments [
7
]. As several species of Thyreophagus have proven to be valuable
factitious prey for the mass rearing of predatory mites, these predatory mites, in turn, play
a crucial role in biological pest control [
26
]. They feed on a variety of agricultural pests
such as spider mites, thrips and other small arthropods that harm crops [
27
]. The signifi-
cance of this approach lies in its potential to reduce our reliance on synthetic pesticides.
Unlike chemical pesticides, Thyreophagus-based biocontrol methods are environmentally
sustainable, as they do not introduce harmful chemicals into the ecosystem. This approach
is highly targeted, focusing solely on the pests without affecting beneficial organisms or pol-
linators. It is also adaptable to various crops and integrated pest management systems [
26
].
Furthermore, the use of Thyreophagus mites to produce predatory mites for biocontrol can
potentially lead to a reduction in pesticide residues on agricultural products, making them
safer for consumption. As the demand for organic and environmentally friendly farming
practices continues to grow, the role of Thyreophagus mites in replacing synthetic pesticides
becomes increasingly significant, offering a promising and sustainable solution for pest
management in agriculture.
Given the economic significance of Thyreophagus, it is important to know its biodi-
versity, habitat and species boundaries based both on morphology and DNA sequences.
However, these aspects remain significantly underexplored. For instance, it is noteworthy
that a substantial number of Thyreophagus species are thought to be yet undescribed [
1
,
8
]
even in regions where extensive biodiversity research has been carried out, notably in
Europe and North America [
4
]. In addition, there is only a single sequence of Thyreophagus
“entomophagus” in GenBank; however, this sequence is likely based on misidentification (see
below). The prevalence of undescribed or poorly characterized species raises important
questions about our understanding of the global Thyreophagus biodiversity. Numerous
Thyreophagus mite species live in subcortical environments, alongside scale insects, or
within the nests of bees and wasps, thus eluding their discovery due to their secretive
lifestyles and cryptic habitats. Understanding and documenting these species is crucial for
achieving a comprehensive picture of the global Thyreophagus biodiversity. Curiously, as
Thyreophagus mites are adapted to live in concealed habitats, which have limited number of
natural predators, this adaptation is one of the biological features that render Thyreophagus
mites useful as factitious prey in industrial settings. These mites, characterized by slow
movement and a lack of many natural defenses, such as long setae, are particularly suitable
as prey for phytoseiid mites when reared industrially. Therefore, as Thyreophagus mites
play an important role in biocontrol applications, a more thorough examination of their
diversity and biology is also essential. Improved knowledge of these mites would not only
facilitate their use in pest management but also potentially uncover new species suitable for
local production of phytoseiid mites, thereby minimizing the risk of introducing non-native
Thyreophagus species into new regions. In light of these considerations, it is clear that a
more concerted effort is needed to study Thyreophagus mites comprehensively. This entails
employing a range of research methods, from intensive fieldwork to laboratory-based
studies. Additionally, molecular techniques can aid in precisely identifying and classifying
these mites, shedding light on their genetic diversity and evolution.
Here, we report the discovery of a new sexual species, Thyreophagus tauricus sp. n.,
and provide thorough analyses of its morphology, key life stages, molecular characteristics,
and its relationship with other species. As we established a pure culture of this new species
in the laboratory, we were able to confidently correlate all its taxonomically important
life stages, males, females and deutonymphs (a dispersal stage). To facilitate molecular
identification and species delimitation, we sequenced the entire COX1 gene, a useful DNA
barcoding gene [
28
] of four Palearctic species: Th. tauricus,Th. corticalis,Th. calusorum and
Th. entomophagus. Of them, the two latter species are used for mass-rearing of phytoseiid
mites [
9
,
15
]. We found that the new species is morphologically close to Th. corticalis,
a widely distributed Palearctic species; however, it differs from Th. corticalis and other
Animals 2023,13, 3546 25 of 26
Thyreophagus species by the following character states: adults of Th. tauricus are distinct
by the presence of flattened, button-shaped or minute spiniform setae sIII-IV, which are
well-developed and spiniform in all other known species of Thyreophagus; heteromorphic
deutonymphs of Th. tauricus are distinct from all other species of Thyreophagus by the
presence of well-developed setae cm on the dorsal part of the subcapitular remnant (absent
in all other species). Despite being very close to Th. corticalis, genetic COX1 K2P distances
were large, 19.8% (Table 1), suggesting the presence of a well-delimited species, Th. tauricus
sp. n., which is distant by both morphology and DNA sequences. COX1 K2P distances
between Th. tauricus and other species (Th. entomophagus,Th. “entomophagus” NC 066986.1,
Th. calusorum,Th. corticalis) ranged between 20.1 and 24.3% (Table 1).
As part of our study, we also verified sequences deposited into the GenBank database.
One such sequence from China (NC 066986.1) was initially identified as ‘Th. entomophagus’.
However, our sequence, derived from a pure, industrially produced European culture
carefully identified by us, displayed a significant 21.16% COX1 K2P distance from this
GenBank sequence (Table 1). This substantial genetic distance strongly suggests that the
GenBank sequence NC 066986.1 was misidentified and does not belong to Th. entomophagus.
This raises questions about the reliability of public databases and the importance of rigorous
verification and validation in biological research.
5. Conclusions
The discovery and comprehensive study of Thyreophagus tauricus underscore the
importance of biodiversity research, taxonomic methods, molecular techniques and the
need for rigorous scientific practices. Understanding the diversity and characteristics of
mite species like Thyreophagus can have broader implications for ecology, agriculture and
biocontrol efforts.
Author Contributions:
Conceptualization, P.B.K. and V.B.K.; methodology, P.B.K. and V.B.K.; val-
idation, P.B.K., V.B.K., V.A.K. and A.A.K.; formal analysis, P.B.K.; investigation, P.B.K. and V.B.K.;
resources, P.B.K. and V.A.K.; data curation, P.B.K. and V.B.K.; writing—original draft preparation,
V.B.K.; writing—review and editing, P.B.K., V.B.K., V.A.K. and A.A.K.; visualization, V.B.K. and
V.A.K.; supervision, P.B.K.; project administration, P.B.K.; funding acquisition, P.B.K., V.B.K., V.A.K.
and A.A.K. All authors have read and agreed to the published version of the manuscript.
Funding:
This research was funded by Ministry of Science and Higher Education of the Russian
Federation within the framework of the Federal Scientific and Technical Program for the Devel-
opment of Genetic Technologies for 2019–2027 (agreement
№
075-15-2021-1345, unique identifier
RF—193021X0012).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are available in a publicly accessible repository.
Conflicts of Interest: The authors declare no conflict of interest.
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