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Molecular characterization of hemocyanin and hexamerin from the firebrat Thermobia domestica (Zygentoma)
Abstract
Hexapods possess a tracheal system that enables the transport of oxygen to the inner organs. Although respiratory proteins have been considered unnecessary in most Hexapoda for this reason, we recently showed the presence of a functional hemocyanin in the stonefly Perla marginata. Here we report the identification and molecular characterization of a hemocyanin from Zygentoma (Thysanura). We obtained the full length cDNA of two distinct subunit types from the firebrat Thermobia domestica, and partial sequences of the orthologs from the silverfish Lepisma saccharina. The native T. domestica hemocyanin subunits both consist of 658 amino acids, but a signal peptide for transmembrane transport is missing in subunit 2. In adult firebrats both hemocyanin subunits represent a substantial proportion of the total hemolymph proteins. Phylogenetic analyses show that the subunit types are orthologous to subunits 1 and 2 of the stonefly Perla marginata. We further identified and sequenced a hexamerin subunit from T. domestica (689 amino acids), which suggests an early emergence of this type of proteins in hexapod evolution. In contrast to most other hexamerins, it does not reveal a high content in phenylalanine and tyrosine, which may be interpreted that the accumulation of aromatic amino acids commenced later in hexamerin evolution. Molecular clock calculations using hexamerins suggest that the divergence of Zygentoma and Pterygota occurred around 387 million years ago, which is in excellent agreement with the available fossil record.
Supplementary resources (5)
Nucleotide Sequence
June 2008
Nucleotide Sequence
June 2008
Nucleotide Sequence
June 2008
Nucleotide Sequence
June 2008
... There were 127 genes co-expressed (r 2 ≥ 0.9, P ≤ 0.05) with Fca(DK)Hx1 (Dataset S1), and their gene ontology (OsiHx1 and OsiHx3) and Lepidocampa weberi (LweHx2 and LweHx3) and four new haemocyanins from the collembolans Orchesella cincta (OciHc1 and OciHc2) and Pogonognathellus sp. AD-2013 (PloHc1 and PloHc2) are compared with three previously reported hexamerins from the zygentoman Thermobia domestica (TdoHx; Pick et al., 2008) and the diplurans Campodea sp. (CspHex1; and Occasjapyx japonicus (OjaHx1; Xie and Luan, 2014), as well as three known haemocyanins from the zygentoman T. domestica (TdoHc1 and TdoHc2; Pick et al., 2008) and the collembolan F. candida (FcaHc2; Xie and Luan, 2014). ...
... AD-2013 (PloHc1 and PloHc2) are compared with three previously reported hexamerins from the zygentoman Thermobia domestica (TdoHx; Pick et al., 2008) and the diplurans Campodea sp. (CspHex1; and Occasjapyx japonicus (OjaHx1; Xie and Luan, 2014), as well as three known haemocyanins from the zygentoman T. domestica (TdoHc1 and TdoHc2; Pick et al., 2008) and the collembolan F. candida (FcaHc2; Xie and Luan, 2014). All sequences were aligned with mafft (Katoh et al., 2017) and visualized using GeneDoc (v. ...
... According to the molecular divergence of hexamerins, the basal hexapods emerged approximately 411 Ma; the stem group of insects arose approximately 359 Ma; and the winged insects first occurred approximately 333 Ma. The estimated dates were similar to those calculated from Pick et al. (2008) and the fossil record (Whalley and Jarzembowski, 1981). When the Palaeozoic oxygen concentration curve (Berner, 2009) was anchored to the time-calibrated phylogenetic tree (Fig. 4), we observed that the origin of hexapod hexamerins coincided with increasing oxygen concentration during the late Silurianearly Devonian period. ...
Hemocyanins constitute a group of copper‐containing respiratory proteins, and hexamerins were derived from hexapod hemocyanin but lost the ability to transport oxygen and serve as storage proteins. Although hexamerins have been reported in most insect species, none of them has been identified in Collembola, one of the most primitive hexapod lineages, thereby preventing us from exploring relevant evolutionary scenarios regarding the origin and evolution of hexamerins in hexapods. Here we report on collembolan hexamerins for the first time, and investigated the temporal expression profiles of hexamerin and hemocyanin in the collembolan Folsomia candida. Hemocyanin was expressed over the entire life cycle, with higher expression at embryonic stage than at other stages, while hexamerin expression was restricted to embryos, unlike insect hexamerins, which are generally expressed from larval to adult stages. The phylogenetic analysis and molecular clock estimation suggested that all investigated hexapod hexamerins have a single and ancient origin (~ 423 MYA), coincident with the rise of atmospheric oxygen levels in the Silurian‐Devonian period, indicating a physiological link between molecular evolution and Palaeozoic oxygen changes.
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... In 2004, a functional Hc was characterized in the haemolymph of the stonefly Perla marginata (Hagner-Holler et al., 2004). Since then, Hc has been found in various ametabolous and hemimetabolous hexapod orders (Fochetti et al., 2006;Pick et al., 2008Pick et al., , 2009Pick et al., , 2010Amore et al., 2009Amore et al., , 2011Yin et al., 2012;Chen et al., 2015). However, Hc appears to be absent in Protura, Diplura, Ephemeroptera, Odonata, Hemiptera and all holometabolous insect orders (Pick et al., 2009;Burmester, 2015). ...
... However, this may not be true for other, phylogenetically more basal insects. At least in the Plecoptera (stoneflies), Archaeognatha (bristletails) and Zygentoma (silverfishes and firebrats), Hc protein occurs in adult stages along with a fully functional developed tracheal system (Hagner-Holler et al., 2004;Pick et al., 2008Pick et al., , 2009. Hc and the tracheal system may work synergistically, with the trachea allowing the diffusion of O 2 into the body and Hc supplying O 2 to the tissues with no connection to the tracheoles (Burmester, 2015). ...
Haemocyanin is the copper-containing respiratory protein present in many arthropods. In the hexapods, respiratory proteins had long been considered unnecessary as sufficient O2 was thought to be obtained via the trachea. Nevertheless, many ametabolous and hemimetabolous hexapod species actually possess haemocyanin. Here we investigated the occurrence of haemocyanin in Collembola (springtails). Haemocyanin was found in 22 collembolan species of the suborders Symphypleona, Tomoceroidea and Entomobryomorpha, demonstrating its widespread occurrence. No haemocyanin was identified in 16 species of these taxa, and it appears to be absent in Poduromorpha. The presence of haemocyanin does not correlate with either the phylogenetic history or lifestyle of the investigated species. We further investigated the function of haemocyanin in Folsomia candida (Entomobryomorpha) by applying different hypoxia regimes. Whereas short-term (1 h) and mild (10% O2 ) hypoxia led to a decrease in haemocyanin mRNA, strong hypoxia (24 h, 1.5% O2 ) resulted in a ∼4300-fold increase in haemocyanin expression. Hypoxia induction of haemocyanin could not be demonstrated in evolutionarily more advanced Hexapoda, where it is restricted to the embryo. The results indicate (1) an important role of haemocyanin in the oxygen supply of F. candida, which may be adaptive in the potentially hypoxic environment in the soil, and (2) a change in haemocyanin function in hexapod evolution.
... Members of the hemocyanin superfamily that evolved independently in Arthropoda are unique and practical evolutionary markers for studying arthropod phylogeny (Burmester, 2002;Ertas et al., 2009;Kusche and Burmester, 2001a, b;Terwilliger et al., 1999). In particular, hexamerins have recently been used in the phylogenetic inference of insects, and the trees constructed from insect hexamerins are consistent with the generally accepted relationships of insect orders (Burmester et al., 1998(Burmester et al., , 2002Hagner-Holler et al., 2007;Pick et al., 2008;Martins et al., 2010). ...
... As a basal group with epimorphosis (Gullan and Cranston, 2005), Diplura undergoes a less extreme metamorphosis than do holo-or hemimetabolous insects. The many dipluran hexamerins found in this study, together with that from a silverfish (TdoHex1, Zygentoma, T. domestica; Pick et al., 2008), documents the occurrence of hexamerins in ametabolous hexapods. In further studies, hexamerins from ametabolous species should be used to explore their essential functions beyond metamorphosis. ...
Hexamerin, as a member of the highly conserved arthropod hemocyanin superfamily, has been shown to be a good marker for the phylogenetic study of insects. However, few studies have been conducted on hexamerins in basal hexapods. The first Diplura hexamerin CspHex1 was reported only recently (Pick and Burmester, 2009). Remarkably, CspHex1 was suggested to have evolved from hexapod hemocyanin subunit type 2, which is very different from all insect hexamerins originated from hexapod hemocyanin subunit type 1. Does this finding suggest double or even multiple origins of hexamerins in Hexapoda? To find more evidence on the evolution of dipluran hexamerins, eight putative hexamerin gene sequences were obtained from three dipluran species, as were three hemocyanin genes from two collembolan species. Unexpectedly, after adding the new sequences into the phylogenetic analyses, all dipluran hexamerins including CspHex1 grouped together and as sister to the insect hexamerins, with high likelihood and Bayesian support. Our analysis supports a single origin of the hexamerins in Hexapoda, and suggests the close relationship between Diplura and Insecta. In addition, our study indicates that a relatively comprehensive taxa sampling is essential to solve some problems in phylogenetic reconstruction.
... Hemocyanin and hemoglobin are both present in the crustaceans, Hymenoptera and Hemiptera. Hemocyanin works as a physiological supplement, compensating for poor oxygen transport in the trachea and assisting insect embryos in aerobic respiration [60] . Hemocyanin expression was dramatically increased in Baifutiao [61] and locust embryos [62] under hypoxia conditions compared to normoxic settings. ...
The genetic recombination of the SARs-CoV-2 virus in bats may result in behaviors comparable to those of certain RNA viruses. This cross-activity helps explain SARs-CoV-2's strange respiratory symptoms and immune evasion abilities. In this present study, the biological roles of SARs-CoV-2 proteins were investigated utilizing bioinformatic techniques involving the search for conserved domains. According to the study, the S and ORF3a proteins of SARs-CoV-2 possess picornavirus/calicivirus capsid domains, can bind hemoglobin, heme, and porphyrin. Both Arg134 of ORF3a and Cys44 of E are iron-binding sites for heme. The ORF3a protein has a region that converts heme into iron and porphyrin. In addition to chitin and polyphenol binding domains, the S protein also contains hemocyanin and phenoloxidase-like domains. The S protein constructs Fe-polyphenol complexes to link the red blood cell membrane, allowing SARs-CoV-2 to hitch a ride on red blood cells for fast delivery to target organs. This type of capsid-like vector delays the immune system but does not significantly alter the function of red blood cells to transport oxygen. Due to the distortion of the cell membrane, red blood cells with an excess of viral particles release hemoglobin to harm the virus. The wbl domains of the S protein respond to nitration, and then phenoloxidase domains oxidize polyphenols, allowing the virus to shed from the red blood cell membrane. ORF3a also attack 1-beta chain of hemoglobin; however, the majority of hemoglobin may retain its native structure. Patients will have variable degrees of respiratory distress and coagulation symptoms, but the hemocyanin domains of the S protein can improve a patient's respiratory status by transporting oxygen.
... Hemocyanin and hemoglobin are both present in the crustaceans, Hymenoptera and Hemiptera. Hemocyanin works as a physiological supplement, compensating for poor oxygen transport in the trachea and assisting insect embryos in aerobic respiration (47). Hemocyanin expression was dramatically increased in Baifutiao (48) and locust embryos (49) under hypoxia conditions compared to normoxic settings. ...
Investigating poor respiratory function and high immunological escape in COVID-19 patients may aid in the prevention of additional deaths. The conserved domain search method was used to evaluate the biological roles of specific SARS-COV-2 proteins in this present study. The research findings indicate that the SARS-COV-2 virus contains domains capable of binding porphyrin and synthesizing heme. S and ORF3a can bind to hemoglobin. The S protein possesses hemocyanin-like function since it contains copper-oxygen binding, immunological agglutination, and phenoloxidase domains. ORF3a's Arg134 and E's Cys44 have heme-iron binding sites, respectively. The ORF3a protein has a region that degrades trapped heme into iron and porphyrin. Hemoglobin that has been attacked by ORF3a may preserve the majority of its native structure but with decreased oxygen delivery function. By targeting hemoglobin and destroying heme, the ORF3a protein caused varying degrees of respiratory distress and coagulation symptoms in COVID-19 individuals. ORF3a of Delta and Omicron variants also retained its capacity to target hemoglobin and heme. But the S protein's hemocyanin-like domain transported oxygen to enhance the patient's respiratory condition. Through a large load of hemocyanin-like proteins, the mutant virus achieved effective oxygen transport and alleviated the symptoms of respiratory distress in patients. Simultaneously, the variant S protein's immunological agglutination and phenol oxidase functions were decreased or eliminated, resulting in a decrease in the strength of the immune response and an increase in immune evasion ability, culminating in increased virus transmission.
... Hemocyanin and hemoglobin are both present in the crustaceans, Hymenoptera and Hemiptera. Hemocyanin works as a physiological supplement, compensating for poor oxygen transport in the trachea and assisting insect embryos in aerobic respiration (47). Hemocyanin expression was dramatically increased in Baifutiao (48) and locust embryos (49) under hypoxia conditions compared to normoxic settings. ...
Investigating poor respiratory function and high immunological escape in COVID-19 patients may aid in the prevention of additional deaths. The conserved domain search method was used to evaluate the biological roles of specific SARS-COV-2 proteins in this present study. The research findings indicate that the SARS-COV-2 virus contains domains capable of binding porphyrin and synthesizing heme. S and ORF3a can bind to hemoglobin. The S protein possesses hemocyanin-like function since it contains copper-oxygen binding, immunological agglutination, and phenoloxidase domains. ORF3a's Arg134 and E's Cys44 have heme-iron binding sites, respectively. The ORF3a protein has a region that degrades trapped heme into iron and porphyrin. Hemoglobin that has been attacked by ORF3a may preserve the majority of its native structure but with decreased oxygen delivery function. By targeting hemoglobin and destroying heme, the ORF3a protein caused varying degrees of respiratory distress and coagulation symptoms in COVID-19 individuals. ORF3a of Delta and Omicron variants also retained its capacity to target hemoglobin and heme. But the S protein's hemocyanin-like domain transported oxygen to enhance the patient's respiratory condition. Through a large load of hemocyanin-like proteins, the mutant virus achieved effective oxygen transport and alleviated the symptoms of respiratory distress in patients. Simultaneously, the variant S protein's immunological agglutination and phenol oxidase functions were decreased or eliminated, resulting in a decrease in the strength of the immune response and an increase in immune evasion ability, culminating in increased virus transmission.
... Hemocyanins (HCNs) are large type-3 metalloproteins that transport oxygen in the blood of invertebrates like arthropods and molluscs (Van Holde and Miller, 1995;Terwilliger, 1998;Burmester, 2002;Pick et al., 2008;Rehm et al., 2012;Marxen et al., 2014;Pinnow et al., 2016). Their binuclear active site coordinates copper as metal and accommodates oxygen as a peroxide ion, due to which copper oxidizes from (I) to (II) oxidation state (Himmelwright et al., 1978(Himmelwright et al., , 1980Magnus and Ton-That, 1991;Jaenicke et al., 2012). ...
Hemocyanin from horseshoe crab in its active form is a homo-hexameric protein. It exists in open and closed conformations when transitioning between deoxygenated and oxygenated states. Here, we present a detailed dynamic atomistic investigation of the oxygenated and deoxygenated states of the hexameric hemocyanin using explicit solvent molecular dynamics simulations. We focus on the variation in solvent cavities and the formation of tunnels in the two conformational states. By employing principal component analysis and CVAE-based deep learning, we are able to differentiate between the dynamics of the deoxy- and oxygenated states of hemocyanin. Finally, our results identify the deoxygenated open conformation, which adopts a stable, closed conformation after the oxygenation process.
... Hexamerins are ubiquitous in insects and are composed of six subunits. 7 Both hexamerin allergens showed 36.5% identity ( Figure S3). This fact corroborated that HLP2 and hexamerin 1B are different allergens, although both belong to the haemocyanin family. ...
Insects are increasingly being considered as occupational and food allergens. Occupational allergy to crickets may be triggered in factory workers who manufacture food for reptiles, in professionals who raise reptiles as pets or in the zoos, and in cooks who make culinary recipes with insects. Additionally, edible insects are gradually being included in the western diet.
... The role hemocyanin plays in insects with the tracheal respiratory system remains mostly a mystery. Recently, however, functional hemocyanin has been identified in the hemolymph of the stonefly Perla marginata (Plecoptera) and the firebrat Thermobia domestica (Zygentoma) (Hagner-Holler et al. 2004;Fochetti et al. 2006;Pick et al. 2008). The presence of hemocyanin in plecopterans was attributed to the semiaquatic lifecycle of these insects (Hagner-Holler et al. 2004). ...
Earwigs (Dermaptera) use different strategies to increase their reproductive success. Most species lay eggs; however, viviparity of the matrotrophic type has been reported in two groups: Hemimeridae and Arixeniidae. In Arixeniidae, offspring develop in two separate places: inside an ovary (the intraovarian phase) and within a uterus (the intrauterine phase). Both morphological and physiological aspects of viviparity in Arixeniidae have begun to be unraveled only recently. Here, we characterize how the first instar larvae of Arixenia esau, developing inside the mother’s reproductive system, manage respiration and gas exchange. Using modern light and electron microscopy techniques as well as immunological approach, we provide a detailed account of the maternal and larval tissue interactions during the intrauterine development. We demonstrate that respiration in the Arixenia first instar larvae relies on the extensive tracheal system of the mother as well as a respiratory pigment (hemocyanin) present within the body cavity of the larvae. Our results indicate that the larval fat body tissue is the likely place of the hemocyanin synthesis. Our study shows that characteristic cone-shaped lobes of the outgrowths located on the larval abdomen are a part of a placenta-like organ and mediate the gas exchange between the maternal and larval organisms. Based on the obtained results, we propose that Arixenia esau evolved a unique biphasic system supporting respiration of the first instar larvae during their development inside the mother’s reproductive tract.
... Cricket (Gryllus bimaculatus) [188] Firebrat (Thermobia domestica) [211] Like other food ingredients derived from plants or animals, insects can cause allergies (Table 4). Three allergens, arginine kinase (AK), hemocyanin (HC), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), have been identified in the muscle of Macrobrachium rosenbergii. ...
... Hemocyanins (Hc) are type-3 copper containing proteins found in blood of invertebrates including arthropods and mollusks, and in larval stages of certain insects (Pick et al. 2008;Terwilliger 1998). Their syntheses in organs vary with respect to different types of organisms. ...
Molecular dynamics simulations were applied to deoxy- and oxy-hemocyanins using newly developed force field parameters for the dicopper site to evaluate their structural and dynamical properties. Data obtained from the simulations provided information of the oxygenation effect on the active site and overall topology of the protein that was analyzed by root-mean-square deviations, b-factors, and dicopper coordination geometries. Domain I of the protein was found to demonstrate higher flexibility with respect to domain II because of the interfacial rotation between domain I and II that was further endorsed by computing correlative domain movements for both forms of the protein. The oxygenation effect on the overall structure of the protein or polypeptide subunit was further explored via gyration radii evaluated for the metal-binding domain and for the whole subunit. The evaluation of hydration dynamics was carried out to understand the water mediated role of amino acid residues of the solvent tunnel facilitating the entry of oxygen molecule to the dicopper site of hemocyanin.
... Cricket (Gryllus bimaculatus) [188] Firebrat (Thermobia domestica) [211] Like other food ingredients derived from plants or animals, insects can cause allergies (Table 4). Three allergens, arginine kinase (AK), hemocyanin (HC), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), have been identified in the muscle of Macrobrachium rosenbergii. ...
The potential of insects as a source of protein for future food and feed is the object of numerous studies. The nutritional value of edible insects is well established, and other aspects of consumption thereof are investigated. In this chapter, we aim to summarize the main features of insects as food. We briefly describe the history of the usage of insects as food for humans and refer to the current acceptance of insects by Europeans based on conducted surveys. We characterize the most common insect species with the biggest potential to be used as food and feed in the EU according to EFSA. We describe the nutritional value of insects and the possibility of application thereof in the food and feed industry, keeping in mind the safety of consumption. In addition, the ecological aspect of insect breeding is discussed. A review of the growing edible insect market in Europe and the USA is also provided. Moreover, we analyze the current legal status of insect intake in Europe. We aim to make this chapter a current conclusion about the consumption of insects.
... Hemocyanins (HMCs) are copper-containing glycoproteins, which mainly transports molecular oxygen to systemic tissues for cellular respiration [10]. HMC is widely distributed in arthropods, molluscs and larval stages of certain insects [11][12][13]. Structurally, arthropod HMCs form hexamers (2 × 6 mer) or oligo-hexamers (8 × 6mer) of identical or related subunits [10]. HMCs are synthesized in hepatopancreas and secreted into the hemolymph, to display function in the homeostasis and immune defenses within marine invertebrates [10,14,15]. ...
... The role of supplying O 2 in embryos of the insect species lacking haemocyanin may be carried out by other respiratory proteins, such as haemoglobins (Hankeln et al., 2002), or other physiological mechanisms (Hoback & Stanley, 2001;Harrison et al., 2006). Mutual complementation of these two respiratory systems is not necessarily limited to embryos and may be extended to later developmental stages, such as the adult stage in stoneflies and firebrats (Hagner-Holler et al., 2004;Pick et al., 2008). In addition to hemimetabolous insects, myriapods (millipedes and centipedes) also have both a tracheal system and circulating haemocyanins (Damsgaard et al., 2013). ...
It remains unresolved how insect embryos acquire sufficient oxygen to sustain high rates of respiratory metabolism during embryogenesis in the absence of a fully developed tracheal system. Our previous work showed that the two distinct subunits (Hc1 and Hc2) of haemocyanin (Hc), a copper-containing protein, display embryo-specific high expression that is essential for embryonic development and survival in the migratory locust Locusta migratoria. Here we investigated the role of haemocyanins in oxygen sensing and supply in the embryo of this locust. Putative binding sites for hypoxia-regulated transcription factors were identified in the promoter region of all of the Hc1 and Hc2 genes. Embryonic expression of haemocyanins was highly upregulated by ambient O2 deprivation, up to 10-fold at 13% O2 content. The degree of upregulation of haemocyanins increased with increasing levels of hypoxia. Compared with low-altitude locusts, embryonic expression of haemocyanins in high-altitude locusts from Tibetan plateau was constitutively higher and more robust to oxygen deprivation. These findings strongly suggest an active involvement of haemocyanins in oxygen exchange in embryos. We thus propose a mechanistic model for embryo respiration in which haemocyanin plays a key role by complementing the tracheal system for oxygen transport during embryogenesis.
... Only a few hemipteran and dipteran species that live in hypoxic environments harbor Hb (Weber and Vinogradov 2001;. However, recent data have demonstrated that Hcs in fact occur in a broad range of ametabolous and hemimetabolous hexapods (Sánchez et al. 1998;Hagner-Holler et al. 2004;Pick et al. 2008Pick et al. , 2009 (Fig. 2). No Hc was found in Protura, Diplura, Ephemeroptera (mayflies), Odonata (dragonflies and damselflies), or Eumetabola (Hemiptera and Holometabola) (Pick et al. 2009). ...
Respiratory proteins enhance the capacity of the blood for oxygen transport and support intracellular storage and delivery of oxygen. Hemocyanin and hemoglobin are the respiratory proteins that occur in the Pancrustacea. The copper-containing hemocyanins evolved from phenoloxidases in the stem lineage of arthropods. For a long time, hemocyanins had only been known from the malacostracan crustaceans but recent studies identified hemocyanin also in Remipedia, Ostracoda, and Branchiura. Hemoglobins are common in the Branchiopoda but have also been sporadically found in other crustacean classes (Malacostraca, Copepoda, Thecostraca). Respiratory proteins had long been considered unnecessary in the hexapods because of the tracheal system. Only chironomids, some backswimmers, and the horse botfly, which all live under hypoxic conditions, were known exceptions and possess hemoglobins. However, recent data suggest that hemocyanins occur in most ametabolous and hemimetabolous insects. Phylogenetic analysis showed the hemocyanins of insects and Remipedia to be similar, suggesting a close relationship of these taxa. Hemocyanin has been lost in dragonflies, mayflies, and Eumetabola (Hemiptera + Holometabola). In cockroaches and grasshoppers, hemocyanin expression is restricted to the developing embryo while in adults oxygen is supplied solely by the tracheal system. This pattern suggests that hemocyanin was the oxygen-transport protein in the hemolymph of the last common ancestor of the pancrustaceans. The loss was probably associated with miniaturization, a period of restricted availability of oxygen, a change in life-style, or morphological changes. Once lost, hemocyanin was not regained. Some pancrustaceans also possess cellular globin genes with uncertain functions, which are expressed at low levels. When a respiratory protein was again required, hemoglobins evolved several times independently from cellular globins.
... However, contrary to this assumption, haemocyanin genes have been found in many insect species since 2004, including ametabolous and hemimetabolous insects (Hagner-Holler et al., 2004). These findings of the widespread occurrence of haemocyanin in insects have implications for their potential association with insect respiration (Burmester, 2001;Burmester & Hankeln, 2007;Pick et al., 2008Pick et al., , 2010. For example, studies with the stonefly Perla marginata demonstrated oxygen-binding properties of haemolymphs containing haemocyanin (Hagner-Holler et al., 2004). ...
Haemocyanins are commonly known as copper-containing oxygen carriers within the haemolymph of arthropods, and have been found in many orders of insects. However, it remains unresolved why haemocyanins persist in insects that possess elaborate tracheal systems for oxygen diffusion to cells. Here we identified haemocyanins in the migratory locust Locusta migratoria that consists of two distinct subunits, Hc1 and Hc2. Genomic sequence analysis indicated that Hc1 and Hc2 have four and three gene copies, respectively, which may have evolved via gene duplication followed by divergent evolution of introns. The two subunits exhibit abundant and embryonic-specific expression at the mRNA and protein level; their expression peaks in the mid-term embryo and is not detectable in the late nymphal and adult stages. A larger proportion of the haemocyanins is present in the yolk compared with that in the embryo. Immunostaining shows that haemocyanins in the embryo are mainly expressed in the epidermis. Knockdown of Hc1 and Hc2 results in significant embryonic developmental delay and abnormality as well as reduced egg hatchability, ie the proportion of hatched eggs. These results reveal a previously unappreciated and fundamental role for haemocyanins in embryonic development and survival in insects, probably involving the exchange of molecules (eg O2 ) between the embryo and its environment.
© 2015 The Royal Entomological Society.
... Hexamerin1B precursor (HEX1B) was found as a novel allergen in G. bimaculatus. Hexamerins consist of six subunits with a molecular mass of 80 kDa and are found in all insects (Pick, Hagner-Holler, & Burmester, 2008). Hexamerins are mainly considered as storage proteins that support nutrition during non-feeding, resting periods, such as molting, and are abundant in the hemolymph (Telfer & Kunkel, 1991). ...
... Hc is distributed within the hemolymph of arthropods, molluscs and larval stages of certain insects (Pick et al., 2008(Pick et al., , 2009Decker and Jaenicke, 2004). Depending on the organism, Hc can be synthesised in organs such as the crustacean hepatopancreas (Lee et al., 2004;Ward et al., 2010) or secreted by specialised cells known as cyanocytes in chelicerates (Fahrenbach, 1970;Kuhn-Nentwig et al., 2014) and rhogocytes in molluscs (Albrecht et al., 2001;Beuerlein et al., 2004). ...
... Hexamerins are synthesized in the fat body, secreted into the larval hemolymph, and taken up again by the fat body shortly before pupation. Within the pupal fat body, these proteins are initially stored in protein granules, and are later proteolytically broken down to supply the amino acids necessary for the completion of adult development (Martins et al., 2008;Pick et al., 2008). Consequently, the 55.3-kDa protein may have been consumed by parasitoid larvae as an amino acid resource for development. ...
Parasitism of lepidopteran larvae by braconid wasps has significant effects on host hemolymph proteins and the physiology of the host insect. In this study the post-parasitism effects of a gregarious idiobiont ectoparasitoid ( Bracon hebetor) on hemolymph plasma proteins in the final instar larvae of the host ( Ephestia kuehniella) were investigated. Hemolymph plasma proteins were analyzed using spectrophotometry and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Total quantity of plasma proteins in the host's hemolymph decreased slightly 24 and 48 h after parasitization; however, SDS-PAGE analysis showed that the quantity of 12 proteins decreased, whereas that of 5 proteins increased in concentration in the hemolymph of parasitized host larvae 24 and 48 h post-parasitism. Therefore, we conclude that host regulation of E. kuehniella by B. hebetor involves only quantitative changes in host plasma proteins and does not lead to upregulation of novel proteins.
... Aquatic Insects Vol. 31, Supplement 1, 2009, 577-583 Gutie´rrez and Bastiani 1998); hemocyanin was found in Thermobia domestica and Lepisma saccarina (Zygentoma) (Pick, Hagner-Holler and Burmester 2008), and in larvae and adults of the stonefly Perla marginata (Hagner-Holler et al. 2004), Perla grandis (Fochetti et al. 2006) and other species of Perloidea (Amore et al. 2009). Recently, Pick, Schneuer and Burmester (2009) have reported the presence of hemocyanin in many hexapod orders, like Collembola, Diplura, Archaeognata, Zygentoma, Orthoptera, Phasmida, Dermaptera, Isoptera and Blattodea. ...
Hemocyanin is a respiratory protein that occurs in the main lineages of Arthropoda. In insects hemocyanin is presently known in many orders. Recently, a functional hemocyanin has also been found in the Plecoptera. Further studies have revealed that hemocyanin seems to be not uniformly distributed within this order. In this paper we report additional data, obtained with RT-PCR sequencing, on the presence of hemocyanin in different stonefly species. In addition, we summarise the present knowledge about the distribution of hemocyanin in the Plecoptera. Biological aspects such as larval size, life cycle length, trophic roles and environmental induction are discussed as possible factors that may be correlated with the presence or absence of hemocyanin in the studied species.
... Sequence analyses have shown that these hemocyanin macromolecules can contain several variants of monomers (Markl 1986, Voit et al. 2000, Hagner-Holler et al. 2004. The presence of the N-terminal signal peptide is typical of arthropod hemocyanins, presumably because they are secreted into hemolymph by hepatopancreas cells or fat body cells (Fochetti et al. 2006;Kusche and Burmester, 2001a, b;Sánchez et al. 1998;Pick et al. 2008), and they are freely dissolved in the hemolymph. Although its O 2binding capacity has earned hemocyanin the common name of "respiratory protein," there is a large body of literature showing that, under experimental conditions, the protein is multifunctional in Chelicerata and Crustacea. ...
Hemocyanins are large oligomeric respiratory proteins found in many arthropods and mollusks. The overall expression of hemocyanin mRNA, revealed by studies on Plecoptera hemocyanin sequencing, has raised the question of whether the protein is expressed or not. In fact, the presence of expressed hemocyanin has only been reported in the literature for one species, Perla marginata (Panzer, 1799). In this paper, we report the presence of hemocyanin and hexamerin proteins in Dinocras cephalotes (Curtis, 1827), a species closely related to P. marginata. To assess the presence of hemocyanin, we used a reproducible and highly sensitive method based on liquid chromatography tandem mass spectrometry. We conclude that regardless of its putative function (respiratory, immune defense, storage protein), the hemocyanin is actually expressed in species in which its mRNA is present.
... Therefore, the presence of specialized oxygen-transport proteins in the circulatory system of insects has been considered generally unnecessary [10,11]. However, a functional hemocyanin has been identified in some basal taxa [12,13]. Hexapod hemocyanins usually consist of two distinct subunit types(Hc1 and Hc2). ...
Hemocyanins are copper-containing (Cu(+)) proteins that transport oxygen in many arthropods hemolymph. We characterized Hc1 gene from the grasshopper species Locusta migratoria manilensis. In particular, we cloned and sequenced the corresponding cDNAs and studied their expression at different developmental stages. The cDNA of Hc1 gene (GenBank accession no.:HQ213937) is 2271 bp in length and the open reading frame is 2016 bp, which encodes a 672 amino acids protein with a calculated molecular mass of 77.9 kD and the isoelectric point of 6.06. Sequence alignment analysis result showed that this gene shares 94.7% identity with Schistocerca americana EHP. In addition, analysis of quantitative RT-PCR indicated that, LmiHc1 was expressed in the embyro (24, 39, 62, 86, 144, and 193 h after hatch), nymphs (1st instar, 2nd instar, 3rd instar, 4th instar and 5th instar) and in adult. These results showed that Hc1 plays an important role in grasshopper, which may be related to an enhanced oxygen supply. Phylogenetic analysis of insecta based on Hc1 are basically consistent with the morphology.
... Each subunit can bind to an O 2 molecule by the virtue of two copper ions that are coordinated by six histidine residues. Hemocyanins have been thoroughly studied in Chelicerata and Crustacea, occur in Myriapoda (Jaenicke et al. 1999; Kusche and Burmester 2001 ), and have recently been identified in Onychophora (Kusche et al. 2002) and Hexapoda (Hagner-Holler et al. 2004; Pick et al. 2008 Pick et al. , 2009). Within Crustacea, hemocyanins have been thought to be confined to Malacostraca (Mangum 1985; Markl and Decker 1992). ...
The Remipedia are enigmatic crustaceans from anchialine cave systems, first described only 30 years ago, whose phylogenetic affinities are as yet unresolved. Here we report the sequence of hemocyanin from Speleonectes tulumensis Yager, 1987 (Remipedia, Speleonectidae). This is the first proof of the presence of this type of respiratory protein in a crustacean taxon other than Malacostraca. Speleonectes tulumensis hemocyanin consists of multiple distinct (at least three) subunits (StuHc1-3; Hc, hemocyanin). Surprisingly, the sequences are most similar to hexapod hemocyanins. Phylogenetic analyses showed that the S. tulumensis hemocyanin subunits StuHc1 and StuHc3 associate with the type 1 hexapod hemocyanin subunits, whereas StuHc2 associates with the type 2 subunits of hexapods. Together, remipede and hexapod hemocyanins are in the sister-group position to the hemocyanins of malacostracan crustaceans. Hemocyanins provide no indication of a close relationship of Myriapoda and Hexapoda but support Pancrustacea (Crustacea + Hexapoda). Our results also suggest that Crustacea are paraphyletic and that Hexapoda may have evolved from a Remipedia-like ancestor. Thus, Remipedia occupy a key position for the understanding of the evolution of hexapods, which are and have been one of the world's most speciose lineage of animals.
Meat analogs are a burgeoning industry, with plant-based meat analogs, insect-based meat analogs, algae-based meat analogs, mycoprotein-based meat analogs, and cell-based meat analogs. However, despite the industry’s growth potential, market expansion faces hurdles due to taste and quality disparities compared to traditional meats. The composition and characteristics of meat analogs currently available in the market are analyzed in this study to inform the development of future products in this sector. The results show that plant-based meat analogs are mainly based on soy protein together with wheat gluten and methylcellulose or spices. Insect-based meat analogs tend to contain processed larvae as the protein source. Seaweed or spirulina is often the main ingredient in algae-based meat analogs. Mycoprotein-based meat analogs all use mycoproteins. Cell-based beef, pork, chicken, and seafood products are already under various stages of development around the world, although many are still at the prototype level.
Respiratory systems are key innovations for the radiation of terrestrial arthropods. It is therefore surprising that there is still a considerable lack of knowledge. In this review of the available information on tracheal systems of hexapods (with a focus on the apterygote lineages Protura, Collembola, Diplura, Archaeognatha and Zygentoma), we summarize available data on the spiracles (number, position and morphology), the shape and variability of tracheal branching patterns including anastomoses, the tracheal fine structure and the respiratory proteins. The available data are strongly fragmented, and information for most subgroups is missing. In various cases, individual observations for one species account for the knowledge of the entire order. The available data show that there are strong differences between but also within apterygote orders. We conclude that the available data are insufficient to derive detailed conclusions on the hexapod ground plan and outline the possible evolutionary scenarios for the tracheal system in this group.
In recent years, an enormous number of naturally occurring biological macromolecules has been reported worldwide due to its antibacterial and anticancerous potential. Among them, in this study, the copper containing respiratory protein namely haemocyanin (HC) was isolated from the haemolymph of mud crab Scylla serrata. The isolated metalloprotein HC was purified using Sepharose column by gel filtration chromatography. The purified HC was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and molecular weight of the protein was identified as 95 kDa. Fourier transform infrared spectrophotometer (FT-IR) and nuclear magnetic resonance (¹H NMR) spectral data revealed the presence of amino acid constituents. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis based mass ion search exposed that the purified protein was HC. HC exhibited an in vitro bacteriostatic effects against the bacterial pathogens and also elevated ROS levels in the treated samples. The half maximal (50%) inhibitory concentration (IC50) of HC was found to be 80 μg/mL against lung cancer cells (A549). Our study collectively addressed the potential antibacterial and anti-cancerous activity of HC. The results obtained from this study suggest that HC can be used for therapeutical application in the near future.
A key role in arthropod phylogeny plays a group of organisms that was already in the focus of taxonomic research of Charles Darwin in the mid of the 19th century, namely the Crustacea. This extremely divers group comprises small species like the Mystacocarida (Derocheilocaris typicus) with only 0.3 mm body size or such big representatives like the Japanese giant crab (Macrocheira kaempferi) with a span width of almost 4 m. Generally accepted are six major crustacean taxa, the Malacostraca (Latreille, 1802), Branchiopoda (Latreille, 1817), Remipedia (Yager, 1981), Cephalocarida (Sanders, 1955), Maxillopoda (Dahl, 1956) and Ostracoda (Latreille 1802). The validity of the taxon Maxillopoda is to date still disputed. The monophyly of some crustacean groups like the Malacostraca and Branchiopoda is generally accepted, but for several other groups unclear. This thesis aims to resolve internal relationships of the major crustacean groups inferring phylogenies with molecular data. The crustaceans are in addition of eminent interest to enlight the question how land was successfully conquered by arthropod taxa. New molecular and neuroanatomical data support the scenario that the Hexapoda might have evolved from Crustacea. The thesis further seeks to address the possible close relationship of Crustacea and Hexapoda. That issue is closely linked to the partly still debated position of crustaceans within arthropods and the supposable sister-group of the Crustacea.
Most molecular studies of crustaceans relied on single gene or multigene analyses in which for most cases partly sequenced rRNA genes were used. However, intensive data quality and alignment assessments prior to phylogenetic reconstructions are not conducted in most studies. Additionally, a complex modeling and the implementation of compositional base heterogeneity along lineages are missing. One methodological aim in this thesis was to implement new tools to infer data quality, to improve alignment quality and to test the impact of complex modeling of the data. Two of the three phylogenetic analyses in this thesis are also based on rRNA genes.
In analysis (A) 16S rRNA, 18S rRNA and COI sequences were analyzed. RY coding of the COI fragment, an alignment procedure that considers the secondary structure of RNA molecules and the exclusion of alignment positions of ambiguous positional homology was performed to improve data quality. Anyhow, by extensive network reconstructions it was shown that the signal quality in the chosen and commonly used markers is not suitable to infer crustacean phylogeny, despite the extensive data processing and optimization. This result draws a new light on previous studies relying on these markers.
In analyses (B) completely sequenced 18S and 28S rRNA genes were used to reconstruct the phylogeny. Base compositional heterogeneity was taken into account based on the finding of analysis (A), additionally to secondary structure alignment optimization and alignment assessment. The complex modeling to compare time-heterogeneous versus time-homogenous processes in combination with mixed models for an implementation of secondary structures was only possible applying the Bayesian software package PHASE. The results clearly demonstrated that complex modeling counts and that ignoring time-heterogeneous processes can mislead phylogenetic reconstructions. Some results enlight the phylogeny of Crustaceans, for the first time the Cephalocarida (Hutchinsoniella macracantha) were placed in a clade with the Branchiopoda, which morphologically is plausible. Unfortunately, the internal relationships of most crustacean groups were still poorly supported. Compared to the time-homogeneous tree the time-heterogeneous tree gives lower support values for some nodes. It can be suggested, that the incorporation of base compositional heterogeneity in phylogenetic analysis improves the reliability of the topology. The Pancrustacea are supported maximally in both approaches, but internal relations are not reliably reconstructed. One result of this analysis is that the phylogenetic signal in rRNA data might be eroded for crustaceans.
Recent publications presented analyses based on phylogenomic data, to reconstruct mainly metazoan phylogeny. Analyzing such a large number of sequences is possible with the “supertree” or “supermatrix” method. The supermatrix method seems to outperform the supertree approach. One main advantage is the possibility to apply modeling for each partition (each gene) separately. Within this thesis crustaceans were collected to conduct EST sequencing projects and to include the resulting sequences combined with public sequence data into a phylogenomic analysis (C). In this analysis the supermatrix approach was applied. New and innovative reduction heuristics were performed to condense the dataset. The strategy of the reduction heuristics relies on the potential relative information content of each gene of each taxon to use a more objective criterion to select taxa and genes. Again, the alignment evaluation and processing was a major aspect for the analysis design. The results showed that the matrix implementation of the reduced dataset ends in a more reliable topology in which most node values are highly supported. In analysis (C) the Branchiopoda were positioned as sister-group to Hexapoda, a differing result to analysis (A), but that is in line with other phylogenomic studies. Unfortunately, important crustacean taxa are still missing to conduct an extensive phylogenomic analysis. Some EST sequencing projects of the collected crustaceans for this thesis were delayed for technical reasons, e.g. the ESTs for Sarsinebalia urgorrii (non-derived malacostracan) and Speleonectes tulumensis (Remipedia) are still in progress. A preliminary result obtained with sequences isolated from remipede tissue is suggesting that remipedes and hexapods are closely related based on homologous hemocyanin subunits.
The conclusion of the analyses conducted in the framework of this thesis is that alignment evaluation and processing improves the resulting inference of the phylogeny. Assessing the quality of the signal or potential conflicts in the dataset is extremely important, also for further decisions on the selection of substitution models and final phylogenetic reconstructions. Complex models can improve the phylogeny reconstruction additionally. This was explicitly demonstrated in analysis B. The supermatrix approach relying on a more objective criterion to select genes and taxa compared to cut-off values is very promising for future studies. However, for the Crustacea it was also demonstrated that this group is problematic regarding the phylogenetic signal of the analyzed single gene data. The hope is, that phylogenomic data with similar complex models as applied in analysis B, in combination with a denser taxon sampling can improve our knowledge about crustacean phylogeny in future studies. This thesis presents essential new methodological but also phylogenetic findings for this challenging task.
Hexamerin was originally identified as a storage protein but later confirmed to be involved in many physiological processes. In the present study, we cloned and characterized a novel hexamerin complementary DNA sequence from the Chinese oak silkworm, Antheraea pernyi (Ap ‐hexamerin), which shows high homology with reported insect methionine‐rich hexamerins. The tissue distribution and time course of expression demonstrated that Ap ‐hexamerin was predominantly synthesized in the fat body and the expression level was significantly increased in response to the microbial challenge, suggesting the relevance of Ap ‐hexamerin to immune responses. In further immune functional studies, Ap ‐hexamerin was confirmed to take part in the upregulation of prophenoloxidase (PPO) activation in A. pernyi haemolymph triggered by pathogen‐associated molecular patterns (PAMPs). Additional molecular interaction analysis revealed that Ap ‐hexamerin is capable of binding the PAMPs used in the phenoloxidase assay, suggesting hexamerin in A. pernyi may positively regulate haemolymph PPO activation, acting as a pattern recognition protein.
Highlights
• A full‐length complementary DNA sequence of a novel hexamerin is identified from Antheraea pernyi .
• Ap ‐hexamerin has detected the immune‐related expression and participates in the prophenoloxidase activation system probably acting as a pattern recognition protein.
Citrobacter freundii is widely distributed in nature as a typical conditional pathogen. In this study, the pathogenicity and histopathology of crayfish (Procambarus clarkii) infected with C. freundii were investigated, and immune-related gene expression profiling was performed. Challenge experiments confirmed that the isolated LJ1 strain of C. freundii had high virulence, leading to significant deaths of otherwise healthy shrimp. Histopathological analysis revealed that the hepatopancreas, intestines, and gills of diseased crayfish exhibited obvious inflammatory responses to C. freundii infection. Quantitative real-time PCR (qRT-PCR) was also performed to investigate the expression pattern of twelve immune-related genes in gill, intestine, hepatopancreas, and hemocyte samples. The results showed various expression profiles and clear transcriptional activation of these immune related genes in the tested tissues. These results will contribute to further study of C. freundii infection and help to understand P. clarkii immune function.
Low temperature frequently influences growth, development, and even survival of aquatic animals. In the present study, physiological and molecular responses to low temperature in Litopenaeus vannamei were investigated. The cDNA sequences of two oxygen-carrying proteins, cytoglobin (Cygb) and neuroglobin (Ngb), were isolated. Protein structure analysis revealed that both proteins share a globin superfamily domain. Real-time PCR analysis indicated that Cygb and Ngb mRNA levels gradually increased during decrease in temperatures from 25 to 15°C and then decreased at 10°C in muscle, brain, stomach, and heart, except for a continuing increase in gills, whereas they showed a different expression trend in the hepatopancreas. Hemocyanin concentration gradually reduced as the temperature decreased. Moreover, the activities of respiratory metabolic enzymes including lactate dehydrogenase (LDH) and succinate dehydrogenase (SDH) were measured, and it was found that LDH activity gradually increased while SDH activity decreased after low-temperature treatment. Finally, damage to gill structure at low temperature was also observed, and this intensified with further decrease in temperature. Taken together, these results show that low temperature has an adverse influence in L. vannamei, which contributes to systematic understanding of the adaptation mechanisms of shrimp at low temperature.
Hemocyanins are large extracellular respiratory proteins distributed within the hemolymph of arthropods, molluscs and larval stages of certain insects. In the present study, we characterized the humoral immune functions of cation specific antibacterial hemocyanin (Ab-Hcy) from mud crab, S. serrata. The bacteriolytic activity of Ab-Hcy was against pathogenic and non-pathogenic to crustaceans which includes, Bacillus sp. N1, B. flexus N3, E. coli, P. aeruginosa, V. harveyi, V. parahaemolyticus and V. vulnificus and also expressed bacteriostatic activity against E. coli, B. flexus N3 and V. harveyi. The agglutination activity of Ab-Hcy ranged from 4-16 against B. flexus N3, E. coli, V. harveyi and V. vulnificus and Ab-Hcy also agglutinated with rat and human O erythrocytes (HA titer: 2). The Ab-Hcy lysed the panel of mammalian erythrocytes and strong hemolytic activity of 1.40, 1.28 and 1.22 units·min− 1·mg protein− 1 against human A, B and O erythrocytes respectively. It was also found that the Ab-Hcy possess phenoloxidase (PO) activity by oxidizing L-DOPA. These results suggest that Ab-Hcy from the serum of mud crab, S. serrata, is capable of performing multiple humoral immune functions in addition to oxygen transportation.
Statement of relevance
This manuscript deals with the humoral immune functions, particularly antibacterial property of hemocyanin in crustacean. In this paper we report the different type of antibacterial activity and other humoral immune functions of a hemocyanin isolated from the serum of mud crab, Scylla serrata. This work will help to understand the role of respiratory molecule in immune system, also will be helpful improve disease free crustacean aquaculture industry.
And we also state this research work is relevant and suitable to publish in the Journal “Aquaculture”.
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.
Copyright © 2014. Published by Elsevier Ltd.
While O(2)-binding hemoglobin-like proteins are present in many insects, prominent amounts of hemoglobin have only been found in a few species. Backswimmers of the genera Anisops and Buenoa (Notonectidae) have high concentrations of hemoglobin in the large tracheal cells of the abdomen. Oxygen from the hemoglobin is delivered to a gas bubble and controls the buoyant density, which enables the bugs to maintain their position without swimming and to remain stationary in the mid-water zone where they hunt for prey. We have obtained the cDNA sequences of three Anisops deanei hemoglobin chains by RT-PCR and RACE techniques. The deduced amino acid sequences show an unusual insertion of a single amino acid in the conserved helix E, but this does not affect protein stability or ligand binding kinetics. Recombinant A. deanei hemoglobin has an oxygen affinity of P(50) = 2.4 kPa (18 torr) and reveals the presence of a dimeric fraction or two different conformations. The absorption spectra demonstrate that the Anisops hemoglobin is a typical pentacoordinate globin. Phylogenetic analyses show that the backswimmer hemoglobins evolved within Heteroptera and most likely originated from an intracellular hemoglobin with divergent function.
Haemocyanins are copper-containing respiratory proteins in the arthropod haemolymph. In hexapods, haemocyanins gave rise to hexamerins, which have lost the ability to bind copper and thus oxygen. Hexamerins are thought to act mainly as storage proteins in nonfeeding periods. So far, hexamerins have only been identified in ectognathan hexapods, but not in Entognatha. Here we report the identification of a putative hexamerin from Campodea sp. (Diplura). The full-length cDNA of Campodea sp. hexamerin 1 (CspHex1) measures 2188 bp and translates into a native polypeptide of 667 amino acids. As in other hexamerins, the six copper-coordinating histidines are not conserved. However, sequence comparison and phylogenetic analyses demonstrated that CspHex1 is not closely related to other hexapod hexamerins, which derive from hexapod type 1 haemocyanin subunits in the ectognathan lineage, but rather resembles a derivative of hexapod type 2 haemocyanin subunits. Hence, haemocyanin-related storage proteins emerged at least two times independently in Hexapoda.
For a long time it had been assumed that specific oxygen transport proteins are absent in insects. Only recently it has been demonstrated that hemocyanins occur in the hemolymph of many ametabolous and hemimetabolous insect taxa, but not in the Eumetabola (Hemiptera+Holometabola). Therefore, the loss of respiratory hemocyanin in insects is not correlated with the evolution of an efficient tracheal system. The specific contribution of hemocyanin to oxygen supply in insects, however, has remained uncertain. Here we investigate the stage-specific expression of hemocyanin in the ovoviviparous cockroach Blaptica dubia (Blattaria), which consists of two distinct subunit types (Hc1 and Hc2). Employing quantitative real-time RT-PCR and Western blotting, we showed that the expression of hemocyanin is restricted to late embryos, thus being detectable also in whole female extracts and oothecae. Hemocyanin protein is also present in 1st instar nymphs, but not in later developmental stages. The ontogeny of hemocyanin in cockroaches is distinct from that known from Zygentoma and Plecoptera, in which hemocyanin occurs in both nymphal and adult stages. Our findings suggest a specific role of hemocyanin in embryonic cockroaches, which may be related to an enhanced oxygen supply in the oothecae. For some reason, the fundamental physiological changes associated to the evolution of holometaboly have made hemocyanin unnecessary.
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.
Hemocyanins are copper-containing, respiratory proteins that occur in the hemolymph of many arthropod species. Here we report
for the first time the presence of hemocyanins in the diplopod Myriapoda, demonstrating that these proteins are more widespread
among the Arthropoda than previously thought. The hemocyanin ofSpirostreptus sp. (Diplopoda: Spirostreptidae) is composed of two immunologically distinct subunits in the 75-kDa range that are most likely
arranged in a 36-mer (6 × 6) native molecule. It has a high oxygen affinity (P
50 = 4.7 torr) but low cooperativity (h = 1.3 ± 0.2).Spirostreptus hemocyanin is structurally similar to the single known hemocyanin from the myriapod taxon, Scutigera coleoptrata (Chilopoda), indicating a rather conservative architecture of the myriapod hemocyanins. Western blotting demonstrates shared
epitopes of Spirostreptus hemocyanin with both chelicerate and crustacean hemocyanins, confirming its identity as an arthropod hemocyanin.
Morphological and molecular data are marshalled to address the question of hexapod ordinal relationships. The combination of 275 morphological variables, 1000 bases of the small subunit nuclear rDNA (18S), and 350 bases of the large subunit nuclear rDNA (28S) are subjected to a variety of analysis parameters (indel and transversion costs). Representatives of each hexapod order are included with most orders represented multiply. Those parameters that minimize character incongruence (ILD of Mickevich and Farris, 1981, Syst. Zool. 30, 351–370), among the morphological and molecular data sets are chosen to generate the best supported cladogram. A well-resolved and robust cladogram of ordinal relationships is produced with the topology (Crustacea ((Chilopoda Diplopoda) ((Collembola Protura) ((Japygina Campodeina) (Archaeognatha (Zygentoma (Ephemerida (Odonata ((((Mantodea Blattaria) Isoptera) Zoraptera) ((Plecoptera Embiidina) (((Orthoptera Phasmida) (Grylloblattaria Dermaptera)) ((((Psocoptera Phthiraptera) Thysanoptera) Hemiptera) ((Neuropteroidea Coleoptera) (((((Strepsiptera Diptera) Mecoptera) Siphonaptera) (Trichoptera Lepidoptera)) Hymenoptera)))))))))))))).
The program MRBAYES performs Bayesian inference of
phylogeny using a variant of Markov chain Monte Carlo.
Availability: MRBAYES, including the source code, documentation,
sample data files, and an executable, is available at http://brahms.biology.rochester.edu/software.html.
Contact: johnh{at}brahms.biology.rochester.edu
The storage hexamers are a family of insect proteins with native molecular weights around 500,000 with six homologous subunits weighing generally between 70,000 and 85,000 daltons. They are synthesized and secreted by the fat body of feeding larvae and nymphs and reach extraordinary concentrations in the hemolymph just prior to metamorphosis. In holometabolous insects, they are partially recaptured by the fat body during the larval to pupal molt and stored in cytoplasmic protein granules, as well as in the hemolymph of the pupa. They disappear from these reservoirs during adult development; in many insects, both hemi- and holometabolous, they are also utilized during the nymphal or larval molts. Their amino acids are primarily incorporated into new tissues and proteins during adult development, but they may also be incorporated into cuticle as intact protein, and in one case were found to be diverted to a small degree into energy metabolism. Finally, some storage hexamers have ligand binding and transport capabilities. Functionally, de- velopmentally, and evolutionarily, they are a complex family of proteins whose analysis promises to be exceedingly useful in the study of many basic problems in the biology of insects.
Crustacean and cheliceratan hemocyanins (oxygen-transport proteins) and insect hexamerins (storage proteins) are homologous gene products, although the latter do not bind oxygen and do not possess the copper-binding histidines present in the hemocyanins. An alignment of 19 amino acid sequences of hemocyanin subunits and insect hexamerins was made, based on the conservation of elements of secondary structure observed in X-ray structures of two hemocyanin subunits. The alignment was analyzed using parsimony and neighbor-joining methods. Results provide strong indications for grouping together the sequences of the 2 crustacean hemocyanin subunits, the 5 cheliceratan hemocyanin subunits, and the 12 insect hexamerins. Within the insect clade, four methionine-rich proteins, four arylphorins, and two juvenile hormone-suppressible proteins from Lepidoptera, as well as two dipteran proteins, form four separate groups. In the absence of an outgroup sequence, it is not possible to present information about the ancestral state from which these proteins are derived. Although this family of proteins clearly consists of homologous gene products, there remain striking differences in gene organization and site of biosynthesis of the proteins within the cell. Because studies on 18S and 12S rRNA sequences indicate a rather close relationship between insects and crustaceans, we propose that hemocyanin is the ancestral arthropod protein and that insect hexamerins lost their copper-binding capability after divergence of the insects from the crustaceans.
We have developed a new method for the identification of signal peptides and their cleavage sites based on neural networks
trained on separate sets of prokaryotic and eukaryotic sequence. The method performs significantly better than previous prediction
schemes and can easily be applied on genome-wide data sets. Discrimination between cleaved signal peptides and uncleaved N-terminal
signal-anchor sequences is also possible, though with lower precision. Predictions can be made on a publicly available WWW
server.
We introduce a graphical method, likelihood-mapping, to visualize the phylogenetic content of a set of aligned sequences. The method is based on an analysis of the maximum likelihoods for the three fully resolved tree topologies that can be computed for four sequences. The three likelihoods are represented as one point inside an equilateral triangle. The triangle is partitioned in different regions. One region represents star-like evolution, three regions represent a well-resolved phylogeny, and three regions reflect the situation where it is difficult to distinguish between two of the three trees. The location of the likelihoods in the triangle defines the mode of sequence evolution. If n sequences are analyzed, then the likelihoods for each subset of four sequences are mapped onto the triangle. The resulting distribution of points shows whether the data are suitable for a phylogenetic reconstruction or not.
Arthropod hemocyanins are members of a protein superfamily that also comprises the arthropod phenoloxidases (tyrosinases), crustacean pseudohemocyanins (cryptocyanins), and insect storage hexamerins. The evolution of these proteins was inferred by neighbor-joining, maximum-parsimony, and maximum-likelihood methods. Monte Carlo shuffling approaches provided evidence against a discernible relationship of the arthropod hemocyanin superfamily and molluscan hemocyanins or nonarthropodan tyrosinases. Within the arthropod hemocyanin superfamily, the phenoloxidase probably emerged early in the (eu-)arthropod stemline and thus form the most likely outgroup. The respiratory hemocyanins evolved from these enzymes before the radiation of the extant euarthropodan subphyla. Due to different functional constraints, replacement rates greatly vary between the clades. Divergence times were thus estimated assuming local molecular clocks using several substitution models. The results were consistent and indicated the separation of the cheliceratan and crustacean hemocyanins close to 600 MYA. The different subunit types of the multihexameric cheliceratan hemocyanin have a rather conservative structure and diversified in the arachnidan stemline between 550 and 450 MYA. By contrast, the separation of the crustacean (malacostracan) hemocyanin subunits probably occurred only about 200 MYA. The nonrespiratory pseudohemocyanins evolved within the Decapoda about 215 MYA. The insect hemocyanins and storage hexamerins emerged independently from the crustacean hemocyanins. The time of divergence of the insect proteins from the malacostracan hemocyanins was estimated to be about 430-440 MYA, providing support for the notion that the Hexapoda evolved from the same crustacean lineage as the Malacostraca.
Arthropod hemocyanins are large, multimeric, (n x 6) copper-containing proteins that deliver oxygen in the haemolymph of many chelicerate, crustacean, myriapod, and also possibly some insect species. The arthropod hemocyanins belong to a large protein superfamily that also includes the arthropod phenoloxidases, certain crustacean and insect storage proteins (pseudo-hemocyanins and hexamerins), and the insect hexamerin receptors. Here I summarise the present knowledge of the origin, functional adaptations, and evolution of these proteins. Arthropod and mollusc hemocyanins are, if at all, only distantly related. As early as in the arthropod stem line, the hemocyanins emerged from a phenoloxidase-like enzyme. The evolution of distinct hemocyanin subunits, as well as the formation of multi-hexamers occurred independently within the arthropod subphyla. Hemocyanin subunit evolution is strikingly different in the Chelicerata, Myriapoda and Crustacea. Hemocyanins individually gave rise to two distinct copper-less storage proteins, the insect hexamerins and the crustacean pseudo-hemocyanins (cryptocyanins). The receptor responsible for the uptake of hexamerin by the larval fat body of the insects emerged from a hexamerin-precursor. Molecular phylogenetic analyses show a close relationship of the crustacean and insect proteins, providing strong support for a pancrustacean taxon, while structural data suggest a myriapod-chelicerate clade.
The velvet worms (Onychophora) are considered living fossils and are closely related to the Euarthropoda. Onychophora possess a tracheal system for respiratory function, but oxygen-transport proteins have been considered unnecessary. Here, we show that the hemolymph of the Epiperipatus sp. (Onychophora: Peripatidae) contains an arthropod-type hemocyanin, demonstrating that such protein exists outside the Euarthropoda. Thus, the evolution of oxygen carriers preceded the divergence of the Onychophora and Euarthropoda and was most likely linked to the evolution of an efficient circulatory system in a low-oxygen environment. The cDNA of the Epiperipatus hemocyanin subunit comprises 2,287 bp and encodes for a protein of 641 aa (73.6 kDa). Phylogenetic analyses of the arthropod hemocyanin sequences show that the Onychophora form a robust sister-group of the Euarthropoda, whereas the monophyly of the Tracheata is not supported.
Insects possess an elaborate tracheal system that enables transport of gaseous oxygen from the atmosphere directly to the inner organs. Therefore, the presence of specialized oxygen-transport proteins in the circulatory system of insects has been considered generally unnecessary. Here, we show for the first time, to our knowledge, the presence of an ancestral and functional hemocyanin (Hc) in an insect. In the hemolymph of nymphs and adults of the stonefly Perla marginata, a hexameric Hc was identified, which consists of two distinct subunit types of 659 and 655 amino acids. P. marginata Hc displays cooperative oxygen binding with a moderately high oxygen affinity [(half-saturation pressure, P(50) approximately 8 torr (1 torr = 133 Pa)]. No evidence was found for the presence of Hcs in the more evolutionarily advanced holometabolan insects, suggesting that this type of respiratory protein was lost later in insect evolution. However, our results demonstrate that, in contrast to the accepted paradigm, certain basal insects have retained an ancestral blood-based mechanism of gas exchange.
The accuracy of multiple sequence alignment program MAFFT has been improved. The new version (5.3) of MAFFT offers new iterative
refinement options, H-INS-i, F-INS-i and G-INS-i, in which pairwise alignment information are incorporated into objective
function. These new options of MAFFT showed higher accuracy than currently available methods including TCoffee version 2 and
CLUSTAL W in benchmark tests consisting of alignments of >50 sequences. Like the previously available options, the new options
of MAFFT can handle hundreds of sequences on a standard desktop computer. We also examined the effect of the number of homologues
included in an alignment. For a multiple alignment consisting of ∼8 sequences with low similarity, the accuracy was improved
(2–10 percentage points) when the sequences were aligned together with dozens of their close homologues (E-value < 10−5–10−20) collected from a database. Such improvement was generally observed for most methods, but remarkably large for the new options
of MAFFT proposed here. Thus, we made a Ruby script, mafftE.rb, which aligns the input sequences together with their close
homologues collected from SwissProt using NCBI-BLAST.
The role of fossils in dating the tree of life has been misunderstood. Fossils can provide good "minimum" age estimates for branches in the tree, but "maximum" constraints on those ages are poorer. Current debates about which are the "best" fossil dates for calibration move to consideration of the most appropriate constraints on the ages of tree nodes. Because fossil-based dates are constraints, and because molecular evolution is not perfectly clock-like, analysts should use more rather than fewer dates, but there has to be a balance between many genes and few dates versus many dates and few genes. We provide "hard" minimum and "soft" maximum age constraints for 30 divergences among key genome model organisms; these should contribute to better understanding of the dating of the animal tree of life.
We characterized two subunits of a putative haemocyanin from the stonefly species Perla grandis. In particular, we cloned and sequenced the corresponding cDNAs and studied their expression in different insect stages. Moreover, using the deduced amino acid sequences, homology studies were performed both on their primary and tertiary structures. 3-D molecular modelling data showed that the residues involved in the oxygen transport and subunits contacts were located in spatial positions preserving the functionality of the molecule. Despite it was paradigmatically affirmed that insects do not have respiratory proteins, our data suggest that the haemocyanin could be involved in the respiratory mechanisms of P. grandis. As far as we know, this is the first haemocyanin 3-D structure described and analyzed in insects.
A new fossil site near Gilboa, New York, is one of only three where fossils of terrestrial arthropods of Devonian age have
been found. The new Gilboan fauna is younger than the other two but richer in taxa. Fragmentary remains and nearly whole specimens
assigned to Eurypterida, Arachnida (Trigonotarbida, Araneae, Amblypygi, and Acari), Chilopoda [Craterostigmatomorpha(?) and
Scuterigeromorpha(?)], and tentatively to Insecta (Archaeognatha) have been found. The centipedes and possible insects may
represent the earliest records known for these groups.
The pioneering work of Munn and colleagues (Munn and Greville, 1969) was the first tangible indication that the larvae of holometabolous insects synthesize large amounts of unusual proteins which accumulate in their hemolymph. These proteins, generally referred to as larval serum proteins (LSPs) or storage proteins, have many common characteristics. They are synthesized by the fat body of actively feeding larvae and their concentrations increase enormously in the last larval instar, making up the major component of the whole larval soluble proteins. They form hexamers in the 5 × 105 Dalton range and dissociate into polypeptides of 7.2 – 9 × 104 Daltons (For review, see Levenbook, 1985). Telfer et al. (1983) suggested that the larval haemolymph proteins which are similar in structure and amino acid composition to calliphorin, the major haemolymph protein of the blowfly, Calliphora vicina, should be called arylphorins to signify that they bear aryl groups.
Archeatelura sturmi gen.n. sp.n. (Zygentoma: Ateluridae) the only fossil representative of the family, is described from the oligocenic Dominican amber (25-40 MY) and compared with the probably closer recent taxa.
Hexamerins are hemocyanin-related haemolymph proteins that are widespread in insects and may accumulate to extraordinarily high concentrations in larval stages. Hexamerins were originally described as storage proteins that provide amino acids and energy for non-feeding periods. However, in recent years other specific functions like cuticle formation, transport of hormones and other organic compounds, or humoral immune defense have been proposed. During evolution, hexamerins diversified according to the divergence of the insect orders. Within the orders, there is a notable structural diversification of these proteins, which probably reflects specific functions. In this paper, the different possible roles of the hexamerins are reviewed and discussed in the context of hexamerin phylogeny.
A detailed description and illustrations of the larva of Remartinia secreta (Calvert) are provided. The main features that distinguish the larva of this species are dark brown coloration; prementum 0.25 times longer than its widest part, with a dorsal row of small spiniform setae close to the lateral margins; length of the lateral spine on abdominal segment six 0.4–0.5 mm; lateral valvae of female gonapophyses shorter than central ones; and female epiproct longer than cerci. Larvae were found living in a lagoon densely covered by aquatic phanerogams such as Eichhornia sp. (Pontedereaceae) and Typha sp. (Thyphaceae). Larva of R. secreta is compared to that of Remartinia luteipennis florida (Hagen), its only sympatric congener.
The recently-developed statistical method known as the "bootstrap" can be used to place confidence intervals on phylogenies. It involves resampling points from one's own data, with replacement, to create a series of bootstrap samples of the same size as the original data. Each of these is analyzed, and the variation among the resulting estimates taken to indicate the size of the error involved in making estimates from the original data, In the case of phylogenies, it is argued that the proper method of resampling is to keep all of the original species while sampling characters with replacement, under the assumption that the characters have been independently drawn by the systematist and have evolved independently. Majority-rule consensus trees can be used to construct a phylogeny showing all of the inferred monophyletic groups that occurred in a majority of the bootstrap samples. If a group shows up 95% of the time or more, the evidence for it is taken to be statistically significant. Existing computer programs can be used to analyze different bootstrap samples by using weights on the characters, the weight of a character being how many times it was drawn in bootstrap sampling. When all characters are perfectly compatible, as envisioned by Hennig, bootstrap sampling becomes unnecessary; the bootstrap method would show significant evidence for a group if it is defined by three or more characters.
Thesis (Ph. D.)--Harvard University, 1950.
A new method for estimating divergence times when evolutionary rates are variable across lineages is proposed. The method, called nonparametric rate smoothing (NPRS), relies on minimization of ancestor-descendant local rate changes and is motivated by the likelihood that evolutionary rates are autocorrelated in time. Fossil information pertaining to minimum and/or maximum ages of nodes in a phylogeny is incorporated into the algorithms by constrained optimization techniques. The accuracy of NPRS was examined by comparison to a clock-based maxi-mum-likelihood method in computer simulations. NPRS provides more accurate estimates of divergence times when (1) sequence lengths are sufficiently long, (2) rates are truly nonclocklike, and (3) rates are moderately to highly autocorrelated in time. The algorithms were applied to estimate divergence times in seed plants based on data from the chloroplast rbcL gene. Both constrained and unconstrained NPRS methods tended to produce divergence time estimates more consistent with paleobotanical evidence than did clock-based estimates.
Arthropodan hemocyanins, prophenoloxidases (PPOs), and insect hexamerins form a superfamily of hemolymph proteins that we propose to call the AHPH superfamily. The evolutionary and functional relationships of these proteins are illuminated by a new embryonic hemolymph protein (EHP) that is expressed during early stages of development in the grasshopper embryo. EHP is a 78-kDa soluble protein present initially in the yolk sac content, and later in the embryonic hemolymph. Protein purification and peptide sequencing were used to identify an embryonic cDNA clone coding for EHP. In situ hybridization identifies hemocytes as EHP-expressing cells. As deduced from the cDNA clone, EHP is a secreted protein with two potential glycosylation sites. Sequence analysis defines EHP as a member of the AHPH superfamily. Phylogenetic analyses with all the currently available AHPH proteins, including EHP, were performed to ascertain the evolutionary history of this protein superfamily. We used both the entire protein sequence and each of the three domains present in the AHPH proteins. The phylogenies inferred for each of the domains suggest a mosaic evolution of these protein modules. Phylogenetic and multivariate analyses consistently group EHP with crustacean hemocyanins and, less closely, with insect hexamerins, relative to cheliceratan hemocyanins and PPOs. The grasshopper protein rigorously preserves the residues involved in oxygen binding, oligomerization, and allosteric regulation of the oxygen transport proteins. Although insects were thought not to have hemocyanins, we propose that EHP functions as an oxygen transport or storage protein during embryonic development.
The distribution of the O2 carrying proteins suggests that the original transport system was a hemoglobin similar to the alpha-chain of hemoglobin A and packaged in a nucleated red blood cell. These molecules, which occur in large open fluid compartments, function as O2 stores for regular periods of hypoxia as well as carriers between sites of gas exchange. When the closed circulatory system first arose, the red blood cell was abandoned in favor of extracellular heme proteins, and the O2 storage function became less important. Alternative O2 carriers, hemerythrins, appear in the blood at about the same phylogenetic level as the intracellular hemoglobins, and their respiratory functions appear to be similar. The presence of hemoglobins instead of hemerythrins in the vertebrates may be an evolutionary accident. Still other O2 carriers, hemocyanins, arose separately in two specialized groups that left no descendants. Their O2 binding has all the adaptive features of vertebrate hemoglobin O2 binding, with unique features also. The respiratory function of the hemocyanins is largely limited to O2 transport, which makes a far greater contribution to aerobic metabolism than the O2 carriers found in simpler systems.
The vertebrate evolution of four proteins (6 and hemoglobins, cytochromec, and fibrinopeptide A) is examined via a maximum likelihood procedure. The fundamental hypothesis is that the process of nucleotide substitution as revealed by the minimum phyletic distance procedure (Fitch, 1971) is Poisson with a constant time average for each protein. The method allows the simultaneous estimation of the relative times of divergence of all common ancestors while utilizing the information from all four proteins. It also affords the possibility of statistically testing several biologically meaningful hypotheses. The results are the following:
1.
The total rate (sum of all proteins) of nucleotide substitution is not constant in time throughout the evolution of vertebrates.
2.
The relative rates (among proteins) of nucleotide substitution are not constant throughout vertebrate evolution.
3.
Despite the variation in the rates of nucleotide substitution the procedure employed provides estimates of the relative time of divergence which correlate well with paleontological dates.
4.
The overall rate of nucleotide substitution within the primates is again found to be less than the rest of the mammals.
Previous methods of heterogeneous ribonucleic acid (HnRNA) extraction yield material which "disaggregates" into small molecules. This could be the fault of either ribonuclease knicks in the polymers sustained during the extraction procedure or disaggregation into real subunits. The present communication distinguishes between these possibilities by describing an RNA extraction procedure which does not yield subunits when HnRNA is denatured. By the criteria of sedimentation through sucrose, formaldehyde, and dimethyl sulfoxide, it is estimated that the majority of the radioactivity of giant HnRNA after a 30-min pulse of [3H]uridine is associated with molecules in the range 5-10 × 106 daltons. In the electron microscope, under denaturing conditions, 84% (mass %) of giant HnRNA has a contour length of 4-9 μ corresponding to a molecular weight of about 5-10 × 106. Giant HnRNA has a "DNA-like" base composition (G + C = 46-54%) and has considerable secondary structure (ca. 60% helix conformation) as judged by its melting profile and reactivity with formaldehyde.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
Dipteran arylphorin receptors, insect hexamerins, cheliceratan and crustacean hemocyanins, and crustacean and insect tyrosinases display significant sequence similarities. We have undertaken a systematic comparison of primary and secondary structures of these proteins. On the basis of multiple sequence alignments the phylogeny of these proteins was investigated. Hexamerin subunits, hemocyanin subunits, and tyrosinases share extensive similarities throughout the entire amino acid sequence. Our studies suggest the origin of arthropod hemocyanins from ancient tyrosinase-like proteins. Insect hexamerins likely evolved from hemocyanins of ancient crustaceans, supporting the proposed sister-group position of these subphyla. Arylphorin receptors, responsible for incorporation of hexamerins into the larval fat body of diptera, are related to hexamerins, hemocyanins, and tyrosinase. The receptor sequences display extensive similarities to the first and third domains of hemocyanins and hexamerins. In the middle region only limited amino acid conservation was observed. Elements important for hexamer formation are deleted in the receptors. Phylogenetic analysis indicated that dipteran arylphorin receptors diverged from ancient hexamerins, probably early in insect evolution.
We have developed a new method for the identification of signal peptides and their cleavage sites based on neural networks trained on separate sets of prokaryotic and eukaryotic sequences. The method performs significantly better than previous prediction schemes, and can easily be applied to genome-wide data sets. Discrimination between cleaved signal peptides and uncleaved N-terminal signal-anchor sequences is also possible, though with lower precision. Predictions can be made on a publicly available WWW server: http://www.cbs.dtu.dk/services/SignalP/.
Phylogenetic inference from amino acid sequence data uses mainly empirical models of amino acid replacement and is therefore dependent on those models. Two of the more widely used models, the Dayhoff and JTT models, are estimated using similar methods that can utilize large numbers of sequences from many unrelated protein families but are somewhat unsatisfactory because they rely on assumptions that may lead to systematic error and discard a large amount of the information within the sequences. The alternative method of maximum-likelihood estimation may utilize the information in the sequence data more efficiently and suffers from no systematic error, but it has previously been applicable to relatively few sequences related by a single phylogenetic tree. Here, we combine the best attributes of these two methods using an approximate maximum-likelihood method. We implemented this approach to estimate a new model of amino acid replacement from a database of globular protein sequences comprising 3,905 amino acid sequences split into 182 protein families. While the new model has an overall structure similar to those of other commonly used models, there are significant differences. The new model outperforms the Dayhoff and JTT models with respect to maximum-likelihood values for a large majority of the protein families in our database. This suggests that it provides a better overall fit to the evolutionary process in globular proteins and may lead to more accurate phylogenetic tree estimates. Potentially, this matrix, and the methods used to generate it, may also be useful in other areas of research, such as biological sequence database searching, sequence alignment, and protein structure prediction, for which an accurate description of amino acid replacement is required.
Rates of molecular evolution vary widely between lineages, but quantification of how rates change has proven difficult. Recently
proposed estimation procedures have mainly adopted highly parametric approaches that model rate evolution explicitly. In this
study, a semiparametric smoothing method is developed using penalized likelihood. A saturated model in which every lineage
has a separate rate is combined with a roughness penalty that discourages rates from varying too much across a phylogeny.
A data-driven cross-validation criterion is then used to determine an optimal level of smoothing. This criterion is based
on an estimate of the average prediction error associated with pruning lineages from the tree. The methods are applied to
three data sets of six genes across a sample of land plants. Optimally smoothed estimates of absolute rates entailed 2- to
10-fold variation across lineages.
A unified understanding of >390 Myr of insect evolution requires insight into their origin. Molecular clocks are widely applied for evolutionary dating, but clocks for the class Insecta have remained elusive. We now define a robust nucleotide and amino acid mitochondrial molecular clock encompassing five insect orders, including the Blattaria (cockroaches), Orthoptera (crickets and locusts), Hemiptera (true bugs), Diptera, and Lepidoptera (butterflies and moths). Calibration of the clock using one of the earliest, most extensive fossil records for insects (the early ancestors of extant Blattaria) was congruent with all available insect fossils, with biogeographic history, with the Cambrian explosion, and with independent dating estimates from Lepidopteran families. In addition, dates obtained from both nucleotide and amino acid clocks were congruent with each other.
Of particular interest to vector biology is the early date of the emergence of triatomine bugs (99.8–93.5 MYA), coincident with the formation of the South American continent during the breakup of Gondwanaland. More generally, we reveal the insects arising from a common ancestor with the Anostraca (fairy shrimps) at around the Silurian-Ordovician boundary (434.2–421.1 MYA) coinciding with the earliest plant megafossil. We explore Tilyard's theory proposing that the terrestrial transition of the aquatic arthropod ancestor to the insects is associated with a particular plant group (early vascular plants). The major output of the study is a comprehensive series of dates for deep-branching points within insect evolution that can act as calibration points for further dating studies within insect families and genera.
Hemocyanins are large oligomeric copper-containing proteins that serve for the transport of oxygen in many arthropod species. While studied in detail in the Chelicerata and Crustacea, hemocyanins had long been considered unnecessary in the Myriapoda. Here we report the complete molecular structure of the hemocyanin from the common house centipede Scutigera coleoptrata (Myriapoda: Chilopoda), as deduced from 2D-gel electrophoresis, MALDI-TOF mass spectrometry, protein and cDNA sequencing, and homology modeling. This is the first myriapod hemocyanin to be fully sequenced, and allows the investigation of hemocyanin structure-function relationship and evolution. S. coleoptrata hemocyanin is a 6 x 6-mer composed of four distinct subunit types that occur in an approximate 2 : 2 : 1 : 1 ratio and are 49.5-55.5% identical. The cDNA of a fifth, highly diverged, putative hemocyanin was identified that is not included in the native 6 x 6-mer hemocyanin. Phylogenetic analyses show that myriapod hemocyanins are monophyletic, but at least three distinct subunit types evolved before the separation of the Chilopoda and Diplopoda more than 420 million years ago. In contrast to the situation in the Crustacea and Chelicerata, the substitution rates among the myriapod hemocyanin subunits are highly variable. Phylogenetic analyses do not support a common clade of Myriapoda and Hexapoda, whereas there is evidence in favor of monophyletic Mandibulata.
Insects are the most diverse lineage of all life in numbers of species, and ecologically they dominate terrestrial ecosystems. However, how and when this immense radiation of animals originated is unclear. Only a few fossils provide insight into the earliest stages of insect evolution, and among them are specimens in chert from Rhynie, Scotland's Old Red Sandstone (Pragian; about 396-407 million years ago), which is only slightly younger than formations harbouring the earliest terrestrial faunas. The most well-known animal from Rhynie is the springtail Rhyniella praecursor (Entognatha; Collembola), long considered to be the oldest hexapod. For true insects (Ectognatha), the oldest records are two apparent wingless insects from later in the Devonian period of North America. Here we show, however, that a fragmentary fossil from Rhynie, Rhyniognatha hirsti, is not only the earliest true insect but may be relatively derived within basal Ectognatha. In fact, Rhyniognatha has derived characters shared with winged insects, suggesting that the origin of wings may have been earlier than previously believed. Regardless, Rhyniognatha indicates that insects originated in the Silurian period and were members of some of the earliest terrestrial faunas.
In 2002, we developed and released a rapid multiple sequence alignment program MAFFT that was designed to handle a huge (up to approximately 5,000 sequences) and long data (approximately 2,000 aa or approximately 5,000 nt) in a reasonable time on a standard desktop PC. As for the accuracy, however, the previous versions (v.4 and lower) of MAFFT were outperformed by ProbCons and TCoffee v.2, both of which were released in 2004, in several benchmark tests. Here we report a recent extension of MAFFT that aims to improve the accuracy with as little cost of calculation time as possible. The extended version of MAFFT (v.5) has new iterative refinement options, G-INS-i and L-INS-i (collectively denoted as [GL]-INS-i in this report). These options use a new objective function combining the weighted sum-of-pairs (WSP) score and a score similar to COFFEE derived from all pairwise alignments. We discuss the improvement in accuracy brought by this extension, mainly using two benchmark tests released very recently, BAliBASE v.3 (for protein alignments) and BRAliBASE (for RNA alignments). According to BAliBASE v.3, the overall average accuracy of L-INS-i was higher than those of other methods successively released in 2004, although the difference among the most accurate methods (ProbCons, TCoffee v.2 and new options of MAFFT) was small. The advantage in accuracy of [GL]-INS-i became greater for the alignments consisting of approximately 50-100 sequences. By utilizing this feature of MAFFT, we also examined another possible approach to improve the accuracy by incorporating homolog information collected from database. The [GL]-INS-i options are applicable to aligning up to approximately 200 sequences, although not applicable to thousands of sequences because of time and space complexities.
For a long time, respiratory proteins have been considered unnecessary in most insects because the tracheal system was thought to be sufficient for oxygen supply. Only a few species that survive under hypoxic conditions were known exceptions. However, recently it has become evident that (1) intracellular hemoglobins belong to the standard repertoire of insects and (2) that hemocyanin is present in many "lower" insects. Intracellular hemoglobins have been identified in Drosophila, Anopheles, Apis and many other insects. In all investigated species, hemoglobin is mainly expressed in the fat body and the tracheal system. The major Drosophila hemoglobin binds oxygen with high affinity. This hemoglobin type possibly functions as a buffer system for oxygen supply at low partial pressures and/or for the protection from an excess of oxygen. Similar hemoglobins, present in much higher concentrations, store oxygen in specialized tracheal organs of the botfly and some backswimmers. The extracellular hemoglobins in the hemolymph of chironomid midges are evolutionary derivatives of the intracellular insect hemoglobins, which emerged in response to the hypoxic environment of the larvae. In addition, several hemoglobin variants of unknown functions have been discovered in insect genomes. Hemocyanins transport oxygen in the hemolymph of stoneflies, but also in the Entognatha and most hemimetabolan taxa. Apparently, hemocyanin has been lost in Holometabola. At present, no physiological or morphological character is known that could explain the presence or loss of hemocyanins in distinct taxa. Nevertheless, the occurrence of respiratory proteins in insects adds further complexity to our view on insect respiration.
Hexamerins are large storage proteins of insects in the 500 kDa range that evolved from the copper-containing hemocyanins. Hexamerins have been found at high concentration in the hemolymph of many insect taxa, but have remained unstudied in relatively basal taxa. To obtain more detailed insight about early hexamerin evolution, we have studied hexamerins in stoneflies (Plecoptera). Stoneflies are also the only insects for which a functional hemocyanin is known to co-occur with hexamerins in the hemolymph. Here, we identified hexamerins in five plecopteran species and obtained partial cDNA sequences from Perla marginata (Perlidae), Nemoura sp. (Nemouridae), Taeniopteryx burksi (Taeniopterygidae), Allocapnia vivipara (Capniidae), and Diamphipnopsis samali (Diamphipnoidae). At least four distinct hexamerins are present in P. marginata. The full-length cDNA of one hexamerin subunit was obtained (PmaHex1) that measures 2475 bp and translates into a native polypeptide of 702 amino acids. Phylogenetic analyses showed that the plecopteran hexamerins are monophyletic and positioned at the base of the insect hexamerin tree, probably diverging about 360 million years ago. Within the Plecoptera, distinct hexamerin types evolved before the divergence of the families. Mapping amino acid compositions onto the phylogenetic tree shows that the accumulation of aromatic amino acids (and thus the evolution of "arylphorins") commenced soon after the hexamerins diverged from hemocyanins, but also indicates that hexamerins with distinct amino acid compositions reflect secondary losses of aromatic amino acids.
Environmental Physiology of Animals A contribution to our knowledge of lepismatid development
- P Willmer
- G Stone
- I Johnston
- Oxford Blackwell
- J T Woodland
Entomol 17, 373–402. Willmer, P., Stone, G., Johnston, I., 2000. Environmental Physiology of Animals. Blackwell, Oxford. Woodland, J.T., 2005. A contribution to our knowledge of lepismatid development. J. Morphol 101, 523–577. Canadian Entomologist 130, C. Pick et al. / Insect Biochemistry and Molecular Biology 38 (2008) 977–983983