Effects of photoperiodism, melatonin and pesticide exposure on Homeobox and N-acetyl transferase gene expression profiles in Spodoptera litura (Insecta: Lepidoptera)

Effects of photoperiodism, melatonin and pesticide exposure on Homeobox and N-acetyl transferase gene expression profiles in Spodoptera litura (Insecta: Lepidoptera) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effects of photoperiodism, melatonin and pesticide exposure on Homeobox and N-acetyl transferase gene expression profiles in Spodoptera litura (Insecta: Lepidoptera) SUBALA P Subramanian, Eliningaya J. Kweka, Shivakumar S Muthugounder This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8547288/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Homeobox (HOX) genes play a major role in shaping the anteroposterior body axis during insect development. Melatonin is a photoperiodic hormone that is involved in insect developmental mechanisms. Here, we demonstrate the presence of Hox and melatonin genes (aanat) in the whole body of S. litu The results showed increased expression of Dfd (4.3 fold) and Lab (8.4 fold) in abamectin exposed 12:12 h light-dark (LD) condition, suggesting the formation of insect head responsive genes. While Abd-A (55 fold), abd-B (182 fold), and aanat(40 fold) expression were high in mel + aba-exposed DD, ubx (137 fold) expression in aba-exposed DD conditions suggests control of abdominal and wing development due to nocturnal synthesis of melatonin, which is confirmed by higher aanat expression in darkness. Our results show that vitellogenin (85 and 120 fold) expression is high under mel and mel + aba exposed light photoperiod (LL), suggesting that vitellogenin production and accumulation requires continuous light. Arylalkalyamine N-acetyl transferase photoperiod melatonin vitellogenin homeobox genes photoperiodic hormone insect development Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Agricultural pests have been a major hindering factor in revolutionizing the horticulture sector productivity in India and tropical Asia of which Lepidopteran pests significantly cause yield loss. Vegetables are among the most profitable crops, and farmers everywhere feel the need to protect these high-value crops from any damage caused by insect pests [ 1 ]. Farmers often use synthetic insecticides randomly, and insecticide resistance has been identified among commonly used pesticides [ 2 ]. Spodoptera litura Fabricus (Lepidoptera: Noctuidae) is a polyphagous insect with about 172 species of host plants from 40 families in India. It is one of the most economically important insect pests in many countries, including India, Japan, China, and other countries in Southeast Asia and Africa [ 3 , 4 ]. In India, S. litura hinders crop production. It has been reported on approximately 112 cultivated plants and causes economic losses ranging from 25.8% to 100%. This nocturnal insect was chosen as a model for this study because of its availability, fast growth, and polyphagous nature [ 5 , 6 ]. Hox genes serve as the main regulators of embryonic development in all animals, including flies, humans, and other animals. It is now possible to examine the molecular mechanisms by which genomic information is subsequently transformed into physiological and morphological processes during the development of many organisms [ 7 , 8 ]. The Antennapedia and Bithorax complexes are two distinct gene clusters in which Hox genes are found in Drosophila [ 9 ]. These genes encode transcription factors (TFs) called homeodomain (HD)-containing TFs, which regulate the expression of numerous downstream target genes by identifying particular enhancers [ 10 – 12 ]. Homeobox genes (Hox) encode a large group of transcription factors involved in developmental processes throughout the animal kingdom [ 9 ]. More than 300 Hox genes and their relatives have been identified in fungi, plants, and animals [ 13 ]. In insects, Hox genes specify the structures along the anteroposterior body axis; three are necessary for the correct development of head segments and appendages: the Labial (lab), Proboscipedia (Pb), and Deformed (Dfd) genes. The Labial (Lab) gene is involved in the proper development of head and mouth structures in insects [ 14 ]. In contrast, the Deformed (Dfd) gene is involved in the formation of head parts, particularly maxillae and mandible,s in insects[ 15 ]. During insect embryogenesis, Hox proteins promote unique morphological patterning events in the abdominal segments, particularly Ultrabithorax (Ubx), Abdominal-A (Abd-A), and Abdominal-B (Abd-B) genes [ 16 , 17 ]. Ultrabithorax(Ubx) is known to suppress wing formation in insects, and the differential development of wings and halteres is dependent on the function of this Hox protein in Drosophila [ 18 ]. In addition, because of the high expression of Ubx and Abd-A proteins in the abdominal segments, they may suppress thoracic leg development during embryogenesis in Drosophila melanogaster [ 19 ]. Vitellogenin (Vg) plays a vital role in oocytes and insect embryo development. It is synthesized in the fat body, moves through the hemolymph, and accumulates in oocytes [ 20 , 21 ]. One of these Hox genes, the cone-rod homeobox (Crx) gene, seems to play an important role in the regulation of aanat transcription [ 22 ]. Melatonin is a circadian rhythm regulator secreted in insects and participates in insect development [ 23 ], but numerous studies have documented its activity as a free radical scavenger [ 24 , 25 ] and antioxidant agent [ 26 ]. In vertebrates, melatonin is synthesized mainly at night because of circadian rhythm modulation. This reaction is mediated by several enzymes, including AANAT [ 27 ]. Based on the available literature, we suggest that hox genes and melatonin are responsible for insect development. In addition, the cone-rod homeobox (Crx) transcription factor regulates the expression of NAT in the mouse pineal gland. LIM homeobox transcription factor Isl1 positively modulates melatonin synthesis by targeting NAT at the (ATTA/TAAT) motif via the ERK signaling pathway of norepinephrine [ 28 ]. Recent studies by Subala and Shivakumar described the impact of circadian rhythm on melatonin levels and redox status under three photoperiods (12L:12D, 0L:24D, and 24L:0D) in the head and hemolymph of S. litura [ 29 ]. Previous research has described that pre-treatment with melatonin decreases abamectin-induced toxicity in the nocturnal insect S. litura [ 6 , 30 ]. It is thought that timing exposure to pesticides may influence insect development, which may be normalized due to melatonin activity [ 31 ]. Moreover, we have previously reported that photoperiod influences the digestive biology of S. litura [ 6 ]. Insects are attractive model systems for studying and elucidating the function of many early developmental genes (including hox genes) because of their short life span, and a large number of animals can be collected from the field and/or reared in laboratories [ 32 ]. Light has been known to affect insect development through subtle unknown mechanisms. Among the genes controlling insect development, the Hox gene complex is known to be very important, as it controls the body plan of insects [ 33 ]. Although Hox genes play a major role in shaping the anterior-posterior body axis during animal development, our understanding of how they act in the presence of melatonin, pesticides, and different photoperiods in insects is rudimentary. As mentioned previously, the major role of Hox genes is in the body patterning of animal development. However, the molecular mechanisms underlying the control of hox by melatonin or vice versa in invertebrates are poorly understood [ 34 , 35 ]. This study aimed to summarize the developmental mechanisms of rhythmic melatonin and Hox gene expression in insects. To achieve this purpose, we investigated (i) the role of hox genes, particularly two antennapedia complex genes ( Dfd , and Lab), and three bithorax complex genes (abd-A, Abd-B, and Ubx), under different photoperiods; and (ii) the vitellogenin (Vg) and N-acetyl transferase ( AANAT ) gene expression profiles on vitellogenin and melatonin synthesis under different photoperiods in the whole body of Spodoptera litura adults. Materials and methods Insect rearing Spodoptera litura eggs were obtained from the National Bureau of Agricultural Insect Resources (NBAIR), formerly the National Bureau of Agricultural Important Insects (NBAII, Bangalore, India) with National Accession No. NBAII-MP-NOC-02. Larvae were maintained on castor leaves as food and maintained at 28 ± 1°C, 70 ± 10% Relative Humidity (RH) under a photoperiod of 12L:12D. Male and female pupae were segregated based on the morphology of the abdominal terminal segments. Virgin males and females were separated into different plastic cups (20×20×30 cm) and fed with a 10% honey solution. Three-day-old adults were placed in 100 ml plastic containers for the experiments. Chemicals Melatonin was purchased from Sigma–Aldrich (India). Abamectin (N-acetyl 2-benzyl tryptamine) was procured from Insecticides Pvt. Ltd, New Delhi (India) and has the commercial grade name of Agrimek with 0.15% EC. The highest dosage of abamectin used was 1.56 ml (28 mg AI)/L, according to previous studies [ 36 ]. Melatonin (4.3×10 − 5 M) was administered to the melatonin group as previously described [ 30 ]. All other chemicals and reagents used in this study were of molecular grade. Experimental Design The experimental larvae were exposed to light using a white fluorescent bulb (27 W, wavelength range 350–650 nm). The light intensity was approximately 0.96W/cm2. Third instar larvae (n = 1080), on day-2 were divided randomly into three photoperiod groups: Normal (LD): 12-h light: 12-h dark cycle (12L:12D; n = 360 larvae) Continuous light (LL): 24-h light cycle (24L:0D; n = 360 larvae) Continuous dark (DD): 24-h dark cycle (0L:24D; n = 360 larvae) Each of these experimental photoperiodic groups (n = 360) of larvae was divided into four subgroups (n = 90) as follows: (a) Control group : 0.1M Phosphate buffered saline containing Na2HPo4 & NaH2Po4; pH 7.5) (b) Melatonin group (Mel) : Melatonin was dissolved in ethanol (> 0.01%)[ 37 ]. (c) Abamectin group (Aba) : An equal volume of cold saline (0.1M PBS, Na2HPo4, and NaH2Po4; pH 7.5) was added to the abamectin [ 36 ]. (d) Melatonin + Abamectin group (Mel + Aba) : Both were incorporated into the larval diet until they reached the fifth instar stage. The experiment was performed in three independent biological replicates. Therefore, from each subgroup (n = 90), we obtained three replicates (30 larvae per replicate) that were kept in plastic containers and fed with 100 ml of artificial diet according to previous studies (Kranthi, 2005). The artificial diet was replaced every three days. RNA extraction and cDNA synthesis Before dissection of the whole moth, the wings were removed and weighed. S. litura adults were dissected under dim light. In this study, Total RNA was manually isolated from the whole bodies of adults kept under three different photoperiods using the TRIZOL method [ 38 ]. RNA concentrations were quantified by measuring the absorbance at A260/280 nm. The purity of RNA obtained was 1.8–2.0, and the yield of RNA was expressed in µg/ml. 1µg of total RNA was reverse transcribed to complementary DNA (cDNA) using a cDNA synthesis kit (Himedia, India) according to the manufacturer’s protocol (Table 1 ). Table 1 The Nanodrop reading of total RNA Photoperiod Treatment Melatonin (µg/ml) Control (µg/ml) Abamectin (µg/ml) Melatonin + Abamectin (µg/ml) 12L:12D 764.3 763.1 477.1 766.8 0L:24D 1072.9 1083.3 751.7 1108.5 24L:0D 1025 1084.2 1056.7 700.6 Note RNA concentrations were quantified by measuring the absorbance at A260/280. The purity of RNA obtained was 1.8–2.0, and the yield of RNA was expressed in µg/ml. Quantitative Real Time PCR (RT-PCR) analysis Quantitative Real Time PCR (RT-PCR) analysis For RT-PCR analysis, primers (Table 2 ) were designed using Primer 3 software (Applied Biosystems, California). The designed primer characteristics were analyzed and confirmed using NetPrimer (Premier Biosoft). SYBR Green PCR was conducted in 0.2 ml PCR tubes with flat 8-cap strips (Axygen, USA) using iQTM SYBR Green Supermix (Bio-Rad, Hercules, USA). Primer alignment was performed with the BLAST database to ensure primer specificity. RT PCR was conducted using a Bio-Rad CFX96 Real-time PCR Detection System (Bio-Rad, Hercules, USA) following the manufactures protocol. RT-PCR was conducted to evaluate the relative gene expression levels in the whole insect body. The RT-PCR program was performed with a melting curve dissociation protocol (from 58 to 95°C) according to the following thermal conditions: initial denaturation at 95°C for 3 min, followed by 46 cycles at 58°C for 1 min and 59°C for 10s. The experiments were performed in duplicate. Gene specificity was determined using the dissociation curve, and validation experiments were conducted to confirm that the efficiencies of the target and reference gene amplifications were approximately equal (80–90%). Gene-specific oligonucleotide primers for the housekeeping gene β-actin were added to the same PCR reaction vial and co-amplified. Table 2 Primers used in Real Time-PCR analysis Gene Genbank Accession Number Primer Sequences Fragment length (bp) References VTG HQ260608 5’-GACTATAAAGATTGTTCCATGTG-3’ 3’-GAGTATGGTGGAGAATCATTAGT-5’ 23 23 Park and Kwak, 2012 Dfd D83534 5’-ATGAGCTCCTTCCTGATG-3’ 3’-TGTGGAAGAGGATGGTAG-5’ 18 18 Parthasarathy and Gopinathan, 2005 Lab AB120761 5’-ACTTCACCAACAAACAGCTC-3’ 3’-TGATCCGTTTCTTCTGCTTC-5’ 20 20 Kimato, et al., 2014 Ubx AB505052 5’-ATGAACTCTTACTTCGAGC-3’ 3’-GTTCAAATGTTTTAATGTTCGG-5’ 19 20 Kimato, et al., 2014 abd-A AB461860 5’-TCTACCTCGCTGTCTAGTTC-3’ 3’-TGCTGCTGCTTCATTCTGTC-5’ 20 20 Kimato, et al., 2014 Abd-B AB461858 5’-CAACCAGCAGTGGTGAATAC-3’ 3’-TTGTTGTTCTGGGCAGCTTG-5’ 20 20 Kimato, et al., 2014 aaNAT1 Y07964 5’-GAAGATTCCGTCGCTGATGG-3’ 3’-CTCCTTGAGGCTTGTAGTCG-5’ 20 20 Hintermann et al., 1996 β-actin AB039726 5’-CATTGTCACCAACTGGGATG-3’ 3’-CTCGAACATGATTTGGGTCA-5’ 20 20 Liu et al., 2006 Results Gene expression studies showed Deformed, Labial, abd-A, Abd-B, Ubx, AA-NAT and Vg genes expression in different photoperiods of Spodoptera litura adults. Among these, Vg gene expression levels did not change during the dark photoperiod. However, exogenous melatonin exposure in the presence of light (LD; 85 fold and LL; 10 fold) led to increased Vg expression levels. Our results show that vitellogenin (120 fold) expression is high at mel + aba exposed light photoperiod (LL), suggesting that vitellogenin production and accumulation require continuous light conditions (Figure.1). The results showed higher expression of Dfd(4.3 fold)in abamectin exposed 12:12 h LD condition, suggesting the formation of insect head responsive genes. Deformed mRNA levels were higher (2 fold) in the LL regim,e followed by the LD photoperiod (4.3 fold) under abamectin treatment (Figure.2). LabialmRNA expression (8.4 fold) was increased in the aba group of LL animal,s followed by the mel group (3.8 fold) under DD conditions (Figure.3). Deformed and labialmRNA levels were changed in response to light conditions. Our results did not show modifications on the Ubx expression in melatonin treated animals in the different photoperiods. However, abamectin exposure in dark photoperiod was higher (137 fold) in aba exposed group (Figure.4). Abdomen-A gene expression levels were higher at LD (55 fold) and DD (43 fold) photoperiods of mel + abagroup. The higher Abd-A levels (84 fold) were observed in the mel and aba groups of DD photoperiod (Figure.5). Abdomen-B gene expression levels were higher at DD photoperiod of mel + abagroup (182 fold) as compared to other photoperiods while melatonin showed 22 folds, abamectin showed 50 fold abd-B expression (Figure.6). AA-NAT level was increased in mel + aba group at DD conditions (40 fold) as compared with other photoperiods (LL, LD; Figure.7). In qRT-PCR, the melt curve analysis and the amplification of hox and melatonin genes by are represented in Figure.8a-c. Data presented are the ratio of transcript level of each gene versus control expressed as fold change. Discussion In insects, the development of front body parts like antennae and mouthparts is controlled by antennapedia (ANT-C) complex genes. Hox genes, which are similar across species, help shape embryos in fruit flies. These Hox clusters are also found in other insects like honeybees, beetles, grasshoppers, mosquitoes, and moths. Scientists use RNAi to study Hox genes like Deformed and maxillopedia in beetles and other early development genes in many insects. Many Hox genes have a homeodomain similar to the Drosophila Antennapedia gene. This study shows that Hox genes affect melatonin production in adult S.litura through AANAT. Vitellogenin levels are much higher in constant light, suggesting it needs continuous light to build up. Also, Hox gene Dfd mRNA levels rise with light, which may help develop insect head segments. Lab mRNA levels are higher in constant light with pesticides and in darkness with melatonin, suggesting melatonin affects head development during dark periods. Bithorax (BX-C) complex genes in S.litura adults In insects, the development of posterior segments is governed by the bithorax (BX-C) complex genes. Research into the developmental basis for Ubx expression has revealed a strong correlation between leg length and the formation of wings and halteres in insects. This gene is known to be responsible for the differential development of wings and halteres in various insects, such as Drosophila [ 18 ]. The study's results indicated no change in Ubx expression levels in either the melatonin groups or the photoperiods, except in the dark photoperiod with the ABA group, which exhibited higher Ubx expression levels (Figure.4). Our findings suggest that melatonin secretion, as well as abdominal and wing development, are regulated by Hox genes through the influence of photoperiod. Recent studies [ 42 , 43 ] have shown that in several orthopterans and dictyopterans, Ubx expression is specifically localized in the insect leg segments. In the lepidopteran insect Preciscoenia, Ubx is necessary for generating eyespots between forewings and hindwings, whereas in dipterans, the differential development of wings and halteres is a function of Ubx [ 18 ]. In Drosophila and other insects, Ultrabithorax and abdominal A proteins are mostly found in the abdominal segments. This study found that Abd-A gene levels were higher in the MEL + ABA groups during 12L:12D and 0L:24D light cycles. Higher Abd-A levels were seen in the MEL and ABA groups during the 0L:24D cycle. Abd-B gene levels were also high in the MEL + ABA group during the 0L:24D cycle compared to other cycles. Abd-A, abd-B, and aanat gene expressions were high in mel + aba exposed DD, while ubx expression was high in aba exposed DD. This suggests that melatonin, made at night, controls abdominal and wing development, confirmed by higher aanat expression in darkness. The study shows that Hox genes control melatonin production, affecting the abdomen and leg development in S. litura. Hox genes and melatonin production in insects involve several Hox genes controlling head development. In vertebrates, some Hox genes mainly work in mature pinealocytes. The cone-rod hox (Crx) gene helps regulate the enzyme for melatonin production. Hox genes play a role in pineal specificity and circadian output in vertebrates. Like vertebrates have photoreceptors in the retina, insect heads also have photoreceptors and internal clocks. The day/night rhythm in melatonin signals reflects light conditions and is independent of behavior. Insect eyes have many ommatidia with photoreceptor cells. Photoreceptor cells send light information to insect organs through brain cells that produce hormones controlling metabolism and behavior. Some hormones are released daily in insects. Light also activates central and peripheral clocks in insects. AA-NAT is a key enzyme during the day and increases at night in rats. AA-NAT is important for making melatonin, the darkness hormone. In vertebrates, the link between light sensing and melatonin production is well-studied. Vertebrates have cells that sense light and produce melatonin at night, guided by daily AA-NAT activity. In contrast, the role of Hox genes, light sensing, and melatonin production is only understood in vertebrates, not insects. In this study, we identified melatonin levels in the entire body of insects as a potential factor influencing developmental and reproductive performance. Consistent with our findings, Coon et al. observed a significant decrease in aanat mRNA expression in Macaca mulatta exposed to continuous light (LL) [ 50 ]. Additionally, other studies [ 51 , 52 ] reported increased aanat mRNA expression in Danio rerio and Carassius auratus under continuous darkness (DD). Our results also demonstrated that continuous darkness led to elevated aanat expression, suggesting that a persistently dark environment regulates melatonin synthesis through a substantial (40-fold) increase in aanat expression. In this study, S. litura larvae subjected to various photoperiods and melatonin treatments exhibited similar patterns of aanat mRNA expression. This may indicate that melatonin synthesis in the whole body of insects can regulate circadian rhythms through AANAT mRNA expression via non-photic cues. The relationship between melatonin and vitellogenin (Vg) transport from haemolymph to oocytes is facilitated by vitellogenin receptors (VgR) located on the oocyte surface. Several VgRs have been characterized in different insect species, including Drosophila, Aedes, Spodoptera, and Nilaparvata [ 20 , 21 , 53 , 54 ]. The results of this study demonstrated that Vg expression levels were directly influenced by the presence of light and melatonin, indicating that melatonin does affect vitellogenin in insects. Conclusion This study suggests that the circadian expression of melatonin is controlled by the Hox genes in S.litura . Vitellogenin, Deformed and Labial mRNA levels increased in response to light photoperiod, which shows that photoperiod regulates the activity Deformed and Labial genes controlling the insect head development. Ultrabithorax, Abdominal-A, Abdominal-B and AANAT gene expressions were higher at dark photoperiod than other photoperiods, suggesting that the melatonin secretion and wing development is regulated with photoperiodic cues. This study will help elucidate the role of photoperiod in the regulation of insect development and reproduction. Abbreviations Deformed ( Dfd ), Labial (Lab), abd-A (Abdomen-A), Abd-B (Abdomen-B), Ubx (Ultrabithorax), arylalkalyamine N-acetyltransferase ( AANAT ), Vitellogenin ( Vg ). Declarations Declaration of interest statement The authors declare that they do not have any conflicts of interest related to this study. Funding: This study was supported by the University Grants Commission, New Delhi, India under grant [F.No.42-201/2013 (SR)]. Ethics declaration: This study was granted ethical approval by Ayya Nadar Janaki Ammal College Data Availability Declaration: Data are available upon request from the authors Consent to Publish declaration: Not applicable Consent to Participate: Not applicable Acknowledgements: The authors wish to thank Dr. B.Ravi Shankar, Assistant Professor& Head, Dr. Balaji, Dr. Sathish, Dr. Sathya, Ph.D., Research scholars from ALMPG Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai for their immediate help for mRNA isolation and Real Time-PCR analysis for this research work. Author Contribution A. and C wrote the main manuscript text and B. did the language correction. All authors reviewed the manuscript. References Sabir N, Singh B: Protected cultivation of vegetables in global arena: A review . The Indian Journal of Agricultural Sciences 2013, 83 (2). Atwell BJ: Well-designed experiments make proteomic studies on stressed plants meaningful . 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Kim D-S, Brooks DJ, Riedl H: Lethal and sublethal effects of abamectin, spinosad, methoxyfenozide and acetamiprid on the predaceous plant bug Deraeocoris brevis in the laboratory . Biocontrol 2006, 51 :465-484. Geihs MA, Vargas MA, Maciel FE, Caldas SS, Cruz BP, Primel EG, Monserrat JM, Nery LEM: Effect of melatonin in the antioxidant defense system in the locomotor muscles of the estuarine crab Neohelice granulata (Decapoda, Brachyura) . General and comparative endocrinology 2010, 166 (1):72-82. Reno C, Marchuk L, Sciore P, Frank C, Hart DA: Rapid isolation of total RNA from small samples of hypocellular, dense connective tissues . Biotechniques 1997, 22 (6):1082-1086. Yasukochi Y, Ashakumary LA, Wu C, Yoshido A, Nohata J, Mita K, Sahara K: Organization of the Hox gene cluster of the silkworm, Bombyx mori: a split of the Hox cluster in a non-Drosophila insect . Development genes and evolution 2004, 214 :606-614. Bucher G, Scholten J, Klingler M: Parental RNAi in tribolium (coleoptera) . Current Biology 2002, 12 (3):R85-R86. Akin Z, Nazarali A: Hox genes and their candidate downstream targets in the developing central nervous system . Cellular and molecular neurobiology 2005, 25 :697-741. Mahfooz N, Turchyn N, Mihajlovic M, Hrycaj S, Popadić A: Ubx regulates differential enlargement and diversification of insect hind legs . PLoS One 2007, 2 (9):e866. Mahfooz NS, Li H, Popadić A: Differential expression patterns of the hox gene are associated with differential growth of insect hind legs . Proceedings of the National Academy of Sciences 2004, 101 (14):4877-4882. Rath MF, Rohde K, Klein DC, Møller M: Homeobox genes in the rodent pineal gland: roles in development and phenotype maintenance . Neurochemical research 2013, 38 :1100-1112. Hardeland R, Pandi-Perumal SR, Cardinali DP: Melatonin . The international journal of biochemistry & cell biology 2006, 38 (3):313-316. Giebultowicz JM: Insect circadian clocks: is it all in their heads? Journal of Insect Physiology 1999, 45 (9):791-800. Hodkova M: Tissue signaling pathways in the regulation of life-span and reproduction in females of the linden bug, Pyrrhocoris apterus . Journal of insect physiology 2008, 54 (2):508-517. Koštál V: Insect photoperiodic calendar and circadian clock: independence, cooperation, or unity? Journal of insect physiology 2011, 57 (5):538-556. Schmutz I, Albrecht U, Ripperger JA: The role of clock genes and rhythmicity in the liver . Molecular and cellular endocrinology 2012, 349 (1):38-44. Coon SL, Del Olmo E, Young III WS, Klein DC: Melatonin synthesis enzymes in Macaca mulatta: focus on arylalkylamine N-acetyltransferase (EC 2.3. 1.87) . The Journal of Clinical Endocrinology & Metabolism 2002, 87 (10):4699-4706. Choi JY, Kim NN, Choi YJ, Park MS, Choi CY: Differential daily rhythms of melatonin in the pineal gland and gut of goldfish Carassius auratus in response to light . Biological Rhythm Research 2016, 47 (1):145-161. Gothilf Y, Coon SL, Toyama R, Chitnis A, Namboodiri M, Klein DC: Zebrafish serotonin N-acetyltransferase-2: marker for development of pineal photoreceptors and circadian clock function . Endocrinology 1999, 140 (10):4895-4903. Shu Y, Wang J, Lu K, Zhou J, Zhou Q, Zhang G: The first vitellogenin receptor from a Lepidopteran insect: molecular characterization, expression patterns and RNA interference analysis . Insect Molecular Biology 2011, 20 (1):61-73. Tufail M, Takeda M: Insect vitellogenin/lipophorin receptors: molecular structures, role in oogenesis, and regulatory mechanisms . Journal of insect physiology 2009, 55 (2):88-104. Additional Declarations No competing interests reported. Supplementary Files SupplementaryfileforRTPCRCalculation.xlsx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 28 Apr, 2026 Reviews received at journal 27 Apr, 2026 Reviewers agreed at journal 19 Apr, 2026 Reviews received at journal 11 Apr, 2026 Reviewers agreed at journal 07 Apr, 2026 Reviewers agreed at journal 06 Apr, 2026 Reviewers invited by journal 05 Apr, 2026 Editor assigned by journal 09 Jan, 2026 Submission checks completed at journal 09 Jan, 2026 First submitted to journal 08 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8547288","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":619705061,"identity":"5ee8519a-1834-4982-8bdf-e518af81b1a0","order_by":0,"name":"SUBALA P Subramanian","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBklEQVRIie3PMUvEMBTA8ZRCXIJzbupXCBwEhZL7IC4vBOrSHoJrh4pwN+ncQfATuDpXCp0OuwZcrrtIQJAbRHztKLR3boL5DyGU9+M1hPh8fzJGyJYQoP3diRjP4KraS6AnaILyIulJcRghSELm6uHbJInK7MlBrpbH0U33zkSrHtY1bsnjszEi7NJwaMwlpUfz2Z14MY8bjaRJsmKM8FRwoJVe4WP4KxJZIQmKepREZTrfwddAwh0Tz0a23TQhNpVcrwZCZ0xUSto9W8TmLTnRtwZJQk9LYUBa3AITb4nWWW3dh9L3101o3adayPa827o8Hv+xn+lhEg4d71v8Ztjn8/n+R9/oFmLv/YZuywAAAABJRU5ErkJggg==","orcid":"","institution":"Ayya Nadar Janaki ammal college (autonomous)","correspondingAuthor":true,"prefix":"","firstName":"SUBALA","middleName":"P","lastName":"Subramanian","suffix":""},{"id":619705062,"identity":"8a143c64-0c61-4cfa-9867-4d350589c7e1","order_by":1,"name":"Eliningaya J. Kweka","email":"","orcid":"","institution":"Tanzania Commission for Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Eliningaya","middleName":"J.","lastName":"Kweka","suffix":""},{"id":619705063,"identity":"787ed9b7-d915-45b1-b56c-0ea9df149606","order_by":2,"name":"Shivakumar S Muthugounder","email":"","orcid":"","institution":"Periyar University","correspondingAuthor":false,"prefix":"","firstName":"Shivakumar","middleName":"S","lastName":"Muthugounder","suffix":""}],"badges":[],"createdAt":"2026-01-08 05:53:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8547288/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8547288/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106536665,"identity":"558d12a9-0b20-4612-94b2-7e179fc6adae","added_by":"auto","created_at":"2026-04-09 15:18:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":21871,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Photoperiod, melatonin, and abamectin exposure on \u003cem\u003evitellogenin\u003c/em\u003e \u003cem\u003e(Vg)\u003c/em\u003e gene expression profiles in the whole body of \u003cem\u003eS. litura \u003c/em\u003eadults by qRT-PCR (\u003cstrong\u003eNote: \u003c/strong\u003eData presented are the ratio of the transcript level of each gene versus the control expressed as fold change).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8547288/v1/66ce7309fe5fd09481f8f359.png"},{"id":106536667,"identity":"0201c8d5-165e-4f70-885e-db9145c30eb5","added_by":"auto","created_at":"2026-04-09 15:18:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":19712,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Photoperiod, melatonin and abamectin exposure in \u003cem\u003eDeformed (Dfd)\u003c/em\u003e gene expression profiles in the whole body of \u003cem\u003eS.litura \u003c/em\u003eadults by qRT-PCR (\u003cstrong\u003eNote\u003c/strong\u003e:Data presented are the ratio of transcript level of each gene versus control expressed as fold change).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8547288/v1/42ad23e242c00708279f0995.png"},{"id":106725214,"identity":"d301bd47-7ecd-4457-a1e0-4fc449e9e8ee","added_by":"auto","created_at":"2026-04-12 18:31:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":18902,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Photoperiod, melatonin and abamectin exposure in \u003cem\u003eLabial (Lab)\u003c/em\u003e gene expression profiles in the whole body of \u003cem\u003eS.litura \u003c/em\u003eadults by qRT-PCR (\u003cstrong\u003eNote\u003c/strong\u003e: Data presented are the ratio of the transcript level of each gene versus control expressed as fold change).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8547288/v1/6d795dc228c25e3aac0ec978.png"},{"id":106536668,"identity":"22f5462d-5cbf-4291-a2e2-3894097325e1","added_by":"auto","created_at":"2026-04-09 15:18:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":17363,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Photoperiod, melatonin and abamectin exposure in \u003cem\u003eultrabithorax (Ubx)\u003c/em\u003e gene expression profiles in the whole body of \u003cem\u003eS.litura \u003c/em\u003eadults by qRT-PCR. (\u003cstrong\u003eNote: \u003c/strong\u003eData presented are the ratio of transcript level of each gene versus control expressed as fold change).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8547288/v1/c287d7eb344751511609dbdb.png"},{"id":106536670,"identity":"774f1c85-d4b5-4d9a-88c6-e73b2bf15625","added_by":"auto","created_at":"2026-04-09 15:18:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":22606,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Photoperiod, melatonin and abamectin exposure in \u003cem\u003eabdomen-A (abd-A) \u003c/em\u003egene expression profiles in the whole body of \u003cem\u003eS.litura \u003c/em\u003eadults by qRT-PCR. Data presented are the ratio of transcript level of each gene versus control expressed as fold change.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8547288/v1/f78929f8b731525f4fc2627d.png"},{"id":106536671,"identity":"ac463ff3-20ff-4977-9687-04fc654f2d98","added_by":"auto","created_at":"2026-04-09 15:18:10","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":17861,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Photoperiod, melatonin and abamectin exposure in \u003cem\u003eAbdomen-B (Abd-B)\u003c/em\u003e gene expression profiles in the whole body of \u003cem\u003eS.litura \u003c/em\u003eadults by qRT-PCR. Data presented are the ratio of transcript level of each gene versus control expressed as fold change.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8547288/v1/7e0ac45163b4e2df91db7425.png"},{"id":106536673,"identity":"95955a33-c064-4823-866d-656e9bdce1c1","added_by":"auto","created_at":"2026-04-09 15:18:10","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":20367,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Photoperiod, melatonin and abamectin exposure in \u003cem\u003eN-acetyl transferase(AA-NAT)\u003c/em\u003e gene expression profiles in the whole body of \u003cem\u003eS.litura \u003c/em\u003eadults by qRT-PCR. Data presented are the ratio of transcript level of each gene versus control expressed as fold change.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8547288/v1/59e033ecad00d9075341deae.png"},{"id":106725717,"identity":"fe966908-7d32-493a-8564-f88db7634a8e","added_by":"auto","created_at":"2026-04-12 18:33:38","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":260922,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Photoperiod, melatonin and abamectin exposure in the melt curve analysis of the whole body of \u003cem\u003eS.litura \u003c/em\u003eadults by qRT-PCR. Data presented are the ratio of transcript level of each gene versus control expressed as fold change.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8547288/v1/348728c291c50ffcbeb7ef34.png"},{"id":106727386,"identity":"9d2bbae8-609e-4eca-b14c-1d2328c8dc32","added_by":"auto","created_at":"2026-04-12 18:38:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3360772,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8547288/v1/a2b71164-301a-4516-b802-902d5c87f9be.pdf"},{"id":106724710,"identity":"2afd60ef-b9c2-4a87-bee5-f5e40e26184a","added_by":"auto","created_at":"2026-04-12 18:29:20","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":54085,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryfileforRTPCRCalculation.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8547288/v1/c8fc9a0b671c513e405d11d8.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of photoperiodism, melatonin and pesticide exposure on Homeobox and N-acetyl transferase gene expression profiles in Spodoptera litura (Insecta: Lepidoptera)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAgricultural pests have been a major hindering factor in revolutionizing the horticulture sector productivity in India and tropical Asia of which Lepidopteran pests significantly cause yield loss. Vegetables are among the most profitable crops, and farmers everywhere feel the need to protect these high-value crops from any damage caused by insect pests [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Farmers often use synthetic insecticides randomly, and insecticide resistance has been identified among commonly used pesticides [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. \u003cem\u003eSpodoptera litura\u003c/em\u003e Fabricus (Lepidoptera: Noctuidae) is a polyphagous insect with about 172 species of host plants from 40 families in India. It is one of the most economically important insect pests in many countries, including India, Japan, China, and other countries in Southeast Asia and Africa [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In India, \u003cem\u003eS. litura\u003c/em\u003e hinders crop production. It has been reported on approximately 112 cultivated plants and causes economic losses ranging from 25.8% to 100%. This nocturnal insect was chosen as a model for this study because of its availability, fast growth, and polyphagous nature [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHox genes serve as the main regulators of embryonic development in all animals, including flies, humans, and other animals. It is now possible to examine the molecular mechanisms by which genomic information is subsequently transformed into physiological and morphological processes during the development of many organisms [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The Antennapedia and Bithorax complexes are two distinct gene clusters in which Hox genes are found in Drosophila [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. These genes encode transcription factors (TFs) called homeodomain (HD)-containing TFs, which regulate the expression of numerous downstream target genes by identifying particular enhancers [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHomeobox genes (Hox) encode a large group of transcription factors involved in developmental processes throughout the animal kingdom [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. More than 300 Hox genes and their relatives have been identified in fungi, plants, and animals [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In insects, Hox genes specify the structures along the anteroposterior body axis; three are necessary for the correct development of head segments and appendages: the Labial (lab), Proboscipedia (Pb), and Deformed (Dfd) genes. The Labial (Lab) gene is involved in the proper development of head and mouth structures in insects [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In contrast, the Deformed (Dfd) gene is involved in the formation of head parts, particularly maxillae and mandible,s in insects[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. During insect embryogenesis, Hox proteins promote unique morphological patterning events in the abdominal segments, particularly Ultrabithorax (Ubx), Abdominal-A (Abd-A), and Abdominal-B (Abd-B) genes [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Ultrabithorax(Ubx) is known to suppress wing formation in insects, and the differential development of wings and halteres is dependent on the function of this Hox protein in \u003cem\u003eDrosophila\u003c/em\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In addition, because of the high expression of Ubx and Abd-A proteins in the abdominal segments, they may suppress thoracic leg development during embryogenesis in \u003cem\u003eDrosophila melanogaster\u003c/em\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Vitellogenin (Vg) plays a vital role in oocytes and insect embryo development. It is synthesized in the fat body, moves through the hemolymph, and accumulates in oocytes [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. One of these Hox genes, the cone-rod homeobox (Crx) gene, seems to play an important role in the regulation of \u003cem\u003eaanat\u003c/em\u003e transcription [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMelatonin is a circadian rhythm regulator secreted in insects and participates in insect development [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], but numerous studies have documented its activity as a free radical scavenger [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and antioxidant agent [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In vertebrates, melatonin is synthesized mainly at night because of circadian rhythm modulation. This reaction is mediated by several enzymes, including \u003cem\u003eAANAT\u003c/em\u003e [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Based on the available literature, we suggest that hox genes and melatonin are responsible for insect development. In addition, the cone-rod homeobox (Crx) transcription factor regulates the expression of NAT in the mouse pineal gland. LIM homeobox transcription factor Isl1 positively modulates melatonin synthesis by targeting NAT at the (ATTA/TAAT) motif via the ERK signaling pathway of norepinephrine [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent studies by Subala and Shivakumar described the impact of circadian rhythm on melatonin levels and redox status under three photoperiods (12L:12D, 0L:24D, and 24L:0D) in the head and hemolymph of \u003cem\u003eS. litura\u003c/em\u003e [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Previous research has described that pre-treatment with melatonin decreases abamectin-induced toxicity in the nocturnal insect \u003cem\u003eS. litura\u003c/em\u003e [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. It is thought that timing exposure to pesticides may influence insect development, which may be normalized due to melatonin activity [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Moreover, we have previously reported that photoperiod influences the digestive biology of \u003cem\u003eS. litura\u003c/em\u003e [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInsects are attractive model systems for studying and elucidating the function of many early developmental genes (including hox genes) because of their short life span, and a large number of animals can be collected from the field and/or reared in laboratories [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Light has been known to affect insect development through subtle unknown mechanisms. Among the genes controlling insect development, the Hox gene complex is known to be very important, as it controls the body plan of insects [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Although Hox genes play a major role in shaping the anterior-posterior body axis during animal development, our understanding of how they act in the presence of melatonin, pesticides, and different photoperiods in insects is rudimentary.\u003c/p\u003e \u003cp\u003eAs mentioned previously, the major role of Hox genes is in the body patterning of animal development. However, the molecular mechanisms underlying the control of hox by melatonin or vice versa in invertebrates are poorly understood [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. This study aimed to summarize the developmental mechanisms of rhythmic melatonin and Hox gene expression in insects. To achieve this purpose, we investigated (i) the role of hox genes, particularly two antennapedia complex genes (\u003cem\u003eDfd\u003c/em\u003e, and Lab), and three bithorax complex genes (abd-A, Abd-B, and Ubx), under different photoperiods; and (ii) the vitellogenin (Vg) and N-acetyl transferase (\u003cem\u003eAANAT\u003c/em\u003e) gene expression profiles on vitellogenin and melatonin synthesis under different photoperiods in the whole body of \u003cem\u003eSpodoptera litura\u003c/em\u003e adults.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eInsect rearing\u003c/h2\u003e \u003cp\u003e \u003cem\u003eSpodoptera litura\u003c/em\u003e eggs were obtained from the National Bureau of Agricultural Insect Resources (NBAIR), formerly the National Bureau of Agricultural Important Insects (NBAII, Bangalore, India) with National Accession No. NBAII-MP-NOC-02. Larvae were maintained on castor leaves as food and maintained at 28\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, 70\u0026thinsp;\u0026plusmn;\u0026thinsp;10% Relative Humidity (RH) under a photoperiod of 12L:12D. Male and female pupae were segregated based on the morphology of the abdominal terminal segments. Virgin males and females were separated into different plastic cups (20\u0026times;20\u0026times;30 cm) and fed with a 10% honey solution. Three-day-old adults were placed in 100 ml plastic containers for the experiments.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eChemicals\u003c/h3\u003e\n\u003cp\u003eMelatonin was purchased from Sigma\u0026ndash;Aldrich (India). Abamectin (N-acetyl 2-benzyl tryptamine) was procured from Insecticides Pvt. Ltd, New Delhi (India) and has the commercial grade name of Agrimek with 0.15% EC. The highest dosage of abamectin used was 1.56 ml (28 mg AI)/L, according to previous studies [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Melatonin (4.3\u0026times;10\u0026thinsp;\u0026minus;\u0026thinsp;5 M) was administered to the melatonin group as previously described [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. All other chemicals and reagents used in this study were of molecular grade.\u003c/p\u003e\n\u003ch3\u003eExperimental Design\u003c/h3\u003e\n\u003cp\u003eThe experimental larvae were exposed to light using a white fluorescent bulb (27 W, wavelength range 350\u0026ndash;650 nm). The light intensity was approximately 0.96W/cm2. Third instar larvae (n\u0026thinsp;=\u0026thinsp;1080), on day-2 were divided randomly into three photoperiod groups:\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eNormal (LD): 12-h light: 12-h dark cycle (12L:12D; n\u0026thinsp;=\u0026thinsp;360 larvae)\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eContinuous light (LL): 24-h light cycle (24L:0D; n\u0026thinsp;=\u0026thinsp;360 larvae)\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eContinuous dark (DD): 24-h dark cycle (0L:24D; n\u0026thinsp;=\u0026thinsp;360 larvae)\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eEach of these experimental photoperiodic groups (n\u0026thinsp;=\u0026thinsp;360) of larvae was divided into four subgroups (n\u0026thinsp;=\u0026thinsp;90) as follows:\u003c/p\u003e\u003cp\u003e \u003cb\u003e(a) Control group\u003c/b\u003e: 0.1M Phosphate buffered saline containing Na2HPo4 \u0026amp; NaH2Po4; pH 7.5)\u003c/p\u003e \u003cp\u003e \u003cb\u003e(b) Melatonin group (Mel)\u003c/b\u003e: Melatonin was dissolved in ethanol (\u0026gt;\u0026thinsp;0.01%)[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e \u003cb\u003e(c) Abamectin group (Aba)\u003c/b\u003e: An equal volume of cold saline (0.1M PBS, Na2HPo4, and NaH2Po4; pH 7.5) was added to the abamectin [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003e(d) Melatonin\u0026thinsp;+\u0026thinsp;Abamectin group (Mel\u0026thinsp;+\u0026thinsp;Aba)\u003c/b\u003e: Both were incorporated into the larval diet until they reached the fifth instar stage.\u003c/p\u003e \u003cp\u003eThe experiment was performed in three independent biological replicates. Therefore, from each subgroup (n\u0026thinsp;=\u0026thinsp;90), we obtained three replicates (30 larvae per replicate) that were kept in plastic containers and fed with 100 ml of artificial diet according to previous studies (Kranthi, 2005). The artificial diet was replaced every three days.\u003c/p\u003e\n\u003ch3\u003eRNA extraction and cDNA synthesis\u003c/h3\u003e\n\u003cp\u003eBefore dissection of the whole moth, the wings were removed and weighed. \u003cem\u003eS. litura\u003c/em\u003e adults were dissected under dim light. In this study, Total RNA was manually isolated from the whole bodies of adults kept under three different photoperiods using the TRIZOL method [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. RNA concentrations were quantified by measuring the absorbance at A260/280 nm. The purity of RNA obtained was 1.8\u0026ndash;2.0, and the yield of RNA was expressed in \u0026micro;g/ml. 1\u0026micro;g of total RNA was reverse transcribed to complementary DNA (cDNA) using a cDNA synthesis kit (Himedia, India) according to the manufacturer\u0026rsquo;s protocol (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe Nanodrop reading of total RNA\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePhotoperiod\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMelatonin\u003c/p\u003e \u003cp\u003e(\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003cp\u003e(\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAbamectin\u003c/p\u003e \u003cp\u003e(\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMelatonin\u0026thinsp;+\u0026thinsp;Abamectin\u003c/p\u003e \u003cp\u003e(\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12L:12D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e764.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e763.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e477.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e766.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0L:24D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1072.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1083.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e751.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1108.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e24L:0D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1025\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1084.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1056.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e700.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eNote\u003c/strong\u003e \u003cp\u003eRNA concentrations were quantified by measuring the absorbance at A260/280. The purity of RNA obtained was 1.8\u0026ndash;2.0, and the yield of RNA was expressed in \u0026micro;g/ml.\u003c/p\u003e \u003c/p\u003e\n\u003ch3\u003eQuantitative Real Time PCR (RT-PCR) analysis\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eQuantitative Real Time PCR (RT-PCR) analysis\u003c/div\u003e \u003cp\u003eFor RT-PCR analysis, primers (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) were designed using Primer 3 software (Applied Biosystems, California). The designed primer characteristics were analyzed and confirmed using NetPrimer (Premier Biosoft). SYBR Green PCR was conducted in 0.2 ml PCR tubes with flat 8-cap strips (Axygen, USA) using iQTM SYBR Green Supermix (Bio-Rad, Hercules, USA). Primer alignment was performed with the BLAST database to ensure primer specificity. RT PCR was conducted using a Bio-Rad CFX96 Real-time PCR Detection System (Bio-Rad, Hercules, USA) following the manufactures protocol. RT-PCR was conducted to evaluate the relative gene expression levels in the whole insect body.\u003c/p\u003e \u003cp\u003eThe RT-PCR program was performed with a melting curve dissociation protocol (from 58 to 95\u0026deg;C) according to the following thermal conditions: initial denaturation at 95\u0026deg;C for 3 min, followed by 46 cycles at 58\u0026deg;C for 1 min and 59\u0026deg;C for 10s. The experiments were performed in duplicate. Gene specificity was determined using the dissociation curve, and validation experiments were conducted to confirm that the efficiencies of the target and reference gene amplifications were approximately equal (80\u0026ndash;90%). Gene-specific oligonucleotide primers for the housekeeping gene β-actin were added to the same PCR reaction vial and co-amplified.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimers used in Real Time-PCR analysis\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenbank Accession Number\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePrimer Sequences\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFragment length (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eReferences\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVTG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHQ260608\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-GACTATAAAGATTGTTCCATGTG-3\u0026rsquo;\u003c/p\u003e \u003cp\u003e3\u0026rsquo;-GAGTATGGTGGAGAATCATTAGT-5\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e23\u003c/p\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePark and Kwak, 2012\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDfd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eD83534\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-ATGAGCTCCTTCCTGATG-3\u0026rsquo;\u003c/p\u003e \u003cp\u003e3\u0026rsquo;-TGTGGAAGAGGATGGTAG-5\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e18\u003c/p\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eParthasarathy and Gopinathan, 2005\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAB120761\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-ACTTCACCAACAAACAGCTC-3\u0026rsquo;\u003c/p\u003e \u003cp\u003e3\u0026rsquo;-TGATCCGTTTCTTCTGCTTC-5\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKimato, et al., 2014\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUbx\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAB505052\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-ATGAACTCTTACTTCGAGC-3\u0026rsquo;\u003c/p\u003e \u003cp\u003e3\u0026rsquo;-GTTCAAATGTTTTAATGTTCGG-5\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e19\u003c/p\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKimato, et al., 2014\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eabd-A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAB461860\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-TCTACCTCGCTGTCTAGTTC-3\u0026rsquo;\u003c/p\u003e \u003cp\u003e3\u0026rsquo;-TGCTGCTGCTTCATTCTGTC-5\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKimato, et al., 2014\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbd-B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAB461858\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-CAACCAGCAGTGGTGAATAC-3\u0026rsquo;\u003c/p\u003e \u003cp\u003e3\u0026rsquo;-TTGTTGTTCTGGGCAGCTTG-5\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eKimato, et al., 2014\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eaaNAT1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eY07964\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-GAAGATTCCGTCGCTGATGG-3\u0026rsquo;\u003c/p\u003e \u003cp\u003e3\u0026rsquo;-CTCCTTGAGGCTTGTAGTCG-5\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHintermann et al., 1996\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ-actin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAB039726\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026rsquo;-CATTGTCACCAACTGGGATG-3\u0026rsquo;\u003c/p\u003e \u003cp\u003e3\u0026rsquo;-CTCGAACATGATTTGGGTCA-5\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLiu et al., 2006\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eGene expression studies showed Deformed, Labial, abd-A, Abd-B, Ubx, AA-NAT and Vg genes expression in different photoperiods of \u003cem\u003eSpodoptera litura\u003c/em\u003e adults. Among these, \u003cem\u003eVg\u003c/em\u003e gene expression levels did not change during the dark photoperiod. However, exogenous melatonin exposure in the presence of light (LD; 85 fold and LL; 10 fold) led to increased \u003cem\u003eVg expression\u003c/em\u003e levels. Our results show that vitellogenin (120 fold) expression is high at mel\u0026thinsp;+\u0026thinsp;aba exposed light photoperiod (LL), suggesting that vitellogenin production and accumulation require continuous light conditions (Figure.1).\u003c/p\u003e\u003cp\u003eThe results showed higher expression of Dfd(4.3 fold)in abamectin exposed 12:12 h LD condition, suggesting the formation of insect head responsive genes. Deformed mRNA levels were higher (2 fold) in the LL regim,e followed by the LD photoperiod (4.3 fold) under abamectin treatment (Figure.2). LabialmRNA expression (8.4 fold) was increased in the aba group of LL animal,s followed by the mel group (3.8 fold) under DD conditions (Figure.3). Deformed and labialmRNA levels were changed in response to light conditions. Our results did not show modifications on the \u003cem\u003eUbx\u003c/em\u003e expression in melatonin treated animals in the different photoperiods. However, abamectin exposure in dark photoperiod was higher (137 fold) in aba exposed group (Figure.4).\u003c/p\u003e\u003cp\u003eAbdomen-A gene expression levels were higher at LD (55 fold) and DD (43 fold) photoperiods of mel\u0026thinsp;+\u0026thinsp;abagroup. The higher \u003cem\u003eAbd-A\u003c/em\u003e levels (84 fold) were observed in the mel and aba groups of DD photoperiod (Figure.5). Abdomen-B gene expression levels were higher at DD photoperiod of mel\u0026thinsp;+\u0026thinsp;abagroup (182 fold) as compared to other photoperiods while melatonin showed 22 folds, abamectin showed 50 fold abd-B expression (Figure.6). AA-NAT level was increased in mel\u0026thinsp;+\u0026thinsp;aba group at DD conditions (40 fold) as compared with other photoperiods (LL, LD; Figure.7). In qRT-PCR, the melt curve analysis and the amplification of hox and melatonin genes by are represented in Figure.8a-c. Data presented are the ratio of transcript level of each gene versus control expressed as fold change.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn insects, the development of front body parts like antennae and mouthparts is controlled by antennapedia (ANT-C) complex genes. Hox genes, which are similar across species, help shape embryos in fruit flies. These Hox clusters are also found in other insects like honeybees, beetles, grasshoppers, mosquitoes, and moths. Scientists use RNAi to study Hox genes like Deformed and maxillopedia in beetles and other early development genes in many insects. Many Hox genes have a homeodomain similar to the Drosophila Antennapedia gene. This study shows that Hox genes affect melatonin production in adult S.litura through AANAT. Vitellogenin levels are much higher in constant light, suggesting it needs continuous light to build up. Also, Hox gene Dfd mRNA levels rise with light, which may help develop insect head segments. Lab mRNA levels are higher in constant light with pesticides and in darkness with melatonin, suggesting melatonin affects head development during dark periods.\u003c/p\u003e\n\u003ch3\u003eBithorax (BX-C) complex genes in S.litura adults\u003c/h3\u003e\n\u003cp\u003eIn insects, the development of posterior segments is governed by the bithorax (BX-C) complex genes. Research into the developmental basis for Ubx expression has revealed a strong correlation between leg length and the formation of wings and halteres in insects. This gene is known to be responsible for the differential development of wings and halteres in various insects, such as Drosophila [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The study's results indicated no change in Ubx expression levels in either the melatonin groups or the photoperiods, except in the dark photoperiod with the ABA group, which exhibited higher Ubx expression levels (Figure.4). Our findings suggest that melatonin secretion, as well as abdominal and wing development, are regulated by Hox genes through the influence of photoperiod. Recent studies [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] have shown that in several orthopterans and dictyopterans, Ubx expression is specifically localized in the insect leg segments. In the lepidopteran insect Preciscoenia, Ubx is necessary for generating eyespots between forewings and hindwings, whereas in dipterans, the differential development of wings and halteres is a function of Ubx [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn Drosophila and other insects, Ultrabithorax and abdominal A proteins are mostly found in the abdominal segments. This study found that Abd-A gene levels were higher in the MEL\u0026thinsp;+\u0026thinsp;ABA groups during 12L:12D and 0L:24D light cycles. Higher Abd-A levels were seen in the MEL and ABA groups during the 0L:24D cycle. Abd-B gene levels were also high in the MEL\u0026thinsp;+\u0026thinsp;ABA group during the 0L:24D cycle compared to other cycles. Abd-A, abd-B, and aanat gene expressions were high in mel\u0026thinsp;+\u0026thinsp;aba exposed DD, while ubx expression was high in aba exposed DD. This suggests that melatonin, made at night, controls abdominal and wing development, confirmed by higher aanat expression in darkness. The study shows that Hox genes control melatonin production, affecting the abdomen and leg development in S. litura. Hox genes and melatonin production in insects involve several Hox genes controlling head development. In vertebrates, some Hox genes mainly work in mature pinealocytes. The cone-rod hox (Crx) gene helps regulate the enzyme for melatonin production. Hox genes play a role in pineal specificity and circadian output in vertebrates. Like vertebrates have photoreceptors in the retina, insect heads also have photoreceptors and internal clocks. The day/night rhythm in melatonin signals reflects light conditions and is independent of behavior. Insect eyes have many ommatidia with photoreceptor cells. Photoreceptor cells send light information to insect organs through brain cells that produce hormones controlling metabolism and behavior. Some hormones are released daily in insects. Light also activates central and peripheral clocks in insects. AA-NAT is a key enzyme during the day and increases at night in rats. AA-NAT is important for making melatonin, the darkness hormone. In vertebrates, the link between light sensing and melatonin production is well-studied. Vertebrates have cells that sense light and produce melatonin at night, guided by daily AA-NAT activity. In contrast, the role of Hox genes, light sensing, and melatonin production is only understood in vertebrates, not insects.\u003c/p\u003e \u003cp\u003eIn this study, we identified melatonin levels in the entire body of insects as a potential factor influencing developmental and reproductive performance. Consistent with our findings, Coon et al. observed a significant decrease in aanat mRNA expression in Macaca mulatta exposed to continuous light (LL) [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Additionally, other studies [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] reported increased aanat mRNA expression in Danio rerio and Carassius auratus under continuous darkness (DD). Our results also demonstrated that continuous darkness led to elevated aanat expression, suggesting that a persistently dark environment regulates melatonin synthesis through a substantial (40-fold) increase in aanat expression. In this study, S. litura larvae subjected to various photoperiods and melatonin treatments exhibited similar patterns of aanat mRNA expression. This may indicate that melatonin synthesis in the whole body of insects can regulate circadian rhythms through AANAT mRNA expression via non-photic cues.\u003c/p\u003e \u003cp\u003eThe relationship between melatonin and vitellogenin (Vg) transport from haemolymph to oocytes is facilitated by vitellogenin receptors (VgR) located on the oocyte surface. Several VgRs have been characterized in different insect species, including Drosophila, Aedes, Spodoptera, and Nilaparvata [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. The results of this study demonstrated that Vg expression levels were directly influenced by the presence of light and melatonin, indicating that melatonin does affect vitellogenin in insects.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study suggests that the circadian expression of melatonin is controlled by the Hox genes in \u003cem\u003eS.litura\u003c/em\u003e. Vitellogenin, Deformed and Labial mRNA levels increased in response to light photoperiod, which shows that photoperiod regulates the activity Deformed and Labial genes controlling the insect head development. Ultrabithorax, Abdominal-A, Abdominal-B and AANAT gene expressions were higher at dark photoperiod than other photoperiods, suggesting that the melatonin secretion and wing development is regulated with photoperiodic cues. This study will help elucidate the role of photoperiod in the regulation of insect development and reproduction.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eDeformed (\u003cem\u003eDfd\u003c/em\u003e), \u003cem\u003eLabial\u003c/em\u003e (Lab), \u003cem\u003eabd-A\u003c/em\u003e (Abdomen-A), \u003cem\u003eAbd-B\u003c/em\u003e (Abdomen-B), \u003cem\u003eUbx\u003c/em\u003e (Ultrabithorax), arylalkalyamine N-acetyltransferase (\u003cem\u003eAANAT\u003c/em\u003e), \u003cem\u003eVitellogenin\u003c/em\u003e (\u003cem\u003eVg\u003c/em\u003e).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of interest statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they do not have any conflicts of interest related to this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the University Grants Commission, New Delhi, India under grant [F.No.42-201/2013 (SR)].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declaration:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was granted ethical approval by Ayya Nadar Janaki Ammal College\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Declaration:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eData are available upon request from the authors\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish declaration:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors wish to thank Dr. B.Ravi Shankar, Assistant Professor\u0026amp; Head, Dr. Balaji, Dr. Sathish, Dr. Sathya, Ph.D., Research scholars from ALMPG Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai for their immediate help for mRNA isolation and Real Time-PCR analysis for this research work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. and C wrote the main manuscript text and B. did the language correction. 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\u003cstrong\u003e55\u003c/strong\u003e(2):88-104.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-tropical-insect-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtis","sideBox":"Learn more about [International Journal of Tropical Insect Science](http://link.springer.com/journal/42690)","snPcode":"42690","submissionUrl":"https://www.editorialmanager.com/jtis/default2.aspx","title":"International Journal of Tropical Insect Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Arylalkalyamine N-acetyl transferase, photoperiod, melatonin, vitellogenin, homeobox genes, photoperiodic hormone, insect development","lastPublishedDoi":"10.21203/rs.3.rs-8547288/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8547288/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHomeobox (HOX) genes play a major role in shaping the anteroposterior body axis during insect development. Melatonin is a photoperiodic hormone that is involved in insect developmental mechanisms. Here, we demonstrate the presence of Hox and melatonin genes (aanat) in the whole body of \u003cem\u003eS. litu\u003c/em\u003e The results showed increased expression of Dfd (4.3 fold) and Lab (8.4 fold) in abamectin exposed 12:12 h light-dark (LD) condition, suggesting the formation of insect head responsive genes. While Abd-A (55 fold), abd-B (182 fold), and aanat(40 fold) expression were high in mel\u0026thinsp;+\u0026thinsp;aba-exposed DD, ubx (137 fold) expression in aba-exposed DD conditions suggests control of abdominal and wing development due to nocturnal synthesis of melatonin, which is confirmed by higher aanat expression in darkness. Our results show that vitellogenin (85 and 120 fold) expression is high under mel and mel\u0026thinsp;+\u0026thinsp;aba exposed light photoperiod (LL), suggesting that vitellogenin production and accumulation requires continuous light.\u003c/p\u003e","manuscriptTitle":"Effects of photoperiodism, melatonin and pesticide exposure on Homeobox and N-acetyl transferase gene expression profiles in Spodoptera litura (Insecta: Lepidoptera)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-09 15:18:05","doi":"10.21203/rs.3.rs-8547288/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-04-28T14:55:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-27T04:25:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"292136546516519502152016721884592182924","date":"2026-04-19T14:02:05+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-12T03:36:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"9128670302107606660071923907998503550","date":"2026-04-07T10:18:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"218307788458917043355057398075371866578","date":"2026-04-06T05:03:23+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-05T12:16:21+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-09T09:39:24+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-09T09:37:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"International Journal of Tropical Insect Science","date":"2026-01-08T05:35:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-tropical-insect-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtis","sideBox":"Learn more about [International Journal of Tropical Insect Science](http://link.springer.com/journal/42690)","snPcode":"42690","submissionUrl":"https://www.editorialmanager.com/jtis/default2.aspx","title":"International Journal of Tropical Insect Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7a00e92c-dd1b-4724-ae9f-6915d26f3853","owner":[],"postedDate":"April 9th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-09T15:18:05+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-09 15:18:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8547288","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8547288","identity":"rs-8547288","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}