World distribution of female flight and genetic variation in Lymantria dispar (Lepidoptera: Lymantriidae).

Female gypsy moths, Lymantria dispar L., from 46 geographic strains were evaluated for flight capability and related traits. Males from 31 of the same strains were evaluated for genetic diversity using two polymorphic cytochrome oxidase I mitochondrial DNA restriction sites, the nuclear FS1 marker, and four microsatellite loci. Females capable of strong directed flight were found in strains that originated from Asia, Siberia, and the northeastern parts of Europe, but flight capability was not fixed in most strains. No flight-capable females were found in strains from the United States or southern and western Europe. Wing size and musculature were shown to correlate with flight capability and potentially could be used in predicting female flight capability. The mtDNA haplotypes broadly separated the gypsy moth strains into three groups: North American, European/Siberian, and Asian. Specific microsatellite or FS1 alleles were only fixed in a few strains, and there was a gradual increase in the frequency of alleles dominant in Asia at both the nuclear and microsatellite loci moving geographically from west to east. When all the genetic marker information was used, 94% of the individuals were accurately assigned to their broad geographic group of origin (North American, European, Siberian, and Asian), but female flight capability could not be predicted accurately. This suggests that gene flow or barriers to it are important in determining the current distribution of flight-capable females and shows the need for added markers when trying to predict female flight capability in introduced populations, especially when a European origin is suspected.

Occurrence of Potato Cyst Nematode, Globodera rostochiensis, on Potato in the Saint-Amable Region, Quebec, Canada.

Potato (Solanum tuberosum L. subsp. tuberosum) is one of the most important food crops in Canada. Potato cyst nematodes, Globodera rostochiensis (Wollenweber, 1923) Skarbilovich, 1959 and Globodera pallida (Stone, 1973) Behrens, 1975, are considered the most economically important nematode pests of potatoes worldwide and are the subject of strict quarantine regulations in many countries including Canada. In Canada, G. rostochiensis was found in the Saanich Peninsula of Vancouver Island, British Columbia and both G. rostochiensis and G. pallida are present on the island of Newfoundland (3). During August of 2006, soil and roots of potato plants collected from a 19.2-ha field in the Saint-Amable region, Quebec, were submitted to the Nematology Laboratory, Canadian Food Inspection Agency, Ottawa. Golden, spherical-shaped cysts were found associated with the roots and were also extracted from the soil. The nematodes recovered were identified by morphological and morphometric analysis and DNA analysis. Measurements and morphological observation of 60 second-stage juveniles were as follows: body length = 427.4 ± 22.0 (395 to 495) µm; stylet length = 21.3 ± 1.2 (18 to 23) µm; distance from stylet base to dorsal esophageal gland opening = 5.5 ± 0.9 (3.5 to 7.0) µm; tail length = 46.7 ± 3.7 (36 to 58) µm; hyaline tail terminus = 21.1 ± 3.1 (13 to 27) µm; and the shape of stylet basal knobs varied from rounded to lateral flattened. Observations for 35 mature cysts were: number of cuticular ridges between anus and vulval fenestra = 21 ± 6 (12 to 31); fenestral length = 18.4 ± 3.6 (11 to 30) µm; distance from anus to the edge of fenestra = 81.1 ± 30.4 (29 to 165) µm; Granek's ratio = 4.4 ± 1.6 (1.8 to 6.0), with parallel wavy cuticular ridges between anus and vulval fenestra. These observations conformed to the published description of G. rostochiensis (2). Additionally, the identification was supported by the PCR-restriction fragment length polymorphism pattern of the ITS-1 region amplified using primers 18S (5'-TTGATTACGTCCCTGCCCTTT-3') and rDNA1.58S (5'-ACGAGCCGAGTGATCCACCG-3') and digested with BstU I and a PCR product specific for G. rostochiensis (1). Sequence of a 1,190-bp PCR fragment of ribosomal DNA amplified using primers rDNA1 (5'-TTGATTACGTCCCTGCCCTTT-3') and rDNA2 (5'- TTTCACTCGCCGTTACTAAGG-3') showed 99.0 to 100% identity with published DNA sequences of the same genomic region for G. rostochiensis (4). The sequence is available from the authors upon request. The origin of the introduction of G. rostochiensis into Saint-Amable is unknown. An intensive soil survey is underway to define the infested area, and strict quarantine measures have been taken to prevent further spread of G. rostochiensis. To our knowledge, this is the first evidence of the occurrence of G. rostochiensis in Quebec, Canada. References: (1) A. Fullaondo et al. Nematology 1:157, 1999. (2) A. M. Golden et al. Proc. Helminthol. Soc. Wash. 39:64, 1972. (3) A. R. Stone et al. Plant Dis. Rep. 61:590, 1977. (4) S. A. Subbotin et al. Nematology 2:591, 2000.

Four methodologies to improve healthcare demand forecasting.

Forecasting demand for health services is an important step in managerial decision making for all healthcare organizations. This task, which often is assumed by financial managers, first requires the compilation and examination of historical information. Although many quantitative forecasting methods exist, four common methods of forecasting are percent adjustment, 12-month moving average, trendline, and seasonalized forecast. These four methods are all based upon the organization's recent historical demand. Healthcare financial managers who want to project demand for healthcare services in their facility should understand the advantages and disadvantages of each method and then select the method that will best meet the organization's needs.

In vitro self-splicing reactions of chloroplast and mitochondrial group-I introns in Chlamydomonas eugametos and Chlamydomonas moewusii.

The self-splicing activity of nine chloroplast group-I introns (CeLSU.1 to CeLSU.6, CepsbC.1, CepsbC.2 and CmpsaB.1) and of one mitochondrial group-I intron (CmmtLSU.1) from the interfertile green algae Chlamydomonas eugametos and C. moewusii was examined using RNA templates produced by in vitro transcription of cloned DNA sequences. All introns, with the exception of the mobile intron CeLSU.5 encoding the site-specific I-CeuI endonuclease, were found to catalyze their own splicing in the absence of proteins. The introns that proved to be the best substrates under the conditions employed are CeLSU.1, CeLSU.3, CeLSU.4, CepsbC.1 and CmmtLSU.1. The implications of our results for the origin and spread of group-I introns in the organellar genomes of green algae are discussed.

PCR amplification of microdissected wheat chromosome arms in a simple 'single tube' reaction.

A novel method for the adaptor-mediated PCR amplification of microdissected chromosome arms is described. This simple and versatile protocol eliminates the need for enzymatic micromanipulation in nanoliter volumes and permits the efficient amplification of as little as two wheat chromosome arms in a 'single tube' reaction.

Group I introns interrupt the chloroplast psaB and psbC and the mitochondrial rrnL gene in Chlamydomonas.

The polymerase chain reaction was used to identify novel IAI subgroup introns in cpDNA-enriched preparations from the interfertile green algae Chlamydomonas eugametos and Chlamydomonas moewusii. These experiments along with sequence analysis disclosed the presence, in both green algae, of a single IA1 intron in the psaB gene and of two group I introns (IA2 and IA1) in the psbC gene. In addition, two group I introns (IA1 and IB4) were found in the peptidyltransferase region of the mitochondrial large subunit rRNA gene at the same positions as previously reported Chlamydomonas chloroplast introns. The 188 bp segment preceding the first mitochondrial intron revealed extensive sequence similarity to the distantly spaced rRNA-coding modules L7 and L8 in the Chlamydomonas reinhardtii mitochondrial DNA, indicating that these two modules have undergone rearrangements in Chlamydomonas. The IA1 introns in psaB and psbC were found to be related in sequence to the first intron in the C. moewusii chloroplast psbA gene. The similarity between the former introns extends to the immediate 5' flanking exon sequence, suggesting that group I intron transposition occurred from one of the two genes to the other through reverse splicing.

Evolution of the fusion protein gene of human parainfluenza virus 3.

The nucleotide sequences of the fusion (F) gene of 15 clinical strains of human parainfluenza virus 3 (HPIV3) isolated between 1959 and 1987 were compared with the F gene sequence of the prototype strain, Wash/47885/57. Nucleotide sequence diversity was greatest in the noncoding regions of the F gene; however, regions believed to function as transcriptional signals were completely conserved. Amino acid sequences were highly conserved and all but a few amino acid substitutions were conservative in nature. Sequence comparisons indicate heterogeneity in HPIV3 F genes; however, a significant proportion of nucleotide changes are maintained after they first appear and seem to be accumulating with time. Phylogenetic analysis suggests that there are 2 lineages of HPIV3 in North America. The two lineages can be distinguished by specific amino acid differences in the F protein, which correlate with differences in antigenic properties and neutralization patterns of HPIV3. The pattern of HPIV3 evolution, based on the analysis of F gene sequences, most closely resembles that of influenza virus B, vesicular stomatitis virus and Newcastle disease virus.

The fusion and hemagglutinin-neuraminidase glycoproteins of human parainfluenza virus 3 are both required for fusion.

Recombinant vaccinia viruses, VF and VHN, expressing the fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins of human parainfluenza virus 3 (HPIV3) were constructed. Infection of HeLa T4 cells with VF and VHN led to the synthesis of glycoproteins, with the correct apparent molecular weights, that were recognized by monoclonal antibodies specific for HPIV3F and HN. The HN glycoprotein was present on the surface of cells infected with VHN and these cells demonstrated both hemadsorbing and neuraminidase activities. The F glycoprotein was present in cleaved and uncleaved forms and was also expressed on the surface of VF-infected cells. Fusion activity, however, as evidenced by syncytium formation and lysis of human erythrocytes, could only be demonstrated when HeLa T4 cells were coinfected with VF and VHN. Fusion events that are mediated by HPIV3, therefore, require both the F and HN glycoproteins.

Rapid synthesis and cloning of complementary DNA from any RNA molecule into plasmid and phage M13 vectors.

We describe several modifications of the Gubler and Hoffman procedure [Gene 25 (1983) 263-269] for complementary DNA (cDNA) synthesis that expand the versatility of this method for the rapid synthesis and cloning of double-stranded (ds) cDNA. These modifications include: (1) The combination of first and second strand synthesis into a single two-step reaction, which reduces the time for synthesis of blunt-ended ds-cDNA to less than 4 h. (2) The use of random hexadeoxyribonucleotide primers (RP) for the synthesis of ds-cDNA, which allows the synthesis of cDNA from any RNA template. (3) The combined use of random primers and DNA ligase treatment of cDNA/RNA hybrids prior to second-strand synthesis, which promotes the production of nearly full length ds-cDNA molecules. (4) The use of gel filtration to size-fractionate ds-cDNA, which allows the selection of specific size classes of ds-cDNA for cloning. (5) The use of blunt-end ligation to insert the ds-cDNA into the vector, which reduces the total time required for the construction of cDNA libraries to less than 24 h.

Nucleotide sequence of the coding and flanking regions of the human parainfluenza virus type 3 fusion glycoprotein gene.

The complete nucleotide sequence of the human parainfluenza virus type 3 (HPIV3) fusion (F) protein gene has been determined. The HPIV3 F gene is 1851 nucleotides long including six U residues in the genomic RNA, which probably direct synthesis of the first few nucleotides in the F mRNA polyadenylate tail. The HPIV3 F gene contains a single long open reading frame coding for 539 amino acids. The predicted molecular weight of the unglycosylated precursor F0 protein was 60031. Four potential carbohydrate acceptor sites were identified. Comparison of the HPIV3 F protein sequence with the F gene sequences of two other paramyxoviruses, Sendai virus and simian virus 5, indicated a very close evolutionary relationship between HPIV3 and Sendai virus. Sequence analysis of HPIV3 F gene flanking regions identified signals which appear to be responsible for polymerase recognition and polyadenylation.

Nucleotide sequence of the coding and flanking regions of the human parainfluenza virus 3 hemagglutinin-neuraminidase gene: comparison with other paramyxoviruses.

The nucleotide sequence of the human parainfluenza virus 3 (HPIV3) hemagglutinin-neuraminidase (HN) gene has been determined using cDNA clones derived from both HPIV3 genomic RNA and mRNA. The HN mRNA contains 1,882 nucleotides, not including the poly(A) tail. Primer extension experiments were carried out to locate the 5' terminal nucleotide of the HN mRNA. The 3' end of the mRNA was located at a putative polyadenylation signal. The HPIV3 HN mRNA has one large open reading frame that codes for 572 amino acids with a deduced molecular weight of 64,178. Potential polymerase recognition signals for the HN and L genes were located in the flanking regions. The HN protein of HPIV3 shares some common features with the previously sequenced HN proteins of Sendai virus and Simian virus 5. The features include: an N-terminal membrane anchor, two regions of highly conserved amino acid sequence and strong conservation in the positions of the cysteine residues. The relationship is closest between Sendai virus and HPIV3.