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.     

Mass Production of the Beneficial Nematode Heterorhabditis bacteriophora and Its Bacterial Symbiont Photorhabdus luminescens

Entomoparasitic nematodes (EPNs) are being commercialized as a biocontrol measure for crop insect pests, as they provide advantages over common chemical insecticides. Mass production of these nematodes in liquid media has become a major challenge for commercialization. Producers are not willing to share the trade secrets of mass production and by doing so, have made culturing EPNs extremely difficult to advance existing technologies. Theoretically, mass production in liquid media is an ideal culturing method as it increases cost efficiency and nematode quantity. This paper will review current culturing methodologies and suggest basic culturing parameters for mass production. This review is focused on Heterorhabditis bacteriophora; however, this information can be useful for other nematode species.     

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.     

Effectiveness of Steinernema spp. and Heterorhabditis bacteriophora against Popillia japonica in the Azores

Steinernema carpocapsae (Breton strain), S. glaseri, and Heterorhabditis bacteriophora were evaluated for their potential to control immature stages of the Japanese beetle, Popillia japonica, on Terceira Island (the Azores). In bioassays carried out at temperatures higher than 15 C, S. glaseri and H. bacteriophora caused 100% mortality of larvae, whereas S. carpocapsae caused 56% larval mortality. At temperatures slightly below 15 C, only S. glaseri remained effective. In field plots, in September, S. glaseri and S. carpocapsae reduced larval populations by 91% and 44%, respectively, when applied at the rate of 10⁶ nematodes/m². In April, S. glaseri caused 31% reduction in numbers of larvae, but S. carpocapsae was ineffective. In colder months (November-February) neither steinernematids nor H. bacteriophora reduced larval populations. Increasing the application rate from 10⁶ to 5 x 10⁶ infective stage S. glaseri per m² increased efficacy from 63% to 79% mortality.     

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.     

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.     

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.      

Variations in Immune Response of Popillia japonica and Acheta domesticus to Heterorhabditis bacteriophora and Steinernema Species

The infectivities of Steinernema carpocapsae, S. glaseri, S. scapterisci, and Heterorhabditis bacteriophora to Japanese beetle larvae, Popillia japonica, and house cricket adults, Acheta domesticus, were compared using external exposure and hemocoelic injection. Only H. bacteriophora and S. glaseri caused high P. japonica mortality after external exposure. When nematodes were injected, P. japonica had a strong encapsulation and melanization response to all species except S. glaseri. Heterorhabditis bacteriophora and S. carpocapsae were able to overcome the immune response, but S. scapterisci was not. All species except S. scapterisci were able to kill and reproduce within the host. Only S. scapterisci and S. carpocapsae caused A. domesticus mortality after external exposure. When nematodes were injected, A. domesticus had a strong immune response to all species except S. scapterisci. Steinernema carpocapsae effectively overcame the strong immune response and caused high host mortality, but S. glaseri and H. bacteriophora did not. Steinernema scapterisci caused high host mortality and reproduced, S. glaseri and H. bacteriophora caused low host mortality but only S. glaseri reproduced, and S. carpocapsae was able to kill the host but reproduced poorly. Most (ca. 90%) of the S. carpocapsae in the hemocoel of P. japonica became encapsulated and melanized within 8 hours postinjection. The symbiotic bacterium, Xenorhabduf nematophilus, was often released before this encapsulation and melanization.     

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.     

Effect of Application Method on Fitness of Entomopathogenic Nematodes Emerging at Different Times

The entomopathogenic nematode species Steinernema feltiae and Heterorhabditis bacteriophora were compared for survival and infectivity of infective juveniles (IJ) collected with a standard White trap (i.e., emerging from hosts and accumulating in water) and later applied to sand (treatment A) to IJ allowed to emerge from hosts into sand (treatment C). Percentage IJ survival and infectivity was compared between treatments for S. feltiae IJ that emerged between days 1 to 3 and days 4 to 6. For H. bacteriophora, percentage IJ survival and infectivity was compared between treatments only for infective juveniles that emerged between days 4 to 6. For S. feltiae IJ percentage survival and infectivity decreased with time (P ≤ 0.05) and was greater (P ≤ 0.05) for IJ from treatment C than for IJ from treatment A. For H. bacteriophora IJ percentage survival decreased (P ≤ 0.05) and percentage infectivity increased (P ≤ 0.05) with time. While percent survival was higher (P ≤ 0.05) for treatment C than for A, percent infectivity was not different between treatments.     

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.     

Behavioural response of the entomopathogenic nematodes Steinernema carpocapsae and Heterorhabditis bacteriophora to oxamyl

Infective juveniles of the entomopathogenic nematode Steinernema carpocapsae show a low level of locomotory activity that is presumed to limit their usefulness as biological insecticides. A 30 μg ml^-1 solution of the carbamate pesticide oxamyl reduced the proportion of nonmobile nematodes by nearly two thirds to 35%, while stimulating a 7.5-fold increase in sinusoidal movement. This increase in activity did not result in a corresponding increase in host-finding. Oxamyl treatment did not enhance infective juvenile pathogenicity to Galleria mellonella larvae. At higher concentrations, oxamyl caused aberrant nematode movement and partial paralysis. Heterorhabditis bacteriophora infective juveniles maintain a high level of locomotory activity. Treatment with 30 μg ml^-1 oxamyl increased the proportion of sinusoidal over nonsinusoidal movements, but infective juvenile host-finding and pathogenicity were significantly reduced. Higher rates impaired movement and induced complete paralysis. We conclude that oxamyl is incompatible with S. carpocapsae and H. bacteriophora. The concept of chemically activating infective juveniles to increased locomotory activity and thereby achieving enhanced efficacy is inconsistent with our results.     

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.     

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.     

嗜菌异小杆线虫H06品系对红棕象甲致病力的时间效应及对血淋巴的影响

【目的】阐明嗜菌异小杆线虫H06品系处理对红棕象甲血淋巴免疫系统的影响。【方法】采用室内生物测定和生理生化测定法,研究了嗜菌异小杆线虫H06品系对红棕象甲不同虫态的致病力及对血淋巴各项指标的调节作用。【结果】嗜菌异小杆线虫H06品系对红棕象甲不同虫态的致病力与处理浓度和处理时间均呈正相关性,经嗜菌异小杆线虫H06品系处理后,红棕象甲血淋巴总量和血淋巴酯酶均呈前期升高、后期降低的趋势,处理后72 h的血淋巴总量和酯酶活性与对照相比分别降低39.73%和53.36%;血淋巴蛋白含量与对照相比呈下降的趋势,虽在60 h时有一个短暂的回升,之后又迅速下降,其中在处理后72 h,血淋巴的蛋白含量与对照相比下降74.96%。【结论】嗜菌异小杆线虫H06品系的侵入破坏了红棕象的血淋巴免疫系统,本研究结果可为揭示昆虫病原线虫对红棕象甲的致病机理提供依据。     

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.