PLoS ONE
The invasive longhorn beetle Xylotrechus chinensis, pest of mulberries, in Europe: Study on its local spread and efficacy of abamectin control
DOI 10.1371/journal.pone.0245527 , Volume: 16 , Issue: 1
Article Type: research-article, Article History
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### Notes

Abstract

The invasive wasp-mimicking Tiger Longicorn Beetle, Xylotrechus chinensis, a potentially lethal pest of mulberry trees (Moraceae: Morus sp.), was first reported in Europe in 2018, although its colonization and establishment were estimated to have occurred during the year 2012 or earlier. In Catalonia the infestation spread from four towns and 44.1 km2 in 2018 to 12 towns and 378.1 km2 in 2020; in the studied town of Barberà del Vallès, infested trees rose from 16.21% in February 2016 to 59.29% in December 2018. Human safety in public parks and avenues is a concern, as beetle infestation increases the risk of falling branches. The main objective of this study was to evaluate how the infestation progresses over time, with and without abamectin treatment, and provide insights into female egg-laying preferences. Such knowledge helps contribute to management efforts to reduce expansion of the range of beetle infestation. Our statistical analysis shows that females prefer laying eggs on larger trees, on the highest part of trunks and on the crown base (this being more preferred than the trunk), and they do so on warmer, SW orientations rather than those facing N, NW and E. Emergence holes and gallery slits predict the spreading of infestations to new trees. An abamectin treatment (trunk injection) carried out at the end of April significantly reduced the number of new infestation. However, for maximum insecticide efficiency, the best time for treating with abamectin would be from mid-July to mid-August, when newly hatched larvae begin feeding on the phloem.

Sarto i Monteys, Costa Ribes, Savin, and Mankin: The invasive longhorn beetle Xylotrechus chinensis, pest of mulberries, in Europe: Study on its local spread and efficacy of abamectin control

## Introduction

### Description of the insect

The invasive wasp-mimicking Tiger Longicorn Beetle, Xylotrechus chinensis (Chevrolat) (Fig 1), is a serious pest of mulberry trees which, if not controlled, causes their death. Its body is relatively large (15–25 mm in length), the antennae are short and widely separated, characteristic bands adorn the elytra and the pronotum [1]. Han and Lyu [2] provided a description of the adult along with colour pictures showing it in dorsal and lateral views, whereas the larvae and eggs were described in [1]. The larvae look typically longhorn, i.e., they have a conical-shaped trunk with very well marked pseudopoda and a whitish colour.

Fig 1
(A) Posing alert on a mulberry bolt. (B) Close-up of male head. (Photos V. Sarto i Monteys).A male of Xylotrechus chinensis.

### Native range and invasion history

This invasive species is native to the East Palaearctic region (NE China, Taiwan, Korean peninsula, and Japan), It was first reported as settled in Europe in 2018, specifically in Catalonia (NE Spain) [1], although the colonization and establishment of this beetle there were estimated to have occurred during the year 2012 or earlier. This beetle had been intercepted before, without establishing, in Germany and the U.S.A. Indeed, in 2007, specifically in Weissenhorn, a town in the Neu-Ulm district in Bavaria, two specimens (a male and a female) appeared on June 15 and 19, 2007 in a wooden packing box from China [3]. And, in June 2011, it was intercepted in the port of Philadelphia (Pennsylvania) by the Customs and Border Protection Agency in non-compliant wood spools that held steel wire rope shipped from China [4]. More recently, in July 2017, Schrader [5] reported an interception in Rhineland-Palatinate of a specimen in a container holding decorative elements (birch and willow) also from China.

Sarto i Monteys & Torras [1] contributed substantial new data on X. chinensis ’ mating behaviour and egg production capacity, seasonality, larval development and damage to mulberries in Catalonia. Based on this evidence, they stated this beetle would likely spread in Europe. Indeed, very shortly after, it was reported that in spring-summer 2017, around 200 mulberry trees near the harbour of the city of Heraklion (island of Crete, Greece) were found to be infested by this beetle. It was most likely introduced to the Heraklion area in 2014 or 2015 with commodities arrived at this port from its area of origin [6]. In July 2018, new outbreaks were detected in the Autonomous Community of València (Spain), specifically in Quartell and Sagunt (València) and Almenara (Castelló de la Plana) [7]. Finally, in France, one cerambycid larva collected in the port village of Sète (Hérault, Mediterranean coast), in October 2017, was molecularly diagnosed as X. chinensis, whereas in June 2018 an adult was collected there. Likewise, in July 2018, an adult beetle was photographed at Le Bouscat (Gironde, Atlantic coast), this town being only three km away from the port of Bordeaux, which might be the entrance point for this beetle. Probably the importation of X. chinensis into these French towns occurred no later than spring 2017, being more likely in 2016 [8]. In Sète, the beetle was clearly established by July 2019 with at least three distinct outbreaks found and evidenced by the presence of numerous adult emergence holes seen on the trunks of mulberries [9]. French authors suppose that, given the dispersal capacities of this beetle, it has certainly already reached neighbouring municipalities and should continue to disperse. See Fig 2 for an updated map gathering all findings of X. chinensis in Europe up to July 2020.

Fig 2
Source map reprinted from https://commons.wikimedia.org/wiki/File:Europe_map.png under a CC BY license, with permission from Wiki-vr, Public domain, via Wikimedia Commons. 30 November 2013.Xylotrechus chinensis in Europe up to July 2020.

In Catalonia, the beetle went from infesting one district and four townships (44.1 km2 ) in 2018 [1] to four districts and 12 townships (378.1 km2 ), including the city of Barcelona, in 2020 (see Fig 3). These are as follows: Vallès Occidental (Cerdanyola del Vallès, Badia del Vallès, Barberà del Vallès, Ripollet, Rubí, Sant Quirze del Vallès, Sant Cugat del Vallès, Sabadell, Terrassa), Vallès Oriental (Santa Eulàlia de Ronçana), Baix Llobregat (Molins de Rei), Barcelonès (Barcelona).

Fig 3
The four townships invaded up to July 2018 are in orange; the eight new townships invaded up to July 2020 are in red. The black asterisk signals the city of Barcelona. Source map of Catalonia obtained from BDLJE CC-BY 4.0 Spanish ‘Instituto Geográfico Nacional’ http://www.ign.es/. The districts and towns were reproduced from the Cartographic and Geological Institute of Catalonia, https://www.icgc.cat/, under a CC BY 4.0 license. The data were assembled using ArcMap 10.3.1 (May 13, 2015) https://desktop.arcgis.com/es/arcmap/10.3/main/map/what-is-arcmap-.htm.Catalonian districts invaded by Xylotrechus chinensis up to July 2020.

In August 2018, the European and Mediterranean Plant Protection Organization (EPPO) added X. chinensis to its Alert List , in order to draw the attention of EPPO member countries about this pest, which might present a risk to them [10]. This means that new outbreaks should be reported, and notice given to either stop or eradicate them. To date, it has not yet been declared a Quarantine pest in Europe, and therefore not subject to mandatory control measures.

### Hosts and impacts

X. chinensis larvae feed and bore on mulberry trees (Morus sp.), but apple, pear and common grape vine have also been recorded in literature as possible host plants (see reviews in [2,11]) which would require confirmation. At least for grape vine, in laboratory conditions, it was concluded that X. chinensis did not use it as an alternative hostplant [1].

The industry of sericulture, and its association to mulberry trees, was economically very important in Spain until the 19th century, especially in the areas of Andalusia and Murcia. However, it virtually disappeared during the first half of the 20th century in Spain and other southern European countries [12]. Mulberry trees used in the past to feed silkworms, mainly Morus alba , are now commonly used in many southern Mediterranean towns to provide shade and ornament to streets and avenues [13]. Their abundance in public and private places are greatly helping the spread and damage caused by X. chinensis . Sarto i Monteys & Torras [1] warned, referring to Catalonia, that the damage to mulberry trees as well as the economic consequences were already substantial. The beetle does not only cause the death of the trees but human safety in public parks and avenues is also a concern because beetle infestation increases the risk of falling branches and the need for rapid response by municipal authorities.

### Description of damage signs and symptoms

Sarto i Monteys & Torras [1] reported the life cycle of Xylotrechus chinensis in Catalonia and the damage caused to mulberry trees. At the end of spring and the beginning of summer adult beetles emerge from mulberry trees, mate, and females lay their eggs on the bark of trees. Infestation that follows can be revealed externally by two consecutive indicators, which add to the previous one constituted by the emergence holes left on the bark. Based on this evidence and new field observations, three types of indicators were selected to document infestation: (1) emergence holes, (2) bark injuries and (3) gallery slits, all visible on the trunk and crown base of trees. These indicators were counted and processed as described in the methods to inform the municipality about the progress of the beetle infestation in the study area.

#### Emergence holes (EH)

They are round holes (ca . 5–6 mm diameter) on the tree periderm made by adult beetles when, after completing metamorphosis, come out from the tree from mid May to mid August. Such holes accumulate summer after summer, and remain visible as long as the periderm stays in place, even on dead trees. Fig 4A shows some of them on the crown base of an infested tree. Fig 6 below shows them along the trunk, here painted in two colours, meaning holes made in two consecutive summers.

Fig 4
(A) Adult beetle emergence holes (most coloured orange) on the trunk of a tree. (Photo G. Torras). (B to D) Bark injury caused by beetle larvae: (B) as seen before removing the dead and dry periderm to discover the feeding gallery underneath, and one beetle larva (C) and (D). (Photos J. Serra). (E-F) Gallery slits caused by beetle larvae: (E) on the trunk, (F) on the crown base. Coloured spots signal emergence holes. (Photos G. Torras (E); V. Sarto i Monteys (F)).X. chinensis infestation indicators on mulberry trees.

#### Bark injuries (BI)

They are produced by active larvae, hatched annually in July-August from eggs laid on the bark, which start making feeding galleries in the tree phloem, hidden from the outside. Bark injuries only become visible when the thin periderm, that covers the cavities the larvae make, dries out and small openings or cracks appear on its surface. This only happens when the larvae have grown enough and have substantially enlarged the cavities under the periderm. Through these cracks, quite often sap oozes and some larval frass is expelled (the latter soon drying out and falling to the ground), helping to spot the injury (Fig 4B–4D). By removing the dry and dead periderm with a punch or knife (where the cracks appear), the feeding galleries can be easily discovered. These show either partly empty cavities where larvae could be found or compacted whitish yellow debris (a mixture of excrement and sawdust from the phloem consumed by the larvae). The latter may occupy, particularly in winter, the entire gallery.

Bark injuries caused by larvae hatched in summer may appear as early as October-November, depending on how quickly the larvae have developed. Occasionally, full-grown larvae have been found overwintering under bark injuries at the end of December. Bark injuries become increasingly more evident towards the end of spring when all larvae have finished growth and have bored into the xylem to pupate. Particularly in the first half of May, many bark injuries still harbour a larva in the cavity underneath (see for instance Fig 4B–4D, with photographs taken on May 10, 2016). Consequently, the presence of bark injuries can be a good indicator of infestation, although their counts underestimate the real number of larval presence in trees. Bark injuries transform into gallery slits usually one year after they appeared. Hence, unlike emergence holes and gallery slits, they are not accumulative on the trees. However, in our study, they are accumulative, since injuries seen in June have not had enough time to transform into gallery slits by December.

#### Gallery slits (GS)

They are formed when the periderm that covers old galleries, i.e. presently with no larvae and filled totally or partly with debris, breaks by itself longitudinally along its full length (10–40 cm). As a result, the galleries become exposed and look like big scars on the bark (Fig 4E and 4F). The debris inside, which in many cases formed a contiguous compact mass along the covered galleries, now exposed, soon dries out in contact with the air, and progressively transforms into disintegrated pieces that end up falling to the ground. If the tree survives the beetle attack, it will eventually cover and close those slits with new phloem tissue that slides, from right and left, over the exposed and hard xylem (which is bored into for pupation but not used as a food source by the beetle larvae).

### Review of within-tree distribution of larvae and pest spread

Huang [14] reported in China about the spatial distribution of overwintering (December to April) larvae of X. chinensis within mulberry trees. He found the larvae were distributed in an aggregation pattern, which fitted a binominal distribution model and, to a smaller extent, a Neyman model. No studies were reviewed that reported how the beetle infestation progressed among the trees in a particular township or area between two consecutive generations, i.e., a statistical analysis of new infestation in relation to past infestation. Nor could we find studies about the degree of infestation with respect to tree size, i.e. based on trunk height and trunk perimeter, or about the geographical orientation of infestation damages and their location on tree trunk height bands and on the crown base. All this would provide important knowledge on the preferred trees for egg laying and where the eggs are most likely to be laid. A goal of the study was to determine such information to assist in scouting for infestations.

### Management options: The past and the future

Chinese [15] and Japanese [16,17] authors dealt in the past with this beetle pest. However, the control methods they proposed are obsolete and impracticable today (see [1] for review). For instance, Aruga [17], in Japan, listed three control measures for X. chinensis , although they are simple and applicable to most coleopteran and lepidopteran tree borers. They are as follows: (1) adults are caught and killed; (2) if frass protrusions reveal the presence of larval cavities, a suitable insecticide should be injected into these cavities; and (3) larvae can be killed by inserting metal wire inside the cavities. And Sun [15], in China, reported that the best method was blocking every (emergence) hole with mud after having injected insecticides into them, and later inserting cotton pellets impregnated with insecticides into these same holes, being the more effective the organophosphate insecticides phoxim and chlorpyrifos.

However, injecting insecticide into the emergence holes seen on mulberries, apart from being very time-consuming, would not be efficient since they are not really well connected to the phloem cavities inhabited by larvae. Obviously, an emergence hole leads to an empty tunnel where the larva pupated and the hole is only made when the adult abandons the tree [1]. And phoxim and chlorpyrifos are presently banned for use on crops in the European Union.

Based on all data gathered, Sarto i Monteys & Torras [1] suggested possible control methods to hinder the spread of this pest. In sum, they were as follows: (a) Physical control. Removal and destruction (burning or grinding) of heavily infested mulberry trees, (b) Chemical control using insecticides, (c) Pheromone control, since the three components of the male-produced aggregation pheromone of X. chinensis are known, and (d) Biological control using parasitoids, since at least one likely parasitoid wasp was found by these authors.

With regard to chemical control, these authors suggested two methods that might complement each other. (1) Targeting ovipositing females, their eggs and first instar larvae, by spraying authorised contact insecticides on barks at the beginning of June, July and August. However, special regulations applying to insecticide use in public areas may hinder its implementation, and (2) Targeting larvae (all instars) feeding in the tree phloem. This would require the use of a systemic insecticide, for instance abamectin, mostly a stomach poison, introduced into the xylem through endotherapy, i.e., the trunk injection of a sap-compatible solution of the insecticide. The xylem sap moves upwards through the vessels and the active ingredient is so transported to the phloem, where beetle larvae feed. This confines the applied insecticide only within the target tree, thereby making it particularly useful in urban situations. Abamectin has shown good results against bark beetles (see for instance [18]).

Since the use of pheromone and biological control methods are not ready yet (research is still necessary in this regard), while removal and destruction (burning or grinding) of heavily infested mulberry trees (the most used measure in Catalonia so far) has been proven to be insufficient and ineffective to control the spread of this pest, some townships tried chemical control injecting abamectin to some of the trees. However, not knowing exactly the best time to treat and with no follow-up of the efficacy of such treatments, no clear guidelines for action have been obtained so far to effectively control this pest. Our results aim to some extent to remedy this gap.

In sum, there is a need for (1) improved management to control this pest, (2) much precise knowledge of spread, (3) knowledge of within-tree distribution of larvae and females’ preferences to lay their eggs on particular trees or sections of trees. Specifically, to aid in scouting, it is important to know if (a) trees with bigger trunk perimeter and height were more attractive to female beetles than smaller ones, and (b) if female beetles showed more preference for particular height bands and compass orientations to lay their eggs on the trees. Finally, (4), there is a need for results concerning the efficacy of insecticide treatments. Providing answers to the four above-mentioned points were our goals in this work.

## Materials and methods

### Surveys: Location, dates and trees

The town of Barberà del Vallès, 8.3 km2, located in the district of Vallès Occidental, 14 km north of Barcelona (Catalonia, Spain), held in February 2016 506 mulberry trees spread over its public avenues, streets and squares. 16.21% of these trees were already infested by X. chinensis [1] (Fig 5). In 2018, two surveys were conducted under permissions granted by the City Council, checking all public trees for beetle infestation. One was in the first half of June, just before the expected emergence of the new annual generation of beetles, and the other in the second half of December, when larvae hatched during the summer (from eggs laid by those beetles) were overwintering (many already fully grown) in the trees.

Fig 5
(Map source: Cartographic and Geological Institute of Catalonia, https://www.icgc.cat/, under a CC BY 4.0 license, accessed 27 April 2020).Satellite map of the town of Barberà del Vallès showing in green the location of its 506 public mulberry trees in 2016.

In May 2018, only 438 public mulberry trees remained, since 68 had been removed (either cut, pulled out or replaced by other non-mulberry trees) by local authorities as they were in poor condition due to beetle attack and there was risk of falling branches. Therefore, the survey in June was done on 438 trees. Between June and December 2018, 30 more mulberry trees dissappeared (16 had been cut, 3 pulled out, and 11 replaced by either Freeman’s maple, Acer × freemanii or Koelreuteria panniculata). Therefore, the December survey was done on 408 trees.

### Data sampling and measurement

All 438 public mulberry trees present in June 2018 were given a numerical code and located on a satellite map, so they could be recognized individually in both June and December surveys. Most trees were on a straight line, along avenues and streets, spaced about 6.5 meters. Two people working together checked each tree, acting in an orderly manner from tree 1 to tree 438, and counted their emergence holes (EH), gallery slits (GS) and bark injuries (BI), noting also their heights above the ground and their compass orientations as one of 8 directions: N, NE, E, SE, S, SW, W, or NW. Position was recorded on consecutive 50 cm-height bands along the trunk (from ground level to its top), plus a final 50 cm band comprising the lowest part of the crown, i.e. the crown base. Above these bands, infestation is generally low and it was not considered. The number of bands along the trunk varies according to its height: the taller the tree, the bigger the number. The top band on the trunk, i.e. the one that reaches the crown base, has obviously a variable height, shorter than 50 cm (Fig 6).

Fig 6
In this tree, the top band on the trunk measures only 25 cm before reaching the crown base from the fourth 50 cm-trunk band. Coloured spots signal emergence holes. (Photo G. Torras).A mulberry tree showing the 50 cm-height bands established for positioning indicators of infestation.

In addition, the trunk perimeter (at a height of 130 cm above ground level) and the trunk height from ground level to the base of the crown, i.e. when the main branches start projecting out from the trunk, were also measured per each tree using a girthing tape.

### Insecticide treatment using abamectin

On April 24 2018, an endotherapy treatment with abamectin was carried out by an external company hired by Barberà del Vallès’ City Council. 107 mulberry trees were treated, nearly all lining up along one row (sometimes two) of a wide avenue heavily infested by the beetle. When two rows were present, they were spaced about 7 m. Within these rows, most trees were injected, regardless of their infestation level; their codes are indicated in S1 Table. The timing and relevance of this treatment had not been decided by us. Neither control group had been taken nor scientific follow up had been planned. This treatment was done as a response to citizen pressure to try to save the trees. Because these 107 trees, among the 438 checked in June 2018, were still present in December, an analysis of the treatment results was included in the study.

The dose of insecticide per drilled hole (trunk injection point) and drillings per tree are both determined according to the trunk perimeter. In this case, 10 ml of a mix containing 5 ml of abamectin 1,8% [EC] w/v (Vertimec®, register # 16784, Syngenta España, Madrid, Spain) and 5 ml of abamectin emulsifier (Endotree Mixable®, Endoteràpia Vegetal, Castelló d’Empúries, Girona, Spain) were injected to the tree per drilling. Typically, there were 2–3 drillings per tree. Specifically, drillings for injecting the insecticide are determined at the rate of one every 30 cm of trunk perimeter at a height of 130 cm. They are performed at the base of the tree trunk, between ground level and the first 20–25 cm, with a 7 mm drill bit, and evenly distributed around the perimetral circumference. Finally, before injecting the insecticide, a special catheter, which acts as a plug (Endoplug®, Endoteràpia Vegetal, Castelló d’Empúries, Girona, Spain) is placed in each hole. This catheter allows the product to enter through a special injection needle (external diam. 1.6 mm, internal diam. 0.8 mm, total length 80.50 mm) and prevents it from exiting when this needle is removed. It also has the function of protecting the hole against the entry of external pathogens. The equipment used for the injection is called Endoplant® (Endoteràpia Vegetal, Castelló d’Empúries, Girona, Spain). It weighs less than 4 kg and consists of three parts: a central body, composed of the mechanical piston dosing pump, a joystick pistol with LCD screen for the control of the application and two bottles containing the products to be injected (see https://endoterapiavegetal.com/ca/equips/equip-endoplant/ for application details).

### Statistical analysis

All data collected for the 438 trees (408 in December) were placed in a table holding 64,824 records (S1 Table), serving as a basis for subsequent analysis. Statistical analyses of field data were performed using R software, v.3.6.2. To test for statistical differences of the distributions of infestation damages (EH, BI, GS), concerning their compass orientation and their location on tree trunk height bands and on the crown base, a non-parametric paired, 2-sample Wilcoxon Test was used that explicitly compares results for the same trees for stochastic dominance and is suitable for data distributed not normally. To test if females preferred the crown base to the trunk for laying their eggs, we used the same paired 2-sample Wilcoxon test. The null hypothesis was that the samples’ mean ranks were the same (there was no difference in the number of damage for trunk and crown base). A P-value below 0.05 leads to rejection of the null hypothesis of no difference between crown base and trunk damage.

A statistical analysis of new infestations in December 2018 was conducted in relation to past infestation, height and perimeter of the tree, as well as the effect of the abamectin treatment. Because the dependent variables are count data with many zeros and conditional means have a value much below their variances, we could not use ordinary least squares (OLS) as the estimation method and opted for a negative binomial regression model (NBRM). NBRM can be considered as a generalization of the Poisson regression since it has the same mean structure and it has an extra parameter to model the over-dispersion. We also run likelihood ratio tests that suggest that NBRM is a better option (than Poisson model) for our data. Furthermore, to ensure better comparability of the control and treatment groups of trees (i.e. those treated with abamectin versus those without the treatment), we applied propensity score matching using nearest neighbour matching algorithm in MatchIt library version 3.0.2.

## Results and discussion

### Infestation of public mulberry trees from 2016 to 2018

A tree was considered to be infested when it showed at least one sign of infestation, i.e. presence of EH, BI, GS or a combination of them. The infestation of a tree can be present or past or both. In February 2016, only 16.21% of the 506 public mulberry trees of Barberà del Vallès showed signs of infestation by X. chinensis [1]. In June 2018, when the present study started, 68 trees had already been cut by the Municipality because of beetle attack, and of those standing (438) nearly half (46.12%) had been infested. This figure would rise to 53.36% if the 68 missing (infested) trees were included. From June to December 2018, 30 more trees were eliminated, and of those standing (408) 49.51% were infested. Again, this figure would rise to 52.97% if the 30 missing trees were included. In sum, in this town, from February 2016 to December 2018, the infestation rose from 16.21% to 59.29%, including removed trees.

The degree of infestation in trees varied notably, from only one sign up to 54 in the worst case. Table 1 and Fig 7 sort the trees checked in this study according to the signs of infestation they bore in June and in December 2018. As mentioned above, the percentage of infested trees increased in December, despite 30 trees, heavily infested in June and removed soon after, were not considered in the December counts.

Fig 7
Graph sorting trees according their sum number of infestation indicators (EH+BI+GS) in June and in December 2018.
Table 1
Trees sorted according their sum number of infestation indicators (EH+BI+GS) in June and in December 2018.
June 2018December 2018
Infestation indicators’ ranges# of trees%# of trees%
[0]23653,88%20650,49%
[1–5]10323,52%10726,23%
[6–10]429,59%389,31%
[11–25]4510,27%4711,52%
[>25]122,74%102,45%
438100%408100%

Fig 8 summarizes all counts for the three established infestation indicators shown separately, comparing the June and December 2018 surveys. Since, as mentioned above, 30 trees present in June were missing in December, regression analysis will only consider the same 408 trees present in both surveys. However, we include in Fig 8 (top table) the 438 initial trees present in June to emphasize these 30 trees were contributing notably to the infestation in June, especially regarding emergence holes and gallery slits (47.05% and 25.03% more, respectively). Should they not have been removed, the December figures would certainly have been much higher. The total accumulative figures for the three infestation indicators used in this study are 2,103. They include all counts made in June 2018, even those on the 30 trees later eliminated, plus counts of (only) new signs seen in December 2018, and distribute as follows: 943 EH (44.84%), 964 GS (45.84%) and 196 BI (9.32%).

Fig 8
For June 2018 figures include the 438 trees initially checked, as well as, again, for only the same 408 trees also present in December 2018, this for comparative reasons (see text).Summary counts for the three established infestation indicators, comparing the June and December 2018 surveys.

Some discussion follows concerning these results. As to emergence holes, since X. chinensis is univoltine and there are no records of specimens enduring two-year cycles as reported for other cerambycids, it must be assumed beetles emerged one summer are offsprings of beetles emerged the previous summer. As to the counts made in June 2018, since holes stay visible on the bark of trees year after year (as long as the bark does not come off), they are in fact accumulative, summing up the holes made by beetles emerged at least in the previous summers of 2015, 2016 and 2017. This is because we estimate the beetle might have colonized the town of Barberà del Vallès in the summer of 2014, so the first emergence holes on trees of this town would have appeared in the summer of 2015.

The December 2018 counts revealed 246 new EH on the same 408 public trees checked in June. Since one new EH equals one new beetle, the latter means 246 beetles emerged in 2018 from those trees (mostly in July), i.e. an increase of accumulative values of 51,90%. If we apply the male/female ratio of 1.16, and 68.2 as the estimated average number of eggs laid per female [1], the estimated new egg load laid by females during the summer of 2018 on the trees of this town would be 7,767. In addition, there is the egg load contributed by beetles emerging from infested trees held in private gardens or compounds of this town, which was not considered. At present, the mortality rate of the different stages of this beetle (eggs, larvae, pupae and adults) and its causative agents are unknown. The finding of one likely parasitoid of beetle larvae [1] seems anecdotal for now. So, if no effective measures of control are taken, the spread of this beetle seems inevitable.

As to BI, those counted in June 2018 are those caused only by larvae hatched in the summer of 2017, since, unlike EH and GS, they do not accumulate year after year on infested trees (instead they transform into GS generally one year after they appeared). This explains why their counts are much lower than those recorded for the other indicators. However, in our study, counts of BI are accumulative from June to December since the injuries seen in June have not had enough time to transform into gallery slits by December, and new BI caused by larvae hatched in the summer of 2018 are added to the count. Indeed, counts made in December 2018 revealed 62 new BI on the same 408 public trees checked in June, i.e. an increase of 50%.

As to GS, the December 2018 counts revealed 36 new slits on the same 408 public trees checked in June, i.e. an increase of accumulative values of only 4,85%. However, their accumulative numbers in June 2018 were 743, by far the highest of the three infestation indicators used. As with EH, GS stay visible on the bark of trees year after year and therefore their counts are accumulative, summing up slits appeared from at least 2015, 2016, 2017 and first half of 2018. The relatively low number of new GS accounted in December 2018 could be explained because many BI seen in June had not yet transformed into GS by December.

### Infestation with respect to tree size based on trunk height and trunk perimeter

The goal was to check if bigger trees, i.e. those with bigger trunk perimeter and height, were more attractive to female beetles (to lay eggs on) than smaller ones. Since our data accumulate damages (EH, BI, GS) attributable to a few years (see above) and we expect females to keep their egg-laying preferences year after year, we included in the analysis all damages found on the 408 trees present in December 2018 plus those found on the 30 trees present in June 2018 but missing in December. All in all, analysis to check the degree of infestation with respect to tree size was carried out on a total of 2,103 damage counts.

#### Infestation with respect to trunk height

The 438 mulberry trees checked in June 2018 had a trunk height between 155 and 335 cm (μ = 240.22), most falling into the 200–250 and 251–300 ranges, as seen in Table 2 where all trees are grouped into four ranges. With regard to numbers of tree infestation, those in the 251–300 cm range were the ones most affected (64.8% showed infestation). Regression analysis was used to test if female beetles prefer to lay eggs in trees of specific heights. Tree height was not found to be a significant factor affecting the distribution of December damage compared to June (see below). Females of this beetle are good flyers, so trunk height does not seem to be important to them when it comes to lay eggs on the bark, unlike trunk perimeter which might be important (see next heading).

Table 2
Tree infestation by X. chinensis based on trunk height (from ground to crown base).
Values accumulated up to December 20181
Trunk heights’ ranges (in cm)Total trees within the rangeNon-infested trees within the rangeInfested trees within the range% of infestation within the range
[155–199]18 (4.11%)11738,89%
[200–250]296 (67.48%)15314348,31%
[251–300]122 (27.85%)437964,75%
[301–335]2 (0.46%)1150,00%
438208230
1 The 30 trees missing in December were incorporated into the counts as if they were present with the same values they had in June.

#### Infestation with respect to trunk perimeter

The 438 trees had a trunk perimeter (at 130 cm above ground level) between 17.5 and 138 cm (μ = 60.0), most (64.38%) falling into the 40–80 cm range (see S1 Fig). Perimeter lengths were grouped into five ranges (Fig 9). The range that gathered most trees (190) was that of 50.1–75 cm; the least abundant range was that of >100 cm with only 21 trees. About 58% of all trees within the range 50,1–75 cm and 75,1–100 cm were infested by X. chinensis, whereas the infestation within the other ranges was clearly lower, especially that of the range 0–25 cm with only 32% of trees infested. Therefore, should many trees be available, females prefer laying eggs on larger trees.

Fig 9
In both graphs the 30 trees missing in December were incorporated into the counts as if they were present with the same values they had in June. The top graph shows the trees infested within each perimeter range. The bottom graph shows the sum of damages (EH+BI+GS) for all trees within their range (total damages being 2,103), and the expected damages if their distribution was neutral, i.e. with no preferences for laying on any trunk perimeter range. Percentage values in red or green tell the difference between the expected and the observed.Tree infestation by X. chinensis based on trunk perimeter.

This is consistent with the regression results (see below), where the tree perimeter length had a positive significant coefficient predicting new EH, BI and GS. Interestingly, if we also add a squared term, it is often found to be significant but negative, implying that starting from a certain value, a tree with a larger perimeter becomes less attractive to the beetle. Fig 10 shows the projected number of EH, BI, GS and the last two combined depending on perimeter size. The projections are done using significant estimates obtained with propensity score matching reported in shaded columns in Tables 4 and 5 below. Note that, as it was shown in [19], the perimeter with maximum infestation signs from the regression can be obtained as $-\beta }{2\alpha }$ where β is the coefficient of perimeter, and α the coefficient of its squared term. As one can see, for all infestation signs except EH (here the relationship is linear) perimeter between 60 and 80 centimeters is the one where the number of new infestations is highest.

Fig 10
BI+GS stands for bark injuries and gallery slits combined.Quadratic functions corresponding to the coefficients identified in the models for perimeter and squared perimeter—with other variables assumed to be zero.

This result does not have an obvious explanation. One could be that trees with largest perimeter stand faraway from other trees and are harder to notice by the beetle. However, by locating these trees on a satellite map, the latter would only apply in a few cases. Another explanation could be that bark injuries become increasingly more difficult to spot on the bark of trees when these are large, i.e. when they have large perimeters. The latter, though, would not apply to GS.

Comparisons were also made of all damages (a total count of 2,103 for all trees) within each established range with the expected damages within this same range (i.e. under a theoretical neutral distribution of damages, where females would show no preference for laying on any trunk perimeter). Interestingly, those trees in the trunk perimeter range of 75.1–100 cm received much more damage (35.5% more) than expected, followed by those in the 50.1–75 cm range (8.8% more). The other three ranges showed less damage than expected, especially in the 25.1–50 cm range (38.3% less). This is again consistent with the inverted U-shape relationship between perimeter size and the probability to be infested stated above.

### Orientation and location of infestation damages

We also checked how all damages (EH, BI, GS) were distributed on the trees, looking at (1) their compass orientation, and (2) their location on consecutive trunk height bands plus crown base as defined above. Results include damages seen on both trunk and crown base, although the count was done separately.

The distributions of the tree types of damage caused by beetle infestation (EH, BI, GS) were plotted in their orientation on trunk and crown base, as well as their distribution on height bands. As mentioned above, all 438 trees were in this analysis, including the December 2018 data and the data contributed by the 30 trees in June that had been removed by December. Furthermore, we aggregated the information on the orientation on trunk and crown base into a single variable of orientation. Lastly, we created two new variables: BI+GS (BI and GS combined) and EH+BI+GS (EH, BI and GS combined). The goal was to form a systemic picture whether the damages are predominantly concentrated on certain orientation or height.

The results are shown in Figs 1114, where they are summarized in two subfigures for each characteristic. The top left subfigure shows the distribution of the respective infestation sign over orientation in averages and error bars depicting +/- 2 s.e. The bottom left subfigure does the same but for the height of the infestation signs. Thus, for example, the distribution of emergence holes on NW orientation in Fig 11 has a mean of approximately 0.3 holes per tree with a confidence interval ≈0.06–0.54. The subfigures on the right side summarize results for stochastic dominance in the distribution of the given damage. All columns of the matrices represent the results of the pairwise Wilcoxon test. The dark shading indicates that the distribution of damage with an orientation or height specified in the respective row dominates the distribution in the corresponding column with a specified significance level. Asterisks ***, **, and * denote 1%, 5%, and 10% significance, respectively. Returning to the example in Fig 11, the number of emergence holes with the SW orientation is statistically higher than the one with the NW orientation, and this result is significant at the 5% significance level.

Fig 11
Distribution of emergence holes (EH) on orientation and height.
Fig 12
Distribution of bark injuries (BI) on orientation and height.
Fig 13
Distribution of gallery slits (GS) on orientation and height.
Fig 14
Distribution of emergence holes, bark injuries and gallery slits combined (EH+BI+GS) on orientation and height.

The compass distribution of EH (Fig 11) indicates that females prefer to lay eggs on the SW side of the tree, both on the trunk and crown base. The 150-200-cm and the 200-250-cm bands have higher values than the others. A somewhat similar pattern emerges in the distribution of BI (Fig 12). SW and NE orientations are more common, while the 0–50 cm, 200–250 cm and 250–300 cm bands are the most preferred. Concerning GS , N and NW side of trees are clearly less preferred, while the 0–50 cm and 200–250 cm bands are the most preferred (Fig 13). When combining EH, BI and GS together (Fig 14), the SW side is the most preferred while N, E and NW the least. The 200–250 cm band shows much higher values than the others. Since larvae, after hatching, do not move much within the tree phloem, the latter means females prefer laying eggs on the highest part of trunks and on the crown base, favoring the SW side and avoiding N, E and NW.

Finally, we checked if females preferred the crown base to the trunk for laying their eggs, since this was our perception in the field. Since trunks as a whole have a lateral surface much bigger than that of the crown base, surface values of these two pieces (trunk and crown base) should be ideally considered when checking the damages they both endured. By knowing the perimeter and height of trunks, their lateral surface can be calculated easily. The lateral surface of crown bases is more difficult to calculate, since big branches project from them, which adds surface value to the crown, yet it could be estimated as the product of trunk perimeter x 50 cm height (as defined above) x 30% more. Then, we measured the number of infestations per 1000 cm2 and the results are shown in Table 3. Clearly, the number of infestation signs per surface on average is always higher for the crown base.

Table 3
Wilcoxon test for the distribution of infestation damages on trunk and crown base (per 1000 cm2).
VariableAverage on trunk (per 1000 cm2)Average on crown base (per 1000 cm2)Wilcoxon test p-value
EH in June0.06800.12790.5265
EH in December0.07620.14640.4804
BI in June0.00920.05210.2226
BI in December0.01520.06830.0396
GS in June0.10760.20810.0159
GS in December0.08730.21170.0043
EH+BI+GS in June0.18480.38810.0012
EH+BI+GS in December0.17870.42630.0001

However, comparing the distribution of infestation signs for the same trees on the trunk and on the crown base (i.e. considering specificity for each tree) we find statistically significant results for BI in December, and for GS and all infestation signs combined (EH+BI+GS) in both months. This suggests that females prefer the crown base to the trunk for laying their eggs.

### Analysis of new infestation in December 2018 as a function of past infestation and other tree variables

By using a NBRM regression analysis, the number of new tree damages in December was explained by the damage the trees showed in June, their height and perimeter, as well as the abamectin treatment 107 trees received on April 24, 2018. In addition, the results were tested for the sum of BI+GS new damage. For proper comparison this analysis is limited to 408 trees present both in June and December 2018. Tables 4 and 5 below summarize the results. The coefficients in these tables should be interpreted as follows: the variable EH_june has a coefficient of 0.223 on the number of new EH in December (ΔEH, see Model 1), which is statistically significant at 1% level. This means that for each one-unit increase in EH_june, the expected log count of ΔEH increases by 0.223. In other words, each extra emergence hole in June leads to e0.235≈1.26 more emergence holes in December.

Table 4
Regression results with NBRM for EH, BI and GS considered separately.
ΔEHΔBIΔGS
Model 1Model 2Model 3Model 4Model 5Model 6Model 7Model 8Model 9Model 10Model 11Model 12
Constant0.6821.793-0.482-0.804-5.991***-10.901***-1.175-6.671-1.723-12.019**-1.545-25.821**
EH_june0.223***0.466***0.185***0.379***-0.355*-0.348-0.401-0.444-0.1120.418-0.5049.378
(EH_june)2-0.009***-0.007**0.006-0.008-0.045-9.291
BI_june0.0740.070-0.038-0.0650.2120.1540.0730.0510.2140.0320.1820.073
GS_june0.190***0.533***0.205***0.306*0.210***0.268*0.130*0.0640.1241.525***-0.0311.381***
(GS_june)2-0.028***-0.009-0.6590.004-0.196***-0.178**
Abamectin-0.624-1.053*-0.644-0.854*-1.316**-1.346**-1.354**-1.298**-0.070-0.288-0.403-1.109*
Perimeter0.026***0.0130.027**0.075*0.019**0.189***-0.0040.174**-0.0010.421**-0.0170.808**
(Perimeter)2-0.001-0.001-0.001**-0.001**-0.004**-0.067*
Height-0.016**-0.020***-0.010-0.016*0.0110.0100.001-0.001-0.004-0.0140.003-1.247
AIC566.76563.90359.60360.32316.60313.23188.63189.11219.87191.21147.86119.64
N obs.408408198198408408198198408408198198
Likelihood ratio test290.64***266.06***30.87***25.56***32.55***9.71***
Asterisks ***, **, and * indicate 1%, 5%, and 10% significance level, respectively. Results on a subsample based on propensity score matching are presented in shaded columns. A significant value of the Likelihood ratio test indicates that NBRM, estimating the dispersion parameter, is more appropriate than the Poisson model.
Table 5
Regression results with NBRM for BI and GS combined.
Δ(BI+GS)
Model 13Model 14Model 15Model 16
Constant-3.528**-9.495***-0.579-9.091**
EH_june-0.224*-0.117-0.448*-0.436
(EH_june)2-0.0010.006
BI+GS_june0.170***0.445***0.116**0.378***
(BI+GS_june)2-0.024***-0.022**
Abamectin-0.803*-1.156**-0.967**-1.350***
Perimeter0.013*0.219***0.0080.273***
(Perimeter)2-0.002***-0.002***
Height0.0050.003-0.001-0.002
AIC443.30424.29265.23253.02
N obs.408408198198
Likelihood ratio test46.12***28.70***
Asterisks ***, **, and * indicate 1%, 5%, and 10% significance level, respectively. Results on a subsample based on propensity score matching are presented in shaded columns. A significant value of the Likelihood ratio test indicates that NBRM, estimating the dispersion parameter, is more appropriate than the Poisson model.

The results indicate that the number of GS in June is a significant predictor of new EH, GS and BI+GS combined in December, while EH in June is a significant predictor only for new EH in December. Thus, the more EH and GS a given tree had in June, the more damage the tree was experiencing in December. BI in June in contrast is not a significant predictor for new damages on the trees. This can be explained since, as mentioned above, bark injuries transform into gallery slits usually one year after they appeared, and this is why their accumulative counts are much lower than those recorded for the other infestation indicators (EH and GS). Therefore, EH and GS can be considered as useful predictors of new coming infestation on trees. The negative and significant coefficient for EH in June on ΔBI and Δ(BI+GS) stands out (models 5 and 13), but since the relation is only weakly significant (and mostly disappears after we apply propensity score matching), we ignore it.

As mentioned above, while perimeter tends to be positively and significantly related with new damages, the tree’s height is insignificant. Moreover, perimeter sometimes has a nonlinear relationship with new damage with the squared term being negative indicating that a tree having a too small or too large perimeter has a lower chance to experience further infestation. A similar “inverse U-shape” relationship is also observed for EH and GS (models 2, 10 and 14) in June. This may be interpreted as beetles reducing infestation of a severely infested tree and possibly moving to a different one, although to confirm it new research is needed about the effects of chemical and visual stimuli. For EH the maximum of the U-shape curve is reached at approximately 25 EH in June, while for GS at 4 GS in June.

#### Effects of abamectin treatment

As mentioned above, on April 24, 2018, a total of 107 trees were injected with abamectin. Most of these trees were already notably infested by X. chinensis and our goal was to check if such a treatment had a significant effect on the larvae finishing their development within the trees, which would transform into adults and emerge soon (from mid-June to mid-August). Therefore, abamectin treatment was included as a predictor in models on the full sample and a robustness analysis was included, in which a propensity score matched treated trees against nearby untreated trees with respect to the five variables listed in Table 4. As a result, we obtained a subsample of 198 trees (99 being treated and 99 being not) that better fits the concept of random treatment of trees and supports credibility of our causal inference on the role of abamectin treatment. S2 Table shows how the trees treated with abamectin compare to the trees selected as a comparison group. After matching of propensity scores the means of the control group in terms of height, perimeter and the number of different infestation signs approach the means of the treated trees. QQ plots in S3 and S4 Figs further illustrate this, not just for the means, but for the entire distribution of the variables in question. It is true, however, that the attack density (particularly for gallery slits) of the treated trees is still higher than for the control group. This is because the number of infestation signs was indeed much higher among the treated trees, and creating a perfectly comparable matching group from the data was not possible. The conclusions are robust (see models 3–4, 7–8, 11–12 and 15–16). Abamectin is significantly reducing the number of new BI at 5% level and for EH and GS considered separately at 10% level, while for BI and GS combined at 1% level. The absolute reduction in the number of new EH/BI/GS for trees treated with abamectin ranges between 2.5 and 4 depending on the infestation indicator.

When the beetle larvae ingest or contact abamectin topically, the chemical interferes with neural and neuromuscular transmission that leads to insect death. Since most X. chinensis larvae stop feeding in May, or earlier, and abandon the phloem (where the abamectin acts) to enter the xylem to pupate, many larvae would not have encountered abamectin, and therefore they would have succeeded emerging as adults in the coming summer, when the injected abamectin will have faded within the tree, facilitating further infestation. The latter implies a much lower efficiency of this end-of-April treatment. Indeed, out of the 107 trees treated, 31 (28,97%) still showed new infestation signs in December, in particular new emergence holes. Yet, as mentioned above, the effects of abamectin were statistically significant, reducing the numbers of new infestations on the treated trees. It is likely that, for maximum insecticide efficiency, the best time for treating with abamectin (trunk injection) would be from mid-July to mid-August, when newly hatched larvae begin feeding on the phloem.

## Conclusions and future prospects

We have evaluated how the infestation progressed as a function of past infestation and provided new relevant insights into female beetle’s preferences to lay their eggs on specific trees or specific areas of trees, hence mapping the larval distributions in them. This mapping was done by locating the infestation damages observed on the trees (their height and compass orientation), therefore providing key information to surveyors and managers on how to deal with this pest. Specifically, our statistical analysis shows that females prefer laying eggs on larger trees, on the highest part of trunks and on the crown base (this being more preferred than the trunk), and they do so on SW orientation avoiding those facing N, E and NW. We have also shown that emergence holes and gallery slits predict new coming infestation on trees. An abamectin treatment (trunk injection) carried out at the end of April significantly reduced the number of new infestations. However, managers should consider that, most likely, for maximum insecticide efficiency, the best time for treating with abamectin would be from mid-July to mid-August, when newly hatched larvae begin feeding on the phloem.

Finally, there are issues which have not been brought up in this work but may be important for the management of this pest. Indeed, based on responses of other invasive beetle pests, research is needed to detect volatiles released by mulberries, because if they were attractive would attract beetles over wide areas. Likewise, since potential attractants are not necessarily tree-based, attractants released by related longhorned beetles might also be of interest.

## Acknowledgements

Barberà del Vallès town hall gave us permission to carry out this study using its public mulberry trees, and its technicians Glòria Torras and Jordi Serra provided advice and some photographs. Lluís Olivet (Endoteràpia Vegetal) provided technical data on the equipment and products used to carry out the endotherapy treatment of mulberry trees.

## References

Sarto i MonteysV, TorrasG. A new alien invasive longhorn beetle, Xylotrechus chinensis (Cerambycidae), is infesting mulberries in Catalonia (Spain). Insects. 2018; 9(2): 52

HanY, LyuD. Taxonomic review of the genus Xylotrechus (Coleoptera: Cerambycidae: Cerambycinae) in Korea with a newly recorded species. Korean J Appl Entomol. 2010; 49: 6982.

BenkerU. Stowaways in Wood Packaging Material. Current Situation in Bavaria. Forstschutz Aktuell. 2008; 44: 3031.

Philadelphia U.S. Customs and Border Protection. Philly CBP Intercepts Nation’s First Xylotrechus chinensis Beetle. 2011 June 13. https://www.cbp.gov/newsroom/local-media-release/philly-cbp-intercepts-nations-first-xylotrechus-chinensis-beetle.

Schrader G. Express PRA zu Xylotrechus chinensis. Julius Kühn Institute for National and International Affairs on Plant Health. 2017; PRA diptych: 2 pp.

LeivadaraE, LeivadaraI, VontasI, TrichasA, SimoglouK, RoditakisE, et al First record of Xylotrechus chinensis (Coleoptera, Cerambycidae) in Greece and in the EPPO region. Bull. OEPP/EPPO. 2018; 48: 277280.

Sarto i Monteys V. El escarabajo-avispa, nueva especie invasora en Europa. Adelantos digital. 2018 Jul 26. https://www.adelantosdigital.com/web/escarabajo-avispa-nueva-especie-invasora-europa/.

CocquempotC, DesblesF, MouttetR, ValladaresL. Xylotrechus chinensis (Chevrolat, 1852), nouvelle espèce invasive pour la France métropolitaine (Coleoptera, Cerambycidae, Clytini). Bull. Soc. entomol. Fr. 2019; 124: 2732.

Valladares L, Brustel H, Cocquempot C. Xylotrechus chinensis: nouvelle espèce invasive arrivée d’Asie. Passion Entomologie. 2019 July 15. https://passion-entomologie.fr/xylotrechus-chinensis-nouvelle-espece-invasive/.

10

EPPO. Xylotrechus chinensis (Coleoptera: Cerambycidae): addition to the EPPO Alert List. EPPO Reporting Service, Pests. 2018; 157 (8): 79.

11

LimJ, JungSY, LimJS, JangJ, KimKM, LeeYM, et al A review of host plants of Cerambycidae (Coleoptera: Chrysomeloidea) with new host records for fourteen cerambycids, including the asian longhorn beetle (Anoplophora glabripennis Motschulsky), in Korea. Korean J Appl Entomol. 2014; 53: 111133.

12

Pelegrin M. Variedades y tipos de moreras en el Levante español. Master’s thesis. Escuela Politécnica Superior de Orihuela. Universidad Miguel Hernández de Elche; 2013. 55 pp.

13

Pérez CañavateJG, González BenaventeA, Fernández HernándezJA, Bañón AriasS. La morera como árbol ornamental. Horticultura. 1992; 76: 1323.

14

HuangC. An investigation of the spatial distribution patterns of Xylotrechus chinensis Chev. larvae. Newsletter of Sericultural Science, 2000 (01).

15

SunYJ. Study on the method for controlling Xylotrechus chinensis Chevrolat. Hubei agricultural Sciences, 2011(10).

16

MurakamiM. Biology and control of Xylotrechus chinensis Chevrolat. Tech. Bull. of Sericulture experimental Station. 1960; 77: 123.

17

ArugaH. Principles of Sericulture. Boca Raton, FL, USA: CRC Press; 1994.

18

FettigCJ, GrosmanDM, MunsonAS. Efficacy of abamectin and tebuconazole injections to protect Lodgepole Pine from mortality attributed to Mountain Pine Beetle (Coleoptera: Curculionidae) attack and progression of Blue Stain Fungi. J. Entomol. Sci. 2013; 48: 270278.

19

MarsonM, SavinI. Ensuring sustainable access to drinking water in sub-Saharan Africa: Conflict between financial and social objectives. World Development. 2015; 76: 2639.

20 Oct 2020

PONE-D-20-28834

The invasive longhorn beetle Xylotrechus chinensis, pest of mulberries, in Europe: study on its local spread and efficacy of abamectin control

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please pay close attention to the comments of the reviewer, with which I concur. Several persons asked to review it previously have turned down the manuscript due to lack of easily accessible information; consequently, the authors should pay close attention to highlighting useful information that other persons addressing the management of this growing pest problem will obtain by reading this manuscript.

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5. Review Comments to the Author

Reviewer #1: This paper presents information about the history of Xylotrechus chinensis detections in Europe, and data on within tree distribution, changes in infestation level as measured by numbers of exit holes, bark injuries and gallery slits on Mulberry street trees that were or were not injected with abamectin in Barbera del Valles, Spain. Although the insecticide treatments were applied under contract by the city as a management operation and not part of a controlled, designed experiment, the results comparing changes in infestation signs compared to untreated trees may still provide valuable information about insecticide efficacy. Within tree distribution results may aid in visual surveys and regression analyses of infestation by tree height and diameter may help in predicting growth of infestations based on tree size. Overall, the paper presents some new and useful information and could be suitable for publication after major revisions.

The manuscript requires considerable editing throughout to improve the scientific English writing. There are many English grammatical errors, long verbose sentence construction, and in many cases colloquial language, such as “ends up killing them” or “settling down.” The manuscript could be greatly condensed by removing redundancies and improving organization.

For instance, description of signs and symptoms of infestation do not belong in the materials and methods but could be more concise and should be in the introduction. I suggest organizing the introduction with a description of the insect, native range, brief invasion history, hosts and impacts, description of damage signs and symptoms, and review of known management options including pesticide efficacy. Then state there is a need for improved management and knowledge of spread, within tree distribution, and efficacy of insecticide treatments. End the introduction with a clear Statement of objectives.

In the methods section there is no need to repeat description of signs and symptoms. Instead, immediately describe the field sites, how sampling was conducted, number of trees/samples, how they were selected, their spacing or experimental design/arrangement, and what variables were measured and analyzed. For the insecticide evaluation, provide greater detail about insecticide product used, application technique (name and supplier of product, type of injection needle/tool (its product name and manufacturer).

How were the 107 trees selected for insecticide injection? How far apart were trees? Since matched controls were not selected at the same time, but were later statistically chosen from the pool of remaining untreated trees of the 438 street trees, it is important to know how they compared to the control trees and their relative spacing and distribution at the site. If the most heavily infested trees were chosen for insecticide treatment their initial attack density may have been higher than the controls. Also, if controls were immediately adjacent to treated trees they may experience some spill over protection if larvae are killed in the treated trees reducing emerging beetles that may disperse and attack nearby trees.

It appears that signs of infestation (emergence holes, bark injuries, gallery slits) were counted per tree and not standardized as attack densities based on tree surface area (height and dbh). How was the sum of indicators determined (was it the total number of exit holes, bark injuries and gallery slits per tree?) Since bark injuries, transform into gallery slits, and are also associated with exit holes, summing and accumulating them may result in overestimating attack density by essentially double-counting.

There is a very large number of tables and figures and some could be dropped or combined. For instance, Table 2 repeats much of the same information as in Figure 8. They could be combined into a single table.

Figure 9 could be dropped since it simply shows 3 values in a pie chart that could be succinctly stated in the text.

Figure 10 may not be needed, since the number of trees by height category is given in the text, and the numbers by trunk perimeter are at least partially given in Table 3 and figure 11. These could be combined into 1 table or figure.

Figure 11 seems to include a table of values that are the same as what is plotted in the stacked bar charts below. Either present the results as a table or as a figure but not both. Also the quality is quite blurry (at least on my computer and printer).

Please improve the explanation for the subfigures in figures 13-16 depicting the matrices for the Mann Whitney test. I was unable to understand what they represent and the quality is also blurry.

It is standard to use tree diameter at breast height (dbh) in 5 or 10 cm dbh classes rather than perimeter.

It would be helpful to include a figure or table showing average ± SE number of exit holes, bark injuries, gallery slits at the time (or soon after, i.e., April or June 2018) of abamectin treatment for treated and control trees and again in December 2018 along with percent reduction and statistical differences.

It would also be helpful to include an overall summary/conclusion. How does the within tree distribution and abamectin treatment evaluation improve recommendations for survey and management?

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

29 Nov 2020

Responses to the Editor and Reviewer

Title: "The invasive longhorn beetle Xylotrechus chinensis, pest of mulberries, in Europe: study on its local spread and efficacy of abamectin control”

Manuscript ID: PONE-D-20-28834 PLOS ONE

We are grateful to the editor and the referee for their positive assessment of our paper and the valuable comments, which helped us to improve the paper. Below we explain how we have addressed each comment.

Response to Journal Requirements:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

We have re-checked the PLOS ONE style templates and believe our manuscript meets the Journal’s requirements.

2. Amended statements about the funding.

The Funding Statement should be updated as follows:

‘Ivan Savin acknowledges financial support from the grant from the President of the Russian Federation for young doctors of science MD-3196.2019.6. The City Hall of Barberà del Vallès (Barcelona) contributed to the costs of organizing and running this Project. The Departament d'Agricultura, Ramaderia, Pesca i Alimentació of the Generalitat of Catalonia contributed 50% of the publication costs.’

All funding-related text have been already removed from the new version of the manuscript.

3. About figures 2, 3 and 5 containing map/satellite images which may be copyrighted.

* The formerly submitted Figure 2 used a map of Europe whose credentials could not be obtained. Therefore, we have replaced it by another one. Now the caption is:

Fig 2. Xylotrechus chinensis in Europe up to July 2020. Source map reprinted from https://commons.wikimedia.org/wiki/File:Europe_map.png under a CC BY license, with permission from Wiki-vr, Public domain, via Wikimedia Commons. 30 November 2013

* The formerly submitted Figure 3 shows a map of Catalonia and a closeup of four invaded subareas within Catalonia. We did not use here any image from the internet, but created these maps ourselves. We used the software ArcMap from Arcgis. Now the caption is:

Fig 3. Catalonian districts invaded by Xylotrechus chinensis up to July 2020. The four townships invaded up to July 2018 are in orange; the eight new townships invaded up to July 2020 are in red. The black asterisk signals the city of Barcelona. Software: ArcMap 10.3 (10.3.1) (May 13, 2015) https://desktop.arcgis.com/es/arcmap/10.3/main/map/what-is-arcmap-.htm

* The formerly submitted Figure 5 shows a satellite map of the town of Barberà del Vallès. It was obtained from the Cartographic and Geological Institute of Catalonia. The geoinformation provided by this Institute is under a Creative Commons Attribution 4.0 International License (CC BY 4.0). Therefore, as stated in its webpage, when reusing its images, only its authorship should be cited; the Institute states that it is not necessary to follow any other procedure or sign any document. Now the caption is:

Fig 5. Satellite map of the town of Barberà del Vallès showing in green the location of its 506 public mulberry trees in 2016. (Map source: Cartographic and Geological Institute of Catalonia, https://www.icgc.cat/, under a CC BY 4.0 license, accessed 27 April 2020)

Since, as a result of the major revision we carried out, some figures were deleted, others were modified and several were renumbered, for the sake of clarity we will upload all figures again -as they are now referenced in the revised version of the manuscript. All figures were previously uploaded to the program PACE which ensured they met PLOS requirements.

Response to Reviewer: (our response is in italics).

Comment 1: This paper presents information about the history of Xylotrechus chinensis detections in Europe, and data on within tree distribution, changes in infestation level as measured by numbers of exit holes, bark injuries and gallery slits on mulberry street trees that were or were not injected with abamectin in Barbera del Valles, Spain. Although the insecticide treatments were applied under contract by the city as a management operation and not part of a controlled, designed experiment, the results comparing changes in infestation signs compared to untreated trees may still provide valuable information about insecticide efficacy. Within tree distribution results may aid in visual surveys and regression analyses of infestation by tree height and diameter may help in predicting growth of infestations based on tree size. Overall, the paper presents some new and useful information and could be suitable for publication after major revisions.

Thank you for your positive evaluation of our work.

Comment 2: The manuscript requires considerable editing throughout to improve the scientific English writing. There are many English grammatical errors, long verbose sentence construction, and in many cases colloquial language, such as “ends up killing them” or “settling down.”

We have improved the scientific English writing as suggested by the Reviewer. All colloquial language has been replaced (for instance “ends up killing them” has become “eventually causes their death” …), grammatical errors have been found and corrected, and sentence construction improved to make it more concise and less verbose. However, please consider that our study is a complex one and looks at different issues. Explaining it sometimes requires compound or complex sentence structures.

Comment 3: The manuscript could be greatly condensed by removing redundancies and improving organization. For instance, description of signs and symptoms of infestation do not belong in the materials and methods but could be more concise and should be in the introduction. I suggest organizing the introduction with a description of the insect, native range, brief invasion history, hosts and impacts, description of damage signs and symptoms, and review of known management options including pesticide efficacy. Then state there is a need for improved management and knowledge of spread, within tree distribution, and efficacy of insecticide treatments. End the introduction with a clear Statement of objectives.

Thank you for this suggestion. The Introduction has been organized as follows: (1) Description of the insect, (2) Native range and invasion history, (3) Hosts and impacts, (4) Description of damage signs and symptoms (moved from Material and Methods), (5) Review of within-tree distribution of larvae and pest spread, and (6) Management options: the past and the future. Also, the Introduction ends with a clear statement of objectives. Throughout the paper we tried to reduce unnecessary repetition of information.

Comment 4: In the methods section there is no need to repeat description of signs and symptoms. Instead, immediately describe the field sites, how sampling was conducted, number of trees/samples, how they were selected, their spacing or experimental design/arrangement, and what variables were measured and analysed. For the insecticide evaluation, provide greater detail about insecticide product used, application technique (name and supplier of product, type of injection needle/tool (its product name and manufacturer).

All Reviewer’s suggestions have been addressed. Now the section ‘Material and Methods’ has four subsections: (1) Surveys: location, dates and trees, (2) Data sampling and measurement, (3) Insecticide treatment using abamectin, and (4) Statistical analysis. Much greater details about the insecticide product used (abamectin + emulsifier), application technique (name and supplier of product, type of injection needle/tool, etc.) have been incorporated into the text.

Comment 5: How were the 107 trees selected for insecticide injection? How far apart were trees? Since matched controls were not selected at the same time, but were later statistically chosen from the pool of remaining untreated trees of the 438 street trees, it is important to know how they compared to the control trees and their relative spacing and distribution at the site. If the most heavily infested trees were chosen for insecticide treatment, their initial attack density may have been higher than the controls. Also, if controls were immediately adjacent to treated trees they may experience some spill overprotection if larvae are killed in the treated trees reducing emerging beetles that may disperse and attack nearby trees.

Nearly all 107 mulberry trees treated were lining up along one row (sometimes two rows) of a wide avenue heavily infested by the beetle, and spaced about 6.5 meters. They were injected, regardless of their infestation level; their codes are indicated in S1 Table. We have added this info to the revised manuscript. Also, as we wrote earlier in the manuscript, Table S2 as well as Figures S1 and S2 (now renamed to Figures S3 and S4) summarize the results on the propensity score matching. In particular, they show how the trees treated by abamectin compare to the trees selected as a comparison group. As we see, after propensity score matching the means of the control group in terms of height, perimeter and the number of different infestation signs become close to the means of the treated trees (Table S2). QQ plots in Figures S3 and S4 further illustrate this not for the means but the entire distribution of the variables in question. It is true, however, that the attack density (particularly for gallery slits) of the treated trees is still higher than for the control group. This is because the number of infestation signs was indeed much higher among the treated trees, and creating a perfectly comparable matching group from the data was not possible. We have now clarified this in the revised manuscript.

Here we list the codes of 99 trees treated with abamectin and selected by propensity score matching:

MA001 MA002 MA003 MA005 MA006 MA007 MA008 MA009 MA010 MA011 MA012 MA013 MA014 MA015 MA016 MA017 MA018 MA019 MA021 MA022 MA023 MA024 MA025 MA026 MA027 MA028 MA029 MA030 MA031 MA032 MA033 MA034 MA035 MA036 MA038 MA040 MA041 MA043 MA044 MA045 MA046 MA047 MA048 MA049 MA053 MA055 MA056 MA058 MA059 MA060 MA062 MA063 MA064 MA066 MA068 MA070 MA072 MA073 MA075 MA076 MA077 MA078 MA079 MA082 MA083 MA084 MA085 MA086 MA087 MA088 MA089 MA091 MA092 MA093 MA094 MA096 MA098 MA099 MA100 MA101 MA103 MA104 MA105 MA106 MA107 MA108 MA111 MA112 MA250 MA273 MA274 MA275 MA276 MA277 MA278 MA279 MA280 MA375 MA389

And here we list the codes of 99 trees selected as a control group by propensity score matching:

MA037 MA042 MA050 MA051 MA052 MA054 MA080 MA095 MA102 MA127 MA138 MA146 MA147 MA154 MA155 MA158 MA162 MA163 MA168 MA171 MA173 MA175 MA179 MA180 MA185 MA187 MA189 MA192 MA194 MA195 MA197 MA201 MA203 MA209 MA213 MA216 MA217 MA220 MA223 MA225 MA234 MA241 MA243 MA244 MA245 MA247 MA251 MA253 MA259 MA261 MA269 MA287 MA290 MA291 MA293 MA298 MA301 MA345 MA356 MA357 MA358 MA359 MA368 MA372 MA374 MA377 MA378 MA379 MA380 MA383 MA384 MA385 MA386 MA388 MA390 MA393 MA394 MA397 MA399 MA402 MA404 MA405 MA406 MA407 MA409 MA412 MA414 MA415 MA416 MA417 MA418 MA419 MA421 MA422 MA428 MA431 MA432 MA433 MA440

Now, we have also listed these two 99-trees groups in a more complete Table S2 and added satellite maps to locate these 99-trees, treated and control (see new Supp. Inform. Figure S2).

Comment 6: It appears that signs of infestation (emergence holes, bark injuries, gallery slits) were counted per tree and not standardized as attack densities based on tree surface area (height and dbh). How was the sum of indicators determined (was it the total number of exit holes, bark injuries and gallery slits per tree?) Since bark injuries, transform into gallery slits, and are also associated with exit holes, summing and accumulating them may result in overestimating attack density by essentially double-counting.

We counted emergence holes, bark injuries and gallery slits per tree. And since we were more interested in knowing where on the tree these indicators appeared (i.e. lower trunk, or mid trunk, or upper trunk, or crown base, and how they were oriented on the compass), we used this system instead of standardizing it as attack densities based on tree surface area (height and dbh). Since we measured the height and perimeter at breast height (130 cm) for all trees, we could easily have calculated the surface areas of trees and then attribute attack densities to them. But we believe the way we did it provides more information about the within-tree distribution of larvae and their associated damage.

However, in one instance (when we wanted to check if females preferred the crown base to the trunk for laying their eggs, since this was our perception in the field) we did use surface areas. Since trunks as a whole have a lateral surface much bigger than that of the crown base, surface values of these two pieces (trunk and crown base) were considered when checking the damages they both endured. In this case we measured the number of infestations per 1000 cm2 and the results are shown in Table 5 of our original manuscript (Table 3 in the revised manuscript).

As for bark injuries, and they transforming into gallery slits, we believe there is a misunderstanding. As we wrote when describing bark injuries: “Bark injuries transform into gallery slits usually one year after they appeared. Hence, unlike emergence holes and gallery slits, they are not accumulative on the trees. However, in our study, they are accumulative, since injuries seen in June have not had enough time to transform into gallery slits by December.” So, we did not overestimate attack density by double-counting. We were very careful about that. Since in our study the three indicators are accumulative from June to December, we can easily know what was new in December by subtracting the June counts to those obtained in December. This can be clearly seen in excel Table S1.

Comment 7: There is a very large number of tables and figures and some could be dropped or combined. For instance, Table 2 repeats much of the same information as in Figure 8. They could be combined into a single table.

Done, we have dropped Table 2 and kept Figure 8, slightly modifying the text to accommodate this change.

Figure 9 could be dropped since it simply shows 3 values in a pie chart that could be succinctly stated in the text.

Done, we have dropped Figure 9, and succinctly stated these 3 values in the text.

Figure 10 may not be needed, since the number of trees by height category is given in the text, and the numbers by trunk perimeter are at least partially given in Table 3 and figure 11. These could be combined into 1 table or figure.

Done, we have dropped Figure 10 from the original manuscript. However, we have kept as Supp. Inform. the graph that showed the distribution of trunk perimeters as we believe it provides useful info to the readers (now as Fig S1).

Values in Table 3 (now Table 2 in the revised manuscript) refer exclusively to trunk height and those in Figure 11 (now Figure 9 in the revised manuscript) refer exclusively to trunk perimeter. Therefore, we believe combining them would be confusing.

Figure 11 seems to include a table of values that are the same as what is plotted in the stacked bar charts below. Either present the results as a table or as a figure but not both. Also, the quality is quite blurry (at least on my computer and printer).

Done, we have removed the table and kept the bar charts in a new figure (now called Figure 9 in the revised manuscript). In this new version we have improved the quality of this figure.

Please improve the explanation for the subfigures in figures 13-16 depicting the matrices for the Mann Whitney test. I was unable to understand what they represent, and the quality is also blurry.

Thank you for the suggestion. We extended the explanation of the figures as follows: (keep in mind that in the revised manuscript Figs 13-16 have become Figs 11-14)

“The results are shown in Figs 11-14, where they are summarized in two subfigures for each characteristic. The first top left subfigure shows the distribution of the respective infestation sign over orientation in averages and error bars depicting +/- 2 s.e. The bottom left subfigure does the same but for the height of the infestation signs. Thus, for example, the distribution of emergency holes on NW orientation in Figure 11 has a mean of approximately 0.3 holes per tree with a confidence interval ≈0.06-0.54. The subfigures on the right side summarize results for stochastic dominance in the distribution of the given damage. All columns of the matrices represent the results of the pairwise Mann-Whitney test. The dark shading indicates that the distribution of damage with an orientation or height specified in the respective row dominates the distribution in the corresponding column with a specified significance level. Asterisks ***, **, and * denote 1%, 5%, and 10% significance, respectively. Returning to the example in Figure 11, the number of emergency holes with the SW orientation is statistically higher than the one with the NW orientation, and this result is significant at the 5% significance level.”

We also improved the resolution of the four figures in the revised manuscript.

Comment 8: It is standard to use tree diameter at breast height (dbh) in 5 or 10 cm dbh classes rather than perimeter.

Indeed, DBH (Diameter at breast height) is one of the most common dendrometric measurements. Yet, the height of an adult's breast is defined differently in different countries and situations. We preferred to do it at 1.3 m above ground, which is the one used in many countries. Also, since we did not have a tree caliper to measure the diameter at breast height of a tree, we used a girthing tape that measured the girth (circumference or perimeter) of the tree at breast height. While doing so we made sure the tape was perfectly level and that the tape was not kinked, so as not to skew the readings. We could have later calculated the diameters from the circumference readings, but decided to keep using the initial readings, i.e the circumference or perimeter of trees. Obviously, this would not affect the results. In Spain, companies applying endotherapy to inject trees also use the trunk perimeter at a height of 130 cm to determine the number of drillings.

Comment 9: It would be helpful to include a figure or table showing average ± SE number of exit holes, bark injuries, gallery slits at the time (or soon after, i.e., April or June 2018) of abamectin treatment for treated and control trees and again in December 2018 along with percent reduction and statistical differences.

We believe that our analysis is misunderstood here. As we describe in the paper, the spread of the invasive species is a dynamic process and depends on many factors such as the number of infestation signs already observed, including those of the different type (e.g. number of new gallery slits depending on earlier number of bark injuries or emergence holes). Hence, it would be incorrect to simply compare the number of infestation signs before and after treatment expecting the numbers to reduce due to the treatment. Instead, what we do in the regression analysis is to compare the spread of infestation signs of the treated trees to those trees that did not receive any abamectin treatment (control group) to infer if the treatment had any significant effect. One would not come to the same conclusion by just looking on the number of infestation signs before and after the treatment.

What we tried to do is to provide the averages (+/-2 s.e.) for the treated and the control group in the table below to see if we observe interesting and significant results. However, as we have many observations with zero infestation signs in June 2018, we cannot calculate percentage changes, but only absolute changes. As you can see from the table below, absolute increases in the number of infestation signs in the control group is always higher than in the treatment group even though in June 2018 the number of infestation signs of all three types was higher in the latter group. Since we do not control for the different starting conditions, the results of the Mann-Whitney test on statistical difference between absolute changes in the treated and the control group here are negative, i.e. no significant difference. But as we have shown in the regression analysis in the manuscript, controlling for the initial conditions and other factors like perimeter or height of the tree show that the abamectin treatment is significant. For the reasons abovementioned we prefer to not include the results from the table below in the manuscript.

Sample of trees treated with abamectin (N=99) Control group created with propensity score matching (N=99) Mann-Whitney’s test p-value

June

2018 December 2018 Absolute change June 2018 December 2018 Absolute change

Emergence holes 2.04

[0.87-3.22] 2.86

[1.36-4.36] 0.82

[0.37-1.27] 0.96

[0.28-1.64] 1.84

[0.92-2.76] 0.88

[0.36-1.40] 1

Bark injuries 0.54

[0.27-0.80] 0.62

[0.33-0.91] 0.08

[0.02-0.14] 0.39

[-0.05-0.83] 0.72

[0.24-1.20] 0.32

[0.11-0.54] 0.12

Gallery

slits 3.61

[2.85-4.36] 3.72

[2.96-4.48] 0.11

[0.03-0.19] 2.17

[1.37-2.98] 2.31

[1.50-3.13] 0.14

[0.02-0.26] 0.64

Note: Results are reported in averages +/- 2 s.e.

Comment 10: It would also be helpful to include an overall summary/conclusion. How does the within tree distribution and abamectin treatment evaluation improve recommendations for survey and management?

We have included a short Conclusion section at the end of the manuscript, just before the Acknowledgments, as follows:

“We have evaluated how the infestation progressed as a function of past infestation and provided new relevant insights into female beetle’s preferences to lay their eggs on specific trees or specific areas of trees, therefore determining the within-tree distribution of larvae. The latter was done considering tree size, the geographical orientation of infestation damages and their location on trees, therefore providing key information to surveyors and managers on how to deal with this pest. Specifically, our statistical analysis shows that females prefer laying eggs on larger trees, on the highest part of trunks and on the crown base (this being more preferred than the trunk), and they do so on SW orientation avoiding those facing N, E and NW. We have also shown that emergence holes and gallery slits predict new coming infestation on trees. An abamectin treatment (trunk injection) carried out at the end of April significantly reduced the number of new infestation. However, managers should consider that, most likely, for maximum insecticide efficiency, the best time for treating with abamectin would be from mid-July to mid-August, when newly hatched larvae start feeding on the tree phloem.”

8 Dec 2020

PONE-D-20-28834R1

The invasive longhorn beetle Xylotrechus chinensis, pest of mulberries, in Europe: study on its local spread and efficacy of abamectin control

PLOS ONE

Dear Dr. SARTO I MONTEYS,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

As the authors have already addressed a thorough review of their manuscript, the editor has examined the revised version and suggests the changes listed below, which can be altered if the editor misunderstood the meaning of the authors:

Line 17 “ a potentially lethal pest of mulberry trees (Moraceae: Morus sp.)

Line 22 “and avenues is a concern, as beetle infestation “

Line 23 “how the infestation progresses over time, with and without Abamectin treatment, and provide insights into female egg-laying preferences.  Such knowledge helps contribute to management efforts to reduce expansion of the range of beetle infestation.”

Line 27 “do so on warmer, SW orientations rather than those facing N, NW and E. Emergence holes and gallery slits predict the spreading of infestations to new trees”

Line 30 “begin feeding on the phloem”

Line 45 Delete “As far as we know . . .described”

Line 53 “This invasive species is native”

Line 75 “into these French”

Line 87 delete “, as we report here”

Line96 “The data were assembled using ArcMap 10.3.1 . .”

Line 112 “Spain and other southern European countries.”

Line 114 Delete “Quite similar . . .countries.”

Line 120 “concern because beetle infestation increases the risk of falling branches and the need for rapid response by municipal authorities.”

Line 128 “observations, three types of indicators were selected to document infestation: (1) “

Line 130 “were counted and processed as described in the methods to inform the municipality about the progress of the beetle infestation in the study area.”

Line 156 “Occasionally, full-grown larvae have been found overwintering under”

Line 157 “Bark injuries become increasingly more evident “

Line 160 “Consequently, the presence of bark injuries can be a good”

Line 172 “which is bored into for pupation”

Line 178 “No studies were reviewed that reported how”

Line 180 “i.e., a statistical analysis of new infestation in relation to past infestationNor could we find studies about the . . . perimeter, or about the geographical”

Line 183 “provide important knowledge on the preferred trees for egg laying and where the eggs are most likely to be laid. A goal of the study was to determine such information to assist in scouting for infestations.

Line 230 “Specifically, to aid in scouting, it is important to know”

Line 234, or perhaps to wait until the discussion. Based on responses of other invasive beetle pests, one might also consider volatiles released by the trees, which, if were attractive would attract beetles over wide areas. The authors don’t bring this up, but many entomologists working in this research area would gravitate towards that possibility immediately. Potential attractants are not necessarily tree-based, so attractants of related longhorned beetles might be of interest.

Line 240 “In 2018, two surveys were conducted under permissions granted by the City Council, checking all public trees for beetle infestation.”

Line 262 “bark injuries (BI), noting also their heights above the ground and their compass orientations as one of 8 directions: N, NE, E, SE, S, SW,W, or NW.

Line 267, delete “To set . . .”

Line 287 “Because these 107 trees, among the 438 checked in June 2018, were still present in December, an analysis of the treatment results was included in the study.

Line 294 “were 2-3 drillings per tree.”

Line 311 “crown base, a non-parametric paired, 2-sample Wilcoxon Test was used that explicitly”

Line 315 delete “here”

Line 316 “A P-value below 0.05 leads to rejection of the null hypothesis of no difference between crown base and trunk damage.”

Line 319 “A statistical analysis of new infestations in December 2018 was conducted in relation to past”

Line 320 “Because the dependent variables are count data with many zeros”

Line 332 “A tree was considered to be infested when”

Line 415” Regression analysis was used to test if female beetles prefer to lay eggs in trees of specific heights. Tree height was not found to be a significant factor affecting the distribution of December damage compared to June (see below). Females of this beetle species are good flyers . . .”

Line 429 “Perimeter lengths were grouped into five ranges “

Line 443 “where the tree perimeter length had a “

Line 458 “This result does not have an obvious explanation”

Line 464 “Comparisons also were made of all damages”

Line 479 “As mentioned above, all 438 trees were in this analysis, including the December 2018 data and the data contributed by the 30 trees in June that had been removed by December.”

Line 484 “The goal was”

Line 490 and elsewhere, including line 496 “emergency” should be “emergence”

Line 506 The compass distribution of EH (Fig 11) indicates that females prefer to lay eggs on the SW side of the tree, both on the trunk and crown base. The 150-200-cm band and the 200-250-cm band” have higher values than the others. A somewhat similar pattern emerges in the distribution of BI”

Line 511 and elsewhere “popular” or “unpopular” should be replaced by “less preferred” or by a statement based on preference rather than popularity. Unfortunately, there are many words like this in the manuscript that should be replaced by more scientifically descriptive terminology or it will reflect negatively on the authors and the editor.

Line 525 Do not start a new paragraph here.

Line 526 new paragraph at “However, comparing the distribution . . “

Line 529 “This suggests that females prefer”

Line 532 and elsewhere, “Wilcox” should be “Wilcoxon”

Line 535 delete “Econometric”

Line 537 “The differences between December and June in the number of new tree damages. . .  was compared for height and perimeter, as well as the “

Line 539 “In addition the results were tested for the sum of BI +GS new damage.”

Line 572 “The results indicate that the number of GS in June is a significant predictor of “

Line 587 “This may be interpreted “. A statement like this is definitely discussion rather than results. While the editor considers this a plausible interpretation, there is not information about effects of chemical or visual stimuli to confirm the interpretation. It seems best to separate Results and Discussion, and then put statements like this only in the discussion. There are several other places also where it would benefit the manuscript to separate results from discussion.

Line 595 “Therefore, abamectin treatment was included as a predictor in models on the full sample and a robustness analysis was included, in which a propensity score matched treated trees against nearby untreated trees with respect to the five variables listed in Table 4.” ? The original sentence was not so clear.

Line 598 “As a result . . .” This sentence probably now should be deleted.

Line 601 Delete “As we see,”  Then say: “After matching of propensity scores, “

Line 602 “become close to” should be “approach”

Line 603 “further illustrate this, not just for the means, but for the entire”

Line 619 “the effects of abamectin were statistically significant, reducing the numbers of new infestations”

Line 627 “The larval distributions were mapped with respect to tree height  . . .”

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24 Dec 2020

Responses to the Editor

Title: "The invasive longhorn beetle Xylotrechus chinensis, pest of mulberries, in Europe: study on its local spread and efficacy of abamectin control”

Manuscript ID: PONE-D-20-28834 PLOS ONE

We do appreciate and are very grateful to the Editor for his good editing and valuable suggestions. Below we explain (in blue letters) how we have addressed such comments/suggestions.

Line 17 “a potentially lethal pest of mulberry trees (Moraceae: Morus sp.) Done!

Line 19 “the infestation spread” Done!

Line 22 “and avenues is a concern, as beetle infestation “ Done!

Line 23 “how the infestation progresses over time, with and without Abamectin treatment, and provide insights into female egg-laying preferences. Such knowledge helps contribute to management efforts to reduce expansion of the range of beetle infestation.” Done!

Line 27 “do so on warmer, SW orientations rather than those facing N, NW and E. Emergence holes and gallery slits predict the spreading of infestations to new trees” Done!

Line 30 “begin feeding on the phloem” Done!

Line 45 Delete “As far as we know . . .described” Done!

Line 53 “This invasive species is native” Done!

Line 75 “into these French” Done!

Line 87 delete “, as we report here” Done!

Line96 “The data were assembled using ArcMap 10.3.1 . .” Done!

Line 112 “Spain and other southern European countries.” Done!

Line 114 Delete “Quite similar . . .countries.” Done!

Line 120 “concern because beetle infestation increases the risk of falling branches and the need for rapid response by municipal authorities.” Done!

Line 128 “observations, three types of indicators were selected to document infestation: (1) “ Done!

Line 130 “were counted and processed as described in the methods to inform the municipality about the progress of the beetle infestation in the study area.” Done!

Line 156 “Occasionally, full-grown larvae have been found overwintering under” Done!

Line 157 “Bark injuries become increasingly more evident “Done!

Line 160 “Consequently, the presence of bark injuries can be a good” Done!

Line 172 “which is bored into for pupation” Done!

Line 178 “No studies were reviewed that reported how” Done!

Line 180 “i.e., a statistical analysis of new infestation in relation to past infestation Nor could we find studies about the . . . perimeter, or about the geographical” Done!

Line 183 “provide important knowledge on the preferred trees for egg laying and where the eggs are most likely to be laid. A goal of the study was to determine such information to assist in scouting for infestations. Done!

Line 230 “Specifically, to aid in scouting, it is important to know” Done!

Line 234, or perhaps to wait until the discussion. Based on responses of other invasive beetle pests, one might also consider volatiles released by the trees, which, if were attractive would attract beetles over wide areas. The authors don’t bring this up, but many entomologists working in this research area would gravitate towards that possibility immediately. Potential attractants are not necessarily tree-based, so attractants of related longhorned beetles might be of interest.

Authors’ response: (Please, see also our response for Line 587 below). Indeed, what the Editor suggests is an important and interesting issue. Unfortunately, we did not work on that in our present work. However, we believe we should briefly mention these topics in our paper as something that should be studied in the future. Therefore, incorporating the Editor’s comments, we have added a brief paragraph at the end of the ‘Conclusions’ section that reads:

“Finally, there are issues which have not been brought up in this work but may be important for the management of this pest. Indeed, based on responses of other invasive beetle pests, research is needed to detect volatiles released by mulberries, because if they were attractive would attract beetles over wide areas. Likewise, since potential attractants are not necessarily tree-based, attractants released by related longhorned beetles might also be of interest.”

Because of this added paragraph, we have also changed the heading “Conclusions” for “Conclusions and future prospects”.

Line 240 “In 2018, two surveys were conducted under permissions granted by the City Council, checking all public trees for beetle infestation.” Done!

Line 262 “bark injuries (BI), noting also their heights above the ground and their compass orientations as one of 8 directions: N, NE, E, SE, S, SW, W, or NW. Done!

Line 267, delete “To set . . .” Done!

Line 287 “Because these 107 trees, among the 438 checked in June 2018, were still present in December, an analysis of the treatment results was included in the study. Done!

Line 294 “were 2-3 drillings per tree.” Done!

Line 311 “crown base, a non-parametric paired, 2-sample Wilcoxon Test was used that explicitly” Done!

Line 315 delete “here” Done!

Line 316 “A P-value below 0.05 leads to rejection of the null hypothesis of no difference between crown base and trunk damage.” Done!

Line 319 “A statistical analysis of new infestations in December 2018 was conducted in relation to past” Done!

Line 320 “Because the dependent variables are count data with many zeros” Done!

Line 332 “A tree was considered to be infested when” Done!

Line 415” Regression analysis was used to test if female beetles prefer to lay eggs in trees of specific heights. Tree height was not found to be a significant factor affecting the distribution of December damage compared to June (see below). Females of this beetle species are good flyers . . .” Done!

Line 429 “Perimeter lengths were grouped into five ranges “Done!

Line 443 “where the tree perimeter length had a “Done!

Line 458 “This result does not have an obvious explanation” Done!

Line 464 “Comparisons also were made of all damages” Done!

Line 479 “As mentioned above, all 438 trees were in this analysis, including the December 2018 data and the data contributed by the 30 trees in June that had been removed by December.”

Line 484 “The goal was” Done!

Line 490 and elsewhere, including line 496 “emergency” should be “emergence” Done!

Line 506 The compass distribution of EH (Fig 11) indicates that females prefer to lay eggs on the SW side of the tree, both on the trunk and crown base. The 150-200-cm band and the 200-250-cm band” have higher values than the others. A somewhat similar pattern emerges in the distribution of BI” Done!

Line 511 and elsewhere “popular” or “unpopular” should be replaced by “less preferred” or by a statement based on preference rather than popularity. Unfortunately, there are many words like this in the manuscript that should be replaced by more scientifically descriptive terminology or it will reflect negatively on the authors and the editor.

Authors’ response: We have addressed this point wherever necessary and believe now it reads much better.

Line 525 Do not start a new paragraph here. Done!

Line 526 new paragraph at “However, comparing the distribution... “Done!

Line 529 “This suggests that females prefer” Done!

Line 532 and elsewhere, “Wilcox” should be “Wilcoxon” Done!

Line 535 delete “Econometric” Done!

Line 537 “The differences between December and June in the number of new tree damages… was compared for height and perimeter, as well as the “

Authors’ response: The suggested sentence does not reflect exactly what we meant since here we did not compare differences between June and December but carried out a NBRM regression analysis. Therefore, we have modified and simplified the sentence as follows (which we believe it reflects exactly what we did): “By using a NBRM regression analysis, the number of new tree damages in December was explained by the damage the trees showed in June, their height and perimeter, as well as the abamectin treatment 107 trees received on April 24, 2018.”

Line 539 “In addition the results were tested for the sum of BI +GS new damage.” Done!

Line 572 “The results indicate that the number of GS in June is a significant predictor of “Done!

Line 587 “This may be interpreted “. A statement like this is definitely discussion rather than results. While the editor considers this a plausible interpretation, there is not information about effects of chemical or visual stimuli to confirm the interpretation. It seems best to separate Results and Discussion, and then put statements like this only in the discussion. There are several other places also where it would benefit the manuscript to separate results from discussion.

Authors’ response: We understand the Editor’s point. However, in this paper we believe it is better to use the option “Results and discussion” instead of the option “Results” and then “Discussion”. This is because this is a rather complex study, where we are dealing with at least six groups of results (*Infestation of public mulberry trees from 2016 to 2018; *Infestation with respect to trunk height; *Infestation with respect to trunk perimeter; *Orientation and location of infestation damages; *Analysis of new infestation in December 2018 as a function of past infestation and other tree variables; *Effects of abamectin treatment), and to our understanding confining specific discussion (if needed) within each specific topic adds clarity to the paper.

Concerning our statement that “This may be interpreted as beetles reducing infestation of a severely infested tree and possibly moving to a different one.“, it is true that we have no information about effects of chemical or visual stimuli to confirm the interpretation. Although this topic is undoubtedly very interesting and we might look into it in the future, for the time being we could not say much more than what is said in this short statement. But we have changed it as follows: “This may be interpreted as beetles reducing infestation of a severely infested tree and possibly moving to a different one, although to confirm it new research is needed about the effects of chemical and visual stimuli.”

Line 595 “Therefore, abamectin treatment was included as a predictor in models on the full sample and a robustness analysis was included, in which a propensity score matched treated trees against nearby untreated trees with respect to the five variables listed in Table 4.” ? The original sentence was not so clear. Done!

Line 598 “As a result . . .” This sentence probably now should be deleted.

Authors’ response: No, we should keep the sentence, because here we explain that we obtained a subsample of 198 trees. Thus, it contains new information. Keep in mind that a total of 107 trees were treated with abamectin, from which only 99 were selected.

Line 601 Delete “As we see,” Then say: “After matching of propensity scores, “Done!

Line 602 “become close to” should be “approach” Done!

Line 603 “further illustrate this, not just for the means, but for the entire” Done!

Line 619 “the effects of abamectin were statistically significant, reducing the numbers of new infestations” Done!

Line 627 “The larval distributions were mapped with respect to tree height . . .”

Authors’ response: We have changed and simplified the original paragraph as follows:

“We have evaluated how the infestation progressed as a function of past infestation and provided new relevant insights into female beetle’s preferences to lay their eggs on specific trees or specific areas of trees, hence mapping the larval distributions in them. This mapping was done by locating the infestation damages observed on the trees (their height and compass orientation), therefore providing key information to surveyors and managers on how to deal with this pest.”

4 Jan 2021

The invasive longhorn beetle Xylotrechus chinensis, pest of mulberries, in Europe: study on its local spread and efficacy of abamectin control

PONE-D-20-28834R2

Dear Dr. SARTO I MONTEYS,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Richard Mankin, Ph. D.

PLOS ONE

8 Jan 2021

PONE-D-20-28834R2

The invasive longhorn beetle Xylotrechus chinensis, pest of mulberries, in Europe: study on its local spread and efficacy of abamectin control

Dear Dr. Sarto i Monteys:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Richard Mankin