Recrystallization in a Freezing Tolerant Antarctic Nematode,Panagrolaimus davidi,and an Alpine Weta,Hemideina maori(Orthoptera; Stenopelmatidae)

The ability of haemolymph from the freezing tolerant weta,Hemideina maori,and supernatant from homogenates of the freezing tolerant nematodePanagrolaimus davidito inhibit the recrystallization of ice was examined using the “splat freezing” technique and annealing on a cryomicroscope stage. There was no recrystallization inhibition in weta haemolymph or in insect ringer controls. Recrystallization inhibition was present in the nematode supernatant but this was destroyed by heating and was absent in controls.P. davidisurvives intracellular freezing and recrystallization inhibition may be important for the survival of this stress.     

The ability of the Antarctic nematode Panagrolaimus davidi to survive intracellular freezing is dependent upon nutritional status

The Antarctic nematode Panagrolaimus davidi is the best documented example of an animal surviving intracellular freezing and the only animal so far shown to survive such freezing throughout its tissues. However, a recent study found that after exposure to a freezing stress that produced intracellular freezing in a proportion of nematodes, the resulting survival levels could be explained if those nematodes that froze intracellularly had died. We have thus re-examined the survival of intracellular freezing in this nematode. The ability to survive a freezing exposure that is likely to produce intracellular freezing (freezing at ?10 °C) declines with culture age. In cultures that are fed regularly, the ability to survive freezing at ?10 °C increases, but in starved cultures freezing survival declines. Survival of intracellular freezing in fed cultures was confirmed using cryomicroscopy, staining of cells with vital dyes and by freeze substitution and transmission electron microscopy. We have thus confirmed that P. davidi can survive intracellular freezing and shown that this ability is dependent upon them being well fed. The effect of culture conditions on the nutrient status of the nematodes should thus be an important factor in the design of experiments.     

Anhydrobiosis and Freezing-Tolerance: Adaptations That Facilitate the Establishment of Panagrolaimus Nematodes in Polar Habitats

Anhydrobiotic animals can survive the loss of both free and bound water from their cells. While in this state they are also resistant to freezing. This physiology adapts anhydrobiotes to harsh environments and it aids their dispersal. Panagrolaimus davidi, a bacterial feeding anhydrobiotic nematode isolated from Ross Island Antarctica, can survive intracellular ice formation when fully hydrated. A capacity to survive freezing while fully hydrated has also been observed in some other Antarctic nematodes. We experimentally determined the anhydrobiotic and freezing-tolerance phenotypes of 24 Panagrolaimus strains from tropical, temperate, continental and polar habitats and we analysed their phylogenetic relationships. We found that several other Panagrolaimus isolates can also survive freezing when fully hydrated and that tissue extracts from these freezing-tolerant nematodes can inhibit the growth of ice crystals. We show that P. davidi belongs to a clade of anhydrobiotic and freezing-tolerant panagrolaimids containing strains from temperate and continental regions and that P. superbus, an early colonizer at Surtsey island, Iceland after its volcanic formation, is closely related to a species from Pennsylvania, USA. Ancestral state reconstructions show that anhydrobiosis evolved deep in the phylogeny of Panagrolaimus. The early-diverging Panagrolaimus lineages are strongly anhydrobiotic but weakly freezing-tolerant, suggesting that freezing tolerance is most likely a derived trait. The common ancestors of the davidi and the superbus clades were anhydrobiotic and also possessed robust freezing tolerance, along with a capacity to inhibit the growth and recrystallization of ice crystals. Unlike other endemic Antarctic nematodes, the life history traits of P. davidi do not show evidence of an evolved response to polar conditions. Thus we suggest that the colonization of Antarctica by P. davidi and of Surtsey by P. superbus may be examples of recent “ecological fitting” of freezing-tolerant anhydrobiotic propagules to the respective abiotic conditions in Ross Island and Surtsey.     

Cold acclimation and cryoprotectants in a freeze-tolerant Antarctic nematode, Panagrolaimus davidi

Panagrolaimus davidi is a freeze-tolerant Antarctic nematode which survives extensive intracellular freezing. This paper describes the development of culture techniques which provide clean samples, with a high degree of freeze tolerance and in sufficient quantities for the analysis of potential cryoprotectants. Cultures grown at 20 °C survived a short-term freezing stress but survival declined with the time spent frozen. Acclimation of cultures at 5 °C enhanced the long-term survival of freezing. Starvation, however, reduced the nematode's ability to survive short-term freezing. The principal cryoprotectants detected by gas chromatography were trehalose and glycerol. The levels of trehalose, but not those of glycerol, increased significantly after acclimation. Trehalose may stabilise membranes and protect them against the dehydrating effects of the osmotic stresses resulting from freeze concentration effects but other factors, such as recrystallisation inhibition, may be involved in long-term survival. Accepted: 7 March 2000     

Molecular evolution in Panagrolaimus nematodes: origins of parthenogenesis, hermaphroditism and the Antarctic species P. davidi

Background  

As exemplified by the famously successful model organism Caenorhabditis elegans, nematodes offer outstanding animal systems for investigating diverse biological phenomena due to their small genome sizes, short generation times and ease of laboratory maintenance. Nematodes in the genus Panagrolaimus have served in comparative development and anhydrobiosis studies, and the Antarctic species P. davidi offers a powerful paradigm for understanding the biological mechanisms of extreme cold tolerance. Panagrolaimus nematodes are also unique in that examples of gonochoristic, hermaphroditic and parthenogenetic reproductive modes have been reported for members of this genus. The evolutionary origins of these varying reproductive modes and the Antarctic species P. davidi, however, remain enigmatic.     

The environmental physiology of Antarctic terrestrial nematodes: a review

The environmental physiology of terrestrial Antarctic nematodes is reviewed with an emphasis on their cold-tolerance strategies. These nematodes are living in one of the most extreme environments on Earth and face a variety of stresses, including low temperatures and desiccation. Their diversity is low and declines with latitude. They show resistance adaptation, surviving freezing and desiccation in a dormant state but reproducing when conditions are favourable. At high freezing rates in the surrounding medium the Antarctic nematode Panagrolaimus davidi freezes by inoculative freezing but can survive intracellular freezing. At slow freezing rates this nematode does not freeze but undergoes cryoprotective dehydration. Cold tolerance may be aided by rapid freezing, the production of trehalose and by an ice-active protein that inhibits recrystallisation. P. davidi relies on slow rates of water loss from its habitat, and can survive in a state of anhydrobiosis, perhaps aided by the ability to synthesise trehalose. Teratocephalus tilbrooki and Ditylenchus parcevivens are fast-dehydration strategists. Little is known of the osmoregulatory mechanisms of Antarctic nematodes. Freezing rates are likely to vary with water content in Antarctic soils. Saturated soils may produce slow freezing rates and favour cryoprotective dehydration. As the soil dries freezing rates may become faster, favouring freezing tolerance. When the soil dries completely the nematodes survive anhydrobiotically. Terrestrial Antarctic nematodes thus have a variety of strategies that ensure their survival in a harsh and variable environment. We need to more fully understand the conditions to which they are exposed in Antarctic soils and to apply more natural rates of freezing and desiccation to our studies.Communicated by: I.D. Hume     

Low cryoprotectant concentrations and fast cooling for nematode cryostorage

Cryopreservation protocols based on slow freezing or vitrification often result in cell injury due to ice formation, cell dehydration and/or toxic concentrations of cryoprotectant (CPA).In this study, we present a cryopreservation technique based on low, non-toxic concentrations of cryoprotectants (≈2–4 M) combined with a rapid cooling rate in the liquid nitrogen phase (−196 °C). Protocols for successfully cryopreserving the plant parasitic nematodes Globodera tabacum tabacum, Heterodera schachtii and Meloidogyne incognita were developed, as demonstrated by the high survival rates and reproducibility of cyst and root-knot nematode species post-cryostorage. This approach for effective cryopreservation of viable plant-parasitic nematodes was developed by inducing an “apparent vitrification” by rapid cooling of the microscopic samples in less than 2 M of cryoprotectant. The extremely thin structure (15–20 μm width, 350–400 μm length) of these nematodes, in combination with a direct and rapid exposure to LN₂, likely prevents the formation of damaging ice crystals. Moreover, this procedure results in viability of both short- and long-cryostorage samples. These techniques could potentially be used for the near-indefinite preservation of thousands of different nematode species. A cryo-nematode collection produced in our lab is available and presented here.     

Osmotic stress effects on the freezing tolerance of the antarctic nematodePanagrolaimus davidi

 The freezing and freezing survival of the Antarctic nematode Panagrolaimus davidi after exposure to solutions of different osmotic concentrations has been examined using a thermoelectric cooling stage and multi-specimen cooling block to see if there is any evidence that freeze-induced desiccation prevents inoculative freezing. The nematodes froze in all the test solutions used (up to 1138 mosmol ⋅ l^-1) and at all cooling rates and nucleation temperatures tested. Freezing survival was at its maximum in 0.1 mol ⋅ l^-1 NaCl in artificial tap water after 1 h exposure to the test solution and in artificial tap water after 24 h exposure. Hyperosmotic and hyposmotic stress adversely affected the nematodes’ ability to survive freezing. In non-frozen controls survival declined with increasing osmolality of the test solution. Measurements of the osmolality of water extracted from a variety of moss samples indicate that the nematodes are exposed to an osmotic concentration of about 9 mosmol ⋅ l^-1 in their natural habitat. This is close to that of artificial tap water. Our experiments, and measurements of freeze concentration effects in the literature, indicate that freeze-induced desiccation is unlikely to prevent inoculative freezing and the survival of nematodes over the winter. Accepted: 5 May 1996     

Relation between nematode communities and trophic state in southern Swedish lakes

The aim of this study was to examine whether littoral nematode community patterns are shaped by lake trophic state. It was hypothesized that trophic level is associated negatively with the proportion of omnivores and positively with the percentages of bacterial feeders, but not at all with the diversity, abundance, and biomass of freshwater nematodes. Sediment samples were taken at littoral sites of eight southern Swedish lakes of different trophy in spring and autumn 2007. Trophic level was found to strongly influence species richness, as oligotrophic and mesotrophic lakes supported the greatest species numbers, whereas nematode abundance, biomass, and Shannon index were unaffected. Furthermore, our results indicated effects on the nematode community’s trophic structure, with a larger proportion of predatory nematodes in oligotrophic and mesotrophic lakes but no differences in the other feeding types (bacteria, algae and suction feeders, omnivorous species). Multivariate analysis indicated a shift in species compositions along the threshold from mesotrophic to eutrophic conditions, with the presence of Tobrilus gracilis, Monhystera paludicola, Brevitobrilus stefanskii, and Ethmolaimus pratensis related to the latter. Nematode communities in oligotrophic and mesotrophic lakes were characterized by a similar species composition, with pronounced occurrences of Eumonhystera longicaudatula, Semitobrilus cf. pellucidus, Prodesmodora circulata, and Rhabdolaimus terrestris. Overall, the results suggested that lake trophic state is a major factor structuring littoral nematode communities, although intra-lake variations might be of importance as well.     

Molecular profiling of nematode associates with Rhynchophorus ferrugineus in southern Italy

A survey of nematodes associated with the red palm weevil Rhynchophorus ferrugineus was conducted in southern Italy in 2015 and 2016 in order to create a species inventory and obtain data about nematode biodiversity. A total of 70 insect samples (pupae and adults) were collected from infested Phoenix canariensis, Phoenix dactylifera, and Chamaerops humilis palms in three Italian Regions: sampling took place at 11 locations in Apulia, 1 in Basilicata, and 1 in Sardinia regions. Individual insects were dissected to determine nematode presence, and different nematode species were also recovered from red palm weevil cocoons collected at the sites in Apulia. Individual nematodes were molecularly identified by sequencing the ITS, D2‐D3 expansion domains of the 28SrRNA gene and the mitochondrial COI and inferring the phylogenetic relationships. The insect‐associated nematofauna identified belonged to the families Rhabditidae, Cephalobidae, and Diplogastridae. Just two nematode species, Teratorhabditis synpapillata and Mononchoides macrospiculum, were always found in association with adult insects and cocoons taken from all sampling sites. This paper reports on the biodiversity of the nematodes associated with R. ferrugineus and on current knowledge of the specific habitat of specialized and divergent entomophilic nematodes.     

Accumulation and metabolism of aldicarb by the free-living nematodes Aphelenchus avenae and Panagrellus redivivus

The accumulation and metabolism of aldicarb has been compared in two species of free-living soil nematodes, Aphelenchus avenae and Panagrellus redivivus, which differ considerably in their sensitivity to this and other pesticides. Similar levels of aldicarb were found to accumulate in the two species. The results showed that although the rate of uptake of aldicarb was greater in P. redivivus, so also was the rate of metabolism and elimination. Levels of toxic metabolites of aldicarb were two to three times higher in the more susceptible species A. avenae after 24 h incubation than in the less susceptible P. redivivus. The toxicological significance of these findings is discussed in relation to previous work with the organophosphorus pesticide phorate and its effects on the same two nematode species.     

Biological control of slugs in winter wheat using the rhabditid nematode Phasmarhabditis hermaphrodita

A field experiment on winter wheat in autumn 1991 investigated the effect of the rhabditid nematode, Phasmarhabditis hermaphrodita, applied to soil at five dose rates (10⁸ - 10¹⁰ infective larvae ha^-1) immediately after seed sowing, on slug populations and damage to seeds and seedlings. The nematode was compared with methiocarb pellets broadcast at recommended field rate immediately after drilling and no molluscicide treatment. Slug damage to wheat seeds and seedlings was assessed 6 and 13 wk after drilling. Seedling survival increased and slug grazing damage to seedlings declined linearly with increasing log nematode dose. These two measures of slug damage were combined to give an index of undamaged plant equivalents, which also increased linearly with increasing log nematode dose. ANOVA showed that, after 6 wk, there were significantly more undamaged plant equivalents on plots treated with the two highest nematode doses (3 × 10⁹ and 1 × 10¹⁰ ha^-1) than on untreated plots, but the number of undamaged plant equivalents on methiocarb-treated plots was not significantly greater than that on untreated plots. Slug populations were assessed by refuge trapping and soil sampling. Deroceras reticulatum was the commonest of several species of slugs recorded. During the first 4 wk after sowing, significantly more slugs were found under refuge traps on plots treated with certain doses of P. hermaphrodita than under traps on untreated plots and more showed signs of nematode infection than expected from the prevalence of infection in slugs from soil samples, suggesting that the presence of P. hermaphrodita altered slug behaviour. Application of P. hermaphrodita had no significant impact on numbers or biomass of slugs in soil during a 27 wk period after treatment, except after 5 wk when slug numbers were inversely related to log nematode dose. However, by this time, numbers in soil samples from untreated plots had declined to levels similar to those in plots treated with the highest dose of nematodes. During the first 5 wk after treatment, c. 20% of slugs in soil samples from untreated plots showed symptoms of nematode infection. It is suggested that this represented the background level of infection in the experimental field rather than spread of infection from treated plots. The apparent lack of impact of P. hermaphrodita on slug numbers and biomass in soil suggests that its efficacy in protecting wheat from slug damage was through inhibition of feeding by infected slugs.     

Distribution and prevalence of the entomopathogenic nematode Steinernema feltiae in Scotland

The distribution of insect parasitic nematodes in Scotland was investigated between April 1988 and April 1989 using the wax moth, Galleria mellonella, larvae as bait. Of the 1014 samples examined only 2.2% were found to be positive and all the nematodes recovered and identified belonged to the one species Steinernema feltiae. A more detailed investigation into the distribution of S. feltiae in a 1 ha block indicated that it had a very aggregated distribution.     

Size-specific susceptibility of the pest slugs Deroceras reticulatum and Arion lusitanicus to the nematode biocontrol agent Phasmarhabditis hermaphrodita

The nematode Phasmarhabditis hermaphrodita is a commercially available biocontrol agent against slugs. This product is especially interesting for use in organic farming, where products containing metaldehyde or carbamates cannot be used for controlling pest slugs. We investigated the potential of P. hermaphrodita for the control of the pest slugs Deroceras reticulatum and Arion lusitanicus. These two species are the most harmful slug pests in Switzerland. At different times of the year, we collected slug specimens of different weight and assessed their susceptibility to P. hermaphrodita in the laboratory. Batches of five slugs were subjected to five different doses of nematodes plus an untreated control and replicated three times. During six weeks, feeding and survival of the slugs were recorded. D. reticulatum was strongly affected by increasing nematode doses, irrespective of the slugs' body weight. In small specimens of A. lusitanicus, feeding and survival were strongly affected by the nematodes, while larger specimens remained almost unaffected. Because A. lusitanicus has an asynchronous development in Switzerland, it seems difficult to control the entire population with a single nematode application. To what extent nematodes will be used in practice for slug control depends on their effectivity against the pest slugs of major importance, on the longevity of the molluscicidal effect and on the price of nematodes.     

Population dynamics of bacteria associated with different strains of the pine wood nematode Bursaphelenchus xylophilus after inoculation in maritime pine (Pinus pinaster)

For a long time it was thought that Bursaphelenchus xylophilus was the only agent of the pine wilt disease. Recently, it was discovered that there are bacteria associated with the nematodes that contribute to the pathogenesis of this disease, mainly through the release of toxins that promote the death of the pines. Among the species most commonly found, are bacteria belonging to the Bacillus, Pantoea, Pseudomonas and Xanthomonas genera.The main objective of this work was to study the effect of inoculation of maritime pine (Pinus pinaster) with four different nematode isolates, in the bacterial population of nematodes and trees, at different stages of disease progression. The monitoring of progression of disease symptoms was also recorded. Also, the identification of bacteria isolated from the xylem of trees and the surface of nematodes was performed by classical identification methods, by the API20E identification system and by sequencing of bacterial DNA.The results showed that for the symptoms progression, the most striking difference was observed for the pines inoculated with the avirulent isolate, C14-5, which led to a slower and less severe aggravation of symptoms than in pines inoculated with the virulent isolates. In general, it was found that bacterial population, inside the tree, increased with disease progression. A superior bacterial quantity was isolated from pines inoculated with the nematode isolates HF and 20, and, comparatively, few bacteria were isolated from pines inoculated with the avirulent isolate. The identification system API20E was insufficient in the identification of bacterial species; Enterobacter cloacae species was identified in 79% of the isolated bacterial colonies and seven of these colonies could not be identified by this method. Molecular identification methods, through bacterial DNA sequencing, allowed a more reliable identification: eleven different bacterial species within the Bacillus, Citrobacter, Enterobacter, Escherichia, Klebsiella, Paenibacillus, Pantoea and Terribacillus genera were identified. General bacterial diversity increased with the progression of the disease. Bacillus spp. were predominant at the earlier stage of disease progression and Klebsiella oxytoca at the later stages. Furthermore, bacterial species isolated from the surface of nematodes were similar to those isolated from the xylem of pines.In the present work new bacterial species were identified which have never been reported before in this type of study and may be associated with their geographical origin (Portugal). P. pinaster, the pine species used in this study, was different from those commonly grown in Japan and China. Furthermore, it was the first time that bacteria were isolated and identified from an avirulent pine wood nematode isolate.     

Food preferences and nematode parasitism in mycophagousDrosophila

Food preferences and nematode parasitism were studied in natural populations of mycophagousDrosophila in and near Sapporo, northern Japan. Species which preferred fresh mushrooms showed species-specific responses toPleurotus mushrooms:D. pirka bred only onPleurotus cornucopiae, D. trivitata onP. cornucopiae andP. ostreatus, D. trilineata on these twoPleurotus mushrooms and some other mushrooms, whileD. sexvittata bred on a wide variety of mushrooms but seldom onPleurotus mushrooms. Species which preferred decayed mushrooms (D. quadrivittata, D. histrioides, D. testacea and species of thequinaria species-group) showed host preferences different from those of the above species. The rate of parasitism by nematodes was generally higher in species which prefer decayed mushrooms than in species which prefer fresh mushrooms. Among species which prefer fresh mushrooms, onlyD. trilineata was parasitized frequently by nematodes. It was not clear what factors determine the rate of parasitism in these mycophagousDrosophila. D. pirka, D. trivittata andD. trilineata passed through three or four generations per year and entered reproductive diapause in early September in and near Sapporo. Bionomics of Drosophilidae (Diptera) in Hokkaido, IX.     

根际效应对大豆田土壤线虫群落组成及多样性的影响

根际作为重要的环境界面是植物与环境之间物质能量交换的场所,关于根际效应的研究已成为土壤生态学的新兴热点领域,然而有关大豆根际效应对土壤动物多样性影响的研究报道并不多见。在三江平原选择连续耕作15a的大豆田,对大豆根际区与非根际区土壤线虫群落结构组成进行了对比分析。结果表明:大豆根际区土壤线虫总数、辛普森多样性指数(Dom)显著高于非根际区,根际区的物种数(S)、物种丰富度指数(SR)显著低于非根际区。说明大豆根际效应增加土壤线虫的丰度,但降低了线虫群落结构的复杂性。大豆根际区植物寄生线虫(PP)、食真菌线虫(FF)和食细菌线虫(BF)数量显著高于非根际区,而PP类群的比例在根际区却显著低于非根际区。这一研究结果表明食微线虫(FF和BF)类群在大豆根际区的比例增加更显著。食真菌与食细菌线虫数量比值(F/B)指示大豆根际区细菌生物量相对高于真菌生物量。研究结果丰富了农田土壤线虫多样性的研究内容,并为我国东北大豆田线虫病害的防治及定制相应的农业管理措施提供参考。     

Effect of nematode Panagrellus redivivus density on growth, survival, feed consumption and carcass composition of bighead carp Aristichthys nobilis (Richardson) larvae

The study aimed to determine the optimum density of free‐living nematodes in feeding bighead carp, Aristichthys nobilis, larvae. In the first experiment, carp stocked at 25 larvae L^?1 were fed varying levels of nematodes (50, 75, 100, 125 and 150 per ml) twice a day for 21 days from the start of exogenous feeding. Final body weight was significantly higher (P < 0.05) in larvae fed 125 and 150 nematodes per ml than in those fed 50 and 75 per ml, but survival was low (61.8 and 63.6%, respectively). Survival rate was highest in larvae fed 100 nematodes ml^?1 (81.3%). Carcass analysis showed that larvae fed 125 and 150 nematodes ml^?1 had significantly lower body protein and higher body lipid than those fed other nematode densities. Carcass ash was similar for larvae fed 50–100 nematodes ml^?1 but it decreased significantly at the higher nematode densities. Carp larvae in a subsequent experiment were given 50, 75 and 100 nematodes ml^?1 per feeding. Newly hatched Artemia was the control feed. Nematode consumption and growth of the larvae were determined. Larvae were sampled at intervals of 2–4 days and the nematodes in the gut were counted and measured. At each nematode density, the number of nematodes present in the gut of the larvae increased significantly with time. At each sampling day, the number of nematodes in the gut did not differ significantly among treatments (P > 0.05) although it tended to increase with nematode density at day 2 and day 4 but decrease at day 7 onward. The carp larvae consumed significantly shorter nematodes on day 2 and day 4 than on the succeeding sampling days regardless of nematode density. However, the length of nematodes in the gut of the larvae did not differ significantly among the nematode densities. The final body weight of larvae increased with increasing nematode density. The body weight of larvae fed 100 nematodes ml^?1 did not differ significantly from that of larvae given Artemia nauplii. Results show that bighead carp larvae should be fed 100 free‐living nematodes per ml at each feeding time.     

Field persistence of the entomopathogenic nematode <Emphasis Type="Italic">Heterorhabditis bacteriophora</Emphasis> in different crops

To investigate nematode establishment and persistence, dauer juveniles (DJs) of Heterorhabditis bacteriophora were applied at 50 cm^-2 in different crops in June and July with conventional spraying equipment and 420 l water ha^-1. Application hardly had any effects on survival and infectivity. The number of DJs reaching the soil was assessed and the establishment and persistence recorded by baiting soil samples with larvae of the wax moth Galleria mellonella. The better the plant canopy was developed the fewer DJs reached the soil during application. Whereas in pasture 77% and in potatoes 78% of the applied nematodes reached the soil, in wheat and peas little less than 50%, in oil-seed rape only 5% and in lupine 6% were recorded. Between 50 and 60% of the soil samples contained H. bacteriophora a month after application with the exception of wheat (>90%) and potatoes (<5%) indicating that the number of nematodes reaching the soil during application had no influence on their establishment in the soil. Probably DJs can survive in the plant canopy and reach the soil later after application. The percentage of nematode-positive soil samples dropped considerably after tillage. In potatoes no nematodes were recovered after two months, which probably was also due to the intensive movement of the soil. Although nematodes are susceptible to freezing, temperatures below 0°C during the winter did not extinguish the H. bacteriophora population. In field crops EPN usually persisted not much longer than one year. The longest persistence of H. bacteriophora was detected 23 months after release in beans followed in rotation by wheat with red clover as cover crop. In this field larvae of the pea weevil Sitona lineatus (Coleoptera: Curculionidae) were detected in soil samples and found infected with the released nematode population. In the laboratory the field soils were tested for persistence of H. bacteriophora at 8°C and a half-life of 24.8 days was recorded in the absence of host insects and plants. Thus long-term persistence in the field was a result of recycling in host insects, which could not be detected in other crops than beans and clover. As H. bacteriophora seems to be restricted in its host potential, this species disappears after release once the host population is not available anymore.     

Long‐term stability of entomopathogenic nematode spatial patterns in soil as measured by sentinel insects and real‐time PCR assays

Quantitative real‐time PCR (qPCR) techniques are being increasingly used to provide accurate and reliable methods to identify and quantify cryptic organisms in soil ecology. Entomopathogenic nematode (EPN) diversity in Florida is known to be extensive and our phylogenetic studies of the D2D3 and ITS regions showed the occurrence of an additional species‐complex in the Steinernema glaseri‐ group in widely separated locations of the peninsula. To address ecological studies, we developed and used qPCR assays to detect and quantify six species of EPN that are naturally distributed in Florida citrus orchards (Steinernema diaprepesi, Steinernema riobrave, Heterorhabditis indica, Heterorhabditis zealandica, Heterorhabditis floridensis and an undescribed species in the S. glaseri group) and an exotic species, S. glaseri. Species‐specific primers and TaqMan® probes were designed from the ITS rDNA region. No nonspecific amplification was observed in conventional or qPCR when the primers and probes were tested using several populations of each of the Florida species and other exotic EPN species. Standard curves were established using DNA from pure cultures. We optimised a protocol for extracting nematodes and DNA from soil samples that can detect one EPN added to nematode communities recovered by conventional extraction protocols. A survey of an 8‐ha orchard in April 2009 compared the EPN spatial patterns derived from qPCR to that obtained by baiting soil samples with Galleria mellonella larvae. The patterns were also compared to those derived from the same site in 2000–01 by repeatedly (12 sampling events) baiting soil in situ with caged larvae of the root weevil Diaprepes abbreviatus. The qPCR assay was more efficient than the Galleria baiting method for detecting the EPN species composition in population mixtures. Moreover, the spatial patterns of EPN in this orchard were remarkably stable over the course of nearly a decade. The pattern of H. zealandica detected at the site 8 years earlier was related to those derived by qPCR (P = 0.002) and from sample baiting (P = 0.02). The spatial pattern of H. indica derived from qPCR, but not that from sample baiting, was also related to the earlier pattern (P = 0.01). The qPCR assay developed here is a fast, affordable and accurate method to detect and quantify these EPN species in soil and offers great potential for studying the ecology of EPN.