Arachnid Hemocyanins.

Hemocyanin (Hc), a copper-containing extracellular multimeric protein, is the major protein component of hemolymph in different arachnid groups. Hc possesses 7 or 8 very well-characterized types of monomers with molecular weights ranging from 70 to 85 kDa, organized in hexamers or multiple of hexamers. The present chapter compiles the existing data with relation to the function of this protein in the arachnids. Hc has as main function the reversible transport of O2, but it shows many secondary though not less important functions. With reference to this, it has been described that Hc can transport hydrophobic molecules (lipid-derived hormones and lipids) to the different organs, having a key role in the lipid transport system. In arachnids, like in other arthropods and invertebrates, Hc has phenoloxidase function which is related to different metabolic processes such as melanin formation and defense against pathogens. In addition, Hc has additional defensive functions since it can serve as precursor for the production of antimicrobial peptides. In short, the evolution of this protein has led to the development of multiple functions essential for organisms possessing this protein.

Patterns of Protein Evolution in Cytochrome c Oxidase 1 (COI) from the Class Arachnida.

Because sequence information is now available for the 648bp barcode region of cytochrome c oxidase 1 (COI) from more than 400,000 animal species, this gene segment can be used to probe patterns of mitochondrial evolution. The present study examines levels of amino acid substitution and the frequency of indels in COI from 4177 species of arachnids, including representatives from all 16 orders and 43% of its families (267/625). It examines divergences at three taxonomic levels-among members of each order to an outgroup, among families in each order and among BINs, a species proxy, in each family. Order Distances vary fourfold (0.10-0.39), while the mean of the Family Distances for the ten orders ranges fivefold (0.07-0.35). BIN Distances show great variation, ranging from 0.01 or less in 12 families to more than 0.25 in eight families. Patterns of amino acid substitution in COI are generally congruent with previously reported variation in nucleotide substitution rates in arachnids, but provide some new insights, such as clear rate acceleration in the Opiliones. By revealing a strong association between elevated rates of nucleotide and amino acid substitution, this study builds evidence for the selective importance of the rate variation among arachnid lineages. Moreover, it establishes that groups whose COI genes have elevated levels of amino acid substitution also regularly possess indels, a dramatic form of protein reconfiguration. Overall, this study suggests that the mitochondrial genome of some arachnid groups is dynamic with high rates of amino acid substitution and frequent indels, while it is 'locked down' in others. Dynamic genomes are most prevalent in arachnids with short generation times, but the possible impact of breeding system deserves investigation since many of the rapidly evolving lineages reproduce by haplodiploidy, a mode of reproduction absent in 'locked down' taxa.

Descriptions of two new, cryptic species of Metasiro (Arachnida: Opiliones: Cyphophthalmi: Neogoveidae) from South Carolina, USA, including a discussion of mitochondrial mutation rates.

Specimens of Metasiro from its three known disjunct population centers in the southeastern US were examined and had a 769 bp fragement of the mitochondrial gene cytochrome c oxidase subunit I (COI) sequenced. These populations are located in the western panhandle of Florida and nearby areas of Georgia, in the Savannah River delta of South Carolina, and on Sassafras Mt. in South Carolina. This range extends over as much as 500 km, which is very large for a species of cyphophthalmid harvestmen and presents a degree of physical separation among populations such that we would expect them to actually be distinguishable species. We examined the morphology, including the spermatopositors of males, and sequences from 221 specimens. We found no discernible differences in the morphologies of specimens from the different populations, but corrected pairwise distances of COI were about 15% among the three population centers. We also analyzed COI data using a General Mixed Yule Coalescent (GMYC) model implemented in the R package SPLITS; with a single threshold, the most likely model had four species within Metasiro. Given this level of molecular divergence, the monophyly of the population haplotypes, and the number of exclusive COI nucleotide and amino acid differences distinguishing the populations, we here raise the Savannah River and Sassafras Mt. populations to species status: M. savannahensis sp. nov., and M. sassafrasensis sp. nov., respectively. This restricts M. americanus (Davis, 1933) to just the Lower Chattahoochee Watershed, which in this study includes populations along the Apalachicola River and around Florida Caverns State Park. GMYC models reconstructed the two main haplotype clades within M. americanus as different species, but they are not exclusive to different areas. We estimate COI percent divergence rates in certain cyphophthalmid groups and discuss problems with historical measures of this rate. We hypothesize that Metasiro began diversifying over 20 million years ago.

Expression and purification of cyto-insectotoxin (Cit1a) using silkworm larvae targeting for an antimicrobial therapeutic agent.

Antimicrobial peptides (AMPs), both synthetic and from natural sources, have raised interest recently as potential alternatives to antibiotics. Cyto-insectotoxin (Cit1a) is a 69-amino-acid antimicrobial peptide isolated from the venom of the central Asian spider Lachesana tarabaevi. The synthetic gene Cit1a fused with the enhanced green fluorescent protein (EGFP) gene was expressed as the EGFP-Cit1a fusion protein using a cysteine protease-deleted Bombyx mori nucleopolyhedrovirus (BmNPV-CP(-)) bacmid in silkworm larva and pupa. The antimicrobial effect of the purified protein was assayed using disk diffusion and broth microdilution methods. The minimum inhibitory concentration of EGFP-Cit1a was also measured against several bacterial strains and showed similar antimicrobial activity to that of the synthetic Cit1a reported earlier. The EGFP-Cit1a fusion protein showed antibiotic activity toward gram-positive and gram-negative bacteria at the micromolar concentration level. These results show that active Cit1a can be produced and purified in silkworm, although this peptide is insecticidal. This study demonstrates the potential of active Cit1a purified from silkworms to use as an antimicrobial agent.

First biosynthetic pathway of 1-hepten-3-one in Iporangaia pustulosa (Opiliones).

Arthropods produce a great variety of natural compounds, many of which have unexplored biosynthesis. Among the armored harvestmen (Arachnida: Opiliones) of the suborder Laniatores, the defensive gland exudates contain vinyl ketones and other constituents of supposed polyketide origin. We have studied the biosynthesis of 1-hepten-3-one in the Neotropical harvestman Iporangaia pustulosa by feeding individuals with ¹³C-labeled precursors, demonstrating its mixed acetate/propionate origin. ¹³C NMR spectroscopy showed an unusual labeling pattern suggesting different propionate sources for starting and extender units. Our analysis also indicates the presence of methylmalonyl-CoA mutase, converting acetate into propionyl-CoA via succinyl-CoA, together with other C3 unit routes. This is the first biosynthetic study of alkyl vinyl ketones in arthropods. Our results shed light on the origin and diversification of chemical compounds in a major arthropod group.

A novel hyaluronidase from brown spider (Loxosceles intermedia) venom (Dietrich's Hyaluronidase): from cloning to functional characterization.

Loxoscelism is the designation given to clinical symptoms evoked by Loxosceles spider's bites. Clinical manifestations include skin necrosis with gravitational spreading and systemic disturbs. The venom contains several enzymatic toxins. Herein, we describe the cloning, expression, refolding and biological evaluation of a novel brown spider protein characterized as a hyaluronidase. Employing a venom gland cDNA library, we cloned a hyaluronidase (1200 bp cDNA) that encodes for a signal peptide and a mature protein. Amino acid alignment revealed a structural relationship with members of hyaluronidase family, such as scorpion and snake species. Recombinant hyaluronidase was expressed as N-terminal His-tag fusion protein (∼45 kDa) in inclusion bodies and activity was achieved using refolding. Immunoblot analysis showed that antibodies that recognize the recombinant protein cross-reacted with hyaluronidase from whole venom as well as an anti-venom serum reacted with recombinant protein. Recombinant hyaluronidase was able to degrade purified hyaluronic acid (HA) and chondroitin sulfate (CS), while dermatan sulfate (DS) and heparan sulfate (HS) were not affected. Zymograph experiments resulted in ∼45 kDa lytic zones in hyaluronic acid (HA) and chondroitin sulfate (CS) substrates. Through in vivo experiments of dermonecrosis using rabbit skin, the recombinant hyaluronidase was shown to increase the dermonecrotic effect produced by recombinant dermonecrotic toxin from L. intermedia venom (LiRecDT1). These data support the hypothesis that hyaluronidase is a "spreading factor". Recombinant hyaluronidase provides a useful tool for biotechnological ends. We propose the name Dietrich's Hyaluronidase for this enzyme, in honor of Professor Carl Peter von Dietrich, who dedicated his life to studying proteoglycans and glycosaminoglycans.

Spatial variation in colour morph, spotting and allozyme frequencies in the candy-stripe spider, Enoplognatha ovata (Theridiidae) on two Swedish archipelagos.

The selective significance, if any, of many invertebrate visible polymorphisms is still not fully understood. Here we examine colour- and black spotting-morph frequencies in the spider Enoplognatha ovata in populations on two Swedish archipelagos with respect to different spatial scales and, in one archipelago, against the background of variation at four putative neutral allozyme marker loci. Every population studied was polymorphic for colour and 28 out of 30 contained all three colour morphs--lineata, redimita and ovata. We found no evidence for a breakdown in the traditional colour morph designation previously suggested for other northern European populations of this species. For colour there is no significant heterogeneity at spatial scales greater than between local populations within islands. Black spotting frequencies show a similar lack of pattern over larger spatial scales except that there are significant differences between the Stockholm and Göteborg archipelagos. Measures of population differentiation (theta) within the Stockholm islands for the two visible systems show them to be significantly more differentiated than the neutral markers, suggesting local selection acting on them in a population-specific manner. On the basis of previous observations and the distribution of spotting phenotypes on a European scale, it is argued that thermal selection might operate on black spotting during the juvenile stages favouring more spots in continental climates. It is not clear what selective forces act on colour.

Distribution of acetylcholinesterase in the central nervous system of harvestmen (Arachnida: Opilionida).

Enzyme histochemical technique (Gomori and Koelle methods, reaction product development according to Lewis) was applied on 10 microm frozen sections of formalin-fixed material to demonstrate the distribution of acetylcholinesterase in the central nervous system (CNS) of harvestmen (Opilionida: Phalangiidae). Distinctly positive reactions were confined to the neuropil, showing strong staining especially in protocerebral brain centres (optic lobes, cerebral ganglia), the cheliceral ganglia (stomodeal bridge, afferent tracts), and the connective ring systems of the subesophageal nerve mass. The results obtained are discussed with regard to central coordinative functions of the respective brain parts.

A sensitive substrate for the clotting enzyme in horseshoe crab hemocytes.

An endotoxin-activated hemocyte lysate from horseshoe crab (Tachypleus and Limulus) was found to hydrolyze specifically BZ-Ile-Glu-Gly-Arg-p-nitroanilide, which was recently introduced as the substrate for assay of the blood coagulation factor, Factor Xa. Further, this amidase activity increased by increasing the concentration of bacterial endotoxin (Salmonella minnesota R595) added to the lysate. Thus, the measurement of the amidase activity in the hemocyte lysate can be very useful to detect and determine the endotoxin.

Hybridization of glyceraldehyde-3-phosphate dehydrogenase in borate.

Conditions for the hybridization of glyceraldehyde-3-phosphate dehydrogenase (GPD) [EC 1.2.1.12] in the presence of dilute borate were examined with horseshoe crab and rabbit GPDs. Hybridization was strongly dependent upon pH, borate concentration, and temperature. The optimum medium for hybridization was 10mM Tris-HCl-1 mM 2-mercaptoethanol-1mM EDTA containing 10-20 mM borate, pH 8.5-9.0. Hybridization was performed by incubation of two electrophoretically distinct GPDs at 30 degrees for 3-15 hr in the above medium at a protein concentration of 1-2 mg/ml. The time course of hybridization was analyzed under the optimized conditions. Symmetrical A2B2-type hybrid appeared only 5 min after incubation for 1 hr. Hybridization of GPDs from 7 different species was examined under the optimal conditions. Hybridization was detected with rabbit-horseshoe crab, yeast-rabbit, yeast-chicken, and chicken-horseshoe crab combinations. Subunit-subunit interaction, the mechanism of hybridization, and the structure of GPD are discussed based on the results obtained.

Glyceraldehyde-3-phosphate dehydrogenase of horseshoe crab (Tachypleus tridentatus).

Glyceraldehyde-3-phosphate dehydrogenase [ED 1.2.1.12] was purified from the horseshoe crab, a living fossil, and its properties were examined. 1 The purified enzyme was homogeneous as judged by various tests. The enzyme, like enzymes from other sources, was a tetramer with a subunit molecular weight of 36,000. The kinetic parameters and pH optimum were also similar to those of other enzymes, though the enzyme was more stable against heat and pH denaturations. 2 Analysis of SH groups showed that there were 4 SH groups per subunit, one of which was essential for the enzyme activity and was highly reactive. 3. CD spectra of the enzyme suggested that the enzyme had a very high content of beta-structure (ca. 45 per cent). 4. The horseshoe crab enzyme could form a hybrid in vitro with the rabbit muscle enzymes in concentrated salt solution at acidic pH. 5. There results indicate that the enzyme has overall structural similarity to other enzymes and that the enzyme is highly conserved during a long period of evolution. Some discussions on the structure and activity of the horseshoe crab enzyme are made in comparison with the enzymes from other sources.

Fructose-diphosphate aldolase of Horseshoe crab (Tachypleus tridentatus).

Fructose-diphosphate aldolase [ED 4.1.2.13] was isolated from horseshoe crab ( living fossil) muscle and some molecular and enzymatic properties were examined. The enzyme was a tetramer with a molecular weight of about 160,000. The enzyme activity was inhibited by reduction with borohydride in the presence of the substrate and was inactivated by carboxypeptidase A [EC 3.4.12.2] digestion. The pH optima for fructose-diphosphate (FDP) and fructose-1-phosphate (F1P) activities were 6.5--8 and 7.5--8.2, respectively. The ratio of FDP/F1P activities was 30 and Km values were 1.7 times 10- minus 5 M and 2.5 times 10- minus 3 M, respectively, for the two substrates. The horseshoe crab aldolase was classified as class 1, type A, based on the results obtained. Extensive homology in various properties of the enzyme was observed when it was compared with enzymes from other sources, though some differences could be found in the amino acid composition and in the kinetic properties.