The brown marmorated stink bug, Halyomorpha halys, native to East Asia, is an invasive pest of economic importance. It has invaded North America and many European countries and is further expanding its range. In Belgium, it was first observed in 2011. Halyomorpha halys is known to cause severe damage in commercial fruit orchards and vegetable crops. A dramatic and unmitigated expansion of H. halys in its adventive range could lead to significant economic implications for agricultural production. In this study, occurrence data of H. halys since its first observation in Belgium was analysed together with molecular information to map the populations and evaluate the genetic diversity of this pest. The genetic diversity of H. halys in Belgium was compared to data from other invaded and native countries reported in previous studies to identify possible invasion routes. The analysis of 1176-bp of mitochondrial DNA cytochrome c oxidase I and II genes (COI and COII) led to the discovery of two novel COI-COII haplotype combinations currently unique to Belgium. The invasion of H. halys in Belgium is likely the result of multiple and ongoing introductions from its native region and from already invaded countries in Europe, particularly Italy. The expansion of the brown marmorated stink bug populations in Belgium is recent and ongoing. Presently, it appears to thrive best in northern Belgium.

brown marmorated stink bug, haplotype diversity, Heteroptera, population genetics

Halyomorpha halys (Hemiptera: Pentatomidae) or the brown marmorated stink bug is a stink bug of economic importance, native to East Asia and an invasive pest species in Europe, North America, and other regions (Hoebeke and Carter 2003; Leskey et al. 2012; Lee et al. 2013; Haye et al. 2015; Zhu et al. 2016; Hamilton et al. 2018). In Europe, H. halys has already been detected as far north as The Netherlands (Aukema et al. 2019), Belgium (Claerebout et al. 2018) and the north of Germany (Hartung et al. 2022). Due to its polyphagous nature, H. halys can feed on virtually all primary fruit and vegetable crops in its invaded areas (Kuhar et al. 2012; Haye et al. 2015). For example, in northern Italy, it has become a key pest in fruit orchards, with an estimated economic impact of €588 million on the production of pear, apple, peach, and kiwi in 2019 (Bulgarini et al. 2020). Halyomorpha halys causes damage through its feeding activity. It inserts its stylets into the plant and injects saliva, pre-digesting the plant tissue before feeding on it (Rice et al. 2014). This feeding behaviour causes scarring, deformities, pitting, and discolouration of the product, rendering it unmarketable or even inedible (Rice et al. 2014; Bulgarini et al. 2020). Since its first record in Belgium in 2011, observations of H. halys on public citizen science databases (Observation International and local partners 2022) only appeared in 2017. Currently, it is assumed that H. halys has established univoltine overwintering breeding populations in Belgium (Claerebout et al. 2018). An observation of H. halys from the Haspengouw region in July 2021 indicated that H. halys is already present in commercial pip fruit orchards in Belgium. However, to date, no dramatic population increases, mass occurrences or damage has been reported in fruit production in Belgium (Berteloot et al. 2021). In 2022, in Belgium, 219.000 t of apples and 366.000 t of pears, some of the most vulnerable fruits to H. halys, were produced (European Commission 2022a, b). Apples and pears represent a combined revenue of more than € 148 million of which pear production is the most significant part with a revenue of € 119 million (Verbond van Belgische Tuinbouwcoöperaties 2022). An unmitigated expansion of H. halys in Belgium could therefore have a considerable impact on fruit production, especially in the Flanders region, which produces most of the apples, pears, and soft fruits in the country.

The increased accessibility and affordability of molecular technologies, as well as the expansion of databases containing publicly available DNA sequence data, have aided in the use of molecular tools to assess the genetic diversity and potential origin of invasive species (Cristescu 2016; Hamelin and Roe 2019). Due to its lack of recombination, relative neutrality and shorter coalescence time, mitochondrial DNA (mtDNA) is a highly popular molecular marker for examining genetic diversity and phylogeography of (invasive) animal species (Ficetola et al. 2008; Rollins et al. 2011; Bras et al. 2019; Ryan et al. 2019). Furthermore, for invasive species, the accumulation of easily reproducible genetic data in public databases facilitates the regular addition of new data from the invasion front to build a global pattern of invasion dynamics. For example, for H. halys, mtDNA sequences (portions of the mitochondrial gene cytochrome c oxidase subunits I and II – COI and COII, respectively) are already available for both native (China, Japan, Korea) and invaded countries (Gariepy et al. 2014; Xu et al. 2014; Cesari et al. 2015; Cesari et al. 2018; Lee et al. 2018; Yan et al. 2021).

In this study, we set out to fill one of the geographical knowledge gaps in Europe on the genetic diversity and distribution of H. halys. First, we investigated the genetic diversity of H. halys in Belgium by sequencing 99 specimens from 18 locations at two mitochondrial genes: COI and COII. Then, we compared those sequences to previously published H. halys sequences to infer the putative invasion routes to Belgium. Lastly, we used public citizen occurrence data to map the distribution of H. halys in Belgium from 2020 to 2022 and gain demographic insights into the Belgian populations.

This study uncovered 9 COI, 5 COII and 12 COI-COII distinct haplotypes from 99 H. halys specimens collected in Belgium. Among these, two new COI-COII haplotype combinations (H03h11 and H08h21), currently unique to Belgium, were observed.

The principal COI haplotypes present in Belgium were H01 (33%), H08 (24%) and H03 (23%). H01 and H03 are the most frequent haplotypes in China and in most invaded countries (Gariepy et al. 2014; Valentin et al. 2017; Cesari et al. 2018; Lee et al. 2018; Yan et al. 2021; Gariepy et al. 2021). As such, their presence in Belgium is to be expected and therefore does not help to determine the origin of H. halys in Belgium. Interestingly, however, H08 is the second most abundant haplotype in Belgium. So far, this haplotype has been exclusively detected in certain European countries: Austria, France, Italy, Switzerland and Serbia (Valentin et al. 2017; Cesari et al. 2018; Gariepy et al. 2021; Yan et al. 2021). This suggests that at least part of the H. halys population in Belgium originates from previously invaded territories in Europe (i.e., bridgehead effect; Lombaert et al. 2010). Remarkably, eight out of the nine COI haplotypes found in Belgium are shared with Italy. Furthermore, half of these haplotypes were reported exclusively in these two countries (H40c, H41c, H42c and H49c) suggesting that Italy is a direct region of origin of H. halys in Belgium. The rare H23 haplotype was only found once in Belgium and has been reported in Japan and the Western USA (Oregon) (Valentin et al. 2017). It was also detected in a shipment in Australia originating from Italy, suggesting that the H23 haplotype is also present in Italy (Horwood et al. 2019; Gariepy et al. 2021). Introductions may thus have originated from Japan or the USA in addition to Italy.

For the COII fragment, h01, h03 and h11 each accounted for 32% of the abundance. The h01 and h03 haplotypes are native to China and Korea (but have not been reported in Japan yet) and are the most frequent COII haplotypes in invaded countries (Xu et al. 2014; Cesari et al. 2018; Yan et al. 2021). The distribution of the h11 COII haplotype is more restricted and has only been reported in Austria, Italy, Japan, and Korea (Xu et al. 2014; Cesari et al. 2018; Yan et al. 2021). Consistent with Italy as a source of H. halys for Belgium, the h21 haplotype was isolated from two specimens collected in Belgium and is only shared with Italy (Cesari et al. 2018).

In this study, the combination of COI and COII fragments did not result in a significantly better geographic resolution to reveal possible origins of the invasion. However, the presence of haplotype H03h21 provides some additional support for Italy as a source of the invasion since this combination is only shared with a sample from the Piedmont region of Italy (Cesari et al. 2018). Additionally, the inclusion of the COII fragment in this study revealed two novel haplotype combinations unique to Belgium: H08h21 and H03h11. The former is likely to originate from Italy since haplotype h21 has been exclusively reported there (Piedmont region, same sample as for H03h21), while H08 has been detected multiple times (Piedmont and Lombardy regions of Italy). H03h11 is more singular because it combines a COI haplotype native to China (H03) and a COII haplotype native to Korea or Japan (h11; see above). Such a combination could arise from recent COI or COII mutations. H03 is only two substitutions from the closest COI haplotype native to Japan (H27), and h11 is only one substitution from three COII haplotypes native to China (h03, h05 and h10). Alternatively, this combination could also exist in the regions of origin (China and/or Japan/Korea) but has not yet been sampled. Belgium’s dominant haplotypes generally overlap with those from previously invaded European countries, such as Italy, Switzerland, Austria, and Hungary. In contrast, rarer haplotypes are exclusively shared with Italy and its native region of Japan (H23).

The genetic diversity found in Belgium is surprisingly high (Hd = 0.79, π = 0.0018, N = 95) compared to other invaded countries previously studied (Table 2), several distinct haplotypes were isolated from single sampling sites (Suppl. material 1). A reduced genetic diversity is usually expected for introduced species because of a limited number of founders associated with early genetic drift. However, genetic bottlenecks associated with introductions can be counteracted if many individuals are introduced at the same time or if repeated introductions occur (Dlugosch and Parker 2008). Similarly, Schuler et al. (2020) found a high haplotype diversity (Hd = 0.68, π = 0.0046, N = 156) in the H. halys population of South Tyrol in Northern Italy. The population in Tyrol was established by a secondary introduction from Eastern USA into the Emilia Romagna region of Italy and the spread of the founder population in Switzerland to the neighbouring countries. In contrast, Valentin et al. (2017) concluded that most invasive populations in North America and Europe were established from a direct introduction of H. halys from China with separate introductions into Eastern and Western USA and Canada, as well as into Switzerland and Greece. The high genetic diversity of H. halys populations in Belgium and the overlap of haplotypes with previously invaded European countries, such as Italy, Switzerland, Austria, and Hungary but also with Japan indicates that the Belgian populations probably originated from multiple invasions from already invaded European countries, mainly Italy, but also directly from Japan through inadvertent human-mediated transportation (often due to global trade of goods) (Valentin et al. 2017). The weak genetic structure detected by our Mantel test could be related to the recent local expansion of these genetically diverse clusters.

Previous studies have shown that citizen science provides valuable data to characterise the spread of H. halys (Maistrello et al. 2016; Stoeckli et al. 2020; Streito et al. 2021). From our data in Belgium, more H. halys observations are recorded in the nosrthern part of Belgium compared to the southern part of Belgium, with more urban areas than agricultural, forested, and rural areas. However, on a national scale, spatial biases are commonly associated with human population density, settlements, and road infrastructure (Kelling et al. 2015; Geldmann et al. 2016; Girardello et al. 2019. The bias towards urban areas in citizen-collected occurrence data and rising popularity of tools to make observations (e.g., smartphones) represents a challenge to infer true spatio-temporal patterns from our occurrence data (Bowler et al. 2022). Nevertheless, it also provides natural experimental gradients to examine the impacts of future environmental scenarios including climate change (Lahr et al. 2018). Urban areas are often seen as heat islands, offering more suitable refugia to survive winters or more favourable conditions throughout the seasons to develop and reproduce. Therefore, these urbanised areas often comprise a larger population of insects (Kaiser et al. 2016; Frank and Backe 2023). According to Kistner (2017), the northern part of Belgium lies near the latitudinal border of the climate in Europe suitable for the winter survival and summer reproduction of H. halys. In contrast, the southern part of Belgium is currently modelled to be an unsuitable eco-climatic region for H. halys, with colder summer and winter temperatures and fewer refugia to survive winter due to the lower urbanisation (Kistner 2017). Niche modelling through climate suitability by Streito et al. (2021) essentially confirms this, with the northern part of Belgium having more suitable areas for H. halys while the southern part is more marginal to unsuitable for its survival and expansion. Despite the possibility of spatio-temporal bias in our occurrence data, some temporal trends can still be derived from citizen-collected occurrence data (Powney et al. 2019; Outhwaite et al. 2020; Sheard et al. 2021; Zattara and Aizen 2021; Bowler et al. 2022). Our observational data shows an almost fifteenfold increase in observations from 2020 to 2022. However, to date, no damage related to H. halys has been reported in agricultural production in Belgium (Berteloot et al. 2021). Based on the occurrence data, it is likely that the population expansion of H. halys in Belgium is very recent, as a relatively high number of observations were only made in 2021, 2022 and 2023. Streito et al. (2021) define three main phases in the invasion of H. halys: (1) the very beginning of the invasion, when populations of H. halys are low, and naturalists and official monitoring services who are excellent observers and expecting the emergence of H. halys can detect its presence, (2) when abundance increases, non-naturalist citizens are able to detect the species and start to provide information on the dynamics of the invasion and indirectly on the level of populations through citizen science platforms, and (3) when populations have expanded and become large enough, agricultural professionals take over and can predict and assess the population density and damages. From our occurrence data, the H. halys population in Belgium is seemingly under expansion, being numerously recorded in the citizen science databases.

Lastly, the univoltine H. halys population in Belgium likely expands in the summer and declines in the winter in current climatic conditions due to Belgium being situated at the northern latitudinal border of climatic suitability for H. halys, with winters cold enough to kill more individuals than winter temperatures in southern European countries like Italy, possibly delaying the fast expansion of its populations. However, both parts of Belgium are modelled to be suitable for the survival and development of H. halys by 2100 (Kistner 2017).

The results of this study provide haplotype information for H. halys from a newly invaded region. The haplotype diversity in Belgium is surprisingly high, with 9 COI, 5 COII and 12 COI-COII haplotypes found. The invasion of H. halys in Belgium likely occurred repeatedly and is assumed to be still ongoing through human-mediated transportation from other invaded European countries and directly from its native regions in Eastern Asia. A significant overlap between Belgian and Italian haplotypes points to Italy as the most probable source for a significant proportion of haplotypes currently present in Belgium. By combining the citizen-collected occurrence data with the molecular data, we assume the population expansion of H. halys is recent and ongoing.

This research was in part supported by funds from VLAIO (Flanders Innovation and Entrepreneurship) through LA-traject HBC.2018.2224 (“SOS Penta”) and by the DATAPESTFRU project (RI 2020-A340) funded by the Belgian Federal Public Service (FPS) Health, Food Chain Safety and Environment. We thank Stéphane Claerebout for providing us one sample collected in 2018 in Mouscron.