Novel primers for the amplification of nuclear DNA introns in the entomopathogenic nematode Heterorhabditis bacteriophora and their cross-amplification in seven other Heterorhabditis species.

We describe 24 novel primers that amplify intron regions in housekeeping and structural genes of Heterorhabditis bacteriophora. The cross-amplification potential of these primers in seven other Heterorhabditis species was determined. The results obtained showed interspecific nucleotide, length and splice site variability in the sequenced introns and for one gene, an intron gain was observed. These primers will be useful tools for studying population genetics, genetic diversity and intron DNA evolution within the genus Heterorhabditis and other genera of rhabditid nematodes.

Dehydration-induced tps gene transcripts from an anhydrobiotic nematode contain novel spliced leaders and encode atypical GT-20 family proteins.

Accumulation of the non-reducing disaccharide trehalose is associated with desiccation tolerance during anhydrobiosis in a number of invertebrates, but there is little information on trehalose biosynthetic genes in these organisms. We have identified two trehalose-6-phosphate synthase (tps) genes in the anhydrobiotic nematode Aphelenchus avenae and determined full length cDNA sequences for both; for comparison, full length tps cDNAs from the model nematode, Caenorhabditis elegans, have also been obtained. The A. avenae genes encode very similar proteins containing the catalytic domain characteristic of the GT-20 family of glycosyltransferases and are most similar to tps-2 of C. elegans; no evidence was found for a gene in A. avenae corresponding to Ce-tps-1. Analysis of A. avenae tps cDNAs revealed several features of interest, including alternative trans-splicing of spliced leader sequences in Aav-tps-1, and four different, novel SL1-related trans-spliced leaders, which were different to the canonical SL1 sequence found in all other nematodes studied. The latter observation suggests that A. avenae does not comply with the strict evolutionary conservation of SL1 sequences observed in other species. Unusual features were also noted in predicted nematode TPS proteins, which distinguish them from homologues in other higher eukaryotes (plants and insects) and in micro-organisms. Phylogenetic analysis confirmed their membership of the GT-20 glycosyltransferase family, but indicated an accelerated rate of molecular evolution. Furthermore, nematode TPS proteins possess N- and C-terminal domains, which are unrelated to those of other eukaryotes: nematode C-terminal domains, for example, do not contain trehalose-6-phosphate phosphatase-like sequences, as seen in plant and insect homologues. During onset of anhydrobiosis, both tps genes in A. avenae are upregulated, but exposure to cold or increased osmolarity also results in gene induction, although to a lesser extent. Trehalose seems likely therefore to play a role in a number of stress responses in nematodes.

An investigation of chemotaxis in the insect parasitic nematode Heterorhabditis bacteriophora.

We tested the chemotactic responses of dauer juvenile stages (DJs) of the insect parasitic nematode Heterorhabditis bacteriophora to a variety of compounds that are known to be highly attractive or highly repellent to Caenorhabditis elegans. While H. bacteriophora DJs respond to alcohols and some aromatic compounds as well as to host metabolites such as uric acid and CO2, the most notable difference in the responses of these two nematodes is that H. bacteriophora DJs are unresponsive to a large number of compounds which C. elegans finds highly attractive. The latter compounds are typical by-products of bacterial metabolism and include aldehydes, esters, ketones and short-chain alcohols. While C. elegans finds long-chain alcohols (e.g. 1-heptanol and 1-octanol) repellent and short-chain alcohols highly attractive, H. bacteriophora DJs are strongly attracted to 1-heptanol, 1-octanol and 1-nonanol and find short-chain alcohols to be only slightly attractive. Parasitic-stage H. bacteriophora nematodes show a very weak chemotactic response to volatile molecules that DJs find highly attractive. Our results suggest that, associated with the adoption of a parasitic mode of life by Heterorhabditis, there was an adaptive change in chemotactic behaviour of the infective stages, resulting in a decreased sensitivity to volatile by-products of bacterial metabolism and an increased sensitivity towards long-chain alcohols and other insect-specific volatiles and possibly also to herbivore-induced plant volatiles.

Detection of changes occurring during recovery from the dauer stage in Heterorhabditis bacteriophora.

Nematodes of the genus Heterorhabditis are insect parasites that are widely used as biological control agents. When conditions are unfavourable for reproduction in H. bacteriophora, a long-lived, non-feeding, survival and dispersal stage, the dauer juvenile (DJ), is formed. This DJ stage is also adapted for host finding and infection. When it infects a suitable host, the DJ recovers and resumes growth and development. We describe a series of methods for improved detection of recovery in H. bacteriophora. We also describe some of the physiological changes that occur immediately after the onset of recovery in these nematodes as revealed using fluorescent nucleic acid binding SYTO dyes. Although recovery could be monitored using morphological changes, we found that observation of the uptake of fluorescent latex microspheres by recovering nematodes was a far more sensitive and efficient means of detecting recovery. SYTO dyes were also found to be useful indicators of recovery, binding to the pharyngeal glands and genital primordia as little as 3 h after the onset of recovery. The use of SYTO dyes also indicated that the pharyngeal glands produce large quantities of RNA following the onset of recovery, implying that these structures may produce proteins important in the infection and/or feeding process of H. bacteriophora.

Development of a diagnostic DNA probe for the fruit flies Ceratitis capitata and Ceratitis rosa (Diptera: Tephritidae) using amplified fragment-length polymorphism.

The AFLP technique (amplified fragment-length polymorphism) was employed to identify and isolate species specific markers in tephritids. We have found that the technique has good potential for this purpose, with the only difficult part being the reamplification of AFLP fragments from silver stained gels. Cloning of putative species-specific markers and genomic dot blot hybridizations resulted in the development of diagnostic probes for tephritid identification. A repetitive DNA sequence from the genome of Ceratitis capitata (Wiedemann) was isolated. This sequence rapidly and reliably identified C. capitata and C. rosa Karsch in a collection of closely related and outgroup species tested in this study. Although this probe has been developed for C. capitata and C. rosa, the proposed methodology can be applied to any group of organisms.

Novel application of PhastSystem polyacrylamide gel electrophoresis using restriction fragment length polymorphism--internal transcribed spacer patterns of individuals for molecular identification of entomopathogenic nematodes.

différences! [editorial] [editorial]onomic way of identifying and assigning nematodes to taxons, which had already been determined either by comparative sequence analysis of nuclear rDNA internal transcribed spacer (ITS) region or by other methods of molecular or conventional taxonomy, is provided. Molecular identification of entomopathogenic nematodes (EPN) can be upgraded by basing it on PhastSystem polyacrylamide gel electrophoresis (PAGE) analysis of restriction fragment length polymorphism (RFLP) patterns of polymerase chain reaction (PCR)-amplified DNA derived from single nematodes of Steinernema or Heterorhabditis spp. Although analysis from single worms has previously been made on agarose gel, the resolution on PhastSystem PAGE gel is much higher. The DNA sequences selected for analysis were those constituting the internal transcribed spacer region between the 18S and 26S rDNA genes within the rRNA operon. RFLP analysis was carried out by gel electrophoresis on the PhastSystem (Pharmacia) as detailed elsewhere (Triga et al., Electrophoresis 1999, 20, 1272-1277. The downscaling from conventional agarose to PhastSystem gels resulted in pattern of DNA fragments differing from those obtained with agarose gel electrophoresis under conventional conditions by increasing the number of detected fragments. The approach supported previous species identifications and was able to identify several unclassified isolates, such as those from Hungary and Ireland, and provides a method for identification of previously unclassified strains. We confirmed that Heterorhabditis "Irish Type", represented by two strains of different geographical origin, comprise a species different from H. megidis. We also confirmed that strain IS5 belongs to the species H. indicus rather than to H. bacteriophora, as had been suggested previously.

Biophysical properties of the surface of desiccation-tolerant mutants and parental strain of the entomopathogenic nematode Heterorhabditis megidis (strain UK211).

Entomopathogenic nematodes (EPN) are useful biological control agents of insect pests. However, the infective juvenile (IJ) stage which is the only stage to occur outside the host is susceptible to environmental extremes such as desiccation. We have isolated desiccation-tolerant strains of the EPN Heterorhabditis megidis. In this paper we describe the surface properties of these desiccation-tolerant mutants. Heterorhabditid IJs retain the sheath of the previous larval stage. The mutant lines possess alterations in the surface properties of the sheath. Differences were observed in fluorescent lipid analogue insertion into the surface of the sheath. Furthermore, cationized ferritin-binding studies demonstrated that the mutant lines possessed an increase in net negative surface charge. Removal of the surface layer of the sheath resulted in the loss of the mutant phenotype and in a reduction in the desiccation tolerance of the parental strain. Therefore, the negatively charged 'surface coat' appears to play an important role in the desiccation tolerance of Heterorhabditis species.

The effect of day of emergence from the insect cadaver on the behavior and environmental tolerances of infective juveniles of the entomopathogenic nematode Heterorhabditis megidis (strain UK211).

Infective juveniles (IJs) of entomopathogenic nematodes (EPNs) are obligate parasites of insect larvae. Inside the host they develop into sexually mature adult stages and complete their life cycle. Two or 3 adult nematode generations can occur in the insect host. The increase in nematode population density in the insect cadaver, together with limiting nutrient conditions, result in the formation of IJs. These IJs emerge into the soil to search for a new host. It typically takes 7-8 days for all IJs to emerge from a parasitized insect. We have investigated the effect of the day of emergence of IJs from insect cadavers on the environmental tolerance and behavior of the EPN Heterorhabditis megidis strain UK211. The IJs that emerge early display good initial host-finding ability and increased temperature tolerance but disperse poorly and have poor tolerance to desiccation. Conversely, the IJs that emerge later display poor initial host-finding ability and poor temperature tolerance but they disperse well and possess increased desiccation tolerance. These phenotypic differences are likely to facilitate early-emerging IJs in locating and infecting hosts in the vicinity of the cadaver, whereas IJs that emerge late are adapted to disperse away from their natal cadaver. We hypothesize that adaptive phenotypic plasticity rather than allelic variability may provide the genetic basis for the different physiological and behavioral phenotypes of the early- and late-emerging IJs.

A phylogenetic analysis of heterorhabditis (nemata: rhabditidae) based on internal transcribed spacer 1 DNA sequence data.

Internal transcribed spacer 1 sequences were used to infer phylogenetic relationships among 8 of the 9 described species and one putative species of the entomopathogenic nematode genus Heterorhabditis. Sequences were aligned and optimized based on pairwise genetic distance and parsimony criteria and subjected to a variety of sequence alignment parameters. Phylogenetic trees were constructed with maximum parsimony, cladistic, distance, and maximum likelihood algorithms. Our results gave strong support for four pairs of sister species, while relationships between these pairs also were resolved but less well supported. The ITS1 region of the nuclear ribosomal repeat was a reliable source of homologous characters for resolving relationships between closely related taxa but provided more tenuous resolution among more divergent lineages. A high degree of sequence identity and lack of autapomorphic characters suggest that sister species pairs within three distinct lineages may be mutually conspecific. Application of these molecular data and current morphological knowledge to the delimitation of species is hindered by an incomplete understanding of their variability in natural populations.

Laparoscopic vs open appendectomy. Prospective randomized study of outcomes.

OBJECTIVE: To compare open appendectomy (OA) with laparoscopic appendectomy (LA) for length of the operation, complications, postoperative pain control, length of hospitalization, postdischarge recovery time, and hospital charges. DESIGN: Prospective randomized clinical trial of patients with acute appendicitis. SETTING: Tertiary care, urban teaching hospital. PATIENTS: A population-based sample of patients (aged > or = 12 years; weight, > 49.7 kg) admitted to a surgical teaching service with a clinical diagnosis of acute appendicitis. Patients were prospectively randomized to either OA or LA during a 20-month period (from April 1, 1994, to December 31, 1995). Fifty-seven patients were initially enrolled in the study; 7 did not complete the study because of a protocol violation. All remaining patients completed the study, including postdischarge follow-up. INTERVENTIONS: Two (7.4%) of the 27 patients in the LA group required conversion to OA because of technical difficulties. One patient (in the OA group) underwent a second surgical procedure for drainage of a pelvic abscess. Three patients (in the LA group) required second surgical procedures. For analysis, no crossovers were allowed and all patients remained in their originally randomized group. MAIN OUTCOME MEASURES: Length of the operation, intraoperative and postoperative complications, postoperative pain control, length of hospitalization, postdischarge recovery time, and hospital charges. RESULTS: Fifty patients (19 women and 31 men) were examined. Twenty-seven patients underwent LA, 2 requiring conversion to an OA. Twenty-three patients underwent an OA. Patient demographics were similar between groups. Statistical differences between the 2 groups were found for (1) length of the operation (median, 81.7 vs 66.8 minutes, LA vs OA groups: P < .002), (2) operating room charges (median, $3191 vs $1514, LA vs OA group; P < .001), and (3) total hospital charges (median, $5430 vs $3673, LA vs OA group; P < .001). No statistical differences between the 2 groups were found for (1) length of hospitalization (median, 1.1 vs 1.2 days, LA vs OA group), (2) pain control (mean, 4 vs 3.7 of 10 [0 indicates least pain; 10, most pain], LA vs OA group), (3) recovery time (time necessary before returning to work or school) (median, 14.0 days for both groups), and (4) complications (5 vs 1, LA vs OA group). CONCLUSIONS: Laparoscopic appendectomies and OAs are comparable for complications, postoperative pain control, length of hospitalization, and recovery time. Patients who underwent an OA had a shorter operative time and lower operating room and hospital charges. Laparoscopic appendectomy does not offer any proved benefits compared with the open approach for the routine patient with acute appendicitis.

The rDNA Internal Transcribed Spacer Region as a Taxonomic Marker for Nematodes.

The ITS region from a wide taxonomic range of nematodes, including secernentean and adenophorean taxa, and free-living, entomopathogenic, and plant-parasitic species, was evaluated as a taxonomic marker. Size of the amplified product aided in the initial determination of group membership, and also suggested groups that may require taxonomic reevaluation. Congeneric species often displayed identically sized ITS regions, but genera such as Pratylenchus and Tylenchorhynchus had species with large differences in size. ITS heterogeneity in individuals and populations was identified in several nematode taxa. PCR-RFLP of ITS1 is advocated as a method of taxonomic analysis in genera such as Helicotylenchus that contain numerous species with few diagnostic morphological characteristics.

Characterization of Heterorhabditis Isolates by PCR Amplification of Segments of mtDNA and rDNA Genes.

Restriction digests of amplified DNA from the mitochondrial genome and the nuclear ribosomal internally transcribed spacer region have been evaluated as genetic markers for species groups in Heterorhabditis. Six RFLP profiles have been identified. These profiles supported groupings determined by cross-breeding studies and were in agreement with less definitive groupings based on other biochemical and molecular methods. Digestion patterns of both amplification products provided strong evidence for the recognition of species groups, which include Irish, NW European, tropical, and a H. bacteriophora complex. The H. bacteriophora complex could be further resolved into three genotypes represented by H. zealandica, the H. bacteriophora, Brecon (Australian) type isolate for H. bacteriophora, and a grouping composed of isolates NC1, V16, HI82, and HP88. All cultures obtained of the H. megidis isolate were identical to the NW European group. These results could be used to aid monitoring of field release of Heterorhabditis as well as allowing a rapid initial assessment of taxonomic grouping.

The effect of acclimation temperature on enzyme activity in Drosophila melanogaster.

1. The response to thermal acclimation of five key rate-limiting enzymes of intermediary metabolism and of six degradative enzymes was measured in tissue extracts of adult Drosophila melanogaster which had been acclimated for 4 days to 15, 25 or 30 degrees C. 2. Three enzymes of intermediary metabolism (HK, alpha-GPDH and CO) showed positive thermal compensation, which is the type of response characteristic of the enzymes involved in energy metabolism in vertebrate ectotherms. 3. The data obtained for CS and G6PDH showed no evidence for increased activity of TCA cycle nor of the pentose phosphate pathway upon cold acclimation in D. melanogaster. 4. Two degradative enzymes, ADH and non-specific esterase, showed inverse thermal compensation which is the type of response characteristic of degradative enzymes in vertebrate ectotherms. 5. In contrast to the situation in vertebrate ectotherms, catalase and the three lysosomal enzymes assayed (APH, acid DNase and acid RNase) displayed positive rather than inverse compensation. 6. The results presented here extend the data on the range of D. melanogaster enzymes which show compensation upon thermal acclimation and on the type of acclimation response which occurs.

Enzyme induction in recovering dauer larvae of the nematode Caenorhabditis elegans in response to increasing concentrations of food source in the recovery medium.

Exposure of recovering dauer larvae of Caenorhabditis elegans to increasing concentrations of Escherichia coli in the recovery medium produced dramatic increases in the enzymes of intermediary metabolism. There was no significant difference between the rates of development of recovering dauer larvae grown on different concentrations of E. coli. When the activity of several key enzymes was assayed after 12, 22 and 32 hours of recovery in 0.5% w/v E. coli it was found that the activities recorded never reached levels observed at 12 hours for larvae grown on the optimum concentration of E. coli. These results imply that enzymes of intermediary metabolism in the nematode C. elegans are capable of being induced in response to changes in nutrient intake, as previously described for mammals and microorganisms.

An investigation of the in vitro inhibition of acetylcholinesterase by the carbamate inhibitor eserine sulphate in eserine resistant strains of Drosophila melanogaster.

1. The kinetics of inhibition of AChE by the carbamate inhibitor eserine sulphate were investigated in five resistant strains of Drosophila melanogaster. 2. The dissociation constant (Kd) was higher and the carbamylation constant was lower relative to the control in four of these. 3. No change was observed in the decarbamylation constants (k3) of the five strains. 4. The Vmax of AChE was higher in the five resistant stocks than in the Canton-S/TM2 controls but no change in the Km of AChE for acetylthiocholine was observed. No electrophoretic variants of AChE were detected. 5. No increase in the activity of nonspecific esterases was detected in the resistant lines. 6. These results indicate that resistance to eserine sulphate may occur in D. melanogaster by a reduction in affinity of AChE for the inhibitor.

The Western Domiciliary Care Service: a report on the development and activities of a home care scheme associated with a teaching hospital.

This article reports the development and first five years experience of the Western Domiciliary Care Service, Adelaide, South Australia. This service began accepting referrals in July 1971. Referrals are accepted without regard to age or degree of disability. Assessments are made in the patients' homes by social workers and therapists, who maintain close contact with the general practitioners. Liaison with The Queen Elizabeth Hospital has been a tremendous advantage. Of the 1,800 active patients, about 700 would be in nursing homes if this service did not exist, and many referrals to The Queen Elizabeth Hospital have been avoided. The scheme has proved very economical.