Received: 7 June 2020

Revised: 30 August 2020

Accepted: 21 September 2020

DOI: 10.1002/elsc.202000042

RESEARCH ARTICLE

A vegetable oil–based biopesticide with ovicidal activity
against the two-spotted spider mite, Tetranychus urticae
Koch
Naoki Takeda1,#
Shimpei Noyama1,3
Dagmar Voigt6

Ayumi Takata1,#

Yuka Arai1

Shigekazu Wakisaka3

Kazuhiro Sasaya1,2Noureldin Abuelfadl Ghazy1,4,5

Takeshi Suzuki1,7

1 Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo, Japan
2 Kanagawa Agricultural Technology Center, Hiratsuka, Kanagawa, Japan
3 OAT Agrio Co., Ltd., Naruto, Tokushima, Japan
4 Agriculture Zoology Department, Faculty of Agriculture, Mansoura University, El-Mansoura, Egypt
5 Japan Society for the Promotion of Science, Chiyoda, Tokyo, Japan
6 Institute for Botany, Faculty of Biology, Technische Universität Dresden, Dresden, Germany
7 Institute of Global Innovation Research, Tokyo University of Agriculture and Technology, Koganei, Tokyo, Japan

Correspondence
Takeshi Suzuki, Graduate School of
Bio-Applications and Systems Engineering, Tokyo University of Agriculture andTechnology, Koganei, Tokyo 184–8588,
Japan.
Email: tszk@cc.tuat.ac.jp# Naoki Takeda and Ayumi Takata sharethe first authorship on this work.
Funding information
JSPS KAKENHI, Grant/Award Number:
18H02203; JSPS Invitational Fellowships
for Research in Japan, Grant/Award Number: L19542; Equal Opportunities Supportof the School of Science at the Technische
Universität of Dresden, Germany

Abstract
A recently developed biopesticide made of safflower and cottonseed oils has
excellent ovicidal activity against the hard-to-control spider mite Tetranychus
urticae Koch (Acari: Tetranychidae). It has attracted attention as a sustainable
treatment for controlling T. urticae because it has low potential for promoting
resistance and little effect on the predatory mite Neoseiulus californicus (McGregor) (Acari: Phytoseiidae), which is an important natural enemy of spider mites.
Here, we investigated the mechanism of its ovicidal activity against T. urticae.
The oil droplets in the oil-in-water emulsion of the biopesticide strongly adhered
to T. urticae eggs, seeped through the chorion being cut during hatching, andinhibited the embryonic rotational movement necessary for cutting and hatching. No adverse effect was observed on N. californicus eggs even in undiluted
biopesticide. We conclude that this biopesticide and N. californicus can be usedsimultaneously in the integrated management of T. urticae in oily biopesticidetolerant plant species.
KEYWORDS

integrated pest management, natural enemies, Neoseiulus californicus, oil droplets, oil-in-water
emulsion
Abbreviations: CI, confidence interval; cryo-SEM, cryo-scanning electron microscopy; LC50 , 50% lethal concentration; O/W, oil-in-water; PDSO,
petroleum-derived spray oil; SEM, scanning electron microscopy
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided theoriginal work is properly cited.
© 2020 The Authors. Engineering in Life Sciences published by Wiley-VCH GmbHEng Life Sci. 2020;20:525–534.

www.els-journal.com

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INTRODUCTION
PRACTICAL APPLICATION

The two-spotted spider mite, Tetranychus urticae Koch
(Acari: Tetranychidae) (Figure 1A), is a cosmopolitan agricultural pest that feeds on more than 1100 plant species[1]. Synthetic pesticides have long been used to control it. However, because of its short developmental timeand high reproductive potential, T. urticae has developed
resistance to 96 active ingredients [2, 3]. Among growing public concern about the environmental and healthrisks associated with the excessive use of synthetic pesticides, the development of alternative treatments which areless prone to the development of resistance and are lessharmful to human and environmental health, is urgently
required.
Food ingredients and food additives (e.g., hydroxypropyl
starch, propylene glycol esters of fatty acids) have beenused as alternative pesticides that physically inhibit respiration and behaviors of pest arthropod herbivores [4,5]. The major advantage of these products over thechemically acting conventional synthetic pesticides isthe elimination of concerns over the development ofresistance because of their physical pesticidal properties.
Although the same advantage can be found in petroleumderived spray oils (PDSOs) [6], food ingredients and foodadditives are less toxic to mammals and can be used
for pest control without compromising environmental
health.
R
is a biopesticide made from 97% v/v safflower
Suffoiland cottonseed oils and 3% v/v emulsifier, and forms oilin-water O/W emulsions. It has acaricidal activity against
all developmental stages of T. urticae. Unlike most pesticides that contain food ingredients or food additives,R
has excellent ovicidal (egg-killing) activity against
SuffoilT. urticae [7], and thus offers effective control. In addition,it has little effect on the predatory mite Neoseiulus californicus (McGregor) (Acari: Phytoseiidae), which is an important natural enemy of spider mites. Neoseiulus californicus
is sold commercially to manage populations of T. urticae
and other spider mites in greenhouses and open fields [8,9]. Therefore, the implementation of both the biopesticideand predatory mites in integrated pest management programs is highly anticipated. However, the mode of action of
the biopesticide and the reason why it has different activities against eggs of T. urticae and N. californicus remain
unclear.
Here, we investigated the mechanism of the ovicidal
activity of the biopesticide against T. urticae and N. californicus eggs by microscopy and experiments.

The high ovicidal activity of a vegetable oil–based
biopesticide against spider mites is due to the inhibition of hatching behavior. Interestingly, no ovicidal effect was observed on the phytoseiid mites
used as biological control agents against spider
mites. This finding will greatly contribute to theuse of the biopesticide in integrated pest management programs. The method used to evaluate ovicidal activity in this study can be used to screenother pesticides that target the hatching behaviorof spider mites.

2
2.1

MATERIALS AND METHODS
Mites

Two populations of T. urticae were used. One population was established in London, Ontario, Canada, in theearly 2000s and was used for whole-genome sequencing[10]. The other was established in Dresden, Saxony, Germany, in 2012 from individuals collected from strawberry
crops in Weixdorf, near Dresden, and was used for the
study of plant–spider mite interactions [11, 12]. The London population was maintained on seedlings of kidneybean (Phaseolus vulgaris L. ‘Hatsumidori #2′, Takii & Co.,
Ltd., Kyoto, Japan) at an air temperature of 24 to 26◦ C, a
relative humidity (RH) of 50% to 65%, and a light periodof 16 h day−1 . The Weixdorf population was maintainedon seedlings of broad bean (Vicia faba L. ‘Witkiem’; ISPInternational Seeds Processing GmbH, Quedlinburg, Germany) at 22 to 24◦ C, 50% to 65% RH, and a light period of
16 h day−1 in wooden, gauze-covered rearing cages. A commercial population of N. californicus was obtained from
Sumika Technoservice Corp. (Takarazuka, Japan) in 2017and maintained under laboratory conditions on detachedkidney bean leaves infested with T. urticae in ventilated
plastic boxes.

2.2

Biopesticide preparationR(OAT Agrio Co., Ltd., Tokyo, Japan) was mixed
Suffoilwith water at a ratio of 1:30, 1:300 (manufacturer’s recommended ratio), 1:3000, or 1:30 000 and emulsified in a
Vortex-Genie 2 mixer (Scientific Industries, Inc., Bohemia,

TAKEDA et al.

NY, USA). Water and paraffin oil (#164-00476; Fujifilm
Wako Pure Chemical Corp., Osaka, Japan), both harmlessto spider mite eggs [13, 14], were used as controls.

2.3
Scanning electron microscopy(SEM) and cryo-SEM
For high-resolution observations of treated T. urticae eggs,
we used SEM with a VHX-D500 microscope (Keyence
Corp., Osaka, Japan) and a JSM-6510 microscope (JEOL
Ltd., Tokyo, Japan), and cryo-SEM with an FE-SEM Supra
40VP-31-79 microscope (Carl Zeiss SMT, Oberkochen, Germany) equipped with an Emitech K250X cryo-preparation
unit (Quorum Technologies Ltd., Ashford, Kent, UK).
Adult T. urticae females were placed onto fresh P. vulgaris ‘Hatsumidori #2′ leaves with a fine tapered artist’s
brush and allowed to lay eggs for 24 h. The eggs laid on theleaves were gently detached and transferred to one side of
a double-sided adhesive carbon tape (Nisshin EM Co.,Ltd.,Tokyo, Japan). The tape with eggs was immersed in waterR
for 1 min and then air-dried(control) or 1:300 Suffoil
for up to 3 h. The side of the tape opposite the eggs was
attached to a metal stub. The samples with no sputtering
treatment were examined by SEM with the VHX-D500
microscope at 1.2 kV. Other treated samples were coatedwith a 20-nm layer of gold in an ion sputter coater (IB-3;
Eiko Engineering, Ltd., Tokyo, Japan) and examined byhigher resolving SEM with the JSM-6510 microscope at
10 kV.
Tetranychus urticae–infested V. faba ‘Witkiem’ leavesRforwere immersed in water (control) or 1:300 Suffoil
1 min and air-dried for up to 3 h. The treated leaves were
cut into 5-mm × 5-mm pieces with micro-scissors andmounted on metal holders in polyvinyl alcohol Tissue-Tek
O.C.T. compound (Sakura Finetek Europe BV, Alphen aan
den Rijn, The Netherlands). They were shock-frozen in liquid nitrogen, transferred to the cryo-preparation chamber
at −140◦ C, sublimed for 30 min at −70◦ C, sputter-coatedwith platinum (∼6 nm), transferred to the cryo-SEM, andthen examined at −100◦ C at 5 kV.

2.4
Biopesticide dose-response of egg
hatching
Adult T. urticae females were placed in an arena fenced
with wet Kimwipes (S-200; Nippon Paper Crecia Co., Ltd.,Tokyo, Japan) in a 35-mm-diameter polystyrene Petri dish (75 mites dish−1 ) and were allowed to lay eggs for
24 h. All females and wet Kimwipes were then removed,
and the deposited eggs were incubated at 25◦ C and 100%
RH for 4 days to synchronize embryonic development just527

before hatching [13]. Adult N. californicus females wereplaced in an arena fenced with Tangle B adhesive (Fuji
Yakuhin Kogyo K.K., Tokyo, Japan) in a 35-mm-diameter
Petri dish (40 to 150 mites dish−1 ) and allowed to lay
eggs for 24 h. Synchronized 5-day-old T. urticae eggs and1-day-old N. californicus eggs were used in the following
experiments.
R
diluted with water at 1:300,
Water (control) or Suffoil
1:3000, or 1:30 000 was added into the Petri dishes in which
the T. urticae or N. californicus eggs were prepared. The
eggs were immersed for 1 min and then drained. Eggs wereincubated at 25◦ C and 50% RH and egg hatch were assessed
after 24 and 48 h with a SZ51 stereomicroscope (Olympus
Corp., Tokyo, Japan). The assay was conducted in three
independent experimental runs.

2.5
Biopesticide effect on embryonicdevelopment
In a preliminary test to confirm the time to eye formationin T. urticae embryos, a single egg immersed in paraffin oil
was photographed with a DFC500 digital camera installed
on a DM2500 light microscope (both from Leica Microsystems GmbH, Wetzlar, Germany) every 30 min.
Adult T. urticae females were placed in an arena
fenced with wet Kimwipes in a 35-mm-diameter Petri dish(25 mites dish−1 ) and allowed to lay eggs for 24 h. After 24 h,
all females and wet Kimwipes were removed from the dish,R
was added.
and 2-mL of water (control) or 1:300 Suffoil
The eggs were immersed for 1 min and then drained. Eggs
were incubated at 25◦ C and 50% RH and embryonic formation of eyes and the hatching of larvae from treated eggs
were observed and recorded every day through the stereomicroscope. The assay was conducted in three independent experimental runs.

2.6
Quantification of biopesticide
coverage of mite eggs
Oil Red O (#154-02072; Fujifilm Wako Pure Chemical
R
atCorp.) used as a tracer dye was added to Suffoil
200 mM w/v to give a final dye concentration of 6.7 ppm
R), 6.7 × 10−1 (1:300), or 6.7 ×(1:30 dilution of Suffoil
−2
10 ppm (1:3000). One-day-old T. urticae and N. californicus eggs were immersed in 2-mL of biopesticide
in a 35-mm-diameter Petri dish for 1 min. The drained
eggs were then photographed with a digital camera(EOS Kiss X7; Canon Inc., Tokyo, Japan) installed on ahigh-magnification stereomicroscope (M205FA; Leica
Microsystems GmbH). The proportion of red in the pixels
of an egg in each image was evaluated in ImageJ 2 software

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[15] to quantify the biopesticide coverage on the surface ofthe egg.

2.7
Effects of biopesticide immersion
and rinsing on egg hatching
Synchronized 5-day-old T. urticae eggs (see section 2.4) and1-day-old N. californicus eggs were immersed in 2 mL parafRin a 35-mm-diameterfin oil (control) or undiluted Suffoil
◦Petri dish and incubated at 25 C. Egg hatch was observedwith the stereomicroscope at 24 and 48 h after treatment.
The assay was conducted in three independent experimental runs.
Synchronized 5-day-old T. urticae eggs were immersedR(see section 2.6) in a 35-mmin 2 mL of dyed 1:300 Suffoil◦
diameter Petri dish at 25 C for 1 min. The liquid was thendrained from the dish; some of the treated eggs were thenrinsed with paraffin oil. The eggs were incubated at 25◦ C
for 24 h, and then egg hatch was observed. The assay was
conducted in three independent experimental runs.

2.8

Data analysis

All data were analyzed in R v. 3.3.2 software [16]. Dose–
response relationships in the pesticide assay were detected
and curves were generated with the two-parameter loglogistic function (drm() function in the R package ‘drc’);they are presented with a 95% confidence interval (CI).
Time-course curves of the proportion of eggs containing
an embryo with visible eye spots and hatched larvae ofT. urticae were plotted with the Kaplan–Meier method(survfit() function in the R package ‘survival’). Differencesin curves between control and biopesticide treatments
were analyzed by the log-rank test (survdiff() function in
the R package ‘survival’). Proportional data on biopesticide coverage of mite eggs and the hatching of larvae fromeggs immersed in undiluted biopesticide and rinsed withparaffin oil were arcsine-square-root transformed, and thenormalized data were analyzed by ANOVA (aov() function in the R package ‘stats’) followed by Tukey’s HSD
test (glht() function in the R package ‘multcomp’) or t-test(t.test() function in the R package ‘stats’) of differences
between species and treatments. Results of the oil-covered
area on eggs and the hatch rate of eggs treated with paraffin oil and biopesticide, or after rinsing with paraffin oil,are presented as overlaid bee-swarm (beeswarm() functionin the R package ‘beeswarm’) and box-and-whisker plots(boxplot() function in the R package ‘graphics’).

F I G U R E 1 (A–F) SEM and (G–L) cryo-SEM images of (A) adultfemale and (B–L) eggs and silk threads of Tetranychus urticae. Eggs
R
were first dipped in (B, E, G) water (control) or (C, D, F, H–L) Suffoil
diluted with water at 1:300 for 1 min. The oils establish liquid bridges
between silk threads, eggs, and the substrate. (J, L) Details of areas
indicated by white arrowheads in images to the left. Scale bars: (A)100 µm, (B–I, K) 50 µm, (J, L) 10 µm

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3

529

RESULTS

3.1
The spider mite egg–oily substanceinterface
SEM and cryo-SEM images clearly demonstrated thebiopesticide covering the surface of eggs and the surrounding silk threads, creating liquid bridges between
threads, eggs, and substrate (Figure 1). Some eggs werecompletely covered; others showed patches (Figure 1F).
The silk threads appeared to act as a scaffold forbiopesticide contact. Obviously, their polymeric structure
altered/denaturated after oil wetting. Also, the biopesticide
accumulated between the egg and the leaf (Figure 1I, J).

3.2
Hatching behavior (without
treatment)
Tetranychus urticae larvae typically rotated inside thespherical egg and probably cut the chorion with their
appendages during the rotational movement before theyhatched (Supplementary Movies S1, S3). Neoseiulus californicus larvae did not; instead, larvae in the ellipsoid egg firstpenetrated the chorion with their dorsal setae Z5 and thenextend their legs for hatching (Supplementary Movie S2).

3.3
Biopesticide dose–response of egg
hatching
The biopesticide caused a dose-dependent increase inlethality to T. urticae eggs (Figure 2). The LC50 value was
359 ± 37 ppm (mean ± SE, n = 31 to 101). In contrast, it
had almost no lethality to N. californicus eggs: the mortality was < 10% (n = 20 to 66), even at the manufacturer’s
recommended dilution ratio of 1:300.

3.4
Biopesticide effect on embryonicdevelopment
Eyes formed (Figure 3D) in > 80% of eggs (n = 169) evenR
(Figure 3E), only slightly less than in
at 1:300 Suffoil
the control (>90%, n = 178). Despite this, the hatchability
was 0% (n = 169), and there was a significant difference inthe time-course curves between the biopesticide treatment
and the control (Figure 3F).

3.5

Biopesticide coverage on mite eggs

A significantly larger area of each egg chorion surface wascovered by the biopesticide in T. urticae (n = 15) than in

F I G U R E 2 Dose–response of hatching in Tetranychus urticae
and Neoseiulus californicus. Eggs were dipped in water (control) orR
diluted with water at 1:30 000 (3.2 × 101 ppm), 1:3000 (3.2 ×
Suffoil
102 ppm), or 1:300 (3.2 × 103 ppm) for 1 min. Drained eggs were incubated at 25◦ C. Data were collected from three independent experimental runs and fitted to a regression curve with a 95% CI (shown bygray area). Numbers of eggs used in each independent experimental
run were 60 to 70 (control), 31 to 101 (3.2 × 101 ppm), 58 to 80 (3.2 ×
102 ppm) and 45 to 86 (3.2 × 103 ppm) for T. urticae, and 30 to 40(control), 35 to 66 (3.2 × 101 ppm), 20 to 41 (3.2 × 102 ppm) and 25 to36 (3.2 × 103 ppm) for N. californicus. The LC50 value for T. urticae
eggs was 359 ± 37 ppm (mean ± SE)

N. californicus (n = 15) at 1:30, 1:300, and 1:3000 dilution
ratios (Figure 4).

3.6
Effects of biopesticide immersion
and rinsing on egg hatching
Almost all eggs of T. urticae (n = 56 to 134) and N. californicus (n = 20 to 26) hatched when immersed in paraffin oil,
although significantly lower hatchability in T. urticae thanin N. californicus (Figure 5A). When immersed in the undiR, although no larvae of T. urticae hatchedluted Suffoil
(n = 53 to 110), almost 100% of N. californicus larvae
hatched (n = 19 to 23). Rinsing with paraffin oil to removeRsignificantly increased the hatchthe dyed 1:300 Suffoil
ability in T. urticae to about 60% (n = 37 to 71) (Figure 5B).

4DISCUSSION

High-resolution observations by SEM and cryo-SEM
Radhered to the surface of eggs
showed that the Suffoil
and to the surrounding web, forming a coat-like layer(Figure 1C, D, F, I–L). Walking larvae to adults of tetranychid mites continuously produce webs by excreting silk
through orifices in their pedipalps which are the second
pair of appendages [17]. The amino acid composition of530

F I G U R E 3 Embryonic development in Tetranychus urticae eggs
immersed in paraffin oil (A) 2, (B) 3, (C) 25, and (D) 53 h after oviposition and held at 25◦ C. (A) Uncleaved egg filled with yolk. (B) Twocell stage established by first cleavage division. (C) Early stage ofembryonic appendage formation. (D) Eye-spot stage. White arrowhead, eyes; black arrowhead, appendages. Scale bar: 50 µm. (E, F)
Time series of (E) proportion of eggs at the eye-spot stage or later and(F) hatchability of T. urticae larvae after being dipped in water (conR
diluted with water at 1:300 for 1 min at 25◦ C. Drainedtrol) or Suffoil
eggs were incubated at 25◦ C. Data were collected from three independent experimental runs. Time-course curves were plotted according
to the Kaplan–Meier method and compared by log-rank test

TAKEDA et al.

F I G U R E 4 Visualization and quantification of oils on mite eggs
by use of a lipophilic red dye. (A) Tetranychus urticae and NeoseiuRlus californicus eggs were dipped for 1 min in Suffoil
containing
200 mM (w/v) Oil Red O, diluted 1:30, 1:300, or 1:3000 with water.
Images of drained eggs were taken with a digital camera attached toa stereomicroscope. (B) The proportion of red in the pixels within asingle egg in each RGB image was evaluated as the amount of biopesticide on eggs. Individual data are displayed as a bee-swarm. In thebox-and-whisker plots, the central line indicates the median, the distance between the box bottom (first quartile) and top (third quartile)
corresponds to the interquartile range, and the whiskers indicate theminimum and maximum values. ***p < 0.001, unpaired t-test of normalized data

the silk is rich in glycine, glutamic acid, alanine, serine,
and aspartic acid, which account for ∼60% of the total,and resembles mammalian pre-keratin, a fibrous protein[18]. The silk fibers of T. urticae are transparent and havea diameter of 54 nm (adult silk) and 23 nm (larval silk)[10], and are thus thinner than the silk fibers of spiders[19] and silkworms [20]. The eggs of the carmine spider
mite, Tetranychus cinnabarinus (Boisduval), a red form of
T. urticae [21], are laid on or under the webbing [17], which

TAKEDA et al.

F I G U R E 5 Effects of oil immersion and rinsing on egg hatch.
(A) Hatching from Tetranychus urticae and Neoseiulus californicus
R
eggs immersed in paraffin oil (control) or undiluted Suffoil◦(biopesticide) in a Petri dish at 25 C. Data were collected fromthree independent experimental runs. Numbers of eggs used ineach independent experimental run were 56 to 134 (paraffin oil)and 53 to 110 (biopesticide) for T. urticae, and 20 to 26 (paraffin oil)and 19 to 23 (biopesticide) for N. californicus. (B) Hatching fromR
(1:300) before (control)
T. urticae eggs treated with dyed Suffoil
and after rinsing with paraffin oil. Data were collected from three
independent experimental runs. Numbers of eggs used in eachindependent experimental run were 37 to 71. The different letter
indicates a significant difference at p < 0.05 by (A) ANOVA followed
by Tukey’s HSD test and (B) paired t-test of normalized data. Figure 4
for explanations of the bee-swarm and box-and-whisker plots

may protect them against the environment and natural
enemies. The strong adhesion of the biopesticide to theT. urticae silk threads (Figure 1C, D, F, I–L) suggests a highaffinity of the oils or the surfactant for the bipolar silk
proteins. This high affinity may support the high ovicidal
activity of the biopesticide, because T. urticae eggs are alsolaid on or under webbing [17].
The manufacturer’s recommended dilution ratio ofR
had high ovicidal activity against T. urticae but
Suffoilalmost no effect on N. californicus eggs (Figure 2). The
area of oily coverage was greater on T. urticae eggs than on
N. californicus eggs (Figure 4), perhaps because N. californicus, unlike T. urticae, does not produce silk threads with531

a high affinity for the biopesticide and the material properties of chorion could differ between phytophagous andpredatory mites. Although the difference in the amount of
biopesticide adhering to eggs could be the reason for thedifference in the ovicidal activity between species, almost
100% of N. californicus larvae hatched, even when theeggs were immersed in the undiluted biopesticide (Figure 5A). Since the biopesticide has no ovicidal activity
Ragainst N. californicus, we conclude that both Suffoil
and N. californicus can be applied simultaneously for theenvironmentally friendly and sustainable management ofT. urticae in oil-tolerant plant species.
Respiration is essential for T. urticae eggs; they die
under anoxic (0% O2 ) conditions at 25◦ C for > 8 h [22,23]. Since two perforation organs penetrate the T. urticae
chorion for taking in air [24], it was initially consideredthat biopesticides would block these organs and suffocate
the eggs. Suffocation by spiracle/stigma blockage was themost accepted theory of the mode of action of PDSOs [25].
To verify this hypothesis, we investigated embryogenesis
in eggs after biopesticide treatment. Spider mites have apair of eyes [26] that are formed late in embryogenesis andare visible through the transparent chorion because of the
red pigmentation (Figure 3D), which is a good indicatorof healthy embryonic development. As eyes were formedin > 90% of eggs in the control and in > 80% eggs treatedwith the biopesticide (Figure 3E), the biopesticide has aslight negative effect on embryogenesis, probably by respiratory inhibition. However, no larvae hatched from thebiopesticide-treated eggs, despite their readiness to hatch(Figure 3F).
Once embryogenesis is completed, the spider miteembryo hatches by cutting the chorion probably withthe appendages while rotating within the egg (Supplementary Movies S1, S3; Figure 6A) [27]. However, in thebiopesticide-treated eggs, no rotational movement wasobserved, or the movement was interrupted (Supplementary Movies S4, S5, S6, Figure 6B). The red dye clearly identified oil droplets (∼10 to 50 µm diameter) in the O/W
emulsion (1:300), which drifted as the moisture evaporated
and adhered to the egg and surrounding silk threads ofwebbing (Supplementary Movie S7). Hatching increased
after the removal of the biopesticide (Figure 5B, 6C), supporting the theory that the oily coverage inhibits the hatching behavior of T. urticae larvae.
Taken together, these results show that oil droplets in theO/W emulsion of the biopesticide effectively adhered toT. urticae eggs and silk threads, establishing liquid bridges
between threads, eggs, and substrate. The oils seeped into
the eggs through the cut chorion as the larvae were hatching and inhibited the embryonic rotational movement
essential for hatching (Figure 6A). Therefore, it may act
mainly by inhibiting the hatching mechanics. We suggest

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F I G U R E 6 Suggested schematic process of hatching from egg in Tetranychus urticae (A) under untreated condition, (B) after treatment
RR, subsequent infiltration into the egg, and silk thread alteration, and (C) after treatment with Suffoil
and subsequent removal ofwith Suffoil
R
Suffoil . The egg may loosely lie on the substrate, or be attached to silk threads which adhere to the substrate. The embryo at the pharate-larva
stage rotates to cut the egg chorion for hatching. Without any treatment (details in the dark blue square), we conjecture friction occurring
between embryo and inner chorion during the rotational movement. The egg is, however, reliably attached to the substrate by frictional forces
acting at the outer chorion-substrate interface and/or the adhesive fixation by silk threads. After treatment with oily fluid (light blue backgroundin B, C, and detail in the light blue square), the fluid not only seeps into the egg through the cut chorion but possibly decomposes silk threads(dotted lines) which loose their adhesive properties. Thus, friction-based repulsion required for the rotational movement towards hatching isinterfered probably by oil lubrication between the chorion and embryo and because the oily fluid leads to silk decomposition and the detachmentof egg-fixing silk threads from the substrate. Interestingly, the impact of oily fluid is recoverable when the oil is subsequently removed (C). Pd,
pedipalps; L1–3, first, second, and third pair of walking legs; L4P, primordia of the fourth pair of walking legs [28]that it causes a loss of repulsion of moving larvae inside
the egg by creating a lubrication layer (Figure 6B). In addition, molecular interaction with the oils could alter theanchoring silk threads, decreasing their adhesion on thesubstrate, and thus, suppressing the internal rotations of
the larvae. Under untreated conditions, the eggs would be
reliably attached to the substrate by silk threads, providing a stable capsule in which larvae may efficiently rotate
because of repulsion generated by friction between the larval integument and the inner chorion.
On the other hand, no adverse effect of the biopesticide
was observed on N. californicus eggs. This may be due tothe fact that the hatching behavior of N. californicus is completely different from that of T. urticae and does not show

any rotational movement inside the eggs (Supplementary
Movie S2).
Further studies are required to fully clarify why N. californicus eggs are resistant to the biopesticide. Also, detailed
understanding of molecular interactions occurring during
diffusion of the biopesticide through the chorion is needed
to evaluate the overall effects in T. urticae eggs under varying environmental conditions.
AC K N OW L E D G M E N T S
This study was partly supported by a JSPS KAKENHI
grant (18H02203) to Takeshi Suzuki. Noureldin Abuelfadl
Ghazy was supported by JSPS Invitational Fellowships for
Research in Japan (L19542). Dagmar Voigt’s contribution

TAKEDA et al.

was co-financed by tax funds using the budget approvedby the Landtag of the Free State of Saxony and by Equal
Opportunities Support of the School of Science at the Technische Universität of Dresden, Germany. We thank Makio
Usui and Naotaka Hayashi for giving us an opportunity tostart this project, Dr. Tetsuo Gotoh (Ryutsu Keizai University) for his advice on the morphology of N. californicus larvae, and to Keyence Corporation for demonstrating SEM
by VHX-D500.
CONFLICT OF INTEREST
This study was funded by OAT Agrio Co., Ltd., which
employs Shimpei Noyama and Shigekazu Wakisaka.
ORCID
Noureldin Abuelfadl Ghazy https://orcid.org/00000001-5911-086X
Dagmar Voigt https://orcid.org/0000-0003-2772-8504
Takeshi Suzuki https://orcid.org/0000-0002-7930-1425
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S U P P O RT I N G I N F O R M AT I O N
Additional supporting information may be found online
in the Supporting Information section at the end of thearticle.

TAKEDA et al.

How to cite this article: Takeda N, Takata A,
Arai Y, et al. A vegetable oil–based biopesticidewith ovicidal activity against the two-spotted spider
mite, Tetranychus urticae Koch. Eng Life Sci.
2020;20:525–534.
https://doi.org/10.1002/elsc.202000042