Arthropod Classes Research Articles

Exploring embryonic germ line development in the water flea, Daphnia magna, by zinc-finger-containing VASA as a marker

VASA is an ATP-dependent RNA helicase belonging to the DEAD-box family that, in many organisms, is specifically expressed in germ line cells throughout the life cycle, making it a powerful molecular marker to study germ line development. To obtain further information on germ line development in crustaceans, we cloned VASA cDNAs from three branchiopod species: water fleas Daphnia magna and Moina macrocopa, and brine shrimp Artemia franciscana. RNA helicase domains in branchiopod VASA were highly conserved among arthropod classes. However, N-terminal RNA-binding domains in branchiopod VASA were highly diverged and, unlike other arthropod VASA reported so far, possessed repeats of retroviral-type zinc finger (CCHC) motifs. Raising specific antibodies against Daphnia VASA revealed that the primordial germ cells (PGCs) in this organism segregate at a very early cleavage stage of embryogenesis in parthenogenetic and sexual eggs. Clusters of PGCs then start to migrate inside the embryo and finally settle at both sides of the intestine, the site of future gonad development. RNA analyses suggested that maternally supplied vasa mRNA was responsible for early VASA expression, while zygotic expression started during blastodermal stage of development.

The fate of isolated blastomeres with respect to germ cell formation in the amphipod crustacean Parhyale hawaiensis

Germ cells may be specified through the localization of germ line determinants to specific cells in early embryogenesis, or by inductive signals from neighboring cells to germ cell precursors in later embryogenesis. Such determinants can be produced and localized during or after oogenesis, either autonomously by oocytes or by associated nutritive cells. In Drosophila, each oocyte is connected to nurse cells by cytoplasmic bridges, and determinants synthesized in nurse cells are transported through these bridges to the oocyte. However, the Drosophila model may not be applicable to all arthropods, since in many species of all four extant arthropod classes, gametogenesis functions without nurse cells. In this paper, I use immunodetection of Vasa protein to study germ cell development in the amphipod crustacean Parhyale hawaiensis, a species whose ovaries lack nurse cells and whose eggs lack obvious polarity. Previous cell lineage analyses have shown that all three germ layers and the germ line are exclusively specified by third cleavage. In the present study, I use a molecular marker to follow germ cell development during P. hawaiensis embryogenesis. I determine the capacity of individual blastomeres to form germ cells by isolating blastomeres at early cleavage stages and provide experimental evidence for localized germ cell determinants at the two-cell stage in P. hawaiensis. These experiments indicate that many aspects of early amphipod development, including timing and symmetry of cell division, the transition from holoblastic to superficial cleavage, and possibly some gastrulation movements, are cell autonomous following first cleavage.

Evidence for occurrence of an organophosphate‐resistant type of acetylcholinesterase in strains of sea lice (Lepeophtheirus salmonis Krøyer)

Acetylcholinesterase (AChE) is the target of a major pesticide family, the organophosphates, which were extensively used as control agents of sea lice on farmed salmonids in the early 1990s. From the mid-1990s the organophosphates dichlorvos and azamethiphos were seriously compromised by the development of resistance. AChE insensitive to organophosphate chemotherapeutants has been identified as a major resistance mechanism in numerous arthropod species, and in this study, target-site resistance was confirmed in the crustacean Lepeophtheirus salmonis Krøyer isolated from several fish-farming areas in Norway and Canada. A bimolecular rate assay demonstrated the presence of two AChE enzymes with different sensitivities towards azamethiphos, one that was rapidly inactivated and one that was very slowly inactivated. To our knowledge this is the first report of target-site resistance towards organophosphates in a third class of arthropods, the Crustacea.

Gene expression in spider appendages reveals reversal of exd/hth spatial specificity, altered leg gap gene dynamics, and suggests divergent distal morphogen signaling

Leg development in Drosophila has been studied in much detail. However, Drosophila limbs form in the larva as imaginal discs and not during embryogenesis as in most other arthropods. Here, we analyze appendage genes in the spider Cupiennius salei and the beetle Tribolium castaneum. Differences in decapentaplegic (dpp) expression suggest a different mode of distal morphogen signaling suitable for the specific geometry of growing limb buds. Also, expression of the proximal genes homothorax (hth) and extradenticle (exd) is significantly altered: in the spider, exd is restricted to the proximal leg and hth expression extends distally, while in insects, exd is expressed in the entire leg and hth is restricted to proximal parts. This reversal of spatial specificity demonstrates an evolutionary shift, which is nevertheless compatible with a conserved role of this gene pair as instructor of proximal fate. Different expression dynamics of dachshund and Distal-less point to modifications in the regulation of the leg gap gene system. We comment on the significance of this finding for attempts to homologize leg segments in different arthropod classes. Comparison of the expression profiles of H15 and optomotor-blind to the Drosophila patterns suggests modifications also in the dorsal—ventral patterning system of the legs. Together, our results suggest alterations in many components of the leg developmental system, namely proximal—distal and dorsal—ventral patterning, and leg segmentation. Thus, the leg developmental system exhibits a propensity to evolutionary change, which probably forms the basis for the impressive diversity of arthropod leg morphologies.

First evidence for photoperiodic regulation of the life cycle in a millipede species, Polydesmus angustus (Diplopoda: Polydesmidae)

The hypothesis that the periods of dormancy previously described in the millipede Polydesmus angustus may be photoperiodically induced diapauses was tested experimentally. In this species, biennial individuals exhibit two successive periods of dormancy: aestivation in the penultimate stadium (stadium VII) and reproductive dormancy in the adults, which emerge in autumn. It was first established that the reproductive dormancy is not a thermally controlled state of quiescence. When adults emerging in autumn were kept at 16 °C under natural photoperiod, their reproduction was delayed for several months in comparison with adults emerging in spring at similar temperatures. This indicates that the reproductive dormancy begins with a period of diapause. Further experiments provided evidence of a photoperiodic induction of the adult diapause. When millipedes were reared under short day length (L:D 12:12 h) throughout their development, they required more time to reproduce than millipedes reared under long day length (L:D 16:8 h) at the same temperatures. Photoperiod influenced reproduction in females, but no significant effects were detected in adult males. On the other hand, stadium VII was markedly longer at L:D 16:8 h than at L:D 12:12 h in both sexes, which strongly suggests that aestivation is also induced by photoperiod. However, the effects on the duration of stadium VII varied among individuals, some of which showed no response to long days. This study is the first to document photoperiodic regulation of the life cycle in the class Diplopoda, a trait common in other classes of terrestrial arthropods.

Thylacocephala (Arthropoda: Crustacea?) from the Cretaceous of Lebanon and implications for thylacocephalan systematics

An intensive study of a collection of arthropods from the Cretaceous of Lebanon, formerly referred to as stomatopod larvae, reveals that these forms belong to the problematical arthropod class Thylacocephala. The species Protozoea hilgendorfi, P. damesi, and Pseuderichthus cretaceus display defining thylacocephalan characters such as a carapace enclosing the entire body bearing a large anterior optic notch; three pairs of large, raptorial appendages; and a posterior battery of small swimming limbs associated with muscle segments. Unique characters of at least the genus Protozoea are the numerous ‘pits’ covering the entire carapace, elongated anterior rostral and posterior spines, and a dorsal hinge indicative of a truly bivalved carapace. We note several anatomical curiosities that provide some insight into the anatomy, ecology and phylogenetic relationships of these and other thylacocephalan species. Structures on the carapace surface of thylacocephalans may represent sensory organs comparable to the receptors seen on thecostracan crustaceans. The affinities of the three species described are still not entirely clear, but a phylogenetic analysis of the entire group may resolve this.

Chelicerate Hox genes and the homology of arthropod segments.

Genes of the homeotic complex (HOM-C) in insects and vertebrates are required for the specification of segments along the antero-posterior axis. Multiple paralogues of the Hox genes in the horseshoe crab Limulus poliphemus have been used as evidence for HOM-C duplications in the Chelicerata. We addressed this possibility through a limited PCR survey to sample the homeoboxes of two spider species, Steatoda triangulosa and Achaearanea tepidariorum. The survey did not provide evidence for multiple Hox clusters although we have found apparent duplicate copies of proboscipedia (pb) and Deformed (Dfd). In addition, we have cloned larger cDNA fragments of pb, zerknullt (zen/Hox3) and Dfd. These fragments allowed the determination of mRNA distribution by in situ hybridization. Our results are similar to the previously published expression patterns of Hox genes from another spider and an oribatid mite. Previous studies compared spider/mite Hox gene expression patterns with those of insects and argued for a pattern of segmental homology based on the assumption that the co-linear anterior boundaries of the Hox domains can be used as markers. To test this assumption we performed a comparative analysis of the expression patterns for UBX/ABD-A in chelicerates, myriapods, crustaceans, and insects. We conclude that the anterior boundary can be and is changed considerably during arthropod evolution and, therefore, Hox expression patterns should not be used as the sole criterion for identifying homology in different classes of arthropods.

Mitochondrial gene arrangement of the horseshoe crab Limulus polyphemus L.: conservation of major features among arthropod classes.

Numerous complete mitochondrial DNA sequences have been determined for species within two arthropod groups, insects and crustaceans, but there are none for a third, the chelicerates. Most mitochondrial gene arrangements reported for crustaceans and insect species are identical or nearly identical to that of Drosophila yakuba. Sequences across 36 of the gene boundaries in the mitochondrial DNA (mtDNA) of a representative chelicerate. Limulus polyphemus L., also reveal an arrangement like that of Drosophila yakuba. Only the position of the tRNA(LEU)(UUR) gene differs; in Limulus it is between the genes for tRNA(LEU)(CUN) and ND1. This positioning is also found in onychophorans, mollusks, and annelids, but not in insects and crustaceans, and indicates that tRNA(LEU)(CUN)-tRNA(LEU)(UUR)-ND1 was the ancestral gene arrangement for these groups, as suggested earlier. There are no differences in the relative arrangements of protein-coding and ribosomal RNA genes between Limulus and Drosophila, and none have been observed within arthropods. The high degree of similarity of mitochondrial gene arrangements within arthropods is striking, since some taxa last shared a common ancestor before the Cambrian, and contrasts with the extensive mtDNA rearrangements occasionally observed within some other metazoan phyla (e.g., mollusks and nematodes).

The “evolution” of Anomalocaris and its classification in the arthropod class Dinocarida (nov.) and order Radiodonta (nov.)

The remarkable “evolution” of the reconstructions of Anomalocaris, the extraordinary predator from the 515 million year old Middle Cambrian Burgess Shale of British Columbia, reflects the dramatic changes in our interpretation of early animal life on Earth over the past 100 years. Beginning in 1892 with a claw identified as the abdomen and tail of a phyllocarid crustacean, parts of Anomalocaris have been described variously as a jellyfish, a sea-cucumber, a polychaete worm, a composite of a jellyfish and sponge, or have been attached to other arthropods as appendages. Charles D. Walcott collected complete specimens of Anomalocaris nathorsti between 1911 and 1917, and a Geological Survey of Canada party collected an almost complete specimen of Anomalocaris canadensis in 1966 or 1967, but neither species was adequately described until 1985. At that time they were interpreted by Whittington and Briggs to be representatives of “a hitherto unknown phylum.”Here, using recently collected specimens, the two species are newly reconstructed and described in the genera Anomalocaris and Laggania, and interpreted to be members of an extinct arthropod class, Dinocarida, and order Radiodonta, new to science. The long history of inaccurate reconstruction and mistaken identification of Anomalocaris and Laggania exemplifies our great difficulty in visualizing and classifying, from fossil remains, the many Cambrian animals with no apparent living descendants.

Ribosomal DNA phylogeny of the major extant arthropod classes and the evolution of myriapods.

The evolutionary relationships among arthropods are of particular interest because the best-studied model system for ontogenetic pattern formation, the insect Drosophila, is a member of this phylum. Evolutionary inferences about the developmental mechanisms that have led to the various designs of the arthropod body plan depend on a knowledge of the phylogenetic framework of arthropod evolution. Based on morphological evidence, but also on palaeontological consideration, the sister group of the insects is believed to be found among the myriapods. Using nuclear ribosomal gene sequences for constructing a molecular phylogeny, we provide strong evidence that the crustaceans and not the myriapods should be considered to be the sister group of the insects. Moreover, the degree of sequence divergence suggests that the diversification of the myriapods occurred during the Cambrian. Our findings have general implications for the course of land colonization by the different arthropod groups, as well as for the interpretation of primitive and derived features of arthropod morphology.

Distribution of transposable elements in arthropods.

Transposable elements of the DNA-mediated and RNA-mediated classes found in arthropods are briefly described and their distribution reviewed. The distribution patterns of DNA-mediated elements are extremely patchy and the principal cause appears to be the horizontal transfer of elements between host lineages. In the best documented case of mariner elements, these hosts can be in different orders of insects, classes of arthropods, and even other phyla of animals. RNA-mediated elements appear to undergo much longer periods of vertical evolution within host lineages, and evidence for their horizontal transfer remains scant. The evolutionary relationships of many of these transposons have recently been illuminated by phylogenetic analyses of the reverse-transcriptase enzymes of the RNA-mediated elements, and the recognition that the transposases of some of the DNA-mediated elements are distantly related to in the integrases of some of the RNA-mediated elements.

Larva lights: A decade of photoaffinity labeling with juvenile hormone analogues

The introduction of photoaffinity labeling into the mode of action of insect hormones and pheromones started 12 yr ago with the photoaffinity labeling of juvenile hormone binding proteins (JHBPs) from cockroaches in the laboratory of the late John K. Koeppe. Applying this technique to Manduca sexta led ultimately to a three-laboratory collaborative project that has begun to dissect the molecular basis for JH transport, metabolism, and nuclear binding and gene activation in Lepidoptera. This review provides (1) a history of the first experiments; (2) an idea of the breadth of the technique in the arthropod classes Insecta, Crustacea, and Arachnida; and (3) evidence for the depth of the technique in unearthing key details about three different types of the molecular action of JH in M. sexta.

Is ecdysone involved in the moult cycle of the terrestrial crustaceanOrchestia cavimana(Amphipoda)?

Summary We have tested several labelled ecdysteroids as possible precursors of moulting hormone biosynthesis in Orchestia cavimana, a terrestrial Amphipoda. Ecdysone, known to be a prohormone in many classes of Arthropods, was never converted into 20-hydroxyecdysone when injected in this animal in vivo, though 20-hydroxyecdysone has been previously shown to be the predominant ecdysteroid. On the contrary, C-2 hydroxylation was evidenced by the conversion of 2-deoxy-ecdysteroids into 2-hydroxylated compounds. The surprising failure of ecdysone conversion was corroborated by its absence of effect on protein synthesis in an ecdysteroid target tissue, the calcium storage organ of O. cavimana. Our results are in agreement with the previously reported findings that 20-hydroxyecdysone is the active moulting hormone in O. cavimana. However, ecdysone does not appear to be its natural precursor.

Developmental regulatory mechanisms in the evolution of insect diversity

ABSTRACT The major architectural differences between most Arthropod classes and orders involve variations in the number, type and pattern of body appendages. We have utilized the emerging knowledge of appendage formation in fruit flies to begin to address the developmental and genetic basis of morphological diversity among insects. Butterflies, for example, differ from fruit flies in possessing larval abdominal limbs, two pairs of adult wings, and a sophisticated system of wing color pattern formation. We have found that the genetic bases for these three major morphological features involve differences between flies and butterflies at three levels of genetic regulation during development. First, we show that the presence of abdominal limbs in butterflies is associated with striking changes in the regulation of specific homeotic genes in the abdominal segments of the butterfly embryo. Second, we suggest that the twowinged state of the fruit fly and the distinct pattern of the butterfly hindwing are the consequence of many accumulated changes in the target genes regulated by the Ultra-bithorax homeotic gene. And finally, we demonstrate that a new genetic program, involving many of the same genes that specify the conserved global patterning coordinates of fruit fly and butterfly wings, has been superimposed onto the butterfly wing to create their unique color patterning system. These findings demonstrate how morphological diversity arises from the different ways in which conserved sets of regulatory genes are deployed during development.

The origin, patterning and evolution of insect appendages

AbstractThe appendages of the adult fruit fly and other insects and Arthropods develop from secondary embryonic fields that form after the primary anterior/posterior and dorsal/ventral axes of the embryo have been determined. In Drosophila, the position and fate of the different fields formed within each segment are determined by genes acting along both embryonic axes, within individual segments, and within specific fields. Since the major architectural differences between most Arthropod classes and orders involve variations in the number, type and morphology of body appendages, the elucidation of the embryology and molecular genetics of the origin and patterning of insect limb fields may help to facilitate an understanding of both the mechanism of appendage formation and some of the major steps in the morphological evolution of the Arthropods. In this review, we will discuss recent studies that have advanced our understanding of both the origin and patterning of Drosophila leg and wing secondary fields. These results provide fresh insights into potentially general mechanisms of how body parts develop and evolve.

Proteins Of crustacean exoskeleton II: Immunological evidence for their relatedness to cuticular proteins of two insects

AbstractThe exoskeletons of crustaceans and cuticles of insects are characterized by an abundance of proteins with acidic pIs and Mrs 31 kDa or smaller. Immunochemical data described here further demonstrate the similarities of such proteins among representatives of these two major classes of Arthropoda. Proteins extracted from the four individual layers of the exoskeletons of Bermuda land crabs Gecarcinus lateralis at different stages of the intermolt cycle and from exuviae, the partially degraded old exoskeleton that is cast at ecdysis, were analyzed on Western blots with polyspecific polyclonal antibodies against either larval cuticular proteins (anti‐LCP) or pupal cuticular proteins (anti‐PCP) of the tobacco hornworm Manduca sexta, or against two pairs of larval cuticular proteins from third instar larvae (anti‐L3CPs) of Drosophila melanogaster. M. sexta anti‐LCP cross‐reacted with many of the crab proteins extracted at all stages of the intermolt cycle. It reacted with more high Mr proteins from late proecdysial exoskeleton and exuviae than with such proteins from anecdysial or early proecdysial exoskeletons, probably because the larger Mr proteins are enriched when the lower Mr proteins are degraded during proecdysis. M. sexta anti‐PCP cross‐reacted hardly at all with the crab proteins. Both D. melanogaster anti‐L3CPs cross‐reacted with crab epicuticular proteins at all stages of the intermolt cycle and from exuviae. Anti‐L3CP 3 + 4 reacted even more intensely and with many more proteins from the three inner layers of the exoskeleton than did anti‐L3CP 1+2. Controls included 2D gels with rabbit anti‐ovalbumin to detect glycoproteins containing mannose and N‐acetyl‐glucosamine; it cross‐reacted with only one protein in membranous layer of an anecdysial crab, while rabbit anti‐fluorescein reacted with none. By contrast, the intensity and specificity of the cross‐reactions of crab exoskeletal proteins with antibodies against cuticular proteins of members of two orders of insects suggest that at least some cuticular proteins in the two arthropod Classes, Crustacea and Insecta, are evolutionarily conserved. Published 1993 Wiley‐Liss, Inc.

Molt-cycle correlated patterns of synthesis of integumentary proteins in the land crab Gecarcinus lateralis

We describe here an in vitro investigation of the formation and dissolution of the crustacean exoskeleton by the integumentary tissues of the Bermuda land crab Gecarcinus lateralis. We focus on the synthesis of proteins during the formation of a new exoskeleton and the degradation of an old one. Tissues of animals at different stages of the molt cycle were labeled in vitro with radioamino acids following which proteins were analyzed by a number of methods. From their patterns of synthesis, crab integumentary proteins (CIPs) may be classified into five groups. Group I CIPs include many over a wide range of molecular weights that are synthesized at a relatively constant rate throughout the molt cycle and are designated “housekeeping” proteins. The synthesis of a subset of such housekeeping CIPs (Group II) increases from two- to fivefold during proecdysis when formation of the epicuticle and exocuticle of the new exoskeleton is in progress. Group III CIPs are synthesized intermittently from very early proecdysis, when degradation of the old exoskeleton begins, and throughout the remainder of proecdysis. Groups IV and V first appear at stages in the molt cycle that suggest that they may be structural components of the new exoskeleton. Group IV CIPs, which are synthesized at the time of epi- and exocuticle formation, have a wide range of sizes. Group V CIPs, which comprise several very large proteins, are synthesized in stage B of metecdysis at the time when the formation of the endocuticle begins. A number of CIPs cross-react strongly with polyclonal antibodies to cuticle proteins of pupae and larvae of the tobacco hornworm Manduca sexta, indicating that exoskeletal/cuticle proteins of these two classes of arthropods, the Insecta and Crustacea, share epitopes. Whether the common epitopes relate to amino acid sequences or structural properties remains to be determined.

Immunoreactive salmon calcitonin-like molecule in crustaceans: High concentrations in Nephrops norvegicus

A salmon calcitonin (CT)-like peptide was characterized in various crustaceans by radioimmunological and radioreceptor assays. The highest levels of the molecule were found in the anterior part of the gut and the hepatopancreas of the Norway lobster: Nephrops norvegicus. Molecular sieving of this molecule suggested an apparent molecular weight of 4500 Da, that is higher than that of calcitonin. This peptide can be refered to as crustacean calcitonin as it inhibits the binding of labeled salmon CT to rat kidney membranes. The high concentration found in crustaceans suggested that this molecule could have an important role in this class of arthropods.

Identification of a Juvenile Hormone-Like Compound in a Crustacean

Juvenile hormone (JH) has central roles in the regulation of insect development and reproduction but has not previously been identified in other arthropod classes. The hemolymph of a crustacean, Libinia emarginata (Leach), has now been analyzed for JH-like compounds. Samples contained 0.003 to 0.030 nanogram of JH III per milliliter and 10 to 50 nanograms of methyl farnesoate per milliliter; methyl farnesoate is a compound structurally related to JH III that has JH bioactivity. Several tissues were examined for synthesis and secretion of JH-like compounds. Of these tissues, only the mandibular organs produced and secreted JH III and methyl farnesoate. However, microchemical analysis revealed that this JH III was racemic, and thus likely an artifactual oxidation product of methyl farnesoate. Secretion of methyl farnesoate was related to reproduction in females, with the highest rates observed in Libinia near the end of the ovarian cycle when oocyte growth and vitellogenesis are greatest. These results indicate that JH-like compounds such as methyl farnesoate have regulatory roles in crustaceans.

Probable occurrence of ecdysteroid fatty acid esters in different classes of arthropods

We investigated the fate of injected [ 3H]ecdysone or [ 3H]20-hydroxyecdysone in various species of ticks, spiders, scorpions, myriapods, crustaceans and insects. Most of these arthropods were able to convert the ecdysteroids to esterase-labile metabolites with a very apolar behaviour in reverse-phase HPLC. Some of them have retention times similar to the apolar conjugates AP2 of the tick, Ornithodoros moubata, which have been identified recently as ecdysteroids esterified as C 22 with palmitic, stearic, oleic or linoleic acid [Diehl et al. (1985a) Int. J. invert. Reprod. Devl. 8, 1–13]. Others are less apolar and could correspond to the AP1 from O. moubata. The possible function of these metabolites remains to be established. They could represent inactivation products and/or a hormone storage-form for embryos.