Storage Hexamerins Research Articles

Diversity, evolution, and function of myriapod hemocyanins

BackgroundHemocyanin transports O2 in the hemolymph of many arthropod species. Such respiratory proteins have long been considered unnecessary in Myriapoda. As a result, the presence of hemocyanin in Myriapoda has long been overlooked. We analyzed transcriptome and genome sequences from all major myriapod taxa – Chilopoda, Diplopoda, Symphyla, and Pauropoda – with the aim of identifying hemocyanin-like proteins.ResultsWe investigated the genomes and transcriptomes of 56 myriapod species and identified 46 novel full-length hemocyanin subunit sequences in 20 species of Chilopoda, Diplopoda, and Symphyla, but not Pauropoda. We found in Cleidogona sp. (Diplopoda, Chordeumatida) a hemocyanin-like sequence with mutated copper-binding centers, which cannot bind O2. An RNA-seq approach showed markedly different hemocyanin mRNA levels from ~ 6 to 25,000 reads per kilobase per million reads. To evaluate the contribution of hemocyanin to O2 transport, we specifically studied the hemocyanin of the centipede Scolopendra dehaani. This species harbors two distinct hemocyanin subunits with low expression levels. We showed cooperative O2 binding in the S. dehaani hemolymph, indicating that hemocyanin supports O2 transport even at low concentration. Further, we demonstrated that hemocyanin is > 1500-fold more highly expressed in the fertilized egg than in the adult.ConclusionHemocyanin was most likely the respiratory protein in the myriapod stem-lineage, but multiple taxa may have independently lost hemocyanin and thus the ability of efficient O2 transport. In myriapods, hemocyanin is much more widespread than initially appreciated. Some myriapods express hemocyanin only at low levels, which are, nevertheless, sufficient for O2 supply. Notably, also in myriapods, a non-respiratory protein similar to insect storage hexamerins evolved from the hemocyanin.

Two-dimensional gel proteome analysis of honeybee, Apis mellifera, worker red-eye pupa hemolymph

Apis mellifera Linnaeus is a holometabolous insect that undergoes complete metamorphosis in its nonfeeding pupal stage before transitioning to the adult stage. Its pupal stages are classifiable by the unique color pigmentation of its compound eyes and thorax; notably, there is a red-eye stage involving an unpigmented body that has a relatively short duration and is easy to recognize. The aim of the current study was to create a proteomic reference map of the worker red-eye pupa hemolymph. Hemolymph was collected from dorsal vessels using glass capillary tubes and was examined using pI 3–10 two-dimensional gel electrophoresis (2DE; 10 and 14 %) and matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)/TOF protein identification. This experimental approach allowed us to identify 129 different proteins organized into orthologous groups. Overall, the predominant category was post-translational modifications, protein turnover and chaperones (23.3 % of the identified proteins). In addition, we identified proteins in the non-orthologous groups of olfaction (2.3 % of the identified proteins) and storage hexamerins (3.1 % of the identified proteins). Quantitatively, the major protein isoforms that were accurately identified via 10 % 2DE were four forms of storage hexamerin: the 110, 70a, 70b, and 70c forms. The most abundant enzymes identified were short-chain dehydrogenases/reductases with pivotal developmental roles in ecdysteroidogenesis and a sigma class glutathione-S-transferase that most likely serves as a major protectant against the by-products of oxidative stress. Many of the identified proteins are known to be involved in the mechanisms of metamorphosis. All of the identified proteins are useful as markers for future comparative physiological and developmental studies.

Linking adaptation, delimitation of evolutionarily significant units (ESUs), and gene function: a case study using hemlock looper ecotypes

Developing genetic markers for the identification of recently diverged groups, such as ecotypes or species complexes, remains difficult due to challenges with incomplete lineage sorting, hybridization and introgression. Genome‐wide scans of single nucleotide polymorphisms (SNPs) have proven useful for inferring patterns of genetic differentiation at the population level. In combination with a new analytical technique, the discriminant analysis of principal components (DAPC), and within the framework of iterative taxonomy, it may also be possible to extract a combination of SNPs as markers for the delimitation of closely related groups. In addition, since DAPC identifies the loci contributing the most to group clustering, it may be possible to link putative biological function to differences that define group boundaries. We tested this technique on two ecotypes of the hemlock looper (Lambdina fiscellaria), which differ in terms of number of larval stadia, developmental rate and fecundity. It was possible to separately cluster the two ecotypes with 95% correct assignment using 27 SNPs. We also determined that a storage hexamerin carried eight of these SNPs, including the two highest contributing loci, of which the top contributor was nonsynonymous. Other studies have found this protein to be highly expressed just before metamorphosis, pointing to a possible connection between its role in clustering ecotypes and its biological function. These SNP markers can now be further developed for high throughput delimitation of individuals of unknown ecotype identity.

Two storage hexamerins from the beet armyworm Spodoptera exigua: Cloning, characterization and the effect of gene silencing on survival

BackgroundIn insects, hemocyanin superfamily proteins accumulate apparently to serve as sources of amino acids during metamorphosis, reproduction and development. Storage hexamerins are important members of the hemocyanin superfamily. Although insects possess storage hexamerins, very little is known about the character and specific functions of hexamerin 1 and storage protein 1 in insect development.ResultsTo gain insight into the function of storage proteins in insects, cDNAs for two storage proteins were cloned from the fat body of Spodoptera exigua. S. exigua hexamerin 1 (SeHex) cDNA contained an open reading frame of 2124 nucleotides encoding a protein of 707 amino acids with a predicted molecular weight of 82.12 kDa. S. exigua storage protein 1 (SeSP1) cDNA contained an open reading frame of 2256 bp encoding a protein of 751 amino acids with a predicted molecular weight of ~88.84 kDa. Northern blotting analyses revealed that SeHex mRNA is expressed in the fat body, cuticle, midgut and Malpighian tubules and SeSP1 in fat body, Malpighian tubules and tracheae. SeHex and SeSP1 mRNAs were expressed in fat body at different levels from first instar larvae to pupae, with expression was much lower from first instar larvae to first-day fifth instar larvae. SeHex transcript expression was high in fat body of wandering larvae (pre-pupae) and steadily decreased to the seventh pupal day. SeSP1 transcript expression was high in fat body of wandering larvae, 2-day-old fifth instar larvae and 2-, 4- and 7-day-old pupae. SeHex and SeSP1 mRNAs levels were expressed lower than control on the condition of starvation at 12 h. Of insects injected with SeHex and SeSP1 dsRNA, 38.7% and 24.3% survived to 204 h after treatment, respectively. This was significantly lower than in the controls groups.ConclusionsThese findings provide new data on the tissue distribution, expression patterns and the function in starvation of storage proteins. RNA interference results revealed that storage protein genes are key in metamorphosis, reproduction and insect development. The results for SeHex and SeSP1 interference reveal that a potential method to control this pest is to disrupt the regulation of storage proteins.

The occurrence of hemocyanin in Hexapoda

Hemocyanins are copper-containing, respiratory proteins that have been thoroughly studied in various arthropod subphyla. Specific O(2)-transport proteins have long been considered unnecessary in Hexapoda (including Insecta), which acquire O(2) via an elaborate tracheal system. However, we recently identified a functional hemocyanin in the stonefly Perla marginata (Plecoptera) and in the firebrat Thermobia domestica (Zygentoma). We used RT-PCR and RACE experiments to study the presence of hemocyanin in a broad range of ametabolous and hemimetabolous hexapod taxa. We obtained a total of 12 full-length and 5 partial cDNA sequences of hemocyanins from representatives of Collembola, Archeognatha, Dermaptera, Orthoptera, Phasmatodea, Mantodea, Isoptera and Blattaria. No hemocyanin could be identified in Protura, Diplura, Ephemeroptera, Odonata, or in the Eumetabola (Holometabola + Hemiptera). It is not currently known why hemocyanin has been lost in some taxa. Hexapod hemocyanins usually consist of two distinct subunit types. Whereas type 1 subunits may represent the central building block, type 2 subunits may be absent in some species. Phylogenetic analyses support the Pancrustacea hypothesis and show that type 1 and type 2 subunits diverged before the emergence of the Hexapoda. The copperless insect storage hexamerins evolved from hemocyanin type 1 subunits, with Machilis germanica (Archeognatha) hemocyanin being a possible 'intermediate'. The evolution of hemocyanin subunits follows the widely accepted phylogeny of the Hexapoda and provides strong evidence for the monophyly of the Polyneoptera (Plecoptera, Dermaptera, Orthoptera, Phasmatodea, Mantodea, Isoptera, Blattaria) and the Dictyoptera (Mantodea, Isoptera, Blattaria). The Blattaria are paraphyletic with respect to the termites.

Presence of a single abundant storage hexamerin in both larvae and adults of the grasshopper, Schistocerca americana

We identified a single hexameric storage protein in the grasshopper, Schistocerca americana, and monitored its abundance through the last larval instar and up until reproductive competence in adults of both sexes. This storage hexamerin, termed Schistocerca americana Persistent Storage Protein (saPSP) was the most abundant soluble protein in both larvae and adults. In both sexes, saPSP abundance started out low at the onset of the last larval instar and accumulated during feeding, peaking just prior to molting. Adults of both sexes contained significant amounts of saPSP after eclosion. In adult males, saPSP content dropped continuously after eclosion and was lowest once individuals reached reproductive maturity. In contrast, adult females depleted saPSP reserves during the first days of adulthood, but subsequently accumulated significant saPSP stores. In adult females, saPSP stores peaked just prior to the completion of egg provisioning. Given the overall patterns of abundance, saPSP has functions in both larvae and adults. In addition, the observed pattern of storage hexamerin accumulation differs from patterns of accumulation in the other known grasshoppers, Locusta migratoria and Romalea microptera, suggesting that significant functional diversity has evolved in storage hexamerins among the grasshoppers.

Complete sequence, expression and evolution of two members of the hexamerin protein family during the larval development of the rice moth, Corcyra cephalonica

Three distinct types of storage hexamerins are expressed in the “last-instar” larvae of the rice moth, Corcyra cephalonica. A cDNA expression library was constructed from fat body-RNA and screened with a polyclonal antibody raised against purified hexamerin (SP2) of Corcyra cephalonica. Two slightly different “full-length” hexamerin cDNA clones (Hex2a and Hex2b) were isolated and sequenced. Both include open reading frames of 2109 bp which are translated into polypeptides of 703 amino acids with 92.5% identity. Signal peptides of 19 amino acids are present at the N-termini. The 684 amino acids native proteins have a high content of aryl groups (17.6%). According to both the criteria for amino acid composition and the phylogenetic analysis, Hex2a and Hex2b belong to the lepidopteran arylphorins. Northern blot studies revealed that the Hex2 genes are species- and tissue-specifically expressed in fat body cells of “last-instar” (= 5th) larvae.

Evolutionary loss of a storage hexamerin

Evolutionary loss of a storage hexamerin

Detection and quantification of a very high density lipoprotein in different tissues of Triatoma infestans during the last nymphal and adult stages

The presence of a very high density lipoprotein (VHDL), an hexameric protein, was explored in different tissues of Triatoma infestans throughout the last nymphal and adult stages, and in egg extracts by Western blot assays. The VHDL was always detected in both, hemolymph and fat body, during the above mentioned stages and it was also observed in the buffer soluble fraction of testis and egg homogenates. An enzyme-linked immunosorbent assay (ELISA) was used to measure the VHDL titer in these tissues. Hemolymph VHDL reaches a maximum value before the last molt, then it abruptly declines in males and females just after emergence, but during adult life it increases again. Fat body VHDL decreases slowly and continuously during the nymph growth reaching a minimum value prior to molting, and in the first week of adult life the values were even two-fold lower; then, it shows a different cycle of accumulation and depletion in males and females. In adult testis the VHDL undergoes a cycle similar to the one observed in male fat body. This protein increases progressively during embryonic development and, at the time of larval hatching it reaches its maximum value. The hexameric protein presents homologies in its N-terminal sequence with storage hexamerins of Diptera, Lepidoptera and Hymenoptera.

Selectivity in storage hexamerin clearing demonstrated with hemolymph transfusions between Hyalophora cecropia and Actias luna.

When Hyalophora cecropia hemolymph was injected into wandering Actias luna larvae, a methionine-rich hexamerin was selectively transferred to the host's fat body, and completely cleared from the hemolymph by the time of pupal eclosion. Donor arylphorin was 30-40% removed from the hemolymph, and riboflavin-binding hexamerin was even less completely cleared. During the pupal-adult molt, these rates were reversed: methionine-rich hexamerin disappeared no faster than bovine serum albumin, while riboflavin-binding hexamerin was rapidly and completely cleared from the hemolymph, even though A. luna hemolymph lacks a homologue of this protein; arylphorin, again, was cleared at an intermediate rate. Selective clearing of the three hexamerins occurred at similar stages in H. cecropia, their species of origin. Developmentally programmed clearing, with selectivity at least partially conserved between genera, was also demonstrated with transfused vitellogenin: in A. luna females that were forming yolk, H. cecropia vitellogenin was cleared more rapidly than bovine serum albumin; but in younger females, and in males at all stages of metamorphosis, this Mr 510,000 molecule was instead an indicator of nonselective, large protein clearing. Nonselective clearing was more complete during adult development than during pupation. It also showed signs of being more effective for small than for large proteins, insensitive to carbohydrate conjugates, and unsaturated at the protein levels used.