DOI: 10.5061/dryad.zcrjdfnjt
title: Study of the overwintering ecology of the hazelnut pest, Palomena prasina (Hemiptera: Pentatomidae) in a perspective of Integrated Pest Management

description: Palomena prasina, the green shield bug (GSB), is widely distributed in the
 Eurosiberian region. In the Southwest of France it is considered a serious
 pest of hazelnuts, its feeding punctures lead to blank hazelnuts and
 kernel necrosis, causing heavy losses in commercial orchards. To date, no
 Integrated Pest Management strategy is available to control P. prasina.
 Control strategies often focus on the pests’ spring-summer ecology, when
 they are in the field or in the vicinity of crops. However, the abundance
 of pest populations in crops is also related to their autumn-winter
 ecology.  The present work focussed on the autumn-winter ecology
 of P. prasina to identify new opportunities for this pest suppression. We
 investigate (i) where P. prasina overwinters, (ii) if it aggregates in its
 overwintering sites, and (iii) if it mates while overwintering. Samples
 were collected over a two-year period in different ecosystems (forests,
 hedges, orchards), in human-made structures, and habitats (litter,
 bushes/trees, dead trees). The reproductive status of GBS individuals was
 monitored in winter, and in spring when they emerged from overwintering
 sites. Our results show that 97% P. prasina adults overwinter  in
 the leaf litter of orchards and natural ecosystems and that 70% overwinter
 individually. The abundance of GSB in those sites is negatively correlated
 with litter temperature and positively correlated with humidity levels.
 Furthermore, adults only mate after leaving their overwintering site.
 Finally, there was an important number of overwintering adults hosting
 endoparasitoids (32%). The fact that GSB overwinters alone in the leaf
 litter means controlling its populations by destroying the overwintering
 sites is not a solution. All the same, our results do open new
 perspectives for the control of P. prasina. First, the emergence traps, in
 particular the cone traps, proved efficient for collecting emerging adults
 and could be used for monitoring. Moreover, our observations point out the
 existence of long-range mating signals that could be exploited for
 trapping. Last but not least, the important number of overwintering
 parasitised adults is a promising biocontrol avenue.
description: <em>Material and methods</em>
 <em>1. Characterisation of overwintering
 sites</em>   A field
 study was conducted in a zone with a radius of 20 km around the city of
 Cancon, France (44°32'09.7"N, 0°37'30.1"E, 160 m
 a.s.l.) from December to February (10 weeks), both in 2021 and
 2022.   In 2021, three
 different types of ecosystems were studied: hazelnut orchards, linear
 hedges and woods. The hazelnut orchards were over 20 years old, at least 1
 ha in size, and most of them were planted with the Pauetet cultivar, which
 is sensitive to<em> P. prasina</em>. They were all commercial
 orchards with no cultural practices or pesticide spraying during the study
 period (the last management practices before that period correspond to
 pesticide spraying in July and harvest in August-September). The linear
 hedges were lines of trees and bushes that separated two fields, and were
 more than 116 m in length. The woods were at least 0.68 ha in size. The
 hedges and the woods were mainly composed of local deciduous trees,
 different species of oak, chestnut, hornbeam, checker tree, and some
 shrubs including dogwood and hawthorn; evergreen bushes included wild
 blackberry, butcher’s broom, and wild madder ( Bonneaud, 2014; Badeau et
 al., 2017). In 2022, apple orchards were added to the above-mentioned
 ecosystems. The apple orchards were at least 3.36 ha in size. All the
 orchards were managed conventionally and most were surrounded by crops,
 mainly hazelnut, plum, and apple orchards. Ten sites representing each
 ecosystem, located at least 8 km apart, were selected for the study. Each
 week, one site of each ecosystem type was prospected (i.e. each site was
 prospected only once per year).  
 Three main types of habitat were sampled in each ecosystem:
 leaf litter, the bark of trees and the foliage of evergreen bushes. Five
 samples were collected at 10-m intervals along a 50 m transect in the
 hedges and at the edges of orchards and woods starting at 0 m and
 continuing to 40 m. A further 5 samples were collected at the centre of
 the orchards and woods, at a distance of between 10 and 50 m from the
 edge.   A total of 600 leaf
 litter samples were collected, of 1 m2 each (250 and 350 samples were
 collected in 2021 and 2022 respectively). For each sample, four abiotic
 parameters were recorded at the surface of the litter using a TR-74Ui
 Series data logger (TandD corporation, Japan): temperature, relative
 humidity, illuminance and UV. The depth of the leaf litter was also
 measured. After the measurements were complete, the leaf litter covering
 each 1 m2 area was collected separately, placed in a black plastic bag and
 transported to the laboratory. The contents of each bag were placed under
 a photo-eclector trap (Soil photo-eclector Ø1m2, ecoTech Umwelt-Meßsysteme
 GmbH, Germany) for 24 h at 25 °C under constant light (Philips Master TDL
 18W/840 Cool white), after which the presence of insects was checked in
 each trap. At the end of the 24-h period, the photo-eclector traps were
 removed, and leaf litter was carefully inspected for any remaining
 insects.   We then defined
 an area with a radius of 2 metres around each leaf litter sampling area.
 In this area, the bark of one dead or standing tree was inspected visually
 for 2 minutes, and one evergreen bush was prospected for 10 seconds using
 a beating tray.   Finally,
 during the same monitoring period, a survey was conducted of potential
 overwintering sites in human-made structures (buildings and houses) based
 on a call for witnesses broadcast by radio, on Internet, and using flyers.
 Witnesses were asked to take photos of stink bugs present in their houses
 and buildings and to send them to the laboratory by email together with
 information on the general geographical location, plus the exact place and
 date of collection. Thirty-seven volunteers in the Cancon region responded
 to the call.   All the
 individuals belonging to the Pentatomoidea collected in the
 above-mentioned samples were identified to species level.
   The abundance of <em>P.
 prasina</em> in the 600 leaf litter samples was analysed. We fitted
 a Generalised Linear Mixed Model (GLMM) with a Poisson error distribution
 and the following fixed effects: type of ecosystem (categorial factors:
 hedge, woods, apple and hazelnut orchards), zone (categorial factors:
 interface and centre; interface corresponds to samples collected in the
 hedges and edges of woods and orchards), abiotic parameters of the leaf
 litter (continuous factors: litter depth, temperature, humidity, light and
 UV), study year (categorial factors: 2021 and 2022). To consider the
 spatial autocorrelation, we added the sampling site as a random
 effect.   The goodness of
 fit was evaluated through the randomised quantile residuals calculated
 using the DHARMa package (version 0.4.6; (Hartig, 2022). The GLMM was
 performed with the HLfit function of the spaMM package (version 3.13.0;
 Rousset and Ferdy, 2014) using R software version: 4.1.2.; (Development
 Core Team, 2021). P-values were calculated using the type II log
 likelihood ratio test (hereafter L.R.). We started with a model including
 all the double interactions between the zone and the other seven
 independent variables and removed them when they were not significant.
 This first model is called “Full model”.
   The same statistical procedure was
 applied to two partial models focused on <em>P. prasina</em>
 abundance in the 250 samples collected in the centre of woods and orchards
 (called the “Centre model”) and in the 350 samples collected at the edge
 of woods and orchards and in the hedges (called the “Interface model”).
 The effect “zone” was removed from each of those GLMM and the remaining
 fixed and random effects were tested. Using the two final partial models
 and the "predict" function in R, we calculated the predicted
 number of <em>P. prasina</em> individuals. We computed
 predictions and their 95% confidence intervals according to each type of
 ecosystem and for the year in which the largest number of individuals was
 recorded, except the apple orchard ecosystem which was only sampled in
 2022. Furthermore, the quantitative predictive variables with no
 significant effect were set to their average value and the random effect
 was not included in the calculation.  
 Next, a qualitative variable with two categories corresponding
 to high and low values of the parameter was created for the quantitative
 abiotic parameters which showed a significant effect in the partial
 models. The threshold for defining these categories was determined by
 selecting a value that minimised the P-value of the effect associated with
 this new qualitative variable in the partial model. In simple terms, this
 threshold represents a value above which the number of <em>P.
 prasina</em> individuals in our leaf litter samples changed
 significantly.  
 <em>2. Reproductive status</em>
   The reproductive status of
 <em>P. prasina </em>was assessed for the overwintering adults
 collected in the leaf litter, as described in the previous section, plus
 adults emerging from overwintering sites at the end of the hibernation
 period, captured as described below. The study was performed in
 2021-2022.   From the end of
 February to the end of April 2022, adults of <em>P.
 prasina</em> newly emerged from overwintering sites were collected
 in two ecosystems: a  2.3 ha hazelnut orchard surrounded by lakes and
 other hazelnut orchards, located near the village of Moulinet
 (44°31'23.9"N, 0°36'00.5"E, 117 m a.s.l.), and a 2.25
 ha wood surrounded by buildings, crop fields, and hazelnut orchards,
 located close to the city of Cancon (44°32’34.6”N, 0°35’34.2”E, 97 m
 a.s.l.). As no information was available concerning the most efficient
 traps to catch P. prasina emerging from overwintering sites, we used two
 different kinds of traps. The first was a homemade cone trap (Raney, 1969)
 adapted from a pop-up mosquito transparent net tent measuring 1.8 m x 2 m
 x 1.5 m (LxWxH, ®GLKEBY). The adaptation consisted of cutting off the
 floor of each tent and placing a plastic cone collector, with a1.5 cm Ø
 entrance at the top of the tent. The tent was kept in place by a 1.50 m
 wooden pole inserted into the ground at the centre of the tent. The second
 trap was a homemade trap based on the Circle trap design (Mulder, Reid,
 Stafne & Grantham, 2012), modified and adapted for P. prasina by
 enlarging the opening at the top of the trap top from Ø 0.80 cm to 1.50
 cm, and by reducing the width of the trap from 81 cm to 66 cm so it fitted
 the circumference of the tree trunks better.
   Ten cone traps and 20 Circle traps
 were installed at each site. The cone traps were spaced about 20 m apart
 and the Circle traps were fixed to trees growing about 10 m apart. The
 presence of <em>P. prasina</em> in the traps was checked three
 times every day: in the morning (at 9 am, at noon, and at 5 pm), to avoid
 leaving emerging individuals together which could bias the evaluation of
 mating status (see below).  
 We checked the sexual maturity of both male and female
 <em>P. prasina </em>collected weekly in the overwintering
 sites between December and February, in 2020-21 and 2021-22, and in the
 cone and Circle traps between the end of February and the end of April
 2022. All parasitised individuals were excluded from the analyses, as they
 could interfere with reproductive status (De Salles, 1992). First, we
 observed the colour of the individuals, which is considered as a proxy for
 reproductive status in the close species Nezara viridula (L.) and Plautia
 stali Scott (Musolin & Numata, 2003; Kotaki & Yagi, 1989):
 russet or reddish brown during the winter reproductive diapause and green
 during the period of active reproduction. The insects were then killed by
 freezing at -20 °C for 2 min, dissected and the sexual organs extracted in
 distilled water under a stereo microscope (Nikon, SMZ1270). Ovarian
 development was assessed using a scale starting at 0 (previtellogenic
 females) and going up to 4 (post-reproductive females) (Kiritani, 1963;
 Nielsen et al., 2017). The mated status of females was evaluated by the
 presence of spermatozoids in the spermatheca (Golec and Hu, 2015; Hamidi
 et al., 2021) observed under the microscope. In males, sexual maturity was
 assessed through the presence of living (i.e. moving) spermatozoids in the
 testes.
description: # Study of the overwintering ecology of the hazelnut pest, *Palomena
 prasina* (Hemiptera: Pentatomidae) in a perspective of Integrated Pest
 Management.
 [https://doi.org/10.5061/dryad.zcrjdfnjt](https://doi.org/10.5061/dryad.zcrjdfnjt) *Six dataset from this paper are available. Data collection is described in the material and methods.* *The files untitled "Overwintering site\_human made structures.xlsx", "Overwintering site\_leaf\_litter.xlsx" , "Overwintering site\_foliage.xlsx" and "Overwintering\_sites\_abiotic\_parameters" contain abundance data and abiotic parameters collected to study the overwintering site of P. prasina in human made structures, leaf litter and evergreen foliage . Then, data set named "Spring\_emergence\_monitoring.xlsx" corresponds to the abundance of P. prasina adults emerging from the leaf litter in spring, that were collected in the Cone traps (20) and the Circle traps (20) at both sites (a wood and a hazelnut orchard). Finally, the dataset "Reproductive\_status.xlsx" is the result of adults dissections collected in their overwintering site and in the spring emergence traps.* ## Description of the data and file structure This is a freeform section for you to describe how the data are structured and how a potential consumer might use them. Be as descriptive as necessary. Keep in mind that users of your data might be new to the field and unfamiliar with common terminology, metrics, etc. Describe relationships between data files, missing data codes, other abbreviations used. Be as descriptive as possible. *"Overwintering site\_human made structures.xlsx":* * year= study year * date= sampling date * volunteer\_ID= identification of the participating volunteers * cities= city where the true bugs were collected * structure= type of human made structures where the bugs were collected * inside\_outside= if the bugs were found inside or outside * site\_structure= which part of the human made structure they were found * true\_bug\_species= species of the collected true bugs * true\_bug\_abundance= their total abundance NA corresponds to "Non Available" data. *"Overwintering site\_foliage.xlsx":* * year= study year * sampling\_date= date of the sampling * site\_ID= identification of the study site * ecosystem\_type= type of ecosystem (described in the Material and methods) * zone= study zone in the study site (described in the Material and methods) * plant\_species= latin name of the sampled plant species * heteropteran\_species= latin name of the collected heteropteran bugs * hetereopteran\_abundnace= their total abundance NA corresponds to "Non Available" data. *"Overwintering site\_leaf\_litter.xlsx":* * year= study year * sampling\_date= date of the sampling * site\_ID= identification of the study site * ecosystem\_type= type of ecosystem (described in the Material and methods) * zone= study zone in the study site (described in the Material and methods) * leaf\_litter\_sampling\_number= the number of the sampling * heteropteran\_species= latin name of the collected heteropteran bugs * hetereopteran\_abundance= their total abundance NA corresponds to "Non Available" data. "Overwintering\_sites\_abiotic\_parameters.xlsx" * year= study year * sampling\_date= date of the sampling * site\_ID= identification of the study site * ecosystem\_type= type of ecosystem (described in the Material and methods) * zone= study zone in the study site (described in the Material and methods) * sampling\_number= the number of the sampling * litter\_depth= leaf litter depth, in centimeters * temperature= temperature of the leaf litter, in Celsius degrees * light= illuminance at the surface of the leaf litter, in Lux * humidity= relative humidity inside the leaf litter, in % * UV= quantity of UV at the surface of the leaf litter, in mW/cm² NA corresponds to "Non Available" data. *"Reproductive\_status.xlsx":* * experiment\_ID= identification of the experiment (Characterisation of overwintering site*s* or monitoring of the spring emergence) * month= month of the sampling * dissection\_date= date on which the dissection of the GSB was done * site\_ID= identification of the study site * sexe= sexe of the collected adult * GSB\_ID= identification of the P. prasina adults dissected * trap= type of trap where *P. prasina* adult was collected in the spring monitoring emergence * trap\_ID= identification of the effective trap * ovarian\_rank= rank in the ovarian development of female (*i. e.* Material and methods) * mating\_status= presence of living spermatozoids found in the female spemateca * spz\_testis\_presence= presence of living spermatozoids found in the male teste * GSB\_colour= colour of the dissected *P. prasina* adult * parasitoid\_presence= presence of endoparasitoids in the dissected adult NA corresponds to "Non Available" data. "Spring emergence monitoring.xlsx": * sampling\_date= date of the sampling * month= month of the sampling * sampling\_hour= monitoring hour of each trap in the day * site\_ID= identification of the study site * trap= type of trap where *P. prasina* adult was collected in the spring monitoring emergence * row= line of trap (i. e. Material and methods) * trap\_number= number of the monitored trap * GSB\_abundance= total abundance of *P. prasina* captured in the trap * male\_abundance= total abundance of male captured in the trap * female\_abundance= total abundance of female captured in the trap NA corresponds to "Non Available" data.