Life Cycle of Steinernema scapterisci Nguyen &Smart, 1990

The life cycle of Steinernema scapterisci Nguyen and Smart, 1990 consists of an egg stage, four juvenile stages, and an adult stage (male and female). The cycle from IJ (third stage infective juveniles) to IJ may proceed by one of two routes. If the nutrient supply is sufficient and the population is not overcrowded, the IJ develop to adult males and females of the first generation. Most eggs from these adult females hatch and the juveniles develop through each life stage to become adult males and females of the second generation. Eggs produced by these females develop to IJ. This cycle takes 8-10 days (long cycle) at 24 C. If the nutrient supply is insufficient or if overcrowded, the IJ develop to adult males and females of the first generation, and eggs produced by the females develop directly to IJ. This cycle takes 6-7 days (short cycle). The nematode is less tolerant of lower temperatures and more tolerant of higher temperatures than are other species of the genus. The sex ratio is influenced by temperature. At 15 and 24 C, females constituted 54% and 60% of the population, respectively, but at 30 C females constituted 47% of the population.     

Degree-day Models for Predicting Egg Hatch and Population Increase of Criconemella xenoplax

A degree-day model was derived to predict egg hatch for Criconemella xenoplax. Eggs collected from gravid females were incubated in distilled water at constant temperatures of 10-35 C. Sixty-six percent of all eggs hatched between 13 and 32 C, and 42% hatched at 10 C. All eggs aborted above 32.5 C. Between 25 and 32 C, 8.5 ± 0.5 days were required for egg hatch. Degree-day requirement for egg hatch at 10-30 C was estimated to be 154 ± 5 with a base of 9.03 ± 0.04 C. This base of 9 C was adopted in studies of the relationship between degree-days and nematode population increase on Prunus seedlings grown 9-11 weeks in a greenhouse. Degree-day accumulations were based upon daily averages from maximum and minimum air temperatures. Ratios of final to initial population densities exhibited an exponential pattern in relation to degree-day accumulations with proportionate doubling increment of 0.100 ± 0.049 every 139 ± 8 degree-days. These results provide a means of predicting nematode population increase under greenhouse conditions and a basis for choosing sampling intervals when evaluating nematode multiplication.     

Stage-specific Population Development and Fecundity of Paratrichodorus minor

A conceptual model of the life cycle of Paratrichodorus minor consisting of the egg stage, four juvenile stages, and the adult stage was proposed. Development of an individual from one stage to the next was described by a probability distribution defined by the mean length of time spent in the stage and the standard deviation associated with the mean duration. Experiments were conducted to estimate stage durations, stage-specific survivorships, and a fecundity rate for females. Eggs hatched on agar plates at a mean time of 53.3 ± 7.3 degree-days using a basal threshold of 10 C (DD₁₀) with a range of 40-64 DD₁₀ after deposition. Forty-five percent of the eggs observed ultimately hatched. Of the eggs that died, 44% died before the nematode form could be observed in the egg and 56% died after movement had been observed. First generation population peaks following inoculation with first-stage juveniles occurred at 28 DD₁₀ for second-stage juveniles, 67 DD₁₀ for third-stage juveniles, 109 DD₁₀ for fourth-stage juveniles, and 143 DD₁₀ for adults. Adult males are rare and were never observed in these studies. The fecundity rate was 0.784 eggs/(female-DD₁₀⁻¹), but the maximum length of the egg-laying period was not determined. The minimum egg-laying period was 73-113 DD₁₀, and minimum egg production was 57-86 eggs per female. The preovipositional period for adult females was estimated to be 79 DD₁₀. In the presence of a host, total population numbers increased, but in the absence of a host, the population declined to 33 % of the initial level after 300 DD₁₀.     

Factors Affecting Egg Hatch of Heterodera mediterranea and Differential Responses of Olive Cultivars to Infestation

The influence of temperature and olive root exudates on Heterodera mediterranea egg hatch and the effects of H. mediterranea on the growth of two olive cultivars (Arbequina and Picual) were investigated. Egg hatch occurred over a temperature range of 10 to 30°C and was optimal at 20 to 25°C. There were no differences in egg hatch between sterile deionized distilled water or root exudate dilutions (undiluted, diluted 1:1, and 1:2) of Arbequina and Picual at 20°C. Heterodera mediterranea reproduced on both olive cultivars in growth chambers at 25°C. Soil and root final nematode populations, as well as total number of cysts per plant and reproduction rate, were significantly higher in Arbequina than in Picual. Shoot dry and root fresh weights as well as increases of shoot height, trunk diameter, and numbers of nodes were significantly suppressed by infection with 10,000 eggs + second-stage juveniles/pot in Arbequina but not in Picual.     

Development of Heterodera glycines Life Stages as Influenced by Temperature

The effects of temperature on rates of development of Heterodera glycines egg and juvenile stages were examined as a basis for predicting generation times of the nematode on soybean. The relationship of temperature to H. glycines embryonic development between 15 and 30 C was described by a linear model, The calculated basal temperature threshold was 5 C. Thermal optimum for embryogenesis and hatch with low mortality was 24 C. Development proceeded to first-stage juvenile at 10 C and to second-stage juvenile at 15-30 C. Hatch occurred at 20-30 C. At 36 C, development proceeded to the four-cell stage, then the eggs died. The range of diurnal soil temperature fluctuation and accumulated degree-days between 5 and 30 C (DD5/30) had an impact on rate of development of juveniles in soybean roots. From early June to early July, H. glycines required 534 + 24 DD5/30 (4 weeks) to complete a life cycle in the field. During the midseason (July and August), life cycles were completed in 3 weeks and 429 ± 24 DD5/30 were accumulated. Late in the season (September to November), declining soil temperatures were associated with generation times of 4 weeks and slower rates of development.     

Heterodera glycines Cyst Components and Surface Disinfestants Affect H. glycines Hatching

We investigated the effects of Heterodera glycines cyst components and surface disinfestants on hatching of H. glycines eggs in vitro. Eggs were incubated in either H. glycines cyst wall fragments, cyst wall and egg rinsate, egg homogenate, or control solutions of soybean root diffusate, sterile distilled water, or zinc sulfate. Hatch in cyst wall and egg rinsate, and egg homogenate, was greater (α = 0.05) than hatch in sterile distilled water; however, it was not different from hatch in zinc sulfate according to Dunnett''s test. Hatch in cyst wall fragments was similar to hatch in sterile distilled water. To determine whether surface disinfestants affected hatch, eggs were treated first with chlorhexidine diacetate, mercuric chloride, sodium hypochlorite, or streptomycin sulfate and then incubated in H. glycines egg homogenate, soybean root diffusate, sterile distilled water, or zinc sulfate. Hatch of eggs treated with chlorhexidine diacetate, mercuric chloride, and streptomycin sulfate was reduced (α = 0.05), and hatch of eggs treated with sodium hypochlorite was increased (α = 0.05) relative to hatch of nontreated eggs in all incubation solutions except zinc sulfate according to Dunnett''s Test. Hatch in zinc sulfate was similar among all surface disinfestants except mercuric chloride, where hatch was reduced relative to hatch of nontreated and other surface disinfestant-treated eggs.     

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.     

Effects of Two Carbamates on Infective Juveniles of Stemernema carpocapsae All Strain and Steinernema feltiae Ume? Strain

Laboratory bioassays were conducted to determine the effects of two carbamates, carbofuran (an acetylcholinesterase inhibitor) and fenoxycarb (a juvenile hormone analog), on survival and infectivity of the infective juveniles (IJ) of Steinernema feltiae Umeå strain and Steinernema carpocapsae All strain. Both insecticides caused mortality of IJ in a dose-related fashion. The two nematode species were equally sensitive to fenoxycarb (LD₅₀ ca. 0.03mg/ml). Whereas IJ of S. feltiae were several orders of magnitude more sensitive to carbofuran (LD₅₀ ≤ 0.2 μg/ml) than to fenoxycarb, S. carpocapsae IJ displayed approximately the same degree of sensitivity to carbofuran (LD₅₀ 0.01-0.03 mg/ml) as they did toward fenoxycarb. Toxicity of the carbamates was the same at all exposure periods from 24 to 168 hours'' duration. Determinations of infective doses of nematodes required to cause 50% mortality of Galleria mellonella larvae showed that the infectivity of IJ that survived exposure to either of the two carbamates was not compromised by treatment.     

Temperature Effects on Race Determination in Heterodera glycines

Currently there are 16 possible races for Heterodera glycines, and these are differentiated based on ability of a nematode population to develop on a set of four differential soybean genotypes. Because results are based on numbers of nematode females that develop to a specific stage rather than on the reproductive capability of these females, race determinations based on female indices may not represent results obtained after several reproductive cycles of H. glycines. Counting numbers of eggs and juveniles, and then developing corresponding indices, would allow reproduction to be considered in making race determinations. Our objectives were to compare the present race identification scheme for H. glycines based on female indices with those using egg and juvenile indices and to examine the effect of temperature on race designations using female, egg, and juvenile indices. Race designations for H. glycines populations from two locations in Illinois were determined at 20, 27, and 30 °C in a water bath. The numbers of females, eggs, and juveniles (at 19 days) were recorded, and an index based on each life stage was calculated. Race determinations based on female, egg, or juvenile indices were inconsistent when conducted at 20 °C, which demonstrates that this temperature is not suitable for identifying races of H. glycines. However race designations at 27 and 30 °C were consistent for all three indices. This indicates that counting females, eggs, or juveniles should be equally reliable when race determinations are conducted at these two temperatures, and choice of method would depend on investigator preference or research objective.     

Effect of Artemisia vulgaris Rhizome Extracts on Hatching,Mortality, and Plant Infectivity of Meloidogyne megadora

The activity of an ethanolic rhizome extract of Artemisia vulgaris against hatching, mortality, host plant infectivity, and galling of the root-knot nematode Meloidogyne megadora was investigated. The extract inhibited egg hatch (50% inhibition by 2.35mg/ml) and caused second-stage juvenile mortality (50% lethality at 12 hours'' exposure to 55.67 mg/ml), both in a dose-dependent manner. Nematode infectivity on Phaseolus vulgaris ''Bencanta Trepar'', a susceptible host, decreased in a dose-responsive manner (50% inhibition at 6.28 hours exposure to extract). When applied directly to the soil, the extract reduced root galling on a susceptible host in a dose-dependent manner (50% inhibition by 32.36 mg/ml). After dilution in distilled water, the extract did not lose activity when stored in the dark at 25°C for 15 days.     

Effect of Temperature on Pratylenchus penetrans Development

Reproduction and development of Pratylenchus penetrans were studied on genetically transformed ladino clover roots. Solitary females developing on transformed roots in nutrient gellan gum medium (pH 5.5) deposited 1.2, 1.5, 1.6, 1.8, and 2.0 eggs per day at the respective temperatures of 17, 20, 25, 27, and 30 °C. The number of eggs deposited was highly correlated with temperature. A reduction in egg-laying rates at the start of hatching was observed at all temperatures. Juvenile mortality was higher at 17 °C (50.4%), 20 °C (50.3%), and 30 °C (58.4%) than at 25 °C (34.6%) and 27 °C (37.6%). Life-cycle (egg deposition to egg deposition) duration was 46, 38, 28, 26, and 22 days at the respective temperatures. The developmental zero degrees (°C) and the effective accumulative temperatures (degree-days) required for hatching, female emergence, and onset of oviposition (completion of one generation) of P. penetrans were estimated to be 2.7 and 200, 4.2 and 548, and 5.1 and 564, respectively. Pratylenchus penetrans reproduces over a wide range of temperatures.     

Feeding and breeding aspects of Stomoxys calcitrans (Diptera: Muscidae) under laboratory conditions

Bionomic aspects of Stomoxys calcitrans (Linnaeus, 1758) (Diptera: Muscidae) were studied under laboratory conditions. For this reason, laboratory-rearing techniques were optimized at the National Veterinary School of Toulouse. The colony was maintained at 25 ± 2 °C, 50 ± 10% RH under a 12-hour light cycle and observed daily. The size of each adult cage is 30 x 30 x 30 cm and designed to house about 500-1,000 flies. The average cycle from egg to adult was 19.2 ± 1.7 days. The mean longevity of imagos was 9.3 ± 5.8 days and not significantly different between sexes. Stable flies were split into two groups; the first was fed with blood, honey and water, and the second was fed only with honey and water. The mean weight of a blood meal was 11.1 ± 3.8 mg with no significant differences between males and females. The mean longevity of non-blood fed flies was found to be significantly higher (10.4 ± 3.9 days) than those fed with blood. The maximum lifespan was shorter for non-blood fed males (17 days) and females (18 days) than for those fed with blood (females: 24 days, males: 23 days). Under these laboratory conditions, S. calcitrans rearing was successfully established. In the end, the number of expected generations of S. calcitrans and the net reproduction rate were estimated to be 11.8 generations/year and 16.2 living females per female respectively.     

Varietal Response and Estimates of Heritability of Resistance to Meloidogyne javanica in Carrots

With methods developed in this study, varietal responses to M. javanica were evaluated and heritability of resistance of two promising carrot cultivars was estimated. More egg masses were found on root systems inoculated with eggs added to the soil in three holes in 250 cm³ cups than by mixing the inoculum with soil in the cups. A resistant breeding line, CNPH 1437, was discriminated from susceptible cultivar Nova Kuroda with inoculum levels higher than 2,000 eggs per cup. Greenhouse and field results suggested that cultivars Nantes Superior and Shin Kuroda were susceptible, Kuronan was somewhat tolerant, and Brasilia and Tropical were resistant to M. javanica. Nantes Superior or Shin Kuroda yielded less in carbofuran-treated soil (3 kg a.i./ha) than Kuronan, Brasilia, and Tropical did in nontreated soil. However, incorporation of the nematicide greatly increased yields of Kuronan (32%), Brasilia (62%), and Tropical (91%). Primary root galling at the seedling stage was an adequate parameter for resistance evaluation. Estimated heritability were 0.48 ± 0.07 for primary root galling and 0.35 ± 0.08 for egg mass production in Brasilia, and 0.16 ± 0.11 for primary root galling and 0.31 ± 0.09 for egg mass production in Kuronan.     

Host Status of Different Potato (Solanum tuberosum) Varieties and Hatching in Root Diffusates of Globodera ellingtonae

Globodera ellingtonae was detected in Oregon in 2008. In order to make decisions regarding the regulation of this nematode, knowledge of its biology is required. We determined the host status of a diversity of potato (Solanum tuberosum) varieties in soil-based experiments and identified hatching stimulants in in vitro hatching assays. ‘Russet Burbank,’ ‘Desiree,’ ‘Modac,’ ‘Norland,’ ‘Umatilla,’ and ‘Yukon Gold’ were good hosts (RF > 14) for G. ellingtonae. Potato varieties ‘Maris Piper,’ ‘Atlantic,’ and ‘Satina,’ all which contain the Ro1 gene that confers resistance to G. rostochiensis, were not hosts for G. ellingtonae. In in vitro hatching assays, G. ellingtonae hatched readily in the presence of diffusates from potato (PRD) and tomato (Solanum lycopersicum; TRD). Egg hatch occurred in an average of between 87% and 90% of exposed cysts, with an average of between 144 and 164 juveniles emerging per cyst, from PRD- and TRD-treated cysts, respectively. This nematode hatched rapidly in the presence of PRD and TRD, with at least 66% of total hatch occurring by day 3 of exposure. There was no dose-response of egg hatch to concentrations of PRD or TRD ranging from 1:5 to 1:100 diffusate to water. When G. ellingtonae was exposed to root diffusates from 21 different plants, hatch occurred in 0% to 70% of exposed cysts, with an average of between 0 to 27 juveniles emerging per cyst. When root diffusate-exposed cysts were subsequently transferred to PRD to test viability, root diffusates from arugula (Eruca sativa), sudangrass (Sorghum bicolor subsp. drummondii), and common vetch (Vicia sativa) continued to inhibit egg hatch compared with the other root diffusates or water in which hatch occurred readily (60 to 182 juveniles emerging per cyst). Previously known hatching stimulants of G. rostochiensis and G. pallida, sodium metavanadate, sodium orthovanadate, and sodium thiocyanate, stimulated some egg hatch. Although, Globodera ellingtonae hatched readily in PRD and TRD and reproduced on potato, the pathogenicity of this nematode on potato remains to be determined.     

Exposure Time to Lethal Temperatures for Meloidogyne incognita Suppression and Its Implication for Soil Solarization

Meloidogyne incognita eggs or J2 were incubated in test tubes containing sand:peat mix and immersed in a water bath heated to 38, 39, 40, 41, 42, 43, 44 and 45°C for a series of time intervals. Controls were maintained at 22°C. Nematodes surviving or hatching were collected from Baermann trays after three weeks of incubation. Regression analyses between percent survival or egg hatch and hours of heat treatment were performed for each temperature. Complete suppression of egg hatch required 389.8, 164.5, 32.9, 19.7 and 13.1 hours at 38, 39, 40, 41 and 42°C, respectively. Complete killing of J2 required 47.9, 46.2, 17.5 and 13.8 hours at 39, 40, 41 and 42°C, respectively. J2 were not completely killed at 38°C within 40 hours of treatment, but were killed within one hour at 44 and 45°C. Effect of temperature on nematode killing is not determined by heat units. Oscillating temperature between cool and warm did not interfere with the nematode suppressive effect by the heat treatment. Six-week solarization in the field during the summers of 2003 and 2004 in Florida accumulated heat exposure times in the top 15 cm of soil that surpassed levels required to kill M. incognita as determined in the water bath experiments. Although near zero M. incognita were detected right after solarization, the nematode population densities increased after a cycle of a susceptible pepper crop. Therefore, future research should address failure of solarization to kill nematodes in the deeper soil layers.     

Influence of Temperature on Multiplication and Egg Hatching of Longidorus africanus

Longidorus africanus multiplication on tomato was highest at 29 °C. Few nematodes were recovered after 6 weeks at soil temperatures of 35 °C or below 23 °C. The time to egg hatching was shortest and the percentage of eggs hatching was highest at 29 °C. The minimum temperature and the heat sum above this temperature required for egg development were calculated to be 14.3 °C and 94.08 degree-days, respectively. The thermal times required for egg development by L. africanus and L. elongatus were nearly identical. For both species the product of the base temperature and the heat sum was near constant, and at a temperature of 22.3 °C the rates of egg development were equal.     

Life Cycle and Mating Behavior of Belonolaimus longicaudatus in Gnotobiotic Culture

The life cycle of Belonolaimus longicaudatus was observed in vitro on excised roots of Zea mays. Roots were cultured on Gamborg''s B5 medium in petri dishes with 1.5% agar adjusted to pH 5.8 and incubated at 28 °C in darkness. Second-stage juveniles (J2) fed on the roots and started the second molt (M2) to the third-stage juveniles 2 days after inoculation (DAI). The third molt (M3) to the fourth-stage juveniles occurred 7 DAI, followed by the fourth molt (M4) to males 13 DAI or to females 14 DAI. Nematode gender differences were observed by the end of the fourth molt. The first male appeared 15 DAI and the first female 17 DAI, after which mating occurred. Males were attracted to females, and mating was observed. Mating was required for reproduction. Fertilized females began to lay eggs 19 DAI and continued egg laying without the further presence of males during a 90-day observation. All of the eggs hatched. Unfertilized females rarely laid eggs, and none of the eggs were able to hatch. Feeding took place between each molt and before egg deposition occurred. The first-stage juveniles molted in the eggs 4 days after deposition, and J2 hatched from eggs 5 days after egg deposition. The life cycle from J2 to J2 was completed in 24 days.     

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.     

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.     

Ultrastructure of Phasmid Development in Meloidodera floridensis and M. charis (Heteroderinae)

Phylogenetic characters for Heteroderinae Luc. et al., 1988 are evaluated in Meloidodera which is believed to have primarily ancestral characters. Phasmid ultrastructure is observed in second-stage juveniles (J2), third-stage juvenile males, fourth-stage juvenile males, and fifth-stage males of Meloidodera floridensis and M. charis. Phasmid secretion occurs inside the egg before the J1-J2 molt. Before J2 hatch, concentric lamellar membranes occur within the sheath and socket cells. Some membranes become lamellae of the sheath cell plasma membrane; others become multilamellar bodies. During early molting, plasma membrane lamellae disappear and a distal dendrite segment appears in a rudimentary canal. After the molt, the distal dendrite is not present within the canal. The phylogenetic utility of phasmid features is discussed. In both species the ampulla shape and size between molts are stable features in juveniles and males. The posthatch J2 sheath cell receptor cavity may vary in a species specific manner, but comparative morphology requires precise timing after hatch.