Characterisation of the myosin light chain kinase (MLCK) gene of Locusta migratoria and the encoded MLCK.

Myosin light chain kinase (MLCK) is a dedicated kinase of myosin regulatory light chain (RLC), playing an essential role in the regulation of muscle contraction and cell motility. Much of the knowledge about MLCK comes from the study of vertebrate MLCK, and little is known about insect MLCK. Here, we identified the single MLCK gene in the locust Locusta migratoria, which spans over 1400 kb, includes 62 exons and accounts for at least five transcripts. We found that the five distinct transcripts of the locust MLCK gene are expressed in a tissue-specific manner, including three muscle-specific isoforms and two generic isoforms. To characterise the kinase activity of locust MLCK, we recombinantly expressed LmMLCK-G, the smallest locust MLCK isoform, in insect Sf9 cells. We demonstrated that LmMLCK-G is a Ca2+/calmodulin-dependent kinase that specifically phosphorylates serine 50 of locust muscle myosin RLC (LmRLC). Additionally, we found that almost all LmRLC molecules in the flight muscle and the hindleg muscles of adult locusts are phosphorylated.

Phase-related differences in egg production of the migratory locust regulated by differential oosorption through microRNA-34 targeting activinß.

Outbreaks of locust plagues result from the long-term accumulation of high-density egg production. The migratory locust, Locusta migratoria, displays dramatic differences in the egg-laid number with dependence on population density, while solitarious locusts lay more eggs compared to gregarious ones. However, the regulatory mechanism for the egg-laid number difference is unclear. Herein, we confirm that oosorption plays a crucial role in the regulation of egg number through the comparison of physiological and molecular biological profiles in gregarious and solitarious locusts. We find that gregarious oocytes display a 15% higher oosorption ratio than solitarious ones. Activinß (Actß) is the most highly upregulated gene in the gregarious terminal oocyte (GTO) compared to solitarious terminal oocyte (STO). Meanwhile, Actß increases sharply from the normal oocyte (N) to resorption body 1 (RB1) stage during oosorption. The knockdown of Actß significantly reduces the oosorption ratio by 13% in gregarious locusts, resulting in an increase in the egg-laid number. Based on bioinformatic prediction and experimental verification, microRNA-34 with three isoforms can target Actß. The microRNAs display higher expression levels in STO than those in GTO and contrasting expression patterns of Actß from the N to RB1 transition. Overexpression of each miR-34 isoform leads to decreased Actß levels and significantly reduces the oosorption ratio in gregarious locusts. In contrast, inhibition of the miR-34 isoforms results in increased Actß levels and eventually elevates the oosorption ratio of solitarious locusts. Our study reports an undescribed mechanism of oosorption through miRNA targeting of a TGFß ligand and provides new insights into the mechanism of density-dependent reproductive adaption in insects.

RNAi-mediated silencing of the neverland gene inhibits molting in the migratory locust, Locusta migratoria.

7-dehydrocholesterol (7-DHC) is a key intermediate product used for biosynthesis of molting hormone. This is achieved through a series of hydroxylation reactions catalyzed by the Halloween family of cytochrome P450s. Neverland is an enzyme catalyzes the first reaction of the ecdysteroidogenic pathway, which converts dietary cholesterol into 7-DHC. However, research on the physiological function of neverland in orthopteran insects is lacking. In this study, neverland from Locusta migratoria (LmNvd) was cloned and analyzed. LmNvd was mainly expressed in the prothoracic gland and highly expressed on days 6 and 7 of fifth instar nymphs. RNAi-mediated silencing of LmNvd resulted in serious molting delays and abnormal phenotypes, which could be rescued by 7-DHC and 20-hydroxyecdysone supplementation. Hematoxylin and eosin staining results showed that RNAi-mediated silencing of LmNvd disturbed the molting process by both promoting the synthesis of new cuticle and suppressing the degradation of the old cuticle. Quantitative real-time PCR results suggested that the mRNA expression of E75 early gene and chitinase 5 gene decreased and that of chitin synthase 1 gene was markedly upregulated after knockdown of LmNvd. Our results suggest that LmNvd participates in the biosynthesis process of molting hormone, which is involved in regulating chitin synthesis and degradation in molting cycles.

Osiris17 is indispensable for morphogenesis of intestinal tract in Locusta migratoria.

The Osiris gene family is believed to play important roles in insect biology. Previous studies mainly focused on the roles of Osiris in Drorophila, how Osiris operates during the development of other species remains largely unknown. Here, we investigated the role of LmOsi17 in development of the hemimetabolous insect Locusta migratoria. LmOsi17 was highly expressed in the intestinal tract of nymphs. Knockdown of LmOsi17 by RNA interference (RNAi) in nymphs resulted in growth defects. The dsLmOsi17-injected nymphs did not increase in body weight or size and eventually died. Immunohistochemical analysis showed that LmOsi17 was localized to the epithelial cells of the foregut and the gastric caecum. Histological observation and hematoxylin-eosin staining indicate that the foregut and gastric caecum are deformed in dsLmOsi17 treated nymphs, suggesting that LmOsi17 is involved in morphogenesis of foregut and gastric caecum. In addition, we observed a significant reduction in the thickness of the new cuticle in dsLmOsi17-injected nymphs compared to control nymphs. Taken together, these results suggest that LmOsi17 contributes to morphogenesis of intestinal tract that affects growth and development of nymphs in locusts.

Syntaxin5 is essential for survival by ensuring midgut epithelial homeostsis and regulating feeding in Locusta migratoria.

Syntaxin5 (Syx5) belongs to SNAREs family, which play important roles in fusion of vesicles to target membranes. Most of what we know about functions of Syx5 originates from studies in fungal or vertebrate cells, how Syx5 operates during the development of insects is poorly understood. In this study, we investigated the role of LmSyx5 in the gut development of the hemimetabolous insect Locusta migratoria. LmSyx5 was expressed in many tissues, with higher levels in the gut. Knockdown of LmSyx5 by RNA interference (RNAi) considerably suppressed feeding in both nymphs and adults. The dsLmSyx5-injected locusts lost body weight and finally died at a mortality of 100%. Furthermore, hematoxylin-eosin staining indicated that the midgut is deformed in dsLmSyx5-treated nymphs and the brush border in midgut epithelial cells is severely damaged, suggesting that LmSyx5 is involved in morphogenesis of the midgut. TEM further showed that the endoplasmic reticulum of midgut cells have a bloated appearance. Taken together, these results suggest that LmSyx5 is essential for midgut epithelial homeostsis that affects growth and development of L. migratoria. Thus, Syx5 is a promising RNAi target for controlling L. migratoria, and even other pests.

Juvenile hormone acts through FoxO to promote Cdc2 and Orc5 transcription for polyploidy-dependent vitellogenesis.

Vitellogenin (Vg) is a prerequisite for egg production and embryonic development after ovipositioning in oviparous animals. In many insects, juvenile hormone (JH) promotes fat body cell polyploidization for the massive Vg synthesis required for the maturation of multiple oocytes, but the underlying mechanisms remain poorly understood. Using the migratory locust Locusta migratoria as a model system, we report here that JH induces the dephosphorylation of Forkhead box O transcription factor (FoxO) through a signaling cascade including leucine carboxyl methyltransferase 1 (LCMT1) and protein phosphatase 2A (PP2A). JH promotes PP2A activity via LCMT1-mediated methylation, consequently triggering FoxO dephosphorylation. Dephosphorylated FoxO binds to the upstream region of two endocycle-related genes, cell-division-cycle 2 (Cdc2) and origin-recognition-complex subunit 5 (Orc5), and activates their transcription. Depletion of FoxO, Cdc2 or Orc5 results in blocked polyploidization of fat body cells, accompanied by markedly reduced Vg expression, impaired oocyte maturation and arrested ovarian development. The results suggest that JH acts via LCMT1-PP2A-FoxO to regulate Cdc2 and Orc5 expression, and to enhance ploidy of fat body cells in preparation for the large-scale Vg synthesis required for synchronous maturation of multiple eggs.

Three alternative splicing variants of Loquacious play different roles in miRNA- and siRNA-mediated RNAi pathways in Locusta migratoria.

RNA interference (RNAi) is a specific post-transcriptional gene-silencing phenomenon, which plays an important role in the regulation of gene expression and the protection from transposable elements in eukaryotic organisms. In Drosophila melanogaster, RNAi can be induced by microRNA (miRNA), endogenous small interfering RNA (siRNA), or exogenous siRNA. However, the biogenesis of miRNA and siRNA in these RNAi pathways is aided by the double-stranded RNA binding proteins (dsRBPs) Loquacious (Loqs)-PB, Loqs-PD or R2D2. In this study, we identified three alternative splicing variants of Loqs, namely Loqs-PA, -PB, and -PC in the orthopteran Locusta migratoria. We performed in vitro and in vivo experiments to study the roles of the three Loqs variants in the miRNA- and siRNA-mediated RNAi pathways. Our results show that Loqs-PB assists the binding of pre-miRNA to Dicer-1 to lead to the cleavage of pre-miRNA to yield matured miRNA in the miRNA-mediated RNAi pathway. In contrast, different Loqs proteins participate in different siRNA-mediated RNAi pathways. In exogenous siRNA-mediated RNAi pathway, binding of Loqs-PA or LmLoqs-PB to exogenous dsRNA facilitates the cleavage of dsRNA by Dicer-2, whereas in endogenous siRNA-mediated RNAi pathway, binding of Loqs-PB or Loqs-PC to endogenous dsRNA facilitates the cleavage of dsRNA by Dicer-2. Our findings provide new insights into the functional importance of different Loqs proteins derived from alternative splicing variants of Loqs in achieving high RNAi efficiency in different RNAi pathways in insects.

The immune response of Locusta migratoria manilensis at different times of infection with Paranosema locustae.

Paranosema locustae is an entomopathogenic microsporidia with promising potential for controlling agricultural pests, including Locusta migratoria manilensis. However, it has the disadvantage of having a slow insecticidal rate, and how P. locustae infection impacts the host immune response is currently unknown. The present study investigated the effect of P. locustae on the natural immune response of L. migratoria and the activities of enzymes that protect against oxidative stress. Infection with P. locustae increased the hemocytes and nodulation number of L. migratoria at the initial stage of infection. The hemocyte-mediated modulation of immune response was also affected by a decrease in the number of hemocytes 12 days postinfection. Superoxide dismutase activity in locusts increased in the early stages of infection but decreased in the later stages, whereas the activities of peroxidase (POD) and catalase (CAT) showed opposite trends may be due to their different mechanisms of action. Furthermore, the transcription levels of mRNA of antimicrobial peptide-related genes and phenoloxidase activity in hemolymph in L. migratoria were suppressed within 15 days of P. locustae infection. Overall, our data suggest that P. locustae create a conducive environment for its own proliferation in the host by disrupting the immune defense against it. These findings provide useful information for the potential application of P. locustae as a biocontrol agent.

Transcription factor CncC regulates the expression of antennal CYP6MU1 gene responsible for trans-2-hexen-1-al and nonanal recognition in Locusta migratoria.

Cytochrome P450 monooxygenases (P450s) are a superfamily of multifunctional heme-containing proteins and could function as odorant-degrading enzymes (ODEs) in insect olfactory systems. In our previous study, we identified a P450 gene from the antennal transcriptome of Locusta migratoria, LmCYP6MU1, which could be induced by a variety of volatiles. However, the regulatory mechanisms of this gene in response to volatiles remain unknown. In current study, we investigated the tissues and development stages expression patterns of LmCYP6MU1 and determined its olfactory function in the recognition of the main host plant volatiles which induced LmCYP6MU1 expression. The results showed that LmCYP6MU1 was antenna-rich and highly expressed throughout the antennal developmental stages of locusts. LmCYP6MU1 played important roles in the recognition of trans-2-hexen-1-al and nonanal. Insect CncC regulates the expression of P450 genes. We tested whether LmCncC regulates LmCYP6MU1 expression. It was found that LmCncC knockdown in the antennae resulted in the downregulation of LmCYP6MU1 and repressed the volatiles-mediated induction of LmCYP6MU1. LmCncC knockdown reduced the electroantennogram (EAG) and behavioral responses of locusts to volatiles. These results suggested that LmCncC could regulate the basal and volatiles-mediated inducible expression of LmCYP6MU1 responsible for the recognition of trans-2-hexen-1-al and nonanal. These findings provide an original basis for understanding the regulation mechanisms of LmCncC on LmCYP6MU1 expression and help us better understand the LmCncC-mediated olfactory plasticity.

NADPH-cytochrome P450 reductase knockdown decreases the response to precocene I in the migratory locust Locusta migratoria.

Precocene I is a juvenile hormone antagonist that needs to be activated via oxidative biotransformation catalyzed by cytochrome P450 (CYP). NADPH-cytochrome P450 reductase (CPR) supplies CYP with electrons in the oxidation-reduction process; however, its functional role in the activation of precocene I remains unexplored. Here, the representative characteristics of CPRs were analyzed in the CPR gene of Locusta migratoria (LmCPR), the result of model docking indicated that the hydrogen bonds were formed between reduced nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) and NADPH-, FAD-, FMN-domains of LmCPR, respectively. Treating the fourth-instar nymphs with precocene I decreased the juvenile hormone titers of nymphs to 0.55-fold of that in acetone-treated controls, and extended the interval time between fourth- and fifth-instar nymphs. 68.75% of the treated fourth-instar nymphs developed into precocious adults in the fifth-instar. LmCPR knockdown decreased the response to precocene I in the nymphs, the occurrence rate of precocious adults induced by precocene I treatment reduced by 23.11%. Therefore, LmCPR may be involved in the activation of precocene I in L. migratoria. In addition, we generated an active recombinant LmCPR protein using a prokaryotic expression system, its activity in reducing cytochrome c was 33.13 ± 11.50 nmol CytCred/min/µg protein. This study lays the foundation for further research on the role of LmCPR in precocene I activation.

Transcription factors, cap 'n' collar isoform C regulates the expression of CYP450 genes involving in insecticides susceptibility in Locusta migratoria.

BACKGROUND: The cap 'n' collar (Cnc) belongs to the Basic Leucine Zipper (bZIP) transcription factor super family. Cap 'n' collar isoform C (CncC) is highly conserved in the animal kingdom. CncC contributes to the regulation of growth, development, and aging and takes part in the maintenance of homeostasis and the defense against endogenous and environmental stress. Insect CncC participates in the regulation of various kinds of stress-responsive genes and is involved in the development of insecticide resistance. RESULTS: In this study, one full-length CncC sequence of Locusta migratoria was identified and characterized. Upon RNAi silencing of LmCncC, insecticide bioassays showed that LmCncC played an essential role in deltamethrin and imidacloprid susceptibility. To fully investigate the downstream genes regulated by LmCncC and further identify the LmCncC-regulated genes involved in deltamethrin and imidacloprid susceptibility, a comparative transcriptome was constructed. Thirty-five up-regulated genes and 73 down-regulated genes were screened from dsLmCncC-knockdown individuals. We selected 22 LmCncC-regulated genes and verified their gene expression levels using RT-qPCR. Finally, six LmCYP450 genes belonging to the CYP6 family were selected as candidate detoxification genes, and LmCYP6FD1 and LmCYP6FE1 were further validated as detoxification genes of insecticides via RNAi, insecticide bioassays, and metabolite identification. CONCLUSIONS: Our data suggest that the locust CncC gene is associated with deltamethrin and imidacloprid susceptibility via the regulation of LmCYP6FD1 and LmCYP6FE1, respectively.

Cuticle protein gene LmCP8 is involved in the structural development of the ovipositor in the migratory locust Locusta migratoria.

The ovipositor comprises the external genitalia of female insects, which plays an important role in the mating and ovipositing process of insects. However, it remains rudimentary of regional gene expression and physiological function in the ovipositor during structural development. Here, we analysed the basic structure and characteristics of the ovipositor in the migratory locust Locusta migratoria. RNA-seq analysis revealed the specialization of chitin metabolism, lipids synthesis and transport, tanning and cuticular protein genes in the ovipositor. Among them, two cuticle protein genes, LmCP8 and LmACP79, were identified, which are specifically expressed in the ovipositor. Functional analysis based on RNA interference showed that deficiency of LmCP8 affected the structural development of the ovipositor resulting in the retention of a large number of remaining unproduced oocysts in the ovary of the locusts. Our results provide a fundamental resource to investigate the structural development and physiological function of the ovipositor in L. migratoria.

The DOMON domain protein LmKnk contributes to correct chitin content, pore canal formation and lipid deposition in the cuticle of Locusta migratoria during moulting.

Insects prevent uncontrolled penetration of water and xenobiotics by producing an impermeable cuticle. The major component of the cuticle is chitin that adopts a crystalline structure thereby contributing to cuticle stability. Our understanding of the contribution of chitin to the cuticle barrier function is limited. Here, we studied the role of the DOMON domain protein Knickkopf (LmKnk) that is involved in chitin organization and cuticle permeability in the migratory locust Locusta migratoria. We show that LmKnk localizes to the chitin layer in the newly produced cuticle. Injection of double-stranded RNA targeting LmKnk (dsLmKnk) in locust nymphs caused failure of moulting to the next stage. Histological experiments revealed that apolysis, i.e., the detachment of the old cuticle from the body surface, was normal; however, the newly synthesized cuticle was thinner than the cuticle of the control insects. Indeed, chitin content dropped after suppression of LmKnk expression. As seen by transmission electron microscopy, crystalline chitin organization was lost in dsLmKnk-treated insects. In addition, the structure of pore canals, which are lipid transporting routes in the cuticle, was abnormal. Consistently, their content was reduced and, probably by consequence, lipid deposition on the cuticle was decreased after injection of dsLmKnk. Suppression of LmKnk transcript levels rendered L. migratoria more susceptible to each of four selected insecticides including malathion, chlorpyrifos, carbaryl and deltamethrin. Overall, our data show that LmKnk is needed for correct chitin amounts and organization, and their changes ultimately affect cuticular permeability in L. migratoria.

Locusta migratoria flight muscle troponin partially activates thin filament in a calcium-dependent manner.

The troponin (Tn) complex, the sensor for Ca2+ to regulate contraction of striated muscle, is composed of three subunits, that is, TnT, TnI and TnC. Different isoforms of TnI and TnC are expressed in the thorax dorsal longitudinal muscle (flight muscle) and the hind leg extensor tibiae muscle (jump muscle) of the migratory locust, Locusta migratoria. The major Tn complexes in the flight muscle and the jump muscle are Tn-171 (TnT1/TnI7/TnC1) and Tn-153 (TnT1/TnI5/TnC3), respectively. Here, we prepared a number of recombinant Tn complexes and the reconstituted thin filaments, and investigated their regulation on thin filament. Although both Tn-171 and Tn-153 regulate thin filament in a Ca2+ -dependent manner, the extent of Ca2+ activation mediated by Tn-171 was significantly lower than that by Tn-153. We demonstrated that TnC1 and TnC3, rather than TnI5 and TnI7, are responsible for the different levels of the thin filament activation. Mutagenesis of TnC1 and TnC3 shows that the low level of TnC1-mediated thin filament activation can be attributed to the noncanonical residue Leu60 in the EF-hand-II of TnC1. We therefore propose that, in addition to Ca2+ , other regulatory mechanism(s) is required for the full activation of locust flight muscle.

CREB-B acts as a key mediator of NPF/NO pathway involved in phase-related locomotor plasticity in locusts.

Gene expression changes in neural systems are essential for environment-induced behavioral plasticity in animals; however, neuronal signaling pathways mediating the effect of external stimuli on transcriptional changes are largely unknown. Recently, we have demonstrated that the neuropeptide F (NPF)/nitric oxide (NO) signaling pathway plays a regulatory role in phase-related locomotor plasticity in the migratory locust, Locusta migratoria. Here, we report that a conserved transcription factor, cAMP response element-binding protein B (CREB-B), is a key mediator involved in the signaling pathway from NPF2 to NOS in the migratory locust, triggering locomotor activity shift between solitarious and gregarious phases. We find that CREB-B directly activates brain NOS expression by interacting with NOS promoter region. The phosphorylation at serine 110 site of CREB-B dynamically changes in response to population density variation and is negatively controlled by NPF2. The involvement of CREB-B in NPF2-regulated locomotor plasticity is further validated by RNAi experiment and behavioral assay. Furthermore, we reveal that protein kinase A mediates the regulatory effects of NPF2 on CREB-B phosphorylation and NOS transcription. These findings highlight a precise signal cascade underlying environment-induced behavioral plasticity.

CYP6FD5, an antenna-specific P450 gene, is potentially involved in the host plant recognition in Locusta migratoria.

Cytochrome P450 monooxygenases (P450s) are a large superfamily of heme-thiolate proteins and play a vital role in the biosynthesis and inactivation of endogenous substances as well as the detoxification of exogenous substances. They also function as odor-degrading enzymes (ODEs) in insect olfactory sensory systems. In the present study, a P450 gene was obtained from the antennae of Locusta migratoria and named as CYP6FD5. Multiple alignment of P450 proteins revealed that LmCYP6FD5 contained five conserved motifs, including the helix C motif, an oxygen-binding site, helix K motif, a meander region, and the haem-binding motif. The expression of LmCYP6FD5 in various tissues and antennal development stages was determined by using RT-qPCR. Our results showed that LmCYP6FD5 was antenna-specific and highly expressed throughout the antennal developmental stages of female and male locusts. Furthermore, the role of LmCYP6FD5 in the perception of host plant volatiles was assessed using RNAi in combination with electroantennogram (EAG) and behavioral responses. Our findings showed that after silencing LmCYP6FD5, the EAG responses of female and male locusts to the main volatiles of gramineous plants, including trans-2-Hexen-1-al, cis-3-Hexenyl acetate, and decanal, were significantly diminished. Moreover, a significant decrease in EAG response of male antennae to benzaldehyde was also observed. In addition, behavioral assay showed that the locust response to single volatile from host plant or wheat remained unchanged after the silencing of LmCYP6FD5. Antenna-specific expression and EAG responses of locusts to host plant volatiles still suggested that LmCYP6FD5 was potentially involved in host plant recognition, although no behavioral changes were observed.

Evaluations of two glutathione S-transferase epsilon genes for their contributions to metabolism of three selected insecticides in Locusta migratoria.

The insect-specific epsilon class of glutathione S-transferases (GSTEs) plays important roles in insecticide detoxification in insects. In our previous work, five GSTEs were identified in Locusta migratoria, and two recombinant GSTEs, rLmGSTE1 and rLmGSTE4, showed high catalytic activity when 1-chloro-2,4-dinitrobenzene (CDNB) was used as a substrate. In this work, we further investigated whether these two GSTEs could metabolize three insecticides including malathion, deltamethrin and DDT. Using ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC/MS) method, we found that rLmGSTE4, but not rLmGSTE1, can metabolize malathion and DDT. Malathion bioassays of L.migratoria after the expression of LmGSTE4 was suppressed by RNA interference (RNAi) showed increased insect mortality from 33.8% to 68.9%. However, no changes in mortality were observed in deltamethrin- or DDT-treated L.migratoria after the expression of LmGSTE4 was suppressed by RNAi. Our results provided direct evidences that LmGSTE4 participates in malathion detoxification in L.migratoria. These findings are important for understanding the mechanisms of insecticide resistance in L.migratoria and developing new strategies for managing the insect populations in the field.

Transcription initiation of distant core promoters in a large-sized genome of an insect.

BACKGROUND: Core promoters have a substantial influence on various steps of transcription, including initiation, elongation, termination, polyadenylation, and finally, translation. The characterization of core promoters is crucial for exploring the regulatory code of transcription initiation. However, the current understanding of insect core promoters is focused on those of Diptera (especially Drosophila) species with small genome sizes. RESULTS: Here, we present an analysis of the transcription start sites (TSSs) in the migratory locust, Locusta migratoria, which has a genome size of 6.5 Gb. The genomic differences, including lower precision of transcription initiation and fewer constraints on the distance from transcription factor binding sites or regulatory elements to TSSs, were revealed in locusts compared with Drosophila insects. Furthermore, we found a distinct bimodal log distribution of the distances from the start codons to the core promoters of locust genes. We found stricter constraints on the exon length of mRNA leaders and widespread expression activity of the distant core promoters in locusts compared with fruit flies. We further compared core promoters in seven arthropod species across a broad range of genome sizes to reinforce our results on the emergence of distant core promoters in large-sized genomes. CONCLUSIONS: In summary, our results provide novel insights into the effects of genome size expansion on distant transcription initiation.

Cuticle Protein LmACP19 Is Required for the Stability of Epidermal Cells in Wing Development and Morphogenesis of Locusta migratoria.

Insect wing consists of a double layer of epidermal cells that produce and secrete the dorsal and ventral cuticular components. It is important for the stability of epidermal cells during wing development and morphogenesis, but its specific gene expression and physiological function during this process remain unclear. In our previous work, a wing cuticle protein gene LmACP19 was identified in Locusta migratoria based on transcriptomic data. Here, we report on its roles in wing development and morphogenesis. LmACP19 encodes a chitin-binding protein belonging to RR-2 subfamily of CPR family, which is highly homologous to CP19-like proteins in other insect species. RT-qPCR analysis revealed that LmACP19 is highly expressed in wing pads of fifth-instar nymphs, and its encoded protein is located in two layers of epidermal cells but not in the cuticle. Suppression of LmACP19 by RNA interference led to abnormal wing pad and wing morphogenesis with curved, unclosed, and wrinkled phenotypes during nymph-to-nymph and nymph-to-adult transition, respectively. Furthermore, deficiency of LmACP19 affected arrangement of epidermal cells, resulting in apoptosis. Our results indicate that LmACP19 is indispensable for wing development and normal morphological structure by maintaining the stability of epidermal cells during L. migratoria molting.

DEAD-Box RNA Helicase DDX47 Maintains Midgut Homeostasis in Locusta migratoria.

Tissue homeostasis is critical for maintaining organ shape, size, and function. The condition is regulated by the balance between the generation of new cells and the loss of senescent cells, and it involves many factors and mechanisms. The midgut, an important part of the intestinal tract, is responsible for digestion and nutrient absorption in insects. LmDDX47, the ortholog of DEAD-box helicase 47 from Locusta migratoria, is indispensable for sustaining a normal midgut in the nymphs. However, the underlying cellular and molecular mechanisms remain to be elucidated. In this study, LmDDX47 knockdown resulted in atrophy of the midgut and gastric cecum in both nymph and adult locusts. After LmDDX47 knockdown, the number of regenerative and columnar cells in the midgut was significantly reduced, and cell death was induced in columnar tissue. LmDDX47 was localized to the nucleolus; this was consistent with the reduction in 18S rRNA synthesis in the LmDDX47 knockdown group. In addition, the acetylation and crotonylation levels of midgut proteins were significantly increased. Therefore, LmDDX47 could be a key regulator of midgut homeostasis, regulating 18S rRNA synthesis as well as protein acetylation and crotonylation in the migratory locust.