Pollen Beetles in Oilseed Rape Fields: Spectrum and Distribution in Czech Republic during 2011–2013

: Oilseed rape is frequently damaged by insect pests. Much attention is paid to the protection of oilseed rape against Brassicogethes aeneus (Coleoptera: Nitidulidae), which is one of the most signiﬁcant pests of spring and winter oilseed rape. The presence of different pollen beetle species was monitored in the Czech Republic in the years 2011–2013. A minimum of 500 individuals were captured at each site. Morphometric characteristics and the morphology of male and/or female genitalia were used to determine species. B. aeneus , B. subaeneus , B. viridescens and B. coracinus were most abundant. Other species presented in oilseed rape were B. coeruleovirens , B. czwalinai , B. matronalis , B. anthracinus , Boragogethes symphyti , Cychramus luteus , Fabogethes nigrescens , Genistogethes carinulatus , Meligethes atratus , Sagitogethes maurus , and Lamiogethes atramentarius . Our main conclusion is that the reason for the presence of the pollen beetle species associated with their development into non-cruciferous plants in oilseeds is the sufﬁciency of pollen as food for beetles. In addition, they may occur here incidentally, as they can be transported relatively long distances by air. Accompanying species of pollen beetles probably also have a positive effect on abundance reduction in species considered to be harmful as they are hosts to parasitoids of the oilseed rape pest.


Introduction
Brassicogethes aeneus Fabricius, 1775 is one of the most significant pests of spring and winter oilseed rape. Treatment against this pest has been used regularly for several decades and is causing problems with resistance to the insecticide's active ingredients. Insecticide resistance testing is carried out in most European countries, including the Czech Republic (CZ). Pollen beetle populations were highly resistant to the pyrethroid lambdacyhalothrin, according to 2014-2015 test results from CZ [1]. Relatively high proportions of the populations also showed resistance to the pyrethroid tau-fluvalinate [2]. At present, especially if the number of registered active ingredients is reduced, a reduction in the level of resistance cannot be expected. Many other pollen beetle species were present in the oilseed rape in addition to Brassicogethes spp. (Coleoptera: Nitidulidae). The larvae of all Brassicogethes species develop on Brassicaceae plants, and adults feed on the pollen of various family plants [3,4]. Usually, the pollen beetle (Brassicogethes aeneus) is considered a pest of oilseed rape, although there are several other pollen beetle species that may inflict damage as well [5].
The pollen beetles, which are the oilseed rape pests, formerly belonged to the genus Meligethes Stephens, 1830. Until recently, the genus Meligethes was one of the most numerous of the Nitidulidae family. Just in the Palearctic region, there are about 250 polinivorous species associated with plants of the families Brassicaceae, Lamiaceae, Fabaceae, and 4 of 23 ment, larvae are associated with Hesperis matronalis L. [31]. It is similar in appearance to B. subaeneus [36]. The distinguishing features are shown in Figure A8.
The distinguishing features are shown in Figure A10. This species is associated with plants of the Boraginaceae family, particularly Symphytum [7]. According to Nunberg [8], larvae develop on Symphytum officinale L.
The Cychramus luteus (Fabricius 1787) adult body is rusty brown; the pronotum and elytra with hairs in visible rows. [35]. The distinguishing features are shown in Figure A11. The larvae of this species develop in fungi of the Agaricaceae and Lycoperdaceae families, they are often present in species of the genus Armillaria and Lycoperdon, and less often, in fungi of the class Ascomycetes [37]. According to Rimšaité [38] and Schigel [39], adults are raised from larvae present in Armillaria spp. Jelínek [34] states that larvae develop in the fruiting bodies of softer wood-destroying fungi and adults are present on flowers, especially of Aruncus dioicus (Walter) Fernald. It is abundant in Europe, adults feed on the pollen on the flowers of plants around the beehives [40]. Neumann and Ritter [41] reported finding adults in hives. However, C. luteus (Fabricius, 1787) did not leave holes in the cells of the honeycomb, and neither oviposition nor the presence of larvae on the combs was detected [41].
Epurea aestiva (Linnaeus, 1758) is 3 mm long, and rusty yellow [35]. The distinguishing features are shown in Figure A12. This species is widespread throughout Europe, where adults are found on plant flowers at the edge of forests [21] and they are abundant in deciduous forests [34]. Kurochkin [32] observed adults most frequently on woody plants of the family Rosaceae-Cerasus fruticosa Pall., Malus domestica (Suckow) Borkh., Prunus avium L., Rosa majalis Herrm., Filipendula ulmaria and F. vulgaris Moench, rarely on Knautia arvensis (L.) J.M. Coult. Majka and Klimaszewski [42] consider adults to be sap-feeders. Larvae develop in the nests of bumblebees and small mammals [19,34] and probably also on moldy substrates [34]. As noted by Arbogast et al. [40], the larvae are mycetophagous. This species is widespread in Europe, Asia, and the USA, and it has also been found in Canada [42].
Fabogethes nigrescens (Stephens, 1830) adults are 1.7-2.5 mm long; black with fine punctuation on pronotum and elytra; antennae and the legs are pitch brown; and the first, second, and fourth teeth of forelegs tibia are bigger [8,11]. The distinguishing features are shown in Figure A13. The larvae are associated with plants of the Fabaceae family, in particular Trifolium spp. [43], Onobrychis spp., Ononis spp., Lotus spp. [5], Medicago spp. [44]. Niezgodziński [45] considers it to be an insignificant pest of Vicia faba L. in Poland. It is found in open habitats [46]. It is an important pollinator of Primula [47,48] and Rubus [29]. Mifsud and Audisio [44] consider it to be a pest of medicinal plants in North America.
Genistogethes carinulatus (Förster, 1849) is 1.4-2.2 mm long, with black antennae and brown legs, and the forelegs tibia are relatively narrow [8]. The distinguishing features are shown in Figure A14. This species is associated with Lotus corniculatus L. by their larval development [11,21]. It is mainly found in meadows [19]. Niezgodziński [45] considers it to be an insignificant bean pest. Duke [48] considers it to be a pest of Onobrychis viciifolia Scop.
Lamiogethes atramentarius (Förster, 1849). The imago is 2.0-2.8 mm long, oval-shaped, and black with metallic blue or green shine. Pronotum and elytra are finely punctuated. Antennae and legs are reddish-yellow. Tibiae of the foreleg are finely toothed [8]. The distinguishing features are shown in Figure A15. The nutrient plants of larvae are Galeobdolon luteum Huds., Lamium album L., and Galeopsis spp., adults are present on the flowers of various woody plants, e.g., Crataegus spp. and Prunus padus L. [49].
Meligethes atratus (Olivier, 1790) is 2.4-3.8 mm long and brownish-black. The first antennal segment is darker [8]. The distinguishing features are shown in Figure A16. It occurs at the edges of forests, road ditches, and grassy areas. Audisio et al. [19] stated its presence in disturbed habitats. Its larvae develop on species of the genus Rosa, including R. canina L. [11]. According to Audisio et al. [7], its larval development is strictly associated with the genus Rosa spp. and Rubus spp. It most likely also develops on Prunus spp., Pyrus spp., and Crataegus spp. [6]. Adults are polyphagous [11] and it is widespread throughout Europe [8,50].
The Pria dulcamare (Scopoli, 1763) imago is 1.6-1.8 mm long and rusty brown. Its body is egg-shaped or oval, less convex, elytra are shortened and 1-3 abdominal tergites are visible. The fourth tarsal segment is always shorter than the others [10,11]. This species is associated with the Solanum dulcamara L. [8,11,51,52], as well as with S. nigrum L. [11] during its larval development. Adults are also found on flower species other than Solanaceae [44].
The Sagitogethes maurus (Sturm, 1845) imago is 2.0-3.0 mm long, black colored, the legs and antennae are dark brown, and the distal part of the tibia of the front legs is distinctly widened [8]. The distinguishing features are shown in Figure A17. All species of this genus are associated with plants of the family Lamiaceae [7]. This species is associated with Salvia spp. [43]. Adults are found on the flower of Salvia pratensis L., S. verticillata L., Sambucus, Taraxacum, and Potentilla [49].
The main contribution of this work was the identification of the species spectrum of the genus Brassicogethes present in oilseed rape stands and its representation in the samples. Determining the proportion of B. aeneus and B. viridescens in the samples was particularly important. The results could help to refine the control of pollen beetles in OSR crops with insecticides, increase their effectiveness, and reduce the selection pressure inducing resistance to insecticides in pollen beetle populations. Until now, only a few authors have dealt with this issue and the available information is not very comprehensive.

Sampling Design and Collection of Beetles
Beetles for species identification and ecological analysis were obtained during the monitoring of pollen beetle populations for insecticide resistance in the Czech Republic. They were collected and tested according to International Resistance Action Committee (IRAC) methods for lambda-cyhalothrin No. 011 [53], BISCAYA 240 OD No. 021 [54], chlorpyrifos No. 025 [55], and indoxacarb No. 027 [56]. Beetles were collected from oilseed rape plants at the BBCH 50-60 growth stage. Samples were taken by digging diagonally across the field to include the edge of the plot and the interior. The sample from a tested site contained at least 500 individuals. The beetles were inserted in polyethylene bags with absorbent paper (to prevent water vapor from condensing on the bag walls) and oilseed rape inflorescence or other pollen sources. After collection, the bags were used to ensure adequate air supply. After laboratory testing for resistance, individuals from each site were preserved in 70% alcohol. The coordinates of the locations are shown in Table 1.

Species Identification
The identification of beetles was performed based on morphometric characteristics and/or using the method of dissection of the genitalia. Individuals that could not be distinguished on the basis of external characteristics were determined according to the species-specific morphology of the genitals. The Olympus SZX-61 stereomicroscope was used for the determination process. The publications by Oliverio et al. [57] and Kirk-Spriggs [11] were used as references for species identification. The scientific names of pests and other animals mentioned in this work are taken from the Fauna Europaea database [58], and the names of the pollen beetles are taken from Audisio [59] and Jelínek [35].

Ecological Analysis
The basic ecological characteristics of the monitored (sub) populations species were the presence and absence of the species in the given collection and are utilized for a simple comparison of individual localities [60]. The frequency and regularity of species occurrence are not taken into account. The basic index calculated for each site is the dominance index (D).
Furthermore, the Jaccard index (Ja'), Shannon-Weiner index (H'), Hill index (N2), E-evennes, and E'-corrected evennes index were calculated. The ComEcoPaC 1.0. software was used to calculate the ecological characteristics [61]. The species were divided into five classes of the Tischler scale determined by the percentage in the collections (localities) [60] Eudominant and subrecedent species predominate in anthropogenically influenced biocenoses [62]. Lasoń [37], in his work on the composition of populations of the families Kateretidae and Nitidulidae, uses an extended range of dominance: this is a six-level scale and includes the following classes of dominance superdominant-over 30%, eudominant-10.0-29.9%, dominant-5.1-9.9%, subdominant-2.1-4.9%, recessive-1.1-2.0% and subrecessive-up to 1% of individuals. In this paper, this scale is only used as an alternative. The key factor in this work is the use of the Tischler scale, which is considered the standard. In the result tables, which are the output of the ComEcoPaC 1.0. software [61], the number of species represented by one (singleton), two (doubleton), or three (tripleton) individuals occurring in the collection at the given locality is also given. The Jaccard index is used to determine the faunistic similarity between pollen beetle samples collected at different localities [60]. The similarity of zoocenoses increases with an increasing index value, which can reach 1 or 100 when converted into percentages.

Species Representation in 2012
A total of 5441 individuals were determined in 2012. Figure 2 shows the proportion of each species in the total sample of individual species. Eudominant species, according to Tischler's classification, were Brassicogethes aeneus and B. subaeneus in all monitored localities, B. coracinus in localities Krhov and Rozsochy, Cychramus luteus in localities Popovice u Rajhradu and Kojetín, Meligethes atratus in localities Vedrovice and Němčice. The  Table 2. The Jaccard index values calculated for individual localities (Table 3) Table 6. As the comparison of the values of the Jaccard index calculated for individual localities (Table 7)

Species Representation in 2013
A total of 5779 individuals were identified in 2013. Figure 3 shows  Table 6. As the comparison of the values of the Jaccard index calculated for individual localities (Table 7)

Discussion
Brassicogethes aeneus (Fabricius, 1775) is considered a key pest of oilseed rape and turnip rape (B. rapa) in Europe [4]; another significant species is B. viridescens (Fabricius, 1787), which is harmful in both Europe and North America. Not only is B. aeneus present in oilseeds, but other species are also common [62][63][64]. To our knowledge, few studies have focused on the species spectrum and abundance of individual pollen beetle species in oilseed rape. In Croatia Gotlin Čuljak and Juran [5] dealt with the species spectrum of pollen beetles in oilseed rape, they reported the presence of Brassicogethes aeneus, B. viridescens, B. coracinus (Sturm, 1845), Clypeogethes lepidii (L. Miller, 1851), and Fabogethes nigrescens (Stephens, 1830). B. aeneus was the eudominant species [5], the same results we obtained in 2011 and 2012. Interestingly, Clypeogethes lepidii was not present in any of the samples we analyzed. The reason for this remains unknown as we were unable to find any relevant information. B. aeneus and B. viridescens are among the most common species in the UK [3,9], both these species are also considered to be important in Sweden [65]. In Estonia, B. viridescens is very common in oilseed rape, more so in winter oilseed rape [66]. As stated by Finch et al. [67], both species mentioned above can also damage cauliflower and broccoli, reducing their market value. On the other hand, none of the listed pollen beetle species are known to be a pest of cruciferous vegetables in the Czech Republic. The species composition of pollen beetle populations present in oilseed rape fields after overwintering was studied in Hungary by Marczali [ [68], they are in fact only one species-Fabogethes nigrescens [59]. As in the work of Marczali [68]

Discussion
Brassicogethes aeneus (Fabricius, 1775) is considered a key pest of oilseed rape and turnip rape (B. rapa) in Europe [4]; another significant species is B. viridescens (Fabricius, 1787), which is harmful in both Europe and North America. Not only is B. aeneus present in oilseeds, but other species are also common [62][63][64]. To our knowledge, few studies have focused on the species spectrum and abundance of individual pollen beetle species in oilseed rape. In Croatia GotlinČuljak and Juran [5] dealt with the species spectrum of pollen beetles in oilseed rape, they reported the presence of Brassicogethes aeneus, B. viridescens, B. coracinus (Sturm, 1845), Clypeogethes lepidii (L. Miller, 1851), and Fabogethes nigrescens (Stephens, 1830). B. aeneus was the eudominant species [5], the same results we obtained in 2011 and 2012. Interestingly, Clypeogethes lepidii was not present in any of the samples we analyzed. The reason for this remains unknown as we were unable to find any relevant information. B. aeneus and B. viridescens are among the most common species in the UK [3,9], both these species are also considered to be important in Sweden [65]. In Estonia, B. viridescens is very common in oilseed rape, more so in winter oilseed rape [66]. As stated by Finch et al. [67], both species mentioned above can also damage cauliflower and broccoli, reducing their market value. On the other hand, none of the listed pollen beetle species are known to be a pest of cruciferous vegetables in the Czech Republic. The species composition of pollen beetle populations present in oilseed rape fields after overwintering was studied in Hungary by Marczali [68],  (Förster, 1849), and B. coracinus (Sturm, 1845). When studying the migration of pollen beetles from hibernacula to oilseed rape fields, Juhel et al. [71] found 99% B. aeneus and the presence of only two individuals of B. viridescens, they did not specify the species affiliation of the remaining proportion.
In other European countries, several pollen beetle species are found in oilseed rape fields like in the Czech Republic [62][63][64]. Brassicogethes aeneus is the eudominant species in the Czech Republic, but in some localities, B. subaeneus is also eudominant [62]. The treatment of oilseed rape against pollen beetles is based on reaching some of the economic threshold values. All specimens of pollen beetles present in oilseed rape are considered to be Brassicogethes aeneus (Fabricius, 1775). However, a whole complex of pollen beetles present in oilseed rape has been identified.
One of the first evaluations of the species spectrum of pollen beetles was carried out by us in 2010 [71], where B. aeneus (Fabricius, 1775) was found to be a eudominant species. B. subaeneus (Sturm, 1845), B. viridescens (Fabricius, 1787), and B. erythropus (Marsham, 1802) were also eudominant species in several localities as well. In most cases, B. subaeneus and B. viridescens were dominant or subdominant species. In the following years, pollen beetle populations were again analyzed to determine their species composition. Several pollen beetle species were present in the samples collected from 2011 to 2013 in oilseed rape in different regions of the Czech Republic. The eudominant species were usually B. aeneus, B. subaeneus, and B. viridescens. These results largely correspond to those obtained in subsequent years [62,64].
In reality, the presence of individual species is not taken into account, although the common occurrence of several pollen beetle species in oilseed rape fields has long been known, e.g., [72,73]. The determination of individual species is very time-consuming, and, with a few exceptions, requires the preparation of male and female genitalia. Knowledge of the species spectrum of pollen beetles and their bionomy could be one of the components of anti-resistance strategies. The degree of resistance of pollen beetles to insecticide active ingredients is investigated for population samples containing different species. Some authors claimed that it is not essential to know the level of resistance of other species [74]. Others, however, have considered this issue in more detail. B. viridescens is not resistant to insecticides [72]. According to Spaar et al. [22], B. aeneus is the most common species in cruciferous oilseeds fields, followed by B. viridescens, B. coracinus (Sturm, 1845), and Fabogethes nigrescens (Stephens, 1830). According to this author, only a few single specimens of B. coeruleovirens (Förster, 1849), Meligethes atratus (Olivier, 1790), Lamiogethes bidens (C. Brisout de Barneville, 1882), Sagittogethes maurus (Sturm, 1849), Meligethes flavimanus (Stephens, 1830), and Lamiogethes pedicularius (Gyllenhal, 1808) were found.
Brassicogethes aeneus is the dominant species together with B. subaeneus and B. viridescens. The genus Brassicogethes was represented in the collections from 81.8 to 90.5% of the pollen beetle population. A similar representation is also mentioned by some authors, e.g., Ouvrard et al. [3], where the representation of B. aeneus is 95%. In Lithuania, the representation is 98.2% to 98.8% [75]. In Romania, Talmaciu and Talmaciu [76] examined the fauna of pollen beetles on oilseed rape, and they reported only B. aeneus. Up to 1% of Brassicogethes coracinus and Fabogethes nigrescens were present in growth, as observed by Ouvrard et al. [3]. The presence of these species in spring and winter oilseed rape growths in Lithuania is reported-0.15% B. coracinus and 0.04% F. nigrescens [77]. In the populations collected from winter oilseed rape fields in the Czech Republic between 2011 and 2013, B. coracinus was represented by 4.21%, 7.72%, and 8.41%, (sorted by years ascending) and F. nigrescens was 3.75%, 2.42%, and 5.11%, 5.45 (sorted by years ascending). Brassicogethes viridescens occurs on all yellow-flowering cruciferous plants, but it is not as abundant as B. aeneus [11]. As stated by Derron et al. [72], its relative representation in the stands is highly variable. Ouvrard et al. [3] stated its representation in oilseed rape stands at 4%. Makūnas [77] showed its representation (average from winter and spring oilseed rape) at 0.35%, but later Makūnas [75] performed a genetic analysis of pollen beetles in Lithuania and stated the representation of this species in spring oilseed rape fields at 1.8%. The proportion of B. viridescens in our collections was 8.66% in 2011, 4.55% in 2012, and 10.51% in 2013. In 2012, it is close to the value that is reported by Derron et al. [72]. Overall, however, our results confirm the variability reported by Derron et al. [72]. In Spain, the presence of B. aeneus, B. viridescens, B. coeruleovirens, and B. coracinus in oilseed rape fields was reported by Moradillo [78].
The presence of adult Meligethes atratus in oilseed rape stands is probably due to the frequent occurrence of rose hips, a nutrient plant for larvae of this species, at field margins. In our collections, M. atratus was represented by 1.97% in 2011, 4.85% in 2012, and 0.48% in 2013. Pollen beetles actively fly into oilseed rape stands, but they also use the wind to occupy fields [79]. The pollen beetles can fly up to 13.5 km and can actively reach a distance of up to 300 m [80]. They are often more abundant at the edges of stands. They are also the first to be colonized by pollen beetles [81]. Pollen beetles are more abundant on the windward side of the field [82] and near overwintering sites [71]. The main reason for the presence of pollen beetle species associated with their development on non-cruciferous plants in oilseed stands is sufficient pollen as a food source for adults. Furthermore, as described by Ouvrard et al. [3], they can also occur here randomly if they can be transported over relatively long distances by air currents.

Conclusions
Several Brassicogethes species occur in oilseed rape stands. Among them, B. aeneus Thus, as our collection analysis shows, the pollen beetle species associated with cruciferous plants clearly predominated in individual years (representation in the collections was 83.7-93.4%), of which more than half were B. aeneus and B. viridescens. The pollen beetle species, whose larvae are not associated with the Brassicaceae family by their development, were represented in the collections between 9.6% and 16.3%.
From the point of view of protection of oilseed rape against pollen beetles, it is possible to ignore the fact that there are several species of pollen beetles and to work with this complex genus Brassicogethes as one species. To determine the critical number of beetles in the stand for protection purposes, it is not necessary to distinguish between pollen beetle species. On the other hand, when pollen beetle populations sampled in distinct localities are tested on susceptibility to insecticides, it would be good to know the differences in the species composition among them.
The presence of accompanying species can be beneficial, as they host some antagonists of harmful pollen beetle species, such as parasitoids, and can also be a reservoir for insect pathogens, thus indirectly contributing to bioregulation.
At the same time, as B. aeneus is treated with insecticides, other pollen beetle species present in the stand are also affected. However, as in the case of B. aeneus or the Brassicogethes species complex, it can be assumed that resistance to the insecticides used is also selected in the associated species.
Nowadays, it is important to monitor the spectrum of pollen beetle species in oilseed rape fields, but also in mustard or poppy fields. In particular, it provides early detection of harmful species of the Nitidulidae family, such as Aethina tumida Murray, 1867, an invasive species harmful to beehives. Species monitoring is also important from an ecological point of view, as it allows us to detect the effects of insecticides on non-target species and to take timely measures against the negative effects of pesticides.

Conflicts of Interest:
The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.   Kirk-Spriggs (1996) [11] and Nunberg (1976) [8].