Microsurgical Obstruction of Testes Fusion in <em>Spodoptera litura</em>

Instead of using genetic methods like RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated ...

South China Normal University-Shipai Campus: South China Normal University

Wing discs of B. mori are transformed to pupal wings during the larva-to-pupa metamorphosis with dramatic morphological and structural changes. To understand these changes at a transcriptional level, RNA-seq of the wing discs from 6-day-old fifth instar larvae (L5D6), prepupae (PP) and pupae (P0) was performed.

Transcriptomic analysis of developmental features of Bombyx mori wing disc during metamorphosis.

20-hydroxyecdysone (20E)-induced expression of the wing disc cuticle protein gene BmWCP4 was mediated by the transcription factor BmPOUM2, which binds to the cis-response elements (CREs) of BmWCP4 gene in Bombyx mori. In this study we report the regulation of BmPOUM2. RT-PCR analysis indicated that in response to 20E, BmPOUM2 was expressed at higher levels in the wing discs during the pre-pupal and mid-pupal stages than other stages and the expression pattern of BmBR-C Z1, BmBR-C Z2 and BmBR-C Z4 was in tandem with the expression of BmPOUM2. BmBR-C Z4 was induced by 20E in the wing discs, whereas BmBR-C Z1 and BmBR-C Z2 were not. Three potential BR-C Z4 cis-response elements (CREs) were identified in the promoter region of BmPOUM2. The expression of BmPOUM2 mRNA and protein was increased by the over-expression of BmBR-C Z4 in BmN cells, which acted at the promoter of BmPOUM2. Electrophoretic mobility shift assay (EMSA) and the luciferase activity analysis under the control of wild-type and mutants of the BR-C Z4 CREs suggested that BmBR-C Z4 protein bound to the predicted BRC-Z4 CRE C (-684 ∼ -660). Taken together the data suggest that BmBR-C Z4 is a direct upstream regulator of BmPOUM2 and regulates the pupal-specific expression of BmWCP4 through BmPOUM2. 

BmBR-C Z4 is an upstream regulatory factor of BmPOUM2 controlling the pupal specific expression of BmWCP4 in the silkworm, Bombyx mori.

BESS domain is a protein binding domain that can interact each other or with other domains. In this study, 323 BESS domain containing (BESS-DC) proteins were identified in 3328 proteomes. These BESS-DC genes pertain to 41 species of five phyla, most of which are arthropod insects. A BESS domain contains two α-helixes linked by a coil or β-turn. Phylogenetic tree and architecture analysis show that the BESS domain seems to generate along with the DNA-binding MADF domain. Two hundred thirty three BESS-DC genes (71.1%) contain at least one MADF domain, while 59 genes (18.2%) had only the BESS domain. In addition to BESS and MADF domains, some of genes also contain other ligand binding domains, such as DAO, DUS and NAD_C. Nineteen genes (5.8%) are associated with other DNA binding domains, such as Myb and BED. The BESS-DC genes can be divided into 17 subfamilies, eight of which have more than one clade. In Bombyx mori, 12 BESS-DC genes that do not contain intron in the BESS domain region were localized to eight chromosomes. Real-time PCR results showed that most of the B. mori BESS-DC genes highly expressed from late larval stage to adult stage. The results of sequence comparison and evolution analyses suggest a hypothesis that the BESS-DC genes may play a role in central nervous system development, long term memory and metamorphosis of insects of different phyla. 

In silico identification of BESS-DC genes and expression analysis in the silkworm, Bombyx mori.

Spodoptera litura is polyphagous pest insect and feeds on plants of more than 90 families. In this study the role of glutathione S-transferase epilson 1 (slgste1) in S. litura in detoxification was examined. This gene was up-regulated in the midgut of S. litura at the transcriptional and protein levels when the insect fed on Brassica juncea or diet containing phytochemicals such as indole-3-carbinol and allyl-isothiocyanate that are metabolic products of sinigrin and glucobrassicin in B. juncea. The SlGSTE1 could catalyze the conjugation of reduced glutathione and indole-3-carbinol and allyl-isothiocyanate, as well as xanthotoxin, which is a furanocoumarin, under in vitro condition. When the expression of Slgste1 in the larvae was suppressed with RNAi, the larval growth and feeding rate were decreased. Furthermore, the up-regulated expression of the SlGSTE1 protein in the midgut of larvae that fed on different host plants was detected by 2-DE and ESI/MS analysis. The feeding adaptation from the most to the least of the larvae for the various host plants was Brassica alboglabra, Brassica linn. Pekinensis, Cucumis sativus, Ipomoea batatas, Arachis hypogaea and Capsicum frutescens. All the results together suggest that Slgste1 is a critical detoxifying enzyme that is induced by phytochmicals in the host plants and, inter alia, may be related to host plant adaptation of S. litura. 

Glutathione S-transferase SlGSTE1 in Spodoptera litura may be associated with feeding adaptation of host plants.

The transcription factor BmPOUM2 interacted with another transcription factor BmAbd-A to regulate the expression of the wing cuticle protein gene BmWCP4 in Bombyx mori. In this study, the binding domains and amino acids for the interaction between BmPOUM2 and BmAbd-A were reported. Two isoforms of BmPOUM2 were identified. The short isoform (BmPOUM2-S) lacks a 114-amino acid sequence containing a POU-homeodomain and a nuclear localization signal peptide (NLS), as compared to the full-length isoform (BmPOUM2). Both BmPOUM2 and BmPOUM2-S proteins bound to the BmAbd-A through the POU-specific domain. When the six amino acids (Lys166, Gly173, Gln176, Ser192, Glu200 and Asn208) that are highly conserved in POU family genes were mutated, BmPOUM2 did not bind to BmAbd-A. BmAbd-A interacted with BmPOUM2 by the homeobox domain or the LCR2 (low complexity region) domain. When seven amino acids (Phe156/248, His158/250, Ala175/263, Cys180/265, Glu190/268, Trp196/274 and Val214/289) that are shared in the homeobox and LCR2 domains were mutated, BmAbd-A did not bind to BmPOUM2. Overexpression of either BmPOUM2 or BmAbd-A or both increased the activity of BmWCP4 promoter in CHO cells. ChIP assay and EMSA showed that BmAbd-A protein bound to the Hox cis-regulatory element in the BmWCP4 promoter, while the BmPOUM2 bound to the nearby POU CRE. A model for the interaction and action of BmPOUM2 and BmAbd-A in regulation of the BmWCP4 expression is proposed.

Identification of the binding domains and key amino acids for the interaction of the transcription factors BmPOUM2 and BmAbd-A in Bombyx mori.

The tobacco cutworm, Spodoptera litura, is among the most widespread and destructive agricultural pests, feeding on over 100 crops throughout tropical and subtropical Asia. By genome sequencing, physical mapping and transcriptome analysis, we found that the gene families encoding receptors for bitter or toxic substances and detoxification enzymes, such as cytochrome P450, carboxylesterase and glutathione-S-transferase, were massively expanded in this polyphagous species, enabling its extraordinary ability to detect and detoxify many plant secondary compounds. Larval exposure to insecticidal toxins induced expression of detoxification genes, and knockdown of representative genes using short interfering RNA (siRNA) reduced larval survival, consistent with their contribution to the insect's natural pesticide tolerance. A population genetics study indicated that this species expanded throughout southeast Asia by migrating along a South India-South China-Japan axis, adapting to wide-ranging ecological conditions with diverse host plants and insecticides, surviving and adapting with the aid of its expanded detoxification systems. The findings of this study will enable the development of new pest management strategies for the control of major agricultural pests such as S. litura.

Genomic adaptation to polyphagy and insecticides in a major East Asian noctuid pest.

The insect fat body is analogous to vertebrate adipose tissue and liver. In this review, the new and exciting advancements made in fat body biology in the last decade are summarized. Controlled by hormonal and nutritional signals, insect fat body cells undergo mitosis during embryogenesis, endoreplication during the larval stages, and remodeling during metamorphosis and regulate reproduction in adults. Fat body tissues are major sites for nutrient storage, energy metabolism, innate immunity, and detoxification. Recent studies have revealed that the fat body plays a central role in the integration of hormonal and nutritional signals to regulate larval growth, body size, circadian clock, pupal diapause, longevity, feeding behavior, and courtship behavior, partially by releasing fat body signals to remotely control the brain. In addition, the fat body has emerged as a fascinating model for studying metabolic disorders and immune diseases. Potential future directions for fat body biology are also proposed herein.

Fat Body Biology in the Last Decade.

Stage-specific gene expression governs metamorphosis of the silkworm,  wing cuticle protein gene 4 () is an essential gene for wing disc development expressed specifically during pupation.  transcription is suppressed at the larval stage by unknown mechanisms, which we sought to elucidate here. Bioinformatics analysis predicted seven potential Forkhead box (Fox) -regulatory elements (CREs) in the  promoter region, and we found that Fox CRE6 contributes to suppression of  expression. Electrophoretic mobility shift (EMSA) and DNA pull-down assays revealed that BmFoxA suppressed activity at the  promoter by specifically binding to the Fox CRE6. The expression level of BmFoxA in the wing discs was higher during the larval stage than at the pupal stage. In contrast, expression of another transcription factor, BmSAGE, increased over the course of development. Of note, the hormone 20-hydroxyecdysone (20E), which governs molting in insects, suppressed  expression in the wing discs and up-regulated that of  EMSA and cell co-transfection assays indicated that BmSAGE interacted with BmFoxA and suppressed its binding to the Fox CRE6, thereby releasing BmFoxA-mediated suppression of  In summary, higher BmFoxA expression during the larval stage suppresses  expression by binding to the Fox CRE6 on the  promoter. During metamorphosis, BmSAGE forms a complex with BmFoxA to relieve this repression, initiating  expression. Taken together, this study reveals a switchlike role for BmFoxA in regulating  expression and provides new insights into the regulatory regulation of wing disc development in insects.

 transcription factors FoxA and SAGE divergently regulate the expression of wing cuticle protein gene 4 during metamorphosis.

Lepidoptera is one group of the largest plant-feeding insects and Spodoptera litura (Lepidoptera: Noctuidae) is one of the most serious agricultural pests in Asia countries. An interesting and unique phenomenon for gonad development of Lepidoptera is the testicular fusion. Two separated testes fused into a single one during the larva-to-pupa metamorphosis, which is believed to contribute to sperm production and the prevalence in field. To study the molecular mechanism of the testicular fusion, RNA sequencing (RNA-seq) experiments of the testes from 4-day-old sixth instar larvae (L6D4) (before fusion), 6-day-old sixth instar larvae (L6D6, prepupae) (on fusing) and 4-day-old pupae (P4D) (after fusion) of S. litura were performed.

Transcriptomic analysis of the testicular fusion in Spodoptera litura.

A large number of in vitro experiments have confirmed that DNA molecules can form i-motif advanced structure when multiple cytosines exist in the sequence. However, whether these structures are present in vivo environment still lacks sufficient experimental evidence.

In vivo visualization of the i-motif DNA secondary structure in the Bombyx mori testis.

G-quadruplex (G4) structures have been predicted in the genomes of many organisms and proven to play regulatory roles in diverse cellular activities. However, there is little information on the evolutionary history and distribution characteristics of G4s. Here, whole-genome characteristics of potential G4s were studied in 37 evolutionarily representative species. During evolution, the number, length, and density of G4s generally increased. Immunofluorescence in seven species confirmed G4s' presence and evolutionary pattern. G4s tended to cluster in chromosomes and were enriched in genetic regions. Short-loop G4s were conserved in most species, while loop-length diversity also existed, especially in mammals. The proportion of G4-bearing genes and orthologue genes, which appeared to be increasingly enriched in transcription factors, gradually increased. The antagonistic relationship between G4s and DNA methylation sites was detected. These findings imply that organisms may have evolutionarily developed G4 into a novel reversible and elaborate transcriptional regulatory mechanism benefiting multiple physiological activities of higher organisms.

Genome-wide analysis of DNA G-quadruplex motifs across 37 species provides insights into G4 evolution.

Krüppel homolog 1 (Kr-h1) is a critical transcription factor for juvenile hormone (JH) signaling, known to play a key role in regulating metamorphosis and adult reproduction in insects. Kr-h1 can also be induced by molting hormone 20-hydroxyecdysone (20E), however, the underlying mechanism of 20E-induced Kr-h1 expression remains unclear. In the present study, we investigated the molecular mechanism of Kr-h1 induction by 20E in the reproductive system of a model lepidopteran insect, Bombyx mori.

Qili Feng

South China Normal University-Shipai Campus: South China Normal University

Microsurgical Obstruction of Testes Fusion in Spodoptera litura