Interaction between Neoaplectana carpocapsae (Nematoda: Steinernematidae) and Apanteles militaris (Hymenoptera: Bracondiae), a parasitoid of the armyworm, Pseudaletia unipuncta

The DD-136 strain of Neoaplectana carpocapsae adversely affected the development of immature stages of Apanteles militaris, a gregarious internal parasitoid of the armyworm, Pseudaletia unipuncta. The adverse effect of the nematode-bacterium complex was indirect, i.e., the infection by the nematode killed the host before A. militaris could complete its development. However, if armyworms containing 10- or 11-day-old A. militaris were exposed to dauer juveniles in Petri dishes, 48.1 and 94.4%, respectively, produced normal cocoons. If hosts containing 9- or 10-day-old A. militaris were fed dauer juveniles, 42.6 and 73.4% of the armyworms, respectively, produced A. militaris which formed normal cocoons. Cocoon-spinning A. militaris larvae were infected by the nematode. After cocoon formation was completed, the dauer juveniles could not penetrate the cocoon and infect the pupa. However, pupae in cocoons which had been deliberately cut open at one end became infected. A. militaris adults were infected when exposed to dauer juveniles in Petri dishes. After 3 days of exposure to dauer juveniles, 25.0, 44.2, and 7.0% of the adults in three trials were alive, whereas 100, 100, and 96.7% of the control adults were alive. Examination of dead adults in the nematode treatment showed that 67.6% contained nematodes. N. carpocapsae developed and reproduced in unparasitized armyworms, in armyworms containing 9-day-old A. militaris, and in those from which A. militaris had emerged. Production of dauer juveniles was significantly higher in unparasitized armyworms and in armyworms containing 9-day-old A. militaris than in those from which A. militaris had emerged.     

Differential susceptibility of lepidopterous pupae to infection by the nematode Neoaplectana carpocapsae

Prepupae of Galleria mellonella, Spodoptera exigua, and Pseudaletia unipuncta were highly susceptible to infection by the nematode Neoaplectana carpocapsae and its associated bacterium, Xenorhabdus nematophilus. On the other hand, pupae of the three species were significantly different in their susceptibility to the nematode. G. mellonella pupae were highly susceptible (100% mortality), S. exigua pupae were moderately susceptible (ca. 75% mortality), and P. unipuncta pupae were least susceptible (ca. 54% mortality). In the latter two species, many pupae died without any nematode development, but the typical signs associated with a nematode infection were evident. Age of the pupae and increasing the dosage of the nematode did not significantly affect mortality of S. exigua or P. unipuncta.     

Susceptibility of Mocis latipes (Lepidoptera: Noctuidae) to Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae)

The susceptibility of larvae, prepupae, and pupae of the grass looper Mocis latipes (Guenée) to the entomopathogenic nematode Heterorhabditis bacteriophora (Poinar) NC strain was evaluated under laboratory conditions. Concentrations of 0, 5, 10, 20, 40, 60, and 120 nematodes per larva, applied in 1 ml of sterile-distilled water, were bioassayed, applying them to groups of 20 individuals of each instar, prepupa or pupa. Mortality was recorded daily for 5 d. All instars and the prepupal stage were the most susceptible to H. bacteriophora. Mortality ranged from 22.5 to 100%. Prepupae had 97.5-100% mortality starting at 10 nematodes per prepupa. Pupal mortality ranged from 27.5 to 41.3% as nematode concentration was increased. H. bacteriophora presented LC50 values that ranged between 5.26 and 37.66 nematodes per larva and LT50 values that ranged between 1.5 and 4.3 d. Results of this study suggest that H. bacteriophora has potential as a biocontrol agent against M. latipes.     

The nematode Neoaplectana carpocapsae and the beet armyworm Spodoptera exigua: Infectivity of prepupae and pupae in soil and of adults during emergence from soil

The nematode, Neoaplectana carpocapsae, infected >90% of the prepupae of Spodoptera exigua in soil even at concentrations as low as five nematodes/cm² of soil surface. Pupae were less susceptible to nematode infection in soil than prepupae, with mortality ranging from 10 to 24% and 10 to 83% for pupae exposed 3–5 days and 6–8 days to the nematode, respectively. Longer exposure (6–8 days) of the pupae to the nematode resulted in higher mortality with a positive relationship with increasing concentrations. Adults of S. exigua were susceptible to nematode infections as they emerged from the soil. The higher nematode concentrations (25 and 50 nematodes/cm²) resulted in higher adult mortality. The majority of nematode-induced mortality occurred within 24 hr after emergence. The susceptibility of emerging S. exigua adults to N. carpocapsae offers a new dimension for insect control.     

Biological control potentials of insect-parasitic nematode Rhabditis blumi (Nematoda: Rhabditida) for major cruciferous vegetable insect pests

Pathogenicity of Rhabditis blumi Sudhaus against major cruciferous insect pests was evaluated in the lab and greenhouse. In Petri-dish tests against the insects, including Artogeia rapae L., Mamestra brassicae L., and Plutella xylostella L., insect mortality by R. blumi and its associated bacteria was dose and time dependent, which increased with dose (0?C80 dauer juveniles/larva) and time increments. Pathogenicity against fourth-instar larvae was higher than the rate of corresponding third-instar larvae. The highest insect mortality rate was observed in fourth-instar larvae of P. xylostella, followed by A. rapae, and M. brassicae, with mortality rates of 93.5, 88.2, and 77.8?%, respectively. Lethal dose values at 50?% (LD₅₀) of R. blumi were 25.7 dauer juveniles/larva on P. xylostella; 28.0 dauer juveniles/larva on A. rapae; and 40.6 dauer juveniles/larva on M. brassicae, respectively. In greenhouse tests, P. xylostella larvae were most susceptible to nematodes, with insect reduction rate of 88.0?%. The rate varied with vegetable species and persistence time of live nematodes on vegetable leaves after spraying. Nematodes established in cadavers showed positive correlation with nematode dose, whereas nematode persistence on the leaf was inversely related to hours after treatment.     

Effect of the Entomogenous Nematode Nemplectana carpocapsae on the Tachinid Parasite Compsilura concinnata (Diptera: Tachinidae)

The entomogenous nematode Neoaplectana carpocapsae and its associated bacterium, Xenorhabdus nematophilus, could not infect the pupal stage of the tachinid Compsilura concinnata through the puparium. N. carpocapsae had an adverse effect on 1-, 2- and 3-day-old C. concinnata larvae within the armyworm host in petri dish tests. All 1-day-old larvae treated with nematodes died in their hosts, whereas 61% and 69% of 2- and 3-day-old larvae treated with nematodes, respectively, died. However, the survivors developed to adults. Nine to thirty-seven percent of adult tachinids which emerged from nematode-treated soil (50 nematodes/cm²) were infected with N. carpocapsae. The nematode adversely affects C. concinnata directly by the frank infection of the tachinid and indirectly by causing the premature death of the host which results in tachinid death.     

Stage-specific mortality of Rhagoletis indifferens (Diptera: Tephritidae) exposed to three species of Steinernema nematodes

Mortality of larval, pupal, and adult western cherry fruit fly, Rhagoletis indifferens (Tephritidae) exposed to the steinernematid nematodes Steinernema carpocapsae, Steinernema feltiae, and Steinernema intermedium, was determined in the laboratory and field. Larvae were the most susceptible stage, with mortality in the three nematode treatments ranging from 62 to 100%. S. carpocapsae and S. feltiae were equally effective against larvae at both 50 and 100 infective juveniles (IJs)/cm². S. intermedium was slightly less effective against larvae than the other two species. Mortalities of R. indifferens larvae at 0, 2, 4, and 6 days following their introduction into soil previously treated with S. carpocapsae and S. feltiae at 50 IJs/cm² were 78.6, 92.5, 95.0, and 77.5% and 87.5, 52.5, 92.5, and 70.0%, respectively, and at 100 IJs/cm² were 90.0, 92.0, 100.0, and 84.0% and 90.0, 50.0, 42.0, and 40.0%, respectively. There was no decline in mortality caused by S. carpocapsae as time progressed, whereas there was in one test with S. feltiae. Larval mortalities caused by the two species were the same in a 1:1:1 vermiculite:peat moss:sand soil mix and a more compact silt loam soil. In the field, S. carpocapsae and S. feltiae were equally effective against larvae. Pupae were not infected, but adult flies were infected by all three nematode species in the laboratory. S. carpocapsae was the most effective species at a concentration of 100 IJs/cm² and infected 11–53% of adults that emerged. The high pathogenicity of S. carpocapsae and S. feltiae against R. indifferens larvae and their persistence in soil as well as efficacy in different soil types indicate both nematodes hold promise as effective biological control agents of flies in isolated and abandoned lots or in yards of homeowners.     

Insecticidal activities of Ginkgo biloba seed coat extracts against Spodoptera exigua (Lepidoptera: Noctuidae)

The present study relates to a methanol extract of the seed coat of Ginkgo biloba, and tested particularly on the third instar larvae of Spodoptera exigua. The extract was found to have an inhibitory effect on the growth of the larvae besides bringing a change in the nutrient reserves in the body of the insect. Topical application of five different doses of the methanol extract resulted in a mortal effect to third instar larvae of S. exigua that is very much dependent on the dose as well as duration of exposure. Lower doses revealed lower mortality after 24 h of application. At doses of 1.00, 2.00, 4.00, 8.00 and 16.00 ng/larva, mortalities were 9.25, 26.07, 50.32, 56.28 and 92.44%, respectively. The dose for 50% mortality (LD₅₀) of methanol extracts by applied by a topical method with 1 µL of acetone solution was 1.92 ng/larva. Nutrient reserves like protein, glycogen and lipid are known to regulate pupation and adult emergence. These reserves have been found to be lower in treated larvae, indicating the insecticidal role of methanol extracts from G. biloba against third instar larvae of S. exigua.     

Evaluation of twenty-eight strains of Heterorhabditis bacteriophora isolated in Azores for biocontrol of the armyworm, Pseudaletia unipuncta (Lepidoptera: Noctuidae)

The armyworm, Pseudaletia unipuncta, is the most important pest in Azorean pastures. Although this pest has some parasitoids and pathogens, additional biological control agents are needed to manage it. Entomopathogenic nematodes, particularly Heterorhaditis bacteriophora, are good candidates because they have been isolated from pastures and crops in almost all islands of the Azorean Archipelago. We tested 28 Azorean isolates of H. bacteriophora in the laboratory against the 6th instar P. unipuncta to determine mortality rates and virulence of each isolate. Plot tests in the field were also conducted to evaluate the best time for application of the selected isolate. All isolates killed the larvae although important differences in the mortality rates were observed. Forty-eight h post exposure (HPE) to the nematode infective juvenile (IJ), insect mortality ranged from 0 to 92.5% and at 96 HPE, mortality ranged from 32.5 to 100%. Based on LC₅₀ and LT₅₀, H. bacteriophora Az29 was the most pathogenic and the remaining 27 isolates were grouped in three classes of virulence. The most virulent class included four isolates with LC₅₀ ranging from 180 to 327 IJs/insect and LT₅₀ from 44 to 62.9 h. These isolates were obtained from three of the nine islands. High intrapopulational variability was detected on isolates in the moderately virulent class suggesting that these isolates are good candidates for genetic improvement. Field tests showed H. bacteriophora Az29 was more effective to control P. unipuncta larvae than Steinernema carpocapsae Az20 and H. bacteriophora Az32, belonging to the less virulent class. These tests also showed that applications performed during May resulted in better control than in July.     

Instar Susceptibility of Simulium vittatum (Diptera: Simuliidae) to the Entomogenous Nematode Neoaplectana carpocapsae

Laboratory bioassays showed that the susceptibility of Simulium vittatum to Neoaplectana carpocapsae increased with successive larval instars. First, second, and third instar larvae were resistant to infection, while seventh instars were highly susceptible. Significant differences in intra-instar susceptibility were also evident, as mortality ranged from 58% for the smallest seventh instar larvae to 97% for the largest. Dissections revealed that the basis for the resistance of early instars was physical exclusion of the comparatively large nematodes. The principle factor regulating the susceptibility of mid and late instars was injury to nematodes caused by larval mouthparts during ingestion. Differences in intra-instar susceptibility were similarly related to nematode injury.     

Parasitism of the mosquito Culex pipiens by the nematode Heterorhabditis bacteriophora

Various concentrations of the nematode Heterorhabditis bacteriophora were added to dishes containing second, third, and fourth larval instars of the mosquito, Culex pipiens, respectively. The infective stage nematodes were ingested by the mosquito larvae, they then penetrated through the alimentary tract in the neck region and entered the hemocoel. A melanization reaction killed many invading nematodes, but heavier concentrations overwhelmed the hosts' defense reaction and 100% mortality of third- and fourth-instar larvae was achieved using between 170 and 200 nematodes per host. Death was either due to the nematode releasing cells of the symbiotic bacterium, Xenorhabdus luminescens, into the hemocoel or to foreign bacteria (mostly Pseudomonas aeruginosa), which were introduced by the penetrating nematodes. The potential use of this nematode as a biological control agent of larval culicine mosquito is discussed.     

Biological Control of the Leafminer Liriomyza trifolii (Burgess): Implications for Intraguild Predation between Diglyphus begini Ashmead and Steinernema carpocapsae (Weiser)

Studies were conducted on the combined use of the eulophid parasitoid wasp Diglyphus begini Ashmead and the entomopathogenic nematode Steinernema carpocapsae (Weiser) for control of the leafminer Liriomyza trifolii (Burgess) on chrysanthemums. Several factors indicated that these two agents were suitable for combined use: adult D. begini were not susceptible to nematode infection, leafminer larvae parasitized by the wasp were less susceptible to nematode infection, adult wasps detected and tended to avoid ovipositing on nematode-infected leafminer larvae, nematode-infected larvae served as host-feeding sources for the adult wasps, and nematodes showed equal orientation toward paralyzed/parasitized leafminer larvae and healthy leafminer larvae. However, interspecific interference and intraguild predation (IGP) between the agents were found. Infection of D. begini larval stages by nematodes was seen in petri dishes and in intact leaf mines. The presence of nematodes in mines with wasp eggs decreased the chance of wasp survival to adulthood. IGP may be minimized through proper timing of natural enemy releases.     

Biology of Octomyomermis muspratti,a parasite of mosquitoes as it relates to mass production

The first- and second-instar larvae of Culex pipiens quinquefasciatus were equally susceptible to parasitism by the mermithid nematode Octomyomermis muspratti; the third-instar hosts were significantly less susceptible. The yield of postparasitic nematodes was higher from hosts exposed as second instars then from hosts exposed as first or third instars at comparable levels of parasitism. Mortality of the host prior to emergence of the nematode occurred most frequently among first- and third-instar hosts. About 4% of the infected hosts exposed as first and second instars and 37% of the infected hosts exposed as third instars pupated prior to emergence of the postparasitic nematodes. The exposure of mosquitoes as second instars was the most efficient for mass rearing and resulted in the highest yields of postparasitic O. muspratti.     

Efficacy of entomopathogenic nematodes against neonate larvae of <Emphasis Type="Italic">Capnodis tenebrionis</Emphasis> (L.) (Coleoptera: Buprestidae) in laboratory trials

The efficacy of five entomopathogenic nematode strains of the families Steinernematidae and Heterorhabditidae was tested against the neonate larvae of Capnodis tenebrionis. The nematode strains screened included two of Steinernema carpocapsae (Exhibit and M137), and one each of S. feltiae (S6), S. arenarium (S2), and Heterorhanditis bacteriophora (P4). Exposure of neonate larvae of Capnodis to 10 and 150 infective juveniles (IJs) per larva (equivalent to 3 and 48 IJs/cm² respectively) in test tubes with sterile sand, resulted in mortality between 60–91% and 96–100%, respectively. At a concentration of 150 IJs/larva, all of the nematode strains were highly virulent. Both S. carpocapsae strains (Exhibit and M137) caused infection and mortality to larvae more quickly than the other strains. However, at a lower concentration assay (10 IJs/larva), S. arenarium was the most virulent strain. The penetration rate as an indicator of entomopathogenic nematode infection was also evaluated. The highest value was recorded for S. arenarium (36%), followed by H. bacteriophora (30.6%), S. feltiae (23.1%), and S. carpocapsae (20.7%).     

Entomopathogenic Nematodes for Control of Codling Moth,Cydia pomonella(Lepidoptera: Tortricidae): Effect of Nematode Species, Concentration, Temperature, and Humidity

The susceptibility of codling moth diapausing larvae to three entomopathogenic nematode species was assessed in the laboratory using a bioassay system that employed cocooned larvae within cardboard strips. The LC₅₀values forSteinernema carpocapsae, S. riobrave,andHeterorhabditis bacteriophorawere 4.7, 4.8, and 6.0 infective juveniles/cm², respectively. When a discriminating concentration of 10 infective juveniles/cm²of each of the three nematode species was evaluated at 15, 20, 25, and 30°C,S. carpocapsaewas the most effective nematode with mortalities ranging from 66 to 90%. Mortalities produced byS. riobraveandH. bacteriophoraat the four temperatures were 2–94 and 25–69%, respectively. Studies were also conducted to test infectivity at 10, 35, and 40°C. No mortality was produced by any of the nematode species at 10°C.S. riobravewas the most infective nematode at 35°C producing 68% mortality which was more than twice that observed forS. carpocapsaeorH. bacteriophora.Codling moth larvae treated with 10 infective juveniles/cm²ofS. carpocapsaeand kept in 95+% RH at 25°C for 0–24 h followed by incubation at 25–35% RH indicated that more than 3 h in high humidity was needed to attain 50% mortality. Trials ofS. carpocapsae, S. riobrave,andH. bacteriophoraat 50 infective juveniles/cm²against cocooned larvae on pear and apple logs resulted in reductions of codling moth adult emergence of 83, 31, and 43%, respectively, relative to control emergence. Trials of the three entomopathogenic nematodes at 50 infective juveniles/cm²against cocooned larvae in leaf litter resulted in 99 (S. carpocapsae), 80 (S. riobrave), and 83% (H. bacteriophora) mortality, respectively. Our results indicate good potential of entomopathogenic nematodes, especiallyS. carpocapsae,for codling moth control under a variety of environmental conditions.     

Nippostrongylus brasiliensis and Haemonchus contortus: Function of the excretory ampulla of the third-stage larva

An improved technique for measuring the water content of nematodes is described using an electronic interferometer. Changes in phase of a laser beam passing through a known pathlength of the nematode have been used to measure the refractive index and hence the water content and relative volume of the animal. Third-stage larvae of Nippostrongylus brasiliensis and Haemonchus contortus which possess an excretory ampulla, differed from second-stage larvae lacking this ampulla in requiring a greater fall in the osmolarity of artificial tap water before there was a significant increase in their water content. Increases in the pulsation frequency of the ampulla also occurred in less hypotonic solutions than those required to increase the water content of the third-stage larvae. The ampulla pulsation frequency of third-stage larvae of N. brasiliensis increased after locomotor activity in hypotonic tap water and locomotory wave frequency of third-stage larvae of N. brasiliensis was independent of the extent of hypotonicity for a range of solutions that reduced wave propogation by its second-stage larva. The results suggest that the ampulla is an adaptation to hypotonic conditions favouring a volume homeostasis that is required for optimal locomotor activity of the third-stage infective larvae of these nematodes.     

Effects of Culture Method and Formulation on the Virulence of Steinernema riobrave (Rhabditida: Steinernematidae) to Diaprepes abbreviatus (Coleoptera: Curculionidae)

The Diaprepes root weevil, Diaprepes abbreviatus, is a pest of vegetables, ornamental plants, sugarcane, and citrus in Florida and the Caribbean. The entomopathogenic nematode, Steinernema riobrave, can reduce larval populations of D. abbreviatus substantially. Efficacy of entomopathogenic nematodes, however, may be affected by culture method and formulation. Using D. abbreviatus as the host, we compared the efficacy of two commercial S. riobrave formulations, a liquid and a waterdispersible granule (WDG), with each other and with in vivo produced S. riobrave. Nematodes in the commercial formulations were produced in vitro through liquid fermentation; the in vivo nematodes were cultured in Galleria mellonella and applied in aqueous suspension. Laboratory experiments measured nematode virulence in plastic cups containing soil and seventh-eighth instar D. abbreviatus. One laboratory experiment was conducted using only fresh nematodes (less than 5 days old); another experiment included WDG nematodes that were stored for 25 days at 10 °C. Two field experiments were conducted in which nematodes were applied either to potted citrus (containing D. abbreviatus larvae) placed beneath mature citrus trees or to soil directly beneath the tree. In the latter experiment, efficacy was determined by measuring mortality of caged D. abbreviatus larvae that were buried beneath the soil surface prior to application. Mortality of D. abbreviatus treated with nematodes ranged from 80-98% and 50-75% in laboratory and field experiments, respectively. In all experiments, we did not detect any significant effects of culture method or formulation.     

Elaphostrongylus rangiferi: Influence of temperature,substrate, and larval age on the infection rate in the intermediate snail host, Aarianta arbustorum

The infection rate of the first stage larval nematodes, Elaphostrongylus rangiferi, was studied experimentally, using the juvenile snail Arianta arbustorum as intermediate host. The nematode showed a linear, fivefold increase in infection rate within the temperature range of 4 to 28 C. The snails were exposed to the larval nematodes on three different substrates. The highest infection rate was recorded when snails were exposed in tap water and significantly slower infection rates were obtained when either lettuce or soil was used as the substrate. First stage larvae of E. rangiferi were infective for at least 2 months when stored at 12 C. Throughout this period, the infection rate showed a significant decline, while the motility of the larvae remained unchanged.     

Granulosis virus in the intestinal lumen of Neoaplectana carpocapsae: Retention of infectivity after treatment with formaldehyde or high pH

High pH values (>11.0) cause the dissolution of occlusion bodies of the granulosis virus (GV) of Pseudaletia unipuncta and subsequent inactivation of the virus within 24 hr. The GV is also inactivated within 48 hr by 0.04% formaldehyde. The GV is found in the intestinal lumen of infective third stage nematodes (dauer juveniles) of Neoaplectana carpocapsae when development occurs in GV-infected hosts. The GV in these dauer juveniles retains its infectivity even when the nematodes are placed into an alkaline solution with pH values of 11.1 or 12.1 or in 0.04% formaldehyde up to 336 hr. However, significant loss of infectivity of GV occurs when the nematodes are in formaldehyde but not at high pH values. The dauer juveniles are ensheathed by the second stage cuticle. This cuticle probably protects the GV in the intestinal lumen of the nematode from the high pH and formaldehyde.     

Effect of humidity and a superabsorbent polymer formulation on the efficacy of Heterorhabditis zealandica (Rhabditida: Heterorhabditidae) to control codling moth,Cydia pomonella (L.) (Lepidoptera: Tortricidae)

Adequate moisture levels are required for nematode survival and subsequent efficacy as entomopathogens. Formulation of nematodes aimed at aboveground applications may assist in maintaining such moisture levels. In this study, we report the effects of a superabsorbent polymer formulation, Zeba® on the performance of an entomopathogenic nematode, Heterorhabditis zealandica Poinar, for controlling diapausing codling moth, Cydia pomonella (L.) larvae in cryptic habitats on trees. Water activity (a_w-value) on bark was considered to be an indication of moisture levels on trees in cryptic habitats where codling moth larvae are known to occur, thereby influencing nematode efficacy. H. zealandica was only able to infect codling moth larvae at a_w≥0.92, with a_w50=0.94 and a_w90=0.96. Laboratory experiments in which nematode concentration was investigated indicated a positive linear relationship between the concentration of nematodes applied and the level of control obtained, with the highest level of mortality recorded at 80 IJs/larva, requiring at least 4 h of conditions conducive to nematode activity to ensure infectivity and subsequent efficacy. Further experimentation showed that the use of the Zeba formulation, together with the nematodes, improved the level of control obtained at 60% and 80% RH in the laboratory and that it also enhanced the survival and infection-ability of the nematodes in the field. The study conclusively illustrates that the tested formulation assisted in maintaining adequate moisture levels on the application substratum, as required for nematode survival and subsequent efficacy.