Disparity of Phoresy in Mesostigmatid Mites upon Their Specific Carrier Ips typographus (Coleoptera: Scolytinae)

Simple Summary This study investigated the phoretic relationship between mites and one of the most aggressive spruce bark beetles from Eurasia. During one season (April–September), bark beetles Ips typographus were collected with a specific synthetic aggregation pheromone. In the lab, we investigated the diversity of mites associated with I. typographus, mite preferences concerning the body parts of the beetles and how phoretic relationships change during the bark beetle’s flight season. Six phoretic mites species were found and 20% of beetles carried mites. Phoretic mite loads and the percent of beetles with mites were highest during the spring flight period. Phoretic mite species had specific preferences regarding their location on the body of the carrier. Abstract Ips typographus Linnaeus, 1758, the most important pest of Norway spruce (Picea abies Linnaeus, 1753) from Eurasia has damaged, in the last decades, a large area of forest in Romania. Associations between beetles and their symbiotic fungi are well known compared to beetle-mite relationships. The objectives of the study are to determine: (i) the diversity of mites species associated with I. typographus in a local outbreak from Central Romania; (ii) the mite’s preferences concerning the body parts of their carriers; and (iii) how phoresy changes during seasonal flight activity of the host. A total of 7896 adult I. typographus were analyzed and six mite species (both adults and immature stages) were found: Dendrolaelaps quadrisetus Berlese,1920, Proctolaelaps fiseri Samsinak, 1960, Trichouropoda polytricha Vitzthum, 1923, Histiostoma piceae Scheucher, 1957, Uroobovella ipidis Vitzthum, 1923, and Uroobovella vinicolora Vitzthum, 1926. Most mites were observed under the carriers’ elytra (46.8%), while 26.7% and 25.8% were seen on the thorax and elytral declivities, respectively. Mite phoresy peaked in the spring corresponding to the dispersal flight of the carrier. A smaller peak in phoresy occurred in the summer during the second beetle generation.


Introduction
Norway spruce (Picea abies) is among the most abundant and economically important tree species in Romania and Europe [1][2][3]. These trees are common attacked by bark beetles, the most aggressive of which being Ips typographus (Coleoptera: Scolytinae) [4][5][6]. In the Romanian Carpathian forests, I. typographus attack together with Pityogenes chalcographus, usually having two generations per year, the female being able to resume the attacks several times causing significant damage [5,[7][8][9][10]. Ips typographus and P. chalcographus attack weakened trees [6] where their offspring develop [11]. Adult beetles introduce fungi that alter the wood and, in some cases, provide nutrients for their Dendrolaelaps quadrisetus and T. polytricha were the most abundant and frequent mite species, together accounting for almost 85% of the total mites identified ( Table 1). The less abundant (subresidual) and frequent (accidental) species were H. piceae, U. vinicolora and P. fiseri. Phoresy rates indicate a significant effect of collection date (df = 19, f = 2.658, p < 0.001), but no effect of phoretic mites on male and female beetles (df = 1, f = 1.367, p = 0.740), even though some differences were noticed at the end of the beetle flight season (Figure 1).   [50].
Dendrolaelaps quadrisetus and T. polytricha were the most abundant and frequent mite species, together accounting for almost 85% of the total mites identified ( Table 1). The less abundant (subresidual) and frequent (accidental) species were H. piceae, U. vinicolora and P. fiseri. Phoresy rates indicate a significant effect of collection date (df = 19, f = 2.658, p < 0.001), but no effect of phoretic mites on male and female beetles (df = 1, f = 1.367, p = 0.740), even though some differences were noticed at the end of the beetle flight season (Figure 1). Of all of collected beetles with mites, 85.5% of beetles had one mite species, 13.5% had two mite species, and 1% carried 3 species. For beetles with only one species, 43.2% had (only) D. quadrisetus; 28.4% had T. polytricha; 13.4% had one of the two Uroobovella species and 0.5% had H. piceae.
The most frequent phoretic mite combinations were those of D. quadrisetus and T. polytricha (6.4% of beetles with mites), and T. polytricha and U. ipidis (4.1% of beetles with mites). Of all of collected beetles with mites, 85.5% of beetles had one mite species, 13.5% had two mite species, and 1% carried 3 species. For beetles with only one species, 43.2% had (only) D. quadrisetus; 28.4% had T. polytricha; 13.4% had one of the two Uroobovella species and 0.5% had H. piceae.
The most frequent phoretic mite combinations were those of D. quadrisetus and T. polytricha (6.4% of beetles with mites), and T. polytricha and U. ipidis (4.1% of beetles with mites).
Male and female beetles did not significantly differ concerning the position occupied by mites over time (df = 1, f = 1.129, p = 0.786).

Ips typographus Flight Activity
Beetle flight activity started when day temperatures (maximum temp. around noon) of 16.5 • were recorded, between 23 and 29 April, when the average capture was 6.13 ± 1.9 beetles per trap. The activity increased until 5 May (25.3 ± 6.8 beetles/trap) and, due to unfavorable conditions (temperature below 16.5 • C and rainy days), reduced to 1.4 beetles/trap. After that, the number of beetles increased considerably until mid-June when 757.3 ± 74.8 beetles/trap were recorded. At this date, we can consider that first flight period, due to the emerging of the overwintering parents (P), ended. After this date, the development of the first generation of the beetles (F1) began, as well as sister broods (S) which was marked with lower catches, up to 145.3 ± 21.6 insects/trap.
The entire development of the first-generation (F1) is marked by low captures, the turning point being recorded at the beginning of July, with captures of 334.46 ± 48.5 insects/trap, which represent the intensification of the activity of the adults (those that have wintered) and will complete the sister brood (S).
The third flight period was recorded between mid-July to September. This last part of the season (F2) is characterized by low captures, from 2.13 ± 0.6 to 152 ± 18.2 insect/trap, with a total of 9486 beetles, 1.53 times lower than the second period (F1), and 3.66 times lower than the first interval (P).
Male beetle numbers were relatively higher (57.1%) at the beginning of the flight and continually decreases until the end of May (38.4% males). Percentages of male beetles were not higher than 35.3%, Insects 2020, 11, 771 6 of 13 except during the sister brood, when the male percentage was around 40%. Differences in the relative abundance of the two sexes were significant different (df = 19, f = 3.750, p < 0.0001). There were no differences between beetle traps (df = 14, f = 1.210, p = 0.328).

Dynamics of Phoresy
Phoretic rates varied over time (df = 19, f = 5.962, p < 0.0001) but there were no significant differences between all 15 traps (df = 14, f = 0.768, p = 0.710). Phoretic rates oscillated between 6.4% and 42.1% (beetles with mites) with two peaks, spring and summer ( Figure 2). The beginning of May coincided with a phoresy rate of 33.1% ( Figure 2) and increased to a rate of 42.1% by mid-May. Phoresy rates then decreased continuously, reaching in July values between 6.4% and 14.3%. After 20 July, the percentage of beetles with phoretic mites gradual increased as beetle trap catches decreased.

Dynamics of Phoresy
Phoretic rates varied over time (df = 19, f = 5.962, p < 0.0001) but there were no significant differences between all 15 traps (df = 14, f = 0.768, p = 0.710). Phoretic rates oscillated between 6.4% and 42.1% (beetles with mites) with two peaks, spring and summer ( Figure 2). The beginning of May coincided with a phoresy rate of 33.1% ( Figure 2) and increased to a rate of 42.1% by mid-May. Phoresy rates then decreased continuously, reaching in July values between 6.4% and 14.3%. After 20 July, the percentage of beetles with phoretic mites gradual increased as beetle trap catches decreased.   The abundance of mites on male and female beetles varied slightly during the season (Figure 4), from a minimum of 0.36 mites/beetle at the beginning of July to a maximum of 3.16 mites/beetle in mid-May. The analysis indicates a significant change in phoretic load during the sampling periods (df = 3, f = 3.850, p < 0.001), but insignificant in terms of host sex (df = 1, f = 1.864, p = 0.740).
T. polytricha ranged from 1.0 to 4.47 mites/beetle with the maximum phoretic loads on 19 May.  Most of U. ipidis and U. vinicolora (80.2%) appeared during May and June. Unlike T. polytricha, the maximum phoretic loads of U. ipidis were recorded at the beginning of July, with 0.52 mites/beetle, after which it appears sporadically. U. ipidis fluctuated between 1.0 and 3.12 mites/beetle with an average value of 1.64 ± 1.38 mites/beetle. Additionally, all 34 U. vinicolora mites were collected from May to June. U. ipidis fluctuated between 1.0 and 3.12 mites/beetle with an average value of 1.64 ± 1.38 mites/beetle. The small number of H. piceae specimens (14 individuals) collected at the beginning of June and July did not allow detailed analysis of phoretic interaction of this species, while for P. fiseri most of the individuals were collected between the beginning of May and the end of June.
The abundance of mites on male and female beetles varied slightly during the season (Figure 4), from a minimum of 0.36 mites/beetle at the beginning of July to a maximum of 3.16 mites/beetle in mid-May.

Diversity and Zoocenological Pattern
The phoresy rates recorded in this study (~5%-45%) varied in time but were within the range of phoretic rates found in Southern Germany [49] (30-36%), Poland (30%) [50], and Sweden (23%) [46]. The significant variation in the percentage of beetles carrying mites in our study likely results from the long sampling period (i.e., throughout flight season) of 22 weeks, a period in which two generations of beetles developed.
The small number of mite species found in this study is in agreement with some authors who discovered six species of mites on I. typographus in Poland [50], five species in Bulgaria [55], and eight species on the same insect in the Czech Republic [56], but represents far less than other studies that revealed between 13-25 mite species on the same host [46,47,49,65]. However, it seems that mite communities differ significantly in Europe and the small number of phoretic mite species may be due to local specificity [50] and sampling length. On the other hand, the reduced diversity can be explained by the excessively large number of mites belonging to D. quadrisetus and T. polytricha. Regardless of the number of phoretic species mentioned in the literature, phoretic mites represent only a small part of the 68 species observed inside the galleries of I. typographus [66].
Two of phoretic mite species (D. quadrisetus and P. fiseri) are believed to be predators. D. quadrisetus have also been reported to consume larvae of bark beetles and nematodes, having a constant presence during the development of the first and second generations of I. typographus, and consuming 9.7% of bark beetles eggs [27,67]. D. quadrisetus has a broad phoretic host range as it has been collected on I. typographus [49,55] and other European bark beetle species: Polygraphus

Diversity and Zoocenological Pattern
The phoresy rates recorded in this study (~5-45%) varied in time but were within the range of phoretic rates found in Southern Germany [49] (30-36%), Poland (30%) [50], and Sweden (23%) [46]. The significant variation in the percentage of beetles carrying mites in our study likely results from the long sampling period (i.e., throughout flight season) of 22 weeks, a period in which two generations of beetles developed.
The small number of mite species found in this study is in agreement with some authors who discovered six species of mites on I. typographus in Poland [50], five species in Bulgaria [55], and eight species on the same insect in the Czech Republic [56], but represents far less than other studies that revealed between 13-25 mite species on the same host [46,47,49,65]. However, it seems that mite communities differ significantly in Europe and the small number of phoretic mite species may be due to local specificity [50] and sampling length. On the other hand, the reduced diversity can be explained by the excessively large number of mites belonging to D. quadrisetus and T. polytricha. Regardless of the number of phoretic species mentioned in the literature, phoretic mites represent only a small part of the 68 species observed inside the galleries of I. typographus [66].
Two of phoretic mite species (D. quadrisetus and P. fiseri) are believed to be predators. D. quadrisetus have also been reported to consume larvae of bark beetles and nematodes, having a constant presence during the development of the first and second generations of I. typographus, and consuming 9.7% of bark beetles eggs [27,67]. D. quadrisetus has a broad phoretic host range as it has been collected on I. typographus [49,55] and other European bark beetle species: Polygraphus polygraphus, Pityogenes chalcographus or Pityokteines curvidens [68][69][70] as well as on the North American bark beetles Ips pini [32,71]. P. fiseri has also been found on at least 25 species of insects [72], among the following bark beetles: Dendroctonus frontalis, D. tenebrans, D. valens, Hylurgops palliatus, I. avulsus, I. grandicolis, I. calligraphus, and Hylastes sp. [32].

Attachment Places
Houck and O'Connor [21] hypothesize that the distribution of mites on the host body is nonrandom, with mite species choosing specific parts of the host body during phoresy. Our phoretic data on host location preferences differ considerably from similar work [46], which located 82.5% of mites under elytra, 10.3% on the thorax, 6.5% on elytral declivity, and less than 1% on other parts of the body. Other studies using pheromone traps [49] found 61.5% of the mites under elytra, 24.3% on the thorax, 10.5% on the elytral declivity, and 3.7% on other parts of the body, which shows a similar pattern of preferences for attachment locations. Differences across studies could result from how beetles were stored (alcohol vs. frozen) as well as host population dynamics, mite species composition, and time of season. Even so, the attachment sites are chosen by the mites to avoid being brushed off by the host [75] or to avoid injury during beetle gallery excavation [76]. Differences in attachment sites could also be due to the space available on the beetle at the time of locating and connecting to the insect. Based on our observations, the abdomen of the beetles is not a good place to attach. In any case, the ontogenetic instar of mites-mainly deutonymphs, in our study-influences the attachment preferences since this stage is critical and reduces vulnerabilities during transportation.
Our findings related to the presence of deutonymphs of D. quadrisetus attached to the internal part of the elytra confirms other studies that noticed a particular preference for this body part [46,49] even on a different beetle host species [42]. The attachment location for H. piceae supports previous findings [46] that indicated the same position on the same host, I. typographus, for all mite specimens belonging to this genus. These findings are somehow surprising since this species was shown to exploit some peculiarity in host morphology of I. typographus and I. cembrae-the mite is observed to "sit "on the host instead of "attaching" [74].

Dynamics of Phoresy
Mites and their potential beetle host must meet each other "at the same time and space" [77] when the beetles are exiting the tree, traveling and entering a new host. As a consequence of this, the existence of phoresy is marked by four crucial moments: (i) initial attachment; (ii) transport to new habitats; (iii) detaching, and resuming the development cycles (for both organisms); and (iv) reattaching to abandon these places after habitat deterioration. For some mite species, diapause is synchronized with beetle developmental stages [78], and maybe this is why in our study the higher quantity of mites was collected from the overwintering parents within the dispersal flight which occurs in early spring. Unlike phoretic mites associated with vertebrate carrion, who have a delay in peak abundances and richness relative to beetle (or host) assemblage [79], in our study the mites reached the peak abundance before peak beetle emergence.
Regardless of the developmental stage of the host, a temporal specificity was observed for all mite species. Even so, the sampling date of the beetles alone could not entirely explain the inconsistencies between our results and previous studies; thus, combined with the "historical status" of the site with respect upon the age of the outbreaks and new sites where the first phoretic loads are smaller than the old ones [80], may give only part of the answer. Another factor that could influence the dynamics of phoretic loads is the seasonal dynamics of their host beetle and their relative densities compared to those of mites within the bark. Studies have found that climate forces the majority of beetle to overwinter in the litter [81], where the probability of the phoretic mite population to growth or survival is small. In our study, we found a higher phoresy rates on beetle populations that overwinter under the spruce bark [82], where mite survival and connection with beetles is much higher.
Morphological instar of the mites, mostly deutonymphs in our study, could be a reason for high peak abundance early in the season, as some species phoresy can only be phoretic during this stage [49]. This is why mites must complete the development when carriers have already finished mating and are creating their maternal galleries, which can last several weeks.
The constancy with which D. quadrisetus was found throughout the host's activity gives this relationship a more intimate character, which would mean more than just a transport relationship (such as a predator-prey association); while the presence of T. polytricha and Uroobovella sp. indicates an exclusive transport relationship, this being mainly associated during the emerging of the overwintering parents (P) and less during the development of the two generations of beetle progeny (F1 and F2). However, in the case of the T. polytricha and both Uroobovella species, the issue regarding the timing of their attachment to beetles is not fully clarified since these species practically disappear by the end of the first generation (F1) and are expected to appear only the following spring. Nevertheless, the high rates of phoresy recorded at the beginning of May indicate that this is the time when the massive attachment of the mites to emerging beetles takes place, and this may be the moment when the mite phoretic stage is most active. Not having data for one autumn and the following spring, we cannot state in our study what causes the difference between the recorded rates. We can only speculate on several factors: the age of the outbreak, the local specificity regarding the quantity and the quality of the resources, the mortality but also the proportion between egg-larvae-pupae and the adults that wintered, all of them potentially influencing the abundance and diversity of the mite species between the cessation of the host's activity within the tree and its emergence the following spring. It was also demonstrated that during the host flight activity, mites could fall from the beetle's body proving the existence of an indirect association between the amount of the mites and flight distances made by the host [80], which represent the distance from an existing outbreak to new sites.

Conclusions
In this study, we found six species associated with I. typographus population, throughout a flight season. Most mites were located under the elytra's host or on the thorax and elytral declivity. The greatest phoretic loads occurred on overwintering beetles during early spring. Unlike D. quadrisetus which was very frequent during the whole season and are predators of nematodes and bark beetle eggs and larvae, T. polytricha and U. ipidis were uncommon and may only associate with beetles for transport. We observed no difference between male and female beetle hosts concerning phoresy rates, although some variation in phoresy rates were noticed mainly during the first and second flights (F1 and F2).
Future studies should monitor phoretic mite loads over multiple years and beetle generations to better understand mite population dynamics and how they may affect bark beetle ecology. Studies should also investigate the potential of predatory mite species like D. quadrisetus which was demonstrated in other studies to consume beetles offspring, for biological control.