Altered tyrosine metabolism and melanization complex formation underlie the developmental regulation of melanization in Manduca sexta

The study of hemolymph melanization in Lepidoptera has contributed greatly to our understanding of its role in insect immunity. Manduca sexta in particular has been an excellent model for identifying the myriad components of the phenoloxidase (PO) cascade and their activation through exposure to pathogen-associated molecular patterns (PAMPs). However, in a process that is not well characterized or understood, some insect species rapidly melanize upon wounding in the absence of added PAMPs. We sought to better understand this process by measuring wound-induced melanization in four insect species. Of these, only plasma from late 5th instar M. sexta was unable to melanize, even though each contained millimolar levels of the putative melanization substrate tyrosine (Tyr). Analysis of Tyr metabolism using substrate-free plasmas (SFPs) from late 5th instar larvae of each species showed that only M. sexta SFP failed to melanize with added Tyr. In contrast, early instar M. sexta larvae exhibited wound-induced melanization and Tyr metabolism, and SFPs prepared from these larvae melanized in the presence of Tyr. Early instar melanization in M. sexta was associated with the formation of a high mass protein complex that could be observed enzymatically in native gels or by PO-specific immunoblotting. Topical treatment of M. sexta larvae with the juvenile hormone (JH) analog methoprene delayed pupation and increased melanizing ability late in the instar, thus linking development with immunity. Our results demonstrate that melanization rates are highly variable in Lepidoptera, and that developmental stage can be an important factor for melanization within a species. More specifically, we show that the physiological substrate for melanization in M. sexta is Tyr, and that melanization is associated with the formation of a PO-containing protein complex.     

Structural features,evolutionary relationships,and transcriptional regulation of C-type lectin-domain proteins in Manduca sexta

C-type lectins (CTLs) are a large family of Ca²⁺-dependent carbohydrate-binding proteins recognizing various glycoconjugates and functioning primarily in immunity and cell adhesion. We have identified 34 CTLDP (for CTL-domain protein) genes in the Manduca sexta genome, which encode proteins with one to three CTL domains. CTL-S1 through S9 (S for simple) have one or three CTL domains; immulectin-1 through 19 have two CTL domains; CTL-X1 through X6 (X for complex) have one or two CTL domains along with other structural modules. Nine simple CTLs and seventeen immulectins have a signal peptide and are likely extracellular. Five complex CTLs have both an N-terminal signal peptide and a C-terminal transmembrane region, indicating that they are membrane anchored. Immulectins exist broadly in Lepidoptera and lineage-specific gene duplications have generated three clusters of fourteen genes in the M. sexta genome, thirteen of which have similar expression patterns. In contrast to the family expansion, CTL-S1∼S6, S8, and X1∼X6 have 1:1 orthologs in at least four lepidopteran/dipteran/coleopteran species, suggestive of conserved functions in a wide range of holometabolous insects. Structural modeling suggests the key residues for Ca²⁺-dependent or independent binding of certain carbohydrates by CTL domains. Promoter analysis identified putative κB motifs in eighteen of the CTL genes, which did not have a strong correlation with immune inducibility in the mRNA or protein levels. Together, the gene identification, sequence comparisons, structure modeling, phylogenetic analysis, and expression profiling establish a solid foundation for future studies of M. sexta CTL-domain proteins.     

Integrated modeling of protein-coding genes in the Manduca sexta genome using RNA-Seq data from the biochemical model insect

The genome sequence of Manduca sexta was recently determined using 454 technology. Cufflinks and MAKER2 were used to establish gene models in the genome assembly based on the RNA-Seq data and other species' sequences. Aided by the extensive RNA-Seq data from 50 tissue samples at various life stages, annotators over the world (including the present authors) have manually confirmed and improved a small percentage of the models after spending months of effort. While such collaborative efforts are highly commendable, many of the predicted genes still have problems which may hamper future research on this insect species. As a biochemical model representing lepidopteran pests, M. sexta has been used extensively to study insect physiological processes for over five decades. In this work, we assembled Manduca datasets Cufflinks 3.0, Trinity 4.0, and Oases 4.0 to assist the manual annotation efforts and development of Official Gene Set (OGS) 2.0. To further improve annotation quality, we developed methods to evaluate gene models in the MAKER2, Cufflinks, Oases and Trinity assemblies and selected the best ones to constitute MCOT 1.0 after thorough crosschecking. MCOT 1.0 has 18,089 genes encoding 31,666 proteins: 32.8% match OGS 2.0 models perfectly or near perfectly, 11,747 differ considerably, and 29.5% are absent in OGS 2.0. Future automation of this process is anticipated to greatly reduce human efforts in generating comprehensive, reliable models of structural genes in other genome projects where extensive RNA-Seq data are available.     

Expression and evolution of hexamerins from the tobacco hornworm,Manduca sexta,and other Lepidoptera

Hexamerins are large hemolymph-proteins that accumulate during the late larval stages of insects. Hexamerins have emerged from hemocyanin, but have lost the ability to bind oxygen. Hexamerins are mainly considered as storage proteins for non-feeding stages, but may also have other functions, e.g. in cuticle formation, transport and immune response. The genome of the hornworm Manduca sexta harbors six hexamerin genes. Two of them code for arylphorins (Msex2.01690, Msex2.15504) and two genes correspond to a methionine-rich hexamerin (Msex2.10735) and a moderately methionine-rich hexamerin (Msex2.01694), respectively. Two other genes do not correspond to any known hexamerin and distantly resemble the arylphorins (Msex2.01691, Msex2.01693). Five of the six hexamerin genes are clustered within ∼45 kb on scaffold 00023, which shows conserved synteny in various lepidopteran genomes. The methionine-rich hexamerin gene is located at a distinct site. M. sexta and other Lepidoptera have lost the riboflavin-binding hexamerin. With the exception of Msex2.01691, which displays low mRNA levels throughout the life cycle, all hexamerins are most highly expressed during pre-wandering phase of the 5th larval instar of M. sexta, supporting their role as storage proteins. Notably, Msex2.01691 is most highly expressed in the brain, suggesting a divergent function. Phylogenetic analyses showed that hexamerin evolution basically follows insect systematics. Lepidoptera display an unparalleled diversity of hexamerins, which exceeds that of other hexapod orders. In contrast to previous analyses, the lepidopteran hexamerins were found monophyletic. Five distinct types of hexamerins have been identified in this order, which differ in terms of amino acid composition and evolutionary history: i. the arylphorins, which are rich in aromatic amino acids (∼20% phenylalanine and tyrosine), ii. the distantly related arylphorin-like hexamerins, iii. the methionine-rich hexamerins, iv. the moderately methionine rich hexamerins, and v. the riboflavin-binding hexamerins.     

A broadly tuned odorant receptor in neurons of trichoid sensilla in locust,Locusta migratoria

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. Odorant receptors (ORs) on the membrane of chemosensory neurons are believed to be key molecules in sensing exogenous chemical cues. ORs in different species of insects are diverse and should tune a species to its own specific semiochemicals relevant to their survival. The orthopteran insect, locust (Locusta migratoria), is a model hemimetabolous insect. There is very limited knowledge on the functions of locust ORs although many locust OR genes have been identified in genomic sequencing experiments. In this paper, a locust OR, LmigOR3 was localized to neurons housed in trichoid sensilla by in situ hybridization. LmigOR3 was expressed as a transgene in Drosophila trichoid olfactory neurons (aT1) lacking the endogenous receptor Or67d and the olfactory tuning curve and dose-response curves were established for this locust receptor. The results show that LmigOR3 sensitizes neurons to ketones, esters and heterocyclic compounds, indicating that LmigOR3 is a broadly tuned receptor. LmigOR3 is the first odorant receptor from Orthoptera that has been functionally analyzed in the Drosophila aT1 system. This work demonstrates the utility of the Drosophila aT1 system for functional analysis of locust odorant receptors and suggests that LmigOR3 may be involved in detecting food odorants, or perhaps locust body volatiles that may help us to develop new control methods for locusts.     

Overview of chitin metabolism enzymes in Manduca sexta: Identification,domain organization,phylogenetic analysis and gene expression

Chitin is one of the most abundant biomaterials in nature. The biosynthesis and degradation of chitin in insects are complex and dynamically regulated to cope with insect growth and development. Chitin metabolism in insects is known to involve numerous enzymes, including chitin synthases (synthesis of chitin), chitin deacetylases (modification of chitin by deacetylation) and chitinases (degradation of chitin by hydrolysis). In this study, we conducted a genome-wide search and analysis of genes encoding these chitin metabolism enzymes in Manduca sexta. Our analysis confirmed that only two chitin synthases are present in M. sexta as in most other arthropods. Eleven chitin deacetylases (encoded by nine genes) were identified, with at least one representative in each of the five phylogenetic groups that have been described for chitin deacetylases to date. Eleven genes encoding for family 18 chitinases (GH18) were found in the M. sexta genome. Based on the presence of conserved sequence motifs in the catalytic sequences and phylogenetic relationships, two of the M. sexta chitinases did not cluster with any of the current eight phylogenetic groups of chitinases: two new groups were created (groups IX and X) and their characteristics are described. The result of the analysis of the Lepidoptera-specific chitinase-h (group h) is consistent with its proposed bacterial origin. By analyzing chitinases from fourteen species that belong to seven different phylogenetic groups, we reveal that the chitinase genes appear to have evolved sequentially in the arthropod lineage to achieve the current high level of diversity observed in M. sexta. Based on the sequence conservation of the catalytic domains and on their developmental stage- and tissue-specific expression, we propose putative functions for each group in each category of enzymes.     

A genome-wide analysis of antimicrobial effector genes and their transcription patterns in Manduca sexta

Antimicrobial proteins/peptides (AMPs) are effectors of innate immune systems against pathogen infection in multicellular organisms. Over half of the AMPs reported so far come from insects, and these effectors act in concert to suppress or kill bacteria, fungi, viruses, and parasites. In this work, we have identified 86 AMP genes in the Manduca sexta genome, most of which seem likely to be functional. They encode 15 cecropins, 6 moricins, 6 defensins, 3 gallerimycins, 4 X-tox splicing variants, 14 diapausins, 15 whey acidic protein homologs, 11 attacins, 1 gloverin, 4 lebocins, 6 lysozyme-related proteins, and 4 transferrins. Some of these genes (e.g. attacins, cecropins) constitute large clusters, likely arising after rounds of gene duplication. We compared the amino acid sequences of M. sexta AMPs with their homologs in other insects to reveal conserved structural features and phylogenetic relationships. Expression data showed that many of them are synthesized in fat body and midgut during the larval-pupal molt. Certain genes contain one or more predicted κB binding sites and other regulatory elements in their promoter regions, which may account for the dramatic mRNA level increases in fat body and hemocytes after an immune challenge. Consistent with these strong mRNA increases, many AMPs become highly abundant in the larval plasma at 24 h after the challenge, as demonstrated in our previous peptidomic study. Taken together, these data suggest the existence of a large repertoire of AMPs in M. sexta, whose expression is up-regulated via immune signaling pathways to fight off invading pathogens in a coordinated manner.     

Cloning and preliminary functional studies of the JAM-A gene in grass carp (Ctenopharyngodon idellus)

Grass carp (Ctenopharyngodon idellus) is a very important aquaculture species in China and other South-East Asian countries; however, disease outbreaks in this species are frequent, resulting in huge economic losses. Grass carp hemorrhage caused by grass carp reovirus (GCRV) is one of the most serious diseases. Junction adhesion molecule A (JAM-A) is the mammalian receptor for reovirus, and has been well studied. However, the JAM-A gene in grass carp has not been studied so far. In this study, we cloned and elucidated the structure of the JAM-A gene in grass carp (GcJAM-A) and then studied its functions during grass carp hemorrhage. GcJAM-A is composed of 10 exons and 9 introns, and its full-length cDNA is 1833 bp long, with an 888 bp open reading frame (ORF) that encodes a 295 amino acid protein. The GcJAM-A protein is predicted to contain a typical transmembrane domain. Maternal expression pattern of GcJAM-A is observed during early embryogenesis, while zygote expression occurs at 8 h after hatching. GcJAM-A is expressed strongly in the gill, liver, intestine and kidney, while it is expressed poorly in the blood, brain, spleen and head kidney. Moreover, lower expression is observed in the gill, liver, intestine, brain, spleen and kidney of 30-month-old individuals, compared with 6-month-old. In a GcJAM-A-knockdown cell line (CIK) infected with GCRV, the expression of genes involved in the interferon and apoptosis pathways was significantly inhibited. These results suggest that GcJAM-A could be a receptor for GCRV. We have therefore managed to characterize the GcJAM-A gene and provide evidence for its role as a receptor for GCRV.     

Diversity at two genetic loci associated with the major histocompatibility complex in the golden snub-nosed monkey (Rhinopithecus roxellana)

Habitat loss via human activity has fragmented populations of the golden snub-nosed monkey (Rhinopithecus roxellana), and thus affected patterns of gene flow. We investigated in-depth a single troop in the Qinling Mountains, central China, two major histocompatibility complex (MHC) class II loci, DQA1 and DQB1, and compared the resulting data with data from troops from the wider Qinling Mountains region. We found that a novel DQB1 allele was only present in the study troop and relatively few divergent alleles at the DQA1 and DQB1 loci were present compared with the wider population. The inbreeding coefficient (F_is) at the MHC region was lower than previous measurements, which may have reflected different sampling strategies. However, R. roxellana has relatively high diversity in MHC genes, even though it has probably experienced serious past population bottlenecks and reduced gene flow between populations. We also found that some alleles present in the wider population had been lost in the study troop, and suggest that conservation management strategies be implemented to increase gene flow between troops in order to increase genetic variation.