Philipp E. Chetverikov

Comparative confocal microscopy of internal genitalia of phytoptine mites (Eriophyoidea, Phytoptidae): new generic diagnoses reflecting host-plant associations

Confocal laser scanning microscopy (CLSM) methods are still rarely used by acarologists although they are very appropriate for studying minute arthropod pests, especially eriophyoid mites. In this paper, the female reproductive system of phytoptines, including the bud mite Phytoptus avellanae, the well-known pest of hazelnut, was studied using CLSM and resulted in new interpretations of the functioning anatomy of phytoptid genitalia. Comparison of cuticle-lined internal genitalia, based on novel CLSM-based 2D and 3D imaging, and multivariate analysis of morphometric measurements, show that two basic types of internal genitalia can be found within Phytoptinae: one type in phytoptines associated with monocotyledoneous hosts (especially Cyperaceae and Asparagaceae), and another one in those associated with various dicotyledoneous hosts. Phytoptines from monocots (genera Oziella and Acathrix and Phytoptus “caricis” sp. group) possess a spherical distal part of the spermathecal tube and a semitriangular transverse genital apodeme, whereas phytoptines from dicots (genus Phytoptus “avellenae” sp. group) possess an elongate distal part of the tube and a trapezoidal apodeme. These differences in the internal genitalic anatomy were used for modifying the diagnosis of phytoptine genera (Phytoptus, Oziella and Acathrix), and reorganizing the Phytoptinae, resulting in new synonymies: 11 species were transferred from genus Phytoptus“caricis” sp. group to the genus Oziella.

Oziella sibirica (Acari: Eriophyoidea: Phytoptidae), a new eriophyoid mite species described using confocal microscopy, COI barcoding and 3D surface reconstruction

Oziella sibirica sp. nov., collected from sedges (Cyperaceae: Carex macroura) in Siberia, Russia, is herein described based on the external morphology of all active instars using primarily conventional phase contrast microscopy, and on the female internal genitalia and prodorsal shield design using confocal laser scanning microscopy (CLSM) imaging and a 3D modelling technique. A partial mitochondrial COI gene sequence of O. sibirica sp. nov. is also provided, through GenBank, and this represents the first published record of any gene sequence data for the family Phytoptidae. We present remarks on the phylogenetic significance of the position of setae 3a in immature instars of eriophyoids and on the ontogenic variability of the empodium morphology of O. sibirica sp. nov. Using this species as a model, we propose a method for describing the internal genitalia of eriophyoids based on CLSM. We advocate the use of CLSM imaging as a new, relatively simple technique for observing and describing the internal genitalia of eriophyoids, as these largely unexplored genitalic structures may provide phylogenetically meaningful information for improving the classification of this poorly understood group of mites. In addition, CLSM may complement conventional light microscopy techniques in facilitating the interpretation of external structures such as body ornamentation or chaetotaxy.

Distal oviduct and genital chamber of eriophyoids (Acariformes, Eriophyoidea): refined terminology and remarks on CLSM technique for studying musculature of mites

The general morphology of cuticle-lined internal genitalia and oviduct is analyzed in intact females of the phytophagous mites, Loboquintus subsquamatus and Trisetacus cf bagdasariani (Acari: Eriophyoidea) using tetramethylrhodamine B isothiocyanate-phalloidin, three anaesthetics (magnesium sulphate, lidocaine and CO2-enriched water) and confocal laser scanning microscopy (CLSM). This is the first protocol adopted for CLSM studying musculature of mites. Revision of the previous terminology of eriophyoid internal genitalia from Nuzzaci and Alberti (Eriophyoid mites: their biology, natural enemies and control. World crop pests 6. Elsevier, Amsterdam, pp 101–150, 1996) resulted in the refinement of the terms “distal oviduct”, “genital chamber” and “spermatheca”. Relative position of the elements of cuticle-lined internal genitalia is discussed and a generalized 3D model and animation (available on-line as supplementary material) of eriophyoid genital apparatus are provided. The wall of eriophyoid oviduct contains strong longitudinal muscles attached to the cuticle genital chamber with folded walls. When the egg is being extruded by contraction of the oviduct muscles, it forms lobes corresponding to the internal topography of the oviduct and genital chamber; these lobes invaginate inward from the gonopore, resulting in the “flower-shaped” figures rarely observed in slide-mounted mites. Gnathosomal muscles (cheliceral muscles and extrinsic muscles of palps) and opisthosomal muscles D1 of Loboquintus mites are attached to the three posterior depressions near the rear prodorsal shield margin. Prospects of CLSM approach for studying different aspects of mite morphology are briefly discussed.

Redescription of an early-derivative mite, Pentasetacus araucariae (Eriophyoidea, Phytoptidae), and new hypotheses on the eriophyoid reproductive anatomy

A unique set of plesiomorphic characters, and its association with an ancient gymnosperm, Araucaria araucana, have made Pentasetacus araucariae a putative relict of a lineage of gymnosperm-associated mites, itself possibly basal to all extant eriophyoids. However, the suboptimal description of this species is impeding morphological comparisons with other species, which are fundamental to eriophyoid systematics. Herein, we designate a female lectotype from syntype specimens and use additional non-type material to redescribe P. araucariae based on external and internal anatomy using different microscopic and 3D reconstruction techniques. Contrarily to statements in the literature, P. araucariae has undivided empodia in all instars, short spermathecal tubes, and large, globose spermathecae in females, as well as rudimentary genital fovea in immatures. In addition, males of P. araucariae were shown to have genitalic attributes similar to a species of Trisetacus studied in parallel, including two reservoir-like structures, which may represent parts of the genital chamber and of the ductus ejaculatorius, respectively, as well as paired testes and ducti deferentes. This is contrary to previous, limited knowledge on eriophyoids indicating that they possess a single testis. Although their short spermathecal tubes weaken the cladistic relationship between P. araucariae (Pentasetacinae) and conifer-associated Nalepellinae (e.g. Trisetacus) having long tubes, the structural similarities in male genitalia may reinforce it.

Gnathosomal interlocking apparatus and remarks on functional morphology of frontal lobes of eriophyoid mites (Acariformes, Eriophyoidea)

Comparative study of eriophyoid mite gnathosomata using different microscopic techniques shows presence of new dorsoproximal structures (here described as “interlocking apparatus”), which could consolidate mouthparts in rest (non-feeding) position and during feeding actions. Four types of the interlocking apparatus are described. Differences in the interlocking mechanisms coincide with the results of recent molecular phylogenetic studies of Eriophyoidea and provide additional support for the major clades revealed in those studies. Comparative analysis on the morphology of the frontal lobe of the prodorsal shield shows that mechanical protection of the motivator surrounding area and interlocking apparatus are possibly the primary function of the frontal lobe. Unusual shapes of the frontal lobe in selected eriophyoids (elongated, broadened, notched lobes) might result from additional functions that the frontal lobe can perform, e.g. providing additional guide for palps and stylets, or hermeticity of the space between dorsoproximal palpcoxae during feeding.

Comprehensive phylogeny of acariform mites (Acariformes) provides insights on the origin of the four-legged mites (Eriophyoidea), a long branch

Eriophyoid, or four-legged mites, represent a large and ancient radiation of exclusively phytophagous organisms known from the Triassic (230 Mya). Hypothesizing phylogenetic relatedness of Eriophyoidea among mites is a major challenge due to the absence of unambiguous morphological synapomorphies, resulting in ten published hypotheses placing eriophyoids in various places in the acariform tree of life. Here we test the evolutionary relationships of eriophyoids using six genes and a representative taxonomic sampling of acariform mites. The total evidence analysis places eriophyoids as the sister group of the deep soil-dwelling, vermiform family Nematalycidae (Endeostigmata). This arrangement was supported by the rDNA and CO1 partitions. In contrast, the nuclear protein partition (genes EF1-α, SRP54, HSP70) suggests that Eriophyoidea is sister to a lineage including Tydeidae, Ereynetidae, and Eupodidae (Eupodina: Trombidiformes). On both of these alternative topologies, eriophyoids appear as a long branch, probably involving the loss of basal diversity in early evolution. We analyze this result by using phylogenetically explicit hypothesis testing, investigating the phylogenetic signal from individual genes and rDNA stem and loop regions, and removing long branches and rogue taxa. Regardless of the two alternative placements, (i) the cheliceral morphology of eriophyoids, one of the traits deemed phylogenetically important, was likely derived directly from the plesiomorphic acariform chelicerae rather than from the modified chelicerae of some trombidiform lineages with a reduced fixed digit; and (ii) two potential synapomorphies of Eriophyoidea+Raphignathina (Trombidiformes) related to the reduction of genital papillae and to the terminal position of PS segment can be dismissed as result of convergent evolution. Our analyses substantially narrow the remaining available hypotheses on eriophyoid relationships and provide insights on the early evolution of acariform mites.

Morphological support for a clade comprising two vermiform mite lineages: Eriophyoidea (Acariformes) and Nematalycidae (Acariformes)

Abstract A morphology-based parsimony analysis (50 taxa; 110 characters) focused on relationships among basal acariform mites places Eriophyoidea (formerly in Trombidiformes) within Nematalycidae (Sarcoptiformes). Although both taxa have worm-like bodies, this grouping is unexpected because it combines obligate plant inhabitants (Eriophyoidea) with obligate inhabitants of deep-soil or mineral regolith (Nematalycidae sensu stricto). The Eriophyoidea + Nematalycidae clade, which is strongly supported (Bremer =5; bootstrap =85%), retains moderately good support (Bremer=3; bootstrap=66%) when three ratio-based characters pertaining to body shape are excluded. A total of eleven unambiguous synapomorphies unite all or some of Nematalycidae with Eriophyoidea. These include an annulated opisthosoma, an unpaired vi seta on the prodorsum, fusion of the palp trochanter with the palp femur, and a large relative distance between the anus and the genitalia. Three of the four Triassic genera of eriophyoid-like mites were also included in our analysis. Although all four genera have been tentatively placed within a new superfamily, we found no support for the monophyly of this group. One other interesting result of the analysis is the placement of a “living fossil”, Proterorhagia oztotloica (Proterorhagiidae), as sister to the rest of Acariformes. However, support for this relationship is weak.

Sierraphytoptines (Eriophyoidea: Phytoptidae) from relict eudicots: reassignment of Sierraphytoptus taiwanensus to a new genus Solenoplatilobus and refinement of generic diagnosis of Austracus

Abstract Mites Solenoplatilobus taiwanensus (Huang 2006) com. nov. (transferred from Sierraphytoptus) of the new monotypic genus Solenoplatilobus n. gen. (Phytoptidae: Sierraphytoptinae: Sierraphytoptini) possess a thin broad frontal lobe of prodorsal shield as well as the tibial solenidion I which were missed by previous authors. Sierraphytoptine mites Austracus havrylenkonis Keifer, 1944, studied in parallel and based on fresh material from Chile, have uniquely organized dorsal annuli which form thin imbricating cuticle plates overlapping one above another resembling roof tiles. The two studied early-derivative sierraphytoptine species inhabit relict eudicots from the families Nothofagaceae and Trochodendraceae which had wider geographic distribution in previous geological epochs. Early-diverging eriophyoids with a complete set of plesiomorphies inhabit arboreous plant species. Combination of plesiomorphic morphology and association with relict hosts justifies treating at least some of the phytoptids as relicts. Searching eriophyoids on relict vascular plants in refugia and biodiversity hotspots may result in finding new representatives of early-derivative taxa of Eriophyoidea which would be important for understanding early steps of evolution of these eriophyoid mites on plants.

Suboral fork: a newly discerned gnathosomal structure from the proboscis of eriophyoid mites (Acari, Eriophyoidea)

The infracapitulum of eriophyoid mites comprises a cone-like basal infracapitulum, containing a pharynx, and a distal infracapitulum, forming a proboscis ensheathing a bunch of stylets. A well-developed basal labral section was observed in all studied specimens. A newly discerned structure, the suboral fork, situated in the ventral part of the proboscis was discovered. It is larger in diptilomiopids and Nalepella and notably smaller in eriophyids and phytoptids. This structure presumably determines the site of piercing and functions in a similar way to the pressure foot of a sewing machine which controls the movements of a needle. In diptilomiopids the suboral fork might have an additional function: it is a stopper which prevents the proboscis from further penetrating into plant tissues. It is possible that the suboral fork is homologous with the labium of early derivative acariform mites. The proboscis might be a fusion product of the infracapitular lateral lips, malapophyses and the labium. The proboscis serves as a feeding structure in eriophyoids; two ways of sucking plant cell sap, depending on shapes of proboscis and labrum, are hypothesized. Further work is needed to draw conclusions on homologies and the function of all gnathosomal structures in eriophyoids.

A study of embryonic development in eriophyoid mites (Acariformes, Eriophyoidea) with the use of the fluorochrome DAPI and confocal microscopy

The embryonic development of four eriophyoid mite species, Cecidophyopsis ribis, Phytoptus avellanae, Oziella liroi and Loboquintus subsquamatus, has been studied with the use of fluorochrome DAPI and confocal microscopy. The first three nuclear divisions occur on the egg periphery (the groups of 2, 4, and 6 nuclei have been recorded), while the biggest part of yolk remains undivided. After four or five nuclear divisions all nuclei are situated only in one sector of the embryo, while other sectors contain only yolk suggesting possible meroblastic cleavage. Later, the formation of superficial blastoderm takes place. A few large yolk cells are situated inside the embryo. Germ band formation initiates as funnel-like cell invagination and leads to formation of a typical stage with four paired prosomal buds (chelicerae, palps, legs I and II). Each palp contains two lobes (anterior and posterior), the adult subcapitulum is presumably a fusion product of the anterior pair of the lobes. Neither rudiments of legs III and IV, traces of opisthosomal segments nor remnants of the prelarval exuvium under the egg shell were detected. Overall, the pattern of embryonic development in eriophyoids re-emphasizes the peculiarity of this ancient group of miniaturized phytoparasitic animals, and invites researches to pursue a deeper investigation of various fundamental aspects of this aberrant group of Acari. Further studies using various fluorescent dyes and transmission electron microscopy are needed to visualize plasma membranes and clarify the pattern of early cleavage of eriophyoids.