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Genes identified in 5th instar molting larvae. A) Gene frequency. B) Gene transcription composition (number, percentage).
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Larval molting and metamorphosis are important physiological processes in the life cycle of the holometabolous insect. We used suppression subtractive hybridization (SSH) to identify genes differentially expressed during larval molting and metamorphosis.
We performed SSH between tissues from a variety of developmental stages, including molting 5th...
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Diapause, a state of arrested development accompanied by a marked decrease of metabolic rate, helps insects to overcome unfavorable seasons. Helicoverpa armigera (Har) undergoes pupal diapause, but the molecular mechanism of diapause initiation is unclear. Using suppression subtractive hybridization (SSH), we investigated differentially expressed g...
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... Comprehensive survey of available protein databases and literature to evaluate the functionalities of identified proteins revealed prominent involvement of multiple proteins with larval cuticle development. Further, hydrolases have been identified to play a key role in insect metamorphosis and larval moulting [45,46] justifying their presence in all the larval stages. String analysis (https://string-db.org/) using the selected protein Ids commonly present in all instars (137 proteins) revealed the protein-protein interaction network of peptide metabolism and amide biosynthesis pathway (Fig. 2B). ...
... In arthropods, apolysis is considered as an initial sign of molting and triggered by rising 20E titer (24,25). Immediately after apolysis, the molting fluid is secreted into the new apolysial space and initiates a series of biological processes such as new cuticle secretion, old cuticle degradation, and accumulation of neuropeptides related to molting (1,(26)(27)(28)(29). These biological processes reveal the existence of a complex and precise regulatory network at the premolt stage. ...
Ecdysis triggering hormone (ETH) plays an important role in molting, reproduction, and courtship behavior in insects. To investigate the potential downstream pathways and genes of ETH in Scylla paramamosain, RNA interference (RNAi) was conducted on crabs at early (D0) and late (D2) premolt substages, and the transcriptome profiles of each group were compared by RNA sequencing. Real-time quantitative polymerase chain reaction (RT-qPCR) and semiquantitative polymerase chain reaction (RT-PCR) results showed a significant knockdown of ETH at D0 stage, whereas a significant increase was shown conversely in crabs at D2 substage after the injection of dsETH. A total of 242,979 transcripts were assembled, and 44,012 unigenes were identified. Transcriptomic comparison between crabs at D2 and D0 substages showed 2,683 differentially expressed genes (DEGs); these genes were enriched in ribosome and pathways related to transcription factor complex and cell part. Twenty DEGs were identified between dsETH-injected and dsGFP-injected crabs at D0 substage; these DEGs were involved in carbohydrate metabolism, one carbon pool by folate, and chitin binding. Twenty-six DEGs were identified between dsETH-injected and dsGFP-injected crabs at D2 substage; these DEGs were involved in calcium channel inhibitor activity, fat digestion and absorption, and cardiac muscle contraction. RT-qPCR verified the differential expression of the selected genes. In conclusion, crabs at D0 substage are more active in preparing the macromolecular complex that is needed for molting. Moreover, ETH has potential roles in carbohydrate metabolism, one carbon pool by folate, and chitin binding for crabs at D0 substage, while the role of ETH turns to be involved in calcium channel inhibitor activity, fat digestion and absorption, and cardiac muscle contraction at D2 substage to facilitate the occurrence of molting. The selected DEGs provide valuable insight into the role of ETH in the regulation of crustacean molting.
... In order to study the differentially expressing genes in resin secreting and non resin secreting stages of lac insects, we have used an approach which combines the power of normalization of expressed genes and subtraction of commonly expressed genes in one step i.e., Suppression Subtraction Hybridization (SSH) as described by Diatchenko et al. (1996). SSH technique was widely used in insects to address various research problems involving different life stages (Dong et al., 2007;Geng et al., 2009;Oppelt et al., 2010). ...
Biosynthesis mechanism of the only animal resin, lac resin remains elusive to the scientific community. An attempt to identify the genes of resin biosynthesis in lac insects was made through SSH (Suppression Subtraction Hybridization) library by subtracting genes of resin non secreting stage (neonate nymphs) from the resin secreting stage (adult female) of the lac insect. SSH library yielded 107 non redundant clones with 61 clones having sequences homologous with known functions (defence, development, metabolism, regulation, transport, cell division, respiration, structural role), 7 clones having hypothetical proteins or uncharacterized proteins and 39 clones did not find any matches. Among the differentially expressed genes, few putative genes of terpene biosynthesis, fatty acid biosynthesis and transcriptional regulation were identified. Out of them, an IDS (Isopentenyl Diphosphate Synthase) type decaprenyl diphosphate synthase and a desaturase gene, acyl-CoA delta desaturase were cloned from lac insects and sequenced. Their expression levels were found to be more in lac resin synthesis stages such as settled nymphs and fertilized female insects in comparison with resin non secreting stage, crawlers (neonate nymphs). It gives a clear indication of their role in lac resin biosynthesis due to the correlation of their expression level with resin biosynthesis in lac insects.
... This could indicate different ligand specificity for the receptor relative to Homam-ASTCR 1 and 2. Indeed, urotensin II receptors from the western clawed frog were reportedly not activated by somatostatin [50]; however, this ligand discrimination does not always appear to be the case, as various somatostatin receptor subtypes can be activated by urotensin II [51]. Alternatively, cell surface trafficking of Homam-ASTCR4 may require the presence of regulatory proteins (e.g., ER chaperones, accessory proteins, and/or receptor activity modifying proteins) present in cells that comprise the CG, but absent in our insect cell system [52,53]. ...
Central pattern generators produce rhythmic behaviors independently of sensory input; however, their outputs can be modulated by neuropeptides, thereby allowing for functional flexibility. We investigated the effects of C-type allatostatins (AST-C) on the cardiac ganglion (CG), which is the central pattern generator that controls the heart of the American lobster, Homarus americanus, to identify the biological mechanism underlying the significant variability in individual responses to AST-C. We proposed that the presence of multiple receptors, and thus differential receptor distribution, was at least partly responsible for this observed variability. Using transcriptome mining and PCR-based cloning, we identified four AST-C receptors (ASTCRs) in the CG; we then characterized their cellular localization, binding potential, and functional activation. Only two of the four receptors, ASTCR1 and ASTCR2, were fully functional GPCRs that targeted to the cell surface and were activated by AST-C peptides in our insect cell expression system. All four, however, were amplified from CG cDNAs. Following the confirmation of ASTCR expression, we used physiological and bioinformatic techniques to correlate receptor expression with cardiac responses to AST-C across individuals. Expression of ASTCR1 in the CG showed a negative correlation with increasing contraction amplitude in response to AST-C perfusion through the lobster heart, suggesting that the differential expression of ASTCRs within the CG is partly responsible for the specific physiological response to AST-C exhibited by a given individual lobster.
... We also identified transcripts that were differentially expressed across most time points (from 12 to 72 h p.i.), and they are listed in Table 2. Of these, we identified 11 transcripts that were induced or upregulated across all times sampled from 12 to 72 h postinfection. These transcripts included HMG176 (a response to Sequence Distribution [Biological Process] cellular protein metabolic process (10) transmembrane transport (11) response to stimulus (12) cellular component organization (12) catabolic process (12) small molecule metabolic process (18) carbohydrate metabolic process (18) cellular nitrogen compound metabolic process (27) biosynthetic process (22) lipid metabolic process (21) single-organism cellular process (19) ...
... REPAT proteins were later identified in other lepidopteran species (17)(18)(19)(20)(21) [see also "(b) REPAT genes" below]. The HMG176 gene of Helicoverpa armigera is normally expressed at high levels in midguts of molting larvae, and the protein is found in the basal lamina of the midgut of molting and feeding larvae, but not in larvae committed to metamorphosis (16,22). The precise function of HMG176 is not known. ...
... Thus far, the precise biochemical or molecular roles of REPAT proteins have not been identified, although they appear to play a variety of roles. While HMG176 is found in the basal lamina of the midgut (16,22), another REPAT protein (MBF2 from B. mori) was identified to be a transcriptional coactivator (20,47). The single identified Drosophila REPAT gene (CG13323) was reported to be upregulated in response to infection with bacteria, fungi, and sigma virus (48), but the function of the encoded protein is unknown. ...
Baculoviruses are virulent pathogens of a number of important insect pest species. In the host Trichoplusia ni , infection begins in the midgut when infectious virions of the occlusion-derived virus (ODV) phenotype enter and subsequently replicate in cells of the midgut epithelium. A second virion phenotype (budded virus [BV]) is produced there, and BV mediates systemic infection of the animal. Most prior detailed studies of baculovirus infections have focused on BV infections of cultured cells. In this study, we examined the transcriptional responses of the T. ni midgut to infection by ODV of the baculovirus AcMNPV and identified a variety of host genes that respond dramatically to viral infection. Understanding the transcriptional responses of the host midgut to viral infection is critically important for understanding the biphasic infection in the animal as a whole.
... Metamorphosis is an essential biological journey of insects characterized by destruction of old tissue and new tissue formation [20], in which hydrolases play key roles in the regulation of insect larval molting and metamorphosis [21,22]. GO analysis identified 28 proteins as being involved in the carbohydrate metabolism process (Supporting Information Table 7). ...
The silkworm is a lepidopteran insect that has an open circulatory system with hemolymph consisting of blood and lymph fluid. Hemolymph is not only considered as a depository of nutrients and energy, but it also plays a key role in substance transportation, immunity response, and proteolysis. In this study, we used liquid chromatography-tandem mass spectrometry to analyze the hemolymph proteins of four developmental stages during metamorphosis. A total of 728 proteins were identified from the hemolymph of the 2(nd) day of wandering stage, the 1(st) day of pupation, the 9(th) day of pupation, and the 1(st) day as an adult moth. Gene Ontology annotations and categories showed that silkworm hemolymph proteins were enriched in carbohydrate metabolism, proteolysis, protein binding, and antibacterial humoral response. The levels of nutrient, immunity-related, and structural proteins changed significantly during development and metamorphosis. Some, such as cuticle, odorant binding, and chemosensory proteins, showed stage-specific expression in the hemolymph. In addition, the expression of several antimicrobial peptides exhibited their highest level of abundance in the hemolymph of the early pupal stage. These findings provide a comprehensive proteomic insight of the silkworm hemolymph and suggest additional molecular targets for studying insect metamorphosis. This article is protected by copyright. All rights reserved.
... McMG176s, as well as McREPAT2, were expressed only in the midgut (Fig. 1); however, SeREPAT genes were also expressed in the hindgut (Herrero et al., 2007) and HMG176 was also expressed in the fat body (Dong et al., 2007;Wang et al., 2007). Upregulation of REPAT genes in response to pathogen challenge and the reduction in virulence of a recombinant baculovirus expressing SeREPAT1 suggest that they have a role in defense against pathogens (Herrero et al., 2007). ...
The peritrophic matrix (PM) is essential for insect digestive system physiology as it protects the midgut epithelium from damage by food particles, pathogens and toxins. The PM is also an attractive target for development of new pest control strategies due to its per os accessibility. To understand how the PM performs these functions, the molecular architecture of the PM was examined using genomic and proteomic approaches in Mamestra configurata (Lepidoptera: Noctuidae), a major pest of cruciferous oilseed crops in North America. Liquid chromatography-tandem mass spectrometry analyses of the PM identified 82 proteins classified as: (i) peritrophins, including a new class with a CBDIII domain; (ii) enzymes involved in chitin modification (chitin deacetylases), digestion (serine proteases, aminopeptidases, carboxypeptidases, lipases and α-amylase) or other reactions (β-1,3-glucanase, alkaline phosphatase, dsRNase, astacin, pantetheinase); (iii) a group polycalin, REPATs, serpin, C-Type lectin and Lsti99/Lsti201 and three novel proteins without known orthologs. The genes encoding PM proteins were expressed predominantly in the midgut. cDNAs encoding chitin synthase-2 (McCHS-2), chitinase (McCHI) and β-N-acetylglucosaminidase (McNAG), enzymes involved in PM chitin metabolism, were also identified. McCHS-2 expression was specific to the midgut indicating that it is responsible for chitin synthesis in the PM, the only chitinous material in the midgut. In contrast, the genes encoding the chitinolytic enzymes were expressed in multiple tissues. McCHS-2, McCHI and McNAG, were expressed in the midgut of feeding larvae, and NAG activity was present in the PM. This information was used to generate an updated model of the lepidopteran PM architecture. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
... It has been observed that the phosphorylation of proteosomes during the larval molting of cotton bollworm (Helicoverpa armigera) (Dong et al. 2007). The upregulation of phosphorylated p in the embryo most likely to signify the participation of proteasomes in tissue remodeling (Mykles 1999) during active embryonic development. ...
... Thus, the BIN identified proteins related to carbohydrate metabolism and energy production (26.7 %), development (17.9 %), protein biosynthesis (14.3 %) cytoskeleton (12.5 %) and protein folding (10.7%) as the most networked groups(Figure 2.6). The results provide a global perspective on56 the protein and the biochemical pathways involved in the honeybee embryo-larvae transition, helping us to better understand honeybee biology. In combination with functional enrichment analysis, these five groups infer as the major modulators for the embryo-larva transition. ...
... The most abundant transcript in each larval dataset was a B. malayi hypothetical protein homologue with similarity to a checkpoint-like protein from Helicoverpa armigera (cotton bollworm) which shows increased expression during larval molting (Dong et al. 2007). Here, the larvae in both treatments were exsheathing during the incubation period and the presence of high levels of this transcript may reflect this process. ...
The studies submitted herein have contributed to our understanding of ruminant immunology, host-parasite interactions during ruminant infection with nematode parasites, and potential vaccine strategies to combat parasitic gastroenteritis (PGE). PGE of sheep and cattle, caused by T. circumcincta and O. ostertagia respectively, is a major problem for the global farming industry both in terms of productivity and animal welfare. To date control of these parasites has relied on the use of anthelmintic drugs however the emergence of widespread anthelmintic resistance is driving the search for alternative methods of control. As ruminants do acquire immunity in the field, vaccination is one such alternative under investigation. The first three papers contributing to this thesis used modern immunological tools alongside a locally developed surgical technique to revisit a model of nematode infection in sheep, investigating the composition and kinetics of the ovine local immune response to infection with Teladorsagia circumcincta via cannulation of the efferent gastric lymph duct. A protective local secondary immune response was observed in sheep which had previously experienced infection with T. circumcincta, but was absent from naive sheep. This immune response consisted initially of a rise in TE and BE cell activity peaking at 3 and 5 days post challenge respectively, followed by a secondary parasiteEspecific IgA response from 5 days post challenge which correlated with stunting of parasite growth. Significant parasite loss occurred by 2 days post challenge, prior to detection of the secondary immune response, suggesting critical early events in the host-parasite interaction and the potential importance of larval antigens in these interactions. No difference was observed in either the manifestations of immunity, or the magnitude and quality of the immune response, between adult sheep and lambs. The fourth and fifth papers describe vaccine trials carried out in bovine and ovine hosts using detergent soluble proteins derived from 4th larval stage Ostertagia ostertagi and Teladorsagia circumcincta respectively as antigens. Substantial reduction in total faecal egg output of up to 85% was observed in the calf trials, but not in the sheep trials which attained a maximum reduction of 29% in total faecal egg output. The sixth paper is a transcriptomic study carried out using the Roche 454 sequencing platform to investigate the immediate responses of Teladorsagia circumcincta upon encountering ovine host tissue of either immune or naive status. Following larval exsheathing and 4 hours of exposure to either immune or naive abomasal environments the transcript level of several genes was observed to differ. Genes which were most upregulated in response to encountering the immune environment included a peptidyl-glycine alpha-amidating mono-oxygenase homologue and a small heat shock protein. The studies described herein represent a body of work carried out using up-to-date tools and technologies. The first three papers confirmed the existence of critical early events in the host-parasite interaction, pointing to the potential use of larval antigens as vaccine candidates described in the trials in papers 4 and 5, and leading to the in-depth transcriptomic analysis described in paper 6. Papers 4 and 5 demonstrated that while Teladorsagia circumcincta and Ostertagia ostertagi have similar life cycles and host-site predilection, and both the ovine and bovine host can develop immunity to incoming parasitic larvae in the field, important differences may exist in either the proteome of the fourth stage larvae and/or the nature of the host response. Paper 6 revealed that changes in T. circumcincta transcript levels in response to ovine-host immune status can be detected early in the host-parasite interaction.
... Up to now, a justified focus on the developmentally earliest expression patterns and control cascades of genes responsible for different aspects of body patterning has resulted in unjustified neglect for the expression patterns and control cascades of developmental genes along the postembryonic segment of life, with few exceptions, as for instance the studies on the metamorphosis of model species among the holometabolous insects (Diptera: Drosophila melanogaster; Lepidoptera: Manduca sexta, Bombyx mori and Helicoverpa armigera) (Arbeitman et al. 2002;Li and White 2003;Riddiford et al. 2003;Dong et al. 2007;Tanaka and Truman 2007;Ando et al. 2011) and a few pioneering studies on the expression of Sex combs reduced in the postembryonic development of hemimetabolous insects (Chesebro et al. 2009;Hrycaj et al. 2010). ...
According to a well-consolidated tradition, the body of arthropods is described in terms of segments and tagmata. Even the oldest names for these animals, Aristotle’s έντομα [entoma, internally (sub)divided] and Linnaeus’ Latin equivalent Insecta, now restricted to one of the major arthropod subgroups, already referred to the modular organization of the body. In the idealistic perspective of the past, this trait, more than the presence of articulated appendages to which the current name of arthropods refers, was considered the defining attribute for the body plan of these animals.
