www.nature.

com/scientificreports

OPEN Morphomolecular characterization

of invasive fruit borer infesting
apple
Danishta Aziz 1, Ishtiyaq Ahad 1, Zahoor Ahmad Shah 2*, Zakir Hussain Khan 1,
Eajaz Ahmad Dar 3*, Bashir Ahmad Alie 2, Aabid Hussain Lone 4, Mumtaz Ahmad Ganie 5,
Lamya Ahmed Alkeridis 6, Laila A. Al‑Shuraym 6, Samy Sayed 7,8 & Mustafa Shukry 9*
Apple, a vital fruit crop worldwide and a major crop grown in northern parts of India, acts as a
backbone for the survival and livelihood of the farming community. However, it is prone to severe
damage from insect pests and diseases. In the past few years, due to erratic weather patterns, there
has been an alarmingly increased infestation of different insect pests, both invasive and non-invasive,
resulting in substantial economic losses to this industry. One similar case was seen in the Himalayan
region of India, where the apple crop in Karewas was heavily damaged by some invasive fruit borer,
feeding on pulp and making galleries to reach the seed locules, thereby destroying the seed material.
To ascertain the true identity of this pest, a study based on morpho-molecular characterization of this
pest was carried out in the Faculty of Agriculture, Wadura, SKUAST Kashmir, India, during the years
2021 and 2022. The invasive fruit samples were collected from apple orchards at different locations
(experimental sites) in North India by installing delta sticky traps @ 5 traps/ha for moth collection.
The fruit-boring larvae and pupa were also collected and reared in confined chambers of the laboratory
with controlled temperature and humidity. All the laboratory investigations were conducted at the
Division of Entomology, FoA, Wadura, SKUAST Kashmir. During the investigation, it was confirmed
that the invasive borer is a codling moth (Cydia pomonella L.), a member of the family Tortricidae,
order Lepidoptera, which was earlier having quarantine importance in India, as it was only present
in the Ladakh region of India. From the phylogenetic analysis of sample sequences, the species of
codling moth present at all experimental sites was more identical to the codling moth sequence
from Leh (the northernmost arid region), India. Further, the study of life cycle and voltinism revealed
that codling moth completes three generations per year in Kashmir, with a single cycle lasting up
to 2.5 months. However, the timing of various generations varies, depending on prevailing weather
conditions at specific locations and times. The number of generations completed by codling moth in
Kashmir conditions during apple growing season was three full and a half overwintering generations.
The study of the life cycle of this important pest was necessary to know the weak link for its effective
management and to prevent the economic loss in apple fruit.

Keywords Apple, Invasive fruit borer, Kashmir, India, Morphomolecular characterization, Life cycle

1
Division of Entomology, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir,
Shalimar, Srinagar 190025, India. 2Advanced Research Station for Saffron and Seed Spices, Sher-e-Kashmir
University of Agricultural Sciences and Technology of Kashmir, Shalimar, Srinagar 190025, India. 3Krishi Vigyan
Kendra Ganderbal, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, Shalimar,
Srinagar 190025, India. 4Mountain Research Centre for Field Crops, Khudwani, Sher-e-Kashmir University of
Agricultural Sciences and Technology of Kashmir, Shalimar, Srinagar 190025, India. 5Faculty of Agriculture,
Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, Shalimar, Srinagar 190025,
India. 6Department of Biology, College of Science, Princess Nourah Bint Abdulrahman University, P.O. Box 84428,
11671 Riyadh, Saudi Arabia. 7Department of Economic Entomology and Pesticides, Faculty of Agriculture, Cairo
University, Giza 12613, Egypt. 8Department of Science and Technology, University College‑Ranyah, Taif University,
P.O. Box 11099, 21944 Taif, Saudi Arabia. 9Department of Physiology, Faculty of Veterinary Medicine, Kafrelsheikh
University, Kafrelsheikh 33516, Egypt. *email: s.zahoor37@gmail.com; darajaz9@gmail.com; Mostafa.ataa@
vet.kfs.edu.eg

Scientific Reports | (2024) 14:11972 | https://doi.org/10.1038/s41598-024-61585-3 1

Vol.:(0123456789)
 www.nature.com/scientificreports/

Apple (Malus × domestica Borkh.) holds a significant commercial position in global fruit production and ranks
4th in production, following bananas, oranges, and ­grapes1. As a significant temperate fruit, it is cultivated in
developing nations worldwide, including India. The global production of apples has touched the 95.96 million-
ton mark, from around 6.93 million hectares in 2022, bearing a productivity of around 13.85 tonnes h ­ a−12. The
top three countries in apple production, China, the USA, and Poland, respectively, are followed by India, which
had a production of 2.59 million tons of apples and productivity of around 8.09 tonnes h ­ a−1, from an area of
2
0.32 million hectares during 2­ 022 . In India, apple cultivation is predominant in Jammu and Kashmir, Himachal
Pradesh, Uttarakhand, and some parts of Arunachal P ­ radesh3.
Out of the total quantity of apple fruit exported from India, Jammu and Kashmir are securing a top position
in contributing more than half of exports from India to outside countries. The area under apple cultivation in
Jammu and Kashmir is believed to rank as the world’s 2nd largest, making it the 2nd largest producer in Asia
and a leading contributor of ­GDP1. In Jammu and Kashmir, the area under apple cultivation was 1.68 lakh hec-
tares in 2022, with a production of 18.98 lakh tonnes and productivity of around 11.26 tonnes ­ha−14. Wearing a
crown, Kashmir is leading in India’s apple production, as almost 89% of horticulture land is dedicated to apple
cultivation. This dominance is wholly attributed to the favorable climatic conditions, awareness of growers, and
the robust adaptability of apple crops to the local ­environment5. Despite having an environment favorable for
apple cultivation, the productivity of this vital crop is not up to mark, reaching a maximum of only 13 tonnes
­ha−1 compared to potential productivity of up to 40 tonnes ­ha−1 in advanced nations of the w ­ orld6. Most of the
state’s income is derived from the apple industry, supporting the livelihood of nearly 3.5 million individuals,
as approximately 5–6 lakh families are directly or indirectly involved in this sector in the ­state7. It is one of the
most widely consumed nutritious fruits globally, and it is immune to the threats posed by invasive pests, whether
insects or disease-causing pathogens. Across the world, various insect pest species and pathogens have wreaked
havoc on apple orchards, leading directly to substantial economic losses and environmental imbalance. From the
insidious spread of codling moths to the devastating impact of fire blight disease, the trending climatic conditions
have increased unpredictability and expansion in the host range of different p ­ ests8.
Consequently, this situation directly influences the outbreak and colonization of invasive pest species into
places previously non-native to their ­attack9. Fire blight, the first bacterial disease identified in pome fruits, caused
by Erwinia amylovara has now widened its range globally, clearly evident by the considerable damage done to
the apple and pear industry by this pathogen in China in the last few years since 2­ 01610. Despite having unique
agro-climatic conditions suitable for temperate fruit production, the productivity of apples in Kashmir is poor
due to various biotic and abiotic factors. Among these factors, the significant biotic stress-causing group is the
insect pests like San Jose scale, Woolly apple aphid, European red mite, and Apple stem ­borer11,12. A continuous
upsurge in the global trade of agricultural commodities has created a favorable path for the entry of invasive pests
to non-native areas. There is a report of around 20% annual yield loss in apple crops in India due to insect p ­ ests13.
In 2019, the northern belt of Kashmir Valley, commonly known as the apple town of Kashmir, India, wit-
nessed considerable premature fruit fall, particularly in Karewas of Baramulla district. To investigate the cause
of this havoc, it was found that some pests bore the apple fruits and fed them inside the pulp and seed cavities.
Feeding of fruit borers leads to early fruit ripening and, hence, premature fruit drop, resulting in yield reduction.
Besides, the infested or bored fruits were prone to secondary infections by different pathogens like bacteria and
fungi, resulting in fruit rotting and severely damaging the economic yield. The feeding behavior damage pattern
and external morphological appearance of this invasive fruit borer resembled that of the codling moth, a pest of
quarantine importance in I­ ndia14. The external appearance of this borer-mimicking codling moth has ash grey
colored body and a wingspan of 15–22 mm. It can be differentiated from other fruit tree moths by their dark
brown wing tips adorned with glossy, copper-colored patterns, enclosed in golden rings, giving an appearance
like little ­mirrors15. The damage caused by this pest to the apple is the same as that caused byodling moth larvae
tutunnelingnto the core of fruits and coconsuming.
Further, the distribution of the codling moth in various continents across the globe was predicted by differ-
ent models like ­CLIMEX16–18 and ­MaxEnt17,19 by multiple researchers, and it was seen the codling moth has a
worldwide distribution, particularly in apple-growing regions except A ­ ntarctica20. In Asia, the majority of areas
that are favorable for codling moth infestation fall in Central and Eastern ­zone20, particularly, the apple-growing
regions of China, Turkey, Azerbaijan, Japan, North Korea, South Korea, India, Iran, Pakistan, and Kazakhstan,
are most suitable for its d ­ evelopment20. However, regarding India, it was only limited to Ladakh region and had
14
quarantine ­importance . The main aim of conducting this research was to ascertain the true identity of the
invasive fruit borer affecting apples in the Himalayan region of India by characterization of this pest at both
morphological and molecular levels, the first of its kind study under Kashmir conditions regarding this pest, so
that efficient management practices could be devised, by knowing its nature vis-a-vis behavior, to prevent the
enormous economic loss to apple industry and secure the livelihood of the farming community.

Results
Morphometric traits of fruit borer
The morphometric measurements of all the body parts revealed no significant difference in dimensions of
the different body parts of invasive fruit borer samples collected from three locations. Head length and width
ranged between 0.67–0.98 mm and 1.01–1.27 mm, respectively. The length of the antenna ranged from 4.43
to 5.59 mm. Proboscis length ranged between 1.01 and 2.06 mm. The length of labial palpi was in the range of
0.95–1.12 mm. The morphometric dimensions of the head and its attached appendages are given in Table 1.
The data on the length of the thorax, abdomen, forewing, hindwing, and frenulum is presented in Table 2. The
thorax length was 3.00–3.98 mm, however, the abdominal length ranged between 5.44–6.17 mm. The forewing
was somewhat longer in size than the hindwing, with lengths ranging between 7.35–8.31 mm and 6.00–7.00 mm,

Scientific Reports | (2024) 14:11972 | https://doi.org/10.1038/s41598-024-61585-3 2

Vol:.(1234567890)
www.nature.com/scientificreports/

Parameter Location *Mean ± S.E (mm) Range (mm) CD (p ≤ 0.05)

Nadihal 1.180a ± 0.026
Head width Chak Gojri 1.186a ± 0.026 1.01–1.27 0.069
Delina 1.191a ± 0.020
Nadihal 0.828a ± 0.033
Head length Chak Gojri 0.797a ± 0.031 0.67–0.98 0.091
Delina 0.797a ± 0.030
Nadihal 5.013a ± 0.119
Antennal length Chak Gojri 5.004a ± 0.085 4.43–5.59 0.326
Delina 5.075a ± 0.129
Nadihal 1.412a ± 0.079
Proboscis length Chak Gojri 1.421a ± 0.048 1.01–2.06 0.197
Delina 1.300a ± 0.072
Nadihal 1.012a ± 0.012
Labial palpi length Chak Gojri 1.011a ± 0.015 0.95–1.12 0.046
Delina 1.018a ± 0.020

Table 1.  Morphometrics of head, antenna, proboscis, and labial palpi of Cydia pomonella L. *Mean of
10 replications. aMeans bearing the same letter within the same column of the same parameter are not
significantly different at p < 0.01.

Parameter Location *Mean ± S.E (mm) Range (mm) CD (p ≤ 0.05)

Nadihal 3.240a ± 0.098
Thorax length Chak Gojri 3.275a ± 0.095 3.00–3.98 0.261
Delina 3.321a ± 0.075
Nadihal 5.687a ± 0.068
Abdomen length Chak Gojri 5.793a ± 0.074 5.44–6.17 0.215
Delina 5.796a ± 0.080
Nadihal 7.825a ± 0.054
Forewing length Chak Gojri 7.846a ± 0.051 7.35–8.31 0.184
Delina 7.790a ± 0.081
Nadihal 6.584a ± 0.094
Hindwing length Chak Gojri 6.561a ± 0.072 6.00–7.00 0.250
Delina 6.565a ± 0.090
Nadihal 1.295a ± 0.024
Frenulum length Chak Gojri 1.296a ± 0.013 1.19–1.42 0.056
Delina 1.279a ± 0.019

Table 2.  Morphometrics of thorax, abdomen, forewing, hindwing, and frenulum of Cydia pomonella L. *Mean
of 10 replications. aMeans bearing the same letter within the same column of the same parameter are not
significantly different at p < 0.01

respectively. The length of the wing coupling apparatus, frenulum, was in the range of 1.19–1.42 mm. The number
of frenulum present per hindwing varied with sex, with males possessing only a single frenulum; however, more
than one (2–3) per hindwing in the case of females. The morphometric measurements of different leg parts are
shown in Table 3. The most significant part of the leg was the femur, and the smallest was the tarsal claw. The
length of coxa, trochanter, femur, tibia, tarsus, and tarsal claw ranged between 1.03–1.14 mm, 0.19–0.22 mm,
1.39–1.50 mm, 0.60–0.67 mm, 1.33–1.40 mm, and 0.08–0.09 mm, respectively. The morphometric data of male
and female genitalia of invasive fruit borer is presented in Table 4. The main parts of male genitalia (Fig. 6A)
include a pair of valva and aedeagus. In contrast, in the case of female genitalia, the main parts are the corpus
bursa, ductus bursa, signa, papillae anales, apophyses anteriores, and apophyses posteriores. The length of valva
(Fig. 6B) and aedeagus (Fig. 6C,D) was 1.53–1.79 mm and 0.85–0.97 mm, respectively. In the case of female
genitalia (Fig. 6E,F), the main parts include corpus bursae, ductus, papillae anales, apophyses posteriores, and
apophyses anteriores, whose length ranged between 1.23–1.89 mm, 0.39–0.67 mm, 0.50–0.58 mm, 0.45–0.56 mm,
and 0.98–1.06 mm, respectively. This invasive borer had holometabolous development, possessing all 4 life cycle
stages: egg, larva, pupa, and adult. The morphometry of different life stages is given in Table 5. The data revealed
a non-significant difference in measuring different life stages of invasive fruit borer samples collected from the
selected locations. The length of the adult, pupa, and larva and the diameter of the egg were found in the range
of 9.93–11.05 mm, 9.00–9.98 mm, 10.89–12.94 mm, and 0.89–1.19 mm, respectively.

Scientific Reports | (2024) 14:11972 | https://doi.org/10.1038/s41598-024-61585-3 3

Vol.:(0123456789)
 www.nature.com/scientificreports/

Parameter Location *Mean ± S.E (mm) Range (mm) CD (p ≤ 0.05)

Nadihal 1.088a ± 0.007
Coxa Chak Gojri 1.085a ± 0.008 1.0–1.14 0.024
Delina 1.091a ± 0.010
Nadihal 0.205a ± 0.003
Trochanter Chak Gojri 0.201a ± 0.004 0.19–0.22 0.014
Delina 0.202a ± 0.007
Nadihal 1.433a ± 0.006
Femur Chak Gojri 1.432a ± 0.010 1.39–1.50 0.022
Delina 1.435a ± 0.007
Nadihal 0.639a ± 0.008
Tibia Chak Gojri 0.632a ± 0.008 0.60–0.67 0.021
Delina 0.637a ± 0.006
Nadihal 1.377a ± 0.005
Tarsus Chak Gojri 1.373a ± 0.006 1.33–1.40 0.015
Delina 1.377a ± 0.005
Nadihal 0.085a ± 0.002
Tarsal claw Chak Gojri 0.084a ± 0.002 0.08–0.09 0.005
Delina 0.084a ± 0.001

Table 3.  Morphometrics of the leg of Cydia pomonella L. *Mean of 10 replications. aMeans bearing the same
letter within the same column of the same parameter are not significantly different at p < 0.01

Parameter Part Location *Mean ± S.E (mm) Range (mm) CD (p < 0.05)
Nadihal 1.654a ± 0.022
Valva Chak Gojri 1.605a ± 0.022 1.53–1.79 0.064
Delina 1.667a ± 0.022
Male genitalia
Nadihal 0.888a ± 0.008
Aedeagus Chak Gojri 0.891a ± 0.010 0.85–0.97 0.027
Delina 0.897a ± 0.009
Nadihal 1.650a ± 0.067
Corpus bursae Chak Gojri 1.621a ± 0.047 1.23–1.89 0.176
Delina 1.659a ± 0.065
Nadihal 0.480a ± 0.026
Ductus bursae Chak Gojri 0.513a ± 0.028 0.39–0.67 0.076
Delina 0.497a ± 0.025
Nadihal 0.530a ± 0.007
Female genitalia Papillae anales Chak Gojri 0.529a ± 0.006 0.50–0.58 0.019
Delina 0.539a ± 0.007
Nadihal 0.501a ± 0.004
Apophyses anteriores Chak Gojri 0.506a ± 0.010 0.45–0.56 0.019
Delina 0.509a ± 0.004
Nadihal 1.013a ± 0.006
Apophyses posteriores Chak Gojri 1.016a ± 0.004 0.98–1.06 0.015
Delina 1.022a ± 0.005

Table 4.  Morphometrics of male and female genitalia of Cydia pomonella L. *Mean of 10 replications. aMeans
bearing the same letter within the same column of the same part of parameter are not significantly different at
p < 0.01.

Molecular characterization
After running the BLASTn tool of NCBI, it was found that the query sequences of the current study (OQ186859,
OQ190153, and OQ187770) showed maximum resemblance of 99.85 percent with two gene sequences,
MW270024 and OQ836351 from Leh, India and Netherlands, respectively, belonging to codling moth species
(Cydia pomonella L.). The results revealed that our query sequences had less intraspecific genetic distance of
0.0015 percent with sequence MW270024 from Leh, India, followed by OQ836351 from the Netherlands and
GU095782 from Canada (0.0016% each) (Table 6). It was evident from the phylogenetic tree (Fig. 1) that the
sequences separated to form two main clusters, and all three query sequences got their placement in the same

Scientific Reports | (2024) 14:11972 | https://doi.org/10.1038/s41598-024-61585-3 4

Vol:.(1234567890)
www.nature.com/scientificreports/

Parameter Location a
Mean ± S.E (mm) Range (mm) CD (p ≤ 0.05)
Nadihal 10.118a ± 0.101
Adult length Chak Gojri 10.176a ± 0.101 9.93–11.05 0.306
Delina 10.224a ± 0.114
Nadihal 9.514a ± 0.083
Pupal length Chak Gojri 9.641a ± 0.074 9.00–9.98 0.228
Delina 9.713a ± 0.078
Nadihal 11.709a ± 0.206
Larval length Chak Gojri 11.989a ± 0.176 10.89–12.94 0.561
Delina 12.141a ± 0.196
Nadihal 1.005a ± 0.023
Egg diameter Chak Gojri 1.035a ± 0.017 0.89–1.19 0.633
Delina 1.061a ± 0.025

Table 5.  Morphometrics of different stages in the life cycle of Cydia pomonella L. *Mean of 10 replications.
a
Means bearing the same letter within the same column of the same parameter are not significantly different at
P < 0.01.

S. no. Place Accession no Intra-specific genetic distance (%) Authors

1 India MW270024 0.0015 Shashank28
2 India MW269987 0.0031 Shashank52
3 India MK759947 0.0031 Shashank et al.53
4 India MW269994 0.0031 Shashank54
5 India MW270030 0.0031 Shashank55
6 Netherlands OQ836351 0.0016 Vossenberg et al.29
7 Canada GU095782 0.0016 Hebert et al.56

Table 6.  COX1 sequences retrieved from GenBank used for building phylogenetic tree (neighbor-joining) and
intraspecific genetic distance Kimura Two Parameter (K2P) analysis.

cluster along with sequence MW270024, which shows the invasive borer samples from three locations Nadi-
hal, Chak Gojri and Delina belong to single species only and identical with the species of codling moth (Cydia
pomonella L.), from Leh, India.
Thus, morphometry, as well as molecular characterization, revealed that invasive fruit borer infesting apples
in karewas of North Kashmir is a codling moth (Cydia pomonella L.) belonging to the family Tortricidae of
order Lepidoptera.

Taxonomic description
The critical characteristics observed during our study revealed that the body is covered by dusky grey scales,
which sometimes appear as small lenses. Two main mouth parts are tusk-shaped labial palpi and unscaled deli-
cate coiled proboscis. A many-segmented long antenna has one row of scales on each segment. Males have an
evident fascicle of long dark hairs in the cubital vein (Cu). The number of frenulum present per hindwing varies
with sex, with males having only one and females having more than one (2–3). The male genitalia has a pair of
valva, bearing straight costa with a round venter. There is a notch in the neck with a ventral spine on each valva,
and the apical part of the valva is covered in dense hairs. Aedaegus has 7 spines-like structures in two groups:
2 at the center and 5 at the apex. Female genitalia contain round corpus bursae, with tiny thorn-like structures
called signa, short ductus bursae, papillae anales, and apophyses anteriores longer than apophyses posteriores.

Feeding behavior
The staging stage of this pest is a larva, which enters the fruit usually through calyx and makes small galleries
inside pulp to reach the seed locules and feed entirely on the seed material, particularly the endosperm. After
reaching maturity, the fully grown larva emerges by making small exit holes covered with frass and excreta, which
are then overwinter under loose bark or other suitable places.

The life cycle of Cydia pomonella L

Codling moth, just like other lepidopterans, possess four stages in its life cycle viz., egg, larva, pupa, and adult.
The life cycle of codling moth, C. pomonella L. is shown in Fig. 2, and the duration of different life stages is given
in Table 7. It was evident from laboratory rearing experiments of codling moths that females laid eggs singly on
the fruits, and few eggs were laid on leaves provided inside the rearing cages. The eggs (Fig. 7A) were somewhat
discoid, resembling a convex lens and having a diameter in the 0.89–1.19 mm range. Freshly laid eggs were
translucent and milk white; however, they changed color to reddish brown while approaching hatching, showing

Scientific Reports | (2024) 14:11972 | https://doi.org/10.1038/s41598-024-61585-3 5

Vol.:(0123456789)
 www.nature.com/scientificreports/

Figure 1.  Phylogenetic tree (Neighbor-joining) depicting evolutionary similarities between the query
sequences and sequences retrieved from GenBank.

a black spot indicating the head of a 1st instar larva. The average duration of egg up to hatching was about
8–10 days. The larval stage is the only damaging stage of codling moth. The color variation was quite visible, as
the 1st instar larva, just after hatching, was creamy white with a brown head wider than the thoracic segments,
while as the mature 5th instar larva, after exiting from fruit, was pinkish red or pinkish brown with a blackish
brown head. After hatching the egg, 1st instar larva usually enters the freshly placed apple fruits through the
calyx end. It makes minute galleries by feeding on the pulp to reach their ultimate destination (seed locules). The
larva ultimately feeds on the seed material and comes out by making exit holes covered with frass and excreta
for overwintering. The average length of mature larva (Fig. 7B) was 10.89–12.94 mm, with a mean duration of
21–30 days from hatching to overwintering. After exiting the fruit, the mature 5th instar larva searches for the
overwintering site, for which it enters the already placed small cardboard strips and spins a white cottony cocoon
inside the galleries in cardboard strips.
After some period, the overwintering larva begins to form a brown-colored pupa—its color changes from
light brown to dark brown as it advances towards adult emergence. There was a difference in the length of the
pupa as per its sex; the female pupa was somewhat longer than the male pupa. However, the length of the pupa
(Fig. 7C) was in the range of 9.00–9.98 mm. The pupal stage was about 12–14 days, and after that, the adult came
out by breaking the pupa from the head end. After emerging from the pupa, the adults appeared similar, covered
by dusky grey scales. There were distinct dark lines on the extreme portion of the forewings. The adult females
laid eggs singly on freshly placed fruits and leaves. The average adult length (Fig. 7D) was 9.93–11.05 mm, and
the adult duration was 14–16 days.

Scientific Reports | (2024) 14:11972 | https://doi.org/10.1038/s41598-024-61585-3 6

Vol:.(1234567890)
www.nature.com/scientificreports/

Figure 2.  Life cycle of Cydia pomonella L.

Life stage Duration in days

Egg 8–10
Larva 21–30
Pupa 12–14
Adult 14–16

Table 7.  Duration of different stages in life cycle of Cydia pomonella L.

Number of generations
The codling moth completed three generations in Kashmir throughout the apple growing season. In addition to
three generations, half of the overwintering larva was observed at the experimental site as temperatures dropped
to minus during harsh winter. There was a difference in the onset of first-generation moths and the end of the
last generation for the consecutive study years, and the comparison is shown in Table 8. During 2021, the first
generation of codling moths commenced from the 18th standard meteorological week (SMW) and ended in
the 26th SMW; the second generation began in the 27th SMW and ended in the 34th SMW. However, the third
generation started in the 35th SMW and ended in the 43rd SMW. In contrast, during 2022, the first generation
of codling moth commenced from 16th SMW and ended in 25th SMW, and the second generation commenced
from 26th SMW and ended in 34th SMW; however, the commencement and end of the third generation were in
35th and 42nd SMW, respectively. The perusal of data on the commencement of the first generation and end of
the third generation of the codling moth for 2021 and 2022 revealed an early emergence of the first generation
and early completion of the last generation in 2022 compared to 2021.

Discussion
In the present investigation, the morphometry of all possible body parts of invasive fruit borer was studied.
However, literature was not available on the characters, such as the head and its appendages, thorax, and leg, and
this type of detailed work on morphometry of codling moth was probably done for the first time. Our results for

Generation Generation
start (SMW) end (SMW)
Generation of codling moth 2021 2022 2021 2022
1st 18th 16th 26th 25th
2nd 27th 26th 34th 34th
3rd 35th 35th 43rd 42nd

Table 8.  Variation in the commencement and end of three generations of Cydia pomonella L. during
consecutive study years.

Scientific Reports | (2024) 14:11972 | https://doi.org/10.1038/s41598-024-61585-3 7

Vol.:(0123456789)
 www.nature.com/scientificreports/

wing dimensions were in close conformity with the reports of ­Pinhey21, who measured the length of the forewing
7–8 mm with a striped appearance with the apical half of the wing medium brown. The number of frenulum
per hindwing helped know the sex of invasive fruit borer samples, as males possessed a single frenulum, while
females more than one (2–3), and this was in line with the findings of ­Common22, who also reported a single
unbranched frenulum in male codling moths, while females possessing more than one (2–6). The size of different
life stages of fruit borer was in agreement with that of B ­ onnemaison23, who recorded the size of an egg, larva,
and pupal stages as 1 mm, 19 mm, and 1 cm, respectively.
Similarly, ­MacKay24 reported the length of the larval stage of codling moth within the range of 15–19 mm.
However, ­Brown25 reported the same in the 14–18 mm range. Another report of codling moth females laying
eggs of 1 mm diameter and larva transforming into pupal stage of average length as 9.64 ­mm26. Similarly, one
more study supported our statement that the size of full-grown last instar larvae of codling moth was 11.98
­mm27. DNA barcoding based on mtCOX1 gene sequences (OQ186859, OQ190153, and OQ187770) of invasive
borer samples revealed that species at all three locations were identical with each other and showed maximum
resemblance of 99.85 percent with two sequences ­MW27002428 and ­O Q8363529 belonging to codling moth,
Cydia pomonella L. from Leh, India and Netherlands, respectively. The least intra-specific distance of query
sequences with the sequences mentioned above and a phylogenetic tree made the identity more apparent, as the
former shared the same clade with sequence M ­ W27002428, which shows the invasive borer samples from selected
locations Nadihal, Chak Gojri, and Delina belong to same species, Cydia pomonella L. and further these were
similar to the codling moth from Leh, I­ ndia28. Therefore, it is evident that the pest that created massive damage
in apple orchards of North Kashmir was a codling moth, which previously was of quarantine importance, as it
was restricted only to Ladakh, ­India14.
The codling moth, C. pomonella L. is a pest of almost all pome fruits belonging to the family Tortricidae of
order Lepidoptera. The moth’s body is covered by dusky grey scales, sometimes appearing as small lenses. Our
results closely conform with those of Hussain et al.26, who reported codling moths with greyish brown color
and coppery wings with dark-colored tips. Mouth parts include a pair of tusk-shaped labial palpi and unscaled
fine-coiled proboscis. R­ entel30 also reported that the head bears tusk-shaped labial palpi and smooth proboscis.
A many-segmented long antenna has a row of scales on each segment. Males have an evident fascicle of long
dark hairs in the cubital vein (Cu). Genitalia, an important character to study while identifying a pest, is also
vital here. The description given about genitalia is in agreement with findings of ­Rentel30, who reported the pres-
ence of a pair of valva with round venter and straight costa, densely covered with hairs and an aedeagus bearing
7 spines-like structures in the male genitalia of codling moth, the female genitalia containing corpus bursae
with two signa on surface and ductus b ­ ursae24. The damaging stage of this pest is a larva, which enters the fruit
usually through calyx and makes small galleries inside pulp to reach the seed locules and feed entirely on the
seed material, particularly the endosperm. Hussain et al.26 and Ju et al.31 validate our finding by mentioning the
codling moth as a direct pest whose larvae bore into the fruits, feed on seeds, and create exit holes by pushing
waste material out. At maturity, the fully grown larva emerges by making small exit holes covered with frass and
excreta, which then overwinter under loose bark or other suitable places. Similar results were obtained by Pajač
et al.15, who stated that the pest overwinters as completely grown larvae within a thick, silken cocoon that can
be found under loose scales of bark and in soil or debris around the tree base, where it pupates after that in early
spring. Our results reported four stages in the life of the codling moth, C. pomonella L. viz., egg, larva, pupa, and
adult, with a duration of 8–10, 21–30, 12–14, and 14–16 days, respectively. Similar results were recorded on the
biology of codling moths by Pajač et al.15, who reported the completion of egg, larval, and pupal stages of cod-
ling moths in 5–12 days, four weeks, and 7–30 days, respectively. In the same way, another researcher recorded
the duration of the egg as 6–14 days while that of the pupal stage as 10–20 ­days3. Similarly, adult longevity was
recorded by Kuyulu and ­Genc32 separately in female and male sex on 10% sugar solution diet as 12.20 ± 0.60 days
and 16.70 ± 1.90 days, respectively.
It was seen that codling moth females lay eggs singly on the fruits and sometimes leaves. The eggs were some-
what discoid, resembling a convex lens and having a diameter of 0.89–1.19 mm. Freshly laid eggs were translucent
and milky white. However, there was a change in color to reddish brown while approaching hatching, showing
a black spot that indicates the head of the 1st instar larva. The findings related to the shape and size of the egg
stage were in accord with the findings of B ­ onnemaison23, who found the eggs of codling moth flat and discoid,
having a diameter of 1 mm, white and later on showing a black head with red ring towards hatching. The fecun-
dity and site of oviposition were confirmed by Pajač et al.15, who found that a female lays 30–70 eggs singly on
the upper surface of leaves or fruit surface. Similar findings were recorded by other researchers too at different
times and ­places3,33–37. Our study revealed that the larval stage is the only damaging stage in the codling moth.
This aligns with the findings of P ­ owell38 and T­ imm39, who reported the larval stage of most tortricids, including
codling moth, as a major threat to the fruit industry. The color variation was quite visible, as the 1st instar larva,
just after hatching, was creamy white with a brown head wider than the thoracic segments.
In contrast, after exiting from fruit, the mature 5th instar larva was pinkish red or pinkish brown with a
blackish brown head. The average length of mature larvae was in the range of 10.89–12.94 mm. Our results are in
­ onnemaison23, who mentioned the color of larva as creamy white with a black head
parallel with the findings of B
initially, which eventually turns to pink towards maturity, and the size of a full-grown larva as around 19 mm
long. Similarly, Hussain et al.3 reported that the 5th instar larva of codling moth is pink and has a brownish head.
The feeding behavior observed in this pest was in agreement with Kuhrt et al.40, who reported that codling moth
larvae feed on the pulp of fruit by making large cavities at the pericarp. The results of Kuyulu and ­Genc32 support
our findings regarding the overwintering of codling moths in the late larval stage and the difference in the length
of pupa concerning sex; female pupa was somewhat longer than male pupa, with an average size of 9.62 mm. At
the same time, results regarding adult description are also supported by Kuyulu and G ­ enc32.

Scientific Reports | (2024) 14:11972 | https://doi.org/10.1038/s41598-024-61585-3 8

Vol:.(1234567890)
www.nature.com/scientificreports/

The number of generations completed by codling moth in Kashmir during apple growing season was three full
and a half overwintering generations. The data is supported by findings of ­Borden33, ­Jackson34 and ­Geier35, who
quoted in their study that codling moth can complete 1–3 generations annually depending on the environmental
conditions and longevity of the growing season in a particular area. Similarly, the findings of ­Chapman41 and
­Audemard42 agree with our results. They have mentioned that codling moth can complete only single generation
in colder areas while 4–5 generations in warm and hot weather conditions. However, the time of onset and end
of the first and last generation for both study years was different. This could be because weather variables directly
influence pest population dynamics, and in certain areas, the growing season is of short duration, while in other
areas, it is quite long. In 2022, the spring months, particularly March, had more hot weather than 2021, which
caused the early emergence of codling moths in 2022 than 2021. Also, there was early ripening and harvesting of
apples during 2022 due to an overall rise in temperature for the entire growing season compared to 2021, which
caused early completion of the life cycle of the last generation moth. All these factors tremendously impact the
activity and emergence of different pests, including codling moths. Hussain et al.14 got the first trap catch of adult
males of codling moth on 21st May while working in Ladakh, India, which is having a short growing season due
to harsh cold climatic conditions. However, in apple orchards of Northern Kyrgyzstan, having hot and humid
weather conditions, Tair and L ­ event43 got their first male trap catch of codling moth in April.

Conclusions
The morphomolecular characterization of invasive fruit borer in apples under Kashmir conditions revealed that
the pest responsible for premature fruit drop and huge yield loss was the codling moth (Cydia pomonella L.),
belonging to the family Tortricidae of order Lepidoptera. The possible route of its first invasion in Kashmir was
from Ladakh, as the phylogenetic analysis of gene sequences (OQ186859, OQ190153, and OQ187770) of this
pest from all three experimental locations resembled the codling moth sequence from Leh Ladakh (MW270024).
Further, the life cycle study revealed that it completes three full and a half overwintering generations under
Kashmir climatic conditions with a duration of the single cycle as 2.5 months. The life cycle study was essential
to know the weak link of this pest for its effective management and to prevent economic loss in Apple.

Materials and methods

Collection of borer sample
The invasive fruit borer samples (adult moths, pupae, and larvae) were collected from apple orchards at three
locations in North Kashmir, India viz., Nadihal (Latitude 34°15′34″N; Longitude 74°21′48″E and 1678 m amsl),
Delina (Latitude 34°14′26″N; Longitude 74°24′58″E and 1606 m amsl)and Chak Gojri (Latitude 34°16′47″N;
Longitude 74°22′42″E and 1618 m amsl), (Fig. 3A). At the experimental site, delta sticky traps (Fig. 3B) procured
from the Division of Entomology, Faculty of Horticulture, Shalimar, SKUAST Kashmir were installed @ 5 traps/
ha for the collection of moths (Fig. 3C). These sticky traps were equipped with a lure containing female phero-
mone to entrap male moths. These delta traps were kept intact at its primary place unless damaged; however, the
sticky white liners were replaced fortnightly with fresh ones if needed, to ensure the effective trapping of male
moths. During the whole study period, 42 traps and 118 liners were used. Simultaneously, fruit boring larvae
and pupae were collected from experimental sites for rearing in a laboratory. All the laboratory investigations
were carried out at the Division of Entomology, FoA Wadura (Latitude 34°20′54″N; Longitude 74°24′02″E and
1606 m amsl), SKUAST Kashmir, between 2021 and 2022.The rearing procedures followed were based on the
study done by Kuyulu and G ­ enc32, and specific climatic conditions were maintained under confined chambers
in the laboratory, with temperature 25 ± 1 °C, relative humidity 60 ± 5%, and 16:8 (light: dark) hour photoperiod.

Microscopy and morphometry of samples

The specimens of invasive fruit borer moths collected from experimental sites, as well as those reared in the
laboratory as per the procedures followed by Dickson et al.44, Geier and ­Briese45, Vetter et al.46 and Kuyulu and
­Genc32, were prepared for microscopy. The live moths were placed in killing jars and anesthetized with ethyl
acetate. Wings and legs were detached first, followed by the dissection of the general body using fine needles,
forceps, and small sharp blades. The male and female genitalia were drawn out from the abdomen very care-
fully with the help of fine brushes. Male genitalia were obtained without any abdominal part attached to it.
However, female genitalia were drawn out along with the 7th sternite, as it is often fused with the sterigma. The
dissected body parts of adult moths were then prepared for microscopy following H ­ orak47, ­Robinson48, ­Park49,
50 51
­Common , Zimmerman and ­Klots procedures. After keeping the dissected parts overnight in 10 (%) KOH
solution (Fig. 3D) for relaxation of the musculature, the specimens were thoroughly washed with distilled water
2–3 times and then immersed in 70 (%) ethanol (Fig. 3E) for about 1 h. This was followed by the overnight
placement of specimens in a clearing agent (xylene and phenol in a 3:1 ratio) (Fig. 3F). The scales were removed
with the help of a fine camel hair brush. Some body parts, completely transparent and hard to describe under
such conditions, were stained by a dye prepared by mixing 1gm of acid fuchsin in 100 ml of 50% ethanol with
5 ml glacial acetic acid. After cleaning, dehydrating, and flattening of genitalia and other body parts, they were
placed and mounted on clean, dry standard microscopic slides in Canada Balsam and covered with a coverslip
for microscopy and morphometry. A stereo zoom microscope with Microview software calibrated with a fixed
known ruler, the stage micrometer, was used to measure minute structures. However, other body parts of large
size, like the abdomen, thorax, and wings, were measured with the help of a normal measuring scale. All the dis-
sected parts were photographed directly using the same stereo zoom microscope, attached with a digital camera
for authentic pest identification.

Scientific Reports | (2024) 14:11972 | https://doi.org/10.1038/s41598-024-61585-3 9

Vol.:(0123456789)
 www.nature.com/scientificreports/

Figure 3.  (A) Satellite map of experimental sites, (B) installation of delta traps, (C) trap catch of adult moths
and (D–F) sample preparation for microscopy.

Morphometric traits of fruit borer studied

The general body parts analyzed for morphometry include the head, thorax, abdomen, appendages, and male
and female genitalia. The length and width of the head (Fig. 4A) were measured from the anterior-most of the
body to the origin of mouth appendages and from one compound eye to the other, respectively. The antennal
length (Fig. 4B) was measured right from the scape to the end of the flagellum. As the fruit borer had completely
coiled proboscis (Fig. 4C), it was somehow unwinded and flattened to measure its length. Measurements of two
important mouth parts, labial palpi and reduced maxillary palpi (Fig. 4D), were also taken. The dimension of the
thorax (Fig. 5A) was taken right from the detachment point of the head to the beginning of the first abdominal
segment. In contrast, the abdominal length (Fig. 5B) was calculated from the end of the thorax to the posterior
end of the moth body.
The length of the forewing (Fig. 5C) and hindwing (Fig. 5D) was calculated from the humeral angle to the
apex. The size of the frenulum and the number of frenuli (Fig. 5E) present per wing concerning sex were also

Scientific Reports | (2024) 14:11972 | https://doi.org/10.1038/s41598-024-61585-3 10

Vol:.(1234567890)
www.nature.com/scientificreports/

Figure 4.  Morphometry of (A) head, (B) antenna, (C) proboscis and (D) labial palpi.

measured. The length of the leg (Fig. 5F) was taken by analyzing different parts viz., coxa, trochanter, femur, tibia,
tarsus, and tarsal claw. Various parts of male and female genitalia (Fig. 6) after processing were measured with
the help of microview software and a stage micrometer. In addition, all life cycle stages were measured (Fig. 7).

Life cycle assessment

For the study of the life cycle and number of generations completed per year by codling moth, fruits of the Red
Delicious apple variety infested with fruit borer (Fig. 8A) were collected from the experimental site (Nadihal) and
brought to the laboratory of Division of Entomology, Faculty of Agriculture, Wadura, SKUAST-K. In addition,
the overwintering stages and pupa were also collected and brought to the laboratory to create the initial culture
(Fig. 8B) of the invasive fruit borer. The infested fruits were placed in plastic/ glass containers along with pieces
of corrugated cardboard strips as pupation sites (Fig. 8C), and the open end of the container was covered with
muslin cloth tied with the help of rubber bands. These containers were monitored daily in the laboratory for
various developmental stages of the pest. Adults emerging from their respective pupae (Fig. 8D) were transferred
to other containers inside the rearing cages and fed with a 10% honey/sugar solution (Fig. 8E), facilitated with
fresh fruits and twigs for oviposition. Eggs laid by female moths (Fig. 8F) were observed daily until the emer-
gence of larva and were then facilitated with fresh apple fruits to penetrate and feed on them. After the entry of
larvae in the fruits through the calyx, infested fruits were observed keenly for the larvae exit, and corrugated
cardboard strips were placed in the same container to facilitate pupation. Pupae and corrugated cardboard strips
were placed in separate containers and keenly supervised until the adult emergence. After this, adults were again
given a 10 percent honey/sugar solution as food soaked on cotton balls. The whole rearing process is shown in
Fig. 8. The time taken for transforming one stage into another developmental stage was noted, and the time taken
to complete the life cycle by fruit borer and the number of generations completed were calculated accordingly.

Statistical analysis
The data obtained was subjected to standard statistical analysis using R software, and significant results of dif-
ferent body parts among the three experimental locations were compared based on critical differences. The data

Scientific Reports | (2024) 14:11972 | https://doi.org/10.1038/s41598-024-61585-3 11

Vol.:(0123456789)
 www.nature.com/scientificreports/

Figure 5.  Morphometry of (A) thorax, (B) abdomen, (C) forewing, (D) hindwing, (E) frenulum and (F) leg.

on morphometry of various body parts of codling moth was expressed as mean ± SE values, and the range was
also calculated for morphometric data across all the locations.

Molecular characterization
The samples of invasive fruit borer were sent to ICAR-National Bureau of Agricultural Insect Resources, Heb-
bal, Bengaluru, where DNA barcoding/molecular characterization and taxonomic description were carried out
in the Division of Genomics. An outsourced agency, Eurofin Genomics, did the sequencing of PCR products.
After obtaining the mitochondrial COX1 sequences of samples of invasive fruit borer from all three locations,
the sequences were submitted to the National Centre for Biotechnology Information (NCBI), and with the help
of the BLASTn tool, further identification of invasive fruit borer was made. A phylogenetic tree was constructed
to evaluate evolutionary similarities, and intra-specific distance was also calculated in MEGA 11 software, bus-
ing the barcodes of all three sample sequences. The accession numbers are given in Table 9. The intra-specific
distance between three query sequences and seven sequences from GenBank was calculated.

Scientific Reports | (2024) 14:11972 | https://doi.org/10.1038/s41598-024-61585-3 12

Vol:.(1234567890)
www.nature.com/scientificreports/

Figure 6.  Morphometry of (A) male genitalia, (B) vulva, (C,D) Aedegus, (E,F) female genitalia.

Scientific Reports | (2024) 14:11972 | https://doi.org/10.1038/s41598-024-61585-3 13

Vol.:(0123456789)
 www.nature.com/scientificreports/

Figure 7.  Morphometry of (A) egg, (B) larva, (C) pupa and (D) adult.

Scientific Reports | (2024) 14:11972 | https://doi.org/10.1038/s41598-024-61585-3 14

Vol:.(1234567890)
www.nature.com/scientificreports/

Figure 8.  Laboratory rearing of codling moth (Cydia pomonella L.), (A) collection of infested fruits, (B) rearing
of larva in protected cages, (C) placing cardboard strips as overwintering sites, (D) adult emergence, (E) feeding
adults on 10% honey solution and (F) egg laying on fruits.

Scientific Reports | (2024) 14:11972 | https://doi.org/10.1038/s41598-024-61585-3 15

Vol.:(0123456789)
 www.nature.com/scientificreports/

Name of the organism Accession number Barcode

Cydia pomonella L., A-1 (sample from Delina) OQ187​770

Cydia pomonella L., A-2 (sample from Chak Gojri) OQ190​153

Cydia pomonella L., A-3 (sample from Nadihal) OQ186​859

Table 9.  Accession numbers and barcode details of invasive fruit borer samples sent to NBAIR for
identification.

Data availability
The datasets used and/or analysed during the current study available from the corresponding author on reason-
able request.

Received: 20 March 2024; Accepted: 7 May 2024

References
1. Shah, Z. A., Dar, M. A., Maqbool, S., Matoo, J. M. & Shah, U. U. F. Media exposure of apple growers about recommended apple
production technology. Ind. J. Ext. Edu. 56(3), 48–53 (2020).
2. FAOSTAT. Food and Agriculture Organization of the United Nations. https://​www.​fao.​org/​faost​at/​en/#​data/​QCL (Accessed 29
Feb 2024).
3. Hussain, B., Buhroo, A. A., War, A. R. & Sherwani, A. Insect pest complex and integrated pest management on apple in Jammu
and Kashmir state, India. In Apple Production and Value Chain Analysis, 261–278. http://​oar.​icris​at.​org/​id/​eprint/​10770 (2018).
4. Directorate of Horticulture. Area and production data of apple. https://​horti​kashm​ir.​gov.​in/​Area%​20Pro​ducti​on%​20data.​html
(Accessed 29 Feb 2024).
5. Ahmad, R., Hussain, B. & Ahmad, T. Fresh and dry fruit production in himalayan Kashmir, sub-Himalayan Jammu and trans-
Himalayan Ladakh, India. Heliyon https://​doi.​org/​10.​1016/j.​heliy​on.​2020.​e05835 (2021).
6. Wani, S. A. et al. Potential of apple cultivation in doubling farmer’s income through technological and market interventions: An
empirical study in Jammu & Kashmir. Ind. J. Agric. Econ. 76(2), 278–291 (2021).
7. Brown, S. Apple, 329–367 https://​doi.​org/​10.​1007/​978-1-​4419-​0763-9_​10 (Springer US, 2012).
8. Andrew, N. R. & Hill, S. J. Effect of climate change on insect pest management. Environ. Pest Manag. Challenges Agron. Ecol. Econ.
Policymakers. https://​doi.​org/​10.​1002/​97811​19255​574.​ch9 (2017).
9. Skendžić, S., Zovko, M., Pajač Živković, I., Lešić, V. & Lemić, D. Effect of climate change on introduced and native agricultural
invasive insect pests in Europe. Insects. 12(11), 985. https://​doi.​org/​10.​3390/​insec​ts121​10985 (2021).
10. Sun, W. et al. Current situation of fire blight in China. Phytopathol. 113(12), 2143–2151. https://​doi.​org/​10.​1094/​PHYTO-​05-​23-​
0170-​RVW (2023).
11. Sharma, J. N. & Verma, K. D. Integrated management of foliar and fruit diseases of apple in Himachal Pradesh. J. Mycol. Plant.
Pathol. 35(1), 48–52 (2005).
12. Sherwani, A., Mukhtar, M. & Wani, A. A. Insect pests of apple and their management. In Insect Pest Management of Fruit Crops,
295–306. https://​www.​resea​rchga​te.​net/​publi​cation/​29005​3683 (Biotech Books, 2016).
13. Khan, A. A., Wani, A. R., Zaki, F. A., Nehru, R. K. & Pathania, S. S. Pests of apple. Pests Manag. https://​doi.​org/​10.​1007/​978-​981-​
10-​8687-8_​14 (2018).
14. Hussain, B., Ahmad, B. & Bilal, S. Monitoring and mass trapping of the codling moth, Cydia pomonella by the use of pheromone
baited traps in Kargil, Ladakh, India. Int. J. Fruit Sci. 15, 1–9. https://​doi.​org/​10.​1080/​15538​362.​2013.​819207 (2015).
15. Pajač, I., Pejić, I. & Barić, B. Codling moth, Cydia pomonella (Lepidoptera: Tortricidae)–major pest in apple production: An
overview of its biology, resistance, genetic structure and control strategies. Agric. Consp. Sci. 76(2), 87–92 (2011).
16. Svobodová, E. et al. Climate variability and potential distribution of selected pest species in south Moravia and north-east Austria
in the past 200 years—lessons for the future. J. Agric. Sci. 152(2), 225–237. https://​doi.​org/​10.​1017/​S0021​85961​30000​99 (2014).
17. Kumar, S., Neven, L. G., Zhu, H. & Zhang, R. Assessing the global risk of establishment of Cydia pomonella (Lepidoptera: Tortri-
cidae) using CLIMEX and MaxEnt niche models. J. Econ. Entomol. 108(4), 1708–1719. https://​doi.​org/​10.​1093/​jee/​tov166 (2015).
18. Guo, S. et al. Projecting the global potential distribution of Cydia pomonella (Lepidoptera: Tortricidae) under historical and RCP4.
5 Climate scenarios. J. Insect Sci. 21(2), 15. https://​doi.​org/​10.​1093/​jisesa/​ieab0​24 (2021).
19. Zhao, L. et al. Mapping the disjunct distribution of introduced codling moth Cydia pomonella in China. Agric. For. Entomol. 17(2),
214–222. https://​doi.​org/​10.​1111/​afe.​12104 (2015).
20. Jiang, D., Chen, S., Hao, M., Fu, J. & Ding, F. Mapping the potential global codling moth (Cydia pomonella L.) distribution based
on a machine learning method. Sci. Rep. 8(1), 13093. https://​doi.​org/​10.​1038/​s41598-​018-​31478-3 (2018).
21. Pinhey, E. C. G. Descriptions and color illustrations of 1183 species. In: Moths of Southern Africa, 37–38. https://​doi.​org/​10.​5555/​
19770​544923 (Tafelberg Publishers, 1975).
22. Common, I. F. B. Tortricoidea. In Moths of Australia, 274–281 (Melbourne University Press, 1990).
23. Bonnemaison, L. Animal enemies of cultivated and forest plants. https://b ​ iblio
​ tecad
​ igita​ l.i​ nfor.c​ l/h
​ andle/2​ 0.5​ 00.1​ 2220/2​ 088 (1964).

Scientific Reports | (2024) 14:11972 | https://doi.org/10.1038/s41598-024-61585-3 16

Vol:.(1234567890)
www.nature.com/scientificreports/

24. Mackay, M. R. Larvae of the Nort american Olethreutidae (Lepidoptera). Mem. Entomol. Soc. Canada 91(10), 5–338. https://​doi.​
org/​10.​4039/​entm9​110fv (1959).
25. Brown, R. L. Tortricidae (Tortricoidea). Immature Insects 1, 419–433 (1987).
26. Hussain, B., Ahmad, F., Ahmad, E., Yousuf, W. & Mehdi, M. Role of pheromone application technology for the management of
codling moth in high altitude and cold arid region of Ladakh. In Moths and Caterpillars. (IntechOpen, 2021).
27. Lacey, L. A. & Unruh, T. R. Biological control of codling moth (Cydia pomonella, Lepidoptera: Tortricidae) and its role in integrated
pest management, with emphasis on entomopathogens. Vedalia. 12(1), 33–60 (2005).
28. Shashank, P. R. https://​www.​ncbi.​nlm.​nih.​gov/​nucco​re/​MW270​024.1/ (2020).
29. Vossenberg, B. T. L. H., Braak, N., Timm, A. E., vanNoort, T. H. & Gilligan, T. M. https://​www.​ncbi.​nlm.​nih.​gov/​nucco​re/​OQ836​
351 (2023).
30. Rentel, M. Morphology and taxonomy of tortricid moth pests attacking fruit crops in South Africa. M. Sc. Thesis submitted to
Faculty of Agri-Science, 1–129. http://​hdl.​handle.​net/​10019.1/​7982 (University of Stellenbosch, 2013).
31. Ju, D. et al. Insecticide resistance in the Cydia pomonella (L): Global status, mechanisms, and research directions. Pestic. Biochem.
Phys. 178, 104925. https://​doi.​org/​10.​1016/j.​pestbp.​2021.​104925 (2021).
32. Kuyulu, A. & Genc, H. Biology and laboratory rearing of codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae) on its
natural host “green immature apple” Malus domestica (Borkh) (Rosales: Rosaceae). Turk. J. Agric. Nat. Sci. 6(3), 546–556. https://​
doi.​org/​10.​30910/​turkj​ans.​595382 (2019).
33. Borden, A. D. Some field observations on codling moth behavior. J. Econ. Entomol. 24, 1137–1145 (1931).
34. Jackson, D. M. Codling moth egg distribution on unmanaged apple trees. Ann. Entomol. Soc. Am. 72(3), 361–368. https://​doi.​org/​
10.​1093/​aesa/​72.3.​361 (1979).
35. Geier, P. W. The life history of codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), in the Australian capital territory.
Austral. J. Zool. 11, 323–367. https://​doi.​org/​10.​1071/​ZO963​0323 (1963).
36. Blomefield, T. L., Pringle, K. L. & Sadie, A. Field observations on oviposition of codling moth, Cydia pomonella (Linnaeus) (Lepi-
doptera: Olethreutidae), in an unsprayed apple orchard in South Africa. Afr. Entomol. 5, 319–336 (1997).
37. Borchert, D. M., Stinner, R. E., Walgenbach, J. F. & Kennedy, G. G. Oriental fruit moth (Lepidoptera: Tortricidae) phenology and
management with methoxyfenozide in North Carolina apples. J. Econ. Entomol. 93, 1353–1364. https://​doi.​org/​10.​1093/​jee/​97.4.​
1353 (2004).
38. Powell, J. A. Biological and Taxonomic Studies on Tortricine Moths, with Reference to the Species in California 317 (University of
California Press, 1964).
39. Timm, A. E. Morphological and molecular studies of tortricid moths of economic importance to the South African fruit industry.
Ph.D. thesis submitted to University of Stellenbosch, 1–127. http://​hdl.​handle.​net/​10019.1/​1404 (2005).
40. Kuhrt, U., Samietz, J. & Dorn, S. Thermoregulation behaviour in codling moth larvae. Physiol. Entomol. 30, 54–61. https://​doi.o ​ rg/​
10.​1111/j.​0307-​6962.​2005.​00431.x (2005).
41. Chapman, P. J. Bionomics of the apple-feeding Tortricidae. Annu. Rev. Entomol. 18, 73–96. https://​doi.​org/​10.​1146/​annur​ev.​en.​
18.​010173.​000445 (1973).
42. Audemard, H. Population dynamics of the codling moth. In Tortricid Pests: Their Biology, Natural Enemies and Control. World
Crop Pests, 329–338. (Elsevier, 1991).
43. Tair, E. & Levent, U. Population development and infestation rate of codling moth (Cydia pomonella) (Lepidoptera: Tortricidae)
in apple orchards in northern Kyrgyzstan. Selcuk J. Agric. Food Sci. 35(2), 108–114 (2021).
44. Dickson, R. C., Barnes, M. M. & Turzan, C. L. Continuous rearing of the codling moth. J. Econ. Entomol. 45(1), 66–68 (1952).
45. Geier, P. W. & Briese, D. T. The demographic performance of a laboratory strain of codling moth, Cydia pomonella (Lepidoptera:
Tortricidae). J. Appl. Ecol. 15(3), 679–696. https://​doi.​org/​10.​2307/​24027​68 (1978).
46. Vetter, R. S., Robert, M. E. & Baker, C. T. Mass rearing of the oriental fruit moth (Lepidoptera: Tortricidae). J. Econ. Entomol. 82(6),
1825–1829. https://​doi.​org/​10.​1093/​jee/​82.6.​1825 (1989).
47. Horak, M. Cydia group. In Olethreutine Moths of Australia (Lepidoptera: Tortricidae) (eds Horak, M. & Komai, F.) 406–422 (CSIRO
Publishing, 2006).
48. Robinson, G. S. The preparation of slides of lepidoptera genitalia with special reference to microlepidoptera. Entomol. Gazeteer
27(2), 127–132 (1976).
49. Park, K. T. Gelechiidae of Taiwan: Review of Anarsia, with descriptions of four new species (Lepidoptera: Gelechioidea). Trop.
Lepidoptera 6(1), 55–66. https://​doi.​org/​10.​5555/​19951​115272 (1995).
50. Common. Lepidoptera. In The Insects of Australia. (CSIRO, Entomology, 1970).
51. Zimmerman, E. C. Microlepidoptera II: Gelechioidae. In Insects of Hawaii (ed. Zimmerman, E. C.) 883–1903 (The University Press
of Hawaii, 1978).
52. Shashank, P. R. https://​www.​ncbi.​nlm.​nih.​gov/​nucco​re/​MW269​987 (2021).
53. Shashank, P. R., Twinkle, S., Anand, H. & Narendra, S. https://​www.​ncbi.​nlm.​nih.​gov/​nucco​re/​MK759​947 (2019).
54. Shashank, P. R. https://​www.​ncbi.​nlm.​nih.​gov/​nucco​re/​MW269​994 (2020).
55. Shashank, P. R. https://​www.​ncbi.​nlm.​nih.​gov/​nucco​re/​MW270​030 (2021).
56. Hebert, P. D. N., Dewaard, J. R. & Landry, J. F. https://​www.​ncbi.​nlm.​nih.​gov/​nucco​re/​GU095​782 (2016).

Acknowledgements
This research was funded by Princess Nourah bint Abdulrahman University Researchers Supporting Project
number (PNURSP2024R82), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

Author contributions
D.A, I.A, Z.A.S, E.A.D, B.A.A, A.H.L, supervised, Designed, Performed experiments, revised the manuscript;
M.A.G, Z.H.K, Performed experiments, analyzed data, L.A.A, L.A, wrote and finalized the manuscript. S.S, M.S
Performed experiments. Performed experiments, wrote, interpreted data, and finalized the manuscript.

Competing interests
The authors declare no competing interests.

Additional information
Correspondence and requests for materials should be addressed to Z.A.S., E.A.D. or M.S.
Reprints and permissions information is available at www.nature.com/reprints.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations.

Scientific Reports | (2024) 14:11972 | https://doi.org/10.1038/s41598-024-61585-3 17

Vol.:(0123456789)
 www.nature.com/scientificreports/

Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, 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 were made. The images or other third party material in this
article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the
material. If material is not included in the article’s Creative Commons licence and your intended use is not
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/.

© The Author(s) 2024

Scientific Reports | (2024) 14:11972 | https://doi.org/10.1038/s41598-024-61585-3 18

Vol:.(1234567890)