Cryopreservation of Steinernema carpocapsae and Heterorhabditis bacteriophora

A method for the cryopreservation of third-stage infective juveniles (IJ) of Steinernema carpocapsae and Heterorhabiditis bacteriophora was developed. Cryoprotection was achieved by incubating the nematodes in 22% glycerol (S. carpocapsae) or 14% glycerol (H. bacteriophora) for 24 hours, followed by 70% methanol at 0 C for 10 minutes. The viability of S. carpocapsae frozen in liquid nitrogen as 20 μl volumes spread over cover slip glass was > 80%. Survival of H. bacteriophora frozen on glass varied from 10 to 60% but was improved to > 80% by replacing the glass with filter paper. Cryopreservation and storage of 1-ml aliqots of S. carpocapsae IJ resulted in > 50% survival after 8 months; pathogenicity was retained and normal in vitro development took place. Trehalose and glycerol levels increased and glycogen levels decreased during incubation of S. carpocapsae IJ in glycerol. Normal levels of trehalose, glycerol and glycogen were restored during post freezing rehydration.     

Life Cycle and Reproductive Potential of the Nematode Heterorhabditis bacteriophora Strain HP88

Development of the entomopathogenic nematode Heterorhabditis bacteriophora strain HP88 was studied in vivo with larvae of the greater wax moth, Galleria mellonella, as host and in vitro. At 25 C in vivo, the duration of the life cycle from egg hatch to egg hatch was 96 hours. Juvenile development took 48 hours, with the duration of each juvenile stage ranging from 8 to 12 hours. Under crowded conditions, development proceeded to the infective juvenile (IJ) stage instead of the third juvenile stage (J3). Life-cycle duration and proportion of the various developmental stages in the population were similar in in vitro and in vivo cultures. When in vivo or in vitro development was initiated from the IJ stage, only hermaphrodites developed in the first generation and males appeared only in the second generation. The average (±SD) number of progeny per hermaphrodite was 243 ± 98. The ratio of males to hermaphrodites in the second generation was 1:9.4 ± 6.8.     

Phenotypic and Genetic Characterization of Two New Mutants of Heterorhabditis bacteriophora

Two new "dumpy" mutants (Hbdpy-2 and Hbdpy-3) of Heterorhabditis bacteriophora were induced and characterized. Mutants (hermaphrodites and males) that hatched from eggs were shorter and wider than the wild-type strain. This phenotype was not discernible in young animals until 24 hours after hatching from eggs or in mutants that developed from infective juveniles. Scanning electron microscopy revealed that the tails of the two mutants are much more slender than in the wild-type. In addition, the vulva of Hbdpy-3 nematodes appeared to be sunken; that of Hbdpy-2 animals was protruding, like in the wild-type. Upon self fertilization, individual Hbdpy-3 hermaphrodites produced fewer progeny than the wild-type. Crosses between virgin Hbdpy-2 and Hbdpy-3 hermaphrodites and wild-type males indicated that the two mutations are recessive. Complementation tests indicated that Hbdpy-1, Hbdpy-2, and Hbdpy-3 affect different genes. The ratio (1.03:1) of wild-type to dumpy phenotype among the F₂ progeny of self-fertilizing heterozygotes suggested linkage among the three genes. The genetic map distance was estimated only between Hbdpy-1 and Hbdpy-2 genes, approximately 29 map units.     

Genetics of the Nematode Heterorhabditis bacteriophora Strain HP88: The Diversity of Beneficial Traits

Genotypic variation among infective juveniles of Heterorhabditis bacteriophora (strain HP88) in heat, desiccation, ultraviolet tolerance, and host-finding ability was assessed by comparing the performance of inbred lines of this entomopathogenic nematode in laboratory assays. Each line consisted of highly homozygous offspring originating from one individual obtained from a natural population. Considerable variation in all four traits was detected among the different inbred lines. The heritability values for heat or ultraviolet tolerance and for host-finding ability were high, indicating that selection should be an efficient way for improving these traits in the population. The results for desiccation tolerance varied considerably within each line. Heritability value was low, indicating that the results were influenced mainly by environmental variation and suggesting that selective breeding for higher desiccation tolerance would be inefficient. Improvement through induction of mutations may be a better alternative in this population.     

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°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.     

Effect of soil type on infectivity and persistence of the entomopathogenic nematodes Steinernema scarabaei, Steinernema glaseri, Heterorhabditis zealandica, and Heterorhabditis bacteriophora

We tested the effect of soil type on the performance of the entomopathogenic pathogenic nematodes Steinernema scarabaei, Steinernema glaseri, Heterorhabditis zealandica, and Heterorhabditis bacteriophora. Soil types used were loamy sand, sandy loam, loam, silt loam, clay loam, acidic sand, and a highly organic potting mix. Infectivity was tested by exposing third-instar Anomala orientalis or Popillia japonica to nematodes in laboratory and greenhouse experiments and determining nematode establishment in the larvae and larval mortality. Infectivity of H. bacteriophora and H. zealandica was the highest in potting mix, did not differ among loamy sand and the loams, and was the lowest in acidic sand. Infectivity of S. glaseri was significantly lower in acidic sand than in loamy sand in a laboratory experiment but not in a greenhouse experiment, and did not differ among the other soils. Infectivity of S. scarabaei was lower in silt loam and clay loam than in loamy sand in a greenhouse experiment but not in a laboratory experiment, but was the lowest in acidic sand and potting mix. Persistence was determined in laboratory experiments by baiting nematode-inoculated soil with Galleria mellonella larvae. Persistence of both Heterorhabditis spp. and S. glaseri was the shortest in potting mix and showed no clear differences among the other substrates. Persistence of S. scarabaei was high in all substrates and its recovery declined significantly over time only in clay loam. In conclusion, generalizations on nematode performance in different soil types have to be done carefully as the effect of soil parameters including soil texture, pH, and organic matter may vary with nematode species.     

Target Host Finding by Steinernema feltiae and Heterorhabditis bacteriophora in the Presence of a Non-Target Insect Host

The ability of Steinernema feltiae or Heterorhabditis bacteriophora infective juveniles (IJ), when applied to the soil surface, to infect a Galleria mellonella larva at the base of a soil-filled cup (276 cm³) was evaluated in the presence and absence of 100 larvae of a non-target insect, the aphid midge Aphidoletes aphidimyza, near the soil surface. In all four trials with either S. feltiae or H. bacteriophora, A. aphidimyza presence did not affect the number of IJ finding and infecting a G. mellonella larva. Steinernema feltiae and H. bacteriophora IJ movement (as measured by the percentage of IJ aggregating on either side of an experimental arena) in the presence of one or many A. aphidimyza larvae was evaluated in agar- and soil-filled petri dishes, respectively. Infective juvenile movement in the presence of A. aphidimyza did not differ from random, indicating that IJ were not attracted to A. aphidimyza. It is suggested, therefore, that A. aphidimyza does not reduce IJ efficacy when these two forms of biological control agent are present together in a field situation even though it is known that A. aphidimyza is susceptible to IJ of these species.     

Infectivity and reproductive potential of the Oswego strain of Heterorhabditis bacteriophora associated with life stages of the clover root curculio,Sitona hispidulus

The infectivity and reproductive potential of the entomopathogenic nematode Heterorhabditis bacteriophora (Oswego strain), at different concentrations, was studied. Seventy to 80.0% mortality to late instar larvae of the clover root curculio, Sitona hispidulus, and 40.0-76.0% mortality to pupae, was observed at concentrations of 15-100 infective juveniles. There were no significant differences in mortality among nematode concentrations. LC(50) levels of 4.0 and 21.4 nematodes were determined for clover root curculio larvae and pupae, respectively. Nematodes did not cause significant mortality to adult or first instar clover root curculio. H. bacteriophora was able to complete its development and reproduce in 74.0-95.0% of clover root curculio late instar larvae and pupae. Reproductive potential in curculio larvae and pupae ranged from 0 to 7040 infective juveniles per host. Larvae exposed to 100 nematodes had a reproductive potential significantly higher than in those larvae exposed to 15 and 50 nematodes. Reproductive potential in pupae decreased with an increased nematode dose, indicating potential crowding effects. Host larval and pupal mass were positively correlated with nematode progeny production.     

Involvement of larvicidal toxins in pathogenesis of insect parasitism with the rhabditoid nematodes,Steinernema feltiae andHeterorhabditis bacteriophora

The insect-parasitic rhabditoid nematodes,Steinernema feltiae andHeterorhabditis bacteriophora, released a compound/s/ toxic to larvae of the greater wax moth,Galleria mellonella, that caused paralysis and death of the insect. Larvicidal substances appeared in wax moth larvae during parasitism and after inoculation with the primary form of the bacterial associates of the nematodes. The nematodeS. feltiae and its associate,Xenorhabdus nematophilus, excreted much less toxic activity within larval body thanH. bacteriophora. The secondary form ofXenohabdus did not produce toxin in parasitized larvae, butX. luminescens, the bacterium associated withH. bacteriophora, released detectable titer of toxin activity in broth cultures. Both nematode toxins were sensitive to heat and produced a specific type of proteolytic activity. Preliminary identification of the compounds responsible for larval toxicity revealed similarities to immune inhibitors produced by some bacterial pathogens of insects.      

Phenotypic and Genetic Analysis of a Mutant of Heterorhabditis bacteriophora Strain HP88

Induction and characterization of a morphological mutant are described for Heterorhabditis bacteriophora strain HP88. A homozygous inbred line was used as the base population for mutagenesis and genetic analysis of mutations. Mutagenesis was induced by exposing young hermaphrodites to 0.05 M ethyl methanesulfonate. A dumpy mutant (designated Hdpy-l) was isolated from the F₂ generation of the mutagenized population. Morphological studies with light and scanning electron microscopy revealed that the head region of the adult stage was compressed. The head region of the infective juvenile was distorted and the mouth open. Backcross with the original population was successful only between mutant hermaphrodites and wild type males; 50-100 percent of the progeny of this cross maintained the dumpy phenotype, indicating that the ratio between self- and external fertilization of the eggs is > 1 and that the dumpy mutation is recessive.     

Morphometrics of Infective Juveniles of Steinernema spp. and Heterorhabditis bacteriophora (Nemata: Rhabditida)

Selected morphometrics of Heterorhabditis bacteriophora and seven species of Steinernema from in vivo culture were compared in relation to time of harvest. In addition, five Steinernema species were reared in vitro and their morphometrics were compared with those from in vivo culture. With in vivo culture, there was generally a negative linear relationship between body length of infective juveniles (IJ) and time of harvest. The distance from the anterior end to the excretory pore (EP) and the tail length (T) of IJ also varied with time of harvest. The E percentage (= EP/T x 100) was the least variable. Body lengths of IJ reared in vitro were much less than those of IJ reared in vivo. The study suggests that IJ harvested from in vivo culture within 1 week of emergence from cadavers are best for species identification. Infective juveniles from in vitro culture should not be used for species identification.     

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.     

Influence of Salinity on Survival and Infectivity of Entomopathogenic: Nematodes

Exposure to NaC1, KCI, and CaCl₂ affected the entomopathogenic nematodes Heterorhabditis bacteriophora and Steinernema glaseri differently. Survival, virulence, and penetration efficiency of S. glaseri were not affected by these salts. At high concentrations, however, all three salts inhibited its ability to move through a soil column and locate and infect a susceptible host. Calcium chloride and KCl had no effect on H. bacteriophora survival, penetration efficiency, or movement through a soil column, but moderate concentrations of these salts enhanced H. bacteriophora virulence. NaCl, however, adversely affected each of these parameters at high salinities (>16 dS/m). Salt effects on S. glaseri are attributed solely to interference with nematode host-finding ability, whereas the NaCl effects on H. bacteriophora are attributed to its toxicity and possibly to interference with host-finding behavior.     

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.     

In vitro micropropagation of the macarronesian evergreen treePersea indica (L.) K. Spreng

Summary Shoot propagation ofPersea indica (L.) K. Spreng was achieved using seedling axillary buds cultured on MS (Murashige and Skoog, 1962) medium with 1 mg/l (2.8 μM) N⁶-benzyladenine (BA). Forty percent of the obtained shoots did not elongate, but showed bud proliferation, which was maximal (three axillary buds per shoot) at the end of the seventh subculture. Sixty percent of the shoots elongated, did not show bud proliferation, and formed calluses at their base. Successful rooting (84.6%) was achieved dipping the base of each elongated shoot in 3 g/l (16.11 mM) indolebutyric acid (IBA) for 1–2 s, and transferring to half strength MS medium without growth regulators. These shoots presented an acclimatization success of 100%. Results suggest that micropropagated elongated shoots ofP. indica can be adequately used in reforestation programs.     

Natural enemies of the pumpkin caterpillarDiaphania indica [Lepidoptera: Pyralidae] in Tamil Nadu

The survey for the natural enemies associated with the pumpkin caterpillar,Diaphania indica revealed the presence of 20 species of parasitoids, predators and pathogens. Of these, 16 were parasitoids belonging to the familiesBraconidae, Ichneumonidae, Bethylidae, Elasmidae andChalcididae. Except for 3 species the remaining parasitoids were new records forD. indica. The predators recorded were ants and spiders. A microsporidia also was recorded for the first time onD. indica.      

Somatic embryogenesis and plant regeneration inAzadirachta indica A. Juss

Summary Media for induction of somatic embryogenesis from immature cotyledonary tissues ofAzadirachta indica (Neem) were determined. Callus was initiated on Murashige and Skoog medium supplemented with 0.5 mg·liter⁻¹ of indol-3 acetic acid, 1.0 mg·liter⁻¹ of 6-benzyl amino purine, and 1000 mg·liter⁻¹ of casein hydrolysate. Effect of kinetin was also studied for embryo induction. Carbohydrate source in the form of sucrose and glucose alone and in combination was tested for embryogenic efficiency. Seventy percent embryos showed germination. Healthy plants were potted in sand and soil. Histologic studies confirmed indirect somatic embryogenesis.     

Efficacy of the Entomopathogenic Nematodes Heterorhabditis megidis and Heterorhabditis bacteriophora for the control of grubs (Phyllopertha horticola and Aphodius contaminatus) in Golf Course Turf

Liquid culture-produced entomopathogenic nematodes, Heterorhabditis megidis and Heterorhabditis bacteriophora, were applied at 0.5 and 1.5 million dauer juveniles m-2 against Aphodius contaminatus and Phyllopertha horticola on a golf course. The reduction of A. contaminatus was found to be between 40 and 62%. P. horticola reduction reached 70% with H. megidis and 83% with H. bacteriophora. Turf damage caused by birds preying on the grubs was successfully prevented.     

Temperature Effects on Heterorhabditis megidis and Steinernema carpocapsae Infectivity to Galleria mellonella

The effect of temperature on the infection of larvae of the greater wax moth, Galleria mellonella, by Heterorhabditis megidis H90 and Steinernema carpocapsae strain All, was determined. For both species, infection, reproduction, and development were fastest at 20 to 24 °C. Infection by both H. megidis and S. carpocapsae occurred between 8 and 16 °C; however, neither species reproduced at 8 °C. Among the nematodes used in experiments at 8 °C, no H. megidis and very few S. carpocapsae developed beyond the infective juvenile stage. Compared with H. megidis, S. carpocapsae invaded and killed G. mellonella larvae faster at 8 to 16 °C. By comparing invasion rates, differences in infectivity between the two nematode species were detected that could not be detected in conventional petri dish bioassays where mortality was measured after a specified period. Invasion of G. mellonella larvae by H. megidis was faster at 24 than at 16 °C.