Insecticidal activity of indole derivatives against Plutella xylostella and selectivity to four non-target organisms

Article abstract of DOI:10.1007/s10646-019-02095-1

The diamondback moth Plutella xylostella (Linnaeus, 1758) (Lepidoptera: Plutellidae) is a destructive pest of brassica crops of economic importance that have resistance to a range of insecticides. Indole derivates can exert diverse biological activities, and different effects may be obtained from small differences in their molecular structures. Indole is the parent substance of a large number of synthetic and natural compounds, such as plant and animal hormones. In the present study, we evaluate the insecticidal activity of 20 new synthesized indole derivatives against P. xylostella, and the selectivity of these derivatives against non-target hymenopteran beneficial arthropods: the pollinator Apis mellifera (Linnaeus, 1758) (Hymenoptera: Apidae), and the predators Polybia scutellaris (White, 1841), Polybia sericea (Olivier, 1791) and Polybia rejecta (Fabricius, 1798) (Hymenoptera: Vespidae). Bioassays were performed in the laboratory to determine the lethal and sublethal effects of the compounds on P. xylostella and to examine their selectivity to non-target organisms by topical application and foliar contact. The treatments consisted of two synthesized derivatives (most and least toxic), the positive control (deltamethrin) and the negative control (solvent). The synthesized compound 4e [1-(1H-indol-3-yl)hexan-1-one] showed high toxicity (via topical application and ingestion) and decreased the leaf consumption by P. xylostella, displaying a higher efficiency than the pyrethroid deltamethrin, widely used to control this pest. In addition, the synthesized indole derivatives were selective to the pollinator A. mellifera and the predators P. scutellaris, P. sericea and P. rejecta, none of which were affected by deltamethrin. Our results highlight the promising potential of the synthesized indole derivatives for the generation of new chemical compounds for P. xylostella management.

Full text of DOI:10.1007/s10646-019-02095-1

Ecotoxicology
https://doi.org/10.1007/s10646-019-02095-1
Insecticidal activity of indole derivatives against Plutella xylostella
and selectivity to four non-target organisms
Ângela C. F. Costa1 Sócrates C. H. Cavalcanti Alisson S. Santana Ana P. S. Lima Thaysnara B. Brito
2 ●
1 ●
1 ●
2 ●
●
2 ●
2 ●
3 ●
4 ●
1,5
Rafael R. B. Oliveira Nathália A. Macêdo Paulo F. Cristaldo Ana Paula A. Araújo Leandro Bacci
Accepted: 31 July 2019
©
Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
The diamondback moth Plutella xylostella (Linnaeus, 1758) (Lepidoptera: Plutellidae) is a destructive pest of brassica crops
of economic importance that have resistance to a range of insecticides. Indole derivates can exert diverse biological
activities, and different effects may be obtained from small differences in their molecular structures. Indole is the parent
substance of a large number of synthetic and natural compounds, such as plant and animal hormones. In the present study,
we evaluate the insecticidal activity of 20 new synthesized indole derivatives against P. xylostella, and the selectivity of
these derivatives against non-target hymenopteran beneficial arthropods: the pollinator Apis mellifera (Linnaeus, 1758)
(
Hymenoptera: Apidae), and the predators Polybia scutellaris (White, 1841), Polybia sericea (Olivier, 1791) and Polybia
rejecta (Fabricius, 1798) (Hymenoptera: Vespidae). Bioassays were performed in the laboratory to determine the lethal and
sublethal effects of the compounds on P. xylostella and to examine their selectivity to non-target organisms by topical
application and foliar contact. The treatments consisted of two synthesized derivatives (most and least toxic), the positive
control (deltamethrin) and the negative control (solvent). The synthesized compound 4e [1-(1H-indol-3-yl)hexan-1-one]
showed high toxicity (via topical application and ingestion) and decreased the leaf consumption by P. xylostella, displaying a
higher efficiency than the pyrethroid deltamethrin, widely used to control this pest. In addition, the synthesized indole
derivatives were selective to the pollinator A. mellifera and the predators P. scutellaris, P. sericea and P. rejecta, none of
which were affected by deltamethrin. Our results highlight the promising potential of the synthesized indole derivatives for
the generation of new chemical compounds for P. xylostella management.
●
●
●
Keywords Chemical control Pest control Pesticides Tryptamine
Introduction
Plants of the Brassicaceae family (e.g., Brassica oleracea:
cabbage, cauliflower, Brussel sprouts, headed cabbage and
broccoli) are important food sources (Higdon et al. 2007) due
to their nutritional properties and anticarcinogenic activities
*
Leandro Bacci
bacci.ufs@gmail.com
(
Hasler 1998; Souza et al. 2003). While such attributes have
motivated the large-scale production of brassicas (Novo et al.
010), the production costs are high because these plants are
1
2
3
Programa de Pós-Graduação em Agricultura e Biodiversidade,
Universidade Federal de Sergipe, São Cristóvão, SE, Brazil
2
attacked by agricultural pests, among which, the diamond-
back moth Plutella xylostella L. (Lepidoptera: Plutellidae), a
brassica specialist insect, stands out. The global economic
losses resulting from the attack of this pest are estimated at
around US$4–5 billion per year (Zalucki et al. 2012; Correa-
Cuadros et al. 2014). Moreover, the inappropriate use of
insecticides to minimize these losses has caused resistance in
P. xylostella to a range of chemical compounds (Bortoli et al.
2013).
Departamento de Farmácia, Universidade Federal de Sergipe,
São Cristóvão, SE, Brazil
Programa de Pós-Graduação em Entomologia Agrícola,
Departamento de Agronomia, Universidade Federal Rural de
Pernambuco, Recife, PE, Brazil
4
5
Departamento de Ecologia, Universidade Federal de Sergipe,
São Cristóvão, SE, Brazil
Departamento de Engenharia Agronômica, Universidade Federal
de Sergipe, São Cristóvão, SE, Brazil

Ângela C. F. Costa et al.
Populations of P. xylostella have shown resistance to
almost all classes of insecticides applied in the field (Bortoli
et al. 2013; Lin et al. 2017), including the pyrethroids
widely used for its control, as well as the recently synthe-
sized products (e.g., chlorantraniliprole and flubendiamide)
(UFS), São Cristóvão, SE, Brazil. Derivatives 2a to 2i were
synthesized according to the procedure of Roszkowski et al.
(2005), wherein tryptamine is reacted with acid chloride
derivatives in the presence of dichloromethane (DCM) and
triethylamine to give substituted 1H-indol-3-ethylamine
(Scheme 1a).
(
Troczka et al. 2012). In addition, pyrethroids have
demonstrated high toxicity to non-target organisms, such as
the agents of biological control (predatory wasps [Galvan
et al. 2002; Fernandes et al. 2008; Bacci et al. 2009]) or
pollinators (bees [Sharma and Abrol 2005; Fernandes et al.
The acylated derivatives 4a to 4j were synthesized from
indole via the regioselective Friedel–Crafts reaction at C3 of
the pyrrole ring in the presence of acyl chloride, DCM and
tin chloride (SnCl ) as the catalyst (Ottoni et al. 2001)
4
2
008]). In order to minimize this problem, several studies
(Scheme 1b). Derivative 5 was obtained from the reaction
of indole in the presence of tosyl chloride, NaOH and
benzyltriethylammonium chloride in DCM. The reactions
were prepared under anhydrous conditions and monitored
by thin layer chromatography, in which the starting material
was analyzed for comparison. Additionally, the reactions
were processed by extraction, drying with sodium sulfate
(Na SO ) and solvent evaporation under reduced pressure.
The obtained compounds were purified by column chro-
matography with silica gel as the stationary phase and
hexane:ethyl acetate (8:2) as the mobile phase. Derivatives
with linear, branched aliphatic chains containing chlorine
atoms and a substituted aromatic derivative were synthe-
sized. Nine compounds derived from tryptamine, and 11
derived from indole, were obtained, totaling 22 compounds,
along with their base substances (tryptamine and indole).
have searched for alternative control products that are
relatively more efficient and environmentally safer. Thus,
synthesized compounds have been highlighted as promising
tools in IPM (Integrated Pest Management) (Alvarenga
et al. 2012; Feng et al. 2012; Lima et al. 2015; Sun and
Zhou 2015; Zhang et al. 2015; Yang et al. 2016).
The synthesis of compounds allows creating new mole-
cules with similar structures to each other, but that can have
different insecticidal activity efficiency. The indole is a parent
substance (e.g., indole and tryptamine) of several derivates.
Indole-based compounds are common in natural products
2
4
(e.g., essential amino acids, plant and animal hormones,
bacteria metabolism) and in small synthetic molecules, pre-
senting a wide range of biological activities (Kaushik et al.
2
013, Song et al. 2016) similar to those observed in plant
defense mechanisms (Ahuja et al. 2015) against insects.
Indole derivatives seem to act in the kynurenine pathway,
which presents intermediate compounds with paralytic and
lethal action on insects (Cerstiaens et al. 2003). In this path-
way, the strong neurotoxic action is associated with the
accumulation of 3-hydroxy-kynurenine (3-HK) in the organ-
ism, causing apoptosis of neuronal cells and motor disorders
in insects (Okuda et al. 2002; Cerstiaens et al. 2003). The
levels of 3-HK in the insect’s physiology is regulated by a
transaminase reaction catalyzed by transaminase 3-HK, which
converts it to the stable xanthurenic acid (Rossi et al. 2006;
Han et al. 2007). Therefore, toxic levels of 3-HK could be
achieved by 3-HK transaminase inhibitors (Rossi et al. 2006),
a role that can be played by the indole derivatives.
Preparation of solutions
All synthesized compounds and the insecticide were pre-
viously weighed in a precision balance with 0.01 mg sen-
sitivity (AUW220D, Shimadzu) and were then diluted in
®
Tween 80 surfactant (Dinâmica , Diadema, SP, Brazil) (4%
v/v), followed by homogenization. Subsequently, dime-
thylsulfoxide (DMSO) (Neon, Suzano, SP, Brazil) (29% v/
v) was added, and a further homogenization was performed.
Finally, distilled water (67% v/v) was added, and the
solution was again homogenized. Preliminary tests were
performed and demonstrated that the solvent components
(Tween + DMSO + distilled water) did not affect the sur-
vival and behavior of insects in the proportions used.
In the present study, we evaluate the insecticidal activity
of new indole derivatives synthesized from indole and
tryptamine against larvae of P. xylostella; as well as their
selectivity to beneficial arthropods (e.g., predator wasps and
pollinator honey bee).
Bioassays
Bioassays were carried out at the Agricultural Entomology
Laboratory of UFS (10°54′S, 37°04′W). The experimental
designs were completely randomized.
Material and methods
Initially, the lethal doses (LDs) of 20 synthesized com-
pounds were evaluated for P. xylostella. Among these
compounds, the most and least toxic were selected for
analysis in the subsequent bioassays since they represented
the range of activity of all the compounds analyzed. All
bioassays consisted of four treatments: two synthesized
Synthesis of the indole derivatives
All indole derivatives were synthesized at the Laboratory of
Medicinal Chemistry of the Federal University of Sergipe

Insecticidal activity of indole derivatives against Plutella xylostella and selectivity to four. . .
Scheme 1 a Preparation of
tryptamine amide derivatives,
and b preparation of indole
derivatives
derivatives (most and least toxic), a positive control (com-
mercial product based on deltamethrin (Decis 25 EC,
females, respectively. The leaves containing the eggs were
used to start a new cycle.
®
Bayer, Gujarat, India) and a negative control (solvent).
For the bioassays, 2nd instar larvae were used, based on
the size and color of the cephalic capsule. The only
exception was made in the avoidance behavior bioassay, in
which 3rd instar larvae were used.
Bioassays with Plutella xylostella
Obtaining the larvae Larvae of P. xylostella were obtained
from rearing, established in the Laboratory of Agricultural
Entomology of UFS. The colony started with eggs, larvae
and pupae collected in cabbage crops (B. oleracea var.
capitata), in the region of Itabaiana, SE, Brazil (10°47′S,
Lethal dose (LD) For calculations of the dosages to be
used in the bioassays, initially, the average mass of 50 P.
xylostella larvae was obtained using a precision analytical
balance (AUW220D, Shimadzu). Preliminary tests were
performed using three doses of all the synthesized indole
derivatives and the insecticide (0.1, 1.0 and 2.5 μg of
substance/mg of insect). From these tests, at least six
doses were determined from the dose–mortality curves.
Four replicates were performed for each treatment
(N = 96).
3
7°22′W, and altitude of 215 m). The eggs and larvae were
kept in plastic containers (2L) covered with organza mesh
under ambient conditions. Cabbage leaves were offered to
larvae. The plants were grown under greenhouse conditions,
without application of agrochemicals. Honey and cabbage
leaves were available for adult feeding and oviposition of

Ângela C. F. Costa et al.
Bioassays were conducted in Petri dishes (Global Trade
Technology, Monte Alto, SP, Brazil) (6.0 cm diam. ×
of the leaf disc was treated with the negative control, and
the other with a solution at the LC50 of the treatments. Each
half of the disk was carefully immersed in the solution for
10 s, and after 30 min, were transferred dry to the Petri dish.
Sixty repetitions were performed for each treatment, total-
ing 240 arenas.
1
.5 cm ht.) covered with moistened filter paper (UniFil,
Alvorada, RS, Brazil) (0.3 mL of distilled water) and
containing a cabbage leaf disc (2.5 cm). Ten larvae, treated
®
with 0.5 μL of solution using a microsyringe (Hamilton ,
Reno, NV, USA), were placed on each leaf disk. The Petri
dishes containing treated larvae were covered with plastic
film (Dispafilm, Guarulhos, SP, Brazil) and kept in a
In the center of each arena, a larva previously marked
with red, non-toxic textile ink (Acrilex Special Tapes S.A.,
São Bernardo do Campo, SP, Brazil) was released 1 h
before starting the bioassay. In each arena, the walking of
larva was recorded for 30 min using a video camera
(Panasonic SD5 Super Dynamic WV-CP504) equipped
with a Spacecom lens (1/3′′ 3–8 mm) coupled to a computer
with EthoVision XT software (version 8.5; Noldus Integra-
tion System, Sterling, VA, USA). Data were analyzed in the
Studio 9 software (Pinnacle Systems, Mountain View, CA,
USA). The duration of insects in the treated and non-treated
areas was recorded.
®
biochemical oxygen demand (B.O.D.) incubator (Biotech ,
Piracicaba, SP, Brazil) at 25 ± 1 °C, >70 ± 10% relative
humidity and 12-h photoperiod (12-h L:12-h D). Evalua-
tions of mortality were performed 24 h after beginning the
bioassays. The insects were considered dead when they
remained immobile after being touched with a fine
paintbrush.
Survival analysis The LD90 obtained for the treatments in
the LD bioassay were used to determine the survival curves
and lethal times (LTs) required to kill 50% of the population
Bioassays with non-target hymenopteran species
(
LT ). The procedures were the same as those adopted in
50
the bioassays of LD; however, 10 repetitions were per-
formed for each treatment (N = 40). The mortality of indi-
viduals was evaluated every 2 h in the first 24 h, followed
by 4-h intervals in the following 24 h, and, after that, every
Obtaining insects Active nests of P. scutellaris, P. sericea
and P. rejecta were collected at the campus of UFS. A.
mellifera foraging bees were collected in the municipality of
Barra dos Coqueiros, SE, Brazil (10°48′S, 36°58′W, and
altitude of 14 m). The wasp nests and the honeybees were
kept, separately, in plastic containers (7L), covered with
organza mesh under ambient condition. The honeybees
were fed with 50% sucrose solution. No food was provided
to wasps.
Bioassays were performed in Petri dishes (9.0 cm diam. ×
1.5 cm ht.) covered by filter paper and containing one glass
vial (1.5 × 0.7 cm) with 0.5 mL of 50% sucrose solution.
Each bioassay repetition consisted of seven treated
individuals for each Petri dish. Insects were considered
dead when they remained immobile after being touched
with a fine paintbrush.
1
2 h until 20% larval mortality was achieved in the negative
control.
Lethal concentration Preliminary tests were performed
using three concentrations (0.1, 1.0 and 10%) of the treat-
ments. Subsequently, six concentrations (between the
minimum and maximum mortality interval) were deter-
mined to obtain the concentration–mortality curves. Four
replicates were performed for each treatment (N = 96).
Bioassays were conducted in Petri dishes, as described in
the LD bioassays. The leaf discs of cabbage were dipped in
solutions for 10 s. After 30 min, the dried discs were
transferred to Petri dishes. The evaluations of mortality
were performed 48 h after beginning the bioassays.
Selectivity by topical application In the pronotun region of
each insect, a 1 µL aliquot of one of the treatments was
applied, using a microsyringe. For all treatments, the solu-
tions were applied at the predetermined LD90 for P. xylos-
tella. The treated individuals were previously kept in a
freezer at −10 °C for 2 min in order to reduce their activity.
The Petri dishes containing the treated insects were covered
by a plastic film with small holes for ventilation. Evalua-
tions of mortality were performed 24 h after beginning the
bioassays.
Feeding behavior Leaf cabbage consumption was deter-
mined following the same procedures used in lethal con-
centration (LC) bioassays, using the lower sublethal
concentration of the treatment’s solution (0.1%). After 48 h,
the leaf discs were collected and photographed. The leaf
area consumed was quantified using NIS Elements software
(
version 4.5).
Avoidance behavior Choice bioassays were performed in
Petri dishes (6.0 cm diam. × 1.5 cm ht.) lined with moist
filter paper (0.3 mL of distilled water) and containing a
cabbage leaf disc (6 cm). Each leaf disc was made so that
the leaf’s central vein delimited two areas of equal size. Half
Selectivity by foliar contact Cabbage leaf discs were
immersed for 10 s in solutions of treatments at the pre-
determined LC90 for P. xylostella. After 30 min, the com-
pletely dry leaf discs were transferred to Petri dishes,

Insecticidal activity of indole derivatives against Plutella xylostella and selectivity to four. . .
followed by the individual insects. Evaluations of mortality
were performed 48 h after beginning the bioassays.
displayed 12.50-fold higher activity. Compounds 4a, 4d
and 4f presented similar potencies to indole (Table 1).
Compound 4e showed higher insecticidal activity than
deltamethrin while three compounds (2d, 2e and 2i) showed
similar potency to deltamethrin. However, the remaining
evaluated derivatives were less active than deltamethrin
(Table 1).
Statistical analysis
Data of mortality were submitted to probit analyses to
determine the dose–mortality and concentration–mortality
curves of each compound, using the PROC PROBIT pro-
cedure. From these curves, the LDs and LCs required to
cause 50% (LD ) and 90% (LD ) of mortality, and their
Survival analysis
5
0
90
respective confidence intervals at 95% of probability (CI ),
The survival of P. xylostella larvae, exposed to the most
(2a) and least toxic (4e) compounds at the LD was sig-
nificantly reduced over time (log-rank test: χ = 205.69; d.f.
= 2; P < 0.001) (Fig. 1).
9
5
were obtained. The LDs and LCs were compared by the
criterion of not overlapping the CI with the origin of the
interval.
9
0
2
The survival analyzes were performed using
Kaplan–Meier estimates from the proportion of surviving
insects at the beginning to the end of the experiment, using
the log-rank test. From these analyzes, survival curves and
LTs to cause mortality in 50% of individuals (LT ) were
Deltamethrin exhibited the lowest lethal time, followed
by compounds 4e and 2a. The survival curves of com-
pounds 2a and 4e differed from each other, whereas, there
was no significant difference between the curves of delta-
methrin and the compound 4e (P < 0.05) (Fig. 1).
5
0
obtained. Differences among these curves were verified by
the Holm–Sidak multiple comparison method (P < 0.05)
Lethal concentration
(
SigmaPlot 11.0).
The response variables, namely, leaf consumption,
Derivatives 2a and 4e were toxic to the P. xylostella larvae.
The LC50 of both substances showed significantly higher
activity than deltamethrin. The compound 4e (LC =
avoidance behavior of P. xylostella larvae and the selec-
tivity of non-target Hymenoptera, were analyzed using
analysis of variance (ANOVA), followed by Tukey’s test
P < 0.05) (PROC GLM with Tukey’s test; SAS). Data
were previously analyzed for conformity with the assump-
tions of normality, using SAS software (SAS 2008).
50
−
1
0.017 mg mL ) was around 8- and 20-fold more potent
−
1
(
than 2a (LC = 0.14 mg mL ) and deltamethrin (LC =
50 50
−
1
0.33 mg mL ), respectively.
Analysis of the LC values revealed that compound 4e
9
1
0
−
(
LC = 0.08 mg mL ) showed higher potency, followed
90
−
1
by deltamethrin (LC = 0.44 mg mL ) and 2a (LC =
0.84 mg mL ) (Fig. 2).
9
0
90
−
1
Results
Bioassays with Plutella xylostella
Feeding behavior
Lethal dose
The leaf consumption by P. xylostella larvae was sig-
nificantly different among treatments (F3,12 = 48.16; P <
0.03). The lowest consumption was observed when the
All indole derivatives were toxic, by contact, to P. xylostella
larvae. Derivative 2a, obtained from tryptamine, exhibited
the lowest toxicity among all tested compounds (LD =
2
plant was treated with compound 4e (154.11 ± 13.44 mm ),
2
followed by deltamethrin (232.75 ± 17.91 mm ) and 2a
5
0
−
1
2
6
.33 µg mg ). Instead, compound 4e, synthesized from
(309.31 ± 34.97 mm ) when compared with the control
2
indole, exhibited the highest toxicity among all compounds
(490.87 ± 0.00 mm ) (Fig. 3).
−
1
(
LD = 0.42 µg mg ) (Table 1).
50
Among the compounds obtained from tryptamine, six
Avoidance behavior
(
2b, 2d, 2e, 2f, 2h and 2i) were more toxic than tryptamine.
The other compounds (2a, 2c and 2g) showed toxicity
Compounds 2a and 4e did not induce avoidance behavior in
P. xylostella larvae, and there was no significant difference
in the time spent by individuals between treated and non-
treated areas (2a: F1,118 = 0.06, P = 0.80; 4e: F1,118 = 0.99,
P = 0.32). However, larvae avoided the areas treated with
deltamethrin (F1,118 = 21.35; P < 0.02), spending 70% of
the time in the non-treated area (Fig. 4).
similar to tryptamine. The highest activity was produced by
−1
compound 2e (LD = 0.70 µg mg ), which was 7.97-fold
50
more active than tryptamine (Table 1).
Eight compounds (4b, 4c, 4e, 4g, 4h, 4i, 4j and 5)
synthesized from indole were more active than indole, with
−
1
emphasis on compound 4e (LD = 0.42 µg mg ), which
50

Ângela C. F. Costa et al.
Table 1 Toxicity by topical application of the indole derivatives on Plutella xylostella after 24 h of exposure
Code Compounds
No of insects LD50 (CI95%) (μg mg−1) LD90 (CI95%) (μg mg−1
)
Slope χ2
P
1
2
2
2
2
2
2
2
2
2
Tryptamine
280
240
320
280
240
240
240
5.58 (4.42–6.84)
6.33 (5.67–7.13)
2.26 (1.86–2.74)
5.12 (3.67–7.19)
0.84 (0.65–1.07)
0.70 (0.44–1.04)
2.46 (1.82–3.35)
4.39 (3.30–5.76)
2.50 (1.95–3.31)
0.85 (0.65–1.10)
43.46 (25.99–117.17) 1.44 0.50 0.78
a
b
c
d
e
f
N-(2-(1H-indol-3-yl)ethyl) acetamide
N-(2-(1H-indol-3-yl)ethyl) propanamide
N-(2-(1H-indol-3-yl)ethyl) butanamide
N-(2-(1H-indol-3-yl)ethyl) pentanamide
N-(2-(1H-indol-3-yl)ethyl) hexanamide
N-(2-(1H-indol-3-yl)ethyl) heptanamide
18.60 (14.67–26.84)
13.10 (9.51–20.20)
2.74 0.45 0.80
1.68 1.39 0.50
111.67 (57.47–311.84) 0.96 0.59 0.75
8.59 (5.49–16.70) 1.27 1.43 0.50
37.18 (15.38–171.97) 0.74 1.85 0.60
39.59 (22.87–86.45) 1.06 1.49 0.52
62.68 (32.53–208.68) 1.11 2.13 0.34
g
h
i
N-(2-(1H-indol-3-yl)ethyl) 2-methylpropanamide 240
2-chloro-N-(2-(1H-indol-3-yl)ethyl) acetamide
240
280
23.94 (14.12–53.64)
13.75 (8.29–28.65)
1.30 0.09 0.95
1.06 2.88 0.58
2,2,2-trichloro-N-(2-(1H-indol-3-yl)ethyl)
acetamide
3
4
4
4
4
4
4
4
4
4
4
5
Indole
240
320
240
240
240
240
280
320
240
280
240
280
240
5.25 (4.70–5.90)
3.82 (3.02–4.91)
1.94 (1.38–2.68)
1.00 (0.68–1.47)
4.53 (4.18–4.93)
0.42 (0.32–0.50)
5.00 (4.57–5.52)
1.27 (0.95–1.65)
3.62 (3.00–4.48)
2.37 (1.88–3.01)
2.77 (2.23–3.32)
2.50 (2.27–2.76)
1.19 (0.90–1.56)
15.21 (12.10–21.54)
2.77 0.80 0.67
a
b
c
d
e
f
1-(1H-indol-3-yl)ethanone
1-(1H-indol-3-yl)propan-1-one
1-(1H-indol-3-yl)butan-1-one
1-(1H-indol-3-yl)pentan-1-one
1-(1H-indol-3-yl)hexan-1-one
1-(1H-indol-3-yl)heptan-1-one
1-(1H-indol-3-yl)-2,2-dimethylpropan-1-one
1-(1H-indol-3-yl)-3-methylbutan-1-one
1-(1H-Indol-3-yl)-3,3-dimethyl-butan-1-one
1-(1H-indol-3-yl)(phenyl)methanone
Tosyl indole
60.29 (35.52–129.50) 1.07 3.49 0.52
38.97 (22.97–80.16)
23.55 (13.28–49.23)
9.51 (8.24–11.59)
2.41 (1.61–5.29)
0.98 0.20 0.90
0.93 0.77 0.68
3.97 0.23 0.89
1.68 2.07 0.36
3.16 1.73 0.63
1.00 3.30 0.51
1.78 0.59 0.75
1.33 1.51 0.53
1.67 1.64 0.55
2.85 2.28 0.52
1.31 1.16 0.56
12.73 (10.78–15.83)
24.10 (15.69–43.23)
18.92 (13.17–31.55)
21.63 (13.81–41.80)
16.20 (11.61–27.26)
7.05 (5.84–9.22)
g
h
i
j
Deltamethrin
11.28 (7.67–18.78)
LD50 = lethal dose required to kill 50% of population
LD90 = lethal dose required to kill 90% of population
CI95% = Confidence intervals at 95%
Bioassays with non-target hymenopteran species
Discussion
Selectivity by topical application
The present study highlights the efficacy of indole deriva-
tives for the control of P. xylostella, emphasizing compound
4e [1-(1H-indol-3-yl)hexan-1-one], which was more effi-
cient than deltamethrin, a product that has been causing
induction of resistance in several populations of this insect
pest. The compound 4e showed high selectivity to target
species rather than different non-target hymenopteran spe-
cies when compared with the synthetic insecticide
deltamethrin.
The survival of A. mellifera, P. scutellaris, P. sericea and P.
rejecta did not differ from the control treatment when
topically treated with the LD90 of compounds 2a and 4e for
P. xylostella. The percentage of surviving individuals
exposed to both derivatives was higher than 85% for all
species, differing significantly from deltamethrin, which
showed only 5% survival of individuals (Fig. 5a).
Synthesized indole derivatives showed insecticidal
activity against P. xylostella larvae. Studies have revealed
that biological activity may be modulated by the presence of
certain chemical chains or groups present in the structure of
a compound (Santos et al. 2011; Barbosa et al. 2012; Oli-
veira et al. 2014; Zhou et al. 2014; Lima et al. 2015;
Nguyen et al. 2015; Gallardo et al. 2016). Some research
also predicts a greater biological activity by molecular
structures with shorter chains than longer chains (Nguyen
et al. 2015; Gallardo et al. 2016). In the present study, all
Selectivity by foliar contact
The survival of individuals of A. mellifera, P. scutellaris, P.
sericea and P. rejecta did not differ from the control after
leaf contact with the LC90 of compounds 2a and 4e. For all
species tested, the percentage of surviving insects exposed
to the two synthesized substances was higher than 85%,
differing significantly from deltamethrin treatment, in which
there were no survivors (Fig. 5b).

Insecticidal activity of indole derivatives against Plutella xylostella and selectivity to four. . .
Fig. 2 Concentration-mortality curves of Plutella xylostella exposed
via ingestion to the more and less toxic derivatives (4e and 2a,
respectively), after 48 h. LC50 = lethal concentration required to kill
Fig. 1 Survival curves and mean lethal time (LT50) of Plutella xylos-
tella exposed via topical application to the lethal dose (LD90) of the
more and less toxic synthesized derivatives (4e and 2a, respectively).
LT50 = lethal time required to kill 50% of the population
5
0% of the population
Fig. 4 Avoidance behavior of Plutella xylostella individuals in the
function of exposure to the more and less toxic derivatives (4e and 2a,
respectively) for 30 min. An asterisk indicates time spent by indivi-
duals between treated and non-treated areas differs by Tukey’s test (P
<
0.05). n.s. = not significant
distinct from that observed by deltamethrin. Although 4e and
deltamethrin are both neurotoxic, they act via different path-
ways or mode of actions. The main target of the pyrethroid
deltamethrin is the sodium channels in the membranes of
nerve cells (Endersby et al. 2011; Du et al. 2016) while
derivative 4e appears to act on the kynurenine pathway by
blocking conversion reactions of kynurenines (neurotoxic
metabolites) to more stable compounds. In insects, kynur-
enine can cause motor dysfunction, paralysis and instant death
(Cerstiaens et al. 2003). Additionally, the differences in
activity between 4e and deltamethrin against P. xylostella
could be related to the insect’s resistance to these compounds.
Under natural conditions, resistance mechanisms of P.
xylostella to indole compounds have been described only for a
restricted group of substances of the defense complex
(glucosinolate–myrosinase or indole glucosinolates) produced
by Brassicaceae plants (Ratzka et al. 2002). Moreover, reports
of P. xylostella resistance to the synthesized indole derivatives
have not appeared in the literature, in contrast to the plethora
of research on deltamethrin.
Fig. 3 Leaf consumption by Plutella xylostella exposed via ingestion
to the solutions (0.1%) of the more and less toxic derivatives (4e and
2
a, respectively) after 48 h. Means followed by the same letters do not
differ by Tukey’s test (P < 0.05)
synthesized compounds exhibited insecticidal activity,
either higher or similar to their respective parent materials
(
indole and tryptamine), but their activities were indepen-
dent of the length of the chains. These variations in the
biological activity of members of a homologous series
could be related to the addition of different chemical groups
that interfere with at least one of the following processes: (i)
specific drug–receptor interaction related to target sites; (ii)
insect metabolism through excretion of bioactive substances
(
Ing 1964); and (iii) changes in physical properties, such as
solubility (Raison and Standen 1955).
The susceptibility of the P. xylostella larvae to 4e was
superior to the other treatments (Table 1). This higher activity
seems to be associated with its mode of action, which is

Ângela C. F. Costa et al.
suggests a possible future use for management. Individuals
of P. xylostella were maintained on the leaves treated with
compound 4e, favoring the contact with the insecticide
while reducing the leaf consumption. Conversely, we
observed that larvae avoided areas treated with deltame-
thrin, which limits their control action through ingestion.
Similar avoidance behavior has also been noticed with other
pyrethroids, such as gamma-cyhalothrin (Nansen et al.
2016).
In addition to presenting high toxicity potential, com-
pounds 2a and 4e were also selective to P. scutellaris, P.
sericea, P. rejecta and A. mellifera, by topical application
and leaf contact (Fig. 5). The levels of kynurenine are
normally elevated in the egg and larval stages (Han et al.
2002), reaching the peak levels in the pupa stage (Cerstiaens
et al. 2003). This compound appears to be strongly asso-
ciated with the paralysis and metamorphosis of insects. In
adult insects, such substances are recognized as causing
motor dysfunctions. Thus, initially, a certain toxic effect
was expected on non-target insects (pollinators and pre-
dators). The observed tolerance may be associated with the
low penetration of the insecticide in the cuticle, due to
differences in solubility of the compounds or by the
increased metabolism of the insecticide and modification at
the target site (Guirado et al. 2009). Instead, deltamethrin
did not show selectivity for the species tested by topical
application or by foliar contact. The wasps and bee mor-
talities were higher than 95% by topical application and
reached 100% by leaf contact (Fig. 5). Previous studies
have also reported high toxicity of deltamethrin (Galvan
et al. 2002; Santos et al. 2003; Sharma and Abrol 2005;
Bacci et al. 2009) and other pyrethroids (Sharma and Abrol
2005; Fernandes et al. 2008; Bacci et al. 2009; Barros et al.
2015) for different species of wasps (Galvan et al. 2002;
Fernandes et al. 2008; Bacci et al. 2009) and bees (Sharma
and Abrol 2005; Fernandes et al. 2008), including P. scu-
tellaris, P. sericea and A. mellifera.
Selectivity studies have great importance to minimize the
negative impact of insecticides on beneficial organisms,
which are essential to ensure ecosystem integrity (Potts
et al. 2010). The industry and public policy have failed in
the past to determine the acceptable insecticides LDs that
are non-harmful to beneficial organisms, such as honey bees
(Sánchez-Bayo et al. 2016). In the present study, we verify
the low toxicity of indole compounds to natural enemies
and pollinator. However, we caution that additional studies
need to be developed in natural populations, including the
lethal and sublethal effects of the compounds in different
castes under field conditions.
Fig. 5 Selectivity of Polybia scutellaris, Polybia sericea, Polybia
rejecta and Apis mellifera exposed via topical application (a) and leaf
contact (b) to the more and less toxic derivatives (4e and 2a, respec-
tively) and deltamethrin after 24 h. Means followed by the same letters
do not differ by Tukey’s test (P < 0.05)
As observed with deltamethrin, derivatives 2a and 4e
showed rapid insecticidal action against P. xylostella larvae
over time (Fig. 1). The higher efficiency of compound 4e
compared with 2a may be the result of the blocking rate of
the kynurenine stabilization reaction, requiring less time to
produce toxicity against the insect. In addition to contact
toxicity, compounds 2a and 4e also showed insecticidal
activity by ingestion (Fig. 2), reduced the leaf consumption
by the insects (Fig. 3) and did not cause escape behavior of
P. xylostella larvae (Fig. 4). Although the food channel of
lepidopteran larvae constitutes a barrier to insecticidal
action, certain toxic agents can overcome this barrier by
morphological alterations in the middle intestine or by the
insensitivity of the detoxifying/digestive enzymes, as
already observed in P. xylostella (Correia et al. 2009;
Ramya et al. 2016). Indole derivatives promote insect
neurotoxicity via the accumulation of kynurenines in the
nervous system (Han et al. 2002; Rossi et al. 2006; Han
et al. 2007), causing paralysis and severe or irreversible
motor dysfunction (Cerstiaens et al. 2003). The reduced
feeding behavior of P. xylostella larvae observed with
compounds 2a and 4e seems to be associated with paralysis,
caused by the accumulation of kynurenine (Chiou et al.
In summary, the synthesized indole derivatives presented
high insecticidal potency against P. xylostella and reduced
the feeding by this insect. In addition, the high selectivity
demonstrated for A. mellifera, P. scutellaris, P. sericea and
1
998). Such toxicity, coupled with the fact that the indole
compounds did not cause avoidance behavior in the larvae,

Insecticidal activity of indole derivatives against Plutella xylostella and selectivity to four. . .
P. rejecta suggests that the synthesized indole derivatives
may have a low negative impact on pollinator insects and
natural enemies under natural conditions. Further studies
should focus on the development of viable formulations for
practical use in the field.
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Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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