Effects of Paenibacillus nematophilus on the entomopathogenic nematode Heterorhabditis megidis

The insect parasitic nematodes Heterorhabditis spp. are mutualistically associated with entomopathogenic bacteria, Photorhabdus spp. A novel association has been detected between H. megidis isolate EU17 and the endospore-forming bacterium Paenibacillus nematophilus. P. nematophilus sporangia adhere to infective juveniles (IJs) of H. megidis and develop in insect hosts along with the nematodes and their symbiont. We tested the effects of P. nematophilus on H. megidis. The yield and quality (size, energy reserves, and storage survival) of IJs were not affected by co-culture in insects with P. nematophilus. Dispersal of IJs in sand and on agar was inhibited by adhering P. nematophilus sporangia: fewer than 2% of IJs with P. nematophilus sporangia reached the bottom of a sand column, compared to 30% of the control treatment. Sporangia significantly reduced infectivity of H. megidis for wax moth larvae in sand, but not in a close contact (filter paper) assay. The results suggest that P. nematophilus may reduce the transmission potential of H. megidis through impeding the motility of IJs.     

Genetic relationships among annual species of Cicer(Fabaceae) using isozyme variation

In order to determine the pattern of genetic diversity within and among the species of Cicer and to estimate interspecific genetic relationships, allelic variation was assayed for 23 isozyme loci in 63 accessions of 11 species of Cicer using starch gel electrophoresis. The total allozymic variation observed in the genus (H _t )was equal to 0.60. When partitioned (G st), 96% of this allelic diversity was found among rather than within species. The allelic diversity among species (D st)and allelic diversity within species (H s)were equal to 0.58 and 0.02, respectively. Cicer reticulatum and C. pinnatifidum had the highest proportion of polymorphic loci (17.39%) and the highest mean number of alleles per locus (1.22 and 1.17, respectively). UPGMA cluster analysis of Nei's unbiased genetic distance revealed four genetic groups. One includes C. reticulatum, C. arietinum and C. echino spermum where the first 2 species represent a putative derivative-progenitor pair. A second cluster contains C. bijugum, C. pinnatifidum and C. judaicum. Cicer yamashitae, C. chorassanicum, C. anatolicum and C. songoricum form a third group. Finally, C. cuneatum, which has a very distinct isozyme profile and peculiar morphological features, is the only member of a fourth species group. This species grouping agrees partially with those obtained from crossability and cytogenetic studies. The results suggest that the annual habit arose from perennial progenitors at least twice in the genus Cicer.     

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.     

Isolation and characterization of microsatellite loci in the entomopathogenic nematode Heterorhabditis bacteriophora

Twenty microsatellite loci were identified from genomic DNA enrichment and expressed sequence tags of entomopathogenic nematode Heterorhabditis bacteriophora. Eight loci were found to be polymorphic in a Northeast Ohio H. bacteriophora population. Levels of polymorphism were evaluated in 31-56 individuals and the number of alleles ranged from two to three. The values of observed and expected heterozygosities ranged from 0 to 0.536 and from 0.223 to 0.616, respectively. All eight loci showed heterozygote deficiencies, but three conformed to Hardy-Weinberg equilibrium at the subpopulation level. This is the first set of microsatellite markers in entomopathogenic nematodes.     

Spatial patterns of the red palm weevil and applied entomopathogenic nematode Heterorhabditis bacteriophora

The distribution and abundance of red palm weevil (Rhynchophorus ferrugineus) within palm tree-infected stems at Ismaelia governorate, Egypt, were investigated. Taylor's power law was fit (P ≤ 0.01) to its larvae, pupae and/or adults. The slope value of this power law indicated the clumped distribution of these insect stages. Sample size optimisation needed to achieve a predetermined level of sampling error for the insect stages was calculated. Also, soil samples were taken after uniform application of entomopathogenic nematode Heterorhabditis bacteriophora strain EG₁ to the soil under citrus tree canopies at the rate of 10⁴ infective juveniles/400 cm² plots, and assayed for the nematode using the Galleria bait method. At the first and second sampling dates, the nematode displayed contagious distribution and attained mean insect mortality of 15.3 and 4, respectively. At the third date of sampling, the nematode showed random distribution according to chi-squared test and caused 1.8 mean insect mortality. Evaluation of H. bacteriophora EG₁ as a biocontrol measure for the weevil was discussed based on their investigated dispersion indices.     

Interactions between isolates of the entomopathogenic fungus Metarhizium anisopliae and the entomopathogenic nematode Heterorhabditis bacteriophora JPM4 during infection of the sugar cane borer Diatraea saccharalis (Lepidoptera: Pyralidae)

Interactions between the nematode Heterorhabditis bacteriophora isolate JPM4 and the fungus Metarhizium anisopliae, isolates LPP45 and LPP39, were studied during dual infections of Diatraea saccharalis. Mortality, production of infective juveniles (IJs) and production of conidia were evaluated. A positive effect was demonstrated for host mortality in duel infections of JPM4 and LPP39, causing 100% mortality with LT₅₀ and LT₉₅ values of 1.8 and 2.8 days, respectively. Higher values were seen when using the nematode or fungi individually. However, a combination of JPM4 + LPP39 caused a significant reduction in IJ production. The results show that faster time to death, a moderately virulent fungal isolate could be combined with the nematode, however at the expense of IJ production.     

Behavioural and physiological responses of infective juveniles of the entomopathogenic nematode Heterorhabditis to desiccation

Infective juveniles (IJs) of entomopathogenic nematodes (EPNs) are susceptible to a wide variety of environmental factors, including desiccation, which limit their usefulness as biocontrol agents. Although EPNs can be subjected to a gradual loss of water in their natural environment they are not full anhydrobiotes, being able to survive only moderate levels of desiccation at high relative humidities (rh). We investigated the desiccation tolerance of IJs of several Heterorhabditisspecies and strains when exposed to fast and slow desiccation regimes. We also investigated the behavioural and biochemical responses of Heterorhabditis IJs when exposed to 98% rh for 4 days. IJs of H. megidis UK211 (but not IJs of H. indica) aggregate into large clumps when desiccated at high rh, but unlike Steinernema spp., neither H. megidis nor H. indica IJs showed any tendency to coil. Preincubation of H. megidis UK211 IJs at high (98%) rh enhances their ability to survive for 150 min at 57% rh. We show that preincubation of H. megidis and H. indica at 98% rh induces the synthesis of glycerol but not of trehalose, whereas identical preincubation conditions do induce trehalose synthesis in Steinernema carpocapsae and Aphelenchus avenae. The biosynthesis of glycerol rather than trehalose by IJs of two species of Heterorhabditis in response to moderate levels of desiccation indicates that Heterorhabditis is unlikely to have the necessary metabolic responses to desiccation required to enable it to enter into a fully anhydrobiotic state.     

Genome size variation and species relationships in the genus Hydrangea

Genome size and base composition in 16 species and subspecies of the Hydrangea, a woody ornamental genus of Hydrangeaceae, were evaluated by flow cytometry in relation to their chromosome number. This is the first such study concerning the genome size of these species together with a karyotype study of the most important species, Hydrangea macrophylla subsp. macrophylla (Hortensia), from an economical point of view. The 2C DNA content ranged from 1.95 pg in Hydrangea quercifolia to 5.00 pg in Hydrangea involucrata. The base composition ranged from 39.9% GC in Hydrangea aspera subsp. sargentiana to 41.1% in Hydrangea scandens subsp. scandens (significant difference at p < 0.05). The smallest genome sizes were those of the three species originating from North or South America. Most of the species studied presented a chromosome number of 2n = 2x = 36, except for those of the section Aspereae which showed 2n = 30, 34 and 36. A primary karyotype has been made for the first time for H. macrophylla subsp. macrophylla. Phylogenetic relationships between species, the origin of chromosome number and an exploration of the genetic diversity within the genus are discussed. Received: 24 July 2000 / Accepted: 31 October 2000     

A novel ascaroside controls the parasitic life cycle of the entomopathogenic nematode Heterorhabditis bacteriophora

Entomopathogenic nematodes survive in the soil as stress-resistant infective juveniles that seek out and infect insect hosts. Upon sensing internal host cues, the infective juveniles regurgitate bacterial pathogens from their gut that ultimately kill the host. Inside the host, the nematode develops into a reproductive adult and multiplies until unknown cues trigger the accumulation of infective juveniles. Here, we show that the entomopathogenic nematode Heterorhabditis bacteriophora uses a small-molecule pheromone to control infective juvenile development. The pheromone is structurally related to the dauer pheromone ascarosides that the free-living nematode Caenorhabditis elegans uses to control its development. However, none of the C. elegans ascarosides are effective in H. bacteriophora, suggesting that there is a high degree of species specificity. Our report is the first to show that ascarosides are important regulators of development in a parasitic nematode species. An understanding of chemical signaling in parasitic nematodes may enable the development of chemical tools to control these species.     

Interaction between halofenozide and the entomopathogenic nematode Heterorhabditis marelatus for control of Japanese beetle (Coleoptera: Scarabaeidae) larvae

Japanese beetle, Popillia japonica Newman is a major pest of turf and ornamentals. Laboratory bioassays were conducted to evaluate the potential interactions between a biological control agent, Heterorhabditis marelatus (Nematoda: Heterorhabditidae) IN strain and the insecticide halofenozide against both overwintered and nonoverwintered 3rd instars of Japanese beetle. Treatments consisted of all combinations of 2 rates of halofenozid with H. marelatus nematodes Imidacloprid was used as a standard. Percentage larval mortality was evaluated at 7, 14, and 21 d after treatment. No deleterious effects were observed. The nematode treatments generally produced significantly greater larval mortality relative to both chemical treatments. Twenty-one days after treatment, both rates of nematodes resulted in 100% mortality, whereas insecticide treatments did not surpass 60% mortality. No synergism was detected in any of the combination treatments. There were no significant differences in nematode reproduction in larvae exposed to halofenozide and nematodes versus larvae exposed to only nematodes.     

Genetic variation in the genus Leiopelma and relationships to other primitive frogs

Allozyme variation was studied the three living species of Leiopelma. L. hamiltoni and L. archeyi are shown to be closely related to each other although L. hamiltoni is slightly more divergent relative to L. hochstetteri. This parallels previous cytoenetic data. The rarity and insularity of L. hamiltoni enables the calculation of a mutation rate based on genetic variance and population size. A mutation rate per generation of 2.7times 10^--⁶ is sufficient to account for the observed levels of variation. Six populations of L. hochstetteri show a pattern of genetic divergence that also closely parallels previously detected cytogenetic variation. L. hochstetteri is genetically distant from its congeneric species while all species of Leiopelma are at an extreme genetic distance from Ascaphus truei, the only other living amphicoelous fro At the limits of resolution of the allozme technique, Ascaphus clusters with the more morphologically advanced frogs, Discogiossus and Bombina, rather than with Leiopelma. Taken with other evidence, this supports recognition of two families, Leiopelmatidae anl Ascaphidae, with Leiopelma the probable sister group of all other frogs.     

Phoresy of the entomopathogenic nematode Heterorhabditis marelatus by a non-host organism, the isopod Porcellio scaber

Entomopathogenic nematodes are widespread in nature and commonly used in the biological control of insect pests. However, we understand little about how these organisms disperse. We show in a laboratory setting that the entomopathogenic nematode Heterorhabditis marelatus is phoretically dispersed by a non-host organism, the isopod Porcellio scaber. These species both inhabit tunnels excavated in the roots and lower stems of bush lupine (Lupinus arboreus) by the nematodes' primary prey, larvae of the ghost moth Hepialus californicus. Phoretic dispersal via P. scaber may play a role in the metapopulation dynamics of this nematode.     

Colorado potato beetle control by application of the entomopathogenic nematode Heterorhabditis marelata and potato plant alkaloid manipulation

Control of the Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), with the entomopathogenic nematode Heterorhabditis marelata Liu and Berry (Nematoda: Heterorhabditidae) was examined in the laboratory and in potato fields in north central Oregon. This research tested the hypothesis that varying nitrogen fertilizer levels would affect foliar alkaloid levels, which would stress the host, and allow increased nematode reproduction and long‐term control of the CPB. Laboratory results indicated that nematodes tended to reproduce more readily in CPB fed on potato plants with high levels of fertilizer. Field trials tested CPB population responses to four treatments: application of nematodes vs. no nematodes, with application of low vs. high rates of nitrogen fertilizer. The higher nitrogen application rate increased field foliar levels of the alkaloids solanine by 35%, and chaconine by 41% over the season. Nematodes were applied twice during the season, causing a 50% reduction in adult CPB populations, and producing six times as many dead prepupae in nematode‐treated soil samples as in the untreated samples. However, no reproducing nematodes were found in the 303 dead prepupae and pupae collected from nematode‐treated plots. Nitrogen fertilizer levels, and their related alkaloid levels, did not affect nematode infection rates or reproduction in the field. Foliar alkaloid levels of plants from the growth chamber were 3–6‐fold as high as those in the field, which may explain the variation in nematode response to nitrogen applications to host plants of the CPB. Heterorhabditis marelata is effective for controlling CPB in the field, and does not have negative non‐target effects on one of the most common endemic CPB control agents, Myiopharus doryphorae (Riley) (Diptera: Tachinidae), but the low rate of nematode reproduction cannot be manipulated through alkaloid stress to the beetle. Until H. marelata can be mass‐produced in an inexpensive manner, it will not be a commercially viable control for CPB.     

Nuclear DNA content variation and species relationships in the genus Lupinus (Fabaceae)

The 2C nuclear DNA content has been estimated by flow cytometry in 18 species and botanical forms of the genus Lupinus (family Fabaceae), using propidium iodide as a fluorescent dye. They represented distinct infrageneric taxonomic groups and differed in somatic chromosome numbers. Estimated 2C DNA values ranged from 0.97 pg in L. princei to 2.44 pg in L. luteus, which gives a more than 2.5-fold variation. Statistical analysis of the data obtained resulted in a grouping that supports the generally accepted taxonomic classification of the Old World lupins. The rough-seeded L. princei turned out to be an interesting exception, getting closer to smooth-seeded species. Results of DNA content analyses are discussed with regards to the phylogenetic relationships among the Old World lupins and some aspects of the evolution of the genus.     

Sex ratios and sex-biased infection behaviour in the entomopathogenic nematode genus Steinernema

In experimentally infected insects, the sex ratio of first generation nematodes of five species of Steinernema was female-biased (male proportion 0.35-0.47). There was a similar female bias when the worms developed in vitro (0.37-0.44), indicating that the bias in these species is not due to a lower rate of infection by male infective juveniles (IJs). Experimental conditions influenced the proportion of males establishing in insects, indicating that male and female IJs differ in their behaviour. However, there was no evidence that males are the colonising sex in any species, contrary to what has previously been proposed. Time of emergence from the host in which the nematodes had developed influenced sex ratios in experimental infections. In three species (Steinernema longicaudum, Steinernema glaseri and Steinernema kraussei), early emerged nematodes had a higher proportion of males than those that emerged later, with the reverse trend for Steinernema carpocapsae and Steinernema feltiae. In a more detailed in vitro study of S. longicaudum, the proportion of males was similar whether or not the nematodes passed through the developmentally arrested IJ stage, indicating that the female bias is not due to failure of males to exit this stage. The sex ratio in vitro was independent of survival rate from juvenile to adult, and was female-biased even when all juveniles developed, indicating that the bias is not explained by failure of males to develop to adults. The female-biased sex ratio characteristic of Steinernema populations appears to be present from at least the early juvenile stage. We hypothesise that the observed female bias is the population optimal sex ratio, a response to cycles of local mate competition experienced by nematodes reproducing within insect hosts interspersed with periods of outbreeding with less closely related worms following dispersal.     

Genetic variation and relationships of seven sturgeon species and ten interspecific hybrids

Background

Sturgeon cultivation is important for both industry and aquaculture in China. To date, more than 17 species or strains have been farmed for fillets and caviar production. Crossbreeding among different sturgeon species is frequent and the F2 hybrids are fertile. However, large-scale farming can have negative impacts on wild populations i.e. escape of exotic sturgeons and must be taken into consideration. Escape of exotic sturgeons can cause severe ecological problems, including threatening native sturgeon species once the exotic varieties become established or hybridize with native individuals. However, little is known about their genetic resources and variation.

Methods

Genetic diversity and introgression of seven sturgeon species were analyzed using mitochondrial DNA cytochrome oxidase subunit I (COI) and nine microsatellite markers. This study included 189 individuals from seven sturgeon species and 277 individuals from ten lineages of F2 hybrid strains.

Results

MtDNA COI sequences (632 bp long) were generated from 91 individuals across the 17 sturgeon strains and produced 23 different haplotypes. Haplotype diversity was high (h = 0.915 ± 0.015) and nucleotide diversity was low (π = 0.03680 ± 0.00153) in the seven sturgeon species and ten interspecific hybrids. Phylogenetic analyses resulted in almost identical tree topologies, and different haplotype structures were mainly related with sturgeons of different female parents. Analysis of molecular variance revealed that 81.73% of the genetic variance was due to matrilineal differences, while 9.40% resulted from strain variation. Pairwise Fst values obtained with POLYSAT software, were high among strains and ranged from 0.031 to 0.164. Admixture analysis assigned seven distinct groups and ten genotypes of admixed clusters composed of hybrid strains using STRUCTURE when assuming K = 7.

Conclusions

The interspecific mtDNA gene tree corresponded to the expected taxonomic divisions. These relationships were also supported by the results from the microsatellite analysis and contributed to unambiguously identify seven sturgeon species and ten F2 hybrid strains from sturgeon farms in China. Moreover, we found that introgressive hybridization is pervasive, exists in both purebred and hybrid sturgeons, and may reflect widespread mismanagement in sturgeon breeding in China.     

A Phosphopantetheinyl transferase homolog is essential for Photorhabdus luminescens to support growth and reproduction of the entomopathogenic nematode Heterorhabditis bacteriophora

The bacterium Photorhabdus luminescens is a symbiont of the entomopathogenic nematode Heterorhabditis bacteriophora. The nematode requires the bacterium for infection of insect larvae and as a substrate for growth and reproduction. The nematodes do not grow and reproduce in insect hosts or on artificial media in the absence of viable P. luminescens cells. In an effort to identify bacterial factors that are required for nematode growth and reproduction, transposon-induced mutants of P. luminescens were screened for the loss of the ability to support growth and reproduction of H. bacteriophora nematodes. One mutant, NGR209, consistently failed to support nematode growth and reproduction. This mutant was also defective in the production of siderophore and antibiotic activities. The transposon was inserted into an open reading frame homologous to Escherichia coli EntD, a 4'-phosphopantetheinyl (Ppant) transferase, which is required for the biosynthesis of the catechol siderophore enterobactin. Ppant transferases catalyze the transfer of the Ppant moiety from coenzyme A to a holo-acyl, -aryl, or -peptidyl carrier protein(s) required for the biosynthesis of fatty acids, polyketides, or nonribosomal peptides. Possible roles of a Ppant transferase in the ability of P. luminescens to support nematode growth and reproduction are discussed.     

Genetic relationships between European and Transcaucasian mice of the genus Apodemus Kaup]

Genetic relations between four European mice species of the genus Apodemus (Apodemus sylvaticus, A. flavicollis, A. microps and A. falzfeini) and five Transcaucasian ones (A. mystacinus, A. microps, and the forms No 2, 3p, 3f) were studied for 37 biochemical loci. Close genetic relations were demonstrated between the mice of subgenus Sylvaemus and A. mystacinus. A. microps and A. falzfeini from the Caucasus were shown conspecific to these species from the Ukraine and the presence of the two new different species--the form No 2 and 3p in the Caucasus was established.