Thyreophagus tauricus sp. n., a New Subcortical Mite Species (Acari: Acaridae), with a COX1 DNA Sequence Analysis of Several Economically Important Species of Thyreophagus

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 s III 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.

Most commonly, mite taxonomists collect and describe females, while other taxonomically 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][17][18].
Because of its economic importance and the presence of potentially interesting biological 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 experiments 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.

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 •  Images were taken from multiple focal planes and assembled in Helicon Focus 7.6.4Pro (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 microphotographs 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).Publicationquality microphotographs were taken using an Axio Imager A2 (Carl Zeiss, Oberkochen, Germany) compound microscope equipped with DIC and phase contrast optics and an Axiocam 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.

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.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 spermatheca.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.
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 s III-IV (Figure 9F) (vs.well-developed spiniform (not much less than v and 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

Discussion
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.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 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 significance 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 pollinators.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 biodiversity, 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 Thyreophagus species by the following character states: adults of Th. tauricus are distinct by the presence of flattened, button-shaped or minute spiniform setae s III-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.

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.