Unfolding the mitochondrial genome structure of green semilooper (Chrysodeixis acuta Walker): An emerging pest of onion (Allium cepa L.)

doi:10.21203/rs.3.rs-1525316/v1

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Unfolding the mitochondrial genome structure of green semilooper (Chrysodeixis acuta Walker): An emerging pest of onion (Allium cepa L.)

https://doi.org/10.21203/rs.3.rs-1525316/v1

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Onion is the most important crop challenged by a diverse group of insect pests in the agricultural ecosystem. The green semilooper (Chrysodeixis acuta Walker), a widespread tomato and soybean pest, has lately been described as an emergent onion crop pest in India. C. acuta whole mitochondrial genome was sequenced in this work. The circular genome of C. acuta measured 15,743 base pairs (bp) in length. Thirteen protein-coding genes (PCGs), 22 tRNA genes, two rRNA genes, and one control region were found in the 37 sequence elements. With an average 395 bp gene length, the maximum and minimum gene length observed was 1749 bp and 63 bp of nad5 and trnR, respectively. Nine of the thirteen PCGs have (ATN) as a stop codon, while the other four have a single (T) as a stop codon. Except for trnS1, all of the tRNAs were capable of producing a conventional clover leaf structure. Conserved ATAGA motif sequences and poly-T stretch were identified at the start of the control region. Six overlapping areas and 18 intergenic spacer regions were found, with sizes ranged 1 to 20 bp and 1 to 111 bp, correspondingly. According to a phylogenetic study, C. acuta belongs to the Plusiinae subfamily of the Noctuidae superfamily, and is closely linked to Trichoplusia ni species from the same subfamily. The current study is the first whole mitochondrial genome sequencing of C. acuta, an emerging onion pest.

Chrysodeixis acuta

emerging pest

mitochondria

next-generation sequencing

onion

phylogenetic analysis

Onion (Allium cepa) of the family Alliaceae has been cultivated for more than 5000 years [1]. Though onion is one of the important vegetables, it is also being used as a spice for flavoring various cuisines globally. [2, 3, 4] Onion being a bulbous crop is mainly grown for its bulbs. However, the green leaves are also consumed worldwide as raw, cooked, semi-cooked, or in processed forms as a condiment, nutritional, nutraceutical, and medicinal purposes. [5, 3, 6] India is the second-largest producer (26738.000 MT), consumer, and exporter of onion globally (https://www.fao.org/faostat/en/#data/QCL). But unfortunately, the productivity per hectare is very low compared to countries where onion is grown commercially. The onion agro-ecosystem has a highly diverse insect pest complex, among which onion thrips. Thrips tabaci is the key pest of onion [7]. Besides thrips, several defoliators of the order lepidoptera are found at damaging levels in onions causing monetary loss globally [2, 1]. These insect pests affect almost every stage of the onion and cause a potential yield loss of 20 to 90% in onion production [8, 9] Insects can coevolve with the host plants, a relatively frequent phenomenon attributed to the prevailing environmental condition and host plant availability [10, 11, 12]. Subsequently, insects pose a serious threat by expanding their geographic distribution and host range.

Green semilooper (Chrysodeixis acuta Walker), an important pest of soybean and tomato across India, is also becoming an emerging onion pest. Onion is commonly intercropped with soybean in India's southern and central agricultural zones to limit insect pests by colonizing biocontrol agents [13]. C. acuta was recorded in onions for the first time in Maharashtra, India, in 2017–2018 [14]. The glossy green larvae are highly polyphagous, multivoltine species with huge potential to spread to new locations and adapt to new climatic or ecological conditions, which is likely why it has recently infested onions [14]. C. acuta has a wide range of hosts, including various vegetables, grains, and fruit crops [15, 16, 17]. C. acuta, like most defoliators, feeds on onion leaves; early instars scrape the leaves' mesophyll tissue, leaving papery white structures, while later instars bore enormous feeding holes. Though this pest currently causes minimal damage to onion crops, significant defoliation by these caterpillars is expected where the soybean-onion cropping system is practiced [14]. Excessive defoliation can lower the net photosynthetic area, resulting in a reduction in bulb size.

There is very little information available on the geographic variability and genetic structure of C. acuta. Chrysodeixis belongs to the Plusiinae subfamily and the Noctuidae family of moths. This subfamily is taxonomically compact and moderately large. More than 500 species have been identified globally [18] with 59 species belonging to 25 genera and three tribes [19] Nonetheless, the NCBI GenBank (https://www.ncbi.nlm.nih.gov/) only contains mitochondrial genome data only for four species from plusiinae subfamily: Diachrysia nadeja (MT916722), Trichoplusia ni (MK714850), Ctenoplusia albostriata (MN495624), and Ctenoplusia limbirena (KM244665). However, no pest species from the genus Chrysodeixis was sequenced to date even though some of the economically important pests under this genus are Chrysodeixis chalcites, Chrysodeixis eriosoma, Chrysodeixis includens, and Chrysodeixis acuta.

Their larvae are usually referred to as semiloopers. They are distinguished by 3 pairs of abdominal legs (prolegs) with biordinal crochets. In contrast, adult moths are robust, small to medium-sized, with a characteristic metallic spot in the center of the forewing. Morphological techniques cannot reliably distinguish these pests. However, taxonomic identification of the particular pest species is the fundamental step to device a suitable management strategy [20]. As a result, the mitochondrial (mt) genome of insects is frequently utilized as a molecular marker to disclose basic information at the genomic level for phylogenetic inference, molecular evolution, identification of species, geographic distribution, and population dynamics [21, 22]. Therefore, the whole mt genome of C. acuta was assembled for the first time using next-generation sequencing (NGS), providing a solution for species evolution and phylogeny. Furthermore, the extensive genetic information obtained from this investigation into the new onion pest may aid in devising effective control or preventative strategies.

Organization of the Genome

The semilooper's circular genome is 15,399 nucleotides in length. There are 37 sequence elements in total, consisting of 13 protein-coding genes (PCGs) which include Cytochrome c oxidase, NADH dehydrogenase, Cytochrome B, ATPase, and as two rRNA genes with 22 tRNA genes, and a control region. Every gene having an average gene length of 395 bp, the maximum and minimum gene lengths were 1749 and 63 bp, respectively (Fig. 1).

Protein-coding genes (PCGs)

The mt genome of C. acuta has 13 genes coding protein scattered in a circular chromosome. These PCGs contain three cytochrome c oxidase (cox1, cox2, and cox3 genes), seven NADH dehydrogenase (nad1, nad2, nad3, nad4, nad5, nad6, and nad4l genes), one cytochrome B (cytB gene), and two ATPase (atp6 and atp8 genes) (Table 1). The H-strand included nine of the 13 PCGs (cox1, cox2, cox3, nad2, nad3, nad6, atp6, atp8, and cytB), whereas the L-strand had the remaining four (nad1, nad4, nad4l, and nad5). In C. acuta, four start codons (ATA, ATT, ATG, and ATC) were found. Seven genes (cox2, cox3, atp6, nad1, nad4, nad4l, and cytB) adopted ATG as the start codons, 3 genes (cox1, atp8 and nad5) used ATT, two (nad2 and nad6) used ATA, while ATC by single gene (nad3). Nine PCGs employed the TAA termination codon (cox3, atp6, atp8, nad1, nad3, nad4l, nad5, nad6, and cytB). The incomplete termination codon T was found in four genes: cox1, cox2, nad2, and nad4 (Table 1). Phenylalanine (Phe, F), Leucine (Leu, L), Isoleucine (Ile, I), Asparagine (Asn, N), and Serine (Ser, S) were found to be the five most commonly occurring amino acids, whereas Cysteine (Cys, C), and Arginine (Arg, R) were found to be the rarest (Table 2). The PCG's covers 71.93% of the circular mt genome of the C. acuta.

Table 1

Summary table for characteristics of the complete mitogenome of *Chrysodeixis acuta*
Name	Start position	Stop position	Strand	Length	Intergenic Spacers	Anticodon	Start codon	stop codon
trnM	1	68	+	68		atg
trnI	69	135	+	67	0	atc
trnQ	133	201	-	69	-2	caa
nad2	276	1149	+	874	74		ata	t
trnW	1261	1327	+	67	111	tga
trnC	1320	1384	-	65	-7	tgc
trnY	1386	1450	-	65	1	tac
cox1	1443	2988	+	1546	-7		att	t
trnL2	2989	3055	+	67	0	tta
cox2	3056	3737	+	682	0		atg	t
trnK	3738	3808	+	71	0	aag
trnD	3818	3886	+	69	9	gac
atp8	3887	4048	+	162	0		att	taa
atp6	4042	4719	+	678	-6		atg	taa
cox3	4730	5515	+	786	10		atg	taa
trnG	5521	5585	+	65	5	gga
nad3	5595	5939	+	345	9		atc	taa
trnA	5939	6003	+	65	-1	gca
trnR	6004	6066	+	63	0	cga
trnN	6067	6133	+	67	0	aac
trnS1	6137	6202	+	66	3	agc
trnE	6203	6268	+	66	0	gaa
trnF	6298	6364	-	67	29	ttc
nad5	6365	8113	-	1749	0		att	taa
trnH	8114	8179	-	66	0	cac
nad4	8252	9590	-	1339	72		atg	t
nad4l	9597	9887	-	291	6		atg	taa
trnT	9890	9955	+	66	2	aca
trnP	9956	10020	-	65	0	cca
nad6	10028	10561	+	534	7		ata	taa
cytB	10581	11732	+	1152	19		atg	taa
trnS2	11735	11800	+	66	2	tca
nad1	11820	12758	-	939	19		atg	taa
trnL1	12760	12828	-	69	1	cta
rrnL	12808	14166	-	1359	-20
trnV	14215	14279	-	65	48	gta
rrnS	14280	15060	-	781	0
Control region	15061	15399	+	339	0

Table 2

Codon usage and relative synonymous codon usage (RSCU) within *Chrysodeixis acuta* mitochondrial genome
Codon	Count	RSCU	Codon	Count	RSCU	Codon	Count	RSCU	Codon	Count	RSCU
UUU(F)	26.3	1.92	UCU(S)	10.2	3.3	UAU(Y)	14.2	1.88	UGU(C)	2.3	2
UUC(F)	1.2	0.08	UCC(S)	0.6	0.2	UAC(Y)	0.9	0.12	UGC(C)	0	0
UUA(L)	35.4	5.27	UCA(S)	4.9	1.59	UAA(*)	0.7	2	UGA(W)	7.1	1.96
UUG(L)	0.6	0.09	UCG(S)	0.1	0.02	UAG(*)	0	0	UGG(W)	0.2	0.04
CUU(L)	2.9	0.44	CCU(P)	6.5	2.69	CAU(H)	4.8	1.85	CGU(R)	1.4	1.33
CUC(L)	0	0	CCC(P)	0.8	0.32	CAC(H)	0.4	0.15	CGC(R)	0	0
CUA(L)	1.4	0.21	CCA(P)	2.4	0.99	CAA(Q)	4.7	1.91	CGA(R)	2.7	2.59
CUG(L)	0	0	CCG(P)	0	0	CAG(Q)	0.2	0.09	CGG(R)	0.1	0.07
AUU(I)	33.8	1.94	ACU(T)	6.4	2.24	AAU(N)	18.7	1.92	AGU(S)	2.4	0.77
AUC(I)	1	0.06	ACC(T)	0.4	0.14	AAC(N)	0.8	0.08	AGC(S)	0	0
AUA(M)	20	1.86	ACA(T)	4.6	1.62	AAA(K)	6.8	1.81	AGA(S)	6.5	2.11
AUG(M)	1.5	0.14	ACG(T)	0	0	AAG(K)	0.7	0.19	AGG(S)	0	0
GUU(V)	5.8	1.99	GCU(A)	6.5	2.67	GAU(D)	4.7	1.94	GGU(G)	4.8	1.22
GUC(V)	0.1	0.03	GCC(A)	0.2	0.06	GAC(D)	0.2	0.06	GGC(G)	0.2	0.06
GUA(V)	5.1	1.75	GCA(A)	2.9	1.21	GAA(E)	5.4	1.87	GGA(G)	9.2	2.36
GUG(V)	0.7	0.24	GCG(A)	0.2	0.06	GAG(E)	0.4	0.13	GGG(G)	1.4	0.35
Average codons = 284

Transfer RNAs (tRNA) and Ribosomal RNAs (rRNA)

The mitochondrial genome of C. acuta had 22 tRNA genes strewn over the circular genome. From the total, 14 tRNAs were present within H- strand, whereas the rest 8 were present within L- strand (Fig. 1 and Table 1). The 22 tRNAs' aggregate sequence was 1,464 bp, 9.51% of the entire mt genome. Except for the trnS1 (AGN) gene, which codes for the serine (Ser, S) amino acid, the secondary structures of the remaining mt tRNA of C. acuta were predicted; all genes revealed the characteristic clover leaf shape (Fig. 2). The two rRNA genes rrnL of 1359 bp and rrnS of 781 bp are found on the L- strand, accounting for 13.90% of the mt genome (Fig. 1).

Overlapping and intergenic spacer regions

The whole circular mitochondrial genome map of C. acuta has six overlapping sections ranging from 1 to 20 bp with a total length of 43 bp. The overlapping area between the trnL and rrnL genes is the longest, l measuring 20 bp. Other overlapping regions include seven bp between trnC and trnW, cox1 and trnY; 6 bp between atp6 and atp 8, 2 bp between trnQ and trnI, and a single base pair between trnA and nad3 genes (Table 1). The mt genome of C. acuta comprises 18 intergenic spacer sequences, ranged 1 to 111 bp which constituted a total 427 bp. The longest intergenic spacer was 111 bp and present between nad2 and trnW an A/T rich region. The largest intergenic spacer sequences are 74 bp and 72 bp situated between trnQ and nad2 genes and trnH and nad4 genes, respectively. The other intergenic spacers are less than 50 bp size (Table 1). The spacer region between trnS2 (UCN) and nad1 includes two motifs ‘ATACTAA’ and ‘TACTAAAAATAAAT’ of 7 and 14 bp respectively (Fig. 3).

The control region

The control region (AT-rich) of C. acuta mt genome comprises 339 bp is positioned between rrnS and trnM genes. It is one of the longest mitochondrial non-coding region which cover about 2.20% of the whole mitochondrial genome and contains 93.51% AT nucleotides. There have been no reports of noticeable extensive repeats in the AT-rich Control region. However, many short repetitive sequences are distributed across the region, including the ATAGA motif, followed by a 19-bp Poly-T stretch, a microsatellite-like (AT) _10, and a 9-bp poly-A stretch downstream of trnM. (Fig. 4). Similar sequence elements were found in the mitochondrial genomes of lepidopteran species, Laelia suffuse [23] and Cerura menciana [22].

Phylogenetic analysis

The phylogenetic tree noctuidae family comprising of 55 Lepidoptera species based on 13 PCGs nucleotide sequence datasets constructed by utilizing Maximum Likelihood (ML), and Bayesian Inference (BI) methods. The phylogenetic tree, as shown in Fig. 4, revealed that the mt genome of C. acuta taxonomic status was closest with T. ni (GenBank accession No. MK 714850), C. limbirena (GenBank accession No. KM 244665), and C. albostriata (GenBank accession No. MN 495624) from the same sub-family Plusiinae. Hadeninae, Noctuinae, Amphipyrinae, Plusiinae, and Heliothinae sub-families of the Noctuidae family are closely related. The sub-family of C. acuta, Plusiinae is closely related to the Heliothinae and Amphipyrinae. However, the Hadeninae and Noctuinae are sister groups from the same superfamily (Fig. 5). The overall results of the phylogenetic analysis show that species belonging to the same family but belonging to separate sub-families are grouped.

Nucleotide composition

The mitochondrial genomes of the insects generally reflect real strand discrimination in nucleotide compositions [24]. The strand dissimilarity is measured as the AT and GC-skews [25]. The J-strand displayed strand bias with negative GC-skew and positive AT-skew pattern (Supplementary Fig. 1) in a comparative study of nucleotide percentages vs skewness of the 55 Noctuidae family members, including C. acuta. The relative synonymous codon usages (RUSC) were evaluated to identify the preference for a specific synonymous (Table 2). The codon usages pattern of C. acuta suggests that the two-fold and four-fold degenerate codons overuse the A and T at the third codon position (Supplementary Fig. 2).

Gene evolution rate

For the analysis of gene evolution rate, the rate of nonsynonymous substitution (Ka, pi modified), synonymous substitution (Ks, pi modified), and the Ka/Ks ratio, as well as the Jukes-Cantor adjusted Ka/Ks (JKa/JKs) ratio, of 13 PCGs from 55 mitogenome sequences from Noctuidae family species, including C. acuta was used. Based on synonymous and nonsynonymous nucleotide substitution analysis, the Ka/Ks values of the 13 PCGs of 55 Noctuidae species ranged from 0.0463 to 1.0947 (Table 3). The Ka/Ks values of only two genes nad2 and cytB were more than one, 1.0947 and 1.0654, respectively, indicating the highest evolutionary rates. While cox1 was recorded lowest (0.0463) among the 13 PCGs. Similarly, Jukes-Cantor adjusted Ka/Ks also showed the cytB and nad2 highest evolutionary rate with JKa/JKs values 2.0443 and 1.6206, respectively. However, cox1 and atp6 with the lowest JKa/JKs values of 0.0349 and 0.0916, respectively. Overall ratios of Ka/Ks and JKa/JKs for the genes cox1, cox2, atp6, nad3, nad4, and nad4L were less than 0.5, indicating that these genes in the Noctuidae family evolved under purifying selects (Supplementary Fig. 3). Though this gene has a relatively slow evolutionary rate, this could be used as the candidate barcoding marker for species identification in Noctuidae family. Nucleotide substitution in the mitogenome indicates the evolution at the molecular level [26]. Nucleotide substitution was previously thought to represent a directional bias between various genes in the mitochondrial genome of insects, according to previous research by Cameron [27].

Table 3

Rates of non-synonymous substitutions (Ka, pi modified), synonymous substitutions (Ks, pi modified) and the Ka/Ks ratio as well as Jukes-Cantor adjusted Ka/Ks (JKa/JKs) ratio in each PCG of 55 mitogenome sequences of Noctuidae family species including *Chrysodeixisacuta* from this stud
Protein-coding genes	Rates of non-synonymous substitutions (Ka)	Rates of synonymous substitutions (Ks)	Ka/Ks ratio	Rates of non-synonymous substitutions Jukes-Cantor adjusted J(Ka)	Rates of synonymous substitutions Jukes-Cantor adjusted J(Ks)	JKa / JKs ratio
nad2	0.33444	0.30550	1.09473	0.65868	0.40644	1.62061
cox1	0.01457	0.31435	0.04635	0.01479	0.42349	0.03492
cox2	0.05820	0.33466	0.17391	0.11659	0.46371	0.25143
atp8	0.15659	0.21912	0.71463	0.18637	0.26938	0.69185
atp6	0.0402	0.3316	0.121261	0.04187	0.4573	0.0916
cox3	0.27558	0.37520	0.73449	0.76770	0.55221	1.39023
nad3	0.10409	0.38134	0.27296	0.18199	0.56431	0.32250
nad6	0.27856	0.28836	0.96601	0.43713	0.37632	1.16159
nad5	0.1939	0.2619	0.740482	0.45273	0.3326	1.3611
nad4	0.04544	0.24551	0.18508	0.04748	0.30501	0.15567
nad4L	0.10840	0.24782	0.43741	0.17589	0.31478	0.55877
cytB	0.40883	0.38370	1.06549	1.15181	0.56342	2.04432
nad1	0.19112	0.26457	0.72238	0.49746	0.33763	1.47339

The mitochondrial genome of C. acuta was sequenced, and its molecular and phylogenetic features, were studied in this work. The semilooper's circular mt genome measured 15,399 bp in size, slightly bigger than Trichoplusia ni (15,239 bp), which belongs to the same Plusiinae subfamily [28]. Similarly, the mt genome of green semilooper is very close to the other defoliator pests infesting onions, such as S. exigua (15,365 bp) and S. litura (15,374 bp) [29, 30]. The protein-coding genes are found scattered on both Heavy and light strands, covered 71.93% of the total mt genome of C. acuta, and were 11,077 bp long. These PCGs include cytochrome c oxidase, NADH dehydrogenase, Cytochrome B, and ATPase, commonly found in most insect species belonging to Lepidopteran order.

Insect species like Ctenoplusia albostriata [31], Diachrysia nadeja [32], Trichoplusia ni [28], Laelia suffuse [23]. Cerura menciana [22], all have the same genes with little differences in size and location. The ATN codon was used to start all of C. acuta's protein-coding genes. The initial codon for cox1 was ATT, instead of "CGA" utilized in several insect species [33]. In C. acuta, two termination codons were discovered. Four genes cox1, cox2, nad2, and nad4, used the incomplete termination codon "T"; nevertheless, other genes were terminated by TAA. Our findings are consistent with those of Liu et al. [34], Dai et al. [22], and Dai et al. [35] who found a single "T" as a termination codon for the cox1 and cox2 genes in the majority of Lepidopteran species. The incomplete termination codons "T" in lepidopteran mt genes might get polyadenylated to TAA codon during translation [36].

Codons having A or T at the third prime position were found to be overused in this study when compared to other similar codons. For example, the valine codons GTC and GTG were rare, whereas GTT and GTA's synonymous codons were prevalent (Table 2). In the mitogenome of Leucoma salicis, a similar tendency was observed [37]. The length of 22 transfer RNA genes in the C. acuta mt genome ranged from 63 bp (trnR) to 71 bp (trnK), which is similar with the mt genomes of L. suffusa [23], D. pyloalis [33], and C. menciana [22]. Except for trnS1 (AGN), which lacks the dihydrouridine (DHU) arm and forms a simple loop, all tRNA secondary structures resembled the conventional clover leaf shape (Fig. 2). Many insect mitogenomes showed similar results, including Bombyx mori [38], Actias selene [39], Spodoptera frugiperda [40], Spodoptera litura [30], Chrysochroa fulgidissima [41], B. zonata [42] and Idioscopus nitidulus [43].

Ten of the tRNA genes were found to contain 14 G-U mismatches in their secondary structures, forming a weak bond. The amino acid acceptor stems of tRNALeu(UUR), tRNAAla, tRNASer1, and tRNASer2 have five U-U mismatches (Fig. 2). Sun et al. [37] observed a similar trend in L. salicis. The control (AT-rich) region varies in size across members of the Noctuidae family, although it includes comparable sequence components in most species. In many Lepidopteron species, the ATAGA motif found at the start of the control region is a conserved motif sequence. Similarly, Li et al. [23] in L. suffuse, and Dai et al. [22] in C. menciana have also reported that the ATAGA motif in the control region followed by the Poly-T stretch and microsatellite A/T repeats elements along with Poly-A stretch at the end of the control region.

The intergenic spacer region between the genes trnS2 (UCN) and nad1 includes the 'ATACTAA' motif, which commonly presents in most lepidopteran species even though the size of the intergenic spacer region varies [44]. This spacer region is generally considered a constitutive synapo-morphic feature of the lepidopteran mitochondrial genomes because this region won't present in non-lepidopteran insects species [45].The similar motif were located in the Dysgonia stuposa [46], L. salicis [37], C. menciana [22] and Ctenoptilum vasava [44]. The motif 'TACTAAAAATAAAT' was located in the intergenic spacer region between the genes trnS2 (UCN) and nad1 of the C. acuta, common in 5 lepidopteran species of the Plusiinae subfamily (Fig. 3). However, this motif was not reported in any other lepidopteran subfamilies.

The phylogenetic analysis discovered that C. acuta is most closely correlated to the cabbage looper, Trichoplusia ni, from the same subfamily Plusiinae, notably in mt genome similarities, 13 PCGs, 22 tRNAs, and two rRNAs. However, the size of the control region appears to be different [28]. The five sub-families comprising Hadeninae, Noctuinae, Amphipyrinae, Plusiinae, and Heliothinae from the Noctuidae family are closely related. The sub-family of C. acuta, Plusiinae is closely related to the Heliothinae and Amphipyrinae. However, the Hadeninae and Noctuinae are sister groups from the same superfamily (Fig. 5). The phylogenetic tree confirmed that the previously characterized species C. albostriata [31], D. nadeja [32], and T. ni [28] belonged to the sub-family Plusiinae and are related to the C. acuta which confirms that the C. acuta has belonged to the Plusiinae sub-family. The phylogenetic relationships were reconstructed based on the concentrated data of the 13 PCGs, which supports the traditional morphology-based view of the relationship within the Noctuidae family.

The mitogenomes of the Noctuidae family have been studied extensively; however, very few reports are available on the Plusiinae subfamily. The first complete mt genome sequence of Chyrsodeixis acuta is revealed in this work. The circular mitogenome of C. acuta is 15,399 bp length and contains 37 sequence elements. It comprises 13 protein-coding genes, ATPase genes, Cytochrome B gene, cytochrome c oxidase gene, NADH dehydrogenase gene, two ribosomal RNA genes, 22 transfer RNA genes, and a control region. The complete mt genome adds to the genetic richness of this species and elucidates crucial information for further evolutionary and phylogenetic studies of the Noctuidae family. In addition, other potential pests of the genus Chrysodeixis, such as Chrysodeixis chalcites, Chrysodeixis eriosoma, and Chrysodeixis includens, infesting various crops, would benefit from the mitochondrial genomic organization of Chyrsodeixis acuta.

Sample collection

Green semilooper, Chrysodeixis acuta larvae were collected from onion plants at the Indian Council of Agriculture Research - Directorate of Onion and Garlic Research (ICAR-DOGR), situated at the latitude (27°19’00.2 N ) and longitude (82°25’00.1E) Pune, Maharashtra, India 553.8 meters above sea level). The insects were initially reared in the laboratory. After completion of the life cycle, newly emerged adults from a single egg mass were collected, one pair was preserved in the insect repository of the Center with voucher specimen number: DOGR Voucher 15 following standard procedures. To further confirm the species identity, larval specimens were analyzed by ‘DNA barcoding’ at ICAR-DOGR, Pune, India and the sequence was submitted under the accession number MT644267 in NCBI GenBank and GBMNC55083-20 in Barcode of Life Data System (BOLD).

Sample preparation and DNA extraction

Laboratory reared larval samples were kept in a refrigerator at -20°C in 100 percent ethanol until the experiment. According to the manufacturer's instructions, total genomic DNA was extracted using the DNeasy Blood and Tissue Kit (QIAGEN, Germany). The quality and amount of extracted DNA were assessed using a 1% agarose gel and with SmartSpec 3000 UV/Visible Spectrophotometer at 260 and 280 nm (Bio-Rad, Hercules, California, USA). By referring to the REPLI-g Mitochondrial DNA Kit (QIAGEN, Germany) protocol, mitochondrial DNA was isolated from genomic DNA.

Sequencing and Mitogenome analysis

The mt genome was sequenced using the Illumina Next Seq 500 sequencing platform and Trimmomatic (v0.38) [47] was used to eliminate sequences of adaptor, ambiguous reads (reads having > 5% unidentified nucleotides), and junk sequences (reads having > 10% quality threshold (QV) 20 phred score) from sequenced raw data to get high-quality clean reads. The high-quality reads aligned to the reference sequences with the help of BWA MEM (version 0.7.17) [48]. The consensus sequence was extracted using SAM toolsmpileup [49]. The consensus sequence was employed to identify protein-coding and RNA genes in the sample. The MITOS algorithm was used for genes prediction from the invertebrates’ mitochondrial genome [50].

The protein-coding and rRNA genes were manually annotated and verified comparing them with four mitogenome sequences of Diachrysia natija (MT916722), Trichoplusia ni (MK714850), Ctenoplusia albostriata (MN495624), and Ctenoplusia limbirena (KM244665) of subfamily Plusiinae available in GenBank. Using tRNAscanSEv 2.0 and a 15.0 covariance limit value, the Mito/Chloroplast model was utilized to recheck the MITOS downloaded two-dimensional tRNA structures [51]. The CGview server was used to generate the circular map of the complete mt genome, GC concentration, and GC skew [52]. Finally, protein-coding sequence regions of mitochondria were assembled and aligned in Mega version X using ClustalW with basic parameters [53].

The A + T contents, codon use, and relative synonymous codon usage (RSCU) of the PCGs were analyzed by using MEGA version X. The GC and AT skews were calculated utilizing the formulas (GC skew = [G-C] / [G + C] and AT skew = [A-T] / [A + T]) given by Perna and Kocher [25]. The overlapping and intergenic spacer region between genes were manually calculated. The synonymous (Ks) and nonsynonymous substitution rates (Ka) for each Protein-coding gene were calculated using the DnaSP 6.0 and Jukes-Cantor adjusted Ka/Ks (JKa/JKs) software programmes [54]. The control region tandem repeats were predicted by using Tandem Repetitions Finder tool with default settings [55]. The assembled and annotated C. acuta mitogenome has been submitted under the accession number OL892047 in NCBI GenBank.

Phylogenetic analysis

All of the available complete Nocutidae mitogenomes (55mitogenomes) were chosen for the phylogenetic study. One of the 55 Nocutidae mitogenomes was sequenced in this study, and the rest samples were obtained from the NCBI database.

The phylogenetic study used concatenated nucleotide sequences from 13 PCGs datasets. The PCG was aligned using MAFFT 7 (https://ma t.cbrc.jp/alignment/server/) based on codons for amino acids [56]. Similarly, to remove ambiguously aligned sites from PCG alignments, GBlocks v.0.91b (http://molevol.cmima.csic.es/castresana/Gblocks/Gblocks documentation.html) was used [57]. MEGA 10.0 was used as the final quality check for all of the alignments. All gene alignments were concatenated using PhyloSuite 1.2.1 [58]. PCG123 matrix with single sequences in the following order: nad2, cox1, cox2, atp8, atp6, cox3, nad3, nad5, nad4, nad4L, nad6, cytb, and nad1. For phylogenetic reconstruction, these thirteen PCGs were concatenated. The best partitioning schemes were chosen using Partition Finder 2.1.1 with the Bayesian Information Criterion (BIC) and greedy algorithm (www.phylo.org) [59]. According to BIC, the GTR + I + G model was ideal for nucleotide alignment analysis. During the research, the PHYML online web server utilized the optimum substitution model obtained from Model Test to infer maximum likelihood (ML) analysis, and model parameter values were calculated [60]. Bayesian analyses were performed by utilizing Markov Chain Monte Carlo (MCMC) method with software, Mr. Bayes v.3.1.2 [61] having two independent runs of 2 × 10⁶ generations with four chains with trees sampled each 100th generation. Mr. Bayes' "sump" command was used to evaluate the similar values for every post-analysis trees and the convergence and burn-in parameters. The top 100 trees from each run were utilized for burn-in, and the remaining trees were used to form a consensus tree with a 50% majority rule.

Acknowledgement: The authors are thankful to the Director ICAR-Directorate of Onion and Garlic Research, Pune, Maharashtra, India, for providing funds. The present study is a part of in-house project, Reference code “IXX14062." The authors further thank the Head of the Division of Entomology, Indian Agricultural Research Institute, New Delhi, India, and Dr. P. R. Shashank, Division of Entomology, IARI, New Delhi, India, for insect species confirmation. We also gratefully acknowledge the editors and anonymous reviewers for their valuable suggestions and comments.

Author's Contribution: SPS, GPP, and AS conceived and designed the analysis; DVS and RK performed molecular analyses and designed figures of the manuscript; PAG facilitated in the sequencing of sample; JSC, NN and DVS performed the molecular analysis and interpreted results; SPS, RK, and GPP drafted the manuscript; SPS, AS, KV and JSC critically reviewed and edited subsequent drafts. MS provided funds and necessary facilities to conduct the present study. All authors read and approved the final manuscript.

Conflict of Interest: All the authors declare that there is no competing conflict of interest.

Permissions: The researchers were granted permission to collect the insect sample as part of the routine monitoring of the experimental trials for emerging and invasive pest species by the Director ICAR-Directorate of Onion and Garlic Research, Pune, Maharashtra, India.

Note: The sequencing data underlying this study have been submitted to NCBI gene bank with accession number OL892047 and will be publicly available after publication.

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Unfolding the mitochondrial genome structure of green semilooper (Chrysodeixis acuta Walker): An emerging pest of onion (Allium cepa L.)

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Abstract

Figures

Introduction

Results

Organization of the Genome

Protein-coding genes (PCGs)

Transfer RNAs (tRNA) and Ribosomal RNAs (rRNA)

Overlapping and intergenic spacer regions

The control region

Phylogenetic analysis

Nucleotide composition

Gene evolution rate

Discussion

Conclusion

Material And Methods

Sample collection

Sample preparation and DNA extraction

Sequencing and Mitogenome analysis

Phylogenetic analysis

Declarations

References

Additional Declarations

Supplementary Files

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