Vol.:(0123456789)
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Journal of Pest Science (2021) 94:1405–1414
https://doi.org/10.1007/s10340-021-01360-9
ORIGINAL PAPER
Impact ofplasma irradiation onTribolium castaneum
WaheedA.A.Sayed1· RedaS.Hassan1· ThanaaM.Sileem1· BirgitA.Rumpold2
Received: 14 December 2020 / Revised: 9 March 2021 / Accepted: 11 March 2021 / Published online: 23 March 2021
© The Author(s) 2021
Abstract
Radio frequency plasma (RFP) could provide reliable, compact, cost-effective irradiation applications against insect pests
of stored food and feed products. Sensitivity of red flour beetle Tribolium castaneum to RFP has been investigated using an
irradiation applicator system with the two types of inert gases, argon (Ar) and helium (He), at 100W for five exposure time
levels of 0, 20, 40, 60 and 90s, respectively. We demonstrated that He RFP was more efficient against T. castaneum than
Ar RFP. In addition, a positive correlation was observed between mortality percentages of treated insect stages and expo-
sure times for both He and Ar RFP. The adult stage showed the highest tolerance to RFP irradiation followed by larvae and
pupae; however, it was more susceptible than larvae within 24h after He RFP treatments. The optimum exposure time was
90s with He RFP, where a full mortality at all tested stages was accomplished, while mortalities of 71.4, 65.3 and 36.7%
were recorded for pupae, larvae and adult stage, respectively, after an Ar RFP treatment of 90s. In case of treated adults,
the reproduction rate was higher than treated larvae and pupae. Our findings indicated that He RFP was an effective method
for inhibiting T. castaneum development and impacting the insect life cycle and could be considered a promising tool for
pest control of stored food.
Keywords Non-thermal plasma· He and Ar gases· Stored product pest· Mortality· Reproduction rate· Red flour beetle
Key message
• Radio frequency plasma (RFP) irradiation is a promising
treatment against pest insects.
• Helium (He) as a precursor gas is more effective against
T. castaneum than argon (Ar).
• Both He RFP and Ar RFP had toxic effects on larvae,
pupae and adults of T. castaneum.
Introduction
The red flour beetle Tribolium castaneum (Herbst) is consid-
ered a destructive pest that attacks most of stored products
and economic commodities (Hagstrum 2016). It is ubiq-
uitous in many regions around the world (Sokoloff 1977).
Chemical fumigants that have been extensively used against
T. castaneum and other stored product pests, in particular in
developing countries, have detrimental effects on the ecosys-
tem and its fauna, in particular ozone layer and human health
(Zettler and Arthur 2000). Due to these environmental and
health concerns, several pest control technologies have been
proposed as alternatives, such as heat treatment (Arbogast
1981), essential oils (Saroukolai etal. 2010), ozone (Xinyi
etal. 2017) and microwaves (Lu etal. 2010). However, draw-
backs of these alternative pest control methods against T.
castaneum include loss of nutrients and deterioration of food
quality, long treatment times, temperature requirements that
limit applications and high costs (Sileem etal. 2017). Over
Communicated by Christos Athanassiou.
* Birgit A. Rumpold
rumpold@tu-berlin.de
Waheed A. A. Sayed
waheed.sa[email protected]g.eg
Reda S. Hassan
Thanaa M. Sileem
1 Biological Applications Department, Nuclear Research
Center, Egyptian Atomic Energy Authority, Cairo, Egypt
2 Department Education forSustainable Nutrition andFood
Science, Technische Universität Berlin, Marchstr. 23,
D-10587Berlin, Germany
1406 Journal of Pest Science (2021) 94:1405–1414
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the last few decades, ionizing radiation has been proposed
to protect commodities and stored products against insect
pests. It is a process, where infested stored products and
commodities are irradiated in order to sterilize, kill or pre-
vent development of insect pests (Hallman etal. 2010; Mor-
rison 1989). Ionizing radiation is successfully and increas-
ingly applied as a disinfestation treatment that is produced
via the disintegration of radioactive atomic nuclei. Although
it is a simple, robust and well-established technology, its
sources are not readily available in many regions and might
become more difficult to obtain in the future as well, because
the irradiation sources are decaying over time. Accordingly,
attempts to find more green and effective pest control strate-
gies have focused on the application of sustainable energy
sources against stored product pests. Electron beam has been
suggested to be used as a source of radiation (Codex Ali-
mentarius Commission 2003), but the high applied doses
of electron beam irradiation required to kill targeted insects
may have unacceptable effects on product quality. Recently,
another form of radiation, cold or non-thermal plasma, has
been suggested to combine properties/effects of irradiation
treatment with a sustainable energy source—which is one
of the major challenges in radiation processes (Kaur etal.
2020). Plasma, the fourth state of matter, is an ionized gas
containing free electrons. Ionized gas is generated by the
addition of energy such as intense radiation, radio frequen-
cies, extreme heat or electrical energy (Eliezer and Eliezer
2001), resulting in a combination of charged and neutral par-
ticles such as electrons, ions, atoms and molecules (Amami
etal. 2008). Depending on the process used to generate the
plasma, the neutral particles, electrons and ions can have dif-
ferent temperatures (Chen 1984). Non-thermal plasmas are
a mixture of cold ions and neutral (< 60°C) and energetic
electrons (≈1eV). They are generated by electric discharge
in a gas at vacuum condition, lower pressure or atmospheric
pressure (Niemira 2012; Thirumdas etal. 2015). Radio fre-
quency cold atmospheric plasma is generated by circulating
radio frequency currents in antennas or coils, applying a
radio frequency voltage across two parallel electrodes and
immersed in the plasma by a dielectric window. Coupling of
the electrons with electromagnetic fields facilitates energy
that transferred them to sustain the plasma. The efficiency
of the coupled power into the charged particles, as well as
the plasma uniformity, is determined by the radio frequency
excitation design (Chabert and Braithwaite 2011).
Many advantages are connected with non-thermal plasma
irradiation such as low heat, short release time and rela-
tively short process time. In addition, it involves few vari-
ables and has comparably low equipment costs. Therefore,
atmospheric plasma operations have been extensively stud-
ied and its applications have gradually increased in vari-
ous fields due to its appreciated economic efficiency (Tyata
etal. 2012). In order to ensure its efficient adoption as a
disinfestation treatment at industrial level as a safe and reli-
able alternative approach compared to ionizing radiation,
significant research effort is still needed. It is difficult to
optimize an optimal dose protocol against microorganisms
and insect pests that infect food products where very varied
equipment and operational environments have been used,
resulting in very different plasma properties (López etal.
2019).
Recently, non-thermal plasma has been increasingly
used in various industrial processes. It has been applied
for medical applications such as sterilization, cancer treat-
ment, wound healing and blood coagulation (Graves 2012).
The effectiveness of non-thermal plasma as a technology
for improving the shelf-life of food products by eliminating
the microbial contamination from fresh and minimally pro-
cessed food has been investigated, and it was observed that
some species of Salmonella and bacterial spores were inac-
tivated on dry food surfaces by non-thermal plasma (Fernán-
dez etal. 2012; Hertwig etal. 2018). It is also suggested as a
promising method to protect dry food against stored product
pest insects (Abd El-Aziz etal. 2014; Donohue etal. 2006;
Keever etal. 2001; Mishenko etal. 2000; Mohammadi etal.
2015). The surface electrostatic excitation of membranes
due to plasma may have a negative effect on nervous and
neuromuscular systems of insects. This phenomenon may be
attributable to the exposure to high voltage discharge and to
the creation of anoxia, which may serve to anesthetize and
immobilize insects (Bures etal. 2005; Donohue etal. 2006).
Another effect of plasma on insects might be the breakdown
of C-H bonds in the lipid layer of the insect cuticle that
causes dehydration of the insect and can lead to its death
(Donohue etal. 2008).
Non-thermal plasma exhibits reactive oxygen species
(ROS) which includes high reactive chemicals such as per-
oxides, superoxide, oxygen and hydroxyl radicals, and nitric
oxide radicals (Ji etal. 2019) that have a negative impact
on pest insects and may intensify the secondary metabo-
lism of plant products that could maximize the economics
of post-harvest treatment (Bußler etal. 2015; Laroussi and
Leipold 2004). The effectiveness of plasma against microor-
ganisms was determined by the type of gas used to generate
the plasma and therefore responsible for free radical forma-
tion. It has been reported that cold plasma had no impact on
the quality of dry stored products. Mortality-responses of T.
castaneum were recorded at 2500V for 1–5min exposure
times without significant changes in the color of the refined
wheat flour (Mahendran etal. 2016). Furthermore, plasma
treatment of wheat grains caused effective decontamina-
tion of bacteria and fungi together with insect pests and
also enhanced the shelf life, germination and initial state of
growth of the grains and resulted in an increased final grain
yield. Moreover, the production of qualitatively better dough
was also observed (Scholtz etal. 2019; Tyata etal. 2012). In
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this context, it was aimed to investigate the susceptibility of
T. castaneum to radio frequency-induced cold atmospheric
plasma (RFP) as an alternative pest control method with
the following main objectives, (1) Confirm the efficacy of
RFP as a killing method (2) Determine the sensitivity pro-
file of different developmental stages exposed to different
RFP doses using two different precursor gases. (3) Examine
the mortality of irradiated T. castaneum where insect devel-
opment was inhibited (4) Determine the reproduction rate
resulting from RFP treated larvae and pupal stages.
Materials andmethods
Insects
The red flour beetle. T. castaneum colony was maintained for
several generations under controlled laboratory conditions
of 27 ± 2°C and 70 ± 2% RH and continuous darkness. It
was initially established from adults collected from infested
storage wheat in Cairo governorate. The colony was fed on a
rearing medium composed of wheat flour and brewer’s yeast
(19:1, w/w) (Ayvaz etal. 2002). The wheat flour was disin-
fested at 60°C for 10h to eliminate possible contaminants
(Tunçbilek and Kansu 1996).
Generation ofradio frequency‑induced cold
atmospheric plasma (RFP) irradiation
An RFP irradiation source was designed as shown in Fig.1.
It consisted of a generator of 13.65MHz radio frequency
(RF) with an automatic tuner, a gas inlet, plasma chamber
and a vacuum system. The RF antenna was fed 0–600W.
Both the RF generator and the auto-matching network
were from T&C Power Conversion Inc, USA. Around the
source chamber (Bottle), two coils allowed an on-axial mag-
netic field which provided 0–200 Gauss. This magnetic field
confined the plasma electrons which increased the plasma
density. The sample holder was a Pyrex bottle (250ml) in
which indirect plasma irradiation was performed in a batch
system. A pressure of 150kPa was used. In the present
experiment, helium (He) and argon (Ar) were utilized as
precursor gases to create plasma, respectively.
RFP treatment andsubsequent bioassay
ofirradiated T. castaneum stages
15- to 17-day-old larvae and one- to three-day-old pupae
and 6- to 7-day-old adults were exposed to the plasma for 0
(control), 20, 40, 60 and 90s at a power level of 100W using
helium and argon gases. For exposure, the larvae, pupae and
adults were transferred separately into the Pyrex bottle, 30
larvae, 20 pupae and 30 adults, respectively, for each repli-
cate. Three replicates were run for each dose (20, 40, 60 and
90s) and for the control groups (0s). The mortality of adults
and larvae was recorded 6, 12, 18 and 24h after exposure to
plasma. Pupal mortality was not recorded because the dead
pupae could not be distinguished during these experimen-
tal times. The remaining plasma-treated larvae, pupae and
adults were then maintained in the laboratory until death to
observe their mortality or development of the treated larval
and pupal stages to pupae and adults, respectively. The accu-
mulative mortality was calculated at the end of adult survival
as well as larval and pupal durations.
Remaining living adults per replicate that emerged from
irradiated larvae and pupae, as well as treated adults, that
had survived RFP treatments, were transferred using a fine
brush to glass jars containing 20g of rearing medium. These
remaining adults were observed for survival and progeny as
an indication of reproductive ability, where the reproduction
rate of surviving adults, defined here as “the ability of adult
previously irradiated as larvae and pupae as well as irradi-
ated adults to produce progeny,” was determined by placing
those adult survivors on a rearing medium and counting the
next generation (F1 progeny) produced at 43–44days. They
are expressed as number of insects per F1 progeny.
Statistical analysis
Data were analyzed using one way analysis of variance
(ANOVA) technique and the means were analyzed using
Duncan’s multiple range test, the ANOVA statistics were
significant (p < 0.01) (Steel and Torrie 1960). The data of
mortality (%) were transformed by arcsine tables, while the
means and standard errors of reproduction rate were from
original data. The reproduction rate averages were calculated
according to Aldryhim and Adam (1999) and Régnière etal.
(2012), with the methodology described by Carey (1993) as
follows: R = Ne/Ns where Ns is the number of adults at the
beginning of generation (P1 generation); Ne is the number
of adults produced in the next generation (F1 generation).
Fig. 1 Schematic diagram of a radio frequency plasma (RFP) source
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The trendline labels were created by Excel 2010. The Lt50
values were estimated from the total mortality- response
data (defined here as the lethal plasma exposure time to kill
half the population) which were expressed as a percentage
transformed using probit analysis (Abbott 1925) to allow for
a direct comparison of insect mortality due to differences in
susceptibility to the plasma discharge by different gases and
different developmental stages of the insects exposed to the
plasma for differing lengths of time.
Results
Effect ofRFP irradiation onT. castaneum stages
Mortality of larval, pupal and adult stages of T. castaneum at
the end of each developmental stage (accumulative mortal-
ity) after exposure to RFP are shown in Fig.2. Results indi-
cated that both Ar and He RFPs have a lethal effect on larval,
pupal and adult stages. Significant mortalities of 1.2 ± 0.00,
11.4 ± 0.00, 18.7 ± 0.00, 43.3 ± 0.02 and 65.3 ± 0.03%
(F4,10 = 191.9, p > 0.0005) were obtained in the case of Ar
RFP treated larvae, mortalities of 3.2 ± 1.59, 35.1 ± 1.92,
38.5 ± 0.28, 48.3 ± 1.88 and 71.4 ± 1.33% (F4,10 = 35.9,
p > 0.0005) for pupae and mortalities of 4.1 ± 0.0, 8.9 ± 1.20,
11.1 ± 1.01, 33.3 ± 1.05 and 36.7 ± 1.45% (F4,10 = 48.2,
p > 0.0005) for adults at 0, 20, 40, 60 and 90s, respectively.
Significant mortalities of 3.7 ± 0.00, 18.4 ± 0.02, 39.5 ± 0.05,
73.8 ± 0.04 and 100% (F4, 10 = 1241.3, p > 0.0005) were also
observed for He RFP treated larvae, mortalities of 6.8 ± 1.59,
47.7 ± 0.33, 47.8 ± 0.72, 89.1 ± 0.60 and 100% (F4,10 = 197.5,
p > 0.0005) for pupae and mortalities of 0, 10.2 ± 0.33,
20.5 ± 0.66, 64.4 ± 0.33 and 100% (F4,10 = 191.9, p > 0.0005)
for adults, respectively. The results indicated that the mor-
talities increased with increasing treatment time for both
gases. Moreover, it was observed that He RFP was more
effective than Ar RFP, where full mortality was obtained
for larvae, pupae and adults after 90s with He RFP, while
relative mortalities were recorded in these stages at the same
exposure times treated with Ar RFP.
Evaluation ofthereproduction rates ofinsect stages
irradiated withRFP
Figures3 and 4 are depicting the effect of Ar and He RFP
treatments, respectively, on the ability of irradiated larvae,
pupae and adults to procreate a next generation compared
to untreated insects as expressed by reproduction rates. The
reproduction rates of larvae amounted to 4.4 ± 0.5, 3.4 ± 0.3,
1.6 ± 0.17 and 1.2 ± 0.16 (F4,10 = 10.9; p = 0.0013) upon Ar
RFP treatment and 3.7 ± 0.76, 2.9 ± 0.65, 1.6 ± 0.57 and
0.0 (F4,10 = 19.9; p = 0.0001) upon He RFP treatment for
20, 40, 60 and 90s, respectively. They were significantly
reduced in comparison with the larvae reproduction rate
of the control treatment (5.1). Similarly, the reduction of
the reproduction rate was significant when the adult stage
was treated with He RFP (7.5 ± 0.60, 4.4 ± 0.58, 4.1 ± 0.10
and 0.0; F4,10 = 27.4, p < 0.0005) and Ar RFP (4.9 ± 0.34,
4.9 ± 0.05, 4.3 ± 0.63 and 3.4 ± 0.10; F4,10 = 10.8; p = 0.0012)
for 20, 40, 60 and 90s, respectively, as compared to control
treatment (8.8). Moreover, the reproduction rate was signifi-
cantly decreased in pupae treated with He RFP (3.1 ± 0.10,
Fig. 2 Motality of the larval, pupal and adult stages exposed to differ-
ent exposure times of RFP using Ar and He gases at the end of each
developmental stage Fig. 3 Reproduction rates (Avg.) of larval, pupae and adult stages
treated to different exposure times of RFP using Ar gas
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2.8 ± 0.33, 2.4 ± 0.29 and 0.0; F4,10 = 10.6, p = 0.0032)
and was also decreased for Ar RFP (4 ± 0.57, 3.3 ± 0.35,
2.6 ± 0.32 and 2.5 ± 0.28; F4,10 = 5.2, p = 0.0153) for 20, 40,
60 and 90s, respectively, as compared to the control treat-
ment (4.5 ± 0.29). The results indicated that the reproduction
rates of treated adults were the highest in comparison with
treated larvae and pupae for both Ar and He RFP treatments.
Moreover, for Ar RFP, the reproduction rates of treated lar-
vae were higher than for pupae at 20 and 40s, while the
rates were higher in case of treated pupae than larvae at 60
and 90s. Similarly, the reproduction rates were lower for
treated pupae than for treated larvae at exposure times of 20
and 40s with He RFP, while it was lower for He RFP treated
larvae than pupae after 60s.
Determination ofthelethal exposure time (LT) ofHe
RFP treatment onT. castaneum stages
The median lethal exposure time (Lt50) in linear trend was
calculated to obtain clear information about the susceptibil-
ity of insect stages to He RFP (Fig.5) Lt50 of pupae (26.3s)
was drastically lower than of larvae and adults (39.0s and
53.8s), respectively. Results indicated that the pupal stage
was susceptible by twofold compared to adults and by 1.5-
fold compared to larvae.
Mortality assay ofT. castaneum stages 24h afterHe
RFP treatments
The stored product pest management efficiency depends on
instantly getting rid of larvae and adults. In this context, the
mortality within 24h upon He RFP treatment was deter-
mined (Fig.6). It was observed that the mortality not only
increased with increasing the exposure times of RFP but
also varied with time up to 24h after treatments. 6h after
treatment for 90s with He RFP, intermediate mortalities of
42.3 ± 1.17 and 38.9 ± 1.31% were observed for larvae and
adults, respectively, while no lethal effects were recorded
at exposure times of 20 and 40s (Fig.6a). This trend con-
tinued 12h after RFP treatment, where the mortality was
drastically increased to 96 ± 2.08 and 98 ± 1.15% for larvae
and adults, respectively, for 90s treatment with He RFP
as compared to mortalities of 11.2 ± 0.91 and 7.5 ± 1.04%
for 60s (Fig.6b). Also, high mortalities of 98.5 ± 1.04 and
100.0% were obtained 18h after He RFP treatment of lar-
vae and adults, respectively, for 90s compared to mortali-
ties of 25.0 ± 1.76 and 27.8 ± 1.85% for 60s. In the same
time frame (18h after treatment), no mortality (0%) was
recorded for larvae treated for 20 and 40s, however, the
mortality percentages were 8.4 ± 0.64 and 9.3 ± 0.38% for
treated adults, respectively (Fig.6c). As shown in Fig.6d,
full mortality (100%) was obtained for both larvae and adults
treated for 90s 24h after treatments compared to mortali-
ties of 53.4 ± 0.65 and 64.4 ± 6.41% for larvae and adults,
respectively, treated for 60s. In the same time frame (24h
after treatment), the mortality of adults exposed for 20 and
40s was 10.2 ± 0.63 and 20.5 ± 0.63%, respectively, while
still no mortality was observed for the larval stage. These
obtained data revealed that the mortality of the adult stage
was higher than that of the larval stage of T. castaneum, both
18 and 24h after plasma treatment. The obtained results for
mortalities of T. castaneum larvae and adults within 24h
after treatment with He RFP indicated that adults were less
Fig. 4 Reproduction rates (Avg.) of larval, pupae and adult stages
treated with different exposure times of RFP using He gas
Fig. 5 LT50 of of larval, pupal and adult stages treated with He RFP
for different exposure times of 0, 20, 40, 60 and 90s
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tolerant than larvae at sub-lethal doses (Fig.6). These differ-
ences in tolerance within 24h after treatment were converted
in the accumulated mortality-responses that were recorded
at the end of the larval duration and adult survival (Fig.1).
Discussion
Results depicted in Fig.2 are in accordance with other stud-
ies. For example, an inactivation of T. castaneum adults
induced by Ar RFP was shown by Sileem etal. (2020) and
also Carpen etal. (2019) who found a varied mortality
induced by Ar/ oxygen and Ar/nitrogen plasmas, where the
nitrogen-containing plasma resulted in a higher mortality
than argon/oxygen plasmas, while the He RFP was effective
for the control of green peach aphids (Bures etal. 2005) and
T. castaneum adults (Sileem etal. 2020). Evidently, plasma
intensity and exposure times have different impacts on dif-
ferent insect stages and species. The power of 2500V for
15min at a 3.7cm distance between electrodes induced full
mortality of larvae and adult T. castaneum stages (Ramanan
etal. 2018). Abd El-Aziz etal. (2014) found that a decrease
in the distance of the plasma jet electrodes to the sample
and increasing pulse numbers lead to increased mortality
of P. interpunctella pupae. Nevertheless, at the maximum
number of pulses and minimum distance, a larvae mortality
of 86% was not exceeded. The impact of non-thermal plasma
on Blattella germanica has been investigated by Donohue
etal. (2008). They observed slightly opened wings, flaccid
antennae, twitched legs and broken abdomen at high plasma
doses, while lower doses caused limited responses such as
lack of normal movement of treated insects. Malformation
of T. castaneum larvae and partial and whole internal mel-
anization of hemolymph has also been reported due to non-
thermal plasma treatment (Ramanan etal. 2018). It was also
suggested that the oxidative stress of plasma may damage
the larval cuticle and epidermis and attract hemocytes to
the affected area resulted from damage signals. The insect
defense response by disintegration of fat bodies to heal the
damage might cause the clotting and melanization. Simi-
lar responses were observed by Ferreira etal. (2016) on D.
melanogaster larvae who reported that the effect of plasma
Fig. 6 Mortality percentages of larval, and adult stages of T. castaneum treated with He RFP for 20, 40, 60 and 90s determined 6 (a), 12 (b), 18
(c), and 24 (d) hours after treatment
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on larval death might be extreme oxidative stress due to
ROS, as well as the melanization caused by the activation
of the immune system.
The various responses to radiation treatment of T. cas-
taneum stages observed in our study were also obtained
by Tunçbilek etal. (2003) in response to gamma rays, who
found that the larvae were more susceptible than the adult
stage. Also, Sileem etal. (2017) observed that the adult
stage of Oryzaephilus surinamensis exposed to low doses
of gamma radiation resulted in less sterility than those previ-
ously irradiated as larvae, while the irradiated pupae showed
the highest sterility. The same trend was recorded by Aldry-
him and Adam (1999) who found that the adults of Sitophi-
lus granarius previously treated as pupae with cold plasma
had a lower reproduction rate than the adults originating
from treated larvae.
Comparing data after Ar and He RFP treatments revealed
that there are differences in the reproduction rates of treated
adults and also the adults that produced from treated lar-
vae and pupae. Our results indicated that a reduction of the
reproduction of treated insects with sub-lethal doses may be
due to physiological changes in particular DNA damage due
to the impact of active ingredients created by plasma such as
ROS and charged particles and could be referred as indirect
effect of plasma treatment. These differences of reproduc-
tion rates might due to differences in reactive species of He
and Ar plasmas. For example, Kwon etal. (2019) found a
different distribution of different ROS in two atmospheric
plasmas. However, they could not confirm the role of ROS
that resulted in insect death after plasma treatments. Fur-
thermore, Kawasaki etal. (2015) found that the ROS amount
varied depending on the equipment used for plasma genera-
tion. It was observed that in case of cold plasma treatments
of T. castaneum, the differences of applied voltage, electrode
distances and exposure time caused differences in mortality
(Mahendran etal. 2016). The insect mortality, reproduc-
tion rate and insect sterility after ionizing radiation treat-
mentshave been investigated (Hallman etal. 2010). These
responses were attributed to the direct effect of ionizing radi-
ation on DNA and their indirect effect due to released free
radicals attacking DNA chains (Hutchinson 1985). These
effects on DNA were also investigated with UV radiation
(Ravanat etal. 2001) and non-ionizing electromagnetic
fields (Saliev etal. 2019). In this context, the mechanism
of cold plasma which creates many active ingredients may
contribute to DNA damage of insect cells. More research
will be required to examine whether non-thermal plasma
is inducing this damage. Our results presented a reduction
of F1 generation (reproduction rate), which indicates latent
effects of plasma on treated insect expressed as negative
impact on insect fertility rather than direct mortality. This
effect may be attributed to the reactive species in particular
the reactive oxygen species (ROS) mediated by plasma that
cause indirect effects on DNA, immune system and circula-
tory system of insects. This hypothesis is going in line with
the impact of ROS produced by chemical treatment (Kumar
etal. 2015), thermal treatment (Lubawy etal. 2019) and low
doses of gamma radiation (Zhikrevetskaya etal. 2015) on
DNA damage of P. interpunctella, Gromphadorinha coquer-
eliana and Drosophila melanogaster, respectively. Another
possible explanation is the direct impact of plasma on the
nervous system (Donohue etal. 2006). Moreover, in order
to maintain homeostasis, the antioxidant enzymes of the
defense system were significantly changed in P. interpunc-
tella exposed to non-thermal plasma due to the free radicals
(Abd El-Aziz etal. 2014). However, to confirm these mecha-
nisms further, biochemical and molecular genetic studies
are needed.
Our results regarding lethal exposure times are contradic-
tory to Keever etal. (2001) who suggested that pupal and
adult stages were more tolerant to plasma because they have
a hard exterior and puparium for protection against plasma
radiation. Their statement only refers to the direct effect of
plasma radiation that was thought to be effective only at
the target’s surface, but this hypothesis does not take into
account the indirect effect due to ROS. It has been shown
that ROS of cold plasma can penetrate into deeper layers of
model bacteria biofilms resulting in DNA damage (Govaert
etal. 2020). This finding supports our hypothesis of inflicted
damage via plasma penetration of pupal cells similar to the
penetration through meat and crevices and cracks of seeds
(Misra etal. 2019). This indirect effect of ROS could explain
the finding by Nasr etal. (2020) who recorded that the adults
of T. castaneum and Sitophilus granaries were the most sus-
ceptible to direct cold plasma treatments of up to 25min.
Although these results contradict our findings, their study
on the tolerance of larval and pupal stages goes in line with
the results of our study.
It is clear that the adult stage was more susceptible for
the direct effect of RFP that was visible within 24h after
treatment than the larval stage, while it was more tolerant
than the larval stage to the indirect effect of RFP caused by
reactive species that could cause free radicals with oxidizing
effects in treated insect cells. These radicals attack the cell
molecules, which may have time-delayed lethal effects on
the insect after the plasma treatment. This effect has been
demonstrated in case of gamma radiation treatments which
caused different mortality-responses during the life span of
treated insects. However, it was observed that the capacity
of total antioxidants of Drosophila melanogaster stages was
nearly constant (Paithankar etal. 2017). Oxidizing effects
in P. interpunctella larvae treated by cold plasma have been
reported that caused a significant reduction of protein and
glutathione contents, as well as high levels of lipid peroxide.
In comparison with the other insect stages, the plasma treat-
ment resulted in a high sensitive reaction of the larval stage
1412 Journal of Pest Science (2021) 94:1405–1414
1 3
(Abd El-Aziz etal. 2014). The response of larvae, pupae
and adults to that time-delayed effect of plasma treatment
will be different by its variance in the intrinsic physiologi-
cal characteristics and the immunity system. To confirm our
hypothesis, further biochemical and molecular research is
required to identify the repair enzymes involved in the dif-
ferent insect stages in relation to the induced DNA damage.
Conclusion
Radio frequency-induced cold atmospheric plasma (RFP)
can be considered a promising alternative for irradiation pro-
cesses against pest insects. The sensitivity of T. castaneum
to the two types He and Ar RFP was investigated and com-
pared. He RFP is more effective in inactivating T. castaneum
at all three developmental stage than Ar RFP. However, both
He and Ar had a toxic effect on larvae, pupae and adults.
The mortality gradually increased with increasing exposure
time for both types of plasma. Moreover, both types Ar and
He RFP had latent effects on insect stages, where they were
effective in reducing the reproduction rate of treated adults
and the adults produced from treated larvae and pupae. The
optimum exposure time of RFP for preventing development
and procreation was 90s applying He RFP. The susceptibil-
ity of insect stages to He RFP treatment differed. The pupal
stage was the most susceptible followed by larval and adult
stages. However, the adult stage was more susceptible than
larvae within 24h after treatments. Our results indicated
that RFP could successfully be used for managing the T. cas-
taneum pest due to both toxic and latent effects. But further
research is required to evaluate the ability of non-thermal
plasma to penetrate the various food products for using as
a phytosanitary treatment rather than surface treatment. In
addition, the effects of cold plasma treatments on insects
and their fertility need to be studied in more detail on a
molecular and a species-specific level in order to design tar-
geted plasma treatments with the desired effects on specific
insects while conserving the stored goods. Research is also
required on plasma treatment of infested food under typical
storage conditions and volumes. Treatment conditions need
to be determined where the respective treated food remains
unaltered while the targeted pest insects are inactivated. In
addition, upscaling and economic as well as environmental
aspects need to be considered.
Acknowledgement The authors are grateful to assent Prof. Amin
M. Hassan from Accelerators and Ion Sources Department, Nuclear
Research Center, Atomic Energy Authority, for providing the necessary
laboratory facilities and assistance in designing the irradiation protocol.
Authors’ contributions WAS, TMS and RSH conceived and designed
research. TMS and RSH conducted experiments. WAS, TMS and RSH
analyzed data. WAS and BAR wrote the manuscript. All authors read
and approved the manuscript.
Funding Open Access funding enabled and organized by Projekt
DEAL.. This research did not receive any specific grant from funding
agencies in the public, commercial or not-for-profit sectors.
Compliance with ethical standards
Conflicts of interest The authors declare that they have no conflict of
interest.
Ethics approval This article does not contain any studies with human
participants or vertebrates performed by any of the authors.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
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permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.
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