Anthonomus spilotus ( Coleoptera : Curculionidae ) : new to the Belgian fauna

1 pcfruit npo (research centre for fruit cultivation), Fruittuinweg 1, 3800 Sint-Truiden, Belgium. 2 Royal Belgian Institute of Natural Sciences, Vautierstraat 29, 1000 Brussels, Belgium. 3 Gembloux Agro-Bio Tech (University of Liège), Passage des Déportés 2, 5030 Gembloux, Belgium. 4 ILVO (Flanders Research Institute for Agriculture, Fisheries and Food), Burg. Van Gansberghelaan 96, 9820 Merelbeke, Belgium. 5 KULeuven University of Leuven, Department of Biosystems, Willem de Croylaan 42, 3001 Leuven, Belgium.

Anthonomus spilotus was first described by RedtenbaCheR (1847), who mentioned it to be not uncommon in fruit trees in Austria (Upper Austria).Most literature on A. spilotus appeared in the context of plant protection in pear growing, with the most reports from France.PussaRd (1930) described the large populations in pear plantations in the Rhône valley and mentioned earlier reports from the south: Montpellier, Marseille, the Pyrenees and Corsica but also from more north: Allier, Nièvre, Seine-et-Marne, Seine-et-Oise (now Essonne, Yvelines and Val-d'Oise), Calvados, Oise, Aube and Somme.Later publications confirmed A. spilotus to be a pest of pear in almost all France [11,10] although rare in the north and Paris Basin [5,9].INRA (French National Institute for Agricultural Research) has specimens in collection from Indre-et-Loire 2010 and barcoded the DNA of the species [12].Anthonomus spilotus is also considered present in Switzerland [11] and bovey & baggiolini (1967) reported it as a pest of pear trees in Romandy although only sporadically, and often overlooked.The weevil is also found in Germany [11].RheinheimeR & hassleR (2010) stated that A. spilotus is rare or even regionally disappeared in Germany, without recorded economic damage in pear orchards.Almost all records are from south and south-west Germany with all recent observations from Baden, Palatinate and Rhineland.The supposed presence of A. spilotus in The Netherlands is based on a species list from 1966 [11,13] but observations are rare or non-existent.A. spilotus is considered absent in Poland [14] but is present in Ukraine (Western Ukraine, Pruto-Dnistrovs'ke Mezhyrichchia) [15], Hungary [11] and Russia [11].In the Czech Republic this weevil is considered present [11] but regionally endangered [16].A. spilotus even occurs as far north as Southern Sweden [11,7], in particular Öland and Scania [14] with a recent specimen in the collection of the Naturhistoriska Riksmuseet [17]. A. spilotus is also reported in Southern Europe and Northern Africa with reports from Italy [11,5], Greece [11,5], Spain [11], Portugal [11], Morocco [3,4,10] and Algeria [3,4,10].In the United Kingdom (Kent), A. spilotus was recorded for the first time in 2017 in pear orchards where it was already considered present for more than three years [8].An overview of the countries with reports of A. spilotus is given in Figure 2. From the above it becomes clear that although widespread in Europe, A. spilotus is considered rare in most countries.This species was considered abundant and an occasional pest of pear only in France and even there recent records are rare.However, A. spilotus has recently appeared in high numbers in the UK and Belgium.While for the UK it is thought to be newly imported [8], it is possible that A. spilotus was a rare and overlooked species in Belgium, hence having a similar status as in Northern France, Northern Germany and The Netherlands.It is, however, remarkable how an overlooked species could become an economically important pest species in a few years.This could indicate that its habitat conditions have somehow become more favourable.These favourable conditions could possibly be explained partially by climate change and/or a shift in insecticide use in pear growing.
In Belgium, A. spilotus was always considered present [11] based on its occurrence in neighbouring countries, without actual reported findings or specimens in collections.Recently the species was observed at nine locations (two natural reserves and seven pear orchards).An overview of the observations is given in Table 1; the sampling locations are depicted in  of its presence were noticed in a second pear orchard in Jeuk (Location d) and in 2017, the population in this orchard was confirmed.At about 1 km from the latter, another pear orchard (Location f) was found to host a population of A. spilotus in 2017.Also in 2017, a pear orchard with signs of A. spilotus was discovered in Les Waleffes (Location e).In 2018, A. spilotus was detected in the other three pear orchards: in Kortessem (Location g), Hoeselt (Location h) and Membruggen (Location i).The latter location contained a large population.
Specimens from Location d and f of 2017 were identified using the key of dieCkmann (1968) and were added to the collections of the Royal Belgian Institute of Natural Sciences.Morphological characteristics for A. spilotus are depicted in Fig. 1: forehead hairs directed towards the eyes and reaching over them, elytra elongated and not widening near the end, pale elytral fascia, inside of protibia strongly curved at the apical half and median lobe slightly asymmetrical and narrowly pointed.Specimens from Location i of 2018 were also molecularly identified as follows: genomic DNA was isolated from individual adults using the Qiagen DNeasy Blood and Tissue Kit (Qiagen Inc, Germany) following the manufacturer's protocol.The COI (Cytochrome c oxidase subunit 1) region was amplified using primer cocktails designed by CRuaud et al. 2010 [18] and geRmain et al. 2013 [19] (Table 2).PCR was carried out in a 50 µl reaction volume including 2 µl of genomic DNA, 22 µl of Milli-Q and 25 µl of BIO-X-ACT™ Short Mix and 0.2 µl of each 10 μM primer cocktail.PCR conditions for COI were: 94°C for three minutes, five cycles of 94°C for 30 seconds, 45°C for 30 seconds and 72°C for 60 seconds, followed by 35 cycles of 94°C for 30 seconds, 51°C for 60 seconds and 72°C for 60 seconds, with a final extension at 72°C for 10 minutes.PCR products were purified and sent for sequencing using M13F (5′-TGTAAAACGACGGCCAGT-3′) and M13R (5′-CAGGAAACAGCTATGAC-3′) primers [20].The Fig. 2 -A.Overview of the geographical distribution of Anthonomus spilotus in Europe, the species is considered (at least regionally) present in the countries marked in black.B. Overview of the Belgian locations where A. spilotus was found, letters referring to the location codes in Table 1.
quality of the Sanger sequencing reads was first assessed in Sequence Scanner v2.0.The sequences were edited and analysed using software packages of Chromas 2.00 (Technelysium, Helensvale, QLD, Australia) and BioEdit 7.0.4.1 [21].The INRA in-house reference sequence of A. spilotus [12] and the sequence of a closely related and most morphologically similar species, Anthonomus ulmi (DeGeer, 1775), available in the GenBank: KM450134.1 [22], were also imported in BioEdit.The alignment of  the sequences showed 99% and 91% similarity between the Belgian specimens and the French (Indreet-Loire) population of A. spilotus and A. ulmi, respectively (Fig. 3).It is therefore both morphologically and molecularly confirmed that A. spilotus is present in Belgian pear orchards.
When rearing adults from collected pupae (Locations d and f in 2017, Location i in 2018), parasitoid wasps (Hymenoptera: Pteromalidae and Hymenoptera: Ichneumonidae) emerged, indicating the presence of antagonists of this species in Belgium.
A tentative phenology of A. spilotus in Belgium can be drawn up from the observations in Table 1.Overall adult activity was observed from end March till June, which is in line with the biology described from neighbouring countries: the end of hibernation/start of oviposition occurred at the end of March [5,3,7] and emergence of the new generation adults took place in June [7,3].The characteristic feeding and oviposition punctures with droplets of plant sap made by the hibernated adults in leaf buds, leaf petioles and spurs and the resulting inhibited sprouting, leaf drop and deformations [3,9,7] were clearly observed.The leaf feeding of the new adults before aestivation [7] could also be seen.The larvae that were observed were mostly feeding on the leaf while sheltered in a leaf that remained rolled up, characterized by necrotic leaf edges.Pupae were found in hard blackened pupal chambers formed on the leaf edges or petioles.All this is analogous to earlier descriptions of this weevil's biology [7,3,9].In 2017 (Locations d, f) and in 2018 (Location g), pupation was noted at the end of April and the beginning of May, respectively, while for both France and Germany it is described as generally occurring at the end of May.
The authors will further investigate the distribution, host plant specificity and phenology of A. spilotus, and to this end, would greatly appreciate receiving information regarding further records of this species in Belgium.
Fig. 1 -Morphological characteristics of Anthonomus spilotus Germar, 1817. A. Elytra, pronotum and head: forehead hairs directed towards the eyes and reaching over them, elytra elongated and not widening near the end and pale elytral fascia.B. Foreleg: inside of protibia strongly curved at the apical half (arrow).C. Median lobe of aedeagus: slightly asymmetrical and narrowly pointed.

TABLE 1
List of observations of Anthonomus spilotus in Belgium.

TABLE 2
PCR primer cocktails used to amplify COI.