An insight into the tribe Hexathrombiini (Actinotrichida: Trombidioidea, Microtrombidiidae, Eutrombidiinae) with new data on host-parasite interaction

Abstract The status of the genera in the small microtrombidiid mite tribe Hexathrombiini is reevaluated. Type specimens representing all genera were studied and diagnostic characters for Hexathrombiini are reviewed, summarized, and new data and a key to the genera in the tribe are provided: Alhamitrombium, Beronium, Hexathrombium and Hoplothrombium. Hexathrombium is the most speciose genus in the tribe and species recorded from South America are compared, as well as those with a divided pygidial plate. A provisional key to species assigned to Hexathrombium is provided. Finally, Hexathrombium abirami was captured in Peru parasitizing a bright metallic tiger beetle (Tetracha fulgida). A total of 361 larvae were removed parasitizing a single carabid host; this is the highest load of parasites reported in terrestrial Parasitengona mites associated with arthropods. This capture represents a new record of Hexathrombiini mites for Peru. A redescription of He. abirami using all specimens available to date is included.


Introduction
The tribe Hexathrombiini Fain & Drugmand, 1993 (see Discussion section for authorship) was erected within Eutrombidiinae Thor, 1935 to accommodate Hexathrombium Cooreman, 1944, Beronium Southcott, 1986and Hoplothrombium Ewing, 1925. Later, Mayoral & Barranco (2005a described Alhamitrombium in Hexathrombiini. Fifteen species, variously distributed within the six zoogeographic regions, have been described in the tribe to date. All remain known exclusively from larvae that parasitize carabids (including formerly distinguished Cincindelidae) and staphylinid beetles. There are two exceptions: Hoplothrombium quinquescutatum Ewing, 1925 reported on a single specimen adhering to an Oribatida mite taken from the stomach of a toad (Ewing 1925), and Hexathrombium southcotti Zheng, 1997 captured from ichneumonid wasps (Zheng 1997;Felska et al. 2018). Haitlinger (1999) considered the latter to be probably an accidental host. Most species are known only from few specimens, and they have been recorded from single localities; as an exception, more than 40 specimens of Beronium laemostenis Mayoral & Barranco, 2005 have been collected from 10 different localities (caves) in Spain (Mayoral & Barranco 2005b;Mayoral 2013).
The genus Beronium Southcott, 1986 was erected by Southcott (1986) to accommodate Hoplothrombium coiffaiti Beron, 1973. This genus is known from the south-western Palaearctic, and comprises Beronium coiffaiti (Beron, 1973), Beronium veronicae Haitlinger, 1994 and B. laemostenis (Beron 1973;Haitlinger 1994;Mayoral & Barranco 2005b;Mayoral 2013). The monotypic genera Hoplothrombium Ewing, 1925, andAlhamitrombium Mayoral &Barranco, 2005 are known from the Nearctic and Palaearctic regions, respectively (Ewing 1925;Vercammen-Grandjean 1967;Mayoral & Barranco 2005a); they include the species Ho. quinquescutatum and Alhamitrombium tetraseta Mayoral & Barranco, 2005. Welbourn (1983 listed Ho. quinquescutatum and Ho. coiffaiti under Hoplothrombium, in addition to the species Trombidium cicindelae Floch & Abonnenc, 1941 and He. spatuliferum. The generic affiliation of "coiffaiti", "cicindelae" and "spatuliferum" was not followed by subsequent authors since those specimens do not comply with the generic diagnosis of the genus and Hoplothrombium has therefore remained monotypic. Here, we redescribe Hexathrombium abirami from new specimens collected in Peru, parasitizing Tetracha fulgida (Klug, 1834) (Carabidae). The load of parasites reported here for T. fulgida is the highest infestation record of terrestrial Parasitengona mites associated with arthropods. The diagnostic traits for the four genera in Hexathrombiini were reviewed and representatives of the different genera were studied (except Hoplothrombium that we relied on published descriptions). An updated list of relevant characters, a key to the genera in Hexathrombiini and a provisional key to Hexathrombium species are provided.
Larvae were detached from the host with an entomological pin and mounted on microscopic slides in Hoyer's medium. The overall number of larvae attached to the host was counted and the measurements were taken using NIS-Elements Br software, under a Nikon Eclipse E600 microscope coupled with DS-Fi1 camera system. Photos were taken with a DS-Fi3 camera attached to a Nikon Eclipse 80i microscope and the images were stacked using Helicon Focus software (© Helicon Soft Ltd., 2000). All measurements are given in micrometers. The terminology follows Southcott (1993), Vercammen-Grandjean (1967) and Wohltmann et al. (2007). The material (slide-mounted and alcohol preserved larvae; host specimen) is deposited in the Department of Invertebrate Systematics and Ecology, Wrocław University of Environmental and Life Sciences, Poland (DISE WUELS). Five larvae (slide-mounted) are deposited at the Museo de Historia Natural, Universidad Nacional Mayor de San Marcos, Lima, Peru (MUSM).
For the purpose of comparison and re-appraisal of characters used in the diagnosis of Hexathrombiini, the type material of He. abirami, He. lubomirae, He. marittae, He. mamerti, B. veronicae, B. laemostenis and A. tetraseta was studied. The references to other members of the tribe are based on original descriptions.
Active postlarval forms. Not known.

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J. Mąkol et al. Type genus. Hexathrombium Cooreman, 1944 Genera included. Hexathrombium Cooreman, 1944;Hoplothrombium Ewing, 1925;Beronium Southcott, 1986;Alhamitrombium Mayoral & Barranco, 2005. Remarks. The position of seta 1a may indistinctly vary in all genera in question, as the seta is inserted close to the coxal plate margin and its actual location in the mounted specimens may be considered dubious. Vercammen-Grandjean (1967) considered 1a, observed within the coxal plate in Ho. quinquescutatum, a "migrated sternal seta". The chaetotaxy of terminal leg segments (Ge -Ta I, Ge -Ta II, Ge -Ta III), with special reference to specialized setae, has been variously interpreted due to the difficulties in their visualization, specially their actual nature and presence. The more detailed close-up to the setae, should be carried out to clarify their actual state in all members of the genus.
The highly modified termination of tarsus III is similar in all Hexathrombiini and differs to those observed in Eutrombidiini. Southcott (1993) described in Hexathrombiini the presence of a branched seta ("penicala" sensu Southcott 1993) on the dorsodistal projection of tarsus III, and also two claws located ventrodistally. From those, the median claw is long, falciform and simple, whereas the posterior claw (i.e. smilum) (anterior claw sensu Southcott 1993) is in the form of a short and thick hook with a dorsal spur-like process. In Eutrombidiini, there is a moustache-shaped seta about 3/4 along the dorsum of tarsus III ("cultala" sensu Southcott 1993), a multipronged seta with several large setules arising dorsally at the distal end of tarsus ("dumala" sensu Southcott 1993), and a thickened setae with setules in the ventrodistal portion of the tarsus ("calcanala" sensu Southcott 1993) (Southcott 1993). Husband and Wohltmann (2011) observed in Eutrombidium the presence of a scopa on tarsus III ("cultala" sensu Southcott, 1993) and lophotrix ("dumala" sensu Southcott 1993). Despite the different position of branched, pectinate seta in Hexathrombiini (seta shifted to a dorsodistal position), its structure is congruent with the lophotrix observed in Eutrombidiini and also in some other microtrombidiid mites. The short and thick inner claw located ventrodistal and covered with few bristles should be considered a smilum. Another modified seta, a scopa, is absent in Hexathrombiini. Cooreman, 1944Hexathrombium Cooreman, 1944: 1. Hexathrombium: Southcott 1993: 945. Diagnosis (after Southcott, 1993.
Type species. Hexathrombium spatuliferum Cooreman, 1944 Species included and country records. He Welbourn (1983) reported two presumably new species of Hoplothrombium from Ecuador; they were never formally described, however, they both should be placed in Hexathrombium (Cal Welbourn, pers. comm.).
Dorsal idiosoma (Figure 2) oval, slightly constricted behind the level of coxae III, rounded at anterior and posterior termination. Integument, except for sclerites, folded in lines. Scutum (Q1) (Figures 2 and 3) pentagonal in outline, rounded anteriorly, bearing paired, non-sensillary setae AM, AL, PL, and a pair of sensilla (S). Antero-lateral parts of scutum with linear pattern, medial part of the sclerite porous. Posterior margin almost straight, bordered with delicate lamellar band. Additionally, small octal-shaped or semi-oval mark, probably representing the less sclerotized part of the sclerite, present antero-laterally in lower layer of scutum (or [?] immediately under the scutum surface), on each side of symmetry axis. Bases of sensilla located between AL and PL bases, closer to PL and slightly shifted to medial position. AM smooth or with 1-2 barbs, AL, PL and S with few barbs along entire stem length. Paired eye lenses (Figure 2), each pair on a common, weakly sclerotized plate located close to the postero-lateral margins of the prodorsal sclerite (Q1). Scutellum (Q2) trapezoidal in shape, with a pair of c 1 setae. Second and third scutellum (Q3, Q4) with a pair of d 1 and e 1 setae, respectively (in one specimen (7802/13) with duplicated seta e 1 on Q4 sclerite); both sclerites rectangular in shape, with rounded corners. Setae e 1 located close to the anterior margin of Q4. The fifth shield (Q5) divided into two separated, oval plates, each bearing a h 1 seta. fD: (2) 4-(2)4-(2)4-6-(1 + 1)2 = 28. Setae in rows C, D, E, F, except for c 1 , d 1 , e 1 , located on small platelets; stems of setae in C-F rows only slightly narrowed at termination and covered with short barbs along the entire length. Setae c 3 , d 3 , e 3 slightly shorter than other setae in the respective rows C-E. Setae h 1 and h 2 longer than the preceding setae, distinctly narrowed apically; h 2 inserted in roundish plates (of diameter similar to the width of h 1 plates, i.e. Q5/2); shafts of h 2 slenderer than those of h 1 and with distinct setules.
Ventral idiosoma (Figure 4). Coxal plates (I -triangular in outline, II -rectangular, rounded at base, III -square-shaped in outline) well sclerotized along the anterior border; the posterior border weakly marked or discontinuous. Anterolateral part of coxa I frame as well as its distal portion strongly sclerotized ( Figure 4); sclerotization of the respective parts of coxae II and III less pronounced. Claparède's organs (clp) present at posterolateral corner of coxa I. Coxa I with two setae, medial coxala (1a) normal, nude, tapering, placed on cuticular band forming the medial extension of the most sclerotized part of coxal frame; lateral coxala (1b) modified, bilobed (Figure 6(a)); coxa II and coxa III with one modified, bilobed seta each (2b and 3b, respectively); all modified coxalae (1b, 2b, 3b) with horizontally diverged processes (lobes); lobes indistinctly narrowing at termination and slightly extending beyond the setal base (widely diverging in 2b, making the seta the widest in comparison with 1b and 3b). Setae 3a (20 long) simple, slender, nude, and located between coxae III. Supracoxalae of coxae I (elc I) not detectable. fV: 4-4u-4-2 = 14. Ventral setae tapering, with thinner shafts than dorsal setae. Anal opening surrounded with membraneous valves, anal sclerites absent. Pre-anal protuberance (tubercle) present, circle-like, similar in diameter to the length of anus, and located anterior to excretory slit. The tubercle, slightly elevated above the idiosoma surface, surrounded with more sclerotized, porous sides.
Legs ( Figure 5(a-c)). Segmentation formula 6-6-6. For leg chaetotaxy see Table II. Normal setae on legs setulated to smooth. Robust, fan-like seta present in distal part of tarsus II. Tarsi I and II terminated with two claws and empodium. Claws similar in length, covered with onychotrichs. Empodium claw-like, slightly spatulate distally. Tarsus III highly modified at termination, with lophotrix in dorsodistal position and two claws located ventrodistal. Lophotrix composed of one branch, with one long, proximal, secondary branch on one side and with several, gradually shortening, secondary and tertiary branches on the other (Figure 7(a)). Inner claw short, robust, with small spurs; medial claw long, falciform.

An insight into the tribe Hexathrombiini
Remarks. The new specimens collected in Peru and studied in this work were compared with the type material of He. abirami, and also with other species of Hexathrombium, with an emphasis on the South American species in the genus. A comparison of the metric data of the Peruvian specimens with the type series is included in Tables I and II. The differences observed in metric data between the type series of He.

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J. Mąkol et al. abirami and specimens collected in Peru (Table I) are attributed to intraspecific variations. These variations may be a result of the differences in geographic locations, ecological factors, and the mounting quality of the type material that may have affected the reliability of some measurements.
Based on the reexamination of the holotype and two paratypes of He. abirami Haitlinger, 1997, some characters redescribed here differ from those reported in the original description. A pair of eyes (each composed of two lenses placed on a common sclerite) are present at each side of scutum (original description, Figure 1, p. 82: one lens present at posterolateral margin of scutum, on each side of symmetry axis). Setae AL = 54 (original description: AL broken in all specimens); fn Fe = 6-5-4 (original description: fn Fe = 5-5-4); fn Ge = 5-2-2 (original description: fn Ge = 5-5-3); fn Ti = 6-5-5 (original description: fn Ti = 8-7-5); fsol Ti = 2-2-0 (original description: 2-1-0); vestigiala on tibia I, 2 eupathidia on tarsus I, famulus on tarsus II present. Preanal tubercle present.   Parasitism of specimens collected in Peru. Altogether, 361 larvae were found parasitizing a single carabid host, T. fulgida (Figures 8(a-d) and 9(a-d)). Eight were attached to the head (including labrum) ( Figure  8(a-d)), 86 to the pronotum (Figure 8(a)), 183 to the elytra (22 under elytra) (Figure 8(a) and 9(a),(c)), 50 to the thorax and venter of abdomen (Figures 8(a-d) and 9(b)), and 26 were distributed among various leg segments (Figure 9(d)); for the remaining eight larvae the attachment sites were not recorded. Parasites seem to show a preference for the dorsal parts of the host and there was no preference for softer cuticle areas (including the dorsal abdomen), and instead the preference was for externally exposed areas that seem to be out of the reach of the host legs. Larvae were relatively firmly attached, and except for eight specimens, the detachment had to be performed with an entomological pin and forceps.
Type species. Hoplothrombium coiffaiti Beron, 1973  Remarks. The absence of eyes, hitherto reported for Beronium, constitutes one of the main differences which allow to distinguish between Beronium spp., Alhamitrombium, spp. and Hexathrombium spp. In B. laemostenis and B. veronicae, ocular sclerites are present and they are elongated in shape, narrowing anteriorly and truncated posteriorly (Figure 10(a)); they are located adjacent to the posterolateral margins of the scutum, and in a similar location to the ocular plates observed in other trombidioid genera (including Hexathrombium). The plates are punctated over the entire surface and surrounded by folded in lines cuticle. The shape of these sclerotized structures resembles the plates surrounding the eye lenses in Hoplothrombium (Vercammen-Grandjean 1967: 5).

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J. Mąkol et al. the main stem; one branch nude, the other one with several (c. 7) secondary branches (Figure 7(d)). Mayoral & Barranco (2005b) in their key to Beronium spp., and followed by Mayoral (2013), referred to the presence of one solenidion on genu I and the lack of solenidia on genu III as characters to differentiate B. veronicae from the other two species in the genus. This was based on the original description of B. veronicae, in which Haitlinger (1994) described one solenidion on genu I and no solenidia on genu III. The present reexamination of the type material of B. veronicae allowed to confirm the presence of one solenidion on Ge I, but there is also a solenidion present on Ge III. Therefore, the presence of one solenidion on genu III should be treated as character shared by all three species assigned to Beronium. It is still possible to separate B. laemostenis from B. veronicae based on the number of solenidia on Ge I (2 vs. 1) (see Table  V). Future reexamination of B. coiffaiti should help to verify the taxonomic status of this species.
The leg chaetotaxy of all three nominal species assigned to the genus is provided in Table V. The differences between the members of Beronium may pertain also to the number of setae in fV formula (B. coiffaiti -19, B. laemostenis -18-23, B. veronicae -22(23)).
presence of 1b which is indistinctly bifid at termination ( Figure 6(b)). This character (seta 1b) should be verified in B. coiffaiti, that according to the description of Beron (1973) is dilated. There are very little data available about the biology and ecology of Hexathrombiini. The infestation of the bright metallic tiger beetle (T. fulgida) by the parasitic mite larvae He. abirami may have taken place on the ground -in wet sand or mud -with sparse or no vegetation. This is the natural habitat of adults of T. fulgida. Remarkably, this beetle has been proposed as a natural predator of the pest mole cricket in golf courses (T. fulgida in GBIF 2019). The number of mite larvae recorded (361) on a single host specimen reported in this study is the highest number of parasites recorded in arthropodassociated terrestrial Parasitengona mites. The next two highest values reported were also recorded for Eutrombidiinae larvae. Severin (1944) reported the presence of 175 larvae of Eutrombidium locustarum on Dissosteira carolina (Orthoptera), and Pérez-Espinoza & Moreno Salas (2016) reported 186 larvae of He. cf. marittae infesting Ceroglossus buqueti (Coleoptera: Carabidae). The preferred attachment site of Hexathrombium spp. larvae varied across different hosts, despite the similarities in the hosts' body (they are closely related species). Around 50% of larvae collected in this study were attached to elytra. No larvae were attached to dorsal abdomen, which is consistent with the observations of Pérez-Espinoza and Moreno Salas (2016). Almada and Cédola (2017) reported that most larvae displayed a preference for the ventral thorax; in our study, only around 14% of larvae were attached to the thorax and venter abdomen.
Several morphological traits (presence of stephanostome facilitating a firm attachment to the host, relatively short legs), behavioral (preference to attachment sites with lower accessibility to hosts' legs) and ecological (relatively narrow host spectrum) are of significant adaptive advantage and may reflect the relatively long co-evolution of Hexathrombium and other Hexathrombiini with their hosts.