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Received: 7 December 2024
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Published: 26 January 2025
Citation: Klimov, P.B.; Kolesnikov,
V.B.; Khaustov, A.A.; Khaustov, V.A.;
Merckx, J.; Duarte, M.V.A.;
Vangansbeke, D.; Geudens, I.; Pepato,
A. Typification of the Economically
Important Species Thyreophagus
entomophagus (Acari: Astigmata:
Acaridae) Used for the Industrial
Production of Predatory Mites: The
Designation of a Neotype with
Detailed Morphological and DNA
Sequence Data. Animals 2025,15, 357.
https://doi.org/10.3390/
ani15030357
Copyright: © 2025 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
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licenses/by/4.0/).
Article
Typification of the Economically Important Species Thyreophagus
entomophagus (Acari: Astigmata: Acaridae) Used for the
Industrial Production of Predatory Mites: The Designation of a
Neotype with Detailed Morphological and DNA Sequence Data
†
Pavel B. Klimov
1,
* , Vasiliy B. Kolesnikov
2,3
, Alexander A. Khaustov
2
, Vladimir A. Khaustov
2
, Jonas Merckx
4,5
,
Marcus V. A. Duarte 4, Dominiek Vangansbeke 4, Ilse Geudens 4and Almir Pepato 6
1Lilly Hall of Life Sciences, Purdue University, G-225, 915 W State St., West Lafayette, IN 47907, USA
2X-Bio Institute, Tyumen State University, 10 Semakova Str., 625003 Tyumen, Russia;
jukoman@yandex.ru (V.B.K.); alkhaustov@mail.ru (A.A.K.); kh4ustov93@yandex.ru (V.A.K.)
3Papanin Institute for Biology of Inland Waters, Russian Academy of Sciences, 152742 Borok, Russia
4
Biobest Sustainable Crop Management, R&D, 2260 Westerlo, Belgium; jonas.merckx@biobestgroup.com (J.M.);
marcus.duarte@biobestgroup.com (M.V.A.D.); dominiek.vangansbeke@ugent.be (D.V.);
ilse.geudens@biobestgroup.com (I.G.)
5Biodiversity Inventory for Conservation NPO (BINCO), Walmersumstraat 44, 3380 Glabbeek, Belgium
6
Laboratório de Sistemática eEvolução de Ácaros Acariformes, Instituto de Ciências Biológicas, Departamento
de Zoologia, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, Pampulha,
Belo Horizonte 31270-901, Brazil; apepato@gmail.com
*Correspondence: pklimov@purdue.edu
†urn:lsid:zoobank.org:pub: 0F9CA490-C9AA-48F0-A724-7DC06DC5FF43.
Simple Summary: The mite Thyreophagus entomophagus is widely used in agriculture
as a food source for breeding predatory mites, which are important for biological pest
control. However, the identity of this species has been uncertain due to its incorrect
identifications involving both morphology and DNA sequence data. To resolve this, we
carefully examined a commercial population, selecting a new type specimen from this
population to standardize its name. We also discovered that a population previously
thought to belong to this species is actually a new species, Thyreophagus holda. These
findings clarify that Th. entomophagus lacks a specialized life stage (deutonymph) in
its life cycle, making it easier and more efficient to mass-produce. Our phylogenetic
analysis shows that this trait, along with asexual reproduction, evolved after the origin
of the genus Thyreophagus. We suggest that these traits—being asexual and lacking the
deutonymph stage—are ideal for effective mass production in biological pest control. Our
study emphasizes the need to explore more mites with these beneficial traits, which could
enhance sustainable agricultural practices and reduce the need for chemical pesticides.
Abstract: The mite Thyreophagus entomophagus is a cosmopolitan species of significant
economic importance in biocontrol applications, serving as a factitious prey for the mass
rearing of predatory mites. This species has been reported from a variety of habitats.
However, the taxonomic reliability of its name is questionable due to inconsistencies in
historical species identifications, the absence of type specimens, and misidentified GenBank
sequences. Here, to address these issues and to standardize the nomenclature, we redescribe
Thyreophagus entomophagus based on a commercial culture with known COX1 barcoding
sequence data and designate a neotype from this culture. As part of delimiting the species
boundaries of Th. entomophagus, the question of whether this species forms heteromorphic
deutonymphs is particularly important. While the literature suggests that most populations
lack them, at least one population in Germany has been reported to produce heteromorphic
deutonymphs. However, after careful examination, we identified this population as a new
Animals 2025,15, 357 https://doi.org/10.3390/ani15030357
Animals 2025,15, 357 2 of 28
species, Thyreophagus holda, indicating that previous identifications of this population as
Th. entomophagus were incorrect. The absence of the heteromorphic deutonymphal stage
is a beneficial trait for mass production, as it simplifies the life cycle by eliminating the
energetically costly heteromorphic deutonymph. Our preliminary molecular phylogenetic
analyses of Th. entomophagus and other species of Thyreophagus indicate that the loss
of heteromorphic deutonymphs and the emergence of asexual reproduction (another
beneficial trait for mass production) are derived traits that arose after the divergence of the
most recent common ancestor of Thyreophagus. These insights enhance our understanding of
the evolutionary traits that increase the effectiveness of Th. entomophagus and related species
in biocontrol settings. Our study points to the need for additional bioprospecting efforts to
identify new candidate species for biocontrol that possess both asexual reproduction and
the absence of heteromorphic deutonymphs.
Keywords: astigmatid mites; prey mites; predatory mites; morphology; redescription;
laboratory culture
1. Introduction
Plant pests, particularly insect pests, pose a significant threat to global agricultural
production. While chemical pesticides are effective in controlling these pests, they have
adverse environmental and health effects, including the development of pesticide-resistant
insect populations. Biological control, which utilizes beneficial insects and mites to prey on
pest species, offers a more sustainable alternative. Predatory mites, such as those in the
family Phytoseiidae, have shown promise in controlling pests like thrips, whiteflies, and
red spider mites [
1
–
4
]. The mass rearing of these beneficial insects and mites is important
for their widespread application in agricultural settings. While early rearing systems relied
on natural food sources, recent technological advances have focused on using factitious
prey mites, such as stored product mites, to provide a consistent and controlled food supply
for predators [
1
–
4
]. Successful mass rearing of beneficial insects and mites is essential for
the continued development and implementation of biological control strategies, reducing
reliance on chemical pesticides, and promoting more sustainable agricultural practices.
Thyreophagus entomophagus (Laboulbène and Robin, 1862) (Acaridae) has gained pop-
ularity as a factitious prey mite due to its favorable characteristics, such as ease of con-
sumption by predators and being less allergenic and less harmful as a stored food pest
compared to other species like Tyrophagus putrescentiae (Schrank, 1781) [
1
–
4
]. Currently, Th.
entomophagus is widely used in the mass production of predatory mites globally [5–7].
Historically, the specific name Thyreophagus entomophagus was proposed as Acarus
entomophagus by Laboulbène in 1852 [
8
]; however, this name was a nomen nudum and
not compliant with ICZN rules. An ICZN-compliant description was made in 1862 under
the name Tyroglyphus entomophagus by Laboulbène and Robin for mites damaging ento-
mological collections in Southern France [
9
]. In 1874, Rondani established a new genus,
Thyreophagus, to include this species, leading to the currently used binomen Thyreopha-
gus entomophagus. Since the type material of Th. entomophagus has been lost [
10
], it was
redescribed from adults collected in the Birmingham region of the United Kingdom, with-
out proposing a neotype by A. Fain in 1982 [
10
]. This species concept continues to be
widely used today, however, deutonymphs remained unknown. Phoretic deutonymphs
of Th. entomophagus were later described from a culture started from specimens collected
in a sparrow nest in Dahlem, Berlin, Germany [
11
], although adults from this German
population have never been described. Remarkably, industrially reared populations of
Animals 2025,15, 357 3 of 28
Th. entomophagus never produce heteromorphic deutonymphs. This suggests that either
cryptic species are involved (one can produce deutonymphs while the other cannot), or
some populations of Th. entomophagus may have permanently lost the ability to produce
heteromorphic deutonymphs. Furthermore (personal observations), GenBank has two
mitochondrial COX1 sequences identified as Th. entomophagus from China (NC_066986)
and Russia (OR640974) that differ by a 21.16% COX1 K2P distance [
7
], indicating that
these samples represent two different species. These findings suggest that the taxonomic
status of the economically important species Th. entomophagus is uncertain in terms of both
morphology and genetics. There is an urgent need to standardize the usage of the name
to ensure taxonomic stability and scientific repeatability of research that uses the name
Thyreophagus entomophagus.
In this study, we carefully examine the German deutonymph-producing population of
Thyreophagus entomophagus to determine if there are morphological differences compared to
a commercial population that never forms heteromorphic deutonymphs and compare it
with specimens from Birmingham region (United Kingdom) described in Fain [
10
]. Addi-
tionally, we provide a detailed morphological description of the commercial population,
supported by COX1 sequences and notes on the phylogeny of Thyreophagus. Since the
German populations were morphologically distinct from specimens in the commercial
culture and the UK population, we propose standardizing the use of the name Thyreophagus
entomophagus by designating a neotype based on the commercial population, where both
the COX1 sequence and morphology are known.
2. Materials and Methods
Commercial cultures were obtained from a European biocontrol company on 20
November 2021 and maintained in the lab. The cultures were reared on a mixture of yeast
and bran in specialized rearing units. The purity of the cultures was verified through
morphological identification of a large number of mites (n = 50). Mites were collected
using a camel brush, preserved in 96% ethanol, cleared in 80% lactic acid for 1–2 days, and
mounted in Hoyer’s medium, followed by a 7-day drying period at 60
◦
C [
7
]. Voucher
specimens were deposited at the Tyumen State University Acarological Collection and the
Zoological Institute, Russian Academy of Sciences, Saint Petersburg, Russia; the neotype
was deposited the Royal Belgian Institute of Natural Sciences, Brussels, Belgium. We also
examined specimens collected from a sparrow nest in Dahlem, a suburban district of Berlin,
by W. Knülle [
11
], as well as specimens from the Birmingham region (United Kingdom)
described in [10] (Figure 1).
DNA extraction, sequencing, and culture collection information was described pre-
viously [
7
]. For each cultured species, genomic DNA was extracted from 150 females
obtained from a pure culture (see above), using a QIAamp DNA Micro kit (Qiagen, Venlo,
The Netherlands) with modifications as described here [
12
]. Illumina sequencing libraries
were generated and sequenced commercially on an Illumina NovaSeq 6000 sequencing sys-
tem. Short Illumina reads were assembled in SPAdes v.3.15.5 [
13
] as follows: metaspades.py
-t 24 -m 240 -1 ${name}_R1_001.fastq -2 ${name}_R2_001.fastq -o ${name}. Full-length cy-
tochrome c oxidase subunit 1 (COX1) sequences were found using a local NCBI BLAST
search [
14
]. Th sequences previously generated by us were used (GenBank accession IDs:
OR640973-OR640976). We also used other GenBank species as detailed in the text and fig-
ures. We used Mesquite version 3.81 [
15
] to create sequence alignments, translate nucleotide
matrices into protein sequences, and store Nexus matrices for both protein and nucleotide
data. Genetic distances were calculated in PAUP v.4a168 [
16
] as follows: begin paup; dset
distance = p; savedist format = tabtext undefined = asterisk file = 1_p_distances.tab; dset dis-
tance = k2p; savedist format = tabtext undefined = asterisk file = 2_k2p_distances.tab; end.
Animals 2025,15, 357 4 of 28
Phylogenetic trees were inferred in IQ-TREE v. 2.3.4 [
17
] using amino acid data (command:
iqtree2 -s $ipf --seqtype AA -T AUTO -m MFP -alrt 1000 -bb 1000 -safe --prefix $ipf_bn),
and nucleotide data (command: iqtree2 -s $ipf -spp partitions.txt -nt 8 -m MFP+MERGE
-rcluster 10 -alrt 1000 -bb 1000 -safe --prefix $ipf_bn). Where $ipf is the input file name (a
nexus matrix) and $ipf_bn is the basename of $ipf.
Animals 2025, 15, x FOR PEER REVIEW 4 of 32
Figure 1. Slides with Fane’s museum specimens examined in this work: (A)—specimens Thyreopha-
gus entomophagus (Laboulbène and Robin, 1862) from the Birmingham region (United Kingdom);
(B–F)—specimens Thyreophagus holda sp. n., collected from a sparrow nest in Dahlem; (F)—slide
with holotype Thyreophagus holda sp. n.
DNA extraction, sequencing, and culture collection information was described pre-
viously [7]. For each cultured species, genomic DNA was extracted from 150 females ob-
tained from a pure culture (see above), using a QIAamp DNA Micro kit (Qiagen, Venlo,
The Netherlands) with modifications as described here [12]. 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 [13] as follows:
metaspades.py -t 24 -m 240 -1 ${name}_R1_001.fastq -2 ${name}_R2_001.fastq -o ${name}.
Full-length cytochrome c oxidase subunit 1 (COX1) sequences were found using a local
NCBI BLAST search [14]. Th sequences previously generated by us were used (GenBank
accession IDs: OR640973-OR640976). We also used other GenBank species as detailed in
the text and figures. We used Mesquite version 3.81 [15] to create sequence alignments,
translate nucleotide matrices into protein sequences, and store Nexus matrices for both
protein and nucleotide data. Genetic distances were calculated in PAUP v.4a168 [16] 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. Phylogenetic trees were inferred in IQ-TREE v. 2.3.4 [17] using
amino acid data (command: iqtree2 -s $ipf --seqtype AA -T AUTO -m MFP -alrt 1000 -bb
1000 -safe --prefix $ipf_bn), and nucleotide data (command: iqtree2 -s $ipf -spp parti-
tions.txt -nt 8 -m MFP+MERGE -rcluster 10 -alrt 1000 -bb 1000 -safe --prefix $ipf_bn).
Where $ipf is the input file name (a nexus matrix) and $ipf_bn is the basename of $ipf.
Images were captured from multiple focal planes and assembled using Helicon Focus
Pro 7.6.4 (algorithm B, occasionally A), with subsequent manual editing (retouching) of
any misaligned regions. Partially overlapping images were merged into a full panorama
in Adobe Photoshop 22.2.0. Line drawings were created in Photoshop 22.2.0 using micro-
photographs as the background. The background images were taken using a Euromex
Color HD-Ultra camera aached to a Bioptic C-400 microscope (Bioptic, Moscow, Russian
Federation) equipped with bright field and differential interference contrast (DIC) optics.
Publication-quality microphotographs were captured using an Axio Imager A2 com-
pound microscope (Carl Zeiss, Oberkochen, Germany) equipped with DIC and phase con-
trast optics, along with an Axiocam 506 color digital camera (Carl Zeiss, Oberkochen, Ger-
many). For scanning electron microscopy (SEM) imaging, alcohol-preserved mites were
Figure 1. Slides with Fane’s museum specimens examined in this work: (A)—specimens Thyreoph-
agus entomophagus (Laboulbène and Robin, 1862) from the Birmingham region (United Kingdom);
(B–F)—specimens
Thyreophagus holda sp. n., collected from a sparrow nest in Dahlem; (F)—slide with
holotype Thyreophagus holda sp. n.
Images were captured from multiple focal planes and assembled using Helicon Focus
Pro 7.6.4 (algorithm B, occasionally A), with subsequent manual editing (retouching) of
any misaligned regions. Partially overlapping images were merged into a full panorama
in Adobe Photoshop 22.2.0. Line drawings were created in Photoshop 22.2.0 using mi-
crophotographs as the background. The background images were taken using a Euromex
Color HD-Ultra camera attached to a Bioptic C-400 microscope (Bioptic, Moscow, Russian
Federation) equipped with bright field and differential interference contrast (DIC) optics.
Publication-quality microphotographs were captured using an Axio Imager A2 compound
microscope (Carl Zeiss, Oberkochen, Germany) equipped with DIC and phase contrast
optics, along with an Axiocam 506 color digital camera (Carl Zeiss, Oberkochen, Germany).
For scanning electron microscopy (SEM) imaging, alcohol-preserved mites were dried in a
JFD 320 freeze dryer (JEOL, Tokyo, Japan), coated with gold, and scanned using a TESCAN
Mira3 LMU SEM microscope. Specimens used for SEM were not preserved.
In the descriptions, idiosomal chaetotaxy follows the system outlined in reference [
18
];
the terminology of coxal setae follows reference [
19
]. For appendages, chaetotaxy and
solenidiotaxy follow Grandjean’s system for palps [
20
] and legs [
21
]. Designations of
tarsal dorsoapical setae on legs III–IV follow reference [
22
]. All measurements are given in
micrometers (µm).
3. Results
3.1. GenBank Data Analysis
Maximum likelihood phylogenetic trees were inferred using protein sequences with
the mtART+I+G4 model (Figure 2A) and nucleotide sequences with a codon model
Animals 2025,15, 357 5 of 28
(
Figure 2B
). The genus Thyreophagus was recovered as monophyletic. The internal relation-
ships within Thyreophagus were resolved as two sister groups: (Th. tauricus,Th. corticalis)
and (Th. “entomophagus” from China [NC 066986], Th. calusorum) (Figure 2). However, the
placement of Thyreophagus entomophagus remained unresolved. In the amino acid phylogeny,
T. entomophagus was recovered as sister to the Th. “entomophagus”
(China) + Th. calusorum
clade, whereas in the nucleotide phylogeny, it was sister to the Th. tauricus +Th. corti-
calis clade. In both phylogenetic trees, the remaining taxa morphologically assigned to
the family Acaridae (Tyrophagus,Sancassania, and Aleuroglyphus) formed a monophyletic
group, corresponding to the subfamilies Tyrophaginae and Acotyledonini (Figure 2). How-
ever, traditional morphological classifications place Sancassania (Acotyledonini) within the
subfamily Rhizoglyphinae, which is typically defined by the presence of smooth dorsal
setae. Interestingly, Fagacarus (Rhizoglyphinae) possesses strongly pectinate setae [
23
,
24
],
while Sancassania exhibits sparse pectinations on some of its setae. These observations
suggest that pectinated dorsal setae, a character subject to evolutionary lability, is un-
reliable for delimiting major groups within Astigmata. In contrast, the presence of the
tarsal seta
aa—although
considered plesiomorphic—appears to be a more robust character
for defining lineages within Astigmata. In addition, our trees indicate that the GenBank
sequence originally identified as Rhizoglyphus robini (NC 038058) actually belongs to the
genus Sancassania (=Caloglyphus) (Figure 2).
Animals 2025, 15, x FOR PEER REVIEW 6 of 32
Figure 2. Phylogenetic relationships of species within the genus Thyreophagus and outgroups based
on cytochrome c oxidase subunit 1 (COX1) sequences, inferred using maximum likelihood analysis
in IQ-TREE. SH-aLTR/UFBootstrap values are shown for all nodes. Uncorrected genetic distances
(p-distances) are provided for the neotype population (PBK20-0101-199.SM38) vs. Chinese popula-
tion: (A) phylogeny based on protein data using the mtART+I+G4 model; (B) phylogeny based on
nucleotide data with a codon model.
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), by mono-
typy.
3.2.2. Thyreophagus entomophagus (Laboulbène and Robin, 1862)
Acarus entomophagus Laboulbène, 1852: LIV (nomen nudum).
Tyroglyphus entomophagus Laboulbène and Robin, 1862: 321, Pl. 10 (female, homeo-
morphic male; types lost [10]).
Material. Cultures were obtained from a European biocontrol company and main-
tained on a mixture of yeast and bran in specialized rearing units by VAK. Cultures were
started on 20 November 2021; specimens were harvested on # COX1 barcoding sequence
GenBank Id: OR640974 (culture PBK 20-0101-199.SM38, from which the neotype was des-
ignated).
Type material. Neotype (female) same data, deposited at the Royal Belgian Institute
of Natural Sciences, Brussels, Belgium.
Female (Figures 3A,B, 4, 5E,F, 6, 7, 9D–I, 10 and 18A,D,E,H). Idiosoma slightly elon-
gate, 400–430 × 210–225 (n = 20), 1.9 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
Figure 2. Phylogenetic relationships of species within the genus Thyreophagus and outgroups based
on cytochrome c oxidase subunit 1 (COX1) sequences, inferred using maximum likelihood analysis
in IQ-TREE. SH-aLTR/UFBootstrap values are shown for all nodes. Uncorrected genetic distances
(p-distances) are provided for the neotype population (PBK20-0101-199.SM38) vs. Chinese popula-
tion: (A) phylogeny based on protein data using the mtART+I+G4 model; (B) phylogeny based on
nucleotide data with a codon model.
3.2. Morphological Description
3.2.1. Genus Thyreophagus Rondani, 1874
Thyreophagus Rondani, 1874: 67 (=Moneziella Berlese, 1897; Monetiella Berlese, 1897;
Animals 2025,15, 357 6 of 28
Monieziella Berlese, 1897; Fumouzea Zachvatkin, 1953; Michaelopus Fain and John-
ston, 1974).
Type species: Thyreophagus entomophagus (Laboulbène and Robin, 1862), by monotypy.
3.2.2. Thyreophagus entomophagus (Laboulbène and Robin, 1862)
Acarus entomophagus Laboulbène, 1852: LIV (nomen nudum).
Tyroglyphus entomophagus Laboulbène and Robin, 1862: 321, Pl. 10 (female, homeomor-
phic male; types lost [10]).
Material. Cultures were obtained from a European biocontrol company and main-
tained on a mixture of yeast and bran in specialized rearing units by VAK. Cultures
were started on 20 November 2021; specimens were harvested on # COX1 barcoding se-
quence GenBank Id: OR640974 (culture PBK 20-0101-199.SM38, from which the neotype
was designated).
Type material. Neotype (female) same data, deposited at the Royal Belgian Institute of
Natural Sciences, Brussels, Belgium.
Female (Figures 3A,B, 4,5E,F, 6,7,9D–I, 10 and 18A,D,E,H). Idiosoma slightly elon-
gate, 400–430
×
210–225 (n = 20), 1.9 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 shield 73–80 long, 75–84 wide, nearly
as long as wide, with setae vi (situated at anterior part of shield, alveoli of vi noticeably
separated—the distance between them almost equal to their width), rounded anterolateral
incisions, and elongate midlateral incisions (insertion points of setae ve). Prodorsal shield
smoothly punctate (with small cells), without distinct lineate pattern (cells may be slightly
elongated or rounded in the posterior part) and with several (3–5) curved longitudinal
lines in posterior part. 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,c
p
,d
2
,e
2
,h
1
,h
3
,ps
3
) smooth, filiform and short, setae se and h
2
twice as long as other idiosomal setae, smooth and filiform; opisthosomal gland openings
between setal bases e
2
and d
2
. Four pairs of fundamental cupules (ia,im,ip and ih) present.
With a pair of additional cuticular pores between setae h
1
. Ventral idiosoma with four pairs
of coxal setae (1a,3a,4a and 4b) and one pair of genital setae (g). Shape of coxal sclerites as
in Figures 3B, 6B and 7B. 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 0.4–0.5 the length of coxal and genital setae. Anal opening
terminal. Copulatory tube present, situated anterodorsally to anus, with distinct opening.
Inseminatory canal of spermatheca long, slender tube-like, leading from copulatory open-
ing to spermatheca, uniformly wide. Base of spermatheca wide, bell-shaped, with a distinct
vestibule. Paired sclerites of efferent ducts elongate, their length approximately 1/2 the
length of spermatheca base, with short stem.
Animals 2025,15, 357 7 of 28
Animals 2025, 15, x FOR PEER REVIEW 8 of 32
Figure 3. Thyreophagus entomophagus (Laboulbène and Robin, 1862), PBK20-0101-199.SM38, neotype
(A,B), not neotype (C,D): (A) female, dorsal view; (B) female, ventral view; (C) male, dorsal view;
(D) male, ventral view.
Figure 3. Thyreophagus entomophagus (Laboulbène and Robin, 1862), PBK20-0101-199.SM38, neo-
type (A,B), not neotype (C,D): (A) female, dorsal view; (B) female, ventral view; (C) male, dorsal
view; (D) male, ventral view.
Animals 2025,15, 357 8 of 28
Animals 2025, 15, x FOR PEER REVIEW 9 of 32
Figure 4. Thyreophagus entomophagus (Laboulbène and Robin, 1862), PBK20-0101-199.SM38, female,
neotype: (A) leg I, dorsal view; (B) tarsus I, ventral view; (C) leg II, dorsal view; (D) tarsus II, ventral
view; (E) leg III, dorsal view; (F) tarsus III, ventral view; (G) leg IV, dorsal view; (H) tarsus IV,
ventral view.
Figure 4. Thyreophagus entomophagus (Laboulbène and Robin, 1862), PBK20-0101-199.SM38, female,
neotype: (A) leg I, dorsal view; (B) tarsus I, ventral view; (C) leg II, dorsal view; (D) tarsus II, ventral
view; (E) leg III, dorsal view; (F) tarsus III, ventral view; (G) leg IV, dorsal view; (H) tarsus IV,
ventral view.
Animals 2025,15, 357 9 of 28
Animals 2025, 15, x FOR PEER REVIEW 10 of 32
Figure 5. Thyreophagus entomophagus (Laboulbène and Robin, 1862), PBK20-0101-199.SM38, neotype
(E,F), non-neotype, but from the same culture (A–D,G): (A) male leg I, dorsal view; (B) male tarsus
I, ventral view; (C) male leg IV, dorsal view; (D) male tarsus IV, ventral view; (E) female gnatho-
soma, ventral view; (F) spermatheca; (G) male genitalia.
Figure 5. Thyreophagus entomophagus (Laboulbène and Robin, 1862), PBK20-0101-199.SM38, neo-
type (E,F), non-neotype, but from the same culture (A–D,G): (A) male leg I, dorsal view; (B) male
tarsus I, ventral view; (C) male leg IV, dorsal view; (D) male tarsus IV, ventral view; (E) female
gnathosoma, ventral view; (F) spermatheca; (G) male genitalia.
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 spiniform, shorter than gT; setae gT I–II and kT III–IV
elongate, somewhat spiniform. Tarsal setation 8-8-8-8; pretarsi consist of hooked empodial
claws attached to short paired condylophores. Tarsi I and II with setae ra,la,fand dfiliform,
e,u,vspiniform, pand qrepresented by very small remnants, sflattened, button-shaped or
minute spiniform (button-shaped in neotype); setae wa absent. Tarsi III and IV with setae f,
d,r,wfiliform, eand sspiniform, uand/or vflattened, button-shaped, pand qrepresented
by very small remnants. Solenidion
ω1
on tarsus I cylindrical, slightly arched, with a slight
narrowing before apical widening. Solenidion
ω1
on tarsus II simple, cylindrical, with
clavate apex, nearly straight. Solenidion
ω2
on tarsus I shorter than
ω1
, cylindrical, with
rounded apex, slightly widened distally, situated slightly anteriad
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 of respective tarsi with ambulacra;
solenidion
ϕ
IV shorter than tarsus IV (with ambulacra). Solenidia
σ
’ and
σ
” on genu I
elongate, tapering,
σ
” longer than
σ
’;
σ
” 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
σ
of genu
III absent.
Animals 2025,15, 357 10 of 28
Animals 2025, 15, x FOR PEER REVIEW 11 of 32
Figure 6. Thyreophagus entomophagus (Laboulbène and Robin, 1862), PBK20-0101-199.SM38, female,
neotype, DIC images: (A) dorsal view; (B) ventral view; (C) prodorsal shield; (D) tarsus III, ventral
view; (E) spermatheca.
Figure 6. Thyreophagus entomophagus (Laboulbène and Robin, 1862), PBK20-0101-199.SM38, female,
neotype, DIC images: (A) dorsal view; (B) ventral view; (C) prodorsal shield; (D) tarsus III, ventral
view; (E) spermatheca.
Animals 2025,15, 357 11 of 28
Animals 2025, 15, x FOR PEER REVIEW 12 of 32
Figure 7. Thyreophagus entomophagus (Laboulbène and Robin, 1862), PBK20-0101-199.SM38, female,
not neotype, DIC images: (A) dorsal view; (B) ventral view.
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 spiniform, shorter than gT; setae gT I–II and kT III–IV
elongate, somewhat spiniform. Tarsal setation 8-8-8-8; pretarsi consist of hooked empo-
dial claws aached to short paired condylophores. Tarsi I and II with setae ra, la, f and d
filiform, e, u, v spiniform, p and q represented by very small remnants, s flaened, buon-
shaped or minute spiniform (buon-shaped in neotype); setae wa absent. Tarsi III and IV
with setae f, d, r, w filiform, e and s spiniform, u and/or v flaened, buon-shaped, p and q
represented by very small remnants. Solenidion ω1 on tarsus I cylindrical, slightly arched,
with a slight narrowing before apical widening. Solenidion ω1 on tarsus II simple, cylin-
drical, with clavate apex, nearly straight. Solenidion ω2 on tarsus I shorter than ω1, cylin-
drical, with rounded apex, slightly widened distally, situated slightly anteriad 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 of respective
tarsi with ambulacra; solenidion ϕ IV shorter than tarsus IV (with ambulacra). Solenidia
σ’ and σ” on genu I elongate, tapering, σ” longer than σ’; σ” slightly not reaching bases of
Figure 7. Thyreophagus entomophagus (Laboulbène and Robin, 1862), PBK20-0101-199.SM38, female,
not neotype, DIC images: (A) dorsal view; (B) ventral view.
Male (n = 5) (Figures 3C,D, 5A–D,G, 8,9A–C,L–J and 11). Idiosoma slightly elongate,
300–360
×
180–200, 1.7–1.8 times longer than wide. Idiosomal cuticle smooth. Gnathosoma
as in female. Prodorsal shield 73–82 long, 73–80 wide, nearly as long as wide, with setae
vi, incisions and ornamentation as in female. Grandjean’s organ (GO) and supracoxal
seta (scx) as in female. All idiosomal setae smooth and filiform, setae se longer and wider
that other setae; setae c
p
,e
2
and h
3
longer than vi,d
2
,h
1
,h
2
. Two pairs of fundamental
cupules (ia and ih) present, im and ip not observed. With a pair of of additional cuticular
pores between setae h
1
. 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). Posterior region of idiosoma with a large rounded lobe extending
posteriorly (45–48
×
80–86, 1.7–1.8 times longer than wide). Shape of coxal sclerites as in
Figures 3D and 8B. Genital region between coxisternal fields IV; genital capsule rounded;
aedeagus short, not protruding beyond anterior edge of genital capsule. Diameter of genital
papillae approximately 0.4–0.5 the length of coxal and genital setae. Anal suckers rounded
in outline. Setae ps1–3 very short.
Animals 2025,15, 357 12 of 28
Animals 2025, 15, x FOR PEER REVIEW 13 of 32
ϕ I. Solenidion σ on genu II more than 6–7 times longer than its width, with rounded tip.
Solenidion σ of genu III absent.
Male (n = 5) (Figures 3С,D, 5A–D,G, 8, 9A–C,L–J and 11). Idiosoma slightly elongate,
300–360 × 180–200, 1.7–1.8 times longer than wide. Idiosomal cuticle smooth. Gnathosoma
as in female. Prodorsal shield 73–82 long, 73–80 wide, nearly as long as wide, with setae
vi, incisions and ornamentation as in female. Grandjean’s organ (GO) and supracoxal seta
(scx) as in female. All idiosomal setae smooth and filiform, setae se longer and wider that
other setae; setae cp, e2 and h3 longer than vi, d2, h1, h2. Two pairs of fundamental cupules
(ia and ih) present, im and ip not observed. With a pair of of additional cuticular pores
between setae h1. 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). Posterior region of idiosoma with a large rounded lobe extending poste-
riorly (45–48 × 80–86, 1.7–1.8 times longer than wide). Shape of coxal sclerites as in Figures
3D and 8B. Genital region between coxisternal fields IV; genital capsule rounded; ae-
deagus short, not protruding beyond anterior edge of genital capsule. Diameter of genital
papillae approximately 0.4–0.5 the length of coxal and genital setae. Anal suckers rounded
in outline. Setae ps1–3 very short.
Figure 8. Thyreophagus entomophagus (Laboulbène and Robin, 1862), PBK20-0101-199.SM38, male,
not neotype, DIC images: (A)—dorsal view; (B)—ventral view.
Figure 8. Thyreophagus entomophagus (Laboulbène and Robin, 1862), PBK20-0101-199.SM38, male, not
neotype, DIC images: (A)—dorsal view; (B)—ventral view.
Animals 2025, 15, x FOR PEER REVIEW 14 of 32
Figure 9. Thyreophagus entomophagus (Laboulbène and Robin, 1862), PBK20-0101-199.SM38, not ne-
otype, DIC images: (A) male prodorsal shield; (B,C) male gnathosoma; (D) spermatheca; (E) female
leg I, dorsal view; (F) female tarsus I, ventral view; (G) female leg I, dorsal view; (H) female tarsi
III–IV, ventral view; (I) female tarsi III–IV, dorsal view; (J) male tarsi III–IV, ventral view; (K) male
tarsus IV, dorsal view; (L) male tarsus I, ventral view.
Figure 9. Cont.
Animals 2025,15, 357 13 of 28
Animals 2025, 15, x FOR PEER REVIEW 14 of 32
Figure 9. Thyreophagus entomophagus (Laboulbène and Robin, 1862), PBK20-0101-199.SM38, not ne-
otype, DIC images: (A) male prodorsal shield; (B,C) male gnathosoma; (D) spermatheca; (E) female
leg I, dorsal view; (F) female tarsus I, ventral view; (G) female leg I, dorsal view; (H) female tarsi
III–IV, ventral view; (I) female tarsi III–IV, dorsal view; (J) male tarsi III–IV, ventral view; (K) male
tarsus IV, dorsal view; (L) male tarsus I, ventral view.
Figure 9. Thyreophagus entomophagus (Laboulbène and Robin, 1862), PBK20-0101-199.SM38, not
neotype, DIC images: (A) male prodorsal shield; (B,C) male gnathosoma; (D) spermatheca; (E) female
leg I, dorsal view; (F) female tarsus I, ventral view; (G) female leg I, dorsal view; (H) female tarsi
III–IV, ventral view; (I) female tarsi III–IV, dorsal view; (J) male tarsi III–IV, ventral view; (K) male
tarsus IV, dorsal view; (L) male tarsus I, ventral view.
Animals 2025, 15, x FOR PEER REVIEW 15 of 32
Figure 10. Thyreophagus entomophagus (Laboulbène and Robin, 1862), PBK20-0101-199.SM38, female,
not neotype, SEM images: (A) posterior part of idiosoma, dorsal view; (B) posterior part of idiosoma,
lateral view; (C) genital area, ventral view; (D) supracoxal seta; (E) Grandjean’s organ; (F) leg I,
dorsal view; (G) tarsus II, ventral view; (H,I) tarsi III, lateral and ventral views.
Figure 10. Thyreophagus entomophagus (Laboulbène and Robin, 1862), PBK20-0101-199.SM38, female,
not neotype, SEM images: (A) posterior part of idiosoma, dorsal view; (B) posterior part of idiosoma,
lateral view; (C) genital area, ventral view; (D) supracoxal seta; (E) Grandjean’s organ; (F) leg I, dorsal
view; (G) tarsus II, ventral view; (H,I) tarsi III, lateral and ventral views.
Animals 2025,15, 357 14 of 28
Animals 2025, 15, x FOR PEER REVIEW 16 of 32
Figure 11. Thyreophagus entomophagus (Laboulbène and Robin, 1862), PBK20-0101-199.SM38, male,
not neotype, SEM images: (A) dorsal view; (B) ventral view; (C) propodosoma, dorsal view; (D)
ano-genital region, ventral view.
Legs I-III as in female, except solenidion ω3 on tarsus I very short, truncated. Tro-
chanter and genu IV without setae, femur IV with wF IV long, filiform, tibia IV with kT IV
elongate, somewhat spiniform. Tarsus IV with 7 or 8 setae (one v or u may be absent), of
them, f, r, w filiform, d and e represented by suckers, s spiniform, u and v flaened, buon-
shaped, p and q represented by very small remnants. Solenidion ϕ on tibia IV short and
wide.
Heteromorphic deutonymph. Absent.
The Birmingham population. The currently used species concept of Th. entomophagus
was established by A. Fain in 1982 [10], who described specimens from the United King-
dom (Birmingham) without designating a neotype. We re-examined two females from the
Birmingham sample and compared them with females from our culture of Thyreophagus
entomophagus (PBK20-0101-199.SM38). These two were conspecific. Particularly, we note
the identical shapes of u and v III, which are flaened and buon-shaped (Figure 18F) and
slightly elongated or rounded cells on the posterior part of the prodorsal shield (Figure
18B), without a lineate paern; A. Fain [10] probably mistook the slightly elongated punc-
tations for a lineate paern, but they are markedly different from the lineate paern seen
in Th. holda sp. n. (Figure 16C). We also note that ventroterminal setae on tarsi III–IV, u
and v III–IV were originally figured by Fain [10] as spiniform, but actually they are
Figure 11. Thyreophagus entomophagus (Laboulbène and Robin, 1862), PBK20-0101-199.SM38, male,
not neotype, SEM images: (A) dorsal view; (B) ventral view; (C) propodosoma, dorsal view; (D) ano-
genital region, ventral view.
Legs I–III as in female, except solenidion
ω3
on tarsus I very short, truncated.
Trochanter and genu IV without setae, femur IV with wF IV long, filiform, tibia IV with kT
IV elongate, somewhat spiniform. Tarsus IV with 7 or 8 setae (one vor umay be absent),
of them, f,r,wfiliform, dand erepresented by suckers, sspiniform, uand vflattened,
button-shaped, pand qrepresented by very small remnants. Solenidion
ϕ
on tibia IV short
and wide.
Heteromorphic deutonymph. Absent.
The Birmingham population. The currently used species concept of Th. entomophagus
was established by A. Fain in 1982 [
10
], who described specimens from the United Kingdom
(Birmingham) without designating a neotype. We re-examined two females from the
Birmingham sample and compared them with females from our culture of Thyreophagus
entomophagus (PBK20-0101-199.SM38). These two were conspecific. Particularly, we note
the identical shapes of uand vIII, which are flattened and button-shaped (Figure 18F) and
slightly elongated or rounded cells on the posterior part of the prodorsal shield (Figure 18B),
without a lineate pattern; A. Fain [
10
] probably mistook the slightly elongated punctations
for a lineate pattern, but they are markedly different from the lineate pattern seen in Th.
holda sp. n. (Figure 16C). We also note that ventroterminal setae on tarsi III–IV, uand v
Animals 2025,15, 357 15 of 28
III–IV were originally figured by Fain [
10
] as spiniform, but actually they are flattened and
button-shaped structures. The shape of the spermatheca base in the specimens described by
A. Fain [
10
] agrees well with that of all the specimens we studied. In some cases, its width
may change, but this is due to deformation during specimen preparation. We were unable
to find any noticeable widening of the inseminatory canal, as depicted in A. Fain [
10
].
Heteromorphic deutonymphs are unknown for the Birmingham sample.
Diagnosis. Adults of Th. entomophagus differ from all other species (except Th. hobe
Klimov et al., 2023) by the presence of flattened, button-shaped setae uand von tarsus
III (vs. spiniform in other species). Thyreophagus entomophagus differs from Th. hobe by
the presence of filiform wIII (minute spiniform in Th. hobe) and the presence of wF IV
(absent in Th. hobe). Also, Th. entomophagus differs from all other species by the bell-shaped
base of spermatheca (vase-shaped, narrow, parallel-sided, broad-arc-shaped, or reduced in
other species).
For Th. entomophagus, three subspecies have been described: Th. entomophagus nomi-
nalis Kadzhaya, 1973, Th. entomophagus ponticus Kadzhaya, 1973 [
25
] and Th. entomophagus
italicus Vacante, 1989 [
26
]. Subsequently, Th. entomophagus ponticus and Th. entomophagus
italicus were elevated to the rank of species [6].
Thyreophagus ponticus Kadzhaya, 1973 has a well-developed opisthosomal projection
in males, as in Th. entomophagus. However, in females of Th. ponticus, setae h
3
are only
slightly longer than ps
3
(more than twice as long in Th. entomophagus), and the anal opening
is approximately equal to the length of setae h
3
(more than twice as long as the anal opening
in Th. entomophagus). Thyreophagus ponticus is insufficiently described and is considered a
species inquirenda [6].
Thyreophagus italicus Vacante, 1989 differs from Th. entomophagus by the shape of the
base of the spermatheca, which is vase-shaped, narrow, with constriction (bell-shaped,
wide, without constriction in Th. entomophagus), very short setae wIII (long, filiform in Th.
entomophagus), spiniform setae uand vIII, the same length as sIII (flattened, button-shaped,
more than twice as short as sIII in Th. entomophagus).
Thyreophagus entomophagus nominalis is an insufficiently described taxon, considered a
species inquirenda [
6
]. It differs from Th. entomophagus by having subequal setae h
1
and ps
1
in females [25] (h1are distinctly longer than ps1in Th. entomophagus).
Neotype. The need for typification of Thyreophagus entomophagus through neotype
designation is based on the following: (i) several morphologically similar species are known
(Th. holda sp. n., Th. leclercqi), but the differences between these nominal species are not
well understood and may represent either interspecific or intraspecific variations; sequence
data are lacking for these species; (ii) specimens from China identified as Thyreophagus “en-
tomophagus” [
27
] (without morphological description) show uncorrected genetic distances
(p-distance) of 18.09% at nucleotide level and 6.36% at the amino acid level (NC_066986 vs.
OR640974), indicating that two genetically distinct species are involved. This suggests that
previous re-descriptions of Thyreophagus entomophagus may not suffice for accurate identifi-
cation of this species and a neotype is needed. Our proposed neotype will provide stability
in the usage of the name Thyreophagus entomophagus, facilitating confident identification
based on both morphology and DNA sequence data. To standardize the current usage of
the name Thyreophagus entomophagus, we propose designating a neotype (female, acces-
sion #) from a commercial culture PBK20-0101-199.SM38 used for the industrial rearing of
predatory mites as described here using light microscopy, SEM, and COX1 sequence data.
3.2.3. Thyreophagus holda Klimov, Kolesnikov, Merckx, Duarte et Vangansbeke sp. n.
Thyreophagus entomophagus Fain et al., 2000: 154, Figures 1–9(heteromorphic deu-
tonymph; misidentification).
Animals 2025,15, 357 16 of 28
Material. Adults and heteromorphic deutonymphs—culture 1, started from adult
specimens collected in a sparrow nest in the suburban district of Berlin (Berlin Dahlem), W.
Knülle [11].
Type material. Holotype (female, slide 29-90a, recognizable by unique shape on slide)
and paratypes (six females, five males and four heteromorphic deutonymph) same data
(slides 29-89, 29-89a, 29-90, 29-90a).
Depository. The holotype and 15 paratypes are deposited in the Royal Belgian Institute
of Natural Sciences, Brussels, Belgium.
Etymology. Holda is a popular figure in German folklore.
Female (n = 7) (Figures 12,14A–D,F,G, 16A,B, 17A,B,D–G,J and 18B,E,I). Idiosoma
slightly elongate, 580–800
×
300–400, 1.7–2.0 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 shield 100–110 long,
110–112 wide
,
1.0–1.1 times longer than wide, with setae vi (situated at anterior part of shield, alveoli
noticeably separated—distance between them almost equal to their width), rounded antero-
lateral incisions, and elongate midlateral incisions (insertion points of setae ve). Prodorsal
shield smoothly punctate (small cells), with lineate cells in posterior part and with several
(3–5) curved longitudinal lines in posterior part. Grandjean’s organ (GO) with seven mem-
branous finger-shaped processes. Supracoxal setae (scx) smooth, sword-shaped, widened
and flattened, tapering at tip. Idiosomal setae (vi,c
p
,d
2
,e
2
,h
1
,h
3
,ps
3
) smooth, filiform
and short, setae se and h
2
twice as long as other idiosomal setae, smooth and filiform;
opisthosomal gland openings between setal bases e
2
and d
2
. Four pairs of fundamental
cupules (ia,im,ip and ih) present. With a pair of additional cuticular pores between setae
h
1
. Ventral idiosoma with four pairs of coxal setae (1a,3a,4a and 4b) and one pair of
genital setae (g). Shape of coxal sclerites as in Figure 12B. 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 0.4–0.5 the length of coxal
and genital setae. Anal opening terminal. Copulatory tube present, situated anterodor-
sally to anus, with developed opening. Inseminatory canal of spermatheca long, slender
tube-like, leading from copulatory opening to spermatheca, slightly widened at junction
with base of spermatheca. Base of spermatheca wide, bell-shaped, with a distinct vestibule.
Paired efferent ducts elongated, their length is approximately 1/2 the length of the base of
spermatheca, with short stem.
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 spiniform, shorter than gT; setae gT I–II and kT III–IV
elongate, somewhat spiniform. Tarsal setation 8-8-8-8; pretarsi consist of hooked empodial
claws attached to short paired condylophores. Tarsus I and II with setae ra,la,fand d
filiform, e,u,vspiniform, pand qrepresented by very small remnants, sflattened, button-
shaped or minute spiniform; setae wa absent. Tarsus III and IV with setae f,d,r,wfiliform,
e,s,uand vspiniform (uand vslightly less than s), pand qrepresented by very small
remnants. Solenidion
ω1
on tarsus I cylindrical, with clavate apex, in front of which there is
a slight narrowing, curved; solenidion
ω1
on tarsus II simple, cylindrical, with clavate apex,
not bent. Solenidion
ω2
on tarsus I shorter than
ω1
, cylindrical, with rounded apex, slightly
widened distally, situated slightly distal to ω1. Solenidion ω3on 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,
Animals 2025,15, 357 17 of 28
extending well beyond apices of respective tarsi with ambulacra; solenidion
ϕ
IV shorter,
shorter than tarsus IV (with ambulacra). Solenidia
σ
’ and
σ
” on genu I elongate, tapering,
σ
” longer than
σ
’,
σ
” 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 = 5) (Figures 13A,B, 14E, 16C,D and 17C,H,I). Idiosoma slightly elongate,
430–470
×
250–300, 1.6–1.7 times longer than wide. Idiosomal cuticle smooth. Gnathosoma
as in female. Prodorsal shield 86–95 long, 90–98 wide, 1.0–1.1 times longer than wide, with
setae vi, incisions and ornamented as in female. Grandjean’s organ (GO) and supracoxal
seta (scx) as in female. All idiosomal setae smooth and filiform, setae se linger and widener
that other, setae c
p
,e
2
and h
3
longer than vi,d
2
,h
1
,h
2
. Two pairs of fundamental cupules
(ia and ih) present, im and ip not observed. Between setae h
1
, there is a pair of additional
cuticular pores. 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). Posterior region of idiosoma with a large rounded lobe extending the body
backwards (45–50
×
78–85, 1.5–1.8 times longer than wide). Shape of coxal sclerites on
Figure 13B. 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 0.4–0.5 the length of coxal and genital setae. Anal suckers rounded
in outline. Setae ps1–3 very short.
Legs I-III as female, except solenidion
ω3
on tarsus I very short, truncated. Trochanter
and genu IV without setae, femur IV with wF IV long, filiform, tibia IV with kT IV elongate,
somewhat spiniform. Tarsus IV with 8 setae, of them, f,r,wfiliform, dand erepresented by
suckers, s,e,uand vspiniform (uand vslightly less than s), pand qrepresented by very
small remnants. Solenidion ϕon tibia IV short and wide.
Heteromorphic deutonymph (n = 4) (Figures 13C,D, 15,16E,F and 17K–M). Body
ovoid, 1.3–1.4 times longer than wide, widest in sejugal region; idiosomal length 240–250
width 170–190. Gnathosoma short, subcapitulum and palps fused, bearing palpal solenidia
(
ω
) apically and filiform apicodorsal setae (sup); setae habsent (their positions marked by
somewhat refractile spots), setae cm absent.
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 68–70) present on prodorsum; lenses and pigmented spots
present; maximum diameter of lenses 9–10. External vertical setae (ve) absent; external
scapular setae se situated below lenses; internal scapular setae (si) distinctly posterior
external scapulars (se). Supracoxal setae of legs I (scx) filiform, with extended base, situated
below si and anterolaterad of ocelli. Sejugal furrow well developed. Prodorsal sclerite 70–75,
hysterosomal shield 158–173, ratio hysterosomal 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 other setae. Opisthonotal gland openings
(gla) situated ventrally on hysterosomal shield, slightly posterior setae c
3
, much closer to
ventral seta c
3
than to dorsolateral seta c
p
(in one specimen approximately equidistant
from setae c
3
and c
p
). Of four fundamental pairs of cupules, three pairs were observed: ia
posteriomediad setae c
2
,im ventral, laterad trochanters IV and ih ventral, laterad posterior
sides of attachment organ.
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,
Animals 2025,15, 357 18 of 28
thin, curved medially. Sternal and ventral shield contiguous. Anterior apodemes of coxal
fields III free, connected by thin transverse sclerotization. Posterior medial apodeme present
in area of coxal fields IV, well-separated from anterior apodemes IV and genital opening.
Posterior apodemes IV absent. Subhumeral setae (c
3
) filiform, situated on ventral surface
between legs II and III, near region separating sternal and ventral shields. Coxal setae
1a and 3a reduced, represented by minute structures. Setae 4b,gfiliform; 4a in form of
small, rounded conoids. Genital region in posterior portion of coxisternal fields IV; genital
opening elongate, with two pairs of genital papillae within genital atrium; genital papillae
two-segmented, with rounded apices. Coxal setae (4b) situated at free ends of anterior
coxal apodemes IV; genital setae (g) laterad genital opening. Attachment organ posterior
to coxal 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 anterolaterad median suckers (ps
3
); lateral conoidal setae of attachment organ (ps
2
)
situated distinctly posterior to line joining centers of median suckers, slightly anteriad
conoidal setae (ps
1
); anterior and posterior lateral and posterior median cuticular conoids
well developed; anus situated between anterior suckers (ad3).
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-II filiform; setae hT II spiniform, setae gT longer
than hT; setae kT III-IV filiform, without a 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,e,ffoliate; seta dfiliform, 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 rfiliform and wfiliform with a distinct prong. Solenidia
ω1
on tarsi I–II
cylindrical, with slightly clavate apices;
ω3
on tarsus I longer than
ω1
, with rounded apex,
situated slightly distal to
ω1
;
ω1
and
ω3
separated by bulbous famulus (
ε
); solenidion
ω2
of tarsus I slightly expanding apically, situated somewhat more basal and posterior to
ω1
+
ε
+
ω3
group; solenidia
φ
of tibiae I–III elongate, tapering;
φ
I and III longer than
tarsus I and III, respectively; solenidion
φ
II shorter than tarsus II; solenidion
φ
IV short;
solenidion
σ
of genu I elongate, slightly tapering, nearly reaching tip of tibia I; solenidion
σ
of genu II much shorter, cylindrical, not reaching midlength of tibia II; solenidion
σ
of genu
III absent.
Diagnosis. Adults of Th. holda are very similar to those of Th. entomophagus, but
differ by the following: apical tarsal setae uand vIII-IV normally developed, spiniform
(vs. flattened and button-shaped in Th. entomophagus) (Figure 18G,I vs. Figure 18D–F,H);
prodorsal shield with a lineate cells in its posterior part (vs. rounded or slightly elongated
in Th. entomophagus) (Figure 18C vs. Figure 18A,B); larger body measurements (females
580–800 and males 430–470 vs. females 400–430 and males 300–360 in Th. entomophagus).
The new species forms heteromorphic deutonymphs (vs. lacking in Th. entomophagus).
Animals 2025,15, 357 19 of 28
Animals 2025, 15, x FOR PEER REVIEW 21 of 32
Figure 12. Thyreophagus holda sp. n., female, holotype: (A) dorsal view; (B) ventral view.
Figure 12. Thyreophagus holda sp. n., female, holotype: (A) dorsal view; (B) ventral view.
Figure 13. Cont.
Animals 2025,15, 357 20 of 28
Figure 13. Thyreophagus holda sp. n., paratypes: (A) male, dorsal view; (B) male, ventral view;
(C) heteromorphic deutonymph, dorsal view; (D) heteromorphic deutonymph, ventral view.
Animals 2025, 15, x FOR PEER REVIEW 23 of 32
Figure 14. Thyreophagus holda sp. n., holotype (A–D,F) and paratypes (E,G): (A) female leg I, poste-
rior view; (B) female leg II, anterior view; (C) female leg III, anterior view; (D) female tarsus III,
posterior view; (E) male leg IV, anterior view; (F,G) spermathecae.
Figure 14. Thyreophagus holda sp. n., holotype (A–D,F) and paratypes (E,G): (A) female leg I, posterior
view; (B) female leg II, anterior view; (C) female leg III, anterior view; (D) female tarsus III, posterior
view; (E) male leg IV, anterior view; (F,G) spermathecae.
Animals 2025,15, 357 21 of 28
Animals 2025, 15, x FOR PEER REVIEW 24 of 32
Figure 15. Thyreophagus holda sp. n., heteromorphic deutonymph, paratype: (A) leg I, antiaxial view;
(B) leg II, anterior view; (C) leg III, anterior view; (D) leg IV, anterior view; (E) gnathosoma, ventral
view.
Figure 15. Thyreophagus holda sp. n., heteromorphic deutonymph, paratype: (A) leg I, antiaxial
view; (B) leg II, anterior view; (C) leg III, anterior view; (D) leg IV, anterior view; (E) gnathosoma,
ventral view.
Animals 2025,15, 357 22 of 28
Animals 2025, 15, x FOR PEER REVIEW 25 of 32
Figure 16. Thyreophagus holda sp. n., phase contrast images: (A) female holotype; (B) female para-
type; (C,D) male paratypes; (E,F) heteromorphic deutonymph paratypes.
Figure 16. Thyreophagus holda sp. n., phase contrast images: (A) female holotype; (B) female paratype;
(C,D) male paratypes; (E,F) heteromorphic deutonymph paratypes.
Heteromorphic deutonymphs of Th. holda sp. n. are similar to Th. leclercqi (Fain,
1982) [
28
] and Th. australis (Clark, 2009) [
29
] in terms of their ovoid bodies, which are
1.3–1.4 times
longer than they are wide, compared to the elongate bodies (more than
1.7 times longer than they are wide) in other species. Th. holda differs from Th. leclercqi
by their short setae hT I, which are more than 2 times shorter than gT I (less than 2 times
shorter in Th. leclercqi). Th. holda differs from Th. australis by their setae kT III, which lack a
prong (with a distinct prong in Th. australis), and the absence of setae wa I-II (present in
Th. australis).
Based on Figure 1in reference [
11
], Barbosa et al. [
30
] and Klimov et al. [
5
] suggested
that one of the diagnostic character states of Th. holda sp. n. heteromorphic deutonymphs
is the position of the opisthonotal gland openings, which are approximately equidistant
from setae c
3
and c
p
. However, our study showed that only one specimen of Th. holda
sp. n. shows this character state, while the remaining specimens from the same culture
have the usual arrangement of these openings (gla is much closer to ventral seta c
3
than to
dorsolateral seta c
p
). Based on these findings, we suggest that this character is variable and
should not be considered diagnostic for Th. holda sp. n.
Animals 2025,15, 357 23 of 28
Animals 2025, 15, x FOR PEER REVIEW 26 of 32
Figure 17. Thyreophagus holda sp. n., holotype (A,F,J) and paratypes (B–E,I,H–M), phase contrast
images: (A,B) female prodorsal shields; (C) male prodorsal shield; (D) female leg I, anterior view;
(E) female leg II, posterior view; (F) female leg III, posterior view; (G) female leg III, anterior view;
(H) male leg III, posterior view; (I) male leg I, anterior view; (J) spermatheca; (K) heteromorphic
deutonymph leg I, anterior view; (L) heteromorphic deutonymph leg II, anterior view; (M) hetero-
morphic deutonymph legs III–IV.
Figure 17. Thyreophagus holda sp. n., holotype (A,F,J) and paratypes (B–E,I,H–M), phase contrast
images: (A,B) female prodorsal shields; (C) male prodorsal shield; (D) female leg I, anterior view;
(E) female leg II, posterior view; (F) female leg III, posterior view; (G) female leg III, anterior view;
(H) male leg III, posterior view; (I) male leg I, anterior view; (J) spermatheca; (K) heteromorphic deu-
tonymph leg I, anterior view; (L) heteromorphic deutonymph leg II, anterior view; (M) heteromorphic
deutonymph legs III–IV.
Animals 2025,15, 357 24 of 28
Animals 2025, 15, x FOR PEER REVIEW 27 of 32
Figure 18. Thyreophagus entomophagus (Laboulbène and Robin, 1862) (A,B,D–F,H), PBK20-0101-
199.SM38, not neotype (A,D,E,H), Birmingham specimens (B,F) and Th. holda sp. n. (C,G,I), phase
contrast images (A–D,F,G), DIC images (E) and lineal drawings (H,I) distinctive morphological
characteristics: (A–C) punctated posterior part of female prodorsal shields; (D–F) female tarsi III
with flaened, buon-shaped setae v, anterior view; (G) female tarsi III with spiniform setae v, an-
terior view. Abbreviations: a—rounded and elongated cells; b—lineate cells; s, v, q—tarsal ventral
setae.
Heteromorphic deutonymphs of Th. holda sp. n. are similar to Th. leclercqi (Fain, 1982)
[28] and Th. australis (Clark, 2009) [29] in terms of their ovoid bodies, which are 1.3–1.4
times longer than they are wide, compared to the elongate bodies (more than 1.7 times
longer than they are wide) in other species. Th. holda differs from Th. leclercqi by their short
setae hT I, which are more than 2 times shorter than gT I (less than 2 times shorter in Th.
leclercqi). Th. holda differs from Th. australis by their setae kT III, which lack a prong (with
a distinct prong in Th. australis), and the absence of setae wa I-II (present in Th. australis).
Based on Figure 1 in reference [11], Barbosa et al. [30] and Klimov et al. [5] suggested
that one of the diagnostic character states of Th. holda sp. n. heteromorphic deutonymphs
is the position of the opisthonotal gland openings, which are approximately equidistant
from setae c3 and cp. However, our study showed that only one specimen of Th. holda sp.
Figure 18. Thyreophagus entomophagus (Laboulbène and Robin, 1862) (A,B,D–F,H), PBK20-0101-
199.SM38, not neotype (A,D,E,H), Birmingham specimens (B,F) and Th. holda sp. n. (C,G,I), phase
contrast images (A–D,F,G), DIC images (E) and lineal drawings (H,I) distinctive morphological
characteristics: (A–C) punctated posterior part of female prodorsal shields; (D–F) female tarsi III with
flattened, button-shaped setae v, anterior view; (G) female tarsi III with spiniform setae v, anterior
view. Abbreviations: a—rounded and elongated cells; b—lineate cells; s,v,q—tarsal ventral setae.
4. Discussion
The mite Thyreophagus entomophagus is an economically important species widely
used as factitious prey in the industrial rearing of phytoseiid mites for biocontrol applica-
tions [
1
,
5
–
7
,
31
]. This is a cosmopolitan species [
32
] that has been reported in the literature
from various habitats: entomological collections [
9
,
10
,
33
]; flour [
34
–
41
]; dry Spanish fly
Lytta vesicatoria harvested for medicinal use [
35
]; vanilla pods and saffron for medicinal
use [
42
]; medicinal plants, including ergot and cardamom; stimulants; spices; food prod-
ucts and fodders (rye and wheat bran) [
33
]; poultry meal [
43
]; on ergot of rye [
42
]; in bird
nests [
33
,
43
]; bracket fungi Fomitopsis betulina [
44
]; and various materials in beehives of
Apis mellifera [
45
,
46
] in association with scale insects [
36
,
47
,
48
], coccids [
48
], and trogid
beetles [49].
Animals 2025,15, 357 25 of 28
However, the reliability of this name may be compromised due to several factors: the
absence of original type specimens, difficulties in interpreting the classical species diagnosis
by A. Fain [
10
], which is not entirely accurate with respect to actual specimens (e.g., the
shape of ventroterminal setae uand vIII–IV), the presence of closely related species such as
Th. holda and Th. leclercqi, and the misidentified and divergent GenBank sequence that lacks
accompanying morphological information. As a result, previous reports in the literature
require verification. Here, to standardize the usage of the name Thyreophagus entomophagus,
we propose designating a neotype for which both detailed morphological and sequence
data are available.
Our research confirms that Th. entomophagus lacks the heteromorphic deutonymph
stage (Figure 1), a trait consistently observed in laboratory populations cultured indepen-
dently across multiple labs and biocontrol facilities (our observations). Earlier reports from
Germany indicated the presence of heteromorphic deutonymphs [
11
], but our study did not
corroborate this, suggesting that the German population represents a different species, Th.
holda sp. n. The absence of heteromorphic deutonymphs is advantageous for mass produc-
tion, as it simplifies the life cycle by eliminating an energetically costly developmental stage.
These deutonymphs are also heavily sclerotized, making them less palatable to predators.
However, Th. entomophagus remains a sexual species, which is a less desirable trait for mass
production since asexual species reproduce more quickly, generating only females. The
only known species that is both asexual and lacks the heteromorphic deutonymph stage is
Th. plocepasseri [22].
Our preliminary phylogenetic analysis suggests that the two traits important for
biocontrol—asexual reproduction and direct development (i.e., absence of the heteromor-
phic deutonymph stage)—are derived, having emerged after the most recent common
ancestor of Thyreophagus (Figure 2). While speculative, asexual reproduction may provide
an advantage in subcortical habitats like fallen branches, where predator activity is low and
the “Red Queen” dynamics [
50
,
51
] are absent. The lack of heteromorphic deutonymphs
might also confer an advantage in vertebrate nests, where animals can transport branches,
facilitating long-distance dispersal. Short-distance dispersal, such as within a nest, can
be accomplished by the mites themselves, particularly the more mobile immature stages.
In contrast, inseminated females tend to burrow into tunnels where they produce large
numbers of eggs as slow-moving, ovigerous individuals.
Our study not only standardizes the nomenclature of Thyreophagus entomophagus, a
species widely utilized in the rearing of predatory mites, but also proposes identifying
species that combine advantageous traits, such as asexual reproduction and a direct life
cycle, for further bioprospecting efforts.
5. Conclusions
The mite Thyreophagus entomophagus is a cosmopolitan mite species with significant
economic importance in biocontrol applications. Its diverse habitat range and frequent
association with various food products and environments suggest its great adaptability.
However, the taxonomic status of this species name is questionable due to inconsistencies in
historical species identifications, the absence of original type specimens, and misidentified
GenBank sequences. To address these issues and to standardize the nomenclature, we
propose the designation of a neotype with both morphological and genetic data available.
Our research shows that Th. entomophagus lacks the heteromorphic deutonymph stage,
an advantageous trait for mass production, streamlining its life cycle. Nonetheless, this is a
sexual species, a feature less desirable for mass-rearing programs. Phylogenetic evidence
suggests that asexuality and the absence of heteromorphic deutonymphs are derived traits,
likely advantageous in specific ecological niches like subcortical habitats or vertebrate
Animals 2025,15, 357 26 of 28
nests. These findings offer important insights into the evolutionary traits that improve
the effectiveness of Th. entomophagus and related species in biocontrol settings. Our study
specifically suggests the need for additional bioprospecting efforts to find new candidate
species for biocontrol applications that exhibit both asexual reproduction and the absence
of heteromorphic deutonymphs.
Author Contributions: Conceptualization, P.B.K. and V.B.K.; methodology, P.B.K., V.B.K., A.A.K.
and V.A.K.; validation, P.B.K., V.B.K., A.A.K., V.A.K., J.M., M.V.A.D., D.V., I.G. and A.P.; formal
analysis, P.B.K. and V.B.K.; investigation, P.B.K., V.B.K., A.A.K., V.A.K., J.M., M.V.A.D., D.V., I.G.
and A.P.; resources, P.B.K., V.B.K., A.A.K., V.A.K., J.M., M.V.A.D., I.G. and D.V.; data curation, P.B.K.
and A.A.K.; writing—original draft preparation, V.B.K.; writing—review and editing, P.B.K., V.B.K.,
A.A.K., V.A.K., J.M., M.V.A.D., D.V., I.G. and A.P.; visualization, V.B.K., A.A.K., V.A.K., J.M., M.V.A.D.,
I.G. and D.V.; supervision, P.B.K.; project administration, A.A.K. and A.P.; funding acquisition, 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 conflicts of interest.
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