Complete Mitochondrial Genome of the Eggplant Fruit and Shoot Borer, Leucinodes orbonalis Guenée (Lepidoptera: Crambidae), and Comparison with Other Pyraloid Moths

Abstract

The eggplant fruit and shoot borer (EFSB) (Leucinodes orbonalis Guenée) is a devastating lepidopteran pest of eggplant (Solanum melongena L.) in the Philippines. Management of an insect pest like the EFSB requires an understanding of its biology, evolution, and adaptations. Genomic resources provide a starting point for understanding EFSB biology, as the resources can be used for phylogenetics and population structure studies. To date, genomic resources are scarce for EFSB; thus, this study generated its complete mitochondrial genome (mitogenome). The circular mitogenome is 15,244 bp-long. It contains 37 genes, namely 13 protein-coding, 22 tRNA, and 2 rRNA genes, and has conserved noncoding regions, motifs, and gene syntenies characteristic of lepidopteran mitogenomes. Some protein-coding genes start and end with non-canonical codons. The tRNA genes exhibit a conserved cloverleaf structure, with the exception in trnS1. Partitioned phylogenetic analysis using 72 pyraloids generated highly supported maximum likelihood and Bayesian inference trees revealing expected basal splits between Crambidae and Pyralidae, and Spilomelinae and Pyraustinae. Spilomelinae was recovered to be paraphyletic, with the EFSB robustly placed before the split of Spilomelinae and Pyraustinae. Overall, the EFSB mitogenome resource will be useful for delineations within Spilomelinae and population structure analysis.

Full text

Citation: Despabiladeras, J.B.;
Bautista, M.A.M. Complete
Mitochondrial Genome of the
Eggplant Fruit and Shoot Borer,
Leucinodes orbonalis Guenée
(Lepidoptera: Crambidae), and
Comparison with Other Pyraloid
Moths. Insects 2024,15, 220.
https://doi.org/10.3390/
insects15040220
Academic Editor: Michael
Kristensen
Received: 18 February 2024
Revised: 17 March 2024
Accepted: 18 March 2024
Published: 25 March 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
insects
Article
Complete Mitochondrial Genome of the Eggplant Fruit and
Shoot Borer, Leucinodes orbonalis Guenée (Lepidoptera:
Crambidae), and Comparison with Other Pyraloid Moths
Joshua B. Despabiladeras and Ma. Anita M. Bautista *
Functional Genomics Laboratory, National Institute of Molecular Biology and Biotechnology, College of Science,
University of the Philippines-Diliman, Quezon City 1101, Philippines; jbdespabiladeras@up.edu.ph
*Correspondence: mmbautista20@up.edu.ph
Simple Summary: Eggplant is an important agricultural produce in the Philippines, but consistent
production is hampered by frequent infestations of the eggplant fruit and shoot borer (EFSB). The
management and monitoring of EFSB infestation requires full understanding of its biology, including
its evolution and adaptations. The use of genomic resources provides a starting point for understand-
ing the biology of EFSB. However, these resources are lacking for EFSB. This study aimed to sequence
and characterize the mitochondrial genome (mitogenome) of the EFSB and use the mitogenome
in reconstructing the evolutionary relationships of the EFSB with respect to other members of the
economically important superfamily, Pyraloidea. The annotated mitogenome of the EFSB contains
the typical elements expected for a lepidopteran mitochondrion, such as gene content, gene syntenies,
and nucleotide composition. Phylogenetic analyses using 72 mitogenomes from other moths robustly
placed the EFSB as basal to its supposed subfamily, Spilomelinae, despite recovering the expected
subfamily relationships of earlier works. The mitogenome resource for the EFSB generated here will
add to the scarce genomic resources for the EFSB and be useful for future phylogenetic reconstruction
of Pyraloidea and Spilomelinae.
Abstract: The eggplant fruit and shoot borer (EFSB) (Leucinodes orbonalis Guenée) is a devastating
lepidopteran pest of eggplant (Solanum melongena L.) in the Philippines. Management of an insect
pest like the EFSB requires an understanding of its biology, evolution, and adaptations. Genomic
resources provide a starting point for understanding EFSB biology, as the resources can be used for
phylogenetics and population structure studies. To date, genomic resources are scarce for EFSB; thus,
this study generated its complete mitochondrial genome (mitogenome). The circular mitogenome
is 15,244 bp-long. It contains 37 genes, namely 13 protein-coding, 22 tRNA, and 2 rRNA genes,
and has conserved noncoding regions, motifs, and gene syntenies characteristic of lepidopteran
mitogenomes. Some protein-coding genes start and end with non-canonical codons. The tRNA genes
exhibit a conserved cloverleaf structure, with the exception in trnS1. Partitioned phylogenetic analysis
using 72 pyraloids generated highly supported maximum likelihood and Bayesian inference trees
revealing expected basal splits between Crambidae and Pyralidae, and Spilomelinae and Pyraustinae.
Spilomelinae was recovered to be paraphyletic, with the EFSB robustly placed before the split of
Spilomelinae and Pyraustinae. Overall, the EFSB mitogenome resource will be useful for delineations
within Spilomelinae and population structure analysis.
Keywords: eggplant fruit and shoot borer; whole genome sequencing; mitochondrial genome;
pest genomics
1. Introduction
The mitochondrion is a fundamental, energy-generating organelle responsible for
oxidative phosphorylation [
1
,
2
]. Mitochondria are believed to have originated from the
Insects 2024,15, 220. https://doi.org/10.3390/insects15040220 https://www.mdpi.com/journal/insects

Insects 2024,15, 220 2 of 22
endosymbiosis of an ancestral Archaeal prokaryote with an alphaproteobacterium [
3
]. The
energy-generating capacity of the mitochondria is fundamental to life that almost all extant
eukaryotes contain fully functional or derivative forms of mitochondria [
4
], with the only
exception to date being the flagellated excavates, Monocercomonoides [
5
]. Although most of
the mitochondrial proteins are encoded by the nuclear genome, most mitochondria preserve
a highly reduced genome encoding 13 protein-coding genes important for the electron
transport chain and respiratory function. Mitochondrial genomes also contain a self-
sufficient translation machinery consisting of a complete set of 22 tRNAs and 2 rRNAs [
6
].
Some mitochondrial genes (e.g., cytochrome oxidase I (COI) subunit) are already widely
established for use in phylogenetics [
7
]. Animal mitogenomes have conserved gene content
and maternal inheritance, properties that enable extensive use not only in phylogenetics
but also in population genetics [
8
], diagnostics and species delineation [
9
], and molecular
evolution [
10
,
11
]. Moreover, the use of multi-gene datasets in phylogenetics has additional
advantages such as reducing sampling error, making it the modern standard in inferring
species relationships between organisms [12,13].
The eggplant fruit and shoot borer (EFSB), Leucinodes orbonalis Guenée, is a devastating
crambid pest of the eggplant, Solanum melongena L. The eggplant is one of the top agricul-
tural products of the Philippines, but consistent production is hampered by frequent EFSB
infestations [
14
]. EFSB is a member of the species-rich lepidopteran superfamily Pyraloidea,
which contains at least 15,500 genera [
15
]. The superfamily is ecologically diverse and
economically significant, having numerous major crop pests [
16
]. Within Pyraloidea, the
EFSB is a member of the largest subfamily, Spilomelinae [
17
]. Obtaining reference mi-
togenomes for this superfamily can aid in pest monitoring and management schemes since
mitochondrial genes have been used for population structure determination [
18
], and the
development of possible control strategies such as the Trojan female [19]. Additionally, as
previously mentioned, mitogenomes are useful in establishing evolutionary relationships
between organisms. Reliable phylogenies are an important tool that could help understand,
for instance, how host-plant ranges evolve within the family [
16
]. Systematics on Spi-
lomelinae has been historically difficult due to morphological heterogeneity, but significant
progress has been made with the help of mitochondrial and nuclear markers [
17
,
20
]. The
use of whole mitogenomes could help resolve the systematics of Spilomelinae. However,
as of 13 March 2024, only 23 whole mitochondrial sequences of Spilomelinae are on the
RefSeq database. Thus, adding more mitogenome sequences and representatives could
improve resolution and taxonomic delineation in this economically important subfamily.
The continued development of high-throughput sequencing (HTS) technologies has
increased accessibility for most laboratories, leading to significant developments in phyloge-
netics, population genetics, barcoding, and biodiversity studies. Philippine EFSB research
could also benefit from genomic resources produced by HTS, which can significantly im-
pact the implementation of pest management. Genetic resources (mitochondrial COI) and
morphological traits have been used by Sagarbarria et al. (2018) to study the population
structure of EFSB in the Philippines [
18
]. The authors concluded that a single, widespread
haplotype predominates Philippine EFSB populations. However, the authors are also aware
of the limitations of using COI as the sole genetic marker for population genetic analysis
and have recommended the use of other markers. The analysis of whole mitogenomes is
attractive since they represent a compromise between single markers and whole genome
analysis. The conserved properties of animal mitogenomes, the advantage of using multi-
gene datasets, and the continued ease of acquisition would provide a promising strategy
for species identification and population genetics using whole mitogenomes [
21
]. Whole
mitogenomes were successfully used to determine the population structure of the Chinese
seabass (Lateolabrax maculatus) in China [
22
] and Mansonia mosquitoes in Brazil [
23
]. Here,
we present a report on the fully annotated sequence of the EFSB mitochondrial genome
from short Illumina sequencing reads and phylogenetic reconstruction of Pyraloidea using
whole mitogenomes. The EFSB mitogenome presented here will contribute to the genetic
resources of EFSB and be useful in future population studies of EFSB in the Philippines.

Insects 2024,15, 220 3 of 22
2. Materials and Methods
2.1. Sample Collection and Genomic DNA Extraction
Eggplants obtained from farmers from different parts of Luzon, Philippines (i.e., Tarlac,
Ilocos Norte, Laguna) were individually dissected and probed for EFSB infestations. The
Institute of Plant Breeding (IPB) at the College of Agriculture and Food Science, University
of the Philippines Los Baños, also provided samples. EFSB samples were stored at
−
80
◦
C
until extraction.
Extraction of EFSB genomic DNA was performed using the Qiagen MagAttract HMW
DNA kit (Qiagen Inc., Mckinley West Park, Taguig, Philippines). The protocol described
by the manufacturer with some modifications recommended by Kingan et al. (2019) was
followed: proteinase K and buffer ATL were pre-mixed, and the resulting solution was the
one used for micropestle homogenization; proteinase K digestion was shortened to 2 h; all
mixing steps were performed via gentle flicking; RNase A incubation time was increased
to 5 min; wide-bore tips were used to transfer all solutions containing nucleic acids; and all
centrifugation steps were performed at 4
◦
C [
24
]. Extracts were run on a 0.7% agarose gel
for 45 min at 70 V and visualized with GelRed (Biotium Inc., Fremont, CA, USA) staining.
Extracts were sent to the Philippine Genome Center for Pulse Field Gel Electrophoresis
(PFGE) analysis.
2.2. Library Preparation and Sequencing
Suitable extracts determined by PFGE were chosen as the starting material for Illumina
library preparation using the TruSeq DNA PCR-Free library prep kit (Illumina Inc., Biopolis
Way, Singapore), with the IDT for Illumina TruSeq DNA UD indexes plate (Illumina
Inc., Biopolis Way, Singapore) serving as the index adapters. Libraries were constructed,
and quality checked using Agilent TapeStation (Agilent Technologies Inc., Yishun Ave. 7,
Singapore). Libraries that were determined to have a 550 bp insert size were then sequenced
on an Illumina NovaSeq 6000 system (Illumina, Biopolis Way, Singapore) using a
2×150 bp
run for 300 cycles at the Philippine Genome Center.
2.3. Pre-Processing, Read Filtering, Mitogenome Assembly, and Annotation
Raw sequences were pre-processed using fastp 0.23.2 [
25
] to remove adapter and
polyG sequences and assessed using FastQC 0.11.9 [
26
]. The processed sequences were then
mapped to reference mitochondrial sequences using Bowtie2 2.3.5.1 [
27
]. Mitochondrial
references were obtained from the NCBI organelle genome database and chosen based on
the following criteria: close phylogenetic relationship with EFSB, tagged as a complete
reference by NCBI, and must be reported or part of a published material. Following said
criteria, the following mitochondrial genomes were chosen: the Glyphodes quadrimaculalis
mitogenome, NC_022699.1 [
28
]; Maruca testulalis mitogenome, NC_024283.1 [
29
]; and
Omiodes indicata mitogenome, NC_039177.1 [
30
]. Mapped sequences were then extracted
and used as the starting input for assembly using metaSPAdes 3.15.4 [
31
] packaged within
MitoZ 3.4 [
32
], with k-mer lengths of 21, 33, 55, and 77. Downstream annotation of the
mitochondrial genes was performed using the annotation module of MitoZ. An additional
annotation was performed using MITOS 2.0.8 [
33
] to confirm the annotations performed by
MitoZ, determine the position of the control region, and determine the structures of the
tRNAs and rRNAs. Secondary structures were verified using tRNAscan-SE 2.0.9 [
34
], and
subsequently drawn using the StructureEditor 1.0 packaged within the RNAstructure 6.4
software package [
35
]. The final annotations were also manually checked to determine if
the gene boundaries were consistent. Coverage analysis was performed by mapping the
reads to the assembled mitogenome using bwa 0.7.17-r1188 [
36
] and assessing the coverage
using samtools 1.16 [
37
] and mosdepth 0.3.2 [
38
]. The final mitogenome of the EFSB has
been deposited and is available in GenBank under accession number PP493058.

Insects 2024,15, 220 4 of 22
2.4. Nucleotide Sequence Composition Analysis
The nucleotide composition, codon usage, and relative synonymous codon usage
(RSCU) of the EFSB mitogenome were calculated using Phylosuite 1.2.3 [
39
]. GC skew
analysis using 100 bp windows and a 20 bp step size was performed using SkewIT [
40
].
GC content in 20 bp sliding windows and sequencing depth per position was calculated
within MitoZ. A Circos plot summarizing gene content and organization, GC content, GC
skew, and coverage across all positions was created using Circos v0.69-8 [41].
2.5. Multiple Sequence Alignment and Phylogenetic Analysis
Reference sequences of representative members of Pyraloidea were obtained from
the NCBI with the following search parameters: “Pyraloidea[ORGN] AND (mitochon-
drion[TITL] or mitochondria[TITL]) AND 10,000:20,000[SLEN]”. The downloaded Genbank
files were imported to Phylosuite [
39
] for sequence cleaning and standardization. Only
sequences flagged as “NC” were retained for analysis. The filtered sequences were then
manually checked for ambiguous bases (R, Y, N, etc.), and sequences containing such bases
were removed. A total of 72 working mitogenome sequences from Pyraloidea were retained
after filtering and standardization. The following subfamilies were represented: Spilomeli-
nae, Pyraustinae, Odontinae, Acentropinae, Schoenobiinae, Glaphyriinae, and Crambinae
of Crambidae; and Gallerinae, Phycitinae, Pyralinae, and Epipaschiinae of Pyralidae. The
full list of organisms used for the alignment is shown in Table 1. Each of the 37 genes was
extracted from the reference sequences and aligned separately using MAFFT 7.505 [
42
],
and uninformative sites were removed using Gblocks 0.91b [
43
]. The cleaned alignments
were then concatenated to form a linear sequence of 13,953 bp for each organism using
Phylosuite [
39
]. Partitioned maximum likelihood and Bayesian inference phylogenetic
analyses were performed using IQ-tree 2.1.2 [
44
,
45
] and MrBayes 3.2.7 [
46
]. For maximum
likelihood phylogenetic analysis, each of the 37 genes forms 1 partition initially. Automatic
model selection was performed using ModelFinder in IQ-tree, and gene partitions were
automatically merged to avoid over-parametrization [
47
]. Branch support was assessed
using the SH-aLRT test with 10,000 replicates. For Bayesian inference, Metropolis-Coupled,
Markov chain Monte Carlo (MC
3
) sampling was performed to estimate the tree with the
highest posterior probability. The parallel version of MrBayes [
46
] was used alongside the
BEAGLE library [
48
] to speed up calculations. In brief, ModelFinder was used to find the
best-of-fit models, with the -mset parameter to restrict the search to models utilized by
MrBayes. Partitions were then merged, and the merged partitions and their respective prior
rates, gamma shape parameter, and proportion of invariant sites were then used as inputs
to MrBayes. A total of three independent MCMC runs were performed, with each run
containing 1 cold and 5 hot chains. The MCMC runs were run for 10,000,000 generations,
with sampling every 1000 generations. Convergence was assessed when the effective
sample size (ESS) for the parameters was greater than 200, and the potential scale reduction
factor (PSRF) approached 1. The first 25% of the trees were discarded as burn-in, and the BI
consensus trees were built using a strict consensus rule (Allcompat). The final consensus
trees for both ML and BI were then drawn and annotated using ggtree [
49
]. Topological
concordance was assessed by plotting both trees side by side and comparing the tips.
3. Results and Discussion
3.1. EFSB Mitogenome Structure and Organization
A total of 975,248 sequences were obtained after Bowtie2 mapping, and FastQC
analysis revealed that the sequences are of high quality (Figure S1), with most of the
sequences having a Phred score of 30 or above. The cleaned sequences were then used
for mitogenome assembly using metaSPAdes and subsequent annotation via MitoZ and
MITOS. The sequencing data obtained here yielded a minimum coverage depth value of
2215
×
and a maximum coverage depth value of 19,989
×
, yielding a mean coverage depth
across all positions of 9571
×
. Cumulative coverage distribution obtained using mosdepth
shows that 100% of the EFSB mitogenome has been covered at least 2215 times (
Figure S2
).

Insects 2024,15, 220 5 of 22
The assembled mitochondrial genome has a total length of 15,244 bp and contains the
37 genes and control region expected for lepidopteran mitochondria (Figure 1).
Insects 2024, 15, x FOR PEER REVIEW 5 of 21
A total of 975,248 sequences were obtained after Bowtie2 mapping, and FastQC anal-
ysis revealed that the sequences are of high quality (Figure S1), with most of the sequences
having a Phred score of 30 or above. The cleaned sequences were then used for mitoge-
nome assembly using metaSPAdes and subsequent annotation via MitoZ and MITOS. The
sequencing data obtained here yielded a minimum coverage depth value of 2215× and a
maximum coverage depth value of 19,989×, yielding a mean coverage depth across all
positions of 9571×. Cumulative coverage distribution obtained using mosdepth shows
that 100% of the EFSB mitogenome has been covered at least 2215 times (Figure S2). The
assembled mitochondrial genome has a total length of 15,244 bp and contains the 37 genes
and control region expected for lepidopteran mitochondria (Figure 1).
Figure 1. Circos plot detailing the assembled EFSB mitogenome and various statistics. (A) Gene or-
ganization of the EFSB mitogenome. Yellow, purple, pink, and grey boxes indicate protein-coding
Figure 1. Circos plot detailing the assembled EFSB mitogenome and various statistics. (A) Gene
organization of the EFSB mitogenome. Yellow, purple, pink, and grey boxes indicate protein-coding
genes, tRNA genes, rRNA genes, and the control region, respectively. Boxes oriented inward are
genes transcribed in the majority strand, while the boxes oriented outward are genes transcribed in
the minority strand. (B) The GC content histogram was calculated in 20 bp sliding windows. Black
and red lines correspond to 50% and 25% GC content, respectively. (C) GC-skew was calculated
using a window size of 100 bp and a step size of 20 bp. Black line corresponds to 0 GC-skew value
and histogram bins colored yellow and red have negative and positive GC skew values, respectively.
(D) Coverage distribution across all positions. The EFSB mitogenome has a mean coverage of 9571
×
.
Positions having coverage values above and below the mean coverage value are colored purple and
green, respectively. The highlighted sector in dotted lines indicates the control region of the EFSB
mitogenome characterized by low GC content and a switch in the sign of the GC skew values.

Insects 2024,15, 220 6 of 22
The EFSB mitogenome contains 13 protein-coding genes, 22 tRNA genes, and 2 rRNA
genes. Of the 37 genes, 23 genes (14 tRNAs and 9 PCGs) are found in the majority strand,
while 14 genes (8 tRNAs, 4 PCGs, and 2 rRNAs) are found in the minority strand. The
obtained mitogenome shows the typical circular double-stranded DNA structure and
exhibits the conserved lepidopteran synteny of trnM-trnI-trnQ-nad2 similarly to other
crambid mitogenomes [
50
,
51
]. This synteny is characteristic of lepidopterans and differs
from the ancestral insect mitogenome synteny of trnI-trnQ-trnM-nad2 [
52
]. The EFSB
mitogenome is highly AT-rich, having an AT% of 80.9%. Lepidopterans consistently have
AT-rich mitogenomes with negative GC skew values (Table 1). The EFSB mitogenome
is consistent with this observation, having a slightly negative AT skew of
−
0.01 and a
negative GC-skew of
−
0.17. The EFSB mitogenome contains a control region with high
AT content and is situated between the s-rna and trnM genes (Figure 1A,B). The control
regions are also characterized by the switch in sign in the GC-skew diagrams (Figure 1C).
The control region contains conserved motifs necessary for mitochondrial replication (see
below) [53].
Table 1. Reference mitochondrial genomes chosen for composition comparison and phylogenetic analysis.
Organism RefSeq ID Taxonomy AT% AT-Skew GC% GC-Skew References
Leucinodes
orbonalis THIS STUDY Crambidae,
Spilomelinae 80.9 −0.013 19.1 −0.172 THIS STUDY
Acrobasis
inouei NC_061244.1 Pyralidae,
Phycitinae 80.3 −0.021 19.7 −0.219 [54]
Aedes
albopictus NC_006817.1 Diptera 79.6 0.008 20.5 −0.181 Direct
submission
Aglossa
dimidiata NC_058009.1 Pyralidae,
Pyralinae 79.1 −0.043 20.9 −0.226 Direct
submission
Amyelois
transitella NC_028443.1 Pyralidae,
Phycitinae 79.6 −0.048 20.4 −0.237 Direct
submission
Botyodes
diniasalis NC_073002.1 Crambidae,
Spilomelinae 80.8 −0.021 19.1 −0.182 Direct
submission
Botyodes
principalis NC_061248.1 Crambidae,
Spilomelinae 80.7 −0.014 19.3 −0.189 [54]
Cataclysta
lemnata NC_050323.1 Crambidae,
Acentropinae 79.5 0.004 20.6 −0.214 Direct
submission
Cathayia
obliquella NC_053657.1 Pyralidae,
Gallerinae 80.6 −0.037 19.4 −0.225 [55]
Chilo saccha-
riphagus NC_029716.1 Crambidae,
Crambinae 81 −0.012 19.1 −0.246 Direct
submission
Chilo
suppressalis NC_015612.1 Crambidae,
Crambinae 80.6 0.008 19.3 −0.235 [56]
Cnaphalocrocis
medinalis NC_015985.1 Crambidae,
Pyraustinae 82 −0.015 18 −0.175 [56]
Cnaphalocrocis
patnalis NC_060868.1 Crambidae,
Pyraustinae 81.8 −0.022 18.2 −0.165 Direct
submission
Conogethes
punctiferalis NC_021389.1 Crambidae,
Spilomelinae 80.6 −0.025 19.4 −0.207 [57]
Organism RefSeq ID Taxonomy AT% AT-Skew GC% GC-Skew References
Crambus
perlellus NC_061606.1 Crambidae,
Crambinae 81.3 −0.008 18.7 −0.197 Direct
submission

Insects 2024,15, 220 7 of 22
Table 1. Cont.
Culex
quinquefasciatus
NC_014574.1 Diptera 78 0.007 22 −0.173 [58]
Cydalima
perspectalis NC_042150.1 Crambidae,
Spilomelinae 80.9 −0.016 19.1 −0.193 [59]
Dausara
latiterminalis NC_056799.1 Crambidae,
Odontinae 80.5 −0.003 19.5 −0.201 [60]
Diatraea
saccharalis NC_013274.1 Crambidae,
Crambinae 80.1 0.021 20 −0.258 [61]
Dioryctria
rubella NC_061242.1 Pyralidae,
Phycitinae 79.8 −0.023 20.1 −0.233 [54]
Drosophila
melanogaster NC_024511.2 Diptera 82.2 0.016 17.8 −0.147 Direct
submission
Dusungwua
basinigra NC_061240.1 Pyralidae,
Phycitinae 80 −0.014 20 −0.213 [54]
Elophila
interruptalis NC_021756.1 Crambidae,
Acentropinae 80.3 −0.011 19.7 −0.229 [50]
Elophila
turbata NC_068592.1 Crambidae,
Acentropinae 81.2 −0.005 18.9 −0.225 Direct
submission
Endotricha
consocia NC_037501.1 Pyralidae,
Pyralinae 79.7 −0.039 20.2 −0.226 [62]
Endotricha
kuznetzovi NC_061642.1 Pyralidae,
Pyralinae 80.7 −0.033 19.2 −0.206 Direct
submission
Ephestia
elutella NC_039716.1 Pyralidae,
Phycitinae 80.7 −0.043 19.4 −0.217 [63]
Ephestia
kuehniella NC_022476.1 Pyralidae,
Phycitinae 79.7 −0.049 20.2 −0.234 [64]
Evergestis
extimalis NC_071781.1 Crambidae,
Glaphyriinae 80.7 −0.018 19.3 −0.168 Direct
submission
Evergestis
junctalis NC_030509.1 Crambidae,
Glaphyriinae 81 −0.015 19 −0.168 Direct
submission
Galleria
mellonella NC_028532.1 Pyralidae,
Gallerinae 80.4 −0.039 19.6 −0.237 Direct
submission
Glyphodes
pyloalis NC_025933.1 Crambidae,
Spilomelinae 80.7 −0.016 19.3 −0.194 Direct
submission
Glyphodes
quadrimaculalis
NC_022699.1 Crambidae,
Spilomelinae 80.8 −0.007 19.2 −0.192 [28]
Heortia
vitessoides NC_056800.1 Crambidae,
Odontinae 80.6 −0.012 19.4 −0.172 [60]
Hypsopygia
regina NC_030508.1 Pyralidae,
Pyralinae 78.7 −0.037 21.3 −0.228 Direct
submission
Lamoria
adaptella NC_062173.1 Pyralidae,
Gallerinae 80.1 −0.012 19.9 −0.24 Direct
submission
Lista
haraldusalis NC_024535.1 Pyralidae,
Epipaschiinae 81.5 −0.007 18.5 −0.171 [65]
Loxostege
sticticalis NC_027174.1 Crambidae,
Pyraustinae 80.8 0.002 19.2 −0.191 Direct
submission

Insects 2024,15, 220 8 of 22
Table 1. Cont.
Maruca
testulalis NC_024283.1 Crambidae,
Spilomelinae 80.8 −0.005 19.2 −0.171 [29]
Maruca
vitrata NC_024099.1 Crambidae,
Spilomelinae 80.7 −0.002 19.3 −0.172 Direct
submission
Meroptera
pravella NC_035242.1 Pyralidae,
Phycitinae 80.5 −0.019 19.3 −0.199 [66]
Nagiella
inferior NC_040973.1 Crambidae,
Spilomelinae 81.5 0.009 18.5 −0.22 Direct
submission
Nomophila
noctuella NC_025764.1 Crambidae,
Spilomelinae 81.4 0.002 18.6 −0.176 [67]
Omiodes
indicata NC_039177.1 Crambidae,
Spilomelinae 81.6 −0.012 18.4 −0.162 [30]
Omphisa
fuscidentalis NC_066444.1 Crambidae,
Spilomelinae 79 0.013 21 −0.274 Direct
submission
Orthaga
euadrusalis NC_061246.1 Pyralidae,
Epipaschiinae 80.2 −0.02 19.8 −0.199 [54]
Organism RefSeq ID Taxonomy AT% AT-Skew GC% GC-Skew References
Orthaga
olivacea NC_046504.1 Pyralidae,
Epipaschiinae 79 −0.043 21 −0.215 Direct
submission
Orthopygia
glaucinalis NC_047304.1 Pyralidae,
Pyralinae 79.2 −0.044 20.8 −0.223 [68]
Orybina
regalis NC_061247.1 Pyralidae,
Pyralinae 81 −0.016 19 −0.205 [54]
Ostrinia
furnacalis NC_056248.1 Crambidae,
Pyraustinae 80.9 0.031 19.1 −0.196 [69]
Ostrinia
kasmirica NC_059846.1 Crambidae,
Pyraustinae 81 0.031 19 −0.192 Direct
submission
Ostrinia
nubilalis NC_054270.1 Crambidae,
Pyraustinae 80.5 0.033 19.4 −0.195 [70]
Ostrinia
scapulalis NC_048887.1 Crambidae,
Pyraustinae 81 0.03 19.1 −0.196 [51]
Ostrinia zealis NC_048888.1 Crambidae,
Pyraustinae 80.9 0.031 19.1 −0.193 [51]
Palpita
hypohomalia NC_039632.1 Crambidae,
Spilomelinae 81 −0.001 18.9 −0.196 Direct
submission
Paracymoriza
distinctalis NC_023471.1 Crambidae,
Acentropinae 82.2 −0.002 17.7 −0.155 [71]
Paracymoriza
prodigalis NC_020094.1 Crambidae,
Acentropinae 81.5 0.002 18.4 −0.183 [72]
Paralipsa
gularis NC_054356.1 Pyralidae,
Gallerinae 79.5 −0.014 20.5 −0.239 Direct
submission
Parapediasia
teterrellus NC_068594.1 Crambidae,
Crambinae 80.5 −0.003 19.5 −0.231 Direct
submission
Parapoynx
crisonalis NC_031151.1 Crambinade,
Acentropinae 82 0.017 18 −0.153 Direct
submission

Insects 2024,15, 220 9 of 22
Table 1. Cont.
Perula sp. NC_066226.1 Pyralidae,
Pyralinae 81 −0.037 19 −0.213 Direct
submission
Plodia
interpunctella NC_027961.1 Pyralidae,
Phycitinae 80.1 −0.05 19.9 −0.233 Direct
submission
Polythlipta
liquidalis NC_073109.1 Crambidae,
Spilomelinae 81 −0.005 19 −0.21 Direct
submission
Prophantis
adusta NC_067853.1 Crambidae,
Spilomelinae 81.5 0.001 18.5 −0.196 Direct
submission
Pseudargyria
interruptella NC_029751.1 Crambidae,
Crambinae 79.4 −0.011 20.6 −0.216 Direct
submission
Pseudonoorda
nigropunctalis NC_056801.1 Crambidae,
Odontinae 81 −0.003 19 −0.201 [60]
Pycnarmon
lactiferalis NC_033540.1 Crambidae
Spilomelinae 81.7 −0.004 18.3 −0.173 [73]
Pygospila
tyres NC_066087.1 Crambidae,
Spilomelinae 81.3 −0.008 18.7 −0.158 Direct
submission
Pyrausta
despicata NC_046050.1 Crambidae,
Pyraustinae 80.9 0.009 19 −0.204 Direct
submission
Scirpophaga
incertulas NC_031329.1 Crambidae,
Schoenobiinae 77.2 0.029 22.9 −0.32 Direct
submission
Sinomphisa
plagialis NC_061243.1 Crambidae,
Spilomelinae 80.6 −0.008 19.4 −0.216 [54]
Sitochroa
verticalis NC_062118.1 Crambidae,
Pyraustinae 80.6 0.005 19.5 −0.203 Direct
submission
Syllepte
taiwanalis NC_061245.1 Crambidae,
Pyraustinae 81.7 −0.009 18.3 −0.182 [54]
Tyspanodes
hypsalis NC_025569.1 Crambidae,
Spilomelinae 81.4 −0.017 18.6 −0.175 [74]
Tyspanodes
striata NC_030510.1 Crambidae,
Spilomelinae 81.3 −0.018 18.7 −0.177 Direct
submission
The EFSB mitogenome is highly compact, containing mostly genes and the control
region with few and small intergenic spacers. The longest spacer is the 69 bp region
between trnQ and ND2. The trnQ-ND2 spacer is considered to be a feature of lepidopteran
mitogenomes [
75
] and is thought to have arisen from partial duplication of the ND2
gene [
76
]. Another spacer in the EFSB mitogenome is the one found between trnS2 and nad1.
This spacer is conserved in all insects and contains the motif “ATACTAA” believed to be the
recognition site for the mitochondrial transcription termination (mTERM) protein [
30
,
75
]
(Figure 2A). In addition, overlapping sequences were also found for the EFSB mitogenome.
In particular, the 7 bp “ATGATAA” overlap found at the interface of ATP6 and ATP8 is
another conserved feature of lepidopteran mitogenomes [
30
,
75
] (Figure 2B). For the control
region, four regions are of interest that are also conserved features of lepidopterans. The
“ATTTA” motif followed by a poly-AT stretch of length 12, and the 13 bp poly-T stretch has
also been documented in other lepidopterans [
30
,
75
,
77
]. The “ATAG” box near the 5’end
of the s-rna is believed to be the origin of light strand replication for the mitogenome [
53
].
Interestingly, for other lepidopteran mitogenomes, the “ATAG” box precedes the poly-T
stretch [78,79], but the motif is “TTAG” in EFSB (Figure 2C).

Insects 2024,15, 220 10 of 22
Insects 2024, 15, x FOR PEER REVIEW 9 of 21
[30,75] (Figure 2A). In addition, overlapping sequences were also found for the EFSB mi-
togenome. In particular, the 7 bp “ATGATAA” overlap found at the interface of ATP6 and
ATP 8 is another conserved feature of lepidopteran mitogenomes [30,75] (Figure 2B). For
the control region, four regions are of interest that are also conserved features of lepidop-
terans. The “ATTTA” motif followed by a poly-AT stretch of length 12, and the 13 bp poly-
T stretch has also been documented in other lepidopterans [30,75,77]. The “ATAG” box
near the 5’end of the s-rna is believed to be the origin of light strand replication for the
mitogenome [53]. Interestingly, for other lepidopteran mitogenomes, the “ATAG” box
precedes the poly-T stretch [78,79], but the motif is “TTAG” in EFSB (Figure 2C).
Figure 2. Intergenic spacers, overlapping regions, and conserved motifs in the EFSB mitogenome.
(A) Intergenic spacers found between trnQ-ND2 and trnS2-ND1. Sequences in black underlines are
the spacers (B) Gene overlap regions between ATP8 - AT P6. Sequences highlighted in grey are the
overlapping regions. (C) Control region motifs found in the EFSB mitogenome. Sequences with the
red line indicate conserved motifs, and the arrow direction shows the transcription direction of the
genes, which are colored for reference.
3.2. Protein-Coding Genes
The EFSB mitogenome contains 13 protein-coding genes with a total concatenated
length of 11,191 bp, comprising 73.41% of the total mitogenome length. All the PCGs are
initiated with ATK codons except for nad2 and cox1, which are initiated by TTG codons
(Table 2). TTG codons are established initiation codons in invertebrate mitochondria [80],
with the TTG codons having relatively high frequencies in the nad2 and cox1 genes of in-
vertebrates [81]. With the PCGs having ATK start codons, ATG (7/13) is slightly preferred
over ATT (4/13) codons (Table 2). On the other hand, some of the PCGs of the EFSB mito-
genome have incomplete stop codons. The genes COX1, ND5, and CYTB have non-canon-
ical T stop codons, while ATP6 and ND4 have non-canonical TA (Table 2). Interestingly,
both MITOS and MitoZ have annotated canonical TAA stop codons for all the PCGs in the
EFSB mitogenomes. However, significant overlap of the annotated stop codons with
downstream genes has been observed for some PCGs. Thus, manual checking of the an-
notated PCG gene boundaries was performed to determine if the gene boundaries were
consistent with downstream genes. The presence of incomplete stop codons has been doc-
umented for invertebrate mitogenomes and are processed into full TAA stop codons post
Figure 2. Intergenic spacers, overlapping regions, and conserved motifs in the EFSB mitogenome.
(A) Intergenic spacers found between trnQ-ND2 and trnS2-ND1. Sequences in black underlines are
the spacers (B) Gene overlap regions between ATP8-ATP6. Sequences highlighted in grey are the
overlapping regions. (C) Control region motifs found in the EFSB mitogenome. Sequences with the
red line indicate conserved motifs, and the arrow direction shows the transcription direction of the
genes, which are colored for reference.
3.2. Protein-Coding Genes
The EFSB mitogenome contains 13 protein-coding genes with a total concatenated
length of 11,191 bp, comprising 73.41% of the total mitogenome length. All the PCGs are
initiated with ATK codons except for nad2 and cox1, which are initiated by TTG codons
(Table 2). TTG codons are established initiation codons in invertebrate mitochondria [
80
],
with the TTG codons having relatively high frequencies in the nad2 and cox1 genes of
invertebrates [
81
]. With the PCGs having ATK start codons, ATG (7/13) is slightly pre-
ferred over ATT (4/13) codons (Table 2). On the other hand, some of the PCGs of the
EFSB mitogenome have incomplete stop codons. The genes COX1,ND5, and CYTB have
non-canonical T stop codons, while ATP6 and ND4 have non-canonical TA (Table 2). In-
terestingly, both MITOS and MitoZ have annotated canonical TAA stop codons for all the
PCGs in the EFSB mitogenomes. However, significant overlap of the annotated stop codons
with downstream genes has been observed for some PCGs. Thus, manual checking of
the annotated PCG gene boundaries was performed to determine if the gene boundaries
were consistent with downstream genes. The presence of incomplete stop codons has been
documented for invertebrate mitogenomes and are processed into full TAA stop codons
post transcriptionally [
82
–
84
]. This observation is supported by the analysis of Donath et al.
(2019), where the authors show that invertebrate COX1,ND5,CYTB,ATP6, and ND4 have
significant frequencies of incomplete stop codons [
81
]. After manual checking, the gene
boundaries of the aforementioned genes were adjusted to end in incomplete stop codons.
Total amino acids encoded shows that the EFSB mitochondrion codes for a higher propor-
tion of hydrophobic amino acids, with leucine (L), isoleucine (I), and phenylalanine (F)
representing three of the top four amino acids used (Figure 3B). This is expected since most
of the proteins encoded by mitogenome are components of membrane-bound, hydrophobic
complexes important for energy generation [
85
]. The relative synonymous codon usage is
shown in Figure 3A. RSCU analysis indicates that AT rich codons are preferentially used
compared to GC rich codons. For example, the EFSB mitochondrion prefers to utilize the

Insects 2024,15, 220 11 of 22
codons UUA, AAU, CAA, and AUU to code for the amino acids leucine (Leu), asparagine
(Asn), glutamine (Gln), and isoleucine (Ile), respectively, compared to their counterpart
codons. The high AT content of the EFSB mitogenome (Table 1) may be correlated to the
bias of using AT-rich codons. Overall, the patterns in codon usage are similar to those
observed in other members of Pyraloidea [51,86].
Table 2. Summary of the genes for the Leucinodes orbonalis Guenée mitogenome.
Gene Location Strand Start Codon Stop Codon Length (bp) Anticodon
trnM 1–69 Majority - - 69 CAU
trnI 70–134 Majority - - 65 GAU
trnQ 132–200 Minority - - 69 UUG
ND2 269–1270 Majority TTG TAA 1002 -
trnW 1285–1351 Majority - - 67 UCA
trnC 1344–1409 Minority - - 66 GCA
trnY 1413–1479 Minority - - 67 GUA
COX1 1493–3026 Majority TTG T 1534 -
trnL2 3027–3093 Majority - - 67 UAA
COX2 3094–3774 Majority ATG TAA 681 -
trnK 3779–3849 Majority - - 71 CUU
trnD 3850–3918 Majority - - 69 GUC
ATP8 3919–4086 Majority ATT TAA 168 -
ATP6 4080–4759 Majority ATG TA 680 -
COX3 4760–5548 Majority ATG TAA 789 -
trnG 5551–5616 Majority - - 66 UCC
ND3 5617–5970 Majority ATT TAA 354 -
trnA 5975–6038 Majority - - 64 UGC
trnR 6039–6102 Majority - - 64 UCG
trnN 6102–6168 Majority - - 67 GUU
trnS1 6171–6236 Majority - - 66 GCU
trnE 6240–6306 Majority - - 67 UUC
trnF 6326–6392 Minority - - 67 GAA
ND5 6393–8124 Minority ATT T 1732 -
trnH 8125–8194 Minority - - 70 GUG
ND4 8195–9534 Minority ATG TA 1340 -
ND4L 9544–9834 Minority ATG TAA 291 -
trnT 9840–9905 Majority - - 66 UGU
trnP 9906–9971 Minority - - 66 UGG
ND6 9974–10507 Majority ATT TAA 534 -
CYTB 10511–11657 Majority ATG T 1147 -
trnS2 11658–11726 Majority - - 69 UGA
ND1 11743–12681 Minority ATG TAA 939 -
trnL1 12683–12754 Minority - - 72 UAG

Insects 2024,15, 220 12 of 22
Table 2. Cont.
Gene Location Strand Start Codon Stop Codon Length (bp) Anticodon
l-rna 12764–14121 Minority - - 1358 -
trnV 14110–14174 Minority - - 65 UAC
s-rna 14175–14983 Minority - - 809 -
Insects 2024, 15, x FOR PEER REVIEW 11 of 21
trnS2 11658–11726 Majority - - 69 UGA
ND1 11743–12681 Minority ATG TAA 939 -
trnL1 12683–12754 Minority - - 72 UAG
l-rna 12764–14121 Minority - - 1358 -
trn
V
14110–14174 Minority - - 65 UAC
s-rna 14175–14983 Minority - - 809 -
Figure 3. Relative synonymous codon usage and total amino acids encoded by the EFSB mitoge-
nome. (A) Relative synonymous codon usage (RSCU) is a measure of the deviation of each codon
from the assumption that all codons are used equally. The bar graph shows the relative distribution
of codon usage across all amino acid codons. The number of codons for each amino acid ranges
from two to eight. The RSCU values are color-coded based on the codons below the amino acid
labels. (B) Amino acids encoded by the EFSB mitogenome. Each amino acid is colored differently,
and the labels in the circles correspond to the one-leer amino acid abbreviations. The number of
times a given amino acid was coded is proportional to the area of the circles and the size of the text
labels.
3.3. Ribosomal and Transfer RNA Genes
The tRNA genes have a total concatenated length of 1479 bp, contributing 9.7% of the
total EFSB mitogenome while the rRNA genes have a total length of 2167 bp, contributing
14.21% of the mitogenome. There are 22 tRNA genes, with serine and leucine having two
isoacceptors, while the other 18 amino acids have 1. All the tRNAs have been predicted
and verified by MITOS [33] and tRNAScanSE [34] except for trnS2, which yielded no ho-
mologous search using tRNAScanSE. This may be due to the four unique unpaired uracils
in the anticodon loop (Figure 4). However, such a case of four unpaired uracils was also
demonstrated in the trnS2 of the hemp borer, Grapholita delineana [87], the trnS2 of Plodia
Figure 3. Relative synonymous codon usage and total amino acids encoded by the EFSB mitogenome.
(A) Relative synonymous codon usage (RSCU) is a measure of the deviation of each codon from the
assumption that all codons are used equally. The bar graph shows the relative distribution of codon
usage across all amino acid codons. The number of codons for each amino acid ranges from two to
eight. The RSCU values are color-coded based on the codons below the amino acid labels. (B) Amino
acids encoded by the EFSB mitogenome. Each amino acid is colored differently, and the labels in the
circles correspond to the one-letter amino acid abbreviations. The number of times a given amino
acid was coded is proportional to the area of the circles and the size of the text labels.
3.3. Ribosomal and Transfer RNA Genes
The tRNA genes have a total concatenated length of 1479 bp, contributing 9.7% of the
total EFSB mitogenome while the rRNA genes have a total length of 2167 bp, contributing
14.21% of the mitogenome. There are 22 tRNA genes, with serine and leucine having two
isoacceptors, while the other 18 amino acids have 1. All the tRNAs have been predicted
and verified by MITOS [
33
] and tRNAScanSE [
34
] except for trnS2, which yielded no
homologous search using tRNAScanSE. This may be due to the four unique unpaired
uracils in the anticodon loop (Figure 4). However, such a case of four unpaired uracils
was also demonstrated in the trnS2 of the hemp borer, Grapholita delineana [
87
], the trnS2
of Plodia interpunctella [
86
], and the trnS2 of five skippers [
53
]. The length of the tRNAs

Insects 2024,15, 220 13 of 22
ranges from 64 bp (trnA and trnR) to 72 bp (trnL1) (Table 2). The secondary structure of
tRNAs is generally conserved, forming the typical three-loop, one-stem cloverleaf structures
consisting of an amino acid acceptor stem, a dihydrouridine loop (D loop), a pseudouridine
loop (
Ψ
U loop), and an anticodon loop. tRNAs also contain a variable region between the
anticodon loop and the
Ψ
U loop. All EFSB mitochondrial tRNAs exhibited this typical
cloverleaf, except for trnS1, where the D loop fails to form a canonical stem–loop structure
(Figure 4). The noncanonical structure of the D-armless trnS1 is a common and conserved
feature of metazoan mitogenomes [
88
]. All the tRNAs have a structurally conserved 7 bp
amino acid acceptor stem, except for trnA which contains a 6 bp stem. The tRNAs trnA and
trnL2 also contain a unique unpaired uracil in the acceptor stem (Figure 4). Similarly, all
the tRNAs contain a conserved 5 bp stem, and a 7 bp loop in the anticodon loop, except
for trnL2, which contains a 4 bp stem, and a 9 bp loop; trnK and trnS1 have a 4 bp stem
with a 7 bp loop; and trnS2 contains a unique anticodon loop with four unpaired uracils
(Figure 4). Noncanonical base pairings also occur in the EFSB mitochondrial tRNAs, with
G-U pairs comprising all such pairings (Figure 4). Despite the conservation of both the
acceptor stem and anticodon loop, the D and
Ψ
U loops show diversity in the number of
stem pairings and the number of nucleotides contained in the hairpin loops. Variability
in the D and
Ψ
U may indicate slight deviations from the canonical D/T loop interactions
important for aminoacylation [
89
], but are still functional. The EFSB mitogenome contains
two ribosomal RNA genes, l-rna and s-rna. The l-rna and s-rna genes are 1358 bp- and
809 bp-long
, respectively. The length of the EFSB mitogenome rRNA genes is similar to
that of other pyraloid moths [30].
3.4. Phylogenetic Relationships
A whole mitogenome phylogenetic analysis was used to determine the placement
of EFSB within Pyraloidea. The organisms, taxonomy, and reference sequence ID of the
mitochondrial genomes used for the analysis are shown in Table 1. For maximum likelihood
(ML) and Bayesian inference (BI) analyses, ModelFinder was first used to determine the
best-of-fit models for the dataset [
47
]. For the ML analysis, the initial 37 partitions were
reduced to 9 partitions, as summarized in Table S1. ModelFinder found that the general
time reversible model (GTR) is the most suitable model for all partitions except partition
3, where the TPM2 model is the most suitable. The GTR model is the model where all
six substitution rates and all four base frequencies are unequal. The TPM2 model, on the
other hand, assumes that the base frequencies are equal, and the substitution rates have the
following relationship: AC=AT, AG=CT, and CG=GT [
45
]. A similar case was observed
when applying ModelFinder for the BI analysis. The initial 37 partitions were also reduced
to 9, and ModelFinder found that the GTR model is the most suitable for all partitions for
BI. In addition, the corresponding parameters for the best model for each partition were
used as the priors for the BI run. All the parameters used as priors are summarized in Table
S2. Upon assessing the individual MCMC runs, all three runs converged, as evidenced by
the similarly shaped violin plots of the total branch length parameter (Figure S3A). The
trace plot for the total branch length across all generations also showed a fairly constant
value with small fluctuations around the mean. Lastly, the diagnostic parameters average
effective sample size (ESS) and the potential scale reduction factor (PSRF) for the total
branch length are 6742.56 and 1.000, respectively (Table S3), indicating that the three MCMC
runs converged.

Insects 2024,15, 220 14 of 22
Insects 2024, 15, x FOR PEER REVIEW 13 of 21
Figure 4. Structures of the EFSB mitogenome tRNAs. All of the EFSB mitogenome tRNAs except for trnS1 follow the canonical conserved 3-loop cloverleaf structure
consisting of the acceptor stem (dark green), dihydrouridine loop (D loop, orange), the pseudouridine arm (
Ψ
U loop, purple), and the anticodon loop (light green).
All tRNAs also contain a variable region (pink). In the case of trnS1, the D loop fails to form a stem–loop structure. The anticodon for each tRNA is indicated in
yellow, while all non-canonical base pairs are indicated by a red bond. All tRNA structures of the EFSB mitogenome were predicted using MITOS, validated using
tRNAScan-SE, and drawn using StructureEditor.

Insects 2024,15, 220 15 of 22
The side-by-side comparison of the obtained phylogenetic trees is shown in Figure 5,
while the consensus ML and BI trees are shown in Figure S4. The ML and BI trees are mostly
concordant with each other. The general subfamily relationships within the Pyraloidea
agree for both trees, with Pyraloidea having the basal split to Crambidae and Pyralidae. The
general relationships across Pyralidae also agree with both trees, having recovered the (Gal-
lerinae, (Phycitinae, (Pyralinae, Epipaschiinae))) relationships. Similarly, the ((Spilomelinae,
Pyraustinae), ((Crambinae, (Schoenobiinae, Acentropinae)), (Odontinae, Glaphyriinae))) re-
lationships in Crambidae were recovered for both trees (Figure 5). The only disagreements
between the ML and BI trees were the order of emergence between Polythlipta liquidalis,
Sinomphisa plagialis, and Conogethes punctiferalis. For the ML tree, the clade containing S. pla-
gialis and C. punctiferalis is basal to P. liquidalis, but the BI tree shows that C. punctiferalis
is basal to the clade containing P. liquidalis and S. plagialis (Figure 5). Nevertheless, on all
other tips, the two trees agree.
The root node for Pyraloidea is highly supported (100% bootstrap support (BS); 1 pos-
terior probability (PP)), indicating the monophyly and shared ancestry for this superfamily.
The expected basal split and monophyly of both Crambidae and Pyralidae were recovered,
as shown in Figure 5and Figure S4. The split is highly supported (95.1% BS; 0.999 PP),
concordant with the earlier works of Regier et al. (2012) [
16
] and Léger et al. (2020) [
17
].
The relationships between the four subfamilies of Pyralidae show high support values. A
split in the clade consisting of Gallerinae and the clade consisting of Phycitinae, Pyralinae,
and Epipaschiinae is moderately supported in the ML tree (77.8% BS) but well supported
in the BI tree (1 PP). Phycitinae was recovered to be a sister clade to the clade containing
Pyralinae and Epipaschiinae, and this is also highly supported (100% BS; 1 PP) and consis-
tent with earlier works [
17
,
54
,
62
]. Interestingly, the analysis failed to resolve the subfamily
relationships between Pyralinae and Epipaschiinae, exemplified by Orthaga olivacea, which
is classified under Epipaschiinae, showing closer relationships to Aglossa dimidiata than
Lista haraldusalis, which are Pyralinae and Epipaschiinae, respectively. Similarly, Perula sp.,
which is Pyralinae, clusters closer to Lista haraldusalis than to other Pyralinae. The relation-
ships between the subfamilies of Crambidae also show relatively good bootstrap support
values. Crambidae was recovered to be monophyletic and showed the split between
the clade consisting of Pyraustinae and Spilomelinae (PS clade) and the clade containing
all other subfamilies (non-PS clade). This expected split is highly supported (95.1% BS;
0.999 PP). The non-PS clade shows a split between the Odontinae-Glaphyriinae (OG) clade
and the Crambinae, Acentropinae, Midilinae, Musotiminae, Schoenobiinae, and Scopari-
inae (CAMMSS) clade, and this split is well-supported (92.8% BS; 0.999 BI). The sister
relationships between CAMMSS and OG, and Odontinae and Glaphyriinae, are consistent
with the findings of Qi et al. (2021) [
60
]. In the CAMMSS, Schoenobiinae was recovered to
be a sister to Acentropinae. This result is consistent with earlier mitogenomic works [
54
,
90
],
but different from others [
60
,
62
], which places Schoenobiinae as a sister to Crambinae. The
subfamilies represented in the non-PS clade were recovered to be monophyletic, and this is
well supported for in both trees (Glaphyriinae: 100% BS; 1 PP, Odontinae: 100% BS; 1 PP,
Crambinae: 100% BS, 1 PP, Acentropinae: 99.9% BS; 1 PP) (Figure 5).

Insects 2024,15, 220 16 of 22
Insects 2024, 15, x FOR PEER REVIEW 15 of 21
Figure 5. Topological concordance between the partitioned maximum likelihood and Bayesian in-
ference phylogenetic trees. The maximum likelihood (ML) and Bayesian inference (BI) phylogenetic
trees are shown on the left- and right-hand side of the labels, respectively. The labeling scheme
follows the tip order from the ML tree as indicated by the doed lines. The subfamilies are coded
according to color. The grey lines connecting the tips of both trees indicate tips that agree for both
trees, while the red lines indicate the tips with a different order and topology between the trees.
SH_aLRT and posterior probability support values are shown for the ML and BI trees, respectively.
The subfamilies included in the analysis are Spilomelinae, Pyraustinae, Acentropinae, Schoenobi-
inae, Crambinae, Odontinae, and Glaphyriinae of Crambidae; and Gallerinae, Phycitinae, Pyralinae,
and Epipaschiinae of Pyralidae. The outgroups chosen for the analysis consists of the dipterans Dro-
sophila melanogaster, Culex quinquefasciatus, and Aedes albopictus. The subfamilies are highlighted ac-
cording to color. The EFSB is highlighted in bold for emphasis. The PS (Pyraustinae-Spilomelinae),
CAMMSS (Crambinae, Acentropinae, Midilinae, Musotiminae, Schoenobiinae, and Scopariinae),
and OG (Odontinae-Glaphyriinae) clades are the clades first defined by Regier et al. (2012) [16].
The root node for Pyraloidea is highly supported (100% bootstrap support (BS); 1
posterior probability (PP)), indicating the monophyly and shared ancestry for this super-
family. The expected basal split and monophyly of both Crambidae and Pyralidae were
recovered, as shown in Figures 5 and S4. The split is highly supported (95.1% BS; 0.999
PP), concordant with the earlier works of Regier et al. (2012) [16] and Léger et al. (2020)
[17]. The relationships between the four subfamilies of Pyralidae show high support val-
ues. A split in the clade consisting of Gallerinae and the clade consisting of Phycitinae,
Pyralinae, and Epipaschiinae is moderately supported in the ML tree (77.8% BS) but well
supported in the BI tree (1 PP). Phycitinae was recovered to be a sister clade to the clade
containing Pyralinae and Epipaschiinae, and this is also highly supported (100% BS; 1 PP)
and consistent with earlier works [17,54,62]. Interestingly, the analysis failed to resolve
the subfamily relationships between Pyralinae and Epipaschiinae, exemplified by Orthaga
olivacea, which is classified under Epipaschiinae, showing closer relationships to Aglossa
dimidiata than Lista haraldusalis, which are Pyralinae and Epipaschiinae, respectively.
Figure 5. Topological concordance between the partitioned maximum likelihood and Bayesian
inference phylogenetic trees. The maximum likelihood (ML) and Bayesian inference (BI) phylogenetic
trees are shown on the left- and right-hand side of the labels, respectively. The labeling scheme
follows the tip order from the ML tree as indicated by the dotted lines. The subfamilies are coded
according to color. The grey lines connecting the tips of both trees indicate tips that agree for both
trees, while the red lines indicate the tips with a different order and topology between the trees.
SH_aLRT and posterior probability support values are shown for the ML and BI trees, respectively.
The subfamilies included in the analysis are Spilomelinae, Pyraustinae, Acentropinae, Schoenobiinae,
Crambinae, Odontinae, and Glaphyriinae of Crambidae; and Gallerinae, Phycitinae, Pyralinae,
and Epipaschiinae of Pyralidae. The outgroups chosen for the analysis consists of the dipterans
Drosophila melanogaster,Culex quinquefasciatus, and Aedes albopictus. The subfamilies are highlighted
according to color. The EFSB is highlighted in bold for emphasis. The PS (Pyraustinae-Spilomelinae),
CAMMSS (Crambinae, Acentropinae, Midilinae, Musotiminae, Schoenobiinae, and Scopariinae), and
OG (Odontinae-Glaphyriinae) clades are the clades first defined by Regier et al. (2012) [16].
The general subfamily relationships in Pyraloidea obtained in Figures 5and S4 are
consistent with earlier works [
16
,
17
]. Interestingly, while Pyraustinae was recovered to be
monophyletic (100% BS; 1 PP), Spilomelinae was recovered to be paraphyletic. The EFSB
is traditionally classified under Spilomelinae, but both ML and BI trees show that EFSB
emerged before the split of Pyraustinae and Spilomelinae (Figure 5). The placement of EFSB
within the PS clade is highly supported (100% BS; 1 PP). Spilomelinae without EFSB is also
highly supported (100% BS; 1 PP), indicating that EFSB may have emerged before the PS
split. Such a case was also documented for the pyralid moth Orybina regalis. Traditionally
placed within Pyralinae, Orybina regalis was recovered to be basal to Gallerinae in the
analysis by Liu et al. (2021) [
54
]. The analysis conducted here also recovered O. regalis
to be basal to Gallerinae in both ML and BI trees (Figures 5and S4). Additionally, while
the subfamily relationships recovered in this study are consistent with earlier works, the

Insects 2024,15, 220 17 of 22
lower-level relationships vary. There are some species which are robustly placed in other
subfamilies. For example, Omphisa fuscidentalis is classified under Spilomelinae but is
robustly placed in Pyraustinae in both ML and BI trees (Figures 5and S4). A similar case
was observed for Syllepte taiwanalis and both Cnaphalocrocis species. Both are classified under
Pyraustinae but were robustly placed in Spilomelinae. It is interesting to note, however, that such
placement for the said taxa was also observed in earlier works [
54
,
90
]. The earlier work by Tang
and Du (2023) shows consistent subfamily relationships with those obtained in Figure 5but
differ greatly in the lower-level relationships within Spilomelinae [
90
]. In Figure 5, the tribe
Spilomelini is a sister to the clade containing Pycnarmon lactiferalis and Syllepte taiwanalis,
and their collective clade is a sister to the tribe Margaroniini, which in Figure 5is the clade
containing the genus Glyphodes and Maruca. This relationship is concordant with Tang and
Du (2023) [
90
]. However, the relationships within Margaroniini are different. For example,
Palpita hypohomalia has close relationships with the genus Glyphodes in Figure 5, but the
same species has a closer relationship with Cydalima perspectalis in Tang and Du (2023) [
90
].
Additionally, while the sister relationships between the tribes Trichaeini (Prophantis adusta)
and Nomophilini (Nomophila noctuella) can be observed, Nagiella inferior clusters close to
these two, which is different from the relationship observed in Tang and Du (2023) [90].
Based on the above analyses, the phylogenetic trees obtained in this work are largely
consistent with earlier works. Both ML and BI trees robustly place Leucinodes orbonalis in
the PS clade but are basal to the split of Pyraustinae and Spilomelinae. However, there
is still the possibility of long-branch attraction. The incorporation of more mitochondrial
sequences from Pyraloidea and the incorporation of nuclear genes may be able to resolve the
lower-level relationships with Spilomelinae and produce a more conclusive phylogenetic
analysis for this economically important subfamily.
4. Summary and Conclusions
The whole mitochondrial genome of the eggplant fruit and shoot borer was assembled
and annotated in this study, producing a complete, circular 15,244 bp-long mitogenome
that contains the 37 genes expected for a pyraloid mitochondrion. The EFSB mitogenome
contains conserved lepidopteran mitogenome features such as the control region motifs, and
the trnM-trnI-trnQ-nad2 synteny. Partitioned phylogenetic analysis using the EFSB sequence
assembled in this study recovered the expected basal split of the families Crambidae and
Pyralidae and the split between the subfamilies Spilomelinae and Pyraustinae. However,
Spilomelinae was recovered to be paraphyletic, as indicated by the robust placing of EFSB
before the split of Spilomelinae and Pyraustinae in both ML and BI trees. The addition
of more mitochondrial sequences from Pyraloidea and the incorporation of nuclear genes
should yield a more resolved phylogeny that details the genus-level relationships within the
economically important Spilomelinae. Overall, the mitogenome produced here contributes
to the scarce genetic resources for the EFSB and will be a great help in future phylogenetic
reconstruction of Spilomelinae and in future population studies and studies that will impact
EFSB management.
Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/insects15040220/s1, Figure S1. Sequence quality of the EFSB
mitogenome reads; Figure S2. Cumulative coverage distribution for the EFSB mitogenome;
Figure S3
.
Estimation of the total branch length parameter across 3 MCMC runs; Figure S4. Partitioned phy-
logenetic analysis of 72 members of the superfamily Pyraloidea and 3 dipterans using maximum
likelihood and Bayesian inference; Table S1. Final gene partitions for the Maximum Likelihood
Phylogenetic analysis; Table S2. Final gene partitions for the Bayesian Inference Phylogenetic analysis;
Table S3. 95% Credibility interval of the sampled parameters during the MCMC run.
Author Contributions: Conceptualization, J.B.D. and M.A.M.B.; methodology, J.B.D. and M.A.M.B.;
formal analysis, J.B.D.; investigation, J.B.D.; resources, M.A.M.B.; writing—original draft preparation,
J.B.D. and M.A.M.B.; writing—review and editing, J.B.D. and M.A.M.B.; visualization, J.B.D.; supervi-
sion, M.A.M.B.; funding acquisition, M.A.M.B. All authors have read and agreed to the published
version of the manuscript.

Insects 2024,15, 220 18 of 22
Funding: The funding for this work was provided by the Department of Science and Technology
Philippine Council for Agriculture, Aquatic and Natural Resources Research and Development
(DOST-PCAARRD) to University of the Philippines Los Baños and University of the Philippines
Diliman Additional funding for the bioinformatics work was also obtained from the Accelerated
Science and Technology Human Resource Development Program (ASTHRDP) of the Department of
Science and Technology Science Education Institute (DOST-SEI). Funding was also provided by the
National Institute of Molecular Biology and Biotechnology, University of the Philippines Diliman
(NIMBB-UPD) in-house funds to sustain further analysis.
Data Availability Statement: The sequence of the final mitogenome assembly has been deposited
into GenBank under accession number PP493058. The final mitogenome assembly, intermediate files,
and scripts used for the analysis of the mitogenome are deposited and openly available in Zenodo at
10.5281/zenodo.10653202.
Acknowledgments: The authors would also like to thank the DNA Sequencing Core Facility (DSCF)
and Core Facility for Bioinformatics (CFB) of the Philippine Genome Center (PGC) for their invaluable
help in sequencing, and preliminary bioinformatics analysis for this work, respectively. Lastly, the
authors would also like to thank the Computing and Archiving Research Environment (COARE) of
the Department of Science and Technology Advanced Science and Technology Institute (DOST-ASTI)
for the HPC services that facilitated the phylogenetic analyses covered in this work.
Conflicts of Interest: The authors declare no conflicts of interest. The funders had no role in the
design of the study; in the collection, analyses, or interpretation of the data; in the writing of the
manuscript; or in the decision to publish the results.
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