Accept e d Preprint

Original Article Integrated Environmental Assessment and Management

DOI 10.1002/ieam.4107
Long-Lasting Insecticide-treated Nets: a new Integrated Pest Management
approach for Popillia japonica (Coleoptera: Scarabaeidae) †

Leonardo Marianellia, Francesco Paolia, Giuseppino Sabbatini Peverieria, Claudia Benvenutia, Gian

Paolo Barzantia, Giovanni Bosiob, Davide Venanziob, Emanuela Giacomettob, Pio Federico Roversia

a
CREA Research Centre for Plant Protection and Certification, Firenze, Italy

b
Settore Fitosanitario e Servizi Tecnico-scientifici - Regione Piemonte, Torino, Italy

*Correspondence: francesco.paoli@crea.gov.it, CREA DC via di Lanciola 12/a 50125 Firenze.

Phone: +39 055 2492246

†
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
[10.1002/ieam.4107]

All Supplemental Data may be found in the online version of this article at the publisher’s website.

This article is protected by copyright. All rights reserved

Submitted 3 August 2018; Returned for Revision 5 November 2018; Accepted 9 November
2018

This article is protected by copyright. All rights reserved

1
Accept e d Preprint
Orcid:

L. Marianelli http://orcid.org/0000-0002-4132-5245

F. Paoli http://orcid.org/0000-0001-7915-3832

G. Sabbatini Peverieri http://orcid.org/0000-0002-3711-1017

C. Benvenuti http://orcid.org/0000-0002-2347-0078

G.P. Barzanti http://orcid.org/0000-0002-8968-8571

P. F. Roversi http://orcid.org/0000-0002-8098-8367

This article is protected by copyright. All rights reserved

2
Accept e d Preprint
Abstract

The Japanese beetle Popillia japonica Newman is a US and EU quarantine insect pest that recently
has invaded Northern Italy. Its ability to rapidly spread in new areas make this insect a threat to
agriculture. In the last decades, several trials on biological control of the Japanese beetle by
entomopathogenic nematodes and fungi have been carried out with variable efficacy. However, the
necessity of an integrated pest management to improve the control has arisen. Long-Lasting
Insecticide-treated Nets (LLINs) have been used to control other agricultural pests using an attract-
and-kill strategy. Here we present results from laboratory evaluation of two LLINs, Storanet®
(BASF™) and ZeroFly® (Vestergaard™), against P. japonica adults. Both of them were effective in
killing the beetles; however, some differences emerged if compared with different exposure times:
ZeroFly® always gave 100% mortality in tests from 5 s to 30 min exposure; Storanet® showed
100% mortality only with 30 min exposure, down to 89–99% mortality for 5 s to 15 min exposure.
A description of the paralysis process occurring at 5 s exposure is given. Possible field application
of LLINs within programs of integrated pest management is discussed. This article is protected by
copyright. All rights reserved

Key words: Japanese beetle, pest control, pyrethroid, invasive species, LLIN

This article is protected by copyright. All rights reserved

3
Accept e d Preprint
Introduction

The Japanese beetle, Popillia japonica, Newman is a serious agricultural pest, especially in newly

invaded areas (Potter and Held 2002). Since the beetle's discovery in Italy in 2014, the infested area

has constantly been spreading [SFR Piemonte 2017]. In 2014, when monitoring activity on this pest

started, about 80 km2 were reported to be infested in the part of Nature Park of the Ticino Valley

situated in Piedmont (North-West Italy). In 2016 the infested area reached about 500 km2, while in

2017 it exceeded 800 km2. Likewise, the number of adults collected by double lure traps has

increased over time. In August and September 2014 in Piedmont, about 28,000 adults were

collected by 64 double lure traps (437 beetles/trap) (Bosio and Venanzio 2015). In 2015 8 million

adults were caught by 564 traps (14,184 beetles/trap), while 14.7 million adults were caught by

2100 traps in 2016 (6,818 beetles/trap), while more than 48 million adults were caught in 2017 by

2100 traps (21,818 beetles/trap).

With the aim to control beetle populations and to limit them spreading into new areas, in 2016 the

Plant Protection Organizations (PPOs) of the regions of Piedmont and Lombardy have treated

hundreds of hectares of heavily infested meadows with entomopathogenic nematodes

(Heterorhabditis bacteriophora) and fungi (Metarhizium anisopliae), resulting in variable extent of

control. Concerning entomopathogenic nematodes an overall reduction of about 45% in the beetle

population was reported in field experiments (Marianelli et al. 2018; Paoli et al. 2017 b). The efforts

required to manage the outbreak in such large and heavily infested areas (in some cases reaching

more than 500 larvae/m2) are extremely high, and new and effective integrated pest management

(IPM) approaches are needed (e.g. Paoli et al. 2017a).

Long-Lasting Insecticide-treated Nets (LLINs) are a technology recently developed to combat

malaria in tropical and sub-tropical areas by killing vector mosquitoes (Curtis et al. 2003; Lencha

2017; Smith Paintain et al. 2014), and other vector borne diseases (Wilson et al. 2014). It is worth

This article is protected by copyright. All rights reserved

4
Accept e d Preprint
mentioning that in 2017 UNICEF provided 23.9 million LLINs that were distributed across 29

countries with a cost of about $ 2 or 3 for each single net [UNICEF 2018]. LLINs are polyester or

polyethylene nets in the shape of a common insect net, in which insecticides as active ingredients

(a.i.) (generally pyrethroids) are incorporated or coated on the surface. The insecticide is protected

from rapid degradation, and it is constantly released over time, so that the nets can be efficient for a

longer period of time (months or years, depending on applications). The release of the a.i. is

absorbed by insects through tarsal contacts; a given dose of acquired a.i. leads to lethal or sub-lethal

effects according, for example, to insect species and exposure time on the nets (Sabbatini Peverieri

et al. 2018a). Compared with common insecticide control methods like nets treated by conventional

dipping used for human health purposes, LLINs have relevant advantages and minimum potential

environmental impact (Guillet et al. 2001): insecticidal activity is long lasting and no efforts are

required to empty the traps.

LLINs were proved to be effective in forest pest control against bark beetles (Wehnert and Müller

2012; John and Zeilhofer, 2013; Skrzecz et al. 2015) and currently are employed in forest protection

and commercialized in some European countries. To date in agriculture only very recent papers

reporting experimental experiences on stink bugs (e.g., Halyomorpha halys), aphids (Aphis

gossypii), witheflies (Bemisia tabaci) and coleopterans (Leptinotarsa decemlineata and

Conotrachelus nenuphar) (Dader et al. 2014; Kuhar et al. 2017; Sabbatini Peverieri et al. 2018a, b;

Gökçe et al. 2018) are available. Therefore LLINs can be regarded as a pioneering strategy for pest

control in agriculture.

The effectiveness of LLINs had not been tested before on P. japonica. In the present work we

evaluated the effectiveness under laboratory conditions of two different LLINs by considering the

number of dead and paralyzed beetles as a function of exposure time on the nets. Moreover, we

presented a description of the paralysis process of the insect occurring upon 5 s exposure. Finally,

This article is protected by copyright. All rights reserved

5
Accept e d Preprint
we discussed the possible use of LLINs in the field for the management of the Japanese beetle

outbreak in Italy.

Materials and methods

Origin of insects

Adults (males and females) of P. japonica, obtained from the infested sites in Piedmont Region

close to the municipality of Cameri were gently hand-picked in the field (45° 30' 53" N - 8° 41' 58"

E) and collected in the last week of June 2017 during the flight peak period. Adults were promptly

transferred to the local entomological laboratory of the Ticino Valley Park located in Villa Picchetta.

Specimens were provided with fresh hazelnut, hornbeam and grapevine leaves, and reared at room

temperature in plastic boxes perforated on the top to allow ventilation for 24h before being tested.

All applicable international, national, and/or institutional guidelines for the care and use of animals

were followed.

Laboratory tests with LLINs

The laboratory experiments were conducted by testing LLINs Storanet® (BASF™, Ludwigshafen a.

R., Germany) and ZeroFly® (Vestergaard™, Lausanne, Switzerland). Storanet® contained α-

cypermethrin at a dose of 1.57 mg a.i./g fiber, usually commercialized to control forest pests (e.g.

Ips typographus L.). The ZeroFly® contained deltamethrin at a dose of 3.85 mg a.i./g fiber. Before

starting tests, adult specimens were individually selected from the rearing units discarding injured

specimens (e.g. with missing limbs and/or antennae), weakened individuals and dead insects.

Alpha-cypermethrin and deltamethrin are pyrethroid insecticide which shows neurotoxic effects

upon contact (Soderlund et al. 2002).

This article is protected by copyright. All rights reserved

6
Accept e d Preprint
Specimens were tested with LLINs in the laboratory, at room temperature (25º C), from 10:00 am to

2:00 p.m. Experimental arenas consisted of plastic boxes (15 cm width x 15 cm length x 7 cm

depth) with perforated tops to allow ventilation. The inner parts of the boxes were entirely covered

by LLINs (Storanet® and ZeroFly®) in order to have insects always in contact with the insecticide.

For each arena one adult was tested.

Once introduced into the arenas, the adults were free to move for the following scheduled time: 5 s,

15 s, 30 s, 1 min, 5 min, 15 min and 30 min. For each treatment, after exposure, specimens were

gently removed from the arenas and introduced into plastic boxes (15 cm width x 15 cm length x 7

cm depth and perforated top for ventilation) and reared providing food as described above for 13

days or until death occurred. On the whole, 50 females and 50 males were used in each treatment,

for a total of 700 specimens in each type of LLIN. The same number of specimens was tested in

arenas without LLINs as a control.

The effects of α-cypermethrin and deltamethrin exposure were monitored daily on reared P.

japonica adults, and their health condition was classified into the following three categories (sensu

Leskey et al. 2012): (i) active (fully able to move, e.g. walking horizontally and vertically; flying

attempts are sometimes observed), (ii) paralyzed (ranging from a partial functional impairment of

legs and/or leg/antennae, to a more advanced stage of paralysis; specimens are often unable to right

themselves if flipped upside down or are completely paralyzed except for subtle movements of legs

or antennae) and (iii) dead (showing complete loss of movement when probed using a fine brush).

With the aim to obtain parameters that weigh the changes of status in adult vitality (active,

paralyzed and dead), a lethality index (LI) was calculated using the formula by Leskey et al. (2012)

and used for statistical analysis. The LI, was selected among indexes because it considers - by

allocating a specific weight within a formula - not only active and dead specimens, but also those

moribund, which are alive but unable to feed or reproduce (Leskey et al. 2012).

This article is protected by copyright. All rights reserved

7
Accept e d Preprint
Description of the paralysis process occurring upon 5 seconds exposure

In accordance with the LI analysis, which showed no significant differences among exposure times

within each LLIN (see results), the insect timing of paralysis process was described by using adults

exposed to Storanet® and ZeroFly® for 5 s. The experiment was carried out on 20 males and 20

females in testing arenas (the same as in the previous experiment). Each treated insect, after

exposure to LLINs, was lab reared and monitored in its health conditions. The following categories

were considered: 1) active, 2) affected and 3) paralyzed (sensu Morrison et al. 2017).

Statistical analysis

Mortality rates and LI were analyzed by chi-square contingency tests followed by pairwise

comparisons (Chi-square test significance level p<0.05) with Bonferroni correction (MacDonald &

Gardner 2000; McDonald 2014; Zar 2010). LI was calculated at day 13 after treatments. Survival

distributions were analyzed by Survival Analysis (Wilcoxon-Gehan). Data for the paralysis process

were analyzed by GLM for main effects and followed by pairwise comparisons (t-tests, sign. level

p<0.05) with Bonferroni correction. To meet assumption of normality, data not normally distributed

(Shapiro-Wilk test) were transformed by x = Log10(xi+1). Statistical analysis was performed by the

software SPSS 20.0.

Results

Small box arena tests carried out with LLINs

The exposure of P. japonica adults to both Storanet® and ZeroFly® had a strong impact on the insect

viability, compared to the control. However, as no significant difference was observed among

numbers of dead females and males (χ2 = 3.01; df = 2; p>0.05), the data were pooled for analysis.

This article is protected by copyright. All rights reserved

8
Accept e d Preprint
Regardless of exposure time (from 5 s to 30 min), with ZeroFly® 100% mortality always occurred

within 10 days after treatments.

On the other hand, using Storanet® the longer the exposure, the higher the mortality rate that was

observed over 13 days. Storanet® resulted in 99% and 100% mortality when insects were exposed

for 15 and 30 min respectively; with exposure times ranging from 30 s to 5 min, cumulative

mortality rates ranged from 92 and 97% (Table 1). Mortality rates < 90% occurred only with brief

(5 s) exposure. In contrast, there was no beetle mortality in the control groups.

Beetle mortality rates among the two LLINs and control were significantly different at 5 s exposure

(χ2=258; df=2; p<0.0001); pairwise comparison showed that ZeroFly® was more effective than

Storanet® (χ2=9.6; df=1; p<0.05). Despite chi-square analysis detecting significant differences

among all treatments (included the control) at the other different exposure times (from 15 s to 30

min), no significant difference was registered between ZeroFly® and Storanet® (see S.1 and S.2).

Data on health conditions of adults on different days after exposure to LLINs are shown (Fig. 1).

For both LLINs and regardless of exposure time all tested beetles were affected after 24 h, i.e. they

showed signs of paralysis or had died. From the 2nd day after treatment, the number of dead

specimens increased with the coincident reduction of paralyzed adults. Just a few adults were able

to recover from paralysis starting from 2nd - 3rd day for exposure timings ranging from 5 s to 1 min,

and from the 3rd-4th day for exposure > 5 min.

Lethality Index ranged from 86.4 and 92.4 for ZeroFly® and from 72.5 to 87.1 for Storanet®. Within

each LLIN, no statistical difference was detected among exposure times (for Storanet ®: χ2=8.2; df =

6; p>0.05; for ZeroFly®: χ2 = 2.2; df = 6; p>0.05) (Table 2). Within each exposure time (from 5 s to

15 min), survival analysis detected significant differences between specimens exposed to Storanet ®

and ZeroFly®, with the exception of 30 min of exposure, where no significant difference among the

two LLINs occurred (χ2 = 0.04, df = 1; p = 0.84) (see S.3).

This article is protected by copyright. All rights reserved

9
Accept e d Preprint
Description of the paralysis process occurring upon 5 seconds exposure

A 5 s exposure caused the following symptoms in the process of paralysis (data are means ± SD):

Storanet®: a mean of 9.2 ± 3.4 min was necessary for males to change from categories (1) to (2) and

4.3 ±2.6 min to change from categories (2) to (3), with a total of 13.5 ± 3.5 min to reach a complete

paralysis of the insect. For females, a mean of 8.9 ±3.9 min was necessary to change from

categories (1) to (2) and 4.2 ±1.9 min to change from categories (2) to (3), with a total of 12.6 ±3.9

mins to reach a complete paralysis of the insect (Fig. 2).

On the whole, to reach complete paralysis ZeroFly® took longer than Storanet®. In fact, in tests with

ZeroFly® males took 6.0 ±3.2 min to change from category (1) to (2) and 10.2 ±13.4 minutes to go

from categories (2) to (3), with a total of 16.2 ±13.9 min to reach a complete paralysis of the insect.

Females took 28.3 ±15.1 min to change from category (1) to (2) and 9.8 ± 7.4 min to go from

category (2) to (3), with a total of 38.1 ±16.0 min to reach a complete paralysis. Statistical

differences were observed between males and females when considering the passage between

category (1) to (2).

Main effects analysis revealed that both gender and type of LLIN had a significant effect on the

time of the paralysis process from active stage (1) to affected (2) (gender: F= 33.9, df = 1, p<0.001;

LLIN: F = 5.7, df = 1, p<0.001). Females took more time to change from active stage (1) to affected

(2) when exposed to ZeroFly® rather than Storanet® (t = 5.6, df = 38, p<0.001) and when compared

to males with exposure on both LLINs (Storanet®: t = 5.8, df = 38, p<0.001; ZeroFly®: t = 7.9, df =

38, p<0.001). On the whole, regarding the passage from affected stage (2) to paralyzed (3), main

effects were significant only for the factor LLINs (F = 5.0, df = 1, p=0.03); specimens exposed to

ZeroFly® took more time to go from affected (2) to paralyzed (3) status than those exposed to

Storanet® (t = 2.2, df = 77, p=0.03).

This article is protected by copyright. All rights reserved

10
Accept e d Preprint
Discussion

Methods to control Japanese beetle outbreaks have often included the use of chemicals (Cowless

and Villani 1996; Grewal et al. 2001; Potter and Held 2002; Ciampitti et al. 2018) and in few cases,

where the environmental conditions were not suitable to the spreading of P. japonica, the use of

insecticide treatments provided local eradication of this pest (Potter and Held 2002).

However, growing concern about the environmental pollution caused by insecticides and their

potential impact on human health necessitates research on biological or low-impact alternatives

(Spadaro and Gullino 2004; Aktar et al. 2009).

Reliance on entomopathogens such as nematodes or fungi alone has not prevented the regional

spread of Japanese beetles despite those agents sometimes giving levels of control comparable to

those provided by chemical insecticides (e.g. Georgis and Gaugler 1991). Effectiveness of microbial

biological agents is affected by heat, soil moisture and texture, and exposure to sunlight, which

sometimes results in suboptimal outcomes (Potter and Held 2002; Shapiro-Ilan et al. 2006; Wraight

et al. 2007).

Integrated pest management, by definition, does not rely on one single strategy, but combines

different approaches to optimize results (Karuppuchamy and Venugopal 2016). In Italy, in territories

with high infestation levels of Japanese beetles, an integrated strategy of biological control with

entomopathogens plus mass trapping has given moderate control on large scale (Paoli et al. 2018b,

Marianelli et al. 2018, Mazza et al. 2017).

Japanese beetles can be mass-trapped using funnel traps baited with double lures consisting of a

food-type attractant (mixture of eugenol, geraniol, and phenethyl propionate) plus synthetic female

sex pheromone. The value of mass trapping with conventional funnel traps in management of this

pest is currently a matter of debate (Potter and Held 2002 and EPPO 2016). In China, Chen et al.

(2014) used funnel traps to mass-trap Popillia quadriguttata, a closely related species to P.

This article is protected by copyright. All rights reserved

11
Accept e d Preprint
japonica, and obtained a reduction of 93% of adults and 90% of soil larvae. Piñero and

Dudenhoeffer (2018) reported that odor-baited traps with larger beetle holding capacity positioned

at the perimeter of blueberry and elderberry orchards collected over 10.3 million P. japonica adults

in a 3-year period. Consistently, beetle densities and levels of feeding damage caused to elderberry

and particularly to blueberry plants were comparatively low for all the years taken into

consideration.

However, the use of standard funnel traps for mass trapping is not without practical difficulties. In

fact, in places where the rate of infestation is very high, such as the Piedmont part of the Ticino

Valley (Italy), during the flight peak period it would be necessary to empty each trap at least twice a

day; indeed, standard funnel traps (e.g. Pherocon® Japanese beetle – Trécé Inc.) may contain about

3,000 individuals (G. Bosio, personal observation).

To overcome this problem, we explored a new possible control strategy for P. japonica by testing

two different LLINs in the laboratory.

Our results showed that the viability of the Japanese beetle was strongly affected by the exposure to

LLINs. On the whole, both Storanet® and ZeroFly® exhibited very good effectiveness in killing P.

japonica. Regardless of exposure duration, after 24 h, all treated individuals of both LLINs were

affected. In the end, ZeroFly® always resulted in 100% mortality of males and females before the

end of the experiment, even when the contact between insect and net was very short, such as 5 s.

Storanet® showed a cumulative mortality rate depending on the insect exposure time ranging from

100% mortality for exposures of 30 min, and between 89–99% mortality for exposure from 5 sec to

15 min. The LI analysis indicated that even a few seconds of exposure to Storanet® was enough to

kill adult beetles landing on the net.

A slight recovery rate was observed in some of the beetles exposed for shorter times. No beetles

were able to recover from paralysis after exposure of 30 min. However, some recovery was evident

This article is protected by copyright. All rights reserved

12
Accept e d Preprint
in samples treated for < 5 min. As expected, the lower the exposure the higher the recovery rate.

However, not all the insects that apparently recovered from paralysis were then able to survive until

the end of the experiment, probably because of stress caused by the pyrethroid to the nervous

system. Nevertheless, it is noteworthy that 5 s contact on this latter net produced about 90%

mortality.

Regarding the paralysis process, our data showed that the effects of the a.i. upon exposure of 5 s

began soon after the contact with the LLINs: an average of about 10 min for Storanet ®, and 5 to 30

min (depending on gender) for ZeroFly® was necessary to consider insects affected. The timing

needed to reach a complete body paralysis was a bit longer in ZeroFly® than in Storanet® but on the

whole it did not exceed the mean value of 38 min.

Our data suggested the possible use of LLINs as part of IPM programs against the Japanese beetle,

as proposed for other insect pests such as Colorado potato beetle, plum curculio, brown marmorated

stink bug and bark beetles (Skrzecz et al. 2015; Gökçe et al. 2018; Sabbatini Peverieri et al.

2018a,b). In fact, to date, the control methods of the Japanese beetle in Italy has mainly been based

on biological control agents with soil applications against larvae [e.g. SFR Piemonte 2016; SFR

Lombardia 2016]. Unfortunately, such biological methods reported variable results and were unable

to limit the pest spreading. A multi-approach system targeting also the adults is therefore desirable.

For what concerns adults, the mass trapping strategy has shown the above mentioned limits of

trapping capability and management cost: in this context LLINs can play a significant role.

The existence of a specific double lure attractant for P. japonica, paired with LLINs could provide

an effective “attract and kill” device. Under this perspective, LLINs may substitute mass trapping

providing useful advantages in the control of adults. However, a non-target study of species

attracted by this pheromone is still missing and will be matter of future studies. In support of this

idea, we conducted preliminary field observations in 2017 (data not shown) aiming to verify the

This article is protected by copyright. All rights reserved

13
Accept e d Preprint
feasibility of the application of LLINs under field conditions. Here, a LLIN was applied using the

system Trinet® (BASFTM), based on Storanet®. These proof-of-concept observations found that the

average time spent by insects walking or grasping on the net was about 1 to 2 min. During

observation times of 20 min repeated three times from 12:00 to 13:00 during sunny days at flight

peak period, means of 65 adults landed on three Trinet® traps and remained there for more than 5 s.

As shown above, this time is enough for the insect to become paralyzed and eventually die.

Potentially, once proven with a dedicated experiment that LLINs are effective under field

application also against P. japonica, the use of lured attract and kill devices, such as Trinet®, would

provide useful advantages in the management of beetle outbreaks: avoiding the need of a

continuous emptying of traps, the system would be effective throughout the season (only

pheromone replacement would be necessary), with a trapping capability that would not be affected

by limitations in volume of collecting jars. Furthermore, this approach would avoid the odor of

collected decaying dead beetles that may interfere with the pheromone attractiveness and eventually

with beetle capture (Alm et al. 1994; Piñero and Dudenhoeffer 2018). In this regard a study on the

attractiveness of a LLIN lured device is currently in progress to explore the potential knock-down

effect on the local population of the Japanese beetle.

In conclusion, the use of LLINs as “attract and kill” devices in agriculture appears to open

interesting perspectives of global importance for the control of insect pests within IPM approaches:

limiting the spreading of chemicals, the environment could benefit from preserving non-target

species and lowering the soil/water pollution.

This article is protected by copyright. All rights reserved

14
Accept e d Preprint
Acknowledgements

We are especially grateful to Prof. D. A. Potter for critical reviewing the first draft of the manuscript

and his helpful suggestions. This work was carried out in the context of the regional project

“COBIPO2 – Biological Control of Popillia japonica” [Piedmont Region (D. n. 1161 –

29/11/2016)]. We thank Georgina Bingham Zivanovic and Vestergaard Frandsen (Lausanne,

Switzerland) for providing a free sample of ZeroFly®, and Gian Luca Tabanelli, Alberto Gasser and

BASF™ Italia (Cesano Maderno, Italy) for their technical support. We thank our colleague

Giuseppe Mazza for his help during the tests.

Disclaimer

All the authors declare that they have no conflict of interest.

Data accessibility

The Supplementary Material file contains the statistical analysis data considered in this paper. Data

on the description of the paralysis process occurring after 5 seconds exposure with LLINs are

available upon request to the corresponding author Francesco Paoli at francesco.paoli@crea.gov.it

This article is protected by copyright. All rights reserved

15
Accept e d Preprint
References

Aktar W, Sengupta D, Chowdhury A. 2009. Impact of pesticides use in agriculture: their benefits
and hazards. Interdisc Toxicol 2: 1-12.

Alm SR, Yeh T, Campo ML, Dawson CG, Jenkins EB, Simeon AE. 1994. Modified Trap Designs
and Heights for Increased Capture of Japanese Beetle Adults (Coleoptera: Scarabaeidae). J Econ
Entomol 87: 775-780.

Bosio G, Venanzio D. 2015. Popillia japonica, un pericoloso insetto esotico di recente comparsa
nell’area del Ticino [Popillia japonica, an exotic insect pest recently introduced into Ticino area
(Italy)]. Agricoltura 87:47-50.

Ciampitti M, Pasqualini E, Bergaglio S, Mori N, Cavagna B, 2018. Verifica dell’efficacia degli

insetticidi per il controllo degli adulti di Popillia japonica: procedura e primi risultati. Poster
session at “Giornate Fitopatologiche”. Chianciano Terme (SI) 6-9 March.

Chen RZ, Klein MG, Li Y and Li QY. 2014. Mass trapping Popillia quadriguttata using Popillia
japonica (Coleoptera: Scarabaeidae) pheromone and floral lures in northeastern China. Environ
Entomol 43:774-781.

Cowles RS, Villani MG. 1996. Susceptibility of Japanese beetle, Oriental beetle, and European
chafer (Coleoptera: Scarabaeidae) to halofenozide, an insect growth regulator. J Econ Entomol
89:1556–65.

Curtis CF, Jana-Kara B, Maxwell CA. 2003. Insecticide treated nets: impact on vector populations
and relevance of initial intensity of transmission and pyrethroid resistance. J Vector Borne Dis 40:1-
8.

Dáder B, Legarrea S, Moreno A, Ambros CM, Fereres A, Viñuela E, Skovmand O, Bosselmann, R.

2014. Insecticide-treated nets as a new approach to control vegetable pests in protected crops. In:

This article is protected by copyright. All rights reserved

16
Accept e d Preprint
Sadka A. (ed) Proc. Intl. CIPA conference 2012 on plasticulture for a green planet, Acta Hortic
1015, ISHS, Tel Aviv, Israel, pp 103-112.

[EPPO] European and Mediterranean Plant Protection Organisation. 2016. PM 9/21(1) Popillia
japonica: procedures for official control. Bulletin OEPP/EPPO Bulletin 46:543-555.

Georgis R, Gaugler R. 1991. Predictability in biological control using entomopathogenic

nematodes. J Econ Entomol 84:713-720.

Guillet P, Alnwick D, Cham MK, Neira M, Zaim M, Heymann D, Mukelabai K. 2001. Bull World
Health Organ 79:998.

Gökçe A., Bingham G.V., Whalon ME. 2018. Impact of long-lasting insecticide-incorporated
screens on Colorado potato beetle and plum curculio. Turk J Agric For 42:38-44.

Grewal PS, Power KT, Shetlar DJ. 2001. Neonicotinoid insecticides alter diapause behavior and
survival of overwintering white grubs (Coleoptera: Scarabaeidae). Pest Manag Sci 57: 852-857.

John R, Zeilhofer S. 2013. Das TriNet der BASF im Praxistes. AFZ-DerWald2/2013: 29-31.

Karuppuchamy P, Venugopal S. 2016. Integrated Pest Management. In: Omkar (ed) Ecofriendly
Pest Management for Food Security, Academic Press, pp 651-684.

Kuhar TP, Short DB, Krawczyk G., Leskey TC. 2017. Deltamethrin-incorporated nets as an
integrated pest management tool for the invasive Halyomorpha halys (Hemiptera: Pentatomidae). J
Econ Entomol 110:543-545.

Lencha B. 2017. Insecticide treated net utilization among underfive children and household net
ownership in Adami Tullu district, Oromia Regional State, Ethiopia: a Community Based Cross-
sectional study. Quality in Primary Care 26:38-44.

This article is protected by copyright. All rights reserved

17
Accept e d Preprint
Leskey TC, Lee D, Short DB., Wright SE. 2012. Impact of insecticides on the invasive
Halyomorpha halys (Hemiptera: Pentatomidae): Analysis of insecticide lethality. J Econ Entomol
105:1726-1735.

Marianelli L, Paoli F, Torrini G, Mazza G, Benvenuti C, Binazzi F, Sabbatini Peverieri G. Bosio G,

Venanzio D, Giacometto E, Priori S, Koppenhöfer A, Roversi PF. 2018. Entomopathogenic
nematodes as potential biological control agents of Popillia japonica (Coleoptera, Scarabaeidae) in
Piedmont Region (Italy). J Appl Entomol 142:311-318.

Mazza G, Paoli F, Strangi A, Torrini G, Marianelli L, Sabbatini Peverieri G, Binazzi F, Bosio G,

Sacchi S, Benvenuti C, Venanzio D, Giacometto E, Roversi PF, Poinar Jr. GO. 2017. Hexamermis
popilliae n. sp. (Nematoda: Mermithidae) parasitizing the Japanese beetle Popillia japonica
Newman (Coleoptera: Scarabaeidae) in Italy. Syst Parasitol 94:915-926.

MacDonald PL, Gardner RC. 2000. Type I error rate comparisons of post hoc procedures for IXJ
chi-square tables. Educ Psychol Meas 60:735-754.

McDonald JH. 2014. Handbook of biological statistics 3rd ed. Baltimore, MD: Sparky House
Publishing.

Morrison WR, Poling B, Leskey TC. 2017. The consequences of sublethal exposure to insecticide
on the survivorship and mobility of Halyomorpha halys (Hemiptera: Pentatomidae). Pest Manag
Sci 73:389-396.

Paoli F, Marianelli L, Torrini G, Mazza G, Benvenuti C, Bosio G, Venanzio D, Tarasco E, Klein M,

Roversi PF. 2017a. Differential susceptibility of Popillia japonica 3rd instars to Heterorhabditis
bacteriophora (Italian strain) at three different seasons. Biocontrol Sci Techn 27:439-444.

Paoli F, Marianelli L, Binazzi F, Mazza G, Benvenuti C, Sabbatini Peverieri G, Bosio G, Venanzio

D, Giacometto E, Klein M, Roversi PF. 2017b. Effectiveness of different doses of Heterorhabditis

This article is protected by copyright. All rights reserved

18
Accept e d Preprint
bacteriophora against Popillia japonica 3rd instars: laboratory evaluation and field application.
Redia 100:135-138.

Piñero JC, Dudenhoeffer AP. 2018. Mass trapping designs for organic control of the Japanese
beetle, Popillia japonica (Coleoptera: Scarabaeidae). Pest Manag Sci, DOI: 10.1002/ps.4862.

Potter DA, Held DW. 2002. Biology and management of the Japanese Beetle. Annu Rev Entomol
47:175-205.

Sabbatini Peverieri G, Binazzi F, Marianelli L, Roversi PF. 2018a. Lethal and sublethal effects of
long-lasting insecticide-treated nets on the invasive bug Halyomorpha halys. J Appl Entomol
142:141-148.

Sabbatini Peverieri G, Bortolotti P P, Nannini R, Marianelli L, Roversi PF. 2018b. Efficacy of long
lasting insecticide nets in killing Halyomorpha halys in pear orchards. Outlooks Pest Manag 29:70-
74.

[SFR Piemonte] Settore Fitosanitario - Regione Piemonte. 2017. Coleottero scarabeide del
Giappone (Popillia japonica Newman). URL, last accessed on August 2nd, 2018,
http://www.regione.piemonte.it/agri/area_tecnico_scientifica/settore_fitosanitario/vigilanza/popillia.
htm

[SFR Piemonte] Settore Fitosanitario - Regione Piemonte. 2016. URL:

http://www.regione.piemonte.it/agri/area_tecnico_scientifica/settore_fitosanitario/vigilanza/dwd/20
16/risultatiPianoAzione2016.pdf. Last accessed on 3rd October 2018.

[SFR Lombardia] Settore Fitosanitario - Ersaf - Regione Lombardia. 2016. URL:

https://agronotizie.imagelinenetwork.com/difesa-e-diserbo/2016/07/04/popillia-japonica-
superlavoro-al-parco-del-ticino-per-bloccarne-l-espansione/49475. Last accessed on 3rd October
2018.

This article is protected by copyright. All rights reserved

19
Accept e d Preprint
Shapiro-Ilan DI, Gouge DH, Piggott SJ, Patterson Fife J. 2006. Application technology and
environmental considerations for use of entomopathogenic nematodes in biological control. Biol
Control 38:124-133.

Skrzecz I, Grodzki W, Kosibowicz M, Tumialis D. 2015. The alpha-cypermetrin coated net for
protecting Norwey spruce wood against bark beetles (Curculionidae, Scolytinae). J Plant Prot Res
55:156-161.

Smith Paintain L, Awini E, Addei S, Kukula V, Nikoi C, Sarpong D, Kwesi Manyei A, Yayemain D,
Rusamira E, Agborson J, Baffoe-Wilmot A, Bart-Plange C, Chatterjee A, Gyapong M, Mangham-
Jefferies L. 2014. Evaluation of a universal long-lasting insecticidal net (LLIN) distribution
campaign in Ghana: cost effectiveness of distribution and hang-up activities. Malaria J 13:71.

Soderlund DM, Clark JM, Sheets LP, Mullin LS, Piccirillo VJ, Sargent D, Stevens JT, Weiner ML.
2002. Mechanisms of pyrethroid neurotoxicity: implications for cumulative risk assessment.
Toxicology 171:3-59.

Spadaro D, Gullino ML. 2004. State of the art and future prospects of the biological control of
postharvest fruit diseases. Int J Food Microbiol 91:185-194.

[UNICEF] 2018. Long-Lasting Insecticidal Nets Price Data. URL:

https://www.unicef.org/supply/index_59717.html. Last accessed 3rd Oct 2018.

Wehnert A, Müller M, 2012. Borkenkäfer-Fangsysteme im Vergleich. AFZ-DerWald 22, S. 22-25.

Wilson AL, Dhiman RC, Kitron U, Scott TW, Van den Berg H, Lindsay SW. 2014. Benefit of
insecticide-treated nets, curtains and screening on vector borne diseases, excluding malaria: A
systematic review and meta-analysis. PLOS Negl Trop Dis 8, e3228.

Wraight SP, Inglis GD, Goettel MS. 2007. Fungi. In: Lacey LA, Kaya H (eds), Field manual of
techniques in Invertebrate Pathology, pp 223-248. Springer.

Zar JH. 2010. Biostatistical analysis, 5th ed. Englewood Cliffs, NJ: Prentice Hall.
This article is protected by copyright. All rights reserved

20
Accept e d Preprint
Figure Legend

Figure 1. Percentages of Popillia japonica adults at different health conditions (active, paralyzed
and dead) upon different exposure times (5, 15, 30 s and 1, 5, 15, 30 min) to LLINs; white = % of
non-affected specimens or recovered from paralysis; grey = % of paralyzed specimens; black = %
of dead specimens (only data till 10th day after treatment are shown here); Z = ZeroFly®, S =
Storanet®

Figure 2. Mean times (±SE) needed for Popillia japonica adults to change from health condition 1
(active) to health condition 2 (affected) to health condition 3 (paralyzed) upon exposure of 5 s to the
different LLINs

This article is protected by copyright. All rights reserved

21
Accept e d Preprint
Table 1 Percentages of mortality in adults of Popillia japonica exposed to LLINs for different times

(n=100 for each time)

time Control (%) ZeroFly® (%) Storanet® (%)

5s 0 100 89

15 s 0 100 96

30 s 0 100 92

1 min 0 100 97

5 min 0 100 93

15 min 0 100 99

30 min 0 100 100

This article is protected by copyright. All rights reserved

22
Accept e d Preprint
Table 2 Lethality indexes of LLINs for different exposure times (n=100 for each time)

Exposure times ZeroFly® Storanet®

5s 86.35 72.54

15 s 86.73 77.04

30 s 88.15 77.50

1 min 89.23 79.50

5 min 89.86 75.27

15 min 92.42 87.07

30 min 90.19 85.50

This article is protected by copyright. All rights reserved

23
Accept e d Preprint

FigurĞ 1

This article is protected by copyright. All rights reserved

24
Accept e d Preprint

FigurĞ 2

This article is protected by copyright. All rights reserved

25