Deep learning reveals a damage signalling hierarchy that coordinates different cell behaviours driving wound re-epithelialisation.

One of the key tissue movements driving closure of a wound is re-epithelialisation. Earlier wound healing studies describe the dynamic cell behaviours that contribute to wound re-epithelialisation, including cell division, cell shape changes and cell migration, as well as the signals that might regulate these cell behaviours. Here, we have used a series of deep learning tools to quantify the contributions of each of these cell behaviours from movies of repairing wounds in the Drosophila pupal wing epithelium. We test how each is altered after knockdown of the conserved wound repair signals Ca2+ and JNK, as well as after ablation of macrophages that supply growth factor signals believed to orchestrate aspects of the repair process. Our genetic perturbation experiments provide quantifiable insights regarding how these wound signals impact cell behaviours. We find that Ca2+ signalling is a master regulator required for all contributing cell behaviours; JNK signalling primarily drives cell shape changes and divisions, whereas signals from macrophages largely regulate cell migration and proliferation. Our studies show deep learning to be a valuable tool for unravelling complex signalling hierarchies underlying tissue repair.

Non-CG DNA methylation marks the transition from pupa to adult in Helicoverpa armigera.

DNA methylation in insects is generally low in abundance, and its role is not well understood. It is often localised in protein coding regions and associated with the expression of 'housekeeping' genes. Few studies have explored DNA methylation dynamics during lifecycle stage transitions in holometabolous (metamorphosing) insects. Using targeted mass spectrometry, we have found a significant difference in global DNA methylation levels between larvae, pupae and adults of Helicoverpa armigera (Lepidoptera: Noctuidae) Hübner, a polyphagous pest of agricultural importance. Whole-genome bisulfite sequencing confirmed these observations and pointed to non-CG context being the primary explanation for the difference observed between pupa and adult. Non-CG methylation was enriched in genes specific to various signalling pathways (Hippo signalling, Hedgehog signalling and mitogen-activated protein kinase (MAPK) signalling) and ATP-dependent chromatin remodelling. Understanding the function of this epigenetic mark could be a target in future studies focusing on integrated pest management.

The molecular basis of scale development highlighted by a single-cell atlas of Bicyclus anynana butterfly pupal forewings.

Butterfly wings display a diversity of cell types, including large polyploid scale cells, yet the molecular basis of such diversity is poorly understood. To explore scale cell diversity at a transcriptomic level, we employ single-cell RNA sequencing of ∼5,200 large cells (>6 µm) from 22.5- to 25-h male pupal forewings of the butterfly Bicyclus anynana. Using unsupervised clustering, followed by in situ hybridization, immunofluorescence, and CRISPR-Cas9 editing of candidate genes, we annotate various cell types on the wing. We identify genes marking non-innervated scale cells, pheromone-producing glandular cells, and innervated sensory cell types. We show that senseless, a zinc-finger transcription factor, and HR38, a hormone receptor, determine the identity, size, and color of different scale cell types and are important regulators of scale cell differentiation. This dataset and the identification of various wing cell-type markers provide a foundation to compare and explore scale cell-type diversification across arthropod species.

Hypoxia delays steroid-induced developmental maturation in Drosophila by suppressing EGF signaling.

Animals often grow and develop in unpredictable environments where factors like food availability, temperature, and oxygen levels can fluctuate dramatically. To ensure proper sexual maturation into adulthood, juvenile animals need to adapt their growth and developmental rates to these fluctuating environmental conditions. Failure to do so can result in impaired maturation and incorrect body size. Here we describe a mechanism by which Drosophila larvae adapt their development in low oxygen (hypoxia). During normal development, larvae grow and increase in mass until they reach critical weight (CW), after which point a neuroendocrine circuit triggers the production of the steroid hormone ecdysone from the prothoracic gland (PG), which promotes maturation to the pupal stage. However, when raised in hypoxia (5% oxygen), larvae slow their growth and delay their maturation to the pupal stage. We find that, although hypoxia delays the attainment of CW, the maturation delay occurs mainly because of hypoxia acting late in development to suppress ecdysone production. This suppression operates through a distinct mechanism from nutrient deprivation, occurs independently of HIF-1 alpha and does not involve dilp8 or modulation of Ptth, the main neuropeptide that initiates ecdysone production in the PG. Instead, we find that hypoxia lowers the expression of the EGF ligand, spitz, and that the delay in maturation occurs due to reduced EGFR/ERK signaling in the PG. Our study sheds light on how animals can adjust their development rate in response to changing oxygen levels in their environment. Given that hypoxia is a feature of both normal physiology and many diseases, our findings have important implications for understanding how low oxygen levels may impact animal development in both normal and pathological situations.

A Novel Low Molecule Peptides-calcium Chelate from Silkworm Pupae Protein Hydrolysate: Preparation, Antioxidant Activity, and Bioavailability.

BACKGROUND: The antioxidant properties of active peptides from silkworm pupae protein hydrolysate are of interest, and it serves as a novel source of calcium supplement. METHODS: Optimize the preparation parameters of silkworm pupae bioactive peptide-calcium chelate, and investigate the mechanism and bioavailability of silkworm pupae active peptide as a transport carrier to promote calcium ion absorption using simulated gastrointestinal digestion and Caco-2 monolayer cell model. RESULTS: The optimal process parameters for preparing peptide calcium chelate were the peptide calcium mass ratio of 3:1, pH of 6.7, a temperature of 35.6°C, and time of 32.8 min by Box-Behnken design, and the calciumchelating rate reached 84.67%. The DPPH radical scavenging activity of silkworm pupae protein hydrolysatecalcium chelate was 79.36 ± 4.31%, significantly higher than silkworm pupae protein hydrolysate (61.00 ± 9.56%). Fourier transform infrared spectroscopy shows that the COO-, N-H, C-H, and C-O groups participated in the formation of silkworm pupae protein hydrolysate-calcium chelate. The particle size of the silkworm pupae protein hydrolysate-calcium chelate was 970.75 ± 30.12 nm, which was significantly higher than that of silkworm pupae protein hydrolysate (253.14 ± 5.72 nm). The silkworm pupae protein hydrolysate-calcium chelate showed a calcium dissolution rate of 71.01 ± 1.91% in the simulated intestinal phase, significantly higher than that of CaCl2 (59.34 ± 1.24%). In the Caco-2 cell monolayers, the silkworm pupae protein hydrolysatecalcium chelate was more favorable for calcium transport. CONCLUSION: A novel silkworm pupa protein hydrolysate-calcium chelate with high antioxidant activity was successfully prepared to improve the bioavailability of calcium.

Mechanistic effects of microwave radiation on pupal emergence in the leafminer fly, Liriomyza trifolii.

Liriomyza trifolii is a significant pest of vegetable and ornamental crops across the globe. Microwave radiation has been used for controlling pests in stored products; however, there are few reports on the use of microwaves for eradicating agricultural pests such as L. trifolii, and its effects on pests at the molecular level is unclear. In this study, we show that microwave radiation inhibited the emergence of L. trifolii pupae. Transcriptomic studies of L. trifolii indicated significant enrichment of differentially expressed genes (DEGs) in 'post-translational modification, protein turnover, chaperones', 'sensory perception of pain/transcription repressor complex/zinc ion binding' and 'insulin signaling pathway' when analyzed with the Clusters of Orthologous Groups, Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes databases, respectively. The top DEGs were related to reproduction, immunity and development and were significantly expressed after microwave radiation. Interestingly, there was no significant difference in the expression of genes encoding heat shock proteins or antioxidant enzymes in L. trifolii treated with microwave radiation as compared to the untreated control. The expression of DEGs encoding cuticular protein and protein takeout were silenced by RNA interference, and the results showed that knockdown of these two DEGs reduced the survival of L. trifolii exposed to microwave radiation. The results of this study help elucidate the molecular response of L. trifolii exposed to microwave radiation and provide novel ideas for control.

CBR1 decreases protein carbonyl levels via the ROS/Akt/CREB pathway to extend lifespan in the cotton bollworm, Helicoverpa armigera.

Reactive oxygen species (ROS) are considered a major cause of ageing and ageing-related diseases through protein carbonylation. Little is known about the molecular mechanisms that confer protection against ROS. Here, we observed that, compared with nondiapause-destined pupae, high protein carbonyl levels are present in the brains of diapause-destined pupae, which is a 'non-ageing' phase in the moth Helicoverpa armigera. Protein carbonyl levels respond to ROS and decrease metabolic activity to induce diapause in order to extend lifespan. However, protein carbonylation in the brains of diapause-destined pupae still occurs at a physiological level compared to young adult brains. We find that ROS activate Akt, and Akt then phosphorylates the transcription factor CREB to facilitate its nuclear import. CREB binds to the promoter of carbonyl reductase 1 (CBR1) and regulates its expression. High CBR1 levels reduce protein carbonyl levels to maintain physiological levels. This is the first report showing that the moth brain can naturally control protein carbonyl levels through a distinct ROS-Akt-CREB-CBR1 pathway to extend lifespan.

Proteomic analysis of pharate pupal molting fluid from the tobacco hornworm, Manduca sexta.

The insect cuticle is a key component of their success, being important for protection, communication, locomotion, and support. Conversely, as an exoskeleton, it also limits the size of the insect and must be periodically molted and a new one synthesized, to permit growth. To achieve this, the insect secretes a solution of chitinases, proteases and other proteins, known collectively as molting fluid, during each molting process to break down and recycle components of the old cuticle. Previous research has focused on the degradative enzymes in molting fluid and offered some characterization of their biochemical properties. However, identification of the specific proteins involved remained to be determined. We have used 2D SDS-PAGE and LC/MS-based proteomic analysis to identify proteins in the molting fluid of the tobacco hornworm, Manduca sexta, undergoing the larval to pupal molt. We categorized these proteins based on their proposed functions including chitin metabolism, proteases, peptidases, and immunity. This analysis complements previous reported work on M. sexta molting fluid and identifies candidate genes for enzymes involved in cuticle remodeling. Proteins classified as having an immune function highlight potential for molting fluid to act as an immune barrier to prevent infections during the cuticle degradation and ecdysis processes. Several proteins known to function in melanin synthesis as an immune response in hemolymph were present in molting fluid. We demonstrated that the bacterium Micrococcus luteus and the entomopathogenic fungus Beauveria bassiana can stimulate activation of phenoloxidase in molting fluid, indicating that the recognition proteins, protease cascade, and prophenoloxidase needed for melanin synthesis are present as a defense against infection during cuticle degradation. This analysis offers insights for proteins that may be important not only for molting in M. sexta but for insects in general.

RNAi-based functional analysis of bursicon genes related to wing expansion in gypsy moths.

Bursicon is a heterodimeric neuropeptide composed of Burs-α and Burs-ß subunits that plays an important role in cuticle tanning and wing expansion in insects. In this study, full-length cDNAs of Burs-α (LdBurs-α) and Burs-ß (LdBurs-ß) genes were identified in gypsy moth (Lymantria dispar) and cloned. The 480 bp and 420 bp open reading frames (ORFs) encode 159 and 129 amino acid polypeptides, respectively. LdBurs-α and LdBurs-ß have 11 conserved cysteine residues, and LdBurs-α and LdBurs-ß genes were expressed during all developmental stages according to quantitative reverse transcription PCR (qRT-PCR), with highest expression in the egg stage. High expression levels were also detected in the haemolymph, cuticle and head. To explore the physiological functions of LdBurs-α and LdBurs-ß, the genes were knocked down in larvae and pupae using RNA interference (RNAi), and expression levels of LdBurs-α and LdBurs-ß were decreased by 42.26-80.09%. Wing defects were observed in L. dispar pupae following Ldbursion silencing, with a phenotypic percentage ranging from 10.17% to 15.00%. RNAi-mediated knockdown of Ldbursicon prevented the expansion of male and female L. dispar adult wings, with malformation rates ranging from 6.38% and 30.00% to 57.69% and 69.23%, but no cuticle tanning defects were observed in pupae or adults. The results indicate that bursicon plays a key role in wing expansion in L. dispar adults, making it a potentially novel molecular target for insecticide-based control of this pest species.

Exploration of Dipeptidyl Peptidase-IV (DPP-IV) Inhibitory Peptides from Silkworm Pupae (Bombyx mori) Proteins Based on In Silico and In Vitro Assessments.

This study aimed at exploring dipeptidyl peptidase-IV (DPP-IV) inhibitory peptides from silkworm pupae proteins by in silico analysis and in vitro assessments. In silico analysis of 274 silkworm pupae proteomes indicated that DPP-IV inhibitory peptides can be released from silkworm pupae proteins. In vitro assessments revealed that pepsin and bromelain led to better production of DPP-IV inhibitory peptides from silkworm pupae protein. Notably, peptide fractions (<1 kDa) from pepsin- and bromelain-treated hydrolysates exhibited more potent DPP-IV inhibitory activities. Two novel DPP-IV inhibitory peptides (Leu-Pro-Pro-Glu-His-Asp-Trp-Arg and Leu-Pro-Ala-Val-Thr-Ile-Arg) were identified by LC-MS/MS with IC50 values of 261.17 and 192.47 µM, respectively. Enzyme kinetics data demonstrated that these two peptides displayed a mixed-type DPP-IV inhibition mode, which was further validated by molecular docking data. Overall, in silico analysis combined with in vitro assessments can serve as an effective and rapid approach for discovery of DPP-IV peptides from silkworm pupae proteins.

Segmental pairs of dermal secretory cells release proteins into the hemolymph at the larval-pupal molt.

At each molt of Manduca, the large dermal secretory cells expel the protein contents of their vacuoles into the hemocoel. The constellation of proteins expelled at the last larval-pupal molt, however, differs qualitatively from those proteins released at earlier larval-larval molts. Secretory cells at the two stages not only have different lectin staining properties but also have different proteins that separate on two-dimensional gels. Numerous physiological changes accompany the termination of the last larval instar, including increased chitin synthesis, diminished oxygen delivery, and reduced humoral immunity. Secretion of trehalase that is essential for chitin synthesis and the release of hypoxia up-regulated protein to ameliorate oxygen deprivation help ensure normal transition from larva to pupa. Proteins released by dermal secretory cells at this last molt could supplement the diminished immune defenses mediated by fat body and hemocytes at the end of larval life. Additional immune defenses provided by dermal secretory cells could help ensure a safe transition during a period of increased vulnerability for the newly molted pupa with its soft, thin cuticle and reduced mobility.

60Co-γ Radiation Alters Developmental Stages of Zeugodacus cucurbitae (Diptera: Tephritidae) Through Apoptosis Pathways Gene Expression.

Radiation is considered as a promising insect pest control strategy for minimizing postharvest yield losses. Among various techniques, irradiation is a method of choice as it induces lethal biochemical or molecular changes that cause a downstream cascade of abrupt physiological abnormalities at the cellular level. In this study, we evaluated the effect of 60Co-γ radiation on various developmental stages of Zeugodacus cucurbitae Coquillett and subsequent carry-over effects on the progeny. For this purpose, we treated eggs with 30- and 50-Gy radiation doses of 60Co-γ. We found that radiation significantly affected cellular antioxidants, insect morphology, and gene expression profiles. Our results indicate that in response to various doses of irradiation reactive oxygen species, catalase, peroxidase, and superoxide dismutase activities were increased along with a significant increase in the malondialdehyde (MDA) content. We observed higher mortality rates during the pupal stage of the insects that hatched from irradiated eggs (50 Gy). Furthermore, the life span of the adults was reduced in response to 50 Gy radiation. The negative effects carried over to the next generation were marked by significantly lower fecundity in the F1 generation of the irradiation groups as compared to control. The radiation induced morphological abnormalities at the pupal, as well as the adult, stages. Furthermore, variations in the gene expression following irradiation are discussed. Taken together, our results signify the utility of 60Co-γ radiation for fruit fly postharvest management.

Enzymatic Hydrolysis of Defatted Antheraea pernyi (Lepidoptera: Saturniidae) Pupa Protein by Combined Neutral Protease Yield Peptides With Antioxidant Activity.

In this study, peptides were prepared from defatted Antheraea pernyi (Lepidoptera: Saturniidae) pupa protein via hydrolysis with combined neutral proteases. Single-factor tests and response surface methodology (RSM) were used to determine the optimal hydrolysis condition suitable for industrial application. Optimal hydrolysis of the defatted pupa protein was found to occur at an enzyme concentration of 4.85 g/liter, a substrate concentration of 41 g/liter, a hydrolysis temperature of 55°C, and a hydrolysis time of 10 h and 40 min. Under these conditions, the predicted and actual rates of hydrolysis were 45.82% and 45.75%, respectively. Peptides with a molecular weight of less than 2,000 Da accounted for 90.5% of the total peptides generated. Some of the peptides were antioxidant peptides as revealed by sequencing and functional analysis. The antioxidant activity of the mixed peptides was subsequently confirmed by an antioxidant activity assay. The results showed that peptides with high antioxidant activity could be obtained from the hydrolysis of A. pernyi pupa protein.

The Ras/MAPK pathway is required for regenerative growth of wing discs in the black cutworm Agrotis ypsilon.

Regeneration is a common phenomenon in various organisms by which tissues restore the damaged or naturally detached parts. In insects, appendage regeneration takes place during the embryonic, larval and pupal stages for individual survival. The wing disc of black cutworm Agrotis ypsilon has the capacity of regeneration after ablation, but understanding of molecular mechanisms in wing disc regeneration is still limited. After ablation of partial or whole wing discs before the fifth instar larval stage, the adult wings appeared to be normal. In the last two larval stages, ablation of the left wing disc led to smaller corresponding adult wing. Cell proliferation was reduced in the ablated wing disc but was gradually recovered two days post ablation. Transcriptome analysis found that genes in the mitogen-activated protein kinase (MAPK) pathway were upregulated. Repression of gene expression in this pathway, including Ras oncogene at 64B (Ras64B), Downstream of raf1 (Dsor1), and cAMP-dependent protein kinase catalytic subunit 3 (Pka-C3) by RNA interference after ablation, led to diminishment of both adult wings, suggesting that the MAPK signaling is essential for wing growth. Additionally, cell proliferation was still decelerated by injecting Ras64B, Dsor, or Pka-C3 dsRNA two days after ablation, indicating that the MAPK signaling-regulated cell proliferation is essential for growth. These results provide molecular clues to the regulation of cell proliferation during regeneration in lepidopteran insects.

COXIV and SIRT2-mediated G6PD deacetylation modulate ROS homeostasis to extend pupal lifespan.

Previous studies have shown that high physiological levels of reactive oxygen species (ROS) in the brain promote pupal diapause, which extends the pupal lifespan. However, the molecular mechanisms of ROS generation are unclear. In this paper, we found that mitochondrial ROS (mtROS) levels in the brains of Helicoverpa armigera diapause-destined pupae (DP) were higher and that the expression of cytochrome oxidase subunit IV (COXIV) was lower than in NP. In addition, downregulating COXIV caused mitochondrial dysfunction which elevated mtROS levels. Protein kinase A (PKA) was downregulated in DP, which led to the downregulated expression of the mitochondrial transcription factor TFAM. Low TFAM activity failed to promote COXIV expression and resulted in the high ROS levels that induced diapause. In addition, low sirtuin 2 expression suppressed glucose-6-phosphate dehydrogenase (G6PD) deacetylation at K382, which led to reduced G6PD activity and low NADPH levels, thereby maintaining high levels of ROS. Two proteins, COXIV and G6PD, thus play key roles in the elevated accumulation of ROS that induce diapause and extend the pupal lifespan.

Label-free quantitative proteomic analysis of insect larval and metamorphic molts.

BACKGROUND: Molting is an essential biological process occurring characteristic times throughout the life cycle of holometabolous insects. However, it is not clear how insects determine the direction of molting to remain status quo or to initiate metamorphosis. To explore the functional factors that determine the direction of molts, liquid chromatography-mass spectrometry was used to identify the molecules involved in larval and metamorphic molting, and the differentially expressed proteins (DEPs) were compared in the two processes. RESULTS: There were 321 and 1140 DEPs identified in larval and metamorphic molting process, respectively. Bioinformatics analyses show that the amino sugar pathway was up-regulated in both processes. The up-regulated protease contributed to the metamorphosis. In addition, several proteins with different expression patterns in larval-larval and larval-pupal transitions, including Endochitinase, GRIM-19 (Genes associated with retinoid-IFN-induced mortality-19), IDE (Insulin-degrading enzyme), Sorcin (Soluble resistance related calcium binding protein), OBP (Odorant-binding protein-2 precursor), TRAP1(Tumor necrosis factor receptor associated protein-1), etc., were further identified by parallel reaction monitoring, which may play diverse functions in larval-larval and larval-pupal transitions. CONCLUSIONS: These results provide a proteomic insight into molecules involved in larval and metamorphic molts, and will likely improve the current understanding of determination of direction of molts.

Identification and profiling of Bactrocera dorsalis microRNAs and their potential roles in regulating the developmental transitions of egg hatching, molting, pupation and adult eclosion.

MicroRNAs (miRNAs) are endogenous small noncoding RNAs (18-25 nt) that are involved in many physiological processes including development, cancer, immunity, apoptosis and host-microbe interactions through post-transcriptional regulation of gene expression. In this study, we measured the profile of small RNAs over the developmental transitions of the oriental fruit fly Bactrocera dorsalis from egg hatching, molting, and pupation to adult eclosion. We identified 250 miRNAs, including 83 known and 167 novel miRNAs, and 47 isomiRNAs. In addition, we identified the miRNAs differentially expressed over the developmental transitions. Interestingly, the miR-309 cluster, the miR-2 cluster/family and the let-7 cluster were among these differentially expressed miRNAs, suggesting a role in the regulation of egg hatching, molting and pupation/adult eclosion, respectively. Moreover, a detailed analysis of the temporal expression patterns of 14 highly expressed miRNAs in the pupal stage revealed three types of expression profiles. Furthermore, injection of a miR-100 mimic in the 3rd instar larvae resulted in a significant decrease in pupation and adult eclosion rates, whereas injection of a miR-317 antagomir resulted in a significant decrease in the pupation rate and a decrease in the pupation time, indicating that miR-100 and miR-317 are involved in the process of pupation. Finally, injection of a miR-100/miR-285 mimic or antagomir in pupae resulted in a significant decrease in the eclosion rate and a significant increase in the prevalence of a partial eclosion phenotype, implying the involvement of miR-100 and miR-285 in the process of adult eclosion. This study identified critical miRNAs involved in the transitions of this important holometabolic model and pest insect B. dorsalis from egg hatching to adult eclosion, thus providing a useful resource for exploring the regulatory role of miRNAs during insect post-embryonic development.

Effects of long-term cadmium exposure on trehalose metabolism, growth, and development of Aedes albopictus (Diptera: Culicidae).

Trehalose is the major blood sugar in insects; it not only serves as an energy source but also plays important roles in physiological responses to adverse conditions. However, only a few studies have explored the effects of heavy metal exposure stress on trehalose metabolism in insects. Therefore, in this study, we examined the effects of cadmium stress on changes in trehalose metabolism in Aedes albopictus. Three concentrations of cadmium (0.005, 0.01, and 0.1 mg/L) were selected for evaluation of long-term stress in Ae. albopictus (from eggs to adults); Ae. albopictus in double-distilled water was used as the control group. The trehalose and glucose contents, trehalase activity, and trehalose metabolism-related gene expression were determined. The effects of long-term cadmium exposure on growth, development, and reproduction were also assessed. Trehalose contents were increased, whereas glucose contents and trehalase activity were decreased in Ae. albopictus following long-term exposure to low concentrations of cadmium compared with those in untreated individuals. Moreover, the expression of trehalose-6-phosphate synthase was upregulated, and that of trehalase was downregulated, indicating that Ae. albopictus may enhance trehalose synthesis to resist cadmium stress. Cadmium exposure also caused Ae. albopictus individuals to become smaller with a longer developmental duration, whereas both reproduction and hatching rates of the offspring were decreased compared with those in the control group. Our findings demonstrated that cadmium exposure affected the morphology, physiology, and biochemistry of Ae. albopictus. These findings also confirmed the role of trehalose in the response of Ae. albopictus to cadmium stress, providing insights into the effects of heavy metal stress on trehalose metabolism in an insect model.

The development of an improved artificial diet for the mass rearing of Eldana saccharina Walker (Lepidoptera: Pyralidae).

BACKGROUND: This study determined the nutrient requirements of Eldana saccharina Walker (Lepidoptera: Pyralidae), a serious sugarcane pest in South Africa, to develop a more efficient artificial diet for mass-rearing purposes for sterile moth production. Diets tested consisted of a minimum specification (MS) diet representing a diet formulated according to the minimum specification of a summary of published diets, which yielded satisfactory results; an ideal amino acid profile (IAAP) diet, where amino acid composition was based on the profile of amino acids in the 2nd (IAAP2) and 5th /6th (IAAP5/6) instar larvae; and lastly two diets based on the nutrient composition of the natural diet of the insects, papyrus (PAP) and sugarcane (SC). Six treatments with 50 replications were randomly allotted to 300 25 mL plastic screw-top vials. The diet (15 mL) was dispensed into each vial and inoculated with two freshly hatched larvae. Larvae, pupae, and moths were harvested at 28 days after inoculation. Overall survivability, pupal weight, sex ratio, and rate of development was determined and compared with the diet currently in use at the South African Sugarcane Research Institute (CON). Physical characteristics of the diets such as the pH and the water-holding capacity of the diets were also determined. RESULTS: The natural diets (PAP and SC) were not viable as they did not yield any results. Survivability was significantly higher (78%) for the MS diet whilst IAAP2 and IAAP5/6 yielded the second highest survivability (74%) compared to CON (68%). There were no differences in male pupal weights between all treatment diets, as was the case for female pupae. Within dietary treatments, female pupae were heavier than male pupae for all treatment diets. CON (1.0: 1.6) produced significantly less male than female pupae with MS (1.0: 1.2), IAAP2 (1.0: 1.0) and IAAP5/6 (1.0: 1.1) all producing equal amounts of male and female pupae. The MS diet (16%) yielded fourfold the number of moths after 28 days compared to CON (4%) and IAAP2 (4%) diets. IAAP5/6 yielded no moths after 28 days. The life stages thus developed fastest in the MS diet. The pH of all treatment diets remained stable for the entire duration of the trial. No biological contamination was observed through all diets. Differences in water-holding capacity were observed between most diets with PAP and SC losing the most moisture whilst the MS and IAAP2 diets retained the most moisture. CONCLUSION: The MS diet most closely represented the nutrient requirements of E. saccharina, leading to its faster development on this formulation, which could be readily applied for large-scale production of this lepidopteran pest as an aid in the mass rearing of sterile males as part of the integrated pest management plan. © 2020 Society of Chemical Industry.

Characterization of an Ecdysteroid-Regulated 16 kDa Protein Gene in Chinese Oak Silkworm, Antheraea pernyi (Lepidoptera: Saturniidae).

A large number of ecdysteroid-regulated 16 kDa proteins (ESR16s) of insects have been isolated and annotated in GenBank; however, knowledge on insect ESR16s remain limited. In the present study, we characterized an ecdysteroid-regulated 16 kDa protein gene isolated in Chinese oak silkworm, Antheraea pernyi Guérin-Méneville ('ApESR16' in the following), an important silk-producing and edible insect. The obtained cDNA sequence of ApESR16 is 1,049 bp, harboring an open reading frame of 441 bp that encodes a polypeptide of 146 amino acids. CD-search revealed that ApESR16 contains the putative cholesterol/lipid binding sites on conserved domain Npc2_like (Niemann-Pick type C-2) belonging to the MD-2-related lipid-recognition superfamily. Sequence comparison revealed that ApESR16 exhibits 51-57% identity to ESR16s of lepidopteran insects, 36-41% identity to ESR16 or NPC2a of nonlepidopteran insects, and 28-32% identity to NPC2a of vertebrates, indicating a high sequence divergence during the evolution of animals. Phylogenetic analysis found that the used sequences were divided into two groups corresponding to vertebrates and invertebrates, and the used insect sequences were also well clustered according to their families. The A. pernyi ESR16 mRNA is expressed during all four developmental stages and in all tested tissues. Injection of 20-hydroxyecdysone (20-E) into A. pernyi diapausing pupae triggering diapause termination induced upregulation of ESR16 mRNA compared to the diapausing pupae, with the highest expression level at day 2 in the ovaries but day 12 in the fat body. Our results suggested that ApESR16 might be a diapause-related gene and plays a vital role in the pupal diapause of A. pernyi.