Establishment, persistence, and introgression of entomopathogenic nematodes in a forest ecosystem.

Entomopathogenic nematodes (EPN) are currently marketed worldwide for use in inundative biological control, where the applied natural enemy population (rather than its offspring) is expected to reduce insect numbers. Unlike classical biological control, in inundative control natural enemy establishment is not crucial in order to achieve pest suppression. Field trials in Irish forestry provided the opportunity to test predictions regarding the establishment of two exotic (Steinernema carpocapsae and Heterorhabditis megidis) and two indigenous (Steinernema feltiae and Heterorhabditis downesi) species. Nematodes were inundatively applied to pine stumps to control populations of pine weevil, Hylobius abietis, on three clearcut sites, and their persistence and spread monitored for up to five years. All species were recovered three years after application but only S. feltiae was recovered in years 4 and 5. Limited horizontal dispersal to 20 cm (but not 100 cm) was observed, but the majority of nematodes were recovered close to the area of application. Steinernema feltiae was also recovered from nearby stumps to which it had not been applied, indicating possible phoretic dispersal by weevils or other stump-associated fauna. EPN were not recovered from stumps outside the treated area, suggesting that such dispersal is quite localized. Two strains of S. feltiae (Irish and exotic) were applied. Amplified fragment length polymorphism (AFLP) analysis on 11 populations isolated from soil four years later showed that all had a much closer affinity to the applied Irish strain, suggesting persistence of this genotype and extinction of the exotic one. Some strains were clustered close together, and this is interpreted in the light of possible population genetic scenarios. The findings from the field study confirm predictions based on background knowledge of the species and demonstrate the importance of medium-term studies, as a 3-year study would have overestimated the risk of establishment of exotic species. Short-term persistence and spread of S. carpocapsae, S. feltiae, and H. downesi was also studied in pine forest mesocosms. Similar trends to field results, such as limited horizontal dispersal, even vertical distribution, and more abundant recovery of S. feltiae than of other species, point to the utility of mesocosm studies as a predictive tool.

Emergence and Dispersal Patterns of Two Isolates of the Entomopathogenic Nematode Steinernema feltiae.

Few studies have addressed the role of the sexes in the emergence and dispersal of entomopathogenic nematodes from host insects. Individuals of two isolates of Steinernema feltiae, UK76 and SBIl, emerging from Galleria mellonella cadavers were classed as Non-Dispersed (remaining on the cadaver for up to nine days) and Dispersed (actively moving away from the cadaver). Sex ratios within both classes were examined in infective (individuals that successfully invaded bait G. mellonella) and entire (infective and noninfective individuals that matured in hanging drops of G. mellonella haemolymph) populations. Sex ratios differed significantly from 1:1 only in the SBIl Non-Dispersed entire population (female bias) and SBIl Non-Dispersed infective population (male bias). For each isolate, Dispersed individuals were significantly more infective than Non-Dispersed. However, only 11% of SBIl and 22% of UK76 Non-Dispersed individuals were found to be mature infective juveniles (IJ) compared with 78% of SBIl and 82% of UK76 Dispersed individuals (based on survival in SDS). Infective juveniles dispersing towards distant radial bait G. mellonella tended to migrate from the head region of the natal cadaver. For each isolate, a higher proportion of males than females arrived early at distant baits. SBIl males survived alone in G. mellonella cadavers for longer periods than did females, which supports the "male colonization" hypothesis.

Persistence of four heterorhabditis spp. isolates in soil: role of lipid reserves.

Infective juveniles of four Heterorhabditis isolates (H. bacteriophora HI, H. megidis UK211 and HF85, and H. downesi M245) were stored in moist (pF 1.7) and dry (pF 3.3) loam soil at 20 degrees C for up to 141 days. Survival, assessed by the number of nematodes extracted by centrifugal flotation, declined over time, reaching fewer than 18% alive by day 141 for all but one treatment (H. bacteriophora HI in dry soil). The infectivity of nematodes in soil for Tenebrio molitor also declined over time, roughly in accordance with the decline in numbers of nematodes. Energy reserves of extracted nematodes were assessed by image analysis densitometry. There were differences among isolates both in survival and in the depletion of reserves, and there was a significant correlation between these two parameters, suggesting that the extent to which energy reserves are depleted affects survival or that a common factor influences both. However, significant nematode mortality occurred while levels of reserves remained high, and the maximum reduction in utilizable body content for any treatment was 51%, well above starvation level. Therefore, the decline in numbers of living nematodes and the reduced nematode infectivity in soil cannot directly result from starvation of the nematodes. Survival and infectivity declined more rapidly in moist than in dry soil; one isolate, H. downesi M245, was less affected by soil moisture content than the other three isolates.

Occurrence and distribution of the entomopathogenic nematodes Steinernema spp. and Heterorhabditis indica in Indonesia.

Soil samples from 79 sites on five islands of Indonesia were baited with insects for the recovery of entomopathogenic nematodes. Heterorhabditis and Steinernema were equally prevalent, and were recovered from 11.7% of samples representing 20.3% of sites sampled. Both genera were recovered from coastal sites only. Entomopathogenic nematodes were more prevalent on the Moluccan islands of Ambon and Seram than on Java or Bali. They were not detected on Sulawesi, where non-coastal sites only were sampled. RFLP analysis was used in the identification of nematode isolates. Heterorhabditis indica was the only heterorhabditid identified. Two RFLP types of Steinernema were identified.

Persistence of heterorhabditis infective juveniles in soil: comparison of extraction and infectivity measurements.

The persistence of Heterorhabditis megidis in soil was studied over a 4-week period. On days 0, 2, 14, and 28, infective juveniles (IJ) were extracted by centrifugal flotation, Baermann funnel, and baiting of soil with Tenebrio molitor larvae, which were then dissected. Extraction efficiencies on day 0 were 82% by centrifugal flotation, 56% by Baermann funnel, and 19.8% by bait insect. The relative efficiency of the three methods changed over time. The relationship between the density of nematodes in the soil and the proportion recovered by dissection was non-linear. Up to a dose of approximately 60 IJ/insect, less than 12% became established, while at higher doses (up to 200 IJ/insect) the invasion efficiency was 23%. Mortality of bait insects increased from day 0 to day 2, but decreased to day 28. A novel method of assessing soil pathogenicity by preparing a soil density series and calculating the dose of soil or IJ that kills 50% of the bait insects gave a similar pattern. This method is recommended as a means of tracking changes in pathogenicity over time when bait insect mortality in undiluted soil is at or near 100%. Two methods of preparing a series of Heterorhabditis IJ densities in soil, either by diluting the soil itself with IJ-free soil or by adding diluted suspensions of IJ to the soil, resulted in the same bait insect mortalities.

Nonradioactive nucleic acid detection by enhanced chemiluminescence using probes directly labeled with horseradish peroxidase.

The use of nucleic acid probes directly labeled with horseradish peroxidase for detection of single copy sequences on Southern blots of human genomic DNA by enhanced chemiluminescence is described. Of the target sequences, 6 x 10(5) molecules (1 amol) have been detected on blue sensitive film using exposures of up to 60 min and probes of 0.3-5.1 kb. The chemiluminescent signal quantified using a cooled charge coupled device (CCD) camera is proportional to probe length for DNA probes in the range 50-3571 bases. The enzyme has no significant effect on the stability of a DNA/DNA hybrid formed with a 3571-base probe and target as determined by increasing the stringency of posthybridization washes by decreasing the concentration of a monovalent cation (NaCl) and by a Tm analysis. The kinetics of DNA hybridization have been analyzed by a cooled CCD camera to provide quantitative data. Ten nanograms per milliliter of probe may be used for an overnight hybridization. Southern blots can be reprobed using a DNA probe for the same or a different sequence without the necessity of stripping off the previously bound probe.

Alzheimer's paired helical filaments share epitopes with neurofilament side arms.

A panel of monoclonal antibodies to neurofilaments have been investigated with regard to the location of their respective epitopes on neurofilament polypeptides and their ability to label the neurofibrillary tangles and paired helical filaments (PHF) which are characteristic of Alzheimer's disease. All of the neurofilament monoclonal antibodies that label tangles and PHF are directed against epitopes in the side arm domains of the two larger neurofilament polypeptides, NF-H and NF-M, and do not recognise the alpha-helical rod domains of these proteins. Immuno-electron microscopy demonstrates that the neurofilament antibodies label the constituent PHF per se and do not simply stain neurofilaments that might be admixed with PHF. These neurofilament epitopes are differentially retained by PHF, following isolation. Thus, antibody labelling of PHF is not simply due to the presence of normal neurofilament polypeptides. We propose that in tangle-bearing neurons, neurofilaments are degraded by proteases and that it is fragments of the side arms which contribute to the composition of PHF.

Determination of nitrite at low level without prior extraction.

A method has been devised for the determination of nitrite at low level that is directly applicable to food or other dried matrices without prior extraction. Nitric oxide released from nitrite through the action of acetic acid is determined using a chemiluminescence analyser. The limit of detection is approximately 0.02 microgram, the coefficients of variation being 5.7 and 8.2% using 0.1 and 0.05 microgram of sodium nitrite, respectively. The chemiluminescence analyser response is diminished when water in excess of 0.5 ml is present in the assay system unless hydrogen bromide in acetic acid is used instead of acetic acid alone. The application of the method to the direct determination of nitrite in freeze-dried cod fish has indicated a content of 0.25 mg NaNO2 per kg, equivalent to 0.050 mg per kg of the original undried material.