Purification, cDNA structure and biological significance of a single insulin-like growth factor-binding domain protein (SIBD-1) identified in the hemocytes of the spider Cupiennius salei

Cupiennius salei single insulin-like growth factor-binding domain protein (SIBD-1), which exhibits an IGFBP N-terminal domain-like profile, was identified in the hemocytes of the spider C. salei. SIBD-1 was purified by RP-HPLC and the sequence determined by a combination of Edman degradation and 5′–3′- RACE PCR. The peptide (8676.08 Da) is composed of 78 amino acids, contains six intrachain disulphide bridges and carries a modified Thr residue at position 2. SIBD-1 mRNA expression was detected by quantitative real-time PCR mainly in hemocytes, but also in the subesophageal nerve mass and muscle. After infection, the SIBD-1 content in the hemocytes decreases and, simultaneously, the temporal SIBD-1 expression seems to be down-regulated. Two further peptides, SIBD-2 and IGFBP-rP1, also exhibiting IGFBP N-terminal domain variants with unknown functions, were identified on cDNA level in spider hemocytes and venom glands. We conclude that SIBD-1 may play an important role in the immune system of spiders.     

Embryonic development and staging of the cobweb spider Parasteatoda tepidariorum C. L. Koch, 1841 (syn.: Achaearanea tepidariorum; Araneomorphae; Theridiidae)

The cobweb spider Parasteatoda tepidariorum (C. L. Koch, 1841; syn.: Achaearanea tepidariorum) has become an important study organism in developmental biology and evolution as well as in genetics. Besides Cupiennius salei, it has become a chelicerate model organism for evo-devo studies in recent years. However, a staging system taking into account the entire development, and detailed enough to apply to modern studies, is still required. Here we describe the embryonic development of P. tepidariorum and provide a staging system which allows easy recognition of the distinct stages using simple laboratory tools. Differences between P. tepidariorum and other chelicerates, primarily C. salei, are discussed. Furthermore, cocoon production and the first postembryonic moulting procedure are described. Schematic drawings of all stages are provided to ease stage recognition.     

Evolution of dorsal-ventral axis formation in arthropod appendages: H15 and optomotor-blind/bifid-type T-box genes in the millipede Glomeris marginata (Myriapoda: Diplopoda)

In Drosophila, the T-box genes optomotor-blind (omb) and H15 have been implicated in specifying the development of the dorso-ventral (DV) axis of the appendages. Results from the spider Cupiennius salei have suggested that this DV patterning system may be at least partially conserved. Here we extend the study of the DV patterning genes omb and H15 to a representative of the Myriapoda in order to add to the existing comparative data set and to gain further insight into the evolution of the DV patterning system in arthropod appendages. The omb gene of the millipede Glomeris marginata is expressed on the dorsal side of all appendages including trunk legs, maxillae, mandibles, and antennae. This is similar to what is known from Drosophila and Cupiennius and suggests that the role of omb in instructing dorsal fates is conserved in arthropods. Interestingly, the lobe-shaped portions of the mouthparts do not express omb, indicating that these are ventral components and thus may be homologous to the endites present in the corresponding appendages in insects. Concerning the H15 gene we were able to identify two paralogous genes in Glomeris. Both genes are expressed in the sensory organs of the maxilla and antenna, but only Gm-H15-1 is expressed along the ventral side of the trunk legs. The expression is more extensive than in Cupiennius, but less so than in Drosophila. In addition, no ventral expression domain is present in the maxilla, mandible, and antenna. Because of this, the role of H15 in the determination of ventral fate remains unclear.     

Female sex pheromone of a wandering spider (Cupiennius salei): identification and sensory reception

Females of the wandering spider Cupiennius salei attach a sex pheromone to their dragline. Males encountering the female dragline examine the silk thread with their pedipalps and then typically initiate reciprocal vibratory courtship with the sexual partner. The female pheromone was identified as (S)-1,1'-dimethyl citrate. The male pheromone receptive sensory cells are located in tip pore sensilla and respond to touching the sensillum tip with female silk or pieces of filter paper containing the synthetic pheromone.     

Fine structural correlates of sensitivity in the eyes of the ctenid spider, Cupiennius salei Keys

We studied fine structural correlates of sensitivity in the principal and secondary eyes of the nocturnal hunting spider Cupiennius salei. In night-adapted eyes the four rhabdomeres of the principal eye photoreceptors are 58 mum long and occupy together 234 mum(2) in cross-section (average), whereas the two rhabdomeres of the secondary eye photoreceptors are about 49 mum long and measure 135-183 mum(2) in cross-section (average). The rhabdoms (photosensitive structures) consist of tightly packed microvilli (diameter 0.1 mum, maximum length 3.5 mum) and occupy up to 63% of the cross-sectional area of the retina. When calculating the amount of light the eyes of Cupiennius are able to capture according to their morphological characteristics, the values for sensitivity S(see Land, 1981, 1985) are between 78 and 109 mum(2). Cupiennius is more sensitive than any other hunting spider examined except Dinopis whose posterior median eyes are the most sensitive ones of all terrestrial arthropod eyes studied. In day-adapted eyes the rhabdomeral microvilli are almost completely degraded. The remaining microvillar surface amounts to only about one-tenth compared with the night-adapted state. Efferent synaptoid terminals have been found to contact the photoreceptors in all eyes of C. salei. The present fine structural data are compared to previous electrophysiological research and underline the significance of vision in Cupiennius.     

Lifespan extension by increased expression of the Drosophila homologue of the IGFBP7 tumour suppressor

Mammals possess multiple insulin-like growth factor (IGF) binding proteins (IGFBPs), and related proteins, that modulate the activity of insulin/IGF signalling (IIS), a conserved neuroendocrine signalling pathway that affects animal lifespan. Here, we examine if increased levels of an IGFBP-like protein can extend lifespan, using Drosophila as the model organism. We demonstrate that Imaginal morphogenesis protein-Late 2 (IMP-L2), a secreted protein and the fly homologue of the human IGFBP7 tumour suppressor, is capable of binding at least two of the seven Drosophila insulin-like peptides (DILPs), namely native DILP2 and DILP5 as present in the adult fly. Increased expression of Imp-L2 results in phenotypic changes in the adult consistent with down-regulation of IIS, including accumulation of eIF-4E binding protein mRNA, increase in storage lipids, reduced fecundity and enhanced oxidative stress resistance. Increased Imp-L2 results in up-regulation of dilp2, dilp3 and dilp5 mRNA, revealing a feedback circuit that is mediated via the fly gut and/or fat body. Importantly, over-expression of Imp-L2, ubiquitous or restricted to DILP-producing cells or gut and fat body, extends lifespan. This enhanced longevity can also be observed upon adult-onset induction of Imp-L2, indicating it is not attributable to developmental changes. Our findings point to the possibility that an IGFBP or a related protein, such as IGFBP7, plays a role in mammalian aging.     

Haemocyanins in spiders, III. Chemical and physical properties of the proteins in Dugesiella and Cupiennius blood.

The haemolymph of the tarantulas, Dugesiella (Eurypelma) californica and Dugesiella (Eurypelma) helluo contains high molecular weight haemocyanin (80-82% of total blood proteins) and a second protein not related to haemocyanin (18-20%). In the Lycosid spider, Cupiennius salei, haemocyanin (75% of total blood protein) occurs in two states of association. The haemocyanins were isolated by ultracentrifugation, gel filtration, isoelectric focusing, or preparative gel electrophoresis. Their sedimentation constants are 36.7 S (both tarantulas), 23.4 S and 15.9 S (Cupiennius). After alkaline dissociation, polypeptides sedimenting at 5.8 S (D. californica) and 4.7 S (Cupiennius) were obtained. The molecular weight of the intact functional subunit is (by sedimentation equilibrium) 70 300 (D. californica) and 69 900 (Cupiennius). Copper analysis results in closely similar values. By sodium dodecylsulphate gel electrophoresis, molecular weights of 71 000 (D. californica), 72 000 (Cupiennius) and 74 000 (D. helluo) were obtained. Denaturation with various agents did not lead to smaller polypeptides. The amino acid composition of the haemocyanins was determined (Table 1). The amino end group is blocked. The haemocyanins contain 1.2-1.5% of neutral carbohydrates and 0.3-0.5% of glucosamine (possibly acetylated). The neutral carbohydrates were identified with glucose, mannose, fucose, and arabinose, glucose being the dominant species. Neuraminic acid was not detected. The haemocyanins of the three species cannot be distinguished by their carbohydrate moieties, while there is a significant difference in amino acid composition between tarantula and Cupiennius haemocyanins. The second, non-respiratory protein isolated from spider blood sediments with 16.1 S (Dugesiella) or 15.9 S (Cupiennius). Its isoelectric point is at pH 5.5 It is stable in weakly alkaline solutions but can be denatured to yield polypeptide chains with molecular weights of 95 000 and 110 000. The amino acid composition is reported. As in the haemocyanins, the N-terminus is blocked. The carbohydrate content is 0.9%, glucose being the only sugar identified.     

Daily patterns of locomotor activity in a wandering spider

ABSTRACT. Cupiennius salei Keys., a lycosid spider, is night-active in its natural habitat in the highlands of Guatemala. Locomotor activity begins at dusk after several transitional states, each of which is correlated with a particular light level. Actograph measurements in controlled conditions (LD 12:12 or reversed cycles) show that the spiders begin walking immediately after dark. The activity maximum is reached within the first 3 h of the scotophase (though some spiders displayed additional lights-on activity in the first hour of the photophase). In DD, Cupiennius shows an endogenous activity rhythm with a mean period of 24.9 h (± 0.31, SE); typically during the first four to six cycles in DD the rhythm 'splits' into two components running at different frequencies. In LL (26 1x) the walking activity became arrhythmic. The results are discussed with reference to field and laboratory observations of Cupiennius behaviour and to the consequences for future physiological research with this species.     

Cupiennin 1a, an antimicrobial peptide from the venom of the neotropical wandering spider Cupiennius salei, also inhibits the formation of nitric oxide by neuronal nitric oxide synthase

Cupiennin 1a (GFGALFKFLAKKVAKTVAKQAAKQGAKYVVNKQME-NH2) is a potent venom component of the spider Cupiennius salei. Cupiennin 1a shows multifaceted activity. In addition to known antimicrobial and cytolytic properties, cupiennin 1a inhibits the formation of nitric oxide by neuronal nitric oxide synthase at an IC50 concentration of 1.3 +/- 0.3 microM. This is the first report of neuronal nitric oxide synthase inhibition by a component of a spider venom. The mechanism by which cupiennin 1a inhibits neuronal nitric oxide synthase involves complexation with the regulatory protein calcium calmodulin. This is demonstrated by chemical shift changes that occur in the heteronuclear single quantum coherence spectrum of 15N-labelled calcium calmodulin upon addition of cupiennin 1a. The NMR data indicate strong binding within a complex of 1 : 1 stoichiometry.     

IGFBP-3 and IGFBP-5 associate with the cell binding domain (CBD) of fibronectin

We have used Surface Plasmon Resonance (SPR) - based biosensor technology to investigate the interaction of the six high affinity insulin-like growth factor binding proteins (IGFBP 1-6) with the cell binding domain (CBD) of fibronectin. Using a biotinylated derivative of the ninth and tenth TypeIII domains of FN (^9-10FNIII), we show that IGFBP-3 and -5 bind to FN-CBD. We show that this binding is inhibited by IGF-I and that, for IGFBP-5, binding occurs through the C-terminal heparin binding domain of the protein. Using site-directed mutagenesis of ^9-10FNIII, we show both the “synergy” and RGD sites within these FN domains are required for maximum binding of both IGFBPs. We discuss the possible biological consequences of our results.     

Immunological correspondence between arthropod hemocyanin subunits. I. Scorpion (Leiurus, Androctonus) and spider (Eurypelma, Cupiennius) hemocyanin

The hemocyanins of the scorpions Leiurus quinquestriatus and Androctonus australis, the tarantula Eurypelma californicum (all 24-mers), and the lycosid spider Cupiennius salei (dodecamer) were dissociated into subunits, the subunits isolated and studied by two-dimensional immunoelectrophoresis for interspecific cross-reactivities. Androctonus hemocyanin yielded a pattern of 8 subunit types in agreement with data from Lamy et al. (1979, Arch. Biochem. Biophys. 193, 140-149). Leiurus hemocyanin is also composed of 8 immunologically distinct subunits which could be assigned to the pattern of Androctonus in a subunit-to-subunit correlation. The subunit designations 1 to 6 of Lamy et al. could be adopted for both scorpion hemocyanins; however, in the present communication, Lamy's subunits 3A/3B are designated as 3'/3", because we could not unequivocally decide if 3' = 3A and 3" = 3B or vice versa. The 7 subunit types a to g of Eurypelma hemocyanin could be correlated with the scorpion hemocyanin subunits as follows: a = 3', b = 5B, c = 3C, d = 5A, e = 6, f = 2, g = 4. Additional cross-reactivities were detected between e/4, and f/5A, respectively. No subunit of Eurypelma hemocyanin is homologous to scorpion 3", which could not be precipitated by anti-Eurypelma antiserum. Antiserum against Cupiennius hemocyanin precipitated subunit f of Eurypelma and subunits 2 and 5A of scorpion hemocyanin. The published models of quaternary structure and a possible subunit phylogeny of arachnidan hemocyanins are discussed in view of the present results.     

The T-box genes H15 and optomotor-blind in the spiders Cupiennius salei, Tegenaria atrica and Achaearanea tepidariorum and the dorsoventral axis of arthropod appendages

SUMMARY Dorsoventral axis formation in the legs of the fly Drosophila melanogaster requires the T-box genes optomotor-blind ( omb ) and H15 . Evolutionary conservation of the patterning functions of these genes is unclear, because data on H15 expression in the spider Cupiennius salei did not support a general role of H15 in ventral fate specification. However, H15 has a paralogous gene, midline ( mid ) in Drosophila and H15 duplicates are also present in Cupiennius and the millipede Glomeris marginata . H15 therefore seems to have been subject to gene duplication opening the possibility that the previous account on Cupiennius has overlooked one or several paralogs. We have studied omb - and H15 -related genes in two additional spider species, Tegenaria atrica and Achearanea tepidariorum and show that in both species one of the H15 genes belongs to a third group of spider H15 genes that has an expression pattern very similar to the H15 pattern in Drosophila . The expression patterns of all omb -related genes are also very similar to the omb expression pattern in Drosophila . These data suggest that the dorsoventral patterning functions of omb and H15 are conserved in the arthropods and that the previous conclusions were based on an incomplete data set in Cupiennius . Our results emphasize the importance of a broad taxon sampling in comparative studies.     

Cupiennius salei and Achaearanea tepidariorum: Spider models for investigating evolution and development

The spiders Cupiennius salei and Achaearanea tepidariorum are firmly established laboratory models that have already contributed greatly to answering evolutionary developmental questions. Here we appraise why these animals are such useful models from phylogeny, natural history and embryogenesis to the tools available for their manipulation. We then review recent studies of axis formation, segmentation, appendage development and neurogenesis in these spiders and how this has contributed to understanding the evolution of these processes. Furthermore, we discuss the potential of comparisons of silk production between Cupiennius and Achaearanea to investigate the origins and diversification of this evolutionary innovation. We suggest that further comparisons between these two spiders and other chelicerates will prove useful for understanding the evolution of development in metazoans.     

Abdominal-B expression in a spider suggests a general role for Abdominal-B in specifying the genital structure.

We have cloned an Abdominal-B (Abd-B) orthologue from the spider Cupiennius salei and have analysed its expression pattern during embryogenesis. An early expression domain is seen in the posterior part of the embryo, with an initial border in the third opisthosomal segment and later in the fifth opisthosomal segment. During mid-stage of germ band extension, two additional spots of expression appear in the posterior parts of the limb buds on the second opisthosomal segment. These coincide with the position of the future genital opening and Cs-Abd-B remains expressed in these regions until the openings are formed. In view of the fact that Abd-B and its orthologous genes are also required for specifying the genitalia in Drosophila and vertebrates, we suggest that this function may constitute an independent and ancestral role of Abd-B that can be separated from its role in specifying the posterior part of the body region.     

Potential involvement of the interaction between insulin-like growth factor binding protein (IGFBP)-6 and LIM mineralization protein (LMP)-1 in regulating osteoblast differentiation

Insulin-like growth factor binding protein (IGFBP)-6 has been reported to inhibit differentiation of myoblasts and osteoblasts. In the current study, we explored the mechanisms underlying IGFBP-6 effects on osteoblast differentiation. During MC3T3-E1 osteoblast differentiation, we found that IGFBP-6 protein was down-regulated. Overexpression of IGFBP-6 in MC3T3-E1 and human bone cells inhibited nodule formation, osteocalcin mRNA expression and ALP activity. Furthermore, accumulation of IGFBP-6 in the culture media was not required for any of these effects suggesting that IGFBP-6 suppressed osteoblast differentiation by an intracellular mechanism. A yeast two-hybrid screen of an osteosarcoma library was conducted to identify intracellular binding partners to account for IGFBP-6 inhibitory effects on osteoblast differentiation. LIM mineralizing protein (LMP-1) was identified as a high affinity IGFBP-6 binding partner. Physical interaction between IGFBP-6 and LMP-1 was confirmed by co-immunoprecipitation. Fluorescent protein fusion constructs for LMP-1 and IGFBP-6 were transiently transfected into osteoblasts to provide evidence of subcellular locations for each protein. Coexpression of LMP-1-GFP and IGFBP-6-RFP resulted in overlapping subcellular localization of LMP-1 and IGFBP-6. To determine if there was a functional association of IGFBP-6 and LMP-1 as well as a physical association, we studied the effect of IGFBP-6, LMP-1 and their combination on type I procollagen promoter activity. LMP-1 increased promoter activity while IGFBP-6 reduced promoter activity, and coexpression of LMP-1 with IGFBP-6 abrogated IGFBP-6 suppression. These studies provide evidence that overexpression of IGFBP-6 suppresses human and murine osteoblast differentiation, that IGFBP-6 and LMP-1 physically interact, and supports the conclusion that this interaction may be functionally relevant.     

Complete hemocyanin subunit sequences of the hunting spider Cupiennius salei: recent hemocyanin remodeling in entelegyne spiders

Hemocyanins are large copper-containing respiratory proteins found in many arthropod species. Scorpions and orthognath spiders possess a highly conserved 4 x 6-mer hemocyanin that consists of at least seven distinct subunit types (termed a to g). However, many "modern" entelegyne spiders such as Cupiennius salei differ from the standard arachnid scheme and have 2 x 6-mer hemocyanins. Here we report the complete primary structure of the 2 x 6-mer hemocyanin of C. salei as deduced from cDNA sequencing, gel electrophoresis, and matrix-assisted laser desorption spectroscopy. Six distinct subunit types (1 through 6) and three additional allelic sequences were identified. Each 1 x 6-mer half-molecule most likely is composed of subunits 1-6, with subunit 1 linking the two hexamers via a disulfide bridge located in a C-terminal extension. The C. salei hemocyanin subunits all belong to the arachnid g-type, whereas the other six types (a-f) have been lost in evolution. The reconstruction of a complex hemocyanin from a single g-type subunit, which commenced about 190 million years ago and was completed about 90 million years ago, might be explained by physiological and behavioral changes that occurred during the evolution of the entelegyne spiders.     

Serum insulin-like growth factor-I, insulin-like growth factor binding protein-3, and the pubertal growth spurt in the female rhesus monkey

While there is good evidence suggesting IGF-I links to pubertal development and crown-rump length growth among rhesus monkeys, linkages between IGF-I and other measures of morphological growth have not been established. In this study, the pubertal growth spurt in a number of morphological characteristics of female rhesus monkeys is related to serum endocrine status of insulin-like growth factor-I (IGF-I) and its binding protein, insulin-like growth factor binding protein-3 (IGFBP-3), to test the hypothesis that elevations in IGF-I and IGFBP-3 coincide with the time of greatest growth rate of different morphological characteristics. A longitudinal study of pubertal growth among four female rhesus monkeys was carried out across a 3-year period. Morphometric measurements included weight, crown-rump length, foot-length, and skinfolds at five sites (biceps, triceps, abdominal, subscapular, and suprailiac). These measures were taken as being representative of total mass, skeletal growth of the trunk and head, limb length, and body fatness, respectively. Measurements were carried out as closely as possible to 3-monthly, with interpolations being performed to standardise the data to exactly 3-monthly intervals for all individuals. Blood samples were taken at time of morphometry. Elevations in serum IGF-I and IGFBP-3 took place in a manner similar to that of humans, and across the period associated with onset of puberty. Mean 3-monthly gain in crown-rump length and foot length showed significant peaks across the measurement period, while mean 3-monthly gains in weight and sum of five skinfolds did not. Greatest foot length gain occurred on average between 3-3.5 years of age, while crown-rump length gain was greatest between 3.75-4 years of age. Periods of greatest gain in crown-rump length and foot length took place across the period of elevated serum IGF-I levels, which was between 3-4.5 years of age. Significant elevations in IGF-I and IGFBP-3 were not coincident with greatest gains in foot length or crown-rump length. Thus the hypothesis does not hold true for the two measures showing significant peaks in 3-monthly gain across the measurement period. The nature of the endocrine impact on macaque morphology remains unclear, although this may be fundamental to the understanding of the variation in the pubertal growth spurt and its influence on morphology at maturity both within and across primate species.     

Neuroarchitecture of the arcuate body in the brain of the spider Cupiennius salei (Araneae, Chelicerata) revealed by allatostatin-, proctolin-, and CCAP-immunocytochemistry and its evolutionary implications

Here we describe the neuronal organization of the arcuate body in the brain of the wandering spider Cupiennius salei. The internal anatomy of this major brain center is analyzed in detail based on allatostatin-, proctolin-, and crustacean cardioactive peptide (CCAP)-immunohistochemistry. Prominent neuronal features are demonstrated in graphic reconstructions. The stainings revealed that the neuroarchitecture of the arcuate body is characterized by several distinct layers some of which comprise nerve terminals that are organized in columnar, palisade-like arrays. The anatomy of the spider's arcuate body exhibits similarities as well as differences when compared to the central complex in the protocerebrum of the Tetraconata. Arguments for and against a possible homology of the arcuate body of the Chelicerata and the central complex of the Tetraconata and their consequences for the understanding of arthropod brain evolution are discussed.     

Natural and synthetic forms of insulin-like growth factor-1 (IGF-1) and the potent derivative, destripeptide IGF-1: biological activities and receptor binding

Insulin-like growth factor-1 (IGF-1), whether recombinant, chemically-synthesised or purified from bovine colostrum, was equipotent in radioreceptor assays with IGF-1 or insulin-like growth factor-2 (IGF-2) as radioligand as well as in its ability to stimulate protein synthesis in L6 myoblasts. The N-terminal truncated, destripeptide derivative of IGF-1 was approximately 7 times more potent than IGF-1 in the protein synthesis bioassay. This increased activity occurred equally with the peptide purified from bovine colostrum as with chemically-synthesised material. The higher potency of the truncated form was not associated with an increased ability to compete for IGF-1 binding to L6 myoblasts.     

The interaction of Insulin-like Growth Factors (IGFs) with Insulin-like Growth Factor Binding Proteins (IGFBPs): a review

The interactions between insulin-like growth factor bindingproteins (IGFBPs) and insulin-like growth factors (IGFs)are important in regulating the availability of IGFs to thetype 1 IGF receptor (IGF1R) but there is still a lack ofinformation regarding the mechanism of IGF binding byIGFBPs. While many studies have defined general regions ofthe IGFBPs that interact with IGFs, only recently havespecific IGF binding residues been described. This reviewwill outline the structural evidence describing residues ofboth the IGFBPs and IGFs involved in the IGFBP-IGFinteraction.