Bacteria belonging to the genus Wolbachia are associated with a variety of reproductive anomalies in arthropods. Allele-specific polymerase chain reaction (= Standard PCR) routinely has been used to amplify Wolbachia DNA from arthropods. While testing the two-spotted spider mite Tetranychus urticae and other arthropods known to be infected with Wolbachia, Standard PCR frequently produced false negatives, perhaps because the DNA from the arthropod host interfered with amplification by Taq DNA polymerase. Long PCR, which uses two enzymes (Taq and Pwo), consistently amplified Wolbachia DNA and a sensitivity analysis indicated that Long PCR was approximately six orders of magnitude more sensitive than Standard PCR in amplifying plasmid DNA spiked into insect genomic DNA. A survey indicated that 76% of sixty-two arthropod species and two subspecies in thirteen orders tested positive for the Wolbachia wsp sequence by Long PCR, which is considerably higher than the rate of 16.9% obtained previously for the ftsZ sequence using Standard PCR (Werren, J.H., Windsor, D. and Gao, L. (1995a) Proc R Soc Lond B 262: 197-204). A subsample of Long PCR products from fourteen arthropod species and two subspecies were sequenced, both directly and after cloning. Two A- and eleven B-Wolbachia strains were detected and their wsp sequences displayed a maximum of 23.7% sequence divergence at this locus. Two new groups (named Fus and Ten) were identified in addition to nineteen reported earlier (Zhou, W., Rousset, F. and O'Neill, S.L. (1998) Proc R Soc Lond B 265: 1-7; van Meer, M.M.M., Witteveldt, J. and Stouthamer, R. (1999) Insect Mol Biol 8: 399-408), because they displayed more than 2.5% sequence divergence from other Wolbachia wsp sequences. PCR products from seventeen of twenty-nine (59%) arthropod species analysed could not be sequenced directly due to apparent infection by multiple Wolbachia strains. The wsp sequences cloned from two such species (Plutella xylostella and Trichoplusia ni) indicated both A- and B-Wolbachia were present in a single individual. Hence, superinfection also may be more widespread than the 1.2% incidence previously estimated.

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Spiders, scorpions, mites and ticks (chelicerates) form one of the most diverse groups of arthropods on land, but their origin and times of diversification are not yet established. We estimated, for the first time, the molecular divergence times for these chelicerates using complete mitochondrial sequences from 25 taxa. All mitochondrial genes were evaluated individually or after concatenation. Sequences belonging to three missing genes (ND3, 6, and tRNA-Asp) from three taxa, as well as the faster-evolving ribosomal RNAs (12S and 16S), tRNAs, and the third base of each codon from 11 protein-coding genes (PCGs) (COI-III, CYTB, ATP8, 6, ND1-2, 4L, and 4-5), were identified and removed. The remaining concatenated sequences from 11 PCGs produced a completely resolved phylogenetic tree and confirmed that all chelicerates are monophyletic. Removing the third base from each codon was essential to resolve the phylogeny, which allowed deep divergence times to be calculated using three nodes calibrated with upper and lower priors. Our estimates indicate that the orders and classes of spiders, scorpions, mites, and ticks diversified in the late Paleozoic, much earlier than previously reported from fossil date estimates. The divergence time estimated for ticks suggests that their first land hosts could have been amphibians rather than reptiles. Using molecular data, we separated the spider-scorpion clades and estimated their divergence times at 397 +/- 23 million years ago. Algae, fungi, plants, and animals, including insects, were well established on land when these chelicerates diversified. Future analyses, involving mitochondrial sequences from additional chelicerate taxa and the inclusion of nuclear genes (or entire genomes) will provide a more complete picture of the evolution of the Chelicerata, the second most abundant group of animals on earth.

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Abstract. Caenorhabditis elegans unc-44 mutations result in aberrant axon guidance and fasciculation with inappropriate partners. The unc-44 gene was cloned by transposon tagging, and verified by genetic and molecular analyses of six transposon-induced alleles and their revertants. Nucleotide sequence analyses demonstrated that unc-44 encodes a series of putative ankyrin-related proteins, including AO49 ankyrin (1815 aa, 198.8 kD), AO66 ankyrin (1867 aa, 204 kD), and AOI3 ankyrin (x<4700 aa, ~<517 kD). In addition to the major set of •6 kb alternatively spliced transcripts, minor transcripts were observed at -,3, 5, 7, and 14 kb. Evidence is provided that mutations in the *14-kb AO13 ankyrin transcript are responsible for the neuronal defects. These molecular studies provide the first evidence that ankyrin-related molecules are required for axonal guidance.TrIOUGH the molecular basis of neural development has been the object of intense study in recent years, the detailed mechanisms of axon guidance remain unknown (for general reviews see Dodd and Jessell, 1988;Jessell, 1988;Takeichi, 1988;Sanes, 1989;Takeichi, 1991;Rathjen et al., 1992;Gumbiner, 1993; for C. elegans reviews see Hedgecock et al., 1987;Wadsworth and Hedgecock, 1992).Mutations in the unc-44 gene affect the direction of axonal outgrowth for many axons (Hedgecock et al., 1985;Siddiqui, 1990;Siddiqui and Culotti, 1991;Mclntire et al., 1992), including the postdeirid (PDE) ~ axon, which normally extends from the postdeirid sensillum on the lateral surface of the nematode to the ventral nerve cord (White et al., 1986). In unc-44 mutants, the initial direction of PDE axon outgrowth along the basement membrane is apparently random, and the misdirected PDE axon fasciculates with inappropriate partners (Hedgecock et al., 1985 The discovery that the C. elegans unc-6 gene encodes a laminin B chain-related product provided evidence that directed axonal outgrowth and cell migration require interactions with the extracellular matrix (Hedgecock et al., 1990), and that these interactions use laminin or related proteins in both invertebrates and vertebrates (Jessell, 1988;Sanes, 1989;Hedgecock et al., 1990;Serafini et al., 1994). The product of the unc-5 gene, which affects dorsalward cell migrations and axon outgrowth, has been proposed to be a cell surface protein which may interact with the extracellular matrix (Leung-Hagesteijn et al., 1992). Thus, it was likely that other mutations affecting axonal outgrowth and guidance were defects in cytoskeletal or extracellular matrix structures. The actin/a-actinin framework of growth cone filopodia or the spectrin/ankyrin network underlying the cytoplasmic surface of the plasma membrane could be the targets for mutations affecting axon outgrowth and growth cone adhesion. In this study, we have discovered that the wild-type unc-44 gene, which is required for proper axonal guidance, encodes a series of putative ankyrin-related proteins.Ankyrin (or bands 2.1 and 2.2) has been most thoroughly studied in erythrocyte "ghost...

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